Skip to main content

Full text of "Report of program activities: National Institute of Allergy and Infectious Diseases"

See other formats


ANNUAL  REPORT 

OF 

PROGRAM  ACTIVITIES 


NATIONAL  INSTITUTES  OF  HEALTH 
1  9  6  0 

NATIONAL  INSOTTE  OF  ALLERGY 

AND  INFECTIOUS  DISEASES 


A'MawAL  •Msravn-s  Of  health 

PUBLIC  HEALTH  SERVICE 
U.  S.  DEPARTMENT  0F  mXm,  EDUCATION,  AND 


•>(■*■*■ 


ANNUAL  REPORT  OF  PROGRAM  ACTIVITIES 
VX.^  National  Institute  of  Allergy  and  Infectious  Diseases 
January  -  December,  1960 


l^0 


INDEX 
ANNUAL  REPORT 
NATIONAL  INSTITUTE  OF  ALLERGY  AND  INFECTIOUS  DISEASES 

Director,  NIAID  l 

Extramural  Programs  

Intramural  Programs  

Laboratory  of  Clinical  Investigation 

Summary  

10  (c)  -  Office  of  the  Chief  5 

12  (c)  -  Respiratory  Viral  Diseases  6 

13  (c)  -  Antimicrobial  Drug  Therapy  1° 

14  (c)  -  Staphylococcal  Disease  13 

15  (c)  -  Hepatitis  and  Mononucleosis  16 

16  (c)  -  Aseptic  Meningitis  17 

17  (c)  -  Allergy-Immunology  19 

18  (c)  -  Enteric  Diseases  28 

19  (c)  -  Clinical  Investigations  on  Helminthic  Diseases  31 

20  (c)  -  Clinical  Investigations  on  Protozoan  Infections 


and  Diseases 


37 


21  (c)  -  Cystic  Fibrosis  of  the  Pancreas  44 

22  (c)  -  Basic  Biochemical  Studies  ^5 

23  (c)  -  Systemic  Fungal  Disease  50 

24  (c)  -  Sarcoidosis 54 

25  (c)  -  Pyelonephritis  55 

26  (c)  -  Urinary  Tract  Viruses  56 

27  (c)  -  Biochemical  Studies  on  Staphylococcal  Cell  Walls  59 

28  (c)  -  Pathogenesis  of  Viral  Infections  61 


Laboratory  of  Cell  Biology 

Summary  

50  -  Biosynthesis  in  Mammalian  Cell  Cultures  3 

51  -  The  Mechanism  of  Production  of  Poliovirus  in  Cultured 

Mammalian  Cells  8 

52  -  Effects  of  Animal  Viruses  on  the  Metabolism  of 

Mammalian  Cell  Cultures  and  the  Mechanism  of  Viral 
Replication  11 

53  -  Mechanism  of  Drug  Resistance  in  Cultured  Mammalian  Cells   14 


54  -  Metabolic  Control  Mechanisms  in  Cultured  Mammalian  Cells  17 

55  -  Application  of  the  Technique  of  Tissue  Cultures  to 

Selected  Genetic  Diseases  19 


Laboratory  of  Infectious  Diseases 

Summary  1 

60  -  Studies  on  Cellular  and  Cell-Free  Staphylococcal 

Penicillinase  44 

61  -  Antibiotic  Activity  of  Sea  Water  47 

62  -  The  Electron  Transport  System  in  an  Autotrophic 

Hydrogenomad  50 

62-A   -  Microbiological  Fractionation  of  the  Hydrogen  Isotopes  .  55 

63  -  Non-specific  Immunity  Associated  with  Serum  Sidero- 

philin  and  the  Iron  Metabolism  of  Both  Pathogen  and 

Host  57 

63-A   -  Chemical  Aspects  of  the  Specific  Binding  of  Iron  to 

Serum  Siderophilin  and  Egg  White  Conalbumin  62 

63-B   -  Biochemistry  of  the  Acquisition  of  Iron  by  Mammalian 
Tissues  as  Mediated  by  the  Iron-Binding  Protein, 
Siderophilin  •  •  •  66 

63-C   -  Development  of  an  Economical  280mu  Light  Source  Suit- 
able for  Detecting  Proteins  in  Effluents  from 
Chromatographic  Columns  69 

64  -  Detoxification  of  Potential  Tuberculostatic,  Fungi- 

static, Parasiticidal  and  Virucidal  Agent  71 

64-A   -  Potential  Fungistatic  and  Parasiticidal  Agents  75 

64-B   -  Isolation  of  Antibacterial  and  Antiviral  Substances 

from  Shellfish  77 

65  -  Longitudinal  Studies  of  Beta-hemolytic  Streptococcal 

Isolations  80 

65-A   -  Streptococcal  M  Protein,  Virulence,  and  Type- 
Specific  Immunity  83 

66  -  Epidemiologic  Studies  of  Illnesses  and  Microbial 

Experience  of  Junior  Village  Nursery  Children  89 

66-A   -  Epidemiologic  Studies  of  Host  Parasite  Disease 

Relationships  93 

66-B   -  Epidemiologic  Studies  of  New  Vaccines  and  Chemo- 

prophylactic  Agents  95 

66-C   -  The  Etiology  of  Respiratory  Disease  on  a  College 

Campus  97 

66-D   -  The  Role  of  Viruses  in  the  Etiology  of  Ear  Disease  99 

66-E   -  A  Study  of  the  Influence  of  Influenza  A  and  B  Mineral 

Oil  Adjuvant  Vaccines  on  Influenza  Antibody  Patterns, 

8-9  Years  after  Vaccination  100 

67-    -  Studies  on  Human  Enteroviruses,  Adenoviruses,  and 

Reoviruses  103 

67-A   -  Studies  on  Viruses  of  the  Enteric  Tract  of  Cattle  107 

67-B   -  Studies  on  the  Etiology  of  Eosinophilic  Meningitis 

in  French  Polynes ia  110 

II 


68  -  Studies  of  Tumor  Viruses  in  Nature  114 

68-A  -  Seroepidemiology  of  Virus  Infections  123 

69  -  Study  of  Viruses  as  Causes  of  Respiratory  Illness 

in  Infancy  and  Early  Childhood  126 

69 -a  -  Viral  Pneumonia:  Etiology,  Therapy,  and  Prevention  130 
69-B  -  Study  of  the  Laboratory  Aspects  of  Respiratory 

Virus  Illness  in  a  Welfare  Orphanage  134 

70  -  Laboratory  Studies  of  Newly  Recognized  Viruses 

Associated  with  Respiratory  Illness 138 

71  -  Studies  of  Tumor-Producing  Viruses  142 

71-A  -  Continuing  Studies  of  Adenoviruses  and  Salivary 

Gland  Viruses  as  Examples  of  Latent  Viruses  147 

71-B  -  Studies  of  Mouse  Viruses  with  Special  Reference 
to  their  Importance  as  Extraneous  Viruses  (back- 
ground noise)  in  Mouse  Tumor  Systems  150 

72  -  Laboratory  Studies  of  Enteroviruses  154 

72-A  -  Study  of  Viral  Respiratory  Disease  in  a  Military 

Population  157 

72-B  -  Study  of  Respiratory  Viruses  in  Human  Volunteers  ...    161 

73  -  Application  of  Fluorescent  Antibody  Staining 

Technique  to  the  Study  of  Respiratory  Viruses  163 

74  -  Study  of  Bovine  Respiratory  Diseases  165 

74-A  -  A  Study  of  Squamous  Cell  Carcinoma  (Cancer  Eye) 

in  Catt  le  170 

75  -  Laboratory  and  Epidemiologic  Studies  of  Viruses 

as  Possible  Etiologic  Agents  of  Acute  Leukemia 

in  the  Pediatric  Age  Group  172 

76-A  -  Ecologic  Studies  of  Fungi  Pathogenic  for  Man  176 

76-B  -  In  vivo  Tests  of  Antimycotic  Drugs  and  Anti- 
biotics     180 

76-C  -  Identification  and  Study  of  New  and  Unusual  Fungi 

from  Mycoses •    183 

78-A  -  Biochemistry  and  Physiology  of  Pathogenic  Fungi 
(In  vitro  Studies  of  the  Action  of  Antifungal 

Agents  on  Pathogenic  Fungi)  186 

78-B  -  Studies  on  the  Physiology  of  Coccidioldes 

immitis  188 

79-A  -  Immunity  Studies  with  Pathogenic  Fungi  191 

79-B  -  Antigenic  Studies  on  Pathogenic  Yeasts  193 

79-C  -  Virulence  and  Pathogenic  Studies  with  Yeasts  196 


III 


Laboratory  of  Biology  of  Viruses 

Summary  ■ • 1 

80  -  Basic  Studies  of  Virus-Host  Cell  Relationships  3 

80-A   -  Rabies  Prophylaxis  6 

81  -  Mechanism  of  Oncogenic  Activity  Polyoma  Viruses  8 

82  -  Cell  Growth  Inhibiting  Substances  11 

83  -  Mutation  in  Animal  Viruses  13 

84  -  Host-Parasite  Relations  15 

85  -  Investigations  of  Animal  Virus  Reproduction  19 

85-A   -  Animal  Virus  Synthesis  22 

86  -  Investigations  of  Interference  Enzymatic  Functioning  of 

Mitochondria  as  a  Mechanism  for  Carcinogenesis  24 

86-A   -  Demonstration  of  Glucose  Metabolism  and  Peptide  Bond 

Formation  by  Isolated  Brain  and  Liver  Mitochondria  ....  27 

87  -  Cytopathogenic  Effect  in  Single  Cells  in  Tissue  Culture  29 
87-A   -  Characterization  of  Mitochondria  31 

88  -  Biology  of  Mitochondria  and  Its  Relation  to  Endogenous  and 

Viral  Diseases  34 

88-A   -  Comparison  of  the  Properties  of  Crude  and  Crystallized 
Coxsackie  A-10  Virus,  of  Cloudman  S  91,  Mouse  Melanoma 

and  of  Mouse  Muscle  Nucleoprotein  36 

89  -  Kinetics  and  Sites  of  Coxsackie  Virus  Multiplication  in 

the  Monkey  Kidney  Cell  38 

89-A   -  Virus  Structure  40 


Laboratory  of  Tropical  Virology 
Middle  America  Research  Unit 
Arthropod-Borne  Virus  Section 

Summary    1 

93     -  Studies  on  Arthropod-Borne  Viruses  in  the  Sub-tropical 

Areas  of  the  United  States  6 

94-A   -  Studies  of  Arthropod-Borne  Viruses  in  Tissue  Culture... 
Part  1.   Evaluation  of  Tissue  Culture  Systems  for  Use 
in  Viral  Isolation,  Identification,  and  in  Serological 
Tests  for  Viral  Antibodies  8 

94-B   -  Studies  of  Arthropod-Borne  Viruses  in  Tissue  Culture. 

Part  2.   Development  of  a  Cell  Culture  System  Utilizing 
Arthropod  Tissue  11 

95-A   -  Studies  on    Antigen- Antibody  Reactions  of  Arthropod- 
Borne  Viruses.   Part  1.   Kinetic  Studies  of  the  Serum 
Neutralization  of  the  Arthropod-Borne  Virus  13 

95-B   -  Studies  on  Antigen- Antibody  Reactions  of  Arthropod- 
Borne  Viruses.   Part  2.   Development  of  a  Practical  and 
Specific  Flocculation  Test  for  the  Demonstration  of 
Arthropod-Borne  Virus  Antibodies  15 


IV 


95-C   -  Typing  of  Viruses  by  Combinations  of  Antiserum  Pools. 

Application  to  Typing  of  Arthropod-Borne  Viruses  ....   17 

96-C   -  Survival  Potential  of  the  Adult  of  Haemagogus  Equinus, 

A  Sylvan  Vector  of  Yellow  Fever  19 

97-A   -  A  Qualitative  Evaluation  of  Experimentally-Induced 
Eastern  Equine  Encephalomyelitis  (EEE)  Virus  Infec- 
tion in  Horses  22 

97-B   -  The  Development  of  an  Inactivated  Vaccine  Against 

Eastern  Equine  Encephalomyelitis  (EEE)  Virus  24 

100-A  -  Virological  Investigation  of  Clinical  Cases  and 

Epidemic  Outbreaks  in  Panama  and  Other  Countries  of 
Middle  America  27 

100-B   -  A  Clinical  and  Virological  Study  of  Oropharyngeal 

Lesions  in  Panamanian  Children  33 

101-A  -  Virological  Aspects  of  a  Cooperative  Investigation 

on  the  Ecology  of  Arthropod-Borne  Viruses  35 

101-B   -  The  Role  of  Chiggers  and  Other  Acarina  of  the  American 
Tropics  in  the  Maintenance  and  Transmission  of  Animal 
Infectious  Agents:   1.  Viral  Aspects  39 

101-C  -  "Jungle  Fever"  in  U.S.  Military  Personnel  41 

102-A  -  Enterovirus  Infections  of  Rural  Guatemalan  Children 

in  Relation  to  Nutrition  43 

102-B  -  Laboratory  Support  of  Phase  1,  National  Program  for 

Poliovirus  Vaccine  Administration  in  Costa  Rica,  1959   45 

102- C  -  Enterovirus  Flora  of  Panamanian  Children:   A  Twelve 

Month  Survey  48 

103-A  -  Use  of  Filter  Paper  Discs  for  Virus  Isolation  and 

Serological  Testing  50 

103-B  -  Eastern  Equine  Encephalomyelitis  (EEE)  Virus  Infec- 
tion in  Panama  53 

103-C  -  Encephalomyocarditis  (EMC)  Virus  Infection.   Studies 
on  Pathogenesis  in  Swine,  Virus  Reservoirs  in  Rodents 
and  Antibody  Status  of  Human  and  Animal  Populations..    55 

108  -  Studies  of  Histoplasmosis  on  the  Isthmus  of  Panama  ..    59 

109  -  Studies  of  Superficial  and  Deep  Mycoses  in  Panama 

and  Central  America  63 


Laboratory  of  Germfree  Animal  Research 

Summary  1 

110  -  Pathogenesis  of  Amoebiasis  5 

111  -  The  Use  of  Germfree  Animal  and  Tissue  for  the  Study 

of  Viruses  8 

112  -  Biology  of  Germfree  Animals  10 

113  -  Origin  of  Anti-Human  Blood  Group  B  Agglutinins  in 

White  Leghorn  Chicks  14 

114  -  Studies  on  Bacterial  Interactions  and  Host  Bacterial 

Relations  in  the  Germfree  Animal  16 

115  -  Behavior  of  Parasitic  Protozoa  in  Germfree  Hosts  ....  18 

116  -  Studies  on  Helminthic  Infections  in  Germfree  Hosts  ..  20 

V 


Laboratory  of  Parasitic  Diseases 

Summary  * 

120  -  Administration  and  Research  Planning  and  Coordination  .  6 
121-A  -  Host  Parasite  Relations  in  Worm  Infections  in  Labor- 
atory Animals  7 

121-B  -  Role  of  Helminths  in  the  Causation  of  Cancer 9 

121-C  -  Destruction  of  Molluscs  by  Chemical  Means  11 

121-D  -  Part  A.   Investigation  of  Intermediate  Hosts  and 

Vectors  of  Human  Diseases  Caused  by  Worms 14 

121-E  -  Development  of  Methods  for  the  Cultivation  of 

Parasitic  Helminths  in  vitro,  and  the  Determination 

of  the  Nutritional  Requirements  of  Such  Organisms 

in  vitro  17 

121-G  -  The  Development  of  Helminths  in  Germfree  Animals  21 

121-1  -  Pathological  Physiology  of  Worm  Infections  23 

121-J  -  Serum  Protein  Studies  on  Germfree  Animals  Infected 

with  Various  Parasites  25 

121-K  -  Effects  of  Nutrition  on  Chemotherapy  of  Parasitic 

Diseases  27 

121-L  -  Effects  of  Improving  the  Nutrition  of  Malnourished 

People  Infected  with  Schistosoma  Mansoni  30 

121-M  -  Ammonia  Metabolism  and  Toxicity  in  Relation  to  Liver 

Disease  33 

121-N  -  Parasitological  Investigations  at  the  Marine  Biological 

Laboratory,  Woods  Hole,  Massachusetts  36 

123-A  -  Fundamental  Physiological  and  Biochemical  Studies  on 

Parasites,  Intermediate  Hosts,  and  Parasitized  Animals  38 
123-B  -  Pathological  Physiology  and  Histochemistry  of  Parasitic 

Diseases  42 

123-C  -  Biochemical  Mechanisms  of  Energy  Metabolism  in  Normal 

and  Parasitized  Animals  45 

123-D  -  Biology  of  Trypanosomes  49 

127-A  -  Studies  on  Toxoplasmosis  52 

127-B  -  Studies  on  Entamoeba  Histolytica  and  Other  Parasitic 

Protozoa  57 

127-C  -  Trichomoniasis  60 

127-D  -  Biochemistry  of  Parasitic  Protozoa  63 


Laboratory  of  Parasite  Chemotherapy 

Summary  1 

130  -  Administration,  Research  Planning  and  Coordination  ....    6 
130-A  -  Studies  in  Human  Malaria  10 

131  -  Drug  Resistance  in  Experimental  Malaria  15 

131-A  -  The  Effect  of  Adrenal  Cortical  Hormone  on  Chemotherapy  of 

Experimental  Malaria  17 

VI 


131-B  -  Antimalarial  Drugs  and  Nucleic  Acid  Synthesis  19 

131-C  -  Experimental  Chemotherapy  of  Helminthic  Diseases  ....  22 
131-D  -  Effects  of  Nutrition  onChemotherapy  of  Parasitic 

Diseases  25 

131-E   -  Comparison  of  Antimalarial  Drugs  Administered  Singly 

and  in  Various  Combinations  28 

131-F  -  Effect  of  Nutritional  Status  of  Host  on  the  Action 

of  Antimalarial  Drugs  30 

132  -  Studies  on  Human  Malaria  32 

132-A  -  Chemotherapy  of  Intestinal  Parasites  37 

132-B  -  Physiology  of  Human  Parasites  Especially  as  Related 

to  Nucleic  Acids  and  Drug  Action  40 

132-C   -  Virus-Parasite  Association  43 

132-D  -  Development  of  Human  Pathogens  in  Immature  Insects  ..  47 
132-E  -  Insect  Tissue  Culture  49 

133  -  Studies  of  the  Biology  of  Exoerythrocytic  Phases  of 

Primate  Malaria  51 

133-A  -  Chemotherapeutic  Studies  of  the  Direct  Effect  of  Drugs 

on  the  Exoerythrocytic  Stages  of  Primate  Malaria  ....  54 
133-B   -  Studies  of  Simian  Malarias  in  Man  to  Determine  If 

Malaria  Is  a  Zoonotic  Disease  in  Areas  in  Which  Man 

and  Monkey  Are  Closely  Associated  57 

133-C  -  In  Vitro  Studies  of  Malaria  Parasites  and  Liver 

Tissue  61 

133-D  -  Immunological  Studies  of  Malaria  Parasites  66 


Laboratory  of  Immunology 

Summary  1 

140  -  Studies  in  the  Transplantation  of  Tissues  4 

141  -  Studies  on  Antibody  Production  and  Mechanism  of  Hyper- 

sensitivity   

142  -  The  Immunology  of  C  h  Mouse  High  Line  Sarcoma  9 

143  -  Characterization  of  Substances  of  Bacterial  Origin 

Affecting  Resistance  to  Infection  11 

144  -  Pathogenesis  of  Allergic  Encephalomyelitis  14 

145  -  Studies  of  Auto-Antibodies  in  Human  Disease  States  and 

in  Experimental  Animals  19 

146  -  The  Resistance  of  Fetal  and  Neonatal  Gonads  to  Damage 

by  Maternal  Immunization  to  Testicular  Antigens  23 

147  -  Immunochemical  and  Immunogenetic  Studies  of  the 

Protein  Isoantigens  in  Serum  25 

147-A  -  Immunochemical  Studies  on  Human  Serum  Antigens  and 

Antibodies  32 

148  -  Basic  Studies  on  the  Cellular  Localization  of  Anti- 

gens, Antibodies,  and  Other  Substances  by  the 
Fluorescent  Antibody  Technique  and  Fluorescence 

Micros  copy  '. 34 

150    -  The  Relationship  of  Delayed  and  Immediate  Hypersensi- 
tivity to  Allergic  Thyroiditis  in  Inbred  Guinea  Pigs    39 

VII 


152  -  The  Chemistry  of  Antibodies  42 

153  -  Basic  Studies  on  the  Chemical,  Physical  and  Skin- 

Reactive  Properties  of  Extracts  of  House  Dust  47 


Rocky  Mountain  Laboratory 

Summary  1 

170  -  Office  of  the  Director  11 

171  -  Rickettsial  Infections 12 

172  -  Q  Fever  13 

173  -  Diseases  Having  a  Reservoir  of  Infection  in  the 

Natural  Environment  (other  than  rickettsioses)  22 

174  -  Transmission  of  Disease  Agents  by  Certain  Vectors  ..  27 

175  -  Allergy  and  Immunology  of  Fungal  Infections  and  the 

Mechanisms  of  Allergic  Phenomena  33 

176  -  Viruses,  Virus  Components,  and  Virus  Surfaces  39 

177  -  Immune  Prophylaxis  of  Mycobacterial  Infections  44 

178  -  Immune  Prophylaxis  of  Salmonella  Infections  48 

179  -  Investigations  of  the  Role  of  Morphological  Elements 

of  Microorganisms  in  Immunity  and  Related  Phenomena  54 

180  -  The  Encephalitides  58 

181  -  Colorado  Tick  Fever  63 

Laboratory  of  Bacterial  Diseases 

Summary 1 

200  -Brucellosis  2 

201  -  Bas  ic  Studies  on  Staphylococcus  5 

202  -  Identification  of  Pseudomonas  7 

203  -  Studies  on  Bacteria  Characterized  by  the  Production 

of  Reproductive  Filterable  Granules  9 

204  -  Intracellular  Parasitism 11 

205  -  The  Role  of  Infection  in  the  Delayed  Deaths  of  Mice 

Following  Extensive  Burn  Injury  13 


VIII 


DIRECTOR  -  NATIONAL  INSTITUTE  OF  ALLERGY  AND  INFECTIOUS 

DISEASE 


Summary 1 


Annual  Report  of  Program  Activities 
National  Institute  of  Allergy  and  Infectious  Diseases 
January  -  December  1960 

OFFICE  OF  THE  DIRECTOR 

1.  The  calendar  year  1960  witnessed  significant  extension  of  both 
intramural  and  extramural  activities  carried  on  or  supported  by  the  National 
Institute  of  Allergy  and  Infectious  Diseases.   These  are  detailed  in  succeed- 
ing sections . 

2.  Of  special  significance  were  the  steps  taken  during  the  year 
toward  the  development  of  International  Centers  for  Medical  Research  and 
Training.   These  are  to  provide  tangible  implementation,  in  addition  to  the 
research  presently  supported  overseas  by  the  various  Institutes,  for  Senator 
Hill's  "International  Health  Research  Act  of  I960"  (Public  Law  86-610). 

These  Centers  have  evolved  from  proposals  submitted  originally  to 
this  Institute  for  research  on  diseases  in  the  tropics.   This  area  of  con- 
cern has  now  been  expanded  to  encompass  most  of  the  research  and  research 
training  interests  supported  by  the  National  Institutes  of  Health.   Each 
Center  represents  a  collaborative  undertaking  between  a  sponsoring  medical 
segment  of  an  American  university  and  a  host  institution  of  medical  learning 
overseas.   Within  this  setting,  it  is  expected  that  faculty  members,  post- 
doctoral trainees,  graduate  students,  and  other  specially  qualified  indivi- 
duals from  this  country  may  work  together  with  foreign  scientists  and  their 
staffs  in  solving  problems  of  universal  medical  interest,  in  extending  scien- 
tific knowledge,  and  in  creating  and  fostering  international  fellowship  and 
good  will . 

While  the  initial  emphasis  is  on  the  medical  and  supporting  sciences, 
it  is  anticipated  and  hoped  that  these  Centers  may  provide  overseas  oppor- 
tunities of  interest  to  the  socio-technical  and  related  research  interests 
supported  by  foundations  or  institutions  outside  the  National  Institutes  of 
Health. 

Four  Centers*  have  been  recommended  for  approval.   Administrative  and 
operational  responsibilities  are  assigned,  for  the  present,  to  the  National 
Institute  of  Allergy  and  Infectious  Diseases.   The  Councils  of  the  other 


*  University  of  California 

Tulane  University 

Johns  Hopkins  University 

University  of  Maryland 


University  of  Malaya,  at  Singapore 
University  of  Malaya,  at  Kuala  Lumpur 

Universidad  del  Valle,  Cali,  Colombia 

All-India  Institute  of  Hygiene, 
Calcutta,  India 

Field  Unit,  Rawalpindi,  Pakistan 


Institutes  and  the  Division  of  General  Medical  Sciences  have  agreed  to  make 
available  to  the  National  Advisory  Allergy  and  Infectious  Diseases  Council 
funds  appropriated  for  International  health  research  in  Fiscal  Year  1960, 
with  the  understanding  that  1)  regular  progress  and  status  reports  will 
be  made  to  the  other  National  Advisory  Councils,  and  2)  that  incorporation 
of  the  lists  of  grants  awarded  in  the  formal  minutes  of  the  other  Councils 
will  constitute  formal  endorsement  of  these  Councils. 

A  standing  committee  has  been  established  to  advise  the  Director, 
National  Institutes  of  Health,  with  reference  to  the  future  conduct  of  the 
Centers,  and  to  evaluate  and  advise  regarding  applications  for  additional 
Centers . 

3.   Staffing,  space,  and  funds  for  the  support  of  the  National 
Institute  of  Allergy  and  Infectious  Diseases  have  been  .reasonably  satisfac- 
tory for  current  needs  this  last  year,  with  one  exception.   The  death  of 
Dr.  Jules  Freund,  since  the  last  Annual  Report  was  written,  has  left  a 
unique  void  in  competent  leadership  in  immunology  and  allergy  that  is 
proving  difficult  to  fill.   Some  eight  outstanding  scientists  and  leaders 
in  this  field  have  been  seriously  considered,  brought  to  the  Institute  to 
see  and  to  be  seen,  and  certain  of  them  have  been  invited  to  take  over  this 
responsibility.   This  activity  has  become  of  special  importance  since  1955, 
when  the  title  of  the  Institute  was  amended  to  include  the  word  "Allergy." 
For  various  reasons,  mainly  satisfaction  with  their  current  situations,  it 
has  not  been  possible  to  persuade  any  of  these  candidates  to  accept  the 
position  as  Chief  of  the  Laboratory  of  Immunology. 

While  the  existing  staff  is  operating  admirably  in  the  discharge  of 

its  responsibilities,  the  lack  of  expert  leadership  is  recognized.   Every 

effort  will  be  made  in  the  forthcoming  year  to  attract  a  competent  individual 
to  this  important  position. 


EXTRAMURAL  PROGRAM 


Summary. 


Annual  Report  of  Program  Activities 

National  Institute  of  Allergy  and  Infectious  Diseases 
January- December,  i960 

Extramural  Programs 


In  spite  of  the  commonly  held  misconception  that  modern 
antibiotics  have  relegated  the  infectious  diseases  to  a  position 
of  relative  unimportance  in  human  health,  recent  statistics  from 
the  U.  S.  National  Health  Survey  indicate  that  Infection  and  al- 
lergy cause  more  than  two-thirds  of  the  acute  illness  in  the 
United  States,  and  furthermore  are  responsible  for  almost  one- 
fifth  of  the  chronic  disease  in  the  Nation.  Fully  cognizant  of 
these  facts,  the  Institute  has  continued  to  build  a  balanced 
program  of  research  and  training  emphasizing  the  fundamental 
nature  of  pathogenic  organisms  and  allergens,  the  mechanisms  by 
which  they  reach  and  damage  their  hosts,  and  the  means  which  may 
be  developed  to  combat  them. 

Research  Grants 

The  distribution  of  research  funds  in  support  of  the  Institute's 
objectives  is  shown  in  Tables  I  and  II.  Data  regarding  the  review 
and  approval  of  research  grants  are  given  in  Table  III. 

The  Council  continued  to  emphasize  and  support  areas  of  special 
research  need.  Under  the  Committee  on  Standardization  of  Allergens 
an  important  program  is  progressing  along  three  fronts:  chemical 
isolation  of  the  allergenic  fractions  of  ragweed  pollen;  immuno- 
logical comparison  and  standardization  of  the  active  fractions;  and 
clinical  testing  to  determine  which  fractions  are  important  in  human 
hay  fever.   Information  exchanged  last  April  during  an  informal  con- 
ference of  the  scientists  working  on  these  three  aspects  indicated 
that  the  substance  known  as  trifidin  apparently  is  the  purest  com- 
ponent yet  isolated,  but  that  at  least  eight  different  ragweed  pollen 
fractions  seem  to  be  active  allergens  in  laboratory  studies. 

To  maintain  continued  stimulation  of  influenza  research  between 
epidemics,  the  Institute  established  a  program  of  "Special  Grants" 
which  the  Council  authorized  under  the  Subcommittee  of  Investigators 
of  the  Committee  on  Influenza  Research.  Selected  scientists  work 
toward  specified  objectives  under  protocols  developed  in  collaboration 
with  the  Subcommittee.  For  this  purpose  the  Council  recommended  a 
reserve  of  $175  .>  000  from  each  of  two  fiscal  years,  with  unused  funds 
reverting  to  the  regular  research  grant  program.  At  the  close  of  the 


-1- 


year  10  such  projects  for  $115,713  are  being  supported.   In  February 
the  Institute  sponsored  a  symposium  on  the  1957-5&  Asian  influenza 
pandemic.  This  conference  emphasized  deficiencies  in  present  know- 
ledge and  thus  suggested  areas  for  intensive  research  stimulation. 
As  a  further  service  to  research  workers,  funds  were  provided  for  the 
preparation  of  an  extensive  annotated  bibliography  of  influenza  research, 
which  has  been  distributed  to  all  medical  libraries  in  the  United  States 
and  to  the  principal  medical  libraries  of  the  world.  In  addition,  the 
Subcommittee  of  Investigators  and  the  Committee  on  Influenza  Research 
promulgated  a  series  of  recommendations  for  dosage  and  criteria  for 
vaccination  against  influenza  in  the  civilian  population. 

Inability  of  the  antibiotics  to  control  resistant  strains  of  staphylo- 
cocci, especially  in  hospital  epidemics,  created  a  critical  and  alarm- 
ing situation  which  prompted  Congress  to  Include  in  the  1959  appropriat- 
ion for  this  Institute  a  one  million  dollar  increase  over  195^  levels 
specifically  for  research  in  this  area.  With  the  impetus  of  these 
additional  funds  the  Institute  so  accelerated  its  grants  program  that 
it  now  supports  102  staphylococcal  research  projects  totalling  $1,922, 
283.  More  than  one-third  of  these  studies  are  directly  related  to 
hospital  problems,  especially  epidemic  outbreaks  in  nurseries  or  post- 
operative Infection  of  surgical  wounds.   One  such  project  concerns  the 
design  of  hospital  nurseries  with  relation  to  the  spread  of  organisms; 
another  is  a  large-scale  coordinated  study  by  five  medical  school 
hospitals  and  the  National  Research  Council  to  evaluate  ultraviolet 
irradiation  of  operating  rooms  in  the  prevention  of  wound  Infections. 
Approximately  one-third  of  the  Institute's  program  in  this  area  deals 
with  basic  studies  on  the  fundamental  nature  of  the  staphylococcus 
organisms  and  their  relationships  to  the  host.   Other  grant- supported 
areas  of  investigation  include  clinical  aspects  of  staphylococcal 
disease,  new  drugs,  and  the  role  of  these  organisms  in  food  poisoning. 

The  Council  pointed  up  special  needs  in  three  other  areas  through 
partial  sponsorship  of  research  conferences,  namely  (l)  the  "Second 
Conference  on  Medical  Mycology,"  (2)  a  "Symposium  on  Immunochemical 
Approaches  to  Problems  in  Microbiology,  "  and  (3)  a  "Symposium  on  Air- 
borne Infection."  Moreover,  consonant  with  the  Institute's  ongoing 
interest  in  tropical  medicine  and  related  fields  the  Council  supported 
the  "Fourth  Conference  on  Research  Needs  in  Tropical  Medicine."  This 
meeting  brought  together  a  group  of  selected  authorities  to  delineate 
the  specific  aspects  which  should  be  emphasized  by  future  research  and 
training  in  the  broad  fields  of  tropical  medicine  and  environmental 
health. 

As  a  consequence  of  the  Council's  interest  and  positive  action 
based  on  a  recommendation  of  the  Virology  and  Rickettsiology  Study 
Section,  an  Adenovirus  Committee  was  established  early  in  the  year 
to  provide  needed  leadership  for  research  on  the  adenovirus  group  of 
filtrable  agents  by  providing  a  generally- accepted  set  of  standards  on 
which  identification  of  these  viruses  can  be  based.  The  most  urgent  of 
the  Committee's  objectives  has  been  the  production  of  standardized 


reagents  which  would  permit  research  workers  to  identify  adenovirus 
strains.  Through  a  technical  subcommittee  specifications  have  "been 
developed  for  the  production  of  type- specif ic  antisera  for  the  first 
2.6   adenovirus  types.  Proposals  have  been  solicited  from  prospective 
producers  and  it  is  expected  that  contracts  can  be  negotiated  soon 
so  that  the  needed  reagents  will  be  available  for  testing  and  certi- 
fication by  the  end  of  1961.  The  Adenovirus  Committee  now  plans  to  move , 
against  other  problems  in  the  standardization  and  characterization  of 
this  group  of  viruses. 

Recognizing  that  significant  research  of  high  quality  is  being 
conducted  at  many  institutions  outside  the  United  States,  the  Council 
has  felt  it  both  justified  and  desirable  to  support  carefully  selected 
projects  wherever  they  may  be  located.  Thus  research  grants  have  been 
awarded  to  scientists  at  foreign  Institutions  whenever  a  competent 
Investigator  proposed  a  project  which  for  geographical  or  other  reasons 
could  best  be  done  abroad,  or  when  the  investigator  was  so  out standing 
that  his  contribution  would  have  general  scientific  significance.  On 
this  basis  some  68  grants  for  $925,829  have  been  supported  during  the 
year. 

The  current  appropriation  for  this  and  several  other  Institutes 
includes  funds  for  the  planning,  negotiating,  and  initial  support  of 
clinical  research  units  spanning  the  interests  of  the  several  sup- 
porting institutes.  Such  centers  are  being  established  in  large  medi- 
cal research  institutions  throughout  the  United  States.  Central  review 
and  administration  of  this  program  is  the  responsibility  of  the  National 
Advisory  Health  Council  and  the  Division  of  General  Medical  Sciences. 
In  addition,  $1,4-54-,  000  from  the  current  appropriation  of  this  Institute 
has  been  reserved  to  establish  the  Institutional  Grants  Program. 

To  provide  more  expeditious  payment  of  continuation  grants  when 
the  appropriation  is  delayed  beyond  the  beginning  of  the  fiscal  year, 
all  research  grants  having  anniversary  dates  in  the  first  two  quarters 
are  being  forward  financed  to  provide  for  renewal  on  December  1  or 
later.  This  process  will  be  completed  before  the  end  of  the  current 
fiscal  year. 

In  conformity  with  the  criterion  regarding  scientific  excellence 
which  the  President  set  forth  in  approving  the  1959  appropriation  bill, 
the  Council  has  continued  to  give  special  consideration  to  all  appli- 
cations falling  within  the  lowest  ten  per  cent  of  the  priority  list  for 
each  study  section. 

Training  Grants 

Following  a  policy  initiated  last  year,  the  Institute  has  con- 
tinued to  develop  its  training  grants  program  along  lines  designed  to 
emphasize  disease-oriented  interests  in  allergy  and  infectious  disorders, 


-3- 


To  Insure  highly  competent  professional  review  of  applications,  two 
panels  of  consultants  were  established  to  cover  the  Institute's  "broad 
training  areas.  These  are  the  Allergy  and  Immunology  Training  Grant 
Committee,  and  the  Infectious  Diseases  and  Tropical  Medicine  Training 
Grant  Committee. 

Table  IV  indicates  the  distribution  of  training  grant  funds  in 
support  of  the  Institute's  objectives.  Table  V  gives  data  regarding 
review  and  approval  of  applications  during  the  calendar  year. 

The  Institute  now  has  completed  the  forward  financing  of  all 
training  grants  so  that  continuation  years  will  begin  on  July  1.  Under 
this  arrangement  it  now  is  possible  to  assure  grantees  of  continuation 
support  several  months  in  advance  of  their  needs,  thus  obviating  the 
uncertainty  and  inconvenience  formerly  caused  by  delays  in  the  appro- 
priation of  funds. 

Certain  foreign  institutions  have  opportunities  for  providing  out- 
standing training  in  special  research  areas  to  American  students  as 
well  as  their  own  nationals.  Recognizing  this  fact  the  Institute  cur- 
rently is  supporting  one  training  grant  at  a  Canadian  institution,  with 
the  proviso  that  stipends  from  the  grant  be  awarded  to  American  citizens 
only.  Two  other  training  grant  applications  from  foreign  institutions 
are  pending  review.  The  Council  has  continued  to  insist  that,  after 
the  first  year,  at  least  half  the  funds  awarded  through  all  training 
grants  be  devoted  to  stipends  and  other  direct  trainee  support  such  as 
tuition,  travel  expenses,  and  consumable  supplies. 

As  the  culmination  of  more  than  two  years  of  planning  by  the 
Council  and  the  Institute's  staff,  a  program  of  tropical  medicine 
training  centers  was  established.  Four  of  the  six  centers  originally 
included  in  this  series  subsequently  have  been  broadened  under  auth- 
ority of  PI  86-610  (see  below).  The  two  remaining  centers  will  pro- 
vide opportunities  for  field  experience  in  tropical  medicine  at  cooper- 
ating research  establishments  in  various  overseas  locations.  Support 
for  five  years  was  recommended,  with  re- evaluation  after  three  years. 
In  recognition  of  the  high  cost  of  this  type  of  training  only  30  percent 
of  the  yearly  budgets  after  the  first  year  must  be  used  for  stipends  and 
direct  trainee  benefits. 

International  Centers  for  Medical  Research  and  Training 

Under  the  International  Health  Research  Act  of  i960  (PL  86-6lO) 
the  Congress  made  funds  available  to  the  NIH  for  the  establishment  of 
International  Centers  for  Medical  Research  and  Training.  The  Council 
of  this  Institute  accepted  responsibility  for  the  review  of  applications 
for  grants  to  support  multidisciplinary  projects  under  this  new  program, 
to  be  financed  from  funds  made  available  under  PL  86-610  to  the  Division 
of  General  Medical  Sciences  and  various  Institutes  of  NIH.  Four  of  the 
programs  already  activated  as  Tropical  Medicine  Training  Centers  (see 
above)  were  appropriately  expanded  into  research  and  training  centers 


encompassing  the  categorical  Interests  of  the  several  Institutes  and 
the  Division  of  General  Medical  Sciences.   In  making  its  recommendations 
the  Council  had  the  technical  advice  of  the  Committee  on  International 
Centers  for  Medical  Research  and  Training.   It  is  anticipated  that  addi- 
tional universities  will  be  considered  as  potential  program  participants 
early  in  the  coming  year. 

Research  Fellowships 

Table  VI  shows  the  distribution  of  awards  during  the  year,  by 
areas  of  study  and  level  of  support.  The  data  regarding  review  and 
approval  of  "applications  .  are  presented  in  Table  VII.  The  fellowship 
program  of  the  Institute  has  followed  the  pattern  set  last  year,  with 
increasing  emphasis  on  awards  at  the  postdoctoral  and  special  levels 
to  produce  increasing  numbers  of  competent  independent  investigators. 
Beginning  July  1,  i960,  the  funding  as  well  as  the  administration  of 
all  predoctoral  awards  was  taken  over  by  the  Division  of  General  Medi- 
cal Sciences.  The  data  given  in  Tables  VI  and  VII  for  predoctoral 
fellowships  apply  to  those  awarded  during  FY  i960  and  funded  by  this 
Institute. 

Three  additional  Fellowships  Programs,  designed  to  support  faculty 
positions  at  different  levels,  are  being  activated  by  the  Institute  and 
will  be  in  operation  during  the  third  and  fourth  quarters  of  FY  1961. 
The  Career  Research  Professor  Grant  Program  has  been  established  to 
support  individuals  of  demonstrated  capacity  to  pursue  with  distinction 
a  professorial  career  in  independent  research  and  teaching.  The  Senior 
Fellowship  Grant  Program,  formerly  limited  to  preclinical  science  depart- 
ments and  administered  only  by  the  Division  of  General  Medical  Sciences, 
has  been  expanded  to  include  clinical  departments  as  well  as  certain 
departments  in  university  graduate  schools  and  other  appropriate  insti- 
tutions. These  awards  are  for  the  support  of  individuals  with  at  least 
five  years  of  relevant  research  experience  beyond  the  doctorate  who  have 
demonstrated  high  potential  for  a  research  or  academic  career.  The 
Special  Fellowship  Grant  Program  will  support  the  promising  young  invest- 
igator not  yet  eligible  for  an  award  at  a  higher  level  but  who  the  appli- 
cant institution  feels  would  be  an  important  adjunct  to  its  teaching  and 
research  staff.  This  program  is  in  addition  to  the  present  Special 
Fellowship  awarded  directly  by  the  Public  Health  Service  to  individuals 
for  advanced  or  special  training. 


-5- 


c 
o 

•H    O 

-P  o 

cd  O 

•H  - 

U  H 

o  o\ 

ISL 


o 

•H  O 
-P   O 

ai  O 
i-t     - 

Pn0\ 

22 
PhCU 
<-«3- 


ai  O 
•H  - 
M  o 

P4C0 
O  00 
U  -> 
Pn-d- 
ft  H 
<-CA- 


<H     -P 

o  u 


3 

co 


t- 

o 


* 

CO 

a 

-p 

ITN 

o 

c 

•H    O 

3 

on 

-P  o 

a 

on 

<a  o 

1 

ro 

v.  j- 

H 

PfOJ 

-«3- 

O  CO 

u      •< 

PhO 

PhH 

<;  -eg- 

• 

C\J 

o 

ON 

co 


NO 

t— 
t- 


on  o  o 
o  c—  no 
itn       o        H 


CvJ        o 

ON         NO 
NO        NO 


£ 


co 
co 
CVI 


o 
co 
0J 


ON 


81    £    ft 

_*  OJ        NO 


NO 
CO 

NO 

H 

8? 

2 

H 

NO 

00 

lf\ 

ITN 

-3" 

•* 

J- 

o 

on 

-* 

O 

co 

H 

ON 

H 

H 

ON 

NO 

t— 

J- 

CVI 

H 

ITN 

-d- 

NO 

■<&■ 

t- 

O 

-d- 

O   -H 

5     <1J 

■P   c 

o 

« 

p 

•H     M 

ft  ai 


_=J- 

CM 

-te- 


n0 


>S 


o 
c\j 

•«e- 


t— 

cvj  a 

Lf\ 

-d- 

CVI 

s 

OJ 

H 

OJ    o 

O 

-^p 

O 

l/N    O 

H 

NO 

t~- 

t—  -H 

O 

H 

^  pq 

p 

CVJ 

on 

>> 

H 

j  i 

-d    i 

a>; 

00 

,a 

-P 

03     L, 

3S 

3  ° 

3  "3 

O     p 

•H   O 

a)    a) 

a 

+>  W 

H  "3 

CQ 

<D     .* 

o  a 

_.   O 

o 

C  R 

U  -H 

<";cn  , 

p  -p 

<u:   *  v 

«l 

^  o 

on 

O   -H 

P  -te- 

©     03 

to      ^ 

S& 

•H      • 

o 

w    W'  o 

ITN    <L> 

Poo 

■*&■& 

r*  fn  o 

■p 

^     rCl         •» 

£  Jh 

M    O 

a)       h 

O  <h 

^iO#3- 

-p 

•H 

a3  O 

b£>.p 

u  o 

C    to 

CO 

O  o 

-9   r> 

?\b 

§ 

aJ^j-   Oj 
►-3  ifN  h1  d 

5h 
tfl 

-3- 

SB 

o 

p    •> 

C   30 


g 


CO 

OS 

rH 


CVI 
-69- 


iB 


CQ 


OH 
S  H 


NO 

tn 

rn 


ON 


On 


Ie± 


a-, 
o 


eg 
-J- 1 


El 


£ 

•H 

oH^n 

-j- 

>J                 0) 

In 
5 

m 

a 

•H 

£ 

•H 

O 

o 

CO" 

CO 

•H 

H 

P 

h 

o 

d 

O 

h 

•H 

u 

<y 

+5 

a 

^1-H 

S 

■H 

to 

Ph 

v 

p^ 

H 

d 

? 

« 

tu 

d 

3 

CO 

to 

u 

o 

oj 

d 

•H 

•H 

PM 

4h 

p 

<D 

-P 

f=M 

O 

d 

to 

to 

0 

-3 

o 

tH 

>i 

1 

2 

ti 

<; 

a 

o 

m 

01 

1 

H 

OJ 

m 

LTN  CM  OJ         OJ  l—  O 

m  OJ  on       m-*  O 
no  oj  on       rnNO  o 


j-  t—  on  O  OJ  rn       oj  m-4- 

oj  f—  -3-  ON  OJ  CO         l/Nt-CO 
ON-*  ON  O  O  OJ        no  rnF- 


H  OJ  CO 
H  J  IA 
COOJ  H 

H  H  H 


ON  O   LTN 

c—  rn  oj 


itn  h  t—  o  on  oj 

t—  CONO   H  ON  OJ 
OJ  H  Hco  ON 


00  NO   LTN        J-   ON  H 

H  OnO  H 


O  ITN  O  ON  NO   LTN 

H-d-  ON       CO  H  OJ 
O  ONCO  fO 


4VOH    LTNOO    H 
l?Nr-j   C-CONO_* 


H   OJNO 
OCOCO 


LTNJ-   t-  ON  NO   O 

t—  OJCO         mj-  O 

rn  ltn  h       ono  ltn 


NO  OJ  CO 
ON  NO  tfN 
NO   t—  NO 


<£££ 


ON  ITN  t— 

no  on  oj 
H 


ON  NO  NO  O  OJ  OJ 

H  O  ONtr-ONO 

H  LTNO 


-*  t—  H    O  OJ  t— CO  COO  On 
a  t-LTN 


M  O  On  itn  no  t—  rn 

NO  O  OJ  OJ  o  t— 

tw  on  t—  OJ  rH  CO  u^ 

o    •»   •«   ~   •»   ->  -> 

t— CO  ltn  o  rnON 
t— NO   OJ  J;  CO 


ir\  rn  rn 
H  rn-* 
t-  oj  rn 


o 


ONHNQ  O  OJ  <M  H  IT\ 

LTN  CO    rn  ITS  r-4^  Oj-* 

ojn3  oj  oj  corn      coo 


CMAH 

H4-ON 

CO  IA        ITN  OJ  H 


JHCM 
ONH  H 


LTN  t— NO  NO   ON  NO  COO  CO 

no  onco  oj  itnno       H  ml— 

HOJLPvC— ITNLfN         NO   t—  IfN 


tfNNO  CO 
ON  NO  O 
ON  H  H 


xi    oj   a)  pd 
o  h  to  to 


OJ 
CO 

a3   - 

^ 

CQ     OJ    T-l 

to 

cq 

•H     tO     O 

to 

cd 

cu 

tO    -H     CJ 

•H 

0) 

w 

O  P    o 
H          CJ 

to 
o 

y 

s 

H    rH     O 
OJ    CO   H 

H 
3 

«d 

•rH 

> 

bO  bO  *>> 

CJ 

CD 

o 

•H    d   ,£ 

u 

-P 

fn 

rd      3      P- 

tu 

cd 

a> 

Eo  £  td 

fl 

H 

p 

p 

CO 

l3 

a) 
U 

£ 

J-    LTN         NO    t- 


^1 


H  g 


a)        d 
§       I 

a     3 


A    A 


p^ 


ph 


0) 

I 


o 

NO 


Vi 


p 
t> 

OJ 
•<-3 

t 


oil     cn| 


H 
•H 

o 

8 

H 


TABLE  III 

RESEARCH  GRANTS  REVIEWED  DURING  CALENDAR  YEAR  i960 


March 

No. 

Received 

Amount 

No. 

Approved 

Amount 

New 

203 

$3,379,967 

130 

$2,008,527 

Continuations 

124 

1,991,573 

105 

1,630,318 

327 

$5,371,540 

235 

$3,638,845 

June 

New 

222 

$4,328,276 

134 

$2,306,799 

Cont  inuat  ions* 

1286 

$19,53^,707 

1261 

$  18,929,517 

1508 

$23,862,983 

1,395 

$  21,236,316 

*  Includes  continuation  grants  for  which  previously  recommended  support 
was  reaffirmed.   (ll86  for  $17,447,083). 

November 

New              204  $3,955,063  133  $2,367,569 

Continuations      110  1,681,293  94  l,34l,294 

314  $5,636,356  227  $3,708,863 

12/15/60 


-8- 


S3 
O 

•H 
-P  O 

n)   O 
•H  O 

U      - 


o 

•H 

-p  o 

ai  O 
•H  O 
fn  - 
P<H 
O  OJ 
*n  NO 
Pi     - 

ftoo 
o 


a 
o 

•H 

-P  o 
ctJ  O 
•H  O 
u  * 
Pit- 

M 

ON 


-3- 

J- 


1 

C 

1 

O 

■H--«, 

-P   H 

P 

ir\ 

t- 

5 

d 

ri 

j± 

t- 

•H   O 

H 

oo 

u  o 

o 

E9 

PtO 

o     - 

1 

US 

On 

1 

u  o 

CM 

H 

&  try 

-e©- 

EH 

&no 

CO 

• 

LT\ 

o 

C\J 

t— 

- 

s 

H 

^ 


^ 


■ft 

ON 
i/n 
ir\j      oo 


^ 

GO 

H 

VO 

O 

NO 

t- 

ON 

& 

-r 

b- 

^ 

col     o 

H|       NO 


•H    O 
<U    O 

s  +> 


"5* 


J? 

a 


o 

-ee- 


o 


O    O  Vt 


<h  NO 
CO 


cu  ,d   cu 


no  H  On  W    • 

iapcacTo 

->  aj  ro  -H  H 

CO  -P  ■«©■  H  no 

J-    qj    cu  73  NO 
-CO-         TJ   -P  CO 

CD    M  H  -3  tfl 

m  q  o       tii 


O 

NO 


■ 


TABLE  V 

TRAINING  GRANTS 

REVIEWED  DURING 

CALENDAR  YEAR 

i960 

1/ 

No. 

March 

Re 

No. 

ceived 

Amount 

Approved 

Amount 

Nev 

31 

$852,681 

18 

$527,304 

Continuations 

0 

31 

._ 

0 

18" 

._ 

$852,681 

$527,304 

June 

New 

25 

$1,791,122 

15 

$1,436,275 

Continuations* 

83 
108 

2,633,605 

$4,424,727 

83 
98" 

2,633,605 
$4,069,880 

*  (includes  continuation  grants  for  which  previously  recommended  support 
was  reaffirmed) . 

November 

New  13     $408,090**         9      $277,730** 

Continuations  0        —  0 


13    $408,090        ~9     $277, 730 

**  (includes  $32,349  to  forward  finance  one  request). 

l/  Amounts  do  not  include  $50,000  approved  as  a  Chairman's  grant,  nor 

$383,162  needed  to  forward  finance  17  grants.  Neither  do  they  include 
awards  for  four  International  Centers  for  Medical  Research  and  Training 
at  a  total  of  $878,342  (lst  year  $478,566;  2nd  year  $399,776). 

12/15/60 


-ID- 


A 


I  I  I  I  I  I  I  I 

I  I  I  I  I  I  I  I 

I  I  I  I  I  I  I  I 

I  I  I  I  I  I  I  I 

I  I  I  I  I  I  I  I 

I  I  I  I  I  I  I  I 


I    I    I    I    I    I    I    I    I    I    I    I 


OJ  I  OOVO  I    CM     I 

co  i  on  oo  i  o   i 

•»  I  •»    •»  I      »v   I 

OJ  I  OOCM  I  J"     I 

H  I  CT)  |                | 


I  I  ON  CM  I  I 

II  -s    -v  l  l 

I     1  iftQ  I  I 

1     I  H  H  |  | 


1 


I     I     I     I     I     I  I     I 


I      I  OJ  I  I    ON 

i    i  lt\  i  i  vo 

I      I  OJ  I  I    LTN 

1  .^  I  I  ~? 

I  I  VO  |  |  CO 

II  I  I    H 


9 


3 


s 


a 


H    i    m  H    I   H    I     I 


OJ  H    i     I 


H    I     I   Oil 


9 


ON 
H 


°  £ 


O  I  Q  ON  I 

o  i  ©vq  i 

vo  i  o  H  i 

•»  i  *   •*  i 

CO  I  r-j   £—  I 

CM  I  OJVO  I 


\o  oo  on  on 
t~  cm  j-  o 
co  o  mco 

•N        «\       •*       •* 

rllAOQ 


I  QQ1A  I  I  I 

I  O  O  C—  I  I  I 

I  O  lACvl  I  I  I 

I  T»    •»_•»  I  I  I 

I  H4-0\  I 

I  H  CM 


H     I      I 


I      I    OJ-=J-  cr, 


I    OIAIA  0OVO  I 

I    O  OO  H   ONCO  I 

I    lArlrl  C— CO  I 

I         •*      *V      *\      *v      •*  I 

i  vo  o\vo  oj4  i 


C— CM  ITNC—    I    H  H  H  CM  t-    1 


H 

-=J- 


I  J" 


8$ 

O  oo   | 


I  VO 
I   H 


l  O  I 

I  LT\  I 

I  VO  I 

1  ,^»  • 

I  VO  I 


I    H    I 


I     I    H  H    I  COI 


1  .ri 
1  <& 

I    ON 

I     •> 

•  S£ 

I  CO 


CM 
US 
VO 

<x> 

•49- 


I     I   ITNl  Q 

HJ    LTN 


°    .2 


I4OC0OW  I 

I    CO   1AO   LT\ J-  I 

|  *V       «\       *V       *V       •»  I 

I     LT\  .sfr   Writ-  I 


I     O   O  VO 

I  t—  Q  ro 
I  OJCO 
I       •»•»•» 

14-rlro 
I 


I    WH4-  HCJ    |H     I     ICMHH 


I  roj-  I  -d-  I  O 

I  •»      -v  I         «\  I  •»  I  •»  I 

I  LTNVO  I  VO  I  OO  I  O  I 

I  H  H  I  I  I  H  I 


I  VO 
I  vo 


I  vo 


I     I    ir\  ir\    i   CM    I    H    l    oo    |     |   OJ 


I     I     I    OOH 
III 


m 
m 

-4- 

-ea- 


IHCOJON 


g 


IHJON 


1    1    1    1 

1  vo 

1     1     1    1 

1    OO 

■    III 

1  CO 

till 

1     1 

1    1    1    1 

1    CM 

•    III 

I 

TABLE  VH 
RESEARCH  FELLOWS  HIPS  REVIEWED  DURING  CALENDAR  YEAR  i960 


Received  (New) 
(Contlnuat ions ) 


Recommended  for 
Approval   (New) 

Inactivated   (New) 

Awarded   (New) 
(Contlnuat ions ) 


Predoctoral 


No.   Amount 


Postdoctoral 


No .   Amount 


77   $269,500   99      $643,500 

68   238,000   43   279,500 

155   $507,500  352   $923,000 


39  $131,687 

20  $  70,000 

19  $  61,687 

19  5^33 

38  $116,120 


53  $328,152 

3  $  19,500 

50  $308, 652 

37  243,920 

~B7  $552,572 


Special 
No.   Amount 


2k 

2* 

13 

1 

12 

12 


$192,000 
$192,000 

$113,127 
$  8,000 

$105,127 
$105,127 


12/15/60 


-12- 


ACCOMPLISHMENT  HIGHLIGHTS 

Important  research  accomplishments  continue  to  be  reported  by  the 
grantees  of  this  Institute  in  increasing  numbers  as  the  extramural  pro- 
grams develop  and  mature.  Results  of  grant- supported  research  are  seen 
not  only  in  the  problems  related  to  diagnosis  and  treatment  of  specific 
diseases,  but  also  in  the  broader  area  of  basic  processes  which  govern 
the  transmission  and  production  of  disease,  and  the  reaction  of  the  host 
to  invading  substances  or  pathogenic  agents.  Such  fundamental  studies 
are  essential  to  a  full  understanding  of  the  disease  process  and  form 
the  basis  on  which  significant  future  research  efforts  can  be  based. 
The  examples  given  below  represent  in  part  the  broad  range  of  interests 
and  approaches  within  the  extramural  programs  of  the  Institute. 

Important  achievements  in  the  related  fields  of  allergy  and  in>- 
munology  have  been  reported  during  the  year.  Grantees  at  the  New  York 
Hospital- Cornell  Medical  Center  carried  out  a  study  which  casts  doubt 
on  the  commonly  held  belief  that  hum an  infants  do  not  begin  to  form 
adequate  circulating  antibodies  to  injected  antigens  until  the  second 
month  of  postnatal  life.  While  the  scientists  feel  that  their  data  are 
not  sufficiently  extensive  to  warrant  sweeping  conclusions,  one  general 
fact  emerged  from  the  study:  newborn  and  premature  infants  naturally 
infected  with  various  types  of  ECHO  viruses  and  adenoviruses  usually 
respond  promptly  with  antibody  formation.  Whether  or  not  detectable 
antibodies  develop  apparently  is  closely  related  to  the  inherent  anti- 
genicity of  the  agent  —  that  is,  to  the  particular  virus  involved  — 
rather  than  to  the  patient's  maturity. 

A  study  of  chronic  asthmatics,  conducted  with  NIAID  grant  support 
at  the  National  Jewish  Hospital  in  Denver,  was  undertaken  to  test  the 
therapeutic  potential  of  negative  and  positive  air-borne  ions.  Studies 
by  several  scientists  on  the  curative  value  of  artificially  ionized  air 
have  been  under  way  since  before  World  War  II,  and  a  report  in  l$A-6  of 
beneficial  effects  from  air-borne  negative  ions  on  paranasal  sinusitis 
and  vasomotor  rhinitis  prompted  inquiry  by  later  investigators  into  the 
influence  of  ionization  in  hay  fever.  Prompt  amelioration  of  symptoms 
appeared  to  be  the  outcome.  Under  the  present  series  of  experiments, 
however,  no  improvement  of  asthmatic  symptoms  was  noted  during  exposure 
to  ionized  air  under  randomized  test  conditions.  The  investigators* 
clear  cut  findings  incline  them  to  discard  ionization  as  therapy  for 
patients  with  asthma. 

A  recently  completed  clinical  trial  by  grantees  at  Northwestern 
University  affirmed  the  usefulness  of  the  drug  SA  97  (homochlorcyclizine) 
in  ameliorating  certain  allergic  symptoms.   In  general,  SA  97  (supplied 
by  Abbott  Laboratories)  was  employed  against  various  allergic  mani- 
festations in  patients  who  failed  to  respond  adequately  to  the  usual 
drugs.   Among  the  asthma  group  about  75  percent  obtained  satisfactory 
results;  most  of  these  cases,  however,  were  not  acute  attacks.  About 
two-thirds  of  the  patients  with  perennial  allergic  rhinitis  obtained 
satisfactory  relief  from  protracted  nasal  blocking.  Most  of  the  sea- 

-1> 


sonal  hay  fever  patients  had  the  ragweed  type  of  hay  fever,  and  the  55 
percent  who  improved  were  mainly  those  failing  to  respond  to  antihista- 
mines.  In  the  urticaria  and  angiodema  group,  three- fourths  of  the 
patients  showed  improvement  ranging  from  relief  of  itching  and  diminished 
swelling  to  complete  regression  of  the  lesions.  The  majority  of  patients 
with  dermatitis,  including  atopic,  contact,  and  unknown  types,  obtained 
effective  relief  of  itching.  The  investigators  conclude  that  SA  97  is 
an  effective  adjunct  to  drugs  already  used  in  the  treatment  of  certain 
allergic  symptoms. 

Studies  on  infection  and  parasitism  form  a  major  portion  of  the 
Institute's  grants  program.   In  the  field  of  tropical  medicine  and 
parasitology,  as  in  other  areas  of  infectious  disease,  achievements 
have  come  from  a  wide  variety  of  projects  ranging  from  basic  studies  on 
pathogenic  organisms  and  the  host-parasite  relationship  to  practical 
applications  of  research  results  in  the  diagnosis,  treatment  or  prevention 
of  diseases. 

Grantees  at  Tulane  University  School  of  Medicine  have  demonstrated 
that  infection  with  Clonorchis  sinensis  can  be  detected  by  the  indirect 
tanned-cell  hemagglutination  test,  thus  adding  a  new  diagnostic  tool 
against  clonorchiasis,  a  human  liver  fluke  disease  highly  endemic  in 
the  Far  East.  The  new  procedure  was  found  to  be  relatively  more  sen- 
sitive than  complement  fixation  in  the  detection  of  Clonorchis  infec- 
tion in  man  and  rabbits. 

Other  Tulane  University  grantees  studying  the  biological  character- 
ization of  venom  from  the  tropical  fire  ant  have  demonstrated  an  anti- 
fungal effect  possessed  by  one  component  of  the  insect's  venom.  The 
crystalline  hemolytic  component  inhibited  the  activity  of  15  fungi, 
mostly  human  pathogens.  The  tropical  fire  ant,  introduced  into  the 
United  States  at  Mobile,  Alabama,  about  1920*  is  not  only  a  growing 
agricultural  problem  in  the  southeastern  United  States  but  a  menace  to 
human  beings.  The  sting  of  this  insect  is  extremely  painful  and  can 
cause  severe  allergic  reactions,  including  anaphylactic  shock.  The 
investigators  caution  that  their  report  is  not  to  be  interpreted  as  a 
study  on  the  antifungal  effects  of  this  material  for  future  clinical 
use  but  rather  an  attempt  to  broaden  methods  for  characterization  of  the 
biological  activity  of  the  crystalline  material  obtained  from  fire  ant 
venom. 

Important  advances  likewise  have  been  reported  for  the  bacterial 
diseases.   In  a  series  of  experiments  jointly  supported  by  the  University 
of  Chicago,  the  Atomic  Energy  Commission  and  this  Institute,  scientists 
at  the  University  of  Chicago  have  clarified  important  details  surround- 
ing the  known  fact  of  increased  mammalian  susceptibility  to  infection 
following  whole  body  irradiation  to  moderate  doses  of  radiation.  In 
mice  experimental  1  y  challenged  intravenously  or  intraperitoneally  with 
Pseudomonas  aeruginosa  after  irradiation,  it  was  found  that  those  in- 
oculated intravenously  displayed  a  higher  susceptibility  to  infection. 
The  difference  was  due  to  the  regular  appearance  of  a  small  focus  of 
infection  at  the  site  of  injection  as  a  result  of  leakage  of  inoculum 
into  the  surrounding  tissue.  Bacterial  multiplication  occurred  in 

-14- 


those  locations  in  irradiated  mice,  but  not  in  unirradiated  mice,  nor 
at  the  site  of  intraperitoneal  inoculation  even  in  irradiated  mice. 

In  studies  conducted  at  Harvard  University,  one  of  our  grantees 
has  shown  that  urinary  tract  infection,  one  of  the  commonest  causes 
of  acute  renal  failure,  can  regularly  be  averted  by  early  recognition 
and  treatment  of  asymptomatic  bacteriuria.   Development  of  a  simple  but 
reliable  test  for  the  detection  of  early  cases  of  pyelonephritis  was 
prompted  by  the  finding  at  autopsy  of  10  to  20  percent  incidence  of 
that  disease,  which  is  often  unsuspected  or  misdiagnosed.  Three- fourths 
of  infected  patients  respond  to  treatment  with  sulfonamides.  Alternative 
therapy  on  the  basis  of  sensitivity  studies  is  used  for  patients  resist- 
ant to  the  sulfonamides.  The  investigator  observes  that  pyelonephritis 
of  pregnancy  occurs  almost  exclusively  in  patients  who  have  bacteriuria 
at  the  time  of  the  first  prenatal  visit.  The  way  is  now  clear,  with 
this  test,  for  the  virtual  elimination  of  pyelonephritis  among  pregnant 
women. 

One  of  our  grantees  at  Washington  University  reported  the  develop- 
ment of  a  modified  urine  hemolytic  test  for  diagnosing  bovine  leptos- 
pirosis  without  some  of  the  limitations  inherent  in  the  currently  used 
methods  of  direct  darkfield  examination,  cultural  procedures  or  animal 
inoculations.   The  new  serodiagnostic  modification  becomes  positive  with- 
in three  weeks  after  infection  and,  in  the  later  stages  of  the  disease, 
continues  to  indicate  the  presence  of  leptospiral  antigen  for  several 
weeks  after  it  is  no  longer  demonstrable  by  laboratory  animal  inocul- 
ations. 

Expecially  significant  findings  have  been  reported  in  the  field  of 
staphylococcal  disease  and  the  special  problems  presented  by  antibiotic- 
resistant  strains  of  staphylococci.  Recent  reports  by  one  of  our  grantees 
at  the  Hospital  for  Joint  Diseases,  New  York  City,  suggest  that  the 
emergence  of  drug  resistance  actually  represents  a  selection  of  pre- 
existing resistant  strains  rather  than  the  development  of  new  ones.   In 
studying  1°^-  staphylococcal  strains  isolated  between  1927  snd   19^7.* 
before  antibiotics  were  widely  used,  this  investigator  found  that  22 
per  cent  belonged  to  the  "phage  type  8o/8l"  which  has  been  particularly 
dangerous  in  hospital  infections  during  recent  years.  This  observation 
dispels  the  apparently  common  belief  that  strains  of  "type  80/'8l"  are 
"new"  staphylococci  of  recent  origin.  Another  grantee,  at  the  University 
of  Texas  Southwestern  Medical  School,  has  shown  that  the  genetic  factor 
responsible  for  resistance  to  streptomycin  can  be  transferred  from 
resistant  to  drug- sensitive  strains  of  staphylococci  by  the  viruses 
(phages)  which  parasitize  them. 

At  Baylor  University  Medical  Center  a  grant- supported  project-  has 
shown  that  normal  human  white  blood  cells,  which  usually  destroy 
bacteria  by  ingesting  them,  actually  provide  protection  against  anti- 
biotics for  pathogenic  staphylococci  ingested  by  the  cells.   In  these 
studies,  10-100  fold  increases  in  the  concentration  of  penicillin  and 
other  antibiotics  failed  to  kill  staphylococci  which  had  been  ingested 
by  the  white  blood  cells.. 

-15- 


Two  groups  have  reported  interesting  findings  on  their  studies 
of  staphylococcal  infections  in  hospital  nurseries.  Working  in  a 
hospital  that  permitted  intermittent  "rooming- in"  of  infants  with  their 
mothers,  investigators  at  the  University  of  California  at  Los  Angeles 
found  that  both  the  infants  and  the  air  of  the  nursery  had  a  very  low 
incidence  of  staphylococci  while  the  mothers  had  a  relatively  high 
incidence  of  bacteria  which  they  did  not  transmit  to  their  rooming- in 
infants.  Another  research  team,  at  the  New  York  Hospital- Cornell 
Medical  Center,  found  that  most  newborn  infants  who  are  infected  with 
staphylococci  have  a  relatively  low  index  of  infectivity,  while  a  small 
minority  are  highly  infectious.  Because  these  latter  infants  literally 
are  surrounded  by  clouds  of  bacteria  they  are  designated  "cloud  babies." 
Follow-up  studies  showed  that  their  explosive  role  in  hospital  epidemics 
continues,  in  the  family  unit,  after  they  leave  the  hospital.  The 
explosiveness  of  the  "cloud  baby"  outbreaks,  the  authors  say, .makes  it 
imperative  to  design  nursery  units  in  such  a  way  as  to  prevent  airborne 
dissemination  of  infection. 

A  great  deal  of  research  progress  has  been  reported  by  our  grantees 
in  the  field  of  virology.  A  research  team  at  Harvard  Medical  School 
revealed  poliovirus  variants  which  resisted  temperatures  generally 
believed  to  be  rapidly  destructive  of  all  three  types  of  virus.  Whereas 
previous  studies  have  shown  that  poliovirus  is  rapidly  destroyed  at 
temperatures  slightly  exceeding  60  degrees  centigrade,  the  present 
series  of  experiments  indicated  that  suspensions  of  the  virus  grown  in 
monkey  kidney  cells  were  not  completely  inactivated  following  exposure 
for  one  hour  to  temperatures  of  60  degrees  and  65  degrees  centigrade. 
In  one  instance  infectious  virus  was  demonstrated  in  Type  II  poliovirus 
after  heating  at  75  degrees  centigrade  for  one  hour.   Progeny  of  virus 
surviving  at  this  temperature  showed  increased  thermoresistance.  These 
results  suggested  that  pasteurization  of  milk  and  other  food  products 
as  now  carried  out  cannot  assure  the  complete  inactivation  of  these 
agents. 

Grantees  at  the  University  of  Pittsburgh  found  that  previously 
recognized  dengue  and  chikungunya  viruses,  as  well  as  two  new  dengue- 
like agents,  apparently  have  an  etiologic  role  in  a  new  and  frequently 
fatal  type  of  human  hemorrhagic  fever  occurring  in  the  Philippine  Is- 
lands and  Thailand.   Aedes  aegypti  mosquitoes  were  incriminated  as  the 
important  vector  in  two  epidemics.   This  work  included  the  first  reported 
instance  of  the  isolation  of  a  dengue  virus  from  wild-caught  mosquitoes. 

Among  young  adults  at  the  University  of  Wisconsin,  the  measure  of 
acute  respirator/disease  which  can  be  identified  etiologically  has  been 
raised  to  over  28  per  cent  by  one  of  our  grantees.   In  previous  work 
this  investigator  had  identified  about  20  percent  of  these  illnesses 
as  specifically  due  to  streptococci,  influenza  virus,  adenovirus,  or 
bacteria  other  than  streptococci.  The  present  studies  on  227  students 
hospitalized  with  respiratory  illness  over  a  two  year  period  showed 


-16- 


that  the  hemadsorption  viruses  are  responsible  for  another  8  to  10  per- 
cent of  the  cases  and  thus  are  the  most  frequent  cause  of  sporadic 
respiratory  infection  yet  found  in  the  student  body.  A  clear  definition 
of  the  relationship  between  specific  viruses  and  clinical  symptomatology 
in  terms  of  age  group,  frequency  of  attack  and  types  of  population 
involved  will  facilitate  the  creation  of  practical  measures  for  the 
prevention  of  respiratory  disease. 

Impressive  evidence  for  the  effectiveness  of  an  attenuated  virus 
vaccine  for  measles  has  been  recently  presented  in  a  series  of  reports 
by  23  investigators  in  11  institutions  working  on  a  coordinated  program 
under  grant  support  from  seven  organizations,  including  this  Institute. 
The  investigators  agree  that  any  proposed  vaccine  for  measles  must  be 
justified  as  both  desirable  and  acceptable.  In  measles,  because  of  the 
significant  mortality,  frequency  of  bacterial  complications,  occasional 
involvement  of  the  central  nervous  system,  widespread  morbidity  of  the 
uncomplicated  disease,  heightened  virulence  of  outbreaks  in  isolated 
populations  and  unfavorable  effects  on  certain  other  pre-existing  ill- 
nesses, a  preparation  conferring  immunity  comparable  to  natural  measles 
seems  desirable.  The  present  experimental  vaccine  may  be  acceptable 
because  of  the  following  properties  delineated  in  the  various  reports: 
ease  of  administration;  lack  of  local  reactions;  absence  of  communicability; 
high  index  of  serological  response;  elimination  of  bacterial  complications; 
and  demonstrated  prophylactic  efficacy.   The  work  specifically  supported 
by  this  Institute  and  carried  out  by  grantees  at  Yale  University  and 
Western  Reserve  University,  respectively,  compared  (l)  the  effectiveness 
of  the  subcutaneous  route  of  injection  with  others  that  might  be  involved 
in  natural  transmission  of  measles  and  (2)  the  clinical,  antigenic  and 
prophylactic  effects  of  the  vaccine  in  institutionalized  and  home- 
dwelling  children. 


-17- 


ASSOCIATE  DIRECTOR  IN  CHARGE  OF  RESEARCH 
INTRAMURAL  RESEARCH  PROGRAM 


Summary 


NATIONAL  INSTITUTE  OF  ALLERGY  AND  INFECTIOUS  DISEASES 
ANNUAL  REPORT  OF  THE  ASSOCIATE  DIRECTOR  IN  CHARGE  OF  RESEARCH 

INTRAMURAL  RESEARCH  PROGRAM 
CALENDAR  YEAR  1960 


The  year  1960  has  seen  both  a  widening  of  the  scope  of  research  activi- 
ties and  an  intensification  of  the  pursuit  of  promising  lines  of  investigation 
in  the  National  Institute  of  Allergy  and  Infectious  Diseases.  The  coming  year 
will  see  even  more  effective  utilization  of  scientific  resources,  as  new  space 
becomes  available  and  programs  initiated  this  year  begin  to  achieve  their 
objectives. 

Clinical  investigations  of  the  Institute  have  advanced  in  several  ways. 
Of  great  significance  for  present  and  future  programs  has  been  the  increased 
utilization  of  prisoner  volunteers  for  clinical  studies,  through  cooperation 
with  the  Federal  Bureau  of  Prisons.   Volunteers  have  been  hospitalized  in  the 
Clinical  Center,  exposed  to  specific  respiratory  viruses,  observed  for  clinical 
manifestations,  and  examined  by  precise  laboratory  techniques  for  evidence  of 
infection.   In  a  short  time,  these  human  volunteer  studies  have  made  available 
a  vastly  greater  amount  of  detailed  information  concerning  the  respiratory 
syncytial  virus  and  the  Eaton  agent  than  would  have  been  possible  by  the  usual 
clinical  and  epidemiological  observations  in  the  general  population. 

In  connection  with  investigations  on  simian  malaria,  prisoner  inmates 
have  been  inoculated  with  various  strains  of  the  parasite  in  the  Atlanta 
Penitentiary.   Some  of  them  were  transferred  to  the  Clinical  Center  for 
precise  clinical  observations.   This  study  also  has  permitted  the  extension 
of  clinical  and  laboratory  observations  not  possible  in  any  other  way.   Other 
clinical  studies  underway  for  some  years  such  as  treatment  of  fungus  infections, 
bacterial  complications  of  cystic  fibrosis,  and  drug  resistance  in  staphy- 
lococcal infection  have  continued  to  add  to  specific  knowledge  of  these 
diseases. 

During  the  year  an  observation  of  great  potential  importance  to  public 
health  was  made  by  Institute  malaria  investigators  and  quickly  substantiated 
by  two  other  laboratories.   Two  scientists  working  with  a  strain  of  monkey 
malaria  were  accidentally  infected,  probably  by  a  mosquito  bite.   This  was  the 
first  clear-cut  evidence  that  certain  strains  at  least,  of  simian  malaria, 
are  pathogenic  for  man.   An  intensive  investigation  using  all  available 
resources  quickly  showed  that  man  could  be  repeatedly  infected  by  this  strain 
through  mosquito  bite,  that  it  could  be  transferred  from  man  to  man  by  blood, 
and  that  monkeys  could  be  infected  by  mosquitoes  fed  on  human  cases.   These 
observations  formed  the  basis  of  a  greatly  expanded  program  on  the  pathology 
and  biology  of  malaria,  including  the  establishment  of  a  field  party  in  Malaya, 
the  origin  of  the  monkey  strain  infectious  for  man. 

The  Middle  America  Research  Unit  in  Panama,  established  three  years 
ago  under  the  sponsorship  of  this  Institute  and  the  Walter  Reed  Army  Institute 
of  Research,  has  intensified  its  studies  of  the  arthropod-borne  viruses  in 


the  tropics.   Laboratory  procedures  have  been  developed  and  diagnostic 
reagents  prepared  for  working  with  a  large  proportion  of  the  more  than  125 
arthropod-borne  viruses.   The  Middle  America  Research  Unit  also  has  extended 
its  studies  to  include  special  epidemiologic  observations  of  poliomyelitis 
in  Panama,  and  investigations  of  the  role  of  mites  in  Central  American  virus 
infections.   In  conjunction  with  Gorgas  Memorial  Laboratory  workers,  Institute 
scientists  recovered  two  strains  ol  vesicular  stomatitis  virus  from  Phlebotomus 
flies.   This  is  the  first  time  this  group  of  insects  has  been  definitely 
incriminated  as  a  possible  vector  of  this  important  virus. 

Increasing  interest  in  immunologic  phenomena  manifests  itself  in 
nearly  all  aspects  of  Institute  research  activities.   The  development  of  new 
immunologic  techniques,  such  as  Immunoelectrophoresis  and  immunofluorescence, 
has  stimulated  new  approaches  to  new  and  old  questions  and  attracted  investi- 
gators into  the  field.   The  result  has  been  a  noticeable  resurgence  of  clinical 
and  scientific  interest  in  allergic  diseases.   The  Institute  initiated  a  new 
program  in  clinical  immunology  during  the  year  which  will  investigate  such 
autoimmune  diseases  as  lupus   erythematosus,  and  thyroiditis,  as  well  as 
certain  clinical  aspects  of  hypersensitivity  and  mechanisms  of  resistance. 

The  very  important  studies  on  respiratory  infections  which  have  been 
underway  for  some  years  continue.   It  is  now  clear  that  the  Eaton  agent  is, 
as  long  suspected,  an  important  cause  of  primary  atypical  pneumonia.   Develop- 
ment of  more  precise  laboratory  techniques  for  isolation  of  viruses  from 
animals,  demonstrated  that  many  laboratory  mouse  stocks  are  grossly  contami- 
nated with  normally  occurring  latent  viruses.   At  least  five  different  viruses 
are  now  known  to  infect  apparently  normal  mice.   This  has  complicated  studies 
on  the  relation  of  viruses  to  cancer,  and  confused  much  of  the  work  in  this 
field  conducted  over  the  last  few  years.   It  seems  unavoidable  that  the 
development  of  pathogen-free  animal  stocks,  particularly  mice,  will  be 
essential  for  future  Investigations  in  cancer  and  in  other  diseases  as  well. 
The  development  of  some  means  to  eliminate  or  control  these  contaminating 
viruses  from  experimental  animals  poses  a  major  and  immediate  problem  to  this 
Institute  and  to  investigators  elsewhere. 

The  year  has  witnessed  also  the  planned  development  of  research 
experience  for  junior  investigators  and  of  opportunities  for  permanent  staff 
members  to  work  in  research  centers  outside  the  National  Institutes  of  Health. 
The  key  to  productive  and  significant  research  is,  as  has  been  pointed  out 
repeatedly,  the  selection  of  imaginative,  energetic  and  well  trained  investi- 
gators and  the  development  of  a  stimulating  environment  in  which  these 
investigators  have  the  freedom  and  resources  to  pursue  their  scientific 
ideas.   One  approach  to  this  goal  is  to  recruit  competent  interested  young 
men  soon  after  completion  of  their  doctoral  training.   During  the  last  year 
three  such  young  men  Joined  this  Institute  as  a  part  of  the  NIH  Research 
Associate  Program  and  seven  as  part  of  the  Clinical  Associate  Program.   Six 
others  were  recruited  to  specialized  research  activities  making  a  total  of 
16  new  scientific  staff  members  at  the  Junior  level.   From  these  no  doubt 
will  come  a  number  of  our  future  permanent  staff  and  all  will  become 
indoctrinated  with  research  experience  of  great  value  to  their  eventual 
scientific  development.   Thus,  the  intramural  activities  perform  an  Important 
role  for  training  in  research  methods  and  goals  to  the  future  benefit  of 
scientific  endeavor,  wherever  these  men  may  work-  in  universities,  hospitals, 
or  in  the  practice  of  medicine.       2 


The  Institute  has  pursued  vigorously  a  policy  of  encouraging  work 
assignments  of  its  permanent  staff  in  other  well  known  research  centers.   One 
scientist  is  working  at  the  Karolinska  Institutet,  Stockholm,  Sweden;  two  are 
at  the  Pasteur  Institute,  Paris,  France;  one  at  the  Max-Planck  Institute  for 
Virus-forshung,  Tubingen,  Germany;  one  in  collaboration  with  the  University 
of  California  and  Institut  de  Recherches  Medicales  de  la  Polynesie  Francaise, 
Papeete,  Tahiti;  one  at  the  Wallaceville  Animal  Research  Station,  Wellington, 
New  Zealand;  and  one  with  the  Virus  Laboratory,  California  State  Health 
Department.   Others  have  spent  shorter  periods  of  time  in  Malaya,  Africa,  and 
Brazil,  pursuing  specific  research  problems.   A  number  of  the  staff  have  served 
on  WHO  Expert  Committees  and  on  special  assignments,  particularly  in  the  areas 
of  tropical  medicine  and  parasitology.   In  this  field,  this  Institute  has 
probably  the  largest  group  of  investigators  of  any  other  center  in  this 
country. 

Research  in  tropical  diseases  will  be  expanded  still  further  under  the 
provisions  of  P.L.  480  which  permits  the  use  of  foreign  currencies  to  support 
specific  intramural  research  projects  in  certain  countries.   Projects  have 
been  formulated  and  implementation  of  them  during  the  coming  year  will  permit 
the  selected  expansion  of  investigations  important  on  a  worldwide  scale. 
These  include  problems  in  such  diseases  as  malaria,  schistosomiasis,  fungus 
infections,  arthropod-borne  viruses,  and  filariasis.   These  international 
activities  emphasize  the  expanding  scope  of  our  research  responsibilities. 

The  Board  of  Scientific  Counselors  met  twice  during  the  year.   The 
first  meeting  considered  Institute  investigations  underway  on  respiratory 
diseases  of  viral  etiology.   The  second  meeting  was  held  at  the  South  Carolina 
State  Hospital,  Columbia,  South  Carolina  where  the  Epidemiology  Section  of  the 
Laboratory  of  Parasite  Chemotherapy  is  located.   Institute  staff  members 
reviewed  studies  on  simian  malaria,  chemotherapy  of  malaria,  and  intestinal 
parasite  infections. 

This  report  arranged  by  laboratory  activities  will  summarize  the 
major  scientific  accomplishments  and  advances  achieved  during  the  year  in 
the  direct  intramural  program. 


LABORATORY  OF  CLINICAL  INVESTIGATION 


Clinical  research  activity  has  expanded  during  the  past  year  largely 
as  a  response  to  enlarging  the  professional  staff  and  cooperation  with  other 
Institute  research  units.   A  further  period  of  growth  will  be  needed  to  staff 
the  clinical  service  in  a  manner  consistent  with  optimum  research  productivity. 

INFECTION  OF  VOLUNTEERS        An  extensive  clinical  study  of  acute  viral 
WITH  RESPIRATORY  VIRUSES       respiratory  disease  was  begun  this  year  in 

association  with  staff  members  of  the 
Laboratory  of  Infectious  Diseases.   The  initiation  of  this  project  required 
much  work  but  with  the  unstinting  assistance  of  the  Clinical  Center,  very 
satisfactory  arrangements  have  been  made  to  transfer  to  the  Clinical  Center, 
Federal  prisoner-volunteers  for  study.   This  has  been  done  with  the  permission 
and  considerable  assistance  of  Mr.  James  Bennett,  Director,  and  Dr.  Harold 
Janney,  Chief  Physician  of  the  Bureau  of  Prisons,  Department  of  Justice. 
A  team  of  custodial  officers  has  also  been  assigned  to  the  Clinical  Center 
by  the  Bureau  of  Prisons  to  oversee  the  volunteers.   The  volunteers  have 
uniformly  cooperated  with  the  program  despite  some  extended  periods  of  room 
isolation,  frequent  blood-letting,  and  other  inconveniences.    Many  adminis- 
trative arrangements  have  been  developed  so  that  the  program  has  worked 
increasingly  smoothly. 

The  results  so  far  justify  the  investment  in  money  and  effort.   It 
has  been  possible  to  produce  in  human  volunteers  a  rather  uniform  "cold" 
with  the  respiratory  syncytial  virus.   It  may  occur  without  respect  to 
preinfection  immunity,  but  the  subsequent  rise  in  complement-fixing  anti- 
body appears  to  correlate  with  severity  of  illness.   Forty-six  men  have  so 
far  participated  in  this  study. 

Approximately  24  other  volunteers  have  participated  in  studies  with 
para-influenza  4  virus  or  Eaton  (primary  atypical  pneumonia)  agent.   Future 
studies  are  planned  with  human  influenza  virus  and  with  the  recently  defined 
group  of  REO  viruses. 

NEW  ANTI-         Beginning  about  four  years  ago,  the  Mycology  Section  of 
FUNGAL  DRUG       the  Laboratory  of  Infectious  Diseases  began  studies  on  an 

antifungal  drug  produced  by  Hoffman  La  Roche,  designated 
as  RO-2.   This  agent,  an  antimicrobial,  was  found  to  be  the  most  active 
material  ever  tested  in  vitro  and  in  animals  against  several  of  the  patho- 
genic fungi,  notably  histoplasmosis  and  blastomycosis. 

In  the  intervening  years,  30  patients  have  been  treated  in  the 
Clinical  Center.   Extremely  favorable  results  have  been  observed  in  several 
patients  severely  ill  with  these  diseases.   From  the  standpoint  of  therapeutic 
activity,  it  appears  that  this  drug  is  the  best  available  for  blastomycosis 
and  histoplasmosis. 


During  the  studies  it  was  noted  that  the  agent  produced  unusual  hepatic 
changes.   It  was  found  that  the  dye,  bromsulphalein,  normally  rapidly  trans- 
ferred from  the  blood  to  the  bowel  by  the  liver,  was  retained  in  high  concen- 
tration in  the  blood  in  patients  treated  with  RO-2.   This  effect  appeared  in 
a  day  or  two  after  start  of  treatment,  before  tissue  changes  would  likely 
occur,  suggesting  competition  of  the  new  drug  for  the  liver  excretory  mechan- 
ism for  bromsulphalein.   After  treatment  was  stopped,  dye  excretion  promptly 
returned  to  normal  or  nearly  normal .   Liver  biopsy  has  revealed  changes  indi- 
cative of  minor  hepatic  damage  in  some  cases.   Because  of  the  great  importance 
of  having  a  drug  in  addition  to  the  relatively  toxic  agent,  amphotericin  B, 
for  the  treatment  of  fungal  diseases,  this  new  agent  continues  under  investi- 
gation. 

VOLUNTEERS  INFECTED      Following  the  demonstration  of  the  infectivity  of 
WITH  SIMIAN  MALARIA      Plasmodium  cynomolgi  bastianellii  for  man,  several 

inmates  at  the  Federal  prison  in  Atlanta  volunteered 
for  exposure  to  this  agent.   The  need  for  careful  clinical  characterization 
of  this  disease  in  man  led  to  the  transfer  of  two  infected  volunteers  to  the 
Clinical  Center.   Both  men  developed  acute  malaria  which  was  carefully  studied 
throughout  its  course.   Significant  alterations  in  urinary  steroid  excretion 
were  detected,  an  unusual  elevation  of  serum  cholesterol  occurred  in  one,  and 
liver  lesions  not  previously  described,  resembling  but  not  identical  with  exo- 
erythrocytic  phase  parasites,  were  demonstrated  in  both  patients. 

PENICILLINS  AND      Previous  work  here  had  defined  nutritional  requirements 
PENICILLINASE        for  penicillinase  production  by  staphylococci  and  many 

parameters  of  its  interaction  with  benzyl  penicillin 
(penicillin  G) .   During  the  year  English  investigators,  working  with  the 
stripped  molecule  of  penicillin,  6-amino-penicillanic  acid,  produced  a  new 
compound  largely  resistant  to  destruction  by  penicillinase.   Working  with 
this  and  several  other  penicillin  derivatives,  it  was  found  that  resistance 
to  penicillinase  is  greatly  influenced  by  steric  positions  of  ethoxy  groups 
on  the  side  chain  and  that  failure  to  be  destroyed  by  penicillinase  is 
associated  with  greater  penicillinase  inducing-capacity .   Perhaps  most 
significantly,  this  work  has  added  to  the  now  substantial  indirect  evidence 
that  the  major  reason  for  present  resistance  of  staphylococci  to  penicillin 
is  the  capacity  of  these  micro-organisms  to  produce  penicillinase  upon  contact 
with  even  low  doses  of  penicillin. 

Clinical  studies  with  the  penicillinase-resistant  penicillin,  dimethoxy- 
phenyl-penicillin,  have  revealed  that  it  possesses  resistance  to  penicillinase 
in  vivo,  and  that  it  is  a  powerful  and  effective  anti-staphylococcal  drug. 
After  treatment  of  some  patients  for  periods  of  three  to  five  months,  no 
penicillin-resistant  staphylococci  have  been  isolated.   It  has  long  been  con- 
sidered that  chronic  staphylococcal  infection  resembles  tuberculosis  and  for 
the  first  time  it  appears  that  an  agent  is  available  which  can  be  employed 
for  extended  periods  of  treatment  without  loss  of  effect,  such  as  isonicotinic 
acid  hydrazide  in  tuberculosis.   If  long-term  therapy  can  thus  be  regularly 
given  an  enormous  benefit  will  accrue  to  thousands  ill  with  chronic  staphy- 
lococcal disease. 


ASCITES  IN  MICE  BY  In  the  past  year  a  staff  member  has  continued 

INJECTION  OF  ADJUVANTS       to  study  ascites  induced  in  mice  by  the  in- 
jection of  adjuvant  mixtures.   This  procedure 
has  provided  a  much  needed  laboratory  method  for  producing  antibodies  of  a 
wide  variety  and  a  means  of  evaluating  antigens.   Recently,  interest  has 
been  focused  on  the  pathology  and  abnormal  physiology  of  the  lesion.   It  has 
been  found  that  strain  differences  are  associated  with  differences  in 
susceptibility  to  ascites.   Since  ascites  was  found  associated  with  a  local 
plasma  cell  reaction,  which  in  some  strains  of  mice  went  on  to  plasma  cell 
tumors,  many  implications  toward  problems  in  neoplasia  have  also  been  raised. 
These  studies  have  become  the  basis  for  biochemical  and  pathologic  studies 
with  other  Institutes. 

HYPOGAMMAGLOBULINEMIA       Staff  members  have  shown  that  patients  with 

hypogammaglobulinemia  possess  low,  but  definitely 
measurable,  levels  of  antibody  to  the  enteric  viruses.   The  implication  of 
this  finding,  in  view  of  the  normal  resistance  of  these  patients  to  viral 
infections,  is  that  extremely  low  levels  of  specific  antibody  may  provide 
adequate  resistance  against  viral  diseases.   Subsequent,  unpublished  studies 
have  indicated  that  these  patients  will  develop  circulating  antibodies  to 
Salk  polio  vaccine. 

BENTONITE  The  modification  of  the  bentonite  flocculation  test 

FLOCCULATION  TEST       for  the  detection  of  gamma  globulin  promises  to 

provide  the  practicing  physician  with  an  accurate, 
convenient  laboratory  aid  in  the  diagnosis  of  hypogammaglobulinemia.   In 
contrast  to  the  electrophoretic  method,  the  results  with  the  bentonite  test 
can  be  known  to  the  physician  within  a  few  minutes  of  arrival  of  the  specimen 
to  the  laboratory.   Other  modifications  of  this  technique  will  also  give  the 
levels  of  albumin  and  other  protein  constituents  of  blood  and  other  fluids 
without  resort  to  the  more  cumbersome  method  of  electrophoresis. 

The  DNA-bentonite  test  for  systemic  lupus  erythematosus  has  also 
been  developed  in  the  past  year.   This  test  measures  the  antibody  in  lupus 
serum  which  is  directed  against  nuclear  material.   The  specificity  of  this 
test  is  greater  than  any  previously  described  test  for  lupus.   This  test, 
however,  is  positive  primarily  in  those  patients  with  active  disease  and 
only  rarely  is  positive  when  the  disease  is  in  remission.   A  modification  of 
this  test,  the  nucleoprotein-bentonite  test,  has  retained  all  the  attributes 
of  the  DNA  test  in  regard  to  specificity,  while  achieving  a  much  higher  level 
of  sensitivity  for  cases  in  remission.   These  promise  to  become  important 
standard  tests  in  the  diagnosis  of  systemic  lupus  erythematosus. 

CYSTIC  FIBROSIS  OF      Despite  the  commonly  accepted  point  of  view  that 

THE  PANCREAS  antibiotics  are  helpful  in  this  disease,  observations 

of  nasopharyngeal  cultures  failed  to  reveal  any 
appreciable  effect  of  antimicrobial  treatment  on  Staphylococcus  aureus. 
This  does  not  negate  a  possible  clinical  benefit  but  does  appear  to  minimize 
its  value.   It  was  coincidentally  observed  that  Escherichia  coli  was  not 
present  in  the  nasopharyngeal  flora  of  any  child  over  the  age  of  eight. 


ROCKY  MOUNTAIN  LABORATORY 


At  the  Rocky  Mountain  Laboratory  research  has  continued  directed 
toward  both  basic  laboratory  investigations  and  field  studies  of  insect- 
and  animal-borne  diseases.   In  the  first  category  are  those  projects  con- 
cerned with  the  chemistry  and  surface  properties  of  viruses,  the  highly 
intriguing  relations  of  hypersensitivity  and  humoral  immunity,  and  the  basic 
relation  of  structural  elements  of  microorganisms  to  the  activity  of  the 
agents,  both  in  vivo  and  in  vitro.   Field  work  is  the  foundation  of  projects 
related  to  studies  of  Q  fever,  tularemia,  Colorado  tick  fever,  and  the  ar?bo 
viruses.   In  addition,  the  combined  efforts  of  the  staff  are  directed  to 
many  other  areas  of  research.   Such  projects  include  Q  fever,  tuberculosis, 
influenza,  poliomyelitis,  and  cryptococcosis. 

HYPERSENSITIVITY       Studies  of  hypersensitivity  at  the  Rocky  Mountain 

Laboratory  have  been  directed  primarily  toward 
clarification  of  the  relations  existing  between  delayed  hypersensitivity 
and  circulating  antibodies  and  determination  of  the  factors  responsible  for 
induction  of  contact  hypersensitivity.   It  has  been  demonstrated  previously 
that  delayed  hypersensitivity  precedes  circulating  antibodies.   This  delayed 
hypersensitivity  is  directed  toward  protein.   When  circulating  antibody 
appears,  as  occurs  with  relatively  large  amounts  of  conjugated  protein,  or 
when  booster  doses  are  administered,  the  specificity  of  the  antibody  response 
becomes  oriented  toward  smaller  configurations  on  the  antigen  molecule. 
Studies  of  immunity  in  neonatal  animals  have  revealed  that  these  animals, 
when  injected  with  an  antigen  12  hours  after  birth,  develop  circulating 
antibodies  but  fail  to  develop  delayed  hypersensitivity.   Additional 
experiments  suggest  that  this  inability  of  neonatal  animals  to  express 
such  reactions  is  due  to  a  deficiency  in  a  skin  reactive  factor  rather  than 
an  inability  to  respond  to  a  primary  injection  of  antigen. 

It  was  shown  that  contact  hypersensitivity  is  directed  toward  a 
hapten  and  that  simple  compounds  rather  than  protein  conjugates  of  these 
compounds  produce  contact  hypersensitivity  when  administered  to  experimental 
animals.   It  is  considered,  however,  that  the  production  of  this  phenomenon 
is  related  to  the  specific  proteins  present  in  the  host  tissues  because  a 
conjugate  containing  soluble  guinea-pig  skin  proteins  and  a  hapten  produces 
in  guinea  pigs  delayed  reactions  and  Arthus  reactions  to  the  conjugate  and 
contact  hypersensitivity  to  the  hapten. 

POLIOVIRUS       Continued  studies  of  poliovirus  have  yielded  considerable 

fundamental  data.   In  cooperation  with  the  group  at  the 
University  of  Minnesota,  it  was  shown  that  agents  such  as  octyl  alcohol- 
chloroform  and  neutral  hydroxylamine  do  not  materially  change  the  physical 
properties  of  purified  infectious  RNA  of  poliovirus  but  destroy  over  99Z 
of  the  infectivity  of  this  material.   The  destruction  of  infectivity  by 
uncoupling  of  a  single  link  in  a  large  particle  (probably  an  acyl  link  of  an 
amino  acid  with  a  phosphate  group  at  the  end  of  an  RNA  chain)  suggests  a 
possible  approach  to  virus  chemotherapy.   Other  studies  have  revealed  that 
only  0.17«  of  RNA  infectivity  could  be  accounted  for  by  residual  protein, 


thus  strengthening  the  concept  that  RNA  does  indeed  constitute  the  infectious 
portion  of  the  poliovirus  moiety.   By  the  use  of  chromatographic  methods,  it 
was  also  demonstrated  that  only  certain  of  the  avirulent  strains  of  polio- 
virus  can  be  differentiated  from  the  virulent  strains  from  which  they  are 
derived.   This  is  in  direct  contrast  to  work  reported  by  others.   In  studies 
of  the  infectivity  of  RNA  it  was  found  that  the  bulk  of  virus  particles  react 
with  susceptible  cells  and  that  the  relatively  poor  correlation  between  virus 
particles  and  PFU  is  due  to  the  poor  efficiency  of  RNA  at  entering  sites 
where  it  can  influence  virus  production.   A  precipitation  test  for  detection 
of  antibodies  against  poliovirus  has  been  developed  which  is  more  sensitive 
than  the  neutralization  test  presently  employed.   The  antigen  used  is  radio- 
active virus. 

ENDOTOXINS  IN  Research  on  endotoxins  derived  from  Gram-negative 

BACTERIAL  FRACTIONS       organisms  has  continued.   As  is  so  frequently  the 

case,  a  fresh  outlook  on  an  old  problem  yields 
results  of  great  value.   The  ideas  held  by  Dr.  Westphal,  which  attributed  the 
activity  of  endotoxins  to  the  presence  of  a  firmly  bound  lipid  ("lipid  A"), 
were  apparently  generally  accepted  until  presentation  of  the  work  done  at  RML. 
The  finding  that  lipid  A  was  not  active  and  that  deproteinized  and  "delipif ied" 
endotoxin  was  active  stirred  considerable  controversy.   In  fact,  the  controversy 
was  so  intense  that  efforts  to  develop  a  vaccine  against  Salmonella  infections 
have  been  diverted  to  settling  this  issue.   Recent  studies  of  the  kinetics 
of  inactivation  of  toxin  by  hydrolysis  with  hot  acid  have  given  data  which 
should  end  this  discussion.   The  old  concept  of  "purified  endotoxins"  must 
be  abandoned  since  there  have  been  no  previous  toxins  as  good  as  those 
obtained  at  RML,  and  these  are  to  be  still  further  purified. 

The  purification  of  Vi  antigen  by  curtain  electrophoresis  is  a  major 
advance  in  the  study  of  this  most  important  antigen.   The  demonstration  that 
certain  labile  acetyl  groups  are  responsible  for  the  activity  of  Vi  antigen 
resulted  in  production  of  material  which  was  ten  times  more  active  than 
purified  preparations  prepared  by  mild  acid  hydrolysis.   Emphasis  should  be 
placed  upon  the  new  chemical,  physical,  and  biologic  methods  that  have  been 
devised  to  solve  these  problems. 

TUBERCULOSIS       The  problem  of  immunity  in  tuberculosis  has  been  studied 
intensively.   Significant  findings  include  the  fact  that 
mice  may  be  satisfactorily  immunized  to  subsequent  pulmonary  infection  with 
virulent  organisms  by  administration  of  small  doses  of  avirulent  organisms 
by  the  aerosol  method.   The  demonstration  that  resistance  is  not  due  to 
interference  strengthens  the  case  for  the  value  of  immunization  with  living 
attenuated  organisms  for  the  prevention  of  tuberculosis.   Since  it  has  been 
shown  that  the  delayed  reactions  elicited  by  protoplasm  of  various  acid-fast 
organisms  are  specific  in  nature,  it  seems  practicable  to  apply  these 
findings  to  certain  diagnostic  problems  in  man.   Use  of  fractions  of  tubercle 
bacilli  in  producing  isoallergic  encephalitis  in  guinea  pigs  shows  that  the 
adjuvant  effect  lies  in  the  cell  walls  and  in  a  water-soluble  protein  prepared 
from  the  walls.   This  latter  finding  is  important  since  it  will  allow  the 
study  of  the  adjuvant  phenomenon  from  a  molecular  level. 


Q  FEVER       It  has  been  demonstrated  that  the  number  of  dairy  cattle 

infected  with  Coxiella  burnetii  is  large  and  is  increasing, 
yet  it  is  extremely  difficult  to  detect  cases  of  clinical  disease  in  man. 
In  Idaho  and  Montana  we  have  shown  that  a  greater  number  of  individuals 
residing  on  infected  premises  have  antibodies  against  this  organism  than 
do  those  living  on  noninfected  premises,  yet  no  difference  can  be  detected 
in  the  number  of  individuals  who  have  symptoms  compatible  with  clinical 
disease.   The  fact  that  organisms  isolated  from  cattle  have  been  uniformly 
of  low  virulence  for  experimental  animals  may  account  for  the  inability  to 
find  clinical  cases  of  disease  in  those  exposed  only  to  cattle. 

By  the  use  of  skin  tests  to  eliminate  allergic  individuals  from  the 
study  group,  190  inmates  of  the  Montana  State  Prison  were  safely  immunized 
without  producing  such  reactions  as  have  been  previously  reported.   It  is 
evident  from  these  results,  as  well  as  from  those  previously  obtained  in 
laboratory  personnel,  that  human  beings  can  be  safely  vaccinated  against 
Q  fever  if  the  precaution  is  taken  to  eliminate  reactors  by  previous 
administration  of  specific  skin-test  antigen. 

Methods  developed  for  growing  rickettsiae  on  modified  Zinsser  tissue 
cultures  yielded  relatively  large  volumes  of  organisms.   These  studies  led  to 
others  involving  purification  of  C.  burnetii  by  sucrose  gradients  and  by 
continuous -flow  centrifugation  in  molar  salt  solution.   These  methods  like- 
wise made  it  possible  to  obtain  certain  chemical  and  physical  fractions  of 
these  organisms.   It  was  found  that  dimethyl  sulfoxide  could  extract  from 
Phase  II  C.  burnetii  a  material  which  acted  only  as  a  hapten,  but  from 
Phase  I  organisms  the  extract  obtained  acted  as  a  complete  antigen.   Lauryl 
sulfate  also  extracts  complete  antigen  from  Phase  I  organisms.   Physically, 
the  cell  walls  of  these  organisms  can  be  separated  from  the  protoplasm,  and 
it  has  been  noted  that  the  cell  walls  are  about  25  times  more  active  in 
producing  immunity  than  is  protoplasm. 

These  studies  on  Q  fever  are  of  considerable  significance.   The 
laboratory  and  related  studies  have  yielded  information  of  both  scientific 
and  applied  interest.   It  is  apparent  now  that  we  can  safely  use  our  present 
vaccines  for  immunization  of  man  and  that  it  is  feasible  to  produce  large 
numbers  of  organisms  which  can  be  purified  and  used  as  vaccine  or  manipulated 
to  give  physical  or  chemical  fractions  which  may  be  less  toxic.   The  failure 
to  find  clinical  cases  of  Q  fever  in  man  in  the  face  of  a  rising  incidence  of 
infection  in  dairy  cattle  is  highly  interesting  even  if  disappointing.   The 
lack  of  virulence  of  strains  of  C.  burnetii  for  laboratory  animals  probably 
is  responsible  for  this  finding. 

OTHER  RICKETTSIOSES       Studies  of  rickettsiae  other  than  C.  burnetii 

have  been  continued.   By  combining  the  methods 
presently  used  for  fluorescent  microscopy,  a  technique  for  sectioning 
arthropods  has  been  developed  which  should  be  of  interest  to  entomologists 
working  in  the  field  of  embryology  and  anatomy  and  to  medical  entomologists, 
since  thin  sections  in  which  the  organs  are  not  displaced  can  be  obtained 
routinely.   By  applying  the  technique  to  the  study  of  ticks  infected  with 
R.  rickettsii  it  was  found  that  the  infection  rate  in  local  ticks  varies 


from  157o  to  287„.   Not  all  of  the  ticks  found  In  ected  by  this  method  are 
infective  for  laboratory  animals.   The  value  of  this  type  of  study  has  yet 
to  be  fully  appreciated. 

The  use  of  specific  toxins  has  resulted  in  clarification  of  many  of 
the  problems  related  to  the  taxonomy  of  rickettsiae  and  has  proved  to  be 
useful  in  ecological  and  epidemiological  studies  of  this  complex  group  of 
diseases.   In  further  studies,  potent  immunogenic  extracts  have  been  obtained 
from  certain  of  the  rickettsiae.   Their  value  as  diagnostic  and  prophylactic 
agents  is  presently  under  consideration. 

BACTERIAL  VACCINES       Studies  have  been  continued  on  vaccines  for  certain 

bacterial  diseases.   It  has  been  found  that  while 
live  Russian  tularemia  vaccine  is  capable  of  protecting  mice  more  effectively 
than  does  ether-extracted  vaccine  derived  from  cell  walls  of  P.  tularensis, 
the  protection  produced  by  live  organisms  was  not  effective  for  long  periods 
of  time.   Continued  studies  have  emphasized  the  value  of  cell  walls  in 
producing  immunity  to  infections  with  Brucella  abortus  in  laboratory  animals. 
It  has  also  been  found  that  live  cells  suspended  in  phosphate  buffer  and 
shaken  with  an  excess  of  ether  are  killed  but  not  disrupted.   These  cells 
constitute  an  excellent  protective  antigen  which  is  less  toxic  (LD50  7.5  mg.) 
than  aqueous  ether  extracts  obtained  by  conventional  methods  (LD50  0-9  to 
2.0  mg.). 

AR-BO  VIRUSES       Studies  of  ar-bo  viruses  have  yielded  results  of  interest 

and  suggest  that  emphasis  on  field  studies  would  greatly 
increase  the  production  of  useful  data.   The  California  strain,  described  by 
Reeves  and  Hammon,  has  been  isolated  from  a  snowshoe  hare  in  Montana,  and 
serologic  studies  of  hares  obtained  from  Michigan  indicate  that  the  majority 
possess  antibodies  against  this  virus.   In  California  it  has  been  demonstrated 
that,  although  most  infections  in  man  with  this  agent  are  of  the  inapparent 
type,  some  infections  result  in  serious  disease.   A  virus  closely  related  to 
Powassan  virus  was  recovered  from  ticks  from  Colorado  and  is  of  importance 
since  viruses  of  this  group  produce  serious  illness  in  man.   Studies  to  date 
indicate  that  ticks  probably  are  not  the  natural  vector,  but  the  relation 
to  Powassan  virus  suggests  that  mosquitoes  would  most  likely  be  the  vector 
in  nature.   In  studies  of  the  complex  relation  of  WEE  virus  with  snakes  and 
mosquitoes  it  has  been  possible  to  demonstrate  that  the  virus  can  be  readily 
overwintered  in  garter  snakes  and  that  mosquitoes  can  be  infected  by  feeding 
on  such  snakes.   While  we  have  not  been  successful  in  isolating  virus  from 
snakes  collected  in  the  field,  the  laboratory  data  suggest  that  these  or 
similar  animals  could  constitute  a  host  suitable  for  overwintering  of  WEE 
virus.   In  Idaho  and  Oregon,  WEE  virus  was  isolated  with  considerable 
frequency  during  the  summer  season,  while  in  North  Dakota  the  virus  did  not 
appear  to  be  active.   Isolations  of  a  considerable  number  of  strains  of 
trivittatus  and  inornata  viruses  were  made. 

Considerable  research  was  performed  to  determine  the  level  of  viremia 
attained  in  wild  and  domestic  birds  infected  with  ar-bo  viruses.   After  in- 
fection with  WEE  or  St.  Louis  viruses,  turkeys,  ducks,  chickens,  and  pheasants 
display  levels  of  viremia  which  should  cause  infection  in  mosquitoes  feeding 
on  them.   It  is  of  interest,  however,  that  in  spite  of  considerable  effort 

10 


we  have  been  unable  to  isolate  ar-bo  viruses  from  the  bloods  of  vertebrates. 
Negative  results  were  obtained  in  examination  of  1,074  specimens  collected 
in  Montana,  North  Dakota,  Oregon,  and  Minnesota  during  the  spring  of  1960. 
These  studies  fail  to  add  weight  to  the  contention  that  latent  infections 
of  birds  are  a  factor  in  overwintering  or  of  introduction  of  virus  into 
endemic  areas. 

COLORADO  TICK      Colorado  tick  fever  continues  to  be  a  problem  in  the 
FEVER  western  United  States.   Without  stimulation  of  physicians 

a  large  number  of  specimens  for  examination  were  received 
this  year  and  virus  was  isolated  from  49  of  them.   Our  interest  in  the 
spectrum  of  symptoms  has  continued  and  we  still  see  severe  cases  of  illness 
due  either  to  encephalitis  or  bleeding  tendencies.   It  was  found  that  the 
complement-fixation  reaction  developed  at  the  Rocky  Mountain  Laboratory  is 
the  simplest  method  for  diagnosis  of  Colorado  tick  fever.   Vaccine  has  been 
prepared  and  has  been  shown  to  be  efficacious  in  mice.   This  type  of  vaccine 
has  been  used  repeatedly  in  man  without  ill  effects,  indicating  that  a 
vaccine  prepared  from  suckling  mouse  brain  is  harmless  to  man  when  repeated 
doses  are  given. 

Ticks  collected  in  Estes  Park,  Colorado,  were  examined  for  the 
presence  of  Colorado  tick  fever  virus.   The  incidence  of  infection  was  found 
to  vary  from  57„  to  217o.   This  high  incidence  of  infection  in  ticks  accounts 
for  the  large  number  of  cases  of  CTF  reported  in  Colorado  annually. 

LABORATORY  OF  TROPICAL  VIROLOGY 

The  activities  of  this  laboratory  are  conducted  at  the  Middle  America 
Research  Unit  in  the  Panama  Canal  Zone  and  at  Bethesda.   The  Middle  America 
Research  Unit  is  a  joint  research  effort  of  the  National  Institute  of  Allergy 
and  Infectious  Diseases  which  has  cognizance  for  studies  on  virus  diseases 
and  the  Walter  Reed  Army  Institute  of  Research  which  has  cognizance  for 
studies  on  fungus  diseases. 

VIRUS  ISOLATES  FROM        During  the  first  12  months  of  a  3-year  project  on 
PANAMANIAN  MOSQUITOES      the  ecology  of  arthropod -borne  viruses  in  the 
AND  SANDFLIES  tropical  rain  forest,  conducted  by  the  Gorgas 

Memorial  Laboratory  with  the  collaboration  of 
MARU,  major  emphasis  has  been  on  virus  isolation  in  suckling  mice  and  hamster 
kidney  cell  cultures.   Fourteen  virus  strains  were  isolated  at  MARU  from  412 
pools  and  63,000  specimens  provided  by  GML.   Virus  isolation  rates  were  for 
Phlebotomus,  1:700  and  for  mosquitoes,  1:7000,  although  the  rates  varied 
greatly  with  species.   Of  the  five  Phlebotomus  isolates,  two  of  broad  host 
range  (including  cell  culture)  and  short  incubation  period  are  serologically 
identical.   These  viruses  have  now  been  identified  as  the  Indiana  type  of 
vesicular  stomatitis  virus.   The  other  three  Phlebotomus  and  nine  mosquito 
viruses  are  being  related  to  each  other,  to  known  virus  groups  and  to  human 
and/or  animal  infection  and  disease. 


11 


EASTERN  EQUINE  ENCEPHALOMYELITIS      The  prevalence  of  EEE  antibodies  in 
VIRUS  INFECTION  IN  PANAMA  horses  and  man  in  two  areas  of  suggested 

EEE  virus  endemicity  has  been  determined, 
allowing  an  evaluation  of  the  relative  usefulness  of  several  serological 
methods  applicable  to  studies  of  this  type.   It  was  found  that  the  incidence 
of  EEE  antibodies  in  460  humans  tested  increased  with  advancing  age  (0.87. 
under  10  years  with  progressive  increase  to  97o  in  the  41-50  year  group)  . 
Complement  fixation  results  on  the  same  sera  indicated  the  probable  presence 
of  other  group  A  viruses. 

Lizards  of  species  common  to  this  part  of  Panama  were  examined  as  a 
possible  virus  reservoir.   Specific  EEE  virus  hemagglutination-inhibitors 
were  found  in  some  of  their  sera.   The  occurrence  of  viremia  and  HI  antibody 
response  following  virus  inoculation  were  experimentally  confirmed  by 
inoculation  of  lizards. 

ENCEPHALOMYOCARDITIS  VIRUS      Previously  this  laboratory  described  an 
INFECTION  outbreak  of  a  fatal  disease  of  swine  caused 

by  the  EMC  virus.   The  outstanding  lesion 
in  pigs  dying  during  the  outbreak  was  acute  myocarditis.   Since  epidemi- 
ological observations  suggested  that  natural  infection  resulted  from  ingestion 
of  contaminated  food,  experiments  were  undertaken  to  reproduce  the  disease  by 
feeding  virus  to  young  pigs.   Viremia  and  virus  excretion  from  the  gastro- 
intestinal tract  were  found  to  occur  following  the  administration  of  brain 
from  EMC  inoculated  mice.   Infected  pigs  developed  high  titers  of  HI  and 
neutralizing  antibody  during  convalescence  and  had  myocardial  fibrosis  at 
autopsy.   Other  studies  included  demonstration  of  EMC  antibodies  in  a  small 
number  of  city  rats  and  rats  caught  on  the  affected  farm,  although  wild 
rodents  were  found  to  be  negative.   Human  sera  were  examined  with  interesting 
differences  in  the  results  depending  on  the  donors'  age:  while  a  substantial 
proportion  of  the  Panamanian  population  has  been  infected  with  EMC  virus, 
the  antibodies  were  found  to  be  more  common  in  persons  of  younger  age. 

ENTEROVIRUS  FLORA  IN  For  a  period  of  12  months  the  enterovirus 

CHILDREN  OF  CENTRAL  AMERICA       flora  of  infants  at  an  outpatient  clinic 

in  Panama  City  was  systematically  explored, 
establishing  a  base  line  of  enterovirus  fluctuation.   The  majority  of  viruses 
isolated  belonged  to  the  ECHO  group,  although  in  late  1959  and  early  1960 
poliovlrus   type  2  had  become  very  prevalent.   This  was  reflected  in  an 
uncommon  occurrence  of  a  small  outbreak  of  paralytic  disease  due  to  type 
2  poliovirus. 

Other  enterovirus  studies  have  included  1)  surveillance  for  the 
presence  of  type  1  poliovirus  in  Panama  in  late  1960  as  a  check  on  dissemi- 
nation and  threatened  spread  of  this  commonly  epidemic  type,  2)  studies  on 
a  major  epidemic  of  ECH0-9  virus  which  swept  through  the  Republic  of  Panama 
and  the  Canal  Zone  and  3)  initiation  of  a  collaborative  project  on  possible 
relation  of  enterovirus  flora  of  Guatemalan  children  to  their  dietary  status. 


12 


MYCOTIC  DISEASES      The  research  program  on  mycotic  diseases  has  markedly 
IN  PANAMA  increased  local  awareness  of  histoplasmosis  in  all  of 

its  clinical  forms,  as  evidenced  by  recognition  of 
three  disseminated  cases,  two  fatal  and  one  successfully  treated,  within  a 
period  of  18  months.   Until  then  only  one  fatal  case  had  been  described 
since  Darling's  original  cases  in  1906.   Ecological  and  epidemiological 
studies  led  to  isolation  of  H^  capsulatum  from  eight  additional  soil  samples, 
bringing  up  to  16  the  total  number  of  recent  isolations  from  Panamanian  soil. 
The  fungus  has  been  repeatedly  recovered  from  the  organs  of  trapped  ground 
mammals,  confirming  its  wide  dissemination  in  nature. 

Histoplasmin  skin  test  continues  to  be  a  major  tool  for  the  study 
of  epidemiology  of  histoplasmosis.   Data  on  9,200  children  between  six 
and  19  years  of  age  have  been  obtained  indicating,  as  expected,  that  the 
percentage  of  reactors  increases  progressively  with  age.   The  rate  of 
histoplasmin  sensitivity  varies  from  13%  to  587»  among  six-year  olds  and  from 
687.  to  927»  among  19  year  olds,  depending  on  location  of  their  residence. 
A  survey  of  631  pre-school  children  (six  months  to  six  years)  in  the  Canal 
Zone  demonstrated  an  increase  in  hypersensitivity  beginning  with  three  years 
of  age.   A  continuing  similar  study  of  Panamanian  children  in  a  city  hospital 
is  now  in  progress  with  information  on  over  800  already  available. 

Projects  on  other  mycotic  diseases  have  included  diagnostic  study 
and  therapy  of  moniliasis,  found  to  be  a  major  superficial  mycosis  among 
both  indigenous  and  transient  population  in  the  tropics. 

ARBOR  VIRUS  STUDIES       At  Bethesda  new  projects  involved  an  interesting 
AT  BETHESDA  application  of  the  technique  of  antiserum  pool 

combinations  to  typing  of  arthropod-borne  viruses, 
a  wealth  of  data  evaluating  experimentally  produced  EEE  virus  infection  in 
horses  and  a  promising  attempt  to  develop  an  inactivated  EEE  virus  vaccine 
for  human  use.   The  infected  horses  yielded  specific  antiserum  which  is 
being  processed  for  prophylactic  use  in  cases  of  human  exposure  under 
laboratory  or  natural  conditions. 

Accidental  labbratory  infection  of  a  staff  member  with  an  arthropod- 
borne  group  C(Apeu)  virus  led  to  the  first  clinical -virological  study  of  a 
syndrome  produced  by  this  important  and  common  group  of  viruses  of  the 
western  hemisphere. 

LABORATORY  OF  BACTERIAL  DISEASES 

The  research  program  of  the  Laboratory  of  Bacterial  Diseases  has 
continued  in  the  same  general  areas  as  last  year. 

INTRACELLULAR       These  studies  deal  with  possible  changes  in  characteristics 
PARASITISM         of  infected  and  immune  cells  as  the  result  of  parasitism, 

and  the  effect  of  intracellular  growth  on  the  parasite. 
One  such  notable  change  of  course  is  the  production  of  specific  antibodies 
by  certain  cells  of  immune  animals.   Effort  has  been  directed  toward  the 
study  of  antibody  production  by  cells  in   vitro  and  the  macrophage  was 

13 


selected  as  a  multi potential  cell  for  such  study.   Macrophages  obtained  from 
the  peritoneal  cavity  of  guinea  pigs  immunized  with  egg  albumin  have  been 
found  to  release  antibody  in  vitro  for  a  period  of  several  days.   This  provides 
a  system  for  further  study  of  the  nutritional  or  other  requirements  for  con- 
tinued antibody  production  in   vitro,  or  even  in  serial  cultures.   Cells  derived 
from  macrophages  have  been  carried  in  serial  tissue  culture  for  several  months, 
retaining  their  phagocytic  ability. 

BRUCELLOSIS       Studies  on  brucellosis  are  conducted  at  a  reduced  tempo. 

There  is  continuing  need  to  collaborate  with  other  brucellosis 
research  centers  throughout  the  world  to  try  and  settle  problems  of  classi- 
fication and  epidemiology  of  the  Brucella.   Currently  we  are  doing  some 
laboratory  testing  of  brucellosis  vaccine  for  human  use  prepared  in  Russia. 
There  is  present  interest  in  this  vaccine  by  the  World  Health  Organization 
for  its  possible  use  in  occupational  and  otherwise  continually  exposed  groups. 

Studies  on  the  Staphylococcus  are  directed  toward  determining  the 
factors  responsible  for  pathogenicity,  and  toward  development  and  standard- 
ization of  tests  for  measuring  relative  pathogenicity  of  strains. 


LABORATORY  OF  CELL  BIOLOGY 

The  activities  of  the  Laboratory  of  Cell  Biology  during  the  calendar 
year  1960  have  been  along  three  major  lines:  (A)  The  continued  exploration 
of  the  metabolism  of  normal  cultured  cells,  and  an  approach  to  the  problem 
of  metabolic  controls;  (B)  the  mechanism  of  viral  synthesis;  and  (C)  the 
study  of  cell  cultures  deriving  from  patients  with  hereditary  metabolic 
disease. 

METABOLISM  OF  NORMAL      A  number  of  significant  observations  have  been 
CULTURED  CELLS  made  with  respect  to  the  amino  acid  metabolism 

of  cell  cultures.   There  has  been  no  further 
elucidation  of  the  pathway  of  serine  synthesis;  but  the  mechanism  of  cystine 
synthesis  has  been  clarified,  in  that  all  the  cell  lines  so  far  studied  have 
been  shown  to  use  the  classical  pathway  involving  the  demethylation  of 
methionine  to  homocysteine,  the  condensation  of  the  latter  with  serine  to 
form  cystathionine,  and  the  cleavage  of  the  latter  to  cysteine  and  homoserine. 
A  dual  pathway  for  proline  synthesis  has  been  indicated,  one  involving 
glutamine  as  the  source  of  the  carbon  skeleton,  and  the  other  involving 
arginine  by  way  of  ornithine. 

An  intriguing  recent  observation  has  been  the  finding  that  a  number 
of  factors  which  are  rigorously  required  by  the  cells  for  survival  and 
growth  can  in  fact  be  synthesized.   Their  nutritional  requirement  reflects 
the  fact  that  they  are  lost  from  the  cellular  pool  to  the  medium  at  rates 
which  exceed  the  biosynthetic  capacity  of  the  cell;  and  with  a  sufficiently 
high  cell  population  density,  when  the  loss  to  the  medium  per  cell  is 
sufficiently  reduced,  the  supposedly  essential  growth  factors  are  in  fact 
not  required  for  survival. 


14 


In  these  cell  cultures,  unlike  bacteria,  the  biosynthesis  of  amino 
acids  is  apparently  not  inhibited  by  the  product  of  the  reaction;  and  this 
mechanism  of  growth  control  is  apparently  not  operative.   Studies  are  in 
progress  as  to  whether  enzyme  repression  or  feedback  inhibition  are 
effective  controls  in  the  biosynthesis  of  pyrimidines.   A  quite  different 
control  mechanism  is  perhaps  indicated  by  the  demonstration  of  a  growth 
inhibitor  in  the  supernatant  medium  of  heavy  cultures.   The  chemical  nature 
of  that  inhibitor  is  under  continuing  study. 

Studies  on  the  mechanism  of  resistance  to  2-deoxyglucose  (2DG)  have 
shown  the  presence  in  the  resistant  variants  of  compounds  which  inhibit  the 
phosphorylation  of  2DG  to  the  metabolically  active  inhibitor,  2DG-phosphate. 
The  relationship  of  that  inhibitor  to ' the  observed  resistance  is  under 
continuing  study. 

MECHANISMS  OF         A  number  of  important  new  observations  have  been  made 
VIRAL  SYNTHESIS       with  respect  to  the  mechanisms  of  viral  synthesis. 
The  puzzling  wide  disparity  between  the  number  of 
physical  particles  in  viral  suspensions,  and  the  number  of  plaque -forming 
units,  i.e.  particles  capable  of  initiating  infection  in  susceptible  cells, 
has  been  partially  resolved  with  the  demonstration  that  after  the  viral 
particle  has  been  absorbed  by  the  cell,  it  may  undergo  several  alternative 
fates.   A  large  proportion  are  rapidly  eluted  into  the  medium,  essentially 
intact  but  no  longer  infectious,  presumably  reflecting  a  minor  alteration 
in  the  protein  coat.   Some  particles  remain  unchanged  within  the  cell. 
Others  are  degraded  intracellular ly,  in  that  the  nucleic  acid  is  exposed 
and  becomes  susceptible  to  intracellular  ribonuclease.   Only  a  small  fraction 
of  the  absorbed  viral  particles  are  stripped  of  their  protein  and  initiate 
infection. 

In  the  case  of  poliovirus  in  the  HeLa  cell,  although  the  viral  protein 
and  RNA  are  synthesized  concomitantly,  a  partial  dissociation  has  been 
achieved  with  appropriate  inhibitors  of  protein  synthesis,  which  completely 
block  the  formation  of  mature  virus,  but  not  of  infectious  RNA.   This  is  of 
particular  importance  in  relation  to  the  supposedly  obligatory  relationship 
between  protein  and  RNA  synthesis  in  growing  cells.   Of  interest  also  is  the 
fact  that  metabolic  inhibitors  which  effectively  block  the  synthesis  of 
cellular  DNA  and  of  DNA  viruses  have  no  effect  on  the  formation  of  poliovirus. 
It  would  therefore  appear  that  poliovirus  RNA  may  be  used  directly  as  a 
template  for  the  formation  of  virus,  without  the  necessity  for  intervening 
DNA  synthesis. 

In  contrast  to  the  situation  with  poliovirus,  in  the  case  of  vaccinia, 
there  was  a  marked  lag  between  the  formation  of  the  viral  nucleic  acid  (DNA) 
and  that  of  the  mature  virus. 

CELL  CULTURES  FROM  PATIENTS      An  exciting  new  development  has  been  the 
WITH  GALACTOSEMIA  successful  cultivation  from  patients  with  a 

hereditary  metabolic  disease  (galactosemia) 
of  cells  which  in  culture  demonstrate  the  metabolic  defect  characteristic 
of  the  disease.   This  suggests  an  entirely  new  experimental  approach  to 
problems  of  human  genetics. 

15 


LABORATORY  OF  GERMFREE  ANIMAL  KESEARCH 

GERMFREE  ANIMAL      A  series  of  observations  has  been  made  on  the  behavior 
STUDIES  of  Entamoeba  histolytica  in  the  germfree  host.   It  is 

to  be  recalled  that,  in  earlier  studies  with  standardized 
techniques,  amoebic  lesions  were  not  produced  in  the  germfree  animal  following 
inoculation.   In  fact,  the  parasite  failed  to  live  in  the  intestine  beyond 
five  days.   Recent  changes  have  been  made  in  the  manner  of  rearing  and 
handling  the  amoebae  in  vitro  prior  to  inoculation  which  seemed  to  result 
in  more  vigorous  organisms.   The  latter  have  produced  lesions  in  the  absence 
of  bacteria,  although  the  type  and  severity  are  still  not  typical  of  those 
encountered  with  a  bacterial  associate.   Thus,  it  would  appear  that  the 
latter  is  not  the  only  determinant  of  the  course  and  the  pathogenesis  of  the 
infection. 

Studies  have  shown  that  the  intestinal  mouse  parasite,  Nematospiroides 
dubius,  does  not  require  a  flora  to  develop  from  an  infective  larva  to  the 
adult  form  in  the  host.   However,  it  apparently  does  require  a  flora  or  its 
products,  to  develop  from  the  egg  to  infective  larva.   These  studies  are 
preparatory  to  those  to  be  undertaken  in  an  analysis  of  the  nature  of  the 
nutritional  effects  observed  in  certain  parasitisms.   One  of  the  interesting 
observations  has  been  the  finding  that  the  sex  of  the  host,  which  has  been 
noted  by  several  workers  to  affect  the  outcome  of  the  infection  in  con- 
ventional (contaminated)  animals,  has  not  appeared  to  be  an  influence  in  the 
germfree  host.   If  these  findings  continue  to  hold  up,  a  hitherto  unrecognized 
role  (either  direct  or  Indirect)  of  the  flora  in  certain  observed  sex  effects 
may  unfold. 

BIOLOGY  OF  GERMFREE      In  studies  on  the  growth  and  biology  of  germfree 
GUINEA  PIGS  guinea  pigs,  a  staff  investigator  has  obtained 

several  advanced  pregnancies  in  animals  maintained 
on  irradiated  diets,  although  no  fetus  was  carried  to  term.   It  is  to  be 
recalled  that  germfree  guinea  pigs  have  not  yet  been  bred  with  success.   The 
importance  of  the  intestinal  flora  to  this  species  was  pointed  up  by  the 
finding  that  conventional  (contaminated)  guinea  pigs  reproduced  normally  on 
this  same  irradiated  diet. 

In  a  collaborative  project  with  an  investigator  at  the  University  of 
Pennsylvania,  it  has  also  been  shown  that  the  use  of  large  dosages  of  a 
cathartic,  or  the  application  of  tourniquet  shock,  increased  the  number  of 
red  cells  of  germfree  chickens  coated  with  human  B-like  antigens  following 
monoinfection  with  Escherichia  coli  0s6 •   These  studies  are  providing 
information  on  the  manner  in  which  red  cells  of  one  type  may  acquire  anti- 
genic characteristics  of  other  cell  types,  especially  B. 

GERMFREE  MOUSE       The  germfree  mouse  colony  has  been  undergoing  an 
COLONY  intensive  serologic  study  including  an  assay  for  the 

presence  of  certain  so-called  "natural  antibodies" 
against  a  variety  of  bacteria.   Such  antibodies  or  antibody-like  reactivities 
for  organisms  like  Staphylococcus ,  E.  coli  and  Salmonella  typhosa  have  been 
found  to  occur  in  a  variety  of  uninoculated  conventional  animals  and  are  pre- 
sumed to  originate  from  encounters  with  the  viable  organisms  or  related 

16 


antigens.   Animals  which  have  lived  for  many  generations  free  from  contact 
with  live  bacteria  are  almost  the  sine  qua  non  for  establishing  finally  the 
validity  of  these  ideas.   Studies  thus  far,  with  the  Communicable  Disease 
Center  and  investigators  in  the  National  Cancer  Institute,  have  shown  the 
germfree  animal  to  be  singularly  free  from  antibody-like  reactivity  toward 
Staphylococcus  and  E.  coli.   Reactivity,  however,  toward  £5.  typhosa  was 
obtained  in  several  instances,  although  no  evidence  of  the  presence  of  the 
latter  was  found  in  the  germfree  colony.   Thus,  this  finding  strengthens 
sporadic  reports  that  non-bacterial  substances  (perhaps  in  this  case  dietary 
components)  can  cause  "cross"  reactions  with  this  organism. 

TUMORS  IN  GERMFREE       The  germfree  animal  colony  and  a  conventional  colony 
ANIMALS  derived  from  the  same  stock  now  has  existed  for 

for  approximately  two  years.   Some  of  our  exbreeders, 
in  spite  of  the  scarcity  of  germfree  unit  animal  space,  are  one  to  two  years 
of  age.   Whenever  a  germfree  or  conventional  animal  not  on  an  experiment  dies, 
especially  if  it  is  six  months  or  more  of  age,  it  is  examined  thoroughly  for 
gross  evidence  of  malformations  or  tumors.   Among  approximately  50  such  animals 
so-called  spontaneous  lung  tumors  have  occurred  in  some  of  the  germfree  as 
well  as  the  conventional  mice.   While  the  numbers  of  animals  are  obviously 
small,  the  incidence  has  been  markedly  higher,  thus  far,  among  the  conventional 
animals  (those  exposed  to  external  contamination)  than  among  the  germfree. 
This  seems  to  be  particularly  true  among  animals  six  to  twelve  months  of  age. 

LABORATORY  OF  PARASITE  CHEMOTHERAPY 

This  country's  commitment  of  38  million  dollars  in  Fiscal  Year  1961 
toward  a  program  of  world-wide  malaria  eradication  and  the  long-term  interest 
in  malaria  by  most  of  the  senior  staff  resulted  in  a  research  effort,  during 
the  past  year,  largely  directed  toward  problems  in  that  field.   Special 
emphasis  was  given  to  the  study  of  simian  malaria  in  man  and  in  monkeys  be- 
cause malaria  in  simians  might  be  a  real  deterrent  to  the  eradication  program. 
Clinical  facilities  for  volunteers  at  the  Atlanta  Penitentiary  were  enlarged 
and  the  staff  increased.   A  laboratory  was  established  at  Kuala  Lumpur,  Malaya, 
in  cooperation  with  the  Malaya  Institute  of  Medical  Research  and  the  United 
States  Medical  Research  Unit.   Studies  on  several  aspects  of  the  simian-human- 
malaria  problem  have  been  in  progress  there  since  mid-August. 

As  a  result  of  the  above  development,  it  was  decided  to  move  the 
Section  on  Cytology,  now  located  at  Memphis,  to  Chamblee,  Georgia,  early  in 
1961.   This  arrangement  will  bring  the  simian  hosts  closer  to  the  human 
volunteers  at  the  penitentiary,  and  the  insectary  maintained  by  the  Section 
will  be  geared  to  accommodate  the  work  at  Chamblee  and  at  the  prison. 

MALARIA  -  HUMAN       Plasmodium  falciparum  (McLendon  strain) :   Chloroquine 
(300  mg,  base)  and  primaquine  (45  mg,  base)  given  to- 
gether beginning  three  days  after  mosquito  bites  and  weekly  thereafter  for  a 
total  of  eight  doses,  resulted  in  suppressive  cure  in  5/5  subjects.   Controls 
were  positive  11  to  15  days  after  infection.   After  two  days  of  parasitemia, 
each  control  was  given  the  above  drug  combination  which  was  repeated  weekly 

17 


for  a  total  of  three  doses.   Parasites  were  removed  promptly  and  cure  was 
obtained  based  on  no  evidence  of  infection  during  22  7  days  of  observation. 

Primaquine,  at  daily  doses  of  0.75  mg,  had  some  sporontocidal  effect 
upon  Plasmodium  falciparum  gametocytes  but  none  against  those  of  P.  vivax 
(one  case).   Therapeutic  doses  (1.4  gm  in  three  days)  of  amodiaquine  had  no 
sporontocidal  effect  against  gametocytes  of  P.  falciparum  (one  case) .   The 
effect  referred  to  is  against  the  development  of  the  malaria  parasites  in  the 
mosquito. 

A  strain  of  Plasmodium  falciparum  from  Colombia,  South  America,  was 
found  to  be  resistant  to  chloroquine.   This  finding  is  of  utmost  importance 
in  terms  of  malaria  eradication. 

Plasmodium  vivax  (Chesson  strain) :   A  drug  combination  of  primaquine 
(45  mg)  and  pyrimethamine  (50  mg)  given  weekly  beginning  seven  days  after 
mosquito  bite  and  continuing  for  a  total  of  four  doses,  gave  suppressive- 
cure  in  4/5  subjects;  the  other  subject  developed  a  patent  infection  240  days 
after  infection.   Pyrimethamine  (50  mg)  given  alone,  as  above,  produced 
suppressive-cure  in  1/4  subjects;  the  other  three  came  down  on  days  82,  83 
and  84.   Five  controls  all  came  down  12  to  13  days  after  infection. 

The  Russian  8-aminoquinoline,  quinocide,  was  compared  with  primaquine 
and  found  to  be  distinctly  inferior  as  a  curative  drug  against  early  and 
late  primary  attacks  of  Chesson  vivax  malaria  particularly  from  the  stand- 
point of  the  occurrence  of  second  and  third  relapses. 

Another  8-aminoquinoline,  Win  5037,  was  studied  in  five  subjects. 
Toxic  effects  and  failure  to  cure  made  further  investigation  unwarranted. 

Plasmodium  malariae:   The  results  of  a  14-year  study  of  the  biology 
of  Plasmodium  malariae  were  drawn  together  for  publication.   The  highest 
infectivity  for  mosquitoes  occurred  during  the  eighth  to  tenth  weeks  of  the 
primary  attack.   Although  the  infection  rate  of  mosquitoes  was  ordinarily  low, 
the  relatively  long  period  during  which  mosquitoes  could  be  infected  may 
explain  the  persistence  of  P.  malariae  in  nature.   The  ability  of  the  symptom- 
free  malarious  patient  to  infect  mosquitoes  at  a  rate  similar  to  that  of  the 
symptomatic  patient  makes  eradication  difficult. 

MALARIA  -  SIMIAN       Plasmodium  cynomolgi  bastianellii:   In  early  May,  two 

accidental  sporozoite-induced  infections  with  Plasmodium 
cynomolgi  bastianellii  occurred  at  our  Memphis  Laboratory.   This  happening 
was  of  signal  importance  because  it  showed  that  simian  malaria,  contrary  to 
the  generally  held  opinion,  was  infectious  to  man.   In  that  light,  full  scale 
study  of  human  infections  was  undertaken  at  our  Atlanta  Penitentiary  installa- 
tion. 

Two  infections  were  induced  in  inmate  volunteers  by  inoculation  of 
infected  blood  obtained  from  one  of  the  accidental  sporozoite-induced  in- 
fections in  man.   Twenty  inmate  volunteers  were  infected  by  bites  of  Anopheles 
quadrimaculatus  or  Anopheles  freeborni  which  had  fed  on  infected  monkeys. 
The  prepatent  period  ranged  from  14  to  29  days  and  the  parasite  density  ranged 

18 


from  5  to  500/cmm.   The  most  constant  symptom  was  headache  and  the  most 
significant  signs  were  fever,  splenomegaly  and  hepatomegaly.   Infections  were 
allowed  to  run  their  course,  generally  without  treatment. 

Anopheles  freeborni  were  infected  from  two  patients  but  attempts  to 
infect  volunteers  by  their  bites  have  yielded  equivocal  results.   The  finding 
that  P.  £.  bastianellii  will  grow  consistently  and  produce  clinical  illness 
in  man  suggested  the  possibility  that  malaria  is  a  zoonotic  disease,  that  is, 
a  disease  which  man  can  acquire  from  animals  with  which  he  is  associated. 
Whether  or  not  such  transfer  occurs  in  nature  is  not  yet  determined,  but 
should  it  occur,  it  would  be  of  greatest  significance  to  the  world-wide 
malaria  eradication  program. 

Plasmodium  cynomolgi  cynomolgi :   Eleven  inmate  volunteers  were  bitten 
by  Anopheles  freeborni  infected  with  P.  c_.  cynomolgi  on  8  September,  and  to 
date  (14  December)  three  have  exhibited  evidence  of  infection  (i.e.,  fever). 
Parasitemia  has  been  demonstrated  in  only  one,  on  the  58th  day  after  mosquito 
bites.   These  results  show  that  this  strain  infects  man  far  less  readily  than 
P.  £.  bastianellii. 

FIELD  STUDIES       Three  staff  members,  Drs .  Eyles,  Dobrovolny,  and  Mr. 

IN  MALAYA  Clinton  S.  Smith,  were  detailed  to  Malaya  during  the  year 

where  they  engaged  in  the  study  of  simian  and  human 
malaria  in  cooperation  with  the  Malayan  Institute  for  Medical  Research  and 
the  U.  S.  Army  Medical  Research  Unit  at  Kuala  Lumpur. 

The  epidemiology  of  monkey  malarias  is  being  studied  and  the  feeding 
habits  of  some  of  the  Anopheles  determined.   By  injection  of  uninfected 
monkeys  with  sporozoites  from  natural  infections,  it  was  determined  that 
Anopheles  hackeri  is  a  natural  vector  of  Plasmodium  knowlesi.   This  is  a  most 
important  discovery,  especially  since  the  vector  of  this  parasite  has  been 
sought  for  repeatedly  during  the  last  25  years. 

Studies  of  malaria  in  aborigenes  associated  with  monkeys  have  been 
made.   Blood  passed  from  aborigenes  to  monkeys  have  thus  far  produced  no 
patent  infection  in  the  monkeys. 

EE  STAGES  AND       Studies  were  continued  on  the  direct  effect  of  drugs  on 
DRUG  ACTION         the  exoerythrocytic  stages  of  primate  malaria.   When  sul- 
fonamides were  used  with  pyrimethamine  to  exploit  the 
possible  synergism  of  the  two  drugs,  monkeys  developed  parasitemia  30  to  40 
days  after  inoculation  with  sporozoites  even  though  all  parasites  observed 
in  liver  biopsies  were  damaged.   The  curative  efficacy  of  quinocide,  the 
Russian  drug,  was  compared  with  primaquine.   Even  when  administered  at  twice 
the  dosage  used  with  primaquine,  quinocide  was  less  effective.   Chloroquine 
had  no  observable  effect  upon  the  liver  forms  of  Plasmodium  cynomolgi.   Young 
parasites  appeared  in  the  blood  in  large  numbers  on  the  8th,  16th,  and  24th 
day  indicating  the  existence  of  secondary  exoerythrocytic  generations. 

INSECT  TISSUE       Blood  cells  from  caterpillers  and  cells  of  the  ovariole 
CULTURE  sheath  of  several  species  of  moth  pupae  have  been  culti- 

vated in  several  different  media.   The  virus  of  St.  Louis 
encephalitis  has  been  maintained  in  cultures  of  hemocytes  from  larvae  of  the 

19 


catalpa  sphinx  for  ten  days.  Oocysts  of  Plasmodium  gallinaceum  attached  to 
the  midgut  of  Aedes  aegypti  have  shown  growth  in  vitro  and  sporozoites  have 
been  produced. 

BIOCHEMICAL  STUDIES       It  was  shown  that  mosquitoes  infected  with  malaria 

have  higher  levels  of  ribonucleic  acid  than  unin- 
fected mosquitoes.   Chromatographically,  the  acid-hydrolysate  of  ribonucleic 
acid  from  a  pyrimethamine -resistant  strain  of  Plasmodium  falciparum  differs 
from  the  acid-hydrolysate  of  ribonucleic  acid  from  a  pyrimethamine-susceptible 
strain.   Bephenium  hydroxynaphthoate  inhibited  glutamic  acid  transaminase  of 
Nippostrongylus  muris .   Bephenium  chloride  and  quinacrine  reduced  the  rate 
of  glucose  absorption  by  the  tapeworm  Hymenolepis  diminuta  but  low  concentra- 
tions of  dithiazanine  iodide  stimulated  glucose  absorption  by  this  cestode. 

INTESTINAL  PARASITES       Epidemiological  studies  on  the  inmates  of  a  mental 

institution  show  a  high  persistence  of  Trichuris 
and  hookworm  for  six  years,  with  an  apparent  decrease  in  Strongyloides .   To 
test  dithiazanine  and  tetrachlorethylene,  alone  and  in  combination,  heavily 
parasitized  mental  patients  were  given  the  drugs  for  about  one  year.   A 
large  number  of  worms  were  removed  but  the  cure  rate  was  low  and  transmission 
was  not  stopped.   Bephenium  hydroxynaphthoate  and  bephenium  chloride  were 
used  with  good  results  against  hookworm,  As car is  and  Trichuris. 

SCHISTOSOMIASIS       The  activity  of  griseofulvin  observed  in  mice  infected 

with  Schistosoma  mansoni  was  not  well  developed  in 
hamsters  or  monkeys.   A  series  of  tetracycline  analogues  which  show  an 
affinity  for  microfilaria  did  not  combine  with  schistosomes  and  were  without 
activity.   One  of  these  analogues  was  significantly  more  active  against  micro- 
filariae of  Dirofilaria  immitis  than  tetracycline. 

In  many  tests,  the  efficacy  of  stibophen  (Fuadin)  therapy  on  mature 
Schistosoma  mansoni  infections  in  mice  was  increased  up  to  16  times  by  feed- 
ing a  balanced  semi -synthetic  diet.   The  toxicity  of  the  drug  was  not 
similarly  increased.   The  enhancement  of  curative  action  by  the  purified 
semi -synthetic  diet  was  thought  to  be  due  to  the  absence  of,  as  yet  un- 
identified, inorganic  salt(s)  that  interfere  with  drug  activity.   It  was 
found  in  mice  fed  on  the  purified  semi -synthetic  diet  that  higher  blood 
levels  of  the  drug  were  maintained  for  a  longer  period  than  when  the  same 
amount  of  Fuadin  was  injected  into  mice  fed  on  the  commercial  pellet  diet, 
suggesting  that  the  increased  cure-rate  was  due  to  higher  blood  drug  level. 
Similar  drug  advantage  was  observed  in  mice  given  tartar  emetic  while  on 
the  purified  diet. 


20 


LABORATORY  OF  PARASITIC  DISEASES 

This  Laboratory  continues  to  emphasize  fundamental  studies  on  para- 
sites and  parasitic  diseases.   No  important  changes  in  the  program  were 
instituted  during  the  year.   The  program  of  the  laboratory  is  well  diversified 
considering  the  size  of  the  staff  and  the  competencies  of  the  various  staff 
members  cover  a  large  proportion  of  the  field  of  parasitology. 

Although  the  emphasis  is  on  basic  studies,  this  does  not  imply  a 
narrow  viewpoint  and  the  laboratory  is  well  aware  of  the  many  practical 
problems  parasitic  diseases  create  throughout  the  world.   The  laboratory  is 
often  called  upon  for  help  and  advice  concerning  prevention  and  control  of 
parasitic  infections  and  so  must  maintain  competence,  and  a  reputation  for 
competence,  to  deal  not  only  with  basic  problems  of  parasitism  but  also  pro- 
blems of  prevention  and  control  of  parasitic  diseases.   Therefore,  the  labor- 
atory continues  to  carry  on  a  variety  of  activities  which  help  it  maintain 
its  international  reputation  and  increase  its  capacity  to  cope  with  problems 
of  parasitism.   Such  activity  also  returns  benefits  in  the  form  of  ideas  for 
laboratory  research  and  clues  which  may  explain  puzzling  laboratory  findings. 

TOXOPLASMOSIS       Studies  on  toxoplasmosis  in  New  Zealand  sheep  have  shown 

that  the  prevalence  is  high.   New  information  has  been 
obtained  concerning  the  distribution  of  the  organisms  in  the  tissues  and 
their  persistence  there.   After  inoculation  the  distribution  of  the  parasite 
in  tissues  is  erratic  and  the  parasites  rapidly  clear  from  tissues  other  than 
the  muscle  and  placenta.   Since  residual  infection  occurs  in  muscle,  mutton 
may  serve  as  a  source  of  human  infection.   Congenital  infection  with  Toxoplasma 
is  an  important  medical  problem,  therefore  it  is  of  special  interest  that  the 
sheep  studies  have  indicated  that  inoculation  of  sheep  60  days  before  preg- 
nancy did  not  result  in  congenital  infection  or  abortion  but  inoculation  at 
30  days  pregnancy  caused  abortion  or  foetal  death  with  absorption.   Infection 
at  90  days  pregnancy  was  less  likely  to  be  dangerous  to  the  foetus. 

The  status  of  resistance  or  immunity  to  Toxoplasma  continues  to  be 
puzzling,  since  living  organisms  fail  to  protect  completely  animals  against 
challenge,  especially  when  the  challenge  is  great,  and  because  low  grade 
parasitemia  may  persist  for  months  in  mice  and  rabbits  in  the  presence  of  high 
serum  antibody  levels.   The  observation  that  cysts  of  Toxoplasma  probably 
form  in  tissue  cultures  provides  a  new  opportunity  to  study  the  manner  of  cyst 
formation  and  the  factors  that  lead  to  cyst  formation. 

AMOEBIASIS       The  work  on  the  preservation  of  living  Entamoeba  histolytica 

and  other  protozoa  has  practical  significance  since  success 
would  permit  retention  of  strains  without  continuous  sub-culturing.   This  is 
a  relatively  new  field  and  techniques  are  still  evolving.   The  work  so  far 
has  shown  that  this  approach  is  feasible  since  four  species  have  been  frozen 
and  stored  for  periods  ranging  from  one  to  four  months  depending  on  the 
species  involved.   E.  histolytica  has  been  kept  at  -197°  C  for  24  hours, 
suggesting  that  almost  indefinite  storage  at  this  temperature  may  eventually 
be  achieved. 


21 


Laboratory  culture  of  E.  histolytica  concinues  to  receive  attention 
since  it  is  so  important  to  learn  more  concerning  its  nutritional  require- 
ments and  its  pathogenicity  in  the  absence  of  other  organisms.   It  is  note- 
worthy that  satisfactory  axenic  culture  of  this  species  has  been  achieved 
for  the  first  time.   The  protozoa  are  cultured  in  a  complex  diphasic  medium 
containing  no  cells  but  including  chick  embryo  extract. 

The  substitution  of  a  species  of  Crithidia  for  Trypanosoma  cruzi  in 
cultures  of  E.  histolytica  provides  a  more  economical  and  rapid  way  of 
producing  large  cultures  of  the  amoeba.   Demonstration  of  the  value  of  the 
Coulter  Counter  for  the  enumeration  of  protozoa  in  suspension  adds  a  valuable 
tool  for  quantitative  work  and  suggests  this  method  may  be  applicable  for 
counting  other  organisms  of  similar  size  such  as  tissue  culture  cells. 

PARASITIC  INFECTIONS  IN       The  use  of  germfree  animals  in  worm-parasite 
GERMFREE  ANIMALS  studies  continues  to  reveal  the  value  of  this 

tool  and  adds  to  our  knowledge  of  the  peculiar 
nature  of  the  germfree  state.   The  technique  seems  to  be  particularly  useful 
for  studying  conditions  that  influence  natural  resistance  and  nutritional 
relationships  of  parasite  and  host.   For  example,  it  was  found  that  the 
roundworm,  Nematospiroides  dubius ,  develops  as  well  in  germfree  as  in 
conventional  mice  but  while  in  conventional  mice  the  worm  recovery  is  much 
higher  from  the  male  animals,  the  recovery  from  germfree  mice  is  the  same 
for  both  host  sexes.   The  cause  of  the  difference  is  unknown.   Also,  it  has 
been  shown  that  the  feces  of  germfree  mice  do  not  support  development  of 
N.  dubius  larvae  and  that  bacteria  in  the  feces  provide  important  factors 
for  larval  development.   There  was  further  evidence  that  the  alteration  in 
levels  of  serum  protein  components  in  germfree  animals  is  due  to  dietary 
factors . 

STERILE  CULTURE       Studies  on  the  sterile  culture  of  worms  continues  to 
OF  WORMS  produce  fundamental  information  on  the  nutritional  re- 

quirements of  the  parasites  and  brings  closer  the  day 
when  we  can  use  the  axenic  animals  for  immunologic  and  therapeutic  studies. 
Survival  studies  using  relatively  advanced  larvae  of  Nippostrongylus  muris 
has  produced  important  results.   The  intent  has  been  to  try,  by  addition  of 
elements  to  the  medium,  to  induce  the  larvae  to  reach  the  adult  stage. 
Starting  with  a  salt  mixture,  dextrose  was  added  until  the  optimal  level  was 
reached.   Then  casein  was  added  and  survival  time  rose  to  11  days,  but  there 
was  not  development  of  the  larvae.   Addition  of  a  yeast  extract  to  this 
mixture  not  only  increased  survival  but  permitted  growth  to  the  adult  stage. 
Thus,  a  much  more  simple  medium  than  used  before  has  been  evolved  and  the 
achievement  of  a  defined  medium  for  culture  of  N.  muris  adults  is  much 
closer.   A  similar  approach  is  being  used  in  attempts  to  culture  micro- 
filariae of  Dirofilaria  immitis. 

NUTRITION  AND  SCHISTOSOMIASIS       Although  the  study  of  the  relation  of 
IN  PUERTO  RICO  nutrition  to  schistosomiasis  in  Puerto 

Rico  is  still  incomplete,  it  appears  that 
enrichment  of  the  diet  does  not  affect  the  number  of  eggs  passed  in  the  feces. 
However,  it  is  interesting  to  note  that  the  enriched  diet  did  cause  a  loss  of 
hookworms  and  whipworms  from  the  intestine.   This  has  a  bearing  on  the 

22 


problem  of  the  existence  of  hookworm  infection  \   ithout  hookworm  disease.   In 
laboratory  studies  conducted  in  Bethesda  the  enhanced  efficacy  of  stibophen 
in  mice  receiving  a  semi-synthetic  diet  was  shown  to  be  due  to  the  absence 
from  this  diet  of  as  yet  unknown  inorganic  salts.   Higher  blood  levels  of 
the  drug  were  maintained  longer  when  the  semi-synthetic  diet  was  used  and 
this  may  explain  the  greater  efficacy.   Demonstration  of  the  influence  of 
simple  salts  on  the  efficacy  of  stibophen  suggests  that  other  drugs  may  be 
similarly  affected  by  diet.   If  the  work  with  the  stibophen-salt  problem 
progresses  satisfactorily  it  is  hoped  that  a  test  of  the  effect  of  human 
diet  on  the  action  of  the  same  drug  may  be  tried  in  Puerto  Rico  before  the 
study  there  is  concluded. 

DUAL  VIRUS  AND  HELMINTH      Interaction  of  two  pathogenic  organisms  in  the 
INFECTIONS  same  host  has  had  relatively  little  attention 

in  spite  of  some  very  provocative  work  done  in 
years  past.   A  study  of  simultaneous  infection  with  encephalomyocarditis 
virus  and  Trichinella  spiralis  in  rats  has  produced  striking  and  significant 
results.   While  the  virus  alone  does  not  injure  adult  white  rats  when  given 
intraperitoneally,  in  the  presence  of  Trichinella  spiralis  infection  many  of 
the  rats  are  crippled  and  die.   This  potentiation  of  virus  pathogenicity  is 
not  due  to  nonspecific  stress  but  seems  to  be  related  to  the  presence  of 
the  worms  on  the  muscles.   The  virus  can  be  recovered  from  the  muscle  of  T. 
spiralis -infected  rats  but  not  from  muscle  of  rats  without  T.  spiralis. 
The  reason  for  the  influence  of  the  worm  infection  on  the  activity  of  the 
virus  is  unknown.   The  phenomenon  offers  an  opportunity  to  study  some  of  the 
fundamental  factors  in  the  pathogenesis  of  both  the  virus  and  the  worm 
parasite.   It  also  provokes  the  question  as  to  what  effect  this  worm  infection 
may  have  on  other  virus  infections. 

AMMONIA  TOXICITY      The  study  of  liver  damage  in  relation  to  ammonia 
IN  MICE  toxicity  in  mice  has  revealed  that  low  oxygen  in 

breathed  air  greatly  enhances  ammonia  toxicity.   The 
mechanism  of  this  effect  is  not  clear.   Though  hepatic  coma  is  usually 
considered  to  be  related  to  ammonia  toxicity  none  of  the  substances  which 
exacerbate  hepatic  coma  in  man  increases  ammonia  toxicity  in  mice.   In  fact, 
six  of  ten  decrease  it.   Ammonia  toxicity  in  mice  was  greatly  reduced  by 
hypothermia  and  this  suggests  that  the  same  measure  may  be  useful  in  treating 
hepatic  coma  in  man.   Finally,  mouse  liver  damage  was  induced  in  eight 
different  ways  but  none  caused  any  change  in  the  animal's  response  to 
intravenous  ammonia.   Thus,  though  high  blood  ammonia  levels  seem  to  be 
related  to  liver  damage,  the  causal  relationships  are  by  no  means  clear. 

BIOCHEMICAL  STUDIES      Fundamental  physiological  studies  have  focused  on 
OF  HELMINTHS  the  calcareous  corpuscles  of  tapeworms  and  on  the 

phospholipids  of  tapeworms.   The  calcareous 
corpuscles  are  amorphous  but,  on  heating,  dolomite,  brucite  or  apatite  may 
be  formed.   Electron  microscope  pictures  of  corpuscles  heated  with  KOH 
reveal  the  presence  of  well-formed  crystals.   The  glycerol  containing 
phospholipids  of  Taenia  taeniaeformis  are  about  half  lecithid  and  half 
cephalin.   Sphingomyelin  is  present  and  more  than  one  cephalin  is  known  to 
occur  in  the  larvae  of  this  tapeworm.   Hexose-containing  phospholipids  occur 
in  both  larvae  and  adults. 

23 


Study  of  the  mechanism  of  energy  metaboxism  of  sub-cellular  elements 
has  dealt,  among  other  things,  with  the  mechanism  by  which  mitochondria 
which  are  depleted  of  high-energy  phosphate  intermediates  are  stimulated  to 
oxidize  substrates  when  ATP  is  added.   This  is  a  complex,  though  fundamental, 
bioenergetic  system  for  which  a  better  understanding  is  needed.   Addition  of 
ATP  not  only  restored  succinate  oxidation  but  also  caused  reduction  of 
intra-mitochondrial  DPN.   The  succinate  oxidation  involves  an  energy-requiring 
reaction  and  this  energy  is  apparently  added  at  one  site  in  the  respiratory 
chain  and  used  at  another  for  reducing  pyridine  nucleotide. 

LABORATORY  OF  BIOLOGY  OF  VIRUSES 

The  basic  objectives  of  this  laboratory  continue  to  be  the  same  as 
last  year.   It  is  obvious  from  this  annual  report  that  four  out  of  five  units 
have  projects  with  the  same  general  objective  --  investigation  of  mechanisms 
and  localization  of  animal  virus  synthesis  within  the  infected  cell.   Each  of 
these  units  is  also  interested  in  the  infectious  nucleic  acid  of  viruses. 
In  view  of  the  complexity  of  this  problem  and  the  important  implications  of 
any  information  that  is  obtained,  this  "duplication"  is  quite  justified. 
Actually,  it  is  not  duplication  since  different  approaches  are  used  and 
different  virus -cell  systems  are  studied. 

The  electron  microscope  has  been  installed  and  is  now  used  not  only 
by  the  Biophysical  Unit  but  also  by  other  units  of  our  laboratory  and  by 
units  of  the  Laboratory  of  Infectious  Diseases.   With  studies  on  the  structure 
of  viruses  and  a  project  concerned  with  the  genetics  of  animal  viruses  added 
to  the  biochemical  and  biological  studies,  there  is  now  fairly  complete  cover- 
age of  the  important  facets  of  basic  virus  biology. 

INTRACELLULAR  LOCATION       By  use  of  radioautographs  and  staining  with 
OF  POLIOVIRUS  fluorescein  tagged  antiviral  antibody,  the  intra- 

cellular location  of  poliovirus  antigen—pre- 
sumably viral  protein--during  the  cycle  of  virus  multiplication  has  been 
determined.   Demonstrable  antigen  first  appeared  one  hour  after  infection  and 
was  diffusely  distributed  through  the  cytoplasm.   At  three  hours,  just  before 
the  appearance  of  new  virus,  it  was  present  throughout  the  nucleus  with  a 
tendency  to  be  concentrated  around  the  periphery  of  the  nucleolus.   At  five 
to  seven  hours,  particulate  accumulation  of  antigen  in  the  cytoplasm  was  noted. 
Incorporation  of  radioactive-tagged  amino  acid  into  cell  protein  ceased 
shortly  after  the  start  of  infection,  whereas  incorporation  of  thymidine  into 
RNA  continued  until  after  three  hours  and  tended  to  localize  in  the  nucleoli. 

MUTANTS  OF       Plaque  type  mutants  of  EMC  virus  have  been  found,  segregated 
EMC  VIRUS        and  characterized.   The  stability  of  the  mutants  has  been 

determined  and  the  plaque  type  shown  to  be  a  function  of  the 
viral  RNA.   It  has  been  shown  that  the  difference  in  the  size  of  the  plaques 
formed  by  these  mutants  is  brought  out  by  an  inhibitor  present  in  the  agar 
overlay  used  on  the  plaque  plates.   This  inhibitor  resides  in  the  agaropectin 
fraction  of  the  agar  and  can  be  separated  from  the  agarose  fraction  which 
then  permits  both  plaque  type  mutants  to  form  similar  sized  plaques. 

24 


POLYOMA  VIRUS       By  the  use  of  a  serum  protection  test  in  newborn  hamsters, 

evidence  was  found  that  polyoma  virus  transforms  normal 
cells  to  tumor  cells  quickly  and  directly  without  extensive  virus  multi- 
plication being  necessary.   Furthermore,  no  evidence  could  be  found  to 
suggest  a  lysogenic  relationship  of  virus  to  tumor  cell.   All  attempts  to 
show  the  presence  of  infectious  or  masked  virus  or  of  virus  antigen  in 
transplantable  polyoma- induced  tumors  have  been  negative.   It  appears  that 
once  the  virus  initiates  the  tumor  it  is  no  longer  required  for  tumor 
growth  and  maintenance. 

TETRACYCLINE  FLUORESCENCE       The  discovery  has  been  made  that  when  the 
LOCALIZED  IN  MITOCHONDRIA       antibiotic  tetracycline  stains  tissues  in 

such  a  way  that  they  fluoresce  under  UV 
light,  this  fluorescence  is  localized  in  the  mitochondria  of  the  cells. 
This  makes  a  convenient  vital  stain  of  these  subcellular  elements  for 
further  studies.   There  appears  to  be  some  similar  localization  of  the 
antibiotic  fluorescence  in  certain  bacteria. 

TMV  MODEL       A  complex  model  of  tobacco  mosaic  virus  has  been  constructed 

on  theoretical  grounds,  and  on  checking  this  model  against 
known  biochemical  and  biophysical  properties  of  the  virus  a  remarkable 
consistency  is  found.   Certain  refinements  of  electron  microscopic  technics 
have  produced  photographs  of  this  virus  which  reveal  previously  not  seen 
fine  structure  also  consistent  with  the  theoretical  model. 


LABORATORY  OF  IMMUNOLOGY 

Since  the  activation  of  the  Laboratory  of  Immunology  in  1957,  the 
program  has  been  concerned,  principally,  with  basic  research.   However,  for 
some  time  an  important  need  has  been  felt  for  the  initiation  of  clinical 
studies  in  immunology  and  allergy.   In  September  1960  the  Clinical  Immunology 
Section  was  activated  and  as  space  permits,  will  be  expanded  and  will  work 
in  close  collaboration  with  the  Laboratory  of  Clinical  Investigation  on 
clinical  studies  involving  immunological  aspects  of  such  diseases  as  lupus 
erythematosus,  nephritis,  and  chronic  thyroiditis,  in  which  an  auto- immune 
basis  is  suspected. 

ALLERGIC  THYROIDITIS       Experimental  allergic  thyroiditis  was  produced 

in  Strain  13,  inbred,  histocompatible  guinea 
pigs  by  immunization  with  a  single  dose  of  guinea  pig  thyroid  extract  in 
complete  Freund's  adjuvant.   Thyroiditis  developed  as  early  as  five  days 
after  immunization,  was  present  in  all  animals  at  16  days,  and  by  seven 
weeks  was  consistently  present  and  generally  severe.   Delayed  skin  test 
hypersensitivity  was  found  as  early  as  five  days  after  immunization  in 
nearly  all  animals,  and  was  present  in  all  animals  with  thyroiditis  at 
seven  weeks.   At  seven  weeks  after  immunization,  anti-thyroid  antibodies 
were  present,  and  antibody  titres  correlated  with  the  presence  and  degree 
of  thyroiditis.   This  correlation  was  not  found  at  certain  other  times 
after  immunization.   The  presence  of  delayed  hypersensitivity  was  correlated 
with  experimental  allergic  thyroiditis,  while  the  presence  of  circulating 
antibody  did  not  correlate  with  thyroiditis.   These  observations  constitute 

25 


the  earliest  production  of  experimental  allergic  thyroiditis  and  the  most 
severe  disease  at  the  time  intervals  studied. 

HOUSE  DUST  ALLERGENS       Studies  on  the  chemical  and  physical  properties 

of  house  dust  extracts  that  are  used  clinically 
for  the  diagnosis  and  treatment  of  house  dust  allergy  have  been  studied  to 
identify  the  components  responsible  for  the  specific  skin  reactions  produced 
in  house  dust  sensitive  individuals.   It  has  been  found  that  the  house  dust 
extracts  consist  of  a  heterogeneous  mixture  of  acidic  polysaccharides.  The 
heterogeneity  has  been  demonstrated  by  electrophoretic  and  ultracentrifuge 
sedimentation  analysis  and  also  by  the  multiplicity  of  cross  reactions  ob- 
tained with  antisera  to  the  various  pneumococcal  polysaccharides.   The  chem- 
ical composition  of  the  various  fractions  has  been  shown  to  be  roughly  5-20% 
polypeptide  and  80-957»  polysaccharide,  containing  about  equal  amounts  of 
uronic  acid  (probably  glucuronic  acid),  D-glucose,  D-galactose,  D-mannose 
with  lesser  amounts  of  L-rhamnose  and  L-arabinose. 

GENETICS  OF  GAMMA  GLOBULIN       Agar-gel  immunochemical  analysis  of  sera 

from  rabbit  litters,  with  precipitating 
antibodies  prepared  in  rabbits,  has  shown  that  seven  antigenic  determinants 
of  the  gamma  globulins  are  genetically  controlled  by  at  least  two  gene  loci 
with  each  specificity  exhibited  when  the  appropriate  allele  is  present.   Since 
the  gamma  globulins  are  soluble  proteins  which  have  properties  of  both  an 
antigen  and  an  antibody,  they  should  be  subject  to  quantitative  estimation 
and  cytological  localization.   This  immunogenetic  system,  therefore,  may  be 
uniquely  suited  for  the  study  of  certain  basic  problems  in  genetics,  embry- 
ology, immunology  and  protein  chemistry. 

In  other  studies,  antibodies  to  human  serum  proteins  were  prepared  in 
monkeys  since  this  animal,  being  a  closely  related  species,  might  be  more 
discriminating  for  minor  antigenic  differences  than  a  distantly  related 
species.   Three  "slow"  gamma  globulins  were  found,  instead  of  the  one  usually 
detected  with  horse  or  rabbit  antibodies.   Two  of  these  were  shown  to  be  re- 
lated to  myeloma  proteins.   The  quantitative  estimation  of  these  gamma  glo- 
bulins in  serum  should  be  helpful  in  the  early  diagnosis  and  study  of  diseases, 
such  as  multiple  myeloma,  which  involve  qualitative  and  quantitative  changes 
in  the  gamma  globulins. 

MECHANISMS  OF         The  genetically  distinct  guinea  pigs  of  inbred  Strains 
HYPERSENSITIVITY       2  and  13  have  proved  to  be  a  very  important  immuno- 
logical tool.   After  studies  established  the  fact  of 
skin  compatibility  in  the  two  strains,  experiments  were  conducted  to  transfer 
cells  with  a  measurable  biological  activity.   Transfers  of  tuberculin  sensi- 
tivity were  undertaken  by  the  intraperitoneal  injection  of  living  lymphoid 
cells  from  compatible  donors.   The  almost  quantitative  transfers  between 
inbred  guinea  pigs  were  a  reflection  of  the  continued  viability  of  the  active 
cells  in  the  recipients. 

Two  models  are  being  developed  to  study  the  mechanisms  of  immediate 
and  delayed  hypersensitivity  in  the  inbred  guinea  pigs;  protracted  anaphy- 
lactic shock  and,  the  massive  local  hemorrhagic  reaction,  respectively.   It 
has  been  shown  that  there  are  differences  in  susceptibility  to  hypersensitivity 

26 


reactions.   Strain  2  guinea  pigs  were  more  resistant  to  death  by  bronchospasm 
and  tended  toward  a  protracted  syndrome  in  anaphylactic  shock.   Both  Strain  2 
and  13  guinea  pigs  required  more  mycobacteria  than  did  random-bred  Hartley 
guinea  pigs  for  inducing  "delayed"  sensitivity  to  egg  albumin,  using  Freund's 
adjuvant. 

HUMAN  SERUM  Fractions  of  human  serum  separated  by  anion- exchange 

AUTO-ANTIBODIES       cellulose  column  chromatography  were  studied  by 

Immunoelectrophoresis .   The  conditions  for  elution 
of  eighteen  immunologically  distinguishable  human  serum  proteins  from  the 
columns  were  determined.   Gamma  globulin  obtained  under  the  appropriate 
conditions  by  this  method  was  found  to  be  pure;  rabbits  immunized  with  this 
fraction  made  antibodies  to  none  of  the  other  serum  proteins.   By  the  use  of 
anion-exchange  cellulose  columns,  it  has  been  found  possible  to  separate  the 
7S  from  the  18-19S  antibody  activities  in  sera  of  patients  with  thyroiditis 
and  lupus  erythematosus.   Initial  results  indicate  that  the  addition  of 
immunoelectrophoretic  characterization  of  these  and  other  sera  will  be 
extremely  helpful  in  our  aim  of  characterizing  the  antibody  activities  found 
in  human  serum. 

FLUORESCENT  ANTIBODY  STAINING      The  fluorescent  antibody  staining  of  the 
OF  MALARIA  PARASITES  human  malaria  parasite,  Plasmodium  vivax, 

has  been  recorded  for  the  first  time.   A 
globulin  fraction  of  convalescent  serum  from  a  patient  having  a  long-standing 
infection  with  P.  vivax  was  labeled  and  the  fluorescent  antibody  applied  to 
thin  blood  films  containing  the  parasite.   The  organism  was  visible  by  virtue 
of  its  specific  immuno-f luorescence.   Fluorescent  antibody  studies  were 
conducted  on  P.  cynomolgi  bastianellii,  the  monkey  malaria  parasite  which, 
recently,  has  been  shown  transmissible  to  man.   Considerable  morphological 
detail  was  observed  at  fluorescence.   Preliminary  studies  on  the  serological 
relationships,  as  based  on  degrees  of  fluorescence,  indicate  that  P.  vivax 
and  P.  cynomolgi  bastianellii  parasites  may  have  common  antigens  and  that 
the  two  species  may  be  closely  related. 


LABORATORY  OF  INFECTIOUS  DISEASES 

In  1960  the  Virus  and  Rickettsial  and  Epidemiology  Sections  of  this 
laboratory  continued  integrated  and  comprehensive  efforts  to  define  the 
importance  of  virus  infections  in  disease.   Field  investigations  of  human 
and  animal  virus  infections  were  made  possible  through  collaboration  with 
a  number  of  other  organizations,  including  the  Bureau  of  Medicine,  USN;  the 
District  of  Columbia  Children's  Hospital  Research  Foundation;  the  District 
of  Columbia  Welfare  Department;  the  New  York  City  Health  Department;  the 
National  Cancer  Institute;  the  National  Institute  of  Allergy  and  Infectious 
Diseases;  the  Laboratory  of  Clinical  Investigation,  NIAID;  and  in  Paris, 
France  the  Laboratoire  des  Virus,  Hopital  Saint-Vincent-de-Paul;  and  Le 
Centre  Claude-Bernard  de  1 'Hopital  Saint  Louis. 


27 


Natural  events  and  opportunities  afforded  by  our  collaborators  shaped 
the  course  of  most  field  studies.   Technical  breakthroughs  in  the  laboratory 
made  it  possible  to  take  fuller  advantage  of  these  opportunities  to  study 
natural  disease  and  thus  acquire  not  only  new  information  about  specific 
virus  infections,  but  also  to  move  nearer  our  ultimate  goal,  namely,  a  clear 
view  of  the  numerous  viral  causes  of  human  diseases  sufficiently  comprehensive 
to  make  concerted  efforts  to  control  them  appear  feasible  and  worthwhile. 

NEW  CAUSES  OF         Pneumonia  and  other  lower  respiratory  tract  infections 
VIRUS  PNEUMONIA       continue  to  represent  major  causes  of  death  and  a 
large  segment,  presumed  to  be  viral  in  origin,  is 
still  uncontrolled.   Until  recently  it  was  wholly  undefined.   During  1958 
and  1959  our  studies  at  Children's  Hospital  and  Junior  Village  helped  define 
the  relative  importance  of  adenoviruses,  para-influenza  viruses,  and 
influenza  viruses  in  causing  lower  respiratory  illnesses  of  childhood.   The 
data  suggested  that  as  much  as  40  percent  of  croup  bronchiolitis  and  pneumonia 
were  explained  by  these  viruses.   In  1960,  using  more  sensitive  methods,  we 
were  able  to  explain  a  much  larger  percentage  of  such  illnesses,  chiefly 
because  we  were  now  able  to  assess  the  very  significant  contributions  of 
respiratory  syncytial  virus  (RS)  to  the  respiratory  disease  problem.   Early 
in  the  year  large  outbreaks  of  RS  virus  were  intensively  studied  both  at 
Children's  Hospital  and  Junior  Village.   Over  80  strains  of  RS  virus  were 
isolated  from  children  with  pneumonia  and  60  percent  with  bronchiolitis 
yielded  RS  virus,  whereas  virus  was  recovered  from  less  than  one  percent 
of  comparable  control  patients  without  respiratory  illness. 

Retrospective  analysis  of  serologic  surveys  of  respiratory  illnesses 
in  Children's  Hospital  since  1957  suggested  that  perhaps  20  percent  of  all 
lower  respiratory  illnesses  observed  during  the  last  three  years  was  due  to 
RS  virus.   Thus,  considering  the  contributions  of  adenoviruses,  para- 
influenza viruses,  influenza  viruses,  and  "PAP"  virus  it  now  appears  that 
50  to  60  percent  of  the  more  severe  respiratory  illnesses  of  young  children 
can  now  be  explained  and,  hopefully,  controlled.   Except  for  influenza  virus 
(which  contributed  probably  less  than  5  percent  of  the  total),  the  LID 
respiratory  virus  unit  personnel  played  key  roles  in  the  discovery  of  the 
first  representatives  of  each  of  the  other  virus  groups  -  adenovirus,  para- 
influenza, and  RS.   Delineation  of  still  undefined  viral  causes  of  the 
respiratory  disease  syndrome  represents  the  major  challenge  to  respiratory 
disease  investigators  for  196i. 

During  1960  several  experimental  but  commercially  prepared  killed 
vaccines  containing  various  combinations  of  adenoviruses  (6  types),  para- 
influenza viruses  (3  types) ,  and  Coxsackie  B  viruses  (5  types)  were  tested 
in  Junior  Village.  The  evidence  suggests  that  while  modestly  antigenic,  the 
vaccines  had  insufficient  potency  to  be  regarded  as  satisfactory  for  larger 
scale  studies. 

PRIMARY  ATYPICAL      The  etio logic  role  of  PAP  (Eaton's  virus)  in  primary 
PNEUMONIA  atypical  pneumonia  suggested  earlier  by  Eaton  and  Liu, 

was  finally  fully  established  in  1960.   In  cooperation 
with  the  Bureau  of  Medicine,  USN,  the  continuing  "epidemic"  of  virus  pneumonia 
in  Marine  recruits  at  Parris  Island  was  studied  in  several  ways.   Serological 

28 


studies  showed  that  51  percent  of  530  pneumonia  cases  had  antibody  rises  to 
PAP  virus;  only  six  percent  revealed  contemporary  infection  with  adeno- 
viruses.  Serologic  studies  of  infection  showed  PAP  virus  to  be  much  more 
common  than  disease;  approximately  30  recruits  were  infected  for  each  case 
of  pneumonia,  information  vitally  important  to  fuller  comprehension  of  the 
natural  history  of  this  important  virus. 

In  1959  treatment  of  Parris  Island  pneumonia  cases  with  broad  spectrum 
antibiotics  (tetracyclines)  appeared  to  reduce  the  severity  and  the  duration 
of  the  Eaton  pneumonias.   In  1960  the  efficacy  of  a  new  tetracycline  drug, 
demethylchlortetracycline,  was  tested  in  a  well-controlled  double  blind  study 
including  290  pneumonia  patients.   The  drug  greatly  reduced  the  severity  and 
duration  of  pneumonitis  and  fever  in  those  shown  to  have  serologic  responses 
to  PAP  virus.   These  findings,  based  on  accurate  laboratory  diagnosis,  fully 
confirm  earlier  but  controversial  reports  of  the  efficacy  of  tetracyclines 
in  atypical  pneumonias.   It  also  adds  further  support  to  the  importance 
of  the  Eaton  virus  as  a  cause  of  virus  pneumonia. 

An  additional  link  in  the  chain  of  evidence  establishing  the  PAP 
virus  as  an  important  cause  of  pneumonia  was  achieved  recently  in  collabora- 
tive studies  with  the  Laboratory  of  Clinical  Investigation,  NIAID.   Volunteers 
inoculated  intranasally  with  PAP  virus  grown  in  tissue  cultures  reacted  with 
a  wide  gamut  of  respiratory  signs  and  symptoms,  including  pneumonitis 
characteristic  of  PAP. 

COMMON  COLDS       Recent  studies  have  served  to  clarify  and  enlarge 
AND  VIRUSES        existing  concepts  of  the  etiology  of  common  mild  respira- 
tory illnesses  in  adults.   It  is  now  quite  clear  that 
instead  of  a  few  specific  closely  related  viruses,  numerous  viruses  belonging 
to  different  groups  each  contribute  in  part  to  the  syndrome  called  the 
"common  cold."   Thus  the  newer  viruses  (adenoviruses,  para-influenza  viruses, 
respiratory  syncytial  virus  and  others),  together  with  older  agents  (influenza 
viruses  and  certain  bacteria),  each  contribute  only  a  small  proportion  of  the 
milder  respiratory  ailments  of  adults.   They  contribute  a  larger  segment  of 
more  serious  diseases,  particularly  in  children.   Very  recent  reports  of 
common  cold  viruses  from  England,  together  with  the  prior  reports  of  agents 
with  somewhat  similar  properties  in  this  country,  served  to  focus  our 
attention  on  these  viruses  in  1960.   Together  with  investigators  elsewhere, 
it  was  found  that  most,  if  not  all,  of  these  agents  -  the  British  HGP  and 
FEB,  the  American  2060,  JH,  Coe  and  PETT  viruses  which  grow  selectively  and 
rather  "fussily"  in  human  epithelial  cell  lines,  really  represent  "fastidious" 
enterovirus  strains  which  have  (as  do  almost  all  Coxsackie  A's  and  some 
ECHO  viruses)  special  growth  requirements.   These  viruses,  as  do  a  number 
of  still  unclassified  agents  found  in  Junior  Village  during  the  past  several 
years,  have  properties  very  similar  to  the  Coxsackie  A  viruses;  indeed, 
several  have  been  shown,  on  the  basis  of  serologic  markers  and/or  by 
suckling  mouse  pathogenicity,  to  be  indistinguishable  from  Coxsackie  A 
viruses. 

NEW  SEROLOGICAL      The  laboratory  section  of  the  Epidemiology  Section 
TEST  PROCEDURES      concentrated  on  the  development,  application  and 

evaluation  of  i_n  vitro  test  procedures  for  the  identi- 
fication of  new  viruses  as  well  as  for  detecting  virus  infection  as  expressed 

29 


in  antibody  responses.   Thus,  using  convention.!  complement  fixation  (CF) 
and  newly  developed  hemagglutination  inhibition  (HI)  procedures  it  has  been 
possible  for  our  group  to  type  thousands  of  virus  isolates  belonging  to  the 
adenovirus,  myxovirus ,  enterovirus,  and  reovirus  groups.   As  was  true  during 
the  past  several  years,  LID  in  1960  again  described  and  characterized  more 
new  representatives  of  these  viruses  than  all  other  virus  laboratories  in 
the  world  combined.   This  was  made  possible  during  1960  because  each  of 
our  various  virus  research  units  contributed  new  diagnostic  techniques. 
One  group  developed  additional  specific  HI  procedures  for  identifying 
adenoviruses  and  adenovirus  infections;  and  for  reoviruses  and  enteroviruses 
as  well.   Similarly,  another  group  developed  tissue  culture  procedures  for 
isolating  Eaton's  PAP  virus,  while  others  not  only  discovered  several  "new" 
mouse  viruses  in  tumor  virus  study  systems,  but  developed  serological 
procedures  for  recognizing  their  presence. 

SEROLOGIC  REAGENTS       But  the  availability  of  simplified  procedures  are 

of  very  little  use  unless  the  necessary  reagents 
are  also  available.   Although  many  virus  research  laboratories  could  do 
the  tests,  few  laboratories  are  able  to  produce  the  necessary  reagents.   The 
magnitude  and  cost  of  producing  and  certifying  them  promises  to  continue  to 
exceed  any  possible  resources  available.   This  fact  has  had  a  very  depressing 
effect  on  research  efforts  aimed  at  the  study  of  viruses  as  causes  of  disease, 
and  serves  as  yet  another  deterrent  to  early  delineation  of  the  common  virus 
diseases  as  public  health  problems.   Consequently,  with  the  help  of  NINDB 
and  Microbiological  Associates,  LID  in  1959  and  1960  accepted  responsibility 
to  develop  and  evaluate  more  than  a  hundred  commercially  produced  virus 
antigens.   LID,  of  course,  has  been  active  in  the  certification  of  virus 
prototypes  and  furnishes  many  to  the  Virus  Registry  of  the  American  Type 
Culture  Collection.   It  is  also  collaborating  with  the  Enterovirus  and 
Adenovirus  national  committees  in  setting  up  standards  for  large  scale 
production  of  certified  antiserums  for  serotyping  and  classification  of 
viruses,  perhaps  the  highest  priority  need  of  all  virus  laboratories  con- 
cerned with  human  infection  and  disease. 

UNOFFICIAL  WORLD  REFERENCE       Wholly  through  the  operation  of  circum- 
LABORATORY  FOR  VIRUSES  stances,  the  Virus  Section  of  LID  has  become 

virtually  the  chief  (in  many  instances  only) 
reference  laboratory  for  many  of  the  newer  viruses,  including  adenoviruses 
(about  30  human  and  several  animal  serotypes),  myxoviruses  (five  new  para- 
influenzas occurring  in  three  species),  reoviruses  (three  serotypes  In  four 
species) ,  many  of  the  newer  and  some  older  enteroviruses  (5  -  10) ,  salivary 
gland  viruses  (from  four  species),  and  new  mouse  viruses  (six),  the  latter 
frequently  found  in  tumor  virus  study  systems. 

Until  virus  reagents  desperately  needed  for  many  extremely  common 
viruses  are  made  available  either  commercially,  through  government  agencies, 
or  both,  LID  as  the  sole  custodian  of  many  of  these  agents  cannot  avoid 
responsibility  for  assisting  other  excellent  virus  laboratories  to  identify 
their  viruses,  and  on  a  pro-tem  basis  at  least  for  keeping  order  in  the 
general  virus  field.   Unfortunately  it  has  no  specific  commitment  to  provide 
such  services  and  even  worse,  no  specific  budget  to  cover  them,  so  that  the 
involuntary,  constantly  growing  and  unavoidable  service  functions  must  be 
done  at  the  expense  of  research  missions. 

30 


However,  it  must  be  admitted  that  the  sampler  virus  diagnostic  tech- 
niques and  the  availability  of  a  complete  supply  of  viral  reagents  in  the 
laboratory  (developed  out  of  necessity)  facilitate  not  only  epidemiologic 
studies  of  naturally  occurring  virus  infection  but  also  enable  it  to  evaluate 
the  significance  of  the  data  furnished  by  other  laboratories  who  come  for 
technical  assistance. 

PROBLEMS  OF  CANCER       Studies  of  cancer  viruses  can  be  subdivided  into 
VIRUSES  several  categories:   (a)  Laboratory  studies  of  the 

properties  of  cancer  viruses  and  development  of 
laboratory  tools  for  detecting  and  working  with  them;  (b)  field  studies  of 
the  behavior  in  nature  of  those  tumor  viruses  for  which  suitable  detection 
tests  are  available;  (c)  studies  of  extraneous  viruses  ("background  noise") 
now  preventing  high  caliber  virologic  practice  in  the  study  of  animal  tumor 
viruses  and  obscuring  interpretation  of  nearly  all  current  observations  on 
them;  and  (d)  the  study  of  general  virus  experiences  in  relation  to  human 
cancer  -  the  "background  noise"  in  the  human  cancer  problem  -  which  must  be 
done  eventually  if  the  role  of  viruses  in  human  cancer  is  to  be  defined. 

The  approach  to  these  various  interdependent  studies  is  based  on  the 
following  beliefs:   1)  That  the  conventional  methods  of  standard  virology 
must  be  applied  to  cancer  virus  research  if  significant  progress  is  to  be 
made;  2)  the  study  of  cancer  viruses  obviously  cannot  be  separated  from 
general  virology;  and  3)  that  the  "biologic  point  of  view"  rather  than 
attitudes  fostered  by  preoccupation  with  categorical  disease,  represents  the 
best  approach  to  a  real  understanding  of  the  natural  history  of  cancer 
viruses  just  as  it  does  to  other  viruses. 

MOUSE  POLYOMA      New  in  vitro  survey  tools  developed  during  1959  (CF,  HI, 
CANCER  VIRUS       and  MAP)  were  evaluated  and  applied  in  1960  in  studies 

of  polyoma  virus  growth  and  excretion,  its  experimental 
epidemiology,  and  its  natural  history.   This  interesting  and  versatile  cancer 
virus  causes  tumors  not  only  in  all  strains  of  Mus  mus cuius,  but  also  in 
hamsters,  rats,  rabbits,  and  guinea  pigs  (Stewart  and  Eddy).   Of  equal 
interest  is  the  fact  that  it  can  be  studied  and  surveyed  with  the  same 
facility  as  ordinary  viruses,  such  as  influenza  and  polioviruses.   Virus 
isolation  and  serologic  procedures,  combined  with  epizootiologic  studies 
have  produced  the  following  interesting  observations: 

Polyoma  virus  was  found  to  be  widely  disseminated  in  mouse  colonies 
nearly  everywhere.   Infection  was  found  to  be  more  commonly  present  than 
absent  in  laboratory  strains  raised  in  experimental  or  commercial  laboratories 
and  in  wild  strains  found  in  city  tenements.   However,  the  basic  ecology  or 
natural  cycle  appears  to  exist  in  rural  areas  -  on  farms  and  in  feed  mills 
in  small  towns. 

A  full  year's  surveillance  of  Mus  mus cuius  infestation  and  polyoma 
infection  of  crowded  tenements  in  Harlem  revealed  that  virus  infections 
persisted  without  exception  in  numerous  separate  foci.   Three  epidemiologic 
factors  seem  most  important,  namely,  large  mouse  populations  capable  of 
furnishing  adequate  supplies  of  young  susceptible  mice,  the  extensive  con- 


31 


tamination  of  the  tenement  environment  (virus  v  as  demonstrated  in  sweepings 
from  areas  showing  signs  of  mouse  activity) ,  and  finally  the  overcrowding 
which  insures  the  continuous  and  extensive  use  of  communal  nesting  areas 
(also  demonstrated  to  be  contaminated  by  virus)  .   Apartment  houses  having 
smaller  and  less  dense  mouse  infestation  were  generally .free  of  infection 
and  remained  so  during  the  study. 

Systematic  studies  of  polyoma  in  rural  environments  were  undertaken 
during  the  last  quarter  of  1960.   However   it  appears  from  preliminary 
data  that  here  may  be  found  the  basic  natural  cycle  of  mouse  polyoma.  Mus 
mus cuius  infestation  and  polyoma  infection  of  Mus  was  found  to  be  most  intense 
in  feed  granaries  on  the  farm  and  in  cereal  grain  storage  elevators  in  mills. 
As  many  as  30  per  cent  of  several  hundred  mice  trapped  in  these  environs 
showed  persistent  evidence  of  polyoma  infection,  many  of  them  apparently 
excreting  virus  in  their  urine.   The  virus  has  been  found  on  cereal  grains 
in  the  vicinity  of  mouse  nesting  areas,  which  appear  to  be  very  numerous  in 
the  granaries  so  far  examined.   The  actual  extent  of  cereal  grain  contamina- 
tion by  mouse  excreta  containing  polyoma  and  no  doubt  other  microbes  must 
still  be  evaluated;  however,  present  evidence  suggests  that  it  probably  is 
very  extensive,  if  not  appalling. 

Since  natural  infection  of  wild  mice  is  not  limited  to  polyoma  virus, 
but  includes  a  number  of  other  viruses  known  or  suspected  to  infect  man  and 
domestic  animals,  the  extension  of  these  preliminary  findings  will  likely 
prove  very  interesting. 

EXTRANEOUS  VIRUSES  IN       In  1960  the  "background  noise"  problem  in  cancer 
CANCER  VIRUS  STUDIES        virus  research  grew  to  almost  "deafening"  propor- 
tions and,  in  the  opinion  of  LID  virologists, 
constitutes  the  number  one  obstacle  to  intelligent  and  truly  effective 
research  on  cancer  viruses. 

Nearly  every  animal  tumor  virus  system  currently  under  study  was 
shown  to  be  contaminated  with  extraneous  agents  and  several  viruses  widely 
proclaimed  as  "tumor"  viruses  turned  out  to  be  fellow  traveling  ordinary 
viruses.   To  list  a  few  examples:   Friend  leukemia  was  found  contaminated 
with  polyoma  and  mouse  adenovirus;  Gross  leukemia  by  polyoma,  K  virus  and 
mouse  adenovirus;  Schwartz  leukemia  with  polyoma,  K  virus  and  mouse  adeno- 
virus; Moloney  leukemia  with  mouse  hepatitis  and  mouse  reovirus;  the  polyoma 
itself  became  contaminated  with  mouse  adenovirus,  hepatitis  and  salivary 
gland  viruses . 

LID  virologists  showed  that  the  "seeds"  of  the  "background  noise" 
viruses  are  commonly  present  in  the  animals  used  for  the  induction  of  tumors, 
and  of  course  in  the  subsequent  passage  materials  as  mentioned  above.   The 
extraneous  viruses  most  commonly  encountered  in  cancer  systems  were  the 
newer  ones,  such  as  polyoma,  K  virus,  mouse  reovirus  and  adenovirus;  but 
this  in  part  may  be  due  to  newly  developed  easily  applied  survey  tools  for 
these  agents.   Other  viruses  encountered  less  often  (perhaps  because  of 
comparatively  less  sensitive  tools)  were  mouse  hepatitis,  mouse  salivary 
gland  virus,  the  newly  discovered  "thymic  agent"  (TA) .   Except  in  newborns, 
most  of  these  viruses  occur  subclinically  and  latently. 

32 


MEDICAL  MYCOLOGY       Investigations  on  pathogenic  fungi  have  included  broad 

fields  of  research  and  although  definitive  goals  have 
been  reached  in  most  of  them,  all  will  be  continued  in  order  to  further 
exploit  productive  lines  of  investigation.   In  most  cases  new  or  additional 
species  of  pathogenic  fungi  will  be  used  in  investigations,  or  techniques 
will  be  altered  to  permit  further  development  of  experimental  studies. 

The  antibiotic  X-5079C  was  found  to  be  fungistatic  but  not  fungicidal 
and  its  apparent  low  degree  of  jin  vitro  activity  due  to  its  decay  in  culture 
medium.   The  yeast  form  of  Histoplasma  capsulatum  is  much  more  sensitive  to 
X-5079C  than  the  mycelial  form  and  an  assay  method,  sensitive  to  1  ug/ml 
using  1U  capsulatum,  was  developed.  X-5079C  has  low  toxicity  for  HeLa  cells 
and  is  active  against  H^  capsulatum  grown  in  HeLa  cells. 

A  second  strain  of  Coccidioides  immitis  has  been  converted  to  serial 
culture  in  the  spherule  form.   Quantitative  measurements  show  the  ability  of 
various  carbon  and  nitrogen  sources  to  support  growth  of  spherule  and  mycel- 
ial forms  of  strain  M-ll  of  C^  immitis .  Only  mannose  is  utilized  as  readily 
as  glucose  by  spherules.  Mannose  and  fructose  support  growth  of  the  mycelial 
form  as  well  as  does  glucose.   A  substrate  which  preferentially  supports 
growth  of  the  spherule  form  was  not  found  in  this  study. 

Spherules  were  utilized  to  immunize  mice.  An  increased  survivor  rate 
in  the  immunized  mice  was  noted  after  challenge  with  a  lethal  infecting  dose 
and  an  earlier  clearance  of  organs  (negative  cultures)  in  the  immunized  mice 
was  observed  after  challenge  with  a  sublethal  dose. 

CRYPTOCOCCUS      By  titrating  and  plating  out  organs  of  experimentally 
NEOFORMANS        infected  mice,  it  was  found  that  several  minutes  after 

Cryptococcus  neoformans  was  injected  either  intravenously 
or  intracerebrally  into  mice,  the  largest  numbers  of  yeast  cells  had  been 
retained  in  the  lungs.   The  fungus  population  in  the  lung  then  decreases  and 
2-3  days  after  infection  multiplication  in  the  brain  is  apparent.   Although 
the  interval  from  infection  to  death  of  infected  mice  varies  with  the  strain 
of  C±   neoformans,  the  numbers  of  yeast  cells  per  gram  of  brain  tissue  are 
approximately  the  same  regardless  of  strain. 

Studies  of  the  saprophytic  occurrence  in  natural  habitats  of  fungi 
which  cause  mycoses  have  continued.   CryptPcoccus  neoformans  has  been  iso- 
lated from  many  additional  collections  of  pigeon  guano.   When  this  material 
is  collected  from  old  pigeon  nests  and  from  roosting  sites  in  hay  mows  of 
barns  and  upper  floors  of  buildings,  Histoplasma  has  never  been  found.  There 
is  increasing  circumstantial  evidence  that  a  presently  unstudied  pneumonic 
form  of  cryptococcosis  has  occurred  in  men  heavily  exposed  to  such  material 
and  that  such  epidemics  have  been  erroneously  diagnosed  histoplasmosis. 

EMMONS I A  CRESCENS       In  collaboration  with  an  investigator  at  the  Rocky 

Mountain  Laboratory  a  new  species,  Emmons i a  crescens, 
was  described.  This  fungus  differs  from  the  first  species  of  Emmonsia 
(E.  parva)  in  vivo  and  Jji  vitro  at  37°  C  by  its  multinucleate  condition 
(instead  of  uninucleate),  its  ability  to  produce  the  in  vivo  form  in  vitro 
at  37°  C,  and  its  greater  size.  E.  parva  conidia  when  inhaled  or  incubated 

33 


at  37°  C  increase  in  diameter  from  2  -  4  u  to  4J0  -  480  u.   This  10  -fold 
increase  in  volume  of  a  single  cell  is  very  unusual  in  the  fungi. 

STAPHYLOCOCCUS  STUDIES       It  has  been  established  that  staphylococcal 

penicillinase  is  associated  with  particulate 
material  in  the  cell  and  thus  an  explanation  has  been  given  for  the  refrac- 
toriness in  preparation  of  this  enzyme  by  conventional  methods.   New  and 
more  potent  inhibition  of  Staph,  penicillinase  have  been  uncovered  and  the 
hope  remains  and  is  heightened  for  the  ultimate  finding  of  a  chemically  use- 
ful inhibitor.   Further,  sea  water  has  been  found  to  possess  strong  inhibi- 
tory activity  against  both  penicillin-sensitive  and  penicillin-resistant 
staphylococci  (phage  type  80/81). 

Real  progress  has  been  reported  in  the  understanding  of  iron  metabolism 
in  the  staphylococci.   As  a  direct  result  of  continuing  work  dealing  with 
mechanisms  of  the  development  of  non-specific  immunity  and  in  particular  the 
function  of  the  iron-transporting  protein  of  plasma,  siderophilin,  fundamen- 
tal observations  on  the  effects  of  iron  deficiency  on  the  growth  and  metabo- 
lism of  S_^   aureus  have  been  reported.   Work  on  the  biology  of  the  staphy- 
lococci so  long  neglected  during  the  "antibiotic  era"  is  cardinal  to  effec- 
tive new  therapy  of  staphylococcal  infections. 

STREPTOCOCCAL  M       Progress  has  also  been  made  on  the  search  for  better 
PROTEIN  methods  of  isolation  and  purification  of  M  protein  of 

streptococci.   These  results  are  of  obvious  importance 
in  the  understanding  of  Group  A  streptococcal  virulence.   Further,  highly 
interesting  observations  have  been  reported  dealing  with  the  mechanism  and 
significance  of  the  long-chain  test  for  determination  of  anti-streptococcal 
immunity . 

BACTERIAL  METABOLISM       Real  understanding  of  the  intimate  mechanisms  of 

energy  metabolism  in  Hydrogenomonas  in  particular 
and  other  bacteria  and  higher  forms  in  general  is  closer  as  a  result  of  work 
performed  in  this  section  this  year.   In  an  enormously  complicated  field, 
progress  has  occurred  in  the  definitions  of  the  essential  reactions. 

Detoxification  studies  on  potentially  useful  chemotherapeutic  agents 
have  continued  and  new  and  promising  leads  have  been  uncovered  for  agents 
active  against  bacteria,  fungi,  parasites  and,  it  should  be  added,  against 
cancer  as  well.   Several  of  the  aforementioned  detoxified  compounds  have 
passed  preliminary  screening  processes  performed  by  the  Cancer  Chemotherapy 
Center. 

Pinpointing  of  the  enzymic  locus  of  discrimination  among  hydrogen 
isotopes  by  pseudomonas  has  been  reported  this  year.  The  area  lies  in 
formic  acid  metabolism. 


34 


LABORATORY  OF  CLINICAL  INVESTIGATION 


Summary 1 

10  (c)  -  Office  of  the  Chief 5 

12  (c)  -  Respiratory  Viral  Diseases 6 

13  (c)  -  Antimicrobial  Drug  Therapy 10 

14  (c)  -  Staphylococcal  Disease 13 

15  (c)  -  Hepatitis  and  Mononucleosis 16 

16  (c)  -  Aseptic  Meningitis 17 

17  (c)  -  Allergy- Immunology 19 

18  (c)  -  Enteric  Diseases 28 

19  (c)  -  Clinical  Investigations  on  Helminthic 

Diseases 31 

20  (c)  -  Clinical  Investigations  in  Protozoan 

Infections  and  Diseases 37 

21  (c)  -  Cystic  Fibrosis  of  the  Pancreas 44 

22  (c)  -  Basic  Biochemical  Studies 45 

23  (c)  -  Systemic  Fungal  Disease 50 

24  (c)  -  Sarcoidosis 54 

25  (c)  -  Pyelonephritis 55 

26  (c)  -  Urinary  Tract  Viruses 56 

27  (c)  -  Biochemical  Studies  on  Staphylococcal 

Cell  Walls 59 

28  (c)    -   Pathogenesis   of  Viral   Infections 61 


Summary  of  Research  Progress 

Calendar  Year  1960 

Laboratory  of  Clinical  Investigations 

This  year  has  seen  some  widening  of  the  scope  of  our  research,  largely 
as  a  response  to  enlarging  the  professional  staff.  We  believe  that  a  further 
period  of  growth  will  be  needed  to  staff  the  clinical  service  in  a  manner 
consistent  with  optimum  research  productivity. 

In  the  past  the  wards  have  operated  at  an  occupancy  rate  of  about  65 
percent.  From  the  service  point  of  view,  a  rate  of  85  percent  would  be  most 
acceptable  i.e.  44  instead  of  34  patients.   It  is  estimated  that  the  unit 
could  quite  easily  provide  material  for  at  least  seven  research  sections  (at 
present  there  are  four).  We  hope  to  have  additional  sections  functioning  as 
soon  as  qualified  investigators  can  be  obtained.   Upon  the  removal  of  the  ad- 
ministration offices  to  the  new  building,  additional  space  will  become  avail- 
able for  a  pediatric  section,  presently  viewed  as  our  most  important  defi- 
ciency in  professional  staffing. 

In  connection  with  staffing,  it  is  worth  noting  that  it  is  difficult  to 
obtain  qualified  investigators  to  head  sections.   I  believe  the  evidence  will 
show  that  civil  service  stipends  for  professional  personnel  are  appreciably 
less  that  those  offered  by  the  best  -  paying  medical  schools,  but  better  than 
the  poorer -paying  ones.  Medical  schools  have  a  double  standard  of  employment, 
however,  and  our  salaries  are  most  competitive  with  the  lower  paying,  non- 
clinical appointments.   I  believe  one  can  employ  without  great  difficulty  bio- 
chemists, microbiologists,  etc.,  but  because  of  the  exceedingly  high  returns 
from  the  practice  of  medicine,  the  medical  schools  have  developed  private 
practice  and  consultation  arrangements  for  the  men  of  their  clinical  faculties 
which  reduce  the  deficiency  between  their  base  pay  and  the  return  obtainable 
from  practice.   This  mechanism  is  not  available  to  us. 

I  believe  the  salary  issue  alone  would  not  be  a  crucial  factor  in 
securing  competent  investigators,  however,  there  is  a  feeling  of  reluctance 
among  many  young  investigators  to  accept  government  employment.   The  attitude 
seems  to  have  developed  from  apprehension  concerning  the  large  size  of  the 
operation,  the  comparative  anonymity  of  the  individual,  the  division  of 
authority  among  departmental  heads,  the  civil  service  organization  and  the 
several  administrative  branches  above  the  operating  unit.   Promotion  is 
usually  slow  and  the  salary  increments  are  not  large.  Good  men  can  advance 
much  more  rapidly  out  of  government.   I  don't  believe  the  government  is 
adequately  authorized  to  recognize  talented  young  investigators  who  in 
university  life  often  achieve  high  faculty  rank.   It  should  also  be  recorded 
that  decisions  concerning  the  salaries  which  can  be  offered  are  also  very 
slowly  reached. 

It  is  my  view  that  the  clinical  sections  of  the  National  Institutes  of 
Health  will  best  achieve  their  goals  by  attracting  the  kind  of  men  who  serve 
on  medical  school  faculties.   I  think  we  should  train  many  who  take  those 
positions.   I  believe  our  senior  staff  should  also  interchange  with  academic 
medicine.  Mechanisms  to  protect  retirement  and  to  permit  such  exchanges 
without  loss  of  security  should  be  made.   Such  a  thing  as  membership  in  the 


Teachers  Insurance  Annuity  Association  as  an  alternative  to  the  civil  service 
annuity  program  would  be  worthy  of  consideration. 

Professional  Staff 

We  are  completing  the  year  with  13  clinical  associates  and  9  senior 
staff  members.   In  July,  Dr.  Donald  Kayhoe  left  the  staff  and  in  October  Dr. 
Howard  Goodman  began  a  joint  appointment  with  a  Section  divided  between  this 
laboratory  and  the  Laboratory  of  Immunology.  Mr.  John  Bozicevich  also  joined 
this  staff  in  July,  by  transfer  from  the  Laboratory  of  Immunology.   (We  regret 
to  announce  the  anticipated  retirement  of  Mr.  Bozicevich  early  in  1961  after 
30  years  of  service). 

Research  Program 

Infection  of  volunteers  with  respiratory  viruses.  An  extensive 
clinical  study  of  acute  viral  respiratory  disease  was  begun  this  year  in  as- 
sociation with  staff  members  of  the  Laboratory  of  Infectious  Diseases.  The 
initiation  of  this  project  required  much  work  but  with  the  unstinting  assi- 
stance of  Dr.  Clifton  Himmelsbach,  Associate  Director,  the  Clinical  Center, 
very  satisfactory  arrangements  have  been  made  to  transfer  to  the  Clinical 
Center,  Federal  prisoner-volunteers  for  study.   This  has  been  done  with  the 
permission  and  considerable  assistance  of  Mr.  James  Bennett,  Director,  and 
Dr.  Harold  Janney,  Chief  Physician  of  the  Bureau  of  Prisons,  Dept.  of  Justice. 
A  team  of  custodial  officers  has  also  been  assigned  to  the  Clinical  Center  by 
the  Bureau  of  Prisons  to  oversee  the  volunteers.   The  volunteers  have  uni- 
formly cooperated  with  the  program  despite  some  extended  periods  of  room 
isolation  to  prevent  spread  of  infection,  frequent  blood-letting,  and  other 
inconveniences.  Many  administrative  arrangements  have  been  developed  so  that 
the  program  has  worked  increasingly  smoothly. 

The  results  so  far  justify  the  investment  in  money  and  effort.   It  has 
been  possible  to  produce  in  human  volunteers  a  rather  uniform  "cold"  with 
respiratory  syncytial  virus.   It  may  occur  without  respect  to  preinfection 
immunity,  but  the  subsequent  rise  in  complement -fixing  antibody  appears  to 
correlate  with  severity  of  illness.  Forty-six  men  have  so  far  participated 
in  this  study. 

Approximately  24  other  volunteers  have  participated  in  studies  with 
para -influenza  4  virus  or  Eaton  (primary  atypical  pneumonia)  virus.   Future 
studies  are  planned  with  human  influenza  virus  and  with  the  recently  defined 
group  of  REO  viruses.  We  believe  that  the  promise  of  this  program  is  great 
and  that  it  may  continue  for  some  years. 

New  antifungal  drug.   Beginning  about  4  years  ago,  Dr.  Chester  Emraone 
began  studies  on  an  antifungal  drug  produced  by  Hoffman  La  Roche,  designated 
as  RO-2.   This  agent,  an  antimicrobial,  was  found  to  be  the  most  active 
material  Dr.  Emmons  had  ever  tested  in  vitro  and  in  animals  against  several 
of  the  pathogenic  fungi,  notably  histoplasmosis  and  blastomycosis. 

In  the  intervening  years,  the  agent  has  been  studied  clinically  by  Dr. 
Utz  and  his  staff.   Now  30  patients  have  been  treated.   Extremely  favorable 
results  have  been  observed  in  several  patients  very  severely  ill  with  these 
diseases.  From  the  standpoint  of  therapeutic  activity,  it  is  the  judgment 
that  this  agent  is  the  best  available  for  blastomycosis  and  histoplasmosis. 

Z 


f>9." 


During  the  studies  it  was  noted  that  the  agent  produced  unusual  hepatic 
changes.   It  was  found  that  the  dye,  bromsulfhalein,  normally  rapidly  trans- 
ferred from  the  blood  to  the  bowel  by  the  liver,  was  retained  in  high  con- 
centration in  the  blood  in  patients  treated  with  RO-2.   This  effect  appeared 
in  a  day  or  two  after  start  of  treatment,  before  tissue  changes  would  likely 
occur,  suggesting  competition  of  the  new  drug  for  the  liver  excretory 
mechanism  for  bromsulfhalein.  After  treatment  was  stopped  dye  excretion 
promptly  returned  to  normal  or  nearly  normal.  Liver  biopsy  has  revealed 
changes  indicative  of  minor  hepatic  damage  in  some  cases.   Because  of  the 
great  importance  of  having  a  drug  in  addition  to  the  relatively  toxic  agent, 
amphotericin  B,  for  the  treatment  of  fungal  diseases,  this  new  agent  continues 
under  investigation. 

Studies  on  volunteers  infected  with  simian  malaria.  Following  the 
discovery  by  Dr.  Eyeles  and  Dr.  Coatney  of  the  infectivity  of  P.  cynomolgi 
bastianellii  for  man,  several  inmates  at  the  Federal  prison  in  Atlanta 
volunteered  for  exposure  to  this  agent  with  the  establishment  of  infection 
in  several.   The  need  for  careful  clinical  characterization  of  this  disease 
in  man  led  to  the  transfer  of  two  infected  volunteers  to  the  Clinical  Center. 
Both  men  developed  acute  malaria  which  was  carefully  studied  throughout  Its 
course.  All  results  are  not  yet  available,  but  significant  alterations  In 
urinary  steroid  excretion  were  detected,  an  unusual  elevation  of  serum 
cholesterol  occurred  in  another,  and  liver  lesions  not  previously  described, 
resembling  but  not  identical  with  exoerythrocytic  phase  parasites,  were 
demonstrated  in  both  patients.  This  interesting  program  will  be  continued. 

Studies  on  penicillins  and  penicillinase.  Previous  work  by  Dr. 
Steinman  had  defined  nutritional  requirements  for  penicillase  production  by 
staphylococci  and  many  parameters  of  its  interaction  with  benzyl  penicillin 
(penicillin  G) .   During  the  year  English  investigators,  working  with  the 
stripped  molecule  of  penicillin,  6-amino-penicillanic  acid,  produced  a  new 
compound  largely  resistant  to  destruction  by  penicillinase.  Working  with 
this  and  several  other  penicillin  derivatives  Dr  .teinman  has  found  that 
resistance  to  penicillinase  is  greatly  influent  j  t   steric  positions  of 
ethoxy  groups  on  the  side  chain  and  that  failure  t  be  destroyed  by  peni- 
cillinase is  associated  with  greater  penicillinase  '.nducing -capacity . 
Perhaps,  most  significantly,  Dr.  Steinman 's  work  has  added  to  the  now  sub- 
stantial indirect  evidence,  that  the  major  reason  for  present  resistance  of 
staphylococci  to  penicillin  is  the  enormous  capacity  of  these  micro-organism 
to  produce  penicillinase  upon  contact  with. even  low  doses  of  penicillin. 

Clinical  studies  with  the  penicillinase-resistant  penicillin, 
dimethoxy phenyl -penicillin,  have  revealed  it  to  possess  resistance  to 
penicillinase  in_  vivo,  and  that  it  is  a  powerful  and  effective  anti- 
staphylococcal  drug.  After  treatment  of  some  patients  for  periods  of  3  to  5 
months  no  penicillin-resistant  staphylococci  have  been  isolated.   It  has  long 
been  considered  that  chronic  staphylococcal  infection  resembles  tuberculosis 
and  for  the  first  time  it  appears  that  an  agent  is  available  which  can  be 
employed  for  extended  periods  of  treatment  without  loss  of  effect  such  as 
isonicotinic  acid  hydrazide  in  tuberculosis.   If  long  term  therapy  can  thu6 
be  regularly  given  an  enormous  benefit  will  accrue  to  thousands  ill  with 
chronic  staphylococcal  disease. 


Ascites  induced  In  mice  by  Injection  of  adjuvant  mixtures.   In  the 
past  year  Miss  Lieberman  has  continued  to  study  the  ascites  induced  in  mice 
by  the  injection  of  adjuvant  mixtures.   This  discovery  provided  a  much  needed 
laboratory  procedure  for  producing  antibodies  of  a  wide  variety  and  a  means 
of  evaluating  antigens.   Recently,  her  interest  has  been  focused  on  the 
pathology  and  abnormal  physiology  of  the  lesion.   It  has  been  found  that 
strain  differences  are  associated  with  differences  in  susceptibility  to 
ascites.   Since  ascites  was  found  associated  with  a  local  plasma  cell  re- 
action, which  in  some  strains  of  mice  went  on  to  plasma  cell  tumors,  many 
implications  toward  problems  in  neoplasis  have  also  been  raised.  These 
studies  have  become  the  basis  for  biochemical  and  pathologic  studies  with 
other  Institutes. 

Allergy  and  Immunology.   Dr.  Nasou  and  Mr.  Bozicevich  have  shown  that 
patients  with  hypogammaglobulinemia  possess  low,  but  definitely  measurable, 
levels  of  antibody  to  the  enteric  viruses.   The  implication  of  this  finding, 
in  view  of  the  normal  resistance  of  these  patients  to  viral  infections,  is 
that  extremely  low  levels  of  specific  antibody  may  provide  adequate  resis- 
tance against  viral  diseases.   Subsequent,  unpublished  studies  have  indicated 
that  these  patients  will  develop  circulating  antibodies  to  Salk  polio 
vaccine. 

The  modification  of  the  bentonite  flocculation  test  for  the  detec- 
tion of  gamma  globulin  promises  to  provide  the  practicing  physician  with  an 
accurate,  convenient  laboratory  aid  in  the  diagnosis  of  hypogammaglobulinemia. 
In  contrast  to  the  electrophoretic  method,  the  results  with  the  bentonite 
test  can  be  known  to  the  physician  within  a  few  minutes  of  arrival  of  the 
specimen  to  the  laboratory.   Other  modifications  of  this  technique  will  also 
give  the  levels  of  albumin  and  other  protein  constituents  of  blood  and  other 
fluids  without  resort  to  the  more  cumbersome  method  of  electrophoresis. 

The  DNA-bentonite  test  for  systemic  lupus  erythematosus  has  also  been 
developed  in  the  past  year.   This  test  measures  the  antibody  in  lupus  serum 
which  is  directed  against  nuclear  material.   The  specificity  of  this  test  is 
greater  than  any  previously  described  test  for  lupus.   This  test,  however,  is 
positive  primarily  in  those  patients  with  active  disease  and  only  rarely  is 
positive  when  the  disease  is  in  remission.  A  modification  of  this  test,  the 
nucleoprotein-bentonite  test,  has  retained  all  the  attributes  of  the  DNA  test 
in  regard  to  specificity,  while  achieving  a  much  higher  level  of  sensitivity  £01 
cases  in  remission.   These  promise  to  become  important  standard  tests  In  the 
diagnosis  of  systemic  lupus  erythematosus. 

Cystic  Fibrosis  of  the  Pancreas.   Despite  the  commonly  accepted  point 
of  view  that  antibiotics  are  helpful  in  this  disease,  observations  of  naso- 
pharyngeal cultures  failed  to  reveal  any  appreciable  effect  of  antimicrobial 
treatment  on  Staphylococcus  aureus .   This  does  not  negate  a  possible  clinical 
benefit  but  does  appear  to  minimize  its  value.   It  was  coincident  ally 
observed  that  Escherichia  coli  was  not  present  in  the  nasopharyngeal  flora 
of  any  child  over  the  age  of  eight.   This  interesting  observation  will 
receive  further  study. 


Serial  No.    NIAID   -    10    (c) 

1.  LCI 

2.  Office  of  the  Chief 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:   Office  of  the  Chief 
Principal  Investigator:   Dr.  Vernon  Knight 
Other  Investigators:   None 
Man  Years  (calendar  year  1960); 


Total: 

7.0 

Professional: 

1.0 

Other: 

6.5 

Project  Description: 

Objectives: 

To  develop,  direct  and  coordinate  the  program  of  the  Laboratory, 
as  defined  in  the  individual  research  projects;  where  necessary,  to 
re-direct  individual  projects  to  meet  current  needs  and  advances  in 
the  field;  to  align  clinical  projects  with  patient  availability;  to 
provide  an  unexcelled  standard  of  patient  care  for  patients  utilized 
in  research. 

Methods  Employed: 

Organize  available  staff  and  recruit  qualified  personnel,  both 
professional  and  sub-professional,  to  develop  the  program  and  carry- 
out  its  aims;  close  liaison  with  area  medical  societies,  institutions 
and  individuals  for  referral  of  patients  whose  diagnoses  fall  withiu 
the  active  or  proposed  disease  research  categories;  the  highest 
standards  of  patient  care  are  maintained  by  selection  of  qualified 
physicians;  the  development  of  necessary  policies  and  continued  close 
and  direct  supervision  of  patient  care  activities;  professional  con- 
sultant services  to  other  institutes  in  the  area  of  infectious 
diseases;  continued  guidance  of  research  projects  undertaken  by 
younger  investigators;  maintenance  of  staff  morale. 


Part  B  included    Yes  /  /    No  /X/ 


Serial  No.  NIAID  -  12(c) 

1.  LCI 

2.  Infectious  and  Pediatric 

Disease  Services 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 


Part  A 


Project  Title:   Respiratory  Viral  Diseases 

Principal  Investigators:   Dr.  John  P.  Utz 

Dr.  Vernon  Knight 

Other  Investigators:   Dr.  Howard  Kravetz 
Dr.  David  Rifkind 

Dr.  Anderson  Spickard  (from  7/1/60) 
Dr.  Robert  Carpenter  (from  7/1/60) 
Dr.  Hugh  Evans  (from  7/1/60) 
Clarence  F.  Szwed 
Margret  A.  Huber 

Cooperating  Units:   Dr.  Robert  Channock,  LID,  NIAID 
Dr.  Karl  Johnson,  LID,  NIAID 

Man  Years  (calender  year  1960): 
Total:        5.5 
Professional:  3.5 
Other:        2.0 

Project  Description: 

Objectives: 

1.   To  define  clinical  entities  in  relation  to  newly  isolated 
respiratory  viruses. 

2.  Further  to  define  diagnostic  criteria,  pathogenesis,  immune 
response,  persistence,  sites  and  effects  on  tissues  of  virus  in  certain 
infections  of  respiratory  passages  and  mouth. 

3.   To  study  host-parasite  relationships  in  reference  to  sus- 
ceptibility to  chronic  or  recurrent  respiratory  diseases. 

4.   To  improve  clinical  laboratory  techniques  in  the  laboratory 
confirmation  of  respiratory  viral  disease  diagnosis. 


Serial  No.  NIAID  -  12(c) 

5.   To  characterize  in  man  the  clinical  course  and  associated 
virological  and  immunological  phenomena  of  infection  with  selected 
respiratory  viral  agents. 

Methods  Employed; 

1.  Results  of  clinical  observations  and  procedures  are  correlated 
with  bacterial,  mycologic  and  viral  isolations  employing  both  animals  and 
tissue  culture  techniques. 

2.  Clinical  observations  are  made  under  carefully  controlled 
isolation  following  intranasal  infection  with  certain  viral  agents. 
Virus  isolation  and  immunologic  studies  performed  by  recognized  pro- 
cedures . 

Patient  Material  and  Major  Findings; 

1.  The  Virology  Unit  of  the  Infectious  Disease  Service  examined 
a  total  of  1245  specimens  from  patients  under  study.   Virus  was  isolated 
in  73  of  these  specimens,  achieving  etiologic  confirmation  of  the  diag- 
nosis in  a  total  of  35  patients. 

2.  The  status  of  treatment  of  viral  pneumonias  has  been  critically 
evaluated  and  reported. 

3.  Inmates  selected  from  volunteers  from  several  Federal  correc- 
tional institutions  were  infected  or  used  as  controls  in  studies  with 
the  following  viral  agents:   Para- influenza  4,  12  volunteers;  respira- 
tory syncytial  virus  infection,  46  volunteers,  Eaton  (primary  atypical 
pneumonia)  virus,  13  volunteers. 

4.  Clinical  and  virological  infection  occurred  in  a  high  pro- 
portion of  patients  given  RS  virus.  Infection  was  less  frequent' with 
the  other  agents,  apparently  as  a  result  of  low  dosage.  This  matter 
is  receiving  further  study. 

Significance  to  Microbiological  Research: 

Studies  in  the  laboratory  and  the  clinic  of  patients  naturally 
infected  with  respiratory  viruses  will  hopefully  provide  information  on 
pathogenesis,  distinctive  clinical  findings,  effects  of  giving  or  with- 
holding antimicrobic  treatment  as  related  to  the  specific,  known  virus 
involved.   This  approach  also  provides  the  only  opportunity  of  discoverir 
and  defining  new  viruses. 

Studies  in  human  volunteers  may  provide  information  on  the  effect 
of  dose,  route  of  administration,  and  kind  of  virus  on  the  induction  of 
clinical  infection.   They  may  also  indicate  the  role  of  immunity  and 
other  protective  mechanisms  as  a  defense  against  infection.   Such  studies 


-  2  - 


Serial  No.  NIAID  -  12(c) 

would  logically  precede  the  development  of  effective  vaccines  against 
these  diseases.   There  are  approximately  100  different  respiratory 
viral  agents  whose  role  in  human  infection  is  not  adequately  defined. 
Moreover,  increasing  evidence  suggests  that  viruses  may  participate 
with  bacteria  in  the  cause  of  infection.   This  may  be  studied  by  the 
present  methods. 

Proposed  Course  of  Project; 

Studies  will  continue  as  outlined  under  "Objectives"  in  patients 
referred  to  the  Clinical  Center  and  in  other  hospital  or  community  sur- 
veys when  indicated.   This  project  should  be  expanded  because  of  the 
great  importance  of  careful  clinical  studies  on  patients  with  respira- 
tory illnesses  in  order  to  link  these  illnesses  with  viral  agents  that 
are  now  being  isolated  in  a  number  of  laboratories. 

Further  studies  with  the  Eaton,  RS,  and  Para- influenza  viruses 
in  human  volunteers  are  planned.   Human  influenza  and  REO  virus  are 
also  being  considered  for  this  program. 


Part  B  included:        Yes  /X/      No  /  / 


Serial  No.  NIAID  -  12(c) 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B:   Honors,  Awards,  and  Publications 
Publications  other  than  abstracts  from  this  project: 

1.  Utz,  J.  P.:  Pneumonias,  Viral.   Current  Therapy,  1960  ed. 

2.  The  studies  with  RS  virus  were  part  of  a  Clinical  Center  Symposium, 

October  20,  1960,  which  will  be  published  in  Annals  of  Internal 
Medicine. 


-  A 


Serial  No.  NIAID  -  13  (c) 

1.  LCI 

2.  Bacteriology 

3.  Bethesda,  Maryland 
PHS-NIH 

Individual  Project  Report 
Calendar  Year  1960 


Part  A. 


Project  Title:  Antimicrobial  Drug  Therapy 

Principal  Investigator:  Dr.  Vernon  Knight 

Other  Investigators:  Dr.  David  Rifkind 
Margret  Huber 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 

Total:  2.4 
Professional:  1.7 
Other:        0.7 

Project  Description: 

Objectives: 

a)   To  evaluate  an  antimicrobic  drug. 

1.  Effectiveness  in  treatment  of  penicillin-resistant 
staphylococcal  infections. 

2.  Effectiveness  in  treatment  of  other  coccal  infections. 

3.  Appropriate  dosages  and  routes  of  administration. 

4.  Toxic  and  allergic  side  effects. 

5.  Effectiveness  in  treatment  of  the  nasal  carrier  state 
of  staphylococci. 

Methods  Employed: 

Hospitalized  patients  with  staphylococcal  and  streptococcal 
infections  were  evaluated  by  clinical  and  laboratory  methods  for 
response  to  antimicrobics.   The  laboratory  methods  included: 

a)  Isolation  and  identification  of  the  etiological  agent. 

b)  Determination  of  the  Jji  vitro  sensitivity  of  the  organism 
to  drug. 

c)  Assay  of  serum  and  urine  for  drug  concentrations  during  the 
course  of  treatment. 

d)  Determination  of  the  inhibitory  effect  of  serum  against  the 
offending  microorganism. 

10 


<■) 


lilt  C  I  I'l    I  nl,"    I  , 

Hi  I       I       l'.l    I      llll       III 


III  I  III 


rial  No.  ni 

'I        It      .   I  |  'II  ,u.l      III I      <llM    I   III        ill.         ,..iii        . 


I'll   I  .ni     Mitl  i'l  I  .1  I     .m.l    M.i  (ni      I'M  mil  hi 

I  .        ,\    i  , .  (  .  i  I     ,  1 1      '  '    1 1 . ,    | .  I  i    ill     .  .  I    |  •  1 1   l  .  1 1 1    ,    i.i.      ii.    1 1  ,  .  I    ...       ii. 

mi. In      I  I  ..il  in. -ni      vs'llli     .1  I  iii.l  Ii.".  \  |'li.  iiv  I      |'in  I  .   I   I  I  I  n  .       '     wIMi     Btfljlliyl 'I 

•..|.|   I  irnill  .1  .      1     With    H  t  AphylOl I       nil'  i.  i' I  •      I.  i.   I  .    i   l    i  I      ....I I  I  I   I  ..  , 

I       Willi      .'.  I   . I  |>tl  V  I  .il    HI    i    :1  I         II    I    III     I    I     I    ••         I  I  i.  I       I'm    I  Mil. 'I  I  I     I   .         '      Wltll      •    In   .'II  I   '  I      l|'li 

,  ...  ,    ,1     ,.-.(  ,-,.,nv.'  IIMi.     II    w  I  I  II        l    i  |'li  v  I  "■  hi  i    i  I       '!•     •  ■  6        'il'    •    .     i   I  • 

llliln-ilri     ni      III.'     i'iii.i.   .in,      I     ..'Mil    Cli.iip    M        I   i  .   pi I      .ml...     n.llllo 

mill      I     wl  I  1 1    III  .iii|.     l»    n  I  I  ■   pi  i  ■•  ni   ■  .i  I     i  I  ii  f .  I  I   I  «  . 

All     |  wi  I  I  .ni  'i     l  i  .    1 1  .  .1    <    I  i  Ii    .1 1  iii.-I  In",  vi'li.  ii',  I     |,.  ii  I  .   I  I  I  I  ii    >,l I 

.  1 1 1. 1 .  .i  i  i-v  i  ,1.11. .    .  ■  i    i  ni|.i . ..'.  iii.  iii   ni   ii,...    «...    no  .i.  1 1  hi       PI    i 

.« j.  v    w.i'i    I'.n  i  I  •  'I  I   ii  I  v    IUOO)    I     fill     III       i    ipli     I  ■•  ■  ■  •  •    i  I      "I'M I   '  .      'i 

.ii  i  iii  i  (  i  ■ .   i  u  i.  in i  i    ind     ubaouti    hud  pi*  In  I   Piulmiti  ill  i  I  h       Therapy  hit 

In    ilitlr    hri'ii    nl      I  hn  n  I  II  f  I  I'll  I     iltilit  I  I  nil    I ,  I  ,.  ii  <•     lull.,      i  urn      nl     \>n i 

Vv'l  Mi    i-ynlli       Ml.i.i'iln     nl      III.      |, i'.-iii.         Il Ii         will.     •  ■   >    ■  •  .  I     I" 

I  iif     ,•  I      ',  I  .i  |,li  v  I .1      I  i  ,,  in    Mi.  ■     n  |  .1 1 1 |'|"    ii      | "i 

I.        (in.      |.  1 1   I  ,  nl      wl  Mi    n  I  .- 1 1  •  I  i  v  I i  I     ,  •     i  .Ill,         i       I    ,|.    |  .1      ,  I  I  .   i 

i. n<-    mi  n  1 1  Ii    nl     Mi.i  .i|",'.        HI  ■  i  I'liv  I I      I  ii  I  .  .   I   I  ,  ,ii        I   III   I  I.,   i      ■!".      I  "    |"  ii  I 

i    I  I  II  ii    Q    iii-iifi  I  I  I pant  I    I  I  M  ii    Q     i  "  I  I  "I    ",l      -I       I I,  .1 

. ',  1 1  i.i  I   I  y     wr  I    I       I  ,,     Mi,       .        I"    l    I  ii I       |„    nl.    I    I    I   In      HI       I  I,,         'I  I     |'  il    I  •    nl   |     wl  I  II 

■:l.,|.|iyl i.M      III  ItM'l   III) ,l,,l.      |/|     .il       /U        II   111     ,,.   nl  .    I    I   I   I  ■•     I.     rtllltll 

I  II  I  «•(   I   I  nil . 

/(  .  Il.ll     I    V       .1.1-1.     r,       M|        II        III       )'l    .III.  Ill  UM I        Ml        I    '■       | U  "■! 

Wi'  I   I      I  ii  I  <-l  .Il  <il     /lln  I      J'.i".       | I      ,    I   I  n  1  .     i  I       Mil.  I  In,        |,   i  I    I  ■    nl       Iii         I 

lifiilnl    wIMi    /,",li    )• n    nl     Mi.     'Iii'r    nVPi     •■•     I"1    «1«y    period 

i'i  ..inn,  i  hi  .  i ,  "  .I  ■  ,i    in. i  prp3  "")■•  d  b] I   Li    ■  I     oi    tlv    ■ 

I  ill,    III  .1  I       |«'lll,    I    I    I    III      .11  n  I      v.l'l      I  in..  I       III      :l  I    I       1 1  n  I    I   ■    nl    |      'ill,    I,         II       "in       I   ,.  I  ■    i 

6 1        All     nl  .i|,liy  I  iii  mi   I     wi   i  .       I I     tO    1 I  "      '     '''     I  " 

,,  i    i  in    ■    i i    i"  ii  1 1  i  i  1 1  u .    ii.  1 1  I.,  i  ng  i  i  "■    i  oi    both  i"  n  i  ■  i  i  Hn 

ru- 1  in  M  I  vi'    unit     l  in  I  nl  aril       ,1  i  <i  I  mi  .       'I'lil  n    iii  ii,      •  |  |        I I    CO   bl       nil- 

Ii.ii   I  .1   I  i    I  -I.i  I       I  II     vl  I  I  II.         M   i  ,    I I      VICt  I         ,1      '•      I I     I  ,.     I..  .   ,,,    I  I    i  vi    . 

Ihiwi- vi   i    ,      .   u  I  .    i  ...  i  ii  i    I      ,  /.    i  .       i  .    I    1 1    I  vi    I  y     i  •   |    I      I    in  I 

/  .        Tin-     Itm  M   I  vl  I   y     nl      p|  nl  .    I   I  I  I  n     i.     i  •   ,  ■  i  ■    I    in l  i    'I'I,  M I      I 

Villi.       til       |M    III    1     I     I     I    I    II      C      -I.     I     I    ..I  M.    .1       wl    Ml        I  ill    )'.  I In.        Im.W.     V,     I     .        Ml.  i|,        I 

i  i  vi  i  y  1 1.  i  in    .  .'|..  i  i  mi  ni  ii  i    | ,1 1. 1 1  i  i  1 1  ii  i ,  in  1 1  ii,  ,i   oon  i  ">i    I*i    thi 

oondltlottii     Thli   I'ln  n.nii,  .I,, I.   i      |.i .    ,,,,,,1.1.'  dui    i"  Mi.    |, i  ...in.  i  i  hi,  ,,i 

|.i  ii  I  '    I   I   I  I  ii/nii       l.y     Mu       'i  I     i  |,|i  I  I -I      wll  I  i  1 1     'I :         |"   nl  '    I    I  I  I  "     I       l)U  I      d 

mil      i  I  I .  .  I     ill  mi  ili„-  /i'ln  n  /  I     pi  i,  I  .  I  I  II  it. 


H.       Ill I     I  ■    "   I         "''      I I    i  "    v.n  /    I  i 

vm  I  y  I  ii  y     I   I  in.   n      I  n  I  I  nwl  ii)'     (III  III  I  II  I  III  I    il  I  on 


L9  /  i "  97   ' i  / 


M 


Serial  No.  NIAID  -  13  (c) 

9.  In  3  cases  of  maculopapular  rash  resulted  from  experimental 
penicillin  treatment.  In  1  case  the  rash  was  severe  enough  to  require 
discontinuance  of  the  medication. 

Significance  to  Biomedical  Research: 

1.  Dimethoxyphenyl  penicillin  is  of  importance,  both  clinically 
and  theoretically  because  of  its  almost  total  resistance  to  penicil- 
linase:  Its  effectiveness  in  the  treatment  of  penicillin-G-resistant 
staphylococcal  infections  is  of  significance  and  utility  in  Itself. 

On  a  more  basic  level,  it  helps  clarify  the  role  of  penicillinase  in 
microbial  resistance  to  penicillin. 

2.  This  drug  holds  promise  for  the  long  term  treatment  of 
chronic  staphylococcal  disease  such  as  osteomyelitis,  recurrent  furunc- 
ulosis,  cystic  fibrosis  of  the  pancreas  and  other  diseases  due  to  peni- 
cillin resistant  staphylococci. 

3.  Dimethoxyphenyl  penicillin  may  prove  to  be  a  useful  agent 
In  the  study  of  penicillin  allergy. 

4.  Dimethoxyphenyl  penicillin,  because  of  its  rather  narrow 
microbial  spectrum  should  provide  information  on  the  role  of  micro- 
organisms in  the  chronic  pulmonary  disease  associated  with  cystic 
fibrosis  of  the  pancreas. 

Proposed  Course  of  Project: 

1.  Continue  present  clinical  studies  with  particular  emphasis 
on  chronic  staphylococcal  infections  and  on  cystic  fibrosis  of  the 
pancreas. 

2.  Extend  preliminary  observations  of  allergic  reactions  to 
this  new  penicillin  and  other  drugs  and  define  the  extent  of  cross 
reactivity  with  penicillin  G. 


Part  B  included    Yes  /_/     No  /X/ 

-3-  12 


Serial  No.    NIAID   -    14(c) 

1.  LCI 

2.  Bacteriology  Section 
PHS-NIH                                   3.      Be the s da,    Maryland 

Individual  Project  Report 
Calendar  Year   1960 


Part  A. 

Project  Title:   Staphylococcal  Disease 

Principal  Investigator:   Miss  Rose  Lieberman 

Other  Investigators:   Mr.  John  Douglas 

Mr.  William  Humphrey 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:        1.7 
Professional:   0.5 
Other:        1.2 

Project  Description: 

Objectives: 

Basic  studies  of  the  pathogenesis  and  immunology  of  staphylococ: 
disease  which  include: 

1.  The  investigation  of  the  antigenicity  and  the  types  of 
antibody  produced  to  staphylococcus  cellular  components  obtained  by 
chemical  and  physical  treatment  of  the  intact  organisms. 

2.  Development  of  techniques  to  study  the  alteration  and 
changes  in  antibody  produced  by  various  components  of  staphylococcus 
administered  to  individual  animals. 

Methods  Employed: 

Staphylococcus  is  fractionated  by  chemical  extraction, 
disintegration  and  sonication.   The  various  components  thus  obtained 
are  investigated  for  their  antigenicity  and  for  the  types  of 
antibodies  produced  to  them.   Induction  of  ascites  in  mice  by 
staphylococcus-adjuvant  mixtures  is  employed  to  provide  a  continuous 
source  of  large  amounts  of  high  titered  antibody  in  individual  anima.  ;  . 
This  method  has  the  advantage  of  being  able  to  study  the  effect  on  the 
types  of  antibody  produced  by  administration  of  two  or  more  different 
antigenic  components  of  staphylococcus  at  different  times  in  an 
individual  mouse  over  long  periods  of  time.   The  antibodies  thus 
produced  are  studied  by  various  means  including  passive  cutaneous 

13 


Serial  No.  NIAID  -  14(c) 


anaphylaxis,  Immunoelectrophoresis,  gel  diffusion,  hemagglutination, 
quantitative  precipitation  and  passive  protection  tests. 

Major  Findings: 

High  titers  of  staphylococcal  antibody  are  present  in  the 
ascitic  fluid  of  immunized  mice.   These  antibodies  are  not  all  alike 
and  appear  to  reach  optimal  levels  at  different  times  after  primary 
immunization.  Different  fractions  of  staphylococcus  vary  in  their 
antigenicity  and  on  immunoelectrophoresis  appear  in  different  areas 
of  the  gamma  globulin  range. 

Significance  to  Microbiological  Research: 

Increase  of  both  primary  and  hospital  infections  with  antibiotic 
resistant  strains  of  staphylococcus  has  become  a  serious  public  health 
problem. 

Research  into  the  pathogenesis,  immunology  and  etiology  of 
staphylococcal  diseases  is  especially  important  because  of  the  lack 
of  any  effective  antimicrobiols. 

Proposed  Course  of  Project: 

To  continue  as  outlined  under  objectives. 


Part  B  Included:  Yes  /X/    No  /_/ 

m 

2 


Serial  No.   NIAID  -   14(c) 


PHS-NIH 
Individual  Project  Report 
Calendar  Year   1960 


Part  B;   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

1.      Lieberman,    R.,   Douglas,   J.   0.   A.   and  Mantel,   N.:      Production 
in  mice  of  ascitic   fluid  containing   antibodies    induced  by 
Staphylococcus   or  Salmonella-adjuvant  mixtures.      J.    Immuno., 
84:514-529,   May,    1960. 


15 


Serial  No.     NIAID  -  15(c) 

1.  LCI 

2.  Infectious  and  Pediatric 

Disease  Services 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 

Part  A. 

Project  Title:   Hepatitis  and  Mononucleosis 


This  project  has  been  temporarily  inactivated  due  to 
lack  of  patient  material.   If  patients  become  available,  the 
project  will  be  reactivated. 


16 


Serial  No.    NIAID  -    16(c) 

1.  LCI 

2.  Infectious  and  Pediatric 

Disease  Services 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 
Part  A 

Project  Title:   Aseptic  Meningitis 

Principal  Investigator:  Dr.  John  P.  Utz 

Other  Investigators:   Clarence  F.  Szwed 

Cooperating  Units:   Children's  Hospital,  Washington,  D.  C. 

Man  Years  (calendar  year  1960): 
Total:        0.4 
Professional:  0.4 
Other:         0.0 

Project  Description: 

Objectives: 

1.  To  develop  methods  and  use  newly  available  methods  to  diagnose 
the  large  group  of  non-bacterial  meningo-encephalitides. 

2.  To  correlate  etiological  findings  with  detailed  historical 
physical  and  clinical  laboratory  observations. 

Methods  Employed: 

Hospitalization  of  patients  for  clinical  observation  and  labora- 
tory studies.   Laboratory  procedures  include:  (a)  tissue  culture  and 
suckling  mouse  inoculation  of  material  for  virus  isolation;  (b)  appro- 
priate serological  procedures  with  acute  and  convalescent  serum  for 
the  known  viral  meningo-encephalitides.   A  number  of  patients  selected 
from  other  hospitals  or  surveyed  groups  will  be  studied  similarly. 

Patient  Material  and  Major  Findings: 

1.   A  remarkably  small  number  of  cases  of  aseptic  meningitis 
occurred  in  1960  in  the  Washington  area  and  only  a  few  of  these  were 
studied.   No  single  viral  agent  predominated. 

2.   A  patient  with  lymphocytic  choriomeningitis  was  intensively 
studied  in  cooperation  with  the  National  Naval  Medical  Center.   Note- 


17 


Serial  No.  NIAID  -  16(c) 

worthy  clinical  aspects  of  his  disease  included  Diphasic  course,  absence 
of  direct  contact  with  mice,  encephalitis,  parotitis  and,  especially, 
a  severe  orchitis.   Virus  was  isolated  from  cerebrospinal  fluid  in  this 
laboratory. 

Significance  to  Microbiological  Research; 

Non-bacterial  meningitis  and  meningo-encephalitis  is  a  common 
and  often  serious  disease  of  whose  etiology  and  pathogenesis  there  is 
insufficient  knowledge.   It  often  results  in  brain  damage  and  behavior 
disturbances.   Increased  knowledge  may  also  clarify  the  pathogenesis  of 
the  encephalitic  complications  of  the  otherwise  minor  diseases  of 
children. 

Proposed  Course  of  Project; 

Continue  above  studies. 


Part  B  included  Yes   /  /    No  /X/ 


2-  18 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Serial  No.  NIAID  -  17(c) 

1.  LCI 

2.  Bacteriology  Section 

3.  Bethesda,  Maryland 


Part  A. 


Project  Title:  Allergy -Immunology 

Sub -project  I:  Diseases  of  Immune  Etiology 


Principal  Investigator: 


Dr.  John  P.  Nasou 
Dr.  Donald  E.  Kayhoe  (from  Jan.  1 
June  30,  I960) 


Other  Investigators: 


Cooperating  Units: 


Mr.  John  Bozicevich  (from  July  1,  1960) 
Dr.  Edward  Eyerman  (to  June  30,  1960) 
Miss  Margret  Huber 
Mr.  Robert  Bowser 
Mr .  Stanley  Ward 

Laboratory  of  Infectious  Diseases,  NIAID; 
The  Laboratory  of  Immunology,  NIAID; 
Division  of  Biological  Standards; 
Laboratory  of  Viral  Products: 
National  Heart  Institute,  Laboratory  of 
Cellular  Physiology  and  Metabolism,  ICPM; 
Georgetown  University  Medical  Center  Clinical 
Laboratories;  and  George  Washington  University 
Hospital  Rheumatology  Group. 

Man  Years  (calendar  year  I960): 
Total:        4.95 
Professional  2.65 
Other        2.3 

Project  Description: 

Ob  jectives: 

1.   To  evaluate  the  role  of  immune  serum  globulin  in  host  response 
to  infectious  diseases. 


2.  To  evaluate  the  role  of  bacterial,  fungal,  or  viral  allergies  in 
recurrent  or  chronic  respiratory  disease. 

3.  To  determine  the  rate  of  metabolism  of  individual  antibodies 
compared  to  that  of  gamma  globulin,  and  to  investigate  the  mechanism 
whereby  the  reticuloendothelial  system  differentiates  between  other- 
wise chemically  homogenous  specific  antibodies. 

19 


4.  To  measure,  quantitatively,  the  rate  of  synthesis  of  specific 
antibody  in  the  human  during  the  primary  and  secondary  immune 
responses. 

5.  To  measure  quantitative  differences  in  the  specific  antibody 
response  of  acute  leukemics,  normal  human  volunteers,  hypo  and 
hyper -gammaglobulinemias. 

6.  To  determine  whether,  in  the  presence  of  a  specific  antigenic 
challenge,  the  specific  antibodies  are  selectively  removed  from  the 
total  exogenous  gamma  globulin  injected  into  agammaglobulinemia 
patients. 

7.  To  determine  the  relationship  of  hormones  to  the  agamma- 
blobulinemic  state. 

8.  To  determine  the  pathogenic  significance  of  the  abnormal  gamma 
globulin  of  patients  with  systemic  lupus  erythematosus. 

9.  To  reproduce  lupus  lesions  in  experimental  animals. 

10.   To  study  the  antinuclear,  anti-DNA  and  antinuclear  protein 
factors  found  in  the  serum  of  patients  with  systemic  lupus  erythema- 
tosus and  to  evaluate  the  relationship  of  the  titer  of  the  substances 
to  the  clinical  status  of  the  patient. 

Methods  Employed: 

Clinical  studies  supplemented  with: 

1.  Plasma  protein  analysis  by  (a)  electrophoresis  both  by  paper 
chromatography  and  moving  boundary  technique,  (b)  chemical  deter- 
minations, and  (c)  immunologic  assays. 

2.  Immunization  and  testing  of  responses  by  serologic  techniques 
or  by  challenge. 

3.  Antibody  and  total  gamma  globulin  alteration  measurement  in 
agammaglobulinemic  patients  following  hormone  administration. 

4.  Pathologic  study  of  animals  following  injection  into  the  cir- 
culating blood  of  suspensions  of  L.  E.  cells  and  L.  E.  bodies. 

5.  Phase  contrast  microscopic  study  of  the  L.  E.  cell  phenomenon. 

Patient  Material  and  Major  Findings: 

la   In  cooperation  with  Dr.  Howard  Goodman  of  the  National  Heart 
Institute  and  Dr.  Richard  Malmgren  of  the  National  Cancer  Institute, 
studies  of  lupus  factor  as  measured  by  the  nuclear  proteins  sen- 
sitized and  red  blood  cell  technique  and  the  fluorescent  antibody 
technique,  and  its  clinical  correlation  have  been  conducted.   This 
data  is  currently  being  prepared  for  publication. 


2.  The  clinical  application  of  the  procedure  devised  by  Mr.  John 
Bozicevich  of  the  Laboratory  of  Immunology,  NIAID,  the  DNA-bentonite 
flocculation  test,  has  been  conducted  within  the  framework  of  this 
laboratory.   This  diagnostic  method  for  systemic  lupus  erythematosus 
has  a  great  specificity.  Further  investigations  with  this  procedure 
(the  DNA-bentonite  flocculation  test)  are  underway  in  an  attempt  to 
evaluate  the  status  of  the  lupus  factor  in  these  patients  over  a 
period  of  time. 

3.  Certain  water  precipitable  proteins  of  the  serum  of  patients 
with  collagen  vascular  diseases  are  providing  interesting  immunologic 
phenomenon.   The  fractions  obtained  in  this  method  of  separation  of 
proteins  are  being  used  in  the  DNA-bentonite  test.   In  doing  this, 

we  have  discovered  that  this  test  assumes  even  greater  importance 
in  the  diagnosis  of  collagen  vascular  diseases. 

A.  We  are  continuing  clinical  studies  of  the  patient  with  lupus 
erythematosus  and  agammaglobulinemia. 

5.  In  cooperation  with  Dr.  Samuel  Baron  and  Dr.  Eugene  Barnett  of 
the  Division  of  Biologic  Standards,  the  study  of  agammaglobulinemia 
patients  and  the  presence  of  antibodies  in  their  serum  to  the 
enteric  viruses  has  been  conducted  by  use  of  very  sensitive 
techniques  to  detect  these  antibodies.   It  has  been  found  that  all 
of  these  patients  have  significant  titers.  Further  studies  are 
being  conducted  in  this  vein  in  the  attempt  to  elicit  antibody 
formation  by  means  of  polio  vaccine  in  those  agammaglobulinemics 
not  showing  a  significant  titer.  These  studies  are  now  completed 
and  are  being  prepared  for  publication. 

6.  A  variation  of  the  bentonite  test  using  nucleoprotein,  instead 
of  DNA,  has  been  devised  by  Mr.  Bozicevich.   This  is  a  much  more 
sensitive  test  and  is  capable  of  detecting  systemic  lupus  erythe- 
matosus regardless  of  the  state  of  activity  of  the  disease.   The 
clinical  evaluation  of  this  test  is  almost  completed. 

7.  A  bentonite  procedure  for  the  detection  of  gamma  globulin  has 
been  devised  by  Mr.  Bozicevich.   This  test  will  make  an  excellent 
screening  procedure  for  agammaglobulinemia  since  it  may  be  done  in 
quantity  and  with  accuracy  at  a  minimun  cost.   Clinical  evaluation 
is  underway. 

8.  Preliminary  studies  are  underway  with  bentonite  flocculation 
procedures  to  detect  albumin, beta-lipoprotein,  and  siderophilin. 

9.  Mr.  Bozicevich  has  been  immunizing  rabbits  with  DNA  and 
nucleoprotein  in  an  attempt  to  produce  antinuclear  antibody.  To 
date  there  is  no  evidence  that  such  antibody  can  be  formed,  though 
additional  studies  are  underway. 


21 


Significance  to  Microbiological  Research: 

Agammaglobulinemia  provides  us  with  a  biological  system  relatively 
free  of  humoral  defense  in  which  we  may  study  infectious  diseases. 
Systemic  lupus,  with  its  attendant  hyperglobulinemia,  is  an  ideal 
disease  for  the  study  of  hypersensitivity  and  autoimmunity. 

Proposed  Course  of  Project; 

Studies  will  be  continued  in  a  wide  variety  of  clinical  disorders 
as  to  the  pathogen-host  factors  involved  in  infectious  disease. 

Study  of  the  present  methods  of  clinical  management  of  agammaglo- 
bulinemia patients  especially  as  to  the  required  frequency  and 
dosage  of  gamma  globulin  injection  and  their  management  with 
antibiotics  will  be  continued. 

The  immune  mechanism  of  agammaglobulinemic  patients  will  be 
studied  further,  especially  as  to  their  response  to  vaccine  and 
other  antigenic  stimulation.  Non-antibody  factors  in  resistance 
to  disease  will  also  be  studied. 

Of  a  special  importance  is  the  continuation  of  the  long  term 
followup  of  patients  with  systemic  lupus  erythematosus.  This  group 
of  patients  has  been  tediously  followed  in  the  clinic  and  with 
frequent  inpatient  admission  and  represents  an  outstanding  gcoup  in 
regard  to  long  term  followup.  During  the  course  of  the  coming  year 
it  is  anticipated  that  the  relationship  of  the  clinical  course  to  the 
level  of  lupus  factor  as  detected  by  the  bentonite  flocculation  test 
will  be  a  major  endeavor. 

The  pathologic  significance  of  the  L.  E.  cell,  especially  when 
found  in  diseases  other  than  lupus,  will  be  studied. 


Part  B  Included:       Yes   /   /      No  /  / 


22 


Serial  No.  NIAID  -  17(c) 
Part  B;   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project! 

1.  Antibodies  to  Enteroviruses  in  Hypogammaglobulinemic  Patients. 
Barnett,  E.  V.,  J.  P.  Nasou,  J.  P.  Utz,  and  S.  Baron.  New 
England  Journal  of  Medicine  262:563-565,  March  17,  1960. 

2.  Desoxyribonucleic  Acid  (DNA)-Bentonite  Flocculation  Test  for 
Lupus  Erythematosus.  Bozicevich,  J.,  J.  P.  Nasou  and  D.  E. 
Kayhoe.  Proceedings  of  the  Society  for  Experimental  Biology 
and  Medicine  103:636-640,  1960. 

3.  Clinical  Evaluation  of  the  DNA  Bentonite  Flocculation  Test  for 
Systemic  Lupus  Erythematosus.   Kayhoe,  D.  E.,  J.  P.  Nasou  and 
J.  Bozicevich.   New  England  Journal  of  Medicine  263:5-10, 
July  7,  1960. 

4.  Naming  the  Bentonite  Procedure  (proposals  relative  to  the 
nomenclature  of  the  bentonite  flocculation  tests).   Nasou,  J.  P. 
J.  Bozicevich  and  D.  E.  Kayhoe.   Journal  of  the  American 
Medical  Association  174  (10) :1348,  1960. 


23 


Sub -project  II;  Ascites  in  Mice 

Principal  Investigator:  Miss  Rose  Lieberman 

Other  Investigators:  Mr.  William  Humphrey 
Mr.  John  Douglas 
Mrs.  Jocelyn  Blakely 

Cooperating  Units:  None 

Man  Years  (calendar  year  I960): 
Total:       1.4 
Professional  0.5 
Other        0 . 9 

Project  Description: 

Objectives: 

1 .  To  determine  the  incidence  of  ascites  in  12  pure  inbred  and  3 
hybrid  strains  of  mice  employing  staphylococcal -adjuvant  mixtures. 

2.  To  observe  the  frequency  of  the  development  of  plasma  cell 
tumors  in  these  various  strains  of  mice. 

3.  To  investigate  the  role  of  the  plasma  cells  in  the  production 
of  specific  antibody. 

Methods  Employed: 

Ascites  inducing  doses  of  staphylococcal -adjuvant  mixtures  are 
administered  to  15  different  strains  of  mice  immunized  with  oval- 
albumin  or  horse  serum.   The  incidence  of  ascites,  reactions  to 
immunizing  agents,  amount  of  ascitic  fluid  produced,  titers  of 
antibody  in  the  ascitic  fluid,  effect  of  single  versus  multiple 
immunizing  doses  on  titer,  appearance  of  persistence  of  antibody, 
and  the  effect  of  "boosters"  on  antibody  titer  are  observed  in 
individual  mice  over  a  long  period  of  time  for  each  strain  studied. 

In  collaboration  with  the  Cancer  Institute,  the  incidence  of 
plasma  cell  tumors  appearing  in  the  different  strains  of  mice  are 
removed  and  transplanted  into  unimmunized  mice  for  studies  on 
appearance  and  production  of  antibodies. 


21 


Major  Findings! 

Specific  strains  of  mice  show  a  higher  incidence  of  ascites  with 
persistent  production  of  large  amounts  of  ascitic  fluid.   Plasma  cell 
tumors  appear  with  some  consistency  in  about  18%  of  the  Balb/c  mice 
treated  with  the  staphylococcal-adjuvant  mixtures.   Several  of  these 
tumors  have  been  successfully  transplanted  through  4-5  generations  of 
mice.   Indications  are  that  a  low  incidence  of  plasma  cell  tumors  may 
appear  in  other  strains  of  mice. 

Significance  to  Microbiological  Research! 

Production  of  ascites  in  mice  is  an  excellent  tool  to  obtain  a  con- 
tinuous source  of  large  amounts  of  potent  antibody  from  individual 
small  animals  over  a  long  period  of  time.   This  method  will  facilitate 
the  study  of  different  antibodies  produced  by  purification  and 
fractionation  of  antigens  and  provide  some  information  on  the  mode  of 
action  and  synthesis  of  antibody. 

The  possibility  of  a  genetic  relationship  to  the  incidence  of 
ascites  in  mice  may  be  ascertained. 

The  role  of  "immune"  plasma  cells  transplanted  into  normal  mice  in 
the  production  of  antibody  may  be  investigated. 

Proposed  Course  of  Project! 

To  continue  as  outlined  under  objectives. 

Sub-project  III;   Interactions  of  Antibodies 

Principal  Investigator:  Miss  Rose  Lieberman 

Other  Investigators:  None 

Project  Description: 

Objectives: 

To  study  the  effect  of  mixtures  of  antibodies  in  conferring 
passive  protection. 

Methods  Employed: 

Mixtures  of  Salmonella  and  of  Proteus  immune  rabbit  sera  are 
separately  investigated  to  determine  the  effect  on  mouse  protection 
of  increasing,  decreasing  or  removing  specific  antibody  components 
present  in  the  antisera  combinations. 

Major  Findings; 

Antibodies  employed  in  combination  are  either  simply  additive, 

25 


enhancing  or  antagonistic  in  conferring  passive  protection  in  mice. 

Significance  to  Microbiological  Research! 

Antibodies  in  combination  behave  differently  from  the  equivalent 
amounts  of  the  same  antibodies  used  separately  and  may  be  more  or 
less  effective  in  conferring  protection. 

Proposed  Course  of  Project: 

To  continue  as  outlined  under  objectives. 


Part  B  Included:         Yes  / /    No   / / 


26 


Serial  No.  NIAID  -  17(c) 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B;   Honors,  Awards  and  Publications 

Publications  other  than  abstracts  from  this  projects 

1.  Lieberman,  R.,  Douglas,  J.  0.  A.  and  Mantel,  N.: 
Production  in  mice  of  ascitic  fluid  containing 
antibodies  induced  by  Staphylococcus  or  Salmonella- 
adjuvant  mixtures.   J.  Immunol.,  84:514-529,  May,  1960. 


27 


Serial  No.   NIAID  -  18(c) 

1.  LCI 

2.  Bacteriology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 
Part  A 

Project  Title:   Enteric  Diseases 

Principal  Investigator:   Dr.  John  P.  Utz 

Other  Investigators:  None 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:         1.1 
Professional:   0.1 
Other:         1.0 

Project  Description: 

Objectives: 

Basic  studies  of  the  etiology,  pathogenesis,  and  immunology  of 
enteric  diseases  which  include: 

1.  The  evaluation  of  antibiotic  immunologic  and  surgical  therapy 
on  the  "carrier  state"  of  salmonellosis. 

2.  Studies  of  cellular  and  humoral  mechanisms  in  active  Salmonella 
infections  as  compared  to  those  present  in  the  "carrier  state." 

Clinical  studies:   To  investigate  the  factors  that  produce  persis- 
tence of  bacteria  in  some  patients  to  test  new  antibiotics  in  such  in- 
fections . 

Methods  Employed: 

1.   Complete  bacteriologic  and  immunologic  survey  of  enteric 
disease  study  patients. 

(a)  In  vitro  determinations  of  synergistic,  or  additive  effects 
of  combinations  of  antibiotics  on  organisms  isolated  from 
"carriers." 

(b)  Determination  of  gamma  globulin,  electrophoretic  pattern, 
and  agglutinating  antibodies  in  acute  and  chronic  enteric 
disease  states. 

28 


Serial  No.  NIAID  -  18(c) 
(c)  Cholecystectomy  or  abscess  irradication  as  Indicated. 
Patient  Material  and  Major  Findings: 

1.  Studies  of  the  Salmonella  carrier  state  in  humans  have  been 
continued  in  2  additional  patients.   Gall  stones  removed  from  these 
patients  have  been  implanted,  as  described  in  last  year's  report,  in 
rabbit  gallbladders.   This  brings  to  a  total  6  patients  who  had  stones 
so  implanted  in  8  rabbits,  7  of  whom  became  carriers.   The  results  of 
this  study  in  abstract  form  were  published. 

2.  Studies  described  in  last  year's  report  on  the  patient  with 
Fusobacterium  septicemia  were  published. 

Significance  to  Microbiological  Research; 

Outbreaks  of  salmonellosis  represent  an  increasingly  serious  public 
health  problem.   The  typhoid  and  other  Salmonella  "carrier  states"  are 
responsible  for  spread  of  the  more  virulent  Salmonella  to  man,  and  pose 
a  serious  problem.   There  is  no  standardized  therapy  for  the  "carrier 
state." 

Proposed  Course  of  Project: 

To  continue  as  outlined  under  "Objectives." 


Part  B  included        Yes   /X_/    No  /_/ 


29 


Serial  No.  NIAID  -  18(c) 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 

Part  B      Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

1.   Tynes,  B.  S.,  and  Utz,  J.  P.:   Fusobacterium  septicemia. 
Am.  J.  Med.  29:  879-887,  1960. 


30 


Serial  No.  NIAID  -  19(c) 
1.   Laboratory  of 

Clinical  Investi- 
gation 
PHS-NIH  2.   Parasitic  Disease 

Individual  Project  Report  Service 

Calendar  Year  1960  3.   Bethesda,  Maryland 


Part  A. 


Project  Title:   Clinical  Investigations  on  Helminthic  Diseases 

Principal  Investigators:   Henry  K.  Beye,  G.  Robert  Coatney,  Leon  Jacobs, 

John  E.  Tobie,  John  Bozicevich 

Other  Investigators:   Steven  Schenker  (since  July  1959),  Donald  Kayhoe 
(until  7/60),  Sanford  Kuvin  (since  7/60),  and 
Elizabeth  Guinn 

Cooperating  Units:   Parasitic  Disease  Service,  LCI,  NIAID 

Laboratory  of  Parasitic  Diseases,  NIAID 
Laboratory  of  Parasite  Chemotherapy,  NIAID 
Laboratory  of  Immunology,  NIAID 
Medical  Division,  Department  of  State, 

Washington,  D.  C. 
British  West  Indian  Labour  Organization 
National  Cancer  Institute 

Man  Years  (calendar  year  1960) 
Total:  3.5 
Professional:  2.0 
Other:  1.5 

Project  Description: 

Objectives: 

The  objectives  of  this  group  of  related  projects  which  include: 
(I)  Filariasis,  (II)  Nematode  Infections,  (III)  Trematode  Infections, 
(IV)  Cestode  Infections,  are  to:  (1)  develop  improved  diagnostic 
techniques,  (2)  better  understand  host-parasite  relationships,  (3)  more 
accurately  describe  diseases  associated  with  these  parasites  and  (4) 
elucidate  the  pathogenesis  of  the  disease  process. 

Methods  Employed: 

Methods  for  patient  procurement  utilized  in  1960  consist  of 
physician  referrals,  a  cooperative  arrangement  with  the  Medical  Division, 
Department  of  State,  whereby  returning  State  Department  employees  are 
screened  for  the  presence  of  intestinal  parasites,  arrangements  with  the 

31 


British  West  Indian  Labour  Organization  ut  1 11 zing  British  West  Indian 
agricultural  migratory  workers,  when  indicated  and  a  diagnostic 
serological  service  for  indicated  special  tests  not  pei I m med  elsewhere. 
Two  new  administrative  aids  have  come  into  existence  this  yen  which 
will  be  very  helpful  in  future  studies.   The  provision  of  funds  for 
aiding  the  transport  of  research  candidates  wi i  ■       il  problems, 
and  the  use  of  volunteers  are  notable  advances. 

The  development  of  a  modest  museum  of  specimens  coming  to  the 
attention  of  the  parasitic  screening  laboratory  Is  beginning  to  be 
fruitful  in  terms  of  investigations  and  training. 

I.   Filariasls 

Patient  Material  and  Major  Findings: 

Probably  the  two  most  important  major  findings  associated  wlili 
this  project  in  1960  were  (1)  the  demonstration  of  the  absorption  of 
tetracycline  by  filarial  worms  with  consequent  fluorescence  when  exposed 
to  ultraviolet  light  and  (2)  the  demonstration  of  the  viability  of 
microfilaria  of  Wuchereria  bancrof tl  for  at  least  18  months  when  ErOEM 
at  -10°C.   The  former  studies  commenced  in  1959  and  involved  the 
administration  of  tetracycline  to  2  patients  exhibiting  cutaneous 
migrations  of  Loa  loa .   These  patients,  when  subsequently  examined  under 
ultraviolet  light,  showed  fluorescence  of  adult  filarial  worms.   During 
the  past  year  fluorescence  has  also  been  demonstrated  to  some  degree  In' 
malaria  parasites,  E.  histolytica  and  tapeworm  proglottlds.   Microfilaria 
used  in  the  viability  studies  were  from  British  West  Indian  Migratory 
workers,  with  filarial  infections  that  were  reported  and  studied  during 
1959  and  discussed  in  last  year's  annual  report.   Specimens  of  heparan- 
lzed  blood  from  these  individuals  containing  microfilaria  wei  ■  placid 
in  the  refrigerator  and  maintained  at  -10°C.   At  approximately  J  oontfa 
intervals,  separate  portions  were  removed  and  allowed  Co  thaw  at  room 
temperature.   As  of  the  present  date,  microfilaria  have  retained 
viability. 

Attempts  were  made  to  develop  a  bentonite  flocculation  test 
for  use  In  the  diagnosis  of  filariasls.   Preliminary  remiltu  on  the  sera 
obtained  from  immunized  animals  gave  encouraging  results.   This  is  to  be 
exploited  on  sera  obtained  from  infected  individuals.   A  paper  was  pre- 
sented at  the  First  International  Congress  on  Trichinosis,  Warsaw,  Poland 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

The  demonstration  of  the  phenomenon  of  tetracycline  c<  ■  i  Ion 

in  filarial  worms  as  well  as  In  other  parasites,  has  much  potential 
significance.   There  has  been  a  great  interest  elicited  since  the 
publication  of  the  original  article  for  using  this  as  a  diagnostic  pro- 
cedure.  At  the  same  time,  interest  has  also  been  expressed  In  the 
utilization  of  this  concentration  of  tetracycline  In  the  development  of 
more  effective  antlfilarial  drugs.   The  viability  studies  are  vary  •- 1  mij>  1  <• 
as  performed  in  our  laboratories  and  iIkt.c  findings  <":<y    ''•'        "Bt 

-2  -  32 


on  the  metabolism  of  these  parasites.   In  time,  such  a  technique  wjuld 
be  extremely  useful  to  medical  schools  providing  viable  microfilaria  for 
class  demonstration  and  study. 

Proposed  Course  of  Project: 

Additional  studies  will  be  undertaken  on  utilizing  and  under- 
standing the  tetracycline  fluorescence  phenomenon  in  filarial  worms.   In 
pursuing  these  studies,  it  is  hoped  to  utilize  staff  and  facilities  in 
British  Guiana  where  a  filariasis  research  and  control  project  is  being 
established,  following  the  recommendations  made  by  the  senior  investigator 
when  he  served  as  a  WHO  consultant  for  that  country  in  filariasis.   In 
addition,  it  is  anticipated  to  pursue  these  studies  by  the  use  of  PL-480 
counterpart  funds  in  various  countries  where  filariasis  is  endemic.   The 
question  of  the  elucidation  of  the  pathogenesis  of  eye  lesions  in 
onchocerciasis  is  a  very  important  and  controversial  one.   It  is  hoped 
that  these  techniques  might  be  applicable  in  this  filaria  infection. 

II.   Nematode  Infections  and  Disease 

Patient  Material  and  Major  Findings; 

1.  Trichostrongylus  infected  patients:   As^  reported  last  year, 
State  Department  employees  continued  to  come  to  our -attention  who  were 
infected  with  trichostrongylus.   Species  have  not  yet  been  determined 
because  of  the  difficulty  of  procuring  adult  worms.   Studies  conducted 
this  year  indicate  that  eggs  of  this  parasite  obtained  from  patients 
treated  with  tetracycline  fluoresce.   Attempts  have  been  made  to  utilize 
this  phenomenon  to  find  adult  worms  after  treatment  but  so  far  these 
attempts  have  been  unsuccessful. 

2 .  Parasites  among  the  British  West  Indian  Migratory  Workers: 
This  group  of  individuals  with  rather  high  prevalence  of  intestinal 
nematodes  is  still  available  to  us  but  during  this  past  year  we  have 
been  unable  to  follow  through  with  this  group. 

3.  Visceral  Larval  Migrans:   A  very  informative  case  referred 

to  the  Clinical  Center  because  of  the  diagnosis  of  eosinophilic  leukemia 
has  been  studied  and  proven  to  have  visceral  larval  migrans.   In  this 
case  the  diagnosis  was  established  through  recovering  larvae  of  probably 
Toxocara  canis  at  liver  biopsy  and  the  adult  worms  were  recovered  from 
the  family  dog  which  died  and  was  posted.   The  patient  has  responded 
well  to  diethy lcarbamazine .   In  summary,  the  case  provides  probably  one 
of  the  best  documented  cases  of  visceral  larval  migrans  which  has  yet 
been  described.   This  patient  was  admitted  to  the  Cancer  Service  and  was 
studied  in  conjunction  with  our  laboratory.  At  the  present  time 
observations  are  still  continuing. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

Probably  the  most  significant  finding  in  this  project  during  1960 
was  the  demonstration  of  this  case  which  had  many  characteristics  of 


3  - 


33 


eosinophilic  leukemia  but  which  responded  well  to  diethylcarbamazine 
therapy.   The  elucidation  of  the  role  of  trichostrongylus  in  returning 
State  Department  people  from  the  Near  East  and  further  development  of  the 
various  nematode  infections  amongst  West  Indian  Migratory  laborers  are 
significant  and  will  be  pursued  when  time  and  facilities  permit. 

Proposed  Course  of  the  Project; 

This  project  will  be  intensified,  particularly  in  respect  to 
investigation  of  trichostrongylus  and  trichinosis.   The  further  ex- 
ploration of  tetracycline  and  autof luorescence;  the  programming  for  our 
activation  of  studies  of  parasitoses  in  1962  in  countries  where  counter- 
part funds  are  available,  will  be  pursued.   The  instrument  section  is 
now  building  a  sigmoidoscope,  utilizing  ultra-violet  light  which  will  be 
of  great  help  in  these  investigations. 

III.  Trematode  Infections  and  Disease 

1.   Schistosomiasis  and  other  diseases  due  to  flukes;   Little 
activity  has  been  carried  on  in  this  area  during  the  past  year,  primarily 
because  of  emphasis  on  simian  malaria  and  tetracycline  studies. 
Dithiazanine  iodide  reported  as  being  effective  against  Clonorchis 
sinensis  has  been  tested  in  this  laboratory.   The  results  of  follow-up 
studies  are  still  in  progress. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute; 

With  the  intense  migration  of  Puerto  Ricans  with  high  infection 
rates  due  to  Schistosoma  mansoni ,  increasing  number  of  Americans  going 
to  infected  areas  and  the  increasing  larger  areas  of  the  world  in  which 
infected  snails  are  found,  makes  schistosomiasis  one  of  the  major  world 
diseases  and  of  increasing  importance  to  the  people  in  the  United  States. 
The  treatment  trials  against  Clonorchis  sinensis  with  dithiazanine  iodide 
is  interesting  and  significant  in  that  no  previous  medication  has  been 
very  effective.   The  most  universally  used  drug,  chloroquine  diphosphate, 
has  not  been  very  effective  in  our  hands  as  well  as  in  other  workers. 

Proposed  Course  of  the  Project: 

Inasmuch  as  facilities  and  staff  allow,  continued  efforts  will 
be  made  to  evaluate  new  drugs.   The  use  of  PL-480  funds  has  made  it 
possible  to  program  a  clinical  study  in  schistosomiasis  in  Brazil  with 
possible  activation  by  July  1961.   This  will  help  provide  a  better 
understanding  of  the  pathogenesis  and  the  manifestations  of  hepatosplenic 
schistosomiasis . 

IV.  Infections  due  to  Cestodes 

Patient  Material  and  Major  Findings: 

A  series  of  patients  with  Taenia  saginata ,  mentioned  in  last  year's 
report,  has  now  increased  to  approximately  26.   During  1960  experimental 

-4-  3H 


administration  has  consisted  of  tetracycline  with  follow-up  studies 
on  tetracycline  fluorescence  plus  oral  atabrine.   In  cases  that  are 
unsuccessful,  the  treatment  of  atabrine  transduodenal ly  is  employed. 
Dithiazanine  has  been  tried  with  inconclusive  results. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

Infections  with  these  parasites  are  difficult  to  treat,  and 
the  success  of  our  method  of  using  the  atabrine  through  a  duodenal 
tube  is  a  rather  significant  contribution. 

Proposed  Course  of  Project: 

It  is  anticipated  that  a  great  deal  of  emphasis  will  be  made  in 
the  next  year  on  Echinococcus  disease,  using  patients  from  Alaska 
or  in  planning  Indies  in  Echinococcus  in  countries  of  high  endemicity, 
utilizing  counterpart  PL-480  funds. 


Part  B  included       Yes       No 


35 


(Attachment   I) 
Serial  No.    NIAID   -   19(c) 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B.   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

1.  Beye,  Henry  K.,  and  Gurian,  Joan:  The  Epidemiology  and  Dynamics  of 
Transmission  of  Wuchereria  bancrofti  and  Wuchereria  ma  lay i.  Indian 
Journal  of  Malariology,  December,  1960. 

2.  Beye,  Henry  K. :   International  Organizations  and  Filarlasis  Control 
(Wuchereria  bancrofti  and  Wuchereria  malayi).   Indian  Journal  of 
Malariology,  December,  1960. 

3.  Tobie,  John  E.  and  Beye,  Henry  K. :   Fluorescence  of  Tetracyclines 
in  Filarial  Worms.   Proc.  Soc.  Exper.  Biol,  and  Med.,  104:137-140, 
1960. 

4.  Kayhoe,  Donald  E.  and  Beye,  Henry  K. :  The  treatment  of  Taenia 
saginata  infections  with  quinacrine  HCL  (Atabrine)  administered 
through  a  duodenal  tube.  Presented  at  the  American  Society  of 
Tropical  Medicine  and  Hygiene  meeting,  November  2-5,  1960. 

5.  Beye,  Henry  K. :   Helminthic  Diseases  and  Anthelminthic  Drugs. 
Clinical  Proceedings  of  The  Children's  Hospital,  In  Press. 

6.  Beye,  Henry  K. :   Filariasis.   Cyclopedia  of  Medicine,  Surgery  and 
Specialties.   In  Press. 


36 


Serial  No.  NIAID  -  20(c) 
1.  Laboratory  of 

Clinical  Investi- 
PHS-NIH  gation 

Individual  Project   Report  2.    Parasitic  Disease 

Calendar  Year    1960  Service 

3.    Bethesda,   Maryland 


Part  A. 


Project  Title:   Clinical  Investigations  in  Protozoan  Infections  and 
Disease 

Principal  Investigators:   Henry  K.  Beye,  G.  Robert  Coatney,  Leon  Jacobs, 

John  E.  Tobie  and  John  Bozicevich 

Other  Investigators:   Donald  Kayhoe  to  July  1960,  Morton  Getz,  Harvey 
Elder  from  7/60,  Steven  Schenker  to  7/60, 
Sanford  Kuvin  since  7/60,  and  Elizabeth  Guinn 

Cooperating  Units:   Parasitic  Disease  Service,  LCI,  NIAID 

Laboratory  of  Parasite  Chemotherapy,  NIAID 
Laboratory  of  Parasitic  Diseases,  NIAID 
Laboratory  of  Immunology,  NIAID 
Medical  Division,  Department  of  State,  Washington, 

D.  C. 
Federal  Bureau  of  Prisons 

Man  Years  (calendar  year  1960) 
Total:  3.5 
Professional:  2.0 
Other:  1.5 

Project  Description: 

Although  the  activities  of  interest  on  specific  protozoan 
infections  vary  from  year  to  year,  fundamental  objectives  and  methods 
remain  essentially  the  same.   Since  April  1960,  a  great  deal  of  the 
emphasis  was  on  the  clinical  investigations  associated  with  simian 
malaria.   Activities  in  relation  to  amebiasis  continued,  but  at  a  re- 
duced activity.   Little  emphasis  was  given  to  trichomoniasis,  toxo- 
plasmosis or  other  protozoan  diseases. 

The  development  of  a  modest  museum  of  specimens  coming  to  the 
attention  of  the  parasitic  screening  laboratory  is  beginning  to  be 
fruitful  in  terms  of  investigations  and  training. 

Objectives: 

(1)  Improve  diagnostic  techniques  (2)  better  understand  host 
parasite  relations  (3)  more  accurately  describe  disease  manifestations 
and  host  response  (4)  elucidate  the  pathogenesis  of  the  disease  process 

37 


in  protozoan  infections  and  (5)  more  accutate  evaluation  of  various 
treatment  regimes. 

Methods  Employed: 

Methods  of  patient  procurement  utilized  in  1960  consisted  of 
physician  referrals,  a  continuation  of  the  cooperative  arrangement 
with  the  Medical  Division,  Department  of  State,  whereby  returning 
State  Department  employees  were  screened  for  the  presence  of  intestinal 
parasites,  the  use  of  inmate  volunteers,  primarily  through  the  Malaria 
Project  at  the  Federal  Penitentiary  in  Atlanta,  Georgia,  a  new  method 
started  this  year  of  the  utilization  of  selected  inmate  volunteers  at 
the  Clinical  Center,  and  the  maintenance  of  a  research  serological 
diagnostic  service  for  protozoan  diseases. 

Methods  for  investigation  and  study  of  these  research  candidates 
remain  the  same,  that  is,  close  communication  and  liason  with  the  basic 
laboratories  and  workers  participating  in  these  investigations,  direct 
activities  through  our  own  Parasitic  Disease  diagnostic  and  investi- 
gative laboratory,  utilization  of  the  Service  activities  provided  by 
the  Clinical  Center  and  finally,  educational  and  program  activities 
related  to  patient  procurement  such  as  lectures  and  talks  at  various 
universities,  exhibits  and  discussions  with  organizations  which  might 
provide  a  source  of  research  candidates. 

I.   Blood  and  Tissue  Protozoan  Diseases 

A.   Simian  Malaria  in  Man 

Patient  Material  and  Major  Findings: 

(1)  Two  blood  induced  infections  with  Plasmodium  cynomolgi 
bastianellii  in  inmate  volunteers  in  May, (2)  twenty  sporozoite  induced 
infections  with  the  same  parasite  in  inmate  volunteers  during  May  and 
June  1960,  the  infective  mosquito  in  this  instance  being  Anopheles 
quadrimaculatus,  (3)  six  sporozoite  induced  infections  amongst  inmate 
volunteers  using  the  same  parasite  but  using  Anopheles  freeborni. 
Two  of  these  patients  were  transferred  to  the  Clinical  Center  immediately 
after  being  bitten  while  the  remainder  stayed  for  observations  at  the 
Unit  at  the  Atlanta  Panitentiary,  (4)  eleven  inmate  volunteers  bitten 
by  Anopheles  freeborni  heavily  infected  with  Plasmodium  cynomolgi.  As 
a  result  of  these  experiments,  a  new  clinical  entity  associated  with 
this  previously  thought  simian  parasite  has  been  first  described. 
These  results  can  be  briefly  summarized  as  follows:   Prepatent  period 
usually  under  18  days,  onset  of  symptoms  ranging  from  14  days  to  2  1/2 
months,  extremely  low  parasitemias  associated  with  rather  severe 
clinical  manifestations  in  a  few  patients,  other  patients  having  in- 
fections but  with  mild  or  asymptomatic  clinical  manifestations.  Mani- 
festations seemingly  more  benign  in  Negroes  than  in  Caucasians.   Changes 
in  certain  laboratory  tests  such  as  increase  in  erythrocyte  sedimentat- 
ion rate,  increase  in  cholesterol  beginning  early  after  the  infection, 
decrease  in  platelet  counts  and  neutropenia,  are  associated  with  the 

38 


infection.   Individuals,  infected  with  regular  cynomolgi  on  the  other 
hand,  do  not  seem  to  develop  the  infection.  A  most  significant  finding 
is  the  demonstration  that  simian  malaria  parasite  is  probably  a  zoonoses, 
the  parasite  has  been  transmitted  from  monkey  to  man  via  the  mosquito, 
and  then  from  man  to  mosquito.   Suggestive  evidence  indicates  the 
transmission  from  man  to  man  by  the  mosquito. 

B.   Chemotherapeutic  studies  using  inmate  volunteers  at  Atlanta 
Penitentiary.   More  than  a  100  volunteers  participated  in  various 
studies  associated  with  the  prophylactic  and  sporozonite  properties  of 
primaquine,  chloroquine  and  a  Russian  preparation,  quinacide.   The 
results  amongst  these  studies  can  be  summarized  as  follows: 

1.  Suppressive  cure  effect  of  a  pyrimethamine -primaquine  com- 
bination:  Pyrimethamine  50  mgm  plus  primaquine  45  mgm  given  once  a 
week  for  four  weeks,  has  protected  five  men  exposed  to  the  bites  of 
mosquitoes  infected  with  Chesson  strain  of  vivax  malaria  for  at  least 
219  days  of  observation. 

2.  Suppressive  cure  effect  of  chloroquine -primaquine  combination: 
Ten  Negro  male  volunteers  were  divided  into  two  groups  of  5  men  each. 
Each  man  in  Group  A  received  chloroquine  300  mg  plus  primaquine  45  mg 

in  a  single  oral  dose  three  days  prior  to  the  date  of  infection,  (0-3), 
and  on  the  same  day  of  each  week  for  a  total  of  four  doses.   The  sub- 
jects in  Group  B  received  no  drugs  and  served  as  controls. 

Both  groups  were  infected  with  the  McLendon  strain  of  P.  falci- 
parum on  1  April  1960,  by  the  bites  of  ten  heavily  infected  A.  quadri- 
maculatus  mosquitoes.   In  an  attempt  to  simulate  field  conditions  the  ten 
volunteers  were  subjected  to  three  subsequent  bitings  at  irregular 
intervals.   At  each  subsequent  biting  time  additional  controls  were 
added. 

Each  of  the  five  men  in  Group  B  developed  parasitemia  on  the  11 
to  13  experimental  day.   After  two  days  of  demonstrable  parasites, 
each  was  given  chloroquine  300  mg  plus  primaquine  45  mg.   This  regimen 
was  repeated  on  the  same  day  of  the  week  for  a  total  of  three   doses. 

None  of  the  five  volunteers  in  Group  A  developed  parasitemia 
during  90  days  of  observation.   None  of  the  man  in  Group  B  has  ex- 
hibited any  evidence  of  infection  following  the  chloroquine-primaquine 
therapy. 

One  control  was  added  for  each  of  the  last  2  biting  episodes. 
Neither  control  developed  malaria  and  the  last  two  bitings  are  con- 
sidered non-infecting  even  though  sporozoites  were  demonstrated  at 
the  post-prandial  dissection  of  the  mosquitoes. 

3.  Curative  effect  of  CN  1115  (WIN  10448,  Quinicide):   To  date, 
8  patients  have  received  Quinicide  therapy  and  10  have  received  Prima- 
quine.  Of  those  patients  who  received  CN  1115,  four  exhibited  early 
primary  attacks  and  four  delayed  primaries.   There  was  a  total  of  6 

39 


recurrences  in  the  early  primary  group  and  two  in  the  delayed  primary 
group . 

To  date,  11  patients  have  received  primaquine  therapy  and  ten 
have  received  CN  1115.   The  dosage  of  each  drug  was  15  mg.  single  dose 
daily  for  14  days.   There  have  been  two  relapses  among  those  who  re- 
ceived primaquine  therapy.   There  have  been  13  relapses  among  those 
given  CN  1115.   Of  these  relapses,  one  man  had  four,  three  had  two,  and 
three  had  one  relapse  each.   Relapse  infections  were  treated  according 
to  the  original  regimen. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

The  significance  of  these  investigations  in  terms  of  the  world- 
wide malaria  eradication  and  in  terms  of  more  adequately  understanding 
the  pathogenesis  of  malaria,  including  the  human  form,  is  very  great. 
For  the  first  time,  reservoirs  of  human  malaria  have  been  definitely 
elucidated  and  perhaps  for  the  first  time,  a  malaria  parasite  with 
clinical  manifestations  similar  in  monkeys  and  man  has  been  found.   This 
will  provide  a  very  useful  tool  in  other  studies  on  malaria. 

Proposed  Course  of  Project: 

This  project  in  clinical  investigation  is  one  aspect  of  an  in- 
tensive program  of  investigating  the  role  of  simian  malaria  in  the 
epidemiology  of  human  malarias  coordinated  through  the  Laboratory  of 
Parasite  Chemotherapy.   It  is  anticipated  that  additional  work  will  be 
done  on  the  clinical  description  of  the  disease  involving  the  use  of 
additional  volunteers  at  the  Clinical  Center.   Studies  of  cross -immunity 
of  the  simian  malarias  to  human  malarias,  particularly  the  regular 
cynomolgi  are  programed.   It  is  anticipated  that  some  activity  may  be 
extended  to  other  countries  through  the  utilization  of  PL-480  funds. 

II.   Amebiasis 

A.   State  Department  Personnel 

Patient  Material  and  Major  Findings: 

As  indicated,  patient  material  has  consisted  of  the  more  than 
6,000  State  Department  employees  who  have  submitted  stool  specimens  to 
the  Parasitic  Disease  Service  for  screening  since  the  inception 
of  this  cooperative  program  with  the  Medical  Division,  Department  of 
State.   This  material  has  been  analyzed,  the  most  interesting  features 
being  that  those  individuals  infected  with  E_.  histolytica  did  not  ex- 
hibit any  additional  clinica 1  manifestations  or  did  they  lose  more  time 
from  work  while  overseas  than  those  individuals  who  were  not  infected. 
This  re -emphasizes  again  the  beginning  appreciation  of  the  benign 
character  of  E.  histolytica  in  normal  healthy  individuals.   At  the  same 
time,  an  occasional  case  was  admitted  to  the  Clinical  Center  with  the 
classical  symptoms  of  acute  amebic  dysentery  and  one  case  of  amebic 
liver  abscess  from  the  Washington  area  was  studied.   Studies  continued 

40 


on  the  development  of  more  definitive  and  more  perfected  serological 
tests  for  amebiasis  including  a  bentonite  flocculation  test.   For  those 
individuals  who  were  accepted  for  research  study,  the  use  of  the  anti- 
amebicide,  Humatin  (Puramycin)  has  been  studied  this  past  year.   This 
drug,  in  our  present  investigations,  seems  to  be  the  most  satisfactory 
amebicide  which  has  been  our  experience  to  study.   The  definitive 
analysis,  however,  of  these  cases,  including  follow-up  observations, 
are  still  in  process. 

B.   Immunology  and  Serology 

Three  series  of  rabbits  were  immunized  to  Entamoeba  invadens , 
12.  terrapinae  and  E.    histolytica  antigens.   The  antigens  were  grown 
in  vitro  on  the  appropriate  media  and  the  animals  immunized  with  the 
supernatant  fluid  contining  the  metabolic  products  and  the  entire 
organisms.   At  intervals,  the  animals  were  bled  and  complement  fixation 
tests  were  conducted  with  the  specific  and  heterologous  antigens.   N0 
cross  reactions  were  found  at  any  time  as  elicited  by  a  positive 
complement  fixation  test.   Additional  immunization  was  given  to  each 
group  with  the  specific  antigen.   Again,  no  cross  reactions  were  en- 
countered with  the  heterologous  antigens  but  specific  reactions  were 
obtained  as  elicited  by  the  complement  fixation  test. 

The  purpose  of  the  above  study  was  to  investigate  the  possibility 
of  utilizing  a  closely  associated  antigen  for  the  complement  fixation 
test  for  the  detection  of  E.  histolytica  infection.   If  this  proves 
to  be  successful,  it  would  facilitate  preparing  antigens  for  the 
diagnosis  of  amebiasis. 

Significance  to  Bio -Medical  Research  and  the  Program  of  the  Institute: 

It  is  only  through  the  understanding  of  the  pathogenesis  of  E. 
histolytica  infections  and  disease  that  this  entity  will  be  placed  in 
proper  prospective.  Work  continues  on  the  elaboration  of  the  patho- 
genesis of  IS.  histolytica  in  man  in  our  basic  laboratories,  particularly 
the  germ-free  laboratory.   The  significance  of  our  observations  is  rather 
large  in  that  it  provides  a  great  deal  of  epidemiologic  data  on  benign 
characters  of  this  infection  among  foreign  service  personnel.   The 
presence  of  the  disease,  however,  in  occasional  cases  only  emphasizes 
the  importance  of  understanding  what  factors  are  responsible  for  these 
manifestations. 

Proposed  Course  of  Project: 

With  adequate  staff  and  time,  it  is  hoped  that  this  project  can, 
in  time,  be  re-oriented  to  concentrate  on  a  few  individual  cases  which 
can  be  followed  with  or  without  treatment  for  long  periods  of  time, 
using  in  association  with  these  studies  newer  techniques  for  the  study 
of  the  upper  GI  tract.   This  would  make  possible  the  description  of  the 
invasiveness  and  the  characteristics  of  the  E.  histolytica  complex  (E. 
hartmani ,  small  and  large  races  of  E_.  histolytica)  along  with  the 
bacterial  associates  in  the  human  GI  tract.   During  this  past  year, 


little  work  was  done  on  trichomona is is,  toxoplasmosis  or  other  pro- 
tozoan diseases  which  have  received  emphasis  in  the  past.   We  hope  to 
reactivate  these  projects  in  the  near  future  as  they  are  of  great  bio- 
medical significance.   This  is  particularly  true  with  toxoplasmosis.   The 
infection  is  wide-spread,  the  disease  manifestations  are  occasionally  very 
severe,  and  the  spectrum  of  host  response  and  clinical  manifestations 
is  as  yet  undescribed. 


Part  B  included        Yes      No 


m 


(Attachment  I) 
Serial  No.  NIAID  -  20  (c) 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B.   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

1.  Beye,  Henry,  Brooks,  Charles  and  Guinn,  Elizabeth:   Intestinal  and 
Blood  Parasitism  among  British  West  Indian  Agricultural  Laborers. 
Review  of  Protozoan  and  Helminthic  Infestations  among  Migratory 
Agricultural  Workers.   Accepted  for  publication  by  the  Journal  of 
American  Public  Health  Association. 

2.  Kayhoe,  Donald  E.,  Beye,  Henry,  Guinn,  Elizabeth  and  George, 
George  P.:   Prevalence  of  Intestinal  Parasites  Among  U.  S.  State 
Department  Employees  and  Their  Dependents.   Presented  at  the 
American  Society  of  Tropical  Medicine  and  Hygiene  Meeting,  Los 
Angeles,  California,  November  2-5,  1960. 

3.  Coatney,  G.  Robert  and  Beye,  Henry  K. :   Simian  Malaria  Infections 

in  Man.   Presented  at  the  American  Public  Health  Association  Meeting, 
San  Francisco,  California,  October  31-November  5,  1960. 

4.  Beye,  Henry  K.  and  Coatney,  G.  Robert:   Preliminary  Report  on 
Sporozoite  Induced  Plasmodium  Infections  in  Man.   Presented  at 
World  Health  Organization  Expert  Committee  on  Malaria. 

5.  Beye,  Henry  K.,  Coatney,  G.  Robert,  Getz,  Morton  and  Eyles,  Donald: 
A  Vivax-Type  Malaria  of  Simian  Origin  in  Man.   Clinical  and 
Parasitological  Characteristics.   Presented  at  the  American  Society 
of  Tropical  Medicine  and  Hygiene  Meeting,  Los  Angeles,  California, 
November  2-5,  1960. 

6.  Getz,  M.E.,  Elder,  H.  A.  and  Todd,  Chas .  S.,  Jr.:   Clinical 
Manifestations  of  Plasmodium  cynomolgi  bastianellii.   Presented 
at  the  National  Institute  of  Allergy  and  Infectious  Diseases 

and  Communicable  Disease  Center  Joint  Conference,  November  17-18,1960. 


k3 


Serial  No.  NIAID  -  21(c) 

1.  LCI 

2.  Pediatric  Service 

3.  Bethesda,  Maryland 
PHS-NIH 

Individual  Project  Report 
Calendar  Year  1960 

Project  Title:  Cystic  Fibrosis  of  the  Pancreas 

Principal  Investigator:   Dr.  George  T.  Bryan 

Other  Investigators:   Dr.  Hugh  Evans  (from  July  1,  1960) 

Dr.  Philip  Fireman  (from  July  1,  1960) 

Dr.  Edward  Eyerman  (to  June  30,  1960) 

Dr.  Lowell  Good  (to  June  30,  1960) 

Dr.  George  Owen  (to  June  30,  I960) 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:   2.85 
Professional:  2.85 

1.  Objectives 

A.  To  study  the  microbial  flora  in  patients  with  cystic  fibrosis  of 
the  pancreas . 

B.  To  evaluate  the  role  of  staphylococcal  immunology. 

C.  To  evaluate  the  effect  of  certain  antibiotic  agents  on  the  bacterial 
flora  of  the  respiratory  tract. 

D.  To  study  the  immunology  of  purified  staphylococcal  penicillinase. 

E.  To  study  the  relationship  of  growth  failure  in  these  patients  to 
their  serum  growth  hormone  levels. 

2.  Patient  Material 

Children  with  proven  cystic  fibrosis  of  the  pancreas  who  are  being  fol- 
lowed regularly  in  this  clinic. 

3.  Major  findings 

The  major  findings  are  limited  to  the  study  of  the  microbial  flora  in 
patients  with  cystic  fibrosis  of  the  pancreas.   These  would  indicate 
that  children  with  this  disease  have  a  relatively  stable  bacterial  flora 
in  spite  of  continuous  therapeutic  antibiotic  therapy.   Investigations 
are  continuing  in  the  study  of  microbial  flora  and  the  role  of  the 
staphylococci  in  cystic  fibrosis  of  the  pancreas. 

Part  B  included  Yes  f~j     No  /X/ 


W 


Serial   No.    NIAID  -    22(c) 

1.  LCI 

2.  Biochemistry  and 

Immunochemistry 
Section 

3.  Bethesda,  Maryland 
PHS-NIH 

Individual  Project  Report 
Calendar  Year  1960 

Project  Title:   Basic  Biochemical  Studies 

Principal  Investigator:   Dr.  Harry  G.  Steinman 

Other  Investigators:   Dr.  Eskin  Huff 

Dr.  Steven  Schenker 

Dr.  Anderson  Spickard 

Dr.  Sheldon  Wolff 

Cooperating  Units:   Hildegard  Wilson,  Laboratory  of  Nutrition  and  Endocrin- 
ology, NIAID 

Ellsworth  R.  Buskirk,  Metabolic  Diseases  Branch,  NIAID 
Ronald  H.  Thompson,  Metabolic  Diseases  Branch,  NIAID 
Howard  Goodman,  Laboratory  of  Immunology,  NIAID 
Emanuel  S.  Hellman,  General  Medicine  Branch,  NCI 

Man  Years  (calendar  year  1960): 
Total:        4.5 
Professional:   2.5 
Other:        2.0 

Project  Description: 

Objectives: 

To  study  the  biochemistry  of  pathogenic  micro-organisms;  the  nature 
of  penicillin  resistance;  the  mechanism  of  action  of  penicillin;  the  bio- 
chemical abnormalities  associated  with  specific  allergic  and  infectious 
disease  processes. 

Methods  Employed: 

1)  Biochemistry  of  pathogenic  micro-organisms.  The  biosynthesis 
of  the  polysaccharides  of  bacterial  cell  wall  material  has  been  investi- 
gated with  emphasis  on  the  role  of  certain  three-carbon  precursors. 

2)  Nature  of  penicillin  resistance.   The  role  of  penicillinase  in 
the  resistance  of  j3^_  aureus  to  penicillin  is  being  investigated  under  a 
variety  of  conditions. 

3)  Mechanism  of  action  of  penicillin.  A  number  of  new  penicillins 
now  available  are  being  compared  in  a  number  of  biological  and  biochemical 
systems  in  order  to  correlate  chemical  structure  with  biologic  activity. 


45 


Serial  No.  NIAID  -  22(c) 

4)   Biochemical  abnormalities  In  allergic  and  infectious  diseases. 
The  biochemical  origins  of  a  rare  clinical  entity  known  as  etiocholano- 
lone  fever  have  been  examined  with  respect  to  the  metabolic  fate  of  spec- 
ific steroid  substances.   The  amino  acid  abnormalities  associated  with 
Familial  Mediterranean  Fever  have  been  studied.   The  effect  of  chloro- 
quine  on  producing  porphyria  is  being  investigated  in  both  animals  p.nd 
patients. 

Patient  Material  and  Major  Findings: 

1)  Biochemistry  of  pathopenic  micro-organisms.   The  study  on  the 
identification  of  lactaldehyde  as  the  oxidation  product  of  1,2-propanediol 
and  the  sterespecificity  of  the  enzymatic  reactions  catalyzed  by  alcohol 
dehydrogenases;  which  was  described  in  a  previous  Project  Report 

(NIAID  -  22(c),  1959)  has  been  summarized  in  a  paper  entitled  "The  Metab- 
olism of  1,2-propanediol"  and  which  is  now  in  press.   Tt '  -  publication 
concludes  the  work  on  the  metabolism  of  1,2-propanediol  and  its  phosphate 
ester. 

2)  Nature  of  penicillin  resistance.  Although  there  is  a  very  high 
degree  of  coincidence  between  the  presence  of  penicillinase  and  the  peni- 
cillin resistance  of  pathogenic  J^  aureus,  the  direct  proof  that  the  enzyme 
is  responsible  for  the  resistance  has  been  lacking.   There  would  appear  to 
be  no  question  as  to  the  role  of  penicillinase  were  it  not  for  the  fact 
that  penicillin  resistance  of  ordinarily  sensitive  strains  of  jj^  aureus 
can  be  readily  achieved  in  the  laboratory  by  a  process  of  selection  of 
natural  mutants.   This  type  of  natural  resistance,  which  does  not  appear 
to  be  stable,  is  not  dependent  on  the  presence  of  the  destructive  enzyme 
penicillinase.   Its  true  basis  remains  unknown.   A  variety  of  experiments 
have  been  performed  which  support  the  thesis  that  penicillinase  is  indeed 
the  primary  factor  effecting  resistance  to  penicillin  in  JL_  aureus.   These 
will  be  reported  at  the  5th  International  Congress  of  Biochemistry  to  be 
held  in  Moscow,  1961. 

3)  Mechanism  of  action  of  penicillin.   As  a  corollary  of  the 
studies  on  penicillinase  the  activities  of  the  reaction  with  several  dif- 
ferent penicillins  had  been  made.   It  became  apparent  that  a  penicillin 
which  was  insensitive  to  the  action  of  the  enzyme  would  be  of  great  value 
to  the  basic  studies  as  well  as  of  clinical  interest.   When  such  a  peni- 
cillin became  available,  as  the  result  of  a  major  breakthrough  in  peni- 
cillin technology,  our  laboratory  techniques  were  able  to  demonstrate 
immediately  the  unique  resistance  to  the  destructive  action  by  staphylo- 
coccal penicillinase  of  the  new  penicillin  and  to  indicate  the  potential 
usefulness  of  the  drug  in  therapy  almost  before  clinical  evidence  of  the 
effectiveness  in  the  treatment  of  penicillin  G-resistant  staphylococcal 
infections  was  at  hand.   These  findings  were  embodied  in  2  papers,  one 
presented  at  a  Symposium  devoted  to  the  new  penicillin  held  in  Syracuse 
on  September  7,  1960  and  the  other  at  the  Conference  on  Anti-Microbial 
Agents  held  in  Washington  on  October  26-28,  1960.   By  examining  a  series 

of  penicillins  in  a  variety  of  biological  and  biochemical  reaction  systems, 

-   2-  1+6 


Serial  No.  NIAID  -  22(c) 

it  has  been  possible  to  ascertain  the  effect  of  various  chemical  substi- 
tuents  in  the  basic  penicillin  molecule  on  a  number  of  specific  biologic 
activities.   These  studies  will  be  reported  at  the  First  International 
Pharmacological  Meeting  to  be  held  in  Stockholm,  1961. 

4)   Biochemical  abnormalities  associated  with  specific  diseac*. 
processes. 

a)  Etiocholanolone  fever.   In  colloboration  with  Dr.  H.  Wilson, 
NIAID,  a  patient  with  the  diagnosis  of  etiocholanolone  fever  has  been 
studied  with  respect  to  the  biochemical  pathogenesis  of  his  disease.   This 
disease  was  characterized  by  bouts  of  fever,  chills,  myalgia  and  anorexia 
lasting  from  a  day  to  several  weeks,  recurring  at  irregular  intervals  and 
associated  with  a  mild  normocytic  normochromic  anemia  and  a  nonspecific 
granulomatous  infiltration  of  the  liver.  At  the  time  of  his  febrile 
attacks,  the  patient  was  found  to  have  elevated  plasma  levels  of  nonconju- 
gated  etiocholanolone  and  dehydroepiandrosterone.   The  i  /.ness  responded 
dramatically  to  steroid  therapy. 

In  order  to  elucidate  the  pathogenesis  of  this  rare  disease,  meta- 
bolic studies  have  been  carried  out  to  determine  1)  the  ability  of  the 
patient  to  conjugate  and  excrete  exogenously  administered  etiocholanolone 
and  2)  the  capacity  of  the  patient  to  synthesize  etiocholanolone  from  its 
precursors.   Twenty  five  milligrams  of  etiocholanolone  were  administered 
intramuscularly  to  the  patient  while  his  own  etiocholanolone  secretion 
was  suppressed  by  steroid  therapy,  and  urine  was  tested  for  etiocholano- 
lone output.   Preliminary  results,  by  the  hot  acid  hydrolysis  method, 
indicate  that  the  patient  excretes  40~507o  of  the  administered  dose  in 
24  hours  comparable  with  normal  patients.   The  urine  will  be  studied  fur- 
ther for  total  quantitative  excretion  of  the  compound  as  well  as  the  amount 
which  is  conjugated  as  a  glucuronide  and  sulfate.   The  patient's  glucuronyl 
transferase  system  was  studied  by  determining  his  ability  to  conjugate  men- 
thol and  this  was  found  to  be  normal.   Subsequently  the  patient  was  given 
doses  of  testosterone  and  hydroxy-androstenedione  and  his  urine  checked  for 
conversion  of  these  substances  to  etiocholanolone.   Initial  results  reveal 
normal  conversion.   Should  the  patient  redevelop  his  symptoms,  it  is 
planned  to  investigate  the  possible  beneficial  role  of  thyroid  analogues 
on  the  course  of  his  illness  as  well  as  to  attempt  to  isolate  a  possible 
thermogenic  principle  from  the  plasma. 

b)  Familial  Mediterranean  Fever.   Preliminary  investigations 
are  under  way  dealing  with  Familial  Mediterranean  Fever.   In  collaboration 
with  Drs.  E.  Buskirk  and  R.  Thompson  of  NIAMD,  we  are  studying  the  physio- 
logic response  of  a  patient  to  cold  stress  and  also  during  an  attack  of 
fever.   Biochemical  studies  of  the  alpha  amino  nitrogen  excretion  and 
chromatographic  analyses  of  the  amino  acid  content  of  blood  and  urine  are 
being  done.   Some  immunologic  studies  in  conjunction  with  Dr.  H.  Goodman, 
NIAID,  are  planned.   Some  therapeutic  trials  are  anticipated  also,  using 
female  hormones. 


-3-  47 


Serial  No.  NIAID  -  22(c) 

c)   Porphyria.   A  patient  with  porphyria  is  also  being  studed. 
Elevations  in  the  patient's  excretion  of  5-hydroxyindoleacetic  acid  has 
been  noted  and  it  is  planned  that  this  finding  be  looked  for  in  other 
patients  and  some  investigation  as  to  why  this  occurs  will  be  undertaken. 
Also  the  effect  of  chloroquine  in  this  disorder  is  being  studied  by 
measuring  urinary  porphyrins  and  delta  aminolevulinic  acid  before  anJ 
after  chloroquine  therapy.   The  latter  study  and  a  study  of  the  effect  of 
chloroquine  in  experimental  porphyria  (in  rats)  is  being  done  in  collabo- 
ration with  Dr.  E.  Hellman  of  NCI. 

Significance  to  Biomedical  Research; 

The  discovery  of  the  high  degree  of  resistance  of  the  new  penicillin, 
2,6-dimethoxyphenylpenicillin,  to  the  destructive  action  of  staphylococcal 
penicillinase  supplied  the  scientific  basis  for  pursuing  the  clinical 
trials  of  the  drug.   Other  laboratory  data  helped  establ  -h  proper  dosage 
and  treatment  schedules.   The  new  antibiotic  shows  great  promise  of 
ameliorating  the  problem  of  hospital-borne  staphylococcal  infections. 
However,  its  intrinsic  chemical  properties  prevent  it  from  being  used 
except  under  hospital  conditions  because  it  is  not  absorbed  into  the 
blood  stream  well  enough  to  be  given  orally,  but  must  be  injected  intra- 
venously (or  intramuscularly).   Our  laboratory  know-how  in  this  field 
will  enable  us  to  evaluate  immediately  and  exploit  any  superior  penicillin 
that  may  appear  in  the  future. 

The  studies  on  the  biochemical  observations  associated  with  such 
obscure  diseases  as  etiocholanolone  fever,  Familial  Mediterranean  Fever, 
and  chloroquine-induced  porphyria  will  be  helpful  in  understanding  the 
pathogenesis  of  these  diseases. 

Proposed  Course  of  Project; 

These  studies  are  being  continued. 


Part  B  included    yes  /X/    no  /  / 

-4-  1*8 


Serial  No.  NIAID  -  22(c) 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 

Part  B    Honors,  Awards,  and  Publications 

1.  Huff,  E. :   The  Metabolism  of  1,2-Propanediol.   Biochem.  et  Bio- 
phys.  Acta.   In  press. 

2.  Steinman,  H.G.:  Factors  modifying  induced  formation  of  penicil- 
linase in  Staphylococcus  aureus.  J.  Bacteriol.   In  press. 

3.  Steinman,  H.G.:   A  biochemical  comparison  of  6-aminopenicillanic 
acid,  benzylpenicillin,  and  2,6-dimethoxyphenylpenicillin.   Proc. 
Soc.  Exptl.  Biol.  Med.   In  press. 

4.  Steinman,  H.G.:   Comparative  activities  of  6-aminopenicillanic 
acid,  benzylpenicillin,  and  2,6-dimethoxyphenylpenicillin.   Paper 
delivered  at  State  University  of  New  York,  Upstate  Medical  Center, 
Syracuse,  New  York,  September  7,  1960;  to  be  published  in 

Bunn,  P. A.  (ed.):   A  Symposium  on  a  New  Synthetic  Penicillin, 
Syracuse  University  Press,  1961. 

5.  Steinman,  H.G.:   A  comparison  of  the  biochemical  activities  of 
6-aminopenicillanic  acid,  benzylpenicillin,  2,6-dimethoxyphenyl- 
penicillin, 2-phenoxyethylpenicillin,  and  phenoxymethylpenicillin. 
Paper  delivered  before  the  Society  for  Industrial  Microbiology, 
Washington,  D.  C. ,  October  27,  1960;  to  be  published  in  Trans- 
actions of  the  Conference  on  Antimicrobial  Agents,  1960. 


49 


Serial  No.  NIAID  -  23(c) 

1.  LCI 

2.  Infectious  Disease  Service 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   Systemic  fungal  disease 

Principal  Investigator:   Dr.  John  P.  Utz 

Other  Investigators:   Dr.  Vincent  T.  Andriole 
Dr.  Michael  W.  Brandriss 
Margret  A.  Huber 

Cooperating  Units:   Laboratory  of  Infectious  Diseases 

Man  Years  (calendar  year  1960): 
Total:       2.1 
Professional:  2.1 
Other:       0.0 

Project  Description: 

Objectives: 

1.  To  study  diagnosis,  pathogenesis  and  natural  course  of  sys- 
temic fungus  infections. 

2.  To  study  the  problem  of  fungus  disease  due  to  overgrowth  dur- 
ing antibiotic  and  steroid  treatment. 

3.  To  evaluate  new  agents  in  antibiotic  and  chemotherapeutic 
treatment  of  systemic  fungus  infections. 

4.  To  set  up  means  of  measuring  blood  levels  of  new  antifungal 
agents  and  to  study  acute  and  long  term  toxic  effects  of  the  drugs. 

5.  To  study  various  immunologic  aspects  of  this  group  of 
diseases . 

Methods  Employed: 

Patients  with  suspected  fungal  infections  are  studied  diagnostical- 
ly  using  the  skin  test,  serologic,  histologic,  and  cultural  methods. 
Family  contacts  are  similarly  investigated  and  environmental  studies 
undertaken,  when  indicated.   Infecting  fungi  from  such  patients  are 


50 


Serial  No.  NIAID  -  23(c) 

screened  for  sensitivity  against  a  considerable  number  of  cheraothera- 
peutic  or  antibiotic  agents  and  therapy  is  undertaken  after  such  studies 
and  where  clinically  advisable. 

Drugs  are  screened  for  activity  against  an  organism  isolated  from 
a  patient  by  Jji  vitro  methods.   Promising  drugs  are  then  tested  in 
animals  for  acute  and  long-term  toxicity  and  for  therapeutic  effectiveness. 

New  methods  are  being  developed  for  detemining  serum  drug  levels, 
rates  of  excretion  of  drug,  and  jji  vitro  screening  of  drugs. 

Patient  Material  and  Major  Findings; 

1.  Studies  were  continued  on  patients  with  a  variety  of  fungal 
diseases.   New  patients  with  culturally  proved  disease  this  year  in- 
cluded cases  of  cryptococcosis  (7),  coccidioidomycosis   3),  histoplas- 
mosis (5),  sporotrichosis  (5),  nocardiosis  (1),  blastomycosis  (3), 
cardidiasis  (1),  aspergillosis  (2),  and  mucormycosis  (1). 

2.  Continued  observation  of  a  total  of  patients  treated  with 
amphotericin  B  has  permitted  more  conclusions  as  to  effectiveness. 
Although  clear-cut  treatment  failures  have  occurred  in  3  cases  of  cryp- 
tococcosis, 7  patients  have  "apparently  recovered"  (clinically  well  with 
normal  cerebrospinal  fluid)  from  their  infections  32,  41,  31,  30,  12,  and 
6  months  after  cessation  of  therapy.   One  patient  died  from  an  unrelated 
disease.   An  additional  6  patients  are  improved  (clinically  well,  but 
with  cerebrospinal  fluid  not  yet  normal).   An  analysis  of  the  failures 

of  amphotericin- treated  patients  was  completed  and  presented  at  the  2nd 
Conference  on  Medical  Mycology,  New  York  Academy  of  Sciences,  January  1960. 

3.  Eleven  new  patients  with  various  culturally  proved  fungus 
diseases,  histoplasmosis  (3),  blastomycosis  (2),  coccidioidomycosis  (1), 
aspergillosis  (1),  mucormycosis  (1),  and  sporotrichosis  (3),  have  been 
treated  with  the  new  antifungal  drug  R0-2-7758,  making  a  total  of  26 
patients  now  treated  with  this  drug.   R0-2  seems  effective  in  histo- 
plasmosis, blastomycosis,  aspergillosis,  maduramycosis ,  mucormycosis, 
and  sporotrichosis. 

A.   An  investigation  has  been  undertaken  of  the  renal  toxic  effects 
of  amphotericin.   Renal  plasma  flow  glomerular  filtration  rate  in  addi- 
tion to  concentrating  ability  in  the  kidney  have  been  determined  in  5 
patients  treated  with  this  drug. 

5.   An  investigation  of  the  toxic  effects  of  RO-2  on  the  liver 
ha?  also  been  undertaken.   Liver  biopsy  and  BSP  determinations  have 
been  evaluated  in  patients  treated  with  this  drug. 


51 


Serial  No.  NIAID  -  23(c) 

6.  An  exhibit  showing  the  results  of  therapy  with  amphotericin 
B  and  RO-2  was  prepared  and  presented  at  the  Annual  Meeting  of  the 
American  Medical  Association  at  Miami  Beach  in  June  1960. 

7.  An  exhibit  showing  the  results  of  RO-2  was  prepared  and 
presented  at  the  Annual  Meeting  of  the  American  Public  Health  Associa- 
tion in  San  Francisco,  California,  in  October  1960,  and  at  the  Clinical 
Meeting  of  the  American  Meeical  Association  in  Washington,  D.  C,  in 
November  1960. 

8.  A  clinical  study  of  Candida  endocarditis  was  undertaken  and 
observations  made  on  6  patients  with  this  disease. 

9.  A  study  is  being  made  of  granulomatous  disease  of  undifferentiated 
cause  occurring  in  man. 

Significance  to  Microbiological  Research: 

The  group  of  fungus  diseases  under  study  is  poorly  understood; 
there  is  no  uniformly  successful  treatment.   Apparently,  certain  of 
them,  such  as  histoplasmosis,  are  being  recognized  as  quite  prevalent;     \ 
others  such  as  candidiasis  create  problems  by  the  very  ubiquity  of  the 
organism,  which  may  grow  more  abundantly  and  cause  systemic  disease  in 
the  presence  of  most  antibiotics  and  steroids,  creating  serious  and 
extensive  clinical  problems.  \ 

Proposed  Course  of  Project; 

To  proceed  as  outlined  under  "Objectives"  and  "Methods." 


Part  B  included:       Yes   /X/    No  /  / 


52 


Serial  No.  NIAID  -  23(c) 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 

Part  B      Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

1.   Utz,  J.  P.,  and  Andriole,  V.  T.:   Analysis  of  amphotericin  treat- 
ment failures  in  systemic  fungal  disease.   Ann.  N.  Y.  Acad.  Sci. 
89:  277,  1960. 


Awards 


Emmons,  C.  W. ,  and  Utz,  J.  P.:   New  chemotherapy  cc  systemic 
mycosis:  Experimental  and  clinical  studies.   An  exhibit  with 
brochure.   Honorable  mention,  Experimental  Medicine  &  Therapeutic 
Section  of  Scientific  Exhibits  at  the  Annual  Meeting  of  the  AMA, 
Miami  Beach,  June  1960. 


-  4  - 


53. 


Serial  No.  NIAID  -  24(c) 

1.  LCI 

2.  Infectious  Disease  Service 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:   Sarcoidosis 


This  project  has  been  temporarily  inactivated  due  to 
lack  of  principal  investigator.   Project  to  be  reactivated  at 
a  proper  time  in  future. 


5H 


Serial  No.  NIAID  -  25(c) 

1.  LCI 

2.  Infectious  and  Pediatric 

Disease  Services 

3.  Bethesda,  Maryland 
PHS-NIH 

Individual  Project  Report 
Calendar  Year  1960 


Part  A. 

Project  Title:   Pyelonephritis 


This  project  has  been  temporarily  inactivated  due  to 
lack  of  investigators.   Project  to  be  reactivated  at  a  proper 
time  in  the  future. 


55 


Serial   No.    NIAID   -    26(c) 

1.  LCI 

2.  Infectious  Disease  Service 

3.  Bethesda,  Maryland 
PHS-NIH 

Individual  Project  Report 
Calendar  Year  1960 
Part  A 

Project  Title:   Urinary  Tract  Viruses 

Principal  Investigator:   Dr.  John  P.  Utz 

Other  Investigators:   Dr.  Hugh  Evans  (from  7/1/60) 
Clarence  F.  Szwed 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960): 
Total:         2.8 
Professional:   0.8 
Other:         2.0 

Project  Description: 

Objectives: 

1.  To  study  the  urine  of  all  patients  with  viral  infection  for 
the  presence  of  viruses  and  to  study  renal  function  concurrently. 

2.  To  investigate  in  animals  the  effect  on  the  kidneys  of  the 
inoculation  of  virus  recovered  from  human  urine. 

3.  To  attempt  to  produce  pyelonephritis  in  animals  by  virus 
inoculation  alone  or  accompanied  by  secondary  physiologic  trauma  or 
bacterial  infection. 

Methods  Employed: 

Clinical  studies:   Patients  with  a  variety  of  viral  infections 
will  be  studied  intensively. 

Laboratory  studies:   Tissue  culture  and  animal  inoculation  tech- 
niques will  be  used. 

Patient  Material  and  Major  Findings: 

1.  Two  additional  viral  isolates  have  been  recovered  from  the 
urine  of  patients  under  study  here. 

2.  In  continuing  animal  studies  on  the  effects  on  the  kidney  of 
viral  infections,  microscopic  lesions,  not  found  in  controls,  have 


56 


Serial  No.  NIAID  -  26(c) 

been  observed  in  the  proximal  convoluted  tubules  of  mice  infected  with 
Coxsackie  B-3  virus.   The  significance  of  these  lesions  is  being  studied 
further  with  fluorescent  antibody  techniques. 

3.  A  direct  comparison  has  been  made  of  tissue  culture  (cytopathic 
effect),  hemadsorption,  and  embryonated  hens  egg  techniques  in  the  isola- 
lation  of  mumps  virus  from  urine  of  patients.  In  20  patients  mumps  virus 
was  recovered  from  urine  by  criteria  of  cytopathic  effect  in  14,  by  egg 
inoculation  in  1  and  by  both  methods  in  2.  In  general  hemadsorption  oc- 
curred when  cytopathic  effect  was  seen  in  tissue  culture  but  occasionally 
one  manifestation  of  infection  was  present  without  the  other. 

4.  Because  of  the  isolated  reports  of  illness  resembling  mumps  in 
dogs  and  of  viral  isolations  from  dogs,  studies  were  undertaken  to  define 
the  matter  by  inoculation  of  quantitative  amounts  of  virus  into  puppies. 
In  4  animals  so  inoculated  no  virus  has  been  recovered  subsequently  and 
no  detectable  disease  has  been  produced.   Presence  or  absence  of  antibody 
response  will  be  determined,  in  addition. 

Significance  to  Microbiological  Research: 

The  recovery  of  a  number  of  viruses  from  the  urine  of  patients 
raises  the  question  of  the  role  they  play  in  disease.   The  relationship 
of  this  infection  to  subsequent  pyelonephritis  needs  to  be  explored. 

Proposed  Course  of  Project: 

As  outlined  in  "Objectives"  and  "Methods." 


Part  B  included:        Yes   /X/       No  /  / 


-  2  -  57 


Serial  No.  NIAID  -  26(c) 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 

Part  B      Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

1.   Habel,  K.  and  Utz,  J.  P.:  Mumps,  Ped.  Clin.  N.  America. 
7(4):  979-988,  1960. 


-  3  - 


58 


Serial  No.    NIAID   -    27(c) 

1.  LCI 

2.  Section  on  Biochemistry 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:   Biochemical  Studies  on  Staphylococcal  Cell  Walls. 

Principal  Investigator:  Dr.  Eskin  Huff 

Other  Investigators:  None 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:  0.4 
Professional:   0.4 

Objectives : 

The  general  objective  of  this  project  is  an  understanding  of  the 
chemical  mechanisms  involved  in  the  synthesis  of  staphylococcal  cell 
walls  and  the  manner  in  which  penicillin  affects  this  synthesis.   It 
is  hoped  that  a  study  of  differences  in  the  chemical  composition  and 
synthesis  of  cell  walls  by  penicillin  sensitive  and  resistant  staphlo- 
cocci  will  throw  some  light  on  the  mechanism  of  antibiotic  resistance 
in  these  organisms. 

Specifically  an  attempt  is  being  made  to  chemically  characterize 
the  organic  phosphate  containing  compounds  occurring  in  the  cell  walls 
of  staphylococcal  species. 

Methods  Employed: 

A  strain  of  Staphylococcus  aureus  (obtained  from  Miss  Rose 
Lieberman)  has  been  grown  in  large  batches  on  trypticase-soy-broth  and 
the  cells  broken  by  shaking  with  glass  beads  in  a  Nossal  Shaker.   The 
cell  walls  have  been  harvested  and  purified  by  differential  centri- 
fugation  using  a  Spinco  preparative  ultracentrifuge.   Lithium  bromide 
solutions  have  been  used  to  extract  organic  phosphate  esters  from  the 
cell  wall  material  and  these  organic  phosphate  esters  are  being  studied 
at  present. 


59 


Major  Findings;  Serial  No.  NIAID  -  27(c) 

No  major  findings  can  be  reported  at  present.   However,  it 
has  been  observed  that  staphylococcal  cell  walls  have  a  high  density 
(specific  gravity  1.5  to  1.6)  as  compared  to  most  proteins  (specific 
gravity  less  than  1.4).   It  has  also  been  noted  that  a  considerable 
amount  of  the  organic  phosphate  containing  compounds  present  in  these 
cell  walls  can  be  extracted  by  means  of  treatment  with  solutions  of 
lithium  bromide. 

Significance  to  Microbiological  Research! 

Badiley  e_t  a_l  have  evidence  for  the  presence  of  polymers  of 
ribitol  phosphate  and  glycerol  phosphate  in  the  cell  walls  of  Bacillus 
subtilis,  Lactobacillus  casei,  and  Staphylococcus  aureus.  These 
workers  have  also  demonstrated  that  In  some  strains  of  S^.  aureus, 
D-ala-nine  and  N-acetyl-glucosamine  are  attached  to  these  polymers 
which  they  call  teichoic  acids.   However,  other  than  the  studies  by 
these  workers  little  is  known  of  the  nature  of  the  phosphate  contain- 
ing compounds  present  in  staphylococci.   In  some  of  the  bacteria 
studied  the  phosphate  containing  compounds  make  up  as  much  as  60%  of 
the  weight  of  the  cell  wall  material.   In  Staphylococcus  aureus  these 
phosphate  containing  compounds  may  account  for  as  much  as  307.  of  the 
weight  of  the  cell  walls.   Although  it  has  been  demonstrated  that 
penicillin  stops  the  synthesis  of  cell  wall  peptides  in  S.    aureus  with- 
out an  effect  on  the  synthesis  of  intracellular  protein  (work  by 
Mandelstam  and  Rogers),  it  is  not  known  what  effect  penicillin  has  on  the 
synthesis  of  the  phosphate  containing  compounds  present  in  these  cell 
walls . 

A  study  of  the  biosynthesis  and  chemistry  of  the  phosphate 
compounds  of  Staphylococcal  cell  walls  appears  to  be  a  reasonable  ap- 
proach to  an  understanding  of  the  manner  in  which  cell  wall  synthesis 
is  affected  by  penicillin. 

Proposed  Course  of  Project: 

At  present  organic  phosphate  containing  compounds  have  been 
prepared  from  a  penicillin  sensitive  strain  of  S_.    aureus .   A  study  of 
the  chemical  nature  of  these  compounds  is  planned  for  the  immediate 
future.   In  the  future,  it  is  also  planned  to  study  the  molecular 
weight,  mode  of  attachment  to  cell  wall,  and  pathway  of  biosynthesis 
of  these  phosphate  containing  compounds.   These  questions  are  being 
pursued  in  the  penicillin  sensitive  strain  of  S_.  aureus  now  under 
study.   Eventually  the  properties  of  the  cell  walls  of  these  penicillin 
sensitive  organisms  will  be  compared  to  the  properties  of  the  cell  walls 
of  penicillin  resistant  organisms. 

Part  B  included  Yes  f~l     No  /X/ 


60 


Serial  No.   NIAID  -   28(c) 

1.  LCI 

2.  Infectious   Disease  Service 
PHS-NIH                             3.      Bethesda,    Maryland 

Individual   Project   Report 
Calendar  Year   1960 


Part  A. 

Project  Title:   Pathogenesis  of  Viral  Infections 

Principal  Investigator:   Dr.  Julius  A.  Kasel 

Other  Investigators:   None 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960) : 
Total:         1.3 
Professional:   1.0 
Other:         0.3 


Project  Description: 
Objectives: 

1.  To  investigate  the  prevalence  of  antigens  separable  from 
infectious  virus  and  their  significance  in  relationship  to 
the  pathogenesis  of  virus  infection. 

2.  To  study  exfoliated  cells  in  the  urine,  buccal  mucosa  and 
blood  cells  from  patients  ill  with  viral  infections  for 
the  presence  of  viral  antigen  by  fluorescent  microscopy 
concurrently  with  virus  isolation  procedures. 

3.  To  determine  sialic  acid  levels  of  serum  and  urine  from 
patients  ill  with  respiratory  viruses. 

Methods  Employed: 

Clinical  studies:   Material  from  patients  with  viral  infections 
and  non-viral  illnesses  will  be  utilized  in  the  projects  under  study. 

Laboratory  studies:   Tissue  culture,  chromatography, 
fluorescent  microscopy,  serologic  and  biologic  techniques  will  be 
utilized. 

Patient  Material  and  Major  Findings: 

1.   Recent  adenovirus  type  1  and  2  isolates  and  prototype 
strains  1,2,4  and  15  were  found  to  possess  the  property  of  removing 


61 


Serial  No.  NIAID  -  28(c) 


erythrocyte  receptor  sites  possibly  by  enzymatic  action,  from  human 
red  blood  cells  for  three  of  five  adenovirus  hemagglutinins.  The 
antigen  responsible  for  this  property  is  separable  from  the  infectious 
virus  particle  and  not  related  to  the  antigens  causing  cell  detachment 
and  early  cytopathogenicity .   Studies  indicated  that  the  adenovirus 
factor  was  a  macromolecular  substance,  heat  stable,  resistant  to 
proteolytic  and  nuclease  enzymes  and  was  neutralized  by  homologous 
but  not  heterologous  serum. 

Virus  suspensions  devoid  of  infectious  virus,  cell-detaching 
and  early  cytopathic  factors  exhibited  an  "inhibiting  property"  for 
certain  adenovirus  serotypes  in  tissue  culture. 

2.  Viruses  sharing  antigenic  properties  of  adenovirus  types 
9  and  15  were  isolated  during  an  investigation  of  an  outbreak  of 
febrile  illness  among  children. 

In  one  patient  convalescing  from  rheumatic  pancarditis,  the 
viral  infection  was  associated  with  signs  and  symptoms  interpreted 
as  consistent  with  re-activation  of  the  rheumatic  process. 

3.  Significant  neuraminidase  activity  was  associated  with 
prototype  adenovirus  type  15  grown  in  HeLa  tissue  cultures.   Screening 
of  type  15  suspensions  were  constantly  negative  for  the  presence  of 
myxovirus  contaminants.   The  enzyme  activity  appeared  to  be 
associated  with  the  virus  particle  since  suspensions  adsorbed  with 
susceptible  erythrocytes  demonstrated  a  marked  decrease. 

Significance  to  Microbiological  Research: 

1.  The  presence  of  viral  antigens  other  than  infectious 
viral  particles  in  infectious  tissue  culture  fluids  raises  the 
question  of  their  significance  in  disease  and  immunity.  Their  role 

in  viral  reproduction  and  possible  effect  on  the  human  host  needs  to  be 
defined. 

2.  Since  prolonged  excretion  in  the  urine  of  mumps  virus 
raises  the  possibility  of  virus  growth  in  the  kidney;  a  study  of 
exfoliated  cells  in  the  urine  for  presence  of  virus  needs  to  be 
studied. 

3.  Determination  of  sialic  acid  levels  in  the  blood  and  urine 
of  patients  ill  with  viral  infections  may  warrant  this  biochemical 
assay  as  a  clinical  tool»   The  further  definition  of  the  enzyme-like 
factor  associated  with  adenoviruses  needs  to  be  further  explored  for 
reaction  products  also,  for  consequent  use  as  a  diagnostic  test. 


62 


Serial  No.   NIAID   -   28(c) 


Proposed  Course   of  Project: 

As   outlined   in   "Objectives   and  Methods." 


Part   B    included:  Yes    /J7  No   AT 

3 


63 


Serial  No.  NIAID  -  28(c) 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 

Part  B:   Honors,  Awards,  and  Publications. 

Publications  other  than  abstracts  from  this  project: 

1.  Kasel,  J.  A.,  Rowe,  W.  P.  and  Nemes,  J.  C:  Modification  of 
Erythrocyte  Receptors  by  a  Factor  in  Adenovirus  Suspensions. 
Virology,  10:  389-391,  1960. 

2.  Cramblett,  H.  G.,  Kasel,  J.  A.,  Langmack,  M.  and  Wilken,  F.  D. : 
Illnesses  in  Children  Infected  with  an  Adenovirus  Antigenically 
Related  to  Types  9  and  15.   Pediatrics,  25:  822-828,  1960. 


6«* 


LABORATORY  OF  CELL  BIOLOGY 


Summary 1 

50  -  Biosynthesis  in  Mammalian  Cell  Cultures..   3 

51  -  The  Mechanism  of  Production  of 

Poliovirus  in  Cultured  Mammalian  Cells...   8 

52  -  Effects  of  Animal  Viruses  on  the 

Metabolism  of  Mammalian  Cell  Cultures 

and  the  Mechanism  of  Viral  Replication...  11 

53  -  Mechanism  of  Drug  Resistance  in  Cultured 

Mammalian  Cells 14 

54  -  Metabolic  Control  Mechanisms  in  Cultured 

Mammalian  Cells 17 

55  -  Application  of  the  Technique  of  Tissue 

Culture   to  Selected  Genetics  Diseases...  19 


SUMMARY 

Research  Projects  from 

The  Laboratory  of  Cell  Biology 

(Serial  Nos.  NIAID  50-55) 

The  activities  of  the  Laboratory  of  Cell  Biology  during  the  calendar 
year  1960  have  been  along  3  major  lines:   (A)  The  continued  exploration  of 
the  metabolism  of  normal  cultured  cells,  and  an  approach  to  the  problem  of 
metabolic  controls;   (B)  the  mechanism  of  viral  synthesis;  and  (C)  the  study 
of  cell  cultures  deriving  from  patients  with  hereditary  metabolic  disease. 

A. 

A  number  of  significant  observations  have  been  made  with  respect  to  the 
amino  acid  metabolism  of  cell  cultures.   There  has  been  no  further  eluci- 
dation of  the  pathway  of  serine  synthesis  since  last  year's  report;  but  the 
mechanism  of  cystine  synthesis  has  been  clarified,  in  that  all  the  cell  lines 
so  far  studied  have  been  shown  to  use  the  classical  pathway  involving  the 
demethylation  of  methionine  to  homocysteine,  the  condensation  of  the  latter 
with  serine  to  form  cystathionine,  and  the  cleavage  of  the  latter  to 
cysteine  and  homoserine.  A  dual  pathway  for  proline  synthesis  has  been 
indicated,  one  involving  glutamine  as  the  source  of  the  carbon  skeleton, 
and  the  other  involving  arginine  by  way  of  ornithine. 

An  intriguing  recent  observation  has  been  the  finding  that  a  number  of 
factors  which  are  rigorously  required  by  the  cells  for  survival  and  growth 
can  in  fact  be  synthesized.   Their  nutritional  requirement  reflects  the  fact 
that  they  are  lost  from  the  cellular  pool  to  the  medium  at  rates  which  exceed 
the  biosynthetic  capacity  of  the  cell;  and  with  a  sufficiently  high  cell 
population  density,  when  the  loss  to  the  medium  per  cell  is  sufficiently 
reduced,  the  supposedly  essential  growth  factors  are  in  fact  not  required 
for  survival. 

In  these  cell  cultures,  unlike  bacteria,  the  biosynthesis  of  amino 
acids  is  apparently  not  inhibited  by  the  product  of  the  reaction;  and  this 
mechanism  of  growth  control  is  apparently  not  operative.   Studies  are  in 
progress  as  to  whether  enzyme  repression  or  feedback  inhibition  are 
effective  controls  in  the  biosynthesis  of  pyrimidines.   A  quite  different 
control  mechanism  is  perhaps  indicated  by  the  demonstration  of  a  growth 
inhibitor  in  the  supernatant  medium  of  heavy  cultures.   The  chemical  nature 
of  that  inhibitor  is  under  continuing  study. 

Studies  on  the  mechanism  of  resistance  to  2-deoxyglucose  (2DG)  have 
shown  the  presence  in  the  resistant  variants  of  compounds  which  inhibit  the 
phosphorylation  of  2DG  to  the  metabolically  active  inhibitor,  2DG-phosphate. 
The  relationship  of  that  inhibitor  to  the  observed  resistance  is  under 
continuing  study. 


-  1  - 


B. 

A  number  of  important  new  observations  have  been  made  with  respect  to 
the  mechanisms  of  viral  synthesis.   The  puzzling  wide  disparity  between  the 
number  of  physical  particles  in  viral  suspensions,  and  the  number  of  plaque- 
forming  units,  i.e.  particles  capable  of  initiating  infection  in  susceptible 
cells,  has  been  partially  resolved  with  the  demonstration  that  after  the 
viral  particle  has  been  absorbed  by  the  cell,  it  may  undergo  several 
alternative  fates.   A  large  proportion  are  rapidly  eluted  into  the  medium, 
essentially  intact  but  no  longer  infectious,  presumably  reflecting  a  minor 
alteration  in  the  protein  coat.   Some  particles  remain  unchanged  within  the 
cell.   Others  are  degraded  intracellular ly,  in  that  the  nucleic  acid  is 
exposed  and  becomes  susceptible  to  intracellular  ribonuclease.   Only  a 
small  fraction  of  the  absorbed  viral  particles  are  stripped  of  their  protein 
and  initiate  infection. 

In  the  case  of  poliovirus  in  the  HeLa  cell,  although  the  viral  protein 
and  RNA  are  synthesized  concomitantly,  a  partial  dissociation  has  been 
achieved  with  appropriate  inhibitors  of  protein  synthesis,  which  completely 
block  the  formation  of  mature  virus,  but  not  of  infectious  RNA.   This  is  of 
particular  importance  in  relation  to  the  supposedly  obligatory  relationship 
between  protein  and  RNA  synthesis  in  growing  cells.   Of  interest  also  is  the 
fact  that  matabolic  inhibitors  which  effectively  block  the  synthesis  of 
cellular  DNA  and  of  DNA  viruses  have  no  effect  on  the  formation  of  polio- 
virus.   It  would  therefore  appear  that  poliovirus  RNA  may  be  used  directly 
as  a  template  for  the  formation  of  virus,  without  the  necessity  for 
intervening  DNA  synthesis. 

In  contrast  to  the  situation  with  poliovirus,  in  the  case  of  vaccinia, 
there  was  a  marked  lag  between  the  formation  of  the  viral  nucleic  acid 
(DNA)  and  that  of  the  mature  virus. 

C. 

An  exciting  new  development  has  been  the  successful  cultivation  from 
patients  with  a  hereditary  metabolic  disease  (galactosemia)  of  cells  which 
in  culture  demonstrate  the  metabolic  defect  characteristic  of  the  disease. 
This  suggests  an  entirely  new  experimental  approach  to  problems  of  human 
genetics. 


2  - 


NIAID  50 

Laboratory  of  Cell  Biology 

Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   Biosynthesis  in  Mammalian  Cell  Cultures 

Principal  Investigator:   Dr.  Harry  Eagle 

Other  Investigators:   Dr„  Jacob  Maizel,  Mr.  Ralph  Fleischman,  Mr„  Aaron 
Freeman,  Miss  Mina  Levy  and  Mr.  Vance  I.  Oyama 

Cooperating  Units:   NIDR,  Dr.  Karl  A.  Piez 

New  York  University,  Dr.  Bernard  Horecker 

Man  Years  (calendar  year  1960): 
Total:   7  1/2 
Professional:   5  1/4 
Other:   2  1/4 

Project  Description: 

Objectives :   This  program  involves  a  broad  exploration  of  the 
nutritional  requirements,  metabolic  activities  and  biosynthetic  pathways  of 
a  variety  of  human  and  animal  cells,  under  varying  conditions  of  culture. 

Methods  Employed:   The  techniques  used  in  the  culture  and  chemical 
fractionation  of  these  cells,  in  the  separation  and  identification  of  their 
metabolic  products,  and  in  the  use  of  isotopically-labeled  precursors,  have 
been  described  in  detail  in  previous  publications. 

Major  Findings; 

1.   Amino  acid  biosynthesis  and  interconversion 

a.   Metabolic  independence  of  serine  and  alanine.   The  so-called 

"glucose  family"  of  amino  acids  (alanine,  serine  and  glycine),  all  of  which 

derive  their  carbon  solely  from  glucose  when  cells  are  grown  in  a  limiting 

minimal  medium,  actually  fall  into  two  sharply  defined  groups,  between  which 

there  is  little  or  no  metabolic  inter-conversion.   Serine  is  the  immediate 

precursor  of  glycine;  but  there  is  no  significant  metabolic  relationship 

between  alanine  and  these  two  amino  acids.  When  cells  are  provided  withc1^- 

labeled  serine  or  glycine,  these  two  amino  acids/are  heavily  labeled,  but 

'  in  the  cell 


Part  B  included. 

-  1  - 


NIAID  50 

protein, 
essentially  none  of  the  isotope  appears  in  the  alanine  residues  of  the  cell/ 

Further,  when  cells  are  grown  on  a  medium  which  contains  ribose  and 

pyruvate  in  lieu  of  glucose,  the  serine  and  glycine  derive  almost  completely 

from  the  ribose,  while  the  alanine  derives  completely  from  the  pyruvate. 

This  finding  provides  a  direct  approach  to  the  as  yet  unknown  mechanisms 

involved  in  the  biosynthesis  of  serine. 

b.  Cystine  synthesis.   It  has  now  been  clearly  established  that  all 
serially  propagated  human  cell  lines  so  far  studied  are  indeed  capable  of 
synthesizing  cystine  from  methionine  and  glucose  by  the  classical  homo- 
cysteine-cystathionine pathway.   The  capacity  to  synthesize  cystine  is 
therefore  not  limited  to  liver  cells,  as  had  previously  been  presumed  to  be 
the  case. 

c.  Proline  synthesis.   In  a  minimal  medium,  the  glutamine  family 

of  amino  acids  (aspartic  and  glutamic  acids,  asparagine  and  proline)  devolve 
primarily  from  glutamine.   In  the  case  of  proline  there  has  however  been  a 
consistent  discrepancy,  in  that  only  half  of  the  carbon  skeleton  derived 
from  glutamine,  about  10  per  cent  additionally  from  glucose,  while  the  rest 
was  unaccounted  for.   This  has  now  been  resolved  with  the  finding  that 
slightly  more  than  1/3  of  the  proline  carbon  derives  from  arginine  by  way 
of  ornithine. 

d.  Negative  feedback.   There  is  now  an  increasing  body  of  evidence 
that  the  biosynthetic  control  mechanisms  operative  in  bacteria,  in  which 
the  product  of  a  given  reaction  serves  to  limit  its  synthesis,  is  not 
operative  in  mammalian  cells,  at  least  with  respect  to  the  amino  acids. 

In  the  examples  so  far  studied  the  provision  of  serine,  cystine,  and 
homocystine  has  not  served  to  curtail  their  continuing  biosynthesis. 

2.  The  anamolous  requirement  by  human  cells  for  metabolites  which 
they  can  synthesize.   The  interesting  observation  has  been  made  that 
cultured  mammalian  cells  frequently  require  metabolites  which  they  are 
actually  capable  of  synthesizing,  and  in  amounts  which  should  suffice  for 
growth.   This  has  been  found  to  be  the  case  for  cystine,  homocystine, 
serine,  inositol,  and  in  the  case  of  certain  cell  lines,  for  pyruvate  as 
well.   This  paradoxical  observation  has  now  been  resolved,  in  that  the 
function  of  the  added  metabolite  is  simply  to  prevent  the  loss  of  the 
newly  synthesized  compound  from  the  cellular  pool  to  the  medium  at  a  rate 
which  exceeds  the  biosynthetic  capacity  of  the  cell.  When  the  population 
density  is  sufficiently  increased,  the  exogenous  metabolite  is  no  longer  . 
required  for  cell  survival  and  growth.   When  the  task  of  "conditioning" 
the  medium  is  then  shared  by  a  sufficiently  large  number  of  cells,  the 
biosynthetic  capacity  of  an  individual  cell  is  no  longer  exceeded. 

3.  Chemostat  studies.   The  development  of  a  chemostat  for  mammalian 
cell  cultures  was  described  in  last  year's  progress  report.   It  has  since 
been  learned  that  when  the  rate  of  growth  is  greatly  reduced  by  appropriately 
limiting  the  rate  at  which  fresh  medium  is  introduced  Into  the  culture,  the 
factor  which  then  limits  the  growth  of  the  cells  is  not  the  depletion  of 


NIAID  50 

medium,  but  instead,  the  elaboration  of  an  active  growth  inhibitor.  This 
growth  inhibitor  is  not  dialyzable  and  is  presumably  associated  with  the 
proteins  of  the  medium.   Attempts  at  its  isolation  and  characterization  are 
now  in  progress. 

4.  Protein  growth  factor.   Most  human  and  animal  cells  in  serial 
culture  have  an  absolute  requirement  for  serum  protein.   That  protein 
however  is  not  used  to  a  significant  degree  for  the  synthesis  of  cell 
protein,  but  instead  has  a  dual  role:   it  promotes  the  adhesion  of  cells 
to  glass  surface,  and  provides  essential  and  as  yet  undefined  growth 
factors.   The  active  factor  has  proved  to  be,  not  the  protein  molecule  as 
such,  but  diffusible  compounds  of  small  molecular  weight  which  are  formed 
from  it  on  its  proteolytic  digestion.   It  is  not  yet  clear  whether  those 
growth  factors  are  peptides,  or  compounds  gf^tmntfel^aaolfce.ular-  wffiighA^ 
initially  bound  to  the  protein  and  released  from  it  as  it  is  digested.   In 
either  case,  the  dialysate  of  such  a  digested  protein  preparation  supports 
the  indefinite  growth  of  a  wide  variety  of  human  cells  in  suspension 
culture.   Studies  on  the  identification  of  the  active  compounds  in  such 
dialysates  are  now  in  progress. 

5.  The  utilization  of  inositol  phosphates.   Dr.  S.  J.  Angyal  of  the 
University  of  New  South  Wales,  Australia,  has  prepared  all  the  inositol 
phosphate  isomers.  All  have  proved  to  be  equally  active  in  supporting  the 
growth  of  human  cells.   It  is  not  yet  clear  whether  the  phosphates  are 
taken  into  the  cell  directly,  or  whether  they  must  first  be  hydrolyzed 
extracellularly. 

Significance  to  the  Program  of  the  Institute:   Studies  on  the  metabolic 
activities  of  human  and  animal  cells  are  of  obvious  basic  significance.   For 
NIAID  research  specifically,  these  studies  are  relevant  to  the  problems  of 
antibody  production,  allergy,  phagocytosis,  inflammation,  virus  propagation, 
and  many  other  problems  which  are  the  concern  of  the  microbiologist 
interested  in  the  study  of  human  disease. 

Proposed  Course  of  Program;   It  is  clear  from  the  foregoing  that  none 
of  these  are  finished  projects.   The  long-range  program  envisages  a  similar 
broad-scale  study  on  the  metabolism  of  tissue  explants.   This  will  have  the 
multiple  purpose  of  pin-pointing  possible  differences  between  the  metabolism 
of  dispersed  and  structurally  organized  cells,  exploring  the  conditions 
necessary  for  the  preservation  of  specialized  functions  ijn  vitro,/ following 
the  chromosomal  aberrations  as  the  cells  multiply  under  varying  conditions 
of  growth. 


-  3  - 


NIAID  50 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B     Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Eagle,  H„   Nutritional  Requirements  for  Cell  Growth  and  Poliovirus 
Propagation.   Perspectives  in  Virology  75-87.   John  Wiley  &  Sons, 
Inc.,  N.  Y.  1959. 

Eagle,  H.   Metabolic  Studies  with  Normal  and  Malignant  Human  Cells 
in  Culture.   The  Harvey  Lectures,  1958-1959.  Academic  Press,  Inc., 
No  Y.  156-175,  I960, 

Eagle,  H.  The  Sustained  Growth  of  Human  and  Animal  Cells  in  a 
Protein-Free  Environment.  Proc.  Natl.  Acad.  Sci.  46:  427-432, 
April  1960. 

Eagle,  H.  and  Piez,  K„  A.   The  Utilization  of  Proteins  by  Cultured 
Human  Cells.   J.  Biol.  Chem.  235:  1095-1097,  April  1960. 

Eagle,  H.,  Oyama,  V.  I.  and  Piez,  K.  A.   The  Reversible  Binding  of 
Half-cystine  Residues  to  Serum  Protein,  and  Its  Bearing  on  the 
Cystine  Requirement  of  Cultured  Mammalian  Cells.   J.  Biol.  Chem. 
235:  1719-1726,  June  1960. 

Eagle,  H.,  Agranoff,  B.  W.  and  Snell,  E.  E.   The  Biosynthesis  of 
meso-Inositol  by  Cultured  Mammalian  Cells,  and  the  Parabiotic  Growth 
of  Inositol-dependent  and  Inositol-independent  Strains.   J.  Biol. 
Chem.  235:  1891-1893,  July  1960. 

Piez,  K.  A.,  Levintow,  L.,  Oyama,  V.  I.  and  Eagle,  H.  Proteolysis 

in  Stored  Serum  and  its  Possible  Significance  in  Cell  Culture.   Nature 

188:  59-60,  October  1960. 

Darnell,  J.  E.,  Jr.  and  Eagle,  H.   The  Biosynthesis  of  Poliovirus  in 
Cell  Cultures.   Advances  in  Virus  Research.   In  press. 

Cohen,  E.  P.,  Nylen,  M.  U.  and  Scott,  D.  B.   Micros true tural  Changes 
Induced  in  Mammalian  Cell  Cultures  by  Omission  and  Replenishment  of 
a  Single  Essential  Amino  Acid.   Exptl.  Cell  Research.   In  press. 

Cohen,  E.  P.  and  Eagle,  H.  A  Simplified  Mammalian  Cell  Chemostat. 
J.  Exptl.  Med.   In  press. 

Levintow,  L.  and  Eagle,  H.   The  Biochemistry  of  Cultured  Mammalian 
Cells.   The  Annual  Review  of  Biochemistry.   In  press. 

Eagle,  H.,  Piez,  K.  A.  and  Oyama,  V.  I.   The  Biosynthesis  of  Cystine 
and  Mammalian  Cell  Culture.   In  preparation.  Q 

-  1  - 


NIAID  50 

Honors  and  Awards  relating  to  this  project: 

Invited  Lectures 

January  22-26,  1960:   Perspectives  in  Virology.   New  York  City. 

February  9,  1960:   NIH  Lecture  Series.  Clinical  Center. 

March  24,  1960:   Bristol  Laboratories.   Syracuse,  New  York. 

March  25-26,  1960:   Symposium  on  the  Phenomena  of  Tumor  Viruses. 
N.Y.C.   Sponsored  by  The  Virology  and 
Rickettsiology  Study  Section,  DRG-NIH. 

June  20-22,  1960:   Kingston,  Rhode  Island.  University  of  Rhode  Island. 
Symposium:   Newer  Chemical  and  Biological  Techniques 
of  the  1960  Medicinal  Chemistry  Section  of  the 
American  Chemical  Society. 

July  5,  1960:   Seminar,  Research  Division  of  Ethicon.   Somerville,  N.  J. 

August  9,  1960:   Long  Island  Biological  Labs.,  Cold  Spring  Harbor,  N.  Y. 

September  12,  1960:  American  Chemical  Society,  New  York  City. 

September  28,  1960:   Brandeis  University  seminar,  Waltham,  Massachusetts, 

November  4,  1960:   University  of  Delaware  seminar,  Wilmington,  Delaware. 

November  5,  1960:   Eastern  Psychiatric  Research  Assoc,  symposium. 
N.  Y.  C. 

November  13-15,  1960:   American  Cancer  Society  Lecturer,  Purdue 
University,  Lafayette,  Indiana. 


-  2  - 


NIAID  51 

Laboratory  of  Cell  Biology 

Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   The  Mechanism  of  Production  of  Poliovirus  in  Cultured 
Mammalian  Cells 

Principal  Investigator:   Dr.  Leon  Levintow 

Other  Investigators:   Dr.  James  E.  Darnell,  Jr.,  Dr.  W.  K.  Joklik, 
Dr.  Jacob  V.  Maizel,  Mrs.  Marilyn  M.  Thoren 
and  Mr.  J.  Leonard  Hooper 

Cooperating  Units:   None 

Man  Years  (Calendar  year  1960) 
Total:   7  1/4 
Professional:   2  1/4 
Other:   5 

Project  Description: 

Objectives:   To  define  the  events  during  the  adsorption  and 
maturation  of  poliovirus  in  biochemical  terms,  and  to  investigate  the 
mode  of  replication  of  viral  protein  and  RNA. 

Methods  Employed:   The  program  is  based  primarily  on  the  following 
techniques:   a)   Infection  of  cells  with  poliovirus  under  controlled 
conditions  in  a  chemically  defined  medium;  b)  Purification  of  the  polio- 
virus so  produced;  c)  Preparation  of  infectious  RNA  from  infected  cultures, 
and  from  purified  virus;  d)  Assays  of  the  infectivity  of  whole  virus  and 
infectious  RNA  by  a  plaque  assay  on  HeLa  cell  monolayers. 

Additional  techniques  include  paper  and  column  chromatography, 
preparative  and  analytical  ultrcentrifugation,  and,  in  collaboration  with 
other  groups,  electron  microscopy. 

Major  Findings:   The  events  early  in  the  infective  cycle  have  been 
studied  with  purified  poliovirus  labeled  with  P32.   Virtually  all  virus 
particles  are  capable  of  being  adsorbed  to  HeLa  cells;  once  adsorbed,  a 
particle  has  several  alternative  fates.   It  may:   1)  be  eluted  into  the 
medium,  essentially  intact  but  no  longer  infectious;  2)  be  degraded  intra- 
cellular ly  so  that  the  P^2  label  becomes  acid-soluble;  3)  be  retained 
essentially  unchanged  within  the  cell;  or  4)  be  stripped  of  its  protein 
coat  and  initiate  infection. 

Part  B  included.  -s 

-i-  3 


N1AID  51 

These  observations  provide  an  explanation  for  the  low  ratio  of 
infectious:   physical  particles  characteristic  of  poliovirus. 

Following  adsorption  of  virus  to  cells,  there  is  a  latent  period 
of  2-2  1/2  hours  during  which  there  is  appreciable  synthesis  of  viral 
material.   The  synthesis  of  viral  RNA  and  protein  then  begin  simultaneously, 
and  mature  virus  begins  to  appear  shortly  thereafter.   Unlike  the  situation 
in  most  other  viral  systems,  there  is  at  no  time  a  sizable  pool  of 
unassembled  viral  macromolecules.   The  synthesis  of  viral  protein  and  RNA 
appears  to  be  more  closely  coordinated  than  in  any  viral  system  heretofore 
studied.   This  coupling  of  RNA  and  protein  synthesis  is  not  obligatory, 
however;  for  in  the  presence  of  p-f luorophenylalanine,  an  inhibitor  of 
protein  synthesis,  the  synthesis  of  viral  RNA  is  initiated  at  the  usual 
time,  but  protein  and  mature  virus  are  not  formed.   When  the  inhibition 
of  protein  synthesis  is  reversed  by  the  subsequent  addition  of  phenylalanine, 
the  synthesis  of  viral  protein  begins,  shortly  followed  by  the  appearance 
of  mature  virus.   These  observations  imply  that  the  synthesis  of  viral  RNA 
is  not  dependent  on  concurrent  protein  synthesis. 

Significance  to  the  Program  of  the  Institute:   Virus  replication 
provides  a  useful  model  of  a  rapidly  reproducing  unit  at  the  cellular  and 
subcellular  level  in  which  protein  and  nucleic  acid  synthesis  can  be 
studied  chemically  and  biologically.   Such  studies  not  only  provide 
insights  into  the  mode  of  the  replication  of  protein  and  nucleic  acid 
molecules,  but  have  an  obvious  and  important  bearing  on  the  evolution  of 
viral  disease  at  both  the  cellular  and  whole  animal  level. 

Proposed  Course  of  the  Projecti 

1.  The  procedure  for  the  purification  of  poliovirus  is  being 
adapted  to  provide  the  larger  quantities  of  pure  virus  necessary  for  the 
physico-chemical  characterization  of  the  protein  subunit. 

2.  Further  investigation  will  be  made  of  the  fate  of  the  infecting 
viral  particle,  a  problem  complicated  by  the  large  number  of  ineffective 
virus  particles. 

3.  The  reasons  for  the  delay  between  adsorption  and  the  initiation 
of  virus  replication  will  be  investigated. 

4.  Further  studies  will  be  carried  out  on  the  chemical,  physical 
and  biological  properties  of  the  infectious  RNA  extracted  from  purified 
virus. 

5.  Experiments  on  the  relationships  between  the  synthesis  of  viral 
protein  and  RNA  synthesis  will  be  continued,  using  appropriate  metabolic 
antagonists. 


-  2  - 


NIAID  51 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Levintow,  L.  and  Darnell,  J.  E.,  Jr.  A  Simplified  Procedure  for 
Purification  of  Large  Amounts  of  Poliovirus:   Characterization  and 
Amino  Acid  Analysis  of  Type  1  Poliovirus.   J.  Biol.  Chem.  235;  70, 
1960. 

Darnell,  J.  E.,  Jr.  and  Levintow,  L.  Poliovirus  Protein:  Source 
of  Amino  Acids  and  Time  Course  of  Synthesis.  J„  Biol.  Chem.  235: 
74,  1960. 

Darnell,  J.  E.,  Jr.,  Levintow,  L.,  Thoren,  M.  M.  and  Hooper,  J.  L. 
The  Time  Course  of  Synthesis  of  Poliovirus  RNA.  Virology.   In  press. 

Joklik,  W.  K.  and  Darnell,  J.  E.,  Jr.  The  Adsorption  and  Early  Fate 
of  Purified  Poliovirus  in  HeLa  Cells.  Virology.   In  press. 

Darnell,  J.  E.,  Jr.  and  Eagle,  H.   The  Biosynthesis  of  Poliovirus  in 
Cell  Cultures.  Advances  in  Virus  Research.   In  press. 


-  1  - 


10 


NIAID  52 

Laboratory  of  Cell  Biology 

Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   Effects  of  Animal  Viruses  on  the  Metabolism  of  Mammalian 
Cell  Cultures  and  the  Mechanism  of  Viral  Replication 

Principle  Investigator:   Dr.  Norman  P.  Salzman 

Other  Investigators:   Mr.  Edwin  D.  Sebring,  Dr.  William  Munyon  and 
Dr.  Chiaki  Nishimura 

Cooperating  Units :   None 

Man  Years  (calendar  year  1960) : 
Total:   3  3/4 
Professional:   1 
Other:   2  3/4 

Project  Description: 

Objectives :   To  determine  the  alterations  in  cell  metabolism 
resulting  from  infection  with  poliovirus  and  vaccinia  virus,  and  to  study 
the  primary  site  of  action  of  the  virus;  to  determine  the  time  course  of 
synthesis  of  the  component  parts  of  vaccinia  virus  (nucleic  acid  and 
protein)  as  related  to  the  formation  of  infectious  virus,  and  to  study  the 
effect  of  inhibitors  on  these  synthetic  processes. 

Methods  Employed:   The  techniques  employed  involve  cell  fractionation, 
isolation  of  the  purified  component  nucleic  acid  bases,  the  use  of  radioactive 
precursors,  the  various  techniques  required  for  the  viral  infection  of  cells, 
the  plaque  assay  for  measurement  of  infectious  virus,  and  chemical  methods  of 
virus  purification. 

Major  Findings:   The  findings  are  in  three  separate  areas. 

1.   Interrelation  between  cellular  RNA,  DNA  and  protein.   Using  a 
specific  inhibitor  of  DNA  synthesis,  5-f luorodeoxyuridine,  the  presence  of 
two  distinct  classes  of  RNA  and  protein  have  been  demonstrated  in  animal 
cells.   When  DNA  synthesis  is  inhibited,  nuclear  RNA  and  nuclear  protein 
synthesis  are  also  inhibited;  under  these  conditions  however,  cytoplasmic 
protein  and  RNA  synthesis  continue  at  optimal  rates.   Because  of  the  linkage 
of  nuclear  RNA  and  protein  synthesis  to  DNA  synthesis,  certain  species  of 
protein  will  be  synthesized  only  in  growing  cells,  for  under  non-growing 
conditions,  there  is  no  increase  in  DNA.   This  may  be  a  fundamental  mechanism 
for  the  control  of  the  levels  of  various  enzymes  in  mammalian  cells. 

li 

i"art:  B  included.  -  1  - 


NIAID  52 

2 .   Incorporation  of  nucleic  acid  analogues  into  pollovlrus.   Our 
previous  studies  established  that  the  presence  of  5-f luorouracil  or  its 
deoxyriboside  in  the  medium  did  not  affect  the  de_  novo  synthesis  of  polio- 
virus.   Since  poliovirus  synthesis  occurred  under  conditions  where  no  DNA 
synthesis  is  possible,  it  seems  likely  that  poliovirus  RNA  is  used  directly 
as  the  template  for  the  synthesis  of  poliovirus  protein.   Changes  in  the 
virus  RNA  template  might  then  be  expected  to  produce  changes  in  the  virus 
protein.   Thus  far  an  altered  template  has  been  produced  by  the  incorporation 
of  5-f luorouracil  into  virus  RNA.   The  degree  of  incorporation  of  5-fluoro- 
uracil  is  concentration  dependent.   Even  when  20  per  cent  of  the  uracil  in 
viral  RNA  has  been  replaced  by  5-f luorouracil,  the  resultant  virus  is  fully 
infectious.  Further  studies  on  the  properties  of  f luorouracil-containing 
poliovirus  are  planned. 

3»  Vaccinia  virus  formation.  The  kinetics  of  formation  of 
vaccinia  virus  DNA  and  of  infectious  virus  was  previously  determined,,  The 
synthesis  of  viral  DNA  was  almost  complete  prior  to  the  formation  of  any 
new  infectious  virus.   It  has  now  been  shown  that  the  viral  protein  is 
synthesized  in  the  time  between  DNA  formation  and  the  appearance  of 
infectious  virus. 

Significance  to  the  Program  of  the  Institute:   The  effect  of  viruses 
on  animal  cells,  the  manner  in  which  viruses  replicate,  and  the  effect  of 
inhibitors  on  these  processes  is  of  obvious  relevance  to  an  understanding 
of  viral  infection,  and  may  conceivably  suggest  new  areas  of  exploration 
in  relation  to  viral  chemotherapy. 

Proposed  Course  of  Projects 

1.   The  properties  of  a  limited  number  of  enzymes  involved  in 
cellular  metabolism  will  be  examined  to  determine  if  they  are  associated 
with  the  nucleus  or  the  cytoplasm.   The  importance  of  these  findings  as  a 
basic  mechanism  in  growth  regulation  will  be  evaluated. 

2.  By  paper  electrophoresis  of  the  RNA  nucleotides  of  purified 
poliovirus,  it  will  be  determined  if  labeled  fluorouracil  is  incorporated 
as  the  nucleotide.   The  percent  replacement  of  uracil  by  fluorouracil  will 
be  measured  by  the  amount  of  radioactive  precursor  incorporated  and  by 
spectrophotometric  determinations  of  the  eluted  nucleotides.   The  chromato- 
graphic analysis  of  the  amino  acid  residues  of  both  fluorouracil-substituted 
and  control  viral  protein  is  planned.   Other  possible  effects  of  fluoro- 
uracil incorporation  into  poliovirus  will  be  studied,  such  as  altered  UV 
inactivation,  heat  inactivation,  specific  infectivity,  and  possible  changes 
in  the  one  step  growth  curve. 

3.  Procedures  for  vaccinia  virus  purification  from  cell  cultures 
are  being  studied.  The  various  protein  components  of  the  virus  will  be 
purified  and  used  to  prepare  specific  precipitating  antisera.   These  will  be 
used  in  studies  on  the  kinetics  of  viral  protein  formation. 

Part  B  included.  12 


NIAID  52 


PHS-NIH 

Individual  Project  Report 
Calendar  Year  1960 


Part  B,   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Salzman,  N.  P.   The  Rate  of  Formation  of  Vaccinia  Deoxyribose 
Nucleic  Acid  and  Vaccinia  Virus.  Virology,  1£:  150  (1960). 

Salzman,  N.  P.  and  Sebring,  E.  D.   The  Source  of  Poliovirus 
Ribonucleic  Acid.  Virology.   In  press. 

Honors  and  Awards  relating  to  this  project:   None. 


-  1  - 


13 


NIAID  53 

Laboratory  of  Cell  Biology 

Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   Mechanism  of  Drug  Resistance  in  Cultured  Mammalian  Cells 

Principal  Investigator:   Dr.  Stanley  Barban 

Other  Investigators:   Mr.  Henry  0„  Schulze 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960) 
Total:   2  3/4 
Professional:   1 
Other:   1  3/4 

Project  Description: 

Objectives:   To  study  the  mechanism  of  resistance  to  a  glucose 
analogue,  2-deoxyglucose  (2DG),  by  a  resistant  strain  of  human  cells  in 
culture. 

Methods  Employed:   By  the  use  of  cloning  procedures,  a  strain  of 
HeLa  cells  has  been  isolated  which  can  grow  in  the  presence  of  high 
concentrations  of  2DGo   Other  techniques  have  included  procedures  for  the 
separation  and  identification  of  the  carbohydrates  and  their  metabolic 
products,  preparation  of  enzymatic  extracts  of  drug-sensitive  and  drug- 
resistant  strains,  and  the  use  of  isotopically  labeled  precursors.  All 
these  procedures  have  been  described  in  detail  in  previous  publications 
relating  to  this  project. 

Major  Findings:   A  strain  of  HeLa  cells  has  been  developed  which 
can  grow  in  the  presence  of  a  molar  ratio  of  2DG:glucose  of  10:1,  while  the 
parent  strain  is  completely  inhibited  at  equimolar  concentrations  of  the 
inhibitor :glucose.   The  resistant  cells  grow  at  approximately  one-half  the 
rate  of  the  parent  strain,  and  the  addition  of  pyruvate  to  the  basal  medium 
is  essential  for  its  continued  propagation. 

2DG  is  rapidly  metabolized  by  HeLa  cells  to  its  phosphorylated 
intermediate,  2-deoxyglucose-6-phosphate.   There  is  a  significant  difference 


Part  B  included. 

1* 


NIAID  53 


in  the  metabolism  of  both  compounds  by  the  sensitive  and  resistant  strains, 
in  that  the  rate  of  2DG  metabolism  in  the  resistant  cells  was  markedly 
reduced  as  much  as  5-fold.   In  cell  extracts  there  was  a  corresponding 
marked  depression  of  the  enzyme  which  phosphorylates  2DG,  as  well  as  the 
enzyme  which  phosphorylates  glucose,  fructose  and  mannose.   The  decreased 
kinase  activity  for  2DG  in  cells  resistant  to  this  compound  may  well  be 
the  primary  basis  of  their  resistance. 

It  has  also  been  found  that  an  extract  of  resistant  cells  inhibits 
the  enzymatic  activity  of  a  sensitive  extract,  suggesting  that  the  resistant 
extracts  contain  an  active  inhibitor  of  hexose  phosphorylation. 

Significance  to  the  Program  of  the  Institute;   The  mechanism  of  the 
development  of  resistance  to  a  glucose  analogue  in  HeLa  cells  may  serve  as 
a  model  for  the  development  of  drug  resistance.   This  is  relevant  to  the 
general  problem  of  chemotherapy,  not  only  of  infectious  diseases,  but  of 
other  disease  areas  (e0g.  cancer,  heart  disease,  etc.)0 

Proposed  Course  of  Project; 

1.  Attempts  will  be  made  to  Isolate  and  characterize  the  nature  of 
the  metabolic  inhibitor  in  resistant  extracts. 

2.  The  enzymatic  differences  between  drug-resistant  and  -resistant 
strains  will  be  further  explored. 

3.  The  nutritional  requirements  of  normal  and  resistant  variants 
will  be  explored,  with  particular  reference  to  the  essential  role  of 
pyruvate  in  the  growth  of  resistant  cells. 

4.  Attempts  will  be  made  to  transform  2DG-sensitive  cells  to  drug 
resistance,  using  the  genetic  material  of  the  2DG-resistant  cells. 


-  2  - 


15 


NIAID  53 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project:   None 

Honors  and  Awards  relating  to  this  project:  Member,  Society  of 
Biological  Chemists. 


16 

-  i  - 


NIAID  54 

Laboratory  of  Cell  Biology 

Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   Metabolic  Control  Mechanisms  in  Cultured  Mammalian  Cells 

Principal  Investigator:   Dr.  Jacob  V.  Maizel,  Jr. 

Other  Investigators:  None 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960): 

Total:   1 

Professional:   1/2 
Other:   1/2 

Project  Description: 

Objective:   To  study  the  intracellular  mechanisms  for  the  control  of 
metabolic  activities  in  mammalian  cell  cultures. 

Methods  Employed:   Enzymatic  assays  by  modifications  of  techniques 
employed  in  other  systems. 

Major  Findings: 

1.  Phosphatases. 

In  Escherichia  coli  alkaline  phosphatase  is  a  repressible  enzyme, 
the  synthesis  of  which  is  affected  by  the  level  of  orthophosphate  in  the 
medium.   In  mammalian  cells  also  the  level  of  alkaline  phosphatase  was  found 
to  be  variable,  in  that  cultures  grown  as  monolayers  attached  to  glass 
regularly  had  at  least  ten  times  as  much  alkaline  phosphatase  activity  as 
suspension  cultures.   However,  the  high  activity  of  monolayer  culture  was  not 
decreased  at  elevated  phosphate  concentrations,  nor  was  the  low  level  in 
suspension  cultures  increased  by  growth  at  low  phosphate  concentrations. 
When  suspension  cultures  were  planted  as  monolayers,  the  alkaline  phosphatase 
levels  increased  ten-fold  in  less  than  two  days,  independent  of  the 
concentrations  of  phosphate  or  calcium,  or  population  density.   These  findings 
suggest  a  control  mechanism  unlike  that  of  bacterial  systems. 

The  phenomenon  cannot  be  explained  as  the  result  of  a  generalized  loss 
of  enzymes  from  cells  in  suspension,  since  the  levels  of  acid  phosphatase 

17 


NIAID  54 


and  aspartate  transcarbamylase  were  the  same  in  suspension  and  monolayer 
cultures. 

II.   Control  of  pyrimidine  synthesis. 

Aspartate  transcarbamylase  is  involved  in  the  biosynthesis  of 
pyrimidines  in  a  number  of  systems,  and  in  Escherichia  coli  is  involved  in 
the  control  of  pyrimidine  synthesis  by  exogenously  supplied  pyrimidine,, 
The  enzyme  was  found  in  extracts  of  HeLa  cells  and  equally  in  both  monolayer 
and  suspension  cultures.   Again  unlike  bateria,  the  enzyme  level  was 
unaffected  by  the  addition  of  pyrimidines  to  the  medium. 

Proposed  Course  of  Program; 

a.  The  question  as  to  whether  the  product  controls  pyrimidine 
biosynthesis  will  be  approached  by  using  appropriate  radioisotope  precursors 
in  the  presence  and  absence  of  added  preformed  pyrimidines. 

b.  The  enzymatic  steps  in  pyrimidine  synthesis  remain  to  be 
elucidated. 

c.  Factors  of  possible  importance  in  the  regulation  of  alkaline 
phosphatase  activity  remain  to  be  investigated. 

Knowledge  of  the  control  of  protein  synthesis  is  of  fundamental 
significance  to  studies  of  antibody  production  and  virus  propagation. 


-  2  - 


ia 


NIAID  55 

Laboratory  of  Cell  Biology 

Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   Application  of  the  Technique  of  Tissue  Culture  to 
Selected  Genetics  Diseases 

Principal  Investigator:   Dr.  Robert  S.  Krooth 

Other  Investigators:  Miss  Mary  Jane  Madden 

Cooperating  Units:   NIAMD,  Dr.  Arnold  W.  Weinberg,  and  Dr.J„  H,  Tjio 
NINDB,  Dr.  Paul  Altrocci 

Man  Years  (calendar  year  1960) : 
Total:  3  3/4 
Professional:   1 
Other:   2  3/4 

Project  Description: 

Studies  to  follow  a  number  of  genetic  diseases  in  tissue  culture 
are  being  continued.   Biopsies  are  performed  on  affected  patients,  and  cell 
lines  are  then  developed  from  the  excised  tissue.   Evidence  bearing  on  the 
persistence  of  the  defect  in  cell  lines  from  affected  persons  (compared  with 
cell  lines  from  controls)  is  obtained.   Ultimately,  mutant  cell  lines  from 
patients  with  inborn  metabolic  errors  should  prove  useful  in  the  study  of 
genetic  exchange  among  human  cells  as  well  as  in  other  aspects.   The  system 
permits  one  to  study  mutant  human  cells  in  a  way  which  hitherto  could  be 
applied  only  to  mutants  of  bacteria  and  fungi.   However,  the  number  of 
genetic  diseases  which  are  susceptible  to  this  approach  is  at  present  quite 
limited. 

Galactosemia  has  been  shown  to  persist  as  a  defect  in  culture,  even 
after  6  months  of  propagation  and  an  increase  in  cell  number  known  to  exceed 
30  billion-fold.   The  defect  can  be  shown  by  growth  studies,  the  ability  of 
the  cells  to  metabolize  galactose-l-C^,  and  direct  enzymatic  assays.   The 
galactosemic  cells  show  a  pattern  of  galactose  sensitivity  similar  to  the' 
transferaseless  mutants  of  E.  coli.  Corresponding  data  have  been  obtained 
for  one  heterozygote,  and  here  also  the  defect  persists. 


Part  B  included. 

-  1  - 


19 


NIAID  55 


Wilson's  disease  and  hypocatalaseraia  are  also  under  study  by  this 
approach. 

Our  efforts  to  follow  Gaucher's  disease  and  certain  other  histiocytic 
states  in  culture  (referred  to  in  the  last  report)  have  not  yet  succeeded. 
Indefinite   propagation  of  recognizably  affected  cells  has  not  been  achieved 
although  the  cells  will  attach  to  glass,  spread,  and  appear  to  increase  in 
number  for  a  few  weeks.   Thereafter,  though  the  cells  can  be  made  to  survive 
for  months,  no  evidence  of  growth  is  found. 

A  search  is  being  made  in  our  laboratory,  as  in  others,  for  new 
chromosomal  abnormalities  in  human  disease.   Cell  lines  have  been  developed 
from  biopsies  on  5  patients  with  congenital  malformations,  in  each  case  of 
a  kind  not  previously  reported.   In  every  instance  thus  far  the  chromosomes 
have  been  normal  in  morphology  and  number. 


-  2  - 


20 


NIAID  55 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 


Krooth,  R.  S.,  and  Weinberg,  A.   (1960)   Properties  of  Galactosemic 
Cells  in  Culture.   Biochem.  Biophys.  Research  Comm.,  in  press. 

Honors  and  Awards  relating  to  this  project:   None. 


21 


LABORATORY  OF  INFECTIOUS  DISEASES 


Summary 1 

60  -  Studies  on  Cellular  and  Cell-Free 

Staphylococcal  Penicillinase 44 

61  -  Antibiotic  Activity  of  Sea  Water 47 

62  -  The  Electron  Transport  System  in  an 

Autotrophic  Hydrogenomad 50 

62-A  -  Microbiological  Fractionation  of  the 

Hydrogen  Isotopes 55 

63  -  Non-specific  Immunity  Associated  with 

Serum  Siderophilin  and  the  Iron 

Metabolism  of  both  Pathogen  and  Host 57 

63-A  -  Chemical  Aspects  of  the  Specific  Binding 
of  Iron  to  Serum  Siderophilin  and  Egg 
White  Conalbumin 62 

63-B  -  Biochemistry  of  the  Acquisition  of  Iron 
by  Mammalian  Tissues  as  Mediated  by  the 
Iron-Binding  Protein,  Siderophilin 66 

63-C  -  Development  of  an  Economical  280mu 
Light  Source  Suitable  for  Detecting 
Proteins  in  Effulents  from  Chromatographic 
Columns 69 

64  -  Detoxification  of  Potential  Tuberculo- 

static, Fungistatic,  Parasitic idal.  and 

Virucidal  Agent „  .  71 

64-A  -  Potential  Fungistatic  and  Parasiticidal 

Agents 75 

64-B  -  Isolation  of  Antibacterial  and  Antiviral 

Substances  from  Shellfish 77 

65  -  Longitudinal  Studies  of  Beta-hemo lytic 

Streptococcal  Isolations 80 

65-A  -  Streptococcal  M  Protein,  Virulence,  and 

Type-Specific  Immunity 83 

66  -  Epidemiologic  Studies  of  Illnesses  and 

Microbial  Experience  of  Junior  Village 

Nursery  Children 89 

66-A  -  Epidemiologic  Studies  of  Host  Parasite 

Disease  Relationships 93 

66-B  -  Epidemiologic  Studies  of  New  Vaccines  and 

Chemoprophy lactic  Agents 95 

66-C  -  The  Etiology  of  Respiratory  Disease  on  a 

College  Campus 97 

66-D  -  The  Role  of  Viruses  in  the  Etiology  of 

Ear  Disease 99 


66-E  -  A  Study  of  the  Influence  of  Influenza 
A  and  B  Mineral  Oil  Adjuvant  Vaccines 
on  Influenza  Antibody  Patterns,  8-9 
Years  after  Vaccination 100 

67  -  Studies  on  Human  Enteroviruses, 

Adenoviruses,  and  Reoviruses 103 

6  7-A  -  Studies  on  Viruses  of  the  Enteric  Tract 

of  Cattle 107 

67-B  -  Studies  on  the  Etiology  of  Eosinophilic 

Meningitis  in  French  Polynesia 110 

68  -  Studies  of  Tumor  Viruses  in  Nature 114 

68-A  -  Seroepidemiology  of  Virus  Infections 123 

69  -  Study  of  Viruses  as  Causes  of  Respiratory 

Illness  in  Infancy  and  Early  Childhood...  126 
69-A  -  Viral  Pneumonia:   Etiology,  Therapy,  and 

Prevention 130 

69-B  -  Study  of  the  Laboratory  Aspects  of 

Respiratory  Virus  Illness  in  a  Welfare 

Orphanage 134 

70  -  Laboratory  Studies  of  Newly  Recognized 

Viruses  Associated  with  Respiratory 

Illness 138 

71  -  Studies  of  Tumor-Producing  Viruses 142 

71-A  -  Continuing  Studies  of  Adenoviruses  and 

Salivary  Gland  Viruses  as  Examples  of 

Latent  Viruses 147 

71-B  -  Studies  of  Mouse  Viruses  with  Special 
Reference  to  their  Importance  as 
Extraneous  Viruses  (background  noise)  in 
Mouse  Tumor  Systems 150 

72  -  Laboratory  Studies  of  Enteroviruses 154 

72-A  -  Study  of  Viral  Respiratory  Disease  in  a 

Military  Population 157 

72-B  -  Study  of  Respiratory  Viruses  in  Human 

Volunteers 161 

73  -  Application  of  Fluorescent  Antibody 

Staining  Technique  to  the  Study  of 
Respiratory  Viruses 163 

74  -  Study  of  Bovine  Respiratory  Diseases 165 

74-A  -  A  Study  of  Squamous  Cell  Carcinoma  (Can- 
cer Eye)  in  Cattle 170 

75  -  Laboratory  and  Epidemiologic  Studies 

of  Viruses  as  Possible  Etiologic  Agents 
of  Acute  Leukemia  in  the  Pediatric  Age 

Group 172 

76-A  -  Ecologic  Studies  of  Fungi  Pathogenic  for 

Man 176 


II 


76-B  -  Iji  vivo  Tests  of  Antimycotic  Drugs 

and  Antibiotics 180 

76-C  -  Identification  and  Study  of  New  and 

Unusual  Fungi  from  Mycoses 183 

78-A  -  Biochemistry  and  Physiology  of 

Pathogenic  Fungi.   (In  vitro  Studies 
of  the  Action  of  Antifungal  Agents  on 
Pathogenic  Fungi) 186 

78-B  -  Studies  on  the  Physiology  of 

Coccidioides  immitis 188 

79-A  -  Immunity  Studies  with  Pathogenic  Fungi.  191 

79-B  -  Antigentic  Studies  on  Pathogenic 

Yeasts 193 

79-C  -  Virulence  and  Pathogenic  Studies  with 

Yeasts 196 


III 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Yoar  I960 

Administrative  Office  -  Edward  F.  Zadai 

Introduction 


The  Administration  of  the  Laboratory  of  Infectious  Diseases 
is  responsible  for  furnishing  all  services,  except  the  actual  conduct 
of  scientific  research,  required  for  the  numerous  scientific  investi- 
gation programs  of  the  4  sections.   In  fulfilling  a  mission  of 
conducting  integrated  laboratory-scientific  research,  investigators 
must  be  provided  with  individual  personal  attention,  an  earnest 
interest  in  their  work,  and  all  the  administrative  support  possible. 
This  responsibility  requires  continuous  evaluation  of  activities  in 
all  of  the  Laboratory  of  Infectiou  the  multiple 

services  needed  both  directly  and  indiroctly. 

Personnel 


During  the  p       nfhs  we  have  been  fortunate  in  almost 
ninating  the  problem  of  carrying  vacant  positions  in  our  laboratoi^ 
We  have  added  two  professional  members  to  our  staff  (Dr.  Shug  -  from 
the  Veterans  Administration  and  Dr.  Huff  -  from  rhe  laboratory  of 
Clinical  Investigation,  NIAID).  We  have  further  employed  two 
additional  Medical  Biology  Technicians  and  2  more  Laboratory  Animal 
Caretakers,  leaving  only  two  nonprofessional  vacancies  in  tho  entire 
laboratory  which  we  hope  to  fill  before  the  completion  of  this 
calendar  year.  Seven  been  promoted  during  the 

year  with  three  promotions  still  pending  final  action • 

Physical  Changes 


This  past  year  has  seen  the  completion  of  planning  and  the 
beginning  of  construction  of  3  major  renovations  in  dosigning  newly 
assigned  laboratory  space.  Rooms  333  and  534  In  Muilding  7  will  be 
converted  from  animal  rooms  into  laboratories  with  work  scheduled 
for  completion  March  5,  1961.  Also  additional  laboratory  space 
will  be  acquired  and  renovated  in  Room  313  for  use  by  a  collaborative 
research  study  with  rhe  National  Institute  Ol  Ni  uiological  Diseases 
and  Blindness.  Lastly  Rooms  204  and  206  in  Building  5  have  been 
vacated  by  the  Division  of  Biologies  Standards  and  will  be  renovated 
for  use  by  our  Medical  and  Physiological  Bac feriology  Section. 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

Scientific  Exchange 


An  exchange  of  scientific  personnel  at  the  international  level 
was  carried  out  during  the  year  by  sending  one  of  our  scientists  to 
Paris,  France,  to  participate  in  research  studies  on  auto-immunity  in 
viral  infections  and  related  virus  research.   In  turn  we  received 
Dr.  Dietrick  Falke,  Senior  Research  Assistant  in  virus  research 
from  the  Hygiene  Institute,  Marburg,  Germany.  Dr.  Falke  is  presently 
spending  a  one  year  stay  with  us  as  a  guest  worker  studying  tumor- 
inducing  viruses. 

Tra  i  n  i  nq 


Courses  in  outside  training  under  sponsorship  of  NiH  were 
completed  by  eight  professional  employees  and  it  is  certainly  anti- 
cipated that  this  will  bring  promising  results  in  our  scientific 
projects. 

Probl ems 


The  ever  present  scarcity  of  good  personnel  and  adequate  space 
has  certainly  affected  our  progress.  Many  paths  toward  completion 
of  good  scientific  experiments  have  been  narrowed  or  blocked  due  to 
the  lack  of  facilities.  This  Administrative  Office  has  just  completed 
preliminary  negotiations  in  purchasing  two  house  trailers  to  be  used 
as  a  field  laboratory.   In  this  manner  we  will  be  able  to  continue 
progress  on  a  virus  study  that  would  have  become  useless  without  a 
means  of  isolating  the  mice  necessary  for  experiments. 

Research  Associates 


In  the  role  of  a  leading  research  facility,  the  Laboratory  of 
Infectious  Diseases  also  conducts  formal  advanced  training  for  young 
scientists  in  certain  specialties.  The  Research  Associate  program 
provides  exposure  to  techniques  and  methodology  in  microbiologic 
research.   In  I960  there  were  several  applicants  from  leading 
universities  and  hospitals  in  this  country  for  these  type  positions 
which  we  were  forced  to  reject.   It  would  be  of  great  value  if  we 
could  expand  our  facilities  to  accept  more  of  these  applications 
because  recognition  of  the  quality  of  experience  in  our  scientific 
programs  is  a  significant  factor  in  the  recruitment  and  retention 
of  staff  members. 


PHS-NIH 
Summary  Statement 
Laboratory  of    Infectious  Diseases,   NIAID 
Calendar  Year    I960 

Cone  I  us  ion 


The  Laboratory  of  Infectious  Diseases  has  earned  the  reputation 
of  a  progressive  organization  in  the  field  of  infectious  disc.) 
research.  There  have  been  many  problems  in  retaining  extremely 
competent  professional,  technical,  and  administrative  personnel.  We 
are  fortunate  in  having  a  devoted  highly  productive  and  intelligent 
senior  staff  made  up  of  internationally  known  young  workers  with 
the  large  part  of  their  careers  still  before  them.  Results  of  signi- 
ficant value  in  the  conquest  of  disease  have  been  achiovod,  and  it 
is  the  desire  of  the  Administrative  Office  to  meet  the  challenge  of 
the  future  with  earnest  and  sincere  efforts.  The  Administrative 
Office  is  fully  aware  of  the  job  whicl.  of  us  if  we  are 

to  support  rather  than  contain  our  scientists.  We  feel  very  strongly 
that  the  time  is  growing  near         will  have  to  expand  our 
facilities  if  we  wish  to  continue  maximum  progress. 

Below  is  a  highlight  summarization  of  some  of  the  honors 
awarded  LID  scientists  during  the  past  year: 

Dr.  Chester  W.  Emmons 

Elected  President,  Mycological  Society  of  America 
Elected  Counc i lor-at-Large,  Society  of  American  Bacteriologists 
Chairman,  Second  Conference  on  Medical  Mycology,  N.Y.  Acad.  Sc i . 
Appointed  Board  Member  of  Amer.  Acad,  of  Micro.  (3  year  term) 
Chairman,  Standards  and  Examination  Committee,  Amer.  Acad.  Micro. 
Convenor  of  Mycological  Symposium,  III  National  Congress  of 

Microbiology,  Mexico 
Honorary  mention  for  exhibit  on  Chemotherapy  of  Systemic 

Mycoses,  AMA  meeting  in  Florida 
Appointed  to  WHO  Expert  Advisory  Panel  on  Parasitic  Diseases 

(5  year  term) 
Member  of  Committee  on  National  Index  of  Fungus  Cultures, 

Quartermaster  Research  and  Development,  National  Academy- 
Research  Council  (3  year  term) 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  N I  AID 

Calendar  Year  I960 

Dr.  Robert  J.  Huebner 

Elected  to  National  Academy  of  Sciences 

Appointed  to  Enterovirus  and  Adenovirus  Committees  of  NIAID,  NCI 
Invited  to  give  several  honorary  lectures,  including  the 
I960  Harvey  Lecture 

Dr.  Wal I  ace  P.  Rowe 

Awarded  Eli  Lilly  Award  at  the  Annual  Meeting  of  the  American 

Society  of  Bacteriologists 
Appointed  to  Research  Advisory  Committee  on  Etiology  of 

Cancer  of  the  American  Cancer  Society 

Dr.  Robert  M.  Chanock 

Elected  to  Society  for  Clinical  Investigation 
Full  membership  to  the  ARD  Commission  of  the  AFEB 

Dr.  Leon  Rosen 

Appointed  to  the  Enterovirus  Committee,  NCI 

Received  a  letter  from  the  Governor  General  of  French  Polynesia 
to  the  Surgeon  General  of  the  United  States  Public  Health 
Service  citing  him  for  his  research  on  viruses  and 
eosinophilic  meningitis  in  French  Polynesia 

Dr.  Arthur  K.  Saz 

Chairman,  President's  Fellowship  Committee,  Society  of  American 
Bacteriologists 

Dr.  Roy  Repaske 

Elected  to  Membership,  American  Society  of  Biological  Chemists 

Dr.  Arthur  L.  Schade 

Invited  to  following  European  universities  to  deliver  lectures: 
Vienna,  Marburg  and  Geissen,  Saar,  Freiburg,  Berne, 
Zurich,  Tubingen,  Frankfurt,  Berlin,  Lund 

Dr.  Robert  C.  Woodworth 

Awarded  NIH  Fellowship  )NHI)  to  spend  a  year  at  Lund  University, 
Ma  I  mo,  Sweden 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  NIAIO 

Calendar  Year  I960 

Virus  and  Epidemiology  Sections 

\ .0   Introduction 

In  I960  the  Virus  and  Rickettsial  and  Epidemiology  Sections 
continued  integrated  and  comprehensive  efforts  to  define  the  importance 
of  virus  infections  in  disease.  Field  investigations  of  human  and 
animal  virus  infections  were  made  possible  through  collaboration  with 
a  number  of  other  organizations,  including  the  Bureau  of  Medicine, 
USN;  the  District  of  Columbia  Children's  Hospital  Research  Foundation; 
the  District  of  Columbia  Welfare  Department;  the  New  York  City  Health 
Department;  the  National  Cancer  Institute;  the  National  Institute 
of  Allergy  and  Infectious  Diseases;  the  Laboratory  of  Clinical 
Investigations,  NIAIO;  and  in  Paris,  France  the  Laboratoire  des 
Virus,  Hopital  Saint-Vincent-de-Paul;  and  Le  Centre  Claude-Bernard 
de  I 'Hopital  Saint  Louis. 

Natural  events  and  opportunities  afforded  by  our  collaborators 
shaped  the  course  of  most  of  our  field  studies.  Technical  breakthroughs 
in  the  laboratory  made  it  possible  for  us  to  take  fuller  advantage 
of  these  opportunities  to  study  natural  disease  and  thus  acquire 
not  only  new  information  about  specific  virus  infections,  but  also 
to  move  nearer  our  ultimate  goal,  namely,  a  clear  view  of  the  numerous 
viral  causes  of  human  diseases  which  is  also  sufficiently  comprehensive 
to  make  concerted  efforts  to  control  them  appear  feasible  and 
worthwhi le. 

In  the  long  run,  a  research  organization  stands  or  falls  on 
its  puolished  work,  so  that  if  we  have  anything  deserving  attention 
it  is  in  the  content  of  our  scientific  reports.  Nearly  50  manuscripts 
were  published  or  accepted  for  publication  and  10  more  papers  are 
either  in  the  Editorial  Board  or  about  to  be  submitted.   It  is  not 
surprising  therefore  that  even  a  summary  makes  for  a  rather  long 
document.  However,  it  may  help  if  I  "highlight"  certain  specific 
findings  which  while  not  necessarily  more  important  have  a  more 
immediate  bearing  on  disease,  and  merely  mention  others. 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases 
Calendar  Year  I960 
2.0  Respiratory  Virus  Disease  Studies 

2. J   New  Causes  of  Virus  Pneumonia 

Pneumonia  and  other  lower  respiratory  tract  infections 
continue  to  represent  major  causes  of  death  and  a  large  segment, 
presumed  to  be  viral  in  origin,  is  still  uncontrolled.  Until 
recently  it  was  wholly  undefined.  During  1958  and  1959  our  studies 
at  Children's  Hospital  and  Junior  Village  helped  define  the  relative 
importance  of  adenoviruses,  para-inf luenza  viruses,  and  influenza 
viruses  in  causing  lower  respiratory  illnesses  of  childhood.  The 
data  suggested  that  as  much  as  40  per  cent  of  croup  bronchiol itis 
and  pneumonia  were  explained  by  these  viruses.   In  I960,  using  more 
sensitive  methods,  we  were  able  to  explain  a  much  larger  percentage 
of  such  illnesses,  chiefly  because  we  were  now  able  to  assess  the 
very  significant  contributions  of  respiratory  syncytial  virus  (RS) 
to  the  respiratory  disease  problem.  Early  in  the  year  large  outbreaks 
of  RS  virus  were  intensively  studied  both  at  Children's  Hospital  and 
Junior  Village.  Over  80  strains  of  RS  virus  were  isolated  from 
children  with  pneumonia  and  60  per  cent  with  bronchiolitis  yielded 
RS  virus,  whereas  virus  was  recovered  from  less  than  one  per  cent 
of  comparable  control  patients  without  respiratory  i  Iness. 

Retrospective  analysis  of  serologic  surveys  of  respiratory 
illnesses  in  Children's  Hospital  since  1957  suggested  that  perhaps 
20  per  cent  of  all  lower  respiratory  illnesses  observed  during  the 
last  three  years  was  due  to  RS  virus.  Thus,  considering  the  contri- 
butions of  adenoviruses,  para-inf I uenza  viruses,  influenza  viruses, 
and  "PAP"  virus  it  now  appears  that  50  to  60  per  cent  of  the  more 
severe  respiratory  illnesses  of  young  children  can  now  be  explained 
and,hopef ul ly,  controlled.  Except  for  influenza  virus  (wLich 
contributed  probably  less  than  5  per  cent  of  the  total),  the  LID 
respiratory  virus  unit  personnel  played  key  roles  in  the  discovery 
of  the  first  representatives  of  each  of  the  other  virus  groups  - 
adenovirus,  para- influenza,  and  RS .  Delineation  of  still  undefined 
viral  causes  of  the  respiratory  disease  syndrome  represents  the 
major  challenge  to  respiratory  disease  investigators  for  1961. 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  NIAID 

Calendar  Year  I960 

A  good  start  toward  meeting  this  challenge  has  already 
been  made.  Preliminary  analysis  of  nearly  1800  enterovirus  infections 
observed  in  our  childhood  study  populations  showed  that  certain  of 
them  such  as  Coxsackie  B  3,  poliovirus  2,  and  ECHO  3  virus  were 
responsible  for  febrile  respiratory  illnesses.  From  recent  results 
obtained  by  other  workers,  and  previously  by  our  own  group,  it  is 
likely  that  newly  recognized  but  extremely  common  enteroviruses 
closely  related  to  the  Coxsackie  A  virus,  will  be  found  to  contribute 
a  significant  proportion  of  still  unexplained  respiratory  illnesses. 

2 12  Respiratory  Virus  Vaccines 

The  fact  that  a  very  significant  proportion  of  the  more 
severe  respiratory  illnesses  occuring  in  childhood  can  now  be 
explained  makes  the  development  of  effective  preventive  vaccines 
against  the  known  agents  a  worthwhile  undertaking.  The  viruses 
involved  (see  above)  are  antigenic  and,  although  reinfection  can 
occur,  evidence  indicates  that  specific  antibodies  provide 
significant  protection  against  the  more  severe  manifestations  of 
these  viruses  during  subsequent  infections.  Unfortunately,  progress 
towards  the  development  of  the  complex  vaccines  containing  many 
different  viruses  can  be  expected  to  be  very  slow  unless  more  interest 
and  attention  can  be  focused  on  the  public  health  importance  of  such 
efforts. 

Unlike  poliomyelitis  and  other  dread  and  dramatic  illnesses, 
common  respiratory  disease,  although  responsible  for  much  more 

illness  and  possibly  more  deaths  as  well,  do  not  have  national 
foundations  devoted  to  their  control  or  eradication.  The  relative 

lack  of  large  scale  goal -oriented  research  activities  contrasts 
vividly  with  the  dimensions  and  technical  needs  of  contemporary 

respiratory  virus  disease  research;  both  exceeding  by  several 
magnitudes  that  of  poliomyelitis. 

During  I960  several  experimental  but  commercial ly  prepared 
killed  vaccines  containing  various  combinations  of  adenoviruses 
(6  types),  para- influenza  viruses  (3  types),  and  Coxsackie  B  viruses 
(5  types)  were  tested  in  Junior  Village.  The  evidence  suggests 
that  while  modestly  antigenic,  the  vaccines  had  insufficient 
potency  to  be  regarded  as  satisfactory  for  larger  scale  studies. 
Additional  studies  of  vaccines  are  planned  for  1961,  but  only  at 
a  pilot  study  level,  since  the  space  and  personnel  available  to  our 
Virus  and  Epidemiological  groups  is  scarcely  sufficient  to  continue 
our  laboratory  and  field  studies  of  the  viruses  as  causes  of 
respiratory  disease. 

8 


PHS-NIH 

Summary  Statement 

Laboratory  of    Infectious  Diseases,   NIAIO 

Calendar  Year   I960 

2.3  Pneumonia  in  Adults 

The  etiologic  role  of  PAP  (Eaton's  virus)  in  primary  atypical 
pneumonia  suggested  earl ier  by  Eaton  and  Liu  and  explored  by  us  in 
1959,  was  finally  fully  established  in  I960.   In  cooperation  with 
the  Bureau  of  Medicine,  USN,  the  continuing  "epidemic"  of  virus 
pneumonia  in  Marine  recruits  at  Parris  Island  was  studied  in  several 
ways.  Serological  studies  showed  that  51  per  cent  of  530  pneumonia 
cases  had  antibody  rises  to  PAP  virus;  only  6  per  cent  revealed 
contemporary  infection  with  adenoviruses.  Serologic  studies  of 
infection  showed  PAP  virus  to  be  much  more  common  than  disease; 
approximately  30  recruits  were  infected  for  each  case  of  pneumonia,' 
information  vital ly  important  to  ful Ier  comprehension  of  the  natural 
history  of  this  important  virus. 

2.4  Treatment  of  Primary  Atypical  Pneumonia  (PAP) 

In  1959  treatment  of  Parris  Island  pneumonia  cases  with  broad 
spectrum  antibiotics  (tetracyclines)  appeared  to  reduce  the  severity 
and  the  duration  of  the  Eaton  pneumonias.   In  I960  the  efficacy  of 
a  new  tetracycline  drug,  demethy Ichlortetracycl ine,  was  tested  in 
a  we  I  I -control  led  double  blind  study  including  290  pneumonia  patients. 
The  drug  greatly  reduced  the  severity  and  duration  of . pneumonitis 
and  fever  in  those  shown  to  have  serologic  responses  to  PAP  virus. 
These  findings,  based  on  accurate  laboratory  diagnosis,  fully  confirm 
earlier  but  controversial  reports  of  the  efficacy  of  tetracyclines 
in  atypical  pneumonias.   It  also  adds  further  support  to  the  importance 
of  the  Eaton  virus  as  a  cause  of  virus  pneumonia. 

An  additional  link  in  the  chain  of  evidence  establishing  the 
PAP  virus  as  an  important  cause  of  pneumonia  was  achieved  recently 
in  collaborative  studies  with  the  Laboratory  of  Clinical  Investiga- 
tions, N I A  ID.  Volunteers  inoculated  intranasal ly  with  PAP  virus 
grown  in  tissue  cultures  reacted  with  a  wide  gamut  of  respiratory 
signs  and  symptoms,  including  pneumonitis  characteristic  of  PAP. 


8 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

2.5  Common  Colds  and  Other  Mild  Respiratory  Illnesses  in  Adults 

Recent  studies  have  served  to  clarify  and  enlarge  existing 
concepts  of  the  etiology  of  common  mild  respiratory  illnesses  in 
adults.   It  is  now  quite  clear  that  instead  of  a  few  specific 
closely  related  viruses,  numerous  viruses  belonging  to  different 
groups  each  contribute  in  part  to  the  syndrome  called  the  "common 
cold."  Thus  the  newer  viruses  (adenoviruses,  para-i nf I uenza 
viruses,  respiratory  syncytial  virus  and  others),  together  with 
older  agents  (influenza  viruses  and  certain  bacteria),  each 
contribute  on  I y  a  small  proportion  of  the  milder  respiratory  ailments 
of  adults.  They  contribute  a  larger  segment  of  more  serious 
diseases,  particularly  in  children.  Very  recent  reports  of  common 
cold  viruses  from  England,  together  with  the  prior  reports  of 
agents  with  somewhat  similar  properties  in  this  country,  served 
to  focus  our  attention  on  these  viruses  in  I960.  Together  with 
investigators  elsewhere,  it  was  found  that  most,  if  not  all,  of 
these  agents  -  the  British  HGP  and  FEB,  the  American  2060,  JH, 
Coe  and  PETT  viruses  which  grow  selectively  and  rather  "fussily" 
in  human  epithelial  cell  lines,  really  represent  "fastidious" 
enterovirus  strains  which  have  (as  do  almost  all  Coxsackie  A's 
and  some  ECHO  viruses)  special  growth  requirements.  These  viruses, 
as  do  a  number  of  still  unclassified  agents  found  in  Junior  Village 
during  the  past  several  years,  have  properties  very  similar  to 
the  Coxsackie  A  viruses;  indeed,  several  have  been  shown,  on  the 
basis  of  serologic  markers  and/or  by  suckling  mouse  pathogenicity, 
to  be  indistinguishable  from  Coxsackie  A  viruses. 

Enteroviruses  as  Specific  Causes  of  Mild  Respiratory  Disease 

In  previous  years  we  have  reported  enteroviruses  in  relation 
to  respiratory  illnesses  in  Junior  Village;  JVI  virus  (now  ECHO  20) 
was  one  such  case  in  point. 


10 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

ECHO  28  (2060-JH  Viruses ) 


Several  years  ago  investigators  at  Great  Lakes  Naval 
Training  Station  and  Johns  Hopkins  reported  viruses  called 
respectively  "2060"  and  "JH"  in  association  with  mild  respiratory 
illness.  Subsequently  these  agents  were  classified  as  ECHO  28 
in  the  enterovirus  family.  During  I960  our  Virus  Section  developed 
a  complement  fixation  test  for  this  virus  as  well  as  for  most  of 
the  58  known  enteroviruses.  Serologic  surveys  of  respiratory 
illnesses  have  revealed  high  prevalence  of  antibodies,  but  thus 
far  rises  in  titer  in  relation  to  illness  have  been  rare. 

ECHO  3 


During  I960  an  outbreak  of  ECHO  3  virus  infections  in 
Junior  Village  was  analyzed  in  relation  to  contemporary  illness. 
The  study  showed  a  temporal  relation  to  mild  respiratory  illness 
attended  by  brief  febrile  responses.  A  report  is  in  preparation. 

Coe  Virus  and  Other  New  Viruses  in  Military  Personnel 

During  I960,  in  cooperation  with  the  Bureau  of  Medicine  and 
Surgery,  USN,  studies  of  mild  respiratory  illness  in  military 
recruits  were  conducted  at  Camp  Lejeune,  North  Carolina.   Initial 
findings  suggested  that  new  para-inf I uenza  viruses  and  respiratory 
syncytial  virus,  in  addition  to  adenoviruses,  were  contributing 
in  part  to  the  syndrome.  Although  the  clinical  importance  of 
these  latter  agents  must  still  be  determined,  the  fact  that  they 
are  encountered  in  adults  is  of  considerable  interest. 

However,  more  recently  (since  October  I960)  we  have  observed 
large  outbreaks  of  Coe  virus  -  over  70  strains  of  virus  were 
isolated  from  as  many  cases  of  mild  respiratory  illnesses. 
Although  identical  serologically  with  the  prototype  Coe  virus, 
these  new  strains  exhibit  a  hemagglutinin  not  previously  reported. 
These  strains  furthermore  produce  effects  in  suckling  mice  which 
are  indistinguishable  from  those  produced  by  Coxsackie  A  viruses. 
Thus  one  more  "new"  virus,  at  first  regarded  as  wholly  unique,  is 
now  found  with  further  study  to  belong  to  a  well  established  virus 
family  -  namely  the  Coxsackie  viruses. 

10 

II 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  N I  AID 
Calendar  Year  I960 

At  the  same  time  and  in  the  same  recruit  population 
numerous  isolations  of  the  so-called  "fussy"  enteroviruses  also 
have  been  recovered  from  persons  with  respiratory  illness,  using 
the  special  techniques  described  last  year  by  British  investigators, 
The  role  of  these  newer  enteroviruses  in  causing  illness  are  now 
under  study. 

Future  Studies  of  Colds  due  to  Enteroviruses 

We  are  making  arrangements  with  the  Bureau  of  Medicine 
and  Surgery,  USN,  not  only  to  continue  but  to  extend  our  studies 
of  the  enteroviruses  as  causes  of  respiratory  illnesses  in  the 
large  military  population  at  Camp  Lejeune.  We  hope  to  include 
observations  on  the  numerous  dependents  and  on  permanent  cadre 
personnel  as  well.  Thus  our  studies  of  enterovirus-caused 
respiratory  illnesses  will  cover  experiences  in  children  as 
well  as  adults,  and  will  include  all  seasons  of  the  year. 


11 


12 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  Nl AID 

Calendar  Year  I960 

3.0  Virus  Diagnostic  Reagents  and  Their  Publ ic  Health  Importance 

The  public  health  importance  of  the  more  than  100  newly 
recognized  viruses  which  commonly  infect  man  is  now  beginning  to  be 
appreciated.  However,  only  a  handful  of  public  health  laboratories 
are  currently  making  systematic  attempts  to  diagnose  viral  infections 
and  even  these  are  limiting  their  surveillance  to  a  pitifully  small 
group  of  viruses,  including  usually  no  more  than  3  pol iovi ruses, 
2  influenza  viruses,  and  several  viral  zoonoses.  This  involuntary 
indifference  to  man's  largest  morbidity  problem  is  largely  due  to 
the  fact  that  they  lack  the  necessary  viral  reagents  and  of  course 
personnel  trained  to  use  them.  Realizing  that  control  of  respiratory 
diseases  must  remain  an  unattainable  objective  unless  and  until 
acute  respiratory  diseases  (see  1957-58  National  Health  Survey)  can 
eventually  be  defined  as  a  public  health  problem,  we  have  made  the 
development  of  simple  and  reliable  virus  diagnostic  procedures  a 
major  part  of  our  research  program. 


12 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  NIAIO 

Calendar  Year  I960 

5.1  New  Serological  Test  Procedures 

The  laboratory  section  of  the  Epidemiology  Section  therefore 
concentrated  on  the  development,  application  and  evaluation  of  in 
vitro  test  procedures  for  the  identification  of  new  viruses  as  well 
as  for  detecting  virus  infection  as  expressed  in  antibody  responses. 
Thus,  using  conventional  complement  fixation  (CF)  and  newly  developed 
hemagglutination  inhibition  (HI)  procedures  it  has  been  possible  for 
our  group  to  type  thousands  of  virus  isolates  belonging  to  the 
adenovirus,  myxovirus,  enterovirus,  and  reovirus  groups.  As  was 
true  during  the  past  several  years,  LID  in  I960  again  described  and 
characterized  more  new  representatives  of  these  viruses  than  all 
other  virus  laboratories  in  the  world  combined.  This  was  made 
possible  during  I960  because  each  of  our  various  virus  research  units 
contributed  new  diagnostic  techniques.  Dr.  Rosen's  group  developed 
additional  specific  HI  procedures  for  identifying  adenoviruses  and 
adenovirus  infections;  and  for  reovi ruses  and  enteroviruses  as  well. 
Similarly,  Chanock,  Johnson  and  Cook  developed  tissue  culture  procedures 
for  isolating  Eaton's  PAP  virus,  while  Rowe  and  Hartley  not  only 
discovered  several  "new"  mouse  viruses  in  tumor  virus  study  systems, 
but  developed  serological  procedures  for  recognizing  their  presence. 

3.2  Serologic  Reagents 

But  the  availability  of  simplified  procedures  are  of  very 
little  use  unless  the  necessary  reagents  are  also  available.  Although 
many  virus  research  laboratories  could  do  the  tests,  few  laboratories 
are  able  to  produce  the  necessary  reagents.  The  magnitude  and  cost 
of  producing  and  certifying  them  promises  to  continue  to  exceed  any 
possible  resources  available.  This  fact  has  had  a  very  depressing 
effect  on  research  efforts  aimed  at  the  study  of  viruses  as  causes  of 
disease,  and  serves  as  yet  another  deterrent  to  early  delineation 
of  the  common  virus  diseases  as  public  health  problems.  Consequently, 
with  the  help  of  NINDB  and  Microbiological  Associates,  LID  in  1959 
and  I960  accepted  responsibility  to  develop  and  evaluate  more  than  a 
hundred  commercially  produced  virus  antigens.  LID,  of  course,  has 
been  active  in  the  certification  of  virus  prototypes  and  furnishes 
many  to  the  Virus  Registry  of  the  American  Type  Culture  Collection. 
We  are  also  collaborating  with  the  Enterovirus  and  Adenovirus  national 
committees  in  setting  up  standards  for  large  scale  production  of 
certified  antiserums  for  serotyping  and  classification  of  viruses; 
perhaps  the  highest  priority  need  of  al I  virus  laboratories  concerned 
with  human  infection  and  disease. 


13 

14 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

3.3    LID  as  the  Unofficial  World  Reference  Laboratory  for  Viruses 

Wholly  through  the  operation  of  circumstances,  the  Virus 
Section  of  LID  has  become  virtually  the  chief  (in  many  instances  only) 
reference  laboratory  for  many  of  the  newer  viruses,  including  adeno- 
viruses (about  30  human  and  several  animal  serotypes),  myxoviruses 
{o   new  para-inf I uenzas  occurring  in  3  species),  reovi ruses  (3  serotypes 
in  4  species),  many  of  the  newer  and  some  older  enteroviruses  (5  -  10), 
salivary  gland  viruses  (from  4  species),  and  new  mouse  viruses  (6), 
the  latter  frequently  found  in  tumor  virus  study  systems. 

Until  virus  reagents  desperately  needed  for  many  extremely 
common  viruses  are  made  available  either  commercially,  through 
government  agencies,  or  both,  LID  as  the  sole  custodian  of  many  of 
these  agents  cannot  avoid  responsibility  for  assisting  other  excellent 
virus  laboratories  to  identify  their  viruses,  and  on  a  pro-tern  basis 
at  least  for  keeping  order  in  the  general  virus  field.  Unfortunately 
we  have  no  specific  commitment  to  provide  such  services  and  even 
worse,  no  specific  budget  to  cover  them,  so  that  our  involuntary, 
constantly  growing  and  unavoidable  service  functions  must  be  done 
at  the  expense  of  our  research  missions. 

However,  it  must  be  admitted  that  the  simpler  virus  diagnostic 
techniques  and  the  availability  of  a  complete  supply  of  viral  reagents 
in  our  laboratory  (developed  out  of  necessity)  facilitate  not  only 
our  own  epidemiologic  studies  of  naturally  occurring  virus  infection 
but  also  enable  us  to  evaluate  the  significance  of  the  data  furnished 
by  other  laboratories  who  come  to  us  for  tec hn Tea  I  assistance.  As 
long  as  LID  continues  its  policy  of  working  on  the  frontiers  of  virus 
disease  problems  we  have  no  way  to  avoid  the  responsibilities  that 
devolve  on  us  as  a  result,  and  for  that  matter  no  real  desire  to  do  so. 
However  the  dimensions  of  this  frontier  have  grown  geometrically  in 
recent  years,  and  unless  LID  and  other  virus  laboratories  can  grow 
with  it  we  cannot  hope  to  continue  to  be  effective  and  to  play  such 
a  decisive  role  in  the  future.  There  may  be  some  justification  to 
the  desire  to  see  this  happen,  but  unless  other  laboratories  step 
into  the  breach  (it  will  take  many  years  to  build  other  groups  with 
the  overall  competence  of  the  Virus  units  in  LID),  the  effect  of  such 
a  policy  will  be  to  slow  up  progress  not  only  in  LID  but  in  many  other 
virus  laboratories  concerned  as  we  are  with  viruses  as  agents  of 
human  disease.  This  may  seem  a  presumptuous  statement,  but  the  facts 
of  the  matter  are  clear  and  objective  enough  to  justify  it. 


15 


11 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

4.0    Sero-epidemioloq ic  Studies  of  Virus  Disease 

Comprehensive  serological  studies  of  over  75  common  viruses* 
became  feasible  in  I960  as  the  result  of  successful  commercial 
production  of  experimental  lots  of  antigens.  This  was  achieved 
collaboratively  with  the  NINDB  Collaborative  Cerebral  Palsy  Study 
Program  and  Microbiological  Associates,  Inc.  (MBA),  with  the  Virus 
Section  of  LID  serving  as  project  director  for  the  production 
items  actually  produced  by  M8A. 

During  I960  all  the  antigens  were  shown  to  have  homologous 
reactivity  and  many  of  them  were  evaluated  in  conventional  complement 
fixation  (CF)  and  hemagglutination  inhibition  (HI)  tests,  and  some 
were  employed  in  routine  diagnostic  tests  in  our  epidemiological 
studies  of  respiratory  disease. 

However  before  definitive  sero-epidemio logic  surveys  of 
virus  infections  in  relation  to  birth  defects  can  be  monitored, 
al I  the  antigens  and  test  procedures  must  be  put  through  "shake 
down"  evaluations  with  standard  human  and  animal  serums  representing 
experiences  with  each  of  the  viruses,  thus  achieving  quantitative 
information  on  sensitivity  and  specificity.  Fortunately  many  of 
these  standard  sera  are  available;  however,  others  must  still  be 
acquired  in  1 961 . 

Costs  and  Micro-techniques. 


The  development  of  meny  antigens  required  concentration  and 
other  special  procedures,  thus  increasing  the  estimated  cost  of 
even  routine  production  to  as  much  as  $10.00  per  ml  -  a  prohibitive 
cost  when  considered  in  relation  to  the  large  amount  of  testing 
which  must  be  done  in  our  sero-epidemiologic  surveys.  Consequently 
we  have  turned  the  attention  and  the  efforts  of  our  serology  teams 
to  the  development  and  evaluation  of  micro-techniques. 


''Influenza,  para-influenza,  poliovirus,  ECHO,  Coxsackie  A,  Coxsackie  B, 
adenovirus,  reovirus,  and  other  new  virus  groups. 


I5 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

The  studies  have  been  postponed  because  of  the  delay  in 
obtaining  the  necessary  precision  instruments.  However  negotiations 
with  an  importer  to  obtain  them  from  Hungary  promises  to  solve 
the  non-scientific  obstacles  to  current  progress.  Preliminary 
studies  suggest  that  the  Takatsy  micro-technique  can  be  applied 
without  modification  in  our  Hi  tests  and  also  suggest  that  slight 
modifications  of  the  equipment  will  permit  micro-CF  tests  as  well. 

Comment 


The  commercial  production  of  numerous  satisfactory  complement- 
fixing  and  hemaggl ut inating  antigens  now  makes  possible  broad  and 
comprehensive  sero-epidemiologic  studies  of  virus  infections  which 
previously  could  not  even  be  contemplated.  However  practical 
considerations,  particularly  their  high  production  cost,  and  the 
lack  of  standard  serums  for  evaluation  (in  which  there  is  also 
a  cost  factor)  will  undoubtedly  slow  up  transition  from  the  stage 
of  possibility  to  that  of  general  feasibility,  -  perhaps  until 
current  concepts  concerning  acceptable  costs  for  such  research 
activities  are  revised  upwards. 


16 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

5.0    Studies  of  Cancer  Viruses 

Our  studies  of  cancer  virus  problems  can  be  subdivided  into 
several  categories:   (a)  Laboratory  studies  of  the  properties  of 
cancer  viruses  and  development  of  laboratory  tools  for  detecting 
and  working  with  them;  (b)  field  studies  of  the  behavior  in  nature 
of  those  tumor  viruses  for  which  suitable  detection  tests  are 
available;  (c)  studies  of  extraneous  viruses  ("background  noise") 
now  preventing  high  caliber  virologic  practice  in  the  study  of 
animal  tumor  viruses  and  obscuring  interpretation  of  nearly  alt 
current  observations  on  them;  and  (d)  the  study  of  general  virus 
experiences  in  relation  to  human  cancer  -  the"background  noise" 
in  the  human  cancer  problem  -  which  we  feel  must  be  done  eventually 
if  the  role  of  viruses  in  human  cancer  is  to  be  defined. 

Our  approach  to  these  various  interdependent  studies  is 
based  on  the  following  beliefs:   I)  That  the  conventional  methods 
of  standard  virology  must  be  applied  to  cancer  virus  research  if 
significant  progress  is  to  be  made;  2)  the  study  of  cancer  viruses 
obviously  cannot  be  separated  from  general  virology;  and  3)  that 
the  "biologic  point  of  view"  rather  than  attitudes  fostered  by 
preoccupation  with  categorical  disease,  represents  the  best  approach 
to  a  real  understanding  of  the  natural  history  of  cancer  viruses 
just  as  it  does  to  other  viruses  (see  Introduction). 


17 

18 


PHS-NIH 
Summary  Statement 
Laboratory  of    Infectious  Diseases,   NIAID 
Calendar  Year    I960 

5.1    Laboratory  Studies  -  Mouse  Polyoma  Cancer  Virus* 

New  in  v i tro  survey  tools  developed  during  1959  (CF,  HI, 
and  MAP)  were  evaluated  and  applied  in  I960  in  studies  of  polyoma 
virus  growth  and  excretion,  its  experimental  epidemiology,  and 
its  natural  history.  This  interesting  and  versatile  cancer  virus 
causes  tumors  not  only  in  all  strains  of  Mus  muscul us,  but  also 
in  hamsters,  rats,  rabbits,  and  guinea  pigs  (Stewart  and  Eddy). 
Of  equal  interest  is  the  fact  that  it  can  be  studied  and  surveyed 
with  the  same  facility  as  ordinary  viruses,  such  as  influenza 
and  pol ioviruses.  Virus  isolation  and  serologic  procedures, 
combined  with  ep izootiologic  studies  have  produced  the  following 
interesting  observations: 

Polyoma  virus  was  found  to  be  widely  disseminated  in  mouse 
colonies  nearly  everywhere.   Infection  was  found  to  be  more 
commonly  present  than  absent  in  laboratory  strains  raised  in 
experimental  or  commercial  laboratories  and  in  wild  strains  found 
in  city  tenements.  However  the  basic  ecology  or  natural  cycle 
appears  to  exist  in  rural  areas  -  on  farms  and  in  feed  mills  in 
smal I  towns. 

In  the  laboratory  the  virus  is  maintained  and  disseminated 
by  experimentally  and  spontaneously  infected  carrier  mice  which 
excrete  virus  in  saliva,  feces  and  urine,  the  latter  appearing 
to  be  the  most  important  vehicle  of  spread.   Infected  infant  mice 
excrete  so  much  virus  (up  to  a  million  ID50's  per  ml  of  urine) 
into  laboratory  environments  that  much  of  the  data  on  polyoma 
acquired  in  such  environs  is  subject  to  question,  particularly 
if  adequate  controls  (uninoculated  mice)  are  not  included  with 
every  animal  experiment.  Polyoma  virus  was  also  shown  to  represent 
one  of  the  more  common  extraneous  agents,  complicating  interpreta- 
tion of  laboratory  experimentation  with  other  mouse  tumor  viruses. 

*  Collaborators  at  the  National  Cancer  Institute  -  Drs.  L.  W.  Law, 
C.  J.  Dawe,  W.  6.  Banfield  and  H.  Kahler. 


18 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

5.2    Natural  Behavior  of  Polyoma 

Studies  of  the  behavior  of  polyoma  in  Mus  muscul us  were 
also  carried  out  in  three  different  field  environments,  in  densely 
populated  tenement  areas  of  a  large  city,  on  farms  producing 
grain  and  livestock,  and  in  grain  mills  located  in  small  rural 
towns.  Each  presents  a  different  ecology  but  in  each  one  focal 
environmental  contamination  appears  to  be  quite  important  to  the 
survival  and  persistence  of  polyoma  infection  in  the  mouse 
populations  observed. 

Harlem  Studies 


A  full  year's  surveillance  of  Mus  muscul us  infestation  and 
polyoma  infection  of  crowded  tenements  in  Harlem  revealed  that 
virus  infections  persisted  without  exception  in  numerous  separate 
foci.  Three  epidemiologic  factors  seem  most  important,  namely, 
large  mouse  populations  capable  of  furnishing  adequate  supplies 
of  young  susceptible  mice,  the  extensive  contamination  of  the 
tenement  environment  (virus  was  demonstrated  in  sweepings  from 
areas  showing  signs  of  mouse  activity),  and  finally  the  over- 
crowding which  insures  the  continuous  and  extensive  use  of  communal 
nesting  areas  (also  demonstrated  to  be  contaminated  by  virus). 
Apartment  houses  having  smaller  and  less  dense  mouse  infestation 
were  generally  free  of  infection  and  remained  so  during  the  study. 


19 

20 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  N I AID 
Calendar  Year  I960 

Rural  Studies 


Systematic  studies  of  polyoma  in  rural  environments  were 
undertaken  during  the  last  quarter  of  I960.  However,  it  appears 
from  preliminary  data  that  here  we  may  have  found  the  basic  natural 
eye le  of  mouse  polyoma.  Mus  musculus  infestation  and  polyoma 
infection  of  Mus  was  found  to  be  most  intense  in  feed  granaries 
on  the  farm  and  in  cereal  grain  storage  elevators  in  mills.  As 
many  as  30  per  cent  of  several  hundred  mice  trapped  in  these 
environs  showed  persistent  evidence  of  polyoma  infection,  many  of 
them  apparently  excreting  virus  in  their  urine.  The  virus  has 
been  found  on  cereal  grains  in  the  vicinity  of  mouse  nesting 
areas,  which  appear  to  be  very  numerous  in  the  granaries  so  far 
examined.  The  actual  extent  of  cereal  grain  contamination  by 
mouse  excreta  containing  polyoma  and  no  doubt  other  microbes  must 
still  be  evaluated;  however,  present  evidence  suggests  that  it 
probably  is  very  extensive,  if  not  appalling. 

The  I iterature  on  the  ecology  of  Mus  muscul us  centers  on 
the  infestations  of  rural  grain  storage  areas  -  particularly  in 
grain  "bins"  and  the  traditional  grain  "ricks."  Natural  ecologic 
arrangements  such  as  are  reported  here  are  no  doubt  much  older 
and  probably  much  more  of  a  factor  in  the  maintenance  of  natural 
mouse  agents  than  the  conditions  that  exist  in  infested  urban 
areas  or,  for  that  matter,  in  production  and  experimental  areas 
housing  laboratory  mice. 

Since  natural  infection  of  wild  mice  is  not  limited  to 
polyoma  virus,  but  includes  a  number  of  other  viruses  known  or 
suspected  to  infect  man  and  domestic  animals,  the  extension  of 
these  preliminary  findings  will  likely  prove  very  interesting. 

Should  our  observations  on  Maryland  farms  prove  not  to  be 
unique  -  and  there  is  no  reason  to  suspect  that  they  would  be  - 
then  extensive  contamination  of  cereal  grains  with  hardy  viruses 
such  as  polyoma  provides  a  logical  vehicle  of  infection  for  labora- 
tory mice  with  polyoma  and  no  doubt  with  some  of  the  other  extraneous 
agents  found  in  most  production  and  experimental  mouse  colonies. 
It  is  interesting  to  consider  the  fact  that  man  and  his  domestic 
animals  have  long  been  exposed  to  foodstuffs  heavily  contaminated 
by  mice  and  other  rodents  and  that  the  viruses  and  the  cancers 
known  to  occur  in  these  species  are  not  remarkably  different. 


20 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

Comment  -  Cancer  a  Possible  Zoonosis? 

The  fact  that  polyoma  virus,  avian  sarcoma  and  bovine 
papilloma,  although  well  established  tumor  viruses,  cross  over 
species  lines  -  polyoma  infecting  rats,  hamsters,  guinea  pigs, 
and  rabbits;  Rous  sarcoma,  turkeys,  ducks;  and  bovine 
papilloma,  horses  -  seriously  challenges  we  think  prevailing 
concepts  about  the  narrow  species  predilection  supposedly  exhibited 
by  cancer  viruses.   It  is  clearly  necessary  now  to  examine  the 
hypothesis  that  some  cancers  in  man  and  domestic  animals  could 
be  due  to  viruses  which  like  the  zoonoses  have  their  basic  natural 
cycle  in  lower  commensal  animals.  Such  studies  can  now  be  done 
on  some  of  the  known  animal  tumor  viruses,  such  as  polyoma. 

Our  field  projects  have  developed  much  information  of 
value  in  defining  not  only  the  natural  behavior  and  the  basic 
cycle  of  polyoma  in  nature,  but  also  the  probable  sources  of  virus 
infections  in  both  experimental  and  production  colonies.  Although 
serologic  studies  do  not  support  the  hypothesis  that  polyoma  virus 
can  infect  man,  widespread  access  of  humans  through  mouse  contam- 
inated environments  and  contamination  of  food-stuffs  with  mouse 
excreta  makes  consideration  of  possible  human  infection  rather 
more  than  academic.  The  lack  of  serologic  correlations  with 
cancer  in  man,  such  as  occurs  in  mice,  may  not  represent  con- 
clusive counter-evidence,  since  hamsters  with  polyoma  induced 
tumors  rapidly  lose  all  evidence  of  infection  with  polyoma 
(Habel),  despite  the  fact  that  the  tumors  persist. 


21 

22 


PHS-NIH 
Summary  Statement 
Laboratory  of    Infectious  Diseases,   NIAID 
Calendar  Year    I960 

Proposed  Course  of  the  Natural  History  Studies 

The  natural  history  of  polyoma  and  other  animal  tumor  viruses 
are  important  subjects  in  their  own  right  and  our  studies  represent 
some  of  the  first  effective  efforts  to  develop  such  information. 
As  noted  above,  there  can  be  no  higher  order  of  information  about 
infectious  agents  than  that  derived  from  nature.   Information 
derived  from  carefully  planned  longitudinal  studies  of  tumor  viruses 
are  relevant  to  any  consideration  of  possible  human  cancer  viruses. 

The  possibility  that  cancer  could  be  the  result  of  a  zoonotic 
infection  no  longer  appears  so  very  unlikely.  We  plan  therefore 
to  study  possible  polyoma  infection  in  various  animal  species, 
particularly  those  having  repeated  exposure  to  environments  and 
to  materials  known  to  contain  tumor  viruses,  such  as  the  polyoma 
and  leukemia  viruses  of  mice,  and  the  leukemia  and  sarcoma  viruses 
of  chickens. 


22 


23 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  N I  AID 
Calendar  Year  I960 

6.0    Extraneous  Viruses  as  "Background  Noise"  in  Cancer  Virus  Studies 

In  I960  the  "background  noise"  problem  in  cancer  virus  research 
grew  to  almost  "deafening"  proportions  and,  in  the  opinion  of  LID 
virologists,  constitutes  the  number  one  obstacle  to  intelligent  and 
truly  effective  research  on  cancer  viruses- 
Near  ly  every  animal  tumor  virus  system  currently  under  study 
was  shown  to  be  contaminated  with  extraneous  agents  and  several 
viruses  widely  proclaimed  as  "tumor"  viruses  turned  out  to  be  fellow 
traveling  ordinary  viruses.  To  list  a  few  examples:  Friend  leukemia 
was  found  contaminated  with  polyoma  and  mouse  adenovirus;  Gross 
leukemia  by  polyoma,  K  virus  and  mouse  adenovirus;  Schwartz  leukemia 
with  polyoma,  K  virus  and  mouse  adenovirus;  Moloney  leukemia  with 
mouse  hepatitis  and  mouse  reovirus;  the  polyoma  virus  itself  became 
contaminated  with  mouse  adenovirus,  hepatitis  and  salivary  gland 
vi  ruses. 

New  "tumor  viruses"  grown  in  tissue  culture  systems  or  propagated 
in  mice  were  reported  and  later  found  to  be  extraneous  agents.  Thus, 
to  give  one  example,  the  tissue  culture  grown  "VL"  strain  of  lympho- 
matosis was  shown  to  be  an  almost  universally  prevalent  adenovirus 
of  chickens  now  called  "GAL"  virus.  LID  virologists  showed  by 
serological  and  virus  isolation  methods  the  lack  of  association 
between  most  of  these  agents  and  the  specific  cancers  with  which 
they  are  associated,  confirming  in  several  instances  similar  findings 
in  other  laboratories. 


23 

24 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 


6. 1 Extraneous  Viruses  in  Tumor  Virus  Study  Systems 

LID  virologists  showed  that  the  "seeds"  of  the  "background 
noise"  viruses  are   commonly  present  in  the  animals  used  for  the 
induction  of  tumors,  and  of  course  in  the  subsequent  passage  materials 
as  mentioned  above.  The  extraneous  viruses  most  commonly  encountered 
in  cancer  systems  were  the  newer  ones,  such  as  polyoma,  K  virus,  mouse 
reovirus  and  adenovirus;  but  this  in  part  may  be  due  to  newly 
developed  easily  applied  survey  tools  for  these  agents.  Other 
viruses  encountered  less  often  (perhaps  because  of  comparatively 
less  sensitive  tools)  were  mouse  hepatitis,  mouse  salivary  gland 
virus,  the  newly  discovered  "thymic  agent"  (TA) .  Except  in  newborns, 
most  of  these  viruses  occur  subc I inical ly  and  latently.  Not  found 
or  seldom  encountered  in  tumor  systems  were  Theiler's  virus,  LCM, 
PVM  and  mouse  pox  (ectromel  ia) ,  all  familiar  to  most  virologists. 
Since  most  of  these  older  agents  generally  produce  very  obvious 
clinical  effects  in  mice,  they  are  perhaps  more  easily  eliminated 
from  production  colonies. 

All  mouse  colonies  used  for  tumor  virus  induction  were  shown 
to  contain  at  least  one  easily  demonstrable  extraneous  virus;  but 
more  than  one  (usually  three  to  five)  was  the  rule.  Discoveries  of 
new  mouse  viruses  and  the  development  of  techniques  for  selecting 
them  enabled  our  scientists  to  survey  and  define  many  mouse  production 
colonies  and  to  some  extent  predict  some  of  the  problems  encountered 
in  current  extensive  efforts  to  establish  cancer  viruses  in  animal 
and  tissue  culture  systems. 


2't 

25 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  Nl AID 

Calendar  Year  I960 

6.2    Proposed  Course  of  "Background  Noise"  Studies 

The  first  principle  of  microbiology  calls  for  "pure  culture" 
of  microbes.  The  extraneous  virus  problem  in  virus  study  systems 
must  be  controlled  before  "good"  virology  can  be  done  on  cancer 
viruses  in  such  systems.  The  extraneous  agents  are  in  most  cases 
slow-growing,  latent,  subclinical  agents.  While  they  do  not  seriously 
handicap  the  study  of  the  acutely  virulent  viruses  such  as  the 
encephalitis  and  Coxsackie  viruses,  they  do  however  pose  serious 
problems  in  studies  of  the  slow-growing  and  also  generally  latent 
cancer  viruses. 

LID  scientists  visualize  several  steps  that  must  be  taken: 
I)  Further  development  and  application  of  sensitive  techniques  for 
detecting  extraneous  viruses;  2)  Surveillance  and  monitoring  of 
existing  mouse  colonies  and  experiments  for  known  agents,  thus  helping 
to  define  current  problems.  This  calls  for  the  production  and 
evaluation  of  diagnostic  reagents;  3)  Establishment  of  relatively 
"clean",  "virus  defined"  colonies  and  animal  research  areas  for 
pilot  studies  aimed  at  the  elimination  of  extraneous  viruses  from 
study  systems;  4)  since  all  cancer  virus  passage  materials  passed 
through  experimental  animals  either  are  known  or  suspected  to  contain 
extraneous  viruses,  they  also  must  be  "cleaned"  up  in  order  to 
utilize  "clean"  animals  in  "clean"  areas  and  finally  achieve  the 
goal  of  "pure  culture";  and  5)  When  sufficient  information  is  available, 
"c lean"animal  production  colonies  and  "clean"  experimental  areas  must 
eventually  be  established  wherever  such  animals  are  to  be  used  for 
study  of  latent  or  tumor  virus  effects. 

Comment .   It  is  clear  that  the  task  described  here  will 
appear  monumental  to  some,  insuperable  to  others,  and  to  still  others 
too  expensive.  However,  it  is  also  clear  that  reluctance  to  meet 
this  problem  squarely  and  failure  to  eventually  achieve  its  solution 
is  to  accept  pre-Pasteurian  concepts  as  guides  for  modern  virology, 
to  waste  uncounted  dollars  and  to  accept  in  the  beginning  of  new 
and  very  expensive  enterprises  on  animal  tumor  viruses  the  probability 
of  f  inal  fai I ure. 

LID  scientists  who  have  helped  to  develop  and  define  this  problem 
recognize  the  importance  of  its  solution  and  accept  responsibility  to 
work  towards  that  necessary  goal.  We  must  do  so  if  we  are  to  satisfy 
our  desire  to  properly  study  the  role  of  latent  viruses  in  chronic  and 
neoplastic  diseases.   It  is  our  opinion  further  that  the  National 
Institutes  of  Health  with  its  large  commitment  to  support  i       i rus 
research  cannot  avoid  this  responsibility  and  that  the  most  effective 
action  towards  the  solution  of  this  critical  problem  depends  on 
well  directed  and  concerted  action  by  N I  AID  and  NCI.  25 

26 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

7.0    Studies  of  Common  Virus  Infections  in  Animals  Likely  to 
be  Important  in  Human  Medicine 

A.  Bovine  Virus  Infections 

During  the  past  several  years  LID  scientists  in  colla- 
boration with  other  groups  have  helped  to  enlarge  current  notions 
of  common  virus  infections  of  dairy  and  beef  cattle.  The  occurrence 
of  several  "new"  viruses,  such  as  reoviruses  types  I,  2  and  3, 
were  reported  in  I960,  and  additional  viruses,  chiefly  bovine 
enteroviruses,  are  currently  under  study.  Observations  on  the 
properties,  clinical  importance  and  prevalence  of  para-inf I uenza  3 
virus,  infectious  bovine  rh i notrachei tis  and  bovine  adenovirus 
were  also  extended,  and  potencies  of  para-inf I uenza  3  vaccines 
were  explored  in  cattle.  A  clear  view  of  the  importance  of  these 
viruses  in  bovine  disease  and  their  significance  for  human  health 
must  await  much  larger  and  comprehensive  efforts.  Such  an  effort 
however  far  exceeds  our  currently  available  facilities  and 
resources.  Consequently,  we  plan  in  1961  to  continue  only  to  a 
limited  degree  to  develop  our  laboratory  and  natural  history 
studies  of  these  agents. 

B.  Virus  Infections  of  House  Mice  (Mus  musculus) 

One  of  the  more  intimate  of  man's  domestic  creatures  is 
the  house  mouse.   It  is  perhaps  no  accident  that  the  known 
viruses  of  mice  closely  resemble  those  of  man;  at  least  13 
mouse  viruses  have  human  counterparts  and  several  are  known  to 
be  identical  with  human  agents.  Thus,  mouse  reoviruses 
types  2  and  3  discovered  during  I960  in  both  laboratory  and  wild 
mice,  and  in  mouse  tumor  passage  materials  (see  6.0  Extraneous 
Viruses  above)  are  indistinguishable  from  the  prototype  human 
strains.  Of  course  LCM  and  Sendai  viruses  of  mice  and  man  were 
previously  shown  to  be  identical. 


26 

27 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

Although  many  of  these  viruses  are  now  important  because 
they  produce  "background  noise"  in  cancer  virus  studies,  they  must 
be  regarded  as  important  in  their  own  right,  both  as  possible 
sources  of  infection  to  man  and  to  other  of  his  more  desirable 
domestic  animals.  They  also  serve  as  interesting  natural  models 
of  similar  human  infectious  disease  processes. 

The  contamination  of  cereal  grains  by  polyoma  virus  have 
been  mentioned  above  (in  5.0).  Since  other  common  viruses 
(such  as  mouse  reoviruses  and  adenoviruses,  etc.)  are  also 
excreted  in  saliva,  urine  or  feces  of  mice,  it  seems  inevitable 
that  they 'will  soon  be  found  on  grain,  which  of  course  if  fed 
uncooked  to  I ivestock. 

Human  infection  with  mouse  viruses  due  to  exposure  to 
cereal  grains  although  less  likely  because  they  are  almost  always 
cooked,  could  also  occur  through  occupational  and  household 
exposure  to  raw  grains  in  the  process  of  grinding,  processing, 
cooking  and  baking  with  flour.  Needless  to  say,  we  plan  sero- 
epidemiological  studies  designed  to  answer  the  questions  posed 
here. 


27 


28 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  Nl AID 
Calendar  Year  I960 

Medical  Mycology  Section 

The  Mycology  Section  reported  8  projects  for  calendar  year 
I960.  All  these  projects  have  included  broad  fields  of  research  and 
although  definitive  goals  have  been  reached  in  most  of  them,  all  will 
be  continued  in  order  to  further  exploti  productive  lines  of  investi- 
gation.  In  most  cases  new  or  additional  species  of  pathogenic  fungi 
will  be  used  in  investigations,  or  techniques  will  be  altered  to 
permit  further  development  of  experimental  studies.  Results  of 
investigations  have  been  published  in  9  papers. 

Dr.  George  W .  Lones,  investigating  antibiotic  X-5079C,  found 
that  it  is  fungistatic  but  not  fungicidal  and  that  its  apparent  low 
degree  of  jm  vitro  activity  is  due  to  its  decay  in  culture  medium. 
The  yeast  form  of  Histoplasma  capsulatum  is  much  more  sensitive  to 
X-5079C  than  the  mycelial  form  and  an  assay  method,  sensitive  to 
I  ug/ml  using  ]H.  capsulatum,  was  developed.  X-5079C  has  low 
toxicity  for  HeLa  cells  and  is  active  against  H.  capsulatum  grown 
in  HeLa  eel  Is. 

Dr.  Lones  has  converted  a  second  strain  of  Coccidioides  immitis 
to  serial  culture  in  the  spherule  form.  He  has  made  quantitative 
measurements  of  the  ability  of  various  carbon  and  nitrogen  sources 
to  support  growth  of  spherule  and  mycelial  forms  of  strain  M-l I  of 
C.  i mm i t i s .  Only  mannose  is  utilized  as  readily  as  glucose  by 
spherules.  Mannose  and  fructose  support  growth  of  the  mycelial 
form  as  well  as  does  glucose.  A  substrate  which  preferentially 
supports  growth  of  the  spherule  form  was  not  found  in  this  study. 

Dr.  Herbert  F.  Hasenclever  utilized  spherules  to  immunize 
mice  and  found  an  increased  survivor  rate  in  immunized  mice  when 
challenged  with  a  lethal  infecting  dose  and  earlier  clearance  of 
organs  (negative  cultures)  in  immunized  mice  challenged  with  a 
sublethal  dose. 

Dr.  Hasenclever  found  two  antigenic  groups  within  the  species 
Candida  albicans.  One  is  simi lar  antigenical ly  to  C.  tropical  is  and 
one  is  antigenically  indistinguishable  from  C.  stel latoidea. 


28 

29  **u 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

Dr.  Hasenclever  found  by  triturating  and  plating  out  organs 
of  experimental ly  infected  mice,  that  several  minutes  after 
Cryptococcus  neoformans  was  injected  either  intravenously  or  intra- 
cerebral ly  into  mice  the  largest  numbers  of  yeast  cells  had  been 
retained  in  the  lungs.  The  fungus  population  in  the  lung  then 
decreases  and  2-3  days  after  infection  multiplication  in  the  brain 
is  apparent.  Although  the  interval  from  infection  to  death  of 
infected  mice  varies  with  the  strain  of  C.  neoformans,  the  numbers  of 
yeast  cells  per  gram  of  brain  tissue  is  approximately  the  same 
regardless  of  strain. 

Dr.  Hasenclever  has  found  that  many  strains  of  Candida 
tropical  is  are  as  pathogenic  for  mice  as  for  rabbits  and  that 
multiplication  in  vivo  is  similar  to  that  of  C.  albicans.  The  kidneys 
are  the  organs  showing  greatest  tissue  damage  and  the  highest  yeast 
eel  I  population. 

Dr.  Chester  W.  Emmons  and  Mr.  Willard  Piggott,  with  the 
assistance  of  Mr.  William  Hill,  have  continued  studies  of  the 
saprophytic  occurrence  in  natural  habitats  of  fungi  which  cause 
mycoses.  Cryptococcus  neoformans  has  been  isolated  from  many 
additional  collections  of  pigeon  guano.  When  this  material  is 
collected  from  old  pigeon  nests  and  from  roosting  sites  in  hay  mows 
of  barns  and  upper  floors  of  buildings,  Histoplasma  has  never  been 
found.  There  is  increasing  circumstantial  evidence  that  a  presently 
unstudied  pneumonic  form  of  cryptococcosis  has  occurred  in  men  heavily 
exposed  to  such  material  and  that  such  material  and  that  such  epidemics 
have  been  erroneously  diagnosed  histoplasmosis. 

Dr.  Emmons  and  Mr.  Piggott  designed  a  simple,  convenient  and 
clean  device  for  exposing  mice  by  inhalation  to  dry  spores  of  patho- 
genic fungi.  This  has  been  used  to  stimulate  natural  conditions  of 
infection. 

Continuing  in  vivo  testing  of  antibiotics  and  of  drugs  prepared 
by  Dr.  Benjamin  Prescott  have  not  yielded  any  chemotherapeutic  agent 
equal  to  X-5079C  in  the  treatment  of  several  mycoses. 


23 

30 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  NIAID 

Calendar  Year  I960 

Or.  Emmons  has  continued  to  collaborate  with  Dr.  William  J. 
Jellison,  Dr.  L ie-Kian-Joe,  Dr.  Charles  Bridges  and  others,  in  the 
study  of  new  or  unusual  mycoses  and  the  fungi  which  cause  them. 
Studies  of  phycomycoses  have  continued.  With  Dr.  Bridges,  a  study 
of  several  cases  of  hyphomycosis  destruens  in  horses  and  of  its 
etiological  agent  has  been  submitted  for  publication.  The  fungus 
is  described  under  the  old  name,  Hyphomyces  destruens.   It  appears 
to  be  a  Phycomycete  but  its  complete  life  history  has  not  been 
determined. 

In  collaboration  with  Dr.  Jellison  a  new  species,  Emmons i a 
crescens,  was  described.  This  fungus  differs  from  the  first  species 
of  Emmons i a  (E_.  parva)  in  vivo  and  _i_n  vitro  at  37°  C  by  its  multi- 
nucleate condition  (instead  of  uninucleate),  its  ability  to  produce 
the  jm  vivo  form  _j_n  vitro  at  37°  C,  and  its  greater  size.  £.  parva 
conidia  when  inhaled  or  incubated  at  37°  C  increase  in  diameter 
from  2  -  4  u  to  400  -  480  u.  This  lO^-fold  increase  in  volume  of 
a  single  cell  is  very  unusual  in  the  fungi. 


30 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  NIAID 

Calendar  Year  I960 

Medical  and  Physiological  Bacteriology  Section 
Dr.  A.  K.  Saz 

Introduction 


Significant  progress  has  been  made  during  the  past  year  on 
all  scientific  problems  under  investigation  by  the  staff  of  the 
Medical  and  Physiological  Bacteriology  Section.  All  professional 
investigators  in  the  Section  have  published  in  the  past  calendar 
year  a  minimum  of  one  paper,  and  in  most  instances  more,  in  reputable, 
first-line  scientific  journals.  The  cohesiveness  of  the  Section 
has  been  further  evidenced  by  the  holding  of  regularly  scheduled 
informal  section  seminars  at  which  the  various  professionals  present 
their  current  raw  data  for  criticism.  The  program  of  the  Section  is 
varied  but  the  one  theme  common  to  all  projects  is  the  pursuit  of 
knowledge  dealing  with  fundamental  biological  activity  of  various 
bacteria.  The  cross-fertilization  which  has  occurred  as  a  result 
of  the  many-faceted  problems  under  investigation  by  the  professional 
personnel  of  the  section  has  been  of  prime  importance  in  progress 
made  and  offers  further  confirmatory  evidence  for  the  validity  of 
the  philosophy  that  team  projects  are  not  the  sole  road  to  scientific 
knowledge. 

It  has  been  established  that  staphylococcal  penici I  I inase  is 
associated  with  particulate  material  in  the  cell  and  thus  an  explana- 

ion  has  been  given  for  the  refractoriness  in  preparation  of  this 
enzyme  by  conventional  methods.  New  and  more  potent  inhibition  of 
Staph,  penic  i I  I inase  have  been  uncovered  and  the  hope  remains  and  is 
heightened  for  the  ultimate  finding  of  a  chemically  useful  inhibitor. 
Further,  sea  water  has  been  found  to  possess  strong  inhibitory 
activity  against  both  penic  i I  I  in-sensitive  and  penici I  I  in-resistant 
staphylococci  (phage  type  80/81). 

Real  progress  has  been  reported  in  the  understanding  of  iron 
metabolism  in  the  staphylococci.  As  a  direct  result  of  continuing 
work  dealing  with  mechanisms  of  the  development  of  non-specific 
immunity  and  in  particular  the  function  of  the  iron-transporting 
protein  of  plasma,  siderophi I  in,  fundamental  observations  on  the 
effects  of  iron  deficiency  on  the  growth  and  metabolism  of  S_.  aureus 
have  been  reported.  Work  on  the  biology  of  the  staphylococci  so 
long  neglected  during  the  "antibiotic  era"  is  cardinal  to  effective 
new  therapy  of  staphylococcal  infections. 


31 

32 


PHS-NIH 

Summary  Statement 

Labc  -atory  of  Infectious  Diseases,  NIAID 

Calendar  Year  I960 

Progress  has  also  been  made  on  the  search  for  better  methods 
of  isolation  and  purification  of  M  protein  of  streptococci.  These 
results  are  of  obvious  importance  in  the  understanding  of  Group  A 
streptococcal  virulence.  Further,  highly  interesting  observations 
have  been  reported  dealing  with  the  mechanism  and  significance  of 
the  long-chain  test  for  determination  of  ant i -streptococcal  immunity. 

Real  understanding  of  the  intimate  mechanisms  of  energy  meta- 
bol ism  in  Hvdroqenomonas  in  particular  and  other  bacteria  and  higher 
form  in  general  is  closer  as  a  result  of  work  performed  in  this  section 
this  year.   In  an  enormously  complicated  field,  progress  has  occurred 
in  the  definitions  of  the  essential  reactions. 

Detoxification  studies  on  potentially  useful  chemotherapeutic 
agents  have  continued  and  new  and  promising  leads  have  been  uncovered 
for  agents  active  against  bacteria,  fungi,  parasites  and  it  should 
be  added,  against  cancer  as  well.  Several  of  the  aforementioned 
detoxified  compounds  have  passed  preliminary  screening  processes 
performed  by  the  Cancer  Chemotherapy  Center. 

Pinpointing  of  the  enzymic  locus  of  discrimination  among 
hydrogen  isotopes  by  Pseudomonas  has  been  reported  this  year.  The 
area  lies  in  formic  acid  metabolism. 

During  the  past  year,  we  were  unfortunate  in  losing  the 
services  of  Dr.  Robert  C.  Woodworth  who  has  left  this  section,  but 
this  was  compensated  for  by  the  selection  of  Dr.  Austin  Shug, 
Project  Associate,  Enzyme  Institute,  University  of  Wisconsin,  to 
replace  him.  Dr.  Shug  will  concern  himself  with  studies  on  the 
iron  metabolism  of  staphylococci. 

We  were  saddened  by  the  passing  of  Mr.  Charles  Offord,  Animal 
Caretaker  in  this  section.  Mr.  Jack  Kelly  has  ably  replaced  Mr.  Offord. 

Dr.  Eskin  Huff,  LCI,  has  been  transferred  to  this  section  and 
will  work  on  the  mode  of  action  of  penicillin  and  the  biochemical 
mechanisms  of  the  development  of  resistance  thereto.  He  is  joined 
by  Miss  Harriet  Milner,  Biologist,  and  will  occupy  the  space  formerly 
assigned  to  Dr.  Repaske,  who  in  turn  will  have  new  quarters  in  the 
space  left  vacant  by  the  departure  of  DBS  personnel  from  the  area. 


32 

33 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  NIAID 

Calendar  Year  I960 

Authorization  still  has  not  been  obtained  for  the  hiring  of 
a  microbial  geneticist.   It  cannot  be  emphasized  too  strongly  that 
this  sectionis  mission  in  bacteriological  research  cannot  be  completely 
fulfilled  unless  a  program  of  genetic  research  is  begun.  This  is  a 
highly  important,  vital  area  of  contemporary  microbiological  research 
and  it  is  incomprehensible  that  an  institute  devoted  to  a  study  of 
the  microbial  world  is  not  represented  in  this  parameter. 

Studies  on  the  Biochemi,~-y  of  Antibiotics . 

Studies  on  the  biochemistry  of  staphylococcal  penicillinase 
are  continuing  on  two  parameters:   I)  search  for  an  effective 
inhibitor  of  the  enzyme,  2)  purification  of  the  enzyme.  Results 
reported  last  year  indicated  that  various  dipeptides,  and  particularly 
those  with  D-val ine  in  the  molecule  were  inhibitory.  Other  inhibitory 
compounds  have  now  been  found.  These  are  an  interesting  group  of 
substances  prepared  by  condensing  various  amino  acids,  primarily  of 
the  D-conf iguration,  into  peptides  with  substances  that  show  per  se 
metal  binding  capacity  but  are  not  in  themselves  inhibitory  to  penicillin. 
On  a  molar  basis,  these  compounds  are  by  far  the  most  inhibitory  of 
any  yet  studied.  The  most  active  are  D-dialanyl-  and  L-dialanyl- 
benzidine,  D-val y I -4-ami nob i phenyl  and  D-phenyl-L-alany l-aminof I uorene. 
Inhibitions  up  to  60  per  cent  have  also  been  observed  with  other 
dipeptides,  i.e.,  L-threony l-D-alanine,  L-al lothreonyl-D-alanine 
and  D-a I  I o  i  so  I eucy l-D-alanine. 

Various  considerations  had  indicated  that  staphylococcal 
penicillinase  conceivably  was  associated  with  particulate  matter 
within  the  cell.  Work  performed  this  year  has  indicated  that  this 
is  indeed  the  case.   It  has  been  possible  to  secure  12-fold  purifica- 
tion of  the  cell-free  enzyme  by  centrifuging  in  the  Spinco  Model  L 
centrifuge  at  144,000  x  g  for  periods  of  5-15  minutes.  All  activity 
is  found  in  the  pellet.   It  is  hypothesized  that  the  marked  stimula- 
tion of  penicillinase  activity  by  various  alcohols  (reported  last  year) 
is  due  to  the  reversible  solubilization  of  the  particle  mediated 
by  the  alcohols.  This  activity  brings  the  enzyme  and  the  substrate 
(penicillin  into  apposition) . 


33 

34 


Mill 

n  i 
Lab-.'  ,   NIAID 

A  corol  lary  ol    Hi     m  ■<  '■    i    portad  >fni  n1  of 

.!  new ,  h  ophotom  i  hod  of   ponl'  1 1  I  i 

i  coloi   of   -i  I'll  Indh  'ii  i 

of   panic!  I  loi<     i'  i 'i  i "i  ma1  Ion  ai  I  ilnfl  from   fni    action  of   panic)  I 

lapti  'i  df  row  1 1  /  U 
of   Di  .Nil  ,   NIAMD. 

it  :i  i  ',i  -  ol  laboi  atl  rlth 

.  aphd    in   1 1 1 .ii  ion  lnv<  -i  Igatoi  i   hha I 
.  .i  mar  ki  /Ity  fa 

man  If  il  n»1  bo1  h  I  pan  fell  i  odm  I  ng( 

pan  id  1 1  In-raala1 

ii  f-.)    Is  hea 

undlml  ■  Uvfty       It  I  col  lab 

; ion  "iii  '  onl Inua. 

pr  ItK  ipal    r>'  I  •  I' 

Bot' 

">ra 

/o 

Y       Q  HD 

Q  H2O  Q  HD 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  Nl AID 

Calendar  Year  I960 

Fractionation  of  deuterium  from  protium  apparently  takes  place 

in  the  step  HCOOH >  CO2  +  Ho'  °^  +fie  *nree  possible  enzyme  systems 

involved  in  equilibrium  reactions  of  atomic  and  molecular  hydrogen, 
viz.  hydrogen  lyase,  dehydrogenase  and  hydrogenase,  only  the  first 
one  is  inhibited  by  deuterium  in  concentrations  equal  or  less  than 
66  per  cent  D2O  or  by  completely  deuterated  formate.  These  findings 
strongly  suggest  that  at  ieast  part  of  the  fractionation  effect 
leading  to  light  hydrogen  gas  can  be  attributed  to  inhibition  of 
specific  enzymes  by  deuterium. 

Studies  with  tritium  have  involved  the  reactions  between 
tritiated  water  and  organic  constituents  in  living  cells.  The 
Pseudomonad  quickly  absorbs  tritiated  water  in  various  media  and 
splits  the  water  molecule  containing  tribium.  Whereas  the  amino 
acids  from  bacterial  protein  show  little  or  no  tritium  bonding,  the 
carbohydrate  fraction  including  polysaccharides  appears  to  contain 
a  large  percentage  of  the  bound  tritium.  Much  of  the  tritium 
entering  the  cell  as  tritiated  water  diffuses  out  later  as  carbo- 
hydrate or  polysaccharide.  Further  studies  are  contemplated  to 
identify  the  tritium-bound  organic  matter.  Since  the  equilibrium 
constant  for  tritium  is  K  =  Q  HTO  Q  H2  =  5.93  +  0.08  @  25°  C 

Q  H20  Q  HD 
which  is  appreciably  higher  than  deuterium,  it  may  be  expected  that 
the  fractionation  factor  involving  atomic  and  molecular  compounds 
of  tritium  will  likewise  be  higher. 

Aside  from  the  demonstration  of  deuterium  and  tritium 
fractionation,  these  studies  have  revealed  the  rapidity  which 
microorganisms  absorb  and  split  or  bind  water.  Tracer  methods  using 
tritiated  water  reveal  that  the  Pseudomonad  pumps  water  molecules 
at  the  rate  of  10 '3  molecules  per  hour  at  25°  C. 

The  implications  from  this  study  include  the  following: 

1)  The  natural  isotope,  deuterium,  appears  to  inhibit  specific 
enzymes  in  living  cells  which  may  affect  the  overall  metabolism. 

2)  Bacteria  in  marine  sediments  may  play  an  important  role 
in  the  concentration  of  deuterium  in  the  marine  environment. 


36 


35 


PHS-NIH 
Summary  Statement 
I  -tboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

3)  Bacteria  may  prove  to  be  effective  agents  for  the  concentra- 
tion of  natural  and  radioactive  isotopes.  The  former  has  practical 
significance  in  the  production  of  fusion  fuel  (HDO)  and  valuable 
trace  elements;  the  latter  may  be  important  in  problems  dealing 
with  water  pollution  from  radioactive  isotopes. 

The  Electron  Transport  System  in  an  Autotrophic  Hydroqenomonad. 

Hydroqenomonas  eutropha  (Bovell)  is  an  autotrophic  bacterium 
which  is  capable  of  obtaining  all  of  its  energy  for  growth,  mainten- 
ance, and  repair  from  the  oxidation  of  hydrogen  gas  by  oxygen  (ZhU  +  C>2 

>  2H2O) .  Although  the  oxidation  of  the  particular  substrate, 

hydrogen,  is  unusual,  the  reaction  is  fundamentally  a  general  one 
in  which  hydrogen  replaces  the  ordinary  organic  substrates  such  as 
glucose,  glycerol  or  other  complex  compounds.  Another  unique  feature 
of  H.  eutropha  is  its  ability  to  synthesize  al I  eel  I  material  from 
carbon  dioxide  with  energy  secured  from  the  oxidation  of  hydrogen. 
Nitrogen  is  obtained  from  ammonium  salts,  sulfur  from  sulfate. 
This  organism  then  is  a  complete  biological  synthetic  machine  which 
manufactures  the  most  complicated  structural  proteins,  enzymes, 
vitamins,  carbohydrates  and  lipids  from  three  gases  (H2,  O2  and  C02) 
and  simple  inorganic  compounds. 

Tremendous  amounts  of  energy  are  needed  for  these  processes, 
yet  the  mechanism  of  energy  production  during  oxidative  metabolism 
is  not  known  for  this  organism  or  any  other  aerobic  organism. 

It  has  been  observed  that  various  biological  systems  share 
many  similar  biochemical  reactions;  this  has  given  rise  to  the  concept 
of  comparative  biochemistry.  Consequently  it  is  assumed  that  the 
mechanism  of  oxidative  phosphorylation  in  H.  eutropha  is  analogous 
to  that  in  other  forms  of  aerobic  biological  life.  Biochemists 
working  with  bacterial,  animal  and  plant  material  have  applied 
themselves  to  the  problem  of  coupled  oxidation  and  phosphorylation 
for  the  past  35  years,  but  the  mechanism  for  trapping  energy  in  the 
form  of  ATP  during  oxidations  of  the  type  mentioned  has  thur  far 
defied  resolution  because  (I)  the  enzymes  of  the  electron  transport 
system  and  the  interacting  energy  yielding  system  are  both  associated 
with  particulate  cellular  elements,  and  (2)  resolution  of  one  or  the 
other  system  into  a  soluble  state  have  generally  resulted  in  loss 
of  both  activities. 


36 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  NIAID 

Calendar  Year  I960 


Hydroqenomonas  eutropha  is  a  bacterium  with  unusual  possibilities 
for  the  study  of  oxidative  phosphorylation.  Present  evidence 
indicates  that  the  electron  transport  system  is  soluble;  consequently, 
classical  enzyme  purification  techniques  should  result  in  separation 
of  the  individual  enzymes  leading  to  an  elucidation  of  their  function. 
It  seems  probable  that  the  enzymes  responsible  for  trapping  the 
resultant  energy  in  the  form  of  ATP  are  also  soluble  and  they  may 
be  similarly  studied. 

Since  an  understanding  of  the  electron  transport  system  is 
prerequisite  to  a  study  of  the  energy  trapping  system,  we  have  begun 
studies  on  the  hydrogen  oxidizing  system.  We  have  found  that  the 
initial  steps  involve  the  activation  of  hydrogen  gas  and  the  passage 
of  electrons  to  the  coenzyme  diphosphopyr idine  nucleotide  (DPN) . 
A  second  coenzyme  requirement,  riboflavin  phosphate  (FMN)  has  also 
been  found,  and  the  data  suggest  that  it  functions  as  the  cof actor 
between  hydrogen  and  DPN.  Extreme  caution  must  be  exercised  in 
handling  the  enzyme  because  of  its  sensitivity  to  oxygen.  This 
has  necessitated  the  design  of  several  new  pieces  of  equipment  and 
of  techniques  which  minimize  oxygen  contamination  during  handling 
of  the  enzyme.  To  date  the  enzyme  has  been  purified  three  to  four 
fold  from  crude,  cell-free  extracts  by  protamine  sulfate  and 
calcium  phosphate  gel  treatments.  Additional  purification  will 
be  necessary  before  investigating  the  next  enzymic  reaction,  which 
is  probably  cytochrome  linked. 

Preliminary  studies  reveal  that  H.  eutropha  contains  but  one 
cytochrome  of  the  "c"  type;  in  contrast,  most  other  aerobic  organisms 
contain  three  of  four  cytochromes.  This  fact  again  indicates  that 
this  organism  possesses  a  less  complicated  electron  transport  system 
than  is  usual ly  encountered. 


37 

38 


PHS-NIH 
Summary  Statement 
Laboratory  of  Infectious  Diseases,  NIAIO 
Calendar  Year  I960 

Detoxification  of  potential  Chemotherapeutic  Agents; 

Further  studies  are  in  progress  to  increase  the  tolerated 
dose  of  various  chemotherapeutic  agents,  such  as  isoniazid  (INH), 
streptomycin  (SM)  and  Kanamycin  for  tuberculosis,  Neomycin  for 
other  infections,  Mystatin  for  fungal  infections;  Miracil  D, 
piperazine  and  complexes  of  tetracycline  for  parasitic  infections 
and  certain  agents  in  use  in  cancer  therapy,  by  concomitant 
administration  of  various  metabolites.  Studies  on  the  tolerance 
of  mice  for  streptomycin  and/or  isoniazid  have  shown  that  a  lethal 
dose  of  either  drug  dissolved  in  glycerol  formal  as  solvent  (25-30 
per  cent)  permitted  90-100  per  cent  survival  in  white  and  DBA  mice. 
Mixed  with  SM-INH,  85  per  cent  of  the  mice  tolerated  a  lethal  dose 
of  10  mg  SM  and  4  mg  INH  and  80  per  cent  survived  with  10  mg  SM 
and  8  mg  INH.   In  addition,  studies  with  steroids  as  adjuvants  showed 
that  a  lethal  dose  of  30  mg  SM  and  4  mg  INH  (5  mg  is  the  tolerated 
dose  of  SM)  administered  simultaneously  with  25  mg  of  sodium 
taurocholate  permitted  55  per  cent  survival  in  two  strains  of  mice. 
With  twice  the  amount  of  sodium  taurocholate,  100  per  cent  of  the 
mice  tested  tolerated  a  30  mg  dose  of  Miracil  D  (6  x  lethal  dose). 

A  number  of  potential ly  non-toxic  new  chemotherapeutic  agents 
against  fungal  and  parasitic  infections  have  been  synthesized. 
Among  these,  long  chain  thiosemicarbazones,  piperazines  and  numerous 
tetracycline  complexes  of  atabrine,  chloroquine,  sulfanilic  acid 
and  naphthoquinones  have  already  demonstrated  high  in  v i vo  activity 
and  yet  drew  negligible  toxicity  for  mice.  Several  of  the  long 
chain  thiosemicarbazones  have  also  passed  initial  tests  in  mice 
when  tested  by  the  Cancer  Chemotherapy  Screening  Center. 

Investigations  of  the  antimicrobial  activity  of  substances 
from  shellfish  are  being  considered.  Two  groups  of  fractions  were 
isolated  from  abalone  juice  by  passage  through  an  an  ion-exchange 
(diethyl  ami  no-ethyl  eel  I ulose)  column  followed  by  elution  with  a 
series  of  Tris-H3P04  buffers.  Antibacterial  activity  of  a  group 
of  the  early  eluates  has  been  reported  (Proc.  Soc.  Exp.  Biol,  and 
Med.,  in  press).  These  fractions  showed  no  inhibition  in  tissue 
culture  against  Japanese  365  strain  of  influenza  A  virus  and 
polyoma  virus.  A  grouping  of  later  eluates  including  a  final  M-NaCI 
wash  from  the  column  contained  no  antibacterial  activity.  However, 
these  combined  eluates  exhibited  definite  inhibition  against 
influenza  virus  and  polyoma  virus.  When  monkey  kidney  cells  were 
treated  for  24  hours  before  virus  inoculation  with  a  fraction  of 
this  group  at  a  concentration  of  50  mg  per  cent,  virus  multiplication 
could  not  be  demonstrated. 


39 


38 


PHS-NIH 

Summary  Statement 

LaLoratory  of  Infectious  Diseases,  NIAID 

Calendar  Year  I960 

Studies  on  the  Growth  and  Metabolism  of  Staphylococci  and  on 

the  Iron  Metabolism  of  the  Host  as  Mediated  by  the  Iron-binding  Serum 
Protein  S iderophi I  in. 

Research  on  this  project  is  oriented  towards  elucidation  of 
the  following  problems: 

1)  The  role  of  siderophilin  as  a  non-specific  immune  factor 
in  the  control  of  bacterial  infection  through  nutritional  iron 
restriction. 

In  the  circulatory  system,  both  in  blood  and  in  the  lymph, 
the  iron  availability  to  the  bacterial  cells  as  well  as  to  the  host 
tissue  cells  is  regulated  by  the  protein  constituent  siderophilin. 
Workers  in  this  section  have  discovered  that  the  growth  of  Staph. 
albus  in  serum  is  completely  inhibited  by  siderophilin  if,  as  occurs 
in  normal  and  in  many  pathologic  sera,  the  protein  is  not  completely 
saturated  with  iron.  Staph,  aureus .  on  the  other  hand,  grows  in 
serum  at  a  rate  which  is  a  function  of  the  percentage  iron  saturation 
of  the  siderophilin.  For  example,  if  the  siderophilin  in  serum  is 
100  per  cent  saturated,  Staph,  aureus  grows  ten  times  faster  than 
when  the  siderophilin  is  only  30  per  cent  iron-saturated  (the 
percentage  saturation  of  normal  serum).  Further,  the  lag  period  of 
growth,  under  the  experimental  physiological  conditions  employed, 
is  extended  from  8  hours  to  27  hours  at  the  lower  percentage 
saturation.  Since,  in  various  pathologic  states,  the  percentage 
iron-saturation  levels  of  serum  siderophilin  vary  in  a  diagnostical ly 
significant  manner  far  above  and  below  the  usual  normal  value  to 
30  per  cent,  the  suitability  of  such  sera  as  in  v i vo  culture  media 
for  the  growth  of  many  pathogens  and,  in  particular,  Staph,  aureus, 
is  a  factor  in  the  defense  mechanism  of  the  host  against  the  establish- 
ment of  a  given  bacterial  infection.  A  phenomenon  associated  with 
infection  and  worthy  of  further  investigation  is  the  response  of 
the  host  to  decrease  the  percentage  iron-saturation  of  its  siderophilin 
by  lowering  the  concentration  of  circulating  serum  iron.  One 
consequence  of  this  response  is  to  provide  a  more  unsuitable 
nutritional  environment  for  the  infecting  bacterium;  another 
consequence  is  to  increase  the  iron  concentration  in  the  reticulo- 
endothelial system.  The  mechanism  by  which  the  response  is  effected 
is  unknown  and  the  significance  of  the  heightened  iron  concentration 
in  the  R.  E.  systen  likewise  requires  elucidation. 

2)  The  effect  of  the  siderophi I i n-control I eo 
iron  concentration  on  the  metabol ic  activities  of  Sj 

33 

40 


PHS-NIH 

Summary  Statement 

Lauoratory  of  Infectious  Diseases,  NIAID 

Calendar  Year  I960 

It  has  been  found  that  the  carbohydrate  metabolism  of  cells 
of  Staph,  aureus  grown  in  medium  restrictive  as  to  available  iron 
(low  percentage  iron-saturation  of  siderophi I i n)  is  greatly  different 
from  those  grown  in  media  providing  5  to  20  times  as  much  free 
iron  (high  percentage  iron-saturation  of  siderophi I  in) .  One  of 
the  most  striking  effects,  among  others,  is  the  failure  of  "low 
iron"  cells  to  oxidize  the  important  pivotal  substrate,  pyruvic 
acid.  The  concentration  of  certain  iron  enzymes,  as  cytochrome 
oxidase  and  catalase,  is  severely  reduced  in  "low  iron"  cells. 

The  biochemical  mechanisms  by  which  iron  controls  the  overall 
metabolism  of  Staph,  aureus  are  being  investigated  and  the  changes 
in  enzymatic  function  and  localization  of  iron-containing  components 
within  various  cellular  structures  are  being  studied. 

3)  A  comparison  of  the  mechanisms  by  which  mammalian  host 
tissues  acquire  iron  from  iron-si derophi I  in  with  that  operating  in 
the  eel  Is  of  Staph,  aureus. 

It  has  been  determined  that  eel  Is  of  Staph,  aureus,  grown 
in  the  presence  of  serum,  obtain  their  iron  from  iron-si derophi I  in 
as  free  ionic  iron.  The  source  of  this  ionic  iron  is  the  natural 
dissociation  of  the  metal  protein  complex  under  physiological 
conditions  of  pH  and  bicarbonate  concentration.  The  relationship 
of  pH  and  bicarbonate  concentration  changes  to  the  dissociation 
rate  of  the  metal  complex  have  been  investigated.   It  has  been 
established  that  bone  marrow  tissue  and  reticulocytes  take  up  iron 
from  i ron-si derophi I  i n  in  serum,  or  from  a  bicarbonate  buffer 
solution  of  the  metal  complex,  not  as  ionic  iron  but  by  some  type 
of  direct  transfer  from  the  complex  to  the  cells.   Investigation 
of  the  basis  for  this  direct  transfer  is  contemplated.  Whether 
other  tissue  ceJIs  than  bone  marrow  and  reticulocytes  depend  for 
their  iron  on  similar  direct  transfer  of  iron  from  iron-si derophi I  in 
complex  or  whether  they  are  similar  to  Staph,  aureus  eel  Is  in 
fulfilling  their  iron  requirements  through  diffusion  of  ionic  iron 
is  a  current  problem  of  active  interest. 

Effective  use  of  Staph,  aureus  and  of  rabbit  reticulocytes  have 
been  made  as  indicators  of  the  integrity  of  isolated,  purified 
s iderophi I  ins  on  the  basis  of  their  efficacy  in  the  donation  of  iron 
to  the  given  cells.  Further  development  of  the  applicability  of 
these  agents  to  such  purpose  is  required. 


HO 

41 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  N I  AID 

Calendar  Year  I960 

Streptococcal  M  Protein,  Virulence,  and  Type-specific  Immunity. 

This  unit  has  been  interested  in  the  bacterial  flora  of  the 
upper  respiratory  tract,  chiefly  the  gram-positive  cocci,  because 
of  their  prevalence  and  pathogenicity.  Chief  among  these  are  the 
beta-hemo lytic  streptococci  of  Group  A.  Despite  more  than  15  years 
of  antibiotic  usage,  these  organisms  appear  to  be  as  ubiquitous  as 
ever.     Widespread  use  of  antibiotics  and  prophylactic  programs  have 
caused,  however,  decreases  in  severity  and  in  non-suppurati ve 
sequelae  such  as  rheumatic  fever  and  acute  glomerulonephritis.  The 
continuing  high  incidence  of  Group  A  streptococcal  infections  is 
probably  caused  by  many  repeat  infections  with  the  same  type,  due 
in  turn  to  the  suppression  of  adequate  type-specific  antibody 
response  by  early  administration  of  antibiotics.  The  role  of  the 
presumptive  virulence  factor,  M  protein,  in  such  infections  is- 
moderately  well-established  by  association,  but  the  mechanism 
of  its  action  is  obscure. 

For  the  past  several  years,  work  in  the  Respiratory  Bacteriology 
Unit  has  emphasized  cultural  studies  in  field  situations,  balanced 
by  increasing  basic  laboratory  investigations  into  the  nature  of 
streptococcal  virulence  and  of  the  protective  type-specific  anti- 
bodies.  It  is  felt  that  such  studies  may  ultimately  aid  also  in 
determining  the  nature  of  virulence  in,  and  of  specific  resistance 
to,  other  gram-positive  cocci  and  particularly  the  staphylococci 
which,  although  they  produce  certain  extracellular  materials  in 
common  with  the  streptococci,  are  less  well  classified  serologically 
or  by  antigenic  dissection. 

Cultural  studies  in  calendar  I960  have  been  limited  to 
serologic  identification  of  beta-hemo lytic  streptococci  isolated 
sequentially  from  personnel  stationed  in  the  Antarctic.  The  study, 
done  in  conjunction  with  a  group  at  the  School  of  Hygiene  of  Johns 
Hopkins  University,  has  shown  that  delayed  isolation  of  beta- 
hemo  lytic  streptococci  from  frozen  glycerine-broth  is  feasible;  and 
that  the  majority  of  streptococci  so  isolated  were  of  Group  A. 
The  correlation  of  serologic  group  with  bacitracin  sensitivity, 
however,  was  low.  The  latter  thus  appears  to  be  a  poorer  screening 
procedure  than  indicated  by  previous  workers.  Continuing  field 
studies  of  this  sort  are  planned,  both  in  the  Antarctic  and  Arctic. 
In  addition,  the  antibody  responses  of  carriers  will  be  studied  by 
new  methods. 


42 


kl 


PHS-NIH 
Summary  Statement 
1 aboratory  of  Infectious  Diseases,  NIAID 
Calendar  Year  I960 

Basic  investigations  have  been  concerned  with  the  development 
of  new  methods  for  extracting  and  purifying  M  protein;  and  with 
improving  tests  for  type-specific  antibody.  M  protein  of  several 
types  have  been  prepared  as  the  acid-alcohol  soluble  picrates. 
These  preparations  are  simple  to  make,  are  antigenic,  and  cross-react 
only  when  the  organism  from  which  they  are  made  is  known  to  contain 
a  cross-reacting  "R"  protein.  Further  purification  and  analysis 
by  antigenicity,  agar  gel  diffusion  and  Immunoelectrophoresis  are 
in  progress.  The  use  of  picrates  and  of  conventionally-prepared 
M  proteins  in  enhancement  of  virulence  or  protection  against 
phagocytosis,  is  being  studied.  Fragmentation  of  M  protein  by 
heat,  pH,  and  enzymes  to  define  the  portion  active  in  virulence, 
as  we  I  I  as  in  other  reactions,  is  under  way. 

Improved  methods  for  the  detection  of  type-specific  antibody 
are  being  pursued.  These  include  the  long  chain  test  and  inhibition 
of  specific  fluorescence.  The  former  has  been  increased  in  simplicity, 
speed,  sensitivity  and  statistical  control  by  using  chi-square 
analyses  of  chain-length  frequency  distributions.  The  importance 
of  time-interval  sampling,  and  the  relation  of  time  to  antibody 
and  antigen  concentrations  used  in  the  test,  have  been  shown.  As 
a  result,  it  is  now  possible  to  titrate  readily  antisera  for  type- 
specific  antibody  to  a  degree  not  previously  possible. 

The  inhibition  of  specific  fluorescence  appears  to  be  a 
sensitive  screening  test  for  type-specific  antibody,  but  its  use 
in  titration  is  hampered  by  subjective  determination  of  the  degree 
of  fluorescence  as  an  end-point.  Type-specific  fluorescent  antisera, 
needed  for  this  test,  were  readily  prepared  by  absorption  of  labelled 
antisera  to  whole  cells,  and  did  not  cross-react.  Such  antisera 
have  also  been  used,  together  with  group  antisera,  to  study  the 
location  of  M  protein  in  the  cell  wall.   It  has  been  shown  that 
type-specific  antibody  will  block  group  antibody,  although  the 
reverse  is  not  true.  These  results  appear  to  verify  the  superficial 
location  of  M  and  the  deeper  position  in  the  eel  I  wal I  of  the  group 
carbohydrate  antigen. 


43 


PHS-NIH 

Summary  Statement 

Laboratory  of  Infectious  Diseases,  N I  AID 

Calendar  Year  I960 

Group  A  streptococci  have  been  shown  to  grow  well  in,  and  to 
form  long  chains  in,  f I uorescein-l abel led  homologous  type-specific 
antiserum.  Once  antibody  in  such  a  system  is  depleted,  however, 
subsequently-formed  portions  of  the  cell  wall  are  not  fluorescent, 
thus  providing  a  sharp  visible  differentiation  of  old  and  new  cell 
wall.  Similar  studies,  using  ferri tin-label  led  antibody  and 
visualization  under  the  electron  microscope,  are  planned. 

Ant i body- I abel I  ing  techniques  are  also  being  used  in  studies 
on  the  nature  of  type-specific  antibody.  Univalent  antibody  has 
been  produced  by  pepsin  and  cysteine  treatment,  and  is  being 
examined  by  a  variety  of  tests. 


k3 

44 


&d 


Serial  No.  NIAID   60 

1.  Infectious  Diseases 

2.  Medical  &  Physiological 

Bacteriology 

3.  Bethesda,  Maryland 


PHS-NLH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:       Studies  on  Cellular  and  Cell-Free 
Staphylococcal  Penicillinase 

Principal  Investigator:       Dr.  Arthur  K.  Saz 

Other  Investigators:  Nona 

Cooperating  Units:  None 

Han  Years  (calendar  year  i960) 
Total:  22/12 

Professional:         8/l2 
Other :  II4/12 

Project  Description: 

Objectives:  To  purify  and  study  penicillinase  from  penicillin- 
resistant  staphylococci.  To  investigate  the  possibility  of  strongly  in- 
hibiting cellular  penicillinase. 

Methods  Employed:     Isolation  and  purification  of  cell-free 
penicillinase  from  staphylococcal  cells  by  standard  biochemical  techniques. 
Measurement  of  penicillinase  activity  manometrically  and  iodome trie ally. 

Major  Findings^       In  continuation  of  one  aspect  of  this  problem 
carried  over  from  last  year,    the  search  for  inhibition  of  penicillinase 
activity  has  continued.     Various  compounds  prepared  by  condensing  amino 
acids  and  metal  binders  have  been  tested  for  anti-penicillinase  activity. 
Of  these,   trie  extremely  insoluble  compounds  D-dialanyl  benzidine  and  L- 
dialanyl  benzidine  show  marked  inhibitory  activity.     Due  to  the  very  low 
solubility     of  these  compounds,   an  accurate  inhibitory  concentration  has 
been  difficult  to  detemine.     However,    these  compounds  are,  on  a  molar  basis, 
by  far  the  most  active  of  all  inhibitors  thus  far  found.     Lesser  activities, 
but  still  potent,    have  been  noted  for  D-valyl  U-aminobiphenyl  and  D-phenyl- 
alanyl  L-aminofluorene.     Inhibitions  of  penicillinase  activity  up  to  60$ 
have  been  observed  with  L-threonyl-D-alanine,  L-allothreonyl-D-alanine  and 
D-alloisoleucyl-D-alanine. 

Efforts  at  purification  of  staphylococcal  penicillinase  have  con- 
tinued.    To  date,   the  most  active  preparations  exhibit  twel"  uri- 

Part  B  included:     Yes  -1-  ^ 


Serial  No.  NIAID  60 

fication.     Various  considerations  indicated  that  the  penicillinase 
as  it  occurs  intracellular  ly,  was  not  a  soluble  enzyme,  but  rather 
was  particulate.     This  has  proved  to  be  the  case.      Cell -free  extracts 
of  the  penicillin-resistant  staphylococci  prepared  by  sonic  or  Nossal  dis- 
integration were  subjected  to  ultracentrifugation  in  the   Spinco  Model  L 
preparative   centrifuge.     After  only  five  minutes  centrifugation  at 
lUit,000  x  g,  all  penicillinase  activity  appeared  in  a  small  pellet  with 
a  concomitant  h  to  6-fold  purification.     Electron  microscopic  observation 
of  the  pellet  showed  particulate  matter,   presumably  though  not  definitively 
associated  with  penicillinase  activity.     This  observation  conceivably 
could  explain  the  marked  stimulation  of  penicillinase  activity,   as  re- 
ported previously,   by  various  alcohols,   and  particularly  n-propanol  and 
n-butanol.     In  this  instance,   the  alcohol  activity  might  parallel  the 
stimulatory  effects  of  various  detergents  and  solvents  on  mitochondria 
derived  from  mammalian  tissue.     The  solvents  presumably  render  the  par- 
ticles more  permeable  to  substrate.     This  possibility  is  under  investi- 
gation. 

Significance  to  bio-medical   research  and  the  program  of  the 
Institute!       If  a  non-metabolizable  non-toxic  inhibitor  of  staphylo- 
coccal penicillinase  could  be  found  it  would  go  a  long  way  toward  solv- 
ing the  penicillin-resistant  staphylococcus  problem.     Any  information 
concerning  penicillinase  is  of  interest  to  this  Institute. 

Proposed  course  of  project;     1.     To  purify  penicillinase  fur- 
ther and  to  study  its  properties.     2.     To  continue  the  search  for  an  ef- 
fective inhibitor.     Animal  studies  will  be  used  as  indicated.     3.     To 
study  the  mechanism  of  the  alcohol  stimulation  of  penicillinase. 


-2- 


k5 


Serial  No.  NIAID  60 


Part  B  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Saz,  Arthur  K.  and  Martinez,  L.  Marina:  Enzymatic  Basis  of  Resistance 
to  Aureomycin.  III.  Inhibition  by  Aureomycin  of  Protein-Stimulated 
Electron  Transport  in  Escherichia  coli.  J.  Bact.,  79,  527-531>  I960* 


Honors  and  awards  relating  to  this  project: 

Chairman,  President's  Fellowship  Committee,  Socieiy  of  American 
Bacteriologists. 

Organizer  and  Convener  of  Panel  on  Mode  of  Action  of  Antibiotics, 
Conference  on  Anti-Microbial  Agents  -  Mayflower  Hotel,  Washington,  D.C. 
Oct.  26-28,  I960. 

Lecturer  (Professor)  in  Botany,  Howard  University,  Washington,  D.  C, 


"6 


Serial  No.  NIAID  61 


1.  Infectious  Diseases 

2.  Medical  &  Physiological 

Bacteriology 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  A, 

Project  Title:   Antibiotic  Activity  of  Sea  Water 

Principal  Investigator:   Arthur  K.  Saz 

Other  Investigators:     Dr.  Stanley  Watson 

Woods  Hole  Oceanographic  Institution 
Woods  Hole,  Massachusetts 

Cooperating  Units:       Woods  Hole  Oceanographic  Institution 

Man  Years  (calendar  year  I960) 
Total:         6/12 
Professional:   6/12 
Other: 

Project  Description: 

Objectives:  To  investigate  the  reported  antibiotic  activity  of 
sea  water.   To  isolate  from  sea  water,  compounds  active  against  fecal 
organisms  and  penicillin-resistant  staphylococci. 

Methods  Employed:  Treatment  of  sea  water  by  various  methods 
(extraction,  chromatography,  electrophoresis)  in  order  to  isolate  in- 
hibitory factors  present.  Standard  bacteriological  techniques. 

Major  Findings:  It  has  been  possible  to  confirm  a  weak  anti- 
coliform  activity  of  sea  water.  Perhaps  of  more  interest,  however,  is 
the  unequivocal  demonstration  for  the  first  time  of  an  antistaphylococcal 
activity  in  sea  water.  The  reputed  strong  anti-coliform  activity  of  sea 
water  is  in  large  part  due  simply  to  the  effect  of  increased  salinity 
in  a  sea  water  environment.  Most  previous  reports  of  this  activity 
failed  to  use  adequate  controls  as  evidenced  by  close  inspection  of  the 
data.  Indeed,  on  occasion,  Escherichia  coli,  inoculated  into  either 
filtered  or  raw  sea  water,  increased  in  numbers  during  the  observation 
period  of  72  hours.  On  other  occasions  and  with  different  specimens  of 
water,  a  weak  activity  (survival  of  $0%   after  72  hours)  was  evident. 

Part  B  included:  Yes 

-1.  »7 


Serial  No.  NIAID  61 


The  problem  of  adequate  controls  for  the  demonstration  of  activity 
was  investigated.  A  small  amount  of  organic  material,  proper  pH, 
temperature  of  20-25°  and  the  use  of  sea  water  in  diluting  inoculated 
samples  for  plate  count  proved  to  be  essential  for  demonstration  of  ac- 
tivity. Further  it  was  essential  to  run  a  control  in  each  experiment 
of  2.5a>  buffered  NaCl  (the  saline  concentration  of  sea  water)  to  eliminate 
the  possibility  of  kill  due  to  salinity. alone. 

Experiments  reported  hereafter  were  performed,  because  of  crowded 
conditions  at  the  Woods  Hole  Oceanographic  Institution,  with  a  strain  of 
penicillin-sensitive  S.  aureus .  However,  preliminary  experiments  per- 
formed at  N.I.H.  indicate  that  a  penicillin-resistant  S taphylococ cus 
(80/81)  is  killed  by  sea  water.  In  a  typical  experiment,  2.6  x  10^ 
staphylococci  per  ml  were  inoculated  with  raw  sea  water.  After  2k   hours, 
5.7  x  loVml  organisms  were  viable  and  in  I4.8  hours,  3.2  x  10^  organisms/ 
ml  survived.  There  were  only  10  viable  organisms/ml  after  72  hours.  In 
contrast,  the  saline  control  for  the  same  periods  showed  1.1  x  10  , 
0.3  x  10  ,  and  3.6  x  lO-3  survivors/ml.  Similar  results  were  obtained  us- 
ing filtered  sea  water.  By  contrast,  when  organisms  were  inoculated  into 
autoclaved  sea  water,  the  anti-staphylococcal  activity  was  completely 
lost.  In  a  preliminary  experiment  raw  sea  water,  diluted  1:8,  exhibited 
as  much  activity  as  whole  sea  water  over  a  2k   hour  period. 

It  was  also  observed  that  not  all  samples  of  sea  water  collected 
exhibited  activity.  To  date,  of  three  samples  studied  two  have  been 
active.  These  results  are  presently  being  written  up  for  publication. 

Significance  to  bio-medical  research  and  the  program  of  the 
Institute;    The  public  health  aspects  of  the  mechanism  of  the  kill- 
ing of  fecal  organisms  in  sea  water  are  obvious.  An  inhibitor  of  peni- 
cillin-resistant staphylococci  would  be  of  prime  importance. 

Proposed  course  of  project:  A  cooperative  arrangement  has  been 
worked  out  with  Woods  Hole  Oceanographic  Institution  for  continuation  of 
the  problem  both  here  at  the  NIH  and  it  is  hoped  at  the  Oceanographic 
Institution.  Isolation  of  the  substance(s)  will  be  attempted.  Since 
all  the  waters  studied  in  this  report  were  surface  (10 'r|)  it  would  be  of 
interest  to  determine  activity  of  deeper  continental  shelf  water  and 
deep,  open  ocean  water.  To  this  end,  it  is  hoped  a  stay  at  W.H.0.1. 
this  summer  can  be  arranged  so  that  use  of  the  Oceanographic  institu- 
tion research  vessels  can  be  utilized  for  this  purpose. 


48 

-2- 


Serial  No.  NIAID  6l 
Part  B    Honors,  Awards,  and  Publications 
Publications  other  than  abstracts  from  this  project: 
none 


Honors  and  awards  relating  to  this  project: 


Appointed  guest  worker  at  the  Oceanographic  Institution, 
Woods  Hole,  Massachusetts. 


-3- 


_      .    ,    >T       lTTATT,     NIAID-62 
Serxal  No.  NIAID 


1.  Infectious  Diseases 

2.  Medical  &  Physiological 

Bacteriology 

3.  Bethesda,  Maryland 


PHS-NIH 


Individual  Project  Report 
Calendar  Year  I960 


Part  A 


Project  Title:    The  electron  transport  system  in  an 
autotrophic  hydro genomad. 

Principal  Investigator:  Dr.  Roy  Repaske 

Other  Investigators:     None 

Cooperating  Units:      None 

Man  Years  (calendar  year  I960) 
Total:  2U/12 

Professional:     12/12 
Other:  12/12 

Project  Description: 

Objectives:  This  research  has  as  its  object  the  identifica- 
tion of  individual  enzymes  and  intermediary  cofactors  which  are  opera- 
tive in  electron  transport.  This  information  may  be  transposable  in 
part  to  mitochondrial  systems  and  provide  important  clues  about  the 
electron  transport  sequence  in  these  particulate  structures.  The  long 
range  plan  will  include  an  investigation  of  the  energy  yielding  reac- 
tions coupled  to  electron  transport. 

Methods  Employed:  The  methods  employed  will  be  those  neces- 
sary for  mass  cultivation  of  organisms  and  for  isolation  and  purifica- 
tion of  the  electron  transport  enzymes.  Growth  of  large  quantities  of 
Hydro genomonas  eutropha  is  technically  difficult  because  of  the  require- 
ment for  large  volumes  of  an  artificial  gas  mixture.  Apparatus  has  been 
designed  and  is  being  built  for  this  purpose.  The  assay  of  the  initial 
enzymatic  reactions  in  cell  free  preparations  also  requires  a  modified 
procedure  because  the  enzymic  activity  must  be  measured  in  a  hydrogen 
atmosphere  in  a  spectrophotometer.  Protein  fractionation  will  be 
achieved  by  procedures  involving  ammonium  sulfate  precipitation,  gel 
adsorption  and  elution,  solvent  precipitation,  and  chromatography. 

Major  Findings:  It  was  readily  apparent  from  the  atypical 
kinetics  and  other  indirect  evidence  that  the  enzymatic  reaction  beingr 

Part  B  included.  Yes  -1- 


Serial  No.  NIAID"62 


studied,  he  oxidation  of  hydrogen  by  diphosphopyridine  nucleotide  (DPN), 
involved  either  a  second  enzyme  or  a  cofactor.  The  cofactor  which  was 
first  found  in  a  boiled  cell  extract  has  been  identified  as  riboflavin 
phosphate  (FMN),  but  its  requirement  could  be  shown  only  when  the  enzyme 
was  preincubated  under  hydrogen  and  reducing  agent  was  added  to  the  assay 
system. 

Enzyme  stability  has  been  controlled  by  storing  and  handling  the 
enzyme  under  an  atmosphere  of  hydrogen  and  in  the  presence  of  cationic 
buffers  of  relatively  high  concentration  (0.05>  to  0.2  M) .  The  pH  opti- 
mum for  stability  is  8.0.  A  sharp  pH  optimum  for  enzyme  activity  was 
found  at  pH  7.5. 

The  enzyme  catalyzing  the  initial  reaction,  i.e.,  DPN  reduction, 
has  been  purified  only  three  fold  by  calcium  phosphate  gel  adsorption 
and  ammonium  sulfate  fractionation.  Unaccountable  losses  of  enzyme 
activity  during  these  procedures  are  at  the  moment  unexplained,  but 
may  reflect  inherently  important  characteristics  of  the  complex.  Con- 
sequently the  reason  for  this  loss  of  activity  is  under  intensive  in- 
vestigation. 

The  most  purified  enzyme  preparation  has  a  very  high  affinity  for 
riboflavin  phosphate  but  it  has  not  been  possible  to  demonstrate  FMN 
reduction  of  added  exogenous  FMN,  indicating  that  the  FMN  is  tightly 
bound  by  the  enzyme  and/is  not  in  equilibrium  with  the  exogenous  FMN 
pool. 

Significance  to  bio-medical  research  and  the  program  of  the  In- 
stitute;  Hydrogenomonas  species  are  unique  organisms  which  obtain 
energy  for  all  of  their  life  processes  through  oxidation  of  hydrogen 
gas  in  the  presence  of  oxygen.  This  is  in  contrast  to  animal  cells 
and  most  bacteria  which  oxidize  organic  substances  for  energy.  Although 
the  initial  substrates  are  different,  there  is  every  reason  to  believe 
that  both  types  of  systems  have  much  in  common.  Both  serve  the  identi- 
cal function  of  providing  energy  for  synthesis,  growth  and  repair,  and 
both  utilize  cytochromes  and  diphosphopyridine  nucleotide  to  mediate 
the  transfer  of  electrons  to  oxygen.  Biochemical  studies  in  the  past 
have  revealed  that  a  common  denominator  exists  between  biochemical  pro- 
cesses of  various  organisms,  and  this  well  supported  concept  of  com- 
parative biochemistry  is  the  rational  basis  for  investigating  a  given 
reaction  in  an  organism  most  amenable  to  study. 

Previous  research  on  this  organism  was  carried  out  at  another 
institution  by  the  principal  investigator.  The  potential  advantage 
of  studying  these  reactions  in  H.  eutropha  lies  in  the  fact  that  the 
system  in  this  organism  is  soluble  and  therefore  it  can  be  fraction- 
ated by  classical  protein  fractionation  procedures. 


-2- 


51 


Serial  No.  NIAID 


-62 


The  electron  transport  system  in  all  other  organisms  studied  to 
date  has  been  associated  with  particulate  elements  of  the  cell.  Ef- 
forts to  dissociate  some  of  these  enzymes  from  the  particles  have 
met  with  limited  success  while  other  enzymes  cannot  be  solubilized. 
Concomitantly  with  the  occurrence  of  electron  transport,  ATP  is  generated; 
of  the  two  processes,  ATP  generating  system  is  the  most  labile  during 
manipulation.  As  a  result  of  these  characteristics,  our  knowledge  of 
electron  transport  and  simultaneous  ATP  formation  is  sketchy  and  indi- 
rect. It  is  felt  that  the  limitations  associated  with  other  systems 
may  not  exist  in  the  H.  eutropha  system. 

Proposed  course  of  Project;  More  extensive  purification  of  the 
DPN  reducing  system  is  planned  to  eliminate  interfering  enzymes  and 
compounds  and  to  demonstrate  with  reasonable  assurance  that  only  one 
enzyme  participates  in  this  reaction.  The  remaining  reactions  between 
reduced  DPN  and  oxygen  will  be  segregated  so  that  the  characteristics 
and  requirements  of  each  individual  reaction  in  the  sequence  will  be 
known.  The  system  reconstructed  with  purified  enzymes  should  have  the 
same  characteristics  as  those  found  in  the  crude  extract.  During  these 
studies,  the  occurrence  of  ATP  formation  during  electron  transport  will 
be  explored  as  a  preliminary  survey  to  an  investigation  of  this  system. 


52 

-3- 


NIAID  -  62 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  B.  Honors, Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Repaske,  R.  and  Seward,  C.:  FHN  as  a  cofactor  in  the  enzymatic  reduction 
of  DPN  by  hydrogen.  Biochem.  Biophys.  Res.  Coram.,  2_,  397-UOl,  I960, 

Litwack,  G.,  Repaske,  R.  and  Myrvik,  Q.:  Lysozyme  as  related  to  problems 
in  Microbiology.   Purdue  University,  I960,  39  p. 

Honors  and  awards  relating  to  this  project: 

Gave  the  following  invited  seminars: 

"Enzymatic  Reduction  of  DPN  by  Hydrogen"  -  Gerontology  Branch,  NHI- 
NIH,  at  City  Hospital,  Baltimore,  Maryland  -  Mar.  23,  I960. 

"Enzymatic  Oxidation  of  Hydrogen  Gas  by  Extracts  of  an  Autotrophic 
Bacterium"  -  Dept.  of  Bacteriology,  Pennsylvania  State  University,  State 
College,  Penn.  -  Oct.  13,  I960. 

"Enzymatic  Reduction  of  DPN  by  Hydrogen  with  Extracts  of  Hydrogeno- 
monas"  -  Dept.  of  Bacteriology,  Indiana  State  University,  Bloomington, 
Indiana,  Nov.  16,  I960. 

Panel  Member  in  a  Symposium  on  Lysozyme  presented  at  Society  of 
American  Bacteriologists  national  meetings  in  Philadelphia,  May  I960. 

Elected  to  membership,  American  Society  of  Biological  Chemists. 

Nominated  as  candidpte  for  Secretary  of  the  General  Division  of  the  So- 
ciety of  American  Bacteriologists. 

Invited  to  participate  in  the  2nd  International  Symposium  on  Fleming? s 
Lysozyme  in  Milan,  Italy  -  April  7-9,  1961. 


53 


_  .  .  ..    NIAID-62 
Serial  No. 


PHS-NIH 

Individual  Project  Report 

Calendar  Tear  I960 


Part  B    Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Repaske,  R.  and  Seward,  C:  FMN  as  a  cofactor  in  the  enzymatic  reduction 
of  DPN  by  hydrogen.  Biochem.  Biophys.  Res.  Comm.,  2,  397-1*01,  I960. 

Litwack,  G.,  Repaske,  R.  and  Myrvik,  Q.:  Lysozyme  as  related  to  problems 
in  Microbiology.  Purdue  University,  I960,  39  p. 


Honors  and  awards  relating  to  this  project: 
Gave  the  following  invited  seminars: 

"Enzymatic  Reduction  of  DPN  by  Hydrogen"  -  Gerontology  Branch,  NHT-NLH, 
at  City  Hospital,  Baltimore,  Maryland  -  Mar.  23,  I960 

"Enzymatic  Oxidation  of  Hydrogen  Gas  by  Extracts  of  an  Autotrophic  Bac- 
terium" -  Dept.  of  Bacteriology,  Pennsylvania  State  University,  State 
College,  Perm.  -  Oct.  13,  I960 

"Enzymatic  Reduction  of  DPN  by  Hydrogen  with  Extracts  of  Hydrogenomonas"  - 
Dept.  of  Bacteriology,  Indiana  State  University,  Bloomington,  Indiana, 
Nov.  16,  I960. 

Panel  Member  in  ^Symposium  on  Lysozyme  presented  at  Soc.  of  American 
Bac^riSlblists1  fiatio'Ka!  fie e t-^0^ Pniladelphia,  May  I960. 

Elected  to  membership  -  American  Society  of  Biological  Chemists. 

Nominated  as  candidate  for  Secretary  of  the  General  Division  of  the  So- 
ciety of  American  Bacteriologists. 

Invited  to  participate  in  the  2nd  International  Symposium  on  Fleming's 
Lysozyme  in  Milan,  Italy  -  Apr.  7-9,  1961. 


-u-  Sij 


Serial  No.  NIAID  -  62-A 

1.  Infectious  Diseases 

2.  Medical  Sc  Physiological 

Bacteriology 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:   Microbiological  Fractionation  of  the  Hydrogen 
Isotopes 

Principal  Investigator:  Dr.  F.  D.  Sisler 

Other  Investigators:     Dr.  Benjamin  Prescott 

Cooperating  Units:      U.  S.  Geological  Survey 

Man  Years  (calendar  year  I960) 
Total:         17/12 
Professional:    9/12 
Other:         8/12 

Project  Description: 

Objectives:  To  determine  the  mechanism  of  hydrogen  isotope 
fractionation  by  microorganisms j  the  divergent  metabolic  pathways 
of  protium,  deuterium  and  tritium;  the  effect  of  the  heavy  hydrogen 
isotopes  on  the  physiology  of  the  simple  biological  systems;  the  eco- 
logical and  evolutionary  significance  of  the  isotope  effect  in  the 
biosphere. 

Methods  Employed:  Pseudomonas  GI4A  and  other  bacterial  cells 
and  cell  products,  from  culture  media  containing  normal  and  enriched 
amounts  of  hydrogen  isotopes  (protium,  deuterium,  tritium)previously 
incubated  under  a  variety  of  conditions  simulating  various  environ- 
mental variables  will  be  analyzed  using  methods  involving  the  mass 
spectrometer,  radioactive  counting  equipment  designed  for  tritium  com- 
pounds, paper  chromatography,  infra  red  and  conventional  biochemical 
analytical  procedures. 

Major  Findings:  The  three  isotopes  of  hydrogen,  viz.  pro- 
tium, deuterium  and  tritium  are  selectively  fractionated  by  micro- 
organisms during  metabolic  processes  involving  hydrogen  compounds. 
Samples  of  gas  from  marine  sediments  and  from  bacterial  fermenta- 
tion analyzed  by  mass  spectrometer  reveal  a  protium  concentration 
in  excess  over  that  expected  from  non-biological  processes.  It 
would  appear  that  in  the  marine  environment  various  microorganisms 

Part  B  not  included.  j*  55 


Serial  No.  HIAID  -  62-A 

particularly  those  containing  hydro genlyase  and  hydrogenase  enzymes 
play  an  important  role  in  hydrogen  isotope  equilibria.  Mechanisms 
by  which  microorganisms  may  fractionate  the  hydrogen  isotopes  involve 
enzyme  specificity,  bond  cleavage  rates,  cell  wall  diffusion  and  the 
formation  of  large  insoluble  molecules,  e.g.  protein-bound  polysac- 
charides. The  latter  process  resembles  a  sequestration  effect  in  that 
the  heavy  isotope  atoms  are  prevented  from  equilibrating  with  the  medium 
so  long  as  the  cell  wall  is  intact.  After  cell  death  and  disintegration, 
predicted  chemical  equilibria  is  reestablished.  Laboratory  studies  irvolv- 
ing  microbial  activity  on  tritium  compounds  reveal  a  multi-staged  process 
by  which  protium  and  tritium  are  fractionated.  It  is  concluded  that 
marine  microorganisms  are  important  geochemical  agents  in  dynamic  processes 
involving  hydrogen  and  other  isotopes  in  the  sea.  The  evidence  accumulated 
to  date  suggests  that  microbiological  fractionation  of  the  hydrogen  iso- 
tope is  a  ubiquitous  phenomenon  throughout  the  biosphere. 

Significance  to  bio-medical  research  and  the  program  of  the 
Ins titute :  A  knowledge  of  hydrogen  metabolism  of  biological  systems  can 
be  considered  as  one  of  the  most  fundamental  of  metabolic  processes.  Hy- 
drogen metabolism  (hydrogen  transfer,  electron  transfer)  is  synonymous 
with  the  energetics  of  living  processes. 

With  increasing  use  of  both  natural  and  radioactive  isotopes  in 
medical  research,  it  is  considered  of  utmost  importance  that  attention 
be  focused  on  fundamental  studies  which  may  contribute  to  the  understanding 
of  the  isotope  effects  in  biological  systems.  This  study  of  the  hydrogen 
isotopes  in  simple  cell  metabolism  should,  therefore,  contribute  basic  in- 
formation of  value  towards  the  solution  of  problems  of  public  health  and 
economic  importance  such  as  implications  in  radioactive  waste  disposal  in 
marine  environments,  toxic  effects  of  deuterium  and  other  isotopes  and 
further  is  of  fundamental  importance  in  studying  new  metabolic  pathways 
in  microbial  and  other  systems. 

Proposed  course  of  project:  To  continue  current  research  with 
the  following  objectives  in  view: 

1.  Isolation  and  identification  of  intracellular  compounds 
primarily  responsible  for  deuterium  and  tritium  fractionation, 

2.  Examination  of  extracellular  products  showing  heavy  isotope 
enrichment. 

3.  Evaluation  of  the  chemical  thermodynamics  involved  in  isotope 
fractionation,  comparing  experimentally  established  values  with  the 
theoretical. 

U.  Further  studies  on  transport  of  isotopes  across  cell  membranes? 
rate  processes  and  steady  state  equilibria  of  all  phases  involved  in 
isotope  fractionation. 

-2- 


Serial  No.  NIAID  63 

1.  Infectious  Diseases 

2.  Medical  &  Physiological 

Bacteriology 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:  Non-specific  Immunity  Associated  with  Serum 
Siderophilin  and  the  Iron  Metabolism  of 
both  Pathogen  and  Host. 

Principal  Investigator:   Dr.  Arthur  L.  Schade 

Other  Investigators:     Dr.  Lathrop  E.  Roberts 

Cooperating  Units:       None 

Man  Years  (calendar  year  1°60) 
Total:  30/12 

Professional:     18/12 
Other:  12/12 

Project  Description 

Objectives:  To  investigate  the  contribution  of  blood  serum 
siderophilin  tc  the  natural  resistance  of  the  host;  to  investigate  the 
iron  requirements  of  selected  pathogens  and  the  effect  of  the  iron- 
chelating  siderophilin  and  conalbumin  on  in  vivo  iron  accumulation, 
growth,  and  metabolism  of  these  pathogens;  to  investigate  the  factors 
leading  to  changes  in  concentration  of  both  serum  iron  and  siderophilin 
in  circulating  blood  as  a  result  of  infection  and  other  abnormal,  condi- 
tions, and  the  consequence  of  such  changes  on  resistance  to  infection, 
iron  absorption  by  the  host,  and  anemia. 

Methods  Employed:  Bacterial  and  cultural  procedures  in  spe- 
cially deironized  raedia;  spectrophotometric,  chromatographic,  and 
radioisotopic  methods;  metabolic  and  enzymatic  analytical  procedures; 
and  methods  particularly  suited  to  estimation  of  iron  chelated 
either  to  siderophilin  or  conalhumin  and  to  their  relative  unsatura- 
tions. 

Part  B  included:  Yes 


57 


Serial  No.  NIAID  63 

Major  Findings:  Previous  findings  have  established  that  some 
bacteria  vail  not  grow  in  serum  unless  the  contained  siderophilin  is 
saturated  with  iron,  while  the  growth  rate  of  many  others  in  serum  is 
a  function  of  the  percentage  iron-saturation  of  the  siderophilin.  We 
have,  for  our  more  intensive  research  investigations,  restricted  our- 
selves to  Staphylococcus  albus,  whose  growth  in  normal  serum  is  negli- 
gible in  the  absence  of  excess  iron,  and  to  Staphylococcus  aureus, 
whose  growth  rate  in  serum  reflects  the  amount  of  bound  serum  iron  com- 
pared with  its  total  iron-binding  capacity. 

A-  Proceeding  from  our  preliminary  studies  on  the  metabolism  of  Staph . 
aureus  cells  grown  in  medium  non-restrictive  with  respect  to  iron  and 
those  grown  in  a  medium  whose  concentration  of  free  ionic  iron  was  se- 
verely limited  by  the  presence  of  either  siderophilin  or  conalbumin  at 
low  iron  saturation  ($%)   we  have  observed  the  following  facts: 

1.  The  rates  of  oxygen  consumption  of  both  high  and  low  iron 
cells  in  the  presence  of  glucose  are  essentially  the  same  but  the 
aerobic  and  anaerobic  glycolytic  rates  of  the  low  iron  cells  are  signifi- 
cantly increased  over  those  of  the  high  iron  cells.  Differences  of 
rates  up  to  three-fold  have  been  observed.  High  iron  cells  oxidize 
pyruvate  at  a  rate  similar  to  that  of  glucose,  while  lactate  is  oxi- 
dized at  approximately  twice  the  rate  as  that  obtained  with  pyruvate. 
Acetate  and  succinate  are  oxidized  at  rates  hP%   and  \2%   that  found  with 
pyruvate  as  substrate.  Low  iron  cells,  on  the  other  hand,  despite  the 
similarity  to  high  iron  cells  in  their  attack  on  glucose,  fail  to  oxi- 
dize pyruvate  but  continue  to  oxidize  lactate,  albeit  at  a  rate  1/3  to 
l/U  that  of  the  high  iron  cells.  Low  iron  cells  fail  also  to  attack 
either  acetate  or  succinate, 

2.  The  extent  of  oxidation  of  several  substrates  by  Staph,  aureus 
cells  cultured  in  media  of  "high"  and  "low"  iron  content  was  determined: 

Molecules  O2  Taken  up  per  Molecule  of  Substrate 

Theoretical  Value  for  Observed  Values* 

Substrate     Complete  Oxidation     "High"  Fe  Cells  "Low"  Fe  Cells 

Glucose  6  3  1 

Formate  0.5  0.5  0 

Acetate  2  2  0 

Pyruvate  2.5  1.5  0 

Lactate  3  1.5  0.5 


■>  Endogenous  oxygen  uptake  subtracted. 

3.  The  rate  of  anaerobic  dismutation  of  2  moles  of  pyruvate  to 
one  mole  each  of  lactic  acid,  acetic  acid,  and  CO2  has  been  studied 


-2- 


58 


Serial  No.  HIAID  63 

manometrically  and  by  chemical  analysis.  High  iron  cells  carry  out 
this  reaction  about  3§  times  as  rapidly  as  low  iron  cells. 

It  is  clear  from  these  metabolic  observations  that  the  carbohy- 
drate metabolism  of  Staph,  aureus  is  profoundly  affected  by  the  avail- 
ability to  them  of  iron  during  their  growth  period.  The  results 
likewise  indicate  that  iron,  either  directly  or  indirectly,  participates 
in  enzymatic  activities  not  generally  appreciated  as  being  mediated  by 
iron  enzjTTies,  e.g.  the  anaerobic  dismutation  of  pyruvate.  We  recall 
that  under  physiological  conditions,  sera  are  often  found  in  which  the 
availability  of  iron  to  this  and  other  pathogens  is  either  greatly 
restricted  or  relatively  unrestricted  depending  upon  the  percentage 
saturation  of  the  siderophilin  of  the  sera.  Many  of  these  results 
present  new  challenges  to  our  understanding  of  the  metabolism  of  Staph. 
aureus  and  require  further  investigation  to  elucidate  the  physiological 
response  of  this  pathogen  to  the  environmental  changes  that  naturally 
occur  in  serum  in  vivo, 

B.  In  an  effort  to  prepare  an  iron -low,  satisfactory  growth  medium  for 
Staph,  aureus,  we  devised  a  simple  procedure  for  rapid  and  efficient 
iron  removal  from  the  complex  culture  medium,  trypticase.  The  procedure, 
to  be  published,  makes  use  of  the  difficultly  water  soluble  bathophenan- 
throline  as  iron  coraplexer  followed  by  extraction  of  the  remaining  traces 
of  bathophenanthrollne  in  the  medium  with  organic,  immiscible  solvents. 
The  method  reduces  the  labor  of  preparing  an  iron-low  medium  from  a  tedi- 
ous, uncertain,  time-consuming  chore  to  a  3  to  h   hour  treatment  to  yield 
large  quantities  of  final  medium  ■whose  iron  content  is  below  the  sensi- 
tivity of  the  most  sensitive  colorimetric  test  available.  Use  of  iso- 
topic  iron  Fe£?  indicates  that  the  iron  concentration  is  below  0.001  u-g 
per  ml  of  final  medium. 

C.  In  association  with  Dr.  Knight  of  the  Laboratory  of  Clinical  Inves- 
tigations we  participated  in  an  extended  study  of  a  case  of  Lupus  Nephrosis 
which,  upon  examination,  disclosed  an  unusually  low  concentration  of 
siderophilin  in  the  serum.  The  serum  iron  was  extremely  low  because  of 
the  severely  reduced  siderophilin  concentration  and  the  low  percentage 
iron-saturation  of  that  present.  We  suggested  the  possibility  of  sider- 
ophilin loss  in  the  urine  and  proceeded  to  analyze  uring  samples  for  this 
protein.  At  the  height  of  the  proteinuria,  we  found  approximately  10$ 

of  the  total  protein  lost  in  a  21;  hour  period  (ca.  20  grams)  to  be  sider- 
ophilin. Iron  was  also  being  lost  through  the  kidney  along  with  the  sider- 
ophilin. Calculation  showed  that  the  patient  was  losing  an  amount  of  her 
iron-binding  protein  in  2U  hours  which  was  equivalent  to  that  in  the  circu- 
lation at  any  given  moment.  The  details  of  this  investigation  and  the  ac- 
companying clinical  data  were  presented  by  Dr.  David  Rifkind,  et  al,  at 
the  recent  (Nov.  $,  i960)  Regional  Meeting  of  the  American  College  of 
Physicians. 


53 


Serial  No.  NIAU)  63 

D.  Our  method  employing  Staph,  aureus  for  the  evaluation  of  the  integri- 
ty of  isolated  siderophilins  has  been  improved  by  the  use  of  the  ultra- 
filtrate  of  normal  human  serum  plus  0.1$  iron-free  trypticase  as  bacterial 
growth  medium.  This  medium  better  approximates  whole  serum  employed  as  a 
control  than  previously  used  media.  Three  new  isolated  siderophilin 
preparations  have  now  been  found  by  this  method  to  be  equivalent  in  their 
iron-donation  capacities  to  the  native  siderophilin  present  in  serum. 
There  is  now  considerable  hematological  interest  in  the  possibility  t 
human  tissues  other  than  bone  marrow  and  reticulocytes  may  obtain  their 
iron  from  the  siderophilin-iron  complex  by  the  mechanism  for  which  Staph. 
aureus  is  the  model. 

Significance  to  the  bio-medical  research  and  the  program  of  the 
Institute ;   The  study  of  the  naturally  occurring  iron-chelating  serum 
protein,  siderophilin,  contributes  to  our  understanding  of  the  non- 
specific immunity  mechanism  available  to  humans  for  protection  against 
microbial  infection.   Investigation  of  the  qualitative  and  quantitative 
effects  of  siderophilin  on  the  growth  and  metabolism  of  the  pathogen 
Staphylococcus  aureus  will  provide  useful  information  on  the  pathogenicity 
of  this  bacterium  especially  under  conditions  closely  approximating  in 
vivo  conditions. 

Proposed  Course  of  Project:  We  shall  investigate  in  detail  the 
growth,  metabolism,  and  enzymatic  capabilities  of  S.  aureus  when  grown 
under  conditions  approximating  the  in  vivo  state  with  especial  regard 
to  the  effect  of  different  serum  iron-saturation  levels  on  such  charac- 
teristics.  Special  emphasis  will  also  be  placed  upon  the  biochemical 
mechanisms  by  which  iron  controls  the  overall  metabolism  of  ihe   orga- 
nism. In  these  studies,  changes  in  the  enzymatic  function  and  the  lo- 
calization of  iron  containing  components  within  various  cellular  struc- 
tures will  be  investigated.  Additional  biological  features  such  as 
hemolysis,  effectiveness  of  phagocytosis,  and  possible  differences  in 
response  to  antibiotics  of  high  and  low  iron -containing  cells  will  be 
studied. 


60 


SERIAL  No.  NIAID  63 

Part  B.  Honors  Awards,  and  Publications 

Publications  other  than  abstracts  frora  this  project: 

Schade,  Arthur  L. :  The  microbiological  activity  of  siderophilin. 
Clinica  Chimica  Acta,  in  press. 

Schade,  Arthur  L.:  Methods  applicable  to  the  study  of  siderophilin. 
Behringwerk  Mitteilungen,  in  press. 

Honors  and  awards  relating  to  this  project: 

Lectures: 

By  invitation,  visited  the  laboratories  and  gave  lectures  ap- 
propriate to  the  interests  of  the  audience  on  different  aspects  of, 
"Iron  metabolism  of  bacterial  pathogens  and  host  tissues  as  mediated 
by  siderophilin",  at  the  following  institutions: 

1.  Univ.  of  Vienna;  Dept.  of  Physiology,  Prof.  Auerswald  -  Oct.  21  &  2lw 

2.  Universities  of  Marburg  and  Giessen;  Medical  School,  Prof.  Schultze. 
Nov.  3  and  U. 

3.  Univ.  of  the  Saar;  Medical  School,  Prof.  Ruranel  -  Nov.  h   and  £. 

U.  Univ.  of  Freiburg;  Medical  School,  Prof.  Heilmeyer.  Nov.  7  and  8. 

$.  Univ.  of  Berne;  Dept.  of  Organic  Chemistry,  Prof.  Nitschmann.  Nov.9  &  10, 

6.  Univ.  of  Zurich;  Medical  School,  Dr.  Hitzig.  Nov.  11  and  12. 

7.  Univ.  of  Tubingen;  Medical  School,  Prof.  Bennhold.  Nov.  lU  and  l£. 

8.  Univ.  of  Frankfurt;  Dept.  of  Pharmacology,  Prof.  Heinz.  Nov.  17  • 

9.  Berliner  Mikrobiologische  Gesellschaft  Meeting;  Prof.  Horing  oflftie 
Free  University  of  Berlin.  Nov.  22. 

10.  Univ.  of  Lund;  Dept.  of  Clinical  Chemistry  (Malmfl),  Prof.  Laurell. 
Nov.  28  and  29. 


61 


Serial  No.  NIAID  63-A 


1.  Infectious  Diseases 

PHS-NIH  2»  Jfedical  &  Physiological 

Individual  Project  Report   ,  _..   .   Bacteriology 

Calendar  Year  I960      3'  Bethesd^  Haiyland 


Part  A. 


Project  Title:     Chemical  Aspects  of  the  Specific  Binding  of 
Iron  to   Serum  Siderophilin  and  Egg  White 
Conalbumin. 

Principal  Investigator:       Dr.  Robert  C.  Woodworth 

Other  Investigators:  Dr.  Arthur  L.   Schade 

Cooperating  Units:  None 

Man  Years   (calendar  year  I960) 
Total:  12/12 

Professional: 
Other:  5/l2 

Project  Description: 

Objectives :     The  goals  of  this  project  are  to  investigate  the 
chemistry  of  the  reactive  groups  in  the  siderophilin  molecule  responsi- 
ble for  the  specific  binding  of  iron  and  to  determine  the  physico-chemical 
means  by  which  the  iron -siderophilin  complex  can  be  dissociated  under 
physiologic  conditions  obtaining  in  pathologic  and  normal  hosts. 

Methods  Employed:     Methods  include  the   employment  of  immunological 
assays  for  purity  and  for  content  of  the  iron-binding  proteins,  radioac- 
tive tracers,   spectral  analyses,    ion-exchange  chromatography,   electropho- 
resis, polarography,   and  polarimetry. 

Major  Findings:     The  previously  reported  technique  of  determining 
the  iron-release  rate  of  the  iron-sidercphilin  complex  by  me£p^lggeJJcor&9 
isotope  exchange  has  been  applied  to  a  detailed  study  of  various/ affect- 
ing the  dissociation  of  iron  from  siderophilin. 

Reaction:   siderophilin-Fe2^     +  2Fe^9  >  siderophilin-Fe2^  +  2Fe^  . 

Assay:  antibody  +  siderophilin-Fe2^9  +  56)..— .>  antibody-siderophi- 
lin-Fe2^  +  ^  '|  .     Coun  Lpitate  for  Fe™  activity. 


Part  B  included.       Yes 


62 


Serial  No.   NIAID  63-A 


After  requisite  mathematical  manipulation,   the  data 
were  plotted  and  consistently  showed  two  intersecting  straight 
lines,  rather  than  a  single  straight  line.     The  occurrence  of 
the  two  intersecting  lines  may  be  interpreted  to  mean  that  the 
two  irons  bound  to  a  siderophilin  molecule  are  attached  to  two 
distinct  sites  whose  properties  differ  in  such  a  way  that  a 
specific  site  must  release  its  iron  first  in  order  that  the 
other  site  may  release  its  iron.     A  mathematical  analysis  of 
the  system  under  study  has  been  generously  provided  by  Dr. 
Clifford  S.  Patlak,   Biometrics  Branch,  NIMH.     By  setting  up 
and  solving  the  applicable  differential  equations,    Dr.   Patlak 
has  provided  us  with  a  method  for  obtaining  from  our  data  the 
different  dissociation  rates  of  the  two  bound  iron  atoms. 

The  effect  of  pH  on  these  dissociation  rates  has  been  of 
considerable  interest  to  us.     Studies  of  the  two  iron-release 
rates  were  made  in  whole  serum  at  various  pH's  which  were 
established  by  varying  the  percentage  composition  of  the  carbon 
dioxide-nitrogen  atmosphere  above  the  serum.     It  appeared  that 
the  first  dissociation  rate  was  dependent  on  the  square  of  the 
hydrogen  ion  concentration,   i£.,   if  the  hydrogen  concentration 
were  doubled,  the  rate  quadrupled;   and  that  the  second  dissoci- 
ation rate  was  independent  of  pH.     A  study  of  the  effect  of 
buffer  concentration  at  constant  pH  revealed,   however,    that  the 
first  dissociation  rate  was  dependent  on  the  first  power  of  the 
concentration  of  the  carbon  dioxide-bicarbonate  buffer  system. 
Since  this  buffer  system  had  been  used  for  the  pH  studies,  it 
became  apparent  that  the  rate  of  dissociation  of  the  first  iron 
was  actually  dependent  on  the  first  power  of  the  hydrogen  ion 
concentration  and  on  the  first  power  of  the  carbon  dioxide-bi- 
carbonate buffer. 

In  an  attempt  to  determine  whether  the  iron  release  rate  is 
subject  to  "general  acid  catalysis"  we  examined  the  concentration 
effects  of  two  other  buffer  systems,  namely  tris-(hydroxyraethyl) 
amino  methane  and  glycylglycinate,  both  in  the  region  of  pH  7* 
Not  only  were  the  isotope  exchange  rates  considerably  slower  in 
these  buffers  than  in  the  carbon  dioxide-bicarbonate   system,  but 
they  were  not  appreciably  affected  by  large  shifts  in  buffer  con- 
centration.    It  would  thus  appear  that  hydrogen  ion  and  carbon 
dioxide-bicarbonate  buffer  are  specific  acid  catalysts  for  the 
dissocation  of  iron  from  iron-siderophilin. 

Associated  with  each  iron  in  the  iron-siderophilin  complex  is  a 
bicarbonate  ion.     We  found  we  could  tag  this  bicarbonate  with  C11*  by  sup- 
plying C-L'^02  as  the  sole  source  of  bicarbonate  when  forming  the  complex 
from  iron   (ferrous)   and  iron-unsaturated  siderophilin.     The  C^  tag  re- 
mained with  the  siderophilin  during  precipitin  and  washing  procedures. 
A   "double-tagging"  experiment  in  which  we  followed  both  the  rate  of 


63 


Serial  No.  NIAID  63-A 

dissociation  of  HCr-^C^"  and  the  rate  of  incorporation  of  Fe'*  showed  that 
the  bicarbonate  may  be  released  from  the  complex  10  to  100  times  faster 
than  the  ir.n  released.  However,  this  experiment  should  be  repeated  tinder 
conditions  more  favorable  to  accurate  determination  of  the  desired  rates. 

The  effect  of  temperature  on  both  iron  dissociation  rates  was 
studied.  Both  rates  appeared  to  decrease  with  decreasing  temperature,  but 
no  quantitative  treatment  of  the  data  has  been  carried  out. 

Significance  to  bio-medical  research  and  the  program  of  the 
Institute:   The  shifts  in  bound  iron  levels  occurring  as  a  result  of 
infectious  diseases,  acute  infections,  and  allergic  reactions  are  recog- 
nized. The  availability  of  plasma  iron  to  bacterial  pathogens  is  a  func- 
tion of  the  siderophilin  molecule's  affinity  for  iron  and  of  its  degree 
of  iron-saturation.  Knowledge  of  the  chemical  bases  of  this  affinity 
should  elucidate  the  manner  in  which  siderophilin  governs  the  transfer  of 
iron  to  the  host  as  well  as  to  the  pathogen. 

Proposed  course  of  project;  Since  Dr.  Woodworth  is  presently 
on  an  N.I.H.  Research  Fellowship  with  Prof.  C.-E.  Laurell  in  Malratt, 
Sweden  and  since  he  may  choose  to  seek  a  University  professorship  rather 
than  apply  for  reassociation  with  N.I.H.  it  appears  that  the  course  of 
this  project  will  be:  first,  to  prepare  in  comprehensive  form  for  pub- 
lication the  accumulated  data  thus  far  obtained,  and,  second  to  pursue, 
as  opportunity  permits  in  his  absence,  the  problem  of  the  rates  of  as- 
sociation of  iron  with  siderophilin  in  serum,  particularly  under  physio- 
logical conditions,  so  that  from  the  rates  of  dissociation  and  association, 
the  concentration  of  free  ionic  iron  in  serum  can  be  approximated. 


-3- 


G*{ 


Serial  No.  NIAID  63-A 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  B.   Honors,  Awards,  and  Publications 
Publications  other  than  abstracts  from  this  project: 

Woodworth,  Robert  C.  and  Schade,  Arthur  L#:  Immunological  Precipitin 
Titrations  based  on  Radioactive  Tagginf  of  the  Iron  Naturally 
Chelated  by  the  Proteins  Siderophilin  and  Conalbumin.  Accepted 
for  publication,  Biochimica  et  Biophysica  Acta. 


Honors  and  Awards  relating  to  this  Project: 

Dr.  Robert  C.  Uoodworth  was  awarded  an  N.I.H.  Fellowship  (National 
Heart  Institute)  to  study  during  1960-61  with  Prof.  Dr.  C.-B.  Laurell, 
Head  of  the  Keraiska  centrallaboratoriet,  Lund  University,  Malmfl,  Sweden, 


-U- 


Serial  No.  NIAID  63-B 

1 .  Infectious  Diseases 

2.  Medical  &  Physiological 
PHS-NIH  Bacteriology 

Individual  Project  Report       3.  Bethesda,  Maryland 

Calendar  Year  I960 

Part  A. 

Project  Title:  Biochemistry  of  the  acquisition  of  iron  by 
mammalian  tissues  as  mediated  by  the  iron- 
binding  protein,  siderophilin 

Principal  Investigator:  Dr.  Arthur  L.  Schade 

Other  Investigators:     Dr.  Robert  C.  Woodworth 

Cooperating  Units:      None 

Man  Years  (calendar  year  I960) 
Total:         17/12 
Professional:    6/12 
Other:         ll/l2 

Project  Description: 

Objectives:  To  investigate  the  mechanism  by  which  mammalian 
tissues  acquire  from  serum  siderophilin  the  iron  required  for  normal 
growth  and  function  and  to  study  the  effect  of  physiological  conditions 
upon  this  exchange. 

Methods  Employed:  Hematological  procedures,  radioactive 
tracer  techniques,  manometric  techniques  and  immunologic  analyses. 

Major  Findings:  Reticulocytes  remove  iron  from  siderophilin 
up  to  67  times  as  fast  as  the  natural  dissociation  rate  of  iron- 
siderophilin  complex  under  physiologic  conditions  of  pH  and  ionic 
strength.  The  rate  of  removal  of  iron  from  siderophilin  by  reticulo- 
cytes is  independent  of  percentage  saturation  of  siderophilin.  Both 
iron  atoms  are  removed  by  reticulocytes  from  this  chelating  protein 
even  in  the  presence  of  excess  ionic  iron  while  iron  chelated  with 
conalbumin  or  complexed  with  ethylenediarnine  tetraacetic  acid  (versene) 
or  in  an  ionic  form  is  not  removed  or  taken  up  at  significant  rates. 
The  absolute  rate  of  iron  uptake  by  reticulocytes  appears  to  be  a  func- 
tion, inter  alia,  of  the  concentration  of  iron-siderophilin  in  such 
manner  as  to  suggest  that  the  cells  possess  a  finite  number  of  specific 
sites  for  accommodating  the  iron-siderophilin  complex.  The  amount  of 
iron  taken  up  by  a  given  blood  sample  is  dependent  on  the  number  of 
reticulocytes  present  and  no  demonstrable  iron  is  absorbed  by  the  mature 
red  cells.  Reticulocyte  uptake  of  iron  is  markedly  restricted  by 
anaerobic  conditions.  This  fact  suggests  either  that  the  iron  exchange 

Part  B  included:  Yes  gg 


Serial  No.  NIAID  63-B 

from  siderophilin  to  the  cell  is  an  oxidative,  energy  requiring  process 
or  that  the  synthesis  of  an  iron  acceptor  system  leading  to  hemoglobin 
production  is  oxygen  dependent.  Attempts  thus  far  to  approximate  the 
iron  uptake  of  the  intact  reticulocytes  by  a  cell-free  hemolysate  pre- 
pared from  reticulocytes  have  not  yet  yielded  definitive  results. 

A  significant  practical  consequence  of  this  work  is  our  development 
of  a  simple  physiological  test  of  the  "integrity"  of  isolated,  purified 
serum  siderophilins  in  terms  of  their  iron-donating  or  iron  exchange 
capabilities.  Progress  in  the  study  of  the  clinical  uses  of  such  isolated 
siderophilins  has  been  stifled  by  the  absence  of  such  a  test.  Now,  the 
employment  of  our  reticulocyte  iron-uptake  method  promises  to  stimulate 
the  production  of  isolated  siderophilin  and  its  application  to  a  great 
variety  of  experimental  and  clinical  investigations.   Our  reticulocyte 
and  Staphylococcus  aureus  iron-uptake  tests  together  have  served  as 
guides  to  the  American  National  Red  Cross  in  its  efforts  to  produce,  on 
a  large  scale,  purified  siderophilin  for  such  investigations.  New  methods 
of  siderophilin  isolation  are  being  sought  so  as  to  preserve  these  phy- 
siological activities  intact. 

Significance  to  bio-medical  research  and  the  program  of  the  Insti- 
tute: As  a  first  approximation,  the  normal  functioning  of  host  tissues 
requires  iron  for  synthesis  of  their  heme  enzymes.  Hence,  the  conditions 
of  availability  of  serum  iron  to  host  tissues  and  the  mechanism  of  its 
transfer  from  siderophilin  are  of  significance  to  host-parasite  relation- 
ships obtaining  in  infections. 

Proposed  course  of  project;  We  propose  to  investigate  with  the 
satisfactorily  active  isolated  siderophilin,  which  heretofore  has  not 
been  available  but  is  now  at  hand  in  small  quantity,  the  relationships 
of  the  absolute  concentrations  of  iron-siderophilin  to  iron  exchange  and 
metabolism  by  reticulocytes  and  Staphylococcus  aureus  cells.  We  wish 
further  to  investigate  the  basis  for  the  indicated  catalysis  of  iron  ex- 
change by  bone  marrow  and  reticulocytes  and  to  determine  whether  such 
a  mechanism  applies  to  non -erythropoietic  tissues  as  well. 


67 

-2- 


Serial  No.  NIAID  63-B 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  B.    Honors,  Avrards,  and  Publications 
Publications  other  than  abstracts  from  this  project: 


Honors  and  Awards  relating  to  this  Project: 

Named  consultant  to  the  American  Red  Cross  to  gather  information 
for  them  on  the  serum  protein  fraction,  siderophilin,  at  Wiesbaden 
and  Bruges  internists1  and  scientists1  meetings  in  the  Spring  of 
I960. 

Gave  invited  lecture  on  "Siderophilin,  Its  Characteristics  and 
Functions"  to  the  research  laboratory  personnel  of  Parke,  Davis  &  Co, 
on  August  3>,  I960. 


-3-  68 


Serial  No.  NIAID  63-C 


1.  Infectious  Diseases 

PHS-NIH  2.  Medical  Sc  Physiological 

Individual  Project  Report    3.  Bethesda,  Maryland ^ 
Calendar  Year  i960 


Part  A. 


Project  Title:  Development  of  an  Economical  280mu.  Light 
Source  Suitable  for  Detecting  Proteins  in 
Effluents  from  Chromatographic  Columns. 

Principal  Investigator:   Dr.  Robert  C.  Wood-worth 

Other  Investigator: 

Cooperating  Units:       None 

Man  Years  (calendar  year  i960) 
Total:         2/12 
Professional:   l/l2 
Other:        1/12 

Project  Description: 

Objectives:  We  desired  to  develop  a  relatively  low-cost  light 
source  for  continuous  photometric  monitoring  of  chromatographic  column 
effluents  at  X  280mu-. 

Methods  Employed:  Spectral  analyses  of  filters  developed  in  the 
course  of  the  research  and  of  the  light  source  itself  with  various  fil- 
ters. 

Major  Findings:  Filter  combinations  have  been  described  by  others 
for  the  isolation  of  various  regions  of  the  ultraviolet  line  spectrum  of 
mercury.  These  filters  provided  a  starting  point  for  our  investigations, 
but  the  combination  suggested  for  the  280mu-  region  provided  too  wide  a 
band-pass  for  our  needs.   This  particular  filter  combination  consists 
of  (1)  a  2mm  Corning  red-purple  Corex  A  filter  No.  9863,  (2)  a  1M  NiSOk 
aqueous  solution  in  a  1  cm  light  path  vycor  or  quartz  cuvette,  and  (3) 
a  0.02^  aqueous  solution  of  2,7-dimethyl-3,6-diazocycloheptadiene-l,6- 
iodide  (hereinafter  referred  to  as  "cyanine"  iodide)  in  a  1  cm  light  path 
vycor  or  quartz  cuvette.  These  three  are  placed  in  series  between  the 
lamp  and  photocell.  The  "cyanine"  iodide  possesses  an  absorption  minimum 
at  263m[i.  Dr.  E.  Kravitz  of  the  National  Heart  Institute  provided  a 
pure  sample  of  phenazine-a-carboxylic  acid  (produced  by  Pseudomonas 
aureofaciens).  A  0.02$  aqueous  solution  of  this  substance  (neutralized 
with  KOH  to  make  it  soluble)  possesses  an  absorption  minimum  at  290n|i.. 
An  equal -weight  mixture  of  "cyanine"  iodide  and  phenazine-a-oarboxylic 
acid  made  to  a  total  of  0.02^  in  water  (with  KOH  just  s 
dissolve  the  acid)  possesses  an  absorption  minimum  at  280m(j..  T 

69 

Part  B  not  included.  -1- 


Serial  No.  NIAID  63-C 

solution  in  a  1  cm  vycor  or  quartz  cuvette,  in  series  with  a  Corning  red- 
purple  Corex  A  filter  and  1  cm  of  1M  NiSOr,  as  described  above,  provides 
a  spectrum  -ath  only  a  single,  narrow  transmission  band  with  a  maximum  of 
280mp..  Appreciable  transmission  appears  again  only  in  the  far  red 
(750mu.),  a  wavelength  region  to  which  commonly-used  phototubes,  i.e., 
No.  93S>>  are  completely  insensitive.  The  filter  has  proved  stable  to 
ultraviolet  radiation  over  an  in-service  period  of  six  months,  i.e.,  the 
transmission  spectrum  of  the  filter  has  remained  unchanged. 

When  the  output  of  a  General  Electric  H3FE  low-pressure  mercury  a 
lamp  is  passed  through  this  filter  system  and  analyzed  with  a  Beckman 
HJ  spectrophotometer,  one  finds  many  mercury  emission  lines  other  than 
280mu..  This  output  spectrum  is  closely  similar  to  that  obtained  from 
the  same  lamp  filtered  by  a  Corning  red-purple  Corex  A  filter  together 
with  a  Baird  Atomic  280mji  interference  filter.   This  latter  filter  system 
and  light  source  are  used  in  a  commercially  available  protein-monitoring 
unit.  A  vast  improvement  in  the  mono chromic ity  of  filtered  light  from 
this  lamp  is  obtained  from  our  filter  system  by  increasing  the  "mixed- 
organic"  filter  ($0%   each  of  "cyanine  iodide~phenazine-a-carboxylic 
acid)  concentration  three-fold  to  0.06$.  If  the  280. hmu.  transmission  is 
now  adjusted  to  lOOJo,  the  only  other  significant  transmission  lines 
found  are  2h%  at  275mu  and  21$   at  289*2mjx,  both  of  which  are  strongly 
absorbed  by  proteins. 

The  "mixed-organic"  filter  has  the  additional  virtue  of  possessing 
a  variable  transmission  maximum  between  263  and  290mu.,  which  is  dependent 
on  the  per  cent  composition  of  the  filter.  Thus,  it  may  be  useful  for 
isolating  other  spectral  lines  than  280mu.  in  this  region. 

The  shops  at  NIH  have  done  preliminary  work  on  a  stable  photometric- 
recording  system  for  use  with  this  light  source,  but  it  is  as  yet  not 
ready  for  use. 

Significance  to  bio-medical  research  and  the  program  of  the  In- 
stitute; The  availability  of  an  economical  photometric  analyzing  system 
for  protein-containing  chromatographic  effluents  makes  possible  the 
release  for  more  important  work  of  an  individual,  professional  or  tech- 
nical, from  the  time-consuming,  routine  task  of  reading  individual  frac- 
tions in  a  manual  spectrophotometer.  Further,  the  use  of  a  light  source 
which  will  be  specifically  absorbed  by  proteins  is  of  paramount  importance, 
in  order  to  provide  adequate  sensitivity  for  minor  components  and  to 
make  possible  quantitative  estimations  of  protein  content,  if  this  should 
be  desired. 

The  25Limu,  line  of  mercury  utilized  in  some  commercially-available 
ultraviolet  monitoring  devices  is  not  suitable  for  use  with  proteins  be- 
cause of  its  demonstrated  denaturing  effects. 

Proposed  Course  of  Project:  A  more  satisfactory  light  source  might 
be  found,  i.e.,  one  with  fewer  emission  bands  close  to  the  280mn  line. 
Such  a  lamp  would  allow  for  a  lower  concentration  of  the  "mixed-organic" 
filter  and  thus  permit  a  higher  intensity  of  usable  light.  Search  iov-, ^ 
such  a  light  source  will  continue.  '  '-' 


Serial  No.  NIAID  6U 


1.  Infectious  Diseases 

2.  Medical  &  Physiological 

Bacteriology 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:  Detoxification  of  Potential  Tuberculostatic, 

Fungistatic,  Parasiticdal  and  Viricidal  Agents. 

Principal  Investigator:   Dr.  Benjamin  Prescott 

Other  Investigators:     None 

Cooperating  Units:       None 

Man  Years  (calendar  year  I960) 
Total:         13/12 
Professional:    3/l2 
Other:         10A2 

Project  Description: 

Objectives:  Further  studies  on  toxicity  and  detoxification 
of  the  tuberculostatic  drugs  streptomycin  (SM),  isoniazid  (INH)  and 
mixtures  of  streptomycin-isoniazid  (SM-INH)  in  two  strains  of  mice. 
In  addition,  to  find  means  of  reducing  the  toxicity  of  the  widely  used 
therapeutic  agent  Neomycin  and  of  several  parasiticidal  drugs. 

Methods  Employed:  The  effect  of  various  chemicals  on  the 
toxicity  of  INH  and/or  SM  and  Neomycin  was  measured  by  the  effect  on 
survival  time  of  normal  mice  following  simultaneous  subcutaneous  ad- 
ministration of  a  given  adjuvant  with  a  lethal  dose  of  drug  (both 
single  and  multiple  administrations).  The  in  vitro  bacteriostatic  action 
of  the  drug-adjuvant  combination  was  also  determined. 

Major  Findings: 

1.  With  glycerol  formal  as  solvent.  Studies  initiated 
with  glycerol  formal  as  solvent  for  INH  and/or  SM  have  shown  that 
with  a  lethal  6  mg  oral  dose  of  INH  in  a  2$%   glycerol  formal  solution 
(in  water)  permitted  95%   survival  of  a  group  of  60  white  mice  and 
90^  survival  in  DBA  mice  (50).  However,  on  repeated  daily  administration, 

Part  B  included:  Yes 

71 


Serial  No.  NIAID  6U 

optimal  results  with  no  toxic  manifestations  were  found  in  mice  given 
a  5tng  dose  (LD_,  )  in  2>S%   solvent. 

The  use  of  this  solvent  with  SM  has  thus  far  shown  that  a  single 
subcutaneous  administration  of  a  lethal  15  mg  dose  of  SM  in  3S%   solu- 
tion permitted  100/!  survival  of  mice.  Decreasing  the  concentration 
of  adjuvant  to  25%   permitted  100%   survival  and  a  1$%   solution  90^. 
The  detoxifying  action  of  this  solvent  was  tested  on  SM-INH  mixtures 
in  two  strains  of  mice.  85$  of  the  mice  tolerated  a  lethal  mixture 
of  10  mg  SM  and  h  mg  INH  and  80%   survival  with  10  mg  SM  and  8  mg  INH. 

2.  With  steroids;  Further  studies  with  steroids  showed 
that  55$  of  both  white  and  DBA  mice  tolerated  a  lethal  dose  of  30  mg  SM 
and  h  mg  INH  when  administered  subcutaneous ly  with  25  mg  of  sodium  tauro- 
cholate.  With  a  lethal  dose  of  20  mg  SM  and  6  mg  INH  or  15  mg  SM  and 

8  mg  INH,  the  same  amount  of  adjuvant  permitted  80  and  90%   survival, 

3.  With  L-argininyl-L-glutamate :  Mice  survived  a  lethal 
15  mg  dose  of  SM  when  it  was  administered  in  combination  with  58  mg  of 
the  peptide  L-argininyl-L-glutamate  in  both  single  and  repeated  doses, 

U.  With  Miracil  D:  Further  studies  with  this  drug  included 
the  use  of  various  steroids  as  possible  detoxifying  agents.  Four 
steroids  (cholic  acid,  sodium  taurocholate,  sodium  glycocholate  and  so- 
dium glycotaurocholate)  were  tested  as  adjuvants  with  various  doses  of 
Miracil  D.  Of  the  four  steroids  tested,  sodium  taurocholate  showed  good 
detoxifying  activity.  100$  of  the  mice  tolerated  a  30  rag  dose  of  Miracil 
D  (6  x  lethal  dose)  when  it  was  administered  simultaneously  with  50  mg  of 
sodium  taurocholate, 

5.  Detoxification  of  Neomycin:  Since  sodium  glucuronate 
and  glycine  proved  effective  in  permitting  mouse  survival  when  administered 
simultaneously  with  toxic  doses  of  INH,  SM  and  mixtures  of  SM-INH,  the 
detoxifying  activities  of  these  adjuvants  were  tested  with  Neomycin  in  two 
strains  of  mice.  When  10  mg  of  Neomycin  per  20  g  mouse  (singly  lethal) 
was  injected  simultaneously  with  a  mixture  of  50  mg  glycine  and  50  mg 
sodium  glucuronate,  99%   of  the  mice  survived.  Even  if  the  Neomycin  dose 
was  increased  to  20  mg,  90%   of  the  mice  survived.  In  chronic  toxicity 
tests,  60$  of  the  animals  tolerated  15  repeated  injections  of  the  10  mg 
dose  of  Neomycin  if  50  mg  glycine  and  50  mg  sodium  glucuronate  were  in- 
jected simultaneously.  No  interference  by  the  detoxifying  agents  tested 
was  observed  on  the  in  vitro  bacteriostatic  action  of  Neomycin  on  one 
strain  of  Staphylococcus  aureus. 


-2- 


72 


Serial  No.  NIAID  6U 


6.  YJith  certain  vitamins;   Of  16  vitamins  similarly 
tested  with  Neomycin,  nicotinic  acid  and  calcium  pantothenate  exhibited 
detoxifying  activity.  The  last  named  in  a  molar  ratio  adjuvant  and 
drug  gave  optimal  results  in  combination  with  10  mg  of  Neomycin.  Sur- 
vival was  100£  in  two  strains  of  mice.  Up  to  1$   daily  doses  of  the 
Neomycin  with  calcium  pantothenate  were  tolerated  by  100%  of  the  mice. 

7.  With  amino  acids:  On  single  administration  of  a  1 
mg  dose  of  Neomycin  per  20  gram  mouse,  D-glutamic,  L-aspartic,  and 
acetyl  DL-methionine  permitted  70  to  86^  survival  of  white  micej  18  other 
amino  acids  tested  showed  little  or  no  activity. 

Significance  to  bio-medical  research  and  the  program  of  the 
Institute:  The  widespread  use  of  the  chemotherapeutic  agents  strepto- 
mycin and  isoniazid  in  the  treatment  of  human  tuberculosis  is  limited 
by  their  toxicity.   Similarly  use  of  Neomycin  is  also  restricted. 
Means  of  increasing  the  tolerance  of  these  toxic  chemotherapeutic  drugs 
should  permit  more  effective  therapy. 

Proposed  course  of  the  Project:  This  study  will  be  extended 
to  include  in  vivo  tests  and  to  a  few  more  adjuvants  that  show  promise. 


73 

-3- 


Serial  No.  NIAID  -  &k 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  B.    Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Prescott,  B.,  Kauffmann,  G.  and  Stone,  H.  J.:  Means  of  increasing 
the  tolerated  dose  of  isoniaaid-streptomycin  mixtures  in  mice. 
II.  Certain  Vitamins.  Antibiotics  and  Chemotherapy  10, 
163-168,  I960 


Honors  and  Awards  relating  to  this  Project: 

Invited  to  participate  in  "Symposium  uber  experimentelle  und 
klinische  Pharmakologie  der  Antibiotika"  to  be  held  in  Aachen,  Germany, 
May  18-19,  1961. 


-k-  v* 


Serial  No.  NIAID  6U-A 


1.  Infectious  Diseases 

2.  Medical  &  Physiological 

Bacteriology 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:   Potential  Fungistatic  and  Parasiticidal  Agents. 

Principal  Investigator:  Benjamin  Prescott 

Other  Investigators:     None 

Cooperating  Units:      Section  on  Mycology,  LID-NIAID 

Dr.  Chester  W.  Emmons  -  Serial  No.   ?6~B 

Section  on  Chemotherapy,  LPC-NIAID 

Dr.  George  Luttermoser  ■  -  Serial  No.  


Man  Tears  (calendar  year  I960) 
Total :         13/12 
Professional:     3/12 
Other:         10/12 

Project  Description: 

Objectives:  Synthesis  of  new  potential  non-toxic  cheraothera- 
peutic  agents  against  fungal  and  parasitic  infections. 

Methods  Employed: 

Antifungal  agents:  A  series  of  30  dithiooxamide  derivatives 
were  synthesized  by  treating  a  solution  of  dithiooxamide  with  hydrazine 
hydrate  forming  a  dihydrazide.  The  resulting  compound  was  then  con- 
densed with  various  aliphatic  and  aromatic  aldehydes  in  $$%   ethyl  alco- 
hol. The  final  recrystallized  products  were  analyzed  for  carbon,  hydro- 
gen, nitrogen  and  melting  points  obtained.  Toxicity  studies  were  per- 
formed in  white  mice.  Since  thiosemicarbazones  have  been  shown  to  have 
chemo therapeutic  activity  (tibione  against  tuberculosis  and  isatin  thio- 
semicarbazone  against  viruses),  a  series  of  50  long  chain  thiosemicarba- 
zones were  synthesized  by  first  preparing  octadecylthiosemicarbazide  and 
condensing  this  compound  with  various  aliphatic,  aromatic  and  heterocyclic 
aldehydes. 


Part  B  not  included. 

75 


Serial  No.  NIAID  6U-A 


Additional  derivatives  of  thymol,  naphthoquinones  and 
U,U'diaminodiphenylsulfone  were  synthesized.  In  mice,  toxicity  of 
these  compounds  was  of  a  very  low  order.  Dr.  Chester  W.  Emmons, 
LID-NIAID,  will  test  the  fungistatic  activity  of  these  compounds. 

Parasiticides;  A  new  series  of  piperazine  derivatives 
were  synthesized  by  condensing  N-aminoethyl  piperazine  with  aldehydes, 
carboxylic  acids,  sulfonic  acids,  isocyanates  and  isothiocyanates. 
In  addition,  H5>  derivatives  of  tetracycline  were  prepared  by  coupling 
tetracycline  with  compounds  like  atabrine,  chloroquine,  piperazine 
monocarboxylic  acid,  sulfanilic  acid,  hetrazan  and  several  naphtho- 
quinones. 

Major  Findings;  Several  of  the  tetracycline  derivatives 
demonstrated  considerable  in  vitro  activity  against  parasites  and 
showed  negligible  toxicity  in  mice. 

Significance  to  bio-medical  research  and  the  program  of 
the  Institute;   Because  of  high  tolerance  and  effectiveness,  the 
tetracycline  derivatives  may  be  useful  in  the  chemotherapy  of  human 
parasitic  infections. 

Proposed  course  of  Project;  Tests  for  usefulness  of  the 
antifungal  and  antiparasitic  compounds  are  to  be  performed  in  vivo 
in  laboratory  animals  (mice  and  dogs)  to  determine  their  effectiveness 
in  experimental  infections.  All  the  compounds  synthesized  for  anti- 
fungal and  antiparasitic  activity  are  also  being  tested  for  anti-tumor 
activity.  About  UOO  compounds  have  been  sent  to  the  Cancer  Chemo- 
therary  screening  program.  Several  long  chain  thiosemicarbazones  have 
passed  the  initial  tests  in  mice. 


76 

-2- 


Serial  No.  NIAID  6U-B 
1.  Infectious  Diseases 


PHS-NIH  2.  Medical  &  Physiological 

Individual  Project  Report         ^   Bethesda,  Ha^land10^ 
Calendar  Year  I960  J  '         * 


Part  A. 


Project  Title:   Isolation  of  Antibacterial  and  Antiviral 
Substances  from  Shellfish 

Principal  Investigator:  Benjamin  Prescott 

Other  Investigators:     None 

Cooperating  Units:      Laboratory  of  Virology  and  Rickettsiology, 

Division  of  Biologies  Standards 
Dr.  Chen  Pien  Li  -  Serial  No.  DBS 

Man  Tears  (calendar  year  I960) 
Total:         16/12 
Professional:     6/l2 
Other:         10/12 

Project  Description: 

Objectives:  To  isolate  and  purify  potential  therapeutic 
agents  from  shellfish. 

Methods  Employed:  Juice  from  fresh  frozen  abalone  and  extracts 
of  oysters  and  clams  were  each  dialyzed  against  distilled  water,  the 
residue  adjusted  to  pH  7.8  and  applied  to  anion-exchange  (diethylaraino- 
ethylcellulose)columns  set  up  on  automatic  fraction  collectors  in  the 
cold  (U°  C).  ELution  of  material  from  the  column  was  carried  out' with  a 
series  of  tris-HoPO^  buffers  of  varying  ionic  strength  and  pH  (3.7  -  7.6) 
and  a  final  wash  with  M-NaCl.  Eluates  were  consecutively  collected. 
Several  pools  were  made  of  various  fractions,  dialyzed  against  distilled 
water  and  lyophilized. 

Major  Findings:  It  was  found  that  the  early  eluate  pool  frac- 
tions contained  antibacterial  activity  against  both  penicillin-sensitive 
and  —resistant  strains  of  Staphylococcus  aureus,  a  beta-hemolytic  strain 
of  Streptococcus  pyogenes,  Salmonella  typhi,  and  S.  paratyphi  A  and  B. 
The  growth  of  10,000  or  wore  organisms  per  ml  was  inhibited  by  these 
fractions  in  a  concentration  of  10  mg  per  cent.  These  fractions  showed 
no  antiviral  activity.  The  fraction  isolated  from  the  final  wash  of 
the  column  with  M-NaCl  showed  no  antibacterial  activity.  However, 
there  was  definite  inhibitory  activity  against  influenza  A  virus  and  poly- 
oma virus  in  tissue  culture. 

Part  B  included:  Yes 

-i.  77 


Serial  Mo.  NIAID  6U-B 


S'  -mif  icance  to  bio-medical  research  and  the  program  of  the 
Institute:   The  abundance  and  variety  of  shellfish  is  a  potential 
source  of  antibacterial  and  antiviral  agents.  Since  fractions  isolated 
fron  this  source  were  found  to  possess  marked  antibacterial  activity, 
they  may  be  valuable  as  therapeutic  and  prophylactic  agents. 

Proposed  course  of  Project:  Preparation  of  large  quantities 
of  active  fractions  and  chemical  identification  of  the  agent  are  in  pro- 
gress. In  addition,  the  therapeutic  efficiency  of  the  agents  is  being 
studied  in  protection  experiments  in  mice  experimentally  infected  with 
streptococci  and  pneumococci. 


-2- 


78 


Serial  No.  NIAID  6U-B 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  B    Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Prescott,  B.  and  Li,  C.  P.:  Abalone  juice:  Fractionation  and  antibac- 
terial spectrum.  Proc.  Soc.  Exp.  Biol.  &  Med.   (in  press) 


Honors  and  Awards  relating  to  this  Project: 
None 


-3-  73 


Serial  No.  NIAID  65 


1.  Infectious  Diseases 

2.  Medical  &  Physiological 

Bacteriology 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:   Longitudinal  Studies  of  Beta-hemolytic 
Streptococcal  Isolations. 

Principal  Investigator:  Roger  M.  Cole 

Other  Investigators:     Edythe  J.  Rose 

Cooperating  Units:      Dept.  of  Pathobiology  . 

School  of  Hygiene  and  Public  Health 
Johns  Hopkins  University 
(Dr.  Wm.  J.  L.  Sladen) 
Bacteriology  Laboratory 
Johns  Hopkins  Hospital 
(Dr.  Jack  Causton 

Man  Tears  (calendar  year  I960): 
Total:         30/12 
Professional:    12/12 
Other:         18/12 

Project  Description: 

Objectives:  An  aspect  of  studies  continued  in  this  laboratory 
for  several  years,  to  extend  knowledge  of  human  experience  with  strepto- 
cocci through  culture,  serologic  and  epidemiologic  methods. 

Methods  Employed:  In  general,  similar  to  laboratory  and  epi- 
demiologic methods  described  in  projects  for  other  years.  In  particular, 
throat  swabs  taken  routinely  from  personnel  in  isolated  situations  in 
Antarctica  were  mixed  in  glycerine  broth  which  was  then  deep-frozen  and 
transported  to  the  United  States.  Some  months  later,  samples  were  plated 
from  the  thawed  fluids  and  colonies  of  beta-hemolytic  streptococci  picked 
for  serologic  identification  and  tested  for  bacitracin  sensitivity  at  a 
level  said  to  distinguish  Group  A  from  other  beta-hemolytic  streptococci. 
Sera  from  the  same  personnel  were  taken  at  intervals  and  kept  frozen  for 
future  reference.  Simultaneous  throat  swabs  were  taken  for       eola- 
tion by  another  group  (Virus  &  Rickettisal  Section,  LID).  In  the  past, 

Part  B  included:  Yes 

_!_  80 


Serial  No.  NIAID  65 

somewhat  similar  studies  have  been  made  in  institutionalized  children 
(Jr.  Village,   D.C.);   and  studies  of  other  methods  of  transport  of 
streptococci  prior  to  culturing  were  made  in  collaboration  with  inves- 
tigators in  California   (U.  Cal.   Berkeley:   see  publication,  Part  B). 

Major  Findings:     Approximately"  2lj.O  cultures  of  beta-hemolytic 
streptococci  have  been  received  from  the  collaborating  laboratory  for 
identification.     Most  of  these  represent  the  same  persons,    repeatedly 
positive  for  streptococci  of  the  same  group  or  type  of  Group  A.     About 
38$  of  the  cultures  were  Group  A,   25$  Group  B,   6%  Group  C,   30$  Group 
G,   and  the  small  remainder  failed  to   grow  or  to  react  serologically. 
About  Gh%  were  bacitracin-sensitive  by  the   test  used:     98$  of  Group 
A  were   sensitive  as  expected,  but  so  were  57$  of  Group  C,   U9$  of  Group 
G  and  31$  of  Group  B.     The  test  as  used  is  obviously  not  an  adequate 
screening  method  for  distinguishing  Group  A  from  other  beta-hemolytic 
streptococci. 

Significance  to  bio-medical  research  and  the  program  of  the 
Institute:       Only  in  isolated  or  semi-isolated  situations  with  limi- 
ted possibilities  for  introduction  of  new  organisms  can  the   persistence 
of  throat  flora  be  adequately  examined.     Whether,  under  such  conditions   of 
carriage,  beta-hemolytic  streptococci  of  Group  A  induce  antibody  forma- 
tion (and  if  so,   how  or  why  they  persist)  is  a  matter  of  considerable  in- 
terest in  evaluating  the  various  aspects  of  streptococcal  virulence  and 
resistance  to  streptococcal  infections. 

Proposed  Course  of  Project:     Continue.     Antibody  responses  will 
be   tested  by  improved  methods  described  under  another  project.     Addi- 
tional culture  and  serum  specimens  are  expected  from  Arctic  areas  within 
the  next  year. 


81 


Serial  No.  NIAID  65 


PHS-NIH 

Individual  Project  Report 
Calendar  Year  I960 


Part  B     Honors,  Awards,  and  Publications 
Publications  other  than  abstracts  from  this  project: 


Hollinger,  N.  F.,  Lindberg,  L.  H. ,  Russell,  E.  L.,  Sizer,  H.  B., 
Cole,  R.  M. ,  Brewne,  A.  S.,  and  Updyke,  E.  L. :  Transport 
of  streptococci  on  filter  paper  strips.  Pub.  Hlth.  Rep., 
75:  251-259,  I960. 


Honors  and  Awards  relating  to  the  project: 
None 


82 


Serial  No.  NIAID  65-A 

1.  Infectious  Diseases 

2.  Medical  &  Physiological 

Bacter  iology 

3.  Be t he s da,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:  Streptococcal  M  protein,  virulence,  and 
type-specific  immunity. 

Principal  Investigator  Dr.  Jerome  J.  Hahn  &  Dr.  Roger  M.  Cole 

Other  Investigators:     None 

Cooperating  Units:      Laboratory  Histology  and  Pathology, 

NIDR  (Dr.  David  Scott) 

(Dr.  Marie  Nylen)  NIDR  "  ** 

Laboratory  Biology  of  Viruses, 
NIAID  (Dr.  C.F.T.  Mat tern) 

Man  Years  (calendar  year  I960): 
Total :         50/12 
Professional :   20/12 
Other:        30 /\2 

Project  Description: 

Objectives:  In  general,  to  study  G,  up  A  streptococcal  M 
protein  and  of  antibody  thereto,  in  order  to  oetter  understand  viru- 
lence and  resistance  to  infection  among  gram-positive  cocci.  Speci- 
fically; (a)  to  improve  methods  of  preparing  antigenic  M  protein  and 
to  determine  the  differences  between  M  proteins  which  account  for  type 
specificity;  (b)  to  investigate  and  improve  methods  for  detecting  anti- 
body to  M  protein;  (c)  to  study  the  relation  between  M  protein,  or  other 
possible  cellular  components,  to  streptococcal  virulence;  and  to  deter- 
mine more  precisely  the  cell  wall  localization  of  M  protein  and  its 
significance. 

Methods  Employed: 

(a)  Neutralized  acid  extracts  of  cells  of  5  strepto- 
coccal types  were  precipitated  with  saturated  picric  acid,  and  the 
precipitates  extracted  with  8o£  acid  ethanol:  supernates  were  pooled 

Part  B  included:  Yes 

-1-  83 


Serial  No.  NIAID  6g-A 

and  further  precipitated  by  acetone.  These  washed  and  dried  precipi- 
tates were  dissolved  in  saline  and  gradually  neutralized,  with  removal 
of  precipitates  as  formed.  Active  material  was  in  the  supernates, 
which  were  analyzed  for  M  protein  immunologically,  for  carbohydrate 
by  the  Molisch  reaction,  and  for  nucleic  acid  by  the  orcinol  method. 
The  immunologic  tests  were  made  by  antigenicity  in  rabbits  and  by  "type- 
specific  precipitation  in  liquid  and  in  agar  gel  diffusion  systems. 

M  proteins,  prepared  by  both  the  conventional  (acid  hydrolysis- 
ammonium  sulfate  precipitation)  method  and  the  picrate  method,  have 
been  subjected  to  Immunoelectrophoresis.  In  addition,  attempts  to 
fragment  M  protein  into  immunologically  reactive  portions  are  being 
made  by  differential  heating  with  pH  control  and  by  fractional  enzy- 
matic degradation. 

(b)  Detection  of  type-specific  antibody  to  M  protein 
has  been  examined  by  two  methods:   (l)  the  long  chain  reaction,  in 
which  streptococcal  chains  are  significantly  longer  in  homologous  anti- 
serum than  in  heterologous  or  normal  serum,  and  (2)  inhibition  by  un- 
labelled  test  antisera  of  fluorescence  on  addition  of  fluorescein- 
labelled  specific  antiserum.  Both  methods  have  been  compared  with  the 
usual  precipitin  and  bactericidal  tests.  The  nature  of  type-specific 
antibody  is  being  examined  by  conversion  to  univalent  fragments  by 
pepsin-cysteine  or  papain  treatment.  Such  univalent  antibody  will  be 
tested  by  blocking  of  precipitation,  blocking  of  fluorescence,  precipitation, 
specific  fluorescence  after  labelling,  bactericidal  activity,  and  the  long 
chain  reaction. 

(c)  Virulence  of  streptococcal  strains  is  tested  by  intra- 
peritoneal injection  of  culture  dilutions  in  mice,  with  subsequent  de- 
termination of  $0%   lethal  doses.  Bactericidal  and  phagocytic  tests  in 
rabbits  or  human  whole  blood  with  or  without  added  specific  sera,  or  with 
mouse  leukocyte  suspensions,  are  also  used  to  detect  presence  in  living 
cells  of  the  presumptive  virulence  factor,  M  protein.  Addition  of  homo- 
logous and  heterologous,  partially  purified,  M  proteins  to  phagocytic 
systems,  has  been  used  to  determine  if  the  extracted  proteins  enhance 
virulence  by  protection  against  phagocytosis. 

Differential  blocking  of  fluorescence  by  group  and  type-specific 
antisera  applied  in  different  sequences,  is  being  used  to  aid  in  localiza- 
tion of  M  and  of  group  carbohydrate  in  the  streptococcal  cell  wall.  Sec- 
tions of  fixed  and  embedded  streptococci  have  been  made  and  examined  under 
the  electron  microscope,  as  a  preliminary  to  the  use  of  ferritin -labelled 
antibody  for  the  electron  microscopic  localization  of  cell  wall  components. 


8'< 


Serial  No.  NIA3D  6g-A 
TTajor  Findings: 

(a)  M  protein  picrates,  made  as  described,  contain 
less  than  0.1$  each  of  carbohydrate  and  nucleic  acid.  During  alka- 
linization  in  their  final  preparation,  picrates  of  Types  1  and  18 
form  precipitates  at  a  lower  pH  (3.7)  than  do  those  of  Types  2,  kf 
and  28  (pH  U.5>).  Picrates  of  Types  1,  h,   and  18  give  precipitin  re- 
actions only  with  their  homologous  type  antisera,  whereas  that  of 
Type  28  reacts  also  with  antisera  to  Types  2,  13  and  Uii.  The  possi- 
bility of  the  presence  of  "R"  protein,  instead  of,  or  in  addition  to, 
M  is  thus  raised;  and  preliminary  studies  in  agar  gels  indicate  that 
at  least  two  reactive  antigens  may  be  present  in  the  picrate  prepara- 
tions. Early  results  from  intramuscular  injection  of  a  Type  1  picrate 
indicate  that  the  material  is  weakly  antigenic,  requiring  h  weekly  in- 
jections of  $  mg.  each  to  produce  antibody  detectable  in  undiluted 
serum  by  the  long  chain  test. 

Immunoelectrophoresis  of  conventionally  prepared  M  proteins  demon- 
strates differences  between  Types  1  and  23.  A  single  precipitin  band 
is  obtained  when  the  former  is  tested  against  homologous  antisera, 
whereas  Type  23  always  shows  two  bands  or  spurs  which  move  together  and 
cannot  be  separated  by  variations  in  buffers,  pH,  voltage,  nor  time  of 
run. 

Boiling  M  protein  at  an  acid  pH  for  between  30  and  60  minutes  ap- 
pears to  produce  a  fragement  which  binds  with  but  does  not  precipitate, 
specific  antibody.  Similar  studies,  using  enzymes,  are  in  progress, 

(b)  The  long  chain  test  for  type-specific  antibody  has 
been  statistically  improved  by  a  more  rapid  and  simpler  method,  uti- 
lizing the  chi-square-analyzed  differences  in  frequency  distributions 
of  chains  above  and  below  a  predetermined  length,  in  test  and  normal 
sera.  Interval  sampling  has  shown  that  time  of  incubation  prior  to 
reading  the  test  is  of  prime  importance :  chains  increase  in  1  ength, 
and  long  chains  increase  in  frequency,  to  a  maximum  with  time  and  then 
decrease.  The  decrease  depends  on  antibody  depletion;  and  maximum 
length  or  frequency  is  reached  sooner  and  is  lower  in  low  concentra- 
tions of  antibody  than  in  high.  As  a  result,  time  interval  sampling 
allows  titration  of  antibody  by  determining  the  highest  dilution  which 
gives  a  positive  result  according  to  predetermined  statistical  criteria. 
The  effect  of  dilution  of  streptococcal  inoculum  in  test  is  opposite 

to  that  of  antibody  dilution.  Total  coccal  growth  is  unaffected  by 
the  presence  of  antibody.  The  Size  Class  Frequency  method  of  determining 
a  positive  long  chain  reaction  has  been  shown  to  be  reproducible  and  more 
sensitive  than  the  previous  method  using  mean  chain  lengths. 

Fluorescein-labelled  group-specific  and  type-spec        ;era  have 
been  successfully  prepared.  No  cross  reactions  among  antisera  to  several 

85 


Serial  No.  NIAID  6g-A 

different  tvpes  have  been  shown  by  this  method.  Inhibition  by  unlabelled 
antisera  of  specific  fluorescent  antisera  appears  to  be  a  simple  and  sen- 
sitive method  for  determining  the  presence  of  type-specific  antibody.  Its 
use  in  titration  is  being  compared  with  the  long  chain  and  bactericidal 
tests,  but  appears  somewhat  limited  by  the  subjective  determination  of  the 
fluorescent  end  point. 

A  method  of  labelling  portions  of  the  streptococcal  cell  wall  by 
growth  in  fluorescent  antiserum  with  subsequent  removal  of  excess  anti- 
body, has  been  devised.  Its  use  in  following  cell  wall  formation  and  in 
analysis  of  the  mechanism  of  the  long  chain  test  is  being  investigated. 

Univalent  antibody,  produced  by  pepsin-cysteine  treatment  of  whole 
antiserum,  has  been  shown  capable  of  blocking  precipitin  and  fluorescent 
reactions,  but  does  not  produce  long  chains.  Additional  studies  of  the 
nature  of  streptococcal  type-specific  antibody  are  under  way. 

(c)  M  protein  of  Type  23  has  been  reported  to  enhance  phago- 
cytosis when  added  to  a  mouse  leukocyte  system  containing  antibody  and 
virulent  Type  23  streptococci.  This  experiment  was  repeated  and  confirmed, 
but  similar  enhancement  in  a  Type  1  system  was  not  found.  No  increase  in 
phagocytosis  by  heterologous  II  protein  occurred.  Because  M,  as  the  pre- 
sumptive virulence  factor,  was  expected  to  prevent  phagocytosis  rather 
than  the  reverse,  the  possibility  of  a  different  virulence  factor  —  at 
least  in  Type  23  —  arose.  To  see  if  removal  of  anti-M  from  a  serum  made 
against  virulent  organisms  would  leave  an  "anti-virulence  factor",  M+ 
avirulent  variants  were  derived  from  Types  1  and  23,  and  then  used  to 
absorb  sera  prepared  against  virulent  organisms.  These  antisera  contained 
" an ti -virulence  factor",  as  shown  by  their  mouse -protective  capacities. 
After  absorption  to  the  point  of  removal  of  type-specific  precipitins,  but 
not  of  long  chain  producing  antibodies,  mouse  protective  antibody  was  still 
present.  Further  absorption  removed  both  long-chain  producing  and  mouse 
protective  antibodies  from  both  Type  1  and  Type  23  antisera.  The  effect 
appears  to  be  a  quantitative  one,  and  this  method  failed  to  distinguish  a 
different  virulence  factor  than  M  protein. 

The  suspected  surface  location  of  M  protein  on  the  streptococcal  cell 
wall  was  verified  by  fluorescence -inhibition  experiments.  Unlabelled 
group-specific  antiserum  failed  to  block  the  fluorescence  of  labelled  type- 
specific  antiserum,  whereas  unlabelled  type-specific  antiserum  inhibited 
the  fluorescence  of  added  group-specific  antiserum.  The  type-specific 
antigen  (M)  therefore  appears  to  be  superficial  to  the  group  antigen,  or 
a  peculiar  steric  relationship  may  exist.  Similar  experiments,  using 
ferritin -labelled  antibody  and  the  electron  microscope,  are  being  initiated. 


.u-  86 


Serial  No.  NIAID  65 -A 


Significance  to  bio-medical  research  and  the  program  of 
the   Instioute:      Group  A  streptococcal  infections  in  man  are  common, 
even  today  with  the  widespread  use  of  antibiotics.     Immunity  to  such 
infections,   shown  in  a  limited  number  of  instances  and  by  use  of  dif- 
ficult and  tedious  methods,    has  been  said  to  be  type-specific.     The 
use  of  rapid  and  improved  methods  such  as  we  have  described  will 
greatly  facilitate  verification  and  expansion  of  these  findings  and 
allow  epidemiological   and  clinical  serologic   studies  on  a  scale  not 
previously  possible. 

The  role  in  virulence  of  the  M  protein  which  stimulates  such 
type-specific  antibodies  is  widely  accepted  but  not  understood.     Mouse 
virulence  is  the  usual  measure,   but  many  11+  strains  freshly  isolated  from 
man  are  not  mouse-virulent  without  passage:     other  discrepancies  occur, 
and  the  possibility  of  other  virulence  factors  requires  further  study. 
Investigations  of  streptococci,  which  are  well  characterized  anti- 
genically,  may  serve  as  useful  models  for  other  gram-positive  cocci  such 
as  staphylococci  which   are  at  present  poorly  defined  antigenically. 

Proposed  course  of  Project:     Continue  along  lines  indicated. 


-5-  87 


Serial  No.  NIAID  «"* 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  B     Honors,  Awards,  and  Publications' 

Publications  other  than  abstracts  from  this  project: 

Kantor,  F.  S.,  &  Cole,  R.  M.:  Preparation  and  antigenicity  of  M  protein 
released  from  Group  A,  Type  1  streptococcal  cell  walls  by  phage- 
associated  lysin.  J.  Exp.  Med.,  112:  77-96,  I960 


Honors  and  Awards  relating  to  the  project: 
None 


.6-  88 


Serial  No.  NIAID  66 


PHS-NIH 
Individual  Project  Report 
Calendar  Tear  I960 


1.  Infectious  Diseases 

2.  Epidemiology 

3.  Bethesda,  Maryland 


Part  A 


Project  Title:  Epidemiologic  Studies  of  Illnesses  and  Microbial 
Experience  of  Junior  Village  Nursery  Children 

Principal  Investigator:  Dr.  Joseph  A.  Bell,  Dr.  Albert  Z.  Kapikian 

Other  Investigators:  Dr.  Francis  M.  Mastrota  and  Dr.  Robert  J.  Huebner 

Cooperating  Units:  Virus  and  Rickettsial  Diseases  Section,  LID, 

(Dr.  Chanock,  Dr.  Johnson),  Epidemiology  Laboratory 
Unit,  (Dr.  Rosen),  Oncolytic  and  Oncogenic  Virus  Unit, 
(Dr.  Rowe),  Virology  Section,  Perinatal  Research  Branch, 
NINDB  (Dr.  Sever),  Parasitic  Disease  Service,  LCI, 
NIAID  (Dr.  Beye),  Infectious  Disease  Service,  LCI, 
(Dr.  Utz),  Pediatrics  Service,  D.  C.  General  Hospital, 
(Dr.  Reichelderfer) 

Man  Tears  (Calendar  year  I960): 
Total:  62/8 
Professional:  19/8 
Otheri  U3/8 

Project  Description: 

This  is  a  long  term,  very  intensive  study  of  the  illness  and 
microbial  experiences  of  nursery  children  at  Junior  Village,  a  District 
of  Columbia  Welfare  Institution.  It  began  in  July  1955  and  provides 
research  material  for  many  projects,  e.g.  66-A,  66-B  and  others. 

(A)  Basic  Objectives:  The  general  objective  is  to  maintain  under  observa- 
tion a  population  group  suitable  for  epidemiologic  study  of  occurrence  of 
infection  and  disease  and  host-parasite-disease-relationships  as  they  occur 
naturally  in  this  group  and  as  they  can  be  altered  by  chemo  prophylaxis 
and  new  vaccines.  One  of  the  chief  interim  objectives  is  the  development 
of  epidemiologic,  clinical  and  laboratory  tools  and  methods  for  studying 
infectious  diseases.  A  concerted  effort  is  being  made  to  find  new  micro- 
biologic agents  which  cause  disease,  methods  of  identification  of  these 
agents,  modes  of  spread  and  methods  for  prevention  and  treatment  of  acute 
illnesses,  particularly  respiratory  illnesses.  The  study  is  designed  to 
shed  light  on  the  nature  and  scope  of  studies  which  should  be  pursued  in 
further  search  for  methods  of  control  of  acute  infectious  diseasea 
particularly,  the  large  mass  of  acute  respiratory  diseases,  including  the 
common  cold. 

Part  B  included  /T7  Tea    /   /  No  83 

1 


Serial  No.  NIAH)  66 

Methods  Employed  and  Patient  Material: 

The  study  children  are  located  in  Southwest  Washington,  D.  C.  The  daily 
population  is  now  close  to  130  white  and  negro  babies  six  to  I|0  months  of  age 
who  are  in  residence  in  Eisenhower  Cottage  and  the  Infirmary  for  domiciliary 
care.  The  mean  duration  of  residence  is  approximately  17  weeks  per  child. 
Children  with  illnesses  are  studied  either  in  their  domicile  or  the  infirmary 
at  Junior  Village,  or  the  Clinical  Center  at  N. I.H.,  or  at  the  D.  c.  General 
Hospital,  depending  upon  the  severity  of  illness  and  study  interest. 

A  full-time  pediatrician,  three  nurses,  and  four  nurses'  assistants 
maintain  constant  medical  surveillance  of  the  children,  record  rectal  tempera- 
tures twice  every  day,  collect  specimens  for  laboratory  study  and  prepare 
clinical  records  on  each  child  each  day,  regardless  of  whether  or  not  ill. 
Both  throat  and  anal  specimens  for  virus  study  are  collected  on  admission, 
and  three  times  weekly  from  every  child,  and  otherwise  when  indicated.  Blood 
specimens  are  collected  on  admission,  on  discharge,  six  weeks  after  admission, 
every  three  months  during  residence,  and  under  special  circumstances. 

Major  Findings: 

Epidemiologic  methods  have  been  devised  for  recognition  of  febrile 
departure  from  normal  health  and  illnesses  have  been  classified  as  (A)  ques- 
tionable fever,  (B)  definite  fever  with  clinical  findings  (FUO)  and  (C)  defi- 
nite illnesses,  i.e.,  fevers  associated  with  clinical  findings. 

Two  major  epidemics  of  interest  have  occurred  in  the  past  year  in  Junior 
Village. 

In  a  period  from  April  2k   to  May  13,  36  or  k0%   of  the  90  residents 
developed  pneumonia.  In  the  Eisenhower  Cottage  where  the  older  children 
resided,  16  (2$%)   of  the  65  children  contracted  pneumonia  while  in  the  infirm- 
ary where  the  younger  children  resided  20  (80$)  of  2$   developed  pneumonia. 
Respiratory  syncytial  virus  isolation  from  throat  swabs  was  associated  with  the 
pneumonia  illness  indicating  an  etiologic  relationship.  Also  91%   of  80  infants 
and  children  tested  showed  U-fold  or  greater  rises  to  RS  antigen  by  complement 
fixation  and/ or  neutralization  tests.  A  four-day  incubation  period  of  RS 
pneumonia  was  also  clearly  shown.  The  pneumonia  illness  was  severe  with  the 
average  of  the  highest  fever  in  each  patient  being  103° F. (Rectal)  and  with  the 
duration  of  a  fever  of  100. 6°F.  or  greater  being  about  k   days. 

An  interesting  outbreak  of  infectious  lymphocytosis  was  recognized  in 
August  I960  when  k   of  the  children  were  discovered  to  have  white  blood  counts 
over  60,000  per  cu.  mm  with  95%  lymphocytes  on  differential  smear.  This 
initiated  an  intensive  effort  to  study  the  epidemiology,  the  clinical  coarse, 
and  the  laboratory  aspects  of  the  disease.  White  blood  counts  were  done  once 
weekly  on  all  the  130  residents.  Appropriate  material  has  been  studied  in 
laboratory  animals  and  in  tissue  culture  in  attempts  to  f-j        Jologic 
agent.  Stools  have  been  tested  for  ova  and  parasites.  Patients  have  been 
admitted  to  the  Clinical  Center  and  D.  C.  General  Hospital  to  study  the 

90 


Serial  No.  NIAID  66 


clinical  findings.  Thus  far,  no  clinical  finding  except  for  the  lymphocytosis 
has  been  sigrificantly  associated  with  the  disease.  Over  25  children  have 
contracted  the  lymphocytosis  with  no  relationship  seen  to  age,  or  length  of 
time  in  residence  at  Junior  Village.  An  incubation  period  of  11-13  days  is 
postulated.  With  the  paucity  of  literature  on  the  subject  of  infectious 
lymphocytosis,  this  study  should  add  measurably  to  the  definition  of  the 
disease. 

Significance  to  3io-medical  Research  and  the  Program  of  the  Institute: 

This  project  provides  a  source  of  specimens  for  finding  new  causes  of 
illness,  for  studying  their  etiologic  significance  and  for  testing  methods 
of  control.  It  is  significant  that  long  term  Junior  Village  studies  of  host- 
parasite-disease-relationships  are  being  carried  out  on  the  bulk  of  diseases 
which  commonly  cause  misery  and  absenteeism  from  schools  and  industry.  The 
studies  are  of  such  a  nature  that  they  are  not  likely  to  be  conducted  by  other 
than  a  government  group.  The  studies  are  progressing  meticulously  and  fairly 
satisfactorily  to  the  investigators. 

Proposed  Course  of  Project: 

Clinical  epidemiological  observations  are  being  continued  together  with 
collection  of  throat  and  anal  specimens  one  to  three  times  weekly  for  virus 
study  and  collection  of  routine  blood  samples.  It  is  planned  to  continue  the 
study  for  as  long  as  it  continues  profitable. 


91 


Serial  No.  NIAID  66 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  B   Honors,  Awards,  and  Publications 

■**  A  Study  of  the  Hemadsorption  ( Para-Influenza)*  And  Other  Viruses 
in  Children  with  and  without  Respiratory  Disease 

Albert  Z.  Kapikian,  M.D.,  Robert  M.  Chanock,  M.D., 

Joseph  A.  Bell,  M.D.,  Dr.  P.H.,  Thomas  E.  Reichelderfer,  M.D.,  M.P.H., 

Robert  J.  Huebner,  M.D. 

**  Published  in  PEDIATRICS  August  1?60 


92 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Serial  No.     NIAID  66-A 

1.  Infectious  Diseases 

2.  Epidemiology 

3.  Bethesda,  Maryland 


Part  A 


Project  Title:  Epidemiologic  Studies  of  Host  Parasite  Disease  Relation- 
ships 

Principal  Investigator:  Dr.  Joseph  A.  Bell 

Other  Investigators:  Dr.  Francis  M.  Mastrota,  Dr.  Albert  Z.  Kapikian, 
Dr.  Robert  J.  Huebner 

Cooperating  Units:  Virus  and  Rickettsial  Diseases  Section,  LID, 

(Dr.  Chanock,  Dr.  Johnson),  Epidemiology  Laboratory 
Unit,  (Dr.  Rosen),  Oncolytic  and  Oncogenic  Virus 
Unit,  (Dr.  Rowe) 

Man  lears (Calendar  year  I960): 
Total:  15/8 
Professional:  5/8 
Other:  10/8 

Project  Description: 

(1)  Objectives:  To  delineate  which  of  the  many  microbial  agents  are 
causing  acute  illness  in  Junior  Village  nursery  and  describe  clinical 
and  epidemiological  nature  of  etiologic  entities  found. 

Patient  Material: 

The  Junior  Village  nursery  studies  are  described  in  project  number  66. 
Epidemiologic  methods  are  being  devised  to  determine  which  of  the  53  typed 
viruses  and  the  many  typed  bacteria,  which  have  been  isolated,  were  causing 
illness. 

The  high  frequency  of  illness  occurrence  (mean  weekly  definite  ill- 
ness attack  rates  of  25  %)   and  the  even  higher  frequency  of  new  and  over- 
lapping infections  with  potentially  pathogenic  microbial  agents  presents 
an  extremely  complex  epidemiological  problem  to  establish  specific  etio- 
logic relationships.  One  of  the  chief  problems  is  serological  determina- 
tion of  susceptibility  and  immunity  to  the  many  specific  infections,  or 
illness  attributable  thereto;  with  the  limited  amounts  of  serums  available. 
To  guide  the  most  profitable  use  of  these  serums  and  for  other  purposes 
preliminary  analyses  of  temporal  relationships  between  onset  of  specific 
infection  and  onset  of  acute  undifferentiated  illnesses  have  been 

Part  B  included   /  J  Yes       /"*~7  u0  q « 


Serial  No,   NIAH)  66-A 

completed  and  top  priority  for  use  of  available  serums  can  be  established 
on  the  basis  of  resolving  the  etiologic  relationship  of  the  more  serious 
illnesses,  study  of  the  many  newly  discovered  viruses  and  study  of  new 
vaccines.  Within  these  limits  the  meticulous  and  time  consuming  analysis 
of  specific  etiologic  relationships  is  in  progress. 

Major  Findings; 

The  preliminary  analyses  of  temporal  relations  between  onset  of 
specific  infection  and  onset  of  acute  undifferentiated  febrile  illness 
using  both  horizontal  and  cross  sectional  controls  have  shown  no  evidence 
that  the  following  agents  were  causing  illness  in  the  nursery  babies: 
Adenovirus  2j  Polio  3;  Coxsackie  B  5;  ECHO  7,  8,  11,  12,  13,  lU,  18,  19, 
20,  25  and  JV  5;  nontypable  Group  A  Streptococci;  Hemophilus  Influenza  B} 
Pneumonia  Type  19,  23 J  Staphylococci  having  Phage  Type  81  in  their  anti- 
genic pattern;  Enteropathogenic  coli  of  various  types,  and  alkalescens 
dispar.  Infection  with  the  following  agents  were  significantly  associated 
with  the  occurrence  of  acute,  febrile,  undifferentiated  illness;  Adeno- 
virus 1,  3,  and  5j  Influenza  virus  Asianj  parainfluenza  virus  1  and  3j 
Poliovirus  2,  Coxsackie  virus  B  3;  Group  A  Streptococci  types  ky   12,  23, 
and  1,  2,  and  5;  and  Shigella  Sonnei.  In  general,  it  is  crudely  estimated 
that  some  h$%   of  the  more  severe  illnesses  of  the  nursery  group  may  be 
accounted  for  by  the  above  agents  and  that  measles  and  adenovirus  infec- 
tions account  for  the  bulk  of  the  more  severe  illness. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

Although  the  study  is  deliberately  limited  to  a  group  of  young 
children  who  have  a  high  proportion  of  susceptibles  to  various  microbial 
infections,  it  shows  some  of  the  potentialities  of  such  infections  in 
older  susceptible  persons.  This  project  represents  a  systematic  approach 
to  determining  the  various  etiologies  of  acute  febrile  illnesses  so  as 
to  guide  research  on  development  of  methods  for  control. 

Proposed  Course  of  Project: 

It  is  planned  to  continue  the  study  for  as  long  as  continued  collec- 
tion of  data  and  data  analyses  appear  profitable. 


sh 


Serial  No.  NIAID  66-B 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


1.  Infectious  Diseases 

2.  Epidemiology 

3.  Bethesda,  Maryland 


Part  A 


Project  Title:  Epidemiologic  Studies  of  New  Vaccines  and  Chemoprophy- 
lactic  Agents 

Principal  Investigator:  Dr.  Joseph  A.  Bell 

Other  Investigators:  Dr.  Francis  M.  Mastrota,  Dr.  Albert  Z.  Kapikian, 
Dr.  Robert  J.  Huebner 

Cooperating  Units:  Virus  and  Rickettsial  Diseases  Section,  LID, 

(Dr.  Chanock,  Dr.  Johnson)  Epidemiology  Laboratory 
Unit,  (Dr.  Rosen),  Oncolytic  and  Oncogenic  Virus 
Unit,  (Dr.  Rowe) 

Man  Years  (Calendar  Year  I960) 
Total:  29/8 
Professional:  l*/8 
Other:  25/8 

Project  Description: 

Objectives:  (A)  To  design  and  have  prepared  monovalent  and  multlpolyvalent 
vaccines  with  inactivated  viruses  which  have  shown  evidence  of  producing 
important  illnesses.  (B)  To  carry  out  preliminary  trials  of  such  vaccines 
for  determination  of  antigenicity  and  untoward  reactions.  (C)  To  observe 
whether  such  vaccines  substantially  reduce  the  occurrence  of  infection 
or  illnesses  in  the  Junior  Village  Institution. 

Patient  Material: 

The  Junior  Village  nursery  studies  are  described  in  project  numbers 
66  and  66-A.  The  rather  intense  daily  clinical  observation  permits  an 
evaluation  of  vaccine  induced  reactions.  The  vaccines  which  have  been 
used  are:  (1)  Adenovirus  types  1,  2,  3,  and  5>  (2)  Adenovirus  types  3, 
h,   and  7;  (3)  Coxsackie  B  virus  types  1,  2,  3>  h,   and  5j  (U)  Rubeola 
virus;  (5)  Respiratory  vaccine  containing  12  virus  strains,  namely, 
monkey  kidney  cell  grown  influenza  Al,  A2,  and  B  viruses;  Adenovirus 
types  1,  3,  hy  5,   7j  Parainfluenza  virus  types  1,  2,  and  3j  (6)  Polio- 
virus  types  1,  2,  3  -  three  different  products;  (7)  Parainfluenza  1  and 
3  -  egg  grown.  In  addition,  a  daily  oral  dose  of  300,000  units  of 
benzathine  Penicillin  has  been  given  to  a  20$  random  sample 
ren.  The  vaccines  are  given  at  time  of  admission,  with  the  second 
three  weeks  later  and  a  third  dose  at  three  months.  The  vacc:.nes  are 

Part  B  included  /  J    Yes    [TJ    No  95 


Serial  No.  NIAID  66-B 


given  in  one  ml  doses  to  preselected  random  samples  of  all  children  admitted 
to  the  Junior  Village  nursery  group.  Blood  sera  for  antibody  studies  is 
collected  on  admission,  at  six  weeks,  and  every  three  months  thereafter  and 
at  discharge. 

Major  Findings; 

Analysis  of  the  previously  mentioned  products  is  in  progress  currently. 
Preliminary  analysis  showed  the  inactivated  rubeola  monkey  kidney  vaccine  to 
offer  no  protection  in  preventing  rubeola.  Serologic  studies  on  this  vaccine 
are  in  progress. 

The  respiratory  vaccine  containing  12  virus  strains  did  not  alter  the 
illness  pattern  in  those  receiving  it  with  the  exception  of  a  period  in 
January  1959  when  Asian  influenza  was  prevalent  at  Junior  Village.  At  that 
time,  those  children  who  had  received  the  vaccine  had  significantly  less 
illness  than  those  without  the  vaccine.  The  ability  of  the  vaccine  to 
produce  CF  antibody  to  Asian  influenza  antigen  was  shown.  Differences  between 
vaccinees  and  controls  at  other  times  of  the  year  were  not  evident. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute; 

It  is  obvious  that  the  many  acute  febrile  respiratory  diseases  and  fevers 
of  undetermined  origin  which  are  a  major  public  health  problem  are  caused  by 
a  great  multiplicity  of  etiologic  agents.  To  prevent  or  ameliorate  these 
illnesses  by  vaccine  prophylaxis  requires  a  multipolyvalent  vaccine  in  as 
much  as  no  one  of  these  known  agents  are  producing  a  very  large  proportion 
of  these  diseases.  It  seems  timely  to  initiate  controlled  epidemiologic 
studies  in  the  use  of  multivalent  vaccines  and  their  possible  enhancement  or 
interference  with  each  other. 

Proposed  Course  of  Project; 

It  is  planned  to  continue  the  study  for  as  long  as  collection  of  data 
and  data  analyses  appear  profitable. 


96 


Serial  No.    NIAID  66-C 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


1.  Infectious  Diseases 

2.  Epidemiology 

3.  Bethesda,  Maryland 


Part  A 


Project  Title:  The  Etiology  of  Respiratory  Disease  on  a  College  Campu^ 

Principal  Investigator:  Dr.  Albert  Z.  Kapikian 

Other  Investigators:  Dr.  Karl  Johnson,  Dr.  Robert  Chanock,  Dr.  Joseph  Bell 
Dr.  Robert  Huebner 

Cooperating  Units:  University  of  Maryland  Infirmary 

Man  Years  (Calendar  Year  I960) 
Total:  0/8 
Professional:  0/8 
Other:  0/8 

Project  Description: 

Objectives :  (1)  To  determine  the  role  of  known  viruses  as  etiologic 
agents  in  a  young  adult  population;  (2)  To  find  new  agents  which  might  be 
associated    etiologically  with  respiratory  disease. 

Patient  Material: 

With  the  cooperation  of  the  University  of  Maryland  Infirmary,  a  study 
was  undertaken  in  October  1958  to  determine  the  role  of  viruses  in  the 
etiology  of  respiratory  diseases  in  a  young  adult  population.  Patients 
who  appeared  with  complaints  referable  to  their  respiratory  tract  were 
examined.  Throat  swabs  and  acute  and  convalescent  bloods  were  also 
obtained.  An  attempt  to  have  a  control  group  of  nonrespiratory  patients 
was  unsuccessful.  The  throat  specimens  were  tested  for  viruses  in  appro- 
priate tissue  culture  and  the  blood  was  tested  for  CF  antibody  against  a 
battery  of  respiratory  virus  antigens. 

Major  Findings: 

The  finding  of  a  new  parainfluenza  virus  type  h  was  presented  previously 

Two  hundred  twenty-four  patients  with  respiratory  disease  were  seen-- 
among  these,  approximately  20$  had  fever  of  99°F.  or  greater.  Virus 
isolation  attempts  were  markedly  unsuccessful,  possibly  a  result  of 
problems  of  storing  of  specimens  enroute  from  Maryland  University  to  N.I.B. 
However,  paried  bloods  were  obtained  from  129  of  the  total.  Thirty  or  2 

Part  B  included  /  J  Yes      /  x    /   No  Q  "J 


Serial  No.  NIAID  66-C 


of  this  latter  group  showed  a  four-fold  increase  tc  one  of  the  following 
respiratory  viral  antigens  by  CF  test:  Parainfluenza  1,  3»  1»,J  Adenovirus; 
Influenza  A  and  Bj  Respiratory  Syncytial  Virus.  Of  the  30  showing  CF  antibody 
rises  over  one-half  ($2%)   were  to  Influenza  B,  17%   to  Respiratory  Syncytial, 
17%   to  Parainfluenza  1,  and  3%   to  each  of  the  other  antigens.  The  illnesses 
of  the  patients  with  the  rises  to  each  of  the  antigens  was  not  associated 
with  any  specific  clinical  syndrome. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

This  project  attempted  to  show  the  etiology  of  respiratory  diseases 
with  respect  to  viruses.  It  is  seen  clearly  that  serologically,  only  23%   of 
the  respiratory  diseases  could  be  associated  with  viruses  and  if  this  had  been 
a  year  when  influenza  B  was  not  prevalent,  the  figure  might  have  been  even 
lower.  The  task  of  further  attempting  to  find  those  agents  which  are  causing 
viral  respiratory  diseases  is  apparent. 

Proposed  Course  of  Project: 

There  are  no  plans  to  renew  this  study  at  the  present  time. 


98 


Serial  No.  NIAID  66-D 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


1.  Infectious  Diseases 

2.  Epidemiology 

3.  Bethesda,  Maryland 


Part  A 


Project  Title:  The  Role  of  Viruses  in  the  Etiology  of  Ear  Disease 

Principal  Investigator:  Dr.  Albert  Z.  Kapikian 

Other  Investigators:  Dr.  Joseph  A.  Bell,  Dr.  Robert  J.  Huebner 

Cooperating  Units:  Washington  Hospital  Center,  Ear,  Nose  and  Throat 
Department 

Man  Years  (Calendar  Year  I960) 
Total:  0/8 
Professional:  0/8 
Other:  0/8 

Project  Description: 

For  many  years,  it  has  been  stated  that  viruses  were  responsible  for 
many  nonbacterial  ear  infections  such  as  serous  otitis  media.  It  was  attempted 
to  study  patients  with  serous  otitis  media  to  determine  the  role  of  viruses  in 
this  disease. 

Patient  Material: 

Patients  at  the  Washington  Hospital  Center  ENT  clinic  with  serous 
otitis  media  were  examined.  Fluids  from  the  middle  ear  were  tested  in  appro- 
priate tissue  culture  lines.  Acute  and  convalescent  bloods  were  obtained. 

Major  Findings: 

Thus  far  only  a  limited  number  of  specimens  from  the  ear  have  been 
tested  with  no  virus  yet  isolated.  Serologic  tests  on  the  sera  are  in  progress. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

The  role  of  viruses  in  ear  disease  is  uncertain.  This  project  attempts 
to  clarify  their  role  in  the  etiology  of  middle  ear  disease,  especially  serous 
otitis  media. 

Proposed  Course  of  Project: 

It  is  planned  to  renew  attempts  to  obtain  appropriate  specimens  dur- 
ing the  year.  J 

Part  B  included  /    J  Yes         /  x  /  No  93 


Serial  No.  NIAID  66-E 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


1.  Infectious  Diseases 

2.  Epidemiology 

3.  Bethesda,  Maryland 


Part  A 


Project  Title:  A  Study  of  the  Influence  of  Influenza  A  and  B  Mineral  Oil 

Adjuvant  Vaccines  on  Influenza  Antibody  Patterns,  8-9  Years 
after  Vaccination 

Principal  Investigator:  Dr.  Joseph  A.  Bell 

Other  Investigator:  Dr.  Robert  J.  Huebner 

Man  Years  (Calendar  Year  I960) 
Total:  0/8 
Professional:  0/8 
Other:  0/8 

Project  Description: 

Objectives:  (1)  To  see  if  Mineral  Oil  Adjuvant  Influenza  A  and  B  vaccines 
influenced  influenza  antibody  patterns,  8-9  years  after  vaccination. 

Patient  Material; 

Approximately  100  N.I.K.  personnel  were  given  one  dose  of  a  mineral 
oil  adjuvant  influenza  vaccine  between  1951  and  1952.  Twenty-five  hundredths 
ml  of  a  100  CCA  unit  vaccine  was  administered.  Influenza  type  A  and  B  products 
were  assigned  by  a  strictly  random  sampling  process  so  that  any  follow-up  of 
the  group  would  give  two  strictly  comparable  groups  of  near  equal  size,  each 
of  which  had  received  one  dose  of  either  type  A  or  type  B  Influenza  adjuvant 
product  8-9  years  previously.  The  A  and  B  groups  should  have  remained  compara- 
bly equal  (within  the  range  of  sampling  variation)  with  respect  to  all  attri- 
butes including  influenza  infection,  disease,  vaccination,  antibody  response, 
etc.,  except  for  the  influence  of  the  different  study  vaccines  given.  The 
100  N.  I.H.  personnel  who  were  given  1  of  the  li  adjuvant  products  and  who  were 
all  pre-bled  before  vaccination  are  in  the  process  of  being  bled  currently. 
Hemagglutination  inhibition  tests  are  to  be  done  to  see  if  the  adjuvant  vaccines 
given  8-9  years  ago  are  still  influencing  the  antibody  pattern. 

Major  Findings:  None 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

The  study  attempts  to  show  the  effectiveness  of  mineral  oil  adjuvant 
vaccines  in  producing  long  lasting  immunity.  This  may  be  of  importance  in 
future  vaccine  programs. 

Part  B  included  /x~7  Yes     1    /  J  No  *^ 


Serial  No.  NIAID  66-E 

Proposed  Course  of  Project; 

No  further  field  work  is  contemplated  currently,  but  the  laboratory 
analysis  will  be  carried  on. 


101 


Serial  No.      NIAID  66-E 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  B 


Honors,  Awards  and  Publications 


Bell,  J. A.,  Craighead,  J.E. ,  and  James,  R.G. 

Epidemiologic  Observations  on  Two  Outbreaks  of  Asian  Influenza  in  a 
Children's  Institution 

American  Journal  of  Hygiene  -  Jan.  1961 


Philip,  R.N.,  Bell,  J. A.,  Davis,  D.J.,  Beem,  M.O.,  Beigelman,  P.M., 
Engler,  J. I.,  Mellin,  G.W.,  Johnson,  J.H.,  Lerner,  A.M. 

Epidemiological  Studies  on  Influenza  in  Familial  and  General  Population 
Groups   2.  Characteristics  of  Occurrence 

American  Journal  of  Hygiene  -  Mar.  1961 


Davis,  D.J.,  Philip,  R.N.,  Bell,  J. A.,  Vogel,  J.E.,  Jensen,  D.V. 

Epidemiological  Studies  on  Influenza  in  Familial  and  General  Population 
Groups   3.  Laboratory  Observations 

American  Journal  of  Hygiene  -  Mar.  1961 


Bell,  J. A.,  Philip,  R.N.,  Davis,  D.J.,  Beem,  M.O.,  Beigelman,  P.M., 
Engler,  J.I.,  Mellin,  G.W.,  Johnson,  J.H.,  Lerner,  A.M. 

Epidemiological  Studies  on  Influenza  in  Familial  and  General  Population 
Groups   U.  Vaccine  Reactions 

American  Journal  of  Hygiene  -  Mar.  1961 


102 


Part  A 


Serial  No.   NIAIO  -  67 


PHS-NIH 

Inoivioual  Project  Report 

Calenoar  Year  1960 


Project  Title: 


Studies  on  human  enteroviruses,  adenoviruses,  and 
reoviruses 


Principal  Investigator:   Dr.  Leon  Rosen 

Other  Investigators:   Dr.  Joseph  A.  Bell,  Dr.  Robert  J.  Huebner,  Dr. 
Albert  B.  Sabin,  Dr.  D.  Mendez-Cash ion,  Mr. 
Jerome  Kern,  and  Mrs.  Janet  Hovis 

Cooperating  Units:   D.  C.  Welfare  Department;  Children's  Hospital 

Research  Foundation,  Cincinnati,  Ohio;  School  of 
Medicine,  University  of  Puerto  Rico,  San  Juan. 

Man  Years:   (Calendar  year  i960) 

Total:  72/12 
Professional:  32/12 
Other:        40/12 

Project  Description: 

Object  i  ves:   To  determine  the  role  in  human  biology  of  entero- 
viruses, ADENOVIRUSES,  AND  REOVIRUSES.   To 
ELUCIDATE  THE  NATURAL  HISTORY  OF  THESE  VIRUSES. 


Methods  Employed! 


The  principal  population  group  under  study 
is  the  Junior  Village  nursery  group  which 
has  been  described  in  detail  in  previous  re- 
PORTS of  the  Epidemiology  Section. 


Recently,  a  collaborative  study  was  institu- 
ted with  the  Department  of  Pediatrics  of  the 
University  of  Puerto  Rico.   In  this  study, 
specimens  from  children  with  a  variety  of 
acute  neurological  disoroers  will  be  stuoied 
IN  Bethesda. 

A  COLLABORATIVE  STUDY  WAS  ALSO  UNDERTAKEN 

with  Dr.  A.  B.  Sabin  of  the  Children's  Hos- 
pital Research  Foundation  of  Cincinnati. 
Dr.  Sabin  has  supplied  several  thousand  non- 
polio  ENTEROVIRUS  ISOLATES  FROM  MEXICO. 


Part  8   Included  Yes 


103 


Serial  No.   NIAID  -  67 
Part  A  Methods  Employeo  (continued) 

These  are  being  studied  for  the  production  of  hemagglutinins  in  an 
attempt  to  obtain  hemagglut i n at  i ng  strains  of  virus  types  which  are 
not  now  known  to  have  this  useful  property. 

Collaborative  studies  with  other  institutions  outside  of  the  National 
Institutes  of  Health  and  with  various  intramural  laboratories  are  un- 
dertaken FROM  TIME  TO  TIME  AS  OPPORTUNITIES  ARE  PRESENTED. 

Major  Findings:   An  extensive  outbreak  of  infection  with  ECHO  type  3 
virus  occurred  in  the  Junior  Village  nursery  population.   This  is  the 
first  known  outbreak  of  infection  with  this  virus  -  the  prototype 
having  been  described  from  a  single  isolate.   preliminary  analysis  of 
data  indicates  that  infection  with  this  agent  was  associated  with  a 
mild  febrile  disease. 

Hemagglutinins  were  found  for  the  first  time  for  ECHO  virus  type  21, 

COXSACKIE  B  VIRUS  TYPES  1  AND  5t  *ND  A  PREVIOUSLY  UNRECOGNIZED  ENTERO- 
VIRUS (JV-9)  WHICH  IS  CYTOPATHOGEN IC  ONLY   IN  HUMAN  TISSUE  CULTURES. 

Simple  H-l  procedures  were  developed  for  identifying  enteroviruses 

AND  ALSO  FOR  PERFORMING  SEROLOGIC  TESTS  WITH  SERA  FROM  PERSONS  SUS- 
PECTED OF  HAVING  ENTEROVIRUS  INFECTIONS. 

TWO  NEW  ENTEROVIRUS  SEROTYPES  (iN  ADDITION  TO  THE  PREVIOUSLY  REPORTEO 
JV-1)  HAVE  BEEN  CHARACTERIZED  AND  ACCEPTED  FOR  NUMBERING  BY  THE  ENTERO- 
VIRUS Committee.   Several  probably  new  serotypes  are  currently  under 

STUDY. 

Four  new  adenovirus  serotypes  have  been  characterized.   Simple  sero- 
logic PROCEDURES  WERE  DEVELOPED  FOR  IDENTIFYING  ADENOVIRUSES  AND  FOR 

performing  serologic  tests  with  sera  from  persons  suspected  of  having 
adenovirus  infections. 

Hemagglutination  of  rhesus  erythrocytes  by  measles  virus  was  demon- 
strated AND  A  SIMPLE  H-l  TECHNIQUE  FOR  WORK  WITH  THIS  VIRUS  WAS 

developed. 

Significance  to  the  Program  of  the  Institute;   The  role  of  the  numerous 
enteroviruses,  adenoviruses,  and  reoviruses  in  human  biology  is  only 
beginning  to  be  studieo.   data  obtained  thus  far  indicate  that  a  num- 
ber of  these  viruses  are  quite  important  in  human  pathology.   further 
progress  in  this  field  depenos  to  a  great  extent  on  the  development  of 
simple  laboratory  procedures  to  cope  with  the  more  than  90  viruses  now 
known  to  belong  to  these  groups. 

Proposed  Course  of  the  Project;   The  study  of  these  three  families  of 
viruses  will  be  continued  both  in  the  field,  as  opportunities  are  pre- 
sented, and  in  the  laboratory.   laboratory  studies  will  be  devoted  pri- 

Part  B   Included  Yes  inii 


Part  A  Proposed  Course  of  the  Project  (continued) 


Serial  No.   NIAID  -  67 


marily  to  the  orderly  classification  of  existing  ano  newly  recognized 
serotypes  and  to  the  development  of  simple  in-vitro  techniques. 


Part  B   Included  Yes 


105 


Serial  No.   NIAIO   -  67 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 

Part  B   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

1.  Rosen,  L.:   Serologic  Grouping  of  Reoviruses  by  Hemagglutinati on- 
Inhibition.   Amer.  J.  Hyg.,  71:2^2-2^9,  1960 

2.  Rosen,  L.,  Hovis,  J.  F.,  Mastrota,  F.  M. ,  Bell,  J.  A.,  and 
Huebner,  R.  J.:   Observations  on  a  Newly  Recognized  Virus  (Abney) 
of  the  Reovirus  Family.   Amer.  J.  Hyg.,  71:258-265,  1960 

3.  Rosen,  L.,  Hovis,  J.  F.,  Mastrota,  F.  M».,  Bell,  J.  A.,  and 
Huebner,  R.  J.:   An  Outbreak  of  Infection  with  a  Type  1  Reovirus 
among  Children  in  an  Institution.   Amer.  J.  Hyg.,  71 • 266-27** » 
1960 

h.      Rosen,  L.:   A  Hemagglut inati on-I nh ib ition  Technique  for  Typing 
Adenoviruses.   Amer.  J.  Hyg.,  71:120-128,  i960 

5.  Rosen,  L.,  Bell,  J.  A.,  and  Huebner,  R.  J.:   Enterovirus  In- 
fections of  Children  in  a  Washington,  0.  C,  Welfare  Institution. 
In  "Viral  Infections  of  Infancy  and  Childhood,"  Rose,  H.  M. ,  ed., 
Hoeber-Harper,  New  York,  1960,  pp.  119-127 

6.  Rosen,  L.:   Hemagglutination  and  Complement  Fixation.   In  "Per- 
spectives in  Pediatric  Virology,"  Thirty-third  Ross  Conference 
on  Pediatric  Research,  Cramblett,  H.  G. ,  eo.,  Columbus,  Ohio, 
Ross  Laboratories,  1959»  pp«  53-56 

7.  Philipson,  L.  and  Rosen,  L.:   Identification  of  a  Cytopathogen ic 
Agent  called  U-Virus  Recovered  from  Patients  with  Non- 
Diphtheritic  Croup  and  from  Day-Nursery  Chiloren.   Archiv.  fur 
die  Gesamte  Vi  rusforschung,  9_;25-30,  1959 

(Not  included  in  1959  report) 

8.  Rosen,  L.:   Hemagglutination  and  Hemagglutinati on-I nhibition 
with  Measles  Virus.   Accepted  for  publication  in  Virology. 

Honors  and  Awards  relating  to  this  project: 

1.  Visiting  lecturer  -jn  Epidemiology,  School  of  Public  Health, 
University  of  California 

2.  Appointeo  to  the  Enterovirus  Committee  of  the  National  Cancer 
Institute 


106 


Serial  No.   NIAID  -  67A 


1.  Infectious  Oiseases 
PHS-NIH  2.  Epidemiology 

Individual  Project  Report      3.  Bethesda,  Maryland 
Calendar  Year  i960 

Part  A 

Project  Title:   Studies  on  viruses  of  the  enteric  tract  of  cattle 

Principal  Investigators:   Dr.  F.  R.  Abinanti  and  Or.  Leon  Rosen 

Other  Investigators:   None 

Cooperating  Units:   Dairy  Husbandry  Department,  University  or 

Maryland;  Home  of  Correction,  Jessup,  Maryland 

Man  Years:   (Calendar  year  1960) 

Total:  6/12 
Professional:  3/12 
Other:        3/12 

Project  Description: 

Objectives:   To  investigate  the  biologic  properties  and  nat- 
ural history  of  the  viruses  inhabiting  the  enteric 
tract  of  cattle 

Methods  Employed:   A  longitudinal  study  of  dairy  cattle  on 

three  different  farms  has  been  carried  out 
for  approximately  two  years.  monthly  fecal 
swabs  and  serum  specimens  are  obtained  from 
each  animal  and  these  are  studied  in  the  lab- 
oratory by  a  variety  of  virus  isolation  and 
serologic  procedures. 

Major  Findings:   Reoviruses  of  three  different  types  serologic- 
ally INDISTINGUISHABLE  FROM  THE  THREE  TYPES 

found  in  humans  have  been  recovereo  on  numer- 
ous occasions  during  this  study.  calves  have 
been  infected  with  each  of  the  three  types  of 
human  origin.  no  evidence  of  disease  was  noted 
in  either  the  naturally  or  experimentally  in- 
fected animals. 

An  apparently  new  hemadsorbing  virus  was  re- 
covered FROM  CATTLE  ON  ALL  THREE  FARMS.   THE 
CHARACTER  OF  THE  HEMADSORPTION  PRODUCED  BY  THIS 
VIRUS  APPEARS  TO  BE  DIFFERENT  FROM  THAT  OF  ANY 
OF  THE  OTHER  HEMADSORBING  VIRUSES. 


Part  8   Incluoed  Yes 


107 


Serial  No.   NIAIO  -  67A 


Part  A  Major  Findings  (continued) 

Several  hundred  isolates  of  enteroviruses  have  been  recovered  from 

CATTLE   IN  THIS  STUDY.    The  RELATIONSHIP  OF  THESE  VIRUSES  TO  EACH 

other  and  to  the  enteroviruses  of  man  is  currently  under  study. 

Significance  to  the  Program  of  the  Institute:   The  recovery  of  vi- 
ruses  (such  a,s  reoviruses)  from  cattle  which  are  ant  i  g  en  i  c  ally 
identical  with  those  which  occur  in  man  is  of  obvious  interest. 
Aside  from  the  possible  importance  of  cattle  as  potential  sources  of 
infection,  a  number  of  aspects  of  the  natural  history  of  these  agents 

CAN  BE  CLARIFIED  BY  THEIR  STUDY  IN  LOWER  ANIMALS.    In  ADDITION,  VI- 
ruses recovered  from  cattle  which  are  analogous,  but  not  identical, 
to  those  of  humans  also  proved  a  mechanism  for  gaining  useful  infor- 
mation about  the  latter  agents. 

Proposed  Course  of  the  Project;   The  animals  in  the  longitudinal 

study  will  be  followed  for  about  another  six  months.   however,  a 

considerable  amount  of  laboratory  work  will  remain  to  be  finished 
after  this  time. 


Part  B   Included  Yes 


108 


Serial  No.   NIAID  -  67A 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B  Honors,  Awards,  ano  Publications 

Publications  other  than  abstracts  from  this  project: 

Rosen,  L.  and  Abinanti,  F.  R.:   Natural  and  Experimental  In- 
fection of  Cattle  with  Human  Types  of  Reoviruses.   Amer.  J.  Hyg., 
71:250-257,1960 


103 


Serial  No.   NIAID  -  67B 


1.  Infectious  Diseases 

PHS-NIH  2.  Epidemiology 

Individual  Project  Report       J>.  Bethesda,  Maryland 
Calendar  Year  1960 


Part  A 


Project  Title:   Studies  on  the  etiology  of  eosinophilic  meningitis 
in  French  Polynesia 

Principal  Investigator:   Dr.  Leon  Rosen 

Other  Investigators:   None 

Cooperating  Units:   School  of  Medicine,  University  of  California,  Los 
Angeles  and  Institut  de  Recherches  Medicales  oe  la 
Polynesie  Franchise,  Papeete,  Tahiti 

Man  Years:   (Calendar  year  i960) 

Total:  V12 
Professional:   3/12 
Other:   1/12 

Project  Description: 

Objecti ves:   To  determine  the  etiology  of  eosinophilic 
meningitis 

Methods  Employed:   A  comprehensive  epidemiologic  study  was  undertaken 
of  this  unusual  disease  which  occurred  on  tahiti. 
Blood,  spinal  fluid,  and  stool  specimens  were  col- 
lected FOR  A  VARIETY  OF  LABORATORY  STUDIES. 

Major  Findings:   Beginning  in  March  1958  many  hundreds  of  cases  of  an 
unusual  type  of  meningitis  of  unknown  etiology  oc- 
curred on  the  island  of  Tahiti  in  French  Polynesia. 
Since  the  most  characteristic  feature  of  this  menin- 
gitis WAS  A  PLEOCYTOSIS  CONSISTING  IN  LARGE  PART  OF 
EOSINOPHILS,  THE  DISEASE  WAS  CALLED  EOSINOPHILIC  MEN- 
INGITIS. 

The  MOST  CHARACTERISTIC  clinical  FEATURES  of  THE  DIS- 
EASE WERE  HEADACHE,  STIFFNESS  OF  THE  NECK  AND  BACK, 
AND  PARESTHESIAS  OF  DIFFERENT  TYPES.    APPROXIMATELY 
FIVE  PERCENT  OF  CASES  HAD  A  FACIAL  PARALYSIS  OF  THE 
PERIPHERAL  TYPE.    ThE  DURATION  OF  ILLNESS  VARIED  FROM 
SEVERAL  DAYS  TO  SEVERAL  MONTHS  AND  REOCCURRENCES  WERE 
COMMON.    No  DEATHS  OCCURRED. 


Part  B   Incluoed  Yes 


110 


Serial  No.   NIAID   -  67B 

Part  A  Major  Finqings  (continued) 

More  than  one-half  of  the  cases  had  a  pleocvtosis  of  500  °"  more 
cells  per  cu.  mm.  of  cerebrospinal  fluid,  and  in  8^  percent  of  all 
cases  eosinophils  accounted  for  more  than  one-fourth  of  the 
pleocytosis. 

Examinations  of  spinal  fluid,  blood,  and  feces  were  negative  for  the 
presence  of  viruses  and  pathogenic  bacteria  and  fungi.   no  helminthic 
parasites  were  found  other  than  those  commonly  infesting  the  inhabit- 
ants of  the  area.   serologic  examination  of  convalescent  sera  against 
a  number  of  different  arthropod-borne  viruses,  enteroviruses,  lepto- 
spira,  and  other  microbial  agents  failed  to  provide  an  indication  of 
the  etiologic  agent. 

the  disease  occurred  primarily  in  adults  and  affected  both  sexes 
equally.   Persons  of  Polynesian  or  part-Polynesian  origin  appeared  to 
have  a  higher  attack  rate  than  europeans  or  chinese. 

There  was  no  sharp  seasonal  distribution  and  cases  occurred  in  every 
month  of  the  year. 

The  geographic  distribution  of  cases  corresponded  roughly  to  the  dis- 
tribution OF  THE  POPULATION  AND  THERE  WAS  NO  DEFINITIVE  EVIDENCE  OF 

a  higher  attack  rate  in  either  rural  or  urban  areas. 

no  evidence  was  found  to  suggest  transmission  of  the  disease  from  per- 
son to  person  or  from  one  geographic  area  to  another.   the  aggregation 
of  cases  by  household  suggested  the  effect  of  a  common  exposure  than 
person-to-person  transmission. 

The  incubation  period  of  the  disease  was  estimated  to  be  between  two 
and  four  weeks. 

Although  the  etiology  of  the  disease  was  not  determined,  the  sum  of 
the  clinical  ano  epidemiologic  evidence  suggested  the  hypothesis 
that  the  oisease  was  caused  by  a  helminthic  parasite  of  the  oceanic 

BONITO  OR  SKIPJACK  TUNA  (KATSUWONUS  PELAMI s)  WHICH   IS  COMMONLY  EATEN 

raw  in  the  area. 

Significance  to  Program  of  the  Institute:   Outbreaks  of  eosinophilic 
meningitis  have  occurred  in  new  caledonia  and  the  caroline  islands, 

AS  WELL  AS   IN  FRENCH  POLYNESIA.    It   IS  OBVIOUSLY  OF  GREAT  PRACTICAL 
INTEREST  TO  OETERMINE  THE  ETIOLOGY  OF  THIS  DISEASE   IN  ORDER  THAT  AP- 
PROPRIATE PREVENTIVE  MEASURES  CAN  BE   INSTITUTED.    FURTHERMORE,   IF. IT 
IS  SHOWN  THAT  THE  OISEASE  IS  INDEED  ACQUIRED  FROM  A  MARINE  FISH,  IT 
WILL  BE  THE  FIRST  INDICATION  THAT  A  PARASITIC  DISEAsE  OF  MAN  CAN  BE 
ACQUIRED  FROM  SUCH  A  SOURCE. 

Part  B   Included  Yes 

Ill 


Serial  No.   NIAIO  -  67B 


Part  A  (continued) 


Proposed  Course  of  Project;   Additional  field  studies  will  be  under- 
taken  early  in  1961  in  an  attempt  to  obtain  further  data  on  the 
etiology  of  the  disease  -  especially  with  regard  to  the  hypothesis 
that  the  disease  is  acquireo  from  eating  raw  fish. 


Part  B   Included  Yes 


112 


Serial  No.   NIAIO  -  67B 


PHS-NIH 
Inoivioual  Project  Report 
Calendar  Year  i960 

Part  B  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

None 

Honors  and  Awaros  relating  to  this  project: 

1.   Letter  from  Governor-General  of  French  Polynesia  to  Sur- 
geon General  of  the  U.S.  Public  Health  Service  expressing 
appreciation  for  the  studies  carried  out  on  Tahiti  by 
Dr.  Leon  Rosen. 


113 


Serial    No.   NIAID-68 

1.  Infectious  Oiseases 

2.  Virus  and  Rickettsial 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual    Project  Report 

Calendar  Year    I960 


Part  A 


Project  Title:  Studies  of  Tumor  Viruses  in  Nature. 

Principal  Investigators:  Dr.  Robert  J.  Huebner  and 

Dr.  Wal lace  P.  Rowe 


Other  Investigators: 


Dr.  Janet  W.  Hartley,  Mr.  William  T.  Lane 
and  Mr.  John  D.  Estes 


Cooperating  Units 


New  York  City  Health  Department 
Dr.  David  Johnson,  Smithsonian  Institute 
Dr.  E.  Baker,  Smithsonian  Institute 
Dr.  B.  Burmester,  Regional  Poultry 
Research  Laboratory,  Agricultural 
Research  Service,  East  Lansing,  Michigan 

Man  Years:   (Calendar  Year  I960) 
Total:  48/12 

Professional :     16/12 
Other:  32/12 

Project  Description: 

Ob jecti ves :  This  project  is  concerned  with  elucidating  natural 
behavior  of  tumor  viruses  in  their  natural  animal  hosts  in  natural 
environments.  We  have  devoted  most  of  our  attention  so  far  to  those 
viruses  for  which  survey  tools  have  been  developed,  namely,  polyoma 
and  papi I loma. 

Methods  Employedit  Survey  tools  -  direct  virus  demonstration 
and  isolation  techniques  plus  serologic  procedures  (CF,  HI,  MAP,  N 
tests)  are  evaluated  and  used  in  examining  specimens  collected  in 
field  studies  for  evidence  of  contamporary  and  prior  virus  infection. 


Part  B  incl uded  : 


Yes 


114 


Serial  No.  NIAID-68 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

Field  Studies :  The  natural  history  of  polyoma  virus  infection 
of  Mus  muscul us,  the  common  house  mouse,  is  under  study  in  a  densely 
populated  urban  area  (Harlem,  N.Y.C.),  and  in  a  rural  area  on  Maryland 
farms  and  grain  mills.  The  distribution  of  rabbit  papilloma  virus 
(independent  of  tumors)  in  cottontails  was  studied  in  an  area  known 
to  be  heavily  infected  (Kansas)  and  in  a  presumably  tumor-free  area 
(Maryland) . 

The  natural  occurrence  of  "VL"  virus  initially  reported 
as  a  tissue  culture  grown  avian  lymphomatosis  virus  was  determined 
by  serologic  surveys  of  commercial  and  experimental  populations. 

Specimens :  Mice  were  live  trapped,  weighed,  sexed,  bled, 
marked,  and  generally  released  (in  same  area  as  caught).  Tissues 
and  urine  were  collected  for  virus  isolations.  Environmental 
materials  contaminated  by  mouse  excreta  such  as  trash  and  grain 
were  collected  in  sterile  containers  for  virus  study.  Rabbits  and 
chickens,  both  with  and  without  tumors,  were  bled  and  the  sera 
studied  for  papilloma  and  "VL"  antibodies. 

Certain  Problems :  Both  virus  demonstration  and  serological 
techniques  required  extensive  evaluation  to  determine  the  reliability, 
accuracy  and  sensitivity  of  the  available  techniques.  For  polyoma 
serology  the  HI  test  was  shown  to  be  more  sensitive  than  the  CF 
test,  and  when  sera  was  treated  with  RDE  and  heat,  almost  entirely 
equivalent  to  the  more  cumbersome  neutralization  tests;  hence  it 
was  selected,  not  as  the  sole  serologic  survey  instrument,  but  as  the 
test  of  choice. 

The  most  severe  laboratory  problem  that  had  to  be  solved 
was  the  problem  of  spontaneous  infection  of  laboratory  mice  with 
laboratory  strains  of  polyoma  virus  in  our  Bethesda  laboratory; 
this,  plus  the  intrusion  of  other  extraneous  agents  in  the  mouse 
study  systems  (see  Project  NIAID-71  B)  invalidated  most  efforts  to 
demonstrate  virus  in  the  MAP  test,  our  most  sensitive  system.  To 
obtain  high  order  information  and  insure  bona-fide  field  isolations, 
we  eventually  were  required  to  set  up  field  laboratories  complete 
with  constant  monitoring  and  adequate  controls.  Fortunately,  we 
were  finally  able  to  obtain  isolations  of  virus  which  surely  originated 
in  the  field  specimens. 


115 


Serial    No.   NIAID-68 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

Major  Findings; 

Po I yoma :  Polyoma  was  found  to  be  widely  distributed  in 
natural  colonies  of  Mus  muscul us,  not  only  in  Harlem  but  on  livestock 
farms  and  grain  mills  furnishing  feed  for  livestock.  As  with  labora- 
tory mice,  virus  was  shown  to  be  excreted  in  saliva  and  excreta 
by  carrier  wild  mice.  Not  only  the  tenement  environments  and  community 
mouse  nests  in  Harlem,  but  even  the  cereal  grains  in  the  feed  mills 
were  found  contaminated;  presumably  the  farm  granaries  where  infected 
mice  also  abound  are  heavily  contaminated  as  well.  Polyoma  virus 
has  been  easily  recovered  from  mice  in  all  three  studies;  however, 
the  relative  extent  of  grain  and  environmental  area  contamination 
must  be  determined. 

Polyoma,  a  ubiquitous  and  persistent  natural  infection 
in  Mus  muscul us  has  now  been  studied  in  three  separate  ecologies  - 
laboratory  breeding  colonies,  urban  tenement  houses,  and  in  rural 
agriculture  establishments.  The  basic  cycle  in  nature  would  appear 
to  be  the  latter,  where  extensive  contamination  of  cereal  grains 
on  the  farm  and  the  feed  mill  not  only  explains  persistent  foci  of 
this  highly  resistant  virus  in  these  areas,  but  also  suggests  that 
the  wide  distribution  (and  occasional  sudden  outbreaks)  of  polyoma 
virus  infection  in  laboratory  colonies  are  induced  and  maintained 
by  uncooked  cereal  grains  commonly  fed  to  such  mice. 

Longitudinal  studies  continued  in  Harlem  tenements 
suggest  that  large  and  very  dense  populations  of  mice,  plus  con- 
taminated foci,  particularly  "community  nests",  are  the  chief 
factors  in  maintaining  continuous  infections  through  several  succeeding 
generations  of  mice.  Polyoma  virus  was  isolated  from  debris  and  dust 
in  one  Harlem  kitchen  cabinet  near  a  nesting  area  over  intervals 
exceeding  eight  months.  During  the  year  of  surveillance  in  New  York, 
negative  premises  tended  to  remain  negative,  and  positive  premises 
without  exception  remained  positive. 

Despite  wide  distribution  and  high  infection  rates,  the 
role  of  polyoma  virus  in  the  genesis  of  naturally  occurring  cancers 
in  mice  is  still  undetermined,  despite  the  fact  that  field  isolates 
are  quite  as  oncogenic  as  established  strains  when  given  to  infant 
laboratory  mice  and  hamsters.  Several  thousand  mice  have  now  been 
I ive  trapped  -  some  of  them  several  times  over  periods  of  several 
months.  To  date  only  one  naturally  occurring  tumor  has  been 
observed  -  a  mammary  tumor  in  a  mouse  negative  for  polyoma.  However, 
it  appears  that  few  wi  Id  mice  I ive  beyond  six  months  of  age  in  the 
urban  environment  where  polyoma  is  prevalent. 

116 


Serial    No.   NIAID-68 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

Additional  Findings:  These  studies  promise  to  provide 
interesting  information  on  the  natural  histories  of  other  latent 
viruses  of  Mus  muscul us  including  mouse  sal ivary  gland  virus,  K  virus, 
and  reovi ruses.   It  is  probable  as  we  intensify  our  laboratory  study 
of  field  specimens  that  other  latent  and  possibly  tumor  viruses  may 
come  to  I ight. 

Observations  on  the  ecology  of  Mus  muscul us  have  led  to 
interesting  findings.   In  the  grain  mill  and  the  feed  barn,  just  as 
in  the  tenement  house,  mice  tend  to  inhabit  the  upper  floors  presumably 
because  rats  are  more  common  in  basements  and  lower  floors;  female 
mice  are  more  numerous  than  males,  apparently  live  longer,  and  as 
adults  are  more  frequently  positive  for  polyoma.  The  latter  presumably 
can  be  explained  by  greater  exposure  of  females  to  infected  communal 
nesting  areas,  which  in  turn  is  due  to  the  much  greater  amounts  of 
virus  excreted  by  mice  infected  as  infants  (as  much  as  10  virus/ml 
of  urine  may  be  excreted  for  several  weeks). 

Rabbit  Papi I loma :  Differential  centrif ugation  combined  with 
several  washings  to  free  antibody  bound  papilloma  virus  and 
complement-fixing  antigens  in  cottontail  papillomas  obtained  from 
Kansas  provided  more  sensitive  tests  for  live  virus  and  CF  antibody. 
Complement-fixing  antibody  was  found  to  be  present  in  50  Kansas 
rabbits  carrying  visible  papillomas;  Kansas  rabbits  free  of 
papillomas  were  most  often  negative  but  occasionally  they  also  were 
positive  in  the  CF  test.  The  CF  antibodies  were  shown  to  correlate 
with  the  presence  of  neutralizing  antibodies  in  the  domestic  rabbit 
test.  Maryland  rabbits  are  reportedly  free  of  papilloma  and  indeed 
none  were  seen  on  some  30  rabbits  live  trapped  in  this  area.  To 
date  all  Maryland  rabbits  have  been  negative  in  the  papilloma 
complement  fixation  test. 

This  test  appears  to  be  very  sensitive  and  quite  specific, 
thus  permitting  surveys  for  virus  prevalence  independently  of  tumor 
production.  Thus  far,  unlike  polyoma  (which  it  resembles  in  many 
physical  and  biologic  traits)  papilloma  virus  would  seem  to  induce 
tumors  in  the  majority  (If  not  all)  Kansas  cottontail  rabbits  which 
are  successful ly  infected. 

Studies  of  modes  of  spread  -  a  search  for  attenuated  or  non- 
tumorigenic  strains  appears  feasible;  similarly,  studies  of  possible 
antigenic  relationships  with  papilloma  agents  of  other  species, Homo 
sapiens  included,  are  indicated. 


117 


Serial    No.   NIAID-68 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


"VL"  (GAL)  Virus:  This  tissue  culture  grown  virus  was  proven 
during  the  year  to  be  an  extremely  prevalent  extraneous  adenovirus- 
like  chicken  virus  and  not  the  agent  of  avian  visceral  lymphomatosis 
as  originally  reported  by  Burmester  and  Sharp  I  ess.  This  dismal 
discovery  was  made  simultaneously  by  Burmester,  Sharp  I  ess  in  labora- 
tory experiments,  and  by  our  own  sero-epizootio logic  studies  of 
the  prevalence  of  antibodies  to  this  agent  in  chicken  flocks. 
Virtually  all  commercial  flocks  and  90  per  cent  of  the  chickens  in 
such  flocks  revealed  neutralizing  antibody  to  the  "VL"  or  "GAL" 
virus  (Gal  I  us  adenovirus- 1  ike) .  Studies  of  isolated  outbreaks  of 
visceral  and  neural  lymphomatosis  revealed  that  while  most  were 
associated  with  GAL  infection,  some  typical  leukemia  outbreaks 
occurred  in  the  complete  absence  of  evidence  for  GAL  infection. 

Surveys  of  Burmester* s  15  I  strains  of  RPL-12  susceptible 
birds  revealed  that  nearly  all  of  them  had  antibodies  to  GAL  virus. 
Fortunately,  additional  surveys  have  revealed  a  substrain  of  15  I 
chickens  free  of  GAL  virus,  which  will  be  used  for  a  re-examination 
of  visceral  lymphomatosis  in  a  system  free  of  an  extraneous  virus 
possessing  many  of  the  properties  of  lymphomatosis  itself. 

Significance  to  the  Program  of  the  Institute:   In  LID  our  approach 
to  cancer  research  is  based  on  a  "biological"  instead  of  the  more 
prevalent  c I  inico-patho logic  point  of  view.  We  do  not  disdain  the 
use  of  laboratory  models  (we  employ  them  as  indicated);  but  our 
major  interest  is  conditioned  by  a  concept  which  we  think  is  funda- 
mental to  an  intelligent  and  logical  approach  to  the  study  of  cancer 
viruses.  We  take  it  as  axiomatic  that  if  a  cancer  is  caused  by  a 
specific  virus,  then  as  is  true  of  all  other  microbial  illnesses,  the 
microbe  is  the  central  issue  and  the  natural  behavior  of  such  a  virus 
the  most  needed  and  highest  order  information  achievable  on  the 
subject. 

This  project  has  already  developed  much  information  of  value 
in  defining  not  only  the  natural  behavior  and  the  basic  cycle  of 
polyoma  in  nature,  but  also  the  probable  sources  of  infection  in 
both  experimental  and  production  colonies. 


118 


Serial    No.   NIAID-68 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


The  demonstration  by  Eddy  and  Stewart  of  the  tumorigenic 
activity  of  polyoma  in  hamsters,  rats,  guinea  pigs,  and  rabbits 
reveals  an  alarming  capacity  for  polyoma  to  cross  species  barriers. 
Although  serologic  studies  do  not  support  definite  infection  of  man 
with  this  virus,  widespread  access  of  humans  through  mouse  contaminated 
environments  and  apparently  extensive  contamination  of  food-stuffs, 
renders  the  question  of  possible  human  infections  rather  more  than 
academic.  The  lack  of  serologic  correlation  with  human  cancer  such 
as  would  be  expected  in  mice  may  not  represent  conclusive  countei — 
evidence,  since  hamsters  with  polyoma  induced  tumors  lose  their 
antibodies  to  polyoma  rapidly  despite  the  persistence  of  eventually 
fata  I  tumors . 

Proposed  Course  of  the  Project:  The  natural  history  and  behavior 
of  polyoma  and  other  animal  tumor  viruses  are  important  subjects 
in  their  own  right  and  studies  of  them  represent  some  of  the  first 
extensive  efforts  to  develop  such  information  about  latent  viruses. 
As  noted  above,  there  can  be  no  higher  order  of  information  about 
infectious  agents  and  such  information  derived  from  careful 
exhaustive  longitudinal  studies  are  directly  relevant  to  the  question 
of  human  cancer  viruses. 

The  possibility  that  cancer  could  be  the  result  of  a  zoonotic 
infection  no  longer  appears  so  very  unlikely.  We  plan,  therefore, 
to  study  possible  polyoma  infection  of  man  and  other  animal  species, 
such  as  domestic  animals  (cattle,  hogs,  dogs,  and  cats),  or  other 
wild  rodents  likely  to  be  exposed  repeatedly  to  infection  with  known 
cancer  viruses  of  mice  (such  as  polyoma)  and  chickens. 


113 


Serial  No.  NIAID-68 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

Part  B   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Huebner,  R.J.:  Some  questions  about  possible  approaches  to 
research  on  viruses  as  a  cuase  of  cancer.  Cancer  Res.,  20 :  669-830. 
I960. 

Huebner,  R.J.:  Viruses  in  search  of  cancer.  Monograph  - 
PERSPECTIVES  IN  VIROLOGY  II,  I960. 

Huebner,  R.J.:  New  possibilities  in  virus  disease  control. 
Medical  Section  Proceedings  of  American  Life  Convention,  I960. 

Halonen,  P.  and  Huebner,  R.J.:  ECHO  and  poliomyelitis  virus 
ant i sera  in  guinea  pigs  with  f I uorocarbon-treated  cell  culture 
antigens.  Proc.  of  Soc.  for  Exper.  Biol,  and  Med.,  105  ;46-49,  I960. 

Honors  and  Awards  relating  to  this  project: 

Participated  in  Variety  Children's  Research  Foundation  Symposium, 
and  dedication  ceremonies  of  new  research  building;  talk:  "Viruses 
in  Children,  I960".  Miami,  Florida,  January  I960. 

Participated  in  the  Gustav  Stern  Symposium  Perspectives  in 
Virology  II.  Talk:  "Viruses  in  Search  of  Cancer."  New  York, 
New  York,  January  I960. 

Participated  in  NCI  Viruses  and  Cancer  Panel.  New  York,  New  York, 
January  I960. 

Invited  speaker,  Epidemiology  for  Veterinarians  course,  -  "New 
Horizons  in  Domestic  Animal  Virology."  Communicable  Disease  Center, 
Atlanta,  Georgia,  February  I960. 

Participated  in  International  Conference  on  Asian  Influenza. 
Discussant,  "Methods  of  Diagnosis."  National  Institutes  of  Health, 
Bethesda,  Maryland,  February  I960. 

Participated  in  Phenomena  of  Tumor  Viruses  Symposium,  New  York, 
New  York,  March  I960. 


120 


Serial    No.   NIAID-68 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

Invited  speaker,  Frederick  County  Tuberculosis  and  Public  Health 
Association,  Frederick,  Maryland.  April  I960. 

Invited  speaker,  American  College  of  Physicians  -  "Viruses  and 
Cancer."  San  Francisco,  California,  April  I960. 

Society  of  American  Bacteriologists.  Talk:  "Viral  Agents  in 
Relation  to  Tumors".  Philadelphia,  Pennsylvania,  May  I960. 

Annual  Health  Conference,  Inc.  Talk:  "The  Growing  Importance 
of  Virology  in  Public  Health.""  New  York,  New  York,  May  I960. 

Invited  speaker,  American  College  of  Obstetricians  and  Gynecologists  - 
"Virus  Infections  in  I960."  Cincinnati,  Ohio,  April  I960. 

Invited  speaker,  The  Medical  Section,  American  Life  Convention  - 
"New  Possi bi I i ties  in  Virus  Disease  Control ."  White  Sulphur  Springs, 
West  Virginia,  May  I960. 

Invited  speaker,  Kansas  Trudeau  Society  -  "Viral  Respiratory 
Diseases."  Kansas  City,  Kansas,  September  I960. 

Invited  speaker,  The  American  Academy  of  General  Practice  -  "What 
is  Non-Paralytic  Polio."  Kansas  City,  Kansas,  September  I960. 

Participant  in  discussions,  University  of  Illinois  Center  for 
Zoonoses  Research.   Urbana,  Illinois,  September  I960. 

Invited  speaker,  NCI  Staff  Conference  -  "Background  Noise  in 
Virus  Study  Systems."  National  Institutes  of  Health,  October  I960. 

Section  chairman,  Southwest  Section  of  American  Association  for 
Cancer  Research.  Talk:  "Viruses  as  a  Cause  of  Cancer."  Galveston, 
Texas,  October  I960. 

Invited  speaker,  Academy  of  Medicine  of  Cincinnati  -  "Viruses  and 
the  Common  Cold."  Cincinnati,  Ohio,  November  I960. 


121 


Serial    No.   NIAID-68 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

Invited  speaker,  The  Harvey  Society  -  "Cancer  as  an  Infectious 
Disease."  New  York,  New  York,  November  I960. 

Invited  speaker,  Chicago  Medical  School,  Abbott  Laboratories 
and  Marquette  University  -  "Respiratory  Disease  due  to  Viruses  - 
A  Comprehensive  View."  Chicago,  Illinois,  November  I960. 

Chairman  and  discussant,  AMA  Clinical  Session  Symposium  on 
Respiratory  Virus  Disease.  Washington,  D.C.,  November  I960. 


Elected  to  membership,  The  National  Academy  of  Sciences, 
April  I960. 


122 


Serial  No.  NIAIO  -  68-A 

1.  Infectious  Diseases 

2.  Virus  &  Rickettsial  Oiseases 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:   Sero-ep idem io logy  of  virus  infections. 

Principal  Investigator:  Or.  John  L.  Sever,  Dr.  Robert  J.  Huebner 

Other  Investigators: 


Cooperating  Units: 


Anita  Ley,  Flora  Wolman,  Renee  Traub, 
Joan  Austin 

NINDB,  Collaborative  study  on  cerebral 
palsy,  mental  retardation,  and  other  neuro- 
logical and  sensory  disorders  of  infancy 
and  childhood  (Dr.  Richard  Masland). 

MBA  -  Or.  Gabriel  Caste  llano 


Man  Years  (calendar  year  I960): 

Total:  74/12 
Professional:  38/12 
Other:        36/12 

Project  Description: 

Objectives:  To  utilize  available  serologic  technique  in  an 
intensive  study  of  newly  recognized  viruses  as  to  their  relation  to 
the  high  incidence  of  acute  undifferentiated  respiratory  diseases, 
chronic  diseases,  birth  defects,  and  cancer.  To  develop,  wherever 
technically  possible,  the  refinements  of  serologic  methods  necessary 
for  a  large  scale  investigation  of  the  natural  course  of  the  disease  as 
caused  by  viral  infections. 

Methods  Employed:   Serologic  techniques,  including  complement 
fixation  tests,  hemagglut inat ion- inh ibit ion,  hemadsorption,  viral 
neutralization,  and  tissue  culture  neutralization  tests  are  now 
developed  for  the  identification  of  over  100  viral  infections. 


Part  B  Included: 


No 


123 


Serial  No.  NIAIO  -  68-A 

Two  major  phases  of  this  project  have  been  pursued.  First,  the 
commercial  production  and  standardization  of  antigens  and  ant i sera 
suitable  for  the  performance  of  the  serological  tests.   Second,  the 
development  of  an  integrated  laboratory  facility,  employing  trained 
technicians,  capable  of  handling  large  scale  testing. 

Human  sera  for  viral  serologic  analysis  and  identification  of 
previous  viral  experiences  are  available  from  studies  of  common  acute 
undifferentiated  respiratory  diseases,  and  various  detailed  studies 
of  special  virus  disease  problems  of  current  interest  to  the  Laboratory 
of  Infectious  Diseases.  A  large  number  of  serial  bleedings  are  being 
obtained  from  mothers  during  the  course  of  pregnancy  and  from  infants 
4  months  after  birth  in  the  Collaborative  Study  on  cerebral  palsy, 
mental  retardation,  and  other  neurological  and  sensory  disorders  of 
infancy  and  childhood  of  NINOB.  These  sera  are  now  becoming  available 
for  testing. 

Major  Findings;  Complement  fixing  antigens  for  more  than  70 
viruses  have  now  been  prepared  by  Microbiological  Associates,   Inc., 
in  consultation  with  the  Virology  Section,  LID  and  NINDB.  The  antigens 
which  have  been  prepared  are  titered  in  both  laboratories  and  when 
found  acceptable  are  produced  in  quantities  of  100  to  1000  ml.  These 
include  complement-fixing  antigens  for:   Adenoviruses  (common  antigen), 
Coxsackie  A  and  B  viruses  (25  types),  Influenza  A,  B,  C,  mumps,  Para- 
influenza (4  types),  Pol  ioviruses  (3  types),  ECHO  viruses  (28  types), 
measles,  and  Herpes  Simplex  and  Respiratory  syncytial.  Viral  antigens 
for  hemagglutination  tests  have  also  been  produced  for  24  adenoviruses, 
4  parainfluenza  viruses,  respiratory  syncytial  virus,  3  Reoviruses, 
mumps,  salivary  gland  virus,  Q  Fever,  Psittacosis,  and  other  viruses 
which  are  utilized  in  routine  tests  in  our  laboratories. 

A  large  scale  program  for  producing  specific  immune  antisera  in 
human  volunteers  has  been  initiated  and  will  be  pursued  during  the 
next  calendar  year.  After  the  completion  of  safety  tests,  groups  of 
volunteers  will  be  given  purified  viral  antigens.  The  pedigreed  stock 
antisera  obtained  in  this  way  will  be  standardized  for  evaluating  the 
potency  of  antigen  preparations. 

The  application  of  a  micro  technique  to  both  hemagglutination  and 
complement  fixation  tests  will  permit  the  use  of  considerably  less 
sera  and  antigen  for  the  performance  of  the  various  tests.  The 
technique  has  now  been  refined  and  developed  to  the  point  where  it  may 
be  applied  in  lieu  of  standard  techniques  whenever  screening  information 
is  desired.  Through  the  use  of  highly  developed  spiral  loops,  accurate 
dilutions  may  be  made  rapidly  in  the  system.  The  technique  should  be 
of  particular  value  in  studying  the  serological  background  of  animals 
from  which  only  a  small  amount  of  sera  is  available. 


124 


Serial  No.  NIAIO  -  68-A 

Significance  to  the  Program  of  the  Institute:  The  availability  of 
reliable  standard  and  micro  serological  techniques  for  a  large  group  of 
new  viruses  provides  an  opportunity  to  investigate  the  course  of  human 
disease  caused  by  viruses  which  are   either  difficult  to  isolate  or  are 
resistant  to  evaluation  because  the  clinical  effects  are  delayed  until 
a  long  time  after  infection  has  subsided.  This  is  particularly  true  in 
the  case  of  birth  defects  and  animal  cancers.  The  application  of  this 
tool  for  analysis  should  provide  considerable  information  on  the 
epidemiological  aspects  of  virus  infections. 

The  program  conducted  in  association  with  the  NINDB  should  provide 
the  tools  necessary  to  help  establish  the  clinical  importance  of  over 
100  new  viruses  of  man. 

Proposed  Course  of  the  Project:  During  the  year  1961,  the  sero- 
logical program  will  be  expanded  both  in  terms  of  antigenic  materials 
and  space  for  the  performance  of  an  increased  testing  program.  The 
space  available  has  already  been  increased  and  will  be  enlarged  to 
include  a  well  equipped  laboratory  and  of f ice  faci I  it ies.  The  con- 
tinuing process  of  evaluating  new  antigens  and  the  volume  production 
of  antigens  will  be  continued.  The  evaluation  of  serial  specimens 
from  the  NINDB  study  will  proceed  in  conjunction  with  information  on 
birth  defects  being  supplied  by  the  Collaborating  Institutions. 


125 


Serial    No.      NIAID  -    69 


1.  Infectious  Diseases 

2.  Virus  &  Rickettsial  Diseases 

3.  Bethesda,  Maryland 


PHS-N I H 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:  Study  of  viruses  as  causes  of  respiratory  illness 
in  infancy  and  early  childhood. 

Principal  Investigator:   Dr.  Robert  M.  Chanock 

Other  Investigators:     Dr.  K.M.  Johnson,  Dr.  R.J.  Huebner 

Cooperating  Units:       Dr.  R.H.  Parrott,  Children's  Hospital 

Research  Foundation,  Washington,  D.C. 

Man  Years  (calendar  year  I960): 
Total:        30/12 
Professional:   5/12 
Other:        25/12 

Project  Description: 

Ob jecti ves:   I)  To  search  for  new  agents  responsible  for 
respiratory  illness  in  infancy  and  childhood.   2)  To  delineate  the 
epidemiology  of  virus  which  have  recently  been  associated  with 
respiratory  illness.   3)  To  continue  the  study  of  certain  well 
established  respiratory  viruses,  i.e.,  their  epidemiology  and 
contribution  to  the  overall  respiratory  disease  experience  during 
nfancy  and  childhood.  4)  To  determine  how  much  pediatric  respiratory 
llness  can  be  associated  with  known  viruses  as  a  guide  to  future 
mmunoprophy I axi  s. 

Methods  Emp I oyed :   Infants  and  children  with  respiratory  illness 
and  suitable  controls  without  such  illness  will  be  studied  at 
Children's  Hospital,  District  of  Columbia.  The  severe  lower  respiratory 
syndromes  will  be  investigated  in  hospitalized  patients,  while  the 
milder  febrile  respiratory  disease  syndromes  will  be  studied  among 
cl  in  ical  patients. 


Part  B  Included:      Yes 


126 


Serial  No.   NIAID  -  69 

The  main  emphasis  will  be  on  the  use  of  various  tissue  culture 
systems  and  the  fluorescent  antibody  technique  for  virus  isolation. 
An  attempt  will  be  made  to  inoculate  specimens  from  patients  directly 
into  tissue  culture  without  prior  freezing  and  thawing  since  such 
treatment  appears  to  rapidly  inactivate  respiratory  syncytial  virus  and 
the  current  strains  of  influenza  B.  Fluorescent  antibody  techniques 
will  be  applied  to  the  search  for  agents  which  may  possibly  grow  in 
tissue  culture  without  causing  cell  destructive  effects. 

After  the  various  isolates  are  identified,  their  contribution 
to  the  different  respiratory  disease  syndromes  will  be  estimated  by 
comparing  the  recovery  rate  in  such  groups  with  that  observed  for 
healthy  children  free  of  respiratory  symptoms.  Children  with  severe 
illness  admitted  to  the  hospital,  as  well  as  control  subjects,  will  be 
studied  serologically  for  evidence  of  infection  with  15-20  known 
respiratory  viruses  as  well  as  any  new  agent  which  may  emerge  as  a 
potentially  important  pathogen. 

Major  Findings; 

Respiratory  syncytial  (RS)  virus;  During  the  past  3  years  RS 
infection  was  detected  by  serologic  means  in  \\%  of  children  with  severe 
lower  respiratory  tract  illness.  The  agent  was  found  to  be  extremely 
labile  and  virus  recovery  was  rarely  accomplished  until  specimens  were 
immediately  inoculated  into  tissue  culture  without  prior  freezing. 
Employing  this  technic,  57  strains  of  RS  virus  were  recovered  from 
children  with  respiratory  illness  from  March  through  July,  I960.  Virus 
was  recovered  most  frequently  from  infants  less  than  7  months  of  age 
who  were  hospitalized  for  pneumonia  (54%)   or  bronchiolitis  (59#).  The 
RS  agent  was  isolated  from  32$   of  children  of  all  ages  with  bronchio- 
litis or  pneumonia  during  this  period.  Virus  was  recovered  significantly 
less  often  (\%)    from  control  subjects. 

Serologic  studies  condirmed  the  virus  isolation  data  and  pro- 
vided additional  evidence  that  the  RS  virus  is  a  major  respiratory 
pathogen  of  infancy  and  childhood.  When  the  serologic  technics  were 
re-examined  in  the  light  of  the  virus  recovery  data,  it  was  found  that 
the  CF  test  was  only  50%   efficient  in  detection  of  infection.  This 
suggested  that  22#  of  the  total  severe  pediatric  respiratory  illness 
seen  in  Washington,  D.C.,  during  the  past  3  years  was  associated  with 
RS  infection. 

Para  influenza  viruses;   Similar  to  the  pattern  established 
during  the  past  2  years  the  para  influenza  types  I  and  3  viruses  were 
active  in  infants  and  children  of  the  Washington,  D.C.,  area  during 
most  months  of  the  current  year.  These  agents  continued  to  play  an 
important  role  in  all  types  of  pediatric  respiratory  disease, 
especially  infantile  croup  which  is  the  most  serious  respiratory 
emergency  of  childhood.  For  the  first  time  evidence  was  obtained  which 

127 


Serial  No.  NIAIO  -  69 


associated  type  2  (CA)  virus  with  illness.  This  agent  was  associated 
with  10  cases  of  croup  hospitalized  during  November  through  January. 

Adenoviruses;  During  the  past  3  years  344  adenoviruses  have 
been  recovered  from  3624  patients  with  respiratory  disease  - 
isolation  rate  =  9.5%.      These  viruses  are  currently  being  typed.  An 
analysis  of  their  contribution  to  pediatric  illness  must  await 
completion  of  typing.   It  is  important  to  assess  the  role  of  each 
adenovirus  type  separately  since  the  recovery  rate  for  all  adenoviruses 
from  control  subjects  is  so  high  (.5%),    and  since  adenoviruses  not 
only  cause  acute  illness  but  persist  for  long  periods  in  a  latent  form 
in  lymphoid  tissues. 

Enteroviruses;   Currently  under  investigation  is  the  importance 
of  the  recently  described  enterovirus- 1  ike  agents  which  grow  only  in 
human  tissue  culture  cells  and  which  have  very  fastidious  conditions 
for  such  growth.  The  agents  have  been  recovered  from  6%  of  children 
with  various  types  of  respiratory  disease.  The  recovery  rate,  however, 
from  control  subjects  was  the  same  {6%).      Numerous  conventional 
enterovirus  infections  have  also  been  observed  in  this  study 
population  during  the  past  few  years.  Analysis  of  their  clinical 
importance  awaits  specific  identification  of  the  many  strains  of 
virus  recovered. 

Significance  to  the  Program  of  the  Institute;  Respiratory 
disease  is  the  most  common  infectious  ailment  of  man.   Such  infections 
occur  commonly  and  in  their  severest  form  during  infancy  and  early 
childhood.  Detailed  knowledge  of  the  agents  responsible  for 
respiratory  disease  and  their  natural  history  are  a  necessary  prelude 
to  attempts  at  either  immunoprophyl axis  or  chemotherapy.  As  has  been 
shown  with  the  respiratory  syncytial  and  para  influenza  viruses,  the  • 
significance  to  be  derived  from  newer  respiratory  agents  discovered 
in  childhood  illness  is  also  increased  because  they  are  associated 
with  infections  which  may  produce  a  general  I y  mi  I der  i I Iness  in  adults. 

Proposed  Course  of  the  Project;  It  is  planned  that  this  study 
will  continue  for  a  period  of  several  years,  since  a  large  segment  of 
childhood  respiratory  illness  remains  to  be  elucidated.  In  addition, 
viruses  responsible  for  respiratory  illness  vary  at  different  times 
and  in  different  localities.  Therefore,  surveillance  should  continue 
in  order  to  comprehend  the  larger  picture. 


128 


Serial  No.  NIAID  -  69 

Part  B.   Honors,  Awards,  and  Publ  ications 

Publications  other  than  abstracts  from  this  project: 

Chanock,  R.M.,  Bell,  J. A.,  and  Parrot,  R.H.:   Natural  history  of 
para  influenza  infection.  Monograph  in:   PERSPECTIVES  IN 
VIROLOGY  II.   In  press,  1961. 

Chanock,  R.M.,  and  Johnson,  K.M.:   Infectious  Diseases 
(respiratory  viruses).   In:  Annual  Review  of  Med.  Pub.; 
Annual  Reviews,  Inc.   In  press,  1961. 


Honors  and  awards  related  to  this  project: 

Invited  to  attend  The  Gustav  Stern  Symposium  on  Perspectives  in 
Virology  II,  and  present  paper  entitled,  "Observations 
on  natural  history  of  infection  with  certain  recently 
recognized  respiratory  viruses."  January  25,  26.,  I960,  New  York 
City,  N.Y. 

Invited  to  participate  at  Asian  Influenza  Conference  (International), 
and  present  paper  entitled,  "Hemadsorption".  February  17,  18, 
19,  I960,  National  Institutes  of  Health,  Bethesda,  Md. 

Invited  to  participate  in  Armed  Forces  Epidemiology  Board  meeting, 
April  4,5,6,  I960,  Kenwood  Country  Club,  Bethesda,  Md. 

Elected  Full  Member,  Armed  Forces  Epidemiology  Board-Commission  on 
Acute  Resp iratory  Disease,  June,  I960. 

Invited  to  participate  in  the  American  Medical  Association  Clinical 
Sessions,  and  present  paper  on  respiratory  virus  infections, 
November  28,  29,  and  December  I,  I960,  Washington,  D-C. 

Invited  to  contribute  chapter  on  Respiratory  Viruses  for  publication 
in  ANNUAL  REVIEW  OF  MEDICINE  -  1961. 


123 


Serial  No.  NIAID  -  69A 

1.  Infectious  Diseases 

2.  Virus  &  Rickettsial 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:   Viral  pneumonia:  etiology,  therapy,  and 
prevention. 

Principal  Investigator:  Or.  Robert  M.  Chanock 

Other  Investigators:     Or.  Karl  M.  Johnson 

Cooperating  Units:       Dr.  J.  Kingston,  Dr.  H.  Bloom, 

Dr.  M.  Mufson,  and  Dr.  F.  Gordon, 
Bureau  of  Medicine  and  Naval  Medical 
Research  Institute,  U.S.  Navy 

Man  Years  (calendar  year  I960): 
Total:        50/12 
Professional:   10/12 
Other:        40/12 

Project  Description: 

Objectives:   I)  To  define  the  etiology  of  viral  pneumonia  as  it 
occurs  in  persons  of  all  ages.   2)  To  determine  the  relative  importance 
of  various  newly  recognized  viruses  in  the  pneumonia  syndrome  in 
different  populations  and  at  different  times.   3)  To  define  the  natural 
history  of  certain  agents  which  appear  to  be  responsible  for  a 
significant  proportion  of  pneumonia  in  the  young  (para  influenza  3, 
respiratory  syncytial,  adenovirus,  and  Eaton  agent)  and  in  the  adult 
(Eaton  agent).  4)  To  continue  the  search  for  other  as  yet  unrecognized 
viral  agents  which  cause  pneumonia.   5)  To  evaluate  the  effect  of 
tetracycline  chemotherapy  in  Eaton  pneumonia. 

Methods  Employed:  Current  emphasis  has  been  placed  on  the  role  of 
Eaton  agent  in  human  pneumonia.  The  presence  and  quantity  of  antibody 
for  the  Eaton  agent  was  determined  by  the  indirect  fluorescent  antibody 
technique,  employing  frozen  sections  of  infected  chick  embryo  lung. 

Epidemiologic  field  studies  were  carried  out  at  a  Marine  Recruit 
Training  Center  (Parris  Island,  S.C.)  which  has  a  high  rate  of  Eaton 
infection.   Patients  with  pneumonia,  febrile  respiratory  illness  without 
pneumonia,  afebrile  respiratory  illness  and  comparable  control  subjects 
free  of  respiratory  illness  were  studied  serologically  for  evidence  of 

Part  B  Included    Yes  13 U 


Serial  No.   NIAIO  -  69A 


infection  with  Eaton  agent,  as  well  as  other  respiratory  viruses. 

The  effect  of  demethylch lortetracycl ine  on  the  course  of  Eaton 
pneumonia  was  determined  in  a  double  blind  therapy  study  in  which  the 
clinical  observers  were  unaware  of  the  patients  diagnostic  or  therapy 
status.   Laboratory  tests  were  performed  without  knowledge  of  which 
patients  received  drug  and  which  patients  received  placebo.  The 
patients  were  given  a  daily  physical  examination,  temperature  was 
recorded  four  times  a  day,  and  chest  X-rays  were  taken  every  third  day. 

Major  Findings; 

A.  Epidemiologic  findings.  Over  a  I -year  period  Eaton  infection 
was  associated  with  5\%  of  the  530  pneumonias  admitted  to  the  Naval 
hospital.  Adenovirus  infection  contributed  significantly  less  to  the 
pneumonia  syndrome  (6%). 

Eighty-three  per  cent  of  the  recruits  lacked  detectable  antibody 
for  Eaton  agent  when  they  entered  recruit  training.   Fifty-three  per 
cent  of  these  sero-negat i ve  men  developed  antibody  during  the  5-month 
training  period.  For  the  group  of  incoming  recruits  as  a  whole,  the 
risk  of  infection  during  training  was  44#.  Over  a  6-month  period  the 
risk  of  pneumonia  during  training  was  2%   and  the  risk  of  an  Eaton 
positive  pneumonia  was  I .5%.      The  infection  to  clinical  pneumonia 
ratio  was  estimated  to  be  30  to  I . 

Infection  was  widely  disseminated  through  the  recruit  center, 
and  occurred  in  almost  every  platoon  of  recruits.  Movement  of 
infection  was  slow;  onsets  of  Eaton  pneumonia  occurred  over  a  9-week 
period  in  certain  platoons.   Long  incubation  period  and  slow  movement 
of  infection  are  attributes  ideally  suited  to  maintain  this  agent  in 
an  ever  changing  recruit  population.  The  fluorescent  antibody  test 
proved  to  be  twice  as  sensitive  as  the  cold  agglutinin  technic  in  the 
diagnosis  of  Eaton  pneumonia. 

B.  Chemotherapy.   In  a  control  led  double  bl  ind  study  involving 
290  patients,  demethy I ch lortetracyc I  ine  significantly  reduced  the 
duration  of  fever,  rales  cough,  malaise,  and  fatigue.  Therapy  stopped 
the  progression  of  pulmonary  infiltration  and  accelerated  its  clearing. 
Fever  did  not  return  when  therapy  was  stopped.  These  findings  strongly 
suggest  that  the  drug  exerted  a  direct  action  upon  the  pulmonary  disease 
process. 

Demethylch lortetracyc I ine  had  no  apparent  effect  on  the  course  of 
a  small  mixed  group  of  illnesses  associated  with  other  known  respiratory 
viruses. 


131 


Serial  No.   NIAIO  -  69A 


Significance  to  the  Program  of  the  Institute;   As  seen  during 
the  past  2  years,  the  consequences  of  infection  with  Eaton  agent  appear 
to  be  of  considerable  public  health  importance  in  both  children  and 
adults.  The  demonstration  of  a  marked  beneficial  effect  of  demethyl- 
chlortetracycl ine  on  Eaton  pneumonia  thus  constitutes  a  finding  of 
major  importance  in  the  therapy  of  viral  pneumonia. 

Proposed  Course  of  the  Project:  The  searcti  for  other  as  yet 
unrecognized  viruses  which  cause  pneumonia  will  continue. 

An  unexpected  finding  in  the  chemotherapy  study  suggests  that 
another  tetracycline  sensitive  agent  was  responsible  for  a  significant 
proportion  of  the  pneumonias  which  were  investigated.  Efforts  to 
recover  this  agent  are  currently  in  progress. 

In  addition  to  the  unexplained  pneumonias  of  early  life,  future 
studies  will  concentrate  upon  the  heretofore  completely  neglected 
problem  of  pneumonia  in  the  aged.  Emphasis  will  be  placed  in  these 
two  areas  since  it  is  here  that  the  problem  of  pneumonia  mortality  is 
greatest. 


132 


Serial  No.  NIAID  -  69A 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  B.  Honors,  Awards,  and  Publ  ications 

Publications  other  than  abstracts  from  this  project: 

Johnson,  R.T.,  Cook,  M.K.,  Chanock,  R.M.,  and  Buescher,  E.L.: 
Family  outbreak  of  primary  atypical  pneumonia  associated  with 
the  Eaton  agent.   New  England  J.  Med.,  v:  262,  817-819,  I960. 

Chanock,  R.M.,  Cook,  M.K.,  Fox,  H.H.,  Parrott,  R.H.,  and  Huebner, 
R.J.:   Serologic  evidence  of  infection  with  Eaton  agent  in 
lower  respiratory  illness  in  childhood.   New  England  J.  Med., 
v:262,  648-654,  I960. 

Cook,  M.K.,  Chanock,  R.M.,  Fox,  H.H.,  Buescher,  E.L.,  Johnson,  R.T., 
and  Huebner,  R.J.:   Studies  on  the  role  of  Eaton  agent  in  lower 
respiratory  tract  illness.  Evidence  for  infection  in  adults. 
Brit.  Med.  J.,  v: I ,  905-911,  I960. 

Chanock,  R.M.,  Mufson,  M.A.,  Bloom,  H.H.,  James,  W.D.,  Fox,  H.H., 
Kingston,  J.R.:   Eaton  agent  pneumonia.   I.   Ecology  of  infection 
in  a  military  recruit  population.   J. A.M. A.   In  press,  I960. 


133 


Serial  No.   NIAIO  -  69B 

1.  Infectious  Diseases 

2.  Virus  &  Rickettsial  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:   Study  of  the  laboratory  aspects  of  respiratory 
virus  illness  in  a  welfare  orphanage. 

Principal  Investigator:   Dr.  Robert  M.  Chanock 

Other  Investigators:     Dr.  K.M.  Johnson,  Dr.  A.Z.  Kapikian, 

Dr.  R.J.  Huebner,  Dr.  J. A.  Bell 

Cooperating  Units:      None 

Man  Years  (calendar  year  I960): 
Total:        62/12 
Professional:   12/12 
Other:        50/12 

Project  Description: 

Objectives:   To  elucidate  the  natural  history  of  certain  newly 
discovered  respiratory  viruses  by  means. of  a  longitudinal  study  of  an 
orphanage  population.  This  study  supplements  our  cross-sectional 
studies  in  pediatric  hospitals,  thus  adding  additional  dimensions 
to  our  observations  and  increasing  the  scope  of  our  comprehensive  study 
of  respiratory  infections. 

Methods  Employed:  The  population  under  study  is  an  orphanage 
nursery  in  the  District  of  Columbia  (s  a  Project  Report  NIAID-66)  whose 
average  population  is  90-100  children  beJween  the  ages  of  6  months  and 
3  years.  The  turnover  of  infants  and  children  is  rather  rapid,  and,  as 
a  result,  a  great  deal  of  respiratory  illness  occurs  in  this  population 
throughout  the  year. 

Rectal  temperatures  are  taken  twice  a  day,  and  the  population 
is  carefully  observed  by  a  full-time  pediatrician  who  also  provides 
medical  care.  Throat  and  rectal  swabs  are  collected  three  times  a 
week.   Serum  specimens  are  obtained  on  admission  and  discharge,  and 
at  various  intervals  between.  Throat  swabs  are  tested  in  monkey  kidney 
tissue  culture  for  the  presence  of  various  myxoviruses  and  in  Hep-2 
cultures  for  respiratory  syncytial  (RS)  and  adenoviruses.  Serum 

Part  B  Included:      Yes 

131 


Serial  No.   NIAIO  -  69B 


specimens  are  tested  for  antibody  to  the  various  myxoviruses  and 
other  respiratory  agents  by  the  neutralization,  hemagglutination- 
inhibition,  or  complement  fixation  technique.   Laboratory  data  are 
then  correlated  with  clinical,  bacteriological,  and  epidemiological 
information  in  an  effort  to  determine  qualitative  and  quantitative 
virus  disease  relationships.   As  was  true  in  previous  years  witti  the 
adenoviruses  and  the  newer  myxoviruses  (para  influenzas)  and  certain 
enteroviruses,  Junior  Village  provided  in  I960  additional  opportuni- 
ties for  defining  the  clinical  importance  of  viruses  in  childhood 
di  sease. 

Major  Findings: 

Respiratory  Syncytial  (RS)  Virus:  An  abrupt  outbreak  of 
pneumonia  occurred  during  the  last  week  of  April  and  the  first  2 
weeks  of  May,  I960.  Thirty-six  (or  40£)  of  the  90  infants  and 
children  in  residence  at  Junior  Village  developed  pneumonia.  The 
usual  rate  of  pneumonia  at  the  nursery  is  I  -  2  cases  per  month. 

The  widespread  occurrence  of  RS  infection  in  the  general 
Washington,  D.C.  community  at  the  same  time,  and  its  association  with 
lower  respiratory  tract  illness, (see  Project  Report  NIAIO  69-B  1959) 
suggested  that  the  nursery  outbreak  might  also  be  caused  by  RS  virus. 
Laboratory  techniques  were  modified  accordingly,  and  throat  swab 
specimens  were  tested  within  a  few  hours  after  collection  without  prior 
freezing.  The  inoculation  of  fresh  specimens  is  necessary  because  of 
the  extreme  lability  of  RS  virus.   Unfortunately  this  type  of  testing 
was  not  initiated  until  the  last  half  of  the  outbreak.  Nevertheless, 
24  strains  of  RS  virus  were  recovered,  18  of  them  from  patients  in 
the  acute  phase  of  a  pneumonia  illness.  An  analysis  of  the  virus 
recovery  and  illness  data  strongly  suggested  that  the  RS  agent  was 
etiological ly  associated  with  pneumonia.   Serologic  tests  indicated 
that  approximately  90£  of  children  in  residence  during  the  outbreak 
developed  a  rise  in  antibody  for  RS  virus. 

Significance  to  the  Program  of  the  Institute:  The  information 
gained  during  the  RS  virus  outbreak  has  yielded  additional  evidence 
that  this  agent  is  a  respiratory  pathogen  of  major  importance  in 
infancy  and  early  childhood.   Study  of  the  RS  pneumonia  outbreak 
provided  an  unusual  opportunity  to  observe  the  clinical  consequences 
of  infection,  and  to  determine  the  risk  of  lower  respiratory  tract 
involvement  in  a  young  population.  The  finding  that  A0%  of  the 
infants  and  children  in  residence  developed  pneumonia  constitutes 
high  order  information  wnich  has  implications  both  in  the  natural 
history  of  infection  with  this  agent,  and  in  attempts  to  control  its 
more  severe  effects  by  immunoprophyl axis. 


135 


Serial  No.   NIAID  -  69B 

Proposed  Course  of  the  Project:  The  project  will  continue  in 
an  effort  to  gain  a  better  understanding  of  the  newer  respiratory 
viruses.  A  retrospective  serological  analysis  of  the  pneumonia 
experience  at  Junior  Village  will  be  undertaken  with  special  emphasis 
on  the  role  of  para  influenza  3  and  respiratory  syncytial  viruses  since 
these  agents  have  been  associated  with  the  majority  of  much  illness  in 
the  past  2  years. 


136 


Serial  No.   NIAID  -  69B 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  B.  Honors,  Awards,  and  Publ ications 

Publications  other  than  abstracts  from  this  project: 

Chanock,  R.M.,  Wong,  O.C.,  Huebner,  R.J.,  and  Bell,  J. A.: 
Serologic  response  of  individuals  infected  with  para 
influenza  viruses.  Am.  J.  Pub.  Hlth.,  I960. 

Johnson,  K.M.,  Chanock,  R.M.,  Cook,  M.K.,  and  Huebner,  R.J.: 
Studies  of  a  new  human  hemadsorption  virus.   I.   Isolation, 
properties,  and  characterization.  Am.  J.  of  Hyg.,  v:7l, 
81-92,  I960. 


137 


Serial  No.   NIAID  -  70 

1.  Infectious  Diseases 

2.  Virus  &  Rickettsial  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:   Laboratory  studies  of  newly  recognized  viruses 
associated  with  respiratory  illness. 

Principal  Investigator:  Dr.  Robert  M.  Chanock 

Other  Investigators:     Dr.  K.M.  Johnson,  Dr.  A.  Kisch, 

Dr.  F.R.  Abinanti 

Cooperating  Units:      None 

Man  Years  (calendar  year  I960): 

Total:  20/12 
Professional:  10/12 
Other:         10/12 

Project  Description: 

Ob  iecti ves:   I)  To  investigate  the  biologic  and  antigenic 
properties  of  the  newly  recognized  para  influenza  viruses.  2)  To  study 
the  properties  of  Eaton  agent  and  its  growth  in  various  types  of  tissue 
culture.   3)  To  develop  simple  procedures  for  the  recovery,  identifi- 
cation, and  serologic  study  of  Eaton  agent. 

Methods  Employed:  The  standard  tools  of  virology — neutralization, 
complement-f ixation,  and  hemagglut ination- inh ibition  are  employed  to 
determine  the  antigenic  specificity  and  existence  of  shared  antigens 
among  the  para  influenza  viruses. 

The  fluorescent  antibody  technique  employing  frozen  sections 
of  chick  embryo  lung  is  used  as  the  standard  of  reference  in  all 
studies  with  Eaton  agent.  Attempts  are  made  to  cultivate  the  agent  in 
various  types  of  tissue  culture  and  laboratory  animals.  Growth  of 
Eaton  agent  is  determined  by  titration  of  tissue  culture  fluid  or 
organ  suspension  in  embryonated  eggs  combined  with  immunof luorescent 
examination  of  embryo  lung  tissue.  Cytopathic  effects,  cytochemical 
changes,  and  inclusion  bodies  are  searched  for  in  the  inoculated  tissue 
cultures.  Fluids  harvested  from  inoculated  tissue  cultures  are  tested 

Part  B  Included:     Yes 

138 


Serial    No.      NIAID  -  70 


for  antigens  which  might  fix  complement  with  potent  convalescent  serum 
from  patients  with  Eaton  pneumonia. 

Major  Findings: 

1 .  Para  influenza  3.  The  hemadsorption  type  I  (HA- I)  virus 
recovered  from  humans  with  respiratory  disease  and  the  SF-4  virus 
recovered  from  cows  with  shipping  fever  were  shown  to  be  distinct 
ant igenical I y.  Previously  ,these  agents  had  been  considered  very 
closely  related  or  identical.  Guinea  pigs  infected  with  the  HA- I 

or  SF-4  viruses  developed  a  hemagglutinat ion-inh ibition  and  neutrali- 
zation antibody  response  which  permitted  differentiation  of  these 
agents.  Complement  fixation  tests  employing  viral  or  soluble  antigens 
indicated  that  the  two  viruses  were  sufficiently  related  to  be 
classified  together  as  sub-types  or  para  influenza  3.  Each  of  23 
human  isolates  from  various  parts  of  the  world  resembled  the  HA- I 
prototype.  Each  of  the  7  bovine  isolates  from  different  parts  of  the 
United  States  were  indistinguishable  from  the  bovine  prototype  SF-4 
virus.  These  findings  suggest  that  these  viruses  do  not  cross  species 
boundaries. 

2.  Eaton  agent.  The  Eaton  agent  was  shown  to  multiply  without 
cytopathic  effect  in  chick  embryo  entodermal,  chick  kidney,  Hep-2, 
human  kidney,  and  monkey  kidney  tissue  cultures.  The  highest  level 

of  replication  occurred  in  monkey  kidney  cultures  where  infected 
fluids  contained  up  to  I04  egg  infectious  doses  of  the  agent.  Antigen 
concentration  within  the  infected  tissue  culture  cells  was  insufficient 
to  permit  visualization  by  immunofluorescence.  Complement  fixing 
antigens  were  not  detected  in  fluids  from  infected  tissue  cultures. 

An  eclipse  phase  was  demonstrated  during  replication  in  monkey 
kidney  cells.  This  suggests  that  the  agent  should  be  classified  as  a 
virus  according  to  a  recent  definition  proposed  by  Burnet. 

In  a  simultaneous  test,  monkey  kidney  tissue  culture  was  found  to 
be  as  sensitive  as  embryonated  eggs  for  the  recovery  of  naturally 
occurring  strains  of  Eaton  agent.   Fourteen  strains  were  recovered  in 
tissue  culture  from  17  patients  with  serologically  positive  pneumonia. 

Preliminary  studies  indicate  that  the  agent  is  inhibited  in 
tissue  culture  by  1.5  micrograms  per  ml.  of  demethylchlortetracycl  ine 
(Dec lomyc in) .  This  concentration  is  approximately  1/2  that  found  in 
the  blood  24  hours  after  oral  administration  of  a  small  dose  (0.5  gm) 
of  the  drug.  These  findings  are  consistent  with  the  therapeutic  effect 
which  we  have  recently  demonstrated  for  demethylchlortetracycl ine  in 
Eaton  pneumonia.   Preliminary  studies  suggest  that  the  drug  inhibits 
intracellular  synthesis  of  Eaton  agent,  and  has  no  effect  upon  the 
agent  when  it  is  in  the  free  extracellular  state. 

X39 


Serial  No.  NIAIO  -   70 


Significance  to  the  Program  of  the  Institute:   Knowledge  of  the 
antigenic  properties  of  the  para  influenza  viruses  is  required  in 
order  to  perform  meaningful  epidemiologic  studies  with  these  agents. 
Information  bearing  upon  the  species  specificity  of  the  para  influenza 
viruses  greatly  adds  to  our  understanding  of  the  natural  history  of 
these  agents. 

The  growth  of  Eaton  agent  in  various  types  of  tissue  culture 
and  the  recovery  of  naturally  occurring  strains  in  monkey  kidney 
culture  represents  a  significant  forward  step  in  the  study  of  this 
respiratory  pathogen.  This  technique  has  made  it  possible  to  recover 
strains  which  were  suitable  for  administration  to  human  volunteers, 
and  to  study  the  behavior  of  the  agent  in  such  infected  individuals. 
Tissue  culture  studies  with  demethylch lortetracycl ine  provide  an 
experimental  basis  for  understanding  the  chemotherapeut ic  effect  of 
this  drug  in  Eaton  pneumonia,  and  offer  a  tool  for  further  analysis  of 
this  phenomenon. 

Proposed  Course  of  the  Study:   Efforts  will  continue  to  maintain 
surveillance  of  the  antigenic  properties  of  the  para  influenza  viruses. 

The  search  for  simpler  laboratory  procedures  for  the  study  of 
Eaton  agent  will  continue.  Various  types  of  tissue  culture  will  be 
tested  for  the  capacity  to  support  multiplication  of  this  agent,  pre- 
ferably with  cytopathic  effect.  Complement  fixing  antigen  and 
hemagglutinin  will  be  sought. 


1U0 


Serial  No.   NIAID  -  70 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  B.   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Chanock,  R.M.,  Johnson,  K.M.,  Cook,  M.K.,  Wong,  O.C.,  and 

Vargosko,  A.:  The  hemadsorption  technique  with  special  reference 
to  the  problem  of  a  naturally  occurring  simian  para  influenza 
virus.  Proc.  of  the  International  Conf.  on  Asian  Influenza. 
In:  AM.  REV.  OF  RESPIR.  DISEASES  (book).   |n  press,  1961. 

Craighead,  J.,  Cook,  M.K.,  and  Chanock,  R.M.:   Infection  of  hamsters 
with  para  influenza  3  viruses.   Proc.  Soc.  Exptl.  Biol.  &  Med., 
v:  104,  301-304,  I960. 

Chanock,  R.M.,  Fox,  H.H.,  James,  W.O.,  Bloom,  H.H.,  Mufson,  M.A.: 
Growth  of  laboratory  and  naturally  occurring  strains  of  Eaton 
agent  in  monkey  kidney  tissue  culture.  Proc.  Soc.  ExptJ .  Biol. 
4  Med.,  v:l05,  371-375,  I960. 

Gordon,  F.B.,  Quan,  A.L.,  Cook,  M.K.,  Chanock,  R.M.,  and  Fox,  H.H.: 
Growth  of  the''Eaton  agent  of  primary  atypical  pneumonia  in  chick 
entodermal  tissue  culture.  Proc.  Soc.  Exptl.  Biol.  &  Med., 
v: 105,  375-377,  I960. 


1U 


Serial 
1  . 
2. 
3. 

No.   NIAIO  -  71 
Infectious  Diseases 
Virus  &  Rickettsial 
Bethesda,  Maryland 

Indi vi 
Cal 

PHS 
dual  1 
endar 

-NIH 

Project  Report 
Year  I960 

Part  A. 

Project  Title:   Studies  of  tumor-producing  viruses. 

Principal  Investigator:   Or.  Wallace  P.  Rowe 

Other  Investigators:     Or.  J.W.  Hartley,  Dr.  R.J.  Huebner, 

Mr.  L.W.  Smith 

Cooperating  Units:       Or.  L.W.  Law  and  C.J.  Dawe,  NCI,  NIAIO 

Man  Years  (calendar  year  I960): 

Total:  45/12 
Professional:  16/12 
Other:        29/12 

Project  Description: 

Object  i  ves:   To  characterize  the  viruses  which  produce  tumors 
in  animals  from  the  standpoints  of  their  laboratory  properties, 
natural  behavior,  and  modes  of  spread. 

Methods  Employed;  Application  of  standard  and  newly  developed 
virologic  procedures  for  detection  and  quantitation  of  animal  tumor 
viruses. 

Major  Findings: 

A.   Mouse  polyoma  virus.   Work  with' this  agent  has  continued 
along  the  I  ines  of  the  past  year.  The  newer  emphasis  has  been 
primarily  on  the  natural  history  in  wild  mouse  populations  as 
described  in  Project  No.  68. 

I.  The  patterns  of  infect  ion i in  individual  animals  have 
been  studied  in  greater  detail.   It  has  been  found  that  the  virus 
produces  a  prolonged  chronic  infection  when  inoculated  into  weanling 
mice.   The  weanling  mice  sporadically  excrete  the  virus  in  the  urine. 


Part  B  included:      Yes 

112 


Serial  No.   NIAIO  71 


2.  Serial  passage  lines  of  the  virus  have  been  carried  in 
newborn  mice  with  passages  made  at  the  peak  of  viral  infectivity. 
In  this  way  strains  have  been  obtained  which  produce  extremely  high 
hemagglutinin  titers  in  suckling  mouse  tissue,  and  which  have  altered 
patterns  of  disease  production  in  newborn  mice,  producing  greater 
tendencies  for  inducing  the  runting  form  of  infection,  and  an  increased 
tendency  to  produce  bone  tumors. 

3.  The  laboratory  aspects  of  the  epidemiological  work  in  wild 
mouse  populations  have  shown  the  existence  of  local  infections  in 
rural  farm  mice  and  in  mice  in  feed  mills.  The  virus  recovered  from 
wild  New  York  mice  was  shown  to  be  oncogenic  in  baby  mice  and  hamsters; 
the  oncogenic  activity  of  rural  polyoma  virus  strains   is  under  study. 

4.  It  has  been  found  that  the  virus  is  frequently  present 
in  transplanted  tumor  lines. 

5.  Production  stocks  of  laboratory  mice  freed  of  infection  by 
rearing  under  conditions  similar  to  specific  pathogen-free  methods. 

6.  Polyoma  virus  in  baby  mice  was  shown  to  be  a  potent 
interfering  agent,  producing  interference  against  the  lethal  effects 
of  vesicular  stomatitis  and  Coxsackie  A  viruses. 

7.  The  reaction  of  mucoprotein  non-specific  inhibitors  with 
the  virus  has  been  further  characterized.  The  reaction  with  the 
inhibitor  was  found  to  induce  marked  aggregation  of  the  virus.  The 
strains  which  have  become  highly  sensitive  to  inhibitors  were  shown  to 
have  lost,  to  a  great  extent,  their  ability  to  induce  tumors  in  mice. 

B.  Mouse  leukemia  viruses.  Studies  of  the  Gross,  Schwartz,  and 
Moloney  mouse  leukemia  viruses  have  centered  primarily  on  reproducing 
the  disease  in  laboratory  mice,  in  attempts  to  free  these  viruses  of 
contaminating  mouse  viruses,  and  on  developing  simpler  assay  pro- 
cedures for  the  leukemia  viruses. 

I.  Moloney  and  Gross  leukemia  viruses  have  been  propagated 
through  several  serial  mouse  passages, produced  a  high  incidence  of 
leukemia  with  relatively  short  incubation  periods.  By  passage  of  the 
Gross  virus  in  the  presence  of  suitable  antiserum,  it  has  been  freed 
of  polyoma  and  K  viruses  for  long  periods,  which  are  contaminants 
of  the  standard  Gross  passage  A  virus.  This  should  make  it  possible 
to  attempt  the  development  of  in  vitro  assay  procedures  which  will  be 
specific  for  the  leukemia  virus.. 


113 


Serial  No.   NIAID  -  71 


C.   Lymphomatosis  virus  of  chickens.  The  sero-ep idem io logic 
studies  of  antibodies  to  the  cytopathic  virus  of  Sharpless  and 
Burmester  indicated  definitely  that  this  virus  is  a  contaminating 
agent  and  not  the  etiologic  agent  of  visceral  lymphomatosis  (see 
Project  Report  68-A). 

Significance  to  the  Program  of  the  Institute:   The  contributions 
which  can  be  made  to  the  knowledge  of  the  biology  of  tumor  viruses, 
such  as  polyoma  Friend  leukemia  and  rabbit  papilloma,  when  studied 
with  standard  virologic  techniques  is  now  becoming  well  recognized. 
The  advantages  of  rapid  indirect  procedures  for  assay  of  tumor 
viruses  (and  extraneous  viruses  as  well)  as  compared  with  the  slow 
unpredictable  tumor  response  endpoints  make  it  imperative  to  attempt 
to  develop  such  procedures  for  additional  animal  tumor  viruses  in 
order  for  reliable  progress  to  be  made  in  clarifying  the  viral 
etiology  of  cancer. 

Proposed  Course  of  the  Project;  The  emphasis  in  the  coming  year 
will  be  to  develop  in  vitro  systems  for  recognition  of  the  mouse 
leukemia  viruses,  particularly  the  Gross  and  Moloney  agents,  with 
special  reference  to  a  propagation  in  tissue  culture.  The  epidemiologic 
studies  of  polyoma  virus  will  be  continued. 


3M 


Serial  No.  NIAIO  -  71 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  B.   Honors,  Awards,  and  Pub  I  ications 

Publications  other  than  abstracts  from  this  project: 

Rowe,  W.P.,  Huebner,  R.J.,  and  Hartley,  J.W.:   The  ecology  of  a 
mouse  tumor  virus.  Monograph  in:   PERSPECTIVES  IN  VIROLOGY  II. 
In  press. 

Huebner,  R.J.:  Viruses  in  search  of  cancer.  Monograph  in: 
PERSPECTIVES  IN  VIROLOGY  II.   In  press. 

Rowe,  W.P.,  Hartley,  J.W.,  Estes,  J.D.,  and  Huebner,  R.J.:  Growth 
curves  of  polyoma  virus  in  mice  and  hamsters.   National  Cancer 
Institute  Monograph  No.  4  -  Symposium  on  Phenomena  of  the  Tumor 
Viruses,  New  York  City,  N.Y. ,  March  25  and  26,  I960. 

Rowe,  W.P.:   The  epidemiology  of  mouse  polyoma  virus  infection. 
Bacteriological  Reviews.   In  press. 

Rowe,  W.P.,  Hartley,  J.W.,  and  Huebner,  R.J.:  The  natural  history 
of  polyoma  virus  infection  -  a  summary.   In:  CANADIAN  CANCER 
CONFERENCE.   In  press. 

Oawe,  C.J.,  Rowe,  W.P.,  and  Law,  L.W.:   Influence  of  age,  species, 
and  immune  factors  on  the  response  of  salivary  gland  to  polyoma 
virus  in  tissue  culture.   Pathologie  et  Biologie.   In  press. 

Law,  L.W.,  Rowe,  W.P.,  and  Hartley,  J.W.:   Studies  of  mouse  polyoma 
virus  infection.  V.  Relation  of  virus  infection  to  lymphocytic 
neoplasms  of  the  mouse.  The  J.  of  Exp.  Med.,  v: I  I  1 ,  517-523, 
I960. 

Honors  and  awards  related  to  this  project: 

Eli  Lilly  Award  -  Meeting  of  the  Society  of  American  Bacteriologists, 
May,  I960,  Philadelphia,  Pa. 

Invited  to  participate  in  Symposium  On  Perspectives  in  Virology  II, 
and  present  paper  entitled  "The  Ecology  of  a  Mouse  Tumor  Virus" 
January  25  and  26,  I960,  New  York  City,  N.Y. 

Invited  to  attend  Symposium  On  Phenomena  of  the  Tumor  Viruses,  and 
present  paper  entitled  "Biologic  Behavior  of  the  Polyoma  Virus 
in  Tissue  Culture"  March  26,  I960,  New  York  City,  N.Y. 


145 


Serial  No.  NIAID  -  71 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Appointed  to  Research  Advisory  Committee  on  Etiology  of  Cancer  of 
the  American  Cancer  Society,  September,  I960. 


1*46 


Serial  No.   NIAIO  -  7IA 

1.  Infectious  Diseases 

2.  Virus  4  Rickettsial 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:  Continuing  studies  of  adenoviruses  and  salivary 
gland  viruses  as  examples  of  latent  viruses. 

Principal  Investigator:  Dr.  Wallace  P.  Rowe 

Other  Investigators:     Dr.  J.W.  Hartley,  Dr.  R.J.  Huebner 

Cooperating  Units:       Dr.  J. A.  Kasel,  LCI 

Man  Years  (calendar  year  I960): 

Total:  5/12 
Professional:  1/12 
Other:        4/12 

Project  Description: 

Objectives:  To  characterize  the  salivary  gland  viruses  and 
adenoviruses,  and  to  determine  their  epidemiology  and  clinical 
importance. 

Methods  Employed:   Serologic  epidemiology  and  virus  isolation 
in  tissue  culture.   Studies  of  the  biology  of  adenoviruses  and 
salivary  gland  viruses  in  lower  animals. 

Major  Findings: 

Complement  fixation  tests  with  the  human  salivary  gland  virus 
have  been  done  on  sera  of  a  number  of  suspected  or  diagnosed  cases 
of  cytomegalic  inclusion  disease  of  the  newborn  submitted  by  workers 
in  various  parts  of  the  world.  The  findings  suggest  that  in  the 
newborn  and  young  infant,  the  test  is  of  I ittle  or  no  value  in 
diagnosis,  in  that  most  cases  have  not  yet  responded  with  antibody 
during  the  period  when  diagnosis  is  necessary. 

The  finding  of  high  prevalence  of  salivary  gland  virus 
infection  in  wild  mice  has  been  extended  by  testing  mice  from  a 
number  of  rural  areas,  and  the  same  high  frequency  of  infection  has 
been  found.   It  has  also  been  found  that  the  infection  in  the  wild 

Part  B  included:     Yes 

147 


Serial  No.  NIAID  -  7IA 

mice  is  of  long  duration,  the  mice  excreting  virus  in  the  saliva  for 
as  long  as  eight  months  after  capture. 

A  new  virus  has  been  isolated  from  Microtus  mice  which  appears 
to  represent  the  Microtus  strain  of  salivary  gland  virus  group  as 
detailed  in  Project  No.  NIA 10-71 B. 

We  have  continued  to  work  with  Or.  Kasel  of  the  LCI  in 
characterization  of  the  enzyme  formed  by  certain  adenovirus  types 
which  destroys  hemagglutination  receptors  on  human  erythrocytes. 
It  has  been  established  that  the  factor  is  neutralized  type  specifi- 
cally by  homologous  rabbit  antiserum,  that  it  is  separable  from  the 
infectious  virus,  and  that  its  activity  shows  many  characteristics  of 
an  enzymatic  reaction. 

Significance  to  the  Program  of  the  Institute:  Th i s  I aboratory 
is  one  of  the  very  few  in  the  world  which  has  competence  in  handling 
the  salivary  gland  viruses.  There  is  great  interest  in  clinical 
fields  in  the  infections  produced  by  this  virus,  and  a  corresponding 
interest  in  laboratory  diagnosis  of  cases.  The  existence  of 
comparable  viruses  in  laboratory  animals  provides  an  excellent 
opportunity  to  study  model  infections. 

Proposed  Course  of  the  Project:  The  diagnostic  studies  and 
the  evaluation  of  the  serologic  tests  will  be  continued.  Epidemiologic 
studies  of  the  spread  of  the  virus  in  laboratory  mice  will  be  carried 
out  by  exposing  mice  through  infected  contacts  under  varying 
conditions. 


148 


Serial    No.    NIAIO  -   7IA 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  B.   Honors,  Awards,  and  Publ  ications 

Publications  other  than  abstracts  from  this  project: 

Hartley,  J.W.,  and  Rowe,  W.P.:   A  new  mouse  virus  apparently 

related  to  the  adenovirus  group.  Virology,  v: 1 1 ,  645-647,  I960. 

Thai  hammer,  0.,  and  Rowe,  W.P.:   Gibt  es  im  Raum  von  Wien 
cytomegal  ie?  Wiener  klinische  Wochenschrl ft.  Sonderabdruck 
aus  72.   Nr.  36,  S.  621-624,  I960. 

Kasel,  J. A.,  Rowe,  W.P.,  and  Nemes,  J.L.:  Modification  of 
erythrocyte  receptors  by  a  factor  in  adenovirus  suspensions. 
Virology,  v: 10,  388-391,  I960. 

Rowe,  W.P.:   Adenovirus  and  salivary  gland  virus  infections  in 
children.   In:  VIRAL  INFECTIONS  OF  INFANCY  AND  CHILDHOOD. 
Ed.  Harry  M.  Rose.   Hoeber-Harper  book,  205-214,  I960. 


143 


Serial  No.   NIAIO  -  7  IB 


1.  Infectious  Diseases 

2.  Virus  &  Rickettsial 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:   Studies  of  mouse  viruses  with  special  reference 
to  their  importance  as  extraneous  viruses 
("background  noise")  in  mouse  tumor  systems. 

Principal  Investigator:   Dr.  Janet  W.  Hartley,  Dr.  Wallace  P.  Rowe 

Other  Investigators:     Mr.  L.W.  Smith,  Dr.  R.J.  Huebner 

Cooperating  Units:       None 

Man  Years  (calendar  year  I960): 
Total:        87/12 
Professional:   30/12 
Other:        57/12 

Project  Description: 

Object  i ves:  To  characterize  and  develop  methods  for 
recognizing  infection  and  determine  the  epidemiological  patterns  of 
the  various  mouse  viruses,  for  the  purpose  of  defining  and  eliminating 
the  problem  of  extrinsic  viruses  as  complicating  factors  in  studies 
of  mouse  tumor  viruses. 

Methods  Employed:   Serologic,  tissue  culture,  and  animal 
pathogenicity  studies;  attempts  to  isolate  agents  from  laboratory  and 
wild  mice  and  from  mouse  tumors.   Studies  of  the  epidemiology  of 
the  agents  under  natural  and  artificial  conditions. 

Major  Findings: 

A.  Mouse  adenovirus.  The  new  virus  recovered  from  General 
Purpose  Swiss  mice  which  produces  myocarditis  and  adrenal  necrosis, 
has  been  identified  as  a  mouse  representative  of  the  adenovirus  group. 
Infection  with  this  agent  is  found  in  many  laboratory  mouse  colonies, 
but,  so  far,  has  not  been  encountered  in  wild  mouse  populations. 


Part  B  Included:      Yes 


150 


Serial  No.  NIAID  -  7  IB 


Virus  is  found  in  urine  of  spontaneously  and  artificially  infected 
mice  for  prolonged  periods  up  to  8  months  or  longer,  and  it  is  con- 
cluded that  the  chief  mode  of  spread  of  this  virus  is  by  urinary 
excretion. 

B.  Thymic  agent  (TA).  A  new  virus  has  been  recovered  from 
the  NIH  General  Purpose  Swiss  mice  and  from  wild  house  mice.  This 
virus  induces  a  unique  necrosis  of  the  thymus  when  inoculated  into 
infant  mice.   It  produces  a  chronic  infection  with  prolonged 
excretion  of  virus  in  saliva. 

C.  Reovi ruses  have  been  found  to  be  very  common  agents  of 
laboratory  mice.  Reovirus  3  has  been  found  in  the  majority  of  mouse 
colonies,  and  suggestive  evidence  of  its  presence  in  germ-free  mice 
has  been  obtained.  The  hepatoencephal it  is  virus  of  Stanley,  which 
was  isolated  in  1953,  and  considered  by  some  workers  to  be  a  mouse 
virus,  has  been  identified  as  a  Reo  3  strain. 

D.  A  new  virus,  presumably  of  mouse  origin,  has  been  isolated 
from  Qrn ithonyssus  bacoti  mites  which  were  feeding  on  laboratory  mice. 
This  virus  induces  an  encephalitis  in  baby  mice. 

E.  In  the  course  of  studies  of  polyoma  epidemiology  in  wild 
rodent  populations,  a  new  virus  isolated  from  the  Microtus  mouse. 
This  virus  perhaps  is  the  Microtus  representative  of  the  salivary 
gland  virus  group,  but  it  differs  from  other  species  salivary  gland 
viruses  by  its  ability  to  grow  in  tissue  cultures  from  many  diffei — 
ent  species.  A  complement  fixation  test  has  been  developed  for  this 
agent. 

F.  "K"  virus.  The  K  virus  of  mice  has  been  found  to  be 
prevalent  in  almost  all  colonies  tested.   Suggestive  evidence  of  its 
presence  in  germ- free  mice  has  been  obtained.  K  virus  has  also  been 
isolated  from  wild  mice,  being  found  in  saliva  or  urine.  A  complement 
fixation  test  has  been  developed  which  currently  is  being  analyzed. 

G.  Mouse  hepatitis  virus.  Studies  have  been  made  of  the  mouse 
hepatitis  virus  and  its  activation  by  Eperythrozoon  coicoides  as 
described  by  Gledhill.  Hepatitis  virus  can  be  activated  in  specific 
pathogen-free  mice  with  the  same  regularity  as  in  conventional  lab- 
oratory mice,  suggesting  that  the  virus  is  transplacental  I y  acquired. 

H.  Mouse  salivary  gland  virus  has  been  found  to  be  prevalent 
in  virtually  all  wild  Mus  musculus  colonies  both  urban  and  rural.   Its 
natural  history  is  currently  under  study. 


151 


Serial  No.  NIAIO  -  7  IB 

Significance  to  the  Program  of  the  Institute:  With  the  con- 
tinual expansion  of  attempts  to  develop  indirect  assay  procedures 
for  mouse  tumor  viruses,  it  has  become  increasingly  evident  that  the 
viral  flora  of  the  mouse  is  a  major  stumbling  block  to  developing 
reliable  study  systems.   In  this  and  other  laboratories,  attempts 
to  develop  such  assays  for  tumor  viruses  have  repeatedly  foundered 
because  of  the  emergence  of  extraneous  mouse  viruses  in  the  indicator 
systems.  Only  by  a  thorough  understanding  of  the  extraneous  agents 
which  are  likely  to  be  encountered,  and  the  ability  to  recognize 
them  can  tumor  virus  studies  proceed  intelligently  with  the  hope  of 
obtaining  reliable  information  of  high  order.   Secondly,  the  increasing 
evidence  of  the  importance  of  the  interference  phenomenon  in  deter- 
mining the  outcome  of  viral  experiments  in  mice  makes  it  important 
to  evaluate  the  importance  of  extraneous  viruses  as  interfering 
factors  in  tumor igenesis  studies.  Thirdly,  the  possibility  must  be 
considered  that  so-called  "non-tumor  viruses"  may  play  a  role  in  the 
etiology  of  neoplasms;  the  mouse  viruses  provide  an  excellent  oppor- 
tunity to  test  this  hypothesis.  Fourthly,  the  viruses  of  mice  have 
long  been  recognized  to  be  useful  models  of  virus  infections  of 
humans  (some  of  them  are  identical  or  similar  to  human  areas)  and  on 
their  own  merit  are  deserving  of  study. 

Proposed  Course  of  the  Project:   Epidemiologic  studies  of  the 
newer  mouse  viruses  will  be  continued,  and  additional  attempts  will 
be  made  to  develop  and  evaluate  serologic  tests  useful  for  detection 
of  infection. 

Extension  of  current  serological  surveys  are  planned  to  detei — 
mine  if  associations  exist  between  certain  mouse  tumors  and  past 
infection  with  the  known  and  newly  discovered  mouse  viruses.  Mice, 
hamsters,  and  other  laboratory  species  will  be  inoculated  with  sub- 
lethal doses  of  the  various  mouse  viruses  and  long-term  observation 
will  be  carried  out  for  possible  tumor  induction. 

Studies  will  be  made  of  the  influence  of  various  mouse  breeding 
practices  such  as  sterilization  of  food  and  rearing  under  specific 
pathogen-free  and  germ-free  conditions  as  they  influence  the 
occurrence  of  mouse  viruses  in  the  hope  that  practices  can  be  designed 
to  eliminate  mouse  viruses  as  an  uncontrolled  variable  in  study 
systems. 


152 


Serial  No.  NIAID  -  7  IB 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  B.   Honors,  Awards,  and  Pub  I  ications 
Publications  other  than  abstracts  from  this  project: 


Hartley,  J.W.,  and  Rowe,  W.P.:  A  new  mouse  virus  apparently 

related  to  the  adenovirus  group.  Virology,  v: I  1 ,  645-647,  I960. 


153 


Serial  No.   NIAID  -  72 


1.  Infectious  Diseases 

2.  Virus  &  Rickettsial  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  A. 

Project  Title:   Laboratory  studies  of  enteroviruses. 

Principal  Investigator:  Dr.  Karl  M.  Johnson 

Other  Investigators:     Dr.  Robert  M.  Chanock,  Dr.  Robert  J.  Huebner 

Cooperating  Units:       Dr.  L.  Rosen,  Epidemiology  Section,  LID 

Man  Years  (calendar  year  I960): 
Total:        14/12 
Professional:  4/12 
Other:       10/12 

Project  Description: 

Objectives:   To  develop  simple  reproducible  methods  for  laboratory 
manipulation  of  enteroviruses,  particularly  the  large  group  of  newly 
recognized  agents  which  appear  to  be  potential  causes  of  respiratory 
disease.   Studies  are  designed  specifically  to  determine  optimum  methods 
of  cultivation  of  the  agents  in  either  tissue  culture  or  small  laboratory 
animals.   In  addition,  efforts  are  made  to  a)  determine  the  optimum 
manner  of  preparing  specific  antisera,  b)  to  evaluate  various  serologic 
procedures  potentially  of  use  in  field  studies,  and  c)  since  many  new 
viruses  are  fastidious  in  their  growth  requirements,  to  study  where 
indicated  certain  fundamental  properties  of  these  viruses. 

Methods  Employed:   These  will  be  described  where  pertinent 
under  consideration  of  results  so  far  obtained  with  certain  of  the  viruses. 

Major  Findings: 

I .  Coe  virus:   Newly  recovered  strains  of  Coe  virus  have  proven 
to  have  properties  differing  from  those  described  for  the  original 
prototype  agent  in  several  important  respects.  The  presence  of  hemagg- 
lutinin is  demonstrable  when  human  type  0  erythrocytes  are  employed 
and  the  test  performed  at  4°  centigrade.  The  reaction  is  temperature 
dependent  and  can  be  reversed  at  34°  C.  Elution,  however,  is  not 
enzymatic  since  the  erythrocyte  receptors  are  not  altered  following 

Part  B  Included:     No 

154 


Serial  No.   NIAID  "  72 


reversal  of  hemagglutination.   The  component  of  the  virus  which 
participates  in  the  hemagglutination  reaction  contains  specific  Coe 
antigen  as  indicated  by  the  inhibitory  effect  of  specific  antiserum. 
The  hemagglutinin  is  quite  stable  at  4°  and  at  -20°  C,  but  is  fairly 
rapidly  inactivated  at  34°  C.   Studies  of  virus  antibody  kinetics  in 
the  hemagglutination  inhibition  (HI)  test  indicate  that  optimal  virus 
antibody  reaction  occurs  following  incubation  at  24°  for  approximately 
2  hours,  or  at  4°  for  14  hours.  Using  the  latter  procedure,  a  rise  in 
HI  antibody  was  demonstrated  for  15  of  19  men  from  whom  the  virus  was 
recovered. 

Comparative  studies  indicate  that  primary  human  embryonic  kidney 
cells  (HK)  are  more  sensitive  than  serially  passaged  human  cells  to 
natural  strains  of  Coe  virus.   In  addition,  HK  cells  produce  a  greater 
quantity  of  hemagglutinin  than  do  continuous  cell  lines  of  human  origin 
regardless  of  the  nutrient  medium  employed.  For  optimum  results  with 
Coe  virus  it  is  necessary  that  the  culture  tube  be  rotated  rather  than 
incubated  in  the  conventional  stationary  position. 

Our  strain  of  Coe  virus,  unl  ike  the  prototype,  has  been  success- 
ful ly  adapted  to  the  suckling  mouse.  Typical  Coxsackie-I  ike  hind  leg 
paralysis  has  been  produced  through  5  consecutive  passages.  This 
observation  is  currently  being  exploited  in  attempts  to  prepare  specific 
viral  antisera,  as  well  as  for  experimental  production  of  a  complement 
fixing  antigen. 

2.   Newly  described  enterovirus- I  ike  agents:   In  January  of  this 
year  English  workers  described  several  strains  of  viruses  with  certain 
properties  of  enteroviruses,  which  they  had  recovered  from  nasal 
secretions  of  volunteers  with  clinical  cold-like  illnesses.  They 
reported  that  an  unusual  set  of  laboratory  conditions  was  required  for 
a  successful  cultivation  of  these  agents.   In  genera  I,, we  have 
succeeded  in  confirming  their  observations  and  have  made  further  progress 
in  efforts  to  adapt  and  manipulate  these  viruses  in  the  laboratory. 
Human  embryonic  kidney  seems  to  be  necessary  for  recovery  of  most  of 
these  agents.   Preliminary  studies  suggest  that  there  is  an  inverse 
relationship  between  the  gestational  age  of  the  fetus  from  whom  the 
cultures  are  derived  and  the  occurrence  of  cytopathic  effects  produced 
by  these  agents.  At  least  two  of  these  viruses  have  been  successfully 
adapted  in  this  laboratory  to  continuous  type  cultures  of  human 
epithelial  origin.   Using  these  cells  as  a  source  of  viral  antigen, 
high-titered  specific  immune  serum  has  been  prepared  in  guinea  pigs 
and  a  reproducible  neutralization  technique  worked  out.  Current  efforts 
are  being  directed  toward  a  search  for  complement  fixing  antigens  and 
hemagglutinins.  Employing  human  embryonic  kidney  tissue  cultures, 
numerous  new  virus  strains  have  been  recovered  which  seem  to  possess 
properties  similar  to  those  of  the  English  viruses.  Sorting  out  these 
new  isolates,  and  the  establishment  of  new  serotypes,  is  currently 
under  way. 

155 


Serial  No.   NIAIO  -  72 


Significance  to  the  Program  of  the  Institute;   These  studies 
provide  new  diagnostic  tools  and  opportunities  for  epidemiologic 
investigation  of  a  large  group  of  viruses  which  are  potentially 
significant  in  human  respiratory  disease. 

Proposed  Course  of  the  Project:   Efforts  will  be  made  to  simplify 
the  identification  of  the  enteroviruses  which  grow  predominantly  in 
human  tissue  culture  cells.  This  includes  sorting  out  of  the  available 
isolates  and  the  recognition  of  new  serotypes.  Fortunately,  methods 
for  CF  and  HI  identification  of  enteroviruses  developed  by  the 
laboratory  unit  of  the  Epidemiology  Section  and  by  the  Serology  Unit 
of  LID,  promises  to  reduce  the  time  and  work  required  to  compare 
presumably  "new"  enteroviruses  with  the  58  serotypes  already  classified. 
Attempts  will  be  made  to  develop  simplified  methods  of  antibody  assay 
for  use  in  epidemiologic  studies.   Until  this  is  done,  understanding  of 
the  role  these  agents  play  in  human  disease  will  proceed  at  a  slow 
pace. 


156 


Serial  No.   NIAID  -  72A 


1.  Infectious  Diseases 

2.  Virus  &  Rickettsial  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title;   Study  of  viral  respiratory  disease  in  a  military 
population. 

Principal  Investigator:  Dr.  Karl  M.  Johnson 

Other  Investigators:   Dr.  Robert  M.  Chanock,  Dr.  Robert  J.  Huebner 

Cooperating  Units:    Dr.  H.H.  Bloom,  Dr.  M.  Mufson,  Naval  Medical 
Field  Research  Laboratory,  Camp  Lejeune, 
North  Carol ina 

Man  Years  (calendar  year  I960): 
Total:         24/12 
Professional:    6/12 
Other:        18/12 

Project  Description: 

Ob ject i ves:   To  study  on  a  continuing  basis  the  role  of  newly 
recognized  viruses  in  adult  respiratory  illness.  This  study  was 
organized  in  order  to  compare  viral  experience  of  men  fresh  from  recruit 
training  with  that  of  individuals  forming  the  stable  cadre  personnel. 
An  opportunity  is  also  provided  to  assay  the  effectiveness  of  certain 
viral  vaccines  administered  to  personnel  during  their  recruit  training 
at  another  mil  itary  center. 

Methods  Employed:   Clinical  records  and  appropriate  specimens  are 
collected  routinely  from  individuals  in  various  dispensaries  at  Camp 
Lejeune,  North  Carolina.   Such  materials  are  also  obtained  from  indivi- 
duals hospitalized  at  this  base,  and  in  certain  instances,  from  children, 
wives  and  mothers  seen  in  dependent  outpatient  clinics.   Special  emphasis 
has  been  placed  upon  the  collection  of  specimens  from  matched  control 
groups. 

CI  inical  specimens  are  tested  in  monkey  kidney  and  Hep-2  tissue 
cultures.  Certain  other  tissue  cultures  are  employed  whenever 
indicated.  Paired  serum  samples  are  available  from  well  over  90#  of 
individuals  in  the  study  and  are  assayed  for  development  of  antibody 
for  various  viral  agents.  Specimens  for  virus  isolation  are  divided 
into  two  portions  and  stored  in  this  laboratory  and  the  Camp  Lejeune 
Part  B  Included:      Yes 

157 


Serial  No.   NIAID  -  72A 

laboratory  respectively. 
Major  Findings; 

1 .  Adenoviruses:   In  February,  March,  and  April  of  I960  adeno- 
viruses were  responsible  for  a  major  outbreak  of  respiratory  disease  in 
the  post  recruit  population  (Infantry  Training  Regiment,  ITR).  Attack 
rates  in  this  unit  attained  a  level  of  nearly  200  per  1000  week. 
Infection  and  apparent  illness  were  also  disseminated  into  the  cadre 
population,  although  there  was  a  clearly  diminishing  incidence  in 
correlation  with  length  of  time  individuals  had  been  in  military  service. 

2.  Myxoviruses  (parainf luenzas) <  During  February  and  March  of 
I960  sporadic  isolations  of  para  influenza  viruses  were  also  recorded. 
Although  insufficient  data  were  obtained  to  permit  epidemiologic 
association  of  para  influenza  virus  infection  with  illness,  several  new 
laboratory  phenomena  were  observed  which  had  not  been  previously 
encountered  with  these  viruses  in  the  study  of  pediatric  populations. 
With  only  one  exception,  all  15  individuals  from  whom  virus  was 
recovered  had  significant  neutralizing  antibody  against  the  infecting 
agents  in  their  acute  phase  sera.  Virus  recovery  proved  difficult,  and 
many  of  the  isolates  were  not  recognized  until  after  two  weeks  of  tissue 
culture  incubation.   Re-isolation  of  the  agents  proved  exceedingly 
difficult.  Appropriate  controls  were  included  in  each  test  to  insure 
that  the  isolates  did  not  represent  contamination  from  the  laboratory 
environment.   Serologic  confirmation  of  infection  was  obtained  in  only 

9  of  the  15  cases  despite  the  utilization  of  neutralization,  hemagg- 
lutination-inhibition,  and  complement  fixation  techniques.  The  lack  of 
serologic  response  to  infection  was  most  common  with  para  influenza  I 
virus.  These  observations  suggest  that  the  infections  were  mild  and 
that  very  smal I  amounts  of  virus  were  excreted  by  the  infected  indivi- 
duals.  Information  derived  from  this  experience,  however,  has  allowed 
a  better  formulation  of  laboratory  procedures  designed  to  elucidate 
the  role  of  these  agents  in  adult  respiratory  illness  in  the  future. 

3.  Coe  virus  outbreak;  During  October  and  November  of  I960,  a 
major  outbreak  of  respiratory  disease  associated  with  Coe  virus,  a 
newly  discovered  enterovirus- 1  ike  agent,  was  observed.  Virus  was 
recovered  significantly  more  often  from  men  with  respiratory  disease 
than  from  comparable  control  subjects  free  of  such  illness.  Attack 
rates  have  not  yet  been  calculated,  but  they  seem  to  have  been 
extremely  high.  Clinical  illness  consisted  of  mild  upper  respiratory 
infection  with  or  without  fever.  Mild  pharyngitis  was  frequently 
observed.  Hospital  surveillance  indicated  no  significant  increase  in 
the  number  of  admissions  for  pneumonia,  nor  were  there  any  clinical 
cases  of  herpangina,  pleurodynia,  or  aseptic  meningitis.  The  occurrence 
of  infection  in  children  during  this  time  has  also  been  documented 

but  the  role  of  this  virus  in  pediatric  illness  remains  to  be  assessed. 

158 


Serial  No.   NIAID  -  72A 


The  current  outbreak  represents  the  first  documented  association  of 

an  enterovirus  with  a  large  scale  occurrence  of  mild  respiratory  illness 

in  adults. 

Significance  to  the  Program  of  the  Institute;   This  project 
represents  a  major  continuing  inquiry  into  the  nature  of  respiratory 
disease  in  adult  populations.   It  has  already  yielded  evidence  for  the 
association  of  a  new  enterovirus  with  respiratory  disease,  as  well  as 
providing  needed  laboratory  information  concerning  para  influenza  virus 
infection  in  adults.  A  well  documented  collection  of  case  histories, 
specimens,  and  paired  sera  provides  an  excellent  source  of  clinical 
material  which  is  available  for  the  evaluation  of  newly  discovered 
agents,  as  we  I  I  as  those  not  yet  known  to  us. 

Proposed  Course  of  the  Project:  Field  surveillance  is  being 
continued.  Extension  of  the  program  to  incorporate  dependents  (both 
adults  and  children)  is  being  undertaken.   In  this  way  a  broader  picture 
of  the  natural  history  of  newly  recognized  respiratory  viruses  can  be 
acqu  ired. 

A  study  designed  to  test  the  effectiveness  of  adenovirus  vaccines 
is  also  being  organized.  Since  all  personnel  at  the  Infantry  Training 
Regiment  represent  immediate  graduates  of  the  recruit  training  base  at 
Parris  Island,  South  Carolina,  and  since  all  these  men  are  currently 
being  given  adenovirus  vaccine  upon  entry  into  the  Marine  Corps  the 
following  procedures  will  be  employed:   A  large  number  of  men  undei — 
going  training  at  Parris  Island  will  be  bled  approximately  4  to  5  weeks 
following  administration  of  adenovirus  vaccine.  Recruits  are  routinely 
bled  when  they  enter  service  and  this  serum  is  available  to  us.  These 
individuals  will  be  observed  during  their  time  at  the  Infantry  Training 
Regiment,  and  a  certain  percentage  of  them  will  also  be  available  for 
study  following  this  period  of  training.   In  this  way  the  immunogenic 
potency  of  the  vaccine  as  well  as  its  effectiveness  in  preventing 
illness  may  be  assessed. 

In  cooperation  with  personnel  at  the  hospital  at  Camp  Lejeune  a 
study  of  the  effectiveness  of  tetracyclines  in  the  therapy  of  febrile 
respiratory  disease  with  and  without  pneumonia  is  also  being  organized. 
Among  the  agents  to  be  included  in  this  study  will  be  pneumonia  caused 
by  the  Eaton  virus  which  has  recently  been  shown  to  respond  to  chemo- 
therapy. 


153 


Serial  No.   NIAID  -  72A 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  B.   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Bloom,  H.H.,  Johnson,  K.M.,  Chanock,  R.M.,  Huebner,  R.J.,  and 
Jacobsen,  R.F.:  Recovery  of  para  influenza  viruses  from  adults. 
J.  of  Clin.  Investigation.   In  press. 


160 


Serial  No.   NIAID  -  72B 

1.  Infectious  Diseases 

2.  Virus  4  Rickettsial  Diseases 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  A. 

Project  Title:   Study  of  respiratory  viruses  in  human  volunteers. 

Principal  Investigator:  Dr.  Robert  M.  Chanock,  Dr.  Karl  M. 

Johnson 

Other  Investigators:     Dr.  V.L.  Knight,  Dr.  H.M.  Kravetz, 

Dr.  D.E.  Rifkind,  Dr.  J. P.  Utz 

Cooperating  Units:       Laboratory  of  Clinical  Investigations, 

NIAID 

Man  Years  (calendar  year  I960): 
Total:        17/12 
Professional:  5/12 
Other:        12/12 

Project  Description: 

Object  i ves:   To  determine  whether  newly  recognized  viruses  of 
potential  respiratory  pathogenicity  are  capable  of  inducing  mild 
illness  in  adults  under  controlled  conditions.  To  examine  the 
experimental  pathogenesis  of  such  viral  infection,  and  to  provide 
new  information  specifically  designed  to  facilitate  the  study  of 
naturally  occurring  mild  respiratory  illness  in  adults. 

Methods  Employed:  Young  adult  male  volunteers  from  federal 
prisons  are  selected  on  the  basis  of  a)  general  good  health,  and  b) 
neutralizing  antibody  status  for  the  particular  virus  to  be  studied. 
Volunteers  are  maintained  in  groups  of  three  in  strict  isolation  on 
a  clinical  service  of  the  NIAID.  Histories  and  routine  physical 
examinations,  as  well  as  certain  basic  laboratory  procedures  are 
performed  upon  admission.  Known  amounts  of  test  viruses  are 
administered  intranasal ly,  and  daily  clinical  observations,  as  well 
as  appropriate  specimens  for  laboratory  analysis,  are  obtained. 
Virus  specimens  are  inoculated  into  appropriate  tissue  cultures  and/or 
animals.   Serologic  tests  are  performed  on  suitably  collected  serum 
samples. 

Part  B  Included:     No 


161 


Serial  No.   NIAID  -  72B 


Major  Findings: 


Para  influenza  4;   In  a  single  experiment,  approximately 
10,000  TCD50  of  monkey  kidney  adapted  para  influenza  4  virus  infected 
only  two  of  six  volunteers  receiving  the  inoculum.  No  clinical 
illness  was  observed. 

Respiratory  Syncytial  Virus:  In  four  experiments,  volunteers 
were  successfully  infected  when  given  a  small  dose  (160  to  640  TCD50) 
of  respiratory  syncytial  (RS)  virus.  One  half  of  the  volunteers 
developed  typical  cold-like  illness  without  fever  or  pharyngitis. 
The  occurrence  of  illness  correlated  with  the  length  of  time  virus 
was  shed  and  the  onset  of  virus  excretion  was  coincident  with  or 
preceded  the  appearance  of  cl  inical  illness  in  every  instance,  pro- 
viding evidence  that  the  observed  colds  were  actually  produced  by  the 
inoculated  virus.  Development  of  RS  virus  complement  fixing  antibody 
correlated  with  the  occurrence  of  illness.   Since  all  volunteers  in 
this  study  possessed  RS  neutralizing  antibody  prior  to  challenge, 
these  experiments  represent  examples  of  mild  illness  produced  by 
viral  reinfection.  The  occurrence  of  illness  was  not  related  to  the 
level  of  neutralizing  antibody  present  prior  to  administration  of 
virus.   Studies  designed  to  investigate  the  circumstances  associated 
with  this  interesting  and  potentially  significant  finding  are 
currently  in  progress. 

Significance  to  the  Program  of  the  Institute:  This  project  has 
resulted  in  the  experimental  demonstration  of  a  significant  new  concept 
concerning  the  pathogenesis  of  mild  respiratory  illness  in  adults.   It 
has  also  provided  valuable  information  pertinent  to  the  design  and 
execution  of  future  field  studies  of  RS  virus  infection. 

Proposed  Course  of  the  Project;   Volunteer  studies  involving 
the  Eaton  agent  (associated  with  all,  or  almost  all,  cold  agglutinin 
positive  atypical  pneumonia  and  a  significant  proportion  of  cold 
agglutinin  negative  pneumonia)  are  in  progress.  This  investigation 
should  yield  important  information  relating  to  the  varying  clinical 
consequences  of  infection  with  this  agent.  Future  studies  are   planned 
to  evaluate  certain  other  potential  respiratory  viruses,  including  a 
para  influenza  virus,  the  family  of  Reoviruses,  as  well  as  certain 
newly  described  enteroviruses. 

Influenza  A  and  B  viruses  grown  in  monkey  and  human  tissue 
cultures  will  be  studied  in  volunteers  as  part  of  a  search  for 
attenuated  variants  suitable  for  inclusion  in  a  live  virus  vaccine. 


162 


Serial  No.  NIAIO  -  73 


1.  Infectious  Diseases 

2.  Virus  A  Rickettsial  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A. 


Project  Title:   Application  of  fluorescent  antibody  staining 
technique  to  the  study  of  respiratory  viruses. 

Principal  Investigator:   Dr.  Alexander  L.  Kisch 

Other  Investigators:     Dr.    Robert  M.  Chanock,  Dr.  Karl  M.  Johnson 

Cooperating  Units:       None 

Man  Years  (calendar  year  I960): 
Total:       8/12 
Professional:  6/12 
Other:       2/12 

Project  Description: 

Ob jecti ves:   I)  To  study  the  sequence  of  intracellular  events 
leading  to  the  production  of  viral  antigen  and  the  production  of 
syncytia  by  respiratory  syncytial  (RS)  virus,  and  to  differentiate 
this  virus  from  others,  such  as  measles,  also  producing  syncytia  in 
tissue  culture.   2)  To  develop,  if  possible,  a  rapid  diagnostic  test 
for  certain  viral  agents  causing  respiratory  disease  in  children 
and  adults  utilizing  fluorescent  antibody  technique.   3)  To  determine 
the  nature  of  the  intracellular  substance  which  is  labelled  by 
f luoresce in-l abel led  antibody  using  cytochemical  methods. 

Methods  Employed:  The  agents  under  study,  specifically  RS  virus 
and  Eaton  agent,  are  grown  in  tissue  culture  using  a  variety  of  tissues. 
At  timed  intervals,  infected  covers  I  ips  are  removed  and  stained  by 
several  methods:  fluorescent  antibody,  Toluidine  Bl ue-Mol ybdate  (for 
RNA),  Feulgen  (for  DNA),  Hematoxyl  in-eosin  (for  inclusion  bodies), 
Sudan  Black  and  PAS  (for  definition  of  "golgi  material").  RS  virus  is 
compared  with  measles  virus, which  also  leads  fo  syncytia  production 
in  the  attempt  to  further  characterize  and  classify  RS  virus. 

Further,  cell-bearing  secretions  from  the  respiratory  tract  of 
volunteers,  some  of  whom  have  been  infected  with  either  RS  or  PAP  virus, 
are  stained  by  indirect  fluorescent  antibody  technique  utilizing 

Part  B  Included:     No 

163 


Serial  No.  NIAID  -   73 

appropriate  controls  in  the  attempt  to  diagnose  infection  early 
and  specifically.   If  this  becomes  possible,  it  would  have  significant 
therapeutic  implications  in  one  of  the  few  virus  diseases  susceptible 
to  broad  spectrum  antibiotics. 

Major  Findings; 

It  was  possible  to  stain  cells  infected  with  RS  virus 
specifically  using  human  convalescent  serum  and  f luorescein-label led 
anti-human  globulin.  Fluorescent  staining  antigenic  material  is 
restricted  to  the  cytoplasm,  and  is  first  demonstrable  after 
approximately  12  hours.   Individual  infected  cells  show  fluorescence 
24  hours  before  the  earliest  characteristic  CPE  is  recognizable  by 
light  microscopy.   No  intranuclear  fluorescence  was  observed  despite 
the  appearance  of  nuclear  changes  occurring  earlier  and  simultaneously 
in  infected  cells  as  demonstrated  by  Toluidine  Blue-Mol ybdate 
staining. 

Attempts  to  demonstrate  specifically-staining  antigen  in  cells 
from  the  nasopharynx  of  volunteers  infected  with  RS  virus  have  been 
unsuccessful  to  date. 

Significance  to  the  Program  of  the  Institute:   Th  i  s  project  has 
resulted  in  a  development  in  this  laboratory  of  a  technically  diffi- 
cult technique  of  wide  applicability  to  the  study  of  viral  infection 
in  tissue  cultures  as  well  as  in  infected  patients.   Should  it  become 
possible  to  diagnose  human  infections  with  RS  virus  and/or  Eaton 
agent  by  this  method,  this  will  be  of  value  in  clinical  and  epidemio- 
logic studies. 

Proposed  Course  of  the  Project:   Further  studies  to  determine 
whether  it  is  the  viral  or  the  soluble  antigen  of  the  RS  virus  which 
is  stained  by  immunofluorescence,  will  be  carried  out.   Studies  on 
secretions  obtained  from  volunteers  infected  with  Eaton  virus  are 
under  way,  and  further  studies  on  subjects  infected  with  RS  virus 
will  be  carried  out  at  a  later  date. 


164 


Serial  No.  NIAID-74 

1.  Infectious  Diseases 

2.  Virus  and  Rickettsial 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual   Project  Report 

Calendar  Year    I960 


Part  A 


Project  Title:  Study  of  Bovine  Respiratory  Diseases. 

Principal  Investigator:  F.  R.  Abinanti 

Other  Investigators:  R.  J.  Huebner  (LID);  Robert  Byrne  (University 
of  Maryland);  Alvin  Hoerlein  (University  of 
I  I  I inois) 

Cooperating  Units:    Veterinary  Science  Department,  University  of 
Maryland;  College  of  Veterinary  Medicine, 
University  of  Illinois 

Man  Years:   (Calendar  Year  I960) 
Total:         6/12 
Professional  :   3/12 
Other:         3/12 

Project  Description: 

Objectives :  This  is  a  continuation  of  a  project  in  effect 
during  the  preceeding  fiscal  year.  Essentially  these  investigations 
include  studies  of  outbreaks  of  respiratory  virus  disease  in  cattle, 
of  experimental  pathogenesis  of  the  viruses  injected  into  cattle, 
and  conducting  sero-epi zootio logic  surveys  of  their  prevalence. 
Experimental  vaccines  are  also  prepared  and  their  antigenic  potency 
tested  in  cattle. 

Specific  Objectives:   (I)  The  isolation  of  viral  agents  from 
the  respiratory  tract  of  normal  cattle  and  cattle  with  respiratory 
disease;  (2)  to  assess  the  role  of  myxovirus  para-inf I uenza  3 
virus  and  other  newly  recognized  viruses  in  respiratory  disease 
of  cattle;  (3)  to  determine  the  relationships  of  the  human  and 
bovine  strains  of  myxovirus  para-inf I uenza  3;  and  (4)  to  evaluate 
antiviral  vaccines. 


Part  B  incl uded :   Yes 


165 


Serial    No.   NIAIO-74 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

Methods  Employed:  Specimens  of  nasal  secretions  were  taken 
from  cattle  and  inoculated  onto  tissue  cultures  of  bovine  and 
monkey  kidney.  The  viruses  isolated  are  characterized  and  compared 
to  known  viral  agents  of  man,  cattle,  and  other  animals.  Serial 
samples  of  blood  are  taken  from  normal  cattle  and  acute  and  con- 
valescent specimens  from  diseased  cattle,  and  hemagglutination 
inhibition,  complement  fixation,  and  neutralization  tests  are  used 
to  obtain  information  concerning  the  prevalence,  spread  and  natural 
history  of  respiratory  viruses.  Groups  of  cattle  are  vaccinated 
with  experimental  vaccines  and  potency  assessed  by  various  serologic 
tests. 

Major  Findings:   (I)  Eighteen  recoveries  of  myxovirus  para- 
influenza 3  virus  have  been  made  from  cattle  suffering  from  respiratory 
disease  and  serologic  evidence  of  a  relationship  of  this  virus  to 
the  outbreaks  of  respiratory  disease  was  found.  Extensive  sero- 
epidemiologic  studies  provided  evidence  that  infection  with  this 
virus  is  widespread.   In  a  preliminary  test,  young  cattle  developed 
good  levels  of  antibody  to  experimental  vaccines  prepared  against 
both  human  and  bovine  strains  of  para-inf I uenza  3;  they  appeared 
to  be  refractory  to  an  aerosol  challenge  by  live  virus. 

(2)  Several  large  groups  of  cattle  were  vaccinated  under 
field  conditions  with  commercially  produced  para-inf I uenza  3  (bovine 
strain)  vaccines.  These  vaccines  also  produced  good  levels  of 
circulating  antibody.  Unfortunately,  in  these  field  trials  little 
or  no  respiratory  disease  occurred. 

(3)  Comparative  studies  of  the  human  and  bovine  strains 
of  the  virus  showed  that  they  can  be  distinguished  by  means  of 
prototype  guinea  pig  sera  in  the  HI  test. 

(4)  Infectious  bovine  rhinotracheitis,  a  virus  usually 
recovered  from  cattle  with  respiratory  disease,  was  recovered  from 
cattle  involved  in  a  large  outbreak  of  bovine  conjunctivitis  - 
attended  by  little  or  no  respiratory  disease.  The  virus  strain 
recovered  produced  conjunc itivtis  and  minimal  respiratory  illness 
in  experimentally  inoculated  calves. 


166 


Serial    No.   NIAID-74 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Significance  to  the  Program  of  the  Institute:  The  discovery 
of  viruses  in  domestic  animals  which  are  similar  or  identical  to 
those  responsible  for  large  amounts  of  human  respiratory  disease 
is  of  obvious  importance.  Furthermore,  the  study  of  respiratory 
disease  in  domestic  animals  and  the  efficacy  of  antiviral  vaccines 
in  controlling  such  illnesses  provide  incomparable  models  of  similar 
problems  in  man.   In  addition,  solutions  to  some  of  the  common 
viral  diseases  of  food  animals  have  great  significance  for  human 
health  in  many  areas  of  the  world  where  animal  protein  is  deficient. 
The  prevalence  of  these  domestic  animal  viruses  suggests  the 
possibility  of  a  widespread  zoonotic  potential. 

Proposed  Course  of  Project:  Work  on  this  project  has  been 
temporarily  discontinued.  We  do  not  have  sufficient  resources  to 
mount  adeguate  field  studies,  and  so  far  we  have  found  it  impossible 
to  find  collaborators  able  to  conduct  studies  designed  to  answer 
definitive  guestions  on  the  etiology  of  bovine  respiratory  diseases. 


167 


Serial    No.    NIAID-74 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  B:   Honors,  Awards,  and  Publications 

Publ ications  other  than  abstracts  from  this  project: 

Abinanti,  F.R.,  Byrne,  R.J.,  Watson,  R.L.,  Poelma,  L.J.,  Lucas, 
F.R.,  and  Huebner,  R.J.:  Observations  on  infection  of  cattle 
with  myxovirus  para-inf I uenza  3.  Amer.  J.  Hyg.,  71  : 52-58, 
I960. 

Abinanti,  F.R.  and  Plumer,  G.J.:  The  isolation  of  infectious 

bovine  rhinotracheitis  (IBR)  virus  from  cases  of  conjunctivitis 
and  observations  on  the  experimental  infection.  A.V.M.A. 
Research  J.  Accepted  for  publication. 

Byrne,  R.J.,  Abinanti,  F.R.,  and  Huebner,  R.J.:  Vaccination  of 
cattle  with  myxovirus  para-inf I uenza  3.  Cornell  Vet. 
Accepted  for  publication. 

Abinanti,  F.R.,  Hoerlein,  A.B.,  Watson,  R.L.,  and  Huebner,  R.J.: 
Serological  studies  of  myxovirus  para-inf I uenza  3  in  cattle 
and  prevalence  of  antibodies  in  bovines.  J.  Immunol.  Accepted 
for  publ ication. 

Book:  ADVANCES  IN  VETERINARY  SCIENCE.  Chapter  on  "'Shipping 

Fever  in  Cattle",  by  S .  K.  Sinha  and  F.R.  Abinanti.   100  pages. 
To  be  included  in  1962  edition. 


168 


Serial    No.   NIAID-74 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Honors  and  Awards  relating  to  this  project: 

Invited  to  participate  in  Annual  Conference  of  Veterinarians. 
Talk:  "Respiratory  Diseases  of  Cattle"  -  University  of  Pennsylvania, 
Phi ladelphia,  May,  I960. 

Program  Chairman  and  participant  in  Maryland  Veterinary  Medical 
Association  Meeting  -  Ocean  City,  Maryland,  June,  I960. 

Participated  in  Conference  on  Zoonoses,  University  of  Illinois, 
Urbana,  September,  I960. 

Invited  to  participate  in  CDC  Biennial  Public  Health  Conference. 
Talk:  "Respiratory  Diseases  of  Man  and  Animals"  -  Atlanta,  Georgia, 
September,  I960. 

Participated  in  Conference  of  Public  Health  Veterinarians, 
American  Public  Health  Association,  San  Francisco,  California, 
November,  I960. 


169 


Serial  No.  NIAID-  74A 

1.  Infectious  Diseases 

2.  Virus  and  Rickettsial 

3.  Bethesda,  Maryland 


PHS-NIH 
ndividual   Project  Report 
Calendar  Year   I960 


Part  A 


Project  Title:  A  Study  of  Squamous  Cell  Carcinoma  (Cancer  Eye) 
in  Cattle. 

Principal  Investigators:  Dr.  F.  R.  Abinanti,  Dr.  W.  Gay  (DRS), 

Dr.  M.  Stanton  (NCI) 

Other  Investigators:      Dr.  R.  J.  Huebner 

Cooperating  Units:       Bay  Manor  Farms  (owner  Mr.  Otis  Smith), 

Lewes,  Delaware;  M.D.  Anderson  Hospital 
(Dr.  Russell),  Houston,  Texas 

Man  Years:   (Calendar  Year  I960) 

Total:  9/12 

Professional :  6/12 

Other:  3/12 

Project  Description: 

Ob.jecti  ves :  There  are  approximately  600  hereford  cattle  on 
the  Bay  Manor  Farms,  Lewes,  Delaware,  and  about  30  per  cent  of  these 
cattle  have  precancerous  or  cancerous  lesions  of  the  eyes  and 
orbital  appendages.  The  principal  efforts  are  to  determine  if  a 
virus  is  responsible  for  this  condition  and  to  study  the  natural 
history  of  the  disease. 

Methods  Employed:  Monthly  inspections  are  made  of  the  herd 
and  a  photographic  record  is  being  made  of  the  progress  of  the 
lesions.  At  this  time  eye  swabs  are  taken  and  blood  for  possible 
future  serological  studies.  Dr.  William  Gay  surgically  removes 
affected  tissues  at  intervals  which  are  examined  histologically 
by  Dr.  M.  Stanton,  and  virological ly  by  Dr.  F.  R.  Abinanti.  Further 
frozen  sections  will  be  examined  by  the  fluorescent  technique  to 
explore  the  possibility  of  there  being  a  specific  antigenic  material 
present  in  the  cancers. 

Part  B  Incl uded  :  No 


170 


Serial    No.   NIAID-  74A 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


A  sero-ep izootio logical  study  of  cattle  with  and  without 
"cancer  eye"  has  been  planned  in  cooperation  with  the  M.D.  Anderson 
Hospital  "Cancer-Eye"  research  group  under  Dr.  William  Russell. 
Several  hundred  sera  will  be  collected  by  this  group  from  cattle 
with  cancer  eye  and  matched  controls.  The  specimens  will  be  tested 
for  antibodies  vs.  bovine  viruses  and  for  some  of  the  mouse,  chicken 
and  rabbit  tumor  viruses. 

Major  Findings:  None.  This  is  a  new  project. 

Significance  of  the  Program  to  the  Institute:  Provides  an 
opportunity  to  explore  the  possible  viral  etiology  of  squamous 
cell  carcinoma.  The  Bay  Manor  herd  provides  excellent  nearby 
material  for  epidemiological  and  natural  history  studies  of  the 
cancer  itself.  Since  the  precancerous  lesions  are  papillomatous, 
the  possible  relation  of  the  lesions  and  papilloma  viruses  is 
currently  under  study. 

Proposed  Course  of  the  Study:  The  continuation  of  this 
project  will  depend  on  whether  practical  leads  concerning  causative 
viruses  can  be  found  in  the  near  future;  in  that  event,  the  termina- 
tion date  of  this  project  cannot  at  this  time  be  predicted. 


171 


Serial    No.   NIAID-  75 

1.  Infectious  Diseases 

2.  Virus  and  Rickettsial 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  A 


Project  Title:  Laboratory  and  epidemiologic  studies  of  viruses 
as  possible  etiologic  agents  of  acute  leukemia 
in  the  pediatric  age  group. 

Principal  Investigator:  Dr.  M.  Katherine  Cook 

Other  Investigators: 


Cooperating  Units: 


Dr.  Robert  J.  Huebner 
Mr.  Horace  C.  Turner 

Dr.  Charles  Chany,  Laboratoire  des  Virus, 
Hopital  Saint-Vincent-de-Paul  ,  Paris; 

Dr.  Jean  Bernard,  Hematology  Service, 
le  centre  Claude-Bernard  de  M Hopital 
Saint  Louis,  Paris 


Man  Years:   (Calendar  Year  I960) 
Total:  14/12 

Professional  :     14/12 
Other :  None 

Project  Description: 

Objectives:   ( I )  To  search  for  viral  agents,  including  unknown 
tumor  viruses,  in  children  with  acute  leukemia;  (2)  To  study  the 
role  of  these  viruses  in  the  etiology  of  acute  childhood  leukemia 
and  to  attempt  to  obtain  some  evidence  to  support  or  disprove  the 
theory  of  the  viral  etiology  of  this  disease;  (3)  To  determine  the 
effect  of  cortisone  on  the  incidence  of  viral  infections  in  children, 
(4)  To  study  the  possibility  of  lysogenic  systems  in  herpes  virus 
and  enterovirus  infections. 


Part  B  Included:   Yes 


172 


Serial    No.   NlAin-     75 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

Methods  Employed:  The  study  population  includes  110  children 
hospitalized  for  acute  leukemia  at  I 'Hopital  Saint  Louis,  Paris; 
the  control  populations  are  (I)  children  hospitalized  for  other 
blood  disturbances  in  the  hematology  wards  with  the  acute  leukemia 
patients;  and  (2)  children  of  similar  age,  economic  background,  sex, 
and  race  hospitalized  in  the  general  pediatric  wards  at  Hopital 
Saint-Vincent-de-Paul,  Paris.  The  children  hospitalized  at  St.  Louis 
were  admitted  from  all  parts  of  France,  and,  on  a  consultation  basis, 
from  all  over  Europe.  The  only  treatments  currently  being  employed 
for  acute  leukemia  at  Saint  Louis  are  X-radiation,  cortisone, 
bone-marrow  transplants,  and  transfusions  with  whole  blood  and/or 
packed  red  eel  Is. 

It  is  postulated  that  if  the  virus  (or  viruses)  responsible 
for  the  leukemia  were  maintained  in  a  "provirus"  state  in  the  cell 
of  the  individual  that  it  should  manifest  itself  by  the  occasional 
production  of  mature  virus  and  that  heavy  cortisone  treatment  may 
enhance  the  possibility  of  recovering  the  agent,  if  present.   It 
has  been  possible  to  study  approximately  70  of  the  leukemic  children 
from  the  day  of  diagnosis  until  their  deaths. 

Conventional  virus  isolation  attempts  in  tissue  cultures 
are  used  on  throat  and  anal  swabs  taken  weekly  from  every  child 
for  the  duration  of  his  hospital ization  and  at  the  time  of  his 
visits  to  the  out-patient  clinic.  Bone-marrow  specimens  are 
obtained  whenever  possible.  Blood  specimens  for  serologic  studies 
are  obtained  every  two  weeks  during  the  period  of  hospitalization 
and  also  when  the  child  reports  to  the  out-patient  clinic.   In 
addition  to  the  standard  isolation  techniques,  the  interference 
technique  using  para-inf luenza  I  (Sendai)  as  an  indicator  virus 
and  the  indirect  fluorescent  antibody  technique  are  employed  to 
study  a  number  of  the  specimens.  Serologic  surveys  with  known 
viruses,  including  known  animal  tumor  viruses,  are  being  performed 
to  obtain  information  on  the  incidence  of  past  infection  in  the 
acute  leukemia  group  as  compared  to  the  two  control  groups. 

Laboratory  data  are  correlated  with  clinical  and  epidemiologic 
information  in  an  effort  to  determine  virus-disease  relationships. 

Major  Findings :  This  is  a  new  project  and  present  information 
is  limited  to  evaluation  of  the  interference  technique  and  indirect 
fluorescent  antibody  techniques  for  demonstration  of  viruses  which 
do  not  cause  cytopathogenic  effects  or  other  changes  in  tissue 
culture,  and  to  the  incidence  of  known  agents  in  the  study  population. 


173 


Serial  No.  NlAin-  75 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

It  can  be  stated,  however,  that  herpes  virus  was  extensively  prevalent 
in  the  leukemic  children  and  that  enteroviruses  were  conspicuously 
absent  as  compared  to  the  control  group. 

Significance  to  the  Program  of  the  Institute:  This  project,  a 
controlled  longitudinal  epidemiologic  and  laboratory  approach  to 
the  study  of  the  viral  etiology  of  acute  lymphocytic  leukemia  in 
the  pediatric  age  group,  represents  a  rather  unique  and  intensive 
effort  to  acquire  a  knowledge  of  the  virologic  experiences  in 
children  with  acute  leukemia.  Such  a  study  is  needed  to  establish 
suitable  baselines  to  interpret  future  laboratory  studies  of  specimens 
from  leukemia  patients.  At  least  one  full  year  of  epidemiologic 
and  laboratory  studies  are  essential  to  account  for  possible  seasonal 
variations  in  the  exacerbation  of  viruses.   It  has  been  reported 
earl ier  this  year  that  two  animal  tumor  viruses,  Rous  sarcoma  virus 
(Prince,  I960)  and  polyoma  virus  (Vogt  and  Dulbecco,  I960)  appear 
to  undergo  something  suggestive  of  lysogeny.   In  view  of  these 
findings  the  potential  importance  of  persistent  and  recrudescent 
viruses,  such  as  recurrent  herpes  and  varicella,  and  incomplete 
enteroviruses  participating  in  a  lysogenic  system  in  the  human 
leukemia  patient  needs  investi gat-ion.  The  effect  of  cortisone 
on  bacterial  infection  in  man  has  been  well  documented  but  little 
information  is  available  on  its  activity  in  viral  infection.  The 
extensive  clinical  use  of  this  drug  is  sufficient  justification  for 
a  control  led  study  to  determine  the  effect  of  cortisone  on  viral 
infections  in  man. 

Proposed  Course  of  Project:  During  the  next  one-two  months 
serologic  studies  with  sera  from  the  leukemia  patients  and  the  two 
control  groups  will  be  completed  to  determine  the  relative  prevalence 
of  viral  infections  in  the  leukemia  group  with  viruses  that  are 
known  to  produce  proliferative  growth  in  tissue  culture,  with  various 
animal  tumor  viruses  and  with  the  more  common  viral  pathogens 
as  compared  with  those  in  control  populations. 

In  collaboration  with  Dr.  Chany,  anal  specimens  will 
continue  to  be  collected  from  leukemia  and  control  children  at 
I'Hopital  St.  Louis  to  determine  if  the  rate  of  enterovirus  isolations 
in  the  study  group  continues  to  remain  low  when  compared  with  the 
control  group.  Serologic  studies  of  the  prevalence  of  the  prevalence 
of  infection  with  the  more  common  respiratory  viruses  in  cortisone 
treated  and  non-cortisone  treated  children  will  be  completed. 


174 


Serial    No.   NIAID-  75 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

Laboratory  investigation  of  incomplete  herpes  virus  and  incomplete 
herpes  virus  and  incomplete  enteroviruses  will  be  pursued  at  the 
Max-Planck  Institute  fur  Virusforschung,  Tubingen,  Germany,  where 
training  received  in  nucleic  acid  work,  high  tension  electrophoresis, 
ul tracentrifugat ion  and  other  physical-biological  techniques  will 
be  applied  to  these  problems. 


PART  B   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Chany,  C.  and  Cook,  M.K. :  Sur  un  facteur  collulaire  induit  par 
le  virus  entrainant  la  formation  de  syncytium  en  culture  de  tissue. 
Ann.  Inst.  Pasteur.   In  press. 


175 


PHS  -  NIH  Serial  No.  NIAID-76-A 


Part  A 


Individual  Project  Report      1.  Infectious  Diseases 
Calendar  Year  I960        2.  Medical  Mycology 

3.  Bethesda,  Maryland 


A.  Project  Title:  Ecologic  studies  of  fungi  pathogenic  for  man. 

Principal  Investigator:    C.  W.  Emmons 

Other  Investigator:       Willard  Piggott 

Cooperating  Units:        Individual  studies  within  this  project 

have  been  conducted  in  cooperation  with 
Dr.  Gordon  Clark,  Patuxent  Wildlife 
Refuge  and  with  Mr.  Charles  Hunt,  U.  S. 
Geologic  Survey,  Denver,  Colorado. 

Man  Years  (calendar  year  I960): 
Total:  20/12 

Professional:       10/12 
Other:  9/12 


Project  Description: 

Objectives: 

To  find  new  environmental  habitats  of  fungi  which  cause  sys- 
temic mycoses;  to  investigate  the  factors  which  limit  growth  of 
these  fungi  to  special  habitats;  to  investigate  methods  of  elimi- 
nating pathogenic  fungi  from  specific  habitats  and  to  apply  the 
information  so  obtained  to  studies  of  the  epidemiology  of  the  sys- 
temic mycoses. 

Methods  Employed: 

Samples  of  soil  were  collected  from  sites  suspected  of  being 
sources  of  infection  in  individual  cases  of  mycoses,  from  types  of 
habitats  known  to  harbor  fungi  in  order  to  extend  experience  in 
this  study  and  repeatedly  in  certain  selected  sites  known  to  harbor 
pathogens  in  intensive  studies  of  their  ecology  and  associated  sap- 


Part  B  included 

176 


Serial  Mo.  NIAID-76-A 


rophytic  microflora.   The  specimens  were  processed  by  conventional 
methods  of  mouse  inoculation  with  soil  suspensions. 

A  laboratory  device  for  exposure  of  mice  by  inhalation  of  dry 
spores  of  fungi  was  designed  in  collaboration  with  Mr.  Willard 
Piggott  and  has  been  used  to  simulate  natural  conditions  of  expos- 
ure in  pursuing  the  objectives  of  this  project. 

Major  Findings; 

The  association  between  Cryptococcus  neoformans  and  pigeon 
excreta  has  been  confirmed  in  many  additional  collections  of  spec- 
imens. Histoplasma  is  not  found  in  this  association.  The  actual 
occurrence  of  an  acute  pneumonic  form  of  cryptococcosis  has  not  yet 
been  proved  because  there  has  been  no  opportunity  to  study  such  an 
outbreak.  However,  circumstantial  evidence  for  the  occurrence  of 
this  type  of  cryptococcosis  continues  to  accumulate. 

Significance  to  bio-medical  research  and  the  program  of  the  Insti- 
tute; 

Environmental  sources  of  infection  urn-elated  to  patients  or 
infected  animals  are  almost  invariably  associated  with  systemic 
human  mycoses.  Some  of  the  diagnoses  in  reported  epidemics  or 
focal  outbreaks  of  mycoses  still  remain  equivocal  until  the  type 
of  information  sought  in  this  project  adds  to  our  knowledge  about 
the  epidemiology  of  the  mycoses. 

Proposed  Course  of  Project; 

This  project  will  be  continued  indefinitely. 


177 


PHS  -  NIH  Serial  No.  NIAID-76-A 


Individual  Project  Report      1.  Infectious  Diseases 
Calendar  Year  I960        2.  Medical  Mycology 

3.  Bethesda,  Maryland 

Part  B.  Honors,  Awards  and  Publications. 

Publications  other  than  abstracts  from  this  project: 

Emmons,  Chester  W.  Prevalence  of  Cryptococcus  neo fornans  in  Pigeon  Habitats. 
Public  Health  Reports,  75:362-365,  Apr.,  I960. 

Piggott,  Willard  R.  and  Ernmons,  Chester  W.  Device  for  Inhalation  Exposure 
of  Animals  to  Spores.  Soc.  Exp.  Biol.  &  Med.,  103:805-806,  I960. 

Honors  and  Awards  relating  to  this  project: 

Presidential  Address  before  the  Mycological  Society  of  America  at 
Stillwater,  Oklahoma,  August,  I960,  entitled:   "The  Jeykll-Hydes  of  Mycology", 

Presentation  of  a  paper  before  the  III  National  Congress  of  Microbiol- 
ogy, Mexico  City,  Oct.  12,  I960,  entitled:   "Inhalation  Route  of  Infection 
in  Experimental  Cryptococcosis". 

Presentation  of  a  paper  before  the  Washington  Speleological  Society, 
Dec.  6,  I960,  entitled:   "The  Occurrence  of  Pathogenic  Fungi  in  Caves". 

Participation  in  "Fireside  Conference",  the  American  College  of  Chest 
Physicians,  Washington,  D.  C,  Nov.  27,  I960.  Geographic  Environment  and 
the  Systemic  Mycoses. 

President,  Mycological  Society  of  America,  Aug.   1959-1960. 

Elected  Councilor-at-Large,  Society  of  American  Bacteriologists,  July 
1,  I960. 

Chairman,  Second  Conference  on  Medical  Mycology,  New  York  Academy  of 
Science,  Jan.  11  and  12,  I960. 

Appointed  Board  Member  of  the  American  Academy  of  Microbiology  for  a 
three -year  term. 

Chairman,  Standards  and  Exaioination  Committee,  American  Academy  of 
Microbiology. 

Convenor  of  Mycological  Symposium,  III  National  Congress  of  Microbiolo- 
gy, Mexico  City,  I960. 


178 


Serial  No.  NIAID-76-A 

Honors  and  Awards  relating  to  this  project  (continued): 

Member  of  Study  Section  on  Mycology  and  Bacterial  Diseases,  NIH. 

Invited  lecture  at  Annual  Meeting  of  the  State  Society  of  Bacteriolo- 
gists, University  of  North  Carolina,  Chapel  Hill,  No.  Carolina,  Apr.,  I960. 


179 


PHS  -  NIH  Serial  No.  NIAID-76-B 


Part  A 


Individual  Project  Report     1.  Infectious  Diseases 
Calendar  Year  I960        2.  Medical  Mycology 

3.  Bethesda,  Maryland 


Project  Title:   I_n  vivo  tests  of  antimycotic  drugs  and  antibiotics. 
Principal  Investigator:    C.  \1.   Emmons 


Other  Investigators:   Prescott,  Benjamin,  Piggott,  Millard, 
Utz,  John  P.  and  Andriole,  Vincent  - 
MPB  Serial  No.  62^-Aj  LCI  Serial  No.  23-C. 


Cooperating  Units:    Medical  Physiological  Bacteriology  Section 
and  Laboratory  of  Clinical  Investigations. 


Man  Years  (calendar  year  I960): 
Total:  22/12 

Professional:        8/12 
Other:  12/12 


Project  Description: 

Objectives: 

To  test  the  efficacy  and  safety  of  new  drugs  and  antibiotics 
in  experimental  mycoses  in  mice  and  cooperate  with  the  Laboratory 
of  Clinical  Investigate  ons  in  clinical  trials. 

Methods  Employed: 

Mice  are  infected  intravenously  with  a  dose  of  fungus  cells 
determined  in  prior  experimental  work  to  be  sufficient  to  kill  un- 
treated (control)  mice  in  1U-21  days.  Treated  mice  are  given  test 
drugs  at  doses  up  to  tolerance  and  effectiveness  of  drug  is  meas- 
ured by  its  ability  to  extend  survival  time  of  treated  mice  and  to 
clear  the  animals  of  infection  as  determined  by  autopsy  and  culture 
of  the  surviving  animals  when  experiment  is  terminated.  Collabora- 
tor has  provided  some  of  drugs  tested  and  has  synthesized  drugs  on 
his  initiative  or  of  types  suggested  by  principal  investigator. 


Part  B  included. 

180 


Serial  No.     NIAID-76-B 

Major  Findings; 

A  new  antibiotic   first  received  by  the  principal  investigator 
four  years  ago  has  now  been  on  clinical  trials  for  1  1/2  years  and 
has  been  found  to  be  superior  to  other  antimycotic  drugs  in  h  myco- 
ses. 

Significance  to  bio-medical  research  and  the  program  of  the  Insti- 
tute; 

Presently  available  antimycotic  drugs  are  too  toxic  and  too 
ineffective  for  safe  and  ideal  clinical  use.     Experimental  thera- 
py in  animals  should  precede  clinical  trial. 

Proposed  Course  of  Project; 

This  project  will  be  continued  indefinitely. 


181 


PHS  -  NIH        Serial  No.  NIAID-76-B 
Individual  Project  Report   1.  Infectious  Diseases 
Calendar  Year  I960      2.  Medical  Mycology 

3.  Bethesda,  Maryland 


Part  B.  Honors,  Awards,  and  Publications, 

Publications  other  than  abstracts  from  this  project: 

Emmons,  C.  W.  Failure  of  Griseofulvin  to  Control  Experimental  Systemic 
Mycoses  in  Mice.  A.M.A.  Arch.  Derm.  81:700-702,  May  I960. 


Honors  and  Awards  relating  to  this  project: 

Presentation  of  a  paper  entitled:   "A  New  Antimycotic  Antibiotic"  at 
the  Fifth  Annual  Meeting  of  VA-Armed  Forces  Coccidioidomycosis  Cooperative 
Study",  Los  Angeles,  Calif.,  Dec.  8  -  9,  I960. 

Honorary  Mention  for  an  Exhibit  on  Chemotherapy  of  Systemic  Mycoses 
at  the  American  Medical  Association  Meeting  in  Miami  Beach,  Florida,  held 
in  June  I960. 

Appointed  to  WHO  Expert  Advisory  Panel  on  Parasitic  Diseases  for  a 
term  of  5  years. 

Presided  at  a  session  on  Antifungal  Agents  at  the  I960  Conference  on 
Antimicrobial  Agents,  sponsored  by  the  Society  for  Industrial  Microbiology, 
Oct.  26,  I960. 

Appointed  on  Scientific  Advisory  Committee,  Institute  of  Microbiology, 
Rutgers  -  The  State  University,  New  Brunswick,  N.  J.  for  a  term  of  three 
years. 

Member  of  Committee  on  National  Index  of  Fungus  Cultures,  Quartermaster 
Research  and  Development,  National  Academy  -  Research  Council,  Natick, 
Massachusetts,  for  a  term  expiring  June  1963. 

Special  Lecturer  on  Medical  Mycology,  George  Washington  University 
School  of  Medicine,  for  Fiscal  Year  beginning  Aug.  I960. 


182 


PHS  -  NIH  Serial  No.     NIAID-76-C 


Part  A 


Individual  Project  Report     1.  Infectious  Diseases 
Calendar  Year  I960       2.  Medical  Mycology 

3.  Bethesda,  Maryland 


C.  Project  Title:   Identification  and  study  of  new  and  unusual  fungi 
from  mycoses. 


Principal  Investigator:    C.  W.  Emmons 


Other  Investigators:  William  L.  Jellison,  RML,  Montana,  Lie-Kian- 
Joe,  Univ.  of  Indonesia,  Djakarta,  and 
Charles  Bridges,  Agricultural  and  Mechanical 
College,  Texas. 


Cooperating  Units:  Rocky  Mountain  Laboratory  and  many  hospitals, 
diagnostic  laboratories  and  individual  physi- 
cians. 


Man  Years  (calendar  year  I960): 
Total:  Ui/12 

Professional:         6/12 
Other:  7/12 

Project  Description: 

Objectives: 

Studies  of  new  mycoses  and  their  etiologic  agents,  of  unusual 
strains  of  pathogenic  fungi,  and  of  the  geographic  distribution  of 
mycoses.  Support  and  encouragement  of  medical  mycology  in  labora- 
tories which  lack  trained  mycologists. 

Methods  Employed: 

Routine  methods  of  fungus  identification,  adaptation  of  such 
methods  when  necessary,  tests  of  pathogenicity  and  virulence  in 
experimentally  infected  animals. 


Part  B  included. 


183 


Serial  No.  NIAID-76-C 

Major  Findings: 

Additional  studies  of  ohycomycosis  have  been  carried  on.  A 
new  phyconycosis  in  horses  has  been  studied  and  a  paper  submitted 
for  publication. 

Significance  to  bio-medical  research  and  the  program  of  the  Insti- 
tute; 

This  combines  a  useful  diagnostic  service  with  an  important 
research  function  because  it  brings  under  scrutiny  unusual,  inter- 
esting and  important  pathogens. 

Proposed  Course  of  Project: 

This  project  will  be  continued  indefinitely. 


18 '4 


PHS  -  NIH  Serial  No.  NIAID-76-C 

Individual  Project  Report 
Calendar  Year  I960 


Part  B:  Honors,  Awards,  and  Publications. 


Publications  other  than  abstracts  from  this  project: 

Joe,  Lie-Kian,  Eng,  Njo-Injo  Tjoei,  Tjokronegoro,  Sutomo,  and  Emmons, 

Chester  W.  Phycomycosis  (Mucormycosis)  in  Indonesia  —  Description  of 
a  Case  Affecting  the  Subcutaneous  Tissue.  A.  J.  Trop.  Med.  &  Hyg. 
9:Ui3-Hj8,  Mar.,  I960. 

Emmons,  C.  W.  and  Jellison,  W.  L.  Emmonsia  Crescens  Sp.  N.  and  Adiaspiro- 
mycosis  (Haplomycosis)  in  Mammals.  N.  Y.  Acad.  Sci.  89:91-101,  Aug. 
I960. 


Honors  and  Awards  relating  to  this  project: 

Invited  lecture  at  the  Agricultural  and  Mechanical  College,  College 
Station,  Texas,  July,  I960. 

Presentation  of  a  paner  entitled:   "Emmonsia  Crescens  Sp.  N.  and 
Adiaspiromycosis  (Haplomycosis)  in  Mammals"  at  the  Second  Conference  on 
Medical  Mycology,  New  York  Academy  of  Science,  Jan.,  I960. 


185 


PHS  -  NIH 

Individual  Project  Report 

Calendar  Year  I960 


Serial  No.     NIAID-78-A 

1.  Infectious  Diseases 

2.  Medical  Mycology 

3.  Bethesda,  Maryland 


A.  Project  Title:  Biochemistry  and  Physiology  of  Pathogenic  Fungi. 

(In  vitro  studies  of  the  acti  on  of  Antifungal  agents 
on  pathogenic  fungi). 


Principal  Investigator:  George  W.  Lones 


Other  Investigators:     None 


Cooperating  Units: 


None 


Man  Years  (calendar  year  I960): 
Total:  1  2/12 

Professional:        9/12 
Other:  5/12 

Project  Description: 

Objectives: 

To  investigate  the  quantitative  relationships  of  antifungal 
drugs  with  respect  to  fungicidal  and  fungistatic  action;  to  study 
certain  nhysical  and  chemical  factors  influencing  this  action;  to 
study  the  effects  of  these  drugs  on  certain  metabolic  activities  of 
fungi;  to  study  the  emergence  of  strains  resistant  to  these  drugs; 
and  ultimately  to  understand  the  mechanism  of  action  of  certain  of 
the  antifungal  antibiotics. 

Methods  Employed: 

Interest  during  the  past  year  has  centered  on  a  new  antibiotic, 
X-5079C  Conventional  methods  are  used  for  determining  the  quanti- 
tative relationships  of  growth  of  Histoplasma  cansulatum  in  the  pres- 
ence of  varying  concentrations  of  the  antibiotic  in  suitable  growth 
media  under  a  variety  of  conditions.  HeLa  cell  cultures  are  infected 
with  H.  capsulatum  in  the  yeast  phase  and  the  effect  of  the  antibiot- 
ic on  the  development  and  persistance  of  the  fungus  determined  by 


Part  B  not  included. 


186 


Serial  No.  NIAID-78-A 


microscopic  examination  of  stained  preparations  and  by  subculture 
following  trypsinization. 

Major  Findings; 

Antibiotic  X-5079C  is  fungistatic  rather  than  fungicidal. 
Contrary  to  early  impressions  the  agent  is  active  against  H.  cap- 
sulatun  in  vitro  as  well  as  in  animals.  The  sensitivity  of  the 
yeast  phase  of  H.  capsulatum  to  antibiotic  X-5079C  is  substantial- 
ly greater  than  that  of  the  mycelial  phase.  An  assay  method  for 
antibiotic  X-5079C  sensitive  to  1  ug/ml  has  been  developed  with  a 
strain  of  H.  capsulatum  as  the  test  organism.  Antibiotic  X-5079C 
is  active  against  H.  capsulatum  in  HeLa  cell  culture,  and  factors 
influencing  activity  in  this  system  have  been  investigated.  It  is 
of  low  toxicity  for  HeLa  cells. 

Significance  to  bio-medical  research  and  the  program  of  the  Insti- 
tute: 

The  need  for  new  and  more  effective  therapeutic  agents  for 
treatment  of  the  systemic  mycoses  continues. 

Antibiotic  X-5079C  has  demonstrated  impressive  therapeutic 
effect  in  experimental  mycoses  in  animals  and  encouraging  results 
have  been  obtained  in  humans.  The  continued  study  of  this  and 
other  antifungal  agents  is  essential. 

Proposed  Course  of  Project: 

It  is  planned  to  continue  these  studies.  The  investigation 
may  well  be  extended  to  pathogenic  fungi  other  than  H.  capsulatum. 
The  effect,  if  any,  of  the  antibiotic  on  respiration  and  assimila- 
tion will  be  determined.  Attempts  will  be  made  to  improve  the 
assay  procedure,  since  a  sensitive  assay  method  is  essential  for  the 
determination  of  serum  levels  and  excretion  rates  in  man  and  animals. 


187 


PHS  -  NIH  Serial  No.  NIAID-78-B 


Part  A 


Individual  Project  Report      1.  Infectious  Diseases 
Calendar  Year  I960         2.  Medical  Mycology 

3.  Bethesda,  Maryland 


B.  Project  Title:   Studies  on  the  physiology  of  Coccidioides  immitis, 
Principal  Investigator:  George  W.  Lones 
Other  Investigator:      None 
Cooperating  Units:       None 


Man  Years  (calendar  year  I960): 
Total:  1  2/12 

Professional:      3/12 
Other:  11/12 

Project  Description: 

Objectives: 

The  biochemistry  and  metabolism  of  this  dimorphic  pathogen 
have  been  little  studied.  It  is  the  purpose  of  this  project  to 
obtain  information  on  the  metabolic  characteristics  of  this 
microorganism,  and  in  particular  to  discover  metabolic  differ- 
ences in  the  two  forms. 

Methods  Employed: 

Shake  culture  techniques  are  employed  to  produce  the  parasitic 
and  saprophytic  morphologic  modifications  of  C.  immitis  in  sizeable 
quantities  for  examination  by  standard  chemical  and  biochemical 
techniques. 

Major  Findings: 

A  second  strain  of  C.  immitis  has  been  successfully  converted 
and  maintained  in  the  spherule  form.  Quantitative  measurements 
have  been  obtained  of  the  ability  of  a  variety  of  potential  carbon 


Part  B  included 


188 


Serial  No.  NIAID-78-B 


and  nitrogen  sources  to  supnort  the  growth  of  the  spherule  phase 
and  the  mycelial  phase  of  strain  M-ll.  No  substrate  has  been  found 
in  this  study  which  preferentially  supports  the  growth  of  the 
spherule  form.  Only  mannose  is  utilized  as  readily  as  glucose  by 
the  spherules.  Mannose  and  fructose  support  growth  of  mycelium  as 
well  as  glucose. 

Significance  to  bio-medical  research  and  the  program  of  the  Insti- 
tute; 

Progressive  coccidioidomycosis  is  a  disease  with  a  high  mor- 
tality. A  better  knowledge  of  the  causative  agent  may  favorably  in- 
fluence our  diagnosis,  prevention  and  treatment  of  the  infection. 

Proposed  Course  of  Project; 

Examination  of  the  metabolism  of  the  two  morphological  forms 
of  the  fungus  will  be  extended.  The  current  nutritional  study  will 
be  completed. 


18S 


PHS  -  NIH 

Individual  Project  Report 

Calendar  Year  I960 


Serial  No.  NIAID-78-B 

1.  Infectious  Diseases 

2.  Medical  Mycology 

3.  Bethesda,  Maryland 


Part  B.  Honors,  Awards,  and  Publications. 


Publications  other  than  abstracts  from  this  project: 


Lones,  G.  W.  and  Peacock,  Carl  L.  Role  of  Carbon  Dioxide  in  the  Dimorphism 
of  Coccidioides  immitis.  J.  3act.  79:308-309,  I960. 

Lones,  G.  W.  and  Peacock,  Carl  L.  Studies  of  the  Growth  and  Metabolism  of 
Coccidioides  immitis.  Annals  N.  Y.  Acad.  Sci.  89:102-108,  I960. 


Honors  and  Awards  relating  to  this  Project: 


Presentation  of  a  paper  entitled:   "Studies  of  the  Growth  and  Metabolism 
of  Coccidioides  immitis",  Second  Conference  on  Medical  Mycology,  New  York 
Academy  of  Science,  January,  I960. 


190 


PHS  -  NIH  Serial  No.  HTAID-79-A 

Individual  Project  Report      1.  Infectious  Diseases 
Calendar  Year  I960  2.  Medical  Mycology 

3.  Bethesda,  Maryland 


A.  Project  Title:  Immunity  Studies  with  Pathogenic  Fungi. 

Principal  Investigator:  H.  F.  Hasenclever 

Other  Investigators:     None 

Cooperating  Units:       None 

Man  Years  (calendar  year  I960): 
Total:  S/12 

Professional:      2/12 
Other:  3/12 

Project  Description: 

Objectives: 

To  obtain  fundamental  knowledge  pertaining  to  the  development 
of  acquired  resistance  in  laboratory  animals  to  coccidioidomycosis. 

Methods  Employed: 

Immunization  procedures  utilizing  spherule  cultures  of  Coccid- 
ioides  immitis  as  vaccines  are  being  studied.  The  vaccines  are 
treated  with  formaldehyde  to  destroy  viability.  Mice  immunized  with 
this  ^reparation  are  challenged  wi th  viable  C.  immitis  arthrospores 
or  spherules. 

Major  Findings: 

Immunized  mice  show  an  increased  survivor  rate  when  compared  to 
normal  mice  challenged  with  similar  infecting  doses  of  C.  immitis. 
In  experiments  where  sublethal  infecting  doses  are  utilized,  the  im- 
munized mice  are  culturally  negative  sooner  than  normal  control  mice. 

Part  B  not  included. 

191 


Serial  No.  NIAID-79-A 


Significance  to  bio-medical  research  and  the  program  of  the  Insti- 
tute; 

Me   are  utilizing  for  this  investigation  cultures  of  spherules 
grown  in  vi tro »  V/hile  mycelial  preparations  have  been  investigated, 
the  "parasitic  form"  of  C.  irriitis,  as  an  immunizing  agent,  has  been 
studied  very  little.  This  project  has  been  undrr  investigation  for 
only  a  short  tine,  but  the  results  thus  far  are  encouraging. 
Coccidioidomycosis  is  a  disease  for  which,  if  a  satisfactory  vaccine 
were  available,  immunization  would  be  of  value  and  practical. 

Proposed  Course  of  Project; 

This  nroject  is  to  be  continued. 


192 


PHS  -  NIH  Serial  No.  NIAID-79-B 


Part  A 


Individual  Project  Report     1.  Infectious  Diseases 
Calendar  Year  I960       2.  Medical  Mycology 

3.  Bethesda,  Maryland 


B.  Project  Title:  Antigenic  Studies  on  Pathogenic  Yeasts. 
Principal  Investigator:  H.  F.  Hasenclever 
Other  Investigators:     None 
Cooperating  Units:       None 
Man  Years  (calendar  year  I960) 


Total: 

9/12 

Professional: 

li/12 

Other: 

5/12 

Project  Description: 


Objectives: 

To  learn  more  about  the  antigenic  interrelationships  of  some 
of  the  provisional  pathogens  with  particular  emphasis  upon  the 
genus  Candida. 

Methods  Employed: 

Antisera  to  the  desired  species  and  strains  of  Candida  are  pre- 
pared in  rabbits.  Homologously  and  heterologously  adsorbed  antisera 
are  utilized  to  show  antigenic  similarities  or  dissimilarities  that 
exist  within  species  or  between  species.  Tube  agglutination  reac- 
tions have  been  used  predominately  and  the  applicability  of  comple- 
ment fixation  and  agar  gel  diffusion  has  been  tested. 

Major  Findings: 

These  studies  have  shown  that  two  antigenic  groups  are  present 
in  the  species  Candida  albicans.  One  of  these  groups  is  antigenical- 
ly  similar  to  C.  tronicalis,  while  the  other  group  is  identical  to 
C.  stella toidea. 


Part  B  included. 

193 


Serial  No.     NIAID-79-B 


Significance  to  bio-medical  research  and  the  program  of  the  Insti- 
tute; 

These   studies  have  contributed  basic  and  fundamental  informa- 
tion about  the  antigenic   structure  of  these  yeast-like  fungi. 

Proposed  Course  of  Project; 

To  be  continued  with  an  effort  to  demonstrate  qualitative  anti- 
genic differences  between  these  two  groups. 


19'* 


PHS  -  NIB  Serial  No.  NIAID-79-B 


Individual  Project  Report      1.  Infectious  Diseases 
Calendar  Year  I960         2.  Medical  Mycology 

3.  Bethesda,  Maryland 

Part  B.  Honors,  Awards,  and  Publications. 

Publications  other  than  abstracts  from  this  project! 

Hasenciever,  H.  F.  and  Mitchell,  William  0.  The  observation  of  Two  Antigenic 
Groups  in  Candida  albicans.  Bact.  Proc.  I960. 


Honors  and  Awards  relating  to  this  Project; 


Convenor  of  Session  on  Medical  Mycology  at  the  I960  Annual  Meeting  of 
the  Society  of  American  Bacteriologists. 

Invitational  lecture  entitled:   "Clinical  and  Mycological  Aspects  of 
Tinea  pedis"  before  the  Annual  Scientific  Convention  of  the  American  Podia- 
try Association,  Nov.,  I960, 


195 


PHS  -  NIH  Serial  No.  NIAID-79-C 


Part  A 


Individual  Project  Report      1.  Infectious  Diseases 
Calendar  Year  I960        2.  Medical  Mycology 

3.  Bethesda,  Maryland 


C.  Project  Title:  Virulence  and  pathogenic  studies  with  yeasts. 

Principal  Investigator:  H.  F.  Hasenclever 

Other  Investigators:     Vincent  T.  Andriole  -  LCI  Serial  #23-C. 

Cooperating  Units:       Laboratory  of  Clinical  Investigations. 

Man  Years  (calendar  year  I960) : 
Total:  18/12 

Professional:     10/12 
Other:  8/12 

Project  Description: 

Objectives: 

To  learn  more  about  the  host-parasite  relationship,  virulence 
and  pathogenicity,  and  the  in  vitro  multiplication  rates  of  several 
yeasts  in  normal  and  physiologically  altered  mice. 

Methods  Employed: 

Virulence  determinations  are  calculated  by  injecting  graded 
doses  of  yeast  cells  into  different  groups  of  mice.  The  fifty  per 
cent  endooint  is  utilized  and  death  or  survival  of  the  test  animals 
is  the  criterion  employed.  In  vivo  growth  rates  are  obtained  by 
determining  the  viable  yeast  cells  in  the  various  organs,  at  differ- 
ent stages  of  disease,  in  animals  infected  with  a  standard  number  of 
yeasts.  Similar  studies  have  been  done  using  mice  with  alloxan  in- 
duced diabetus  mellitus. 


Part  B  included. 


196 


Serial  No.  HIAID-79-C 

Major  Findings: 

Studies  with  several  strains  of  Cryntococcus  neoformans  have 
shown  that  several  minutes  after  injection  the  largest  number  of 
yeast  cells  are  found  in  lung  tissue,  regardless  of  whether  the 
mice  were  injected  intravenously  or  intracerebrally.  Soon  the  pop- 
ulation in  the  lungs  decreases,  and  by  2-3  days  after  injection, 
multiplication  in  the  brain  is  apparent.  The  time  required  to  pro- 
duce terminal  cryptococcosis  after  injection  in  mice  varies  with 
the  strain.  In  moribund  mice,  however,  the  number  of  viable  yeast 
cells  per  0.1  gm  of  brain  tissue  is  about  the  same  regardless  of 
strain. 

Studies  with  Candida  tropicalis  have  shown  that  most  strains 
of  this  yeast  are  quite  pathogenic  for  mice  but  not  for  rabbits. 
Multiplication  in  vivo  is  quite  similar  to  that  of  Candida  albicans, 
i.e.,  the  kidneys  are  the  organs  showing  the  greatest  amount  of 
tissue  destruction,  and  the  highest  yeast  cell  population. 

An  effective  dose  of  alloxan  monohydrate  which  would  produce 
constant  glycosuria  without  a  great  number  of  fatalities  was  deter- 
mined. 

Lethality  studies  revealed  that  certain  strains  of  Candida 
albicans  and  C_.  tropicalis  caused  death  in  alloxanized  mice  at  a 
faster  rate  than  in  normal  control  animals.  This  was  not  true  for 
certain  strains  of  C_.  parap  silo  sis  and  C.  guilliermondii. 

Plating  experiments  revealed  that  the  course  of  C.  albicans 
infection  in  the  mouse  was  more  severe  when  the  animal"  had  been 
physiologically  altered  with  alloxan  monohydrate. 

Significance  to  bio-medical  research  and  the  program  of  the  Insti- 
tute: 

These  investigations  have  contributed  to  knowledge  about  the 
parasite  multiplication  within  host  tissues.  We  have  shown  that 
mice  are  quite  susceptible  to  infections  due  to  £.  tropicalis. 

Proposed  Course  of  Project: 

To  be  continued  with  further  study  to  be  given  to  the  effects 
of  physiological  alteration  of  the  host  in  relation  to  increased 
or  decreased  susceptibility  to  fungus  infections. 


197 


PHS  -  NIH  Serial  Ho.  HIAID-79-C 


Individual  Project  Report      1.  Infectious  Diseases 
Calendar  Year  I960         2.  Medical  Mycology 

3.  Bethesda,  Maryland 


Part  B.  Honors,  Awards  and  Publications. 
Publications  other  than  abstracts  from  this  project: 


Hasenclever,  H.  F.  and  Mitchell,  William  0.  Virulence  and  Growth  Rates  of 
Crvptococcus  neoformans  in  Mice.  Annals  N.  Y.  Acad.  Sci.  89:1^6-162, 
i960: 

Kasenclever,  H.  F.  and  Mitchell,  Uilliam  0.  Pathogenicity  of  Candida  albi- 
cans and  Candida  tropicalis.   Sabouraudia  (in  press). 


Honors  and  Awards  relating  to  this  project: 

Presentation  of  a  paper  entitled:   "Comparative  Pathogenicity  of 
Candida  albicans  and  Candida  tropicalis,"  at  the  Annual  Meeting  of  the 
Mycological  Society  of  America,  American  Institute  of  Biological  Sciences, 
I960,  Stillwater,  Oklahoma. 

Presentation  of  a  paper  entitled:   "Virulence  and  Growth  Rates  of 
Cryotococcus  neoformans  in  Mice",  at  the  Second  Conference  on  Medical  Mycol- 
ogy, Hew  York  Academy  of  Science,  Jan.,  I960. 


198 


LABORATORY  OF  BIOLOGY  OF  VIRUSES 


Summary  1 

80  -  Basic  Studies  of  Virus-Host  Cell  Rela- 

t ionships  3 

80-A  -  Rabies  Prophylaxis  6 

81  -  Mechanism  of  Oncogenic  Activity  Polyoma 

Viruses  8 

82  -  Cell  Growth  Inhibiting  Substances  11 

83  -  Mutation  in  Animal  Viruses  13 

84  -  Host-Parasite  Relations  15 

85  -  Investigations  of  Animal  Virus  Repro- 

duction    19 

85-A  -  Animal  Virus  Synthesis  22 

86  -  Investigations  of  Interference  Enzymatic 

Functioning  of  Mitochondria  as  a 

Mechanism  for  Carcinogenesis  24 

86-A  -  Demonstration  of  Glucose  Metabolism  and 
Peptide  Bond  Formation  by  Isolated  Brain 
and  Liver  Mitochondria  27 

87  -  Cytopathogenic  Effect  in  Single  Cells  in 

Tissue  Culture  29 

87-A  -  Characterization  of  Mitochondria  31 

88  -  Biology  of  Mitochondria  and  Its  Relation 

to  Endogenous  and  Viral  Diseases  34 

88-A  -  Comparison  of  the  Properties  of  Crude 
and  Crystallized  Coxsackie  A- 10  Virus, 
of  Cloudman  S  91,  Mouse  Melanona  and  of 
Mouse  Muscle  Nucleoprotein  36 

89  -  Kinetics  and  Sites  of  Coxsackie  Virus 

Multiplication  in  the  Monkey  Kidney  Cell  38 
89-A  -  Virus  Structure  .. ...  40 


PHS-NIH 

Summary  Statement 

Office  of  Chief 

Laboratory  of  Biology  of  Viruses 

Calendar  Year  i960 


This  laboratory  has  been  anticipating  a  physical  shift  in  most 
of  its  activities  to  space  in  Building  5.  Although  scheduled  to  take 
place  during  the  past  calendar  year,  it  is  now  obvious  that  it  will 
not  take  place  until  well  into  1961.  Because  of  this  delay,  space 
problems  have  been  severe.  For  instance,  it  has  been  impossible  to 
establish  our  planned  central  tissue  culture  production  unit  although 
the  subprofessional  responsible  for  this  program  has  been  on  duty 
since  March  i960.  She  has  become  well  acquainted  with  our  technics, 
and  equipment  necessary  for  the  operation  has  been  collected.  A 
second  severe  limitation  of  space  involves  the  newly  established 
Physical  Biology  Unit.  Dr.  Mattern  has  had  to  install  his  electron 
microscope  in  one-half  of  a  basement  room  measuring  8  x  l'+  and  this 
has  been  the  total  working  space  for  a  whole  year  for  him  and  one 
professional  assistant. 

During  this  year  one  whole  unit,  The  Rickettsial  Biology  and 
Metabolism  Unit  involving  3  professional  and  2  subprofessional  staff 
members,  was  transferred  from  this  laboratory  to  the  Division  of 
Biologies  Standards.  No  replacement  personnel,  budget  or  space  was 
made  available  for  this  loss. 

One  new  research  associate  joined  the  staff  this  year,  making 
two  such  now  a  part  of  this  laboratory.  One  of  these  associates  has 
been  recruited  to  become  a  part  of  the  permanent  staff  at  the  end  of 
this  fiscal  year. 

The  basic  objectives  of  this  laboratory  continue  to  be  the 
same  as  last  year.   It  is  obvious  from  this  annual  report  that  four 
out  of  our  five  units  have  projects  with  the  same  general  objective — 
investigation  of  mechanisms  and  localization  of  animal  virus  synthesis 
within  the  infected  cell.  Each  of  these  units  is  also  interested  in 
the  infectious  nucleic  acid  of  viruses.   In  view  of  the  complexity  of 
this  problem  and  the  important  implications  of  any  information  that 
is  obtained,  we  feel  that  this  "duplication"  is  quite  Justified. 
Actually,  it  is  not  duplication  in  so  far  as  different  approaches  are 
being  used  and  different  virus-cell  systems  studied. 

The  electron  microscope  has  been  installed  and  is  now  being 
used  not  only  by  the  Biophysical  Unit  but  also  in  collaboration  with 
other  units  of  our  laboratory  and  units  in  the  Laboratory  of  Infectious 
Diseases.  With  studies  on  the  structure  of  viruses  and  a  project 


Summary  Statement — LBV/NIAJD  (Cont.) 


concerned  with  the  genetics  of  animal  viruses  added  to  our  biochemical 
and  biological  studies,  we  feel  that  ve  now  have  fairly  complete  cover- 
age of  the  important  facets  of  basic  virus  biology. 

By  the  use  of  radioautographs  and  staining  with  fluorescein 
tagged  antiviral  antibody,  the  intracellular  location  of  poliovirus 
antigen — presumably  viral  protein — during  the  cycle  of  virus  multi- 
plication has  been  determined.  Demonstrable  antigen  first  appeared 
one  hour  after  infection  and  was  diffusely  distributed  through  the 
cytoplasm.  At  3  hours,  just  before  the  appearance  of  new  virus,  it 
was  present  throughout  the  nucleus  with  a  tendency  to  be  concentrated 
around  the  periphery  of  the  nucleolus.  At  5  to  7  hours,  particulate 
accumulation  of  antigen  in  the  cytoplasm  was  noted.   Incorporation  of 
radioactive-tagged  amino  acid  into  cell  protein  ceased  shortly  after 
the  start  of  infection,  whereas  incorporation  of  cytidine  into  RNA 
continued  until  after  3  hours  and  tended  to  localize  in  the  nucleoli. 

Plaque  type  mutants  of  EMC  virus  have  been  found,  segregated 
and  characterized.  The  stability  of  the  mutants  has  been  determined 
and  the  plaque  type  has  been  shown  to  be  a  function  of  the  viral  RNA. 
It  has  been  shown  that  the  difference  in  the  size  of  the  plaques  formed 
by  these  mutants  is  brought  out  by  an  inhibitor  present  in  the  agar 
overlay  used  on  the  plaque  plates.  This  inhibitor  resides  in  the 
agaropectin  fraction  of  the  agar  and  can  be  separated  from  the  agarose 
fraction  which  then  permits  both  plaque  type  mutants  to  form  similar 
sized  plaques. 

By  the  use  of  a  serum  protection  test  in  newborn  hamsters, 
evidence  was  found  that  polyoma  virus  transforms  normal  cells  to  tumor 
cells  quickly  and  directly  without  extensive  virus  multiplication  being 
necessary.  Furthermore,  no  evidence  could  be  found  to  suggest  a  lyso- 
genic  relationship  of  virus  to  tumor  cell.  All  attempts  to  show  the 
presence  of  infectious  or  masked  virus  or  of  virus  antigen  in  trans- 
plantable polyoma- induced  tumors  have  been  negative.   It  appears  that 
once  the  virus  initiates  the  tumor  it  is  no  longer  required  for  tumor 
growth  and  maintenance. 

The  discovery  has  been  made  that  when  the  antibiotic  tetracycline 
stains  tissues  in  such  a  way  that  they  fluoresce  under  UV  light,  this 
fluorescence  is  localized  in  the  mitochondria  of  the  cells.  This  makes 
a  convenient  vital  stain  of  these  subcellular  elements  for  further 
studies.  There  appears  to  be  some  similar  localization  of  the  anti- 
biotic fluorescence  in  certain  bacteria. 


Summary  Statement — LBV/NIAID  (Cont.) 


A  complex  model  of  tobacco  mosaic  virus  has  been  constructed 
on  theoretical  grounds,  and  on  checking  this  model  against  known 
biochemical  and  biophysical  properties  of  the  virus  a  remarkable 
consistency  is  found.  Certain  refinements  of  electron  microscopic 
technics  have  produced  photographs  of  this  virus  which  reveal 
previously  not  seen  fine  structure  also  consistent  with  the  theoreti- 
cal model. 


Serial  No.   NIAID-80 

1.  Biology  of  Viruses 

2.  Viral  Growth 

3.  Bethesda,  Maryland 


FHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A 


Project  Title:  Basic  Studies  of  Virus-Host  Cell  Relationships 
Principal  Investigator:  Dr.  Karl  Hahel 
Other  Investigators:  Rosalie  Silverherg 
Cooperating  Units:  None 


Man  Years: 

Total: 

1 

Professional: 

5/12 

Other: 

7/12 

Project  Description: 

Objectives: 

To  determine  mechanism  of  and  factors  influencing  attachment, 
invasion,  multiplication  and  release  of  viruses  from  susceptible  and 
resistant  cells. 

Methods  Employed: 

Various  strains  of  tissue  culture  cells  either  suspended  or  on 
glass  are  exposed  to  viruses  and  at  different  stages  of  infection  the 
cells  are  studied  for  viability  and  virus  content.  All  quantitation  is 
by  plaque  methods. 

Major  Findings: 

After  rapid  adsorption  of  poliovirus  to  HeLa  cells  the  virus 
exists  in  h   different  relationships  with  the  cell: 

(1)  Virus  loosely  bound  that  will  wash  off, 

(2)  Virus  firmly  bound  but  still  available  to  extracellular 
antibody, 

(3)  Virus  firmly  bound  but  not  available  to  antibody, 
(h)   Virus  already  eclipsed. 

Part  B  included:  Yes 


Serial  No.  NIAID-80 

Although  all  four  states  may  exist  at  one  time,  the  normal 
sequence  of  events  seems  to  be  (l),  (2)  and  (4).  The  virus  in  (3)  may- 
go  to  (U)  and  thus  initiate  infection  but  this  may  be  a  relatively 
inefficient  process  compared  to  the  other  sequence. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

These  findings  suggest  that  effective  initiation  of  the  infectious 
cycle  can  occur  rapidly  but  that  under  certain  circumstances  virus  is 
firmly  attached  to  the  cell  before  being  eclipsed.  Understanding  of 
these  events  occurring  at  the  start  of  infections  at  the  cell  surface 
may  provide  a  logical  target  for  attempts  at  inhibition  of  infection 
■without  serious  deleterious  effects  upon  the  cell. 

Proposed  Course  of  the  Project: 

These  studies  will  be  continued  with  attempts  to  determine 
morphological  and  biochemical  cellular  components  responsible  for  the 
phenomena  already  demonstrated. 


Serial  No.   NIAID-80 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Habel,  Karl  and  Utz,  John  P.  Mumps.  Pediatric  Clinics  of 
North  America,  Vol.  7,  No.  k,   November,  i960. 


Honors  and  Awards  relating  to  this  project: 
None 


Serial  No.  NIAID-80A 

1.  Biology  of  Viruses 

2.  Viral  Growth 

3.  Bethesda,   Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:  Rabies  Prophylaxis 

Principal  Investigator:  Dr.  Karl  Habel 

Other  Investigators:  Rosalie  Silverberg 

Cooperating  Units:  Expert  Committee  on  Rabies,  World  Health 
Organization 

Man  Years: 

Total:  1  6/12 
Professional:  6/12 
Other:        1 

Project  Description: 

Objectives: 

To  improve  methods  of  rabies  prophylaxis  in  man  and  to  reduce 
non-specific  reactions  caused  by  vaccines. 

Methods  Employed: 

This  year  's  activity  has  been  chiefly  aimed  at  developing  a 
tissue  culture  source  of  virus  for  vaccine  production. 

Major  Findings: 

A  practical  tissue  culture  system  consisting  of  normal  chicken 
embryo  cells  in  medium  199  has  been  infected  with  a  fixed  strain  of 
rabies  virus  and  propagated  through  10  serial  passages.  Some  passage 
fluids  have  had  an  infectious  titer  as  high  as  105  and  with  more 
concentrated  inocula  produced  a  cytopathic  effect  which  was  neutral- 
izable  with  antirabies  serum. 


Part  B  included:  No 


Serial  No.  NIAJD-80A 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

A  practical  tissue  culture  source  of  inactivated  rabies  vaccine 
would  represent  a  tremendous  improvement  in  the  type  of  biological  product 
used  for  rabies  prophylaxis  in  man. 

Proposed  Course  of  the  Project: 

Tissue  culture  as  a  possible  source  of  vaccine  will  be  further 
explored. 


Serial  No.  WIAID-81 

1.  Biology  of  Viruses 

2.  Viral  Growth 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:  Mechanism  of  Oncogenic  Activity  of  Polyoma  Virus 

Principal  Investigator:  Dr.  Karl  Habel 

Other  Investigators:  Rosalie  Silverberg  and  Dr.  Lowell  Glasgow 

Cooperating  Units:  None 

Man  Years: 

Total:  3  5/12 

Professional:  2 

Other:  1  5/12 

Project  Description: 

Objectives: 

To  determine  biological  factors  involved  in  the  induction  of 
tumors  by  oncogenic  viruses. 

Methods  Employed: 

By  studying  the  virus-tumor  cell  relationships  of  polyoma  virus 
in  animal  and  tissue  culture  systems,  the  events  occurring  in  newborn 
versus  adult  mice  and  hamsters  on  injection  of  virus  are  being  compared. 
Attempts  are  being  made  to  induce  malignant  characteristics  in  cells 
in  tissue  culture  and  in  adult  animals  by  varying  the  physiological 
state  during  infection  with  polyoma  virus. 

Major  Findings: 

Tumors  produced  in  newborn  hamsters  by  polyoma  virus  when  passive 
antibody  is  present,  occur  only  at  the  site  of  virus  inoculation,  contain 
no  demonstrable  virus  and  frequently  do  not  result  in  an  active  anti- 
viral antibody  response. 


Part  B  included:  Yes 


Serial  No.  NIAID-81 

Tumors  can  be  produced  in  adult  hamsters  by  applying  virus  to 
the  granulation  tissue  resulting  from  scarification  but  not  by  simple 
intradermal  inoculation  of  virus. 

Attempts  to  "transform"  normal  mouse  and  hamster  embryo  tissue 
culture  cells  to  tumor  by  establishing  in  them  a  continuing  infection 
with  polyoma  virus  have  been  negative. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

Any  information  concerning  factors  responsible  for  oncogenic 
properties  of  tumor  viruses  may  well  apply  to  the  oncogenic  effects  of 
other  agents  and  provide  leads  for  understanding  tumor  development 
under  natural  conditions  in  man. 

Proposed  Course  of  the  Project; 

Attempts  will  be  made  to  find  factors  that  will  enhance  the 
ability  of  polyoma  virus  to  induce  tumors  in  adult  animals  and  to 
inhibit  this  process  in  suckling  animals. 


Serial  No.  NIAID-81 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B    Honors,  Avards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Habel,  Karl  and  Silverberg,  Rosalie  J.  Relationship  of  Polyoma 

Virus  and  Tumor  in  Vivo.  Virology  12:  I+63-I+76,  November  i960. 


Honors  and  Awards  relating  to  this  project: 
None 


10 


Serial  No.  NIAID-82 

1.  Biology  of  Viruses 

2.  Viral  Growth 

3.  Bethesda,  Maryland 


FHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:   Cell  growth  inhibiting  substances 

Principal  Investigator:  Dr.  John  W.  Hornibrook 

Other  Investigators:  None 

Cooperating  Units:  Viral  Biochemistry  Unit,  LBV. 

Man  Years: 

Total:  2  7/12 

Professional:  1 

Other:  1  7/12 

Project  Description: 

Objectives: 

To  find  and  investigate  substances  and  mechanisms  which  prevent 
the  growth  of  cells  in  the  adult  animal. 

Methods  Employed: 

Isolation  from  serum  and  tissues  of  substances  which  will  inhibit 
the  growth  of  mammalian  cells  in  tissue  culture. 

Major  Findings: 

An  inhibitor  from  serum  has  been  partially  purified.  It  is 
apparently  not  a  carbohydrate  and  does  not  adsorb  U.V.  radiation  at 
280  or  260  mu.   It  is  active  at  approximately  0.3  mg/ml. 

Methods  of  isolating  and  purifying  this  material  are  being 
improved  and  this  work  will  continue. 


Part  B  included:  No 


11 


Serial  No.  NIAID-82 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

Information  concerning  the  existence  and  mode  of  action  of  mitotic 
inhibitors  in  tissues  or  body  fluids  is  of  fundamental  biological 
importance  and  of  practical  significance  to  those  engaged  in  tissue 
culture  and  cancer  research. 

Proposed  Course  of  the  Project: 

Attempts  will  continue  to  purify  and  identify  these  substances 
and  study  their  mode  of  action. 


12 


Serial  No.   NIAID-83 

1.  Biology  of  Viruses 

2.  Viral  Growth 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:  Mutation  in  Animal  Viruses 

Principal  Investigator:  Dr.  K.  K.  Takemoto 

Other  Investigators:  Dr.  Harvey  Liebhaber 

Cooperating  Units:  None 

Man  Years: 

Total:  3  6/12 

Professional:  2  l/l2 

Other:  1  5/12 

Project  Description: 

Objectives: 

1.  To  isolate. and  characterize  virus  mutants  and  to  investigate 
the  chemistry  and  biology  of  these  mutants. 

2.  To  investigate  various  methods  for  concentration  and  purifi- 
cation of  viral  mutants  in  order  to  obtain  sufficient  quantities  of 
virus  for  chemical  as  well  as  biological  investigations. 

Methods  Employed: 

Genetically  pure  lines  of  virus  are  isolated  by  plaque  tech- 
niques. Mutants  forming  plaques  which  differ  from  the  parental  type 
are  isolated  and  studied  further. 


Part  B  included:  No 

13 


Serial  No.  NIAID-83 
Major  Findings: 

1.  Encephalomyocarditis  (EMC)  virus  produces  plaques  which  are 
small  and  ragged  with  diffuse  boundaries.  A  mutant  of  this  virus  has 
been  isolated  which  forms  large  plaques  with  sharply  defined  boundaries. 
Both  virus  types  are  immunologically  identical  and  do  not  differ  in 
their  growth  rates,  thermal  stability  or  mouse  virulence.  However, 

the  large  plaque  mutant  differs  in  its  hemagglutinating  properties, 
having  a  significantly  higher  HA/pfu  ratio. 

2.  The  basis  for  plaque  size  differences  has  been  found  to  be 
due  to  an  inhibitory  factor  in  the  agar  used  in  the  overlay  medium. 
The  primary  effect  of  the  inhibitor  appears  to  be  interference  with 
adsorption  of  virus. 

3.  Infectious  RNA  extracted  from  both  types  of  virus  have 
yielded  progeny  which  produce  plaques  identical  to  those  from  which 
the  RNA  was  obtained,  proving  that  genetic  information  determining 
plaque  type  is  carried  solely  by  the  viral  nucleic  acid. 

h.     Preliminary  experiments  have  indicated  that  large  volumes 
of  EMC  virus  can  be  concentrated  and  purified  by  a  simple  procedure 
which  utilizes  the  partition  coefficient  of  the  virus  between  two 
phases  of  aqueous  solutions  of  high  molecular  weight  polymers. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

Studies  on  variations  and  mutations  of  animal  viruses  not  only 
lead  to  further  knowledge  and  understanding  of  the  nature  of  viruses 
but  have  practical  implications  in  the  development  of  vaccines  in 
virus  diseases. 

Proposed  Course  of  the  Project: 

1.  Utilizing  the  genetic  markers  of  plaque  type  and  hemaggluti- 
nation, experiments  are  planned  to  demonstrate  various  types  of  genetic 
interaction  such  as  recombination  and  reactivation. 

2.  Procedures  are  being  developed  for  large  scale  purification 
of  virus  so  that  sufficient  quantities  of  relatively  pure  virus  will 
be  available  for  studies  on  viral  nucleic  acid  and  protein. 


14 


Serial  No.  NIAID-8^ 

1.  Biology  of  Viruses 

2.  Virus  Host  Relationship 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A 


Project  Title:   Host-Parasite  Relations 

Principal  Investigator:  Victor  H.  Haas 

Other  Investigators:  None 

Cooperating  Units:  Miss  Delta  Uphoff  (NCl) 

Man  Years: 

Total:  2 

Professional:  1 

Other:  1 

Project  Description: 

Objectives: 

(a)  To  continue  studies  on  relationships  between  the  virus  of 
lymphocytic  choriomeningitis  (LCM)  and  the  transmissible  ascitic  tumor 
P288  in  mice,  particularly  in  mice  immune  to  the  virus. 

(b)  To  study  the  effect  of  various  antigens,  including  tumor 
material  and  a  virus  strain  derived  from  LCM-tumor  passage,  on  immune 
response  to  the  P288  tumor. 

(c)  To  conclude  studies  on  LCM  immunity  in  X-rayed  mice  kept 
alive  by  marrow  and  spleen  transplants . 

Methods  Employed: 

A  line  of  P288  tumor  carrying  LCM  with  it  during  passage  was 
maintained  in  CDBA  mice,  some  of  them  previously  immunized  against  LCM. 
A  strain  of  LCM  virus  derived  from  the  tumor  passage  line  was  separated 
from  the  tumor  and  maintained  by  serial  passage  in  non-immune  mice. 


Part  B  included:  Yes 


15 


Serial  No.  NIAID-8^ 

The  P288  tumor  was  passaged  in  CDBA  mice  and  from  time  to  time  in  general 
purpose  mice,  the  latter  being  treated  with  amethopterin.  Detection  of 
LCM  in  various  passage  mice  was  by  demonstration  of  viremia  or  by  immunity 
tests .  Bone  marrow  and  spleen  transplants  were  done  by  Miss  Uphof f  in 
mice  previously  prepared  by  me  in  respect  to  immune  status  against  LCM, 
and  irradiation  was  obtained  from  Mr.  Meyer  of  NCI. 

Major  Findings: 

Two  years  ago,  I  established  a  passage  strain  of  LCM  virus  that 
was  carried  with  the  ascites  tumor  P288  through  mice  immune  to  LCM.  For 
the  virus  to  survive,  it  was  necessary  to  treat  the  mice  with  amethopterin. 
After  nearly  a  year  of  such  passage,  I  derived  from  it  a  strain  of  LCM 
which  survived  in  immune  mice,  when  passed  with  the  tumor  P288,  even 
though  no  amethopterin  was  given.  During  the  current  year  I  have  passed 
this  latter  strain  of  LCM  through  non- immune,  non-tumor  bearing  mice  and 
found  that  it  produced  a  benign,  transmissible  ascites  of  a  type  which  I 
have  not  encountered  in  my  ordinary  passage  strains  of  LCM.  White  (general 
purpose)  mice  recovering  from  this  ascitic  infection  frequently  have  been 
resistant  to  P288  tumor,  when  the  tumor  was  given  during  amethopterin 
treatment,  a  combination  generally  fatal  to  these  mice.  No  other  instances 
of  tumor -immunizing  capability,  and  none  of  tumor-producing  capability,  of 
this  virus  strain  have  been  detected,  despite  repeated  efforts. 

Earlier  work  in  this  laboratory  demonstrated  that  general  purpose 
mice  are  normally  refractory  to  the  P288  tumor,  but  that  fatal  ascitic 
tumors  developed  if  they  were  treated  with  amethopterin  after  tumor 
injection.  During  this  year,  I  have  found  that  injections  of  tumor  given 
without  amethopterin  treatment  made  the  mice  immune  to  later  challenge 
with  the  same  tumor  plus  amethopterin.  Attempts  to  immunize  with  disrupted 
tumor  cells  (freezing-thawing)  have  indicated  that  such  preparations  may 
have  an  immunizing  effect  but  these  experiments  are  not  as  yet  complete. 
A  similar  effect  seemed  to  occur  when  normal  tissues  of  the  CDBA  strain  of 
mice  (the  strain  in  which  tumor  P288  originated  and  is  maintained)  were 
used  in  lieu  of  tumor  preparations  for  immunizing  the  white  mice. 

Experiments  on  immunity  to  LCM  in  X-rayed  mice  kept  alive  by 
marrow  and  spleen  transplants  have  been  completed.  The  immune  status  of 
the  X-rayed  mouse  before  radiation  and  tissue  transplant  determined  its 
response  to  later  LCM  challenge .  Marrow  and  spleen  from  immune  donors  did 
not  confer  immunity  on  the  recipients  which  had  not  been  previously 
immunized. 

Significance  of  the  Program  to  the  Institute; 

A  significant  part  of  the  Institute  's  program  on  basic  research  in 
virology  concerns  the  alterations  produced  in  the  virus-infected  animal, 
as  an  entity  and  on  a  cellular  basis.  One  such  alteration  could  be  the 
induction  of  tumor  growth,  another  could  be  the  induction  of  resistance 

16 


Serial  No.  NIAID-8U 

to  tumors.  These  experiments  have  yielded  information  on  how  a  particular 
virus — LCM — and  a  transplantable  tumor  —  P288 — become  inter-related  during 
passage  together.  The  alteration  of  the  general  purpose  mouse's  response 
to  the  P288  tumor  by  amethopterin  provides  an  opportunity  for  studying 
the  immune  reaction  to  various  antigens  as  measured  by  resistance  to 
challenge  with  the  tumor. 

Proposed  Course  of  the  Project: 

This  project  comes  to  an  end  this  year  because  of  my  retirement. 


17 


Serial  No.  NIAID-8^ 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Haas,  Victor  H.  Serial  passage  of  a  lymphocytic  tumor  and 
choriomeningitis  virus  in  immune  mice.  Jour.  Natl. 
Cancer  Inst.  25:  75-83  (i960). 

Uphoff,  Delta  E.  and  Haas,  Victor  H.   Immunologic  response 
to  lymphocytic  choriomeningitis  virus  in  lethally 
irradiated  mice  treated  with  bone  marrow.  Jour.  Natl. 
Cancer  Inst.  25:  779-786  (i960). 


Honors  and  Awards  relating  to  this  project: 
None 


18 


Serial  No.  NIAID-85 

1.  Biology  of  Viruses 

2.  Viral  Growth 

3-  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:   Investigations  of  Animal  Virus  Reproduction 

Principal  Investigator:  Dr.  Hilton  B.  Levy 

Other  Investigators:  Dr.  Frank  De  Filippes 

Cooperating  Units:  None 

Man  Years: 

Total:  2  11/12 
Professional:  11/12 
Other:        2 

Project  Description: 

Objectives: 

To  gain  information  relative  to  the  relationship  that  exists 
between  the  infected  cell  and  the  virus  reproducing  therein.  More 
particularly,  to  determine  at  a  molecular  level  the  mechanisms  by  which 
a  virus  is  reproduced  by  the  infected  cell.  Knowledge  of  either  the 
details  of  altered  cell  metabolism  or  mechanisms  of  virus  reproduction 
might  be  useful  in  the  development  of  chemotherepeutic  agents  to  inhibit 
virus  growth  and  to  aid  the  host.  Comparison  of  oncogenic  viruses  such 
as  Rous  Sarcoma  with  cytocidal  viruses  such  as  poliovirus  should  give 
insight  into  growth  controlling  mechanisms. 

Methods  Employed: 

The  program  for  this  year  sought  the  intracellular  localization 
of  the  infecting  virus  particle  during  the  course  of  the  infection,  and 
also  where  the  components  of  the  new  virus  were  made.  High  resolution 
autoradiography  and  fluorescent  antibody  techniques  were  used.  Nucleic 
acid  bases  containing  the  weak  beta  particle  emitter  tritium  were  used 
to  study  nucleic  acid  metabolism  and  to  prepare  labelled  poliovirus. 
Tritium  labelled  histidine  was  used  for  protein  studies.  Rabbit  anti- 
serum to  highly  purified  poliovirus  was  prepared  for  the  fluorescent 
antibody  work. 

Part  B  included:  Yes 

13 


Serial  No.  NIAID-85 

Major  Findings; 

The  first  detected  change  in  poliovirus  infected  HeLa  cells  occurs 
about  an  hour  after  infection,  when  there  is  seen  increased  turnover  of 
RNA,  particularly  and  almost  exclusively  in  the  nucleoli,   (it  is  about 
this  time  that  our  earlier  work  detected  increased  glycolytic  energy 
production.)  This  increased  RNA  turnover  continues  for  3  l/2  to  k   hours 
after  which  it  greatly  decreases.  At  about  1  l/2  to  2  hours  after 
infection,  there  appears  a  virus  specific  antigen  in  the  cytoplasm,  even 
though  total  cell  protein  metabolism,  as  measured  by  tritiated  histidine, 
has  declined  markedly.  By  3  to  3  l/2  hours  after  infection,  viral 
antigen  appears  in  the  nucleus.  This  nuclear  antigen  does  not  occur  in 
the  nucleolus,  which  latter  structure  has  a  bright  thin  ring  of  stained 
antigen  around  it.   It  might  be  that  this  nuclear  antigen  is  made  at 
the  periphery  of  the  nucleolus,  or  is  made  in  the  nucleolus  but  is  not 
susceptible  to  reaction  with  fluorescent  antibody  until  released  at  its 
surface.  By  about  h   to  5  hours  after  infection  there  is  a  decline  in 
the  number  of  cells  showing  nuclear  antigen  and  the  appearance  of 
brightly  staining  antigen  in  the  cytoplasm,  suggesting  that  the  nuclear 
material  has  migrated  there.  Since  parallel  viral  growth  studies  show 
the  first  appearance  of  new  virus  at  this  time,  it  would  suggest  that 
this  nuclear  to  cytoplasmic  migration  is  the  step  that  forms  new  virus. 
Whatever  nucleic  acid  or  specific  protein  synthesis  was  implicated  in 
the  increased  nucleolar  RNA  turnover  decreases  at  this  time.  Shortly 
thereafter,  the  increased  energy  production  stops. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

Increased  research  in  biochemistry  along  the  lines  of  protein  and 
nucleic  acid  biosynthesis  has  resulted  in  the  synthesis  of  at  least  two 
specific  proteins  in  cell  free  systems.  The  way  seems  clear  to  do  this 
with  virus  protein.  The  intracellular  localization  of  where  specific 
virus  protein  is  made  indicates  which  subcellular  components  to  use  in 
such  an  attempt. 

Comparable  studies  with  other  cytocidal  as  well  as  with  oncogenic 
viruses  will  add  to  the  Institute 's  program  directed  to  understanding 
mechanisms  by  which  host  cells  are  diverted  from  normal  cell  behavior  to 
the  production  of  new  virus  or  to  tumor  characteristics. 

Proposed  Course  of  the  Project; 

Attempts  will  be  made  to  utilize  the  information  obtained  this 
past  year  to  synthesize  some  viral  components  in  cell  free  systems. 
Comparison  with  other  viruses  will  be  made.  Further  studies  on  the 
nature  of  the  increased  nucleolar  activity  will  be  made  to  see  if  viral 
nucleic  acid  is  being  made. 


20 


Serial  No.  NIAID-85 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 

Part  B   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Levy,  Hilton  B.  and  Sober,  Herbert.  A  Simple  Chromatographic 
Method  for  Preparation  of  Gamma  Globulin.  Proc.  Soc. 
Exptl.  Biol. &  Med.  103:  250-252,  i960. 

Levy,  Hilton  B.  and  Snellbaker,  LeRoy.  Phosphorus  Metabolism 
in  Infection  vith  Murine  Leukemia  Virus.  Proc.  Soc. 
Exptl.  Biol.  &  Med.  103:  503-506,  i960. 

Levy,  Hilton  B.  and  Lynt,  R.  K.  Heterogeneity  in  Cytoplasmic 
RNAs  of  Mouse  Spleen  and  Effect  Thereon  of  a  Leukemia 
Virus.  Submitted  for  publication. 


Honors  and  Awards  relating  to  this  project: 

The  work  on  the  Friend  leukemia  virus  was  chosen  to  be 
published  in  the  M.  D.  Anderson  Hospital  annual  publication  of 
significant  reports  in  cancer  research. 


21 


Serial  No.   NIAID-85A 

1.  Biology  of  Viruses 

2.  Viral  Growth 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:  Animal  Virus  Synthesis 

Principal  Investigator:  Dr.  Frank  M.  DeFilippes 

Other  Investigators:  Dr.  Hilton  B.  Levy 

Cooperating  Units:  None 

Man  Years: 

Total:  1  1/12 

Professional:  1  1/12 

Other:  0 

Project  Description: 

Objectives: 

1.  To  investigate  the  synthesis  of  an  animal  virus  in  a  tissue 
culture  system  by  studying  subcellular  particles  which  are  involved  in 
protein  synthesis  in  many  animals. 

2.  To  increase  the  efficiency  of  infection  of  monkey  kidney  cells 
with  RNA  extracted  from  purified  poliovirus  to  a  level  similar  to  that 
obtainable  with  whole  virus. 

Methods  Employed: 

1.  HeLa  cells  infected  with  poliovirus  are  grown  in  a  radioactive 
medium.  The  cells  are  collected  at  different  times  and  cell  fractions 
are  isolated  with  particular  attention  being  given  to  the  ribosomal 
material.  The  specific  radioactivity  of  the  ribosomes  is  followed  during 
the  increase  of  intracellular  virus  and  compared  to  ribosomes  isolated 
from  uninfected  cells.  The  ribosomal  material  is  identified  by  its 
spectrum  and  also  by  electron  microscopy.  The  variation  of  radioactivity 
of  other  cell  fractions  isolated  during  the  purification  of  the  ribosomes 
is  also  under  investigation. 

Part  B  included:  No 

22 


Serial  No.  NIA3D- 85A 

2.  Monkey  kidney  cell  monolayers  are  infected  vith  RNA  extracted 
from  purified  poliovirus  under  a  variety  of  ionic  and  pH  conditions. 

Major  Findings; 

1.  A  procedure  has  been  worked  out  which  consistently  allows  the 
isolation  of  at  least  1C$  of  the  ribosomal  material  from  HeLa  cells  in 
a  relatively  pure  state.  The  ribosomal  ribonucleoprotein  particles 
may  be  separated  from  the  viral  ribonucleoprotein  particles  by  passage 
through  an  ECTEOLA-cellulose  column. 

The  specific  activity  of  ribonucleoprotein  particles  from 
infected  cells  is  less  than  that  of  the  ribosomal  material  of  uninfected 
cells  7  hours  after  the  addition  of  virus  under  conditions  of  high  and 
low  multiplicity  of  infection.  With  a  high  multiplicity,  the  cellular 
protein  sedimented  at  15,000g  for  10  minutes  shows  a  dramatic  and 
continuous  decline  in  specific  activity. 

2.  The  efficiency  of  infection  with  extracted  RNA  has  been  brought 
to  a  level  of  about  0.5$  that  obtainable  with  whole  virus. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

The  program  is  designed  to  lead  to  a  general  picture  of  the 
synthesis  of  an  animal  virus  by  following  events  at  a  molecular  level. 
It  is  hoped  that  the  key  steps  involved  in  the  conversion  of  the  cellular 
machinery  from  normal  metabolic  activity  to  a  virus  producing  system  will 
be  elucidated.   Interference  with  these  key. steps  may  lead  to  new  and 
general  methods  of  arresting  viral  disease. 

Proposed  Course  of  the  Project; 

Investigation  of  cellular  fractions  and  especially  ribosomal 
material  will  be  continued  under  conditions  where  the  cells  are  infected 
with  different  virus  multiplicities.   It  is  hoped  that  infection  with 
very  high  multiplicities  may  remove  all  remnants  of  normal  activity  and 
clarify  the  situation  with  respect  to  virus  growth.  Also  ribosomes  from 
cells  actively  synthesizing  new  protein  during  infection  will  be  compared 
to  cells  which  are  infected  while  they  are  in  a  resting  state. 

Infection  of  monkey  kidney  cells  with  RNA  extracted  from  polio- 
virus  and  the  subsequent  isolation  and  purification  of  ribosomal  material 
will  also  be  attempted  to  study  the  role  of  the  viral  genetic  material. 


23 


Serial  No.   NIAID-86 

1.  Biology  of  Viruses 

2.  Virus-Host  Relationship 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:   Investigations  of  interference  with  enzymatic 
functioning  of  mitochondria  as  a  mechanism  for 
carcinogenesis 

Principal  Investigator:  Dr.  Marie  L.  Hesselbach 

Other  Investigators:  None 

Cooperating  Units:   Laboratory  of  Pathology  &  Histochemistry,  NIAMD 
Laboratory  of  Pathology,  NCI 

Man  Years: 

Total:  10/12 

Professional:  9/12 

Other:  l/l2 

Project  Description: 

Objectives: 

To  determine  whether  interference  with  the  functioning  of  mito- 
chondrial enzymes  leads  to  neoplasia.  Specifically,  it  is  desired  to 
find  a  single  dye  which:   l)  is  adsorbed  by  mitochondria;  2)  effects 
the  functioning  of  mitochondrial  enzymes,  and  3)  induces  neoplasia. 
Then,  perhaps,  a  connection  between  these  two  functions  of  a  single 
agent  can  be  demonstrated. 

Methods  Employed: 

Conventional  Warburg  manometry  for  metabolic  study  of  induced  and 
transplanted  tumors,  also  for  analysis  of  interaction  of  the  dyes  under 
study,  with  mitochondrial  enzymes. 

Different  methods  of  preparation  of  tissue  for  metabolic  studies: 
slicing,  homogenization,  and  differential  centrifugation. 


Part  B  included:  No 

2k 


Serial  No.  NIAID-86 

Chemical  analyses  for  total  nitrogen,  lactic  acid  and  inorganic 
phosphorus . 

Histological  preparation  and  examination  of  treated  areas,  tumors, 
and  organs . 

A  study  of  vehicles  which  would  allow  repeated  injections  of 
Janus  green  B  over  a  long  period  has  been  made.   Non-aqueous  media  were 
found  to  be  necessary.   Prolonged  testing  has  made  it  possible  to  choose 
the  best  of  these . 

Major  Findings: 

Absence  of  glucolysis  in  most  of  the  Fast  Green-  and  Light  Green- 
induced  tumors  and  their  early  transplant  generations  was  found  to 
correlate  with  the  large  amounts  of  enzymatically  inert  collagen  present 
in  them.  Age  of  tumor  was  found  not  to  play  a  role  in  absence  of 
glucolysis.   In  later  generations  glucolysis  was  more  commonly  seen  and 
collagen  decreased.  Mitochondrial  preparations  which  glucolyzed  could 
be  prepared  from  the  most  metabolically  active  tumors. 

It  has  been  demonstrated  that  Fast  Green  and  Light  Green  can  be 
added  to  total  rat  brain  homogenates  at  concentrations  which  inhibit  and 
at  other  concentrations  which  stimulate  oxygen  uptake  with  added  glucose 
as  substrate.  The  same  concentrations  which  stimulate  with  glucose, 
fail  to  do  so  with  fructose-diphosphate .  The  dyes  do  not  appear  to  be 
uncouplers  of  oxidative  phosphorylation. 

Repeated  injection  of  Janus  Green  B  has  induced  gross  changes 
suggestive  of  tumor  formation,  but  these  have  not  yet  been  checked 
histologically. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

Both  viruses  (exogenous)  and  altered  subcellular  particles 
(endogenous)  have  been  implicated  as  the  cause  of  cancer.  This  study  is 
an  effort  to  determine  the  relation  of  mitochondria  to  these  possible 
oncogenic  agents. 

Proposed  Course  of  the  Project: 

Further  studies  of  the  Fast  Green- induced  tumor  transplant  line 
will  be  made  to  see  whether  the  homogenates  acquire  glucolysis  "spontaneously," 
or  by  changes  in  preparative  procedure,  or  the  cofactors  added.   It  will 
also  be  determined  which  of  the  3  enzymes  which  convert  glucose  to  fructose- 
diphosphate  are  destroyed  by  homogenization. 


25 


Serial  No.  NIAID-86 

The  study  of  the  interaction  of  Fast  Green  and  Light  Green  with 
mitochondrial  enzymes  will  be  extended  to  include  all  possible  enzyme 
systems.  The  question  of  adsorption  and  physical  interaction  of  these 
dyes  with  the  mitochondrial  substance  will  be  taken  up. 

Now  that  it  is  possible  to  give  repeated  doses  of  Janus  Green  B, 
an  experiment  will  be  set  up  to  see  whether  this  dye  is  cancerogenic. 
The  treated  areas,  and  any  tumors  formed,  will  be  analyzed  histologically. 
Any  induced  tumors  will  be  transplanted,  and  studied  metabolically.  The 
interaction  of  Janus  Green  B  and  mitochondrial  enzymes  will  be  examined 
in  detail. 


26 


Serial  No.  NIA3D-86A 

1.  Biology  of  Viruses 

2.  Virus-Host  Relationship 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:   Demonstration  of  Glucose  Metabolism  and  Peptide 
Bond  Formation  by  Isolated  Brain  and  Liver 
Mitochondria 

Principal  Investigator:     Dr.  Marie  L.   Hesselbach 

Other  Investigators:  Dr.  H.  G.  du  Buy 

Cooperating  Units:  Analytical  Chemistry  Section,  NIAMD 

Man  Years: 

Total:  V12 

Professional:  3/12 

Other:  1/12 

Project  Description: 

Objectives: 

To  demonstrate  that  isolated  mitochondria  participate  in  protein 
metabolism  and  that  not  only  isolated  brain  mitochondria  but  also  liver 
mitochondria  contain  the  complete  enzyme  systems  to  metabolize  glucose. 

Methods  Employed: 

Conventional  Warburg  manometry,  chemical  lactic  acid  determinations, 
centrifugal  separation  of  subcellular  elements,  biochemical  and  bio- 
physical approach  to  choice  of  materials  for  a  suspension  medium  vhich 
will  keep  mitochondria  structurally  and  functionally  intact,  biochemical 
approach  to  determining  materials  to  be  added  to  isolated  mitochondria 
to  restore  in  vivo  enzymatic  activity.  For  demonstration  of  peptide- 
bond  formation:  one -dimensional  paper  chromatography. 

Major  Findings: 

Work  was  continued  on  trying  to  demonstrate  glucose  utilization 
by  isolated  rat  liver  mitochondria.  Many  variations  in  medium  composition 

Part  B  included:  No 

27 


Serial  No.  NIAID-86A 

were  used.  These  involved  physico-chemical  agents  such  as  methocel, 
salts,  chelating  agents,  protein  derivatives,  ribonucleic  acid,  and 
phospholipid  derivatives.  The  effects  of  the  hormones  epiniphrine  and 
glucagon  were  studied,  as  well  as  the  enzymes  0!-  and  (3-amylase  and 
hexokinase.  The  reducing  agent  and  cof actor,  glutathione,  and  the 
diabetogenic  agent  phlorizin,  were  also  tried.  Some  of  these  substances 
were  used  in  the  isolation  medium,  while  others  were  added  to  the 
reaction  vessels. 

At  times  hexokinase  greatly  increased  glucose  utilization,  at 
others  it  had  little  or  no  effect.  Some  of  the  other  materials,  such 
as  glutathione,  seemed  to  increase  glucose  utilization  very  slightly, 
but  not  to  a  significant  level. 

Addition  to  liver  mitochondria  of  the  natural  "fat, "  collected 
from  the  surface  of  these  aqueous  preparations,  was  more  successful  in 
increasing  glucose  utilization  than  anything  except  the  specific  enzyme, 
hexokinase. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

The  relation  of  viruses  to  mitochondria  (site  of  virus  repro- 
duction, origin  of  viruses,  site  of  neoplastic  change)  can  only  be  fully 
understood  when  the  physical  and  chemical  characteristics  of  mitochondria 
become  known.  It  would  be  strange  if  liver  tissue  could  not  metabolize 
glucose,  since  it  stores  it  (as  glycogen),  etc.   It  would  also  be 
strange  if  this  essential,  energy- producing  metabolism  were  not  located 
on  the  mitochondria  in  liver  cells  as  it  is  in  brain  cells. 

If  isolated  mitochondria  can  be  shown  to  synthesize  the  peptide 
bond,  it  will  add  significantly  to  our  knowledge  of  the  role  of  mito- 
chondria in  the  living  cell. 

Proposed  Course  of  the  Project; 

It  is  desired  to  obtain  liver  mitochondria  which  will  readily 
oxidize  glucose  in  significant  quantity,  and  will  synthesize  the  peptide 
bond.  Further  studies  will  be  made  to  obtain  better  media  and  to 
determine  what  other  chemical  or  physical  agents  can  be  added  to  obtain 
sustained  enzyme  activity  of  a  number  of  complete  enzyme  sequences  on 
mitochondria  in  vitro. 


28 


Serial  No.  NIAID-87 

1.  Biology  of  Viruses 

2.  Virus-Host  Relationship 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:  Cytopathogenic  effect  in  single  cells  in  tissue 
culture. 

Principal  Investigator:  JansShowacre 

Other  Investigators:  None 

Cooperating  Units:  None 

Man  Years: 

Total:  6/12 

Professional:  V12 

Other:  2/12 

Project  Description: 

Objectives: 

To  study  the  morphologic  and  metabolic  effects  of  virus  infection 
on  individual  virus  infected  cells  in  tissue  culture. 

Methods  Employed: 

Primary  and  established  cell  lines  were  cultured  on  coverslips 
and  then  mounted  in  a  special  tissue  culture  flow  chamber  which  permits 
direct,  phase  and  darkfield  microscopy  under  conditions  of  cell  growth. 
Cells  growing  in  the  chamber  were  then  infected  with  virus  and  changes 
in  morphology  studied.  Phase  and  fluorescent  microscope  observations 
were  made  in  rapid  succession  on  identical  living  cells  immediately 
after  mounting  on  regular  slide  mounts. 


Part  B  included:  No 

29 


Serial  No.  NIAID-87 

Major  Findings: 

Studies  with  EMC,  HA  1  and  M  25  infected  tissue  cultures  have 
continued.  Marked  changes  in  cell  morphology  have  not  been  observed 
in  initial  stages  of  infection.  Fluorescent  antibodies  against  HA  1 
and  M  25  gave  readily  recognizable  peripheral  staining  of  living  infected 
cells  in  late  stages  of  infection,  similar  to  those  reported  by  0 'Dea 
and  Dineen  with  Herpes  simplex.  Antibody  against  EMC  is  being  obtained 
to  determine  whether  increased  titer  and  purification  will  improve  the 
efficiency  of  the  technique  in  early  stages  of  infection.  Primary 
cultures  of  embryonic  and  adult  mouse  brain  have  been  obtained  for  an 
in  vivo  study  of  the  effects  of  neurotropic  viruses  on  Nissl  substance. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute; 

Morphological  evidence  of  early  specific  effects  of  virus  invasion 
of  a  cell  may  indicate  the  intracellular  locus  of  virus  activity. 

Proposed  Course  of  the  Project: 

Further  study  of  morphological  and  physiological  changes  of 
subcellular  elements  following  the  introduction  of  viruses  and  other 
pathogenic  micro-organisms. 


30 


Serial  No.  NIAID-87A 

1.  Biology  of  Viruses 

2.  Virus-Host  Relationship 

3.  Bethesda,  Maryland 


PHS-NU 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:  Characterization  of  mitochondria 

Principal  Investigator  :  Jane  Showacre 

Other  Investigators:  Dr.  H.  G.  du  Buy 

Cooperating  Units:  None 

Man  Years: 

Total:  8/12 

Professional:  7/12 

Other:  1/12 

Project  Description: 

Objectives: 

To  study  the  localization  of  non-toxic  fluorophors  in  living 
cells. 

Methods  Employed: 

Living,  unfixed  tissues  vere  examined  by  phase  contrast  and 
fluorescence  microscopy  following  exposure  to  fluorescent  compounds. 
Preparations  were  made  from  animal  organs,  a  number  of  tissue  cultures 
including  monkey  kidney,  HeLa  and  strain  L  and  from  cultures  of  micro- 
organisms. The  results  were  recorded  photographically. 

Major  Findings: 

Of  the  fluorescent  compounds  studied  the  most  promising  have  been 
the  tetracyclines.   These  antibiotics,  tetracycline,  oxytetracycline, 
and  chlor tetracycline,  were  found  to  specifically  combine  with  mito- 
chondria of  living  cells  in  tissue  cultures  or  in  fresh  preparations 
from  various  organs  of  mice,  and  in  bacteria  such  as  Salmonella  typhosa. 

Part  B  included:  Yes 

31 


Serial  No.  NIAID-87A 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

Tetracyclines  can  now  serve  as  an  additional  vital  stain  for  the 
characterization  of  mitochondria.  As  such  these  compounds  may  aid  in 
the  determination  of  subcellular  changes  under  different  conditions, 
e.g.,  viral  infection.   In  this  connection  the  specific  localization 
suggests  that  mitochondria  are  implicated  in  the  fatty  degeneration 
occurring  in  liver  following  prolonged  tetracycline  therapy.  Further, 
the  fluorescent  properties  may  serve  to  identify  the  site  of  antibiotic 
action  in  bacteria  and  elucidate  differences  in  antibiotic  effectiveness 
under  different  environmental  conditions. 

Proposed  Course  of  the  Project; 

Studies  are  continuing  on  factors  influencing  the  retention  of 
tetracyclines  by  mitochondria  as  a  preliminary  to  investigations  of 
possible  change  in  the  staining  properties  of  mitochondria  during 
infection  of  cells  with  viruses,  during  cell  division  and  following 
cell  fractionation.  Bacteria  under  different  environmental  conditions 
are  also  being  examined. 


32 


Serial  No.  NIAID-87A 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

duBuy,  H.  G.  and  Showacre,  J.  L.  Selective  localization 
of  tetracycline  in  the  mitochondria  of  living  cells. 
Accepted  for  publication  in  Science. 


Honors  and  Awards  relating  to  this  project: 
None 


33 


71    3.1 

9'' 


Serial  No.  NIAID-88 

1.  Biology  of  Viruses 

2.  Virus -Host  Relationship 

3.  Bethesda,   Maryland 


FHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:  Biology  of  mitochondria  and  its  relation  to 
endogenous  and  viral  diseases. 

Principal  Investigator:  Dr.  H.  G.  duBuy 

Other  Investigators:  Dr.  M.  L.  Hesselbach  and  J.  L.  Shovacre 

Cooperating  Units:  Analytical  Services  Unit,  Mr.  H.  G.  McCann 

Man  Years: 

Total:  1  Vi2 

Professional:  10/12 

Other:  6/12 

Project  Description: 

Objectives: 

Biological  definition  of  normal  versus  pathogenic  or  virus- 
altered  mitochondria. 

Methods  Employed: 

The  methods  encompass  the  applications  of  the  cytochemical 
findings  reported  under  project  No.  NIAID-87A.  They  also  include 
Warburg  metabolic  techniques  and  determinations  of  oxidative  phos- 
phorylation in  order  to  define  different  mitochondria  metabolically. 
The  results  are  applied  to  in  vitro  cultivation  of  isolated  mito- 
chondria. 

Major  Findings: 

A  manuscript  is  in  preparation  on  evidence  that  the   ~,yme 
behavior  of  mitochondria  obtained  by  the  sucrose  gradient  .   .iainly 
due  to  the  unavoidable  dilution  of  mitochondria,  when  this  t  chnique 
of  separation  is  used.  The  results  are  applied  specifically  to  the 
loss  of  enzymes  by  melanized  mitochondria  of  the  Cloudman  S  91  mouse 
melanoma. 

Part  B  included:  No 

34 


Serial  No.   NIA33-88 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

The  maintenance  or  cultivation  of  mitochondria  in  vitro  when 
accomplished  should  facilitate  the  investigation  of  many  metabolic 
activities  of  normal  cells  as  compared  to  tumor  cells  or  those  infected 
vith  various  types  of  viruses. 

Proposed  Course  of  the  Project: 

Further  studies  of  mitochondria  from  different  sources  will  be 
carried  out  in  order  to  learn  more  about  the  characteristics  of  these 
elements.  Additional  experiments  will  be  done  to  explore  further  the 
complete  enzymatic  complement  of  mitochondria,  especially  as  this 
relates  to  synthetic  activities.  All  information  obtained  will  be 
applied  to  continued  attempts  at  in  vitro  cultivation  of  mitochondria. 


35 


Serial  No.  NIAID-88A 
1.     Biology  of  Viruses 


2.  Virus-Host  Relationship 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:  Comparison  of  the  properties  of  crude  and 

crystallized  Coxsackie  A- 10  virus,  of  Cloudman  S  91* 
mouse  melanoma  and  of  mouse  muscle  nucleoprotein. 

Principal  Investigator:  Dr.  H.  G.  duBuy 

Other  Investigators:  H.  Sasame 

Cooperating  Units:  None 

Man  Years: 

Total:  1  k/12 

Professional:  3/12 

Other:  1  1/12 

Project  Description: 

Objectives: 

To  compare  preparations  of  purified  virus  with  those  of  mouse 
muscle  and  melanoma  nucleoprotein,  chemically  and  immunologically. 

Methods  Employed: 

For  virus  purification:  Coxsackie  A- 10  virus  of  suckling  mouse 
origin  is  purified  and  concentrated  "by  chemical,  physical  and  ultra- 
centrifugal  means.  Purified  virus  is  analyzed  for  protein  and  nucleic 
acid  content. 

For  muscle  and  S  91  nucleoprotein  preparation  available  methods 
vere  not  applicable  to  the  materials  under  study.  Some  steps,  used 
for  the  preparation  of  so-called  ribosomes,  followed  by  modifications 
of  existing  purification  procedures,  have  given  promising  results. 


Part  B  included:  No 

36 


Serial  No.  NIAID-88A 

Major  Findings; 

Melanin  granules,  isolated  by  selective  centrifugation,  contained 
30  to  kO   percent  of  the  total  ribose  nucleo-protein  of  the  melanoma  cell. 
This  supports  the  view  that  the  granules  are  modified  mitochondria.  The 
antigenic  activity  of  this  material  is  determined  by  the  number  of 
"takes"  of  transplanted  melanoma  cells  in  mice  which  have  previously 
been  injected  with  immunizing  doses  of  the  melanoma  nucleoprotein,  as 
compared  with  non- immunized  mice. 

Purified  Coxsackie  A- 10  virus  has  been  introduced  into  normally 
resistant  cells  by  cellular  uptake  of  glass-adsorbed  virus. 

Significance  to  Bio-medical  Research  and  the  program  of  the  Institute: 

The  introduction  of  virus  into  cells  which  are  normally  not 
susceptible  to  this  virus,  except  in  its  nucleic  acid  stage,  might 
throw  further  light  on  the  conditions  which  govern  virus  multiplication 
in  host  cells. 

The  introduction  of  a  self -duplicating  portion  of  mitochondria 
from  cancer  cells  into  susceptible  hosts  might  lead  to  formation  of 
this  specific  neoplasm,  and  thus  bridge  the  virus  and  the  mitochondrial 
theories  of  carcinogenesis. 

Proposed  Course  of  the  Project;  . 

To  obtain  sufficient  quantities  of  the  three  nucleoproteins,  each 
with  a  standard  nucleic  acid-protein  ratio  as  an  index  of  purity,  to 
allow  quantitative  antigenic  studies.  At  this  time,  purified  muscle 
nucleoprotein  to  be  used  for  control  studies  has  not  yet  been  obtained. 


37 


Serial  No.  NIAID-89 


1.  Biology  of  Viruses 

2.  Viral  Growth 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:  Kinetics  and  Sites  of  Coxsackie  Virus  Multiplication 
in  the  Monkey  Kidney  Cell 

Principal  Investigator:  Dr.  C.  F.  T.  Mattern 

Other  Investigators:  Lotta  Chi 

Cooperating  Units:  None 

Man  Years: 

Total:  1 

Professional:  1 

Other:  0 

Project  Description: 

Objectives: 

To  establish  the  intracellular  site  or  sites  of  viral  RNA  and 
protein  synthesis. 

Methods  Employed: 

Coxsackie  A-9  virus  is  cultivated  in  monkey  kidney  cells  for 
various  time  periods.  Cells  are  fractionated  by  the  Dounce  Citric  Acid 
Procedure  and  by  conventional  homogenization.  Two  major  fractions  are 
now  being  investigated  for  mature  virus  content,  namely  the  nuclear 
fraction  and  the  remainder  of  the  cell,  called  the  "cytoplasmic"  fraction. 
It  has  been  previously  shown  by  this  group  that  cold  phenol  will  not 
extract  RNA  from  purified  Coxsackie.  Also  the  suckling  mouse  inoculated 
I.M.  is  an  excellent  assay  system  for  the  RNA,  whereas  tissue  culture  is 
poor.  These  cell  fractions  are  assayed  for  mature  virus  and  extracted 
with  cold  phenol  for  a  virus  precursor,  whether  free  RNA  or  a  RNP  other 
than  mature  virus. 


Part  B  included:  No 

38 


Serial  No.  NIAID-89 
Major  Findings: 

1.  Mature  virus,  that  is  virus  refractory  to  cold  phenol  extraction 
of  its  RNA,  is  clearly  associated  with  the  cytoplasmic  fraction  at  all 
time  periods  from  2  to  12  hours  post- inoculation. 

2.  There  is  evidence  of  a  cold  phenol  extrac table  precursor.  This 
precursor  has  been  extracted  from  whole  cells  and  cell  homogenates  and 
appears  maximally  produced  by  6  hours,  remaining  constant  in  quantity 
thereafter.  The  titer  of  mature  virus,  on  the  other  hand  appears  to 
continue  to  increase  until  9-12  hours  post- inoculation.  Most  of  this 
"precursor"  appears  associated  with  the  "nuclear"  fraction,  thus  show- 
ing a  distribution  within  the  cell  that  is  different  from  that  of 
"mature"  virus. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

This  study  is  intended  to  contribute  to  our  knowledge  of  the 
nature  of  the  processes  by  which  viruses  multiply.  It  has  been  proposed 
by  others  that  viral  RNA  is  synthesized  in  the  nucleus  on  the  basis  of 
indirect  evidence.  The  studies  herein  described  would  be  the  first 
direct  evidence  of  viral  RNA  synthesis  in  the  nucleus. 

Proposed  Course  of  the  Project: 

It  is  proposed  to  continue  this  project  in  order  to  evaluate  the 
significance  of  our  findings  to  date,  especially  with  respect  to  whether 
they  are  representative  of  actual  intracellular  events  or  artifacts  of 
cell  fractionation.  Other  cell  fractionation  techniques  will  be  employed. 
Our  first  "control"  experiments  indicated,  for  example,  that  this 
distribution  of  "precursor"  is  not  the  result  of  selective  absorption 
of  "cytoplasm  precursor"  by  nuclei.  In  addition  a  collaborative  project 
with  Dr.  Hilton  Levy  is  planned  in  which  these  events  will  be  followed 
by  autoradiography  in  the  Coxsackie-monkey  cell  system.  Dr.  Levy  has 
been  utilizing  a  poliovirus-HeLa  cell  system  and  has  made  observations 
which  seem  compatible  with  our  interpretation  of  our  infectivity  data. 

Since  there  is  reason  to  believe  that  this  "precursor"  is  a  RN 
protein,  we  plan  to  attempt  to  isolate  and  characterize  this  component. 


33 


Serial  No.  NIAID-89A 

1.  Biology  of  Viruses 

2.  Viral  Growth 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A 


Project  Title:  Virus  Structure 

Principal  Investigator:  Dr.  C.  F.  T.  Mattern 

Other  Investigators:  Lotta  Chi 

Cooperating  Units:  None 

Man  Years: 

Total:  1 

Professional:  1 

Other:  0 

Project  Description: 

Objectives: 

This  project  involves  the  construction  of  "biological  and  related 
biochemical  models  of  viruses  by  a  new  model  approach.  The  inmediate 
objective  is  to  test  the  "reality"  of  the  virus  models  by  electron 
microscopy  and  X-ray  diffraction. 

Methods  Employed: 

1.  Electron  Microscopy.  The  substructure  of  viruses  is  being 
examined  by  several  techniques:  conventional  shadowing,  negative  stain- 
ing with  phosphotungstic  acid,  and  positive  staining  with  uranium  salts. 
In  addition,  a  new  shadowing  procedure  has  been  developed  and  its 
potentialities  in  revealing  substructure  are  being  studied. 

2.  X-ray  diffraction.  This  involves  an  indirect  approach  in  which 
theoretical  diffraction  patterns  may  be  calculated  and  compared  with 
patterns  obtained  from  viruses  by  others. 


Part  B  included:  No 

40 


Serial  No.  NIAID-89A 
Major  Findings; 

1.  A  new  model  for  Tobacco  Mosaic  Virus  has  been  constructed 
which  contains  a  substantial  number  of  those  structural  elements  known 
from  a  wide  variety  of  biochemical  and  biophysical  data  of  others. 

2.  The  model  has  predicted  several  gross  features  which  differ 
from  currently  accepted  models  and  which  should  be  demonstrable  by 
electron  microscopy.  Our  electron  micrographs  taken  with  metal  shadow- 
ing and  negative  staining  are  remarkably  compatible  with  the  model. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

This  project  is  an  effort  to  elucidate  the  three  dimensional 
structure  of  viruses,  beyond  the  subunit  organization.  The  selection 
of  TMV  for  constructing  a  detailed  model  was  necessary  because  it  is 
the  most  thoroughly  studied  virus  and  the  only  one  about  which  there 
is  sufficient  structural  data  to  attempt  to  construct  a  detailed  model. 
The  structure  of  other  viruses,  in  particular  animal  viruses,  will 
also  be  investigated  by  this  approach  as  additional  data  accumulates. 

Proposed  Course  of  the  Project: 

Because  of  the  controversial  nature  of  this  project,  it  is 
felt  advisable  to  proceed  to  accumulate  a  substantial  amount  of 
experimental  data  before  challenging  the  currently  held  views  on  virus 
structure,  and  in  particular  TMV.  A  purely  hypothetical  paper  describ- 
ing the  model  building  system,  without  detailed  reference  to  specific 
structures,  is  in  the  process  of  being  written. 


41 


LABORATORY  OF  TROPICAL  VIROLOGY 
Middle  America  Research  Unit 
Arthropod-Borne  Virus  Section 


Summary 


93    -  Studies  on  Arthropod-Borne  Viruses  in  the  Sub- 
Tropical  Areas  of  the  United  States  6 

94-A  -  Studies  of  Arthropod-Borne  Viruses  in  Tissue 

Culture.   Part  1.   Evaluation  of  Tissue  Culture 
Systems  for  Use  of  Viral  Isolation,  Identifica- 
tion, and  in  Serological  Tests  for  Viral 
Antibodies  8 

94-B  -  Studies  of  Arthropod-Borne  Viruses  in  Tissue 
Culture.   Part  2.   Development  of  a  Cell  Cul- 
ture System  Utilizing  Arthropod  Tissue  11 

95-A  -  Studies  on    Antigen- Antibody  Reactions  of 
Arthropod-Borne  Viruses.   Part  1.   Kinetic 
Studies  of  the  Serum  Neutralization  of  the 
Arthropod-Borne  Virus  13 

95-B  -  Studies  on  Antigen- Antibody  Reactions  of 

Arthropod-Borne  Viruses.   Part  2.   Develop- 
ment of  a  practical  and  Specific  Flocculation 
Test  for  the  Demonstration  of  Arthropod -Borne 
Virus  Antibodies  15 

95-C  -  Typing  of  Viruses  by  Combinations  of  Antiserum 
Pools.   Application  to  Typing  of  Arthropod- 
Borne  Viruses 17 

96-C  -  Survival  Potential  of  the  Adult  of  Haemagogus 

Equinus,  A  Sylvan  Vector  of  Yellow  Fever  19 

97-A  -  A  Qualitative  Evaluation  of  Experimentally 
Induced  Eastern  Equine  Encephalomyelitis 
(EEE)  Virus  Infection  in  Horses  22 

97-B  -  The  Development  of  an  Inactivated  Vaccine 

Against  Eastern  Equine  Encephalomyelitis  (EEE) 

Virus 24 

100-A  -  Virological  Investigation  of  Clinical  Cases 
and  Epidemic  Outbreaks  in  Panama  and  Other 
Countries  of  Middle  America 27 

100-B  -  A  Clinical  and  Virological  Study  of  Oro- 
pharyngeal Lesions  in  Panamanian  Children  33 

101-A  -  Virological  Aspects  of  a  Cooperative  Investi- 
gation on  the  Ecology  cf  Aithropod-Borne 
Viruses  35 


101-B  -  The  Role  of  Chlggers  and  Other  Acarlna  of 
the  American  Tropics  in  the  Maintenance 
and  Transmission  of  Animal  Infectious 
Agents:   1.  Viral  Aspects  39 

101-C  -  "Jungle  Fever"  in  U.S.  Military  Personnel  41 

102-A  -  Enterovirus  Infections  of  Rural  Guatemalan 

Children  in  Relation  to  Nutrition  43 

102-B  -  Laboratory  Support  of  Phase  1,  National  Program 
for  Poliovirus  Vaccine  Administration  in  Costa 
Rica,  1959  45 

102-C  -  Enterrovirus  Flora  of  Panamanian  Children:   A 

Twe  lve  Month  Survey  48 

103-A  -  Use  of  Filter  Paper  Discs  for  Virus  Isolation 

and  Serological  Testing  50 

103-B  -  Eastern  Equine  Encephalomyelitis  (EEE)  Virus 

Infection  in  Panama  53 

103-C  -  Encephalomyocarditis  (EMC)  Virus  Infection. 
Studies  on  Pathogenesis  in  Swine,  Virus 
Reservoirs  in  Rodents  and  Antibody  Status  of 
Human  and  Animal  Populations  55 

108  -  Studies  of  Histoplasmosis  on  the  Isthmus  of 

Panama  59 

109  -  Studies  of  Superficial  and  Deep  Mycoses  in 

Panama  and  Central  America  63 


II 


PHS-NIH 

Summary  Statement 

Office  of  Chief 

Laboratory  of  Tropical  Virology 

Calendar  Year  i960 

I.   ADMINISTRATIVE  ASPECTS 

The  second  year  of  the  Laboratory's  administrative  existence  was  event- 
ful and  stormy.   Activities  of  the  Arthropod  Borne  Virus  Section  (ABVS)  in 
Bethesda  have  been  affected  by  the  uncertainties  in  connection  with  Dr.  William 
Pond's  resignation  from  the  position  of  Section  Head  and  Assistant  Chief, 
LTV.   Difficulties  were  compounded  by  the  resignation  of  Dr.  Herbert  T.  Dalmat, 
who  temporarily  was  Acting  Head  of  the  Section.   Mr.  Clarence  J.  Gibbs,  Jr. 
then  became  Acting  Head  under  difficult  administrative  circumstances  aggra- 
vated by  loss  of  several  technicians  and  the  usual  problems  in  communications 
with  Office  of  the  Chief  physically  located  in  Panama.  The  Section  has  weath- 
ered the  storms,  carried  on  the  active  research  program  and  is  now  anticipat- 
ing assignment  of  replacements  for  Drs.  Pond  and  Dalmat. 

As  all  other  NIH  components  in  Bethesda,  the  ABVS  is  plagued  with  a 
shortage  of  space.   Besides  a  modest  area  allotment  in  square  feet,  the  lab- 
oratory is  located  in  a  somewhat  uninviting  basement  of  Building  5.   Sched- 
uled expansion  into  several  rooms  finally  vacated  by  the  Division  of  Biologic 
Standards  will  alleviate  the  work  space  shortage.  However,  such  plans  as 
the  creation  of  a  third  section  of  LTV  to  be  located  in  Bethesda  (proposed  a 
year  ago)  are  hardly  realistic. 

The  Panama  Laboratory  has  also  had  space  problems  -  but  in  reverse: 
for  the  past  ten  months  the  headaches  were  due  to  planning  and  execution  of 
reconstruction  to  convert  some  of  the  ample  space  into  a  functional  research 
laboratory  and  supporting  service  areas.  Although  the  job  is  not  finished, 
sometime  early  in  1 96 1  the  virus  research  area  will  be  more  than  doubled. 
Especially  needed  are  the  several  isolation  cubicles  constructed  on  the  second 
floor  and  provision  of  air-conditioned  desk  space  for  Investigators  near 
their  own  working  areas. 

With  clarification  of  the  MARU  mission  and  commitment  to  several  import- 
ant projects  there  has  been  a  commensurate  gradual  increase  in  personnel, 
particularly  at  the  sub-professional  level. 

The  next  calendar  year  will  bring  many  more  changes  in  the  Laboratory 
of  Tropical  Virology.  At  MARU  there  will  be  a  new  Director,  a  new  Head  of 
Virus  Section  and  a  new  Head  of  Mycology  Section;  in  Bethesda,  a  new  Head 
of  the  Section  and  physically  relocated  Laboratory  Chief  from  Panama.   Un- 
doubtedly, these  factors  will  affect  administration  as  well  as  the  research 
program  of  the  laboratory. 


I.   RESEARCH  HIGHLIGHTS 

MARU.  C.  Z.  -  VIRUS  SECTION 

1 .  Virus  Isolates  from  Panamanian  Mosquitos  and  Sandflies 

During  the  first  12  months  of  a  3-year  project  on  the  ecology  of 
arthropod  borne  viruses  in  the  tropical  rain  forest,  which  is  being  con- 
ducted by  GML  with  the  collaboration  of  MARU,  major  emphasis  has  been 
on  virus  isolation  in  suckling  mice  and  hamster  kidney  cell  cultures. 
Fourteen  virus  strains  were  isolated  at  MARU  from  412  pools  and  63,000 
specimens  provided  by  GML.   Virus  isolation  rates  were  for  Phlebotomus 
1:700  and  for  mosquitoes  1:7000,  although  the  rates  varied  greatly  with 
species.   Of  the  five  Phlebotomus  isolates,  two  of  broad  host  range 
(including  cell  culture)  and  short  incubation  period  are  serologically 
identical.   These  viruses  have  now  been  identified  as  the  Indiana  type 
of  vesicular  stomatitis  virus.   The  other  three  phlebotomus  and  nine 
mosquito  viruses  are  being  related  to  each  other,  to  known  virus  groups 
and  to  human  and/or  animal  infection  and  disease. 

2.  Eastern  Equine  Encephalomyelitis  Virus  Infection  in  Panama 

The  prevalence  of  EEE  antibodies  in  horses  and  man  in  two  areas  of 
suggested  EEE  virus  endemicity  has  been  determined,  allowing  an  evalua- 
tion of  the  relative  usefulness  of  several  serological  methods  applic- 
able to  studies  of  this  type.   It  was  found  that  the  incidence  of  EEE 
antibodies  in  460  humans  tested  increased  with  advancing  age  (0.8%  under 
10  years  with  progressive  increase  to  9%  in  the  41-50  year  group). 
Complement  fixation  results  on  the  same  sera  indicated  the  probable  pre- 
sence of  other  group  A  viruses. 

Lizards  of  species  common  to  this  part  of  Panama  were  examined  as 
a  possible  virus  reservoir.  Specific  EEE  virus  hemagglutination- 
inhibitors  were  found  in  some  of  their  sera.  The  occurrence  of  viremla 
and  HI  antibody  response  following  virus  inoculation  were  experimental ly 
confirmed  by  inoculation  of  lizards. 

3.  Etiology  of  "Jungle  Fever" 

About  200  paired  specimens  were  collected  from  military  students 
participating  in  jungle  warfare  training  courses  in  the  Canal  Zone. 
From  one  of  the  specimens  obtained  during  an  episode  of  fever  following 
known  exposure  to  jungle  environment,  a  virus  was  isolated.  This  agent 
as  yet  has  not  been  related  to  viruses  known  to  be  active  on  the  Isthmus 
of  Panama. 

4.  Encephalomyocard? tis  Virus  Infection 

Previously  this  laboratory  described  an  outbreak  of  a  fatal  disease 
of  swine  caused  by  the  EMC  virus.  The  outstanding  lesion  in  pigs  dying 
during  the  outbreak  was  acute  myocarditis.   Since  epidemiological  obser- 


vations  suggested  that  natural  infection  resulted  from  ingestion  of  con- 
taminated food,  experiments  were  undertaken  to  reproduce  the  disease  by 
feeding  virus  to  young  pigs.   Viremia  and  virus  excretion  from  the  gas- 
trointestinal tract  were  found  to  occur  following  the  administration  of 
brain  from  EMC  inoculated  mice.   Infected  pigs  developed  high  titers  of 
HI  and  neutralizing  antibody  during  convalescence  and  had  myocardial 
fibrosis  at  autopsy.   Other  studies  included  demonstration  of  EMC  anti- 
bodies In  a  small  number  of  city  rats  and  rats  caught  on  the  affected 
farm,  although  wild  rodents  were  found  to  be  negative.   Human  sera  were 
examined  with  interesting  differences  in  the  results  depending  on  the 
donors*  age:  while  a  substantial  proportion  of  the  Panamanian  population 
has  been  infected  with  EMC  virus,  the  antibodies  were  found  to  be  more 
common  in  persons  of  younger  age. 

5.  Enterovirus  Flora  in  Children  of  Central  America 

For  a  period  of  12  months  the  enterovirus  flora  of  Infants  at  an  out- 
patient clinic  in  Panama  City  was  systematical ly  explored  establishing  a 
base  line  of  enterovirus  fluctuation.  The  majority  of  viruses  isolated 
belonged  to  the  ECHO  group,  although  in  late  1959  and  early  I960  polio- 
virus  type  2  had  become  very  prevalent.  This  was  reflected  in  an  un- 
common occurrence  of  a  small  outbreak  of  paralytic  disease  due  to  type 
2  pol iovirus. 

Other  enterovirus  studies  have  Included  1)  surveillance  for  the  pre- 
sence of  type  1  pol iovirus  in  Panama  in  late  I960  as  a  check  on  dissemina- 
tion and  threatened  spread  of  this  commonly  epidemic  type,  2)  studies  on 
a  major  epidemic  of  Echo-9  virus  which  swept  through  the  Republic  of 
Panama  and  the  Canal  Zone  and  3)  initiation  of  a  collaborative  project 
on  possible  relation  of  enterovirus  flora  of  Guatemalan  children  to 
their  dietary  status. 

6.  Mite  Virus  Project 

Ors.  J.  M.  Brennan  and  C.  E.  Yunker  of  Rocky  Mountain  Laboratory 
staff  have  been  assigned  to  LTV  component  in  Panama  to  conduct  a  two- 
year  study  on  the  possible  role  of  chiggers  and  other  Acarina  of  the 
American  tropics  in  the  maintenance  and  transmission  of  animal  viruses. 
To  our  knowledge  this  is  the  first  serious  attempt  to  explore  this  im- 
portant area. 

MYCOLOGY  SECTION,  MARU 

The  research  program  of  the  Section  has  markedly  increased  local 
awareness  of  histoplasmosis  in  all  of  its  clinical  forms,  as  evidenced 
by  recognition  of  three  disseminated  cases  (2  fatal  and  one  success- 
fully treated)  within  a  period  of  18  months  (until  then  only  one  fatal 
case  had  been  described  since  Darling's  original  cases  in  1906) . 
Ecological  and  epidemiological  studies  led  to  isolation  of  H.  capsulatum 
from  eight  additional  soil  samples  bringing  up  to  sixteen  the  total 
number  of  recent  isolations  from  Panamanian  soil  (while  only  a  single 


positive  soil  sample  was  recorded  until  this  Section  was  established). 
The  fungus  has  been  repeatedly  recovered  from  the  organs  of  trapped 
ground  mammals  confirming  its  wide  dissemination  in  nature. 

Histoplasmin  skin  test  continues  to  be  a  major  tool  for  the  study 
of  epidemiology  of  histoplasmosis.   Data  on  9,200  children  between  6 
and  19  years  of  age  have  been  obtained  indicating,  as  expected,  that 
the  percentage  of  reactors  increases  progressively  with  age.  The  rate 
of  histoplasmin  sensitivity  varies  from  13  to  58%  among  six-year  olds 
and  from  68  to  92%  among  19  year  olds,  depending  on  location  of  their 
residence.   A  survey  of  631  pre-school  children  (6  months  to  6  years) 
in  the  Canal  Zone  demonstrated  an  increase  in  hypersensitivity  beginning 
with  three  years  of  age.   A  continuing  similar  study  of  Panamanian  child- 
ren in  a  city  hospital  is  now  in  progress  with  information/over  800 
already  available. 

Projects  on  other  mycotic  diseases  have  included  diagnostic  study 
and  therapy  of  moniliasis,  found  to  be  a  major  superficial  mycosis  among 
both  indigenous  and  transient  population  in  the  tropics. 

LTV.  BETHESDA  -  ABV  SECTION 

In  spite  of  the  difficult  administrative  and  working  conditions, 
the  research  staff  pursued  the  several  important  projects  initiated 
during  the  preceding  calendar  year.   New  projects  Involved  an  interest- 
ing application  of  the  technique  of  antiserum  pool  combinations  to  typing 
of  arthropod  borne  viruses,  a  wealth  of  data  evaluating  experimentally 
produced  EEE  virus  infection  in.  horses  and  a  promising  attempt  to  develop 
an  inactivated  EEE  virus  vaccine  for  human  use.  The  Infected  horses 
yielded  specific  antiserum  which  is  being  processed  for  prophylactic  use 
in  cases  of  human  exposure  under  laboratory  or  natural  conditions. 

Accidental  laboratory  infection  of  a  staff  member  with  an  arthropod 
borne  group  C  (Apeu)  virus  led  to  the  first  cl inical-virological  study 
of  a  syndrome  produced  by  this  important  and  common  group  of  viruses  of 
the  western  hemisphere. 

III.   PERSONNEL 

ABVS.  Bethesda  -  Dr.  W.  L.  Pond  and  Dr.  H.T.  Dalmat  resigned 
during  second  half  of  the  year;  not  replaced  at  the  end  of  Calendar 


Year. 


Virus  Section,  MARU: 


Dr.  J.  E.  Craighead  and  Dr.  C.  G.  Dobrovolny  resigned  in  midyear. 
Dr.  Craighead's  position  at  MARU  is  now  occupied  by  Or.  E.  A.  Bruckner 
transferred  from  Bethesda  to  the  Canal  Zone.   Dr.  J.  V.  Ordonez,  staff 
member  of  Bacteriology  Section  of  Instituto  de  Nutricion  de  Centro 
America  y  Panama  (Guatemala)  began  a  one  year  fellowship  in  virology 
(under  the  sponshorship  of  Parke,  Davis  &  Co.)  in  July  i960.   Drs.  J.  M. 
Brennan  and  C.  E.  Yunker  transferred  from  RML  to  LTV-MARU  for  a  period 


of  2  years  to  initiate  a  project  on  the  role  of  Acarina  in  transmission 
of  infectious  diseases. 

Mycology  Section,  MARL); 

No  professional  personnel  changes.  The  Research  and  Development 
Command  of  US  Army  has  accepted  Mrs.  M.  Shacklette  and  Mr.  J.  Fuentes 
by  transfer  from  NIH  to  US  Army  Caribbean  payroll. 


Serial  No.  NIAID  -  93 


1.  Tropical  Virology 

2.  Arthropod-borne  Virus 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 


Part  A 


Project  Title:   Studies  on  arthropod-borne  viruses  in  the  sub- 
tropical AREAS  OF  THE  UNITED  STATES 

Principal  Investigator:  Robert  M.  Pennington 

Other  Investigators:   Clarence  J.  Gibbs,  Jr. 
William  L.  Pond 

Cooperating  Units:  University  of  Miami  School  of  Medicine 
Southwest  Blood  Banks,  Inc. 

Man  Years  (calendar  year  1960) 
Total:        2-3/4 
Professional:   1-1/4 
Other:  1/2 

Project  Description: 

Object ives: 

Testing  of  sera  from  human  beings  residing  in  the  Southern 
United  States  has  been  in  progress  to  establish  a  baseline  for 
the  interpretation  of  tests  used  in  investigations  of  viral  diseases 
in  non-temperate  climates.  Moreover,  these  tests  can  be  expected  to 
give  an  indication  as  to  which  arthropod-borne  viruses  are  present 
and  which  viruses  are  probably  absent  from  this  area. 

Methods  Employed: 

Neutralization,  hemagglutination  inhibition,  and  complement 
fixation  tests  of  the  arthropod-borne  viruses  are  being  carried 
out  on  sera  obtained  from  residents  of  subtropical  areas  of  the 
United  States. 

Major  Findings: 

Neutralization  and  hemagglutination  inhibition  tests  on  125 
sera  from  Miami  residents  indicate  activity  of  St.  Louis  enceph- 
alitis, encephalomyocardit is,  and  possibly  Ilheus  virus.  There 


Serial  No.  NIAID  -  93 


ARE  ALSO  STRONG  INDICATIONS  THAT  ANOTHER  GROUP  "B"  VIRUS  IS  ACTIVE  OR  HAS 


BEEN  ACTIVE  IN 
INVEST  IGATED. 


the  Miami  area.  The  identity  of  this  agent  is  being 


Significance  to  Program  of  the  Institute: 

Potential  and  actual  public  health  importance  of  arthropod-borne 
viruses  for  residents  of  the  Southern  United  States  should  be  determined 
as  part  of  investigations  of  tropical  viral  diseases. 

Proposed  Course  of  Project: 

Neutralization,  hemagglutination  inhibition,  and  complement  fixation 
tests  will  be  carried  out  on  sera  not  already  tested  and  the  serum 
collections  on  hand  will  be  augmented  with  additional  sera  from  other 
subtropical  areas  of  the  united  states. 


Part  B  included 


No 


Part  A 


Serial  No.  NIAID  -  94-A 

1 .  Trop ical  Virology 

2.  Arthropod-borne  Virus 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Project  Title:  Studies  of  arthropod-borne  viruses  in  tissue  culture. 
Part  1.  Evaluation  of  tissue  culture  systems  for  use 
in  viral  isolation,  identification,  and  in  serological 
tests  for  viral  antibodies. 

Principal  Investigator:   Charles  R.  Rosenberger 

Other  Investigators:  Walter  L.  Newton 

Cooperating  Units:   Laboratory  of  Germfree  Animal  Research, 
NIAID-118 

Man  Years  (calendar  year  1960) 
Total:       1-7/8 
Professional:   7/8 


Other: 


1 


Project  Description: 

Objectives: 

Tissue  culture  studies  are  being  applied  to  arthropod-borne 
viruses  to  (a)  develop  a  more  efficient  system  for  the  successful 

ISOLATION  OF  VIRUSES  FROM  VECTORS  AND  NATURALLY  INFECTED  HOSTS,  (b) 

aid  in  classification,  identification,  and  characterization  of  these 
viruses  through  development  of  new  techniques  utilizing  cell  cul- 
tures, and  (c)  provide  a  source  of  virus  material  suitable  for  use 
in  vaccine  development. 

Methods  Employed: 


:  studied  in  a 
l  and  human 
infection  and 


Propagation  and  cytopathogen ic ity  of  viruses  af 
variety  of  cell  cultures  prepared  from  selected  anim' 
tissues.  Maximum  sensitivity  of  cell  lines  to  virus 
proliferation  is  determined  by  type  and  degree  of  ci  opathic  changes 
as  well  as  by  titers  of  virus  obtained  in  cell  cultui  es  and  in  mice. 
Viruses  are  identified  and  classified  into  serological  groups  by 


Serial  No.  NIAID  -  94-A 


neutralizing,  with  specific  antiserum,  the  ability  of  the  virus  to 
cause  cytopathic  changes,  form  plaques,  or  produce  hemadsorption. 
Strains  of  virus  which  show  reduced  pathogenicity  in  animals,  greater 
growth  or  cytopathogen ic ity  in  cell  cultures,  or  other  desired 
characteristics,  are  selected  by  serial  passage  in  cell  cultures  or 
by  selecting  individual  virus  particles  through  plaque  techniques. 

Major  Findings; 

Hamster  kidney  cell  cultures  (HKTC)  have  been  found  to  be  partic- 
ularly USEFUL  IN  PROPAGATING  ARTHROPOD-BORNE  VIRUSES.   PRESENTLY,  MORE 
THAN  20  OF  THESE  VIRUSES  HAVE  BEEN  GROWN  AND  OBSERVED  TO  PRODUCE 
CYTOPATHIC  CHANGES  IN  THIS  TYPE  CELL  LINE. 

A  CONTAMINATING  NON-VIRAL  ORGANISM  WAS  ISOLATED  FROM  APPARENTLY 

normal  HKTC.  This  organism  appears  to  be  a  bacterial  L  form.   It 
produces  a  "hemonuclear  adsorption  reaction1'  in  cell  cultures  which 
results  in  dissolution  of  the  cytoplasm  of  nucleated  chick  erythro- 
cytes leaving  nuclei  adsorbed  onto  cells  of  infected  cultures.  the 
organism  will  propagate  in  a  suspension  of  chick  erythrocytes  in 
balanced  salt  solution.  the  "hemonuclear  adsorption*.'  phenomenon  has 
proven  to  be  reliable  and  is  used  routinely  in  our  laboratory  as  a 
test  to  detect  the  presence  of  this  organism  in  cell  cultures. 

in  collaboration  with  dr .  walter  l.  newton,  laboratory  of  germ- 
free  Animal  Research,  many  cell  lots  have  been  prepared  from  germ- 
free  animals.  Comparative  tests,  with  kidney  cell  cultures  prepared 
from  germfree  and  conventional  mice,  have  not  shown  differences  in 
growth  or  cytopathogen ic ity  with  the  following  viruses:   yellow  fever 
(French  neurotropic  strain),  Anopheles  A,  Murray  Valley  encephalitis, 
and  Oriboca.  Previously  reported  preliminary  results  of  greater 
cytopathogen  ic  ity  with  dengue  type  1  virus  (moch i zuk i  strain)  in 
kidney  cells  prepared  from  germfree  mice  than  in  cells  from  conven- 
tional mice  have  not  been  consistent  on  additional  investigations. 

Significance  to  the  Program  of  the  Institute: 

These  studies  shall  provide  additional  methods  for  more  success- 
ful isolation,  identification,  and  classification  of  ARBOR  viruses. 
They  will  facilitate  investigations  into  the  mechanism  of  virus-cell 
interrelationships. 


Serial  No.   NIAID  -  94-A 

Proposed  Course  of  the  Project: 

The  study  or  ARBOR  viruses  in  tissue  culture  systems  will  con- 
tinue WITH  GREATER  UTILIZATION  OF  PLAQUE  TECHNIQUES. 

a  study  of  the  bunyamwera  group  of  viruses  has  been  initiated. 
Cell  line  susept ib i l ity,  growth  characteristics,  plaque  production, 
and  antigenic  relationship  between  the  viruses  of  this  group  will  be 
investigated.  these  studies  will  provide  definitive  tests  for  the 
identification  and  classification  of  these  viruses. 


Part  B  included  -  No 


10 


Serial  No.   NIAID  -  94-B 


1  .   TROP ICAL  VlROLOGr 

2.  Arthropod-borne  Virus 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 


Part  A 


Project  Title:  Studies  of  arthropod-borne  viruses  in  tissue  culture, 
Part  2.  Development  of  a  cell  culture  system  utili- 
zing ARTHROPOD  TISSUE. 

Principal  Investigator:  Charles  R.  Rosenberger 

Other  Investigators:   None 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960) 


Total: 

1/8 

Profess ional: 

1/8 

Other: 

None 

Project  Description: 

Objectives: 

To  develop  methods  of  preparing  cell  cultures  from  arthropod 
tissues.  To  provide  a  sensitive  and  more  suitable  cell  culture 
system  for  physiological  studies  of  arthropod-borne  virus  cell-host 
interact  ions. 

Methods  Employed: 

Specific  tissues  are  dissected  from  arthropods  and  prepared  as 
cell  cultures.  the  blood  of  the  arthropod  is  sometimes  collected 
and  added  to  the  culture  media.  various  culture  media  and  culture 
techniques  are  used  in  attempts  to  provide  a  system  suitable  for  a 
particular  tissue.  certain  arthropods  are  reared  under  sterile 
conditions  to  eliminate  contaminating  organisms  when  cell  cultures 
are  prepared. 

Major  Findings: 

Treatment  of  silkworm  ovarian  tissue  with  a  balanced  salt  solu- 
tion EXTRACT  OF  THE  CROP  OF  THE  BLUE  CRAB  CAUSES  D I SASSOC  I  AT  I  ON  OF 

cells.  This  treatment  provides  a  suspension  of  cells  rather  than 

TISSUE  FRAGMENTS  OR  CLUMPS  OF  CELLS.    In  CULTURE,  CELLS  ATTACH  TO  THE 
GLASS  SUBSTRATUM  BUT  FAIL  TO  PROLIFERATE  IN  WYATT'S  MEDIUM  WHEN  HELD 
AT  A  VARIETY  OF  TEMPERATURES  OF  INCUBATION. 


11 


Serial  No.  NIAID  -  94-B 

Significance  to  the:  Program  or  the  Institute; 

Cell  cultures  of  arthropod  tissues  would  provide  a  completely 
new  approach  for  studies  of  viruses,  host  cells,  and  their  inter- 
ACTIONS.  In  the  field  of  arthropod-borne  virus  research,  insect 

CELL  CULTURE  MAY  PROVIDE  A  SENSITIVE  AND  MORE  SUITABLE  CULTURE 
MED IUM. 

Proposed  Course  of  the  Project: 

Basic  studies  in  the  field  of  cell  culture  of  arthropod  tissues 
will  be  continued.  the  possibility  of  maintaining  tissue  and  cell 
suspension  type  cultures  prepared  from  mosquitoes  reared  under 
sterile  conditions  will  be  investigated. 


Part  B  included  -  No 


12 


Serial  No.  NIAID  -  95-A 


1 .  Trop ical  Virology 

2.  Arthropod-borne  Virus 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   Studies  on  antigen-antibody  reactions  of  arthropod- 
borne  viruses.  Part  1.  Kinetic  studies  of  the  serum 
neutralization  of  arthropod-borne  viruses. 

Principal  Investigator:   Robert  M.  Pennington 

Other  Investigators:  William  L.  Pond 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960) 

Total:         1 
Professional:   1/2 
Other:        1/2 

Project  Description: 

Object i ves: 

To  investigate  the  variables  of  the  neutralization  test. 
Specifically  time  (as  an  independent  variable)  and  the  relative 
degree  of  reaction  (amount  of  virus  neutralization)  due  to  prior 
incubation  of  the  virus-serum  reactants.  Such  information  is 
expected  to  give  insight  into  the  kinetics  of  virus-antibody 
combination.  The  rate  of  reactivity  of  the  virus-antibody 
systems  may  indicate  if  the  instantaneous  reaction  of  homologous 
bacterial  antigen-antibody  systems  is  also  applicable  to  homologous 
virus-antibody  systems. 

Methods  Employed: 

The  rapid  in  vitro  combination  of  virus  with  homologous  antibody 
and  the  comparatively  slow  speed  of  reaction  (avidity)  with  heter- 
ologous ANTIBODY  HAS  BEEN  SUCCESSFULLY  APPLIED  TO  MARKEDLY  INCREASE 

the  specificity  of  the  arthropod-borne  virus  neutralization  tests. 
Project  type  serological  tests,  results  of  which  are  un  interpretable 
because  of  broad  cross-reactive  antigenic  relationships,  were 
studied  by  kinetic  neutralization  tests. 


13 


Serial  No.  NIAID  -  95-A 

Major  Findings; 

The  concept  of  "instantaneous"  neutralization  of  viruses  with 
homologous  antibody  was  tested  using  group  "b"  moderately  reactive 
sera  obtained  from  residents  of  guatemala  and  group  "b"  broadly 
reactive  sera  obtained  from  indigenous  residents  of  southeast  asia. 

the  data  indicate  that  the  neutralization  of  virus  by  homologous 
antibody  is  an  instantaneous  phenomenon.  the  guatemalan  sera,  as 
expected,  due  to  their  moderate  cross  reactivity,  as  demonstrated  in 
the  conventional  neutralization  test,  were  differentiated  by  neutral- 
ization index  using  the  "mod  i  f  l_edm  neutralization  test  ( no  incubation 
of  the  virus-serum  reactants)  /.discussed  in  this  reporj/.  the  southeast 
Asian  sera  however,  remained  broadly  cross  reactive,  although  to  a  lesser 
degree  in  the  modified  neutralization  test  than  in  the  conventional 
neutralization  test. 

Significance  to  Program  of  the  Institute: 

The  kinetic  neutralization  studies  have  shown  that  the  specificity 
of  the  mouse  neutralization  test  may  be  increased  by  eliminating  the 
prior  incubation  of  virus-serum  reactants.  wlth  cross  reactivity  at  a 
minimum,  a  specific  virus  may  be  identified  as  causing  a  once  broadly 
cross  reactive  serum  antibody.  this  study  demonstrates  that  the  basic 
phenomenon  of  instantaneous  homologous  antigen-antibody  combinat-ion 
existing  in  bacterial  antibody  systems  functions  in  the  same  manner 
with  virus  antibody  systems. 

Proposed  Course  of  Project: 

The  instantaneous  neutralization  of  viruses  with  homologous  and 
heterologous  antibody  is  to  be  studied  using  sera  on  hand  collected  from 
human  beings  residing  in  the  miami  area.  these  studies  will  be  enlarged 
to  include  additional  serological  tests  designed  to  elicit  serological 

SURVEY  SPECIFICITY.    In  ADDITION,  ANIMAL  SERA,  PREPARED  BY  SINGLE  AND  DUAL 
INFECTIONS,  WILL  BE  STUDIED  TO  DETERMINE  THE  CORRELATION  OF  SEROLOGICAL 
TEST  RESULTS  OBTAINED  WITH  THE  MlAMI  SERA  AND  THE  LABORATORY  ANIMAL  SERA. 


Part  B  included  -  No 


14 


Serial  No.  NIAID  -  95-B 


1 .  Trop ical  Virology 

2.  Arthropod-borne  Virus 

3.  Bethesda,  Maryland 


PHS-NIH 
ndividual  Project  Report 
Calendar  Year  1960 


Part  A 


Project  Title:   Studies  on  antigen-antibody  reactions  of  arthropod- 
borne  viruses.  Part  2.  Development  of  a  practical 
and  specific  flocculation  test  for  the  demonstration 
of  arthropod-borne  virus  antibodies. 

Principal  Investigator:   Robert  M.  Pennington 

Other  Investigators:   None 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960) 
Total:         1/2 
Professional:   1/2 
Other:        None 

Project  Description: 

Object  i  ves: 

Development  of  a  rapid  flocculation  test  for  the  detection  of 
arthropod-borne  virus  antibodies  and  its  evaluation  as  a  worthwhile 
laboratory  procedure.        ' 

Methods  Employed: 

Arthropod-borne  viruses,  contained  in  cell  culture  supernatant 
fluids,  are  adsorbed  onto  clay  particles  in  the  manner  previously 
described  by  bozicevich  of  the  laboratory  of  clinical  investigations 
for  the  bentonite  flocculation  test  for  trichinosis  and  for  other 
diseases.  Flocculation  of  the  viral  ant igen-coated  Bentonite 
particles  is  demonstrated  in  the  presence  of  the  specific  viral 
ant ibody. 

Major  Findings: 

The  Bentonite  Flocculation  Test  using  Eastern  equine  encephalo- 
myelitis (EEE),  and  St.  Louis  encephalitis  (SLE)  viruses  as  proto- 
types AGAINST  HOMOLOGOUS  ANTISERA  HAS  GIVEN  SPECIFIC  HIGH  TITERED 
REACTIONS.    NO  CROSS  REACTIVITY  WAS  DEMONSTRABLE   IN  FLOCCULATION 


15 


Serial  No.   NIAID  -  95-B 


tests  with  related  group  "a"  sera  or  related  group  "b"  sera.  however, 
antibody  titers,  using  the  same  immune  sera  and  different  lots  of 
infective  hktc  fluid,  have  been  variable.  this  variability  is  most 
likely  a  function  of  virus  antigen  concentration  and/or  the  amount 
of  adsorption  of  virus  to  the  bentonite  clay  particles. 

Significance  to  the  Program  of  the  Institute; 

The  Bentonite  Flocculation  Test,  as  applied  to  the  arthropod- 
borne  VIRUSES,  MAY  CONSTITUTE  ANOTHER  POSSIBLY  VALUABLE  SEROLOGICAL 

test  to  be  used  in  conjunction  with  the  standard  complement  fixation, 
hemagglutination  inhibition,  and  neutralization  tests.  moreover, 
preliminary  observations  indicate  that  the  ant  i gen-benton  i te  combina- 
tion may  be  of  value  in  subsequent  adsorption  of  selected  antibody 
from  serum. 

Proposed  Course  of  the  Project; 

Evaluation  of  the  Bentonite  Flocculation  Test  (as  applied  to 
arthropod-borne  virus  systems)  in  terms  of  (1)  improved  antigen 
adsorption  to  Bentonite,  (2)  reproducible  results,  (3)  stability  of 
the  test  reagents,  (4)  improvements  in  the  methods  for  preparing  test 
reagents  and  techniques  of  the  test. 


Part  B  included  -  No 


16 


Serial  No.   NIAID  -  95-C 


1  .  Trop ical  Virology 

2.  Arthropod-borne  Virus 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   Typing  of  viruses  by  combinations  of  antiserum  pools. 
Application  to  typing  of  arthropod-borne  viruses. 

Principal  Investigator:   Clarence  J.  Gibbs,  Jr. 

Other  Investigators:   None 

Cooperating  Units:   None 

Man  Years:  (calendar  year  1960) 
Total:         1 
Professional:   1/2 
Other:        1/2 

Project  Description: 

Object i ves: 

The  purpose  of  this  investigation  is  to  develop  methods  by  which 
individual  tests  of  an  unknown  ARBOR  virus  against  a  number  of  typing 
sera  can  be  replaced  by  tests  against  a  small  number  of  pools  of 
these  sera.  the  serum  pools  must  yield  combinations  of  results 
specific  for  each  type  according  to  the  distribution  of  the  sera  in 
the  pools. 

Methods  Employed: 

Viruses  thus  far  employed  in  these  studies  are  prototype  strains 
of  representative  arthropod-borne  viruses  of  serological  groups  A,  B, 
and  C.  These  are  Eastern  equine  encephalomyelitis,  Japanese  B 

ENCEPHALITIS,  AND  APEU,  CARAPARU,  ORIBOCA,  MARITUBA,  MURUTUCU,  AND 
Itaqui  viruses.   In  ADDITION,  AN  UNKNOWN  VIRUS  ISOLATED  FROM  A 

laboratory  investigator  and  suspected  of  belonging  to  group  c,  was 
employed.  Without  exception,  the  antisera  employed  in  these  studies 
were  prepared  by  hyper  i mmun i z at  i  on  of  rabbits  with  mouse  adapted  virus 
strains.  Neutralization  tests  were  carried  out  by  intracerebral 
inoculation  of  5-7  day  old  suckling  mice.   In  all  tests  equal 
volumes  of  inactivated  undiluted  serum,  or  sometimes  sera,  were  mixed 
with  equal  volumes  of  varying  dilutions  of  viruses.  combination  serum 


17 


Serial  No.  NIAID  -  95-C 


POOLS  WERE  PREPARED  BY  MIXING  KNOWN  SPECIFIC  ANTISERA  ON  A  1:1  RATIO 
WITHOUT  REGARD  TO  QUANTITATIVE  LEVELS  OF  NEUTRALIZING  ANTIBODY. 

Combination  pools  of  group  C  antisera  were  prepared  on  the  basis  of 

serological  sub-groups.  controls  consisted  of  mixtures  of  equal 

volumes  of  virus  with  undiluted  normal  rabbit  serum  or  ]0%  normal 
rabbit  serum  in  borate-kcl  buffer  solution  at  ph  9.0. 

Major  Findings; 

Prototype  viruses  were  neutralized  to  a  greater  extent  when 
tested  against  their  homologous  antiserum  alone  or  against  pools 
which  contained  such  antiserum.  heterologous  neutralization  of 
prototype  viruses  occurred  only  between  members  of  the  same 
serological  group.  there  was  no  serological  crossing  over  between 
groups  a,  b,  or  c.  an  unknown  viral  isolate,  suspected  of  being 
apeu,  a  member  of  serological  group  c,  when  tested  against  combination 
serum  pools  was  neutralized  by  combination  of  group  c  antisera  but  not 
by  pools  of  groups  a  or  b.  furthermore,  pools  containing  apeu  anti- 
serum showed  greater  degrees  of  neutralization  than  did  pools  that  did 
not  contain  specific  apeu  antisera.  thus,  our  data  show  that  this 
method  can  be  applied  successfully  in  group  typing  of  an  unknown 
virus  if  the  virus  does  not  cross  react  to  any  great  extent  with 
heterotypic  antisera  and  if  the  potency  of  the  antisera  permits 
their  mutual  dilution  when  mixed  together. 

Significance  to  the  Program  of  the  Institute; 

the  method  described  in  this  report  reduces  the  number  of  tests 
that  have  to  be  done  in  identifying  and  classifying  an  unknown  virus. 
With  further  refinements,  it  should  allow  a  central  laboratory  to 
supply  other  laboratories  with  pools  of  antisera  representing  all 
serological  groups  of  arbor  viruses  made  and  tested  in  bulk.  without 
the  necessity  of  having  to  carry  stocks  of  specific  antisera  to  all 
types,  any  field  laboratory  can  type  an  unknown  virus  as  a  member  of 
a  serological  group.   it  will  facilitate  handling  of  epidemics  due  to 
arbor  viruses  as  the  group  reactivity  of  several  isolates  should 
point  to  where  emphasis  must  be  placed. 

Proposed  Course  of  the  Project; 

These  studies  are  to  be  continued  along  the  lines  described  and 

will  be  broadened  to  include  all  of  the  known  serological  groups  of 

arthropod-borne  viruses.  Methods  of  combining  specific  antisera  in 
order  to  provide  broadly  reactive  but  group  specific  pools  of 

ANTISERA  WILL  BE  DEVELOPED.   Th I S  TECHNIQUE  WILL  BE  EMPLOYED  AS  AN 

aid  in  classifying  and  further  identifying  unknown  viruses  submitted 
to  this  Section  for  study. 

Part  B  included  -  No 

18 


Serial  No.  NIAID  -  96-C 


1 .  Tropical  Virology 

2.  Arthropod-borne  Virus 

3.  Bethesda,  Maryland 


PHS-MIH 
Individual  Project  Report 
Calendar  Year  1960 


Part  A 


Project  Title:   Survival  potential  of  the  adult  of  Haemagogus  equinus, 
a  sylvan  vector  of  yellow  fever. 

Principal  Investigator:   Paul  A.  Woke 

Other  Investigators:   None 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960) 

Total:  1 
Professional:  1 
Other:        0 

Project  Description: 

Objectives: 

To  improve  the  opportunities  for  more  individuals  in  caged 
populations  of  adults  to  live  out  their  potential  life  spans, 
thus  providing  healthier  and  longer  lived  experimental  insects 
for  use  in  studies  on  the  interrelationships  between  viruses, 
vectors,  and  the  environment,  for  studies  on  the  spread  of  virus 
diseases  among  vertebrate  hosts  in  normal  situations  and  in 
studies  directed  toward  the  control  of  arthropod-borne  virus 
d  iseases. 

Methods  Employed: 

Haemagogus  equinus  is  used  as  the  experimental  species  for  which 
IT  IS  suited  by  reason  of  its  importance  as  a  vector  and  its  adapt- 
ability to  laboratory  experimental  conditions.  Populations  were  set 
up  under  conditions  for  survival  that  were  the  best  possible  at  the 
time.  The  mosquitoes  were  closely  observed  throughout  their  lifespans 
in  order  to  learn  all  preventable  causes  of  deaths.  Corrections  were 
applied  and  means  devised  by  which  to  reduce  and/or  eliminate  the 
current  causes  of  mortality.  The  influence  of  parental  age,  age  of 
eggs  at  the  time  of  hatching,  and  conditions  under  which  the  larvae 
were  reared  were  determined  by  trial.  survival  served  as  a  criterion 
of  suitability.  j  q 


Serial  NIAID  -  96-C 


Proposed  Course  of  the  Project: 

Continue  to  improve  conditions  for  the  maintenance  of  experimental 
stocks  of  haemagogus  equ  inus  and  to  apply  the  findings  to  other  species 
of  vectors  and  potential  vectors  of  arthropod-borne  diseases. 

Utilize  the  methods  and  findings  in  studies  on  the  interrelationships 
OF  viruses,  vectors,  and  the  environment. 

Apply  the  findings  in  studies  directed  toward  the  development  of 
control  measures  for  arthropod  vectors  of  virus  diseases. 


Part  B  included  -  No 


20 


Serial  No.  NIAID  -  96-C 

Major  Findings: 

Survival  of  Haemagogus  equinus  adults  is  greatest  in  those  popu- 
lations THAT  ARISE  FROM  EGGS  LAID  BY  YOUNG  FEMALES  FERTILIZED  BY  YOUNG 

males,  from  surviving  eggs  of  batches  that  have  been  held  for  periods 
of  time  up  to  near  the  maximum  periods  of  survival  under  adverse 
conditions,  and  from  larvae  that  developed  at  lower  temperatures,  and 
is  greatest  in  those  populations  of  adults  that  are  maintained  in 
varied  rather  than  constant  temperatures  and  humidities.  survival  is 
reduced  by  higher  temperatures,  by  excessive  activity  induced  by  high 
intensities  of  white  light,  and  by  air-borne  vaporized  oil  of  exceed- 
ingly low  concentration. 

Under  present  laboratory  conditions  the  70#  survival  point  for 
Haemagogus  equ  inus  adults  is  50  days  for  males  and  64  days  for  females; 
the  90#  survival  point  is  33  days  for  males  and  44  days  for  females. 
The  maximum  life  span  has  been  increased  from  11  days  for  males  and  39 
days  for  females  to  107  days  for  males  and  108  days  for  females.  known 
circumstances  indicate  that  the  survival  potentials  and  potential  life 
spans  are  still  above  these  values. 

Significance  to  the  Program  of  the  Institute: 

Haemagogus  equinus  adult  mosquitoes  can  now  be  chosen  for  labora- 
tory EXPERIMENTS  WITH  REASONABLE  CERTAINTY  OF  SURVIVAL  ACCORDING  TO  A 

predictable  distribution  when  maintained  within  a  certain  generally 
useful  set  of  conditions.  the  life  expectancy  is  such  as  to  permit  the 
completion  of  experiments  involving  all  normal  events;  the  survivorship 
distribution  probably  comes  near  to  the  potential  for  the  species. 
Long  survival  is  accompanied  by  health  and  vigor.  Needed  laboratory 
investigations  can  now  be  planned  intelligently  in  numerous  areas  on 
the  basis  of  the  known  lifespan  of  the  species  when  maintained  by  the 
methods  that  have  been  developed.  thus,  insects  cultured  according  to 
the  methods  now  available  are  suitable  for  laboratory  investigations  of 
virus-vector-environment  interrelationships  and  of  factors  important 
in  the  transmission  of  the  virus  and  spread  of  the  viral  disease,  and 
for  investigations  directed  toward  the  development  of  control  measures 
against  the  vector.  analytical  studies  of  the  effects  on  survival  of 
specific  environmental  factors  are  now  possible.   information  that  has 
been  and  can  be  derived  from  laboratory  experimentation  can  be  used  in 
field  studies  on  the  spread  of  arthropod-borne  virus  diseases  and  on 
means  by  which  to  control  the  vectors.  the  spread  of  viruses  and 
control  of  vectors  in  the  natural  habitat  are  influenced  by  the  envi- 
ronment, and  efficiency  as  a  vector  depends  in  part  on  longevity  of  the 
vector.  Methods,  experience,  and  information  which  has  been  gained  in 
this  study  of  Haemagogus  equ  inus  will  be  useful  in  similar  work  with 
other  vectors  of  arthropod-borne  viral  diseases. 


21 


Serial  No.  NIAID  -  97-A 


1 .  Trop ical  Virology 

2.  Arthropod-borne  Virus 

3.  Bethesda,  Maryland 


PHS-NIH 
ndividual  Project  Report 
Calendar  Year  1960 


Part  A 


Project  Title:   A  qualitative  evaluation  of  experimentally  induced 
Eastern  equine  encephalomyelitis  (EEE)  virus 

INFECTION  IN  HORSES 

Principal  Investigator:   Clarence  J.  Gibbs,  Jr. 

Other  Investigators:  William  L.  Pond 

Robert  J.  Byrne  (University  of  Maryland) 
Charles  R.  Rosenberger 

Cooperating  Units:  Grayson  Laboratory 

University  of  Maryland 

Man  Years  (calendar  year  1960) 
Total:  3 

Professional:   1-1/2 
Other:        1-1/2 

Project  Description: 

Objectives: 

An  immunological  investigation  of  experimentally  induced  EEE 
VIRUS  infection  in  horses  designed  to  (1)  elicit  the  formation, 
development,  and  persistence  of  viremia  and  hemagglut inat  ion- 
inhibiting  (HAI ),  complement  fixing  (CF)  and  neutralizing  (Neut) 
antibodies;  (2)  to  establish  a  reservoir  of  standardized  reference 
EEE  antiserum  for  use  in  immunological  and  serological  investiga- 
tions of  EEE  and  related  arthropod-borne  viruses. 

Methods  Employed: 

Each  of  three  horses  free  of  serologically  detectable  EEE  anti- 
bodies was  pre-bled  and  then  injected  with  10,000  MOUSE  intracerebral 
(IC)  LDc-n  doses  of  infectious  EEE  virus.  Following  inoculation,  small 
volume  BLEEDINGS  (50  ml.)  were  taken  on  the  first  6  days  and  large 

VOLUME  (500  ML.)  BLEEDINGS  APPROXIMATELY  EVERY  5  DAYS  THEREAFTER  FOR 
A  TOTAL  OF  50  DAYS.   AFTER  DAY  50,  BLEEDINGS  WERE  PERFORMED  AT  90 
DAY  INTERVALS.   BLEEDINGS  ON  EACH  OF  THE  FIRST  6  DAYS  WERE  PROCESSED 


22 


Serial  No.      NlAID  -  97-A 


IMMEDIATELY  FOR  VIREMIA  STUDIES.   SUBSEQUENT  BLEEDINGS  WERE  PROCESSED 
FOR  SERUM  24  HOURS  AFTER  STORAGE  ON  THE  CLOT  AT  4-8  C.   VlREMIA 
DETERMINATIONS  WERE  PERFORMED  ON  A  COMPARATIVE  BASIS  IN  ONE  DAY  OLD 
CHICKS,  HAMSTER  KIDNEY  CELL  CULTURES,  AND  IC  IN  3  DAY  OLD  AND  21  DAY 
OLD  MICE  TO  ELICIT  THE  BEST  SYSTEM  TO  BE  USED  IN  FIELD  ISOLATIONS  OF 

EEE  virus.  HAI,  CF,  and  Neut  tests  are  being  carried  out  on  all 
serum  samples  collected. 

Major  Findings: 

Following  the  exposure  of  horses  to  EEE  virus,  viremia  is 
detectable  within  24  hours  and  persists  at  a  significant  level 
for  72  hours.  The  1  day  old  chick  is  the  most  efficient  system 
for  the  detection  of  eee  virus  in  the  horse  blood.  significant 
levels  of  hai  antibody  are  present  on  the  10th  day  after  inoculation 
(1:160,  1:80,  1:80)  and  are  maintained  at  a  high  level  30  days  post- 
inoculation  (1:1280,  1:640,  1:320).  Complement-fixing  antibodies  are 
detectable  10  days  after  inoculation  (1:4,  1:256,  1:8)  and  reach  a 
maximum  level  between  days  30  and  35  (1:1024,  1:2048,  1:1024).  High 
levels  of  cf  antibodies  are  detectable  through  day  50.   neutralizing 
antibodies  also  are  detectable  at  a  significant  level  10  days  after 
inoculation  (logs  protection  1.3,  2.0,  1.7),  reach  a  high  level 
between  the  21 and  30 days  (2.5,  3.8,  3.0)  and  persist  through 

THE  204 DAY  AFTER  I NOCULAT  I  On(3  .4,  3.6,  2.7). 

Significance  to  Program  of  the  Institute: 

This  study  is  providing  detailed  serological  and  immunological 
data  of  EEE  virus  infection  in  horses.  The  North  American  enceph- 

ALITIDES  HAVE  BEEN  CAPABLE  OF  PRODUCING  EXPLOSIVE  EPIDEMICS  IN 
ANIMALS  AND  HUMAN  BEINGS;  E.G.,  MASSACHUSETTS,  NEW  JERSEY,  AND 

Panama,  with  high  mortality  in  animals  and  men  infected  with  EEE 
virus.  After  being  properly  checked  for  safety  and  sterility,  the 
serum  obtained  during  this  study  will  provide  a  post-exposure 
prophylactic  serum  for  inoculation  of  personnel  exposed  to  the  virus. 
It  also  provides  the  Public  Health  Service  with  standardized  reference 
antiserum  as  part  of  arbor  virology  investigations  throughout  the 

WORLD. 

Proposed  Course  of  Project: 

It  is  intended  that  HAI,  CF,  and  neutral i zat ing  antibody  persis- 
tence WILL  BE  FOLLOWED  ON  A  CONTINUING  BASIS  IN  AT  LEAST  ONE  OF  THE 
THREE  HORSES.    In  ADDITION,  THE  CRITICAL  NEED  FOR  STANDARDIZED 
REFERENCE  POLYVALENT  ANTISERUM  NECESSITATES  INOCULATION  OF  EEE  IMMUNE 
HORSES  WITH  OTHER  GROUP  A  VIRUSES  IN  ATTEMPTS  TO  BROADEN  THE  SPECTRUM 
OF  SEROLOGICAL  REACTIVITY. 


Part  B  included  -  No 


23 


Serial  No.  NIAID  -  97-B 


1  .  Trop ical  Virology 

2.  Arthropod-borne  Virus 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 


Part  A 


Project  Title:   The  development  of  an  inactivated  vaccine  against 
Eastern  equine  encephalomyelitis  (EEE)  virus. 

Principal  Investigator:   Clarence  J.  Gibbs,  Jr. 

Other  Investigators:   Charles  R.  Rosenberger 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960) 
Total:       1-1/2 
Professional:   1 
Other:       1/2 

Project  Description: 

Object  i  ves: 

The  purpose  of  this  study  is  to  develop  an  ant igen ically  potent 
and  safe  inactivated  vaccine  suitable  for  immunizing  animals  and 
human  beings  against  eee  virus  infection. 

Methods  Employed: 

Hamster  kidney  cell  cultures  (HKC)  are  inoculated  with  cell 
culture  propagated  eee  virus.   infected  cultures  are  maintained 
in  medium  199  free  of  serum  at  35  c.  until  a  viral  cytopathic 
effect  on  the  cells  of  3+  or  greater  (4+  =  100$  cellular  destruction) 
has  been  observed  microscopically.  the  supernatant  material, 
containing  virus  and  cellular  debris,  is  asept1cally  harvested, 
pooled,  and  clarified  by  centr  i  fugat i  on  at  an  r.c.f.  of  1 070xg .  in 
an  International  refrigerated  centrifuge.  The  supernatant  material 
is  separated  and  aliquots  removed  for  virus  infectivity  titration 
in  hkc  and  suckling  mice  as  well  as  for  bacteriological  sterility 
checks  on  blood  agar  plates  and  in  th  i ogl ycollate  broth.  the 
remainder  of  the  supernatant  material  is  formalinized  to  a  final 
concentration  of  0.1  percent  neutral  formalin  by  volume.  the 
formalinized  preparation  is  held  at  37  c.  for  72  hours  and  at  4-6  c. 
for  an  additional  6  days  prior  to  testing  for  viable  virus. 

2k 


Serial  No.  NIAID  -  97-B 


Safety  tests  consist  of  inoculating  400  gram  guinea  pigs  intra- 
cerebrally  (ic)  with  0.1  ml.  of  undiluted  vaccine.  our  safety 
requirements  also  include  ic  inoculation  of  8-10  gram  mice  with 
0.03  ml.  of  vaccine  undiluted  and  diluted  1:10,  1:100,  1:1000  with 
survival  of  all  animals  as  the  criterion  of  safety. 

Antigenic  potency  of  the  vaccine  is  determined  by  injecting  600 
gram  guinea  pigs  i ntradermally  with  two  0.1  ml.  doses  at  7  day 
intervals.  Fourteen  days  after  the  last  dose  of  vaccine,  animals 
are  challenged  IC  WITH  100-1000  LD,-0  doses  (per  0.1  ML.)  OF  AN  EEE 
STRAIN  DIFFERENT  FROM  THAT  USED  TO  PREPARE  THE  VACCINE.    In  ORDER  TO 

meet  the  minimum  requirements  of  the  bureau  of  animal  industry, 

Department  of  Agriculture,  at  least  2/3  of  the  vaccinated  guinea 

pigs  must  survive  the  challenge.  there  are  no  prescribed  minimum 
requirements  for  use  in  human  beings. 

Pre  and  post-vacc inat  ion  bleedings  are  done  on  all  test  animals 
to  determine  serologically  detectable  response  TO  THE  vaccine. 

Major  Findings: 

So  FAR,  TWO  LOTS  OF  EEE-HKC  VACCINE  HAVE  BEEN  PREPARED  AND 

assayed.  Our  data  show  that  neutral  formalin,  in  the  concentration 
employed,  is  capable  of  completely  inactivating  detectable  viable 
virus  without  destroying  the  antigenicity  of  the  product.  vaccinated 
animals  have  been  able  to  survive  ic  challenge  with  as  many  as  1000 
ic  guinea  pig  ldc0  doses  of  virus.  serological  data  show  the  vaccine 
to  be  capable  of  eliciting  neutralizing  antibodies  (log.  of  neut. 
indices  1.5  -  2.2),  but  that  hemagglut i  nat  i  on- i nh i b i t i ng  antibodies 
are  not  detectable.  tests  to  determine  complement  fixing  antibody 
response  have  not  been  done. 

Significance  to  the  Program  of  the  Institute: 

eee  virus  constitutes  a  serious  veterinary  and  human  public 
health  problem  in  many  areas  of  the  world.  the  virus  is  also 
hazardous  to  handle  in  the  laboratory.  no  licensed  eee  vaccine  is 
available  for  immunizing  human  beings  at  risk  in  the  laboratory  or 
in  an  epidemic  area  because  a  safe  and  potent  vaccine  has  not  been 
developed. 


25 


Serial  No.  NIAID  -  97-B 


Proposed  Course  of  the  Project: 

Additional  lots  of  vaccine  will  be  prepared  employing  EEE 

INFECTED  HKC  CULTURES  AS  THE  SOURCE  OF  ANTIGEN.    INASMUCH  AS 
FORMALIN  MAY  REDUCE  SOME  OF  THE  ANTIGENICITY  OF  THE  VACCINE, 
OTHER  METHODS  OF  INACTIVATION  WILL  BE  STUDIED;  E.G.,  ETHYLENE 
OXIDE  VAPORS  AND  INACTIVATION  BY  USE  OF  HIGH  INTENSITY  LIGHT 
SOURCES  (PHOTO  INACT I  VAT  ION)  OTHER  THAN  ULTRA-VIOLET.   STUDIES 
TO  DETERMINE  THE  EFFECTS  OF  FILTRATION  ON  THE  VACCINE  WILL  BE 
CARRIED  OUT.   ADDITIONAL  SAFETY  AND  STERILITY  STUDIES  ARE  PLANNED. 


Part  B  included 


No 


26 


offer  viral  diagnostic  services  to  the  community  and  public   health  agen- 
cies. 

Methods  Employed: 

Clinical  case  specimens  are  either  submitted  by  practicing  physicians 
or  public  health  officials  in  Panama,  the  Canal  Zone  and  the  neighboring 
countries,  or  are  collected  by  MARU  medical  personnel.   In  the  case  of 
epidemic  outbreaks  already  in  progress  (or  when  leads  of  epidemic  import- 
ance are  uncovered  in  the  course  of  testing  clinical  case  specimens)  ar- 
rangements for  field  activities  are  initiated  either  by  appropriate  of- 
ficials or  by  Director  of  MARU.   In  the  Republic  of  Panama  such  arrange- 
ments are  made  through  Director  of  Gorgas  Memorial  Laboratory  and  in  the 
Canal  Zone  through  Health  Director's  office.   For  other  Middle  America 
countries  two-way  communications  between  MARU  and  the  National  Governments 
are  always  through  Office  of  the  Representative,  Zone  1 1  I  of  Pan-American 
Sanitary  Bureau,  Pan-American  Health  Organization,  WHO. 

Laboratory  procedures  with  specimens  from  either  individual  clinical 
cases  or  from  epidemic  outbreaks  are  generally  the  same;  appropriate  cell 
cultures  (as  available)  and  laboratory  animals  are  inoculated  for  virus 
isolation,  while  serological  testing  of  blood  specimens  for  the  presence 
of  specific  antibodies  is  performed  by  standard  techniques. 

Major  Findings: 

1.  CI inical  Cases.  A  variety  of  viral  agents  has  been  recovered 
with  the  predominance  of  enteroviruses  (pol iovi ruses, Coxsackie  A  &  B, 
ECHO)  from  throat  and  rectal  swabs,  cerebrospinal  fluid  and  patients*  sera. 
Strains  of  myxoviruses,  adenoviruses  and  Herpes  simplex  have  also  been 
recovered.   In  many  cases  the  virus  isolates  were  related  to  the  clinical 
illness  by  serological  tests. 

2.  CA  Virus  Infection  in  Adults.   Croup-Associated  (parainfluenza  2) 
virus  infections  were  demonstrated  in  two  young  adults.   The  virus  was  re- 
covered from  throat  swabs  taken  at  the  time  of  acute  illness,  utilizing 
human  amnion  cultures  and  the  hemadsorption  test;  significant  rises  in  HI 
and  neutralizing  antibodies  during  convalescence  were  demonstrated.   The 
findings  indicate  that  CA  virus  can  cause  clinical  illness  in  adults. 

3.  Epidemic  Influenza,  1959.   Last  year's  report  referred  to  etiol- 
ogical studies  on  an  epidemic  of  influenza  in  Panama  due  to  influenza 
B-virus  and  a  major  epidemic  in  British  Guiana  during  the  same  months  of 
mid-1959  due  to  the  Asian  influenza  virus  (A2) •   A  report  describing 
these  findings  has  been  accepted  for  publication  in  the  Am.  ^J.  Trop.  Med. 
&  Hyg. 

k.      Viral  CNS  Disease  in  a  Guatemala  Nn-sery.   Last  year's  report 
referred  to  a  tragic  occurrence  of  fatal  pai  jlytic  poliomyelitis  cases 
and  many  aseptic  meningitis  cases  due  to  po  o  and  non-pol iovi ruses  in  a 
Guatemala  City  charity  nursery.   The  laboratory  findings  could  not  be 
fully  reported  at  the  time.  A  total  of  kh   <  iterovirus  strains  were   00 

Serial  No.  NIAID-100-A 


offer  viral  diagnostic  services  to  the  community  and  public   health  agen- 
cies. 

Methods  Employed: 

Clinical  case  specimens  are  either  submitted  by  practicing  physicians 
or  public  health  officials  in  Panama,  the  Canal  Zone  and  the  neighboring 
countries,  or  are  collected  by  MARU  medical  personnel.   In  the  case  of 
epidemic  outbreaks  already  in  progress  (or  when  leads  of  epidemic  import- 
ance are  uncovered  in  the  course  of  testing  clinical  case  specimens)  ar- 
rangements for  field  activities  are  initiated  either  by  appropriate  of- 
ficials or  by  Director  of  MARU.   In  the  Republic  of  Panama  such  arrange- 
ments are  made  through  Director  of  Gorgas  Memorial  Laboratory  and  in  the 
Canal  Zone  through  Health  Director's  office.   For  other  Middle  America 
countries  two-way  communications  between  MARU  and  the  National  Governments 
are  always  through  Office  of  the  Representative,  Zone  1 1  I  of  Pan-American 
Sanitary  Bureau,  Pan-American  Health  Organization,  WHO. 

Laboratory  procedures  with  specimens  from  either  individual  clinical 
cases  or  from  epidemic  outbreaks  are  generally  the  same;  appropriate  cell 
cultures  (as  available)  and  laboratory  animals  are  inoculated  for  virus 
isolation,  while  serological  testing  of  blood  specimens  for  the  presence 
of  specific  antibodies  is  performed  by  standard  techniques. 

Major  Findings; 

1.  CI inical  Cases.  A  variety  of  viral  agents  has  been  recovered 
with  the  predominance  of  enteroviruses  (pol iovi ruses, Coxsackie  A  £■  8, 
ECHO)  from  throat  and  rectal  swabs,  cerebrospinal  fluid  and  patients'  sera. 
Strains  of  myxoviruses,  adenoviruses  and  Herpes  simplex  have  also  been 
recovered.   In  many  cases  the  virus  isolates  were  related  to  the  clinical 
illness  by  serological  tests. 

2.  CA  Virus  Infection  in  Adults.   Croup-Associated  (parainfluenza  2) 
virus  infections  were  demonstrated  in  two  young  adults.   The  virus  was  re- 
covered from  throat  swabs  taken  at  the  time  of  acute  illness,  utilizing 
human  amnion  cultures  and  the  hemadsorption  test;  significant  rises  in  HI 
and  neutralizing  antibodies  during  convalescence  were  demonstrated.   The 
findings  indicate  that  CA  virus  can  cause  clinical  illness  in  adults. 

3.  Epidemic  Influenza,  1959.   Last  year's  report  referred  to  etiol- 
ogical studies  on  an  epidemic  of  influenza  in  Panama  due  to  influenza 
B-virus  and  a  major  epidemic  in  British  Guiana  during  the  same  months  of 
mid-1959  due  to  the  Asian  influenza  virus  (A2) .   A  report  describing 
these  findings  has  been  accepted  for  publication  in  the  Am.  ^J.  Trop.  Med. 
£•  Hyg. 

k.      Viral  CNS  Disease  in  a  Guatemala  Ni-sery.   Last  year's  report 
referred  to  a  tragic  occurrence  of  fatal  pai  jlytic  poliomyelitis  cases 
and  many  aseptic  meningitis  cases  due  to  po  o  and  non-pol iovi ruses  in  a 
Guatemala  City  charity  nursery.   The  labora'  >ry  findings  could  not  be 
fully  reported  at  the  time.  A  total  of  kk  >   iterovirus  strains  were   00 

Serial  No.  NIAID-100-A 


isolated  from  103  individuals  tested  during  five  months  of  the  study. 
Two  or  more  specimens  were  available  from  the  majority  of  affected  in- 
fants.  Twenty-two  attendant  nursing  personnel  were  found  to  be  negative 
for  any  enterovirus  isolations. 

From  the  two  initial  paralytic  fatal  cases  Type  1  poliovirus  strains 
were  isolated  from  several  sources  (in  one  -  from  the  throat  and  rectal 
swabs,  cerebrospinal  fluid  and  brain  sections,  and  in  the  other  from  se- 
veral areas  of  the  brain).   Two  nonparalytic  cases  occurring  during  the 
early  part  of  the  outbreak  also  yielded  Tl  poliovirus.   The  only  other 
Tl  isolate  was  from  an  infant,  undoubtedly  suffering  from  non-polio  viral 
meningitis,  who  was  infected  with  Tl  after  many  days  in  a  hospital  ward. 
No  strains  of  poliovirus  T2  were  found,  but  2  strains  of  poliovirus  T3 
and  3  strains  of  Coxsackie  A  were  isolated  from  asymptomatic  infants. 
The  remaining  3^  viruses  apparently  belonged  to  the  ECHO  group:  9  ECHO 
types  2,  11  and  12  and  17  which  were  either  untypable  or  were  not  typed. 
Work  on  the  remaining  8  strains,  recovered  during  post-epidemic  spread, 
was  abandoned. 

5.  Poliovirus  Type  2,  Panama.  A  small  outbreak  of  paralytic  polio- 
myelitis caused  by  T2  virus  was  investigated.   Of  the  15  clinical  cases, 
11  were  under  2k   months  of  age.   Type  2  viruses  were  recovered  from  12 
cases.   During  the  epidemic  (October  1959  to  March  I960)  a  survey  of  en- 
terovirus flora  in  Panamanian  children  was  conducted  as  part  of  another 
study.   It  indicated  a  wide  dissemination  of  the  virus  in  the  community. 
The  emergence  of  poliovirus  T2  during  the  past  two  years  as  an  epidemic 
virus  in  Central  America  will  be  closely  followed  by  MARU  staff. 

6.  ECH0-9  Virus  Epidemic,  Panama.   A  major  epidemic  of  ECH0-9  virus 
disease  swept  through  Panama  and  the  Canal  Zone  from  July  to  October  I960. 
To  date  isolations  have  been  made  from  20  patients.   In  these  clinical 
cases  ECHO  9  virus  was  isolated  most  commonly  from  cerebrospinal  fluid, 
then  throat  swabs  and  least  often  from  rectal  swabs. 

During  the  epidemic  (but  apparently  after  its  peak)  189  Panamanian 
children  at  the  Hospital  del  Nino  Outpatient  Clinic  were  surveyed  with 
throat  swabs  and  rectal  swabs  taken  from  all  and  98  venous  blood  speci- 
mens drawn  at  the  same  time  as  finger  puncture  blood  was  absorbed  on 
filter  paper  discs.   The  purpose  was  to  document  occurrence  of  ECHO  9 
virus  (and  possibly  pol iovi ruses)  and  to  study  the  practical  applications 
of  filter  paper  discs  as  a  serological  tool.   In  contrast  to  clinical 
cases,  the  virus  was  much  more  frequently  recovered  from  rectal  swabs 
than  from  throat  swabs.  Relevant  serological  results  with  filter  paper 
disc  eluates  are  discussed  under  a  separate  project  heading. 

7.  Measles  Epidemic,  Panama.   In  June  i960  the  Ministry  of  Health 
of  Panama  requested  MARU  assistance.   A  staff  medical  officer  accompanied 
two  Panamanian  physicians  in  a  U.S.  Air  Force  helicopter  to  Las  Barretas 
village  in  the  interior  of  Panama  where  a  serious  epidemic  of  measles 
had  occurred:  between  late  April  and  early  June  35  deaths  were  reported 
in  a  population  of  1 500.   During  the  2  days'  stay  the  sick  were  treated 
and  specimens  collected.  <^o 

Serial  No.  NIAID-100-A 


The  area  is  not  endemic  for  measles  and  the  last  epidemic  occurred 
in  1952.   Of  33  fatal  cases  whose  age  was  known,  27  were  seven  years  old 
and  less.   Forty-seven  sera  were  tested  for  complement  fixing  antibodies: 
The  majority  of  individuals  over  seven  years  of  age  with  no  history  of  a 
recent  characteristic  rash  had  the  CF  titer  of  less  than  1:4.   Studies 
by  others  In  areas  where  measles  is  endemic  revealed  that  CF  titer  per- 
sists in  a  fashion  similar  to  neutralizing  antibody  titers.   By  using 
the  neutralization  test  on  sera  from  communities  where  measles  has  been 
absent  for  known  intervals,  we  hope  to  clarify  the  immunological  status 
of  populations  in  non-endemic  areas. 

8.  Epidemic  Poliomyelitis,  Nicaragua.   A  severe  epidemic  of  polio- 
myelitis occurred  in  Nicaragua  in  late  December  1959  through  March  I960. 
Nicaraguan  public  health  authorities  and  the  Pan-American  Sanitary  Bureau 
invited  the  laboratory  participation  of  MARU.   Specimens  for  virus  isola- 
tion were  sent  to  both  MARU  and  the  Lederle  laboratories.   The  epidemic 
was  of  special  interest  since  Nicaragua  had  suffered  an  epidemic  of  un- 
precedented severity  due  to  poliovirus  Type  2  in  1958  and  most  of  the 
children  had  received  the  Lederle  live  poliovirus  vaccine.   Unfortunately, 
the  initial  campaign  concentrated  on  manovalent  T2  vaccine  with  much 
sparser  coverage  by  T3  and  Tl  vaccine  strains. 

Of  66  paralytic  cases  from  whom  specimens  were  received,  49  virus 
isolates  were  made  -  47  of  Tl  and  2  of  T2.   The  findings  were  reported 
to  the  Government  of  Nicaragua  and  the  Pan-American  Sanitary  Bureau. 
Representatives  of  both  of  these  agencies  made  a  report  on  the  joint  find- 
ings at  the  Second  International  Conference  on  Live  Poliovirus  Vaccines 
held  in  Washington,  D.  C.  in  June  i960. 

9.  Community  Spread  of  Poliovirus  Type  1,  Panama.   Following  the 
isolation  of  poliovirus  Tl  from  two  paralytic  cases  during  the  first  week 
of  November  I960,  a  survey  of  enterovirus  flora  was  conducted  in  Panama 
and  the  Canal  Zone.   From  children  at  four  Canal  Zone  locations  on  the 
Pacific  Coast  112  rectal  swab  specimens  were  collected  and  90  rectal  swabs 
were  procured  from  the  out-patient  clinic  of  Hospital  del  Nino. 

Testing  of  these  in  monkey  kidney  cell  cultures  failed  to  support  the 
suspicion  of  wide  dissemination  and  spread  of  Tl  poliovirus  strains  in 
the  community.  A  most  striking  finding  was  the  abundance  of  enteroviruses 
among  children  of  the  poorer  families  in  Panama  and  the  sparsity  of  isola- 
tions from  the  Canal  Zone,  regardless  of  the  community,  ethnic  background 
or  relative  income. 

10.  Another  Outbreak  of  Fatal  Swine  Disease,  Panama.  An  epizootic 
of  fatal  disease  at  a  major  commercial  pig  farm,  similarly  affected  two 
years  ago,  was  investigated  in  October  jointly  with  other  agencies.   The 
clinical  picture  was  different  from  the  EMC  syndrome  described  by  our 
group  for  the  I958  outbreak  (Science  131 :  498-499,  i960).   Fever,  lethargy, 
difficulty  in  standing  and  walking,  coughing  and  nasal  discharge,  labored 
breathing  and  diarrhea  were  now  common.   Miscellaneous  specimens  were 
collected  from  17  live  pigs;  organs  from  two  autopsied  animals.   Serum, 
rectal  swabs  and  a  few  urine  and  nasal  swabs  were  tested  for  virus  issjte- 

4  Serial  No.  NIAID-100-A 


lation  in  suckling  mice,  monkey  kidney  and  hamster  kidney  cell  culture 
tubes,  and  chick  embryo  cells  in  bottles.   No  virus  isolations  were  ac- 
complished in  these  systems.   Subsequently,  it  was  established  by  patho- 
logical and  bacteriological  studies  at  Laboratorio  Veterinario  and  Gorgas 
Memorial  Laboratory  that  this  was  an  outbreak  of  hog  cholera  (swine  fever) 
complicated  by  Pasteurel la  suis  infection.   Unfortunately,  the  outbreak 
continued  to  spread  involving  many  animals.   Our  study  was  terminated 
early. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute; 

Besides  providing  viral  diagnostic  services  to  the  medical  community, 
this  project  represents  in  essence  epidemic  intelligence  indispensable  to 
proper  functioning  of  this  field  station.  With  but  a  few  exceptions, 
such  as  Bocas  del  Toro  study,  the  virus  projects  have  been  an  outgrowth 
of  careful  consideration  of  leads  discovered  in  the  course  of  our  willing 
cooperation  with  the  health  agencies  and  individual  physicians  in  Panama 
and  nearby  countries. 

Proposed  Course  of  the  Project: 

Individual  subprojects  will  be  either  terminated  or  developed  into 
planned  major  projects  as  indicated.   Participation  in  work-up  of  epidemic 
outbreaks  and  of  promising  individual  cases  will  continue. 


31 

Serial  No.  NIAID-100-A 


Serial  No. 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 

Part  B;    Honors,  Awards  and  Publications 

Publications  other  than  abstracts  from  this  project: 

1.  Craighead,  J.E.,  Shelokov,  A.,  Vogel  J.E.,  Peralta,  P.H. 
An  Outbreak  of  Influenza  B  in  Panama.   Accepted  for  publication  in 
Amer.  J.  Trop.  Med.  S-  Hyg. 

2.  Craighead,  J.  E.,  Shelokov,  A.,  Peralta,  P.H.,  Vogel,  J.E. 
Croup  Associated  Virus  Infection  in  Adults:  Report  of  Two  Cases. 
Accepted  for  publication  in  New  Eng.  J.  Med. 

3.  Chi,  L.,  Vogel,  J.E.,  Shelokov,  A.,  Selective  Phagocytosis  of 
Nucleated  Erythrocytes  by  Cytotoxic  Amebae  in  Cell  Culture.   Science, 
130;  1763  (Dec.)  1959  (LISTED  LAST  YEAR  AS  ACCEPTED  FOR  PUBLICATION) 


Honors  and  Awards  relating  to  this  project: 
None 


?2 


Serial  No.  NIAID-100-A 


Serial  No.  NIAID-100-B 

1.  Tropical  Virology 

2.  Middle  America  Research  Unit 

3.  Panama  Canal  Zone 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A: 


Project  Title:  A  Clinical  and  Virological  Study  of  Oropharyngeal 
Lesions  in  Panamanian  Children 

Principal  Investigator:   Dr.  J.  V.  Ordonez 

Other  Investigators:     Dr.  S.de  Leon  (H.del  N.),  Dr.  J.  A.  Brody, 

Dr.  E.  Bruckner 

Cooperating  Units:       Hospital  del  Nino,  Panama  City 

Man  Years  (calendar  year  i960) 
Total:  3/12 

Professional:     2/12 
Other:  1/12 

Project  Description 

Objectives: 

Clinical  and  virological  evaluation  of  oropharyngeal  lesions 
in  Panamanian  children. 

Methods  Employed: 

Swabs  are  collected  in  skim  milk  medium  from  oropharyngeal 
lesions  in  children  examined  in  a  pediatric  outpatient  clinic. 
Each  group  of  specimens  (collected  over  a  half-day  period)  is 
kept  frozen  until  inoculation  into  the  test  systems  (monkey  kidney, 
hamster  kidney  and  chicken  fibroblast  cell  cultures,  as  well  as 
suckling  and  weanling  mice). 

Patient  Material: 

Children  with  observed  oropharyngeal  lesions  attending  the 
outpatient  clinic  of  Hospital  del  Nino  in  Panama. 

Major  Findings: 

This  study  is  still  in  its  initial  stages  and  consequently  no 
data  are  as  yet  available  for  presentation. 

Part  B  Included     No  ~ ,, 

~~  ,  33 


Significance  to  Blo-medical  Research  and  the  Program  of  the  Institute; 

Distressing  and  even  painful  lesions  of  the  oropharynx  are  not  un- 
common among  children  seen  in  the  outpatient  clinics  of  Panama.  To  our 
knowledge,  no  virological  investigation  regarding  the  etiology  of  such 
lesions  has  ever  been  carried  out  in  the  American  tropics. 

Proposed  Course  of  the  Project: 

Work  has  been  initiated  as  proposed  and  will  be  carried  out  for 
at  least  the  next  six  months. 


34 

Serial  No.  NIAID-100-B 


Serial  No.  NIAID-101-A 

1.  Tropical  Virology 

2.  Middle  America  Research  Unit 

3.  Panama  Canal  Zone 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A: 


Project  Title:  Virological  Aspects  of  a  Cooperative  Investigation 
on  the  Ecology  of  Arthropod  Borne  Viruses. 

Principal  Investigator:    Dr.  A.  Shelokov 

Other  Investigators  GML:   Dr.  P.  Galindo,  Dr.  E.  de  Rodaniche  and 

Dr.  C.  M.  Johnson 
MARU:   Dr.  P.H.  Peralta,  Dr.  C.G.  Dobrovolny, 

D. .  Longfellow,  J.  Vogel,  Dr.  E.A.  Bruckner, 
Dr.  J.V.  Ordonez 

Cooperating  Units:   Gorgas  Memorial  Laboratory,  Panama  City 

Hospital  Div.,  Chiriqui  Land  Co.,  Almirante,  R.de  P. 
Arthropod  Borne  Virus  Section,  LTV  (Bethesda) 

Man  Years:   (calendar  year  I960) 
Total:  5-9/12 

Professional:       3-1/12 
Other:  2-8/12 

Project  Description 

Objectives: 

1)  To  isolate  and  identify  arthropod-borne  viruses  occurring 
in  the  tropical  rain  forest  area  near  Almirante,  Bocas  del  Toro, 
Province,  R.de  P.;  2)  To  investigate  the  role  of  the  virus  isolates 
in  human  and  domestic  animal  disease;  3)  To  study  the  role  of  arth- 
ropod vectors  and  animal  reservoirs  in  the  epidemiology  of  infec- 
tions due  to  the  arthropod  borne  viruses  in  the  study  area  and  other 
parts  of  the  Isthmus. 

Methods  Employed: 

In  accordance  with  the  original  plan  for  the  joint  project, 
Gorgas  Memorial  Laboratory  (GML)  has  provided  MARU  with  arthropods 
collected  by  standard  techniques  and  identified  as  to  genus  and  in 
most  cases  species.   MARU  has  also  received  aliquots  of  all  human 
sera  jointly  collected  at  Almirante  in  October  i960  and  from  the 
small  mammals  bled  by  GML  collectors  earlier  in  the  year.  An  ex- 

35 

Part  B  included     no  ^  Serial  No.  NIAID-101-A 


perimental  method  for  the  collection  of  bird  blood  by  saturation  of  filter 
paper  discs  has  been  utilized  for  virus  isolation  attempts  at  MARU.   All 
birds  caught  or  shot  by  the  GML  team  beginning  October  are  being  thus 
sampled. 

Standard  methods  have  been  used  for:  1)  isolation  attempts  in  suck- 
ling mice  (SM)  and  hamster  kidney  cell  cultures  (HKTC) ;  2)  passages  of 
isolates  in  these  and  other  host  systems  for  characterization  and  adapt- 
ation; 3)  desoxycholate  and  ether  sensitivity  tests;  h)    preparation  of 
viral  antigens  and  specific  antisera  and  5)  serological  tests  -  comple- 
ment fixation  (CFT),  hemagglutination  (HA),  hemagglutination- inhibition 
(HI),  and  neutralization  (NT).   Preparation  of  "immune"  ascitic  fluid 
in  laboratory  animals  has  been  initiated  on  a  small  scale. 

Major  Findings: 

1.   Isolation.   The  results  of  virus  isolation  from  mosquito  and 
phlebotomus  pools  during  the  first  year  at  MARU  and  GML  are  shown  by 
species   in  the  attached  table.   In  the  new  year  of  operations  a  few 
additional  isolations  have  been  made  at  MARU,  including  three  from 
Psorophora  ferox  and  1  from  Aedes  (Ochlerotatus)  spp.   Isolation  attempts 
with  bird  bloods  eluted  from  discs  were  begun  late  in  the  year,  without 
positive  results  so  far. 

2.  Characterization  and  Identification,  a)  Phlebotomus  Isolates: 
Three  of  these  are  similar  in  that  they  infect  SM  i.e.  only,  with  a 
somewhat  irregular  incubation  period  (3-5  days)  even  after  repeated  pass- 
age; titers  of  SM  brain  pools  have  been  low,making  them  difficult  to 
work  with.   A  sufficiently  high  titer  has  been  obtained  with  one  of  these 
to  permit  testing  by  NT  against  antisera  to  the  two  types  of  Sandfly  fe- 
ver virus  with  negative  results. 

The  other  two  sandfly  agents  appear  indistinguishable  in  their  bio- 
logical characteristics  and  serological  cross-reactivity  (both  CFT  and 
NT).   BT-78  (chosen  as  prototype),  a  DCA  and  ether-sensitive  agent,  was 
not  neutralized  in  HKTC  by  antisera  to  the  two  types  of  Sandfly  Fever, 
Herpes-B,  EMC  or  the  New  Jersey  type  of  Vesicular  Stomatitis  virus  (VSV). 
It  was  neutralized  by  an  antiserum  for  the  Indiana  type  VSV,  with  which 
It  also  was  reactive  by  CF  test. 

b)  Mosquito  Isolates:  The  twelve  mosquito  isolates  fall  into  sever- 
al groups  according  to  their  behavior  in  mice  and  MKTC.   The  only  definite 
serological  cross-relationship  so  far  demonstrated  (both  CFT  and  NT)  was 
between  the  two  virus  strains  from  pools  of  C.vomerifer.   None  of  the 
several  isolates  tested  has  been  cross-reactive  with  Eastern  or  St.  Louis 
encephalitis,  llheus  or  Mayaro  (known  or  suspected  in  the  area)  or  the 
viruses  isolated  from  Panamanian  mosquitoes  collected  in  1958-59. 

3.  Antibody  Survey.  Preliminary  testing  of  human  sera  on  hand  from 
several  areas  in  Panama  against  phlebotomus  virus  isolate  BT-78  indicated 
that  20-35%  of  the  population  in  certain  areas  possess  neutralizing  anti- 
bodies.  Convalescent  sera  from  27  Panama  City  and  Canal  Zone  patients 

2  3QSerial  No.  NIAID-101-A 


with  inf luenza-1 ike  illnesses,  suspected  CNS  disease  or  FUO  were  negative 
by  NT. 

Among  domestic  animals,  4/28  pigs  (from  two  areas)  and  19/63  horses. 
and  mules  (from  5  localities  on  the  isthmus)  were  shown  to  have  neutral- 
izing antibodies,  with  65%  of  the  equines  positive  in  one  area. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

The  stated  objective  of  the  Virus  Section  of  MARU  has  been  "to  eval- 
uate the  significance  of  viral  agents  found  among  the  inhabitants  of 
Middle  America  as  related  to  causation  of  human  and  animal  disease.  Spe- 
cial emphasis  is  placed  on  arthropod  borne  viruses,  their  natural  reser- 
voirs and  vectors  as  well  as  other  agents  which  may  be  of  potential  dan- 
ger to  the  population  of  other  American  countries,  including  the  United 
States".  The  Bocas  collaborative  project  is  a  major  endeavor  to  fulfill 
this  mission  in  a  most  thorough,  efficient  and  economical  way  by  combin- 
ing and  supplementing  the  efforts  and  skills  of  Gorgas  Memorial  Laboratory 
and  Middle  America  Research  Unit. 

Proposed  Course  of  the  Project: 

This  field  and  laboratory  project  is  now  entering  the  second  of  its 
three  scheduled  years.   Isolation  attempts  will  be  continued  as  the  GML 
field  team  proceeds  with  arthropod  collection.  Cul icoides  midges  will 
be  collected  during  the  second  year.  Bird  and  small  animal  blood  and  or- 
gans will  be  obtained  in  increasing  numbers  for  virus  isolation  and  sero- 
logical testing.  Sentinel  suckling  mouse  techniques  are  being  adapted 
for  field  use  in  Panama.  Characterization  and  identification  procedures 
will  be  accelerated  with  the  increasing  availability  of  immune  sera  to 
project  isolates,  type  specific  and  groupings  sera  (obtained  with  the 
collaboration  of  ABVS-LTV).   Plans  include  the  use  of  additional  tech- 
niques (such  as  TC  plaques,  agar-gel  diffusion)  as  they  become  feasible 
and  desirable  for  the  execution  of  the  project. 

The  second  and  third  objectives  of  the  project  will  be  actively  pur- 
sued with  arthropod  borne  virus  isolates  for  which  the  vector- reservoir 
relationships  and  public  health  significance  should  be  sought. 


37 

Serial  No.NIAID-101-A 


BOCAS  DEL  TORO 

PROJECT 

First  Year's  Summary 

'  of  Mosquito  and  Sandfly 

Col  lections" 

*  and 

Virus 

solations  at  GML  and  MARU 

1  Sept.  1959  -  31  Aug.  I960 

/Data  f 

rom  Dr.  P.  G< 

al indo, 

GML/ 

Pools   Inoc. 

Specimens 

Inoculated 

Virus 

Isol. 

Species 

GML 

MARU 

GML 

MARU 

GML 

MARU 

Haemagogus  spp.         , 

15 

12 

884 

787 

Aedes  (Ochlerotatus)  spp. 

69 

59 

9,670 

8,199 

1 

leucocelaenus  clarki 

4 

3 

168 

116 

leucotaeniatus 

1 

22 

(Finlaya)  spp. 

1 

6 

(Howard ina)  spp. 

3 

2 

85 

40 

Psorophora  albipes 

13 

15 

1,651 

2,032 

1 

ferox 

12 

8 

1,401 

1,079 

3 

2 

lutzii 

1 

1 

101 

97 

1 

cingulata 
spp. 

6 

4 

543 

420 

36 

38 

4,932 

5,142 

1 

1 

Mansonia  venezuelensis 

67 

67 

9,849 

9,291 

t i ti 1 lans 

20 

19 

2,400 

2,257 

arribalzagae 

4 

2 

248 

227 

indubi  tans 

3 

54 

nigricans 

1 

34 

Culex  nigri palpus-^ 

223 

139 

23,296 

20,058 

1 

1 

declarator 

5 

4 

432 

410 

coronator 

8 

5 

552 

598 

cornlger 

4 

1 

234 

105 

vomer  if er 

15 

15 

1,782 

1,749 

2 

taeniopus 

9 

7 

816 

627 

elevator 

6 

6 

558 

555 

chrysonotum 

5 

4 

418 

351 

spp. 

9 

8 

704 

653 

Jranotaenia  spp. 

3 

4 

95 

89 

i> ichoprosopon  spp. 

7 

7 

609 

585 

Wye  omyia  spp. 

15 

14 

1,531 

1,513 

Sabethes  chloropterus 

17 

13 

1,344 

1,311 

1 

Sabethes  spp 

5 

5 

283 

175 

Anopheles  neivai 

3 

1 

68 

27 

Anopheles  spp. 

14 

13 

1,408 

1,366 

1 

1 

Phlebotomus  spp. 

18 

18 

2,721 

3,391 

2 

5 

Totals: 

1 _ — 

622 

494 

68,899 

63,160 

12 

13 

*  BY  G0RGAS  MEMORIAL  LABORATORY  STAFF 


1. 

Includes: 

A. 

2. 

Includes: 

A. 
P. 

3. 

Includes: 

C. 

serratus,  A.  angustiv? ttatus,  A.  hastatus,  A.  tormentor, 
ol  igopistus,  A.  fulvus.  _Q 

ferox,  £.  albipes  and  £.  lutzi  1  OO 

inf 1  ictus. 

L  Serial  No.  NIAID-101-A 


Serial    No.    NIAID-101-B 

1.  Tropical    Virology 

2.  Middle  America   Research  Unit 

3.  Panama  Canal    Zone 


PHS-NIH 
Individual    Project  Report 
Calendar  Year   1 960 


Part  A: 


Project  Title:   The  Role  of  Chiggers  and  Other  Acarina  of  the 
American  Tropics  In  the  Maintenance  and  Trans- 
mission of  Animal  Infectious  Agents:  X»Viral  Aspects. 

Principal  Investigator:   Dr.  J.  M.  Brennan 

Other  Investigators:     Dr.  C.  E.  Yunker 

Cooperating  Units:       Rocky  Mountain  Laboratory,  NIAID 

Gorgas  Memorial  Laboratory,  R.de  P. 

U.S.  Army  Malaria  Control  &  Survey  Branch,  C.Z, 

Veterinary  Division,  Health  Bureau,  C.Z. 

Man  Years  (calendar  year  i960): 
Total:  1-2/12 

Professional:     1 
Other:  2/12 

Project  Description: 

Objectives: 

To  explore  the  role  of  chiggers  and  possibly  other  Acarina, 
such  as  mites  and  ticks,  in  the  maintenance  and  transmission  of 
viral  agents  of  actual  or  potential  importance  in  Panama  and  nearby 
tropical  areas. 

Methods  Employed: 

The  problem  will  be  approached  by  trapping,  bleeding,  preparing 
and  identifying  hosts  of  parasitic  Acarina,  collecting  their  mites 
and  processing  for  inoculation  (by  the  staff  of  Virus  Section,  MARU) 
into  laboratory  animals  and  tissue  culture.   If  viral  agents  are 
isolated,  they  will  be  identified,  related  to  possible  occurrence 
of  viremia  or  the  presence  of  antibodies  in  the  donor  host  by  stand- 
ard virological  procedures. 


Part  B  included     No  33 


Major  Findings  (during  the  calendar  year): 

This  a  is  new  collaborative  project  in  a  field  setting  which  has 
taken  several  months  to  initiate.   The  many  anticipated  and  some 
unexpected  problems  have  been  largely  resolved  and  the  project  should 
be  in  full  operation  by  the  beginning  of  the  new  calendar  year.   In 
the  meantime,  approximately  *f00  animals,  including  bats,  snakes,  birds 
and  rodents,  have  been  examined  for  parasitic  mites. 

In  the  course  of  initial  exploratory  studies  a  discovery  was  made 
of  two  new  genera  and  species  of  chiggers  parasitic  In  the  nasal  passages 
of  Panamanian  bats.   Although  intranasal  chiggers  have  been  recovered 
from  rodents  in  Africa  and  Malaysia,  to  our  knowledge  no  chiggers  util- 
izing an  intranasal  habitat  have  ever  been  recorded  for  Chlroptera. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

The  role  of  chiggers  and  other  Acarina  in  transmission  of  certain 
diseases  is  well  known,  (e.g.,  scrub  typhus,  rickettsialpox,  Russian 
spring-summer  encephalitis,  etc.).   However,  no  serious  attempt  has 
been  made  so  far  to  define  their  role  in  transmission  of  classical  and  the 
newly  recognized  arthropod  borne  viruses.   Hence  positive  or  negative 
information  will  constitute  a  distinct  contribution  to  our  limited 
understanding  of  the  ecology,  epidemiology  and  epizootiology  of  this 
important  group  of  pathogens. 

Proposed  Course  of  the  Project: 

The  field  project  will  continue  as  proposed  until  leads  Indicate 
the  desirability  of  faunal  or  geographical  specialization.   In  the  event 
of  bona  fide  isolation  of  viral  agents,  emphasis  will  be  placed  on  a 
particular  species  relative  to  its  abundance,  distribution,  host  rela- 
tionships, disease  transmission  potential,  including  field  ecological 
studies  and  laboratory  colonization  attempts.   To  determine  the  poten- 
tial public  health  significance  of  the  viral  isolates  attempts  will  be 
made  to  relate  them  serologically  to  human  and  animal  infection. 

As  opportunities  arise,  it  is  planned  to  explore  further  the  role 
of  Acarina  in  maintenance  and  transmission  of  other  pathogenic  agents, 
including  rickettsiae,  protozoa,  bacteria  and  fungi. 


40 

Serial  No.  NIAID-101-B 


Serial  No.  NIAID-101-C 

1.  Tropical    Virology 

2.  Middle  America   Research   Unit 

3.  Panama  Canal    Zone 


PHS-NIH 
Individual    Project  Report 
Calendar  Year   i960 


Part  A: 


Project  Title:  Viral  "Jungle  Fever"  in  U.S.  Military  Personnel 

Principal  Investigator:   Dr.  J.  A.  Brody 

Other  Investigators:     D.  Longfellow,  Dr.  J.  V.  Ordonez, 

Dr.  E.  Bruckner  and  Dr.  A.  Shelokov 

Cooperating  Units:       Lt.  Col.  J.  E.  Goldoni,  CO, 

Jungle  Warfare  Training  Center  (JWTC)  U.S.  Army 
Col.  H.  B.  Leach,  MC,  Chief  Surgeon,  USARCARIB 

Man  Years  (calendar  year  I960) 
Total:  8/12 

Professional:     3/12 
Other:  5/12 

Project  Description 

Objectives: 

1.  Isolation  of  viral  agents  from  the  blood  of  men  with  minor 
illness  following  known  exposure  to  jungle  environment. 

2.  To  determine  the  rate  of  human  infection  with  the  newly 
isolated  as  well  as  viruses  known  to  be  active  on  the  Isthmus. 

Methods  Employed: 

Blood  samples  are  drawn  from  military  students  participating  in 
a  3-week  course  at  JWTC.  Specimens  are  secured  by  corpsmen  from 
men  reporting  to  the  aid  station  with  other  than  traumatic  complaints. 

Specimens  are  tested  for  virus  isolation  in  suckling  mice  (SM) 
and  hamster  kidney  cell  cultures.  Three  successive  cycles  of  classes 
at  JWTC  were  bled  before  and  after  the  3-week  course.   Some  200  paired 
specimens  were  collected. 


Part  B  included     No 


m 


Major  Findings: 

Because  of  reticence  to  be  bled  only  18  specimens  for  Isolation 
have  been  secured  from  the  first  900  candidates.   These  have  yielded 
one  definite  virus  isolate  ( JW- 10).   The  agent  was  isolated  and  re- 
isolated  from  a  soldier  with  fever  of  101  F  and  malaise.   The  virus  pro- 
duces illness  in  SM  inoculated  intracerebral ly  in  3  days  with  100%  mor- 
tality in  5  days.   When  inoculated  intraper i tonea 1 ly  the  illness  and 
death  are  delayed  1  to  2  days.   It  Is  pathogenic  for  weanling  mice  i.e. 
but  not  i.p.   Virus  pools  have  been  prepared  and  an  Immune  mouse  serum 
is  available.   Adequate  CF  and  HA  antigens  are  being  worked  out.   Pre- 
liminary results  with  an  HA  antigen  at  low  pH  (5.2  -  5.7)  are  promising. 
To  date  attempts  to  adapt  this  agent  to  various  cell  culture  lines  have 
not  been  successful. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

The  likelihood  that  isolates  from  human  blood  are  of  pathogenic 
significance  makes  information  from  this  study  important  in  defining 
not  only  the  local  health  problems  but  viral  infections  of  importance 
to  the  military  training  program  of  the  area. 

Proposed  Course  of  the  Project: 

Although  the  numbers  of  specimens  for  isolation  are  small,  each 
isolate  is  significant.   Therefore,  attempts  at  Isolation  will  continue. 
Paired  bloods  will  be  tested  for  antibody  rises  against  known  isolates 
by  suitable  means  and  will  also  be  tested  against  other  MARU  virus 
isolates  as  indicated. 


hi 

Serial  No.  NIAID-101-C 


Serial  No.  NIAID-102-A 

1 .  Tropical  Vi  rology 

2.  Middle  America  Research  Unit 

3.  Panama  Canal  Zone 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1 960 


Part  A: 


Project  Title:   Enterovirus  Infections  of  Rural  Guatemalan 
Children  in  Relation  to  Nutrition 

Principal  Investigators:   Dr.  A.  Shelokov  &  Or.  W.  Ascol i  (INCAP) 

Other  Investigators  MARU:  Dr.  J.  V.  Ordonez,  Dr.  J.  A.  Brody, 

Dr.  P.  H.  Peralta,  Dr.  E.  Bruckner 

INCAP:  Miss  V.  Pierce,  Mr.  H.  Bruch, 

Dr.  N.  Scrimshaw  £■  Dr.  J.  Gordon 

Cooperating  Units:   Instituto  de  Nutricion  de  Centro  America 
y  Panama  (INCAP),  Guatemala 

Man  Years  (calendar  year  I960) 
Total:  1/12 

Professional:        1/12 
Other:  None 

Project  Description: 

Objectives: 

1.  To  compare  the  incidence  and  nature  of  viral  flora  among 
the  children  in  three  carefully  controlled  villages  under  constant 
survei 1  lance. 

2.  To  explore,  if  possible,  the  relationship  of  certain  entero- 
viruses to  the  occurrence  of  clinical  cases  of  diarrhea. 

Methods  Employed: 

Each  village  comprises  a  population  of  approximately  1,000 
with  200  children  under  age  5.   Socio-economic,  climatic,  and 
topographical  aspects  are  comparable.   One  village  serves  as  con- 
trol, in  the  other  all  children  under  age  5  receive  liberal  diet 
supplements,  while  in  the  third  the  diet  is  unchanged  but  sanitary, 
prophylactic,  and  therapeutic  measures  to  prevent  and  ameliorate 
cases  of  diarrhea  are  enforced. 


Part  B  included     No 


43 


The  entire  study  group  population  of  each  village  will  be  surveyed 
every  six  weeks  by  rectal  swabs  for  isolation  of  cytopathogenic  entero- 
viruses.  These  will  be  identified  and  grouped  as  far  as  possible.   Vi- 
rus isolates  will  be  related  to  clinical  cases  of  diarrhea  by  epidem- 
iological and  serological  techniques. 

Major  Findings  (during  the  calendar  year): 

This  is  a  new  collaborative  project  between  INCAP  and  MARL)  which 
has  been  planned  during  the  calendar  year.   The  INCAP  group  is  engaged 
in  a  3-year  epidemiological  study  of  the  relationship  between  infectious 
diseases  and  nutrition  in  children  under  5  years  of  age  in  these  three 
rural  Guatemalan  communities.   This  has  presented  an  unusual  opportunity 
for  a  concurrent  investigation  of  the  viral  flora  during  the  second  year 
of  the  INCAP  study.   Dr.  J.  V.  Ordonez,  staff  member  of  INCAP' s  Bacter- 
iology section  was  assigned  to  MARL)  in  August  i960  on  a  1-year  fellow- 
ship (Parke,  Davis  &  Company)  to  participate  in  the  laboratory  work  and 
to  provide  liaison. 

The  first  group  of  specimens  should  be  received  before  the  end  of 
the  calendar  year,  but  no  findings  can  be  listed  as  yet. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

Diarrhea  in  young  children  ranks  high  among  serious  public  health 
problems  and  causes  of  death  in  Guatemala  and  all  other  countries  of 
Central  America.  Any  and  all  information  resulting  from  this  study  will 
provide  much  needed  background.   The  unusual  opportunity  to  explore  the 
epidemiological  aspects  of  enterovirus  infections  in  relation  to  the  nu- 
tritional status  of  controlled  population  groups  would  in  itself  justify 
this  laboratory  project. 

Proposed  course  of  Project: 

It  is  anticipated  that  collection  of  appropriate  specimens  from 
children  in  the  study  groups  and  their  testing  will  continue  for  one 
year.   Another  year  will  be  devoted  to  identification  of  virus  isolates, 
serological  studies  (if  possible)  and  correlation  of  laboratory  and 
epidemiological  information. 


Serial  No.  NIAID-102-A 


Serial  No.  NIAID-102-B 

1.  Tropical  Virology 

2.  Middle  America  Research  Unit 

3.  Panama  Canal  Zone 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  A: 


Project  Title:   Laboratory  Support  of  Phase  I,  National  Program  for 

Poliovirus  Vaccine  Administration  in  Costa  Rica,  1959* 

Principal  Investigator:   Or.  A.  Shelokov 

Other  Investigators:     Dr.  J.  A.  Brody,  D.  Longfellow  &  J.  E.  Vogel 

Cooperating  Units:       Ministry  of  Public  Health,  Costa  Rica 

Pan-American  Sanitary  Burea/PAHO/WHO 
San  Juan  de  Oios  Hospital,  San  Jose,  Costa  Rica 
PASB-TC  Lab.  at  Univ.  del  Valle,  Cali,  Colombia 
Division  of  Biological  Standards,  NIH 
Epidemiology  Branch,  CDC,  BSS 

Man  Years  (calendar  year  I960): 
Total:  2/12 

Professional:        1/12 
Other:  1/12 

Project  Description: 

Objectives: 

1.  To  support  surveillance  of  neurological  illnesses  during 
the  administration  of  live  poliovirus  vaccine  in  San  Jose,  Costa 
Rica. 

2.  To  provide  virological  information  on  a  series  of  well 
studied  cases  of  neurological  illness  in  a  Central  American  country. 

Methods  Employed: 

All  cases  of  neurological  illness  during  the  oral  vaccination 
program  in  the  capital  city  were  investigated  by  Dr.  Brody  on  duty 
in  Costa  Rica.   From  almost  every  patient  throat  and  rectal  swabs 
and  paired  blood  samples  were  obtained. 

Specimens  for  isolation  were  tested  at  MARU  in  monkey  kidney 
cell  cultures  and  suckling  mice.   Duplicate  specimens  were  tested 
by  Dr.  H.  Doany  (PASB-TC  Laboratory,  Cali)  in  HeLa  cells  and  suck- 
ling mice.   Serological  testing  of  acute  and  convalescent  blood 

Part  B  included     Yes  1  ^^ 


specimens  was  performed  at  the  Cali  laboratory,  Division  of  Biological 
Standards  in  Bethesda  and  to  a  limited  extent  at  MARU. 

Major  Findings: 

Most  of  the  work  was  accomplished  during  the  preceding  calendar  year. 
In  summary,  51  cases  or  siblings  were  examined.   Six  of  these  had  clinical 
poliomyelitis  and  in  6  more  the  final  diagnosis  of  poliomyelitis  could 
not  be  ruled  out.   There  were  no  cases  in  which  the  vaccine  could  be 
clearly  related  to  the  clinical  illness. 

Eleven  viral  agents  from  10  individuals  were  isolated  by  testing 
8h   specimens.  The  isolations  included  one  strain  of  poliovirus  Tl ,  two 
poliovirus  T2,  two  poliovirus  T3,  two  Coxsackie  Qk,    two  ECHO  ]k,    one 
ECHO  8  and  one  unidentified  virus.   Serological  studies  against  homologous 
virus  isolates  supported  the  isolation  findings.   Determinations  of  anti- 
bodies for  the  3  types  of  poliovirus  revealed  occasional  vaccine  failures. 

Among  the  cases  of  clinical  poliomyelitis  only  2  were  seen  early 
enough  in  the  course  of  illness  to  make  specimens  meaningful.   Poliovirus 
T2  was  isolated  from  one  and  no  virus  was  isolated  from  the  other. 
Neither  patient  had  been  vaccinated.   Of  the  6  cases  in  which  the  diag- 
nosis of  poliomyelitis  could  not  be  ruled  out,  the  following  poliovirus 
isolations  were  made:   T3  -  2  cases,  T2  -  1  case,  Tl  -  1  case.  Coxsackie 
Qk   was  recovered  from  two  cases:  from  one  Tl  poliovirus  was  also  recovered 
and  the  other  had  a  serological  rise  to  T3  poliovirus.  The  latter 
patient  had  not  been  vaccinated,  while  the  others  had  received  at  least 
one  dose  of  oral  vaccine. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

This  study  failed  to  raise  serious  doubts  as  to  the  safety  of  the 
Lederle  oral  live  poliovirus  vaccine.   It  emphasized  the  difficulties 
in  diagnosis  and  interpretation  of  laboratory  results  on  hospitalized 
patients  with  neurological  symptoms,  especially  when  fed  live  virus  vac- 
cine.  It  also  revealed  that  in  the  absence  of  a  clear  cut  syndrome  the 
virus  yield  in  this  hospitalized  population  was  small. 

Proposed  Course  of  Project: 

The  project  has  been  terminated  and  the  findings  were  reported  to 
the  Government  of  Costa  Rica  and  the  Pan-American  Sanitary  Bureau. 


46 

Serial  No.  NIAID-102-B 


Serial  No.  NIAID-102-B 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  B: 


Publications  other  than  abstracts  from  this  project: 

1.  Quirce,  J.  M.,  Vargas  Mendez,  0.,  Nunez  J.,  Montoya,  J.  A., 
Brody  J. ,  Henderson,  0.  A.,  and  Martins  Da  Silva,  M.  "Vaccination 

with  Attenuated  Polioviruses  in  Costa  Rica"  in  Live  Poliovirus  Vaccines: 
Papers  Presented  and  Discussions  Held  at  the  First  International  Con- 
ference on  Live  Poliovirus  Vaccines,  Washington,  D.  C,  Pan  American  Sani- 
tary Bureau/WHO,  1959,  713  pp. 

2.  Quirce,  J.  M. ,  Nunez,  J.,  Guevara,  E.  C,  Montoya,  J.  A., 
Doany,  H.,  and  Shelokov  A.  "Vaccination  with  Attenuated  Polioviruses  in 
Costa  Rica.   Second  Progress  Report,  Section  II:  Surveillance  Program" 
in  Live  Poliovirus  Vaccines:  Papers  Presented  and  Discussions  Held  at 
the  Second  International  Conference  on  Live  Poliovirus  Vaccines.  Wash- 
ington, D.  C,  Pan  American  Health  Organizat ion/PASB/WHO,  i960,  63*+  pp. 


Honors  and  Awards  relating  to  this  project: 
None 


47 


Serial  No.  NIAID-102-C 

1.  Tropical  Virology 

2.  Middle  America  Research  Unit 

3.  Panama  Canal  Zone 


PHS-NIH 

Individual    Project  Report 

Calendar  Year   I960 


Part  A: 


Project  Title:   Enterovirus  Flora  of  Panamanian  Children:  A  Twelve- 
Month  Survey. 

Principal  Investigator:   Dr.  J.  E.  Craighead 

Other  Investigators:     Dr.  A.  Shelokov 

Cooperating  Units:       Hospital  del  Nino 

Man  Years  (calendar  year  1980) : 
Total:  6/12 

Professional:     2/12 
Others  VI 2 

Project  Description: 

Objectives: 

This  study  was  undertaken  with  the  purpose  of  gaining  informa- 
tion on  the  enteric  virus  flora  of  infants  and  young  children  re- 
siding in  a  tropical  environment.   Surveying  the  population  at  re- 
gular intervals  for  a  year  should  reveal  fluctuations  in  the  inci- 
dence and  type  of  virus  infections  and  contribute  to  a  better  un- 
derstanding of  community  outbreaks  of  clinical  disease  due  to 
enteroviruses. 

Methods  Employed: 

During  the  12  months  of  the  study,  30-35  rectal  swab  specimens 
were  obtained  every  two  weeks  by  the  same  MARU  investigator  from 
children  under  the  age  of  three  years  in  the  out  patient  clinic  of 
Hospital  del  Nino.   Specimens  were  tested  for  virus  isolation  in 
MKTC.   Identification  of  cytopathogenic  agents  was  accomplished 
by  neutralization  tests  employing  antiserum  for  the  three  types  of 
poliovirus,  five  Coxsackie  B  types  and  Coxsackie  Ag.   No  attempt 
was  made  to  identify  agents  not  neutralized  by  one  of  these  specific 
antisera. 


Part  B  included     No 


48 


Major  Findings; 

A  total  of  805  specimens  was  collected  during  the  12  months  of  the 
study;  of  these  256  were  obtained  in  the  months  of  January,  February, 
March  and  April  of  1 960.  The  results  during  I960  followed  the  trends 
established  in  1 959-   Forty  six  cytopathogenic  agents  were  recovered 
during  the  four  months  of  i960  (if  contaminated  with  bacteria  specimens 
were  excluded  from  the  final  computations).   Twenty  per  cent  of  rectal 
swabs  satisfactorily  tested  in  i960  and  25%  of  specimens  tested  over  the 
12  months  of  the  study  were  positive  for  enterovirus  isolation.   In 
January  19°0,  19%  yielded  viruses,  in  February  20%,  in  March  16%  and  in 
April  25%. 

Throughout  the  survey  surprisingly  few  Coxsackie  8,  Coxsackie  An  and 
poliovirus  type  1  and  3  strains  were  isolated.   The  majority  of  agents 
recovered  were  not  identified  by  the  techniques  used  (presumably  they 
are  miscellaneous  ECHO  viruses).   In  late  1959  and  the  early  months  of 
I960,  a  substantial  proportion  of  children  tested  yielded  poliovirus 
Type  2.  The  appearance  of  this  virus  in  the  survey  was  associated  with 
an  outbreak  of  paralytic  poliomyelitis  due  to  the  same  virus  type  In 
Panama  City.  The  outbreak  is  reported  under  another  heading.   In  this 
study  intestinal  viral  infection  was  not  associated  with  a  recognizable 
clinical  syndrome.  Viruses  were  recovered  as  frequently  from  well  child- 
ren as  from  children  with  diarrhea,  fever  and  respiratory  symptoms.  No 
significant  seasonal  variations  in  the  incidence  of  enterovirus  infection 
were  demonstrated. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute; 

Infantile  diarrhea  and  severe  respiratory  infections  have  been  among 
the  most  serious  health  problems  in  Central  and  South  America.  Viruses 
are  often  blamed  but  while  in  North  America  and  Europe  enterovirus  flora 
has  been  carefully  investigated,  practically  no  information  is  available 
on  the  frequency  and  nature  of  enterovirus  infection  in  the  American 
tropics.  The  recognized  role  of  non-polio  enteroviruses  in  the  causation 
of  aseptic  meningitis  (and  occasionally  muscle  paresis)  underscores  the 
need  for  long-range  evaluation  of  local  enterovirus  flora. 

Proposed  Course  of  Project; 

Specimen  collections  for  this  12-month  survey  (which  developed  from 
an  earlier  6-week  exploratory  study  in  1958)  were  completed  in  May  i960. 
Interesting  and  significant  information  is  emerging  from  analysis  of  the 
data,  providing  background  for  subsequent  studies.  One  such  investiga- 
tion on  the  community  spread  of  poliovirus  type  1  is  already  in  progress. 


43 

Serial  No.NIAID-102-C 


Serial  No.  NIAID-103-A 

1.  Tropical  Virology 

2.  Middle  America  Research  Unit 

3.  Panama  Canal  Zone 


PHS-NIH 

Individual    Project  Report 

Calendar  Year    I960 


Part  A: 


Project  Title:   Use  of  Filter  Paper  Discs  for  Virus  Isolation 
and  Serological  Testing. 

Principal  Investigator:   Dr.  J.  A.  Brody 

Other  Investigators:     Dr.  J.  V.  Ordonez  and  D.  Longfellow 

Cooperating  Units:       Staff  of  Gorgas  Memorial  Laboratory  and 

Hospital  del  Nino 

Man  Years  (calendar  year  I960) 
Total:  6/12 

Professional:     3/12 
Other:  3/12 

Project  Description 

Objectives: 

To  develop  a  field  tool  for  virus  isolation  and  for  sero- 
logical testing. 

Methods  Employed: 

Discs  produced  by  Carl  Schleicher  and  Schnel 1  Co.  (cat.  No. 
7^+0-E)  are  used  in  all  experiments.   Humans  are  bled  by  finger 
puncture  and  small  animals,  particularly  suckling  mice  (SM),  by 
opening  the  chest  and  absorbing  the  blood  onto  the  discs. 

For  virus  isolation,  Eastern  equine  encephalitis  (EEE)  virus 
has  served  as  a  model.   Virus  is  inoculated  into  SM  which  are  bled 
in  36  hours.   Whole  blood  and  discs  are  collected  and  treated  under 
varying  conditions.   At  definite  intervals  the  dried  discs  are  re- 
suspended  in  diluent  and  titered.   Virus  determinations  are  con- 
ducted in  hamster  kidney  cell  cultures  and  occasionally  in  SM.   In 
addition  St.  Louis  encephalitis  (SLE),  llheus  and  yellow  fever 
viruses  have  been  studied  to  a  limited  extent. 

Serological  experiments  have  employed  standard  neutralization 
procedures  in  monkey  kidney  cell  cultures  using  ECHO-9  and  polio- 
viruses.   Discs  are  resuspended  overnight  before  use  and  compared 

Part  B  included     No  1  50 


with  sera  obtained  by  venipuncture  from  same  subjects. 

Major  Findings: 

EEE  virus,  titering  10   in  whole  SM  blood,  was  absorbed  on  discs  and 
left  in  the  refrigerator  and  at  ambient  temperatures.   After  12  days  in  the 
refrigerator  the  titer  on  the  disc  was  10  and  after  32  days  10^*5.   The 
environmental  temperature  disc  titered  10^-5  at  k   days  and  the  virus  was 
recoverable  (in  SM)  at  8  days.   Crude  attempts  to  dehumidify  the  discs  did 
not  seem  to  enhance  preservation  of  the  virus.   Various  methods  of  resus- 
pension  have  not  improved  on  the  basic  method  of  O.occ  of  diluent  per  disc 
left  overnight  at  k   C.   Freezing  wet  and  dry  discs  did  not  alter  titers 
appreciably.   SLE  virus  is  fairly  stable  on  dried  discs.  V/hen  viremia  ti- 
tered 10°*-'  in  SM,  the  discs  left  at  26°C  contained  10  '"'  logs  of  virus 
at  21  days  and  the  discs  left  k   days  at  ambient  temperatures  contained 
10',-J  logs  of  virus.   Ilheus  and  yellow  fever  viruses  are  more  fragile  on 
discs.   Both  were  recoverable  from  discs  after  k   days  at  k°,    but  not  from 
discs  at  environmental  temperatures. 

Because  of  the  practical  advantages  of  the  disc  method  for  isolation 
of  viruses  from  small  wild  mammals  and  birds,  Gorgas  Memorial  Laboratory 
is  cooperating  in  a  field  trial.   Birds  shot  at  Bocas  del  Toro  are  being 
bled  by  cutting  the  jugular  vein  to  saturate  the  discs  then  sent  to  MARU 
under  refrigeration.   Three  discs  are  combined,  resuspended  and  inoculated 
into  SM.   To  date  no  virus  isolations  have  been  made.   (GML  reports  that 
no  viruses  have  been  isolated  by  standard  methods  from  sera  of  the  same 
birds). 

Serological  studies  have  been  performed  using  finger  puncture  discs 
and  venous  blood  sera  from  the  same  individuals.   We  have  established  that 
blood  resuspended  from  discs  can  not  be  titered  out  and  can  not  be  stored 
at  room  temperature  for  more  than  ^-h   weeks. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

The  original  work  by  Karstad  and  Hanson  at  University  of  Wisconsin 
showed  that  EEE  could  be  recovered  from  discs.   We  are  amplifying  this  find- 
ing by  exploring  the  stability  of  viruses  dried  on  discs.   The  potential 
value  of  discs  as  a  field  tool  is  considerable:   increasing  emphasis  is 
being  placed  on  cycles  involving  nestling  birds  and  small  mammals  in  the 
ecology  of  arthropod  borne  viruses;  study  of  these  cycles  is  limited  be- 
cause their  bloods  are  difficult  to  collect;  the  discs  may  offer  a  partial 
solution  to  the  problem  of  virus  isolations  in  the  field. 

The  possibilities  of  a  reliable  serological  screening  tool  which  does 
not  require  a  syringe  and  vein  are  enormous.   In  tropical  areas,  where  con- 
version rates  are  high  in  the  young  children  (who  are  difficult  to  bleed) 
this  procedure  should  be  especially  helpful. 

Proposed  Course  of  Project: 

1)  To  continue  field  trials  for  virus  isolations;  2)  To  explore  fur- 

51 
2  *«*■     Serial    No.   NIAID-103-A 


ther  the  use  of  discs  as  a  reliable  serological  method  for  human  surveys; 

3)  To  experiment  with  CF  and  HAI  antibodies  on  disc-eluted  bloods;  k)   To 

attempt  serological  procedures  for  arthropod  borne  viruses  on  disc-absorbed 
blood  specimens  from  wild  life. 


52 


Serial  No.NIA|D-103-A 


Serial  No.  NIAID-103-B 

1.  Tropical  Virology 

2.  Middle  America  Research  Unit 

3.  Panama  Canal    Zone 


PHS-NIH 
Individual    Project  Report 
Calendar  Year   i960 


Part  A: 


Project  Title:   Eastern  Equine  Encephalomyelitis  (EEE) 
Virus  Infection  in  Panama 

Principal  Investigator:   Dr.  J.  E.  Craighead 

Other  Investigators:     Dr.  P.  H.  Peralta,  D.  Longfellow 

J.  E.  Vogel  and  Dr.  A.  Shelokov 

Cooperating  Units:       Gorgas  Memorial  Laboratory  (GML) 

Laboratorio  Veterinario,  Panama 
U.S.  Army  Mission  to  Panama 
Vet.  Div.,  WRAIR,  Washington,  D.  C. 

Man  Years  (calendar  year  I960) 
Total:  9/12 

Professional:     5/12 
Other:  VI 2 

Project  Description: 

Objectives: 

The  I960  objectives  were  an  outgrowth  of  our  investigation  of  a 
1958  EEE  outbreak  in  horses:  1)  To  determine  the  prevalence  of  EEE 
antibody  in  horses  and  man  in  two  areas  of  probable  EEE  virus  en- 
demicity;  2)  To  evaluate  the  possible  role  of  lizards  as  reservoirs 
of  EEE  virus  in  Panama;  3)  To  evaluate  the  relative  usefulness  of 
serological  methods  applicable  to  studies  of  this  type. 

Methods  Employed: 

Neutralization  (NT)  in  mice,  complement  fixation  (CFT)  and  hemag- 
glut ination-inhibi tion  (Hi)  were  used  as  classical  techniques  or 
with  recently  described  but  widely  accepted  modifications.   EEE  virus 
isolate  (1958)  from  a  Panamanian  horse  brain  was  used  both  as  in- 
fectious agent  and  antigen. 

Major  Findings: 

1.   Human  Population:   Four  hundred  sixty  serum  specimens  col- 
lected by  GML  and  MARU  in  Darien  and  Panama  provinces  of  the  Republic 

53 

Part  B  included     No  1 


were  surveyed  for  EEE  antibodies  by  HI  test.   Reactive  sera  were  tested 
in  mouse  NT.  Thirty-two  sera  (7-6%)  were  reactive  by  HI  test  at  >  1:20; 
12(3%)  neutralized  ^  1.7  logs  of  virus  in  the  adult  mouse  test  and  k 
additional  sera  neutralized  ;>,  1.7  logs  of  virus  when  tested  l.P.  in  suck- 
ling mice.  The  incidence  of  EEE  antibodies  in  humans  increased  with  ad- 
vancing age  (0.8%  under  10  years  with  progressive  increase  to  9%  in  the 
41-50  year  group).  The  mean  percentage  of  positive  sera  was  3-7%.  Anti- 
bodies were  equally  common  in  both  sexes.  Complement  fixation  tesir 
were  carried  out  on  all  k60   sera,  but  the  results  were  difficult  to  inter- 
pret, suggesting  heterologous  antibodies  to  other  group  A  viruses. 

2.  The  Virus  Reservoir:  Many  lizards  of  several  species  were  ob- 
served on  the  ranches  where  the  outbreak  had  occurred.  Other  investigat- 
ors (RML)  have  indicated  the  possible  role  of  reptiles  in  the  ecology  of 
ARBOR  viruses.  Because  of  these  considerations,  2^6  wild  lizards  caught 
in  the  suspecL  area  were  examined  by  the  HI  test.  Specific  EEE  virus 
hemagglutination  inhibitors  were  found  in  a  small  proportion  of  these 
sera. 

Several  experiments  were  carried  out  with  lizards  of  one  of  the 
three  species  common  in  Panama  (Ameria  festiva)  confirming  the  occurrence 
of  viremia  and  HI  antibody  response  following  virus  inoculation. 

3.  Evaluation  of  Methods:  The  horse  serum  HI  results  were  compared 
with  CFT  and  NT  results  on  same  sera.   Kaolin  and  acetone  procedures  for 
removal  of  non-specific  serum  hemagglutination  inhibitors  were  also  com- 
pared.  It  was  concluded  that  the  HI  test  is  useful  for  rapid  evaluation 
of  sera  for  antibodies,  since  HI  negative  sera  were  consistently  nega- 
tive by  NT  and  CFT.   Further,  large  numbers  of  specimens  can  be  quickly 
tested  by  surveying  sera  at  a  single  dilution.  However,  a  titer  can  be 
considered  only  suggestive  of  specific  EEE  antibodies  and  the  NT  is 
needed  for  confirmation,  particularly  in  areas  of  likely  activity  of 
other  group  A  viruses  (6  of  the  13  sera  tested  were  shown  to  have  VEE 
neutralizing  antibodies  by  a  cooperating  laboratory).  Our  studies  sug- 
gest that  kaolin  treatment  of  sera  is  not  as  efficient  as  acetone  ex- 
traction for  routine  removal  of  inhibitors,  but  acetone  may  also  remove 
much  heterologous  antibody,  which  can  be  disadvantageous  in  some  survey 
situations. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

EEE  virus  continues  to  cause  not  only  epizootics,  but  occasional 
epidemics  associated  with  human  fatalities  and  permanent  sequelae. 
Studies  on  the  ecology  and  epidemiology  of  the  disease  utilizing  improved 
sero-diagnost ic  procedures  should  contribute  to  control  of  this  public 
health  problem  common  to  both  temperate  and  tropical  areas. 

Proposed  Course  of  Project: 

Experimental  work  on  this  project  was  concluded  in  June  I960  and  the 
results  are  being  prepared  for  publication. 

54 

2         Serial  No.  NIAI0-103-B 


Serial  No.NIAID-103-C 

1.  Tropical  Virology 

2.  Middle  America  Research  Unit 

3.  Panama  Canal  Zone 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 


Part  A: 


Project  Title:  Encephalomyocardl tls  (EMC)  Virus  Infection.  Studies  on 
pathogenesis  In  swine,  virus  reservoirs  In  rodents  and 
antibody  status  of  human  and  animal  populations. 

Principal  Investigator:  Dr.  J.  E.  Craighead 

Other  Investigators:     Ma j .  T.  G.  Murnane,  VC,  USA,  Dr.  P.  H.  Peralta 

Dr.  A.  Shelokov 

Cooperating  Units:       Laboratorio  Veterinario,  R.de  P. 

U.  S.  Army  Mission  to  Panama 

Man  Years  (calendar  year  1 960) 
Total:  8/12 

Professional:     4/12 
Other:  VI 2 

Project  Description: 

Objectives: 

1)  To  conclude  a  basic  investigation  on  the  pathogenesis  of 
EMC  infection  in  swine;  2)  To  examine  small  wild  rodents  for  EMC 
antibodies  as  possible  reservoir  hosts;  3)  To  determine  the  pre- 
valence of  EMC  infection  in  the  Panamanian  population  by  testing 
randomly  collected  human  sera  for  antibodies. 

Methods  Employed: 

Laboratory  methods  developed  during  the  preceeding  calendar 
year  were  employed  in  these  studies.   Relevant  details  are  mentioned 
under  Major  Findings. 

.Major  Findings: 

1.  Biology  and  Pathogenesis  of  EMC  Infections.   Following 
the  feeding  of  virus,  3  pigs  without  pre-existing  antibodies  devel- 
oped viremia  for  2-k   days  and  excreted  virus  in  the  feces  for  as 
long  as  10  days.   During  the  period  of  active  infection  the  pigs 
exhibited  no  evidence  of  illness.  All  3  developed  substantial  in- 
crease in  antibody  by  hemagglutination  inhibition  (Hi)  and  neutral- 


Part  B  included      Yes 


55 


ization  (NT)  tests.   The  2  autopsied  pigs  had  histological  evidence 
of  myocardial  fibrosis  compatible  with  earlier  myocarditis.   A  fourth 
pig,  wi  th  pre-existing  antibody,  developed  viremia  transiently,  excreted 
virus  for  only  two  days  and  exhibited  an  antibody  rise  for  only  a  brief 
period.   A  fifth  pig  was  fed  a  large  amount  of  virus  after  only  one  mouse 
passage.   This  pig  died  (in  arrythmia?)  5  days  later  with  histologic  evi- 
dence of  acute  myocarditis.   The  virus  titer  of  the  heart  tissue  was 
greater  than  10°»  other  organs  yielded  lesser  amounts. 

2.  Virus  Reservoirs:  In  an  attempt  to  find  possible  virus  reservoirs 
In  nature,  wild  rodents  were  trapped,  bled  and  tested  for  EMC  antibodies. 
Two  of  53  rats  (Rattus  rattus)  caught  in  or  in  the  vicinity  of  Panama 
City  were  found  to  have  antibodies,  but  all  sera  from  42  other  small  ro- 
dents of  several  species  were  negative. 

3.  Human  Serological  Studies:  Eighteen  percent  of  158  sera  from  4 
localities  in  Panama  were  found  to  contain  significant  levels  of  EMC  an- 
tibodies by  NT  in  HeLa  cell  cultures  (TC).   There  were  interesting  differ- 
ences in  the  results  with  age.   Thus,  in  the  group  under  10  years  23% 
were  positive,  10-19  years  -  23%,  20-29  years  -  17%,  30-49  years  -  17%, 
while  only  7%  of  persons  over  the  age  of  50  possessed  antibodies.   These 
studies  were  carried  out  employing  diluted  serum  and  varying  dosages  of 
virus.   Sera  considered  positive  neutralized  2.5  logs  or  more  of  virus. 
Many  of  the  positive  sera  were  evaluated  in  the  diluted  state  against  ap- 
proximately 100  TCDcjq  doses  of  virus.   More  than  half  of  the  sera  tested 
in  this  way  had  demonstrable  antibodies  at  a  dilution  of  1:4  or  greater. 

A  large  number  of  NT  negative  and  positive  sera  were  tested  by  HI.   Twenty- 
nine  of  40  NT  positive  sera  were  also  positive  at  a  dilution  of  1:4  or 
greater  in  the  HI  test,  whereas  only  4  of  36  NT  negative  were  positive. 
Many  of  the  HI  positive  sera  had  demonstrable  antibodies  at  high  dilu- 
tions.  Limited  studies  were  carried  out  in  an  attempt  to  compare  the 
results  of  HeLa  TC  and  mouse  NT  tests.  While  many  sera  were  positive  by 
both  tests,  TC  positive  sera  frequently  were  negative  in  mouse  NT.   These 
data  suggest  that  a  substantial  proportion  of  the  Panamanian  population 
has  been  infected  with  EMC  virus. 

The  explanation  for  the  relatively  high  incidence  of  antibodies  in 
the  younger  persons  is  obscure.   Possible  explanations  are:  a)  the  virus 
has  been  recently  introduced,  b)  infection  occurs  early  in  life  and  an- 
tibodies disappear  with  time,  c)  the  virus  infection  ultimately  contri- 
butes to  mortality  at  a  young  age,  d)  the  "antibodies"  are  actually  non- 
specific neutralizing  substances  (this  is  highly  unlikely). 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

This  project  was  based  on  our  demonstration  of  natural  infection  of 
swine  with  EMC  virus  causing  the  most  extensive  outbreak  of  EMC  infection 
in  man  or  animals  in  which  the  virus  was  recovered.   Our  subsequent  stud- 
ies suggest  that  rats  may  constitute  a  natural  source  of  infection,  while 
pigs  contract  the  disease  by  ingestion  of  rats  and/or  droppings.   Improved 
laboratory  diagnostic  procedures  developed  during  this  study  allowed  ready 
demonstration  of  specific  antibodies  in  many  Panamanians,  especially  In 


56 


Serial  No.NIAID-103-C 


the  younger  age  groups.   These  findings  may  contribute  to  a  definition 
of  not  only  another  veterinary  problem,  but  of  possibly  an  important 
zoonosis,  especially  In  the  Caribbean  area  and  Southern  United  States. 

Proposed  Course  of  the  Project: 

The  project  has  been  terminated  and  the  findings  are  being  reported 
in  medical  and  veterinary  research  journals. 


57 

Serial  No.  NIAID-103-C 


Serial  No.  NIAID-103-C 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  B:     Honors,  Awards  and  Publications 


Publications  other  than  abstracts  from  this  project: 

Murnane,  T.  G.,  Craighead,  J.  E.,  Mondragon,  H.,  Shelokov,  A, 
Fatal  Disease  of  Swine  Due  to  Encephalomyocardi tls  Virus, 
Science,  131:498-*f99  (Feb.)  I960  /reported  as  "accepted  for 
publication"   last  year/. 


Honors  and  Awards  relating  to  this  project: 
None 


58 


Serial  No.  NIAID-108 

1.  Tropical  Virology 

2.  Mycology  Section,  MARU 

3.  Panama  Canal  Zone 

PHS-NIH 
Individual    Project  Report 
Calendar  Year   i960 

Part  A; 

Project  Title:  Studies  of  Histoplasmosis  on  the  Isthmus  of  Panama 

Principal  Investigator:  Capt.  R.  Taylor,  MSC 

(R&D  Command,  U.S.  Army) 

Other  Investigators:     Dr.  F.  Abildgaard,  Coco  Solo  Hospital 

Dr.  C.  G.  Dobrovolny,  MARU 

Cooperating  Units:       Walter  Reed  Army  Institute  of  Research 

Gorgas  Memorial  Laboratory,  R.de  P. 
Hospital  del  Nino,  R.de  P. 
Gorgas  and  Coco  Solo  Hospitals,  C.  Z. 
Ministry  of  Health,  R.de  P. 
Canal  Zone  Health  Bureau 
Army,  Navy  and  Air  Force  Surgeons 
U.S.  Army  Malaria  Control  and  Survey  Branch 
U.S.  Army  Preventive  Medicine  Officer 
Veterinary  Health  Offices,  C.  Z. 

Man  Years  (calendar  year  1 960) 
Total:        3-1/2 
Professional:     3/k 
Other:         2- 3 A 

Project  Description: 

Objectives: 

1.  To  determine  the  extent  of  histoplasmosis  on  the  Isthmus  of 
Panama  and  the  significance  of  this  disease  In  the  military  person- 
nel stationed  In  an  area  of  histoplasmosis  endemlclty. 

2.  To  explore  the  epidemiology  and  ecology  of  HIstoplasma 
capsulatum  on  the  Isthmus  of  Panama  and  Central  America. 

3.  To  provide  diagnostic  aids  and  adequate  clinical  follow-up 
on  all  recognized  cases  of  histoplasmosis  In  this  area. 

k.     To  evaluate  experimental  therapy  of  histoplasmosis  (In  co- 
operation with  Gorgas  Hospital). 


Part  B  Included     Yes 


5S 


Methods  Employed; 

In  cooperation  with  military  and  civilian  health  authorities  obtain 
data  on  hypersensitivity  to  histoplasmin  in  individuals  arriving  for  duty 
on  the  Isthmus,  as  well  as  long  time  residents  of  the  Canal  Zone  and  the 
Republic  of  Panama.   Provide  diagnostic  assistance  to  Isthmian  medical 
facilities  in  locating  and  following  all  clinical  cases  of  histoplasmosis. 

Collect  soil  samples  from  areasof  suspected  endemicity  as  suggested 
by  patient  interviews  to  demonstrate  the  presence  of  H.  capsulatum  and 
the  foci  of  infection.  Determine  the  extent  of  naturally  occurring 
histoplasmosis  in  the  wild  animal  population  as  possible  reservoirs  of 
H.  capsulatum  and  define  the  local  areas  of  endemicity. 

Major  Findings: 

In  the  first  eleven  months  of  1 960,  forty-three  cases  of  acute  his- 
toplasmosis in  the  Canal  Zone  were  serologically  confir  ^d.   In  addition, 
one  case  of  disseminated  histoplasmosis  in  an  11  month  old  infant  was 
culturally  proven  and  the  child  successfully  treated  at  Coco  Solo  Hos- 
pital. The  first  known  case  of  cavitary  histoplasmosis  in  this  area  oc- 
curred in  a  North  American  employee  in  the  Canal  Zone,  who  was  evacuated 
to  the  Clinical  Center  NIH  for  therapy. 

Histoplasmin  skin  testing  was  completed  in  the  Canal  Zone  School 
system  yielding  data  on  9»200  children  between  6  and  19  years  of  age. 
This  survey  provides  data  on  the  entire  school-age  population  permitting 
comparison  of  hypersensitivity  rates  according  to  location  and  length 
of  residence,  race,  age  and  sex.  As  expected,  the  percentage  of  react- 
ors increases  progressively  from  the  younger  to  older  children.  A  si- 
milar increase  occurs  in  the  same  age  group,  as  length  of  residence  in- 
creases. The  rate  of  histoplasmin  sensitivity  in  life-residents  varies 
from  13  to  58%  among  6  year  olds  and  68  to  92%  among  19  year  olds,  de- 
pending on  location  of  residence.   The  Central  area  of  the  Isthmus 
showed  the  highest  sensitivity  rates  followed  by  the  Pacific  and  Atlantic 
areas. 

A  completed  survey  of  631  pre-school  children  on  the  Atlantic  side 
of  the  Isthmus  provides  data  on  children  from  6  months  to  six  years  of 
age.   The  results  of  this  study  indicate  a  steady  increase  in  the  rate 
of  hypersensitivity  to  histoplasmin  from  approximately  3  years  to  6 
years  of  age  when  the  results  dovetail  with  the  results  obtained  in 
the  school  system  on  the  Atlantic  side.   A  similar  study  is  currently 
under  way  at  Hospital  del  Nino  to  further  augment  the  data  on  the  younger 
children.   This  survey  has  been  in  progress  for  11  months,  and  results 
on  approximately  800  children  are  available. 

Ecological  and  epidemiological  studies  resulted  in  the  isolation 
of  _H.  capsulatum  from  8  soil  samples:  one  was  from  the  residence  of  a 
clinical  case  of  histoplasmosis;  one  from  a  U.S.  Army  training  area; 
another  from  the  sparsely  populated  remote  province  of  Darien,  and  four 

60 

2  Serial  No.  NIAID-108 


from  below  bat  roosts  in  a  cave  frequented  by  Canal  Zone  residents.   The 
remaining  isolation  was  from  a  repeatedly  positive  tree  buttress  and  con- 
firms the  ability  of  H.  capsulatum  to  propagate  in  this  site  through 
both  rainy  and  dry  seasons.   This  brings  the  total  number  of  isolations 
from  soil  up  to  sixteen,  representing  six  widely  separated  locations  on 
the  Isthmus  of  Panama. 

The  trapping  of  wild  animals  continued  this  year  and  H.  capsulatum 
v/as  recovered  from  the  livers  and  spleens  of  5  spiney  rats  and  9  oppos- 
sums  trapped  on  the  Atlantic  side.  The  area  of  trapping  is  extensively 
utilized  by  the  U.S.  Army  as  a  training  site  and  is  the  same  area  where 
H.  capsulatum  was  isolated  from  soil. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

As  would  be  expected  the  research  program  has  resulted  In  a  greater 
local  awareness  of  histoplasmosis  in  all  of  its  clinica;  forms,  as  evi- 
denced by  recognition  of  three  disseminated  cases  (two  fatal  and  one 
successfuly  treated)  within  a  period  of  eighteen  months.   Previously, 
only  one  fatal  case  had  been  described  since  Darling's  original  cases 
in  1906. 

It  is  hoped  that  the  current  studies  on  ecology  and  epidemiology 
will  elucidate  the  clinical  cases  of  this  disease,  frequently  misdiag- 
nosed as  tuberculosis,  carcinoma  and  FUO,  among  military  and  civilian 
personnel  and  their  dependents  in  this  strategically  important  area. 

The  study  offers  an  opportunity  to  study  the  organism  in  a  tropical 
climate  where  the  ecology  may  be  considerably  different  from  the  United 
States. 

Proposed  Course  of  the  Project; 

Clinical,  ecological  and  epidemiological  investigations  will  be 
continued  utilizing  methods  and  techniques  now  employed,  as  well  as 
those  being  developed  in  this  laboratory  and  elsewhere,  until  either  the 
major  objectives  are  achieved  or  the  studies  become  obviously  unproductive. 


61 

Serial  No.  NIAID-108 


Serial  No.  NIAIO-108 

1.  Tropical  Virology 

2.  Mycology  Section,  MARU 

3.  Panama,  Canal  Zone 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  I960 

Part  B:     Honors,  Awards,  and  Publications 


Publications  other  than  abstracts  from  this  project: 

Abildgaard,  C.  F.  and  Taylor  R.  L. 

Generalized  Histoplasmosis  in  a  Panamanian  Infant:  Case  Report 

Am.  Jour,  of  Trop.  Med.  &  Hyg.  %:   400-01,  (July)  i960 

Taylor,  R.  L.  and  Oobrovolny,  C.  G. 

The  Distribution  of  Histoplasmin  Sensitivity  in  Guatemala 

Am.  Jour,  of  Trop.  Med.  &  Hyg.  2.:   518-22  (Sept.)  i960 


Honors  and  Awards  relating  to  this  project: 
None 


62 


Serial  No.  NIAID-109 

1.  Tropical  Virology 

2.  Mycology  Section,  MARU 

3.  Panama  Canal  Zone 


PHS-NIH 
Individual    Project  Report 
Calendar  Year   i960 


Part  A: 


Project  Title:   Studies  of  Superficial  and  Deep  Mycoses  In  Panama 
and  Central  America 

Principal  Investigator:  Capt.  L.  Taylor,  MSC,  R&D  Command,  U.S.  Army 

Other  Investigators:     Capt.  A.  W.  McFadden,  MC,  Gorgas  Hospital 


Cooperating  Units: 


Gorgas  Hospital,  C.Z. 

U.  S.  Military  Dispensaries,  C.Z. 

Veterinary  Division,  Health  Bureau,  C.Z. 

Raymond  Clinic,  R.de  P. 

Santo  Tomas  Hospital,  R.de  P. 

Man  Years  (calendar  year  I960) 
Total:  1/2 

Professional:     \/k 
Other:  \/k 

Project  Description: 

Objectives: 

To  establish:  1)  the  nature  of  common  superficial  mycoses  In 
the  tropics  and  the  efficacy  of  newer  therapeutic  regimens;  2)  the 
extent  and  types  of  deep  mycoses  and  3)  to  correlate  the  abundant 
airborne  mold  spores  to  respiratory  allergy. 

Methods  Employed: 

Standard  cultural  diagnostic  assistance  has  been  made  avail- 
able to  local  physicians  and  medical  facilities  where  patients 
with  mycoses  are  commonly  seen.  A  survey  of  the  airborne  molds  of 
Panama  was  conducted  by  regularly  exposing  Petri  dishes  with  medium 
at  six  specified  locations  for  one  year.  Hairbait  cultures  were 
utilized  for  recovery  of  keratophilic  fungi  from  soil  samples  which 
are  being  processed  for  Histoplasma  capsulatum. 


Part  B  Included: 


No 


63 


Major  Findings: 

1.  Airborne  fungi  in  Panama.   A  one  year  survey  of  low  altitude 
mold  spores  was  completed  this  year.   Plates  were  exposed  twice  weekly 
at  six  locations  on  the  Pacific  side  of  the  Isthmus.   Over  3,000  colo-. 
nies  were  identified:   Hormodendrum  predominated,  followed  by 

Asper igi 1 lus  and  Penici 1 1 ium  species.   The  total  counts  showed  a  seasonal 
variation  with  a  peak  in  July  and  August  and  a  low  from  November  to 
February.   Meteorological  data  were  obtained  and  a  correlation  of  total 
counts  with  relative  humidity,  temperature,  rainfall  and  wind  velocity 
wtl 1  be  attempted. 

2.  Griseofulvln  Therapy.  Therapy  of  chronic  derma tophytos is  with 
griseofulvin  is  being  conducted  by  Dr.  A.  W.  McFadden,  dermatologist, 
Gorgas  Hospital,  with  MARU  furnishing  cultural  diagnosis  and  follow-up. 
The  patients  selected  are  primarily  those  with  recalcitrant  nail  Infec- 
tions. The  causative  agent  Is  determined  prior  to  therapy  and  the  pa- 
tient is  followed  by  a  series  of  clinical  laboratory  tests  and  additional 
cultures  to  determine  toxicity  and  efficacy  of  the  drug.   Excel  lent "cl I- 
nical  results  have  been  obtained  to  date;  however,  no  new  findings  on 
the  use  of  the  drug  are  anticipated. 

3.  Therapy  of  Moniliasis.   A  "blind"  comparative  clinical  evalua- 
tion of  Amphotericin  B  and  Mycostatin  for  monilial  infections  is  being 
conducted  by  Dr.  A.  W.  McFadden  and  the  physicians  at  the  military  dis- 
pensaries.  All  cultural  material  is  submitted  to  MARU  for  identifica- 
tion of  the  causative  agent.   Preliminary  results  indicate  one  of  the 
two  drugs  to  be  more  effective  (the  code  has  not  been  broken).   This 
study  has  brought  out  the  major  importance  of  moniliasis  in  a  population 
living  in  the  tropics. 

k.      Hair-bait  Culture  of  Soils.   Microsporum  gypseum  has  been  isol- 
ated repeatedly  from  soil  using  the  hair-bait  culture  technique.   No 
clinical  correlation  has  been  attempted. 

5.  Epidemiology  of  Animal  and  Human  Dermatophytosis.   The  occur- 
rence of  dermatophytosis  in  animals  and  their  role  in  human  infection 

is  being  studied  in  collaboration  with  the  Canal  Zone  Veterinary  Clinic. 
Several  isolations  have  been  made  from  clinical  infections  in  animals  with 
only  one  highly  suspicious  but  unproved  case  of  transmission  to  humans. 

6.  Deep  Mycoses.   The  utilization  of  MARU's  cultural  diagnostic 
facilities  by  Dr.  C.  Calero  (Raymond  Clinic  in  Panama)  resulted  in  con- 
firmation of  one  case  of  cervico-facial  actinomycosis.   This  success 
may  arouse  further  interest  in  diagnosis  of  other  mycoses  previously 
unrecognized  in  this  area. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 

As  a  by-product  of  the  long-range  major  histoplasmosis  project 
there  are  opportunities  to  explore  other  mycological  problems.  While 
much  of  this  information  can  be  classified  as  "geographical  pathology", 
some  of  it  may  be  obviously  important  to  public  health  authorities  of 

2  Q l|  Serial  No.  NIAID-109 


the  area  while  Interesting  leads  of  general  concern  may  be  uncovered. 
Proposed  Course  of  the  Project; 

The  course  of  these  studies  is  determined  by  the  time  and  personnel 
available  as  well  as  the  relative  importance  of  the  findings  to  National 
public  health  authorities  of  Panama  and  the  U.S.  military  personnel  and 
dependents  in  the  area. 


65 

Serial  No.  NIAID-109 


LABORATORY  OF  GERMFREE  ANIMAL  RESEARCH 


S  ummary . 


1 


110  -  Pathogenesis  of  Amoebiasis 5 

111  -  The  Use  of  Germfree  Animal  and  Tissue  for 

the  Study  of  Viruses 8 

112  -  Biology  of  Germfree  Animals 10 

113  -  Origin  of  Anti-Human  Blood  Group  B 

Agglutinins  in  White  Leghorn  Chicks 14 

114  -  Studies  on  Bacterial  Interactions  and 

Host  Bacteria  Relations  in  the  Germfree 
Animal 16 

115  -  Behavior  of  Parasitic  Protozoa  in 

Germfree  Hosts 18 

116  -  Studies  on  Helminthic  Infections  in 

Germfree  Hosts 20 


Laboratory  of  Germfree  Animal  Research 

National  Institute  of  Allergy  and  Infectious  Diseases 

Summary  statement  of  research  progress,  calendar  year  1960 

Program  developments  and  trends: 

During  the  year,  Laboratory  plans  to  broaden  the  scope  of  its 
research  activities  showed  considerable  and  rapid  progress.  Authorization 
to  occupy  completely  the  south  end  of  the  third  floor  of  Building  8, 
following  the  departure  of  DBS  from  this  space,  was  finally  clarified.   The 
contract  for  the  remodeling  of  this  area  was  awarded  and  renovations  got 
underway  early  in  the  Fall.   It  is  anticipated  that  this  space  will  be 
ready  for  occupancy  (according  to  the  schedule)  around  March  or  April  of 
1961.   Following  the  transfer  of  LGAR  personnel  to  the  south  end,  alterations 
in  the  north  end,  converting  the  entire  area  into  space  for  germfree  animal 
units  and  maintenance,  will  commence.   This  will  be  effecte-l  slowly  by  NIH 
personnel,  with  the  view  of  disrupting  normal  activities  as  little  as 
possible.   When  this  is  accomplished,  additional  germfree  animal  units  will 
be  obtained  and  put  into  operation. 

As  a  part  of  the  broadening  in  research  scope,  it  was  deemed  advisable 
to  strengthen  the  program  by  the  addition  of  pathology  and  virology  activities, 
The  staffing  for  this  has  proceeded  well.   Of  the  5  new  positions  authorized 
by  the  Scientific  Director  up  through  the  fiscal  year  ending  July  1961, 
personnel  for  4  are  already  committed.   The  new  personnel  will  enter  on  duty 
in  the  Spring  when  the  space  is  available.   For  the  pathology  group, 
Dr.  Edwin  Lerner,  Senior  Surgeon,  will  transfer  from  NIAMD  to  our  Laboratory. 
Dr.  Fred  Gorstein,  S.  A.  Surgeon,  who  has  had  training  in  pathology  will 
enter  on  duty  about  July  1.   Technical  assistance  for  these  people  has  been 
arranged  for.   The  only  position  outstanding  for  which  a  candidate  is  not 
yet  lined  up  is  the  virologist.   Efforts  are  currently  being  made  to  locate 
one  at  the  level  of  a  GS-11  or  12. 

The  alteration  of  the  air  conditioning  system  for  other  institutes 
on  the  first,  and  especially  the  second,  floor  during  the  last  five  or  six 
months  of  the  year  has  caused  some  inconvenience  especially  with  noise, 
vibration  and  occasional  shutting  down  of  a  utility.   However,  the  research 
program  continued  at  a  satisfactory  pace  in  spite  of  some  of  these  drawbacks. 
As  was  true  of  last  year,  the  program  reflected  an  increased  variety  of 
interests  and  an  increase  in  the  number  of  cooperative  projects  planned  or 
underway  with  other  laboratories,  and  even  other  institutes.  As  examples, 
we  have  recently  undertaken  on  a  limited  scale  with  Dr.  Rowe,  LID,  serologic 
and  tissue  studies  for  the  possible  presence  of  viruses  in  our  germfree 
animal  colony.   Such  data  will  establish  baselines  for  future  studies  with 
virus-free  animals,  or,  if  it  turns  out  that  way,  animals  containing  a  few 
known  viruses.   We  have,  in  collaboration  with  Dr.  Landy  of  the  NCI, 
initiated  studies  on  the  origin  and  specificity  of  natural  antibodies  to 
enteric  bacteria,  and  similar  studies  in  connection  with  Staphylococcus  with 
the  CDC.   Experiments  in  germfree  animals  would  appear  to  provide  the  crucial 


-  1 


information  in  these  areas.  Also,  such  studies  can  provide  information  on 
the  nature  of  the  mechanism  whereby  endotoxin  alters  resistance  to  infection 
with  gram  negative  pathogens.   With  Doctors  Kelly  and  O'Gara,  also  of  the 
NCI,  we  have  initiated  on  a  small,  but  satisfactory,  scale  a  study  to 
ascertain  whether  the  incidence  of  chemically- induced  lung  tumors  is  the 
same  in  germfree  mice  as  in  conventional  mice  of  the  same  strain.   In  view 
of  the  large  numbers  of  viruses  demonstrated  as  occurring  in  experimental 
mice,  (Huebner,  Rowe) ,  the  question  arises  whether  the  chemicals  induce  the 
tumors  by  activating  viruses,  or  some  other  living  agents  that  are  present 
in  the  animal.   Such  studies  in  an  organism- free  host  could  provide  rather 
significant  leads  in  the  approach  to  understanding  tumor-virus  relations. 

The  apparent  increase  in  the  interest  in  germfree  animals  in  medical 
research  stimulated  a  request  by  Dr.  Peterson  to  have  a  film  made  on  this 
subject  for  use  by  medical  schools,  universities,  research  institutions, etc. 
The  Audio-Visual  Aids  section  of  the  CDC  came  to  the  Laboratory  and  we  made 
a  19-minute  color  movie.  The  popularity  of  the  subject  matter  is  pointed 
up  by  the  fact  that,  recently,  all  28  copies  of  the  film  which  CDC  had  in 
its  library  were  booked  up  on  loan  for  a  month  in  advance. 

Significant  scientific  advances: 

With  regard  to  the  progress  in  some  of  the  research  projects,  an 
interesting  series  of  observations  has  been  made  by  Mr.  Phillips  on  the 
behavior  of  Entamoeba  histolytica  in  the  germfree  host.   It  is  to  be  recalled 
that,  in  earlier  studies  with  standardized  techniques,  amoebic  lesions  were 
not  produced  in  the  germfree  animal  following  inoculation.   In  fact,  the 
parasite  failed  to  live  in  the  intestine  beyond  5  days.  Recent  changes  have 
been  made  in  the  manner  of  rearing  and  handling  the  amoebae  In  vitro  prior 
to  inoculation  which  seemed  to  result  in  more  vigorous  organisms.   The  latter 
have  produced  lesions  in  the  absence  of  bacteria,  although  the  type  and 
severity  are  still  not  typical  of  those  encountered  with  a  bacterial  associate- 
Thus,  it  would  appear  that  the  latter  is  not  the  only  determinant  of  the 
course  and  the  pathogenesis  of  the  infection. 

In  collaborative  studies  with  Dr.  Weinstein  of  LPD,  we  have  shown 
that  the  intestinal  mouse  parasite,  Nematospiroides  dubius,  does  not  require 
a  flora  to  develop  from  an  infective  larva  to  the  adult  form  in  the  host. 
However,  it  apparently  does  require  bacteria,  or  their  products,  to  develop 
from  the  egg  to  infective  larva.   These  studies  are  preparatory  to  those  to 
be  undertaken  in  an  analysis  of  the  nature  of  the  nutritional  effects 
observed  in  certain  parasitisms.   One  of  the  most  interesting  observations 
has  been  our  finding  that  the  sex  of  the  host,  which  has  been  noted  by 
several  workers  to  affect  the  outcome  of  the  infection  in  conventional 
(contaminated)  animals,  has  not  appeared  to  be  an  influence  in  the  germfree 
host.   If  these  findings  continue  to  hold  up,  a  hitherto  unrecognized  role 
(either  direct  or  indirect)  of  the  flora  in  certain  observed  sex  effects 
may  unfold. 

In  studies  on  the  growth  and  biology  of  germfree  guinea  pigs, 
Dr.  Horton  has  obtained  several  advanced  pregnancies  in  animals  maintained 


on  irradiated  diets,  although  no  fetus  was  carried  to  term.   It  is  to  be 
recalled  that  germfree  guinea  pigs  have  not  been  bred  with  any  success. 
The  importance  of  the  intestinal  flora  to  this  species  was  pointed  up  by 
the  finding  that  conventional  (contaminated)  guinea  pigs  reproduced  normally 
on  this  same  irradiated  diet. 

In  a  collaborative  project  with  Dr.  Springer  of  the  University  of 
Pennsylvania,  Dr.  Horton  has  also  shown  that  the  use  of  large  dosages  of  a 
cathartic,  or  the  application  of  tourniquet  shock,  increased  the  number 
of  red  cells  of  germfree  chickens  coated  with  human  B-like  antigens  following 
mono-infection  with  E.  coli  086-  These  studies  are  providing  information 
on  the  manner  in  which  red  cells  of  one  type  may  acquire  antigenic  character- 
istics of  other  cell  types,  especially  B. 

Our  germfree  mouse  colony  has  been  undergoing  an  intensive  serologic 
study  from  several  points  of  view.  One  of  the  most  interesting  has  been 
an  assay  for  the  presence  of  certain  so-called  "natural  antibodies"  against 
a  variety  of  bacteria.  Such  antibodies  or  antibody- like  re? -t:ivities  for 
organisms  like  Staphylococcus,  E.  coli  and  S_.  typhosa  have  been  found  to 
occur  in  a  variety  of  uninoculated  conventional  animals  and  are  presumed 
to  originate  from  encounters  with  the  viable  organisms  or  related  antigens. 
Animals  which  have  lived  for  many  generations  free  from  contact  with  live 
bacteria  are  almost  the  sine  qua  non  for  establishing  finally  the  validity 
of  these  ideas.   Studies  thus  far,  with  the  CDC  and  with  Dr.  Landy  of  NCI, 
have  shown  the  germfree  animal  to  be  singularly  free  from  antibody-like 
reactivity  toward  Staphylococcus  and  E.  coli,  but  reactivity  toward  S_.  typhosa 
was  obtained  in  several  instances.   We  are,  of  course,  unable  to  find 
evidence  of  the  presence  of  the  latter  in  the  germfree  colony.  Thus,  this 
finding  strengthens  sporadic  reports  that  non-bacterial  substances  (perhaps 
in  this  case  dietary  components)  can  cause  "cross"  reactions  with  this 
organism. 

We  have  now  had  our  germfree  animal  colony  and  a  conventional  colony 
derived  from  the  same  stock  for  approximately  two  years .   Some  of  our  ex- 
breeders  which  we  try  to  keep,  in  spite  of  the  scarcity  of  germfree  unit 
animal  space,  are  of  the  order  of  1-2  years  of  age.  We  have  made  it  a 
practice  that  whenever  a  germfree  or  conventional  animal  not  on  an  experiment 
dies,  especially  if  it  is  6  months  or  more  of  age,  it  is  checked  thoroughly 
for  gross  evidence  of  malformations  or  tumors.  As  of  now,  we  have  informa- 
tion on  a  total  of  more  than  50  animals .   It  is  of  interest  that  we  have 
found  so-called  spontaneous  lung  tumors  in  the  germfree  as  well  as  the 
conventional  mice.  Also,  while  the  numbers  of  animals  are  obviously 
relatively  small,  the  incidence  has  been  markedly  higher,  thus  far,  among 
the  conventional  animals,  i.e.,  those  exposed  to  external  contamination, 
than  among  the  germfree.   This  has  been  particularly  true  among  animals 
6-12  months  of  age.  These  observations  have  been  discussed  with  Doctors 
Kelly  and  O'Gara  of  the  NCI  who  find  them  of  considerable  interest.   Such 
data  will  continue  to  be  collected  and  will  serve  as  corollary  information 
along  with  experiments  in  which  such  tumors  are  induced  chemically  in  both 
germfree  and  contaminated  animals.   If  this  difference  in  incidence  continues, 
it  could  point  up  further  the  importance  of  microbial  agents  per  se,  or 


3  - 


stresses   -oduced  by  such  agents,  in  the  induction  of  these  tumors.  While 
it  is,  v-   ourse,  too  soon  to  say  much  in  this  regard,  the  possibilities 
suggests   jy  even  this  limited  information  are  very  intriguing. 


Serial  No.   NIAID-110 

1.  Germfree  Animal  Research 

2.  Pathogenesis 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:   Pathogenesis  of  amoebiasis 

Principal  Investigator:   Bruce  P.  Phillips 

Other  Investigators:   None 

Cooperating  Units:   NIAID-127-B 

Man  Years  (calendar  year  1960): 
Total:  1  3/4 

Professional:  3/4 

Other :  1 

Project  Description: 

Objectives: 

To  study  the  etiology,  pathogenesis,  and  pathology  of 
intestinal  amoebiasis  with  a  view  toward  ascertaining  the  role  of 
the  associated  intestinal  flora. 

Methods  Employed: 

Guinea  pigs  are  inoculated  intracecally  with  Entamoeba 
histolytica  trophozoites  cultivated  in  vitro  without  bacteria. 
Conventional,  germfree  and  ex-germfree  animals  with  various  known 
single  or  multiple  bacterial  infestations  are  used,  and  the  disease 
processes  produced,  if  any,  are  compared  grossly  and  histologically. 
Modifications  of  techniques  for  cultivating  and  harvesting  the 
amoebae  are  also  employed  in  an  effort  to  obtain  maximum  virulence. 

Major  Findings: 

Amoebic  lesions  did  not  occur  in  germfree  animals  which 
received  amoebic  inocula  cultivated  and  prepared  by  previously 
standardized  procedures,  even  though  such  inocula  regularly  pro- 
duced amoebic  ulceration  in  conventional  hosts.   Amoebic  ulceration 
varying  in  extent  and  severity  did  occur  following  introduction  of 
similar  amoebic  inocula  into  ex-germfree  animals  harboring  each  of 
the  following  bacteria  as  a  monocontaminant:   Escherichia  coli, 
Aerobacter  aerogenes,  Streptococcus  faecalis,  Bacillus  subtilis, 

-  1  -  * 


Serial  No.  NIAID-110 

Lactobacillus  acidophilus,  Staphylococcus  aureus,  Micrococcus  sp. 
(from  conventional  guinea  pig) .  More  recently,  amoebic  lesions 
have  been  produced  in  germfree  animals  following  intracecal 
inoculation  of  E.  histolytica  cultivated  and  harvested  by  newer 
procedures  developed  as  a  part  of  these  investigations .  However, 
these  lesions  were  not  typical  of  those  observed  in  animals 
harboring  bacteria. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

The  wide  range  of  host-parasite  relationships  which  characterize 
enteric  amoebic  infection  have  long  been  a  matter  of  considerable 
scientific  interest.  The  demonstration  of  bacterial  influence  on 
development  of  the  disease,  although  as  yet  incomplete,  may  provide 
at  least  partial  explanation  for  the  diverse  manifestations  of 
amoebic  infection  which  range  from  asymptomatic  infestation  to  acute, 
sometimes  fatal  ulcerative  enteritis.  Recent  success  in  producing 
enteric  lesions  in  germfree  animals  following  inoculation  of  very 
large  members  of  vigorous  amoebae  has  not  altered  the  concept  that 
bacterial  participation  is  essential  for  the  development  of 
symptomatic  intestinal  amoebiasis.   It  does  provide,  however,  an 
opportunity  to  study  intestinal  tissue  changes  resulting  from  the 
activity  of  E_.  histolytica,  alone,  disassociated  from  all  bacterial 
influence. 

Proposed  Course  of  the  Project: 

With  reference  to  the  recent  finding  that  amoebic  lesions  can 
be  produced  in  germfree  hosts,  there  are  two  areas  in  particular 
that  require  clarification.  First,  since  the  lesions  appear  to 
emanate  possibly  from  the  site  of  the  puncture  wound  resulting  from 
injecting  inoculum  through  the  cecal  wall,  it  may  be  that  such  trauma 
is  a  prerequisite.   Secondly,  since  E.  histolytica  has  not  been 
cultivated  axenically,  our  amoebic  inocula  contain  invertebrate  forms 
of  Trypanosoma  cruzi,  with  which  amoebae  are  grown  ±n  vitro.  The 
possibility  exists,  therefore,  that  the  flagellates  may  participate 
in  the  development  of  amoebic  lesions  in  the  germfree  animals.   Efforts 
will  be  directed  toward  ascertaining  whether  either,  or  both,  of 
these  points  is  a  factor. 

Part  B  Included:   Yes 


2  - 


Serial  No.   NIAID-110 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B.   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  Project: 

Phillips,  Bruce  P.,  and  Wolfe,  P.  A.:   Pneumonic  disease  in  germfree 
animals.   J.  Inf.  Dis .   (In  press) 


Honors  and  Awards  relating  to  this  project:   None 


3  - 


Serial  No.   NIAID-111 

1.  Germfree  Animal  Research 

2.  Pathogenesis 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:   The  use  of  germfree  animals  and  tissues  for  the 
study  of  viruses 

Principal  Investigator:   Walter  L.  Newton 

Other  Investigators:   Charles  Rosenberger,  Laboratory  of  Tropical 
Virology 
Wallace  P.  Rowe,  Laboratory  of  Infectious 
Diseases 

Cooperating  Units:   NIAID- 

Man  Years  (calendar  year  1960) : 
Total:  1 

Professional:  1/4 

Other:  3/4 

Project  Description: 

Objectives: 

To  explore  the  possibility  that  germfree  animals  and  tissues 
might  serve  as  particularly  favorable  experimental  tools  for  certain 
aspects  of  virus  study.   To  ascertain  whether  viruses  are  present 
in  the  germfree  mouse  colony. 

Methods  Employed: 

Comparative  studies  are  carried  out  on  the  relative  susceptibili- 
ties of  germfree  and  conventional  mice  to  infection  with  certain 
viruses.   Cultures  prepared  from  germfree  animal  tissues  are  also 
used.   Sera  are  examined  for  evidence  of  antibodies  to  viruses. 
Standard  virologic  techniques  are  employed. 

Major  Findings: 

1.   Preparations  containing  Dengue  type  I  virus  (Mochizuki 
strain)  continued  to  cause  much  more  CPE  in  kidney  tissue  cultures 
prepared  from  germfree  mice  than  in  cultures  prepared  from 
conventional  mouse  kidney.   However,  in  recent  animal  tests  to 

8 
-  i  - 


Serial  No.  NIAID-111 

ascertain  whether  an  increase  in  viral  growth  was  associated  with 
the  difference  in  CPE,  no  evidence  of  such  growth  was  obtained. 
Furthermore,  essentially  no  CPE  was  obtained  following  inoculation 
of  the  same  material  into  either  cell  type.   Efforts  are  being 
directed  toward  resolving  this  apparent  inconsistency. 

2.   Sera  from  several  germfree  mice  1  year  or  more  of  age  have 
been  examined  for  evidence  of  the  presence  of  certain  mouse  viruses. 
Numbers  are  too  few  for  negatives  to  be  conclusive,  but  thus  far  no 
positives  for  polyoma  and  mouse  adenovirus  have  been  obtained. 
Evidence  for  the  presence  of  Reo  3  is  questionable.   However,  there 
is  good  evidence  that  K  virus  may  be  present. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

The  study  of  viruses  in  animals  without  the  poss  ioility  of 
influence  of  other  concomitant  infections,  or  with  known  infections, 
should  provide  special  insight  into  virus-host  relations.  Also, 
with  the  increase  in  the  number  of  viruses  shown  to  occur  in  the 
conventional  mouse,  animals  with  a  defined  viral  status  and  for 
which  exposure  to  infection  can  be  controlled  become  increasingly 
more  valuable.  This  is  particularly  true  in  view  of  the  current 
interest  in  tumor-virus  studies. 

Proposed  Course  of  the  Project: 

Efforts  will  be  directed  toward  clarifying  the  variation  in  the 
behavior  of  dengue  virus  in  the  germfree  animal  tissue  culture.  Also, 
further  serological  and  tissue  analysis  of  the  viral  state  of  the 
germfree  mouse  colony  will  continue. 

Part  B  Included:   No 


• 


2  - 


Serial  No.   NIAID-112 

1.  Germfree  Animal  Research 

2.  Biology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  A: 

Project  Title:   Biology  of  germfree  animals 

Principal  Investigator:  Richard  E.  Horton 

Other  Investigators:   Walter  L.  Newton 

William  B.  DeWitt,  Laboratory  of  Parasitic 

Diseases 
N.  A.  Jaworski  and  John  L.  S.  Hickey,  Division 

of  Research  Services 

Cooperating  Units:   DRS  6.4 

Man  Years  (calendar  year  1960) : 
Total:  2% 

Professional:         1% 
Other:  1 

Proiect  Description: 


To  obtain  descriptive  bioj.ugn.cii  aaca  on  germfree  animals  for 
the  purposes  of  determining  in  what  manner  they  may  differ  from  the 
conventional  animal,  and  establishing  baseline  values  for  other 
studies  employing  these  animals. 

Methods  Employed: 

Comparative  studies  are  made  on  germfree,  specifically- 
contaminated,  and  conventional  animals  in  such  areas  as  hematology, 
blood  chemistry,  serology,  gross  and  microscopic  anatomy,  growth 
curves,  longevity,  etc.   Standard  laboratory  techniques  are  employed. 

Major  Findings: 

In  efforts  to  improve  germfree  guinea  pigs  by  maintaining  them 
on  diets  sterilized  by  irradiation  instead  of  steam,  some  success 
was  obtained  with  the  use  of  irradiated  semisynthetic  diet.   However, 
neither  conventional  nor  germfree  animals  on  irradiated  diet  grew 
as  well  as  conventionals  on  non-irradiated  diet.   It  was  observed 


10 


Serial  No.   NIAID-112 

that  the  growth  rate  of  germfree  animals  reared  on  a  semisynthetic 
diet  sterilized  by  2  million  rad  was  greater  than  that  of  animals 
reared  on  the  same  diet  sterilized  by  3  million  rad.   Irradiation 
has  an  obvious  detrimental  effect  on  the  nutrition  adequacy  of  the 
diet. 

Five  pregnancies  have  been  observed  in  adult  germfree  guinea 
pigs  maintained  on  the  irradiated  ration.  None  of  the  females  was 
able  to  carry  the  fetuses  to  term;  they  usually  aborted  near  the 
5th  or  6th  week  of  gestation.   In  most  instances,  procidentia  of 
the  uterus  was  a  sequela  that  eventually  led  to  the  death  of  the 
animal.  The  administration  of  additional  amounts  of  Vitamins  K  and 
E  to  the  last  two  pregnant  animals  did  not  appear  to  alter  the  course 
of  their  pregnancies.  This  failure  to  reproduce  has  not  been 
observed  in  the  colony  of  conventional  guinea  pigs  maintained  on  the 
same  sterilized  diet  for  four  generations.  Histology :al  examination 
of  several  of  the  older  germfree  guinea  pigs  (approximately  a  year 
old)  reared  on  the  irradiated  diet  has  revealed  fatty  degeneration 
of  the  liver. 

Further  study  of  the  serum  proteins  in  germfree  and  conventional 
mice  has  provided  data  similar  to  those  reported  earlier  with  germ- 
free guinea  pigs:   Gamma  globulin  levels  in  the  germfree  were  the 
same  as  those  in  mice  from  a  conventional  colony  maintained  on  the 
same  sterilized  diet,  but  both  groups  showed  values  lower  than  those 
of  mice  from  the  NIH  conventional  colony  which  is  maintained  on  a 
non-sterilized  diet. 

Data  are  being  accumulated  on  the  incidence  of  tumors  (especially 
lung)  in  our  germfree  and  conventional  mice  (the  same  genetic  stock) 
dying  or  sacrificed  after  6  months  of  age.  Lung  tumors  have  been 
found  in  the  germfree  mice,  but  there  is  some  evidence  that  the 
incidence  is  lower  than  in  their  counterparts  exposed  to  the  outside 
environment.   The  possibility  of  this  interesting  difference  will 
continue  to  be  explored: 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute; 

Studies  of  this  nature  provide  baseline  data  for  current  and 
future  experiments  that  utilize  the  germfree  animal.   Also,  they 
provide  an  opportunity  to  study  the  role  that  the  "normal"  flora  may 
play  in  establishing  hematological  and  serological  values  and  general 
growth,  longevity,  and  fecundity. 

Proposed  Course  of  the  Project; 

Studies  will  continue  along  essentially  the  same  lines.   Where 
differences  between  the  germfree  and  the  conventional  "contaminated" 
animals  are  encountered,  efforts  will  be  made  to  establish  whether 

11 


Serial  No.  NIAID-112 

fundamental  principles  are  involved,  and  whether  a  particular 
difference  is  worth  further  exploitation.   Also,  attempts  will  be 
made  (through  the  use  of  hormones,  vitamins,  etc.)  to  determine 
reasons  for  the  germfree  guinea  pig's  failure  to  reproduce 
satisfactorily. 

Part  B  Included:  Yes 


12 


Serial  No.  NIAID-112 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B.   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  Project: 


Horton,  R.  E.  and  Hickey,  J.  L.  S.:   Irradiated  Diets  for  Rearing  Germfree 
Guinea  Pigs.   Proc.  of  Animal  Care  Panel.   (In  press) 

Newton,  Walter  L . ,  Pennington,  Robert  M.  and  Lieberman,  Jacob  E.:   Compara- 
tive Hemolytic  Ccnolement  Activities  of  Germfree  and  Conventional 
Guinea  Pig  Serum.   Proc.  Soc.  for  Exper .  Biol,  and  Med.,  1960,  Vol.  104, 
Pp.  486-488. 

Baer,  Paul  N.,  and  Newton,  Walter  L.:   Studies  on  Periodontal  Disease  in 
the  Mouse.   III.   The  Germ-Free  Mouse  and  Its  Conventional  Control.  Oral 
Surg.,  Oral  Med.  and  Oral  Path.,  Vol.  13,  No.  9,  Pp  1134-1144,  Sep.  1960. 


Honors  and  Awards  relating  to  this  project: 

Dr.  Newton  was  invited  to  present  a  paper  on  the  work  on  gamma  globulin  in 
germfree  guinea  pigs  at  a  special  session  of  the  Fifth  International 
Congress  on  Nutrition. 


13 


Serial  No.   NIAID-113 

1.  Germfree  Animal  Research 

2.  Biology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:   Origin  of  Anti-Human  Blood  Group  B  Agglutinins  in 
White  Leghorn  Chicks 

Principal  Investigator:   Richard  E.  Horton 

Other  Investigators:   G.  F.  Springer,  University  of  Pennsylvania 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960) : 
Total:  1 

Professional:  1/4 

Other:  3/4 

Project  Description: 

Objectives: 

To  determine  if  the  blood  group  B  active  polysaccharide  of 
E.  coli  Oq/-  B:7  is  absorbed  onto  the  erythrocytes  of  monocontaminated 
chicks  harboring  the  organism  in  the  intestinal  tract. 

Methods  Employed: 

Groups  of  germfree  and  conventional  White  Leghorn  chicks  are 
inoculated  orally  with  E.  coli  086  B:7  at  selected  ages.  Techniques 
which  increase  permeability  of  bacterial  endotoxin  across  the 
intestinal  wall  are  employed:   the  induction  of  chemical  enteritis 
by  prolonged  feeding  of  drastic  cathartics;  and  the  induction  of 
tourniquet  shock.   Blood  samples  are  taken  shortly  after  the  period 
of  physical  stress  by  cardiac  puncture.   Standard  serological 
testing  procedures  are  used  to  determine  the  presence  of  group  B 
active  polysaccharide  on  erythrocytes  and  the  titer  of  anti  B 
agglutinins  in  blood  samples. 

Major  Findings: 

In  vitro  studies  have  shown  that  erythrocytes  of  White  Leghorn 
chicks  are  easily  and  irreversibly  coated  with  blood  group  B  active 
bacterial  products.  We  have  found  that  erythrocytes  of  the  germfree 

14 


Serial  No.   NIAID-113 

chicks  used  in  our  studies  do  not  contain  blood  group  B-like  antigen. 
When  healthy  germfree  chicks  are  infected  with  E.  coli  086>  only  a 
minority  of  the  animals  acquire  B-like  antigens  on  the  red  cells. 
When  a  series  of  large  doses  of  a  cathartic  (cascara  sagrada)  is 
fed  to  the  E.  coli  Ogg  infected  chicks,  the  proportion  of  animals 
having  antigen  coated  red  cells  is  increased.  Tests  made  on  blcod 
samples  taken  several  hours  after  E.  coli  0g6  infected  chicks  have 
been  subjected  to  tourniquet  shock  show  that  almost  all  chicks 
possess  varying  amounts  of  antigen-coated  erythrocytes.  Two  to  three 
days  after  application  of  tourniquet  shock,  the  anti-B  titer  of 
these  animals  was  found  to  rise  considerably,  but  there  was  no 
demonstrable  decrease  in  coated  erythrocytes. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

Studies  of  this  nature  may  help  explain  the  mec.i.mism  by  which 
human  patients  with  severe  intestinal  disorders  (e.g.  cancer  of  the 
colon,  incarcerated  hernia,  etc.)  acquire  a  transitory  B  antigen  on 
their  erythrocytes  and  the  altered  polyagglutinability  of  those  red 
cells  which  accompany  this  phenomenon. 

Proposed  Course  of  the  Project: 

In  vivo  studies  will  be  conducted  to  determine  extent  of  coating 
of  the  erythrocytes  with  B  antigen  when  germfree  chicks  and  chicks 
monocontaminated  with  E.  coli  0g6  are  given  purified  B-substance 
parenterally .  When  these  results  have  been  obtained,  further  work 
on  this  project  will  be  terminated. 

Part  B  Included:   No 


15 


Serial  No.   NIAID-114 

1.  Germfree  Animal  Research 

2.  Biology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:  Studies  on  bacterial  interactions  and  host 
bacteria  relations  in  the  germfree  animal 

Principal  Investigator:  Norman  S.  Ikari 

Other  Investigators:   Walter  L.  Newton 

Maurice  Landy,  National  Cancel,  institute 

Cooperating  Units:   CDC,  Atlanta,  Georgia 

Man  Years  (calendar  year  1960): 
Total:  1  1/4 

Professional:  1/2 

Other:  3/4 

Project  Description: 

Objectives: 

To  determine  whether  certain  bacterial  phenomena  observed  in 
vitro,  e.g.  genetic  recombination  between  two  enteric  strains  of 
bacteria,  occur  within  the  intestine  of  the  living  mammalian  host. 
To  evaluate  host  responses  to  particular  bacterial  species  without 
interference  from,  and  in  association  with,  other  known  organisms. 

Methods  Employed: 

In  vitro  studies  have  shown  that  the  ability  to  produce  colicine, 
an  antibiotic-like  substance  lethal  to  some  enteric  bacteria,  can  be 
genetically  transferred  from  one  bacterial  strain  to  another.   In  our 
studies,  E.  coli  K  12  Row  (streptomycin-resistant,  colicine-sensitive) 
is  established  in  the  germfree  mouse  intestinal  tract  by  inoculation 
of  the  water  bottles.  Later,  Paracolon  CA62  (streptomycin-sensitive, 
colicine-positive  (col+))  is  fed  by  mouth  tube,  and  fecal  pellets  are 
collected  at  intervals  thereafter.   Saline  dilutions  of  these  pellets 
are  assayed  for  col+  colonies  by  plating  onto  streptomycin  agar  plates 

Sera  of  germfree  and  variously  contaminated  mice  are  compared 
for  levels  of  antibody  activity  towards  specific  organisms  using 
Ouchterlony  and  CF  techniques. 

16 


Serial  No.  NIAID-114 
Major  Findings: 

Col+,  streptomycin-resistant  hybrids  resembling  the  E.  coli 
K  12  parent  were  obtained  at  every  sampling  for  periods  ranging  from 
24  hours  to  one  month  after  the  Paracolon  feeding.   Further  examina- 
tion of  the  col+  colonies  confirmed  the  stability  of  this  transfer 
and  revealed  the  possibility  of  an  additional  change(s)  in  these 
hybrids  not  previously  shown  in  _in  vitro  studies.   Sharp  dichotomy 
was  noted  between  germfree  and  conventional  animals  with  respect  to 
staphylococcal  antibody.  No  antigen-antibody  lines  were  noted  in 
the  germfree  serum  diffused  against  Cowan  I  soluble  antigens,  whereas 
one  or  more  lines  were  obtained  with  conventional  mouse  serum. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute; 

Studies  on  whether  genetic  recombination  and  ouar  bacterial 
interactions  observed  in  the  test  tube  occur  in  natural  ecological 
surroundings  can  provide  information  on  ways  in  which  a  host  may 
affect  these  phenomena.  Also,  since  backgrounds  of  "normal"  antibodies 
(e.g.,  to  organisms  like  staphylococcus)  that  are  often  observed  in 
conventional  animals  can  complicate  attempts  at  serologic  typing  and 
fluorescent  antibody  study,  the  potential  value  of  the  germfree 
animal  for  such  studies  is  well  worth  exploring. 

Proposed  Course  of  the  Project; 

Other  known  jin  vitro  genetic  recombination  systems  will  be 
attempted  in  the  germfree  animal.   The  finding  of  large  numbers  of 
very  mucoid  variants  completely  different  from  either  parental  type 
or  the  col+  hybrids  has  suggested  possible  studies  in  a  different 
direction. 

Thorough  analysis  of  the  germfree  mouse  for  the  presence  of 
antibody  or  antibody-like  reactivity  to  a  variety  of  organisms, 
especially  gram  negatives,  will  continue. 

Part  B  Included:   No 


17 

2  - 


Serial  No.  NIAID-115 

1.  Germfree  Animal  Research 

2.  Pathogenesis 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:   Behavior  of  parasitic  protozoa  in  germfree  hosts 

Principal  Investigator:   Walter  L.  Newton 

Other  Investigators:   Bruce  P.  Phillips 

Lucy  V.  Reardon,  Laboratory  of  Parasitic 
Diseases 

Cooperating  Units:   NIAID-127-C 

Man  Years  (calendar  year  1960); 
Total:  1% 

Professional:  ^ 

Other:  3/4 

Project  Description: 

Objectives: 

To  determine  whether  the  presence  of  bacteria  affect  the  course 
of,  and  response  of  the  host  to,  protozoan  infections  other  than 
those  caused  by  E.  histolytica. 

Methods  Employed: 

Germfree,  monocontaminated,  and  conventional  guinea  pigs  are 
inoculated  with  axenically-reared  or  micro-isolated  species  of 
Trichomonas,  Trypanosoma,  or  Giardia.   The  inoculated  animals  are 
examined  for  the  presence  and  type  of  lesions,  time  of  death,  etc. 

Major  Findings: 

It  has  been  shown  previously  that  T.  vaginalis,  when  injected 
subcutaneous ly,  soon  disappears  in  conventional  guinea  pigs,  but 
multiplies  and  produces  a  severe  lesion  in  germfree  animals.   However, 
when  the  germfree  animal  is  orally  contaminated  with  even  a  single 
species  of  bacteria,  its  response  is  like  the  conventional  animal.   In 
recent  studies,  the  infection  has  been  followed  in  the  germfree  animals 
until  subsidence  --  often  requiring  several  weeks.   When  such  animals 
are  later  re-exposed  to  the  parasite,  the  pattern  has  been  like  that 
in  the  conventional  animal.   The  encounter  with  the  infection,  even 

.  i-  13 


Serial  No.  NIAID-115 

though  it  eventually  disappeared  (and  the  animal  became  "germfree" 
again),  seemed  to  activate  a  defensive  response  which  persisted. 

Techniques  to  inoculate  germfree  animals  with  sterile  Giardia 
cysts  have  been  worked  out.   Preliminary  attempts  at  infection  have 
not  been  successful,  although  the  numbers  of  organisms  inoculated 
have  been  small,  thus  far. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

Studies  of  this  type  can  lead  to  a  better  understanding  of  the 
possible  effects  of  the  "normal  flora"  in  maintaining  a  host's 
natural  defensive  mechanisms.   It  would  appear  that  this  system 
(involving  a  host  and  an  organism  to  which  it  is  resistant  in  the 
conventional  but  not  the  germfree  state)  provides  a  good  tool  for 
such  studies.  Also,  the  role  of  the  intestinal  flor*  in  the  patho- 
genesis of  a  variety  of  protozoal  diseases  has  yet  to  be  established. 

Proposed  Course  of  the  Project: 

Additional  experiments  involving  the  "conditioning"  of  germfree 
animals  with  non-living  stimuli  such  as  dead  bacteria,  egg  albumen, 
etc.,  prior  to  challenging  with  T.  vaginalis  are  planned.  A  compara- 
tive study  of  the  tissue  responses  in  the  conditioned  and  unconditioned 
germfree  animal  is  planned.   Attempts  at  establishing  other  parasitic 
protozoa  in  germfree  animals  will  continue. 

Part  B  Included:   No 


« 


Serial  No.  NIAID-116 

1.  Germfree  Animal  Research 

2.  Pathogenesis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  A: 

Project  Title:   Studies  on  helminthic  infections  in  germfree  hosts 

Principal  Investigator:   Walter  L.  Newton 

Other  Investigators:   Paul  P.  Weinstein,  Laboratory  of  Parasitic 
Diseases 

Cooperating  Units:   NIAID-121-G 

Man  Years  (calendar  year  1960) : 
Total:  1% 

Professional:         % 
Other :  1 

Project  Description: 

Objectives: 

To  ascertain  what  effect  the  bacteria  and  other  organisms 
normally  present  in  the  conventional  animal  have  upon  the  course  of 
helminth  infections,  and  upon  the  host's  response  to  these  parasites. 

Methods  Employed: 

Germfree  animals  are  fed  or  inoculated  with  sterilized  eggs,  or 
axenically-reared  infective  larvae,  of  various  parasitic  helminths. 
The  development  of  these  parasites  is  followed  by  established  pro- 
cedures, and  lungs,  intestine,  and  other  appropriate  tissues  are 
examined  histologically  for  the  study  of  host  response.   The  findings 
are  compared  with  those  obtained  in  conventional  controls. 

Major  Findings: 

Mature,  fertile  adult  worms  were  obtained  in  germfree  as  well  as 
conventional  mice,  inoculated  with  infective  larvae  of  Nematospiroides 
dubius.   This  indicated  that  the  lack  of  bacteria  appeared  to  have 
little  or  no  effect  on  the  development  of  the  parasite  from  the 
infective  larva  to  the  adult  stage  in  the  host. 

However,  development  of  the  parasite  from  the  egg  to  infective 
larva  in  feces  from  germfree  mice  was  extremely  poor.   Fatty  degenera- 

20 
-  i  - 


Serial  No.  NIAID-116 

tion  not  unlike  that  associated  with  B-vitamin  deficiency  occurred. 
If  living  bacteria  from  a  conventional  animal  were  added  to  the 
germfree  feces,  development  of  the  larvae  progressed  normally. 
Apparently,  feces  without  bacteria  fail  to  provide  certain  essentials 
for  development  of  the  larvae. 

Of  special  interest  was  the  apparent  indication  that  the  sex  of 
the  host,  which  is  a  factor  in  parasitism  in  the  conventional  animal, 
was  unimportant  among  the  germfree  animals.   In  the  conventional 
animals,  males  had  2-3  times  the  worm  burden  at  necropsy  that  the 
females  had.  Among  the  germfree  animals,  however,  average  worm  counts 
were  about  the  same  in  both  sexes. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute; 

The  fact  that  bacteria,  per  se,  are  apparently  ™ot  required  for 
full  development  of  some  parasites  opens  the  way  for  other  studies  in 
the  areas  of  nutrition  and  parasitism,  chemotherapy,  in  vitro  culti- 
vation, and  physiologic  studies.  Also,  opportunity  is  provided  for 
the  study  of  eggs  and  larvae  in  fresh  (non-sterilized)  feces  without 
bacteria,  and  perhaps  to  ascertain  factors  contributed  by  the  latter 
toward  their  proper  development.   If  the  sex  phenomenon  holds  up  in 
future  tests,  interesting  and  potentially  important  relationships 
may  be  uncovered. 

Proposed  Course  of  the  Project: 

Of  course,  the  validity  and  significance  of  the  difference  in 
the  host  sex  effect  between  germfree  and  conventional  mice  will  be 
studied.   Efforts  to  complete  the  entire  life  cycle  of  the  parasite 
in  a  sterile  environment  will  be  continued.   Attempts  may  be  made  to 
ascertain  the  role  of  bacteria  in  certain  parasite-diet  relationships 
noted  in  conventional  mice. 

Part  B  Included:   No 


21 


LABORATORY  OF  GERMFREE  ANIMAL  RESEARCH 


Summary 1 

110  -  Pathogenesis  of  Amoebiasis 5 

111  -  The  Use  of  Germfree  Animal  and  Tissue  for 

the  Study  of  Viruses 8 

112  -  Biology  of  Germfree  Animals 10 

113  -  Origin  of  Anti-Human  Blood  Group  B 

Agglutinins  in  White  Leghorn  Chicks 14 

114  -  Studies  on  Bacterial  Interactions  and 

Host  Bacteria  Relations  in  the  Germfree 
Animal 16 

115  -  Behavior  of  Parasitic  Protozoa  in 

Germfree  Hosts 18 

116  -  Studies  on  Helminthic  Infections  in 

Germfree  Hosts 20 


Laboratory  of  Germfree  Animal  Research 

National  Institute  of  Allergy  and  Infectious  Diseases 

Summary  statement  of  research  progress,  calendar  year  1960 

Program  developments  and  trends: 

During  the  year,  Laboratory  plans  to  broaden  the  scope  of  its 
research  activities  showed  considerable  and  rapid  progress.  Authorization 
to  occupy  completely  the  south  end  of  the  third  floor  of  Building  8, 
following  the  departure  of  DBS  from  this  space,  was  finally  clarified.  The 
contract  for  the  remodeling  of  this  area  was  awarded  and  renovations  got 
underway  early  in  the  Fall.   It  is  anticipated  that  this  space  will  be 
ready  for  occupancy  (according  to  the  schedule)  around  March  or  April  of 
1961.   Following  the  transfer  of  LGAR  personnel  to  the  south  end,  alterations 
in  the  north  end,  converting  the  entire  area  into  space  for  germfree  animal 
units  and  maintenance,  will  commence.  This  will  be  effected  slowly  by  NIH 
personnel,  with  the  view  of  disrupting  normal  activities  as  little  as 
possible.   When  this  is  accomplished,  additional  germfree  animal  units  will 
be  obtained  and  put  into  operation. 

As  a  part  of  the  broadening  in  research  scope,  it  was  deemed  advisable 
to  strengthen  the  program  by  the  addition  of  pathology  and  virology  activities, 
The  staffing  for  this  has  proceeded  well.   Of  the  5  new  positions  authorized 
by  the  Scientific  Director  up  through  the  fiscal  year  ending  July  1961, 
personnel  for  4  are  already  committed.   The  new  personnel  will  enter  on  duty 
in  the  Spring  when  the  space  is  available.   For  the  pathology  group, 
Dr.  Edwin  Lerner,  Senior  Surgeon,  will  transfer  from  NIAMD  to  our  Laboratory. 
Dr.  Fred  Gorstein,  S.  A.  Surgeon,  who  has  had  training  in  pathology  will 
enter  on  duty  about  July  1.   Technical  assistance  for  these  people  has  been 
arranged  for.   The  only  position  outstanding  for  which  a  candidate  is  not 
yet  lined  up  is  the  virologist.   Efforts  are  currently  being  made  to  locate 
one  at  the  level  of  a  GS-11  or  12. 

The  alteration  of  the  air  conditioning  system  for  other  institutes 
on  the  first,  and  especially  the  second,  floor  during  the  last  five  or  six 
months  of  the  year  has  caused  some  inconvenience  especially  with  noise, 
vibration  and  occasional  shutting  down  of  a  utility.   However,  the  research 
program  continued  at  a  satisfactory  pace  in  spite  of  some  of  these  drawbacks. 
As  was  true  of  last  year,  the  program  reflected  an  increased  variety  of 
interests  and  an  increase  in  the  number  of  cooperative  projects  planned  or 
underway  with  other  laboratories,  and  even  other  institutes.   As  examples, 
we  have  recently  undertaken  on  a  limited  scale  with  Dr.  Rowe,  LID,  serologic 
and  tissue  studies  for  the  possible  presence  of  viruses  in  our  germfree 
animal  colony.   Such  data  will  establish  baselines  for  future  studies  with 
virus-free  animals,  or,  if  it  turns  out  that  way,  animals  containing  a  few 
known  viruses.   We  have,  in  collaboration  with  Dr.  Landy  of  the  NCI, 
initiated  studies  on  the  origin  and  specificity  of  natural  antibodies  to 
enteric  bacteria,  and  similar  studies  in  connection  with  Staphylococcus  with 
the  CDC.   Experiments  in  germfree  animals  would  appear  to  provide  the  crucial 


-  1 


information  in  these  areas.  Also,  such  studies  can  provide  information  on 
the  nature  of  the  mechanism  whereby  endotoxin  alters  resistance  to  infection 
with  gram  negative  pathogens.   With  Doctors  Kelly  and  O'Gara,  also  of  the 
NCI,  we  have  initiated  on  a  small,  but  satisfactory,  scale  a  study  to 
ascertain  whether  the  incidence  of  chemically- induced  lung  tumors  is  the 
same  in  germfree  mice  as  in  conventional  mice  of  the  same  strain.   In  view 
of  the  large  numbers  of  viruses  demonstrated  as  occurring  in  experimental 
mice,  (Huebner,  Rowe) ,  the  question  arises  whether  the  chemicals  induce  the 
tumors  by  activating  viruses,  or  some  other  living  agents  that  are  present 
in  the  animal.   Such  studies  in  an  organism- free  host  could  provide  rather 
significant  leads  in  the  approach  to  understanding  tumor-virus  relations. 

The  apparent  increase  in  the  interest  in  germfree  animals  in  medical 
research  stimulated  a  request  by  Dr.  Peterson  to  have  a  film  made  on  this 
subject  for  use  by  medical  schools,  universities,  research  institutions, etc. 
The  Audio-Visual  Aids  section  of  the  CDC  came  to  the  Laboratory  and  we  made 
a  19-minute  color  movie.   The  popularity  of  the  subject  matter  is  pointed 
up  by  the  fact  that,  recently,  all  28  copies  of  the  film  whx^h  CDC  had  in 
its  library  were  booked  up  on  loan  for  a  month  in  advance. 

Significant  scientific  advances: 

With  regard  to  the  progress  in  some  of  the  research  projects,  an 
interesting  series  of  observations  has  been  made  by  Mr.  Phillips  on  the 
behavior  of  Entamoeba  histolytica  in  the  germfree  host.   It  is  to  be  recalled 
that,  in  earlier  studies  with  standardized  techniques,  amoebic  lesions  were 
not  produced  in  the  germfree  animal  following  inoculation.   In  fact,  the 
parasite  failed  to  live  in  the  intestine  beyond  5  days.  Recent  changes  have 
been  made  in  the  manner  of  rearing  and  handling  the  amoebae  in   vitro  prior 
to  inoculation  which  seemed  to  result  in  more  vigorous  organisms.   The  latter 
have  produced  lesions  in  the  absence  of  bacteria,  although  the  type  and 
severity  are  still  not  typical  of  those  encountered  with  a  bacterial  associate. 
Thus,  it  would  appear  that  the  latter  is  not  the  only  determinant  of  the 
course  and  the  pathogenesis  of  the  infection. 

In  collaborative  studies  with  Dr.  Weinstein  of  LPD,  we  have  shown 
that  the  intestinal  mouse  parasite,  Nematospiroides  dubius,  does  not  require 
a  flora  to  develop  from  an  infective  larva  to  the  adult  form  in  the  host. 
However,  it  apparently  does  require  bacteria,  or  their  products,  to  develop 
from  the  egg  to  infective  larva.   These  studies  are  preparatory  to  those  to 
be  undertaken  in  an  analysis  of  the  nature  of  the  nutritional  effects 
observed  in  certain  parasitisms.  One  of  the  most  interesting  observations 
has  been  our  finding  that  the  sex  of  the  host,  which  has  been  noted  by 
several  workers  to  affect  the  outcome  of  the  infection  in  conventional 
(contaminated)  animals,  has  not  appeared  to  be  an  influence  in  the  germfree 
host.   If  these  findings  continue  to  hold  up,  a  hitherto  unrecognized  role 
(either  direct  or  indirect)  of  the  flora  in  certain  observed  sex  effects 
may  unfold. 

In  studies  on  the  growth  and  biology  of  germfree  guinea  pigs, 
Dr.  Horton  has  obtained  several  advanced  pregnancies  in  animals  maintained 


-  2 


on  irradiated  diets,  although  no  fetus  was  carried  to  term.   It  is  to  be 
recalled  that  germfree  guinea  pigs  have  not  been  bred  with  any  success. 
The  importance  of  the  intestinal  flora  to  this  species  was  pointed  up  by 
the  finding  that  conventional  (contaminated)  guinea  pigs  reproduced  normally 
on  this  same  irradiated  diet. 

In  a  collaborative  project  with  Dr.  Springer  of  the  University  of 
Pennsylvania,  Dr.  Horton  has  also  shown  that  the  use  of  large  dosages  of  a 
cathartic,  or  the  application  of  tourniquet  shock,  increased  the  number 
of  red  cells  of  germfree  chickens  coated  with  human  B-like  antigens  following 
mono- infection  with  E.  coli  086-  These  studies  are  providing  information 
on  the  manner  in  which  red  cells  of  one  type  may  acquire  antigenic  character- 
istics of  other  cell  types,  especially  B. 

Our  germfree  mouse  colony  has  been  undergoing  an  intensive  serologic 
study  from  several  points  of  view.  One  of  the  most  interesting  has  been 
an  assay  for  the  presence  of  certain  so-called  "natural  antibodies"  against 
a  variety  of  bacteria.  Such  antibodies  or  antibody- like  re£'_^ivities  for 
organisms  like  Staphylococcus,  E.  coli  and  S.  typhosa  have  been  found  to 
occur  in  a  variety  of  uninoculated  conventional  animals  and  are  presumed 
to  originate  from  encounters  with  the  viable  organisms  or  related  antigens. 
Animals  which  have  lived  for  many  generations  free  from  contact  with  live 
bacteria  are  almost  the  sine  qua  non  for  establishing  finally  the  validity 
of  these  ideas.   Studies  thus  far,  with  the  CDC  and  with  Dr.  Landy  of  NCI, 
have  shown  the  germfree  animal  to  be  singularly  free  from  antibody- like 
reactivity  toward  Staphylococcus  and  E.  coli,  but  reactivity  toward  S_.  typhosa 
was  obtained  in  several  instances.   We  are,  of  course,  unable  to  find 
evidence  of  the  presence  of  the  latter  in  the  germfree  colony.  Thus,  this 
finding  strengthens  sporadic  reports  that  non-bacterial  substances  (perhaps 
in  this  case  dietary  components)  can  cause  "cross"  reactions  with  this 
organism. 

We  have  now  had  our  germfree  animal  colony  and  a  conventional  colony 
derived  from  the  same  stock  for  approximately  two  years .   Some  of  our  ex- 
breeders  which  we  try  to  keep,  in  spite  of  the  scarcity  of  germfree  unit 
animal  space,  are  of  the  order  of  1-2  years  of  age.  We  have  made  it  a 
practice  that  whenever  a  germfree  or  conventional  animal  not  on  an  experiment 
dies,  especially  if  it  is  6  months  or  more  of  age,  it  is  checked  thoroughly 
for  gross  evidence  of  malformations  or  tumors .  As  of  now,  we  have  informa- 
tion on  a  total  of  more  than  50  animals .   It  is  of  interest  that  we  have 
found  so-called  spontaneous  lung  tumors  in  the  germfree  as  well  as  the 
conventional  mice.  Also,  while  the  numbers  of  animals  are  obviously 
relatively  small,  the  incidence  has  been  markedly  higher,  thus  far,  among 
the  conventional  animals,  i.e.,  those  exposed  to  external  contamination, 
than  among  the  germfree.   This  has  been  particularly  true  among  animals 
6-12  months  of  age.  These  observations  have  been  discussed  with  Doctors 
Kelly  and  O'Gara  of  the  NCI  who  find  them  of  considerable  interest.   Such 
data  will  continue  to  be  collected  and  will  serve  as  corollary  information 
along  with  experiments  in  which  such  tumors  are  induced  chemically  in  both 
germfree  and  contaminated  animals.   If  this  difference  in  incidence  continues, 
it  could  point  up  further  the  importance  of  microbial  agents  per  ^e,  or 


stresses  produced  by  such  agents,  in  the  induction  of  these  tumors.  While 
it  is,  of  course,  too  soon  to  say  much  in  this  regard,  the  possibilities 
suggested  by  even  this  limited  information  are  very  intriguing. 


4  - 


Serial  No.   NIAID-110 

1.  Germfree  Animal  Research 

2.  Pathogenesis 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:   Pathogenesis  of  amoebiasis 

Principal  Investigator:   Bruce  P.  Phillips 

Other  Investigators:   None 

Cooperating  Units:   NIAID-127-B 

Man  Years  (calendar  year  1960): 
Total:  1  3/4 

Professional:         3/4 
Other:  1 

Project  Description: 

Objectives: 

To  study  the  etiology,  pathogenesis,  and  pathology  of 
intestinal  amoebiasis  with  a  view  toward  ascertaining  the  role  of 
the  associated  intestinal  flora. 

Methods  Employed: 

Guinea  pigs  are  inoculated  intracecally  with  Entamoeba 
histolytica  trophozoites  cultivated  jLn  vitro  without  bacteria. 
Conventional,  germfree  and  ex-germfree  animals  with  various  known 
single  or  multiple  bacterial  infestations  are  used,  and  the  disease 
processes  produced,  if  any,  are  compared  grossly  and  histologically. 
Modifications  of  techniques  for  cultivating  and  harvesting  the 
amoebae  are  also  employed  in  an  effort  to  obtain  maximum  virulence. 

Major  Findings: 

Amoebic  lesions  did  not  occur  in  germfree  animals  which 
received  amoebic  inocula  cultivated  and  prepared  by  previously 
standardized  procedures,  even  though  such  inocula  regularly  pro- 
duced amoebic  ulceration  in  conventional  hosts .   Amoebic  ulceration 
varying  in  extent  and  severity  did  occur  following  introduction  of 
similar  amoebic  inocula  into  ex-germfree  animals  harboring  each  of 
the  following  bacteria  as  a  monocontaminant:   Escherichia  coli, 
Aerobacter  aerogenes,  Streptococcus  faecalis,  Bacillus  subtilis, 

5 


Serial  No.  NIAID-110 

Lactobacillus  acidophilus,  Staphylococcus  aureus,  Micrococcus  sp. 
(from  conventional  guinea  pig).  More  recently,  amoebic  lesions 
have  been  produced  in  germfree  animals  following  intracecal 
inoculation  of  E.  histolytica  cultivated  and  harvested  by  newer 
procedures  developed  as  a  part  of  these  investigations.  However, 
these  lesions  were  not  typical  of  those  observed  in  animals 
harboring  bacteria. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

The  wide  range  of  host-parasite  relationships  which  characterize 
enteric  amoebic  infection  have  long  been  a  matter  of  considerable 
scientific  interest.  The  demonstration  of  bacterial  influence  on 
development  of  the  disease,  although  as  yet  incomplete,  may  provide 
at  least  partial  explanation  for  the  diverse  manifestations  of 
amoebic  infection  which  range  from  asymptomatic  infestation  to  acute, 
sometimes  fatal  ulcerative  enteritis.  Recent  success  in  producing 
enteric  lesions  in  germfree  animals  following  inoculation  of  very 
large  members  of  vigorous  amoebae  has  not  altered  the  concept  that 
bacterial  participation  is  essential  for  the  development  of 
symptomatic  intestinal  amoebiasis.   It  does  provide,  however,  an 
opportunity  to  study  intestinal  tissue  changes  resulting  from  the 
activity  of  E.  histolytica,  alone,  disassociated  from  all  bacterial 
influence. 

Proposed  Course  of  the  Project: 

With  reference  to  the  recent  finding  that  amoebic  lesions  can 
be  produced  in  germfree  hosts,  there  are  two  areas  in  particular 
that  require  clarification.  First,  since  the  lesions  appear  to 
emanate  possibly  from  the  site  of  the  puncture  wound  resulting  from 
injecting  inoculum  through  the  cecal  wall,  it  may  be  that  such  trauma 
is  a  prerequisite.   Secondly,  since  E.  histolytica  has  not  been 
cultivated  axenically,  our  amoebic  inocula  contain  invertebrate  forms 
of  Trypanosoma  cruzi,  with  which  amoebae  are  grown  ±n  vitro.  The 
possibility  exists,  therefore,  that  the  flagellates  may  participate 
in  the  development  of  amoebic  lesions  in  the  germfree  animals.   Efforts 
will  be  directed  toward  ascertaining  whether  either,  or  both,  of 
these  points  is  a  factor. 

Part  B  Included:   Yes 


-  2 


Serial  No.  NIAID-110 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B.   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  Project: 

Phillips,  Bruce  P.,  and  Wolfe,  P.  A.:  Pneumonic  disease  in  germfree 
animals.   J.  Inf.  Dis .   (In  press) 


Honors  and  Awards  relating  to  this  project:  None 


Serial  No.  NIAID-111 

1.  Germfree  Animal  Research 

2.  Pathogenesis 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:   The  use  of  germfree  animals  and  tissues  for  the 
study  of  viruses 

Principal  Investigator:   Walter  L.  Newton 

Other  Investigators:   Charles  Rosenberger,  Laboratory  of  Tropical 
Virology 
Wallace  P.  Rowe,  Laboratory  of  Infectious 
Diseases 

Cooperating  Units:   NIAID- 

Man  Years  (calendar  year  1960) : 
Total:  1 

Professional:  1/4 

Other :  3/4 

Project  Description: 

Objectives: 

To  explore  the  possibility  that  germfree  animals  and  tissues 
might  serve  as  particularly  favorable  experimental  tools  for  certain 
aspects  of  virus  study.   To  ascertain  whether  viruses  are  present 
in  the  germfree  mouse  colony. 

Methods  Employed: 

Comparative  studies  are  carried  out  on  the  relative  susceptibili 
ties  of  germfree  and  conventional  mice  to  infection  with  certain 
viruses.   Cultures  prepared  from  germfree  animal  tissues  are  also 
used.   Sera  are  examined  for  evidence  of  antibodies  to  viruses. 
Standard  virologic  techniques  are  employed. 

Major  Findings: 

1.   Preparations  containing  Dengue  type  I  virus  (Mochizuki 
strain)  continued  to  cause  much  more  CPE  in  kidney  tissue  cultures 
prepared  from  germfree  mice  than  in  cultures  prepared  from 
conventional  mouse  kidney.   However,  in  recent  animal  tests  to 

8 


Serial  No.  NIAID-111 

ascertain  whether  an  increase  in  viral  growth  was  associated  with 
the  difference  in  CPE,  no  evidence  of  such  growth  was  obtained. 
Furthermore,  essentially  no  CPE  was  obtained  following  inoculation 
of  the  same  material  into  either  cell  type.   Efforts  are  being 
directed  toward  resolving  this  apparent  inconsistency. 

2.   Sera  from  several  germfree  mice  1  year  or  more  of  age  have 
been  examined  for  evidence  of  the  presence  of  certain  mouse  viruses. 
Numbers  are  too  few  for  negatives  to  be  conclusive,  but  thus  far  no 
positives  for  polyoma  and  mouse  adenovirus  have  been  obtained. 
Evidence  for  the  presence  of  Reo  3  is  questionable.   However,  there 
is  good  evidence  that  K  virus  may  be  present. 

Signif icanct:  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

The  study  of  viruses  in  animals  without  the  possJ-ility  of 
influence  of  other  concomitant  infections,  or  with  known  infections, 
should  provide  special  insight  into  virus-host  relations.  Also, 
with  the  increase  in  the  number  of  viruses  shown  to  occur  in  the 
conventional  mouse,  animals  with  a  defined  viral  status  and  for 
which  exposure  to  infection  can  be  controlled  become  increasingly 
more  valuable.  This  is  particularly  true  in  view  of  the  current 
interest  in  tumor-virus  studies . 

Proposed  Course  of  the  Project; 

Efforts  will  be  directed  toward  clarifying  the  variation  in  the 
behavior  of  dengue  virus  in  the  germfree  animal  tissue  culture.  Also, 
further  serological  and  tissue  analysis  of  the  viral  state  of  the 
germfree  mouse  colony  will  continue. 

Part  B  Included:   No 


Serial  No.   NIAID-112 

1.  Germfree  Animal  Research 

2.  Biology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  A; 

Project  Title:   Biology  of  germfree  animals 

Principal  Investigator:  Richard  E.  Horton 

Other  Investigators:  Walter  L.  Newton 

William  B.  DeWitt,  Laboratory  of  Parasitic 

Diseases 
N.  A.  Jaworski  and  John  L.  S.  Hickey,  Division 

of  Research  Services 

Cooperating  Units:   DRS  6.4 

Man  Years  (calendar  year  1960) : 
Total:  2\ 

Professional:         1% 
Other :  1 

Proiect  Description: 


To  obtain  descriptive  bioiogicai  aaca  on  germfree  animals  for 
the  purposes  of  determining  in  what  manner  they  may  differ  from  the 
conventional  animal,  and  establishing  baseline  values  for  other 
studies  employing  these  animals. 

Methods  Employed: 

Comparative  studies  are  made  on  germfree,  specifically- 
contaminated,  and  conventional  animals  in  such  areas  as  hematology, 
blood  chemistry,  serology,  gross  and  microscopic  anatomy,  growth 
curves,  longevity,  etc.   Standard  laboratory  techniques  are  employed. 

Major  Findings: 

In  efforts  to  improve  germfree  guinea  pigs  by  maintaining  them 
on  diets  sterilized  by  irradiation  instead  of  steam,  some  success 
was  obtained  with  the  use  of  irradiated  semisynthetic  diet.   However, 
neither  conventional  nor  germfree  animals  on  irradiated  diet  grew 
as  well  as  conventionals  on  non- irradiated  diet.   It  was  observed 


10 


Serial  No.   NIAID-112 

that  the  growth  rate  of  germfree  animals  reared  on  a  semisynthetic 
diet  sterilized  by  2  million  rad  was  greater  than  that  of  animals 
reared  on  the  same  diet  sterilized  by  3  million  rad.   Irradiation 
has  an  obvious  detrimental  effect  on  the  nutrition  adequacy  of  the 
diet. 

Five  pregnancies  have  been  observed  in  adult  germfree  guinea 
pigs  maintained  on  the  irradiated  ration.   None  of  the  females  was 
able  to  carry  the  fetuses  to  term;  they  usually  aborted  near  the 
5th  or  6th  week  of  gestation.   In  most  instances,  procidentia  of 
the  uterus  was  a  sequela  that  eventually  led  to  the  death  of  the 
animal.  The  administration  of  additional  amounts  of  Vitamins  K  and 
E  to  the  last  two  pregnant  animals  did  not  appear  to  alter  the  course 
of  their  pregnancies .  This  failure  to  reproduce  has  not  been 
observed  in  the  colony  of  conventional  guinea  pigs  maintained  on  the 
same  sterilized  diet  for  four  generations.  Histolog-!  ral  examination 
of  several  of  the  older  germfree  guinea  pigs  (approximately  a  year 
old)  reared  on  the  irradiated  diet  has  revealed  fatty  degeneration 
of  the  liver. 

Further  study  of  the  serum  proteins  in  germfree  and  conventional 
mice  has  provided  data  similar  to  those  reported  earlier  with  germ- 
free guinea  pigs:   Gamma  globulin  levels  in  the  germfree  were  the 
same  as  those  in  mice  from  a  conventional  colony  maintained  on  the 
same  sterilized  diet,  but  both  groups  showed  values  lower  than  those 
of  mice  from  the  NIH  conventional  colony  which  is  maintained  on  a 
non-sterilized  diet. 

Data  are  being  accumulated  on  the  incidence  of  tumors  (especially 
lung)  in  our  germfree  and  conventional  mice  (the  same  genetic  stock) 
dying  or  sacrificed  after  6  months  of  age.  Lung  tumors  have  been 
found  in  the  germfree  mice,  but  there  is  some  evidence  that  the 
incidence  is  lower  than  in  their  counterparts  exposed  to  the  outside 
environment.   The  possibility  of  this  interesting  difference  will 
continue  to  be  explored: 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

Studies  of  this  nature  provide  baseline  data  for  current  and 
future  experiments  that  utilize  the  germfree  animal.   Also,  they 
provide  an  opportunity  to  study  the  role  that  the  "normal"  flora  may 
play  in  establishing  hematological  and  serological  values  and  general 
growth,  longevity,  and  fecundity. 

Proposed  Course  of  the  Project: 

Studies  will  continue  along  essentially  the  same  lines.   Where 
differences  between  the  germfree  and  the  conventional  "contaminated" 
animals  are  encountered,  efforts  will  be  made  to  establish  whether 

11 


Serial  No.  NIAID-112 

fundamental  principles  are  involved,  and  whether  a  particular 
difference  is  worth  further  exploitation.  Also,  attempts  will  be 
made  (through  the  use  of  hormones,  vitamins,  etc.)  to  determine 
reasons  for  the  germfree  guinea  pig's  failure  to  reproduce 
satisfactorily. 

Part  B  Included:  Yes 


12 

3  - 


Serial  No.  NIAID-112 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B.   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  Project: 


Horton,  R.  E.  and  Hickey,  J.  L.  S.:   Irradiated  Diets  for  Rearing  Germfree 
Guinea  Pigs-   Proc .  of  Animal  Care  Panel.   (In  press) 

Newton,  Walter  L . ,  Pennington,  Robert  M.  and  Lieberman,  Jacob  E.:   Compara- 
tive Hemolytic  Complement  Activities  of  Germfree  and  Conventional 
Guinea  Pig  Serum.  Proc.  Soc.  for  Exper .  Biol,  and  Med.,  1960,  Vol.  104, 
Pp.  486-488. 

Baer,  Paul  N.,  and  Newton,  Walter  L.:   Studies  on  Periodontal  Disease  in 
the  Mouse.   III.  The  Germ-Free  Mouse  and  Its  Conventional  Control.  Oral 
Surg.,  Oral  Med.  and  Oral  Path.,  Vol.  13,  No.  9,  Pp  1134-1144,  Sep.  I960. 


Honors  and  Awards  relating  to  this  project: 

Dr.  Newton  was  invited  to  present  a  paper  on  the  work  on  gamma  globulin  in 
germfree  guinea  pigs  at  a  special  session  of  the  Fifth  International 
Congress  on  Nutrition. 


13 


Serial  No.   NIAID-113 

1.  Germfree  Animal  Research 

2.  Biology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:   Origin  of  Anti-Human  Blood  Group  B  Agglutinins  in 
White  Leghorn  Chicks 

Principal  Investigator:   Richard  E.  Horton 

Other  Investigators:   G.  F.  Springer,  University  of  Pennsylvania 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960) : 
Total:  1 

Professional:  1/4 

Other:  3/4 

Project  Description: 

Objectives: 

To  determine  if  the  blood  group  B  active  polysaccharide  of 
E.  coli  0q£  B:7  is  absorbed  onto  the  erythrocytes  of  monocontaminated 
chicks  harboring  the  organism  in  the  intestinal  tract. 

Methods  Employed: 

Groups  of  germfree  and  conventional  White  Leghorn  chicks  are 
inoculated  orally  with  E.  coli  086  B:7  at  selected  ages.  Techniques 
which  increase  permeability  of  bacterial  endotoxin  across  the 
intestinal  wall  are  employed:   the  induction  of  chemical  enteritis 
by  prolonged  feeding  of  drastic  cathartics;  and  the  induction  of 
tourniquet  shock.   Blood  samples  are  taken  shortly  after  the  period 
of  physical  stress  by  cardiac  puncture.   Standard  serological 
testing  procedures  are  used  to  determine  the  presence  of  group  B 
active  polysaccharide  on  erythrocytes  and  the  titer  of  anti  B 
agglutinins  in  blood  samples. 

Major  Findings: 

In  vitro  studies  have  shown  that  erythrocytes  of  White  Leghorn 
chicks  are  easily  and  irreversibly  coated  with  blood  group  B  active 
bacterial  products.  We  have  found  that  erythrocytes  of  the  germfree 

14 


Serial  No.  NIAID-113 

chicks  used  in  our  studies  do  not  contain  blood  group  B-like  antigen. 
When  healthy  germfree  chicks  are  infected  with  E.  coli  086>  only  a 
minority  of  the  animals  acquire  B-like  antigens  on  the  red  cells. 
When  a  series  of  large  doses  of  a  cathartic  (cascara  sagrada)  is 
fed  to  the  E.  coli  Ogg  infected  chicks,  the  proportion  of  animals 
having  antigen  coated  red  cells  is  increased.  Tests  made  on  blood 
samples  taken  several  hours  after  E.  coli  086  infected  chicks  have 
been  subjected  to  tourniquet  shock  show  that  almost  all  chicks 
possess  varying  amounts  of  antigen-coated  erythrocytes.  Two  to  three 
days  after  application  of  tourniquet  shock,  the  anti-B  titer  of 
these  animals  was  found  to  rise  considerably,  but  there  was  no 
demonstrable  decrease  in  coated  erythrocytes. 

Significance!  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

Studies  of  this  nature  may  help  explain  the  mec'.rnism  by  which 
human  patients  with  severe  intestinal  disorders  (e.g.  cancer  of  the 
colon,  incarcerated  hernia,  etc.)  acquire  a  transitory  B  antigen  on 
their  erythrocytes  and  the  altered  polyagglutinability  of  those  red 
cells  which  accompany  this  phenomenon. 

Proposed  Course  of  the  Project: 

In  vivo  studies  will  be  conducted  to  determine  extent  of  coating 
of  the  erythrocytes  with  B  antigen  when  germfree  chicks  and  chicks 
monocontaminated  with  E.  coli  Ogg  are  given  purified  B-substance 
parenterally .  When  these  results  have  been  obtained,  further  work 
on  this  project  will  be  terminated. 

Part  B  Included:   No 


15 


Serial  No.   NIAID-114 

1.  Gennfree  Animal  Research 

2.  Biology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:   Studies  on  bacterial  interactions  and  host 
bacteria  relations  in  the  gennfree  animal 

Principal  Investigator:  Norman  S.  Ikari 

Other  Investigators:   Walter  L.  Newton 

Maurice  Landy,  National  Cancel  Institute 

Cooperating  Units:   CDC,  Atlanta,  Georgia 

Man  Years  (calendar  year  1960): 
Total:  1  1/4 

Professional:  1/2 

Other:  3/4 

Project  Description: 

Objectives: 

To  determine  whether  certain  bacterial  phenomena  observed  in 
vitro,  e.g.  genetic  recombination  between  two  enteric  strains  of 
bacteria,  occur  within  the  intestine  of  the  living  mammalian  host. 
To  evaluate  host  responses  to  particular  bacterial  species  without 
interference  from,  and  in  association  with,  other  known  organisms. 

Methods  Employed: 

In  vitro  studies  have  shown  that  the  ability  to  produce  colicine, 
an  antibiotic-like  substance  lethal  to  some  enteric  bacteria,  can  be 
genetically  transferred  from  one  bacterial  strain  to  another.   In  our 
studies,  E.  coli  K  12  Row  (streptomycin-resistant,  colicine-sensitive) 
is  established  in  the  germfree  mouse  intestinal  tract  by  inoculation 
of  the  water  bottles.   Later,  Paracolon  CA62  (streptomycin-sensitive, 
colicine-positive  (col+))  is  fed  by  mouth  tube,  and  fecal  pellets  are 
collected  at  intervals  thereafter.   Saline  dilutions  of  these  pellets 
are  assayed  for  col+  colonies  by  plating  onto  streptomycin  agar  plates, 

Sera  of  germfree  and  variously  contaminated  mice  are  compared 
for  levels  of  antibody  activity  towards  specific  organisms  using 
Ouchterlony  and  CF  techniques. 

16 
-  i  - 


Serial  No.   NIAID-114 
Major  Findings: 

Col+,  streptomycin-resistant  hybrids  resembling  the  E.  coli 
K  12  parent  were  obtained  at  every  sampling  for  periods  ranging  from 
24  hours  to  one  month  after  the  Paracolon  feeding.   Further  examina- 
tion of  the  col+  colonies  confirmed  the  stability  of  this  transfer 
and  revealed  the  possibility  of  an  additional  change(s)  in  these 
hybrids  not  previously  shown  in  j^n  vitro  studies.   Sharp  dichotomy 
was  noted  between  germfree  and  conventional  animals  with  respect  to 
staphylococcal  antibody.   No  antigen-antibody  lines  were  noted  in 
the  germfree  serum  diffused  against  Cowan  I  soluble  antigens,  whereas 
one  or  more  lines  were  obtained  with  conventional  mouse  serum. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

Studies  on  whether  genetic  recombination  and  o:.i  ar  bacterial 
interactions  observed  in  the  test  tube  occur  in  natural  ecological 
surroundings  can  provide  information  on  ways  in  which  a  host  may 
affect  these  phenomena.   Also,  since  backgrounds  of  "normal"  antibodies 
(e.g.,  to  organisms  like  staphylococcus)  that  are  often  observed  in 
conventional  animals  can  complicate  attempts  at  serologic  typing  and 
fluorescent  antibody  study,  the  potential  value  of  the  germfree 
animal  for  such  studies  is  well  worth  exploring. 

Proposed  Course  of  the  Project: 

Other  known  jLn  vitro  genetic  recombination  systems  will  be 
attempted  in  the  germfree  animal.  The  finding  of  large  numbers  of 
very  mucoid  variants  completely  different  from  either  parental  type 
or  the  col+  hybrids  has  suggested  possible  studies  in  a  different 
direction. 

Thorough  analysis  of  the  germfree  mouse  for  the  presence  of 
antibody  or  antibody-like  reactivity  to  a  variety  of  organisms, 
especially  gram  negatives,  will  continue. 

Part  B  Included:   No 


17 


Serial  No.   NIAID-115 

1.  Germfree  Animal  Research 

2.  Pathogenesis 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:   Behavior  of  parasitic  protozoa  in  germfree  hosts 

Principal  Investigator:   Walter  L.  Newton 

Other  Investigators:   Bruce  P.  Phillips 

Lucy  V.  Reardon,  Laboratory  of  Parasitic 
Diseases 

Cooperating  Units:   NIAID-127-C 

Man  Years  (calendar  year  1960); 
Total:  Ik 

Professional:         % 
Other:  3/4 

Project  Description: 

Objectives: 

To  determine  whether  the  presence  of  bacteria  affect  the  course 
of,  and  response  of  the  host  to,  protozoan  infections  other  than 
those  caused  by  E.  histolytica. 

Methods  Employed: 

Germfree,  monocontaminated,  and  conventional  guinea  pigs  are 
inoculated  with  axenically-reared  or  micro- isolated  species  of 
Trichomonas,  Trypanosoma,  or  Giardia.  The  inoculated  animals  are 
examined  for  the  presence  and  type  of  lesions,  time  of  death,  etc. 

Major  Findings: 

It  has  been  shown  previously  that  T.  vaginalis,  when  injected 
subcutaneous ly,  soon  disappears  in  conventional  guinea  pigs,  but 
multiplies  and  produces  a  severe  lesion  in  germfree  animals.  However, 
when  the  germfree  animal  is  orally  contaminated  with  even  a  single 
species  of  bacteria,  its  response  is  like  the  conventional  animal.   In 
recent  studies,  the  infection  has  been  followed  in  the  germfree  animals 
until  subsidence  --  often  requiring  several  weeks.   When  such  animals 
are  later  re-exposed  to  the  parasite,  the  pattern  has  been  like  that 
in  the  conventional  animal.   The  encounter  with  the  infection,  even 


1  - 


13 


Serial  No.  NIAID-115 

though  it  eventually  disappeared  (and  the  animal  became  "germfree" 
again),  seemed  to  activate  a  defensive  response  which  persisted. 

Techniques  to  inoculate  germfree  animals  with  sterile  Giardia 
cysts  have  been  worked  out.   Preliminary  attempts  at  infection  have 
not  been  successful,  although  the  numbers  of  organisms  inoculated 
have  been  small,  thus  far. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

Studies  of  this  type  can  lead  to  a  better  understanding  of  the 
possible  effects  of  the  "normal  flora"  in  maintaining  a  host's 
natural  defensive  mechanisms.   It  would  appear  that  this  system 
(involving  a  host  and  an  organism  to  which  it  is  resistant  in  the 
conventional  but  not  the  germfree  state)  provides  a  good  tool  for 
such  studies.  Also,  the  role  of  the  intestinal  flora  in  the  patho- 
genesis of  a  variety  of  protozoal  diseases  has  yet  to  be  established. 

Proposed  Course  of  the  Project: 

Additional  experiments  involving  the  "conditioning"  of  germfree 
animals  with  non-living  stimuli  such  as  dead  bacteria,  egg  albumen, 
etc.,  prior  to  challenging  with  T.  vaginalis  are  planned.  A  compara- 
tive study  of  the  tissue  responses  in  the  conditioned  and  unconditioned 
germfree  animal  is  planned.   Attempts  at  establishing  other  parasitic 
protozoa  in  germfree  animals  will  continue. 

Part  B  Included:   No 


19 

2  - 


Serial  No.  NIAID-116 

1.  Germfree  Animal  Research 

2.  Pathogenesis 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A: 


Project  Title:   Studies  on  helminthic  infections  in  germfree  hosts 

Principal  Investigator:   Walter  L.  Newton 

Other  Investigators:   Paul  P.  Weinstein,  Laboratory  of  Parasitic 
Diseases 

Cooperating  Units:   NIAID-121-G 

Man  Years  (calendar  year  1960) : 
Total:  1% 

Professional:         % 
Other:  1 

Project  Description: 

Objectives: 

To  ascertain  what  effect  the  bacteria  and  other  organisms 
normally  present  in  the  conventional  animal  have  upon  the  course  of 
helminth  infections,  and  upon  the  host's  response  to  these  parasites. 

Methods  Employed: 

Germfree  animals  are  fed  or  inoculated  with  sterilized  eggs,  or 
axenically-reared  infective  larvae,  of  various  parasitic  helminths. 
The  development  of  these  parasites  is  followed  by  established  pro- 
cedures, and  lungs,  intestine,  and  other  appropriate  tissues  are 
examined  histologically  for  the  study  of  host  response.   The  findings 
are  compared  with  those  obtained  in  conventional  controls. 

Major  Findings: 

Mature,  fertile  adult  worms  were  obtained  in  germfree  as  well  as 
conventional  mice,  inoculated  with  infective  larvae  of  Nematospiroides 
dubius.  This  indicated  that  the  lack  of  bacteria  appeared  to  have 
little  or  no  effect  on  the  development  of  the  parasite  from  the 
infective  larva  to  the  adult  stage  in  the  host. 

However,  development  of  the  parasite  from  the  egg  to  infective 
larva  in  feces  from  germfree  mice  was  extremely  poor.  Fatty  degenera- 

20 
-  i  - 


Serial  No.  NIAID-116 

tion  not  unlike  that  associated  with  B-vitamin  deficiency  occurred. 
If  living  bacteria  from  a  conventional  animal  were  added  to  the 
germfree  feces,  development  of  the  larvae  progressed  normally. 
Apparently,  feces  without  bacteria  fail  to  provide  certain  essentials 
for  development  of  the  larvae. 

Of  special  interest  was  the  apparent  indication  that  the  sex  of 
the  host,  which  is  a  factor  in  parasitism  in  the  conventional  animal, 
was  unimportant  among  the  germfree  animals .   In  the  conventional 
animals,  males  had  2-3  times  the  worm  burden  at  necropsy  that  the 
females  had.  Among  the  germfree  animals,  however,  average  worm  counts 
were  about  the  same  in  both  sexes. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the  Institute: 

The  fact  that  bacteria,  per  se,  are  apparently  "ot  required  for 
full  development  of  some  parasites  opens  the  way  for  other  studies  in 
the  areas  of  nutrition  and  parasitism,  chemotherapy,  ^n  vitro  culti- 
vation, and  physiologic  studies.  Also,  opportunity  is  provided  for 
the  study  of  eggs  and  larvae  in  fresh  (non-sterilized)  feces  without 
bacteria,  and  perhaps  to  ascertain  factors  contributed  by  the  latter 
toward  their  proper  development.   If  the  sex  phenomenon  holds  up  in 
future  tests,  interesting  and  potentially  important  relationships 
may  be  uncovered. 

Proposed  Course  of  the  Pro.ject: 

Of  course,  the  validity  and  significance  of  the  difference  in 
the  host  sex  effect  between  germfree  and  conventional  mice  will  be 
studied.   Efforts  to  complete  the  entire  life  cycle  of  the  parasite 
in  a  sterile  environment  will  be  continued.  Attempts  may  be  made  to 
ascertain  the  role  of  bacteria  in  certain  parasite-diet  relationships 
noted  in  conventional  mice. 

Part  B  Included:   No 


21 


LABORATORY  OF  PARASITIC  DISEASES 


Summary  1 

120   -  Administration  and  Research  Planning  and 

Coordination  6 

121-A  -  Host  Parasite  Relations  in  Worm  Infections 

in  Laboratory  Animals  7 

121-B  -  Role  of  Helminths  in  the  Causation  of 

Cancer 9 

121-C  -  Destruction  of  Molluscs  by  Chemical  Means  11 

121-D  -  Part  A.   Investigation  of  Intermediate 
Hosts  and  Vectors  of  Human  Diseases 
Caused  by  Worms  14 

121-E  -  Development  of  Methods  for  the  Cultiva- 
tion of  Parasitic  Helminths  in  vitro,  and 
the  Determination  of  the  Nutritional  Re- 
quirements of  Such  Organisms  in  vitro  ...  17 

121-G  -  The  Development  of  Helminths  in  Germ- 
free  Animals  21 

121-1  -  Pathological  Physiology  of  Worm  Infec- 
tions    23 

121-J  -  Serum  Protein  Studies  on  Germfree  Animals 

Infected  with  Various  Parasites 25 

121-K  -  Effects  of  Nutrition  on  Chemotherapy  of 

Parasitic  Diseases  27 

121-L  -  Effects  of  Improving  the  Nutrition  of 
Malnourished  People  Infected  with 
Schistosoma  Mansoni  30 

121-M  -  Ammonia  Metabolism  and  Toxicity  in  Rela- 
tion to  Liver  Disease  33 

121-N  -  Parasitological  Investigations  at  the 

Marine  Biological  Laboratory,  Woods  Hole, 
Massachusetts  36 

123-A  -  Fundamental  Physiological  and  Biochemical 
Studies  on  Parasites,  Intermediate  Hosts, 
and  Parasitized  Animals  38 

123-B  -  Pathological  Physiology  and  Histochemis- 
try of  Parasitic  Diseases  42 

123-C  -  Biochemical  Mechanisms  of  Energy  Metabo- 
lism in  Normal  and  Parasitized  Animals  ..  45 

123-D  -  Biology  of  Trypanosomes  49 


127-A  -  Studies  on  Toxoplasmosis  52 

127-B  -  Studies  on  Entamoeba  Histolytica  and  Other 

Parasitic  Protozoa  57 

127-C  -  Trichomoniasis  60 

127-D  -  Biochemistry  of  Parasitic  Protozoa  63 


II 


Laboratory  of  Parasitic  Diseases 
National  Institute  of  Allergy  and  Infectious  Diseases 

Summary  statement  of  research  activities,  calendar  year  1960 

Program  development  and  trends: 

Two  of  the  regular  staff  members,  Dr.  Jacobs  and  Dr.  Weinbach,  have 
been  on  research  assignment  outside  the  country  for  more  than  half  of  the  year. 
On  the  other  hand,  we  have  had  two  persons  on  assignment  to  the  Laboratory  from 
other  countries,  Mr.  Ian  Sommerville  from  the  McMaster  Laboratory,  Sydney, 
Australia;  and  Dr.  Iain  R.  Bowman  recently  from  the  University  of  Aberdeen, 
Scotland.  Mr.  Sommerville  has  been  working  with  Dr.  Weinstein  on  problems  re- 
lated to  sterile  culture  of  worms,  while  Dr.  Bowman  has  been  conducting  bio- 
chemical studies  with  Dr.  von  Brand.   In  July  Dr.  Allen  W.  Cheever  was  assigned 
to  the  Laboratory  as  a  Research  Associate.  He  has  been  placed  in  the  Section 
on  Helminthic  Diseases  and  is  conducting  research  on  schistosome  pathology,  and 
on  fundamental  problems  related  to  immunity  and  strain  differences  in  suscepti- 
bility to  the  liver  tapeworm  of  small  rodents,  with  Dr.  Olivier. 

The  Laboratory  continues  to  emphasize  fundamental  studies  on  parasites 
and  parasitic  diseases.  No  important  changes  in  the  program  were  instituted 
during  the  year.  The  program  of  the  laboratory  is  well  diversified,  consider- 
ing the  size  of  the  staff,  and  the  competencies  of  the  various  staff  members 
cover  a  large  proportion  of  the  field  of  parasitology. 

Although  the  emphasis  in  on  basic  studies,  this  does  not  imply  a  nar- 
row viewpoint  and  the  Laboratory  is  well  aware  of  the  many  practical  problems 
parasitic  diseases  create  throughout  the  world.  The  Laboratory  is  often  called 
upon  for  help  and  advice  concerning  prevention  and  control  of  parasitic  infec- 
tions and  so  must  maintain  competence,  and  a  reputation  for  competence,  to  deal 
not  only  with  basic  problems  of  parasitism  but  also  problems  of  prevention  and 
control  of  parasitic  diseases.  Therefore,  the  Laboratory  continues  to  carry 
on  a  variety  of  activities  which  help  it  maintain  its  international  reputation 
and  increase  its  capacity  to  cope  with  problems  of  parasitism.   Such  activity 
also  returns  benefits  in  the  form  of  ideas  for  laboratory  research  and  clues 
which  may  explain  puzzling  laboratory  findings. 

The  Laboratory  is  prepared  to  carry  out  field  studies  when  these  are 
logical  and  reasonable  extensions  or  applications  of  laboratory  work.  The  pro- 
ject in  Puerto  Rico  on  the  relation  of  nutrition  to  schistosomiasis  is  a  case 
in  point.  Also,  during  the  year  steps  were  taken  to  extend  laboratory  findings 
and  interests  into  foreign  laboratories  and  into  foreign  field  situations  through 
the  use  of  Public  Law  480  funds  in  various  countries.  A  series  of  project  pro- 
posals has  been  made  for  work  in  Israel,  Poland,  Yugoslavia,  India,  and  Brazil. 
Negotiations  for  two  PL  480  projects  in  Brazil  are  actively  under  way  and  it 
is  expected  that  both  will  be  started  in  1961. 


The  PL  480  projects  should  return  substantial  dividends,  not  only  in 
new  data  which  could  not  be  obtained  in  the  Bethesda  Laboratory,  but  also  in- 
valuable experience  and  sophistication  for  the  staff  members  involved.  The 
projects  are  designed  so  as  to  cause  minimal  interference  with  essential  lab- 
oratory activities.   If  the  Laboratory  is  to  consider  more  than  a  very  small 
number  of  PL  480  projects  it  would  have  to  add  personnel  to  staff  them  and 
would  probably  need  additional  funds  to  cover  incidental  expenses. 

The  Laboratory  has  continued  to  maintain  liaison  with  international 
agencies  interested  in  health  problems.  Two  of  the  staff  (Olivier  and  Berry) 
have  been  chosen  to  serve  on  the  WHO  Expert  Panel  on  Parasitic  Diseases.  These 
same  staff  members  were  loaned  for  short  periods  to  WHO  as  consultants  on 
schistosomiasis  research  and  control  activities.   Four  staff  members  (Berry, 
von  Brand,  Olivier,  Weinstein)  made  important  contributions  to  international 
symposia  or  conferences  dealing  with  research  on  human  disease. 

The  development  of  cooperative  clinical  studies  with  the  Laboratory  of 
Clinical  Investigation  has  been  disappointing.  Relatively  little  significant 
cooperative  clinical  work  was  done  during  the  year.  This  did  not  result  from 
lack  of  desire  on  the  part  of  either  the  Laboratory  of  Clinical  Investigations 
or  the  Laboratory  of  Parasitic  Diseases  since  as  excellent  rapport  has  been 
maintained,  but  rather  because  of  problems  inherent  in  procurement  of  useful 
patients  and  also,  to  some  extent,  to  failure  to  form  a  productive  "team". 
It  is  hoped  that  progress  along  this  line  can  be  made  in  1961. 

Scientific  advances  -  made  in  1960: 

The  Laboratory  has  produced  a  number  of  noteworthy  advances  in  knowl- 
edge during  the  year.   Many  of  these  can  be  classified  as  being  additions  to 

"basic"  knowledge  but  some  have  "practical"  implications.   Selection  of  items 
for  emphasis  is  sometimes  presumptuous  and  always  risky  since  the  importance 

of  an  individual  item  is  hard  to  judge  and  often  the  "small"  contribution  may, 

in  the  long  run,  turn  out  to  be  "large".  Nevertheless,  attention  is  called 
to  the  following: 

The  studies  on  toxoplasmosis  in  New  Zealand  sheep  (127-A)  have  shown 
that  the  prevalence  is  high.   Considerable  new  information  has  been  obtained 
concerning  the  distribution  of  the  organisms  in  the  tissues  and  their  persis- 
tence there.   After  inoculation  the  distribution  of  the  parasite  in  tissues 
is  erratic  and  the  parasites  rapidly  clear  from  tissues  other  than  the  muscle 
and  placenta.   Since  residual  infection  occurs  in  muscle,  mutton  may  serve  as 
a  source  of  human  infection.   Since  congenital  infection  with  Toxoplasma  is 
an  important  medical  problem,  it  is  of  special  interest  that  the  sheep  studies 
have  indicated  that  inoculation  of  sheep  60  days  before  pregnancy  did  not  re- 
sult in  congenital  infection  or  abortion  but  inoculation  at  30  days  pregnancy 
caused  abortion  or  foetal  death  with  absorption.   Infection  at  90  days  preg- 
nancy was  less  likely  to  be  dangerous  to  the  foetus. 

The  status  of  resistance  or  immunity  to  Toxoplasma  continues  to  be 
puzzling,  since  living  organisms  fail  to  completely  protect  animals  against 


challenge,  especially  when  the  challenge  is  great,  and  because  low  grade  para- 
sitemia may  persist  for  months  in  mice  and  rabbits  in  the  presence  of  high  ser- 
um antibody  levels.   The  observation  that  cysts  of  Toxoplasma  probably  form  in 
tissue  cultures  provides  a  new  opportunity  to  study  the  manner  of  cyst  forma- 
tion and  the  factors  that  lead  to  cyst  formation  (127 -A). 

The  work  on  the  preservation  of  living  Entamoeba  histolytica  and  other 
protozoa  (127-B)  has  practical  significance  since  success  would  permit  reten- 
tion of  strains  without  continuous  sub-culturing.  This  is  a  relatively  new 
field  and  techniques  are  still  evolving.  The  work  so  far  has  shown  that  this 
approach  is  feasible  since  four  species  have  been  frozen  and  stored  for  periods 
ranging  from  one  to  four  months  depending  on  the  species  involved.  Entamoeba 
histolytica  has  been  kept  at  -197°  C  for  24  hours,  suggesting  that  almost  in- 
definite storage  at  this  temperature  may  eventually  be  achieved. 

Laboratory  culture  of  Entamoeba  histolytica  continues  to  receive  atten- 
tion since  it  is  so  important  to  learn  more  concerning  its  nutritional  re- 
quirements and  its  pathogenicity  in  the  absence  of  other  organisms.   It  is 
noteworthy  that  satistactory  axenic  culture  of  this  species  has  been  achieved 
for  the  first  time  (127-B).   The  protozoa  are  cultured  in  a  complex  diphasic 
medium  containing  no  cells  but  including  chick  embryo  extract.  This  is  a  long 
and  very  important  step  forward. 

The  substitution  of  a  species  of  Crithidia  for  Trypanosoma  cruzi  in 
cultures  of  E.  histolytica  provides  a  more  economical  and  rapid  way  of  pro- 
ducing large  cultures  of  the  amoeba.  Demonstration  of  the  value  of  the  Coulter 
Counter  for  the  enumeration  of  protozoa  in  suspension  adds  a  valuable  tool  for 
quantitative  work  and  suggests  this  method  may  be  applicable  for  counting  other 
organisms  of  similar  size  such  as  tissue  culture  cells  (127-B). 

The  use  of  germ  free  animals  in  worm-parasite  studies  continues  to  re- 
veal the  value  of  this  tool  and  adds  to  our  knowledge  of  the  peculiar  nature 
of  the  germ  free  state.  The  technique  seems  to  be  particularly  useful  for 
studying  conditions  that  influence  natural  resistance  and  nutritional  rela- 
tionships of  parasite  and  host.  For  example,  it  was  found  (121-G)  that  the 
roundworm,  Nematospiroides  dubius,  develops  as  well  in  germ  free  as  in  con- 
ventional mice  but  while  in  conventional  mice  the  worm  recovery  is  much  higher 
from  the  male  animals,  the  recovery  from  germ  free  mice  is  the  same  for  both 
host  sexes.  The  cause  of  the  difference  is  unknown.   Also,  it  has  been  shown 
that  the  feces  of  germ  free  mice  do  not  support  development  of  N.  dubius  larvae 
and  that  bacteria  in  the  feces  provide  important  factors  for  larval  develop- 
ment (121-G).  There  was  further  evidence  that  the  alteration  in  levels  of 
serum  protein  components  in  germ  free  animals  is  due  to  dietary  factors  (121-G). 

Studies  on  the  sterile  culture  of  worms  continues  to  produce  fundamen- 
tal information  on  the  nutritional  requirements  of  the  parasites  and  brings 
closer  the  day  when  we  can  use  the  axenic  animals  for  immunologic  and  thera- 
peutic studies.   Survival  studies  using  relatively  advanced  larvae  of  Nippos- 
tronqylus  muris        has  produced  important  results  (121-E).   The  intent  has 
been  to  try,  by  addition  of  elements  to  the  medium,  to  induce  the  larvae  to 
reach  the  adult  stage.   Starting  with  a  salt  mixture,  dextrose  was  added  until 

3  3 


the  optimal  level  was  reached.  Then  casein  was  added  and  survival  time  rose 
to  11  days,  but  there  was  not  development  of  the  larvae.  Addition  of  a  yeast 
extract  to  this  mixture  not  only  increased  survival  but  permitted  growth  to 
the  adult  stage.  Thus,  a  much  more  simple  medium  than  used  before  has  been 
evolved  and  the  achievement  of  a  defined  medium  for  culture  of  N.  muris  adults 
is  much  closer.  A  similar  approach  is  being  used  in  attempts  to  culture  mi- 
crofilariae of  Dirof ilaria  immitis  (121-E). 

Although  the  study  of  the  relation  of  nutrition  to  schistosomiasis  in 
Puerto  Rico  is  still  incomplete,  it  appears  that  enrichment  of  the  diet  does 
not  affect  the  number  of  eggs  passed  in  the  feces.  However,  it  is  interesting 
to  note  that  the  enriched  diet  did  cause  a  loss  of  hookworms  and  whipworms 
from  the  intestine  (121-L).  This  has  a  bearing  on  the  problem  of  the  exist- 
ence of  hookworm  infection  without  hookworm  disease.   In  laboratory  studies 
conducted  in  Bethesda  the  enhanced  efficacy  of  stibophen  in  mice  receiving 
a  semi-synthetic  diet  was  shown  to  be  due  to  the  absence  from  this  diet  of  as 
yet  unknown  inorganic  salts  (121-K).  Higher  blood  levels  of  the  drug  were 
maintained  longer  when  the  semi-synthetic  diet  was  used  and  this  may  explain 
the  greater  efficacy.  Demonstration  of  the  influence  of  simple  salts  on  the 
efficacy  of  stibophen  suggests  that  other  drugs  may  be  similarly  affected  by 
diet.   If  the  work  with  the  stibophen-salt  problem  progresses  satisfactorily 
it  is  hoped  that  a  test  of  the  effect  of  human  diet  on  the  action  of  the  same 
drug  may  be  tried  in  Puerto  Rico  before  the  study  there  is  concluded. 

Interaction  of  two  pathogenic  organisms  in  the  same  host  has  had  rela- 
tively little  attention  in  spite  of  some  very  provocative  work  done  in  years 
past.  A  study  of  simultaneous  infection  with  encephalomyocarditis  virus  and 
Trichinella  spiralis  in  rats  has  produced  striking  and  significant  results 
(121-A).  While  the  virus  alone  does  not  injure  adult  white  rats  when  given 
intraperitoneally,  in  the  presence  of  Trichinella  spiralis  infection  many  of 
the  rats  are  crippled  and  die.  This  potentiation  of  the  virus  pathogenicity 
is  not  due  to  non-specific  stress  but  seems  to  be  related  to  the  presence  of 
the  worms  on  the  muscles.  The  virus  can  be  recovered  from  the  muscle  of  T. 
spiralis-infected  rats  but  not  from  muscle  of  rats  without  T.  spiralis.  The 
reason  for  the  influence  of  the  worm  infection  on  the  activity  of  the  virus 
is  unknown.  The  phenomenon  offers  an  opportunity  to  study  some  of  the  funda- 
mental factors  in  the  pathogenesis  of  both  the  virus  and  the  worm  parasite. 
It  also  provokes  the  question  as  to  what  effect  this  worm  infection  may  have 
on  other  virus  infections. 

Continued  study  of  the  hepato-splenic  syndrome  in  mice  infected  with 
Schistosoma  mansoni  has  added  new  evidence  to  show  that,  in  mice  at  least, 
the  schistosome  eggs  are  the  prime  cause  of  liver  damage  and  therefore  the 
chief  cause  of  fibrosis,  portal  hypertension,  and  collateral  circulation  which 
are  so  often  the  cause  of  morbidity  and  mortality  in  human  schistosome  infec- 
tion (121-1).  Diet,  dead  worms,  and  toxins  produced  by  the  worms  seem  to  be 
less  important  in  contributing  to  liver  damage  related  to  schistosome  infec- 
tion. 


The  study  of  liver  damage  in  relation  to  ammonia  toxicity  in  mice  has 
revealed  that  low  oxygen  in  breathed  air  greatly  enhances  ammonia  toxicity 
(121-M).  The  mechanism  of  this  effect  is  not  clear.  Though  hepatic  coma  is 
usually  considered  to  be  related  to  ammonia  toxicity  none  of  the  substances 
which  exacerbate  hepatic  coma  in  man  increases  ammonia  toxicity  in  mice.   In 
fact,  6  of  10  decrease  it.  Ammonia  toxicity  in  mice  was  greatly  reduced  by 
hypothermia  and  this  suggests  that  the  same  measure  may  be  useful  in  treating 
hepatic  coma  in  man.  Finally,  mouse  liver  damage  was  induced  in  eight  differ- 
ent ways  but  none  caused  any  change  in  the  animal* s  response  to  intravenous 
ammonia.  Thus,  though  high  blood  ammonia  levels  seem  to  be  related  to  liver 
damage,  the  causal  relationships  are  by  no  means  clear. 

Fundamental  physiological  studies  have  focused  on  the  calcareous  cor- 
puscles of  tapeworms  and  on  the  phospholipids  of  tapeworms  (123-A).  The  cal- 
careous corpuscles  are  amorphous  but,  on  heating,  dolomite,  brucite  or  apatite 
may  be  formed.  Electron  microscope  pictures  of  corpuscles  heated  with  KOH  re- 
veal the  presence  of  well-formed  crystals.  The  glycerol  containing  phospholi- 
pids of  Taenia  taeniaef ormis  are  about  half  lecithid  and  half  cephalin.   Sphin- 
gomyelin is  present  and  more  than  one  cephalin  is  known  to  occur  in  the  larvae 
of  this  tapeworm.  Hexose-containing  phospholipids  occur  in  both  larvae  and 
adults. 

Study  of  the  mechanism  of  energy  metabolism  of  sub-cellular  elements 
has  dealt,  among  other  things,  with  the  mechanism  by  which  mitochondria  which 
are  depleted  of  high-energy  phosphate  intermediates  are  stimulated  to  oxidize 
substrates  when  ATP  is  added.  This  is  a  complex,  though  fundamental,  bioener- 
getic  system  for  which  a  better  understanding  is  needed.  Addition  of  ATP  not 
only  restored  succinate  oxidation  but  also  caused  reduction  of  intra -mitochon- 
drial DPN.  The  succinate  oxidation  involves  an  energy-requiring  reaction  and 
this  energy  is  apparently  added  at  one  site  in  the  respiratory  chain  and  used 
at  another  for  reducing  pyridine  nucleotide  (127-D). 


Serial  No.    NIAID-120 

1.  Parasitic  Diseases 

2.  Office  of  the  Chief 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:  Administration  and  research  planning  and  coordination. 

Principal  Investigator:  Leon  Jacobs 

Other  Investigators:  Louis  J.  Olivier 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:  2)4 

Professional:         \ 
Other:  2 

Project  Description: 

This  project  furnishes  supervisory  and  administrative  services  to 
all  research  projects  in  the  Laboratory,  as  folows: 

Over-all  evaluation  of  research  plans  and  initiation  of  field 
projects;  integration  of  laboratory  research  activities  with  clinical 
studies  of  the  Clinical  Center;  editing  scientific  and  technical  reports 
and  manuscripts;  preparation  of  reports,  budget  estimates,  and  exhibits; 
supervision  over  personnel,  travel,  correspondence,  and  maintenance; 
requisitioning  and  supervision  of  equipment  and  supplies;  maintenance  of 
reference  library,  etc. ;  and  consultatory  services  to  individuals, 
academic  and  other  organizations,  and  liaison  activities  with  other 
branches  of  the  Service  and  the  Federal  Government. 

Research  projects  are  reviewed  and,  in  consultation  with  Section 
heads,  changes  in  research  program  are  considered  and  initiated.  The 
emphasis  is  placed  on  fundamental  aspects  of  medical  parasitology; 
extensions  of  projects  into  the  fields  of  clinical  or  practical  preventive 
medicine  are  attempted  only  when  the  laboratory  work  has  progressed  to  a 
point  where  a  sound  basis  exists. 


Serial  No.   NIAID-121-A 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:  Host  parasite  relations  in  worm  infections  in 
laboratory  animals. 

Principal  Investigator:  Louis  J.  Olivier 

Other  Investigators:  Allen  W.  Cheever 

Cooperating  Units:  Laboratory  of  Viral  Products,  DBS 

Man  Years  (calendar  year  1960): 
Total:  2& 

Professional     ]H 
Other:  114 

Project  Description: 

Objectives: 

To  study  the  characteristics  of  infection  in  laboratory  animals; 
to  learn  how  resistance  to  infection  may  be  enhanced  and  reduced;  to 
learn  how  the  host  defense  mechanisms  act  to  resist  infections;  to  study 
variations  in  resistance  of  host  strains;  to  study  the  mechanism  of 
natural  resistance  to  parasites;  to  study  how  two  simultaneous  infections 
may  affect  each  other. 

Methods  Employed: 

Infect  animals  in  such  a  way  as  to  produce  predictable  infections; 
treat  with  hormones  and  antigens  and  so  forth;  challenge  with  infective 
worms  or  other  agents;  observe  the  host  and  parasite;  and  determine  the 
degree  of  resistance  to  infection,  the  host  reaction,  and  the  effect  of 
one  agent  on  another. 

Major  Findings: 

Encephalomyocarditis  virus  is  pathogenic  to  very  young  rats,  but 
is  not  ordinarily  pathogenic  to  older  ones.  However,  this  virus  is 
highly  pathogenic  in  older  rats  if  the  latter  harbor  new  infections  with 
Trichinella  spiralis.  The  rats  are  crippled  by  the  infection  and  many 
die.  Moreover,  the  virus  is  recoverable  from  trichinous  muscle  in 
greater  quantity  than  from  non-trichinous  muscle.   The  enhanced  patho- 


Serial  No.  NIAID-121-A 


genicity  is  not  due  to  non-specific  stress  since  other  stresses,  such  as 
chilling,  fighting,  and  so  forth,  do  not  give  this  result.   This  potenti- 
ation of  virus  pathogenicity  by  T.  spiralis  is  very  striking,  but  not 
well  understood  as  yet.   Schistosome  infection  does  not  potentiate  the 
virus. 

Significance  to  the  Program  of  the  Institute: 

Problems  of  resistance  and  immunity  are  basic  to  understanding  of 
host  parasite  relationships  and  pathogenicity  of  parasites.   Natural 
resistance  is  an  elusive  phenomenon,  but  worm  infections  provide  a  use- 
ful and  attractive  system  for  its  study  since  infective  doses,  antigenic 
mass,  and  lesion  size  can  be  controlled  and  measured  quite  accurately. 
Some  parasites  do  not  multiply  in  the  host  and  are  not  attacked  by 
phagocytes.   These  facts  simplify  the  study  of  the  effects  of  hormones 
and  other  agents  on  infections. 

The  revelation  of  the  potentiation  of  EMC  by  T.  spiralis  may  pro- 
vide a  wonderful  opportunity  to  study  the  pathways  of  host  damage  by 
both  the  virus  and  the  worms. 

Proposed  Course  of  the  Project: 

To  test  whether  mice  develop  acquired  immunity  to  the  liver 
parasite  of  rats;  to  study  paths  of  transfer  of  immunity  of  rats;  to 
study  further  the  affect  of  hormones  on  this  resistance. 

To  study  natural  resistance  to  Cysticercus  fasciolaris  in  mice 
and  other  rodents. 

To  follow  the  EMC-T.  spiralis  relationship  and  to  study  other 
possible  virus  worm  associations.  To  determine  the  role  of  the  reticulo- 
endothelium  system  of  the  liver  in  resistance  through  the  use  of  reticulo- 
endothelial system  stimulating  and  depressing  agents.  To  determine 
whether  immunologic  tolerance  to  Cysticercus  fasciolaris  can  be  induced 
in  rodents. 


Part  B  included     No 


3 


Serial  No.   NIAID-121-B 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:  Role  of  helminths  in  the  causation  of  cancer 

Principal  Investigators:  Elmer  G.  Berry  and  Louis  J.  Olivier 

Other  Investigators:  None 

Cooperating  Units:  Pathologic  Anatomy  Branch,  National  Cancer  Institute, 
NCI— 525 
Laboratory  of  Viral  Products,  DBS 

Man  Years  (calendar  year  1960) 
Total:  1/4 

Professional:        1/4 
Other:  0 

Project  Description: 

Objectives: 

To  test  whether  worm  parasites  can  induce  malignant  growths  in  their 
hosts.  To  test  whether  Schistosoma  haematobium  infections  in  hamsters 
cause  tumors  of  the  digestive  or  urinary  tract.  To  test  whether  the  ma- 
lignant tumors  produced  in  rat  liver  by  larval  tapeworms  (Taenia  taeniae- 
formis)  are  virus-induced.  To  test  whether  cats,  the  host  of  the  adult 
tapeworm,  have  a  virus  which  can  be  related  to  these  tumors. 

Methods  Employed: 

Schistosoma  haematobium.  Hamsters  are  infected  with  S.  haematobium. 
The  animals  are  allowed  to  live  as  long  as  possible  to  determine  whether 
the  schistosome  infection  induces  malignant  growths. 

Taenia  taeniaeformis.  Rats  are  infected  and  the  liver  cysts  are 
ground  and  injected  into  rats  and  other  laboratory  animals.  Material 
from  cats  and  rats  is  studied  by  virological  and  immunological  methods. 

Major  Findings: 

Rat  virus  has  been  isolated  from  two  rats  having  the  liver  sarcomas. 


Serial  No.   NIAID-121-B 


Significance  to  the  Program  of  the  Institute: 

Cancer  of  the  bladder  appears  to  be  much  higher  in  Egypt  and 
Mozambique  than  it  is  in  other  areas.  Although  this  has  been  attributed 
to  the  particular  strain  of  S.  haematobium  which  occurs  in  these  countries 
the  actual  cause  for  these  differences  is  not  known.   It  is  hoped  that 
this  study  might  help  to  solve  the  problem. 

The  problem  of  virus  etiology  of  cancer  is  at  the  center  of 
concern  in  the  cancer  field. 

Proposed  Course  of  the  Project; 

Termination  of  the  S.  haematobium  study  when  the  last  infected 
animals  die. 

Continued  study  of  the  rat  tumor  project  and  possible  addition  of 
similar  projects. 


Part  B  included      No 


10 


Serial  No.   NIAID-121-C 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  A. 

Project  Title:  Destruction  of  molluscs  by  chemical  means. 

Principal  Investigator:  Louis  J.  Olivier 

Other  Investigators:  None 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960) 


Total: 

1 

Professional: 

1/2 

Other: 

1/2 

Project  Description: 

Objectives: 

To  discover  more  effective  means  for  killing  snail  vectors  of 
schistosomiasis.  To  study  the  mode  of  action  of  chemicals  used  as  mol- 
luscicides.  To  develop  more  effective  means  for  study  of  snail  poisons 
in  the  laboratory. 

Methods  Employed: 

Snail  vectors,  or  their  eggs,  are  exposed  to  chemicals  and  the  de- 
structive efficacy  of  the  chemicals  is  recorded.  The  optimal  conditions 
for  use  of  chemicals  against  snails  are  sought.  By  quantitative  methods 
the  optimal  duration  of  exposure,  concentration,  temperature,  etc.  are 
determined. 

Major  Findings: 

Using  standardized  methods  the  sodium  pentachlorophenate  LD50  was 
determined  for  eggs  and  adults  of  Australorbis  qlabratus.   Eggs  are  more 
susceptible  to  the  chemical  than  adults  by  a  chemical  concentration  fac- 
tor of  about  5.   Strain  differences  in  susceptibility  were  not  great. 
Very  small  differences  were  found  between  young  and  old  post-enbryonic 
snails. 


11 


Serial  No.  NIAID-121-C 


Following  failure  of  NaPCP  to  kill  snails  as  predicted  in  some 
field  situations  it  was  shown  that  ultra-violet  light  distroys  the  com- 
pound rapidly  and  that  this  could  explain  some  of  the  field  failures. 
Quantitative  laboratory  data  showed  the  half-life  of  NaPCP  solutions  in 
sunlight  may  be  less  then  an  hour  whereas  the  duration  of  an  effective 
field  concentration  probably  has  to  be  8  or  more  hours. 

Significance  to  the  Program  of  the  Institute: 

For  the  present,  at  least,  the  best  means  to  control  schistoso- 
miasis is  interruption  of  transmission  from  man  to  man  by  destruction  of 
the  snail  intermediate  hosts.  The  molluscicides  now  available  are  not 
ideal.  The  project  attempts  to  find  better  and  more  economical  ways  to 
use  the  available  molluscicides.   The  evidence  from  the  work  done  so  far 
suggests  the  possibility  that  more  efficient  use  may  be  made  of  mollusci- 
cides if  they  are  directed  against  the  snail  eggs  than  against  the  adults. 
The  project  puts  the  Institute  in  a  better  position  to  take  part  in 
measures  directed  against  the  schistosomes  which  are  rapidly  coming  to 
prominence  as  dangerous  parasites  of  man. 

Proposed  Course  of  the  Project: 

To  continue  with  efforts  to  standardize  the  laboratory  method  for 
study  of  molluscicides  with  the  aim  of  perfecting  a  method  for  highly 
analytical  quantitative  work  with  snail  poisons  and  also  with  the  aim  of 
providing  laboratories  interested  in  chemical  screening  with  a  better 
tool.   To  investigate  the  action  of  chemicals  already  studied  upon  other 
species  of  vector  snails.  To  try,  under  the  same  conditions,  other  mol- 
luscicides of  high  promise  so  that  there  may  be  accurate  and  quantitative 
data  by  which  molluscicides  can  be  compared.  To  devise  a  method  for 
screening  for  compounds  which  may  be  active  but  insoluble. 


Part  B  included     Yes 


12 


Serial  No.   NIAID-121-C 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B     Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Olivier,  L.  and  Haskins  W.  T.  The  effects  of  low  concentrations  of  sodium 
pentachlorophenate  on  the  fecundity  and  egg  viability  of  Australorbis  qlabratus. 
Amer.  Jour.  Trop.  Med.  Hyg.  9;   199-205  (1960) 

Olivier,  L.  Factors  affecting  the  survival  of  aestivating  pulmonate  vectors 
of  schistosomiasis.  Anniversary  Volume  for  Dr.  Caballero  y  Caballero.  pp.  215- 
225  (1960) 

Hiatt,  C.  W. ,  Haskins,  W.  T. ,  and  Olivier,  L.  The  action  of  sunlight  on  so- 
dium pentachlorophenate.  Amer.  Jour.  Trop.  Med.  Hyg.  9;  527-531.   (1960) 

Awards:  None 

Honors:  Dr.  Louis  J.  Olivier  served  in  Geneva  as  Consultant  to  the  WHO  Section 
on  Endemic  Diseases  from  18  September  to  10  October  1960.  During  this  time  he 
took  part  in  a  meeting  of  the  WHO  Expert  Comittee  on  Bilhorziasis  which  met 
from  26  September  to  10  October  1960. 

Dr.  Louis  J.  Olivier  was  invited  to  present  one  of  the  papers  in  the  Fourth 
Conference  of  the  Industrial  Council  for  Tropical  Health  which  met  on  July 
20  -  22  in  the  Harvard  School  of  Public  Health. 

Under  the  sponsorship  of  WHO  Dr.  Louis  J.  Olivier  visited  three  laboratories 
studying  chemicals  useful  as  animal  toxins  in  water  in  order  to  gather  infor- 
mation and  increase  exchange  of  information  and  ideas  among  those  interested 
in  the  subject. 

Dr.  Louis  J.  Olivier  has  been  chosen  to  serve  on  the  WHO  Expert  Advisory 
Panel  on  Parasitic  Diseases. 


13 


Serial  No.   NIAID-121-D 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  Investigation  of  intermediate  hosts  and  vectors  of 
human  diseases  caused  by  worms. 

Principal  Investigator:  Elmer  G.  Berry 

Other  Investigators:  None 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:  1  3/4 

Professional:        3/4 
Other:  1 

Project  Description: 

Objectives: 

To  investigate  the  distribution,  life  history,  and  ecology  of 
snail  intermediate  hosts  and  to  develop  a  rational  and  useful  system  of 
classification  and  means  of  identification. 

Methods  Employed: 

Colonies  of  snails  which  serve  as  intermediate  hosts  of  schisto- 
somes are  maintained  in  the  laboratory  where  they  are  available  for 
intensive  study.  Laboratory-reared  specimens  are  exposed  to  miracidia 
to  evaluate  the  susceptibility  and  to  determine  whether  strain  differences 
are  present. 

Major  Findings: 

Additional  specimens  belonging  to  the  genera  Bulinus  and  Biompha- 
laria  were  collected  from  the  Belgian  Congo,  Mozambique,  Kenya,  and  the 
Transvaal.  Colonies  of  these  species  are  now  established  in  the  labora- 
tory. Continued  progress  has  been  made  toward  clarification  of  taxonomic 
problems. 


n 


Serial  No.  NIAID-121-D 


Significance  to  the  Program  of  the  Institute; 

This  knowledge  is  essential  in  understanding  the  epidemiology  of 
bilharziasis  and  its  control. 

Proposed  Course  of  the  Project: 

Continuance  of  the  anatomical  studies  of  the  snails  which  serve 
as  intermediate  hosts  and  to  compile  this  information  into  a  manual. 


Part  B  included      Yes 


15 


Serial  No.   NIAID-121-D 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B.      Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project:  None 

Awards:  None 

Honors:  Dr.  Elmer  G.  Berry  served  as  a  Consultant  and  Leader  of  Discussion 
on  Snail  Control  by  Chemical  Means  at  the  African  Symposium  on  Bilharziasis 
which  met  in  Lourenco  Marques,  Mozambique,  30  March  to  8  April  1960. 

Dr.  Elmer  G.  Berry  served  as  Consultant  on  bilharziasis  research  at  the  Expert 
Committee  on  Bilharziasis  (Molluscicides)  which  met  in  Geneva,  Switzerland 
26  September  to  1  October  1960. 

Dr.  Elmer  G.  Berry  was  appointed  to  serve  on  the  World  Health  Organization 
Expert  Advisory  Panel  on  Parasitic  Diseases  for  a  period  of  five  years, 
beginning  June  1,  1960. 


1G 


Serial  No.   NIAID-121-E 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  Development  of  methods  for  the  cultivation  of  parasitic 
helminths  ki  vitro,  and  the  determination  of  the  nutri- 
tional requirements  of  such  organisms  in  vitro. 

Principal  Investigator:  Paul  P.  Weinstein 

Other  Investigators:   I.  R.  Sommerville  (visiting  scientist) 
T.  K.  Sawyer 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:  4 

Professional:        2  1/4 
Other:  1  3/4 

Project  Description: 

Objectives: 

To  develop  media  and  physical  conditions  suitable  for  the  in  vitro 
cultivation  of  helminths  throughout  their  life  cycle,  and  to  obtain  infor- 
mation on  their  specific  nutritional  requirements. 

Methods  Employed: 

Worm  eggs,  infective  larvae,  or  partially  matured  worms  are  iso- 
lated from  charcoal  cultures,  rodent  hosts,  or  mosquitoes,  depending  on 
the  helminths  involved.  These  are  inoculated  into  culture  media  to  be 
tested  for  growth -promoting  properties,  and  are  observed  for  development 
and  differentiation. 

Major  Findings: 

1.  Cultivation  of  trichostronqylids.   Survival  of  fourth  stage 
larvae  of  Nippostronqylus  muris  was  studied  in  salt  solutions  to  which 
nutrients  were  added.   In  a  modified  Krebs-Ringer  salt  solution,  survi- 
val was  limited  to  3  or  4  days,  but  the  addition  of  dextrose  increased 
the  survival  time  to  6-8  days.  The  optimal  concentration  of  dextrose  was 
0.014  M.  Change  in  phosphate  concentration  was  without  effect.  When 

IT 


Serial  No.  NIAID-121-E 

casein  was  added  to  the  solution  of  salts   and  dextrose,    survival  time  in- 
creased to  11  days,    but  no  developmental  changes  were  observed.     Replace- 
ment  of  casein  with  either  enzymic   or  acid  hydrolysates   of  casein  gave 
inferior  results,    and   acid  hydrolysates   appeared  to  be  toxic.     The  addition 
of  a  water-soluble  extract   of  yeast  to  a  solution  containing  casein,   dex- 
trose and   salts  not   only  enhanced   survival  but  yielded   adult  worms.     Better 
survival,   but  no  development  was   obtained  when  a  mixture  of  L-amino  acids 
in  the  proportions   in  which  they  occur   in  casein  was  used   in  place  of 
casein. 


13 


Serial  No.  NIAID-121-E 


Although  N.  muris  does  not  mate  when  grown  in  a  complex  medium 
composed  of  chick  embryo  extract,  vitamin  mixture  and  serum,  it  was 
found  that  the  third,  fourth,  and  early  fifth  stages,  if  taken  from 
such  cultures  and  put  directly  into  the  small  intestine  of  rats  will 
mate  and  produce  viable  eggs. 

The  closely  related  nematode,  Nematospiroides  dubius,  was  placed 
in  the  same  medium  which  was  not  conducive  to  the  mating  of  N.  muris. 
Under  these  conditions,  N.  dubius  was  observed  to  mate.  This  makes  more 
likely  the  possibility  that  a  complete  generation  of  a  parasitic  nematode 
may  be  obtained  in  vitro. 

2.  Cultivation  of  Dirofilaria  immitis  microfilariae.  Survival 
in  balanced  salt  solutions  alone  is  very  short.  Addition  of  dextrose 
increased  survival  time  about  fourfold.  There  is  a  direct  relation 
between  concentrations  of  magnesium  and  dextrose  and  survival  time. 
With  sodium  to  potassium  ratios  near  that  of  mammalian  blood  (29:1)  or 
close  to  that  of  some  insect  fluids  (5:1  or  less),  survival  is  good, 
whereas  at  intermediate  ratios  (20:1,  10:1)  it  is  relatively  poor. 

3.  Nematode  coelomocytes  and  vitamin  B]?.  This  vitamin  added 

to  a  defined  medium  resulted  in  the  development  of  a  deep  rose-pink  pig- 
ment in  the  coelomocytes  of  the  dog  hookworm  Ancylostoma  caninum. 
similar  to  what  was  previously  reported  for  N.  muris.   It  is  suspected 
that  this  pigment  represents  a  specific  concentration  of  Bi2» 

Significance  to  the  Program  of  the  Institute: 

The  growth  jji  vitro  of  parasitic  helminths  will  provide  an  impor- 
tant tool  with  which  to  study  the  physiology  and  metabolism  of  these 
organisms,  and  should  facilitate  the  isolation  and  preparation  of  meta- 
bolic antigens  involved  in  the  development  of  functional  immunity. 

Proposed  Course  of  the  Project: 

Work  will  continue  on  the  specific  nutritional  requirements  of 
N.  muris  and  N.  dubius  in  vitro. 

The  study  of  the  interaction  of  B^  and  coelomocytes  will  be 
extended  to  other  nematodes. 

Dirofilaria  uniformis  infections  in  rabbits  have  now  been  es- 
tablished in  the  laboratory,  and  coupled  with  D.  immitis  from  dogs, 
studies  will  continue  on  the  growth  requirments  of  filariids  in.  vitro. 

Part  B  included     Yes 

13 


Serial  No.   NIAID-121-E 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B.      Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Weinstein,  Paul  P.:  Excretory  mechanisms  and  excretory  products  of  nematodes: 
An  appraisal.  In  "Host  Influence  on  parasite  physiology."  Rutgers  University 
Press:  65-92.   (1960). 

Weinstein,  Paul  P. :  The  current  status  of  the  axenic  cultivation  of  helminths. 
(In  press). 

Honors:  Dr.  Weinstein  was  invited  to  participate  in  the  Annual  Conference  on 
Protein  Metabolism  entitled,  "Host  Influence  on  Parasite  Physiology,"  sponsored 
by  Rutgers  University,  January  1960. 

Dr.  Weinstein  was  invited  to  present  the  "Annual  Address"  to  the  Annual 
Meeting  of  the  Midwestern  Conference  of  Parasitologists  held  in  conjunction 
with  the  International  Symposium  on  Growth  at  Purdue  University  to  commemorate 
the  dedication  of  the  Life  Sciences  Building,  June  1960. 

Dr.  Weinstein  was  invited  by  the  First  Pan-American  Congress  on  Biology  and 
Experimental  Pathology  to  participate  in  a  symposium  entitled,  "Parasitic 
Biodynamics,"  held  in  Caracas,  Venezuela,  September  1960. 

Awards:  None 


20 


Serial  No.   NIAID-121-G 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  The  development  of  helminths  in  germfree  animals. 

Principal  Investigator:  Paul  P.  Weinstein 

Other  Investigators:  I.  R.  Sommerville,  (visiting  scientist),  LPD 
Thomas  K.  Sawyer,  LPD 

Cooperating  Units:  Laboratory  of  Germfree  Animal  Research,  NIAID  Serial 
No.  NIAID-116 

Man  years  (calendar  year  1960): 
Total:         Vt 
Professional:  \ 
Other:         J£ 

Project  Description: 

Objectives: 

To  establish  helminth  infections  in  germfree  animals  so  that  prob- 
lems involving  host-parasite  relations,  nutrition,  immune  response,  and 
pathology  can  be  studied  in  the  absence  of  other  complicating  microorganisms. 

Methods  Employed: 

Larvae  are  reared  to  the  infective  stage  under  axenic  conditions, 
and  eggs  and  larvae  are  also  isolated  from  natural  sources  and  rendered 
axenic;  these  are  used  to  infect  germfree  animals. 

Major  Findings: 

Axenically  reared  Nematospiroides  dubius  larvae  will  consistently 
mature  in  germfree  mice.  Total  worm  recoveries  from  germfree  animals  have 
been  equivalent  to  those  from  conventional  ones.  While  in  conventional 
animals  there  is  a  considerable  higher  worm  recovery  from  male  than  from 
female  mice,  in  germfree  mice  worm  recovery  was  essentially  the  same  from 
both  sexes.  Apparently,  diet  does  not  affect  this  difference. 

N.  dubius  larvae  which  hatched  from  eggs  passed  in  axenic  feces  did 
not  develop  normally  in  such  feces  maintained  axenically.  When  feces  from 

21 


Serial  No.  NIAID-121-G 


conventional  animals  containing  bacteria  was  added  to  such  cultures,  rapid, 
normal  larval  development  to  the  infective  stage  occurred,  indicating  that 
bacteria  in  feces  provide  important  components  for  larval  development. 

Significance  to  the  Program  of  the  Institute. 

These  experiments  demonstrate  the  feasibility  of  using  the  axenic 
helminth  parasite  and  host  in  studying  factors  which  concern  the  host  par- 
asite relationship.  This  technique  has  value  particularly  where  intesti- 
nal parasites  are  concerned  in  studying  conditions  which  influence  natural 
resistance,  host  specificity,  and  nutritional  relationships  of  parasite 
and  host. 

Proposed  Course  of  the  Pro.ject: 

Further  work  will  be  done  to  compare  the  susceptibility  to  infection 
of  male  and  female  conventional  and  germfree  mice  with  a  helminth  parasite. 
It  is  also  planned  to  attempt  to  establish  the  infection  on  a  continuing 
basis  in  the  germfree  environment  through  successive  generations  of  host 
and  parasite,  and  to  follow  the  course  of  the  infection  under  such  condi- 
tions. Nutritional  factors  relating  to  infection  are  to  be  studied.   It 
is  also  planned  to  use  worms  developing  in  axenic  hosts  as  inocula  for  cul- 
tures for  in  vitro  study  under  axenic  conditions. 


Part  B  included      No 


22 


Serial  No.   NIAID-121-I 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:  Pathological  physiology  of  worm  infections 

Principal  Investigators:  Kenneth  S  Warren  and  Allen  W.  Cheever 

Other  Investigators:  None 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:  1)4 

Professional:         3/4 
Other:  J£ 

Project  Description: 

Objectives: 

To  study  the  dynamic  relationships  existing  between  worms  and 
their  hosts.  Specifically  to  study  the  syndrome  in  mice  which  resembles 
human  hepato-splenic  schistosomiasis  mansoni  and  learn  its  causes;  to 
use  this  syndrome  as  a  tool  for  the  study  of  liver  diseases;  to  study 
the  pathogenesis  of  schistosome  infection,  using  mice  as  tools. 

Methods  Employed: 

Use  unisexual  and  bisexual  infections  to  determine  the  cause  of 
schistosome  liver  disease;  evaluation  by  use  of  portal  pressure  deter- 
minations, study  of  tissues,  surgical  procedures,  nutritional  changes, 
liver  function  tests,  and  physiological  studies.  Study  vascular  damage 
in  liver  by  injection  of  colloidal  carbon  or  carmine.  Study  importance 
of  eggs,  dead  worms,  etc.  as  factors  in  schistosome  liver  damage. 

Major  Findings: 

In  mice  hepato-splenic  schistosomiasis  occurs  in  the  presence  of 
good  nutrition  and  is  not  exacerbated  by  poor  nutrition;  unisexual  in- 
fections do  not  cause  typical  hepato-splenic  schistosomiasis,  but  in 
male  infections  there  is  a  moderate,  transient  splenomegaly  and  an 
increase  in  portal  pressure.  The  production  of  eggs  by  the  worms  appears 

23 


Serial  No.  NIAID-121-I 


to  be  necessary  before  hepato-splenic  schistosomiasis  develops.  There  is 
no  lung  shift  of  worms  following  the  use  of  high  doses  of  Fuadin.  There 
is  some  evidence  that  the  liver  recovers  rapidly  from  damage  due  to  dead 
worms. 

Significance  to  the  Program  of  the  Institute: 

Much  controversy  exists  concerning  the  relative  roles  played  by 
ova,  toxins,  dead  worms,  and  immunity  in  the  production  of  schistosoma 
liver  disease.  The  dispute  is  of  practical  importance  since  those  who 
believe  dead  worms  to  be  the  chief  pathogenic  agent  do  not  treat 
infected  patients.   In  general,  pathogenesis  of  worm  parasites  needs  more 
attention.   Its  study  should  return  fundamental  information  of  broad 
usefulness. 

Proposed  Course  of  the  Project: 

To  continue  to  study  the  relations  of  schistosomes  to  their  host 
by  studying  the  role  of  male  schistosomes  in  the  cause  of  liver  damage; 
by  injecting  schistosome  eggs  into  the  portal  system  in  order  to  observe 
the  damage  they  cause  alone;  by  attempting  to  produce  immunologic  tolerance; 
by  studying  the  role  of  immunity  in  host  damage;  by  testing  further  the 
role  of  dead  worms  in  causing  liver  damage;  and  other  similar  approaches 
to  the  problem. 

It  is  also  intended  to  study  the  pathogenesis  of  tapeworm  in- 
fections in  rat  liver. 


Part  B  included     No 


24 


Serial  No.   NIAID-121-J 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  Serum  protein  studies  on  germfree  animals  infected  with 
various  parasites. 

Principal  Investigator:  William  B.  De  Witt 

Other  Investigators:  None 

Cooperating  Units:  Laboratory  of  Germfree  Animal  Research,  NIAID-112. 

Man  YearsCcalendar  year  1960): 
Total:  1/4 

Professional:       0 
Other:  1/4 

Project  Description: 

Objectives: 

To  determine  the  effects  of  a  germfree  environment  on  the  relative 
distribution  of  various  protein  components  of  germfree  guinea  pigs,  mice, 
and  other  small  laboratory  animals.  This  work  is  being  done  to  obtain 
base  line  values  in  preparation  for  contemplated  serologic  studies  on  the 
pathogenesis  of  parasitic  infections  in  germfree  hosts. 

Methods  Employed: 

Serum  from  animals  maintained  in  germfree  tanks  on  a  sterilized 
ration  is  compared  with  that  obtained  from  animals  housed  in  conventional 
cages  and  given  the  same  ration.  A  third  group  of  animals,  housed  in 
conventional  cages  and  fed  a  commercial  pellet  diet  is  also  studied. 
Paper  electrophoretic  studies  are  carried  out  according  to  standard 
techniques. 

Major  Findings: 

This  project  has  been  relatively  inactive  during  the  past  year. 
However,  further  confirmatory  evidence  was  obtained,  indicating  that 
previously  observed  alterations  in  levels  of  serum  protein  components 
in  the  germfree  mouse  was  due  mainly  to  dietary  factors  rather  than  to 
the  germfree  state. 

25 


Serial  No.  NIAID-121-J 


Significance  to  the  Program  of  the  Institute: 

Before  the  effects  of  parasitic  infections  on  the  relative  distri- 
bution of  serum  protein  components  of  the  gerrafree  animal  can  be  studied, 
it  is  first  necessary  to  establish  baseline  values  in  the  axenically 
reared  animal.   It  is  hoped  that  serum  protein  studies  on  animals  experi- 
encing for  the  first  time  the  invasion  and  development  of  parasites  will 
shed  light  on  the  role  played  in  the  defense  of  the  host  by  circulating 
antibodies  associated  with  the  various  globulin  components. 

Proposed  Course  of  the  Project: 

The  present  study  is  to  be  extended  to  determine  the  effect  of  age 
and  various  nutritional  factors  on  the  relative  distribution  of  the  serum 
protein  components.  Later  the  response  to  infection  with  a  variety  of 
parasites  will  be  measured. 


Part  B  included     No 


26 


Serial  No.   NIAID-121-K 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  Effects  of  nutrition  on  chemotherapy  of  parasitic  diseases 

Principal  Investigator:  William  B.  DeWitt 

Other  Investigators:  None 

Cooperating  Units:  Laboratory  of  Parasite  Chemotherapy,  NIAID-131-D. 

Man  Years  (calendar  year  1960): 
Total  1  3/4 

Professional:  1/2 

Other  1  1/4 

Project  Description: 

Objectives: 

To  determine  whether  the  nutrition  of  the  host  can  influence  the 
efficacy  of  parasite  chemotherapy. 

Methods  Employed: 

Animals  maintained  on  defined  diets  either  enriched  or  deficient 
in  certain  components  are  exposed  to  a  single  species  of  parasite  and  the 
developing  infection  is  treated  either  in  the  prepatent  or  patent  periods. 
The  influence  of  the  diet  on  treatment  is  determined  by  (1)  the  reduction 
of  the  number  of  parasites,  (2)  condition  of  the  parasites,  (3).  growth 
and  survival  of  the  host,  and  (4)  pathologic  conditions  developing  in  the 
host. 

Major  Findings: 

The  efficacy  of  stibophen  (Fuadin)  therapy  on  mature  Schistosoma 
mansoni  infections  in  mice  was  increased  up  to  16  times  by  feeding  a 
balanced  semi-synthetic  diet.  The  toxicity  of  the  drug  was  not  similarly 
increased.  The  enhancement  of  curative  action  by  the  purified  semi- 
synthetic diet  was  found  to  be  due  to  the  absence  of  as  yet  unidentified 
inorganic  salt(s)  that  interfere  with  drug  activity.   It  was  found  in 
mice  fed  on  the  purified  semi-synthetic  diet  that  higher  blood  levels  of 
the  drug  were  maintained  for  a  longer  period  than  when  the  same  amount 

27 


Serial  No.   NIAID-121-K 


of  Fuadin  was  injected  into  mice  fed  on  the  commercial  pellet  diet, 
suggesting  that  the  increased  cure-rate  was  due  to  the  higher  blood 
drug  level. 

Significance  to  the  Program  of  the  Institute: 

Since  parasitic  infections  occur  most  frequently  in  backward 
areas  where  malnutrition  is  also  prevalent,  it  is  necessary  to  determine 
the  relation  between  the  nutritional  status  of  the  people  and  the  results 
obtainable  by  chemotherapy.   Failure  of  chemotherapy  is  a  common  experi- 
ence in  such  areas. 

Pronosed  Course  of  the  Project: 

The  present  study  is  to  be  extended  to  determine  the  specific 
nutritional  factors  involved  in  the  enhancement  of  Fuadin  efficacy  in 
schistosomiasis.   Other  drugs  used  for  the  treatment  of  this  and  other 
parasitic  diseases  will  be  investigated  under  similar  experimental 
conditions. 


Part  B  included     Yes 


28 


Serial  No.   NIAID-121-K 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B.     Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Luttermoser,  George  W.,  and  DeWitt,  William  B. :  Studies  on  interrelations 
of  nutrition  and  treatment  of  schistosomiasis  I.  Enhancement  of  stibophen 
(Fuadin)  activity  against  Schistosoma  mansoni  in  mice  by  feeding  purified 
semi-synthetic  diets.   (In  press) 

Honors:  None 

Awards:  None 


25 


Serial  No.  NIAID-121-L 
1.  Parasitic  Diseases 
raS-NIH  2.  Helminthic  Diseases 

Individual  Project  Report       3#  Bethesda,  Maryland 
Calendar  Year  1960  '   ' 3 


Part  A. 


Project  Title:   Effects  of  improving  the  nutrition  of  malnourished 
people  infected  with  Schistosoma  mansoni. 

Principal  Investigator:  William  B.  De  Witt 

Other  Investigators:  None. 

Cooperating  Units:   School  of  Medicine,  San  Juan,  P.  R. 

Rio  Piedras  Hospital,  Rio  Piedras,  P.R. 

Man  Years  (calendar  year  1960): 
Total:  1 

Professional:        1/2 
Other:  1/2 

Project  Description: 

Objectives: 

To  evaluate  the  effects  of  providing  malnourished  people  infected 
with  Schistosoma  mansoni  with  an  enriched  diet  containing  an  abundance 
of  animal  protein. 

Methods  Employed: 

Malnourished  people  suffering  from  schistosomiasis  are  given 
high-protein,  high-caloric,  vitamin-enriched  food  over  an  extended 
period  so  that  the  effects  of  the  improved  diet  on  their  well-being  and 
on  the  parasitic  infections  may  be  determined.   Such  factors  as  the 
following  are  observed  before  and  at  various  intervals  after  the  patients 
are  given  the  enriched  diet:   (1)  Nutritional  status  as  determined  by 
physical  examinations  and  biochemical  tests;  (2)  egg-production  and  via- 
bility of  eggs  of  the  parasite;  (3)  antibody  levels;  (4)  skin  sensitivity 
to  schistosome  antigens;  (5)  intestinal  uptake  of  essential  nutrients; 
(6)  liver  function,  as  determined  by  BSP  and  thymol  turbidity  measure- 
ments; and  (7)  liver  biopsy  examinations.  After  the  effects  of  the 
improved  diet  on  the  health  of  the  patients  have  been  determined,  the 
patients  will  be  given  a  standard  course  of  treatment  so  that  the  effects 
of  the  improved  nutrition  on  the  efficiency  of  the  drug  can  be  determined. 
Suitable  control  patients  are  likewise  to  be  studied. 


30 


Serial  No.   NIAID-121-L 


Major  Findings; 

The  first  part  of  this  project  has  been  completed  and  preliminary 
analysis  of  the  data  indicates:   (1)  Malnourished  people  suffering  from 
schistosomiasis  respond  favorably  to  an  enriched  diet.  Weight  gain  and 
improvement  in  general  well-being  were  particularly  marked.   (2)  The 
effects  of  the  enriched  diet  on  passage  of  schistosome  eggs  did  not 
appear  to  be  significant.  However,  with  some  patients  a  decrease  was 
noted  in  the  number  of  schistosome  eggs  passed.   (3)  Several  patients 
with  concomitant  hookworm  and  whipworm  infections  apparently  underwent 
self-cure  while  on  the  enriched  diet.  Similar  cures  were  not  observed 
among  the  control  subjects.   (4)  Biochemical  studies  made  on  patients 
before  they  received  the  improved  diet  revealed  a  complete  absence  of 
urinary  vitamin  C  in  more  than  half  of  those  examined.  Most  of  those 
studied  also  had  low  serum  vitamin  A  levels. 

Significance  to  the  Program  of  the  Institute: 

This  project  is  a  direct  extension  of  some  of  our  basic  labora- 
tory studies  which  indicate  that  the  normal  growth  and  development  of 
schistosomes  is  prevented  when  the  experimental  host  is  given  a  deficient 
diet.  Many  of  the  worms  remain  sexually  immature  and  are  unable  to  pro- 
duce eggs,  which  are  important  factors  in  the  pathogenic  mechanism  of 
schistosomiasis.  No  information  is  available  concerning  the  interrela- 
tion of  diet  and  schistosomiasis  in  humans.   If,  as  has  been  shown  in 
animal  experiments,  the  efficacy  of  treatment  of  schistosomiasis  in 
humans  can  be  greatly  enhanced  by  providing  an  enriched  diet,  it  would 
be  a  major  accomplishment.  As  to  date,  no  satisfactory  treatment  of  the 
disease  has  been  discovered. 

Proposed  Course  of  the  Project: 

The  second  phase  of  the  project  is  concerned  with  determining  the 
effects  of  dietary  improvement  on  the  efficacy  of  drugs  against  schisto- 
somiasis. The  curative  action  of  the  drug  (Fuadin)  will  be  evaluated 
when  different  regimens  are  employed  and  the  occurrence  and  severity  of 
side  reactions  will  be  noted. 


Part  B  included      Yes 


31 


Serial  No.   NIAID-121-L 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B.     Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project:  None 

Honors:  Dr.  William  B.  De  Witt  was  made  editor  of  Tropical  Medicine 
and  Hygiene  News. 

Awards:  None 


32 


Serial  No.   NIAID-121-M 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  Ammonia  metabolism  and  toxicity  in  relation  to  liver 
disease. 

Principal  Investigator:  Kenneth  S  Warren 

Other  Investigators:  None 

Cooperating  Units:  Laboratory  of  Clinical  Investigations,  NIAID 


Man  Years  (calendar  year  1960): 
Total:  lfc 

Professional:        1 
Other:  J£ 

Project  Description: 

Objectives: 

To  study  the  biochemical  pathway  of  ammonia  toxicity  and  the 
possible  relation  between  ammonia  toxicity  and  hepatic  coma.  More  speci- 
fically, to  determine  whether  drugs  which  exacerbate  hepatic  coma  or  are 
used  in  its  treatment  effect  ammonia  toxicity;  to  test  whether  the  sus- 
ceptibility of  animals  to  ammonia  toxicity  is  altered  by  different  types 
of  liver  disease. 

Methods  Employed: 

Ammonia  toxicity  to  mice  is  established  by  determining  the  intra- 
venous LD50.  The  modification  of  the  LD50  by  drugs,  anti-metabolites 
and  liver  disease  is  then  determined.  Blood  and  brain  ammonia  concen- 
trations, glutamine  concentrations,  and  blood  pH  are  also  studied. 

Major  Findings: 

1.  Low  oxygen  in  breathed  air  enhances  ammonia  toxicity,  but 
hypoglycemia  and  cyanide  have  no  such  effect.  Uncoupling  agents  have  a 
slight  effect  and  substances  which  effect  the  lower  part  of  the  Embden- 
Myerhoff  cycle  and  the  Krebs  cycle  up  to  succinate  increase  ammonia 
toxicity.  Ammonia  toxicity  bears  no  relation  to  brain  acetyl-choline 

33 


Serial  No.   NIAID-121-M 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B.       Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Warren,  K.  S,  Iber,  F.  L.,  Dolle,  W.,  and  Sherlock,  S:  The  effect  of 
alterations  in  blood  pH  on  the  distribution  of  ammonia  from  blood  to 
cerebrospinal  fluid  in  patients  in  hepatic  coma.  J.  Lab.  Clin.  Med.  56:  687 

Warren,  K.  S,  and  Schenker,  S. :  Hypoxia  and  amonia  toxicity.  Am.  J.  Physiol. 
(Dec.  1960). 

Honors:  None 

Awards:  None 


35 


Serial  No.  NIAID-121-N 

1.  Parasitic  Diseases 

2.  Helminthic  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:   Parasitological  investigations  at  the  Marine  Biological 
Laboratory,  Woods  Hole,  Mass. 

Principal  Investigator:   Paul  P.  Weinstein 

Other  Investigators:  None 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:         Y4 
Professional:   54 
Other:         None 

Project  Description: 

Objectives: 

A.  To  study  the  ectocommensal  associates  of  the  horseshoe  crab, 
Limulus  polyphemus.  and  the  factors  which  influence  the  host-ectocommensal 
relationship. 

B.  To  study  the  little-understood  life  cycles  of  trypanorhynchid  and 
tetraphyllidean  tapeworms. 

Methods  employed: 

A.  Various  stages  of  Limulus  were  collected  and  the  parasites  were 
studied  in  vivo  and  in.  vitro. 

B.  Usual  biological  methods  were  used  to  study  life  cycles  of  the 
tapeworms. 

Major  Findings: 

A.   A  new  species  of  oncholaimid  nematode  was  discovered  as  an  ecto- 
commensal on  the  ventral  surface  of  Limulus.   Its  entire  life  cycle  has 
been  found  to  take  place  in  this  habitat,  and  various  stages  from  the  egg 
to  the  adult  have  been  studied.   Information  has  been  obtained  on  seasonal 
changes  in  growth  of  the  worm,  prevalence  rate  on  different  instars  of 
Limulus,  and  the  relationship  of  molting  of  Limulus  to  continued  "infestation. 

3G 


Serial  No.  NIAID-121-N 

The  turbellarian  ectocommensals,  Bdelloura  Candida.  B.  propinqua  and 
Syncoelidium  pellucidum  were  studied  "in  vitro",  and  information  was  ob- 
tained on  feeding  response  to  various  substances.   Cocoon  deposition,  which 
ordinarily  only  occurs  on  the  gills  of  Limulus.  was  obtained  hi  vitro,  and 
these  cocoons  were  successfully  embryonated  and  hatched. 

Studies  made  on  Limulus  from  the  trilobite  stage  to  the  adult  gave 
some  information  on  the  sequence  of  "infestation"  of  this  host  with  its 
nematode  and  turbellarian  ectocommensals,  and  has  clarified  somewhat  the 
"epidemiological"  picture. 

B.   Studies  done  with  the  trypanorhynch,  Lacistorhynchus  tenuis  indi- 
cate that  certain  copepods  will  support  rapid  development  of  procerci  after 
exposure  to  coracidia.  No  procerci  were  observed  following  exposure  of  co- 
pepods to  the  eggs  of  two  species  of  Calliobothrium  (tetraphyllidean),  nor 
were  procerci  of  either  genus  found  in  the  exposed  benthic  fauna. 

Significance  to  the  Program  of  the  Institute: 

A.  The  ectocommensals  of  Limulus  have  a  strict  host  specificity;  they 
are  found  nowhere  else  in  nature.  Delineating  the  factors  underlying  such 

a  relationship  should  contribute  toward  an  understanding  of  ectocommensalism, 
which  is  one  of  the  interesting  categories  of  animal  association,  and  is  in 
an  evolutionary  sense  a  forerunner  to  parasitism. 

B.  The  tapeworms  studies  have  given  information  on  the  first  inter- 
mediate hosts  of  a  relatively  primitive  group  of  tapeworms,  whose  life  cycles 
are  essentially  unknown.  The  trypanorhynchid  cycle  may  prove  to  be  similar 
to  that  of  Diphyllobothrium  latum,  the  fish  tapeworm  of  man. 

Proposed  Course  of  the  Project: 

This  project  has  been  terminated  to  permit  time  to  develop  plans  for 
the  contemplated  filariasis  program  under  PL  480. 


Part  B  included     No 


37 


Serial  No.   NIAID-123-A 

1.  Parasitic  Diseases 

2.  Physiology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  Fundamental  physiological  and  biochemical  studies  on 

parasites,  intermediate  hosts,  and  parasitized  animals. 

Principal  investigator:  Theodor  von  Brand 

Other  investigators:  Eleanor  J.  Tobie,  Patricia  A.  McMahon,  Iain  B. 
Bowman  (Guest  worker). 

Cooperating  Units:  Laboratory  of  Histology  and  Pathology,  NIDR. 

Man  Years  (calendar  year  1960) 
Total:  4  1/2 

Professional        3 
Other  1  1/2 

Project  Description: 

Objectives: 

The  objective  is  to  gain  knowledge  on  the  chemical  composition  and 
the  metabolism  of  parasites,  intermediate  hosts,  and  parasitized  animals. 

Methods  Employed: 

Trypanosoma  cruzi,  T.  qambiense  and  T.  rhodesiense  are  cultivated 
in  wholly  fluid  media  and  trehalose  utilization  is  studied  by  quantita- 
tive methods.   Insight  into  a  major  metabolic  pathway  of  Trypanosoma  cruzi 
is  sought  by  estimating,  with  the  help  of  radioactive  CO2,  the  carbon  di- 
oxide fixation  into  succinic  acid.  The  aerobic  and  anaerobic  metabolism 
of  Taenia  taeniaef ormis  are  studied  by  determining  quantitatively  the 
elimination  of  various  metabolic  end-products  and  the  utilization  of  car- 
bohydrate. The  calcareous  corpuscles  of  the  same  tapeworm  are  isolated 
by  various  means,  treated  in  a  muff lefurnace,  and  then  turned  over  to  the 
collaborating  laboratory  for  diffraction  and  electronmicroscopic  studies. 
The  phospholipids  of  larval  and  adult  Taenia  taeniaef ormis  are  fraction- 
ated and  studied  by  electrophoretic,  chromatographic,  and  chemical 
methods,  stress  being  laid  on  the  quantitative  relationships  between  var- 
ious components. 


o 


3 


Serial  No.  NIAID-123-A 


Major  Findings: 

The  trypanosome  species  studied  are  not  capable  of  utilizing 
trehalose  and  the  presence  of  this  disaccharide  does  not  render  cultures 
of  African  trypanosomes  infective.   The  production  of  succinic  acid  by 
the  culture  form  of  T.  cruzi  is  dependent  on  the  presence  of  carbon 
dioxide  in  its  surroundings.   Taenia  taeniaeformis  produces  anaerobically 
much  more  succinic  acid  than  it  does  aerobically.  Stored  lipids  are  not 
utilized  by  this  worm  during  short  periods  of  aerobic  or  anaerobic 
starvation.  Upon  heating  the  calcareous  corpuscles  of  larval  or  adult 
Taenia  taeniaeformis  to  300°  C  diffraction  lines  appear  that  are  very 
close,  if  not  identical,  to  those  of  dolomite.  Upon  heating  to  higher 
temperatures,  the  dolomite-like  substance  is  decomposed  to  calcium  oxide 
and  magnesium  oxide.   Upon  treating  of  corpuscles  with  KOH  or  NaOH,  no 
dolomite  is  formed,  but  brucite  and  apatite  are  produced.  Highly  magni- 
fied electron-microscope  photographs  of  KOH-isolated  corpuscles  reveal 
the  presence  of  well  formed  crystals.  The  glycerol-containing  phospho- 
lipids of  both  larval  and  adult  Taenia  taeniaeformis  are  approximately 
half  lecithin  and  half  cephalin.  Electrophoretic  studies  confirmed  the 
presence  of  sphingomyelin  and  indicated  two  cephalin  fractions  in  larval 
T.  taeniaeformis  as  well  as  a  lecithin  differing  from  that  of  the  adult 
tapeworm.  Hexose-containing  phospholipids  have  been  found  in  both  the 
larval  and  the  adult  worms. 

Significance  to  the  Program  of  the  Institute: 

The  studies  on  metabolism  and  chemical  composition  of  parasites, 
intermediate  hosts,  and  parasitized  animals  are  essential  for  an  under- 
standing of  the  pathogenesis  of  parasitic  diseases.  They  also  lay  a 
foundation  for  a  rational  approach  to  chemotherapy. 

Proposed  Course  of  the  Project: 

The  studies  on  the  metabolism  of  trypanosomes  will  be  continued 
with  emphasis  on  tracer  studies.  The  studies  on  the  overall  metabolism 
of  Taenia  taeniaeformis  will  be  terminated  within  the  next  few  months. 
A  study  of  the  influence  of  oxygen  tension  and  carbon  dioxide  tension  on 
the  metabolism  of  tapeworms  will  be  initiated.  The  studies  on  the 
calcareous  corpuscles  will  be  continued,  but  may  be  terminated  some  time 
during  the  coming  year.  The  studies  on  phospholipids  will  be  continued 
with  emphasis  on  electrophoretic  studies. 


Part  B  included     Yes 


39 


Serial  No.   NIAID-123-A 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B    Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

von  Brand,  Theodor:  Recent  advances  in  carbohydrate  biochemistry  of  helminths. 
Helminthological  Abstracts  29:  97-111  (1960). 

von  Brand,  Theodor:  Der  Stoffwechsel  der  Trypanosomen.  Ergejibnisse  der 
Bio  log ie  _12:  30-46  (1960) 

von  Brand,  Theodor:   Influence  of  oxygen  on  life  processes.   In:  Sasser,  J.N., 
and  Jenkins,  W.R.  (Editors):  Nematology.  The  University  of  North  Carolina 
Press,  pp  242-248.  (1960). 

von  Brand,  Theodor:  Influence  of  size,  motility,  and  age  on  metabolic  rate. 
In:  Sasser,  J.N.,  and  Jenkins,  W.R.  (Editors):  Nematology.  The  University 
of  North  Carolina  Press,  pp.  233-241.  (1960). 

von  Brand,  Theodor:  Influence  of  pH,  ions,  and  osmotic  pressure  on  life 
processes.   In:  Sasser,  J.N.  and  Jenkins,  W.R.  (Editors):  Nematology. 
The  University  of  North  Carolina  Press,  pp.  249-256.  (1960). 

von  Brand,  Theodor:   Influence  of  temperature  on  life  processes.   In:  Sasser, 
J.N.  and  Jenkins,  W.R.  (Editors):  Nematology.  The  University  of  North 
Carolina  Press,  pp.  257-266.   (1960). 

von  Brand,  Theodor:  Introductory  remarks.  In:  Stauber,  L.A.  (Editor): 
Host  influence  on  parasite  physiology.  Rutgers  University  Press,  pp.  1-3. 
(1960). 

von  Brand,  Theodor,  Mercado,  Teresa  I.,  Nylen,  M.U.,  and  Scott,  D.B. : 
Observations  on  function,  composition,  and  structure  of  cestode  calcareous 
corpuscles.  Exper.  Parasitol.  9:  205-214.  (1960). 

Thompson,  M.J.,  Mosettig,  E.,  and  von  Brand,  T. :  Unsaponif iable  lipids  of 
Taenia  taeniaeformis  and  Moniezia  sp.  Exper.  Parasitol.  9:   127-130.   (1960). 

Bowman,  Iain  B.R.,  von  Brand,  Theodor,  and  Tobie,  Eleanor  J.:  The  cultivation 
of  trypanosomes  in  the  presence  of  trehalose  with  observations  on  trehalase 
in  blood  serum.  Exper.  Parasitol.  (In  press). 

von  Brand,  Theodor:  The  metabolism  of  trypanosomes  with  special  reference  to 
Trypanosoma  cruzi.   Proc.  1.   Internat.  Congr.  Chagas  Dis.   (In  press). 


Serial  No.  NIAID-123-A 


von  Brand,  Theodor:  Influencia  del  tamano,  motilidad,  ayuno  y  edad  sobre  la 
actividad  metabolica.  Biologia  28:  117-128.  (1959).   (Issued  April  1960  and 
therefore  not  reported  as  published  in  1959). 

Awards:  None 

Honors:  Theodor  von  Brand  presented  the  introductory  remarks  on  the  occasion 
of  the  symposium  "Host  influence  on  parasite  physiology,"  Rutgers  University's 
16th  Annual  Conference  on  Protein  Metabolism,  New  Brunswick,  January  1960. 

Theodor  von  Brand  served  as  coordinator  of  the  panel  "Impact  of  modern 
instrumentation  on  medicine  in  the  tropics."  Fourth  Conference  on  Research 
Needs  in  Tropical  Medicine.  New  Orleans,  La.  April  1960. 

Theodor  von  Brand  served  as  chairman  of  the"Symposium  sobre  biodinamia  para- 
sitaria".   I.  Congreso  Panamericano  de  Biologia  y  Patologia  Experimental, 
Caracas,  Venezuela,  September  1960. 

Theodor  von  Brand  gave  a  series  of  seven  lectures  on  parasite  physiology 
before  the  Instituto  de  Medicina  Tropical,  Universidad  Central  de  Venezuela, 
September  1960. 

Theodor  von  Brand  was  nominated  "Huesped  de  Honor"  by  the  Medical  Faculty 
of  the  Universidad  Central  de  Venezuela,  September  1960. 


11 


Serial  No.   NIAID-123-B 

1.  Parasitic  Diseases 

2.  Physiology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  Pathological  physiology  and  histochemistry  of  parasitic 
diseases. 

Principal  Investigator:  Teresa  I.  Mercado 

Other  Investigators:  Theodor  von  Brand 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:  2)4 

Professional:    1# 
Other:  1 

Project  Description: 

Objectives: 

To  gain  a  better  understanding,  by  means  of  studies  at  the 
cellular  level,  of  the  pathological  physiology  of  parasitic  diseases 
as  related  to  interaction  between  parasite  and  host. 

Methods  Employed: 

Normal  rats  and  rats  infected  with  Plasmodium  berqhei  are  fed 
large  amounts  of  hytakerol  (dihydrotachysterol)  for  4,  5,  or  6  days. 
They  are  then  killed  and  various  organs  are  fixed.   Sections  are 
stained  for  calcium,  primarily  by  the  von  Kossa  method,  and  for 
mucopolysaccharides  by  the  alcian  blue  procedure.  The  slides  are  then 
studied  in  respect  to  the  possible  presence  of  quantitative  and  quali- 
tative calcification  differences  between  normal  and  infected  animals. 

Major  Findings: 

In  most  organs  studied,  such  as  kidney,  heart,  lung,  and  other 
essentially  the  same  type  of  calcification  occurred  in  normal  and 
parasitized  animals;  the  question  of  possible  occurrence  of  quantitative 
differences  (either  in  respect  to  the  number  of  animals  showing  calci- 
fications, or  in  respect  to  the  degree  of  calcification)  requires 
further  analysis  of  the  prepared  slides.  A  significant  qualitative 
difference  between  malarious  and  control  rats  was  observed  in  stomach 
calcification.  The  majority  of  infected  animals  showed  pronounced  l|2 


Serial  No.  NIAID-123-B 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B    Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Mercado,  Teresa  I.,  and  von  Brand,  Theodor:  Histocheraical  studies  of 

liver  glycogen  and  lipid  in  some  parasitic  infections.   J.  Infect.  Dis.  106: 

95-105.   (1960). 

Honors:  None. 

Awards:  None. 


43 


Serial  No.  NIAID-123-B 


calcium  deposits  between  the  glands  of  the  stomach  mucosa,  resulting 
in  a  mosaic-like  pattern.  This  was  found  only  in  a  few  non-infected 
animals;  when  the  latter  showed  calcification,  the  deposits  were  seen 
more  frequently  in  the  muscular  coat. 

Significance  of  the  Program  to  the  Institute: 

Histocheraical  studies  concerned  with  the  pathological  physiology 
of  parasitic  diseases  allow  the  study  of  some  facets  of  the  complicated 
interaction  between  parasite  and  host  that  cannot  be  investigated  by 
purely  chemical  means.   As  such,  they  may  be  used  to  throw  light  on 
such  fundamental  questions  as  the  elusive  question  concerning  the 
possible  production  of  specific  parasite  substances  damaging  the  host, 
the  intimate  distribution  of  enzyme  patterns,  and  others;   all  of  them 
important  for  the  advancement  of  modern  chemotherapeutic  and  immuno- 
logical concepts. 

Proposed  Course  of  the  Project; 

The  calcification  studies  will  be  terminated  during  the  coming 
year.   H  is  proposed  to  study  by  modern  histochemical  procedures  cer- 
tain enzymes,  such  as  succinoxidase  or  cytochrome  oxidase,  in  the  liver 
of  normal  and  parasitized  rats. 


Part  B  included  Yes 


44 


Serial  No.  NIAID-123-C 
1.  Parasitic  Diseases 
PHS-NIH  2.  Physiology 

Individual  Project  Report       3.  Bethesda,  Maryland 
Calendar  Year  1960 


Part  A. 


Project  Title:  Biochemical  mechanisms  of  energy  metabolism  in 
normal  and  parasitized  animals. 

Principal  Investigator:  E.  C.  Weinbach 

Other  Investigators:  None 

Cooperating  Units:  Laboratory  of  Parasite  Chemotherapy;  Wenner-Grens 
Institute  Stockholm,  Sweden. 

Man  Years  (calendar  year  1960) 
Total:  ]% 

Professional:        1 
Other:  1/4 

Project  Description: 

Objectives: 

The  objective  is  to  conduct  fundamental  studies  on  the  mechanism 
of  energy  metabolism  in  normal  and  parasitized  animals. 

Methods  Employed: 

Mammalian  tissues  are  fractionated  by  mechanical  disruption  of 
the  cells  and  the  subcellular  elements  are  isolated  by  differential 
centrifugation.  Mitochondria  are  fragmented  further  by  various 
chemical  and  mechanical  means  to  obtain  membranes  bearing  the  electron 
transport  system.  These  cellular  and  subcellular  fractions  are  employed 
as  biological  catalysts  to  study  electron  transport,  oxidative  phos- 
phorylation, and  related  exergonic  reactions.   Intramitochondrial  sub- 
strates and  cofactors  of  metabolism  are  determined  by  suitable  enzymatic, 
chemical,  radiochemical,  and  spectrophotometric  techniques.   Manometric 
procedures  and  oxygen  electrode  recordings  are  used  to  measure  oxidation 
rates.  Radioisotope  techniques  are  used  to  investigate  subtle  metabolic 
changes.  Similar  studies  are  conducted  with  tissues  obtained  from 
parasitized  animals. 

Major  Findings: 

The  stability  of  isolated  liver  mitochondria  is  related  to  the 
endogenous  oxidative  phosphorylation.  Oxygen  electrode  recording  of 

45 


Serial  No.   NIAID-123-C 


the  respiratory  control  index  has  revealed  that  liver  mitochondria  are 
more  stable  when  they  exhibit  a  high  endogenous  metabolism. 

Phosphoenolpyruvate  is  formed  from  endogenous  substrates  in 
isolated  liver  mitochondria. 

Work  at  the  Wenner-Grens  Institut: 

Studies  are  conducted  on  the  oxidation  of  f lavosubstrates  by  the 
mitochondrial  respiratory  chain  and  on  the  regulation  by  f lavosubstrates 
of  the  redox  state  of  the  intramitochondrial  pyridine  nucleotides. 
Specifically,  the  present  study  concerns  the  mechanism  by  which  the 
addition  of  ATP  to  mitochondria  which  are  depleted  of  their  endogenous 
high  energy-phosphate  intermediates  stimulates  the  oxidation  of  a 
flavosubstrate  such  as  succinate  and,  simultaneously,  reduce  pyridine 
nucleotides.   In  effect,  this  is  a  "reversal"  of  oxidative  phosphory- 
lation and  it  appears  to  be  the  most  powerful  tool  to  date  for  studying 
the  mechanism  of  this  complex  bioenergetic  process. 

Major  Findings: 

Mitochondria,  depleted  of  endogenous  high-energy  phosphate, 
exhibit  a  marked  diminution  in  the  capacity  to  oxidize  succinate. 
Addition  of  ATP  restored  the  succinate  oxidation  and,  simultaneously, 
initiated  reduction  of  intramitochondrial  DPN.   Since  the  ATP  effect 
was  obtained  in  the  presence  of  uncoupling  agents,  it  must  be  exerted 
at  a  site  which  is  not  accessible  to  mitochondrial  ATPase.  This 
suggests  compartmentalization  in  the  mitochondria  and  preliminary 
studies  with  P3^  supports  this  concept.  The  important  fact  is  that  the 
oxidation  of  succinate  is  linked  to  an  energy-requiring  reaction,  and 
that  this  energy  is  invested  at  one  site  of  the  respiratory  chain  and 
then  utilized  at  another  site  for  reducing  pyridine  nucleotide. 

Significance  to  the  Program  of  the  Institute: 

This  investigation,  because  of  its  fundamental  nature  in  eluci- 
dating one  of  the  vital  processes  in  all  living  cells,  has  obvious 
significance  to  biochemistry  in  general,  and  provides  a  firm  basis  for 
our  understanding  of  the  subtle  biochemical  changes  in  energy  metabolism 
associated  with  parasitism.   In  addition,  it  provides  a  solid  foundation 
for  future  studies  on  the  complex  bioenergetic  mechanisms  of  parasitic 
forms. 

Proposed  Course  of  the  Project: 

Efforts  aimed  at  obtaining  additional  information  on  the  basic 
metabolism  of  mitochondria,  and  an  increased  understanding  of  the 
fundamental  mechanism  of  oxidative  phosphorylation  will  be  continued. 


Serial  No.  NIAID-123-C 


Specifically  at  the  Wenner-Grens  Institut; 

We  intend  to  measure  the  requirement  for  high-energy  phosphate 
of  different  systems  capable  of  succinate  oxidation.  Since  the  ATP 
effect  is  not  found  with  non-phosphorylating  submitochondrial  prepa- 
rations, we  plan  to  study  the  aerobic  oxidation  of  succinate  in  sub- 
mitochondrial fragments  which  have  different  degrees  of  phosphorylating 
capacity.  An  intense  effort,  employing  radioisotopes,  will  be  made  to 
examine  the  compartmentalization  concept  of  mitochondrial  organization. 


Part  B  included        Yes 


4? 


Serial  No.   NIAID-123-C 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B.     Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Weinbach,  E.C. :  Biochemical  changes  in  mitochondria  associated  with  age. 
In:  Strehler,  B.L.  (Editor):  The  Biology  of  Aging.  A  Symposium.  American 
Institute  of  Biological  Sciences,  Washington,  D.C.  Publication  No.  6: 
pp.  328-331.   (1960). 

Weinbach,  E.C. :  Oxidative  phosphorylation  with  endogenous  mitochondrial 
substrates.  Acta  Chem.  Scand.   (In  press). 

Schellenberg,  K.A.,  and  Weinbach,  E.C:  The  endogenous  formation  of 
phosphoenolpyruvate  by  rate  liver  mitochondria.  Biochem.  et  Biophys.  Acta. 
(In  press). 

Honors:  None 

Awards:  None 


H8 


PHS-NIH 

Individual  Project  Heport 

Calendar  Year   1960 


Part  A. 


Serial  No.  NIAID-123-D 

1.  Parasitic  Diseases 

2.  Physiology 

3.  Bethesda,  Maryland 


Project  Title:  Biology  of  trypanosomes. 

Principal  Investigator:  Eleanor  J.  Tobie 

Other  Investigators:  None 

Cooperating  Units:  Applied  Immunology  Section,  Laboratory  of  Immunology, 
NIAID.  Serial  No.  NIAID-148. 

Man  Years  (calendar  year  1960): 
Total:  1  3/4 

Professional:  3/4 

Other:  1 

Project  Description: 

Objectives: 

To  clarify  the  life  cycle  of  Trypanosoma  ranqeli  in  relation  to 
its  vertebrate  and  invertebrate  hosts;  to  study  ways  of  maintaining  and 
enhancing  infectivity  of  strains. 

Methods  Employed: 

Young  nursling  rats  and  Rhodnius  prolixus  are  used  as  hosts.  A 
colony  of  R.  prolixus  is  maintained.  One  strain  of  T.  ranqeli  is 
maintained  in  R.  prolixus- and  several  are  maintained  in  vitro.  Rats  and 
R.  prolixus  are  infected,  both  by  artificial  and  natural  means.   Infec- 
tions in  rats  are  determined  by  microscopic  examination  of  blood  or  by 
in  vitro  cultivation  of  blood.   Infections  in  R.  prolixus  are  proved  by 
microscopic  examination  of  fecal  material  and  hemo lymph,  feeding  on 
fresh  rats,  and  by  dissection,  with  microscopic  examination  of  body  fluids 
and  organs.   Fresh  frozen  sections  are  made  of  infected  R.  prolixus, 
stained  and  examined  microscopically.  Culture  material  is  grown  and 
harvested  for  immunizing  of  rabbits. 

Major  Findings: 

Various  strains  of  T.  ranqeli  react  differently  in  the  white  rat 
as  well  as  in  R.  prolixus.   Evidence  suggests  that  R.  prolixus  may  not  be 


Serial  No.  NIAID-123-D 


the  natural  invertebrate  host  for  all  strains  of  T.  ranqeli  or  that 
trypanosomes  designated  as  T.  ranqeli  actually  represent  more  than  one 
species.  Passage  through  R.  prolixus.  following  inoculation  into  the 
hemocoele  enhanced  the  ability  of  one  strain  to  invade  the  salivary 
glands,  making  it  possible  to  maintain  the  strain  by  cyclical  trans- 
mission. The  infectivity  of  i_n  vitro  strains  can  be  extended  by 
repeated  isolation  in  vitro  and  passage  through  the  rat.  R.  prolixus 
does  not  develope  resistance  to  T.  ranqeli  when  the  infection  does  not 
pass  beyond  the  digestive  tube.  Artificial  transfer  of  parasites  from 
fecal  material  to  the  hemocoele  resulted  in  completion  of  the  cycle. 

Significance  of  the  program  to  the  Institute: 

Intensive  biological  studies  of  this  species  may  provide  new  and 
valuable  information  general  to  trypanosomes.  This  is  a  valuable 
laboratory  tool. 

Proposed  Course  of  the  Project: 

Studies  will  be  directed  to  determining  the  life  cycle  of  the 
parasite  within  the  insect  host  by  localizing  the  organism  during  its 
stages  of  development  in  the  various  organs  by  means  of  the  fluorescent 
antibody  technique. 


Part  B  included        Yes 


r 


0 


Serial  No.   NIAID-123-D 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B    Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

von  Brand,  Theodor,  and  Tobie,  Eleanor  J:  The  mechanism  of  elimination  of 
certain  strains  or  species  of  trypanosomes  when  mixed  in  experimental 
infections.  J.  Parasitol.  46  (2):  129-136.   (1960). 

Tobie,  Eleanor  J. :  Experimental  transmission  and  biological  comparison  of 
strains  of  Trypanosoma  ranqeli.  Exp.  Parasitol.   (In  press). 

Honors:  None 

Awards:  None 


51 


Serial  No.   NIAID-127-A 

1.  Parasitic  Diseases 

2.  Protozoal  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  Studies  on  toxoplasmosis 

Principal  Investigator:  Leon  Jacobs 

Other  Investigators:  Milford  N.  Lunde,  Marjorie  L.  Melton, 
Anastasia  S.  Stanley 

Cooperating  Units:  Ophthalmology  Branch,  NINDB 

Man  Years  (calendar  year  1960): 
Total:  5% 

Professional:        3J£ 
Other:  2 

Project  Description: 

Objectives: 

The  accumulation  of  data  on  the  occurrence  of  toxoplasmosis  as  an 
acute  and  chronic  infection  of  man  and  animals;  the  study  of  the  biology 
of  the  proliferative  parasite  and  the  cyst,  with  the  aim  of  evaluating 
their  importance  in  the  epidemiology  of  toxoplasmosis  and  in  the  pro- 
duction of  chronic  disease;  the  determination  of  the  usefulness  of 
immunological  tests  in  diagnosis,  and  the  identification  of  antigens  and 
antibodies;  the  description  of  factors  related  to  the  virulence  of 
Toxoplasma,  and  of  those  involved  in  susceptibility  and  immunity  of  the 
host,  and  in  the  occurrence  of  congenital  disease;  the  elucidation  of  the 
mechanism  of  cyst  formation,  relative  to  factors  in  the  host;  the  critical 
study  of  toxoplasmicidal  agents  against  proliferative  parasites  and  cysts; 
the  study  of  the  role  of  hypersensitivity  in  ocular  toxoplasmosis. 

Methods  Employed: 

Standard  procedures  of  animal  inoculation  plus  special  techniques 
for  injection  into  the  anterior  or  posterior  chambers  of  the  eye.  A 
microisolation  apparatus  is  used  for  handling  cysts.  Histochemical  pro- 
cedures are  being  initiated  to  complement  electron-microscopic  work. 


52 


Serial  No.  NIAID-127-A 


Tissue  cultures  are  used  for  morphological,  histochemical,  and 
electron-microscopic  studies  and  for  attempts  at  growing  Toxoplasma  cysts 
in  vitro.  The  dye  and  hemagglutination  tests  are  used  for  serological 
studies,  and  the  antigens  are  investigated  by  biochemical  and  electro- 
phoretic  techniques. 

Major  Findings: 

Sheep  Studies  in  New  Zealand:  The  prevalence  of  Toxoplasma  in 
New  Zealand  sheep  is  very  high.   The  distribution  of  parasites  in  the 
tissues  of  sheep  is  erratic.   Parasitemia  was  found  only  in  the  first 
week  of  infection  with  two  strains  of  Toxoplasma  given  by  various  routes. 
Toxoplasma  in  the  sheep  is  rapidly  cleared  from  tissues  except  for  muscle 
and  placenta.   Residual  infection  in  sheep  is  more  likely  to  persist  in 
muscle  than  in  brain.   Mutton  can  therefore  definitely  serve  as  a  source 
of  human  infection. 

The  following  statements  concerning  the  circumstances  of  con- 
genital transmission  appear  justified  from  data  obtained,  although  not 
yet  complete:   Infection  60  days  prior  to  onset  of  pregnancy  does  not 
result  in  congenital  infection  or  abortion.  Abortion  or  foetal  death 
and  resorption  occurs  following  infection  of  the  ewe  at  30  days 
pregnancy.   Infection  at  90  days  pregnancy,  by  various  routes,  sometimes 
results  in  abortion  or  death  of  the  lamb  soon  after  birth.   In  other 
cases,  the  lamb  survives,  although  the  foetal  cotyledons  are  infected. 
Natural  immunity  or  active  immunity  produced  by  vaccination  with  live 
parasites  prior  to  mating  does  not  protect  ewes  from  Toxoplasma  abortion 
when  the  challenge  inoculum  is  high.  When  congenital  transmission  does 
occur,  only  one  of  twin  lambs  may  be  affected.  The  foetal  cotyledons  are 
more  consistently  infected  than  are  the  tissues  of  the  lambs. 

Studies  at  N.I.H:  The  failure  of  immunization  with  live  organisms 
completely  to  protect  animal  from  a  challenge  infection  has  been  demon- 
strated in  further  tests  in  guinea  pigs. 

Further  studies  have  been  conducted  on  parasitemia  in  chronically 
infected  mice  and  additional  work  has  been  done  on  rabbits.  Both  species 
may  exhibit  low  grade  parasitemia  for  months.   Since  these  animals  have 
high  antibody  levels,  it  is  difficult  to  explain  the  persistent  activity 
of  the  parasite. 

Various  domestic  animals  have  a  heat-labile,  non-specific,  anti- 
Toxoplasma  activity  in  their  serum.   It  has  been  found  also  in  human 
sera.  The  use  of  citrate  in  the  collection  of  accessory  factor  sera  for 
the  dye  test  diminishes  or  eliminates  this  non-specific  activity. 

A  serological  survey  of  cattle  sera  indicates  that  in  these  animals 
there  is  considerable  difference  in  hemagglutination  and  dye  test  titers. 

53 


Serial  No.  NIAID-127-A 


Ultra  violet  absorption  at  260  and  280  millimicrons  shows  a 
relatively  large  amount  of  nuclear  protein  material  in  the  hemagglutina- 
tion antigen.  Agar  gel  diffusion  with  the  hemagglutination  antigen  and 
its  rabbit  antiserum  indicates  at  least  two  separate  antigen-antibody 
systems  are  present. 

The  complement-fixation  test  for  toxoplasmosis  has  been  set  up 
as  an  additional  diagnostic  procedure. 

Use  of  pyrimethamine  in  mice  with  chronic  infections  has  reduced 

the  number  of  cysts.  This  has  important  implications,  possibly,  relative 

to  control  of  toxoplasmic  chorioretinitis.  The  same  effect  has  been 

observed  with  sulfadiazine. 

Cysts  of  Toxoplasma  have  been  found  at  all  levels  of  the  intes- 
tinal tract  and  in  the  lung  of  chronically  infected  mice  three  to  four 
months  after  infection.  There  is  evidence  that  cysts  of  Toxoplasma  form 
in  tissue  cultures.  Suspensions  of  cultures  have  infected  mice  after 
pepsin-HCl  digestion,  a  treatment  which  kills  proliferative  forms. 

Significance  to  the  Program  of  the  Institute: 

Knowledge  of  the  transmission  of  Toxoplasma  in  nature  is  requisite 
as  a  basis  for  recommendations  for  prevention  of  the  infection.  Continued 
work  is  necessary  to  explain  mechanisms  of  transmission  to  herbivores 
and  vegetarian  human  beings. 

The  analysis  of  antigens  derived  from  Toxoplasma  has  importance 
in  relation  to  an  understanding  of  the  course  of  the  disease  and  the 
action  of  various  antibodies,  as  well  as  diagnosis.  Continued  work  on 
the  hemagglutination  test  may  establish  it  as  the  best  practicable  pro- 
cedure for  diagnosis  of  human  toxoplasmosis. 

Extensive  and  intensive  basic  study  of  Toxoplasma  infection  will 
furnish  explanations  for  clinical  observations  and  contribute  to  a 
general  understanding  of  chronic  infections.  This  is  of  great  importance 
in  relation  to  ocular  disease  and  may  also  reveal  instances  of  chronicity 
in  systemic  infections. 

Proposed  Course  of  the  Project: 

Attention  will  be  paid  to:  the  epidemiology  of  toxoplasmosis  in 
herbivorous  animals;  the  circumstances  of  cyst-formation  and  rupture  in 
chronic  infections;  the  relation  of  cysts  and  dormant  parasites  to 
chronic  disease;  the  cultivation  of  cysts  in  tissue  culture;  the  mechanism 
of  proliferation  of  Toxoplasma  in  cells  in  tissue  cultures;  the  relation 
of  antibodies  to  particular  antigenic  components  and  to  persistence  of 

5h 


Serial  No.   NIAID-127-A 


chronic  infections;  the  effect  of  drugs  on  cyst  and  on  activity  of  the 
parasite  during  chronicity;  the  relation  of  local  tissue  immunity  to 
the  spread  of  challenge  infections;  and  the  significance  of  non-specific 
ant i -Toxoplasma  activity  of  serum. 


Part  B  included     Yes 


55 


Serial  No.   NIAID-127-A 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Jacobs,  Leon,  Remington,  Jack  S.,  and  Melton,  Marjorie  L.   The  resistance  of 
the  encysted  form  of  Toxoplasma  gondii.   J.  Parasitol.  46:   11-21.   (1960) 

Jacobs,  Leon,  Remington,  Jack  S.,  and  Melton,  Marjorie  L.   A  survey  of  meat 
samples  from  swine,  cattle,  and  sheep  for  the  presence  of  encysted  toxoplasma. 
J.  Parasitol.  46:  23-28  (1960) 

Remington,  Jack  S. ,  Jacobs,  Leon,  and  Kaufman,  Herbert.  Toxoplasmosis  in  the 
adult.   New  England  Journal  Medicine  262:   180-186,  237-241.   (1960) 

Frenkel,  J.  K.,  Weber,  R.  W.,  and  Lunde,  M.  N.  Acute  toxoplasmosis.   Effective 
treatment  with  pyrimethamine,  sulfadiazine,  leucovorin  calcium,  and  yeast. 
J.A.M.A.   173:  1471-1476.   (1960) 

Jacobs,  Leon.  Ocular  toxoplasmosis:  Laboratory  contributions  to  diagnosis 
and  chemotherapy.  Reprinted  from  Human  Toxoplasmosis  Copenhagen:  Munksgaard. 
(1960) 

Remington,  Jack  S.,  Jacobs,  Leon,  and  Melton,  Marjorie  L.  Chronic  toxoplasma 
infection  in  the  uterus,   (in  press)  J.  Lab  and  Clin.  Med. 

Remington,  Jack  S.,  Jacobs,  Leon,  and  Melton,  Marjorie  L.  Congenital  transmis- 
sion of  toxoplasmosis  from  mother  animals  with  acute  and  chronic  infections. 
J.  Inf.  Dis.   (in  press) 

Honors  and  Awards: 

Dr.  Leon  Jacobs  was  awarded  a  Fulbright  Fellowship  for  study  of  problems 
in  the  epidemiology  and  pathology  of  toxoplasmosis  in  New  Zealand.   He  departed 
on  this  assignment  May  1. 

Dr.  Leon  Jacobs  visited  Brisbane,  Sydney,  and  Melbourne  from  10  to  24 
October  on  a  visiting  lecturer  under  the  Australian  Fulbright  Program.  He 
lectured  in  universities,  hospitals  and  veterinary  research  institutions. 

Dr.  Leon  Jacobs  was  awarded  a  Guggenheim  Fellowship  which  will  permit  him 
to  visit  a  number  of  Asian  and  European  parasitology  laboratories  on  his  return 
trip  from  New  Zealand. 


56 


Serial  No.  NIAID-127-B 

1.  Parasitic  Diseases 

2.  Protozoal  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:  Studies  on  Entamoeba  histolytica  and  other 
parasitic  protozoa. 

Principal  Investigator:  Louis  S.  Diamond 

Other  Investigators:  Harry  D.  Baernstein 
Lucy  V.  Reardon 

Cooperating  Units:  Biophysics  Laboratory,  Naval  Institute  for 
Medical  Research. 

Man  Years  (calendar  year  1960): 
Total  2% 

Professional:        1J£ 
Other:  1 

Project  Description: 

Objectives: 

To  study  metabolism  of  E.  histolytica,  methods  of  diagnosis, 
mechanisms  of  pathogenesis,  factors  in  host  susceptibility,  methods  of 
culture,  immunology,  and  variation.  To  study  the  cultural  requirements 
of  axenically  cultivated  Entamoeba  spp.  of  lower  animals  in  order  to 
extend  our  knowledge  of  the  biology  of  these  important  parasites.  To 
devise  a  method  for  the  axenic  cultivation  of  Trichomonas  tenax,  the 
oral  trichomonad  of  man.  To  develop  techniques  for  the  preservation  of 
parasitic  protozoa  by  freezing  and  storage  at  low  temperatures  and  by 
freezing  and  storage  in  the  dry  state. 

Methods  Employed: 

Usual  methods  for  axenic  studies  and  for  use  of  single  bacterial 
or  protozoan  associates;  micro-isolation  methods;  immunological  methods, 
including  adsorption  and  extraction.   For  preservation  studies,  protozoa 
are  suspended  in  a  variety  of  suitable  agents  and  cooled  slowly  from 
ambient  temperatures  to  -25°  C,  then  stored  at  this  temperature  or  at 
temperatures  of  -79°  C  and  -197°  C;  or  the  protozoa  are  frozen  rapidly 
in  vacuo  at  a  temperature  of  -30°  C,  then  stored  at  -197°  C;  or  protozoa 
are  supercooled,  frozen  and  dried  i_n  vacuo. 

57 


Serial  No.   NIAID-127-B 


Major  Findings; 

Axenic  cultivation  of  E.  histolytica  and  T.  tenax  was  achieved  in 
an  anaerobic  medium  supplemented  with  a  cell  free  extract  of  chick  embryo. 
Neither  species  grew  in  the  absence  of  the  extract.  Furthermore,  growth 
of  E.  histolytica  occurred  only  when  the  medium  was  made  up  in  a  diphasic 
form,  i.e.,  a  solid  agar  slant  with  a  liquid  overlay. 

A  substitute  for  T.  cruzi  as  an  associate  in  the  monoxenic  culti- 
vation of  E.  histolytica  was  found  in  the  form  of  a  new  species  of 
Crithidia  isolated  from  the  gut  of  a  hemipteran.  The  new  associate  in- 
creases the  yield  of  amoebae  by  a  factor  of  4,  does  not  require  a  separate 
medium  for  maintenance  of  stock  cultures,  and  is  not  known  to  be  a  para- 
site of  man. 

Several  batches  of  antigen  have  been  prepared  from  monoxenic  E. 
histolytica  -  T.  cruzi  cultures. 

Studies  on  the  cultural  requirements  of  E.  inyadens  resulted  in 
the  following:  a)  development  of  a  technique  for  counting  the  amoebae 
with  a  Coulter  Electronic  counter;  b)  the  finding  that  the  amoebae  re- 
acted adversely  to  any  change  in  concentrations  of  trypticase,  yeast 
extract  or  horse  serum;  c)  that  yeast  extract  could  not  be  replaced  by 
Eagle's  vitamin  B  mixture. 

There  has  been  progress  toward  development  of  a  method  for  preser- 
vation of  E.  histolytica  and  other  protozoan  species  by  freeze-drying,  but 
so  far  it  has  not  been  possible  to  store  the  freeze-dryed  material  success- 
fully and  still  preserve  viability. 

Attempts  to  preserve  the  protozoans  by  freezing  without  drying 
had  been  more  successful.  The  slow  freezing  and  subsequent  storage  at 
-79°  C  of  T.  vaginalis,  T.  hominis,  T.  gallinae,  and  E.  histolytica 
suspended  in  dimethyl  sulfoxide  has  resulted  in  preservation,  to  date, 
of  these  protozoa  for  periods  of  4  months,  3  months,  30  and  29  days 
respectively.  E.  histolytica  cooled  to  a  temperature  of  -197°  C  by  a 
similar  technique  has  been  stored  successfully  at  this  temperature  for 
a  period  of  24  hours.   This  last  finding  is  most  encouraging,  since 
theory  predicts  that  a  cell  capable  of  withstanding  exposure  to  -197°  C, 
even  for  such  a  short  time,  should  be  capable  of  almost  indefinite 
storage  at  such  temperatures. 

Significance  to  the  Program  of  the  Institute: 

The  development  of  a  technique  for  growing  E.  histolytica  under 
axenic  conditions  should  open  the  way  to  the  solution  of  many  unsolved 
problems  in  amoebiasis.   It  should  be  possible,  for  example,  to  determine 
whether  or  not  £.   histolytica,  by  itself,  is  capable  of  initiating  the 

53 


Serial  No.  NIAID-127-B 


lesions  of  araoebiasis;  to  prove  or  disprove  the  existence  of  lytic 
substances  of  amoebic  origin;  and  to  determine  growth  requirements  in 
vitro.   Solution  of  the  technical  problems  associated  with  axenic  culti- 
vation of  the  amoebae  and  T.  tenax  should  find  application  in  solving 
similar  problems  met  with  in  the  cultivation  not  only  of  other  parasites, 
but  mammalian  cells  as  well. 

Techniques  developed  for  the  preservation  of  parasitic  protozoa 
by  freezing  and  freeze-drying  should  be  applicable  to  preservation  of 
other  types  of  cells. 

Development  of  a  technique  for  enumerating  protozoa  in  culture 
with  the  aid  of  the  Coulter  Electronic  counter  has  extended  the  use  of 
this  instrument,  which  was  originally  designed  to  count  blood  cells, and 
gives  indication  that  this  instrument  could  be  utilized  profitably  in 
counting  mammalian  cells  from  tissue  culture. 

Proposed  Course  of  Project: 

Efforts  will  be  made  to  define  and  improve  media  employed  in 
axenic  cultivation  of  E.  histolytica  and  T.  tenax;  to  develop  methods 
for  axenic  cultivation  of  other  parasitic  protozoa  of  man;  to  develop 
more  sensitive  antigens  for  serological  diagnosis;  to  study  factors 
associated  with  invasiveness  and  pathogenicity  of  strains  of  E.  histo- 
lytica in  experimental  hosrs;  to  study  cytochemical  changes  during 
division  of  E.  histolytica;  to  refine  techniques  for  preservation  of 
protozoa  by  freezing  and  freeze-drying. 


Part  B  included     No 


5S 


Serial  No.  NIAID-127-C 

1.  Parasitic  Diseases 

2.  Protozoal  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:  Trichomoniasis 

Principal  Investigator:  Lucy  V.  Reardon 

Other  Investigators:  Louis  S.  Diamond 

Cooperating  Units:  Laboratory  of  Clinical  Investigations,  NIAID 
Laboratory  of  Germfree  Animal  Research,  NIAID 
Laboratory  of  Pathology  and  Histochemistry,  NIAMD 
Clinical  Investigations  Branch,  NIDR 

Man  Years  (calendar  year  1960): 
Total:  1-3/4 

Professional:         3/4 
Other:  1 

Project  Description: 

Objectives: 

Continued  study  of  the  pathogenesis  of  Trichomonas  vaginalis; 
transfer  of  genetic  characters  in  strains  of  T.  vaginalis;  antibody  for- 
mation and  hypersensitivity  in  relation  to  resistance  and  pathogenesis; 
the  action  of  purported  trichomonacides  (as  well  as  amoebacides)  as 
determined  by  in  vitro  tests;  the  possible  relation  of  oral  protozoa  to 
peridontal  disease. 

Methods  Employed: 

Pathogenesis/trichomonads  is  tested  in  mice  by  intraperitoneal 
injection  of  concentrated  organisms.  Microbe-free  cultures,  maintained 
without  use  of  antibotics,  furnish  the  inocula. 

Pathogenesis  is  also  studied  in  germfree  guinea  pigs.  Microbe- 
free  material  is  injected  subcutaneously  into  germfree  pigs,  monocon- 
taminated  pigs,  and  conventional  pigs.  The  effect  of  the  sera  from  the 
above  guinea  pigs  on  washed  concentrates  of  trichomonads  is  observed 
microscopically. 


60 


Serial  No.  NIAID-127-C 


For  studies  on  genetic  transfer,  organisms  of  the  non-virulent 
R  strain  are  associated  with  horaognates  of  the  virulent  C-l  strain 
processed  to  contain  DNA  of  the  strain  and  then  injected  into  mice  for 
evidence  of  alteration  of  characters.  Genetic  transfer  is  also  attempted 
through  efforts  to  produce  an  aureomycin  resistant  strain  of  the  organism. 
The  haemagglutination  test  is  used  for  evidence  of  antibody  production. 
Human  sera  and  vaginal  exudates  are  likewise  tested.   Evaluation  of  pur- 
ported trichomonacides  (as  well  as  amoebacides),  is  made  by  i_n  vitro 
tests.  Diseased  and  normal  tissues  from  patients  with  peridontal 
disease  are  examined  for  oral  protozoa,  both  by  direct  examination  of 
the  material  and  by  cultures. 

Major  Findings: 

Virulent  and  avirulent  T.  vaginalis  were  retested  in  mice  after 
having  been  in  continuous  in  vitro  cultivation  4  and  5  years  respectively. 
No  change  in  virulence  was  observed. 

Experimental  studies  of  germfree  guinea  pigs,  monocontaminated 
pigs  (as  well  as  conventional  pigs)  showed  as  in  previous  experiments, 
large,  palpable  swellings  or  abscesses  occurring  at  the  site  of  in- 
jection in  germfree  pigs,  less  marked  swelling  in  the  monocontaminated 
pigs,  and  none  in  conventional  pigs.   Germfree  pigs,  rechallenged  with 
trichomonads  after  subsidence  of  swellings,  failed,  in  one  experiment, 
to  develop  new  swellings.   Sera  from  germfree  or  monocontaminated  pigs 
appeared  to  have  a  deleterious  effect  on  the  trichomonads.   Sera  from 
the  conventional  pigs  had  no  effect  on  the  trichomonads  other  than  to 
cause  agglutination. 

Additional  experiments  on  transformation  of  virulence  by  assoc- 
iation of  the  non-virulent  strain  with  DNA  of  the  virulent,  showed  no 
evidence  of  such  transformation. 

The  preparation  of  T.  vaginalis  antigens  was  continued.   Pro- 
duction in  guinea  pigs  of  high  titers  of  haemagglutinins  against  T. 
vaginalis  demonstrated  the  usefulness  of  whole  organisms  as  a  source  of 
antigen.   Haemagglutinins  against  an  enzyme  preparation  of  sonicated 
T.  vaginalis  were  also  produced  in  guinea  pigs.   These  enzyme  haemag- 
glutinins tested  in  assay  system  for  malic  dehydorgenase  showed  decided 
inhibitory  action  against  this  enzyme. 

Humatin,  (Paromomycin  sulfate),  failed  to  cause  complete  inhi- 
tition  of  activity  of  T.  vaginalis  in  concentrations  of  the  drug  up  to 
800  ug/ml  of  medium.   E.  histolytica  in  association  with  Trypanosoma 
cruzi  was  not  inhibited  in  concentrations  of  the  drug  up  to  64  ug/ml. 
However,  in  association  with  a  Crithidia  (an  insect  flagellate)  as  its 
supporting  organism,  E.  histolytica  appeared  inhibited  in  concentrations 
of  32  and  64  ug/ml  but  not  in  lower  concentrations.   The  Crithidia  was 
likewise  not  inhibited  in  the  lower  concentrations  of  the  drug. 

61 


Serial  No.   NIAID-127-C 


Significance  to  the  Program  of  the  Institute. 

Such  studies  should  give  new  basic  information  on  the  trichomonads 
and  lead  to  a  better  understanding  of  their  pathogenecity,  variation, 
transmission,  life  history,  etc. 

Proposed  Course  of  Project: 

Attempts  will  be  made  to  induce  haemagglutinis  against  individual 
enzymes  of  T.  vaginalis  and  to  develop  a  hemagglutination  test  for  tri- 
chomoniasis. Transformation  and  strain  studies  will  be  continued. 


Part  B  included      No 


62 


Serial  No.   NIAID-127-D 

1.  Parasitic  Diseases 

2.  Protozoal  Diseases 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  Biochemistry  of  parasitic  protozoa. 

Principal  Investigator:  Harry  D.  Baernstein 

Other  Investigators:  L.  V.  Reardon 
L.S.  Diamond 

Cooperating  Units:  None 

Man  Years  (calendar  year  1960): 
Total:  3/4 

Professional:        3/4 
Other:  None 

Project  Description: 

Objectives: 

To  study  the  biochemistry  of  the  parasitic  protozoa:  especially 
the  intracellular  enzymes  and  biochemical  problems  related  to  axenic  and 
monoxenic  cultivation. 

Methods  Employed: 

Cultivation  of  various  species  and  evaluation  of  growth  rates  in 
relation  to  composition  of  the  medium  by  use  of  the  Coulter  Counter. 

The  isolation  and  purification  of  intracellular  enzymes  by  con- 
ventional precipitation  methods  and  by  electrophoresis,  and  spectro- 
photometry methods  to  evaluate  activities  related  to  the  concentration 
of  protein  present.  Relations  of  the  organisms  to  their  hosts  are 
studied  by  analysis  for  antienzymes  and  other  antibodies  developed  in 
serum  against  purified  antigens. 

Major  Findings: 

Studies  on  the  enzymatic  activation  of  acetate  in  T.  vaginalis 
showed  the  presence  of  such  activation  as  determined  by  hydroxamic  acid 
production.  However,  the  type  of  hydroxamic  acid  produced  must  await 
purification  of  the  enzyme  and  identification.  Preliminary  experiments 
on  acetylation  of  sulfanilamide  were  negative. 

63 


Serial  No.   NIAID-127-D 

Significance  to  the  Program  of  the  Institute: 

The  elucidation  of  biochemical  properties  of  these  parasitic 
organisms  should  help  us  understand  their  pathogenicity  and  their 
susceptibility  to  drugs.   Specific  enzyme-inactivating  antibodies  can 
be  useful  in  the  study  of  enzyme  function  and  differentiation  and  identi- 
fication of  enzymes.  The  techniques  developed  for  producing  these  anti 
enzymes  should  be  applicable  in  the  study  of  enzymes  of  a  variety  of  cells. 

Proposed  Course  of  Project: 

Analysis  of  the  complex  components  of  media  employed  in  cultiva- 
tion of  parasitic  protozoa.  Analysis  of  the  enzyme  systems  present  in 
the  organisms.   Better  preparations  with  fewer  interfering  enzymes 
should  result  by  application  of  electrophoresis.   The  problem  of  multiple 
enzymes  having  the  same  substrate  specificity  (isoenzymes)  will  be 
studied.   The  special  biochemical  problems  associated  with  anaerobiasis 
such  as  electron  transport  will  be  explored. 


Part  B  included    Yes 


Oh 


Serial  No.    NIAID-127-D 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B.       Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Baernstein,  H.  D. :   Malic  dehydrogenase  of  Trichomonas  vaginalis. 
J.  Parasitol.   (in  press). 

Honors:  None 
Awards:  None 


LABORATORY  OF  PARASITE  CHEMOTHERAPY 


/-" 


/ 


Summary 1 

130  -  Administration,  Research  Planning  and 

Coordination 6 

130-A  -  Studies  in  Human  Malaria 10 

131  -  Drug  Resistance  in  Experimental 

Malaria 15 

131-A  -  The  Effect  of  Adrenal  Cortical 

Hormone  on  Chemotherapy  of 

Experimental  Malaria 17 

131-B  -  Antimalarial  Drugs  and  Nucleic  Acid 

Synthesis 19 

131-C  -  Experimental  Chemotherapy  of 

Helminthic  Diseases 22 

131-D  -  Effects  of  Nutrition  on  Chemotherapy 

of  Parasitic  Diseases 25 

131-E  -  Comparison  of  Antimalarial  Drugs 

Administered  Singly  and  in  Various 

Combinations 28 

131-F  -  Effect  of  Nutritional  Status  of  Host 

on  the  Action  of  Antimalarial  Drugs..  30 

132  -  Studies  on  Human  Malaria 32 

132-A  -  Chemotherapy  of  Intestinal  Parasites.  37 
132-B  -  Physiology  of  Human  Parasites 

Especially  as  Related  to  Nucleic 

Acids  and  Drug  Action 40 

132-C  -  Virus-Parasite  Association 43 

132-D  -  Development  of  Human  Pathogens  in 

Immature  Insects 47 

132-E  -  Insect  Tissue  Culture 49 

133  -  Studies  of  the  Biology  of 

Exoerythrocytic  Phases  of  Primate 
Malaria 51 

133-A  -  Chemotherapeutic  Studies  of  the 
Direct  Effect  of  Drugs  on  the 
Exoerythrocytic  Stages  of  Primate 
Malaria 54 

133-B  -  Studies  of  Simian  Malarias  in  Man 

to  Determine  if  Malaria  is  a  Zoonotic 
Disease  in  Areas  in  which  Man  and 
Monkey  are  Closely  Associated 57 

133-C  -  In  Vitro  Studies  of  Malaria  Parasites 

and  Liver  Tissue  61 

133-D  -  Immunological  Studies  of  Malaria 

Parasites  66 


Laboratory  of  Parasite  Chemotherapy,  NIAID 
Summary  Statement  of  Research  Activities,  Calendar  Year  1960 

This  Country's  commitment  of  38  million  dollars  in  Fiscal  Year  1961 
toward  a  program  of  world-wide  malaria  eradication  and  the  long-term  interest 
in  malaria  by  most  of  the  senior  staff  resulted  in  a  research  effort,  during 
the  past  year,  largely  directed  toward  problems  in  that  field.   Special 
emphasis  was  given  to  the  study  of  simian  malaria  in  man  and  in  monkeys  be- 
cause malaria  in  simians  might  be  a  real  deterrent  to  the  eradication  program. 
Clinical  facilities  at  the  Atlanta  Penitentiary  were  enlarged  and  the  staff 
increased.   A  laboratory  was  established  at  Kuala  Lumpur,  Malaya  in  cooperation 
with  the  Malaya  Institute  of  Medical  Research  and  the  United  States  Medical 
Research  Unit.   Studies  on  several  aspects  of  the  simian-human-malaria  problem 
have  been  in  progress  there  since  mid-August. 

As  a  result  of  the  above  development,  it  was  decided  to  move  the  Section 
on  Cytology,  now  located  at  Memphis,  to  Chamblee,  Georgia,  early  in  1961. 
Alterations  necessary  to  accommodate  this  move  are  nearing  completion.   This 
arrangement  will  bring  the  simian  hosts  closer  to  the  human  volunteers  at  the 
penitentiary,  and  the  insectary  maintained  by  the  Section  will  be  geared  to 
accommodate  the  work  at  Chamblee  and  at  the  prison. 

Dr.  Jeffery  of  the  Section  on  Epidemiology  is  spending  a  year  in 
graduate  study  at  Yale  University  and  therefore  the  Section  has  been  without 
his  services  since  July.   The  program  of  the  Laboratory  was  reviewed  by  the 
Board  of  Scientific  Councilors  who  visited  the  Laboratory  at  Columbia,  November 
9-10. 

The  work  of  the  Section  on  Chemotherapy  has  been  curtailed  due  to  two 
resignations,  Drs .  Gaudette  and  Schellenberg,  during  the  past  year.   During 
this  period,  Dr.  Jacobs  joined  the  staff  of  that  Section  and  has  initiated  a 
program  of  research  aimed  at  an  assessment  of  the  place  of  nutrition  in 
chemotherapy. 

MALARIA  -  HUMAN    Plasmodium  falciparum  (McLendon  strain) :   Chloroquine 

(300  mg,  base)  and  primaquine  (45  mg,  base)  given  together 
beginning  3  days  after  mosquito  bites  and  weekly  thereafter  for  a  total  of  8 
doses,  resulted  in  suppressive  cure  in  5/5  subjects.   Controls  were  positive 
11-15  days  after  infection.  After  2  days  of  parasitemia,  each  control  was 
given  the  above  drug  combination  which  was  repeated  weekly  for  a  total  of  3 
doses.   Parasites  were  removed  promptly  and  cure  was  obtained  based  on  no 
evidence  of  infection  during  227  days  of  observation. 

Primaquine,  at  daily  doses  of  0.75  mg,  had  some  sporontocidal  effect 
upon  Plasmodium  falciparum  gametocytes  but  none  against  those  of  P.  vivax 
(one  case).   Therapeutic  doses  (1.4  gm  in  3  days)  of  amodiaquine  had  no 
sporontocidal  effect  against  gametocytes  of  P.  falciparum  (one  case).   The 
effect  referred  to  is  against  the  development  of  the  malaria  parasites  in  the 
mosquito. 


A  strain  of  Plasmodium  falciparum  from  Colombia,  South  America,  was 
found  to  be  resistant  to  chloroquine.  This  finding  is  of  utmost  importance 
in  terms  of  malaria  eradication. 

Plasmodium  vivax  (Chesson  strain):   A  drug  combination  of  primaquine 
(45  mg)  and  pyrimethamine  (50  mg)  given  weekly  beginning  7  days  after  mosquito 
bite  and  continuing  for  a  total  of  4  doses,  gave  suppressive-cure  in  4/5 
subjects;  the  other  subject  developed  a  patent  infection  240  days  after  infec- 
tion.  Pyrimethamine  (50  mg)  given  alone,  as  above,  produced  suppressive-cure 
in  1/4  subjects;  the  other  3  came  down  on  days  82,  83  and  84.   Five  controls 
all  came  down  12  to  13  days  after  infection. 

The  Russian  8-aminoquinoline,  qulnocide,  was  compared  with  primaquine 
and  found  to  be  distinctly  inferior  as  a  curative  drug  against  early  and  late 
primary  attacks  of  Chesson  vivax  malaria  particularly  from  the  standpoint  of 
the  occurrence  of  second  and  third  relapses. 

Another  8-aminoquinoline,  Win  5037,  was  studied  in  5  subjects.   Toxic 
effects  and  failure  to  cure  made  further  investigation  unwarrented. 

Plasmodium  malar iae:   The  results  of  a  14-year  study  of  the  biology 
of  Plasmodium  malariae  were  drawn  together  for  publication.   The  highest 
infect ivity  for  mosquitoes  occurred  during  the  8th  to  10th  weeks  of  the 
primary  attack.   Although  the  infection  rate  of  mosquitoes  was  ordinarily  low, 
the  relatively  long  period  during  which  mosquitoes  could  be  infected  may  ex- 
plain the  persistence  of  P.  malariae  in  nature.   The  ability  of  the  symptom- 
free  malarious  patient  to  infect  mosquitoes  at  a  rate  similar  to  that  of  the 
symptomatic  patient  makes  eradication  difficult. 

MALARIA  -  SIMIAN    Plasmodium  cynomolgi  bastianellii:   In  early  May,  two 

accidental  sporozoite-induced  infections  with  Plasmodium 
cynomolgi  bastianellii  occurred  at  our  Memphis  Laboratory.   This  happening 
was  of  signal  importance  because  it  showed  that  simian  malaria,  contrary  to 
the  generally  held  opinion,  was  infectious  to  man.   In  that  light,  full  scale 
study  of  human  infections  was  undertaken  at  our  Atlanta  Penitentiary  installa- 
tion. 

Two  infections  were  induced  in  inmate  volunteers  by  inoculation  of  in- 
fected blood  obtained  from  one  of  the  accidental  sporozoite-induced  infections 
in  man.   Twenty  inmate  volunteers  were  infected  by  bites  of  Anopheles 
quadrimaculatus  or  Anopheles  freeborni  which  had  fed  on  infected  monkeys.   The 
prepatent  period  ranged  from  14  to  29  days  and  the  parasite  density  ranged  from 
5  to  500/cmm.   The  most  constant  symptom  was  headache  and  the  most  significant 
signs  were  fever,  splenomegaly  and  hepatomegaly.   Infections  were  allowed  to 
run  their  course,  generally  without  treatment. 

Anopheles  freeborni  were  infected  from  two  patients  but  attempts  to 
infect  volunteers  by  their  bites  have  yielded  equivocal  results.   The  finding 
that  P.  c.  bastianellii  will  grow  consistently  and  produce  clinical  illness  in 
man  suggested  the  possibility  that  malaria  is  a  zoonotic  disease,  that  is,  a 
disease  which  man  can  acquire  from  animals  with  which  he  is  associated.   Whether 

2 


or  not  such  transfer  occurs  in  nature  is  not  yet  determined,  but  should  it 
occur,  it  would  be  of  greatest  significance  to  the  world-wide  malaria  eradica- 
tion program. 

Plasmodium  cynomolgi  cynomolgi:   Eleven  inmate  volunteers  were  bitten 
by  Anopheles  freeborni  infected  with  P.  c_.  cynomolgi  on  8  September,  and  to 
date  (14  December)  three  have  exhibited  evidence  of  infection  (i.e.,  fever). 
Parasitemia  has  been  demonstrated  in  only  one,  on  the  58th  day  after  mosquito 
bites.   These  results  show  that  this  strain  infects  man  far  less  readily  than 
P.  c.  bastianellii. 


FIELD  STUDIES    Three  staff  members,  Drs.  Eyles,  Dobrovolny,  and  Mr.  Clinton 
IN  MALAYA        S.  Smith,  were  detailed  to  Malaya  during  the  year  where  they 
engaged  in  the  study  of  simian  and  human  malaria  in  coopera- 
tion with  the  Malayan  Institute  for  Medical  Research  and  the  U.  S.  Army  Medical 
Research  Unit  at  Kuala  Lumpur. 

The  epidemiology  of  monkey  malarias  is  being  studied  and  the  feeding 
habits  of  some  of  the  Anopheles  determined.   By  injection  of  uninfected 
monkeys  with  sporozoites  from  natural  infections,  it  was  determined  that 
Anopheles  hackeri  is  a  natural  vector  of  Plasmodium  knowlesi.   This  is  a  most 
important  discovery,  especially  since  the  vector  of  this  parasite  has  been 
sought  for  repeatedly  during  the  last  25  years. 

Studies  of  malaria  in  aborigenes  associated  with  monkeys  have  been 
made.   Blood  passed  from  aborigenes  to  monkeys  have  thus  far  produced  no 
patent  infection  in  the  monkeys. 

EE  STAGES  AND    Studies  were  continued  on  the  direct  effect  of  drugs  on  the 
DRUG  ACTION      exoerythrocytic  stages  of  primate  malaria.   When  sulfonamides 

were  used  with  pyrimethamine  to  exploit  the  possible  syner- 
gism of  the  two  drugs,  monkeys  developed  parasitemia  30  to  40  days  after 
inoculation  with  sporozoites  even  though  all  parasites  observed  in  liver 
biopsies  were  damaged.   The  curative  efficacy  of  quinocide,  the  Russian  drug, 
was  compared  with  primaquine.   Even  when  administered  at  twice  the  dosage 
used  with  primaquine,  quinocide  was  less  effective.   Chloroquine  had  no 
observable  effect  upon  the  liver  forms  of  Plasmodium  cynomolgi.   Young  para- 
sites appeared  in  the  blood  in  large  numbers  on  the  8th,  16th  and  24th  day 
indicating  the  existence  of  secondary  exoerythrocytic  generations. 

INSECT  TISSUE    Blood  cells  from  caterpillers  and  cells  of  the  ovariole 
CULTURE  sheath  of  several  species  of  moth  pupae  have  been  cultivated 

in  several  different  media.  The  virus  of  St.  Louis  encepha- 
litis has  been  maintained  in  cultures  of  hemocytes  from  larvae  of  the  catalpa 
sphinx  for  10  days.  Oocysts  of  Plasmodium  gallinaceum  attached  to  the  midgut 
of  Aedes  aegypti  have  shown  growth  in  vitro  and  sporozoites  have  been  produced. 


BIOCHEMICAL  STUDIES    It  was  shown  that  mosquitoes  infected  with  malaria 

have  higher  levels  of  ribonucleic  acid  than  uninfected 
mosquitoes.   Chromatographically,  the  acid-hydrolysate  of  ribonucleic  acid 
from  a  pyrimethamine-resistant  strain  of  Plasmodium  falciparum  differs  from 
the  acid-hydrolysate  of  ribonucleic  acid  from  a  pyrimethamine-susceptible 
strain.   Bephenium  hydroxynaphthoate  inhibited  glutamic  acid  transaminase  of 
Nippos  trongy lus  muris .   Bephenium  chloride  and  quinacrine  reduced  the  rate  of 
glucose  absorption  by  the  tapeworm  Hymenolepis  diminuta  but  low  concentrations 
of  dithiazanine  iodide  stimulated  glucose  absorption  by  this  cestode. 


INTESTIONAL  PARASITES    Epidemiological  studies  on  the  inmates  of  a  mental 

institution  show  a  high  persistence  of  Trichuris 
and  hookworm  for  six  years,  with  an  apparent  decrease  in  Strongyloides .   To 
test  dithiazanine  and  tetrachlorethylene,  alone  and  in  combination,  heavily 
parasitized  mental  patients  were  given  the  drugs  for  about  one  year.  A  large 
number  of  worms  were  removed  but  the  cure  rate  was  low  and  transmission  was 
not  stopped.   Bephenium  hydroxynaphthoate  and  bephenium  chloride  were  used  with 
good  results  against  hookworm,  Ascaris  and  Trichuris . 

SCHISTOSOMIASIS    The  activity  of  griseofulvin  observed  in  mice  infected  with 

Schistosoma  mansoni  was  not  well  developed  in  hamsters  or 
monkeys.   A  series  of  tetracycline  analogues  which  show  an  affinity  for  micro- 
filaria did  not  combine  with  schistosomes  and  were  without  activity.   One  of 
these  analogues  was  significantly  more  active  against  microfilariae  of  Diro- 
f ilaria  immitis  than  tetracycline. 

In  many  tests,  the  efficacy  of  stibophen  (Fuadin)  therapy  on  mature 
Schistosoma  mansoni  infections  in  mice  was  increased  up  to  16  times  by  feeding 
a  balanced  semi-synthetic  diet.   The  toxicity  of  the  drug  was  not  similarly 
increased.   The  enhancement  of  curative  action  by  the  purified  semi-synthetic 
diet  was  thought  to  be  due  to  the  absence  of,  as  yet  unidentified,  inorganic 
salt(s)  that  interfere  with  drug  activity.   It  was  found  in  mice  fed  on  the 
purified  semi-synthetic  diet  that  higher  blood  levels  of  the  drug  were  main- 
tained for  a  longer  period  than  when  the  same  amount  of  Fuadin  was  injected 
into  mice  fed  on  the  commercial  pellet  diet,  suggesting  that  the  increased 
cure-rate  was  due  to  higher  blood  drug  level.   Similar  drug  advantage  was 
observed  in  mice  given  tartar  emetic  while  on  the  purified  diet. 

DRUG  COMBINATIONS    Various  combinations  of  primaquine,  chloroquine,  amodia- 

quine,  and  pyrimethamine  proved  to  be  no  more  effective 
than  single  drugs  against  blood  induced  Plasmodium  berghei  in  mice. 

NUTRITION  AND  MALARIA    An  intensive  study  of  the  influence  of  nutritional 

deficiencies  on  the  activity  of  antimalarials  has  been 
initiated. 


ADRENAL  HORMONE    The  influence  of  cortisone  on  antimalarial  activity  was 
AND  MALARIA        investigated  in  Plasmodium  berghei  and  Plasmodium  gallina- 

ceum.   The  drugs  used  were  primaquine,  chloroquine,  and 
pyrimethamine.   The  only  combination  in  which  cortisone  exerted  an  adverse 
effect  on  antimalarial  action  was  primaquine  and  Plasmodium  gallinaceum. 
In  Plasmodium  berghei,  cortisone  has  a  slight  synergistic  effect  when  given 
concurrently  with  antimalarial  drugs. 


Serial  No.  NIAID-130 

1.  Parasite  Chemotherapy 

2.  Office  of  the  Chief 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual   Project   Report 

Calendar  Year   1960 


Part  A 


Project  Title:   Administration,  research  planning  and 
coordination 

Principal  Investigator:   G.  Robert  Coatney 

Other  Investigators:   None 

Cooperating  Units:   None 

Man  Years  (calendar  year  1960) : 
Total:        1  3/4 
Professional:   3/4 
Other:        1 

Project  Description: 

This  project  furnishes  certain  technical,  supervisory,  and  administrative 
services  to  all  research  projects  in  the  Laboratory,  as  follows: 

(a)  Over-all  planning  of  research  and  the  coordination  of  research 
activities  in  the  various  Sections  of  the  Laboratory. 

(b)  Integration  of  Laboratory  research  activities  with  clinical  studies 
of  the  Clinical  Center. 

(c)  Supervision  over  personnel,  maintenance,  travel,  and  correspondence. 

(d)  Requisitioning  and  supervision  of  equipment  and  supplies, 

(e)  Preparation  of  reports,  budget  estimates,  and  exhibits  and  other 
public  relations  materials. 

(f)  Maintenance  of  reference  library,  reprint  files,  card  catalogues, 
and  specimen  collections. 

(g)  Consul tatory  services  to  individuals,  commercial  organizations, 
nongovernmental  organizations,  Government  agencies  including  liaison  activities 
with  other  branches  of  the  Service,  foreign  governments,  and  international 
agencies . 


Serial  No.  NIAID-130 


(h)   Reviewing,  editing,  and  revising  scientific  and  technical  reports  and 
manuscripts. 

Progress  on  research  projects  is  reported  quarterly  and,  when  necessary, 
are  discontinued  or  revised  in  order  to  meet  current  objectives.   During  the 
past  year  some  changes  have  been  made  in  the  research  program  of  the  laboratory, 
impetus  given  to  others,  and  certain  activities  directed  into  new  channels. 
The  discovery  that  simian  malaria  was  infective  to  man  brought  about  changes 
in  three  directions:   (1)  Plans  were  laid  for  moving  the  Memphis  activities  to 
Chamblee,  Georgia  in  order  to  have  a  flourishing  insectary  and  the  simian  hosts 
close  to  the  inmate  volunteers  at  the  Atlanta  prison,  (2)  A  laboratory  was 
established  at  Kuala  Lumpur,  Malaya,  to  carry  out  investigations  in  the  field 
relative  to  all  aspects  of  the  simian-human-malaria  chain,  and  (3)  Studies 
in  human  volunteers  at  the  Atlanta  prison  were  tailored  to  an  all-out  study 
of  simian  infections  in  man.   Continued  emphasis  was  placed  on  the  fundamental 
aspects  of  other  problems  in  tropical  parasitology  and  chemotherapy.   These 
included  the  culture  of  insect  tissues,  intensive  and  extensive  study  of  the 
development  and  chemotherapy  of  fixed  tissue  stages  of  certain  mammalian 
malarias,  the  tissue  culture  of  mammalian  malarias,  the  mechanism  of  drug 
action  on  parasites  and  the  inhibiting  effect  of  antimetabolites,  the  effect 
of  nutrition  on  chemotherapy,  drug  resistance  by  parasites,  chemotherapy  of 
intestinal  parasites,  and  the  carriage  of  viruses  by  parasites. 

The  cooperative  study  with  Dr.  John  Tobie,  LI  (Ser.  No.  NIAID-148),  on  the 
application  of  fluorescent  antibody  techniques  to  the  staining  of  human  Plas- 
modia for  mass  screening  with  a  mechanical  scanner  was  continued. 


Part  B  included  -  Yes 


Serial  No.  NIAID-130 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B    Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Gaudette,  Leo  E.  and  Coatney,  G.  R.   Stability  of  primaquine  diphosphate 
under  various  conditions.   Am.  J.  Trop.  Med.  &  Hyg.  9_:  532-535,  1960. 

Coatney,  G.  R. ,  Elbel,  R.  E.,  and  Kocharatana,  P.   Some  blood  parasites 
found  in  birds  and  mammals  from  Loei  Province,  Thailand.   J.  Parasitol.  46: 
701-702,  1960. 

Coatney,  G.  R.  and  Greenberg,  J.   The  effect  of  a  diet  deficient  in 
Factor  3  on  the  course  of  Plasmodium  berghei  infection  in  mice.   Proc. .  VI 
Internatl.  Congress  Trop.  Med.  &Mal.  (in  press). 

Coatney,  G.  R.  and  Hinman,  E.  H.   Malaria.   Cyclopedia  of  Medicine, 
Surgery  and  Specialties  (in  press). 

Honors  and  Awards  relating  to  this  project: 

Visiting  Professor,  Department  of  Preventive  Medicine  and  Public  Health, 
School  of  Medicine,  Howard  University. 

Visiting  Lecturer  on  Tropical  Public  Health,  Harvard  School  of  Public 
Health. 

Visiting  Lecturer  on  chemotherapy,  Malaria  Eradication  Training  Center, 
Kingston,  Jamaica. 

Consultant  to  Chief,  Section  on  Malaria  Eradication,  Pan  American 
Sanitary  Bureau. 

Vice  President,  American  Society  of  Tropical  Medicine  and  Hygiene. 

President-Elect,  American  Society  of  Tropical  Medicine  and  Hygiene. 

Vice  Chairman  for  the  meeting  of  the  Expert  Committee  on  Malaria  held  in 
Geneva,  Switzerland,  25-30  July  1960. 

Chairman,  Technical  Meeting  on  Malaria,  Geneva,  Switzerland,  14-19 
November  1960. 

Member  of  the  Expert  Advisory  Panel  on  Malaria  of  the  World  Health 
Organization. 


Serial  No.  NIAID-130 

Consultant  to  the  Director,  Division  of  Malaria  Eradication,  World  Health 
Organization,  Geneva,  Switzerland,  on  the  place  of  drugs  in  malaria  eradication 
in  Africa,  and  in  that  capacity,  visited  Liberia,  French  Equitorial  Africa, 
Belgian  Congo,  Zanzibar,  Tanganyika,  Southern  Rhodesia,  and  Geneva,  during 
March  and  April,  1960. 


Serial  No.  NIAID-130-A 

1.  Parasite  Chemotherapy 

2.  Office  of  the  Chief 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   Studies  in  human  malaria 

Principal  Investigator:   G.  Robert  Coatney 

Other  Investigators:   Morton  E.  Getz,  Henry  K.  Beye,  Harvey  Elder, 
Don  E.  Eyles,  and  Martin  D.  Young 

Cooperating  Units:   Department  of  Justice,  Bureau  of  Prisons,  and 

Institute  for  Medical  Research,  Christ  Hospital, 
Cincinnati,  Ohio 

Man  Years  (calendar  year  1960) : 
Total:         3  3/4 
Professional:   1  1/4 
Other:        2  1/2 

Project  Description: 

Ob  jectives : 

1.  To  evaluate  new  antimalarial  drugs  and/or  drug  combinations  and 
methods  of  administration  applicable  to  mass  chemotherapy  as  applied  to 
malaria  eradication. 

2.  To  study  the  clinical  manifestations  and  parasitology  of  simian  malaria 
infections  in  man  with  special  emphasis  on  Plasmodium  cynomolgi  bastianellii. 

Methods  Employed:  Normal  inmate  volunteers  at  the  Federal  Penitentiary, 
Atlanta,  Georgia,  are  screened  before  they  are  admitted  to  the  project. 
Volunteers  are  under  the  care  of  the  resident  investigators  whether  handled 
as  outpatients  or  as  inpatients.   They  take  drugs  as  prescribed,  submit  to 
necessary  laboratory  procedures  and  to  infection  by  bites  of  infected  mosquitoes. 
Participation  may  be  for  6  weeks  to  2  months  or  up  to  one  year  depending  on  the 
nature  of  the  project. 

Major  Findings: 

McLendon  strain  of  falciparum  malaria. 

Chloroquine  (300  mg,  base)  and  primaquine  (45  mg)  given  together  in  a 


10 


Serial  No.  NIAID-130-A 


single  oral  dose  beginning  day  0-3,  and  weekly  thereafter  for  a  total  of  8 
doses,  resulted  in  suppressive  cure  of  5/5  subjects.   Controls  were  positive 
11-15  days  after  infection.  After  2  days  of  parasitemia,  each  control  was 
given  the  above  drug  combination  as  a  single  dose  and  then  weekly  for  a  total 
of  3  doses.   Parasites  were  removed  promptly  and  cure  was  obtained  based  on  no 
evidence  of  infection  following  227  days  of  observation. 

Chesson  strain  of  vivax  malaria. 

Primaquine,  given  30  mg  weekly  (single  dose)  beginning  day  0-1  and  con- 
tinuing on  the  same  day  each  week  and,  to  another  group,  at  3  mg  daily  starting 
day  of  infection,  failed  to  prevent  or  suppress  infection.   Treated  and  control 
patients  all  exhibited  patent  infections  9  to  12  days  after  infection. 

A  drug  combination  of  primaquine  (45  mg)  and  pyrimethamine  (50  mg)  given 
in  a  single  dose  weekly,  beginning  on  day  0+7,  and  weekly  thereafter  for  a 
total  of  4  doses,  gave  suppress ive-cure  to  4/5  subjects;  the  other  subject 
developed  a  patent  infection  240  days  after  infection.  Pyrimethamine  (50  mg) 
given  alone,  as  above,  produced  suppress ive-cure  in  1/4  subjects;  the  other  3 
came  down  on  day  82,  83,  and  84,  respectively,  after  infection.  The  controls 
(5)  all  came  down  12  to  13  days  after  infection. 

An  8 - amino quino line  allied  to  primaquine  and  known  as  CN1115  and  Win 
10,448  in  the  United  States  and  as  quinocide  by  the  Russians  was  purported  to 
produce  radical  cure  and  result  in  less  toxicity  than  primaquine.   The  effec- 
tive dosage  of  each  drug  was  supposed  to  be  the  same,  15  mg  daily  x  14. 

Primaquine  compared  with  quinocide  against  sporozoite-induced 
Chesson  strain  P.  vivax;  each  drug  at  15  mg  daily  x  14  after 


600 

mg  base  chloro 

quine 

Type  of 
attack 

1st  relapse 
(Days) 

2nd  relapse 
(Days) 

3rd  relapse 
(Days) 

Days  of 
observation* 

PRIMAQUINE 

E.  P. 
D.  P. 

1/5  (48) 
1/5  (35) 

1/5  (250) 

281-446 
401-593 

QUINOCIDE 

E.  P. 
D.  P. 

3/4  (7-356) 
2/4  (32-140) 

3/4  (21-218) 
1/4  (161) 

2/4  (46-68) 

405-446 
443-446 

11 


Serial  No.  NIAID-130-A 


E.  P.  =  early  primary  attack 

D.  P.  =  delayed  primary 

(   )   =  days  to  relapse  since  last  Rx 

*     =  as  of  1  December  1960 

The  results  show  that  primaquine  is  distinctly  superior  to  the  other 
compound. 

Reports  on  a  new  8 - amino quino line,  6  methoxy-8(5  propylaminoamylamino) 
quinoline  phosphate,  Win  5037,  indicated  that  this  drug  was  as  therapeutically 
effective  as  chloroquine  and  as  effective  as  primaquine  in  producing  cure. 
At  dosages  of  20  mg  b.i.d.  for  2  days,  and  then  10  mg  daily  x  12,  and  at  30 
mg  b.i.d.  x  2,  and  then  20  mg  daily  x  12,  the  parasites  were  removed  and  the 
fever  subsided  almost  as  rapidly  as  following  600  mg  of  chloroquine,  but  the 
entire  regimen  did  not  produce  cure.   Those  on  regimen  1  (3  pts)  relapsed 
after  35  to  45  days  and  were  re-treated  on  two  occasions;  at  neither  time  was 
the  blood  cleared  of  parasites.   Those  on  regimen  2  (2  pts.)  each  relapsed  on 
day  103  and  were  cured  with  chloroquine  and  primaquine.   Because  of  toxic 
effects,  failure  to  cure,  and  drug  resistance  after  the  initial  trial,  further 
investigation  was  not  considered  warranted. 

Simian  malaria,  Plasmodium  cynomolgi  bastianellii. 

In  early  May,  two  infections  were  induced  in  inmate  volunteers  by  inocula- 
tion of  infected  blood  obtained  from  a  sporozoite-induced  accidental  infection. 
Following  these  infections,  individuals  (whites  and  Negroes)  have  been  infected 
by  bites  of  infected  Anopheles  quadrimaculatus  or  Anopheles  freeborni  fed  on 
infected  monkeys.   In  cases  where  typical  parasites  could  be  demonstrated,  the 
prepatent  period  ranged  from  14  to  29  days.   The  parasite  density  ranged  from 
5  to  500/cmm.   In  some  patients,  parasitemia  was  demonstrated  before  the  onset 
of  symptoms.   The  most  frequent  symptom  was  headache,  followed  in  decreasing 
frequency  by  anorexia,  abdominal  pain,  joint  pain,  nausea,  myalgia,  back  pain, 
vomiting,  chest  pain,  chills,  and  cramping.   Headache  was  generally  described 
as  an  aching,  bilateral,  frontal  pain,  fairly  persistent  and  often  extending 
to  the  back  of  the  head  and  down  the  back  and  the  lateral  aspects  of  the  neck. 
There  was  often  muscle  tenderness  in  the  head  and  neck.  Abdominal  pain  was 
often  in  the  right  and  left  lower  upper  quadrants.   It  occasionally  was  in- 
creased on  deep  inspiration.   The  most  significant  findings  were  splenomegaly 
and  hepatomegaly  usually  associated  with  tenderness.   In  those  patients  ex- 
hibiting a  tertian  fever,  the  pattern  was  initiated  between  the  19th  and  23rd 
day;  there  was  no  correlation  between  parasitemia  and  febrile  response. 
Observations  to  date  would  show  no  febrile  response  after  the  75th  day  of 
infection  (based  on  24  patients). 

Anopheles  freeborni  were  infected  after  feeding  on  two  different  patients 
with  gametocytes  as  evidenced  by  finding  normal  looking  oocyts  on  the  gut  and 
'normal'  sporozoites  in  the  salivary  glands,  but  attempts  to  infect  volunteers 

12 


Serial  No.  NIAID-130-A 


by  mosquito  bite  have  so  far  yielded  equivocal  results.  One  patient  became 
patent  after  58  days  but  the  source  of  the  circulating  parasites  is  open  to 
question. 

Plasmodium  cynomolgi  cynomolgi. 

Eleven  inmate  volunteers  were  bitten  by  large  numbers,  up  to  40,  of  in- 
fected mosquitoes  (A.f_.)  on  8  September,  and  to  date  three  patients  have  ex- 
hibited evidence  of  infection  (i.e.,  fever);  parasitemia  has  been  demonstrated 
in  only  one  and  that  on  the  58th  day  after  infection.   Blood  was  drawn  from 
each  man  on  day  8,  and  subsequently  when  there  was  clinical  evidence  of  infec- 
tion, and  inoculated  into  clean  monkeys  at  Dr.  Schmidt's  laboratory,  Cincinnati. 
So  far  no  infections  have  developed  in  monkeys  except  in  those  inoculated  with 
blood  from  the  one  patient  shown  to  have  had  parasites  on  smear.   These  results 
show  that  P.  £.  cynomologi  will  produce  infection  in  man  but  that  it  is  far 
less  infective  than  P.c_.  bastianellii. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
This  country,  along  with  other  countries  of  the  world,  is  committed  to  a 
program  of  world-wide  eradication  of  malaria  and  to  that  end  the  Congress 
appropriated  thirty-eight  million  dollars  for  fiscal  year  1961.   It  was 
considered  by  malar iological  consultants  that  non-human  reservoirs  would  not 
be  a  problem  in  this  effort,  and   this  may  still  be  true.   However,  the  ease 
with  which  P.  c.  bastianellii  of  monkeys  can  be  transmitted  to  man  raises  an 
important  question  about  the  place  of  simian  hosts  in  human  disease.   Studies 
in  volunteers  have  a  two-fold  purpose  in  terms  of  malaria  eradication: 

(1)  clinical  and  parasitological  studies  of  simian  malarias  in  man,  and 

(2)  evaluation  of  drugs  and  drug  combinations.   It  is  expected  that  the  know- 
ledge obtained  can  be  applied  in  this  field. 

Proposed  Course  of  the  Project:   This  project  is  being  enlarged  in  order 
to  include  studies  on  simian  malarias  in  man  along  with  the  assessment  of 
methods  of  mass  chemotherapy  and  the/parasite  resistance  in  terms  of  malaria 
eradication.  stuay  of 


Part  B  included  -  Yes 


13 


Serial  No.  NIAID-130-A 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B.   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Eyles,  Don  E.,  Coatney,  G.  R. ,  and  Getz,  M.  E.  Vivax-type  malaria 
parasite  of  Macaques  transmissible  to  man.   Science  131 ;1812-1813,  1960. 


1'* 


Serial   No.    NIAID-131 

1.  Parasite  Chemotherapy 

2.  Chemotherapy 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 


Part  A 


Project  Title:   Drug  resistance  in  experimental  malaria 

Principal  Investigator:   William  F.  Cantrell 

Other  Investigators:   None 

Man  Years  (calendar  year  1960) : 
Total:       1  1/2 
Professional:   1/2 
Other:  2/3 

Project  Description: 

Objectives :   To  obtain  strains  of  malaria  resistant  to  a  variety  of  anti- 
malarial drugs  and  to  study  the  genetics  of  drug  resistance  by  producing 
hybridization  of  these  strains  in  the  mosquito  phase  of  the  life  cycle. 

Methods  Employed:   Large  populations  of  Plasmodium  gallinaceum  are  ex- 
posed to  the  pyrimethamine,  chloroquine  or  primaquine  by  treating  infected 
chickens.  After  one  to  three  days,  0.5  ml  of  blood  is  transferred  to  a  new 
chicken  and  treatment  is  repeated. 

Major  Findings :   Repeated  exposure  of  large  populations  of  malaria  para- 
sites resulted  in  resistance  only  in  the  case  of  pyrimethamine.   It  appears 
that  even  in  very  large  populations  parasites  resistant  to  chloroquine  or  prima- 
quine do  not  occur.  An  attempt  was  made  to  select  a  chloroquine  resistant 
strain  from  the  pyrimethamine  resistant  strain  without  success. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  development  of  resistance  to  chloroquine  or  primaquine  is  a  greatly  feared 
possibility  in  the  world-wide  malaria  eradication  program.   Fundamental  know- 
ledge about  the  process  by  which  malaria  parasites  become  resistant  may  lead 
to  means  for  preventing  drug  resistance  or  combating  drug  resistant  strains. 

Proposed  Course  of  the  Project:   Other  drugs  will  be  tried  in  Plasmodium 
gallinaceum  in  order  to  obtain  genetic  markers.   Experiments  will  be  made  with 
Plasmodium  berghei  in  which  species  chloroquine-resistant  strains  are  known. 
Here  the  mosquito  phase  of  the  work  will  be  more  difficult  and  will  require 
special  study. 

15 


Serial  No.  NIAID- 131 

The  possibility  of  using  X-ray  to  speed  up  the  development  of  resistant 
strains  will  be  explored. 

The  pyrimethamine  resistance  characteristic  will  be  employed  in  studies 
directed  toward  the  prevention  of  the  development  of  resistance  by  simultaneous 
administration  of  drugs  and  other  means. 


Part  B  included  -  No 


16 


Serial  No.    NIAID-131-A 

1.  Parasite  Chemotherapy 

2.  Chemotherapy 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   The  effect  of  adrenal  cortical  hormone  on 
chemotherapy  of  experimental  malaria 

Principal  Investigator:   William  F.  Cantrell 

Other  Investigators:   None 

Cooperating  Units :   None 

Man  Years  (calendar  year  1960) : 
Total:        1  1/2 
Professional:    1/2 
Other :        1 

Project  Description: 

Objectives :   To  determine  whether  adrenal  cortical  hormones  affect  the 
action  of  antimalarial  drugs  adversely  or  otherwise. 

Methods  Employed:  Chickens  infected  with  Plasmodium  gallinaceum  or  mice 
infected  with  Plasmodium  berghei  are  treated  simultaneously  with  cortisone  and 
one  of  the  antimalarials  and  the  effect  is  compared  with  the  effect  of  the 
antimalarial  alone  and  cortisone  alone. 

Major  Findings :   In  the  case  of  P.  gallinaceum,  cortisone  (or  hydrocorti- 
sone) alone  was  without  effect  on  the  course  of  the  infection.   In  the  case  of 
P.  berghei,  cortisone  (or  hydrocortisone)  alone  has  a  weak  but  significant 
antimalarial  effect.   In  the  case  of  P.  gallinaceum  treated  with  primaquine, 
cortisone  has  an  adverse  effect  on  antimalarial  action.   In  the  case  of  P. 
gallinaceum,  treated  with  chloroquine  or  with  pyrimethamine,  cortisone  is 
without  effect  even  in  high  doses.   In  the  case  of  P.  berghei,  the  antimalarial 
action  of  cortisone  (or  hydrocortisone)  is  combined  with  the  antimalarial 
action  of  chloroquine,  primaquine  or  pyrimethamine  giving  a  slightly  increased 
antimalarial  effect. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  adverse  effect  of  cortisone  on  host  defenses  in  a  number  of  experimental 
infections  and  in  a  few  human  diseases  suggested  that  more  information  with 
regard  to  malarial  infections  would  be  useful.   The  diverse  responses  found 
with  different  drugs  and  different  host-parasite  systems  indicates  that  no 
direct  application  should  be  made  to  human  malaria.   On  the  other  hand,  the 

17 


Serial  No.  NIAID-L31-A 


finding  that  there  is  system  in  which  cortisone  has  a  favorable  effect  on 
therapy  is  of  some  value  from  the  point  of  view  of  experimental  chemotherapy 
as  a  science.   The  finding  that  cortisone  interferes  with  chloroquine  activity 
against  P.  gallinaceum  but  not  with  primaquine  nor  pyrimethamine  suggests 
that  primaquine  activity  depends  more  on  a  cooperative  action  by  the  host 
defense  system. 

Proposed  Course  of  the  Project:  No  further  exploration  of  this  phase  of 
the  work  is  planned,  but  studies  of  other  physiological  factors  affecting  the 
response  of  malaria  to  chemotherapy  will  be  made. 


Part  B  included  -  No 


18 


Serial  No.  NIAID-131-B 

1.  Parasite  Chemotherapy 

2 .  Chemo  ther  apy 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A 


Project  Title:   Antimalarial  drugs  and  nucleic  acid  synthesis 

Principal  Investigator:   Karl  A.  Schellenberg 

Other  Investigator:   G.  Robert  Coatney 

Cooperating  Units :   None 

Man  Years  (calendar  year  1960) : 
Total:        1  1/3 
Professional:    2/3 
Other:  2/3 

Project  Description: 

Objectives :   To  determine  the  effects  of  antimalarial  drugs  on  nucleic 
acid  synthesis  in  experimental  malaria. 

Methods  Employed:   The  incorporation  of  radioactive  phosphate  into  ribo- 
nucleic acid  and  deoxyribonucleic  acid  was  measured  and  the  influence  of 
several  antimalarial  drugs  and  other  substances  was  determined  in  Plasmodium 
gallinaceum  and  Plasmodium  berghei. 

Major  Findings:   Quinine,  chloroquine,  and  quinacrine  inhibited  the  in- 
corporation of  P32  into  both  RNA  and  DNA,  whereas  pyrimethamine  and  the  tria- 
zine  metabolite  of  chloroguanide  specifically  inhibited  incorporation  of  P32 
into  DNA.   The  inhibition  by  pyrimethamine  was  not  reversed  by  folic  acid, 
folinic  acid,  thymidine,  deoxyuridine,  uracil,  thymine,  glycine  or  adenine. 
Chloroguanide  was  active  in  vivo  but  not  in  vitro. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  highly  specific  effect  of  pyrimethamine  on  deoxyribonucleic  acid  metabolism 
occurring  at  concentrations  which  may  be  reached  in  actual  therapy  affords  an 
explanation  of  drug  action  on  a  more  fundamental  level  than  is  available  for 
most  drugs.   This  linking  of  drug  action  to  synthesis  of  the  genetic  material 
of  a  parasitic  organism  affords  an  approach  to  the  discovery  of  new  drugs 
through  advances  in  the  highly  active  field  of  nucleic  acid  enzymology. 


13 


Serial  No.  NIAID-131-B 


Proposed  Course  of  the  Project:   Dr.  Schellenberg  has  resigned  in  order 
to  continue  advanced  study  in  biochemistry.   Continuation  of  this  important 
line  of  research  depends  on  finding  a  suitably  trained  person. 


Part  B  included  -  Yes 


20 


Serial  No.  NIAID-131-B 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 

Part  B    Honors,  Awards,  and  Publications 

Schellenberg,  K.  A.  and  Coatney,  G.  R.   The  Influence  of  antimalarial 
drugs  on  nucleic  acid  synthesis  in  Plasmodium  gallinaceum  and  Plasmodium 
berghei.   Biochem.  Pharmacol,  (in  press). 

Schellenberg,  K.  A.  and  Weinbach,  E.  C.  The  endogenous  formation  of 
phosphoenolpyruvate  by  rat-liver  mitochondria.  Biochim.  et  Biophys.  Acta 
45:593-595,  1960. 


21 


Serial  No.   NIAID-131C 

1.  Parasite  Chemotherapy 

2.  Chemotherapy 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  A. 


Project  Title:  Experimental  chemotherapy  of  helminthic  diseases 

Principal  Investigator:  George  V.  Luttermoser 

Other  Investigators:  None 

Cooperating  Units:  Dr.  B.  Prescott,  LID,  NIAID-6lAand  Dr.  J.  Tobie,  LI, 
NIAID-1U8 

Man  Years  (calendar  year  I960): 
Total:       1  1/2 
Professional:   l/2 
Other*       1 

Project  Description: 

Objectives:  To  discover  new  and  more  effective  schistosomacides  and 
compounds  active  against  other  helminth  parasites.  To  learn  how  the  chemicals 
selectively  kill  those  parasites. 

Methods  Employed:  Mice  are  infected  with  cercariae  of  Schistosoma  mansoni 
and  treatment  is  begun  (l)  immediately  (prophylaxis)  or  (2)  after  35  days 
(curative).  The  mice  are  examined  $0   days  or  more  after  exposure  to  infection 
or  for  the  presence  of  dead  worms.  In  critical  evaluation  of  drug  activity, 
the  liver  and  portal  system  are  perfused  and  the  live  or  dead  worms  are  counted. 
Live  mature  worms  removed  from  treated  and  untreated  animals  are  observed  in 
maintenance  culture  for  activity  and  survival.  Drugs  are  added  to  some  of  the 
cultures.  Monkeys  are  infected  with  the  same  parasite  and  dogs,  with  8. 
japonicum  (Formosan).  A  test  for  either  prophylactic  or  curative  activity  of 
promising  compounds  was  conducted  in  the  larger  animals  in  a  similar  fashion 
to  the  mouse  test  except  that  fecal  egg  counts  were  also  utilized  as  an  indi- 
cator of  the  status  of  the  infection. 

For  studies  on  nematode  parasites,  dogs  naturally  infected  with  Diro- 
filaria  immitis  and  mice  infected  with  pinworms  were  utilized.  Observations 
were  made  on  the  parasiticidal  effects  of  the  drugs  in  vivo  and  in  vitro. 
Fluorescence  of  the  helminth  was  read  in  UV  light  which  indicated  whether  the 
drugs  were  absorbed. 


22 


Serial  No.  NIAID-  131C 

Major  Findings;  Completion  of  the  test  for  prophylactic  activity  of 
the  antibiotic  S1629  (Griseofulvin  or  Fulvicin)  indicated  that  oral  adminis- 
tration of  50  mg/kg  of  the  drug  daily  starting  the  fourth  day  before  exposure 
to  S.  iransoni  and  continuing  for  12  days  brought  about  a  reduction  in  the 
numbers  of  schistosomes  developing  in  these  animals.  This  was  not  found  in 
similar  experiments  with  hamsters.  There  was,  however,  an  indication  of  weak 
prophylactic  activity  in  two  monkeys  started  on  the  drug  two  days  before 
exposure  and  continued  en  a  daily  dose  for  9  days. 

Since  filariids  and  other  helminths  absorb  tetracycline  and  will 
fluoresce  (Tobie  and  Beye,  I960),  a  study  was  made  of  the  effects  of  tetra- 
cycline and  12  analogues  of  tetracycline  on  Dirofilaria  iremitis  and  on  mouse 
pinworms.  A  comparison  was  made  of  the  longevity  of  the  larvae  and  adults 
of  these  nematodes  in  maintenance  media  with  and  without  drugs  added  at  final 
concentrations  of  from  1  ug  to  1  mg  per  ml.  One  of  the  analogues  (1686-7) 
killed  the  microf ilariae  of  D.  immitis  at  in  -vitro  concentrations  as  low  as 
10  ^ug/ml  in  a  period  of  h   to  9  hours,  while  tetracycline  itself  did  not  do 
so.  The  oral  administration  of  15  daily  oral  doses  of  20  mg  of  this  analogue 
per  kg  body  weight  likewise  caused  a  marked  reduction  in  the  microfilarial 
blood  count.  Nevertheless,  many  of  the  micro-  and  macrofilaria  which  fluoresced 
after  in  vitro  exposure  to  other  of  these  "tetracyclines",  continued  to  live 
for  several  hours  afterward.  In  mice  with  pinworm  inlection,  the  analogue 
1686-7  was  not  more  active  than  tetracycline  itself  in  clearing  the  animals  of 
pinworms.  However,  a  2  day  regimen  of  another  of  the  analogues  (168°)  cleared 
the  pinworm  infections  from  most  of  the  mice  while  a  similar  regimen  of  an 
active  piperazine  did  not.  Weak  fluorescence  was  only  observed  in  small  areas 
of  the  upper  intestine  and  of  the  genital  system  of  the  pinworm  whereas  strong 
fluorescence  was  observed  throughout  the  body  wall  and  intestine  of  the 
filariids. 

The  analogues  of  tetracycline  tested  have  not  been  found  to  be  schisto- 
somacidal  nor  have  they  been  found  to  cause  the  schistosome  to  fluorece. 

Significance  to  the  program  of  the  Institute;  New  information  with 
regard  to  a  reaction  between  a  chemical  and  a  parasite  may  be  a  "lead"  for 
the  development  of  better  drugs  needed  for  control  of  parasitic  infections 
such  as  schistosomiasis  and  filariasis.  Current  treatments  for  these 
infections  are  inadequate. 

Proposed  Course  of  the  Project;  The  principal  investigator  will  be 
Visiting  Professor  at  the  American  University  of  Beirut,  Lebanon,  during 
196l.   Studies  of  effects  of  new  drugs  on  schistosomes  will  be  continued  by 
Dr.  William  Cantrell. 


23 


Serial  No.  NIAID-131C 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 

Part  B    Honors,  Awards,  and  Publications 

Honors  and  Awards  relating  to  this  project: 

President  of  the  Helminthological  Society  of  Washington,  I960. 

Appointed  consultant  on  schistosomiasis  to  WHO  and  accepted  assignment 
to  Ministry  of  Health  of  Venezuela  for  the  period  22  January  to  18  March, 
I960. 

Named  Visiting  Research  Professor  in  the  Department  of  Tropical  Health, 
American  University,  Beirut,  Lebanon,  for  the  year  196l, 


24 


Serial  No.  NIAID-131-D 

1.  Parasite  Chemotherapy 

2.  Chemotherapy 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  A 


Project  Title:  Effects  of  nutrition  on  chemotherapy  of  parasitic 
diseases. 

Principal  Investigator:  George  W.  Luttermoser 

Other  Investigators:  None 

Cooperating  Units:  Dr.  W.  DeWitt,  LPD,  NIAID-121K 

Man  Years  (calendar  year  I960) : 
Total:       1  1/2 
Professional:   l/2 
Other:       1 

Project  Description: 

Objectives:  To  determine  whether  the  nutrition  of  the  host  can  influence 
the  chemotherapy  of  parasitic  infections. 

Methods  Employed :  Animals  maintained  on  defined  diets  either  enriched  or 
deficient  in  certain  components  are  exposed  to  a  single  species  of  parasite 
and  the  developing  infection  is  treated  in  either  the  prepatent  or  patent 
period.  The  influence  of  the  diet  on  treatment  is  determined  by  (l)  the 
reduction  of  the  number  of  parasites  (2)  the  condition  of  the  parasites  (3) 
growth  and  survival  of  the  host  and  (Li)  the  pathologic  conditions  developing 
in  the  host. 

Major  Findings:  In  many  tests,  the  efficacy  of  stibophen  (Fuadin)  therapy 
on  mature  Schistosoma  mansoni  infections  in  mice  was  increased  up  to  16  times 
by  feeding  a  balanced  semi-synthetic  diet.  The  toxicity  of  the  drug  was  not 
similarly  increased.  The  enhancement  of  curative  action  by  the  purified  semi- 
synthetic diet  was  thought  to  be  due  to  the  absence  of,  as  yet  unidentified, 
inorganic  salt(s)  that  interfere  with  drug  activity.  It  was  found  in  mice 
fed  on  the  purified  semi-synthetic  diet  that  higher  blood  levels  of  the  drug 
were  maintained  for  a  longer  period  than  when  the  same  amount  of  Fuadin  was 
injected  into  mice  fed  on  the  commercial  pellet  diet,  suggesting  that  the 
increased  cure-rate  was  due  to  higher  blood  drug  level.  Similar  drug  advantage 
was  observed  in  mice  given  tartar  emetic  while  on  the  purified  diet. 


25 


Serial  No.  NIAID-131-D 


Significance  to  the  Program  of  the  Institute :  Since  parasitic  infections 
occur  most  frequently  in  backward  areas  where  malnutrition  is  also  prevalent, 
it  is  necessary  to  determine  the  relationship  between  the  nutrition  of  the 
people  and  the  results  obtainable  by  chemotherapy.  Failure  of  chemotherapy 
is  a  common  experience  in  such  areas.  Projects  for  screening  drugs  for 
schistosomacidal  activity  might  be  improved  if  more  optimum  dietary  conditions 
for  experimental  chemotherapy  are  found. 

Proposed  Course  of  the  Project;  During  the  next  year  the  Principal 
Investigator  will  be  Visiting  Professor  at  the  American  University,  Beriut, 
Syria.  During  his  absence  Dr.  DeWitt  (LPD)  will  continue  the  investigation 
to  determine  the  specific  nutritional  factors  involved  in  the  enhancement  of 
the  efficacy  of  stibophen  in  schistosomiasis.  Other  drugs  used  for  the 
treatment  of  this  and  other  parasitic  diseases  will  be  investigated  under 
similar  conditions  with  different  animals. 


Part  B  included  -  Yes 


2C 


Serial  No.  NIAID-131-D 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B    Honors,  Awards,  and  Publications 

Publications   other    than  abstracts    from  this   project: 

Luttermoser,  G.  W.  and  DeWitt,  W.  B.   Studies  on  interrelations  of 
nutrition  and  treatment  of  schistosomiasis  I.   Enhancement  of  stibophen 
(Faudin)  activity  against  Schistosoma  mansoni  in  mice  by  feeding  purified 
semi-synthetic  diets.   Am.  J.  Trop.  Med.  &  Hyg.  (in  press). 


27 


Serial  No.     NIAID-131-E 

1.  Parasite  Chemotherapy 

2  .  Cheruo  therapy 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  A 


Project  Title:  Comparison  of  antimalarial  drugs  administered  singly  and 
in  various  combinations. 

Principal  Investigator:  Richard  L.  Jacobs 

Other  Investigators:  William  F.  Cantrell 

Cooperating  Units :  None 

Man  Years  (calendar  year  I960) : 
Total :        1/3 
Professional:  l/6 
Other:       l/6 

Project  Description: 

Objectives:  To  determine  the  dosage-level  of  chloroquine,  primaquine, 
amodiaquine  and  pyrimethamine  required  to  give  any. desired  degree  of 
suppression  of  parasitemia  in  mice.  To  compare  the  effectiveness  of  various 
combinations  of  these  drugs  to  single-drug  treatment. 

Methods  Employed:  NIH  Swiss  female  mice,  averaging  20  gm/each,'  were  in- 
fected by  tail-vein  injection  of  one  million  RBC  infected  with  P.  berghei 
NYU-2  strain.  Drugs  were  administered  orally  at  various  levels  for  five 
consecutive  days,  beginning  on  day  of  infection.  Blood  smears  were  made  on 
four  consecutive  days,  beginning  on  the  third  day  after  infection,  for  evalu- 
ation of  the  effect  of  different  treatments  on  the  course  of  parasitemia. 
Each  trial  involved  five  animals  per  group  and  all  trials  were  repeated  at 
least  one  time. 

Major  Findings:  Chloroquine,  primaquine,  and  amodiaquine,  when  reduced 
to  base  equivalents,  were  equally  effective  in  suppressing  parasitemia  in  mice 
by  the  method  described.  Approximately  50  percent  suppression  in  parasitemia 
resulted  from  administering  U0  /ig/mouse/day.  Pyrimethamine  is  equally  effective 
at  one-third  the  dosage  level  of  the  above  drugs. 


28 


Serial  No.  NIAID-131-E 

Various  combinations  of  these  four  drugs  were  no  more  effective  than 
single-drug  treatment.  When  combined,  results  indicated  consistently  that 
the  effect  was  additive,  or  less  than  additive. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  practice  of  combining  drugs  has  been  used  extensively,  often  with 
favorable  results.  Reports  have  indicated  that  certain  combinations  of  anti- 
malarial drugs  exhibit  a  synergistic  effect  in  suppression  of  parasitemia 
(quinine  and  pamaquine  in  chicks) .  Also,  clinical  trials  have  indicated  that 
antimalarial  combinations  are  more  effective  in  preventing  relapse  in  vivax 
malaria.  The  results  of  this  study  indicate  that  combinations  of  the  drugs 
tested  offer  no  advantage  over  single-drug  therapy  in  suppressing  erythro- 
cytic forms  of  P.  berghei.  Suppressive  levels  of  drugs  have  been  established 
for  use  in  other  studies. 

Proposed  Course  of  the  Project;  This  project  has  been  completed. 
Part  B  included  -  No 


23 


Serial  No.     NIAID-131-F 

1.  Parasite  Chemotherapy 

2.  Chemotherapy 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  A 


Project  Title:  Effect  of  nutritional  status  of  host  on  the  action  of 
antimalarial  drugs. 

Principal  Investigator:  Richard  L.  Jacobs 

Other  Investigators:  William  F.  Cantrell 

Cooperating  Units:  None 

Man  Years  (calendar  year  l°6o)» 
Total:        2/3 
Professional:  1/3 
Other :        l/3 

Project  Description: 

Objectives:  To  study  the  effect  of  the  nutritional  status  of  the  host  ani- 
mal on  the  course  of  parasitemia  in  experimental  malarial  infection,  both  with 
suppressive  levels  of  antimalarial  drugs  and  in  the  absence  of  drug  therapy. 
It  is  hoped  that  the  results  of  this  study  will  yield  information  regarding 
the  mode  of  action  of  antimalarial  drugs. 

Methods  Employed:  The  parasite  P.  berghei,  NYU-2  strain  is  being  studied 
in  mice  of  altered  nutritional  status.  Young  NIH  Swiss  female  mice  are 
obtained  averaging  nine  to  ten  grams  each  and  are  maintained  on  various  test 
diets  for  approximately  four  weeks  prior  to  infection.  An  alternate  method 
consists  of  the  administration  of  antimetabolites  prior  to,  or  at,  time  of 
infection.  Effects  are  studied  both  in  absence  of  and  in  suppressive  levels 
of  chloroquine. 

Major  Findings:  Preliminary  results  indicate  that  alterations  in  the 
course  of  parasitemia  in  mice  by  dietary  deficiencies  are  variable.  The  vari- 
ability is  due,  primarily,  to  the  degree  of  the  deficiency.  Also,  the  possi- 
bility of  multiple-deficiencies  cannot  be  neglected  when  the  semi -purified 
diet  is  employed. 

In  tests  employing  folic  acid  deficient  diets,  the  effect  on  the  course 
has  been  variable^  however,  a  striking  suppression  of  parasitemia  has 

30 


Serial  No.  NIAID-131-F 

consistently  been  observed  when  aminopterin  is  administered. 

The  effect  of  nutritional  deficiencies  appears  to  be  influenced  by 
suppressive  drug  treatment.  Pyridoxine-deficient  animals  without  drug  treat- 
ment showed  an  increased  parasitemia;  when  a  $0%   suppressive  dose  of  chloro- 
quine  was  administered,  the  pyridoxine -deficiency  appears  to  inhibit  para- 
sitemia. 

Vitamin  deficiency  states  that  appear  to  favor  the  course  of  parasitemia 
include:  Pantothenic  acid,  biotin,  riboflavin  and  choline. 

Deficiencies  that  suppress  parasitemia  include:  ascorbic  acid  and  niacin. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
It  is  believed  that  the  results  of  this  study  may  give  some  insight  into  the 
mode  of  action  of  antimalarial  drugs. 

Proposed  Course  of  the  Project »  Investigations  along  the  lines  outlined 
above  will  be  continued  and  the  use  of  antimetabolites  will  be  extended. 
Further  work  will  be  initiated  to  clarify  promising  leads. 

Part  B  included  -  No 


31 


Serial  No.  NIAID-132 


1.  Parasite  Chemotherapy 

2.  Epidemiology 

3.  Columbia,  South  Carolina 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  A. 


Project  Title:  Studies  on  Human  Malaria 

Principal  Investigator:  Martin  D.  Young 

Other  Investigators:  None 

Cooperating  Units: 

Man  Years  (calendar  year  l°60)j 
Total :        3 
Professional:    l/2 
Other:        2  l/2 

Project  Description: 

Objectives: 

To  add  to  the  biological  knowledge  of  malaria  by  observations 
and  experiments  on  induced  malaria.  To  determine  the  sporontocidal 
activity  of  antimalarial  drugs  and  how  developed  resistance  influ- 
ences transmission  by  mosquito  vectors.  To  study  the  development 
and  inheritance  of  resistance  to  drugs  by  the  parasites.  To 
determine  if  the  development  of  resistance  to  insecticides  by 
mosquitoes  alters  their  ability  to  transmit  malaria.  To  serve  as 
probably  the  only  source  in  the  nation  for  established  strains  of 
malaria  for  use  on  a  nationwide  basis  for  therapy  of  neurosyphilis, 
nephrosis,  Parkinsonism,  and  for  experimental  malaria  studies. 

Methods  Employed: 

Malaria  is  induced  in  neurosyphilitic  patients  and  in  volun- 
teers under  normal  and  experimental  conditions.  The  patent  infec- 
tions are  challenged  by  drugs.  When  resistance  occurs  measurements 
are  made  of  its  characteristics.  Mosquitoes  are  allowed  to  bite 
patients  with  induced  infections  before  and  at  intervals  after 
drug  administration.  The  resulting  infections  in  the  mosquitoes 
are  correlated  with  the  presence  or  absence  of  drug  resistance 
of  the  malaria  infection  in  the  human  host.  Insecticide -resistant 
and  normal  mosquitoes  are  fed  simultaneously  for  comparison  of 
ability  to  transmit  malaria. 

32 


Serial  No.  NIAID-132 


Major  Findings; 

Primaquine  given  1.5  mg.  daily  had  sporontocidal  effect 
upon  Plasmodium  vivax  and  P.  falciparum  but  little  effect  upon 
the  asexual  parasites.  Daily  doses  of  0.75  mg.  had  some  sporontocidal 
effect  upon  P.  falciparum  but  none  against  one  P.  vivax  case. 
Therefore,  1.5  mg.  daily  appears  to  be  the  lowest  amount  that  is 
reliably  sporontocidal  against  both  P.  falciparum  and  P.  vivax. 

Therapeutic  doses  (I.I4  gm  in  3  days)  of  amodlaquine  had 
no  sporontocidal  effect  upon  one  P.  falciparum  case. 

P.  falciparum  from  South  Rhodesia  was  susceptible  to 
pyrime  thamine . 

Preliminary  tests  indicate  no  diffprence  in  vectorial 
ability  of  insecticide -susceptible  and  insecticide-resistant 
mosquitoes. 

P.  vivax  was  maintained  in  a  viable  condition  for  five 
years  at  -70°  C.  The  adding  of  glycerine  to  blood  containing 
chicken  and  rat  malaria  aids  in  its  preservation  at  low 
temperatures. 

A  lU-year  study  of  the  biology  of  P.  malariae ,  during 
which  time  it  had  been  passaged  through  neurosyphilitic  patients 
mainly  by  blood  transfusions,  revealed  the  following:  there 
was  no  apparent  reduction  in  viabilityj  the  infection  and  com- 
pletion of  the  sporogonous  cycle  in  Anopheles  aztecus;  the  com- 
parative susceptibilities  of  the  mosquitoes  tried,  in  decreasing 
order,  were:  A.  freeborni,  A_.  punctipennis,  A.  aztecus,  and  A» 
quadrimacula tu s ;  no  infections  were  found  in  A.  crucians  and  A. 
albimanus;  the  length  of  the  sporogonous  cycle  in  A.  quadrima- 
culatus  ranged  from  19  to  23  days  when  incubated  at  7o°  F.j 
A.  freeborni  when  paired  with  A.  quadrimacula tus  also  had  an 
identical  23-day  cycle;  of  7,3~5U  A.  quadrimaculatus  dissected, 
3.1  per  cent  were  infected;  the  oocysts  per  infected  gut 
averaged  3.0;  only  3*9   per  cent  of  the  guts  had  10  or  more 
oocysts;  transmission  of  the  malaria  to  six  patients  was 
accomplished,  three  by  the  bites  of  infected  mosquitoes  and  three 
by  the  intravenous  injection  of  the  glands  of  these  mosquitoes; 
there  was  wide  variation  in  the  infectiousness  of  different 
malarious  patients  to  mosquitoes;  mosquitoes  became  infected 
in  nearly  every  week  of  the  first  six  months  with  the  highest 
inf ectivity  occurring  during  the  eighth  to  tenth  weeks  of  the 
primary  attack;  the  asymptomatic  parasitemias  infected  mosqui- 
tces  at  about  the  same  rate  as  the  symptomatic  parasitemias. 


33 


Serial  N0.  NIAID-132 


an  autochthonous  chronic  asymptomatic  case  of  P.  malariae  of 
at  least  18  months  duration  infected  mosquitoes;  although 
the  infection  rate  of  mosquitoes  in  ordinarily  low,  the  relatively 
long  period  of  infection  during  which  mosquitoes  can  be  infected 
may  explain  the  persistence  of  P.  malariae  in  certain  areas; 
the  ability  of  the  symptom-free  malarious  patient  to  infect 
mosquitoes  at  a  rate  similar  to  that  of  the  symptomatic  patient 
makes  case  finding  more  difficult,  especially  in  important 
areas  of  malaria  eradication. 

A  strain  of  P.  falciparum  induced  into  neurosyphilitic 
patients  was  resistant  to  normal  and  above  normal  doses  of 
chloroquine . 

Significance  to  Bjo-Hedical  Research  and  the  Program  of  the 
Institute ; 

The  finding  of  resistance  of  malaria  parasites  to 
chloroquine  is  of  importance  to  the  malaria  eradication  pro- 
grams in  many  parts  of  the  world  as  drugs  are  being  used  as 
supplemental  measures  in  some  programs.  It  is  important  to 
determine  if  there  is  cross-resistance  between  the  U-amino- 
quinoline  drugs.  Additional  information  is  needed  on  the 
biology  of  the  parasite  especially  in  relationship  to  drugs. 

The  comparative  ability  of  insecticide-susceptible  and 
insecticide -resistant  mosquitoes  to  transmit  drug-susceptible 
and  drug -resistant  parasites  needs  to  be  determined,  especially 
to  indicate  whether  the  change  in  the  vector  or  parasite  has 
significance  in  the  epidemiology  of  malaria. 

The  low  but  persistent  infectivity  of  P.  malariae  to 
mosquitoes  helps  explain  its  epidemiology  and  especially  its 
persistence  in  nature. 

Proposed  Course  of  the  Project; 

To  determine  more  fully  the  nature  and  extent  of  the 
chloroquine  resistance  in  human  malaria,  as  well  as  cross- 
resistance  to  other  drugs  such  as  the  h-aminoquinolines.  Further 
investigate  the  ability  of  insecticide-susceptible  and  insecticide- 
resistant  mosquitoes  to  transmit  drug-susceptible  and  drug- 
resistant  malaria  parasites.  Continue  to  obtain  basic  informa- 
tion, especially  epidemiological  and  host-parasite  relation- 
ships on  induced  human  malarias,  both  domestic  and  foreign. 
Important  strains  of  malaria  will  be  added  to  those  now  in  the 
local  repository  which  apparently  is  the  only  existing  one  for 
the  preservation  of  malaria. 

Part  B  included  -  Yes  „  °  ' 


Serial  No.  NIAID-132 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  B. 


Honors,  Awards,  and  Publications. 

Publications  other  than  abstracts  from  this  project: 

Young,  M.  D.  The  Effect  of  Snail  Doses  of  Primaquine  Upon 

Malaria  Infections.  Ind.  J.  Mai.  13:69-7h.  I960. 

Young,  M.  D.  MALARIA  -  A  Study  of  Scientific  Exploration 

and  Achievement.  Bull.  S.  C.  Acad.  Sci.  21:32-37. 
I960.  — 

Young,  M.  D.  Malaria  (Chapter  in  Manual  of  Tropical  Medicine, 
third  edition,  by  Hunter,  Frye,  and  Swartzwelder. 
Saunders).  I960. 

Jeffery,  G.  M.  Infectivity  to  Mosquitoes  of  Plasmodium  vivax 

and  Pla  sm odium  falciparum  Under  Various  Conditions. 
Am.  J.  Trop.  Med.  and  Hyg.  9:315-320.  I960.  May. 

Jeffery,  G.  M.  Book  Review:  The  Ecology  of  Human  Diseases, 

by  Jacques  M.  May.  Am.  J.  Trop.  Med.  and  Hyg.  9: 
350-351.  I960. 

Young,  M.  D.  Book  Review:  Parasitology  (Protozoology  and 

Helminthology)  in  Relation  to  Clinical  Medicine,  by 
K.  D.  Chatterjee.  Am.  J.  Trop.  Med.  and  Hyg.  9: 
351. 

Young,  M.  D.  Chemo therapeutic  agents  and  malaria  eradication. 

Proceedings  of  Sixth  International  Congresses  on  Trop. 
Med.  and  Mai.   (In  press).  I960. 

Garnham,  P.  C.  C,  Jeffery,  G.  M.,  and  Young,  M.  D.  Preservation 

of  strains  of  malaria  parasites.  Proceedings  of  Sixth 
International  Congresses  on  Trop.  Med.  and  Mai.  (In 
Press.)  I960. 

Jeffery,  G.  M.  Inoculation  of  Human  Malaria  into  a  Simian  Host, 
Macaca  mulatta.  J.  Parasit.  (In  Press.) 

Young,  M.  D.,  and  Burgess,  R.  W.  The  Infectivity  to  Mosquitoes 

of  Plasmodium  Malariae.  Am.  J.  and  Hyg.   (in  press). 
I960. 


35 


Serial  No.  NIAID-132 

Honors  and  Awards  Relating  to  this  Project: 

For  Dr.  Martin  D.  Young: 

Visiting  Lecturer,  Department  of  Microbiology, 
Meharry  Medical  School. 

Visiting  Lecturer  and  Temporary  Advisor  on  Chemotherapy 
PAHO  Malaria  Eradication  Training  Center,  Kingston, 
Jamaica. 

Member,  City  Board  of  Health,  Columbia,  South  Carolina. 

Member,  Expert  Advisory  Panel  on  Malaria,  World  Health 
Organization. 

Member,  Editorial  Board,  American  Journal  of  Tropical 
Medicine  and  Hygiene. 


3C 


Serial  No.     NIAID-132-A 

1.  Parasite  Chemotherapy 

2.  Epidemiology 

3.  Columbia,  South  Carolina 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  A. 

Project  Title:  Chemotherapy  of  intestinal  parasites. 

Principal  Investigator:  Martin  D.  Young 

Other  Investigators:     Geoffrey  M.  Jeffery 

Cooperating  Units:       South  Carolina  State  Hospital 

Man  Years  (calendar  year  1960) x 

Total:  2  5/6 
Professional:  5/6 
Other:        2 

Project  Description: 

Objectives: 

To  find  adequate  drugs  for  the  prophylaxis  and  treatment 
of  parasitic  infections  which  constitute  serious  health  problems 
in  certain  types  of  mental  patients  as  well  as  in  more  normal 
populations;  to  determine  the  relationship  of  parasite  load  to 
psychological  or  psychiatric  considerations  in  the  patients; 
also  to  investigate  factors  influencing  parasite  spread  and 
persistence  among  patients. 

Methods  Employed: 

Various  segments  of  the  institutional  population  are 
examined  to  determine  qualitatively,  and  quantitatively  when 
possible,  their  parasitic  infections.  Promising  compounds  in 
varying  dosages  and  regimens  are  tried  against  selected  cases; 
post-treatment  evaluation  determines  the  effectivity  of  the 
drug  against  the  various  parasites.  Occasionally  careful  pre- 
and  post-treatment  psychological,  psychiatric  and  physical 
evaluations  are  done  to  determine  the  possible  effect  of 
parasite  removal.  The  results  are  based  upon  thousands  of 
patient-drug  (individual  regimens)  trials. 

37 


Serial  No.  NIAID-132-A 


Major  Findings; 


Epidemiological  studies  on  a  defined  population  show  a 
high  persistence  of  Trichuris  and  hookworm  for  six  years,  with 
an  apparent  decrease  in  Stronqyloides.  The  yearly  studies 
show  fluctuations  in  protozoal  infections  indicating  that 
transmission  is  occurring. 

To  test  the  therapeutic  and  prophylactic  effect  of 
dithiazanine  and  tetrachloroethylene  alone  and  in  combination, 
a  group  of  mental  patients  heavily  parasitized  were  given  drugs 
at  various  intervals  for  about  one  year.   A  large  number  of 
worms  were  removed  but  the  cure  rate  was  low.  Treatments  sub- 
sequent to  the  initial  one  had  reduced  effectiveness.  Trans- 
mission was  not  stopped.  These  drugs  do  not  appear  to  be 
adequate  prophylactic  agents  under  conditions  of  high  exposure 
such  as  represented  in  this  study. 

Bephenium  hydroxynaphthoate  continued  to  exhibit 
excellent  results  against  hookworm  and  Ascaris  infections 
found  in  mental  patients.  A  retrial  of  bephenium  chloride 
was  begun.  The  results  so  far  indicate  that  it  may  be  more 
effective  in  single  comparative  doses  than  the  bephenium 
hydroxynaphthoate.  It  is  showing  promising  activity  against 
Trichuris.  Combined  with  an  anti-nausea  drug,  Marezine,  the 
side  effects  of  nausea  and  vomiting  appear  to  be  reduced. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the 
Institute; 

Currently  available  drugs  in  the  regimens  tried  have 
not  been  shown  to  be  adequate  in  the  prevention  of  trans- 
mission of  Trichuris  and  hookworm  parasites  in  areas  of  high 
exposure  risks.  Better  drugs,  or  better  regimens  of  the 
present  ones,  are  needed.   Information  obtained  should  be  of 
value  in  mental  hospitals  and  in  many  tropical  areas. 

Proposed  Course  of  the  Project: 

The  evaluation  of  promising  compounds  for  better  prophy- 
lactic and  parasiticidal  agents  will  be  continued  with  especial 
interest  on  different  regimens  of  bephenium  chloride.  Parallel 
investigations  are  underway  on  the  mode  of  action  of  drugs  on 
parasites.   Efforts  are  being  made  to  explain  the  failure  of 
effective  drugs  to  produce  radical  cures,  the  reduced  effective- 
ness of  drugs  after  the  initial  treatment,  and  similar  problems. 
Further  investigations  on  the  epidemiology  of  parasitic  infections 
in  mental  hospital  populations  will  be  continued. 

Part  B  included:   Yes.  °  " 


Serial  No.   NIAID-132-A 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B.    Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Jeffery,  G.  M.   A  Three-Year  Epidemiologic  Study  of  Intestinal 
Parasites  in  a  Selected  Group  of  Mental  Patients. 
Am.  J.  Hyg.  71:1-8.   1960. 

Young,  M.  D.,  Jeffery,  G.  M.,  Morehouse,  W.  G.,  Freed,  J.  E.,  and 
Johnson,  R.  S.   The  Comparative  Efficacy  of  Bephenium 
Hydroxynaphthoate  and  Tetrachloroethylene  against  Hookworm 
and  other  Parasites  of  Man.   Am.  J.  Trop.  Med.  &  Hyg. 
9:488-495. 


3S 


Serial  No.    NIAID-132-B 

1.  Parasite  Chemotherapy 

2.  Epidemiology 

3.  Columbia,  South  Carolina 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:     Physiology  of  human  parasites  especially  as 
related   to  nucleic  acids  and  drug  action. 

Principal   Investigator:     Kenneth  0.  Phifer 

Other  Investigators:  None 

Cooperating  Units:  None 

Man  Years    (calendar  year  I960): 

Total:  1 

Professional:       1 
Other*  0 

Project  Description: 

Objectives: 

To  determine  the  place  of  action  of  drugs  upon  malaria 
and   intestinal  parasites  of  man.     The  study  of  the  action  of 
drugs  on  malaria  includes  the  mechanism  and   locale,   the  develop- 
ment of  resistance  and   a   search  for  methods  to  avoid  the 
development  of  resistance  or  how  to  overcome  it.     Similar 
studies   are  planned  on  the  developmental   chemotherapy  of  other 
parasites,    especially  the  worms  of  the  intestinal  tract. 

The  nucleic  acids  of  malaria  parasites  are  being 
examined   qualitatively  and   quantitatively  in  order  to  determine 
whether  drug-resistance  affects  the   synthesis  of  these  sub- 
stances*    To  ascertain  whether  malaria-infected   mosquitoes  have 
higher  nucleic  acid   levels  than  non-infected  mosquitoes.     To 
determine  what  effect  anthelmintics  have  on  the  in  vitro  meta- 
bolism and  in  vivo  distribution  of  helminths. 

Methods  Employed: 

Malaria-infected  red  blood   cells   and   infected  mosquitoes 
are  homogenized   and   fractionated   into  ribonucleic  acid;     this 

40 


Serial  No.  NIAID-132-B 


fraction  is  then  measured  colorimetrically.  Qualitative 
analysis  of  the  nucleic  acid  fraction  is  carried  out  through 
the  use  of  paper  chromatography  and  paper  electrophoresis. 

Helminths  are  incubated  both  in  the  presence  and  in 
the  absence  of  anthelmintics  and  several  measures  of  metabolism 
are  utilized;  e.g.,  transaminase  reaction  and  rate  of  glucose 
uptake.   Also  noted  is  the  effect  of  anthelmintics  on  egg  pro- 
duction and  distribution  of  the  worms  in  the  intestine  of  the 
host. 

Major  Findings: 

It  was  shown  that  mosquitoes  infected  with  malaria 
parasites  tend  to  have  higher  levels  of  ribonucleic  acid 
than  do  uninfected  mosquitoes. 

Chromatographically,  the  acid-hydrolysate  of  ribonucleic 
acid  from  a  pyrimethamine-resistant  strain  of  Plasmodium  falci- 
parum appears  to  be  different  than  the  acid-hydrolysate  of 
ribonucleic  acid  from  a  pyrimethamine-susceptible  strain  of 
this  species.  Preliminary  experiments  point  toward  similar  findings 
in  the  case  of  chloroquine  resistance. 

Bephenium  hydroxynaphthoate  inhibited  glutamic  acid-alanine 
transaminase  and  glutamic  acid-aspartic  acid  transaminase  in 
Nippostrongylus  muris  in  vitro.  This  drug  also  appears  to  remove 
more  worms  from  the  anterior  part  of  the  intestine  of  infected 
rats  than  from  more  posterior  segments  of  the  gut. 

Bephenium  chloride  and  atabrine  reduced  the  rate  of 
glucose  absorption  by  the  tapeworm,  Hymenolepis  diminuta,  from 
30  to  50  per  cent.  At  low  concentrations  the  cyanine  dye, 
dithiazanine  iodide  stimulated  glucose  absorption  by  this  cestode. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the 
Institute: 

Since  several  antimalarial  agents  have  chemical  structures 
similar  to  those  of  components  of  nucleic  acids  it  seems  advis- 
able to  determine  whether  these  drugs  affect  the  parasite's 
basic  nuclear  compounds.   It  is  felt  that  if  it  were  demonstrated 
that  nucleic  acids  are  altered  by  antimalarials,  this  clarifi- 
cation of  mechanism  of  drug  action  would  provide  a  more  logical 
rationale  for  the  synthesis  of  new  drugs. 


Serial  No.  NIAID-132-B 


Resistance  of  parasites  to  drugs  is  a  serious  world 
problem  with  respect  to  malaria  eradication.  Since  resistance 
has  been  shown  to  be  under  genetic  control  in  many  instances, 
demonstration  of  changes  in  nucleic  acid  composition  in 
response  to  exposure  to  a  drug  should  be  of  value  in  determin- 
ing mechanisms  of  resistance.  Once  mechanisms  of  resistance 
are  delineated,  the  problems  associated  with  combatting  drug 
resistance  might  be  more  readily  solved. 

A  corollary  aim  of  this  project  is  the  attempt  to 
develop  a  biochemical  means  of  detection  of  infection  of  mos- 
quitoes before  morphological  stages  in  the  parasite's  life 
cycle  are  evident.   If  such  a  means  proved  reliable,  some 
insight  could  be  gained  as  to  the  changes  occurring  and  the 
experimental  procedures  would  be  shortened. 

Study  of  the  effect  of  anthelmintics  on  helminth 
metabolism  will  aid  in  a  clearer  understanding  of  drug  action 
and  might  be  valuable  in  the  design  of  new  drugs. 

Study  of  the  effect  of  anthelmintics  on  the  distribution 
of  helminths  in  the  host  intestine  is  important  since  in  human 
cases  of  helminthiasis,  a  few  worms  often  remain  after  therapy 
and  the  question  of  whether  this  is  due  to  resistance  to  the 
drug  or  a  re-location  in  the  gut  should  be  answered. 

Proposed  Course  of  the  Project; 

Studies  of  nucleic  acid  composition  of  malaria  parasites 
from  drug-resistant  and  drug-susceptible  strains  will  be  con- 
tinued using  both  paper  chromatography  and  absorption  spectrum 
analysis  via  spectrophotometry. 

Infected  mosquitoes  will  be  examined  for  ribonucleic 
acid  level  to  determine  conclusively  whether  increased  nucleic 
acid  levels  can  be  reliable  indices  of  infection. 

Studies  with  respect  to  the  permeation  of  drugs  into  the 
malaria  parasite  will  be  carried  out,  comparing  the  uptake  of 
drug  in  susceptible  and  resistant  strains. 

Animals  infected  with  helminths  will  be  drugged  with 
anthelmintics  and  the  worms  will  be  counted,  position  and  size 
of  worm  noted,  and  relative  metabolic  activity  assayed.  Both 
cestodes  and  nematodes  will  be  utilized. 

Part  B  included:   No  ^*- 


Serial  No.    NIAID-132-C 


1.  Parasite  Chemotherapy 

2.  Epidemiology 

3.  Columbia,   South  Carolina 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  A. 

Project  Title:  Virus-Parasite  Association 

Principal  Investigator:  Geoffrey  M.  Jeffery 

Other  Investigators:     None 

Cooperating  Units:       None 

Man  Years  (calendar  year  1960): 

Total:  1  1/3 
Professional:  l/3 
Other:        1 

Project  Description: 

Objectives: 

To  determine  experimentally  if  parasites  might  be 
associated  with  some  of  the  virus  diseases  of  man  or  animals 
as  vectors  or  in  some  other  capacity.  To  study  such  relation- 
ships under  natural  conditions  and  to  investigate  known  virus 
infections  and  known  populations  to  determine  existing  re- 
lationships, including  the  study  of  possible  intestinal  viruses 
among  institutionalized  mental  patients.   In  view  of  current 
findings,  further  study  the  relationships  of  virus-malaria 
and  virus-endoparasite  associations  under  experimental  conditions. 
Investigate  the  possibility  that  certain  parasites  may  them- 
selves be  afflicted  with  harmful  virus  infections.   Investigate 
the  effects  of  parasitic  infections  upon  virus  infections  and 
vice  versa. 

Methods  Employed: 

Small  animals  and  their  common  parasites  are  used  in 
attempts  at  transmission  of  viruses.  Viruses,  such  as  lympho- 
cytic choriomeningitis  (LCM),  polio,  St.  Louis  encephalitis  (SLE), 
rabbit  papilloma  and  fibroma  are  maintained  in  small  animals  by 
serial  transmission,  providing  infective  virus  material  for 


*»3 


Serial  No.  NIAID-132-C 


experimental  work.  Besides  the  common  helminth  parasites  of 
mice,  Nippostronqylus  muris,  Nematospiroides  dubius,  Hymenolepis 
nana,    and  others,  the  malaria  parasites  Plasmodium  berqhei  and 
P.  gallinaceum  are  also  maintained  as  experimental  parasites. 
Viruses  and  parasites  are  brought  together  in  various  ways,  such 
as  by  simultaneous  infection  in  animals,  during  cultivation  of 
the  free-living  stages,  or  by  simultaneous  exposure  of  the  host 
to  infective  parasite  stages  and  virus  suspensions.  Tissue 
culture  or  small  animals  are  used  for  detection  of  virus  infec- 
tion carried  by  parasites  from  virus-infected  animals,  for 
virus  detection  in  parasites  suspected  of  harboring  viruses  in 
nature,  and  for  detection  of  virus  infection  in  arthropods. 
Incidence  of  intestinal  viruses  and  their  possible  association 
with  parasites  in  institutionalized  mental  patients  is  studied 
with  animal  inoculation,  tissue  culture,  and  other  means. 

Major  Findings; 

In  mice  inoculated  with  SLE  virus  intraperitoneal^,  the 
presence  of  P.  berqhei  aided  in  the  transport  of  the  virus  to 
the  central  nervous  system  as  shown  by  symptoms  and  by  death 
of  the  mice.  The  same  occurred  when  LCM  virus  and  P.  berqhei 
were  combined.  The  virus  appeared  earlier  and  persisted  longer 
when  malaria  was  present.  SLE  virus  and  P.  gallinaceum  com- 
bined  caused  death  earlier  in  some  chicks  and  enhanced  the 
viremia  in  others  over  that  found  when  malaria  alone  was 
present. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the 
Institute: 

Many  problems  associated  with  the  transmission  of  viruses 
in  human  and  animal  populations  remain  unsolved.   New  viruses 
in  mammals  and  arthropods  are  continually  being  discovered. 
There  are  suggestions  in  the  literature  and  theoretical  possi- 
bilities that  parasites  may  act  as  reservoirs  and  agents  of 
transmission  for  viruses  of  importance  to  man.  Elucidation  of 
association  of  viruses  and  parasites,  both  experimental  and 
natural,  will  do  much  toward  bringing  about  a  more  complete 
understanding  of  the  epidemiology  of  diseases  produded  by  such 
organisms,  alone  or  in  combination.  The  possible  role  of  malaria 
in  transmitting  virus  from  host  to  host  either  by  extending  the 
viremia  or  by  actually  transporting  the  virus  in  the  sporozoite 
is  of  epidemiological  importance. 

4  H 


Serial  No.  N1AID-132-C 


Proposed  Course  of  Projectt 


The  general  course  of  the  project  will  be  changed  little. 
Repetition  and  expansion  of  the  experiments  combining  nematode 
parasites  and  viral  agents  in  the  vertebrate  host,  during  free- 
living  stages,  and  in  culture,  will  be  carried  out.  An  increased 
amount  of  effort  will  be  directed  toward  the  association  of 
viruses  and  malaria  parasites,  and  possibly  other  protozoans. 
The  detection  of  viruses  in  malaria  parasites,  especially  the 
sporozoites  by  fluorescent  antibody  techniques  and  by  inoculation 
will  be  attempted.  Further  refinement  of  viral  detection  methods 
will  be  investigated  and  incorporated  in  the  work  when  practicable. 
Most  of  the  experimental  work  planned  is  in  the  realm  of  artifi- 
cial viral-parasite  associations,  but  the  possibility  of  extension 
of  the  studies  to  naturally  occurring  associations  will  be  always 
in  mind  and  such  studies  will  be  pursued  as  opportunities  arise. 

Part  B.  included*  Yes. 


45 


Serial  No.  NIAID-132-C 

PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 

Honors,  Awards,  and  Publications 

Honors  and  Awards  Relating  to  this  Project: 

Member  Council,  South  Carolina  Academy  of  Science. 

Editor,  ASB  Bulletin,  published  by  the  Association  of 
Southeastern  Biologists. 

Recipient  of  Jefferson  Award  of  the  South  Carolina 
Academy  of  Science. 


46 


Serial  No.     NIAID-132-D 

1.  Parasite  Chemotherapy 

2.  Epidemiology 

3.  Columbia,    South  Carolina 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  A. 


Project  Title:  Development  of  human  pathogens  in  immature 
insects. 

Principal  Investigator:  William  E.  Collins 

Other  Investigators:     None 

Cooperating  Units:       None 

Man  Years  (calendar  year  I960): 

Total:  1  1/2 
Professional:  l/2 
Other :        1 

Project  Description: 

Objectives : 

To  determine  experimentally  to  what  extent  immature 
stages  of  arthropods  will  support  growth  of  pathogenic 
organisms  with  particular  emphasis  on  the  viruses  and 
protozoans  in  mosquito  larvae.   If  experimental  evidence 
indicates  the  growth  or  maintenance  of  these  microorganisms 
in  insect  larvae,  investigations  will  be  made  to  determine  the 
possible  role  larval  infection  plays  in  the  transmission  of 
these  infections  to  man. 

Methods  Employed: 

The  larvae  of  mosquitoes  belonging  to  several  different 
genera  are  exposed  to  virus  suspensions  under  varying  con- 
ditions. The  quantity  of  virus  present  in  larvae,  pupae,  and 
adults  subsequent  to  exposure  is  determined  by  small  animal 
inoculations.  Transmission  of  the  virus  by  the  adult  mosqui- 
toes is  made  using  small  animals  and  blood  pools. 


^ 


Serial  No.  NIAID-132-D 


Major  Findinqst 

Further  studies  confirmed  the  results  of  the  prior  year 
where  the  virus  of  St.  Louis  encephalitis  was  taken  up  by  the 
larvae  of  Aed e s  aeqypti  and  an  increase  in  virus  titer  found 
in  subsequent  larval  and  pupal  mosquitoes.  The  larvae  of  Culex 
quinquef asciatus  exposed  under  similar  conditions  take  up  the 
virus  to  a  lesser  extent,  and  the  adults  have  not  demonstrated 
the  virus.  Transmission  of  the  virus  to  baby  chicks  has  been 
confirmed  using  mosquitoes  9  to  26  days  after  adult  emergence. 
The  rate  of  adult  infection  and  virus  transmission  using  St. 
Louis  encephalitis  virus  and  Aedes  aeqypti  mosquitoes  has  been 
less  than  one  per  cent  under  these  experimental  conditions. 

Significance  to  Bio-Medical  Research  and  the  Program  of  the 
Institute: 

The  presence  of  viruses  and  protozoans  in  mosquito 
breeding  pools  as  a  result  of  improper  sanitation  procedures 
or  by  death  and  decomposition  of  infected  animals  and  arthro- 
pods poses  the  possibility  that  these  organisms  may  be  taken 
up  by  the  mosquito  larvae  and  subsequently  transmitted  by  the 
adults.  Information  on  such  larval  infection  will  add  to  a 
more  complete  understanding  of  the  epidemiology  of  a  number 
of  arthropod -borne  diseases. 

Proposed  Course  of  the  Project i 

The  general  course  of  the  project  will  be  changed 
little.  The  relative  ability  of  mosquito  larvae  of  different 
genera  to  take  up  the  virus  of  St.  Louis  encephalitis  and  the 
effect  of  initial  virus  titer,  incubation  temperature  and 
larval  instar  on  the  rate  of  infection,  virus  titer  and 
adult  transmission  will  be  tested. 

Part  B  included:  No. 


43 


Serial  No.  NIAID-132-E 

1.  Parasite  Chemotherapy 

2.  Epidemiology 

3.  Columbia,  South  Carolina 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  I960 


Part  A. 


Project  Title:  Insect  Tissue  Culture 

Principal  Investigator:  William  E.  Collins 

Other  Investigators:  None 

Cooperating  Units:  None 

Man  Years  (calendar  year  I960): 

Total :       1 
Professional:   l/2 
Other:         1/2 

Project  Description: 

Objectives: 

To  establish  insect  tissues  in  culture  in  order  to  study  better 
those  factors  necessary  for  insect  growth  aid  metamorphosis  and  the  effect 
of  microorganisms  on  insect  tissues.  To  determine  which  of  the  viruses, 
protozoans  or  helminths  will  persist,  develop,  or  multiply  in  the  different 
insect  tissues  grown  in  vitro  using  both  natural  vectors  and  other  species. 

Methods  Employed} 

Tissues  and  cells  from  moth  larvae  and  pupae  and  from  mosquito 
larvae,  pupae  and  adults  are  removed  and  set  up  in  experimental  culture 
media  using  several  different  culture  methods.  The  presence  of  mitosis, 
tissue  contractility  or  general  cell  condition  are  used  as  indications  of 
tissue  growth  or  maintenance.  Viruses  are  added  to  aich  cultures  and  periodic 
samples  collected  and  titrated  in  small  animals.  Protozoans  are  added  and 
microscopic  examinations  made  to  determine  persistence  or  development  of  the 
parasites.  Guts  of  mosquitoes  previously  infected  with  Plasmodium  sp.  are 
removed  and  set  up  in  culture.  Periodic  examination  and  measurement  of  the 
parasite  development  are  made. 


43 


Serial  No.  MIA  ID-  132 -E 


Major  Fjnding3t 


Blood  cells  from  caterpillars  and  cells  of  the  ovariole  sheath  of 
several  species  of  moth  prepupae  and  pupae  have  undergone  growth  in  culture 
in  several  different  experimental  media.  The  virus  of  St.  Louis  encephalitis 
has  been  maintained  in  cultures  of  third,  fourth,  and  fifth  instar  hemocytes 
from  the  catalpa  sphinx  (Ceratomia  catalpae)  for  10  days  with  little  drop  in 
titer  over  that  which  occurred  during  the  first  2k   hours.  The  oocysts  of 
Plasmodium  gallinaceum  attached  to  mosquito  midguts  in  vitro  have  exhibited 
growth  and  on  several  occasions,  sporozoites  have  been  produced. 

Significance  to  Bio-Iledical  Research  and  the  Program  of  the  Institute: 

Insects  serve  as  alternate  hosts  for  a  number  of  virus  and 
protozoan  diseases  of  man.  Insect  tissue  culture  would  offer  the  opportunity 
to  study  these  parasites  and  their  growth  in  insect  cells  in  vitro,  and  thus 
quite  possibly  shed  new  light  on:  (l)  parasite  growth  requirements  (2) 
arthropod  tissue  specificity  and  (3)  reasons  why  many  human  pathogenic 
viruses  will  multiply  in  the  insect  with  no  visible  or  apparent  pathology. 
These  studies  may  yield  information  on  those  factors  controlling  the  ability 
of  insect  vectors  to  transmit  virus  and  protozoan  diseases  whereas  closely 
related  species  of  insects  do  not. 

Proposed  Course  of  the  Project? 

Various  tissues  of  mosquito  larvae,  pupae,  and  adults,  and  moth 
larvae  and  pupae  will  be  established  in  culture  and  tested  for  their  ability 
to  support  the  virus  of  St.  Louis  encephalitis.  Microscopic  studies  will  be 
made  in  efforts  to  detect  any  cytopathology  of  the  tissue  as  a  result  of 
virus  infection.  In  addition,  fluorescent  antibody  studies  will  be  made  to 
determine  in  which  cells  or  tissues  the  virus  is  present.  Guts  from  mosquitoes 
infected  with  Plasmodium  sp.  will  be  set  up  in  different  culture  media  in  an 
attempt  to  determine  those  factors  necessary  for  the  development  of  sporozoites 
in  vitro.  Further  studies  on  the  effect  of  virus  on  caterpillar  blood  cells 
and  ovariole  tissue  will  be  made. 

Part  B  included:  No. 


50 


Serial  No.   NIAID-133 

1.  Parasite  Chemotherapy 

2.  Cytology 

3.  Memphis,   Tennessee 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:  Studies  of  the  biology  of  exoerythrocytic  phases  of 
primate  malaria. 

Principal  investigator:  Don  E.  Eyles 

Other  investigators:  Robert  L.  Ingram,  Nell  C.  Owen,  Frances  E.  Jones 
and  C.  S.  Smith 

Cooperating  Units:  None 

Man  Years  (calendar  year  i960): 
Total        3 
Professional:  1  1/2 
Other:       1  1/2 

Project  Description: 

Objectives:  To  extend  the  knowledge  of  the  exoerythrocytic  stages  of  pri- 
mate malaria,  determine  the  type  of  cells  parasitized,  and  trace  the  development 
from  sporozoite  to  mature  schizont.  To  determine  if  the  liver  is  the  sole 
tissue  parasitized.  To  determine  the  origin  and  nature  of  the  late  tissue 
stages  and  to  determine  their  relationship  to  relapse.  To  define  in  detail 
quantitative  relationships  in  the  exoerythrocytic  cycle.  To  study  the 
exoerythrocytic  cycle  as  it  is  related  to  the  immune  processes  of  the  monkey 
host.  To  determine  factors  influencing  the  degree  of  exoerythrocytic 
infection.  To  provide  materials  and  correlative  information  to  accompany 
projects  on  growth  of  malaria  parasites  in  vitro  and  the  chemotherapy  of 
malaria. 

Methods  Employed:  Plasiiiodium  cynornolgi,  which  is  closely  related  to 
P.  vivax  of  man,  is  maintained  in  Macaca  mulata  monkeys.   Strains  of  P. 
ponderi  (also  a  simian  vivax-type  parasite)  and  P.  inui  (related  to  human 
P.  malariae)  are  also  maintained.  Anopheles  mosquitoes  of  several  species 
are  produced  and  mosquitoes  infected  in  order  to  produce  very  large  numbers 
of  infected  mosquitoes.   Salivary  glands  are  dissected  from  these  mosquitoes 
and  injected  intravenously  in  order  to  produce  patent  liver  infection. 


51 


Serial  No.  NIA3D-  133 

At  predetermined  intervals,  liver  specimens  are  taken  by  laparotomy. 
Histological  studies  are  made  on  living  and  stained  material.  Flourescent- 
antibody  as  an  aid  in  locating  parasites  is  employed,  and  other  labelling 
methods  will  be  used  as  necessary. 

Major  Findings;  Continued  study  was  made  on  quantitative  aspects  of  the 
problem  such  as  the  relationship  of  inoculm  size  to  the  development  of  liver 
parasites  and  subsequent  parasitemia.  Other  studies  were  made  which  indicated 
that  secondary  generations  of  exoerythrocytic  parasites  in  the  liver  do  occur. 
Experiments  on  route  of  inoculation  (ie.  portal  circulation  versus  femoral 
vein)  indicated  no  difference  in  degree  of  liver  infection. 

Work  on  this  project  was  curtailed  due  to  the  urgency  of  work  on  malaria 
as  a  zoonosis. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute; 
Confirmation  of  the  liver  cycle  of  malaria  puts  work  on  chemotherapy  on  a 
sound  basis.  Unelucidated  aspects  of  the  cycle  may  have  significance  in 
chemotherapy  and  basic  biology  of  the  parasites.  Study  of  simian  species  is 
especially  appropriate  as  the  close  relationship  to  the  human  species  indicates 
that  any  findings  may  be  directly  applied  to  human  infection  with  few 
reservations.  The  present  project  is  closely  correlated  with  chemotherapy  and 
tissue  culture  projects,  and  is  another  phase  of  the  integrated  malaria  study 
of  the  Laboratory  of  Parasite  Chemotherapy. 

Proposed  Course  of  the  Project;  During  the  coming  year  special  emphasis 
will  be  placed  on  study  of  the  exoerythrocytic  stages  of  newly  isolated  strains 
of  simian  malaria  and  on  comparative  biology  of  the  parasites  of  different 
species  of  primate  malaria.  Work  is  again  proposed  on  the  influence  of 
immunity  on  exoerythrocytic  parasites,  and  the  project  will  continue  to  be  a 
source  of  material  for  tissue  culture  work  and  for  chemotherapy  studies. 


Part  B  included  -  Yes 


52 


Serial  No.   NIAID-133 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 

Part  B«  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Eyles,  D.  E.  Anopheles  freebcrni  and  A.  quadrimaculatus  as  experimental 
vectors  of  Plasmodium  cynomolgi  and  P.  inui«  J.  Parasit.,  U6:5UO.  I960. 

Eyles,  D.  E.  The  exoerythrocytic  cycle  of  Plasmodium  cynomolgi  and 
P.  cynomolgi  bastianellii  in  the  rhesus  monkey.  Araer.  J.  Trop.  Med.  &  Hyg., 
9 :5U3-555."  I9oo; 

Eyles,  D.  E.  Susceptibility  cf  Anopheles  alblmanus  to  primate  and  avian 
malarias.  Mosquito  News,  In  press.  I960. 

Eyles,  D.  E.  and  Coleman,  N.  The  effect  cf  metabolites  on  the  antitoxo- 
plasmic  action  of  pyrimethamine  and  sulfadiazine.  Am.  J.  Trop.  Med.  &  Hyg., 
9:277-283 

Eyles,  D.  E.  The  treatment  of  toxoplasmosis.  From  Human  Toxoplasmosis, 
Munksgaard,  Copenhagen,  pp.  127-1U5.  I960. 

Gibson,  C.  L.  and  Jumper,  J.  R.  The  prevalence  of  canine  toxoplasmosis 
in  Memphis,  Tennessee.  J.  Parasit.,  U6:55>9-565.  I960 

Note:  Last  three  papers  not  directly  associated  with  project  but  repre- 
sent previous  projects  not  reported  this  year. 


53 


Serial  No.   NIAID-133-A 

1.  Parasite  Chemotherapy 

2.  Cytology 

3.  Memphis,  Tennessee 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  A. 


Project  Title:  Chemotherapeutic  studies  of  the  direct  effect  of  drugs 
on  the  exoerythrocytic  stages  of  primate  malaria. 

Principal  investigator:  Don  E.  Eyles 

Other  Investigators:  Robert  L.  Ingram,  Nell  C.  Owen, 
Clinton  S.  Smith 

Cooperating  Units:  None 

Man  Years  (calendar  year  1°60) : 
Total:       2  l/2 
Professional:  1  l/2 
Other :       1 

Project  Description: 

Objectives:  To  determine  the  direct  effects  upon  exoerythrocytic  parasites 
of  primate  malarias  of  established  antimalarial  drugs.  To  describe  morpho- 
logical and  histochemical  changes  and  relate  these  to  mode  of  action.  To 
determine  dosages  necessary  for  effect.  To  s  tudy  effect  as  related  to  time  of 
administration;  that  is,  before  infection  and  at  various  times  during  infection. 
To  study  possible  synergists.  To  study  the  reaction  of  the  primate  host  to 
killed  or  damaged  parasites.  In  the  case  of  incomplete  effects,  to  determine 
how  parasites  survive  treatment  and  produce  relapse. 

To  study  the  direct  effect  of  various  substances  of  known  metabolic  or 
antimetabolic  activity  on  primate  exoerythrocytic  stages.  To  relate  findings 
to  physiological  processes  of  the  parasites,  with  the  ultimate  objectives  of 
obtaining  leads  toward  development  of  more  effective  drugs.  It  is  quite  possi- 
ble that  incomplete  effects,  not  detectable  by  indirect  studies  of  relapse, 
might  be  seen  directly,  and  provide  information  useful  in  future  work. 

Methods  Employed:  Plasmodium  cynomoigi,  the  vivax-type  parasite  of 
macaques,  is  the  principal  parasite  used;  however,  we  plan  to  use  the  malar iae- 
type,  Plasmodium  inui,  as  suitable  techniques  are  developed.  For  chemothera- 
peutic studies,  infections  are  established  in  the  livers  of  monkeys  by  the 
intravenous  inoculation  of  massive  numbers  of  sporozoites.  Patent  infections 
can  ue  produced  consistently.  Drugs  or  other  substances  are  administered  either 
before  or  after  infection  is  produced.  In  the  event  of  prior  drug  administration, 

5^ 


Serial  No.  NIAID-133-A 

controls  are  established  using  identical  inocula.  When  treatment  is  begun 
after  the  infection  is  established,  pre-treatment  and  post-treatment  biopsies 
are  made  and  the  parasites  compared  with  those  found  in  control  animals. 
The  infections,  both  erythrocytic  and  exoerythrocytic,  are  followed  after 
treatment  to  obtain  information  on  relapse. 

Parasites  affected  by  the  drugs  are  studied  morphologically  and  histo- 
chemically  by  the  application  of  various  staining  techniques.  Drug  levels 
are  obtained  when  necessary.  A  flexibility  of  procedure  is  maintained  so  that 
the  methods  may  be  modified  to  apply  to  specific  problems  as  they  arise. 

Major  Findings;  Further  study  of  pyrimethamine  confirmed  the  fact  that 
when  administered  on  the  sixth  day  of  sporozoite  induced  infection  all  para- 
sites seen  on  subsequent  biopsy  were  severely  damaged  or  killed,  but  para- 
sitemia developed  30  to  U0  days  later  indicating  some  parasites  escaped  the 
action  cf  the  drug.  When  sulfonamides  were  used  with  pyrimethamine  to  exploit 
the  possible  synergistic  interaction  of  the  two  drugs,  similar  results  were 
obtained — monkeys  developed  parasitemia  30  to  U0  days  after  infection  even 
though  all  parasites  seen  were  damaged. 

Quinocide,  the  Russian  8-aminoquinoline  drug,  was  compared  with  Prima- 
quine. Even  when  administered  at  twice  the  dosage  used  with  Primaquine  the 
drug  was  less  effective.  Treatment  on  the  sixth  day  did  not  prevent  the 
appearance  of  large  numbers  of  blood  forms  on  the  ninth  day,  but  the  liver 
parasites  after  five  days  of  treatment  did  appear  damaged. 

Untreated  parasites  and  parasites  treated  with  pyrimethamine  were  fixed  in 
Carnoy's  and  stained  with  toluidine  blue  before  and  after  exposure  of  sections 
to  the  action  of  ribonuclease .  Preliminary  conclusions  are  that  the  synthesis 
of  DNA  is  markedly  inhibited  by  the  drug. 

Chloroquine  had  no  observable  effect  upon  the  liver  forms  of  Plasmodium 
cynomolgi,  normal  exoerythrocytic  stages  being  observed  after  treatment.  Ring 
stage  parasites  appeared  in  the  blood  in  large  numbers  on  the  8th,  l6th  and 
about  the  2Uth  day  after  inoculation  indicating  the  existence  of  secondary 
exoerythrocytic  stages. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
This  project  is  part  of  an  intergrated  program  of  the  Laboratory  of  Parasite 
Chemotherapy  to  study  this  subject  in  all  of  its  aspects.  The  methods  used 
allow  direct  evaluation  of  antimalarial  drugs  as  illustrated  by  the  tests  with 
Quinocide,  and  provide  a  means  for  studying  drug  action  histochemically.  Since 
the  exoerythrocytic  stages  are  those  responsible  for  relapse  and  must  be  elimi- 
nated for  radical  cure,  the  studies  have  significance  on  the  use  of  drugs  in 
malaria  eradication. 


55 


Serial  No.  NIAID-133-A 


Proposed  Course  of  Project:  The  project  was  somewhat  curtailed  due  to 
the  urgent  investigation  of  malaria  as  a  zoonosis.  During  the  coming  year 
it  is  expected  to  increase  the  emphasis  along  lines  proposed  in  the  previous 
year's  reportj  that  is,  studies  on  Plasmodium  inui  and  studies  on  mode  of 
action  using  metabolites  and  cytochemical  methods.   Studies  of  resistant 
strains  are  also  contemplated. 


Part  B.  included  -  No. 


56 


Serial  No.    NTATD-1  ^-b 

1.  Parasite  Chemotherapy 

2.  Section  on  Cytology 

3.  Memphis,  Tennessee 


PHS-NIH 

Individual  Project     Report 

Calendar  Year   1960 


Part  Aj. 

Project  title:  Studies  of  simian  malarias  in  man  to  determine 

if  malaria  isazoonotic  disease  in  areas  in  which 
man  and  monkey  are  closely  associated. 

Principal  investigators:  Don  E.  Eyles,  Charles  G.  Dobrovolny 

Other  investigators:  Clinton  S.  Smith,  local  Malayan  staff 

Cooperating  units:  Institute  for  Medical  Research 

Federation  of  Malaya,  Kuala  Lumpur 

U.  S.  Army  Medical  Research  Unit 
Kuala  Lumpur,  Federation  of  Malaya 

Man  Years  (Calendar  year  1960) 

Total  4 

Professional       1 
Other  3 

Project  Description: 

Objectives: 

To  determine  the  degree  to  which  established  strains  of  monkey 
malaria,  particularly  Plasmodium  cynomolqi.  will  grow  in  man.  To  isolate 
new  strains  of  monkey  malaria  for  comparison  with  established  strains 
and  for  study  in  man.  To  work  out  the  epidemiology  and  transmission  of 
monkey  malarias  in  the  field,  and  apply  knowledge  obtained  to  the  problem 
of  inter-relationship  between  human  and  monkey  malarias.  To  determine  if 
monkey  malarias  are  acquired  by  man  in  the  field  and  if  they  can  maintain 
themselves  in  man.  To  collect  and  correlate  all  information  on  distri- 
bution and  host  relationships  of  primate  malarias. 

Methods  employed: 

Strains  of  monkey  malaria  are  maintained  in  captive  monkeys  in  the 
laboratory.  Using  insectary  reared  mosquitoes,  these  strains  are 

57 


Serial  No.  NIAID-133-B 

passed  from  monkey  to  monkey  and  from  monkey  to  man.  Clinical  study  of 
the  malaria  produced  in  man  is  described  in  a  separate  project  at  the 
Atlanta  Federal  Penitentiary.  Strains  of  malaria  are  compared  before 
and  after  passage  through  man  and  attempts  are  made  to  transmit  the 
infections  man  to  man. 

A  field  party  is  operating  in  the  Federation  of  Malaya  cooperating 
with  the  Malayan  Institute  for  Medical  Research  and  the  U.  S.  Army 
Medical  Research  Unit.  A  systematic  study  is  being  made  of  the  malarias 
present  in  far  eastern  macaques  and  other  monkeys.   Isolation  of  strains 
is  being  attempted  from  selected  monkeys  from  a  variety  of  species  and 
from  several  geographical  areas.  Studies  are  being  made  of  the  vectors 
of  monkey  malaria  as  compared  with  the  vectors  of  human  malaria.  The 
prevalence  of  human  malaria  is  studied  in  aborigines  groups  closely 
associated  with  monkeys. 


Major  findings: 

It  was  found  that  laboratory  personnel  bitten  by  mosquitoes  infected 
with  Plasmodium  cvnomolqi  subspecies  bastianellii  will  become  infected 
and  clinically  ill  with  malaria.  This  finding  was  confirmed  immediately 
by  experimental  inoculation  of  prison  inmates.  Clinical  and  parasitological 
characteristics  of  the  infections  in  man  are  being  studied  (separate  project). 

It  was  found  that  the  malarias  could  be  passed  back  to  monkey  easily 
and  identified  by  cross-immuno logical  experiments.   It  was  found  possible 
to  infect  mosquitoes  on  human  infections,  and  one  instance  of  man  to  man 
passage  by  mosquitoes  has  been  seen,  the  malaria  being  then  reintroduced 
into  monkey  successfully.  Man  to  man  blood  passage  was  accomplished 
readily. 

In  the  field  notable  progress  has  been  made  in  delineating  the 
prevalence  of  malarias  of  various  types  in  monkeys.  A  number  of  strains 
have  been  isolated  or  are  in  the  process  of  being  isolated.  Other 
infected  animals  are  on  hand  for  future  isolation  attempts.  Many  new 
findings  on  host  distribution  and  on  geographic  distribution  of  monkey 
malarias  have  been  made.  Studies  of  malaria  in  aborigines  associated  with 
monkeys  have  been  made  in  Malaya  revealing  many  malaria  infections. 
Blood  passed  from  aborigines  to  monkeys  have  thus  far  produced  no  patent 
infections  in  the  monkeys. 

Studies  of  the  epidemiology  of  monkey  malarias  have  been  initiated 
and  the  feeding  habits  of  some  of  the  Anopheles  determined.  By  injection 
of  uninfected  monkeys  with  sporozoites  from  natural  infections  it  was 
determined  that  Anopheles  hackeri  is  a  natural  vector  of  Plasmodium  knowlesi. 


58 


Serial  No.  NIAID-133-B 

Significance  to  Bio-medical  Research  and  the  Program  of  the.  Institute; 

The  finding  that  Plasmodium  cynomolgj  subspecies  bastianellii  will 
grow  consistently  and  produce  clinical  illness  in  man  suggests  the 
possibility  that  malaria  is  a  zoonotic  disease;  that  is,  a  disease 
which  man  can  acquire  from  animals  with  which  he  is  associated.  Whether 
or  not  such  transfer  of  infection  occurs  in  nature  is  not  yet  determined, 
but  should  it  occur  it  would  be  of  greatest  significance  to  the  worldwide 
malaria  eradication  program. 

More  basically,  the  findings  of  the  study  taken  in  conjunction  with 
previous  findings  indicate  that  there  is  not  the  strict  specificity  of 
primate  malarias  previously  supposed  but  rather  that  the  specificity  is 
a  matter  of  degree  and  varies  within  the  species.  The  possibility  of 
variants  with  greater  infectivity  to  man  occuring  naturally  is  a  matter 
of  concern. 

The  full  significance  of  the  study  cannot  be  completely  evaluated 
until  study  has  been  carried  further,  but  the  potential  importance  makes 
it  essential  that  the  project  be  prosecuted  vigorously. 


Proposed  Course  of  Project; 

Isolation  of  strains  from  different  monkeys  and  from  different  geo- 
graphical areas  shall  be  continued,  with  the  view  of  studying  these  in 
man  and  comparing  them  immunologically  and  morphologically.  Work  in  Asia 
at  present  should  eventually  be  extended  to  Africa  and  the  new  world 
tropics. 

Studies  shall  be  continued  to  determine  if  transmission  from  monkey 
to  man  occurs  in  nature,  and  if  so  whether  or  not  man  is  a  "deadend"  host 
or  can  pass  the  malaria  on  man  to  man.  This  study  requires  further  study 
of  populations  associated  with  monkeys  and  the  study  of  the  epidemiology 
of  malaria  in  monkeys,  a  subject  which  has  not  been  at  all  well 
elucidated. 


Part  B  included  —  Yes. 


5S 


Serial   No.  NIATD-] 


PHS-NIH 
Individual  Project  Report 
Calendar     Year  1960 


Part  B.  Honors,   Awards,   and  Publications 


Publications  other  than  abstracts  from  this  project: 


Eyles,  D.  E.,  Coatney,  G.   R.   and  Getz,   M.  E.     Vivax-type  malaria 
parasite  of  macaques  transmissible  to  man.     Science,    131:1812- 
1813.      1960. 


Honors  and  awards   relating  to  this  project; 


None. 


60 


Serial  No.    NTATT)-m-n 

1.  Parasite  Chemotherapy 

2.  Section  on  Cytology 

3.  Memphis,  Tennessee 


PHS-NIH 

Individual  Project  Report 

Calendar  Year   1960 


Part  Aj. 


Project  title:  In  vitro  studies  of  malaria  parasites  and 
liver  tissue. 

Principal  investigator:  Robert  L.  Ingram 

Other  investigators:  John  R.  Jumper 

Cooperating  units:  None 

Man  years  (Calendar  Year  1960): 


Total: 

2-3/4 

Professional: 

1 

Other: 

1-3/4 

Project  Description: 

Objectives: 

To  develop  techniques  of  maintaining  adult  liver  tissue 
in  vitro  in  order  to  study  the  biology  of  the  exoerythrocytic  stages 
of  malarias,  to  test  antimalarial  drugs  and  antimetabolites  on  the 
course  of  the  infection  of  the  tissue  and  other  forms  of  the 
organism  and  to  investigate,  if  possible,  certain  liver  metabolic 
pathways  and  ways  in  which  the  parasite  may  interfere  with  normal 
metabolism  of  the  hepatic  cells.  Also,  to  study  the  degradation 
of  various  therapeutic  antimalarial  agents  by  the  liver,  and  the 
parasites  which  inhabit  the  hepatic  cells.  And  to  trace  the 
cycle  of  the  organism  in  the  host. 

Methods  employed : 

Routine  tissue  culture  methods  are  used,  but  the  mediums 
employed,  accessory  factors,  conditions  of  the  cultures,  methods  of 
preparation,  etc.,  are  devised  to  produce  the  desired  effects. 

61 


Serial  No.  NIAID-133-C 

These  include  considerations  of  the  composition  of  the  medium, 
accessory  factors  such  as  glycolytic  intermediates,  hormones  which 
stimulate  growth  or  otherwise  affect  metabolic  pathways,  gas 
requirements  for  the  proper  oxygen  tension,  buffering  capacity, 
etc.  The  effect  of  various  agents  against  sporozoites  of  the 
malaria  parasite  were  determined  by  bioassay  using  P_s_  gallinaceum 
and  baby  chicks.  Standard  bacteriological  procedures  were  used  to 
assess  the  effect  of  the  compounds  on  the  microflora  of  the 
mosquito. 

Histological  sections  were  made  and  material  inoculated  into 
experimental  animal  to  determine  the  results  of  the  studies. 
Staining  of  the  material  with  fluorescein  labelled  antibodies  and 
tagging  with  radioactive  isotopes,  using  the  autoradiographic 
techniques  for  detection  of  the  parasite  were  also  used. 

32      3       14 
P  ~~   and  H  -  and  C  -  labelled  thymidine  were  obtained  from 

commercial  sources.  At  first  approximately  5/Lic  of  the  H  and  C*4 

compounds  were  injected  into  the  body  cavity  of  infected  mosquitoes. 

This  produced  a  high  mortality  which  did  not  appear  to  be^due 

entirely  to  traumatization  of  the  insect.  Thereafter,  p   and 

tritrated  thymidine  was  added  to  the  food  of  the  mosquitoes.  The 

mosquitoes  were  examined  later  for  oocysts  and  in  some  experiments 

dissections  were  made  for  sporozoites.  After  14  days  the  mosquitoes 

were  offered  a  blood  meal  on  the  experimental  animal  and  the  glands 

or  whole  ground  mosquitoes  were  injected  into  the  animal.  Tissue 

sections  were  monitored  and  film  strip  radioautographs  were  made. 

Chemical  fraction  of  the  nucleic  acids,  nucleotides  and 
purine  and  pyrimidine  bases  were  carried  out  by  methods  of  Schmidt 
and  Thannhauser,  Schneider,  and  others.  Separation  and  identification 
of  the  nucleotides  and  bases  have  been  attempted  by  ionexchange  and 
paper  chromatography  and  spectrophotometry  analysis. 

Fetal  human  material  was  obtained  from  a  local  hospital. 


'1ajor  findings; 

Slight  improvements  have  been  made  for  maintaining  the  liver 
tissue  over  that  reported  last  year.  However,  many  additional 
factors  and  conditions  which  needed  to  be  tested  have  been  studied 
for  their  effect  on  liver  maintenance.  Also,  greater  success  has 
been  had  with  outgrowth  of  "hepatic"  cells  which  meet  a  single 
criterion  of  hepatic  cells,  e.g.  the  selective  uptake  of  neutral 
red  dye  as  reported  in  the  literature.  Sufficient  quantities 
of  material  have  not  been  available  to  do  further  studies,  espec- 
ially of  a  biochemical  nature,  to  characterize  the  cells. 

62 


Serial   No.    NIAID-133-C 

We  have  continued  to  grow  the  HE    stages  of  the  parasite 
whenever  these   are     available  in   sufficient  numbers  in  the    livers 
of  Pj;.  cvnomolgi  bastianellii   infected  monkeys.      Also,  we  have 
demonstrated  by  tissue   culture  that  the  parasite   is  in  the   liver 
four  days  after   sporozoite  inoculation.     This  represents  a   some- 
what closer  elucidation  of  the  cycle  of  the  parasite  in  the 
animal. 

Sulfapyrazine,   6-mercaptopurine,   and  pyrimethamine,   at   lOfj. 
M  per  ml  adversely  affected  the    in.  vitro   survival  of  organized 
liver  tissue,   especially  when  chick  plasma  was  used  as   substrate 
(in  a  tissue  culture   sense,  not  in  a  biochemical   sense)    for  the 
tissue   fragments.      Aminoptein  at  the    same   level   showed    little 
deleterious  effect.     Human  plasma  as  substrate  greatly  counter- 
acted the  inimical  effect  of  the  drugs  on  the   survival  of  the 
tissue. 

Penicillin  and    streptomycin  at  500-1000  units  and  micro- 
gram per  ml   respectively   showed  very  definite  parasiticidal 
activity.     Sulfa  drugs  such  as  sulfathiozole,  Gantricin,   and 
Elkosin  at  500  micrograms  per  ml  also   showed   some  parasiticidal 
activity,  but   less  than  that   shown  by  the  penicillin  and 
streptomycin  combination.     The  broad   spectrum  antibiotic,   Achromycin, 
had   less  effect  on  the   sporozoites  than  did  the  sulfa  drugs.     The 
addition  of  250  micrograms  of  "tycostatin,   a   fungicidal  drug,   to 
certain  of  the  other  drugs  did  not  increase  their  effect  on  the 
survival  of  the    sporozoites. 

Achromycin  in  combination  with  Dow  A,  a   fungicidal  compound, 
or  '.Tycostatin  eliminated  or  greatly   inhibited   the    growth  of 
most  of  the  microorganisms  associated  with  the  mosquito.     Penicillin 
and    streptomycin  and  the   sulfa  drugs  gave  only  partial   control  of 
the  micro  flora.     The  studies  indicate  that  in  attempts  to  infect 
organized  tissue,    cellular  outgrowths,   or  cell   lines,  with 
sporozoites,  penicillin  and   streptomycin,   the  commonly  used   anti- 
biotics to  combat   contamination     in  tissue   cultures,    should  not 
be  used   —  first,  because  they  will   adversely  affect  the   sporozoites 
and   secondly  they  are      ineffective  against  the  mosquito  flora. 

32 

The  mosquitoes  which  were  given  p   showed  fewer  oocysts  and 

in  some  cases  the  oocysts  at  14  days  was  the  size  of  a  normal  7 
day  old  oocyst.  To  date,  no  sporozoites  have  been  demonstrated  in 
these  mosquitoes  either  microscopically  or  by  the  mosquitoes  being 
able  to  infect  an  experimental  animal. 

3 
Mosquitoes  which  were  given  the  H  -  thymidine  showed  more  oocysts 

than  did  those  which  received  the  p^2  which  were  allowed  to  feed  upon 

the  same  infected  monkey.  About  90 per  cent  of  Anopheles  f reeborni 

63 


Serial  No.  NIAID-133-C 

3 

H  fed  mosquitoes  were  positive  for  oocysts;  many  had  over  100 
oocysts  on  the  gut.  They  also  showed  high  sporozoite  infections. 
Experimental  animals  inoculated  with  gliffls  from  these  mosquitoes 
became  infected.  However,  because  of  faulty  film  and  other 
technical  difficulties  radioautographs  of  the  parasite  has  not, 
as  yet,  been  demonstrated.  However,  we  have  promising  slides 
in  the  process  of  being  exposed. 

The  nucleic  acid  studies  are  in  the  formative  stages  with 
the  greatest  effort  at  present  being  directed  toward  separation  and 
identification  using  commercially  available  known  compounds. 


Significance  to  Bio-medical  Research  and  the  Program  of  the 
Institute: 

The  liver  has  such  a  varied  and  interesting  function  that  any 
possibility  of  maintaining  it  in  vitro  would  be  of  interest, 
especially,  to  biochemists;  also,  to  tissue  culture  workers.  The 
results  of  the  in  vitro  maintenance  of  the  liver  would  be  of 
interest  to. other  workers  who  are  making  studies  with  malaria  similar 
to  ours.  The  finding  should  be  of  interest  to  workers  who  are 
studying  infectious  hepatitis  and  other  liver  infections,  of  which 
there  are  a  large  number  of  parasitic  ones. 

The  importance  of  the  drugs,  tested  for  their  effect  on  the 
survival  of  organized  liver  tissue  in  vitro,  as  antimetabolites  and 
antimalarial  are  such  that  any  information  concerning  their  mode 
of  action  would  be  of  general  interest  to  biochemists,  pharma- 
cologists, 3nd  malariologists.   Also,  any  clues  to  substances  which 
might  counter  their  actions  such  as  competitive  substrates,  etc., 
would  be  of  general  interest  to  workers  studying  chemotherapeutic 
agents  and  to  biochemists.  Many  bio-medical  scientists  are  attempt- 
ing to  isolate  viruses  from  mosquitoes  and  other  Diptera  by  means 
of  tissue  culture.  This  is  especially  true  of  the  encephalitides, 
dengue  and  related  viruses.  The  findings  reported  here  concerning 
the  effect  of  antibiotics  against  the  microflora  of  mosquitoes 
should  be  useful  to  these  investigators.  The  results  should  be 
especially  useful  to  those  who  attempt  to  infect  tissue  culture 
material  with  sporozoites  or  who  attempt  embryonic  inoculations 
with  sporozoites  and  hope  to  eliminate  the  possible  concomitant 
bacterial  and  fungal  infections. 

If  we  are  successful  in  tracing  the  cycle  of  the  malaria 
parasite  in  the  host  by  the  radioisotope  technique  this  will  ad- 
vance our  knowledge  of  the  parasite  and  should  be  a  very  useful 
finding.   It  will  probalby  encourage  others  to  use  the  method 
in  similar  studies.  Biochemical  knowledge  related  to  malaria 
organisms  is  extremely  fragmentary  and  it  is  bdieved  that  our 
approach  to  the  subject  will  produce  fruitful  results.  Nucleic 


Serial  No.   NIAID-133-C 

acid   studies  are     among  the   forefront  of  bio-medical    studies 
at  the  present  time;   this  apparently  indicates    the    sugnificance 
with  which  biological  scientists  regard  these  compounds. 


Proposed   course  of  the  project: 

We   intend  to  make  use  of  the  results  reported  here   to  make 

a   number  of   studies  concerning  the  biology  of  the  EE  stages  of 

malaria  if  we  discover  ways  in  which  to  produce  high  infections  in 
the    liver. 

Work  will  be  extended  with  the  antimalarial  and  antimetabolite 
drugs.     We  especially  wish  to  screen  a     number  of  these  drugs  for 
their  antimalarial  potency  by     means  of  tissue   culture. 

The  tracer  work  will  be  oontinued  unless  it  is  found  that 
the  organism  will  not  incorporate   some  of  the  radioactive   label; 
this  seems  highly  unlikely.     We    shall  also   look  for  mutagenic 
effects  of  the     isotopes  on  the  parasite.     Hopefully  this  method 
can  be  used  to  determine  the  mode  or  site  of  action  of  some  of  the 
drugs  to  be  tested. 


Part  B.   included   —  No. 


G5 


Serial  No.  NTATn-133-n 

1.  Parasite  Chemotherapy 

2.  Section  on  Cytology 

3.  Memphis,  Tennessee 


PHS-NIH 
Individual  Project  Report 
Calendar  Year   1960 


Part  kj_ 

Project  title:  Immunological  studies  of  malaria  parasites. 

Principal  investigator:  Robert  L.  Ingram 

Other  investigators:  John  R.  Jumper 

Cooperating  units:  None. 

Man  years  (Calendar  Year  1960): 


Total 

1 

Professional 

1 

Other 

0 

Project  Description: 
Objectives: 

To  develop  ways  of  distinguishing  closely  related  malaria  organisms 
to  devise  means  of  detecting  the  organisms  in  the  tissue  phase  as  soon 
as  possible  after  infecting  the  experimental  animal  so  as  to  delineate 
the  cycle  of  the  organism,  and  basic  immunochemical  studies  concerning 
antigen-antibody  reactions  of  protozoa. 

Method s  employed: 

Antigenic  material  was  prepared  from  both  the  erythrocytic  and  the 
sporozoite  stages  of  the  malaria  parasites.  White  rabbits  were  given 
a  series  of  injections  of  these  preparations  and  after  a  suitable 
time,  serums  were  collected.  These  were  pooled,  the  gamma  globulin  was 
fractionated  and  conjugated  with  fluorescein  isothiocyanate  (F.I.C.). 
This  preparation  was  used  to  stain  the  parasites  which  were  then 
examined  using  fluorescent  microscopy  equipment. 

66 


Serial  No.  NIAID-133-D 
Major  findings; 

Blood  forms  of  Ejl  gallinaceum  were  stained  with  a  serum  prepared 
by  using  sporozoites  of  this  species  as  the  antigenic  material. 
P.  fallax  and  F_j_  lophurae  also  were  stained  with  this  labeled  serum. 
Blood  forms  of  P_t.  cvnomolqi  bastianellii  failed  to  stain  with  this 
preparation.  However,  F.I.C.  labeled  serum  from  experimentally 
infected  convalescent  monkeys  was  used  to  stain  P^  cvnomolqi  bastian- 
ellii. P.  gallinaceum,  P.  fallax  and  P^.  lophurae  failed  to  stain  with 
this  preparation. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute ; 

The  use  of  the  fluorescent  antibody  technique  has  in  the  past  few 
years  received  much  attention  by  bio-medical  investigators.  The 
reported  findings  that  malarias  can  be  studied  by  this  method  will 
find  utility  in  a  number  of  laboratories  where  malaria  is  being 
studied.  The  fact  that  one  stage  of  an  organism  was  used  to  stain  a 
different  stage  of  the  same  organism  should  facilitate  the  study  by 
this  technique  of  many  parasitic  diseases  in  which  the  organism 
undergoes  several  stages  of  development. 

Proposed  course  of  the  study ; 

As  other  species  of  malarias  become  available  to  us  and  experimental 
infection  with  these  become  possible,  we  shall  study  immunological  cross 
reactions  among  the  various  species.  We  also  intend  to  use  this  method 
to  help  trace  the  cycle  of  the  organism  through  the  host.  Attempts 
may  be  made  to  partially  purify  the  antigen  or  antigens  involved  in 
the  reaction. 


67 


Serial  No.    NIAID-133-D 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 

Part  B    Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Ingram,  Robert  L.,  Otken,  Luther  B.,  Jr.  and  Jumper,  John  R. 
Staining  of  malaria  parasites  by  the  fluorescent  antibody  technique. 
In  press.   Proc .  Soc.  Exp.  Biol,  and  Med. 


63 


LABORATORY  OF  IMMUNOLOGY 


Summary 1 

140  -  Studies  in  the  Transplantation  of 

Tissues 4 

141  -  Studies  on  Antibody  Production  and 

Mechanism  of  Hypersensitivity 7 

142  -  The  Immunology  of  Coh  Mouse  High 

Line  Sarcoma * 9 

143  -  Characterization  of  Substances  of 

Bacterial  Origin  Affecting  Resistance 

to  Infection 11 

144  -  Pathogenesis  of  Allergic  Encephalo- 

myelitis  14 

145  -  Studies  of  Auto-Antibodies  in  Human 

Disease  States  and  in  Experimental 
Animals 19 

146  -  The  Resistance  of  Fetal  and  Neonatal 

Gonads  to  Damage  by  Maternal 
Immunization  to  Testicular  Antigens. ...  23 

147  -  Immunochemical  and  Immunogenetic 

Studies  of  the  Protein  Isoantigens  in 

Serum 25 

147-A  -  Immunochemical  Studies  on  Human  Serum 

Antigens  and  Antibodies 32 

148  -  Basic  Studies  on  the  Cellular 

Localization  of  Antigens,  Antibodies 
and  other  Substances  by  the  Fluorescent 
Antibody  Technique  and  Fluorescence 

Microscopy 34 

150   -  The  Relationship  of  Delayed  and 

Immediate  Hypersensitivity  to  Allergic 
Thyroiditis  in  Inbred  Guinea  Pigs 39 

152  -  The  Chemistry  of  Antibodies 42 

153  -  Basic  Studies  on  the  Chemical, 

Physical  and  Skin  Reactive  Properties 

of  Extracts  of  House  Dust 47 


LABORATORY  OF  IMMUNOLOGY 
SUMMARY  STATEMENT 
CALENDAR  YEAR  i960 


During  the  past  year  there  has  been  a  tremendous  resurgent  interest  in 
the  field  of  immunology.  This  has  been  brought  forcefully  to  our  attention 
by  a  variety  of  events  which  occurred  during  the  Fall  of  i960.  A  very  high 
percentage  of  the  many  Research  Associate  applicants,  which  were  interviewed 
over  a  period  of  several  weeks,  chose  the  Laboratory  of  Bnmunology  as  being 
the  one  in  which  they  desired  to  conduct  research  for  a  two  year  period. 
The  interest  of  these  physicians  in  immunology  stems  from  the  possibilities 
that  they  envisage  in  the  application  of  immunology  and  its  newer  techniques 
to  the  study  of  human  disease.  Many  of  these  exceptionally  bright  young  men 
have  been  stimulated  by  their  professors  at  medical  schools  such  as  Harvard, 
N.Y.U.,  etc.  During  the  course  of  their  medical  studies  they  have  had  the 
opportunity  to  carry  on  immunological  investigations,  which  have  made  them 
realize  the  importance  of  immunology  as  a  fruitful  area  of  research. 

For  several  years  the  Laboratory  of  Immunology  has  conducted  a  bi- 
weekly immunology  seminar.   In  the  past,  this  was  attended  by  the  members  of 
our  staff  and  a  few  investigators  from  several  other  Institutes.   In  view  of 
the  ever-increasing  interest  in  immunology,  and  in  anticipation  of  this,  the 
Laboratory  scheduled  a  weekly  seminar  which  is,  at  the  time  of  this  writing, 
so  well  attended  that  it  has  been  necessary  to  abandon  the  NIAID  conference 
room  and  to  hold  these  lectures  and  discussions  in  the  Clinical  Center 
Auditorium  (l^t-th  Floor).  The  investigators  attending  these  seminars  are  from 
all  of  the  Institutes  and  represent  a  variety  of  disciplines.   It  is  evident 
that  these  scientists  have  found  the  need  for  incorporating  immunology  in 
their  studies  in  order  that  they  may  attain  their  research  goals.  This  is 
another  example  of  the  resurgence  of  immunological  research  at  NIH  and  points 
out  the  necessity  for  the  development  of  a  truly  strong  and  outstanding 
laboratory.  At  the  present  time  we  possess' a  small  nucleus  of  competent 
immunologists  and  from  this  nucleus  it  will  be  possible  to  develop  a  labora- 
tory which  potentially  can  be  a  national  and  international  leader  in  the 
field. 

During  the  year  the  demands  on  the  scientific  staff  of  the  Laboratory 
for  consultation  and  collaboration  have  been  increasing.  Many  of  the  demands 
on  their  time  center  around  the  performance  of  the  newer  techniques  and 
methods  of  immunological  research,  such  as  immuno-electrophoresis,  fluorescent 
antibody,  passive  cutaneous  anaphylaxis,  immunization  with  adjuvants,  etc. 
This  almost  daily  demand  has  been  of  a  proportion  as  to  interfere  at  certain 
times  with  the  scientists'  own  research  program. 

Since  the  activation  of  the  Laboratory  of  Dmnunology  in  1957*  the  pro- 
gram has  been  concerned,  principally,  with  basic  research.  However,  for  some 
time  an  important  need  has  been  felt  for  the  initiation  of  clinical  studies  in 
immunology  and  allergy.   In  September  i960  the  Clinical  Immunology  Section 
was  activated  with  the  appointment  of  Dr.  Howard  c.  Goodman  as  Head  of  this 


Section.  For  some  years  he  has  been  engaged  in  clinical  studies  and  more 
recently  has  been  concerned  with  the  importance  of  auto-antibodies  in  the 
■j  vthogenesis  of  certain  disease  states.  Dr.  Goodman  spent  the  year  preced- 
ing his  appointment  at  the  Pasteur  Institute  where  he  was  engaged  in  studies 
on  serum  proteins  involving  the  use  of  the  technique  of  Immunoelectrophoresis. 
As  space  permits,  the  Section  will  be  expanded  with  the  addition  of  two  investi- 
gators at  the  doctoral  level.  Dr.  Goodman's  section  will  be  working  in  close 
collaboration  with  Dr.  Vernon  Knight,  Clinical  Director,  NIAID,  on  clinical 
studies  involving  the  immunological  aspects  of  such  diseases  as  lupus  erythem- 
atosus, nephritis,  chronic  thyroiditis,  and  others  in  which  an  auto-immune 
basis  is  suspected.  In  order  that  clinical  studies  are  executed  properly,  it 
has  become  apparent  that  the  clinician  must  have  intimate  contact  with  the 
basic  scientist  and  the  laboratory  methods  involved  in  immunological  research. 
The  newly  activated  Clinical  Immunology  Section,  being  an  integral  part  of 
our  Laboratory,  affords  us  a  unique  opportunity  to  accomplish  first-rate  and 
complete  clinical  studies. 

Due  to  lack  of  space  since  the  formation  of  the  Laboratory,  it  was 
necessary  to  place  several  of  our  permanent  professional  staff  on  contract 
with  universities.  Two  of  these  scientists  resigned  during  the  year  and 
accepted  positions  at  institutions  of  medical  research.  The  exact  reasons 
for  their  leaving  is  not  completely  understood  but,  in  part,  it  was  due  to 
better  salary  opportunities  and  our  present  lack  of  facilities.  The  loss  of 
these  established  scientists  leaves  a  gap  in  certain  areas  of  our  research 
program.  Another  scientist  working  on  tissue  transplantation  resigned  during 
the  year  to  enter  training  in  surgery.   It  seems  imperative  that  this  person 
e  replaced  in  the  near  future  in  order  that  this  important  segment  of  immu- 
njlogical  research  be  represented  in  our  Laboratory. 

The  activities  of  the  staff  have  resulted  in  certain  significant  scien- 
tific advances* 

Aller^J-C  Thyroiditis 

Experimental  allergic  thyroiditis  was  produced  in  Strain  13,  inbred, 
histocompatible  guinea  pigs  by  immunization  with  a  single  dose  of  guinea  pig 
thyroid  extract  in  complete  Freund's  adjuvant.  Thyroiditis  developed  as  early 
as  5  days  after  immunization,  was  present  in  all  animals  at  16  days,  and  by 
7  weeks  was  consistently  present  and  generally  severe.  Delayed  skin  test 
hypersensitivity  was  found  as  early  as  5  days  after  immunization  in  nearly  all 
animals,  and  was  present  in  all  animals  with  thyroiditis  at  7  weeks.  Circu- 
lating anti-thyroid  antibody  was  absent  at  5  days  in  animals  with  thyroiditis 
and  with  delayed  hypersensitivity.  At  7  weeks  after  immunization,  anti-thyroid 
ntibodies  were  present,  and  antibody  titres  correlated  with  the  presence  and 
.egree  of  thyroiditis.  This  correlation  was  not  found  at  certain  other  times 
after  immunization.  To  intensify  the  formation  of  antibody  without  producing 
delayed  hypersensitivity,  guinea  pigs  were  immunized  with  thyroid  extract  in 
Freund's  incomplete  adjuvant.  These  animals  at  7  weeks  showed  no  thyroiditis 
and  no  delayed  hypersensitivity,  although  they  did  develop  low  levels  of  circu- 
lating antibody.  The  presence  of  delayed  hypersensitivity  was  correlated  with 
experimental  allergic  thyroiditis,  while  the  presence  of  circulating  antibody 
did  not  correlate  with  thyroiditis.  These  observations  constitute  the  earliest 


production  of  experimental  allergic  thyroiditis  and  the  most  severe  disease 
at  the  time  intervals  studied. 

House  Dust  Allergens 

Studies  on  the  chemical  and  physical  properties  of  house  dust  extracts 
that  are  used  clinically  for  the  diagnosis  and  treatment  of  house  dust  allergy 
have  been  studied  to  identify  the  components  responsible  for  the  specific  skin 
reactions  produced  in  house  dust  sensitive  individuals.  It  has  been  found  that 
the  house  dust  extracts  consist  of  a  heterogeneous  mixture  of  acidic  poly- 
saccharides. The  heterogeneity  has  been  demonstrated  by  electrophoretic  and 
ultracentrifuge  sedimentation  analysis  and  also  by  the  multiplicity  of  cross 
reactions  obtained  with  antisera  to  the  various  pneumococcal  polysaccharides. 
The  chemical  composition  of  the  various  fractions  has  been  shown  to  be  roughly 
5-20$  polypeptide  and  80-95$  polysaccharide,  containing  about  equal  amounts 
of  uronic  acid  (probably  glucuronic  acid),  D-glucose,  D-galactose,  D-mannose 
with  lesser  amounts  of  L-rhamnose  and  L-arabinose.  Starch  block  electrophore- 
sis experiments  at  low  pH  and  in  the  presence  of  7M  urea  have  shown  that  the 
polypeptide  components  are  covalently  linked  to  the  polysaccharides.  A 
number  of  different  kinds  of  experiments  have  shown  that  the  colored  compo- 
nents may  be  separated  from  the  bulk  of  the  material  and  the  skin  test  data 
suggest  that  the  color  is  not  associated  with  skin  reactivity.  The  skin  test 
data  also  indicate  that  more  than  one  allergen  specificity  is  present  in  the 
dust  extracts.  By  starch  block  electrophoresis  it  has  been  shown  that  certain 
isolated  fractions  have  greater  skin  reactivity  than  the  original  extract; 
however,  it  should  be  appreciated  that  the  skin  test  assay  is  exceedingly 
inaccurate  so  it  is  not  possible  to  determine  the  degree  of  concentration  of 
activity. 

Immuno chemistry  and  Genetics  of  Gamma  Globulin 

Agar-gel  immunochemical  analysis  of  sera  from  rabbit  litters,  with 
precipitating  antibodies  prepared  in  rabbits,  has  shown  that  seven  antigenic 
determinants  of  the  gamma  globulins  are  genetically  controlled  by  at  least 
two  distinct  gene  loci  with  each  specificity  exhibited  when  the  appropriate 
allele  is  present.   Since  the  gamma  globulins  are  soluble  proteins  which  have 
properties  of  both  an  antigen  and  an  antibody,  they  should  be  subject  to 
quantitative  estimation  and  cytological  localization.  This  immunogenetic 
system,  therefore,  may  be  uniquely  suited  for  the  study  of  certain  basic 
problems  in  genetics,  embryology,  immunology  and  protein  chemistry.   Since 
gamma  globulin  should  be  expected  to  pass  through  maternal- fetal  barriers, 
of  considerable  interest  would  be  the  study  of  gamma  globulin  synthesis  in 
fetal  and  neonatal  rabbits  uncomplicated  by  the  problem  of  distinguishing 
maternally  derived  gamma  globulin.  In  other  studies,  antibodies  to  human 
serum  proteins  were  prepared  in  monkeys  since  this  animal,  being  a  closely  re- 
lated species,  might  be  more  discriminating  for  minor  antigenic  differences 
than  a  distantly  related  species.  Three  "slow"  gamma  globulins  were  found, 
instead  of  the  one  usually  detected  with  horse  or  rabbit  antibodies.  Two  of 
these  were  shown  to  be  related  to  myeloma  proteins.  The  quantitative  estima- 
tion of  these  gamma  globulins  in  serum  should  be  helpful  in  the  early  diagnosis 
and  study  of  diseases,  such  as  multiple  myeloma,  which  involve  qualitative  and 


■^antitative  changes  in  the  gamma  globulins. 

M ;  hanisms  of  Hypersensitivity 

The  genetically  distinct  guinea  pigs  of  inbred  Strains  2  and  13  have 
proved  to  be  a  very  important  immunological  tool.  After  studies  established 
the  fact  of  skin  compatibility  in  the  tvo  strains,  experiments  were  conducted 
to  transfer  cells  with  a  measurable  biological  activity.  Transfers  of  tuber- 
culin sensitivity  were  undertaken  by  the  intraperitoneal  injection  of  living 
lymphoid  cells  from  compatible  donors.  The  almost  quantitative  transfers 
between  inbred  guinea  pigs  were  a  reflection  of  the  continued  viability  of 
the  active  cells  in  the  recipients.  It  was  found  that  transfers  could  be 
made  soon  after  active  sensitization  of  the  donor;  early  transfers  merely 
required  a  longer  waiting  period  for  full  sensitivity  to  appear  in  the  re- 
cipient. This  contrasted  with  the  need  to  test  as  soon  as  possible  after 
transfer  in  random-bred  animals  in  face  of  a  dwindling  population  of  trans- 
ferred lymphoid  cells  in  a  non- compatible  host.  Two  models  are  being  develop- 
ed to  study  the  mechanisms  of  immediate  and  delayed  hypersensitivity  in  the 
inbred  guinea  pigs;  protracted  anaphylactic  shock  and,  the  massive  local 
hemorrhagic  reaction,  respectively.  It  has  been  shown  that  there  are  differ- 
ences in  susceptibility  to  hypersensitivity  reactions.  Strain  2  guinea  pigs 
were  more  resistant  to  death  by  bronchospasm  and  tended  toward  a  protracted 
syndrome  in  anaphylactic  shock.  Both  Strain  2  and  13  guinea  pigs  required 
more  mycobacteria  than  did  random-bred  Hartley  guinea  pigs  for  inducing  "de- 
layed" sensitivity  to  egg  albumin,  using  Freund's  adjuvant. 

..  aracterization  of  Human  Serum  Auto-antibodies 


Fractions  of  human  serum  separated  by  anion- exchange  cellulose  column 
chromatography  were  studied  by  Immunoelectrophoresis .  The  conditions  for 
elution  of  eighteen  immunologically  distinguishable  human  serum  proteins 
from  the  columns  were  determined.  Gamma  globulin  obtained  under  the  appro- 
priate conditions  by  this  method  was  found  to  be  pure;  rabbits  immunized  with 
this  fraction  made  antibodies  to  none  of  the  other  serum  proteins.  By  the 
use  of  anion- exchange  cellulose  columns,  it  has  been  found  possible  to  sep- 
arate the  7S  from  the  18-19S  antibody  activities  in  sera  of  patients  with 
thyroiditis  and  lupus  erythematosus.   Initial  results  indicate  that  the  addi- 
tion of  imraunoelectrophoretic  characterization  of  these  and  other  sera  will 
be  extremely  helpful  in  our  aim  of  characterizing  the  antibody  activities 
found  in  human  serum. 

Fluorescent  Antibody  Staining  of  Malaria  Parasites 

The  fluorescent  antibody  staining  of  the  human  malaria  parasite, 
.  asmodium  vivax,  has  been  recorded  for  the  first  time.  A  globulin  fraction 
o  convalescent  serum  from  a  patient  having  a  long-standing  infection  with  ■ 
P.  vivax  was  labeled  and  the  fluorescent  antibody  applied  to  thin  blood  films 
containing  the  parasite.  The  organism  was  visible  by  virtue  of  its  specific 
immuno-fluorescence.   If  blood  films  can  be  stained  in  such  a  manner  that, 
essentially,  only  the  parasites  are  visible,  this  could  provide  a  method 
whereby  numerous  blood  films  could  be  examined  in  a  single  day  by  means  of 
t.n  automatic  scanning  microscope.  Fluorescent  antibody  studies  were  conduct- 
ed on  P.  cynomolgi  bastianellii,  the  monkey  malaria  parasite  which,  recently, 


has  been  shown  to  be  transmissible  to  man.  Considerable  morphological  detail 
was  observed  at  fluorescence.  Preliminary  studies  on  the  serological  relation- 
ships, as  based  on  degrees  of  fluorescence,  indicate  that  P.  vivax  and  P. 
cynomolgi  bastianellii  parasites  may  have  common  antigens  and  that  the  two 
species  may  be  closely  related.  This  is  of  importance  in  the  program  of  the 
world-wide  eradication  of  malaria  which  has  become  more  complex  with  the 
possibility  that  monkeys  may  serve  as  reservoirs  for  human  infection. 


Submitted  by: 


c  .  ^Atf-^Q 


JohnT;.  Tobie,  Ph.D.,  Acting  Chief, 
Laboratory  of  Immunology,  NIAID 


Form  No.  ORP-2  Serial  No.  NIAID  -  lto 

Nov.  i960  1.  Laboratory  of  immunology 

PHS-NIH  2.  Natural  &  Acquired 

Individual  Project  Report  Resistance 

Calendar  Year  i960  3«  Bethesda,  Maryland 


Part  A: 


Project  Title;  Studies  in  the  Transplantation  of  Tissues 

Principal  Investigator:  Dr.  Sanford  H.  Stone 

Other  Investigators;  Dr.  Joseph  A.  Bauer,  Jr. 

Cooperating  Units;  None 

Man  Years:  Patient  Days;  None 

Total:  1.25 
Professional:  0.75 
Other:  0.5 

Project  Description;  Isologous  and  Homologous  Lymphoid  Cell  Trans- 
plants 

Objectives: 

To  compare  the  relative  efficiency  of  passive  transfer  of  tuberculin 
sensitivity  and  antibody  producing  activity  between  histocompatlble 
guinea  pigs  with  that  between  random-bred  animals. 

Methods  Employed; 

Inbred  guinea  pigs  are  sensitized  with  egg  albumin  and  sheep  erythro- 
cytes in  Freund's  complete  adjuvant  (containing  mycobacteria).  Lym- 
phoid cell  transfers  are  made  by  intraperitoneal  route  into  inbred  and 
random-bred  recipients.  Cell  recipients  are  tested  for  skin- reactivity 
and  circulating  antibody. 

Major  Findings; 

1.  The  passive  transfer  of  delayed  hypersensitivity  and  antibody  syn- 
thesizing capacity  appears  to  be  more  effective  between  inbred 
guinea  pigs  than  between  random-bred  animals. 

2.  Soni cation  or  freezing  and  thawing  of  lymphoid  cells  obliterates 
their  capacity  for  successful  transfer. 

Significance  to  the  Program  of  the  Institute; 

Passive  transfer  with  viable  lymp^  i  cells  represents  an  important 


Serial  No.  NIAID  -  lto 

tool  in  the  study  of  immune  phenomena.  In  histocompatible  animals,  the 
variable  of  transplantation  immunity  is  eliminated,  the  transferred 
cells  thrive,  and  go  on  to  immune  maturity  in  the  recipient  (if  the 
lymphoid  cells  are  harvested  soon  after  sensitization  of  the  donors, 
a  longer  waiting  period  will  intervene  before  full  sensitivity  is 
manifested  in  the  recipient). 

Proposed  Course  of  Project: 

F,  generation  guinea  pigs  of  the  inbred  Strain  13  -  inbred  Strain  2 
crosses  will  be  used  to  study  problems  of  host  vs.  graft  and  graft  vs. 
host  reactions  during  transfers  of  lymphoid  cells. 


Part  B  included:  Yes   X      Ho 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


(Attachment  I) 
Serial  No.  NIAID  -  lliO 


Part  B:  Honors,  Awards,  and  Publications 

Publications: 

Bauer,  J.  A.,  Jr.  and  Stone,  s.  H. :  Isologous  and  Homologous 
lymphoid  Transplants  I.  The  Transfer  of  Tuberculin  Hypersensitivity 
in  Inbred  Guinea  Pigs.  J.  Immunol.  85:  (in  press),  i960. 


Form  No.  ORP-2  Serial  No.  NIAID  -  1^1 
Nov.  i960              PHS-NIH  1.  Laboratory  of  Immuno logy- 
Individual  Project  Report  2.  Natural  and  Acquired 
Calendar  Year  i960  Resistance 


3.  Bethesda,  Maryland 


Part  A: 


Project  Title;  Studies  on  Antibody  Production  and  Mechanism  of  Hyper- 
sensitivity 

Principal  Investigator;  Dr.  Sanford  H.  Stone 

Other  Investigators;  None 

Cooperating  Units;  None 

Man  Years;  Patient  Days;  None 

Total;  1.0 
Professional;  0.5 
Other;  0.5 

Project  Description; 

Objectives; 

To  study  the  mechanisms  of  "Immediate"  and  "delayed"  hypersensitiv- 
ity. 

Methods  Bnployed; 

Inbred  histocompatible  guinea  pigs  permit  experimentation  on  genetic 
factors  involved  in  hypersensitivity.  The  experimental  models:  random- 
bred  or  inbred  guinea  pigs  injected  with  antigen  in  complete  (for  "de- 
layed" hypersensitivity  studies)  or  incomplete  (for  "immediate"  hyper- 
sensitivity studies)  Freund's  adjuvants,  and  challenged  by  the  subcu- 
taneous route  with  homologous  antigen.  Methods  of  quantitation  of  the 
manifestations  of  "immediate"  (survival  time  in  protracted  anaphylaxis) 
and  "delayed"  (amount  of  mycobacterium  necessary  to  sensitize)  become 
feasible.  The  local  massive  hemorrhagic  reaction  described  in  previous 
reports  is  mot  versatile  for  manifestation  of  "delayed"  hypersensitivity. 

Major  Findings; 

If  quantitative  methods  are  to  be  applied  to  the  field  of  hyper- 
sensitivity research,  it  becomes  important  to  know  if  all  guinea  pigs 
react  similarly  to  the  conditions  set  up  in  an  assay.  It  was  found 
that  the  NIH  inbred  strains  2  and  13  differ  from  the  random-bred  Hartley 
guinea  pigs  in  several  significant  areas  concerned  with  hypersensitivity. 
Strain  2  guinea  pigs  are  relatively  resistant  to  the  lethal  bronchospasa 

7 


Serial  Wo.  NIAID  -  lH 

of  anaphylactic  shock,  and  tend  toward  the  protracted  type  of  syndrome 
involving  the  gastrointestinal  system.  Both  Strains  2  and  13  are  more 
difficult  to  sensitize  for  "delayed"  type  reactivity  (local  hemorrhagic 
reaction ),  requiring  from  10  to  50  times  as  much  mycobacteria  in  the 
Freund  adjuvant  emulsion.  Questions  of  genetic  variance  in  such  areas 
as  mast  cell  concentration,  affinity  of  antibody  for  tissue,  competence 
of  mononuclear  cells  to  sensitize  or  of  the  skin  to  react,  etc.,  come  to 
the  fore. 

Significance  to  the  Program  of  the  Institute: 

The  genetic  influences  on  hypersensitivity  and  its  manifestations 
have  been  relatively  unstudied.  The  significance  of  the  availability  of 
the  N.I.H.  inbred  strains  of  guinea  pigs  is  best  stated  by  quoting  from 
Dr.  Freund' s  summary  statement  of  1959:  "The  most  important  of  these 
(new  methods  involves)  the  introduction  of  genetically  distinct,  highly 
inbred  guinea  pigs  into  Immunologic  research  work.  The  initiation  of  such 
studies  is  of  particular  importance  in  the  field  of  delayed  type  of  hyper- 
sensitivity. The  guinea  pig  is  unique  in  its  suitability. . .high  degrees 
of  local  and  systemic  reactions  can  be  produced." 

Proposed  Course  of  Project: 

The  mechanisms  of  protracted  anaphylaxis  and  the  local  hemorrhagic 
reaction  will  be  investigated  using  random-bred  and  inbred  guinea  pigs. 


Part  B  included:  Yes         No 


Form  No.  ORP-2  Serial  No.  NIAID  -  1^+2 

Nov.  i960                PHS-NIH  1.  Laboratory  of  Immunology 

Individual  Project  Report  2.  Applied  Immunology 

Calendar  Year  i960  3.  Paris,  France 


Part  A: 


Project  Title:  The  Immunology  of  Cob.  Mouse  High  Line  Sarcoma 

Principal  Investigator;  Dr.  Philip  R.  B.  McMaster 

Other  Investigators;  None 

Cooperating  Units; 

Dr.  Pierre  Grabar,  Laboratorie  de  Chimle  Microbienne,  mstitut 
Pasteur,  Paris,  France 

Dr.  Georges  Barsky,  The  Hospice  Paul  Brousse,  Paris,  France 

Man  Years;  Patient  Days;  None 

Total;  O.J* 
Professional;  0.2 
Other:  0.2 

Project  Description: 

Objectives: 

To  attempt  to  find  an  antigen  in  the  C3I1  mouse  high  line  sarcoma, 
developed  by  Earle  and  others  at  the  NIH,  which  is  not  present  in  the 
normal  mouse  tissue,  as  well  as  to  determine  the  most  effective  method 
of  immunization  to  prevent  the  growth  of  subsequently  inoculated  tumor. 

Methods  Employed: 

To  compare  by  immunoelectrophoretic  and  other  methods,  the  nature 
and  number  of  antigens  in  normal  mouse  tissue  and  the  above  named 
sarcoma,  as  well  as  to  try  various  methods  of  immunization  to  determine 
which  is  the  more  effective  in  the  prevention  of  the  growth  of  sub- 
sequently injected  tumor. 

Major  Findings: 

Study  in  its  initial  stages.  No  significant  findings  to  date. 

Significance  to  the  Program  of  the  Institute: 

The  continued  search  for  antigens  in  tumors  not  present  in  normal 
tissue  may  yield  results  which  would  seem  to  offer  diagnostic  and 


Serial  HO.  NIAID  -  lk2 
therapeutic  possibilities. 
Proposed  Course  of  Project; 

To  be  continued  along  the  lines  outlined  above. 


Part  B  included  :  Yes       Ho  X 


10 


Form  No.  ORP-2  Serial  No.  NIAID  -  1^3 

Nov.  i960                PHS-NIH  1*  laboratory  of  Immunology 

Individual  Project  Report  2.  Iramunochemistry  Section 

Calendar  Year  i960  3«  New  York  City,  New  York 


Part  A: 


Project  Title;  Characterization  of  Substances  of  Bacterial  Origin 
Affecting  Resistance  to  Infection 

Principal  Investigator:  Dr.  Curtis  A.  Williams,  Jr. 

Other  Investigators;  None 

Cooperating  Units;  Dr.  Rene  J.  Dubos,  Rockefeller  Institute,  N.Y.,  N.Y. 

Man  Years;  Patient  Days;  None 

Total:  1.0 
Professional:  0.5 
Other:  0.5 

Project  Description; 

Objectives: 

1.  Further  examination  of  biologically  active  materials  extracted  from 
tubercle  bacilli ^  Their  isolation  and  composition. 

2.  To  distinguish  between  the  mechanisms  of  specific  and  non-specific 
acquired  resistance  to  infectious  diseases. 

Methods  Employed; 

1.  Animal  protection  tests  are  performed  in  albino  mice.  They  are 
studied  by  bacterial  enumeration  from  infected  organs  and  by  survival 
time* 

2.  Biologically  active  substances  from  tubercle  bacilli  are  compared 
with  bacterial  endotoxins  from  gram  negative  organisms,  which  have 
similar  chemical  composition  and  most  of  the  same  activities. 

3.  Clearance  of  microorganisms  from  the  blood  stream  of  mice. 

k.     Chromatography,  zone  electrophoresis,  ultracentrifugation,  standard 
immunochemical  tests. 

Major  Findings 

1.  There  are  many  variables  to  be  carefully  controlled  in  the  prepara- 
tion of  active  substances  from  tubercle  bacilli.  Solvent  ratios, 

11 


Serial  No.  NIAID  -  1*»3 

water  content  of  organic  solvents,  and  temperature  of  Importance  In 
terms  of  recoverable  activity  in  products. 

2.  Strains  of  mice  differ  in  their  response  to  these  products.  C57/6 
mice  and  Rockefeller  Swiss  behaved  roughly  as  predicted  from  their 
respective  responses  to  BCG. 

3.  Studies  with  Serratia  plymuthica  have  demonstrated  that  the  use  of  so- 
called  "non-pathogens"  to  infectious  disease  may  be  very  fruitful.  The 
usual  serological  reactions  and  Immunological  responses  are  operative. 
The  cause  of  death  is  apparently  toxemia.  Survival  2b  hours  usually 
leads  to  complete  recovery  and  a  high  level  of  immune  resistance. 

k.   The  blood  clearance  mechanism  has  been  examined  with  reference  to 
Immune  and  non-immune  resistance,  to  strain  of  mouse,  to  physiologic 
and  genetic  variants  among  bacteria.  It  has  become  clear  that  bacterial 
variation  plays  a  more  significant  role  in  the  function  of  this  mecha<- 
nism  in  resistance  than  many  host  treatments  or  the  strain  of  host. 

Significance  to  the  Program  of  the  Institute; 

Relating  recognized  resistance  mechanisms  in  experimental  animals  to 
the  outcome  of  infectious  diseases  is  of  obvious  importance.  The  study 
of  bacterial  variation  with  respect  to  these  mechanisms,  however,  could 
lead  to  an  eventual  understanding  of  the  terms  pathogenicity,  virulence, 
susceptibility,  and  resistance. 

Proposed  Course  of  Project; 

Terminated  as  NIAID  Project  June  31,  i960,  with  the  resignation  of 
principle  investigator. 


Part  B  included:  Yes  X       No 


12 


(Attachment  i) 

Serial  No.  NIAID  -  1^3 
PHS-NIH 
Individual  Project  Report 
Calendar  year  i960 

Part  B:  Honors,  Awards,  and  Publications 

Publications: 

Williams,  jr.,  Curtis  A.:  Immunoelectrophoresis.  Scientific 
American.  130:lU0,  i960. 


13 


Form  No.  ORP-2  Serial  No.  NIAID  -  ikk 

Nov.  i960                PHS-NIH  1.  Laboratory  of  Immunology 

Individual  Project  Report  2.  Clinical  Immunology 

Calendar  Year  i960  3-  Bethesda,  Maryland 


Part  A: 


Project  Title;  Pathogenesis  of  Allergic  Encephalomyelitis 

Principal  Investigator;  Dr.  Philip  Y.  Paterson 

Other  Investigators; 

Miss  Jennifer  Bell  and  Mr.  S.  Martin  Harwin,  medical  students  at 
N.Y-U.  School  of  Medicine  and  Mr.  N.  C.  Didakow,  laboratory 
technician. 

Cooperating  Units;  None 

Man  Years;  Patient  Years;  None 

Total;  2 

Professional;  0.5 
Other;  1.5 

Project  Description; 

The  allergic  encephalomyelitis  (AE)  induced  in  rats  and  other 
animals  by  injection  of  normal  nervous  tissue  combined  with  Freund 
type  adjuvants  is  under  study  at  New  York  University  College  of 
Medicine  under  contract  with  the  National  Institute  of  Allergy  and 
Infectious  Diseases  (Contrac-;  Serial  #SA-^3«PH  3027). 

Project  Subtitle  I:  Transfer  of  Allergic  Encephalomyelitis  (AE)  by 
means  of  Lymph  Node  Cells 

Objectives; 

To  transfer  AE  from  diseased  donors  to  normal  recipients  using 
lymph  node  cells. 

Methods  Employed; 

Adult  donor  rats  are  sensitized  to  nervous  tissue  by  injection  of 
guinea  pig  spinal  cord  emulsified  in  Freund' s  complete  adjuvant  (oil, 
emulsifier  and  killed  mycobacteria) .  At  varying  times  thereafter, 
the  lymph  node  cells  (LNC)  from  these  donors  are  collected  and  used 
for  2  different  types  of  transfer: 

(a)  homologous  type  of  transfer — the  LNC  are  injected  intra- 
cerebrally  into  rats  of  a  genetically  different  strain. 


Serial  No.  NIAID  -  1^ 

(b)  isologous  type  of  transfer— the  LNC  are  injected  intra- 
venously into  rats  of  the  same  genetic  strain. 

Major  Findings; 

1.  Homologous  type  transfers  -  AE  lesions  may  be  identified  in 
approximately  10$  of  recipients  if  the  donor  cells  are  injected 
via  the  intracerebral  route.  Transfer  of  cells  by  other  routes 
(viz.,  intravenous,  intraperitoneal)  has  not  resulted  in  transfer. 

2.  Isologous  type  transfers  -  AE  lesions  may  only  rarely  (less  than 
5$)  be  found  in  recipients. 

Significance  to  the  Program  of  the  Institute; 

These  means  of  transferring  AE  by  LNC,  in  addition  to  the  technique 
described  in  Annual  Report  for  1959>  provides  further  support  for  the 
cellular  nature  of  the  disease  and  offers  a  means  for  ultimately 
defining  the  morphogenesis  of  the  AE  lesion  in  precise  terms.  The 
information  obtained  should  prove  helpful  in  transfer  studies  of 
analogous  auto-immune  disease  states,  e.g.,  thyroiditis,  aspermato- 
genesis. 

Proposed  Course  of  Project; 

Contract  terminated  July  1,  i960. 

Project  Subtitle  II;  Role  of  Freund's  adjuvant  components  in  induction 
of  Allergic  Encephalomyelitis  (AE). 

Objectives; 

To  define  which  component (s)  of  Freund's  adjuvant  is  required  for 
rapid  and  regular  induction  of  AE  in  rats  and  guinea  pigs. 

Methods  Employed; 

Groups  of  rats  and  guinea  pigs  are  given  a  single  intracutaneous 
injection  of  guinea  pig  or  rat  spinal  cord  alone  or  combined  with 
Freund's  adjuvant  components  in  varying  combinations.  Occurrence  of 
AE  is  determined  by  presence  or  absence  of  AE  lesions  in  brains  and 
spinal  cords  of  animals  about  3  weeks  post-sensitization. 

Major  Findings; 

AE  may  be  regularly  induced  in  the  rats  and  infrequently  induced  in 
the  guinea  pig  by  injection  of  nervous  tissue  combined  with  Freund's 
incomplete  adjuvant,  i.e.,  paraffin  oil,  emulsifying  agent  but  no 
killed  mycobacteria.  The  paraffin  oil  is  the  important  component  of 


2 


15 


Serial  No.  NIAID  -  1U4 

the  adjuvant  for  rapid  induction  of  the  disease  in  the  rat.   In 
contrast,  both  paraffin  oil  and  killed  mycobacteria  are  essential  for 
rapid  and  regular  induction  of  disease  in  guinea  pigs.  An  occasional 
rat  may  exhibit  lesions  of  AE  if  injected  with  nervous  tissue  alone; 
such  is  not  the  case  in  the  guinea  pig.  In  both  the  rat  and  the  guinea 
pig,  heterologous  nervous  tissue  is  more  active  as  sensitizing  antigen 
than  is  homologous  nervous  tissue. 

Significance  to  the  Program  of  the  Institute: 

In  addition  to  providing  a  less  complex  technique  for  induction  of 
the  disease  at  least  in  the  rat,  these  findings  provide  additional 
clues  to  understanding  the  mode  of  action  of  Freund  type  adjuvants  and 
role  of  cellular  and  humoral  factors  in  development  of  experimental 
auto-immune  disease.  This  work  underscores  the  long  appreciated  poten- 
tial hazard  of  using  any  vaccine  consisting  of  nervous  tissue  for 
immuni zation  of  man. 

Proposed  Course  of  Project: 

Contract  terminated  July  1,  i960. 

Project  Subtitle  III:  Antibodies  against  autologous  brain  in  rats  with 
Allergic  Encephalomyelitis  (AE) . 

Objectives: 

To  determine  whether  rats  sensitized  to  homologous  or  heterologous 
nervous  tissue-adjuvant  produce  antibodies  specifically  directed 
against  their  own  (autologous)  brain. 

Methods  Employed: 

Rats  are  sensitized  to  nervous  tissue  by  a  single  injection  of  rat 
or  guinea  pig  spinal  cord  in  adjuvant.  About  3  weeks  later  the  rats 
are  bled,  sacrificed  and  their  sera  tested  (by  complement  fixation)  for 
antibodies  using  both  autologous  and  homologous  brain  extracts  as  the 
antigens.  Appropriate  controls  are  included  in  every  test. 

Major  Findings: 

More  than  half  of  the  rats  tested  produce  antibodies  against  their 
own  (autologous)  nervous  tissue.  The  antibody  appears  organ-specific; 
it  does  not  react  with  kidney  tissue  and  does  not  appear  in  response 
to  sensitization  with  kidney-adjuvant. 

Significance  to  the  Program  of  the  Institute: 

These  results  add  weight  to  the  presumed  auto-immune  theory  of  AE 
and  analogous  forms  of  experimental  tissue  damage. 

18 
3 


Proposed  Course  of  project; 

Contract  terminated  July  1,  i960. 


Serial  No.  NIAID  -  Ikk 


Part  B  included:  Yea    X        No 


17 


(Attachment  I) 

Serial  No.  NIAID  -Ihk 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  1960 

Part  B:  Honors,  Awards,  and  Publications 

Publications ; 

Paterson,  P.  Y. :  Transfer  of  Allergic  Encephalomyelitis  in  Rats 
by  Means  of  Lymph  Node  Cells.  J.  Exper.  Med.  111:119,  i960. 

Bell,  J.,  and  Paterson,  P.  Y- :  Rapid  Induction  of  Allergic 
Encephalomyelitis  in  Rats  Without  the  Use  of  Mycobacteria. 
Science.  131:lWt8,  i960. 

Harwin,  S.M.,  Paterson,  P.  Y.,  and  Didakow,  N.C:  Antibodies 
Against  Autologous  Brain  in  Rats  With  Allergic  Encephalomyelitis. 
Nature  (in  press). 


18 


Form  No.  ORP-2  Serial  No.  NIAID  -  1^5 

Nov.  i960  PHS-NIH  1.  Laboratory  of  immunology 

Individual  Project  Report     2.  Clinical  Immunology 
Calendar  Year  i960       3.  Paris,  France  and 

Betheada,  Maryland 


Part  A: 


Project  Title;  Studies  of  Auto-Antibodies  in  Human  Disease  States 
and  in  Experimental  Animals. 

Principal  Investigator:  Dr.  Howard  C  Goodman 

Other  Investigators;  None 

Cooperating  Units; 

Dr.  John  L.  Fahey,  GM.,  NCI,  and  Dr.  Richard  A.  Malmgren,  PA., NCI. 

Dr.  James  H.  Baxter,  LC.,  NOT,. 

Dr.  Leonard  Laster,  A&R.,  NIAMD. 

Man  Years;  Patient  Days;  60 

Total:  2.0 
Professional:  1.0 
Other:  1.0 

Project  Description; 

Objectives; 

1.  To  detect  the  factors  in  human  sera  vhich  react  with  human  tissue 
antigens . 

2.  To  characterize  these  factors,  i.e.  show  whether  they  are  actually 
gamma-globulins  and  whether  the  reactions  with  tissue  antigens 
behave  like  typical  antigen-antibody  reactions. 

3.  To  attempt  to  produce  similar  auto-antibodies  in  experimental 
animals. 

k.     To  determine  whether  animals  with  experimentally  produced  auto- 
antibodies develop  tissue  damage. 

Methods  Employed; 

The  tannic  acid  hemagglutination  test  of  Boyden,  gel-diffusion 
antigen-antibody  precipitation  tests  (Ouchterlony  and  Oudln), 
Immunoelectrophoresis  (Grabar  and  Williams),  and  DEAE  cellulose 
column  chromatography  (Sober  and  Peterson)  techniques  are  used  to 

19 


Serial  No.  NIAJD  -  l*+5 

detect  and  characterize  the  serum  factors  which  react  with  tissue 
antigens . 

To  produce  auto-antibodies  in  animals  experimentally,  rabbits, 
guinea  pigs  and  rats  are  Immunized  with  tissue  extracts  incorporated 
in  Freund's  adjuvant. 

Patient  Material; 

Patients  with  lupus  erythematosus  and  idiopathic  nephrotic  syndrome 
are  admitted  to  the  clinical  center  for  these  studies.  Kidney  biopsies 
are  performed  and  the  clinical  response  to  steroid  administration  is 
studied;  lipid  metabolism  studies  on  patients  with  the  nephrotic  syn- 
drome have  been  performed  in  conjunction  with  Dr.  James  Baxter,  NEE. 

Major  Findings: 

During  the  past  year  at  the  Pasteur  Institute  with  Professor  Pierre 
Grabar,  the  technique  of  imaiunoelectrophoresis  was  used  to  analyze  the 
fractions  obtained  from  normal  human  serum  after  DEAE  cellulose  column 
chromatography.  The  information  gained  about  conditions  for  elution 
of  some  20  of  the  immunologically  distinguishable  serum  proteins  from 
the  cellulose  columns,  particularly  the  gamma,  beta-2  A,  and  beta-2  M 
(the  macrogammaglobulins),  will  be  immensely  useful  this  year  when  we 
attempt  to  see  into  which  of  these  classes  of  "immune»globulins"  auto- 
antibodies fall.  A  specific  example  of  what  will  be  a  general  usefulness 
of  Immunoelectrophoresis  in  characterization  of  serum  proteins  in  other 
diseases  (not  necessarily  caused  by  immune  reactions)  is  the  demonstra- 
tion with  Dr.  Leonard  Laster  (NIAMD)  and  Dr.  Donald  Frederickson  (NET) 
of  the  absence  of  beta  lipoproteins  from  the  serum  of  their  patient, 
thus  confirming  the  diagnosis  of  "a-betalipoproteinenda",  a  newly 
described  disease  syndrome. 

Attempts  to  detect  auto-antibodies  in  patients  with  nephritis  and 
nephrosis  and  to  produce  auto-antibodies  to  kidney  tissue  and  renal 
disease  in  animals  have  not  been  successful  so  far,  although  these 
studies  continue.  Auto-antibodies  are  detectable  in  patients  with 
lupus  erythematosus,  chronic  thyroiditis,  and  a  few  patients  with 
scleroderma  and  rheumatoid  arthritis.  In  patients  with  thyroiditis, 
the  serum  factors  which  react  with  thyroglobulin  have  been  shown  to  be 
gamma-globulins  and  to  belong  to  both  the  7S  (mol.  wt.  about  160,000) 
and  IBs  (mol.  wt.  about  1,000,000)  gamma-globulins.  In  the  sera  of 
patients  with  lupus  erythematosus,  auto-antibodies  reacting  with  com- 
ponents of  cell  nuclei  have  been  studied.  The  L.  E.  cell  factor  has 
been  shown  to  be  in  the  TS  gamma-globulins.  Some  of  the  other  anti- 
nucleus  factors  were  found  to  be  7S  gamma-globulins,  and  were  demon- 
strated for  the  first  time  to  be  among  the  l8s  gamma  globulins. 

Rabbits  injected  with  calf  thymus  and  human  liver  nucleoprotein 
extracts  have  been  shown  to  develop  antibodies  not  only  to  the  injected 

20 

2 


Serial  No.  NIAID  -  1^5 

nuclear  material,  but  also  auto-antibodies  to  their  ovn  cell  nuclei. 
So  far  no  associated  tissue  damage  has  been  detected. 

Significance  to  the  Program  of  the  Institute: 

Obese  studies,  as  veil  as  those  to  be  developed  in  our  ovn  Labora- 
tory of  Immunology  and  vith  the  cooperating  units  listed,  represent 
the  approach  of  the  section  on  clinical  immunology  to  the  study  of  the 
role  of  immune  mechanisms  in  human  disease  states.  The  question  of 
the  importance  of  auto-antibodies  in  the  pathogenesis  of  a  great  number 
of  diseases  deserves  critical  study.  The  finding  that  anti-nucleus 
auto-antibodies  in  lupus  erythematosus  can  be  macro-globulins  raises 
nev  questions  about  the  reasons  for  the  production  of  high  molecular 
weight  antibodies.  The  production  of  anti-cell  nucleus  auto-antibodies 
in  experimental  animals  should  enable  us  to  determine  whether  these 
antibodies  can  produce  a  disease  state  in  these  animals,  and  thus  help 
decide  whether  the  auto-antibodies  are  important  in  the  pathogenesis  of 
the  human  disease  states  where  they  are  found. 

Proposed  Course  of  Project; 

Attempts  to  detect  auto-antibodies  in  different  disease  states  vill 
continue  (e.g.  ulcerative  colitis  with  Dr.  Laster).   The  auto-anti- 
bodies detected  will  be  characterized  by  immunoelectrophoresis  and 
cellulose  column  chromatography.  A  logical  extension  of  our  studies 
on  serum  antibodies  in  disease  states  will  be  to  apply,  in  conjunction 
with  Dr.  Wilton  E.  Vannier,  the  technique  of  DEAE  cellulose  column 
chromatography  and  immunoelectrophoresis  to  the  characterization  of  re- 
agins  in  serum  of  patients  with  clinical  allergy. 

Animal  studies  on  the  production  of  and  characterization  of  auto- 
antibodies and  production  of  tissue  damage  will  continue,  with  emphasis 
on  the  antibodies  to  components  of  cell  nuclei  to  thyroid  tissue  and  to 
kidneys.  In  patients  and  in  experimental  animaiw  where  tissue  damage 
is  associated  with  auto-antibodies,  attempts  will  be  made  to  determine 
whether  tissue  damage  is  caused  by  hypersensitivity  of  the  delayed  type 
or  due  to  circulating  antibodies.  Cell  and  serum  transfer  experiments 
can  help  decide  this  question,  and  we  are  in  a  position  to  do  these 
studies  using  the  histocompatlble,  pure  strains  of  guinea  pigs  develop- 
ed at  NIH. 


Part  B  included:  Yes  X        No 


21 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


(Attachment  i) 
Serial  No.  NIAID  -  1^5 


Part  B:  Honors,  Awards,  and  Publications 

Publications; 

Baxter,  James  H.,  Goodman,  Howard  C.  and  Havel,  Richard  J.  Serum 
Lipid  and  Lipoprotein  Alterations  in  Nephrosis.  J.  Clin.  Inv. 
39:^55,  I960. 

Fahey,  John  L-,  Dutcher,  Tftomas  E.  and  Goodman,  Howard  C.  Cell 
Nuclei  and  Gamma  Macroglobulins .  Royal  Soc.  Med.  Vol.  53  No.  8,  i960 

Fahey,  John  L.  and  Goodman,  Howard  C.  Characterization  of  Anti- 
HSiyroglobulin  Factors  in  Human  Serum.  J.  Clin.  Inv.  39:1259,  i960. 

Goodman,  Howard  C.,  Fahey,  John  L.  and  Malmgren,  Richard  A.  Serum 
Factors  in  Lupus  Erythematosus  and  Other  Diseases  Reacting  With 
Cell  Nuclei  and  Nucleoprotein  Extracts:  Electrophoretic,  Ultra- 
centrifugal  and  Chromatographic  Studies.  J.  Clin.  Inv.  39:1595, 
i960. 


22 


Form  No.  ORP-2  Serial  No.  NIAID  -  1^6 

Nov.  i960                PHS-NIH  1.  Laboratory  of  Immunology 

Individual  Project  Report  2.  Applied  Immunology 

Calendar  Year  i960  3«  Bethesda,  Maryland 


Part  A: 


Project  Title:  The  Resistance  of  Fetal  and  Neonatal  Gonads  to 
Damage  by  Maternal  Immunization  to  Testicular 
Antigens . 

Principal  Investigator;  Dr.  Philip  R.  B.  McMaster 

Other  Investigators:  None 

Cooperating  Units:  None 

Man  Years:  Patient  Days:  None 

Total:  0.1* 

Professional:  0.2 
Other:  0.2 

Project  Description: 

Objectives: 

To  demonstrate  that  maternal  Immunization  to  testicular  antigens  Is 
innocuous  to  the  development  of  the  offspring  in  contrast  to  the  report- 
ed results  of  maternal  immunization  to  lense  and  brain,  and  thereby 
lend  support  to  the  hypothesis  that  delayed  hypersensitivity  is  a  re- 
quisite for  the  development  of  allergic  aspermatogenesis,  and  also  to 
provide  a  control  model  for  the  study  of  vhite  cell  transmission  across 
the  placenta. 

Methods  Employed: 

Adult  female  outbred  Hartley  strain  guinea  pigs  were  immunized  with 
testicular  extract  in  complete  adjuvant  in  two  groups,  one  shortly 
before  mating  and  the  other  shortly  after  mating.  These  animals  were 
later  tested  for  delayed  hypersensitivity  and  serum  antibodies  to 
testicular  extract,  whereas  the  offspring  were  examined  for  the  same 
serum  antibodies  as  well  as  for  histologic  and  physiologic  abnormalities 
of  the  reproductive  system. 

Major  Findings: 

All  the  actively  Immunized  animals  developed  circulating  antibody 
and  delayed  hypersensitivity  to  testicular  extract.  All  the  off- 
spring had  circulating  antibody  in  their  serum,  but  nevertheless  their 
reproductive  organs  were  normal  physiologically  and  histologically. 

23 


Serial  NO.  NIA3D  -  lM? 

Significance  to  the  Program  of  the  Institute; 

!Riis  study  extends  the  previous  work  of  Dr.  Freund,  who 
demonstrated  a  correlation  between  aspermatogenesis  and  delayed  hyper- 
sensitivity to  testicular  antigens,  and  a  lack  of  such  a  correlation 
between  the  disease  and  serum  antibody  in  adult  animals.  Ifcese 
experiments  on  the  development  of  offspring  of  immunized  females  show 
that  circulating  anti-testicular  antibody  does  not  cause  detectable 
damage  even  when  present  early  in  life  during  the  development  of 
barrier  membranes  and  organ  differentiation.  This  study  also  indicates 
a  lack  of  a  transfer  of  prolonged  nature  across  the  placental  membrane 
of  outbred  animals,  even  when  a  high  degree  of  delayed  hypersensitivity 
is  present  in  the  maternal  parent. 

Proposed  Course  of  Project: 

Hiis  study  may  serve  as  a  model  control  for  the  study  of  white  cell 
transfer  across  the  placenta,  by  comparing  the  above  with  a  similar 
study  in  inbred  animals,  in  which  transferred  white  cells  would  survive. 


Part  B  included  :  Yes        Ho 


2H 


Form  No.  ORP-2  Serial  No.  NIAID  -  lVf 
Nov.  I960              PHS-NIH  1«  Laboratory  of  Immunology 

Individual  Project  Report    2.  immunochemistry  Section 
Calendar  Year  i960       3«  Bethesda,  Maryland 


Part  A: 


Project  Title;  Immunochemical  and  Immunogenetic  Studies  of  the  Protein 
Isoantigens  in  Serum 

Principal  Investigator;  Dr.  Sheldon  Dray 

Other  Investigators;  Miss  Glendowlyn  0.  Young 

Cooperating  Unit; 

Project  Subtitle  I  -  Drs.  Charles  W.  Mcpherson  and  Donald  Bailey, 
Animal  Production  Section,  Serial  No. 

Project  Subtitle  V  -  Dr.  T.  N.  Harris,  Children's  Hospital  of 
Philadelphia,  Philadelphia,  Pennsylvania 

Man  Years;  Patient  Days;  None 

Total:  3.0 
Professional:  1.5 
Other:  1.5 

Project  Description; 

Project  Subtitle  I:  Genetic  Control  of  Serum  Isoantigens 

Objectives; 

To  establish  the  genetic  basis  for  the  presence  of  serum  isoantigens 
and  to  develop  lines  of  animals,  (rabbits)  with  genetically  defined  iso- 
antigens . 

Methods  Employed; 

1.  Qualitative  tests  using  primarily  agar-gel  methods  for  the  identifica- 
tion of  isoantigens  in  families  of  rabbits  and  in  various  rabbit 
populations . 

2.  Selective  breeding  of  rabbits  to  test  genetic  hypotheses  and  estab- 
lish genetically  defined  lines. 

Major  Findings; 

The  genetic  control  for  two  gamma-globulin  allotypes  have  been  estab- 
lished. A  genetic  theory  for  five  additional  gamma  globulin  allotypes  is 

25 


Serial  NO.  NIAID  -  3A7 

"being  tested  utilizing  the  serum  bank  of  rabbit  families  available  from 
earlier  studies. 

Significance  to  the  Program  of  the  Institute; 

To  know  the  genetic  mechanism  for  the  control  of  isoantigens  is 
essential  for  the  usefulness  of  these  isoantigens  in  studies  of  basic 
immunology  and  hypersensitivity.  The  molecules  having  antigenic  sites 
under  the  genetic  control  postulated  are  soluble  proteins,  namely,  gamma 
globulins.  Some  of  these  molecules  may  also  react  as  antibodies.  As 
antigens,  precipitable  by  antibodies,  they  should  be  subject  to  quanti- 
tative estimation.  Moreover,  these  molecules  may  be  expected  to  pass 
through  maternal- fetal  barriers.  Thus,  this  immunochemical  genetic 
system  may  be  uniquely  suited  for  studies  of  some  basic  problems  in 
Immunology,  hypersensitivity,  embryology,  and  protein  chemistry. 

Proposed  Course  of  Project: 

1.  To  establish  genetically  defined  lines  of  rabbit  gamma-globulin 
types  so  that  they  may  be  available  for  other  studies. 

2.  To  study  the  genetic  control  of  new  gamma  globulin  isoantigens  or 
other  isoantigens  as  they  are  discovered. 

Project  Subtitle  II;  Chemical  Studies  of  Serum  Isoantigens 

Objectives; 

1.  To  identify  the  antigens  in  serum  which  induce  isoantibodies. 

2.  To  study  the  chemical  nature  of  these  isoantigens. 
Methods  Employed; 

1.  Immunochemical  analysis  by  diffusion  in  agar-gel;  simple  diffusion 
in  plates,  and  immunoelectrophoresis . 

2.  Passive  cutaneous  anaphylaxis,  tanned-cell  hemagglutination  and 
other  Immunological  tests  for  antibody. 

3.  Cellulose  ion-exchange  chromatography  and  other  protein  fractiona- 
tion and  purification  methods. 

k.     Enzyme  (papain)  digestion  of  isoantigens  to  produce  smaller  frag- 
ments for  study. 

Major  Findings; 

Investigations  have  shown  that  components  of  sera  from  individual 
rabbits  are  antigenic  in  certain  other  rabbits  and  that  isoprecipitins 
can  be  produced  which  react  with  serum  antigens  having  electrophorettah 

2 


Serial  Ko.  NIAID  -  IkJ 

mobilities  corresponding  to  alpha-,  beta-,  and  gamma-globulins. 

Significance  to  the  Program  of  the  Institute; 

1*  Differences  in  serum  proteins  between  individuals  of  the  same  species 
may  be  of  genetic  and  clinical  significance  similar  to  that  of  the 
blood  groups.  The  serum  isoantigens  may  be  implicated  in  some  of  the 
unexplained  transfusion  reactions  in  man. 

2.  Serum  isoantigens  may  be  useful  in  the  study  of  "immune  tolerance" 
and  perhaps  serve  as  a  somewhat  analagous  experimental  model  for  the 
study  of  supposed  tolerance  to  Rh  antigen* 

3*  Serum  protein  isoantigens  may  be  particularly  suitable  for  study  of 
some  basic  problems  in  immunology  concerning  the  nature  of  antigen 
and  antibody  sites,  antigenicity  of  antibodies,  chemical  structure 
of  gamma-globulin,  the  antigen-antibody  reaction,  etc. 

Proposed  Course  of  Project; 

1.  Chemical  characterization  of  the  gamma-globulin  isoantigens  in 
rabbits.  Development  of  methods  of  assay  for  each  of  the  iso- 
antigens. 

2.  Studies  on  the  chemical  structure  of  gamma-globulin  by  papain  diges- 
tion studies  and  quantitative  assay  of  antigenic  sites. 

3*  Search  for  additional  gamma-globulin  isoantigens  and  additional 
alpha-  and  beta-globulin  isoantigens. 

k.  Specificity  of  rabbit  isoantigens  and  antigenicity  in  other  species. 

5.  Isoantigens  in  other  species,  particularly  mice  and  man., 

6.  isoantigens  In  other  tissues. 

7.  Antibody  production  in  rabbits  of  different  gamma-globulin  types. 

Project  Subtitle  III;  Application  of  serum  isoantlgen-isoantlbody 
systems  to  study  of  "Immune  Tolerance." 

Objectives; 

To  investigate  the  question  as  to  whether  the  isoantigens  would  be 
useful  in  the  study  of  so-called  "immune  tolerance." 


27 


Serial  No.  NIAID  -  1^7 

Methods  Employed; 

1.  Selective  breeding  of  rabbits  on  the  basis  of  known  genetics  of 
gamma-globulin  isoantigens* 

2.  Immunochemical  methods  to  evaluate  production  of  isoantibodies  and 
presence  of  isoantigens. 

2.     Quantitative  assay  of  isoantigens  by  agar-gel  methods  and  labeling 
with  tracers* 

Major  Findings; 

un 
Results  of  preliminary  experiments  suggest  that  "immunologic Respon- 
siveness" to  gamma-globulin  isoantigens  may  be  induced  in  rabbit  off- 
spring in  the  following  ways.  The  offspring  may  be  exposed  to  the 
"foreign"  isoantigen  naturally  during  pregnancy  as  a  result  of  the 
"foreign"  isoantigen  of  the  mother  passing  through  the  maternal- fetal 
barrier.  The  offspring  may  also  be  exposed  artificially  by  trans- 
fusion of  the  mother  with  the  "foreign"  isoantigen  during  the  last  week 
of  pregnancy. 

Significance  to  the  Program  of  the  Institute; 

"Immunologic  unresponsiveness"  has  been  one  of  the  most  interesting 
problems  in  hypersensitivity  during  the  last  few  years  as  a  result  of 
new  developments  in  our  knowledge  of  tissue  grafting.  Should  the  iso- 
antigen- isoantibody  system  be  effective  in  the  induction  of  "tolerance," 
this  system  may  very  well  be  most  suited  for  a  study  of  some  of  the  basic 
mechanisms  involved  in  "tolerance."  The  isoantigen-antibody  system  has 
the  advantage  that  one  may  work  with  soluble  proteins  (rather  than  tissue) 
and  with  the  precipitating  antibodies  (rather  than  tissue  rejection) . 

Proposed  Course  of  Project; 

Discontinued  at  present  because  of  lack  of  sufficient  animals  and 
facilities. 

Project  Subtitle  IV;  Synthesis  of  Gamma-Globulins  in  The  Fetus  and 
Newborn 

Objectives; 

To  determine  when  and  where  gamma-globulins  are  first  produced  by  the 
fetal  or  neonatal  rabbit  and  the  rate  at  which  the  gamma-globulin 
synthesized  by  the  offspring  appears  in  the  serum. 

Methods  Employed; 

1.  Selective  breeding  of  rabbits  on  the  basis  of  the  known  genetics  of 

28 


Serial  Ho.  NIAID  -  1^7 

gamma-globulin  antigens. 

2.  Immunochemical  methods  to  assay  quantity  of  gamma-globulin  iso- 
antigen  appearing  in  the  serum. 

3*  Fluorescent  antibody  to  localize  time  and  site  of  appearance  in 
tissues. 

Major  Findings: 

Preliminary  findings  have  shown  that  the  six-week  neonatal  rabbit 
lacks  some  of  the  isoantlgens  it  later  produces. 

Significance  to  the  Program  of  the  Institute: 

1.  The  isoantigen-isoantibody  system  in  rabbits  provides  the  best  means 
for  study  of  gamma-globulin  formation  in  the  newborn  animals  since 
the  gamma- globulin  produced  by  the  offspring  can  be  clearly  dis- 
tinguished from  maternal  gamma-globulins  which  pass  the  placental 
barriers . 

2.  This  study  may  lead  into  an  experimental  system  analagous  to  the 
Bh  system* 

3.  Serve  as  a  pilot  study  for  the  question  of  transfusion  reactions  to 
plasma. 

Proposed  Course  of  Project; 

1.  Study  the  production  of  gamma  globulin  in  offspring  of  mothers 
Immunized  to  the  gamma-globulin  produced  by  the  fetus  in  analogy  to 
the  Bh  system. 

2.  Use  of  isoantlgens  as  cell  markers  in  connection  with  studies  of  skin 
homograft  rejection  in  cooperation  with  Dr.  Joseph  Bauer,  Peter  Bent 
Brigham  Hospital,  Boston,  Massachusetts. 

3.  One  of  the  interesting  by-products  would  be  the  possibility  for  the 
study  of  the  rate  of  gamma-globulin  synthesis  per  cell. 

Project  Subtitle  V;  Use  of  Isoantigens  as  Cell  Markers  in  Cell-Transfer 
Studies. 

Objectives: 

To  determine  whether  donor  white  cells  from  an  immunized  rabbit 
produce  antibody  in  an  irradiated  recipient. 


29 


Serial  No.  NIAID  -  IkJ 
vfethods  Employed; 

1.  Use  of  gamma- globulin  typed  rabbits  as  donors  and  recipients. 

2.  Fluorescent  antibody  to  identify  the  source  of  gamma-globulin  anti- 
body found  in  the  recipient. 

Major  Findings;  None 

Significance  to  the  Program  of  the  Institute; 

The  isoantigen-isoantibody  system  provides  the  means  to  definitively 
determine  the  above  objective,  and  would  demonstrate  the  applicability 
of  this  system  to  other  problems  in  immunology  involving  cell  transfer. 

Proposed  Course  of  Project; 

Evaluate  the  question  as  to  whether  one  or  two  gamma-globulin  iso- 
antigens  may  be  produced  in  the  same  cell.  This  is  a  question  of  con- 
siderable theoretical  interest  with  regard  to  theories  of  antibody  produc- 
tion. 


Part  B  included:  Yes  X     No 


30 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


(Attachment  i) 
Serial  No.  NIAID  -  Hf7 


Part  B:  Honors,  Awards,  and  Publications 

Publications; 

Dray,  Sheldon  and  Young,  Glendovlyn,  0.:  Genetic  Control  of  Two 
Gamma-Globulin  Isoantlgenic  Sites  in  Domestic  Rabbits.  Science, 
131:738-739,  i960. 


31 


Form  No.  ORP-2  Serial  No.  NIAID  -  1^7-A 

Nov.  i960               PHS-NIH  1.  Laboratory  of  Immunology 

Individual  Project  Report  2.  Immuno chemistry  Section 

Calendar  Year  i960  3.  Bethesda,  Maryland 


Part  A: 


Project  Title:  Immunochemical  Studies  on  Human  Serum  Antigens  and 
Antibodies 

Principal  Investigator;  Dr.  Sheldon  Dray 

Other  Investigators:  None 

Man  Years;  Patient  Years:  None 

Total:  2.0 
Professional:  0.5 
Other:  1.5 

Project  Description: 

Objectives: 

1.  Identification,  fractionation,  purification,  and  chemical  properties 
of  antigens  in  human  serum. 

2.  Significance  of  serum  antigens  and  antibodies  in  disease. 
Methods  Employed: 

1.  Immunochemical  analysis  by  diffusion  in  agar-gel;  simple  diffusion 
in  tubes,  double  diffusion  in  plates,  and  Immunoelectrophoresis. 

2.  Cellulose  ion-exchange  chromatography,  zone  electrophoresis,  and 
other  methods  of  protein  fractionation  and  purification. 

3>  Use  of  primates  for  immunization  with  human  serum  antigens. 

Major  Findings: 

1.  Primates  were  selected  for  immunization  with  human  serum  antigens 
with  the  idea  that  a  closely  related  species  to  man   might  yield 
antibody  to  components  of  human  serum  not  readily  obtainable  in 
other  ways.  Immunization  of  Rhesus  monkeys  with  various  antigens 
from  human  serum  has  yielded  precipitating  antibody  to  many  compo- 
nents of  human  serum  (albumin,  alpha-,  beta-,  and  gamma-globulins). 
Precipitating  antibodies  were  found  which  were  specific  for  three 
normal  gamma-globulins  of  relatively  slow  electrophoretic  moMllty 
instead  of  the  one  usually  found  with  rabbit  or  horse  antisevi. 
Furthermore,  the  immunochemical  relationship  between  the  thj«?e 
normal  7S  gamma-globulin  antigen  and  two  myeloma  gamma-globi .  .ins 

32 


Serial  No.  HIAID  -  lVf-A 

were  investigated  and  it  was  found  that  one  of  the  myeloma  gamma- 
globulins corresponded  to  one  of  the  normal  gamma-globulins  while  a 
second  myeloma  gamma-globulin  corresponded  to  the  second  myeloma 
gamma-globulin.  It  was  further  found  that  these  globulins  have 
antigenic  determinants  in  common. 

2.  Nine  chimpanzees  have  been  immunized  with  components  of  human  serum. 
The  results  thus  far  have  revealed  two  antisera  to  human  alpha- 
globulin  and  several  antisera  to  human  gamma-globulin. 

Significance  to  the  Program  of  the  Institute; 

The  availability  of  precipitating  antibodies  specific  for  three 
normal  human  gamma-globulins  should  facilitate  many  studies  of  consider- 
able interest  concerning  these  gamma-globulins,  such  as:  quantitative 
estimation  in  serum  and  other  body  fluids;  fractionation  and  purifica- 
tion; chemical  structure,  particularly  in  the  analysis  of  fragments  re- 
sulting from  enzyme  digestion;  antibody  properties  in  infectious  dis- 
eases and  diseases  of  supposed  Immunologic  etiology;  cytological  locali- 
zation by  fluorescent  antibody;  and  possible  genetic  differences.  Of 
immediate  clinical  interest,  the  quantitative  estimation  of  these  gamma- 
globulins in  serum  should  be  useful  for  early  diagnosis  and  study  of 
diseases  which  involve  qualitative  and  quantitative  changes  in  the 
gamma-globulins,  such  as  in  multiple  myeloma. 

Proposed  Course  of  the  Project: 

1.  Continue  the  immunization  of  primates  with  various  fractions  of 
human  serum  and  to  study  the  antigens  which  then  may  be  detected. 

2.  Survey  30  multiple  myeloma  sera  (in  collaboration  with  Dr.  John 
Fahey,  Metabolism  Branch,  NCI)  with  respect  to  their  reactions  with 
monkey  antisera. 

3.  immunization  of  monkeys  with  myeloma  proteins,  rheumatoid  factor, 
and  other  proteins  of  pathological  origin. 

k.     In  cooperation  with  Dr.  Edward  C.  Franklin  (New  York  University 
College  of  Medicine)  to  investigate  the  antigenic  determinants  of 
papain  fragments  of  human  gamma-globulins. 


Part  B  included         Yes        No    X 

33 


Form  No.  ORP-2  Serial  No.  NIAID  -  lh8 

Nov.  i960               PHS-NIH  1.  Laboratory  of  Immunology 

Individual  Project  Report  2.  Applied  Immunology 

Calendar  Year  i960  3«  Bethesda,  Maryland 


Part  A: 


Project  Title;  Basic  Studies  on  the  Cellular  Localization  of  Antigens, 
Antibodies  and  other  Substances  by  the  Fluorescent 
Antibody  Technique  and  Fluorescence  Microscopy. 

Principal  Investigator:  Dr.  John  E.  Tobie 

Other  Investigators:  None 

Cooperating  Units: 

Project  Subtitle  II  -  Dr.  G.  Robert  Coatney,  LPC,  Serial  NO.  NIAID- 
130-B 

Project  Subtitle  III  -  Mrs.  Eleanor  J.  Tobie,  LPD,  Serial  No.  NIAID- 
123-D 

Man  Years:  Patient  pays:  None 

Total:  3*0 
Professional:  1.0 
Other:  2.0 

Project  Description: 

Project  Subtitle  I:  Antibody  Formation  in  Neonatal  Rabbits 

Objectives: 

To  perform  studies  on  the  types  of  cells  involved  in  antibody  forma- 
tion and  to  determine  the  capacity  of  neonatal  animqlg  to  synthesize 
antibody. 

Methods  Employed; 

Neonatal  and  adult  rabbits  were  given  a  single  injection  of  bovine 
serum  albumen,  crystalline  egg  albumin  or  killed  typhoid  organisms, 
combined  vith  hydrocarbon  adjuvants,  into  the  footpad.  The  npitngin 
were  bled  frequently  and  tested  by  hemagglutination  or  agglutination 
reactions  in  an  effort  to  determine  the  earliest  time  at  which 
circulating  antibodies  first  appear.  In  certain  animals  the  regional 
popliteal  lymph  node  was  removed  and  the  node  fluid  tested  for 
presence  of  antibody.  Fluorescent  antibody  methods  will  be  employed 
to  determine  the  lymph  node  cells  involved  in  antibody  synthesis. 

3k 


Serial  No.  NIAID  -  lUQ 

Major  Findings; 

Using  the  antibody  responses  after  a  single  injection  of  bovine 
serum  albumen  as  an  example ,   neonatal  animals  (1-3  days  old  when  inject- 
ed) were  very  slow  in  producing  antibody.  None  of  the  rabbits  produced 
detectable  antibody  earlier  than  3-4  weeks  while  in  individual  animals 
it  took  as  long  as  100  days.  Certain  animals  failed  completely  to  re- 
spond to  a  single  injection  when  followed  for  as  long  as  1*00  days. 
One  animal  which  became  positive  at  102  days  continued  to  produce  anti- 
bodies through  k&l   days,  at  which  time  the  animal  was  sacrificed.  In 
contrast  to  the  antibody  response  in  neonatals,  antibody  could  be 
detected  consistently  in  adult  rabbits  7-9  days  after  a  single  injection. 
In  several  animals  no  circulating  antibody  could  be  detected  in  spite 
of  the  presence  of  antibody  in  lymph  node  fluid. 

Significance  to  the  Program  of  the  Institute: 

A  study  of  the  nature  of  the  antibody  response  in  neonatal  animals 
may  lead  to  a  better  understanding  of  the  mechanisms  involved  in 
"immunological  immaturity"  in  the  infant.  The  influence  of  age  upon 
antibody  formation  is  an  important  factor  in  the  capacity  of  infants 
to  be  actively  immunized. 

Proposed  Course  of  Project: 

Fluorescent  antibody  studies  will  be  conducted  on  lymph  nodes  from 
the  neonatal  rabbit  because  the  slow  antibody  formation  in  the  neonatal, 
as  compared  with  adults,  seems  to  offer  a  better  opportunity  to  study 
the  cellular  aspect  of  antibody  synthesis. 

Project  Subtitle  II;  Fluorescent  Antibody  Studies  on  Malaria 
Parasites 

Objectives: 

To  specifically  stain  human  malaria  parasites  by  the  fluorescent 
antibody  technique  in  such  a  manner  that  essentially  only  the  malaria 
parasites  are  visible  on  a  blood  film. 

To  feed  such  blood  films  through  an  automatic  scanning  microscope 
for  the  rapid  detection  of  parasites  in  large-scale  surveys. 

Methods  Employed: 

Globulin  fractions  of  sera  were  prepared  from  inmate  volunteers  who 
had  long-standing  infections  with  Plasmodium  vivax.  The  globulin  was 
conjugated  with  fluorescein  isothiocyanate  and  applied  to  thin  blood 
films  which  had  been  dehemoglobinized  and  the  films  examined  at  fluores- 
cence microscopy. 

35 


Serial  No.  NIAID  -  lW? 

Globulin  from  a  patient  infected  with  the  vivax-like  malaria 
parasite  (P.  cynomolgi  bastianellii)  of  rhesus  monkeys  was  labeled 
and  applied  to  individual  blood  films  containing  either  parasites  of 
human  origin  or  those  of  monkey  origin.   The  cross  reactions  between 
these  two  malaria  species  were  determined  by  the  fluorescent  light 
emitted. 

Major  Findings; 

The  first  recorded  instance  of  the  fluorescent  antibody  staining  of 
human  malaria  parasites,  Plasmodium  vivax,  and  that  of  the  monkey 
parasite,  P.  cynomolgi  bastianellii,  has  been  accomplished.  Prelim- 
inary studies  on  the  serological  relationships  of  these  Plasmodia,  as 
based  on  the  degree  of  fluorescence,  indicate  that  the  two  species  may 
be  closely  related.  The  separate  application  of  P.  vivax  labeled  anti- 
body to  individual  blood  films  containing  the  parasites  of  rodent 
malaria,  P.  berghei,  or  human  malaria,  p.  vivax,  resulted  in  the  latter 
fluorescing  approximately  3  times  as  bright  suggesting  that  these  two 
species  probably  are  not  as  closely  related. 

Significance  to  the  Program  of  the  Institute; 

The  fluorescent  antibody  studies  on  malaria  could  lead  to  a  method 
whereby  hundreds  of  blood  films  could  be  scanned  in  a  single  day.  A 
study  of  the  serological  relationships  between  malaria  Plasmodia  of 
human  and  animal  origin  are  of  great  importance  in  connection  with  the 
program  of  the  worldwide  eradication  of  malaria  which  has  become  more 
complex  with  the  possibility  that  monkeys  may  serve  as  reservoirs  for 
human  infection. 

Proposed  Course  of  Project; 

To  continue  the  studies  on  the  serological  relationships,  explore 
methods  of  producing  high-titered  antisera  in  rabbits  by  sporozoite 
immunization  and  investigate  the  application  of  fluorescent  stained 
malaria  films  to  rapid  scanning  in  an  automatic  microscope. 

Project  Subtitle  III;   localization  of  Trypanosoma  rangeli  in  the 
Insect  Host 

Objectives; 

To  determine  the  organ  localization  of  Trypanosoma  rangeli  in  the 
arthropod  host  and  to  study  the  developmental  relationships  between 
the  parasite  and  the  insect  vector. 

Methods  Employed; 

The  insect  vector,  Rhodnius  prolixus,  has  been  experimentally 
has  been  experimentally  infected,  either  artificially,  by  injection 
of  trypanosomes  into  the  hemocoel  or  naturally,  by  allowing  the  on 

3 


Serial  No.  NIA3D  -  1^8 

insect  to  take  "blood  meals  on  an  infected  animal.  Fresh-frozen  sections 
of  the  whole  insect  were  prepared  and  the  sections  conventionally 
stained.  Fluorescent  antibody  methods  also  will  be  employed  for 
discrete  localization  in  the  arthropod  host. 

Major  Findings: 

The  trypanosomes  have  been  localized  in  the  salivary  glands  of  the 
insect  after  artificial  infection.  After  certain  intervals,  the 
developmental  stages  of  the  trypanosome  also  were  found  in  the  intes- 
tinal tract  of  the  arthropod. 

Significance  to  the  Program  of  the  Institute; 

The  successful  localization  of  Trypanosoma  rangeli  in  fresh-frozen 
sections  of  whole  insects  opens  up  the  possibility  of  the  precise 
localization  of  other  infectious  agents  in  arthropod  hosts.  Detailed 
studies  on  the  developmental  relationships  between  the  parasite  and  the 
insect  vector  are  now  within  the  realm  of  possibility. 

Proposed  Course  of  Project: 

Fluorescent  antibody  studies  have  been  initiated  in  an  effort  to 
elucidate  the  mechanisms  involved  in  the  insect  transmission  of  this 
trypanosome. 


Part  B  included:       Yes  X    No 


37 


PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


(Attachment  I) 
Serial  NO.  NIAID  -  Ihd 


Part  Bt  Honors,  Awards,  and  Publications 

Publications; 

Milch,  Robert  A.,  Tobie,  John  E.  and  Robinson,  Robert  A.: 
A  Microscopic  Study  of  Tetracycline  Localization  in  Skeletal 
Neoplasms.  Jour.  Histochem.  Cytochem.,  In  press. 

Tobie,  John  E.  and  Beye,  Henry  K. :  Fluorescence  of  Tetra- 
cyclines in  Filarial  Worms.  Proc.  Soc.  Exp.  Biol.  Med., 
10U:137-l»»O,  i960. 

Tobie,  John  E.,  Burgdorfer,  Willy  and  Larson,  Carl  L.:  Frozen 
Sections  of  Arthropods  for  Histological  Studies  and  Fluorescent 
Antibody  Investigations.  Experimental  Parasitology,  In  press. 

Tobie,  John  E.  and  McCullough,  Norman  B.:  Serological  Evidence 
of  Leptospira  pomona  Infections  in  Meat  Inspectors.  Jour.  Am. 
Vet.  Med.  Assoc.  In  press. 


38 


Form  NO.  ORP-2  Serial  NO.  NIAID  -  150 

Nov.  i960              PHS-NIH  1.  Laboratory  of  immunology 

Individual  Project  Report  2.  Applied  Immunology 

Calendar  Year  i960  3.  Bethesda,  Maryland 


Part  At 


Project  Title;  The  Relationship  of  Delayed  and  Immediate  Hypersensi- 
tivity to  Allergic  Thyroiditis  in  Inbred  Guinea  Pigs. 

Principal  Investigator;  Dr.  Philip  R.  B.  McMaster 

Other  Investigators;  None 

Cooperating  Units;  Dr.  Edwin  M.  Lerner,  LPH,  Serial  NO.  NIAMD  •  k 

Man  Years;  Patient  Days;  None 

Total:  1.2 
Professional:  0.6 
Other:  0.6 

Project  Description: 

Objectives: 

To  determine  the  relative  importance  of  delayed  and  Immediate  hyper- 
sensitivity to  thyroid  extract  in  the  etiology  and  pathogenesis  of 
allergic  thyroiditis. 

Methods  Employed: 

Two  groups  of  strain  13  histocompatible  guinea  pigs,  one  Immunized 
with  the  aid  of  complete  adjuvant,  the  other  with  the  aid  of  incomplete 
adjuvant,  were  compared  with  respect  to  the  development  of  circulating 
antibody,  delayed  hypersensitivity  to  thyroid  extract,  and  thyroiditis. 

Major  Findings; 

A  close  correlation  between  the  appearance  of  delayed  hypersensi- 
tivity to  thyroid  extract  and  allergic  thyroiditis  has  been  found.  The 
discovery  that  the  disease  could  appear  as  early  as  five  days  after 
the  start  of  immunization  allowed  the  investigators  to  show  an  inverse 
relation  between  serum  antibody  and  the  disease  could  exist  as  well  as 
a  direct  correlation  between  the  two,  depending  on  the  interval  of  time 
after  immunization.  This  suggests  the  possibility  of  a  similar  relation- 
ship between  delayed  hypersensitivity  to  the  thyroid  and  Hashimoto's 
disease. 


33 


Serial  HO.  NIAID  -  150 

Significance  To  The  Program  of  The  Institute; 

As  a  result  of  these  studies,  a  close  correlation  has  been  demon- 
strated "between  delayed  hypersensitivity  to  the  thyroid  and  experimental 
allergic  thyroiditis.  This,  therefore,  provides  experimental  evidence 
for  the  role  of  delayed  hypersensitivity  in  the  pathogenesis  of  experi- 
mental allergic  thyroiditis  and  suggests  the  possibility  of  a  similar 
role  in  the  development  of  allergic  thyroiditis  in  humans. 

Proposed  Course  of  Project; 

The  long  term  course  of  this  disease  In  guinea  pigs  is  now  being 
followed  in  preparation  for  a  study  of  the  effects  of  desensitization 
to  an  auto-antigen  upon  an  auto-allergic  disease,  for  which  this 
particular  condition  is  most  admirably  suited. 


Part  B  included;  Yes  X     Bo 


40 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


(Attachment  I) 
Serial  no.  NIAID  -  150 


Part  Bt  Honors,  Awards,  and  Publications 

Publications: 

McMaster,  Philip  R.B.:  Autoantibodies  and  Autosensitivity,  in 
Fundamentals  of  Modern  Allergy,  Edited  by  Samuel  J.  Prigal,  51-69* 
i960. 

McMaster,  Philip  R.B.:  Decreased  Aqueous  Outflow  in  Rabbits  With 
Hereditary  Bupthalmia.  A.M. A.  Arch.  Ophth.  6Jm 388-391,  i960. 

McMaster,  Philip  R.B.,  Lerner,  Edwin  M.,  and  Exum,  Eurmal  D.:  The 
Relationship  of  Delayed  Hypersensitivity  and  Circulating  Antibody 
to  Experimental  Allergic  Thyroiditis  in  Inbred  Guinea  Pigs.  J.  Exp. 
Med.  (in  Press) 


u 


Form  No.  ORP-2  Serial  No.  NIAID  -  152 

Nov.  i960               PHS-NIH  1.  Laboratory  of  Immunology 

Individual  Project  Report  2.  Applied  immunology 

Calendar  Year  i960  3.  Pasadena,  California  and 


Bethesda,  Maryland 


Part  A: 


Project  Title;  The  Chemistry  of  Antibodies 

Principal  Investigator;  Dr.  Wilton  B.  Vannier 

Other  Investigators:  None 

Cooperating  Units: 

Project  Subtitle  I  -  Dr.  Ernest  M.  Heimlich  of  the  Department  of 
Pediatrics  of  the  University  of  California  at  Los  Angeles 

Project  Subtitle  II  -  Dr.  James  Miller  of  the  Department  of  Infec- 
tious Diseases  of  the  University  of  California  at  Los  Angeles 

Project  Subtitle  III  -  Dr.  Anil  Saha  of  the  California  Institute  of 
Technology  and  Dr.  Jean-Marie  Dubert  of  the  Pasteur  Institute  in 
Paris 

Man  Years:  Patient  Days :  None 

Total:  1.16 
Professional:  0.66 
Other:  0.5 

Project  Description: 

Project  Subtitle  I:  The  Chemistry  of  the  Skin  Sensitizing  Antibodies 
of  Atopic  Human  Allergy 

Objectives: 

To  study  the  sedimentation  properties  of  the  skin  sensitizing  anti- 
bodies of  the  sera  of  individuals  with  atopic  allergies. 

Methods  Employed: 

Sera  from  allergic  individuals  were  fractionated  by  sedimentation 
in  an  angle  rotor  in  the  preparative  ultracentrifuge,  by  sedimenta- 
tion in  the  Waugh  partition  cell  in  the  analytical  ultracentrifuge 
and  by  sedimentation  in  salt  density  gradients  in  a  swinging  bucket 
rotor  in  the  preparative  ultracentrifuge.  The  fractions  obtained 
have  been  characterized  by  analytical  ultracentrifugatlon,  electro- 
phoresis and  passive  transfer  skin  reaction. 

42 


Serial  No.  NIAID  -  152 
Major  Findings: 

As  previously  reported,  our  data  show  that  the  skin  sensitizing 
activity  cannot  be  exclusively  associated  with  the  S19  component  and 
that  probably  the  S19  component  is  not  active  at  all.  Our  data  and  more 
recent  experiments  carried  out  by  Dr.  Alec  Sehon,  Dr.  Julius  Gordon  and 
Dr.  Ladislas  Gyenes  at  Montreal  are  all  consistent  with  the  hypothesis 
that  the  skin  sensitizing  activity  is  associated  with  a  serum  component 
or  components  of  intermediate  sedimentation  coefficient,  i.  e.  between 
7S  and  18s.  The  salt  density  gradient  studies  have  shown  that  the 
sensitizing  antibody  appears  to  be  associated  with  serum  proteins  of 
hydrated  density  of  about  I.36  rather  than  being  present  in  the  lipo- 
protein fractions  of  density  1.2  or  lower.  During  the  course  of  the 
salt  density  gradient  studies,  it  has  been  observed  that  there  is  a 
considerable  loss  in  antibody  activity  associated  with  the  fairly  high 
salt  concentrations  used. 

Significance  to  the  Program  of  the  Institute; 

The  physical  properties  of  the  skin  sensitizing  antibody  of  atopic 
allergy  are  of  fundamental  importance  in  understanding  allergic  re- 
actions. The  skin  sensitizing  antibody  is  qualitatively  different  from 
most  ordinary  precipitating  antibody  in  its  electrcphoretic  and  sedimen- 
tation properties  and  in  that  it  is  retained  locally  when  injected  intra- 
dermally  rather  than  diffusing  away.  The  process  of  antibody  fixation 
to  tissues  is  an  important  problem  in  all  allergic  reactions  and  any 
differences  that  can  be  demonstrated  in  the  chemical  or  physical  prop- 
erties between  ordinary  precipitating  and  skin  sensitizing  antibody  may 
be  important  in  this  process. 

Proposed  Course  of  Project; 

A  more  detailed  study  of  the  salt  inactivation  of  the  skin  sensitiz- 
ing antibody  will  be  carried  out. 

Project  Subtitle  II;  The  Chemistry  of  the  Antibodies  of  Syphilis. 

Objectives: 

To  study  the  physical  properties  of  some  of  the  antibodies  associated 
with  syphilitic  infections  and  to  study  a  macroglobulin  antibody  system 
in  which  a  specific  purification  of  antibody  may  possibly  be  achieved. 

Methods  Employed; 

Sera  from  individuals  with  various  stages  of  syphilitic  infection  are 
being  fractionated  by  starch  block  electrophoresis  and  ultracentrifuga- 
tion  methods  and  the  fractions  tested  for  VDRL  flocculation  antibody  and 
for  TPI  antibody. 

1*3 


Serial  No.  NIAID  -  152 


Major  Findings : 


The  work  has  not  progressed  far  enough  for  any  definite  conclusions 
to  be  reached. 

Significance  to  the  Program  of  the  Institute; 

There  is  confusion  in  the  literature  with  regards  to  the  inter- 
relationship between  the  antibodies  in  syphilis  as  measured  by  the 
various  tests  available.  Studies,  such  as  this,  involving  the  prepara- 
tion of  purified  antibody  fractions  may  help  to  clarify  the  problem. 
The  study  of  the  chemical  and  physical  properties  of  specifically 
purified  macroglobulin  antibody  may  yield  basic  information  regarding 
the  structure  of  these  antibodies  and  their  role  in  immune  processes. 

Proposed  Course  of  Project: 

The  studies  will  be  continued  as  indicated.  In  the  future  further 
fractionation  will  be  attempted  using  ion  exchange  chromatography. 

Project  Subtitle  III:  The  Fractionation  and  Specific  Purification  of 
Antibody 

Objectives: 

1.  To  explore  possible  specific  methods  of  antibody  purification. 

2.  To  achieve  the  chromatographic  fractionation  of  specifically 
purified  antibody  and  study  changes  in  the  distribution  of  specific 
bonding  affinities  of  antibodies  during  the  course  of  immunization. 

Methods  Employed: 

Specific  antigen-antibody  aggregates  are  dissociated  at  low  pH  and 
the  antibody  recovered.  Cellulose  coupled  with  p-arsanilic  acid  through 
tyramine  is  being  used  as  a  solid  adsorbent  to  purify  and  fractionate 
antibody  directed  against  the  p-arsanilic  acid  hapten. 

Major  Findings: 

1.  Purified  antibody  fractions  have  been  obtained  by  the  low  pH 
dissociation  of  BSA  antiBSA  precipitates  and  the  elution  of  anti- 
body from  the  cellulose-hapten  adsorbent  with  simple  hapten  (sodium 
p-arsanilate) . 

2.  Cellulose-tyramine-p-arsanilic  acid,  cellulose-histamine-p-arsanilic 
acid  and  cellulose-lysine-p-arsanilic  acid  adsorbents  have  been  pre- 
pared and  it  has  been  found  that  all  three  will  adsorb  antibody  from 
sera  obtained  by  immunization  with  p-arsanilic  acid  coupled  hemo- 
cyanin. 

3 


Serial  No.  NIAID  -  152 

Significance  to  the  Program  of  the  Institute; 

The  general  problem  of  specific  purification  of  antibody  is  one  that 
is  of  basic  importance  for  chemical  work  in  immunology.  The  presence 
of  large  amounts  of  nonspecific  protein  with  antibody  complicates  or 
prevents  a  study  of  the  chemical  reactions  of  antibodies,  the  structure 
of  antibodies  or  the  physical  properties  of  antibodies.  A  study  of  the 
heterogeneity  of  antibodies  and  especially  the  influence  of  degree  or 
route  of  immunization  on  the  distribution  of  antibodies  found  will  pro- 
vide additional  basic  information  about  the  nature  of  the  immune  process. 

Proposed  Course  of  Project; 

The  yield,  purity  and  immunochemical  characteristics  of  antibody  re- 
moved by  the  specific  adsorbents  and  purified  by  dissociation  of  specific 
precipitates  will  be  further  investigated.  The  preparation  of  cold  and 
C^  labeled  haptens  for  equilibrium  dialysis  studies  with  antihapten 
antibody  fractions  will  be  continued. 


Part  B  included  :    yes  /x/   no  /  / 


KS 


(Attachment  I) 
Serial  No.  NIATD  -  152 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B:  Honors,  Awards,  and  Publications 

Publications : 

Heimlich,  Ernest  W.,  Vannier,  Wilton  E.  and  Campbell,  Dan  H.: 
Sedimentation  Studies  of  Skin  Sensitizing  Antibody.  J.  Allergy 
31:361+  (i960). 


46 


Form  No.  ORP-2  Serial  No.  NIAJD  -  153 

Nov.  i960               PHS-NIH  1.  Laboratory  of  Immunology 

Individual  Project  Report  2.  Applied  Immunology 

Calendar  Year  i960  3.  Pasadena,  California 


and  Bethesda,  Maryland 


Part  A: 


Project  Title;  Basic  Studies  on  the  Chemical,  Physical  and  Skin 
Reactive  Properties  of  Extracts  of  House  Dust 

Principal  Investigator:  Dr.  Wilton  E.  Vannier 

Other  Investigators:  None 

Cooperating  Units: 

Dr.  Ernest  M.  Heimlich  of  the  Department  of  Pediatrics  of  the 
University  of  California  at  Los  Angeles  and  Dr.  A.  M.  Targov,  a 
practicing  allergist  in  Los  Angeles  have  tested  various  house  dust 
fractions  for  skin  reactivity  in  specifically  sensitive  individuals. 

Man  Years;  Patient  Days:  None 

Total:  O.83 
Professional:  O.33 
Other:  O.50 

Project  Description: 

Objectives: 

1.  To  study  the  chemical  and  physical  properties  of  the  materials  used 
clinically  for  the  diagnosis  and  treatment  of  house  dust  allergy. 

2.  To  try  to  identify  the  components  responsible  for  the  specific  skin 
reactivity. 

Methods  Employed: 

1.  Fractions  of  aqueous  house  dust  extracts  have  been  prepared  by 
starch  block  electrophoresis  and  evaluated  for  activity  by  direct 
skin  test  and  analyzed  chemically. 

2.  Studies  are  in  progress  to  determine  the  effect  of  chemical  or 
enzymatic  modification  of  the  dust  fractions  on  the  specific  skin 
reactivity. 

Major  Findings: 

1.  Previously  it  vas  shown  that  the  dust  allergen  fractions  consist 

47 


Serial  No.  NIAID  -  153 

of  a  heterogeneous  mixture  of  acidic  polysaccharides  containing  5-20$ 
polypeptide.  Starch  block  electrophoresis  studies  at  low  pH  and  in 
7M  urea  have  indicated  that  the  polypeptide  and  polysaccharide  com- 
ponents are  linked  through  covalent  bonds  rather  than  being  present 
as  separate  components  or  linked  "by  ionic  bonds  or  multiple  hydrogen 
bonds. 

2.  Preliminary  experiments  involving  the  chemical  modification  and 
enzyme  treatment  of  active  dust  fractions  have  been  carried  out. 
Partial  methyl  esteriflcation  of  the  free  carboxyl  groups  of  the 
polypeptide  portion  did  not  affect  the  activity;  however,  the  ex- 
tent of  conversion  to  ester  was  rather  low  and  it  will  be  necessary 
to  test  materials  in  which  a  more  complete  methyl  esteriflcation 
has  been  achieved  in  order  to  be  sure  that  the  carboxyl  groups  are 
not  involved  in  determining  the  allergen  activity.  Treatment  of 
active  dust  allergen  fractions  with  pepsin  has  had  no  influence  on 
their  skin  reactivity. 

Significance  to  the  Program  of  the  Institute; 

1.  About  one- third  of  all  allergic  individuals  give  a  positive  skin 
reaction  to  house  dust  extracts.  These  extracts  have  been  used 

for  many  years  in  the  diagnosis  and  treatment  of  house  dust  allergy. 
It  is  of  importance  to  investigate  the  chemical  nature  of  these 
materials  and  to  find  out  what  chemical  groups  are  involved  in  the 
skin  reactions. 

2.  A  rational  standardization  of  the  potency  of  house  dust  allergen 
extracts  might  well  be  based  on  a  knowledge  of  the  chemical  nature 
of  the  active  materials. 

Proposed  Course  of  Project: 

The  study  of  chemically  modified  and  enzyme  treated  du3t  fractions 
will  be  continued. 


Part  B  included:        Yes/X"/      No/ — f 


48 


(Attachment  I) 

PHS-NIH         Serial  No.  NIAID  -  153 
Individual  Project  Report    '  ~~ 

Calendar  Year  i960 

Part  B;  Honors,  Awards,  and  Publications 

Publications: 

Vannier,  Wilton  E.  and  Campbell,  Dan  H. :  A  Starch  Block 
Electrophoresis  Study  of  Aqueous  House  Dust  Extracts. 
J.  Allergy,  in  press. 


1*9 


ROCKY  MOUNTAIN  LABORATORY 


Summary p  i 

170  -  Office  of  the  Director 11 

171  -  Rickettsial  Infections 12 

172  -  Q  Fever 13 

173  -  Diseases  Having  a  Reservoir  of 

Infection  in  the  Natural  Environment 
(other  than  rickettsioses) 22 

174  -  Transmission  of  Disease  Agents  by 

Certain  Vectors 27 

175  -  Allergy  and  Immunology  of  Fungal 

Infections  and  the  Mechanisms  of 
Allergic  Phenomena 33 

176  -  Viruses,  Virus  Components,  and  Virus 

Surfaces 39 

177  -  Immune  Prophylaxis  of  Mycobacterial 

Infections 44 

178  -  Immune  Prophylaxis  of  Salmonella 

Infections 48 

179  -  Investigations  of  the  Role  of 

Morphological  Elements  of  Micro- 
organisms in  Immunity  and  Related 
Phenomena 54 

180  -  The  Encephalitides 58 

181  -  Colorado  Tick  Fever 63 


PHS-NIH 

Summary  Statement 

Rocky  Mountain  Laboratory 

Hamilton,  Montana 

Calendar  Year  i960 

The  accompanying  annual  report  summarizes,  as  well  as  such  a  report 
can,  the  activities  of  the  staff  of  the  Rocky  Mountain  Laboratory  for  the  year 
i960.  We  have  presented  our  projects  in  the  same  manner  as  we  have  in  previous 
years.  This  is  necessary  because  of  the  frequent  cooperation  of  workers  in 
various  sections  in  the  solution  of  mutual  problems  and  because  of  the  types 
of  problems  in  which  we  are  engaged. 

Our  research  has  continued  to  be  directed  toward  both  basic  laboratory 
investigations  and  field  studies  of  insect-  and  animal-borne  diseases.  In  the 
first  category  are  those  projects  concerned  with  the  chemistry  and  surface 
properties  of  viruses,  the  highly  intriguing  relations  of  hypersensitivity  and 
humoral  immunity,  and  the  basic  relation  of  structural  elements  of  microorgan- 
isms to  the  activity  of  the  agents,  both  in  vivo  and  in  vitro.  Field  work  is 
the  foundation  of  projects  related  to  studies  of  Q  fever,  tularemia,  Colorado 
tick  fever,  and  the  ar-bo  viruses.  In  addition,  the  combined  efforts  of  the 
staff  are  directed  to  many  other  areas  of  research.  Such  projects  include  Q 
fever,  tuberculosis,  influenza,  poliomyelitis,  and  cryptococcosis. 

We  are  also  engaged  in  studies  of  a  cooperative  nature  with  members  of 
the  staff  of  the  National  Institutes  of  Health,  Communicable  Disease  Center, 
University  of  Minnesota,  University  of  Georgia,  Purdue  University,  University 
of  Michigan,  New  York  University,  Buffalo  University,  and  other  institutions. 
The  cooperators  have  sought  our  aid  because  of  special  methods,  techniques,  or 
ideas  which  have  been  developed  at  RML.  As  a  result  of  our  past  accomplishments, 
63  visitors  have  spent  some  time  here  during  the  last  year.  I  believe  this 
reflects  in  some  measure  the  solid  contributions  the  staff  has  made  in  both  the 
recent  and  distant  past,  for  these  visitors  represent  scientists  interested  not 
only  in  entomological  and  biological  problems  but  also  those  interested  in 
chemical,  physical,  and  immunological  problems  related  to  many  fields  of 
microbiology. 

Hypersensitivity.  Studies  of  hypersensitivity  have  been  directed 
primarily  toward  clarification  of  the  relations  existing  between  delayed  hyper- 
sensitivity and  circulating  antibodies  and  determination  of  the  factors  respon- 
sible for  induction  of  contact  hypersensitivity.   It  has  been  demonstrated 
previously  that  delayed  hypersensitivity  precedes  circulating  antibodies.  This 
delayed  hypersensitivity  is  directed  toward  protein.  When  circulating  antibody 
appears,  as  occurs  with  relatively  large  amounts  of  conjugated  protein,  or  when 
booster  doses  are  administered,  the  specificity  of  the  antibody  response  becomes 
oriented  toward  smaller  configurations  on  the  antigen  molecule.  Studies  of 
immunity  in  neonatal  animals  have  revealed  that  these  animals,  when  injected 
with  an  antigen  12  hours  after  birth,  develop  circulating  antibodies  but  fail 
to  develop  delayed  hypersensitivity.  Additional  experiments  suggest  that  this 
inability  of  neonatal  animals  to  express  such  reactions  is  due  to  a  deficiency 


in  a  skin  reactive  factor  rather  than  an  inability  to  respond  to  a  primary 
injection  of  antigen. 

It  was  shown  that  contact  hypersensitivity  is  directed  toward  a  hapten 
and  that  simple  compounds  rather  than  protein  conjugates  of  these  compounds 
produce  contact  hypersensitivity  when  administered  to  experimental  animals. 
It  is  considered,  however,  that  the  production  of  this  phenomenon  is  related 
to  the  specific  proteins  present  in  the  host  tissues  because  a  conjugate  con- 
taining soluble  guinea-pig  skin  proteins  and  a  hapten  produces  in  guinea  pigs 
delayed  reactions  and  Arthus  reactions  to  the  conjugate  and  contact  hyper- 
sensitivity to  the  hapten. 

Poliovirus.  Continued  studies  of  poliovirus  have  yielded  considerable 
fundamental  data.   In  cooperation  with  the  group  at  the  University  of  Minnesota, 
it  was  shown  that  agents  such  as  octyl  alcohol-chloroform  and  neutral  hydroxyl- 
amine  do  not  materially  change  the  physical  properties  of  purified  infectious 
RNA  of  poliovirus  but  destroy  over  99$  of  "the  infectivity  of  this  material. 
The  destruction  of  infectivity  by  uncoupling  of  a  single  link  in  a  large 
particle  (probably  an  acyl  link  of  an  amino  acid  with  a  phosphate  group  at  the 
end  of  an  RNA  chain)  suggests  a  possible  approach  to  virus  chemotherapy.  Other 
studies  have  revealed  that  only  0.1$  of  RNA  infectivity  could  be  accounted  for 
by  residual  protein,  thus  strengthening  the  concept  that  RNA  does  indeed  con- 
stitute the  infectious  portion  of  the  poliovirus  moiety.  By  the  use  of  chromo- 
tographic  methods,  it  was  also  demonstrated  that  only  certain  of  the  avirulent 
strains  of  poliovirus  can  be  differentiated  from  the  virulent  strains  from  which 
they  are  derived.  This  is  in  direct  contrast  to  work  reported  by  others.  In 
studies  of  the  infectivity  of  RNA  it  was  found  that  the  bulk  of  virus  particles 
react  with  susceptible  cells  and  that  the  relatively  poor  correlation  between 
virus  particles  and  PFU  is  due  to  the  poor  efficiency  of  RNA  at  entering  sites 
where  it  can  influence  virus  production.  A  precipitation  test  for  detection 
of  antibodies  against  poliovirus  has  been  developed  which  is  more  sensitive 
than  the  neutralization  test  presently  employed.  The  antigen  used  is  radio- 
active virus. 

Endotoxins  in  bacterial  fractions.  Research  on  endotoxins  derived  from 
Gram-negative  organisms  has  continued  to  occupy  much  of  our  attention.  As  is 
so  frequently  the  case,  a  fresh  outlook  on  an  old  problem  yields  results  of 
great  value.  The  ideas  held  by  Dr.  Westphal,  which  attributed  the  activity  of 
endotoxins  to  the  presence  of  a  firmly  bound  lipid  ("lipid  A"),  were  apparently 
generally  accepted  until  presentation  of  the  work  done  at  RML.  The*  finding 
that  lipid  A  was  not  active  and  that  deproteinized  and  "delipified"  endotoxin 
was  active  stirred  considerable  controversy.   In  fact,  the  controversy  was  so 
intense  that  our  efforts  to  develop  a  vaccine  against  Salmonella  infections 
have  been  diverted  to  settling  this  issue.  Recent  studies  of  the  kinetics  of 
inactivation  of  toxin  by  hydrolysis  with  hot  acid  have  given  data  which  should 
end  this  discussion.  The  old  concept  of  "purified  endotoxins"  must  be  abandoned 
since  there  have  been  no  previous  toxins  as  good  as  those  obtained  at  RML,  and 
we  feel  these  are  to  be  still  further  purified.  Many  of  our  studies  would  have 
been  difficult  to  pursue  without  the  active  participation  of  Dr.  Landy  and 
others,  especially  certain  commercial  concerns  which,  because  of  interest  in 
our  results,  provided  us  with  mass  cultures  of  organisms  grown  in  commercial  lots. 


The  purification  of  Vi  antigen  by  curtain  electrophoresis  is  a  major 
advance  in  the  study  of  this  most  important  antigen.  The  demonstration  that 
certain  labile  acetyl  groups  are  responsible  for  the  activity  of  Vi  antigen 
resulted  in  production  of  material  which  was  ten  times  more  active  than  purified 
preparations  prepared  by  mild  acid  hydrolysis.  Emphasis  should  be  placed  upon 
the  new  chemical,  physical,  and  biologic  methods  that  have  been  devised  to  solve 
these  problems. 

Many  of  these  methods  and  ideas  have  been  used  in  studies  of  other 
problems  raised  here  and  by  visiting  scientists.  Contributions  made  to  the 
study  of  immunology  of  brucellosis  and  of  Q  fever  will  be  discussed  elsewhere. 
The  fine  structure  of  bacterial  spores  has  been  studied  in  conjunction  with 
Dr.  Gerhardt.  These  studies  show  that  the  coat  is  made  up  of  two-dimensional 
crystals.  Results  of  a  study  with  the  group  from  Purdue  University  revealed 
that  the  fine  structure  of  the  walls  of  Penicillium  chrysogenum  contained  much 
chitin  and  very  little  glucan  in  contrast  to  the  previous  finding  of  this  group 
of  workers.  These  studies  substantiate  the  results  with  Histoplasma  capsulatum 
previously  reported  from  EML. 

While  these  results  may  be  considered  to  be  of  only  theoretical  interest, 
many  questions  have  been  solved  by  methods  applied  here.  It  is  believed  that 
these  studies  are  of  fundamental  importance  and  will  serve  as  guidelines  in 
future  studies  of  the  relation  of  morphological  elements  to  various  in  vitro 
and  in  vivo  activities  of  microorganisms. 

Tuberculosis.  The  problem  of  immunity  in  tuberculosis  has  been  studied 
intensively.  Significant  findings  include  the  fact  that  mice  may  be  satisfac- 
torily immunized  to  subsequent  pulmonary  infection  with  virulent  organisms  by 
administration  of  small  doses  of  avirulent  organisms  by  the  aerosol  method. 
The  demonstration  that  resistance  is  not  due  to  interference  strengthens  the 
case  for  the  value  of  immunization  with  living  attenuated  organisms  for  the 
prevention  of  tuberculosis.  Since  it  has  been  shown  that  the  delayed  reactions 
elicited  by  protoplasm  of  various  acid-fast  organisms  are  specific  in  nature, 
it  seems  practicable  to  apply  these  findings  to  certain  diagnostic  problems  in 
man.  Use  of  fractions  of  tubercle  bacilli  in  producing  isoallergic  encephalitis 
in  guinea  pigs  shows  that  the  adjuvant  effect  lies  in  the  cell  walls  and  in  a 
water-soluble  protein  prepared  from  the  walls.  This  latter  finding  is  important 
since  it  will  allow  us  to  study  the  adjuvant  phenomenon  from  a  molecular  level. 

Q  fever.  Our  studies  on  Q  fever  in  many  ways  have  been  most  productive 
and  in  other  ways  most  frustrating.   It  has  been  demonstrated  that  the  number 
of  dairy  cattle  infected  with  Coxiella  burnetii  is  large  and  is  increasing,  yet 
it  is  extremely  difficult  to  detect  cases  of  clinical  disease  in  man.  In  Idaho 
and  Montana  we  have  shown  that  a  greater  number  of  individuals  residing  on  in- 
fected premises  have  antibodies  against  this  organism  than  do  those  living  on 
noninfected  premises,  yet  no  difference  can  be  detected  in  the  number  of  indi- 
viduals who  have  symptoms  compatible  with  clinical  disease.  The  fact  that 
organisms  isolated  from  cattle  have  been  uniformly  of  low  virulence  for  experi- 
mental animals  may  account  for  our  inability  to  find  clinical  cases  of  disease 
in  those  exposed  only  to  cattle. 


More  satisfying  experiences  were  noted  with  other  phases  of  the  studies. 
By  the  use  of  skin  tests  to  eliminate  allergic  individuals  from  the  study  group, 
190  inmates  of  the  Montana  State  Prison  were  safely  immunized  without  producing 
such  reactions  as  have  been  previously  reported.   It  is  evident  from  these 
results,  as  well  as  from  those  previously  obtained  in  laboratory  personnel, 
that  human  beings  can  be  safely  vaccinated  against  Q  fever  if  the  precaution 
is  taken  to  eliminate  reactors  by  previous  administration  of  specific  skin-test 
antigen. 

Methods  developed  for  growing  rickettsiae  on  modified  Zinsser  tissue 
cultures  yielded  relatively  large  volumes  of  organisms.  These  studies  led  to 
others  involving  purification  of  C.  burnetii  by  sucrose  gradients  and  by  con- 
tinuous-flow centrifugation  in  molar  salt  solution.  These  methods  likewise 
made  it  possible  to  obtain  certain  chemical  and  physical  fractions  of  these 
organisms.   It  was  found  that  dimethyl  sulfoxide  could  extract  from  Phase  II 
C .  burnetii  a  material  which  acted  only  as  a  hapten,  but  from  Phase  I  organisms 
the  extract  obtained  acted  as  a  complete  antigen.  Lauryl  sulfate  also  extracts 
complete  antigen  from  Phase  I  organisms.  Physically,  the  cell  walls  of  these 
organisms  can  be  separated  from  the  protoplasm,  and  it  has  been  noted  that  the 
cell  walls  are  about  25  times  more  active  in  producing  immunity  than  is 
protoplasm. 

In  addition,  considerable  effort  was  expended  in  evaluating  Q  fever 
vaccines  and  in  assembling  data  which  might  be  used  for  promulgating  minimum 
standards  for  production  of  Q  fever  vaccines. 

These  studies  on  Q  fever  are  of  considerable  significance.  The  labora- 
tory and  related  studies  have  yielded  information  of  both  scientific  and  applied 
interest.   It  is  apparent  now  that  we  can  safely  use  our  present  vaccines  for 
immunization  of  man  and  that  it  is  feasible  to  produce  large  numbers  of  organ- 
isms which  can  be  purified  and  used  as  vaccine  or  manipulated  to  give  physical 
or  chemical  fractions  which  may  be  less  toxic.  The  failure  to  find  clinical 
cases  of  Q  fever  in  man  in  the  face  of  a  rising  incidence  of  infection  in  dairy 
cattle  is  highly  interesting  even  if  disappointing.  The  lack  of  virulence  of 
strains  of  C.  burnetii  for  laboratory  animals  probably  is  responsible  for  this 
finding. 

Other  rickettsioses.  Studies  of  rickettsiae  other  than  C_.  burnetii  have 
been  continued.  By  combining  the  methods  presently  used  for  fluorescent  micro- 
scopy, we  have  developed  with  Dr.  Tobie  a  technique  for  sectioning  arthropods 
which  should  be  of  interest  to  entomologists  working  in  the  field  of  embryology 
and  anatomy  and  to  medical  entomologists,  since  thin  sections  in  which  the 
organs  are  not  displaced  can  be  obtained  routinely.  By  applying  the  technique 
to  the  study  of  ticks  infected  with  R.  rickettsii  it  was  found  that  the  infec- 
tion rate  in  local  ticks  varies  from  15$  to  28$.  Not  all  of  the  ticks  found 
infected  by  this  method  are  infective  for  laboratory  animals.  The  value  of 
this  type  of  study  has  yet  to  be  fully  appreciated. 

By  application  of  methods  developed  at  RML  we  have  been  able  to  produce 
a  vaccine  for  immunization  against  Rocky  Mountain  spotted  fever  which  is  10  to 
100  times  more  potent  than  those  presently  manufactured  on  a  commercial  basis. 


The  use  of  specific  toxins  has  resulted  in  clarification  of  many  of  the  prob- 
lems related  to  the  taxonomy  of  rickettsiae  and  has  proved  to  he  useful  in 
ecological  and  epidemiological  studies  of  this  complex  group  of  diseases.   In 
further  studies,  potent  immunogenic  extracts  have  been  obtained  from  certain 
of  the  rickettsiae.  Their  value  as  diagnostic  and  prophylactic  agents  is 
presently  under  consideration. 

Tick  paralysis.  Studies  of  the  incidence  of  ticks  capable  of  producing 
tick  paralysis  in  hamsters  have  shown  that  2^$  to  90$  of  lots  from  Colorado 
induce  disease;  from  0$  to  50$  of  those  from  the  Bitterroot  Valley;  and  75$ 
from  adjoining  Missoula  County.  The  incidence  of  ticks  with  ability  to  produce 
disease  does  not  appear  to  be  related  to  the  number  of  cases  of  tick  paralysis 
reported  in  man. 

Pasteurella  novicida.  Considerable  debate  has  been  caused  by  our  de- 
scription of  P.  novicida  as  a  new  species.  Application  of  the  Ovary  reaction 
(passive  cutaneous  anaphylaxis)  has  given  weight  to  our  description. 

Bacterial  vaccines.  Studies  have  been  continued  on  vaccines  for  certain 
bacterial  diseases.   It  has  been  found  that  while  live  Russian  tularemia 
vaccine  is  capable  of  protecting  mice  more  effectively  than  does  ether-extracted 
vaccine  derived  from  cell  walls  of  P.  tularensis,  the  protection  produced  by 
live  organisms  was  not  effective  for  long  periods  of  time.  Continued  studies, 
in  conjunction  with  Dr.  Foster,  have  emphasized  the  value  of  cell  walls  in 
producing  immunity  to  infections  with  Brucella  abortus  in  laboratory  animals. 
It  has  also  been  found  that  live  cells  suspended  in  phosphate  buffer  and  shaken 
with  an  excess  of  ether  are  killed  but  not  disrupted.  These  cells  constitute 
an  excellent  protective  antigen  which  is  less  toxic  (ID50  7*5  rog»)  than  aqueous 
ether  extracts  obtained  by  conventional  methods  (LD50  0.9  to  2.0  mg.).   It  is 
planned  that  Dr.  Foster  will  continue  these  studies  in  Georgia. 

Investigations  of  other  bacterial,  fungal,  and  viral  infections  common 
to  man  and  animals  have  been  continued.  P.  multocida  is  found  as  a  contaminant 
of  the  oral  cavity  of  many  species  of  animals,  and  this  situation  is  a  potential 
source  of  human  infection.  Leptospira  pomona  has  been  shown  to  be  present  in 
cattle  in  western  United  States,  and  the  organism  was  obtained  from  cerebro- 
spinal fluid  of  cattle  for  the  first  time.  The  organism  causing  haplomycosis 
in  animals  was  demonstrated  in  tissues  of  animals  from  a  number  of  different 
countries  and,  in  conjunction  with  Dr.  Emmons,  a  new  name,  "adiaspiromycosis," 
was  applied  to  this  type  of  parasitism.  It  has  been  found  that  massive  doses 
of  this  organism  are  lethal  for  monkeys  and  sheep.  The  possible  relation  of 
this  agent  to  hemorrhagic  fever  has  been  suggested. 

Rabies  in  bats.  The  study  of  rabies  in  bats  has  been  continued  and, 
although  the  number  of  infected  bats  has  not  been  high,  it  continues  as  a 
problem.  Progress  has  been  made  in  maintaining  bat  colonies  in  the  laboratory, 
and  it  is  now  practicable  to  commence  laboratory  investigations  of  pathogenesis 
and  other  aspects  of  rabies  infections  in  bats. 

Cryptococcosis.  An  antigen  isolated  from  Cryptococcus  neoformans  is 
now  being  tested  in  man  to  determine  the  incidence  of  cryptococcosis  in  New  York, 


an  area  in  vhich  pigeons  have  been  shown  to  be  heavily  infected  with  this 
fungus. 

Ar-bo  viruses.  Studies  of  ar-bo  viruses  have  yielded  results  of  inter- 
est and  suggest  that  emphasis  on  field  studies  would  greatly  increase  the  pro- 
duction of  useful  data.  The  California  strain,  described  by  Reeves  and  Hammon, 
has  been  isolated  from  a  snowshoe  hare  in  Montana,  and  serologic  studies  of 
hares  obtained  from  Michigan  indicate  that  the  majority  possess  antibodies 
against  this  virus.   In  California  it  has  been  demonstrated  that,  although  most 
infections  in  man  with  this  agent  are  of  the  inapparent  type,  some  infections 
result  in  serious  disease.  A  virus  closely  related  to  Powassan  virus  was  re- 
covered from  ticks  from  Colorado  and  is  of  importance  since  viruses  of  this 
group  produce  serious  illness  in  man.  Studies  to  date  indicate  that  ticks 
probably  are  not  the  natural  vector,  but  the  relation  to  Powassan  virus  sug- 
gests that  mosquitoes  would  most  likely  be  the  vector  in  nature.   In  studies 
of  the  complex  relation  of  WEE  virus  with  snakes  and  mosquitoes  it  has  been 
possible  to  demonstrate  that  the  virus  can  be  readily  overwintered  in  garter 
snakes  and  that  mosquitoes  can  be  infected  by  feeding  on  such  snakes.  While 
we  have  not  been  successful  in  isolating  virus  from  snakes  collected  in  the 
field,  the  laboratory  data  suggest  that  these  or  similar  animals  would  consti- 
tute a  host  suitable  for  overwintering  of  WEE  virus.   In  Idaho  and  Oregon, 
WEE  virus  was  isolated  with  considerable  frequency  during  the  summer  season, 
while  in  North  Dakota  the  virus  did  not  appear  to  be  active.   Isolations  of  a 
considerable  number  of  strains  of  trivittatus  and  inornata  viruses  were  made. 

Considerable  research  was  performed  to  determine  the  level  of  viremia 
attained  in  wild  and  domestic  birds  infected  with  ar-bo  viruses.  After  infec- 
tion with  WEE  or  St.  Louis  viruses,  turkeys,  ducks,  chickens,  and  pheasants 
display  levels  of  viremia  which  should  cause  infection  in  mosquitoes  feeding 
on  them.   It  is  of  interest,  however,  that  in  spite  of  considerable  effort  we 
have  been  unable  to  isolate  ar-bo  viruses  from  the  bloods  of  vertebrates. 
Negative  results  were  obtained  in  examination  of  1,07^+  specimens  collected  in 
Montana,  North  Dakota,  Oregon,  and  Minnesota  during  the  spring  of  i960.  These 
studies  fail  to  add  weight  to  the  contention  that  latent  infections  of  birds 
are  a  factor  in  overwintering  or  of  introduction  of  virus  into  endemic  areas. 

Colorado  tick  fever.  Colorado  tick  fever  continues  to  be  a  problem  in 
the  western  United  States.  Without  stimulation  of  physicians  we  still  received 
a  large  number  of  specimens  for  examination  this  year  and  isolated  virus  from 
U9  of  them.  Our  interest  in  the  spectrum  of  symptoms  has  continued  and  we 
still  see  severe  cases  of  illness  due  either  to  encephalitis  or  bleeding  tend- 
encies.  It  was  found  that  the  complement-fixation  reaction  developed  at  the 
Rocky  Mountain  Laboratory  is  the  simplest  method  for  diagnosis  of  Colorado 
tick  fever.  Vaccine  has  been  prepared  and  has  been  shown  to  be  efficacious 
in  mice.  This  type  of  vaccine  has  been  used  repeatedly  in  man  without  ill 
effects,  indicating  that  a  vaccine  prepared  from  suckling  mouse  brain  is  harm- 
less to  man  when  repeated  doses  are  given. 

Ticks  collected  in  Estes  Park,  Colorado,  were  examined  for  the  presence 
of  Colorado  tick  fever  virus.  The  incidence  of  infection  was  found  to  vary 
from  5$  to  21$.  This  high  incidence  of  infection  in  ticks  accounts  for  the 


large  number  of  cases  of  CTF  reported  in  Colorado  annually. 

Publications.  At  the  time  this  report  was  written  we  had  published  k$ 
papers  and  had  27  accepted  for  publication  during  the  calendar  year  i960.  This 
compares  favorably  with  those  published  in  previous  years,  and  the  diversity 
of  subjects  dealt  with  reflects  the  varying  interests  and  capabilities  of  the 
members  of  the  staff  of  the  Rocky  Mountain  Laboratory. 

Comment.  Although  the  RML  has  developed  a  broad  research  program  and 
a  staff  capable  of  dealing  adequately  with  the  laboratory  problems  assigned  to 
it  and  those  arising  from  public  health  needs,  consideration  should  be  given 
to  certain  physical  needs  of  the  plant.  These  needs  include  a  building  suitable 
for  housing  experimental  animals  and  facilities  for  housing  the  library  and  the 
administrative  staff.  These  are  matters  that  call  for  additions  to  our  present 
facilities  or  radical  changes.  The  availability  of  funds  to  modernize  certain 
of  our  laboratories  would  result  not  only  in  some  consolidation  of  space,  but 
also  would  lead  to  better  safeguards  for  the  health  of  our  staff. 

Difficulties  were  experienced  again  this  year  in  operating  the  Rocky 
Mountain  Laboratory  on  a  budgetary  apportionment  of  80$  for  personal  services 
and  20$  for  other  expenses.  Even  though  some  of  our  allotted  positions  have 
not  been  filled,  20$  of  our  budget  has  not  been  adequate  to  provide  essential 
materials  and  equipment.   In  view  of  the  proposed  reactivation  of  the  pathology 
unit  and  approved  addition  of  personnel  in  the  areas  of  epidemiology  and  immu- 
nology, a  material  increase  in  the  amount  allotted  for  other  expenses,  in 
addition  to  that  allotted  for  staff  increases,  is  essential  for  continued  pro- 
ductive research  and  for  the  morale  of  our  present  staff. 

The  quality  and  quantity  of  our  field  work  is  a  specific  weakness  which 
we  hope  to  correct  by  the  addition  of  a  qualified  epidemiologist  to  our  staff. 
The  application  of  some  of  our  basic  findings  to  limited  human  experience  would 
establish  whether  or  not  the  products  developed  in  the  laboratory  might  be  use- 
ful for  control  or  diagnosis  of  disease.  The  recent  study  of  Q  fever  vaccine 
in  humans,  previously  skin  tested,  to  detect  and  eliminate  reactors  shows  that 
vaccine  can  be  used  with  impunity  if  proper  precautions  are  taken.  Similar 
studies  with  typhoid  fever  vaccine  (based  on  observations  by  Ribi  and  Milner) 
should  surely  be  attempted  on  the  above  basis  and  followed  by  challenge  studies 
in  cooperation  with  Woodward' s  group.  Another  vaccine  that  should  be  studied 
in  man  is  prepared  from  B.  pertussis  and  described  by  Ribi  and  Munoz.   In 
addition,  purified  products  of  C.  burnetii  should  be  examined,  and  the  value 
of  various  fractions  of  acid-fast  organisms  as  diagnostic  agents  should  be 
investigated.  Studies  of  this  nature  are  essential  in  order  to  prove  the  value 
of  basic  research  and  to  shorten  the  temporal  gap  which  exists  between  discovery 
in  the  laboratory  and  application  to  man.  This  is  a  legitimate  field  for  en- 
deavor in  this  institute  and  in  no  way  interferes  with  the  programs  of  CDC. 

It  should  be  emphasized  that  the  scientific  stature  of  the  RML  has  not 
suffered  from  the  partial  shift  in  the  research  aims  of  the  group.  Exceptional 
interest  and  competence  have  continued  to  be  manifested  by  those  members  of 
the  staff  who  have  followed  their  studies  of  insect-  and  animal-borne  diseases 
through  field  and  laboratory  investigations.  The  recent  assignment  of 


Drs.  Brennan  and  Yunker  to  MARU  to  study  the  role  of  mites  in  the  transmission 
of  disease  is  a  move  in  the  direction  of  establishing  the  necessary  type  of 
entomological  field  investigation. 

Increases  in  our  staff  during  the  past  few  years  have  allowed  us  to 
develop  programs  of  significance  in  areas  new  to  us.  These  areas,  specifically 
viral  proliferation,  allergy,  and  the  relation  of  morphological  elements  of 
microorganisms  to  their  immunologic  activity,  have  been  highly  productive  and 
even  have  created  international  interest.  It  should  be  emphasized  that,  in 
spite  of  the  relative  isolation  of  this  institution,  new  ideas,  new  methods, 
and  important  scientific  results  are  forthcoming.  As  a  matter  of  fact,  it  is 
my  opinion  that  much  of  our  productiveness  may  be  a  result  of  the  relative 
remoteness  of  the  laboratory. 


Carl  L.  Larson,  M.D. 

Director 

December  1,  i960 


ROCKY  MOUNTAIN  LABORATORY,   HAMILTON,   MONTANA 
ANNUAL  REPORT,   i960 

NLAED-I70  OFFICE  OF  THE  DIRECTOR 

C.   L.    Larson,    Director 

C.   B.  Philip,   Acting  Director 

H.  G.   Stoenner,   Assistant  to  the  Director 

NIAID-171  RICKETTSIAL  INFECTIONS 

Principal  investigator:      C.   B.  Philip 

Others:     E.   J.   Bell,  W.   Burgdorfer,  C.  M.  Clifford, 

G.  M.  Kohls,   D.   B.   Lackman,   R.  A.  Ormsbee, 

H.  G.  Stoenner 

NIAID-I72         Q  FEVER 

Principal  investigators:      L.   Luoto,   H.  G.   Stoenner 
Others:      B.   H.   Hoyer,  D.   B.   Lackman,   R.  A.   Ormsbee, 
E.   Ribi 

NIAID-I73  DISEASES  HAVING  A  RESERVOIR  OF  INFECTION  IN  THE  NATURAL 

ENVIRONMENT  (OTHER  THAN  RICKETTSIOSSS") 
Principal  investigator:      J.  F.  Bell 
Others:     W.   L.  Jellison,   D.   B.   Lackman,  M.  T.  McKee, 
C.   R.  Owen,   H.  G.   Stoenner 

NIAID-17U         TRANSMISSION  OF  DISEASE  AGENTS  BY  CERTAIN  VECTORS 
Principal  investigator:     C.   B.  Philip 
Others:      J.  F.   Bell,   J.  M.   Brennan,  W.   Burgdorfer, 

C.  M.  Clifford,  W.   L.   Jellison,  G.  M.  Kohls, 

D.  B.   Lackman,  V.  F.  Newhouse 

NIAID-175  ALLERGY  AND  IMMUNOLOGY  OF  FUNGAL  INFECTIONS  AND  THE 

MECHANISMS  OF  ALLERGIC  PHENOMENA 

Principal  investigator:      S.   B.   Salvin 
Others:      M.   B.   Gregg,    R.   p.   Smith 

NIAID-I76         VIRUSES,   VIRUS  COMPONENTS,   AND  VIRUS  SURFACES 
Principal  investigator:      B.   H.   Hoyer 
Others:      R.  K.  Gerloff,   F.  G.   Jarvis*,   R.  A.  Ormsbee, 
D.    B.   Ritter 


NIAID-I77  IMMUNE  PROPHYLAXIS  OF  MYCOBACTERIAL  INFECTIONS 

Principal   investigator:      E.    Ribi 
Others:     W.  T.   Haskins,   C.   L.   Larson 

NIAID-I78  IMMUNE  PROPHYLAXIS  OF  bAITDNELLA  INFECTIONS 

Principal   investigator:      E.    Ribi 
Others:      W.   T.    Haskins,   F.   G.   Jarvis*,   K.   C.  Milner 


NIAID-179  INVESTIGATIONS  OF  THE  ROLE  OF  MORPHOLOGICAL  ELEMENTS 

OF  MICROORGANISMS   IN  IMMUNITY  AND  RELATED  PHENOMENA 


Principal  investigator:      E.   Ribi 
Others:      R.   L.  Anacker,   J.  E.  Coe,  C. 
K.  C.  Milner 


L.   Larson, 


NIAID-180         THE  ENCEPHALITIDES 


Principal  investigator:      C.  M.   Eklund 
Others:      W.   Burgdorfer,   D.   B.   Lackman,   V.  F.  Newhouse, 
L.  A.  Thomas 


NIAID-181         COLORADO  TICK  FEVER 


Principal  investigator:      C.  M.  Eklund 
Others:     W.   Burgdorfer,   G.  M.  Kohls,   D.   B.   Lackman, 
L.  A.  Thomas 


*Resigned  July  29,   i960 


10 


Serial  No.  NIAID-170 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Rocky  Mountain  Laboratory 
Hamilton,   Montana 


Part  A. 


Project  Title:   Office  of  the  Director 
Principal  Investigator:  C.  L.  Larson,  Director 

Other  Investigators: 


Cooperating  Units: 


C.  B.  Philip,  Acting  Director 

H.  G.  Stoenner,  Assistant  to  the  Director 

None 


Man  Years  (calendar  year  i960): 
Total:        31.5 
Professional:   1.5 
Other:        30.0 

Project  Description: 

The  over-all  direction  and  supervision  of  the  scientific  research 
program  of  the  Rocky  Mountain  Laboratory;  the  direction  of  the  adminis- 
trative aspects  of  the  Laboratory  including  all  fiscal,  supply,  house- 
keeping, and  maintenance  activities;  and  the  providing  of  scientific 
services  as  required  by  the  research  staff,  including  laboratory  animal 
care  and  breeding,  sterile  glassware  and  media  preparation,  special  shop 
services,  library,  graphic  arts,  etc. 


Part  B  included:  No 


11 


Serial  No.  NIAID-171 

Rocky  Mountain  Laboratory 
Hamilton,   Montana 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:   Rickettsial  Infections 

Principal  Investigator:  C.  B.  Philip 

Other  Investigators:     E.  J.  Bell,  W.  Burgdorfer,  C.  M.  Clifford, 

G.  M.  Kohls,  D.  B.  Lackman,  R.  A.  Ormsbee, 
H.  G.  Stoenner 

Cooperating  Units:       None 

Man  Years  (calendar  year  i960): 
Total:        11.0 
Professional:    3«0 
Other:         8.0 

Project  Description: 

Objectives: 

This  project  is  concerned  with  elucidation  of  problems  created  by 
rickettsial  agents  (exclusive  of  Coxiella  burnetii)  causing  human  and 
animal  diseases.   In  the  main,  attention  has  continued  to  be  directed 
toward  the  spotted  fever  group  of  diseases,  their  relationships,  and 
classification. 

Methods: 

The  customary,  standardized  procedures  for  study  of  rickettsial 
agents  have  been  followed  with  particular  attention  to  methods  of  purifi- 
cation, cultivation,  and  use  of  specific  rickettsial  toxins  in  study  of 
relationships  of  the  spotted  fever  group. 

Major  findings: 

1.  The  value  of  the  mouse  protection  test  against  specific  toxin 
has  been  demonstrated  for  measuring  immunogenic  activity  of  spotted  fever 
vaccines  and  for  correlation  with  other  methods  of  potency  assay. 


Par:  I  included:  Yes 


12 


Serial  No.  NIAID-171 

2.  By  improved  standardized  procedures,  yolk-sac  vaccines  prepared 
at  RML  were  10-  to  100-fold  more  potent  than  commercial  products.  It  was 
a  surprise  to  find  that  some  vaccines  prepared  from  infected  ticks  by  the 
old  methods  and  stored  at  5  C.  for  from  10  to  15  years  exhibited  immuno- 
genic potency  values  equivalent  to  those  of  the  commercial  vaccines. 

3.  Factors  affecting  optimum  toxin  production  from  R.  conorii  and 
R.  rickettsii  grown  in  fertile  hen's  eggs  have  been  defined.  Temperature 
of  incubation  (33*5°  C.)  before  and  after  death  of  the  embryos  is  im- 
portant, and  yolk  sacs  should  be  harvested  from  36  to  k&   hours  after 
death.  However,  a  better  yield  is  obtained  from  eggs  injected  with 
rickettsias  of  Kenya  and  Siberian  tick  typhus  and  South  African  tick-bite 
fever  if  eggs  are  held  U8  to  60  hours  after  death.  Snyder's  solution, 
which  inhibits  growth  of  organisms,  is  contraindicated  for  use  as  a 
rickettsial  seed  diluent. 

k.   An  agent  consistently  nonpathogenic  for  guinea  pigs  and  mice 
was  isolated  from  local  ticks.  The  identity  and  relationships  of  this 
isolate,  which  resembles  rickettsia,  are  under  investigation.  The 
relationship  of  an  atypical  rickettsial  isolate  from  a  patient,  who  died 
of  an  undiagnosed  disease,  is  also  being  studied. 

5.  Dimethyl  sulfoxide  will  extract  a  complement-fixing  antigen 
from  purified  R.  rickettsii  but  not  from  R.  prowazekii. 

6.  The  fluorescent  antibody  technique  was  found  to  provide  a  re- 
liable specific  diagnostic  tool  for  detection  of  R.  rickettsii  in 
sections  of  infected  ticks  which  had  been  held  for  3  days  at  37°  C. 
Conditions  were  adequately  controlled  by  comparison  with  preparations  of 
uninfected  ticks,  by  inhibition  with  unlabeled  immune  serums,  or  by  use 

of  heterologous  conjugates.  Furthermore,  untreated  samples  of  fluorescent- 
positive  ticks,  experimentally  infected,  caused  infection  in  guinea  pigs. 
As  demonstrated  by  this  technique,  natural  infection  rates  among  2^5 
ticks  from  k   localities  in  the  Bitterroot  Valley  varied  from  15-5$  to 
28$.  However,  only  a  small  percentage  of  ticks  representative  of  the 
group  yielded  strains  when  tested  in  eggs  or  guinea  pigs. 

7.  In  a  study  of  transovarial  transmission,  100$  of  585  eggs  and 
205  larvae  from  5  female  ticks  were  fluorescent  positive. 

8.  Sexual  transmission  of  R.  rickettsii  from  infected  male  ticks 
to  the  progeny  of  noninfected  females  appears  to  be  through  the  agency  of 
"contaminated"  fluid  surrounding  the  spermophores  received  by  the  female, 
rather  than  through  the  medium  of  rickettsia-bearing  sperm. 

Significance: 

Continued  refinement  of  methods  of  preparation  of  potent  ricket- 
tsial vaccines,  particularly  in  areas  of  purified  antigens  and 

13 


Serial  No.  NIAID-171 

standardized  potency  assays,  should  benefit  commercial  producers.  The 
more  precise  characterization  of  different  isolates  of  R.  rickettsii  and 
relatives  within  the  group  should  provide  tools  for  improved  investi- 
gation of  other  rickettsial  entities;  for  example,  the  rather  bewildering 
variation  observed  in  the  tsutsugamushi  (scrub  typhus)  agents.  By  these 
methods  additional  knowledge  of  variations  in  the  properties  of  strains 
from  various  geographic  locations,  both  here  and  abroad,  should  become 
available . 

Likewise,  techniques  developed  for  study  of  R.  rickettsii  in  D. 
andersoni  by  fluorescent  microscopy  should  be  adaptable  to  investigations 
of  other  rickettsia-vector  relationships.  A  better  insight  into  vector- 
parasite  relationships,  for  example  the  difference  in  ability  of  acarine 
vectors  and  insect  vectors  to  pass  rickettsiae  through  their  eggs,  should 
also  be  facilitated.  This  new  technique  revealed  that  natural  infection 
in  ticks  from  the  west  side  of  the  Bitterroot  Valley  was  about  double  the 
highest  infection  rate  observed  following  inoculation  of  ticks  into 
guinea  pigs  during  a  5-year  study  in  this  area  in  the  1950' s.  However, 
it  should  be  noted  that  a  considerable  proportion  of  fluorescent-positive 
ticks  failed  to  cause  infection  when  untreated  samples  were  injected  into 
embryonated  eggs  and  guinea  pigs.  This  raises  questions  of  quantitative 
or  qualitative  factors  involved. 

The  occurrence  of  fluorescing  R.  rickettsii  in  100$  of  progeny  of 
5  female  ticks  is  in  contrast  to  former  reports  of  only  incomplete  trans- 
ovarial  passage. 

Proposed  course; 

Completion  of  spotted  fever  assay  studies  is  expected.  Lacunae  in 
our  knowledge  of  the  relationships  of  the  spotted  fever  group  will 
continue  to  be  filled  by  results  of  studies  of  rickettsial  toxins. 
Initial  observations  by  fluorescent  microscopy  on  development  of  R. 
rickettsii  in  ticks  will  be  elaborated. 


11 


Serial  No.  NIAID-171 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Bell,  E.  J.  and  Stoenner,  H.  G.:   Immunologic  relationships  among  the 
spotted  fever  group  of  rickettsias  determined  by  toxin  neutralization 
tests  in  mice  with  convalescent  animal  serums.  J.  Immunol.  8^(2): 
171-182.  Feb.  i960. 

Philip,  C.  B.:   Rickettsial  diseases.   In  A  Manual  of  Tropical  Medicine, 
3rd  ed.  W.  B.  Saunders  &  Co.,  Philadelphia,  i960,  pp.  6I-69,  82-IO3. 

Burgdorfer,  W.  and  Lackman,  D.:   Identification  of  Rickettsia  rickettsii 
in  the  wood  tick,  Dermacentor  andersoni,  by  means  of  fluorescent  antibody. 
J.  Inf.  Dis.  107(2):  241-2M+.  Sept. -Oct.  i960. 

Philip,  C.  B.:  Microtatobiotes,  Rickettsiae  and  Tick-borne  Diseases. 
McGraw-Hill  Encyclopedia  of  Science  and  Technology.  McGraw-Hill  Book 
Co.,  Inc.,  New  York.  i960.  Vol.  8,  pp.  I1O3-U0U;  Vol.  11,  pp.  567-570; 
Vol.  13,  pp.  63O-63I. 

In  press: 

Kohls,  G.  M.:   Rocky  Mountain  spotted  fever.  History  of  Medical  Depart- 
ment of  Army  in  World  War  II. 

Philip,  C.  B.:   Scrub  typhus.  History  of  Medical  Department  of  Army  in 
World  War  II. 

Tobie,  E.  G.  and  Burgdorfer,  W.:  Cryostat  technique  for  sectioning 
arthropods.  Exp.  Parasitol. 

Honors  and  Awards  relating  to  this  project: 

Dr.  D.  B.  Lackman 

Appointed  lecturer  in  Microbiology,  University  of  Montana,  Missoula, 
Montana. 

Dr.  H.  G.  Stoenner 

Invited  to  rewrite  chapter  on  Rocky  Mountain  spotted  fever  for  Tice's 
Practice  of  Medicine  with  the  collaboration  of  Dr.  C.  M.  Eklund  and 
Glen  M.  Kohls. 


15 


Serial  No.  NIAID-172 

Rocky  Mountain  Laboratory 
Hamilton,  Montana 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:  Q  fever 

Principal  Investigators:   L.  Luoto,  H.  G.  Stoenner 

Other  Investigators:      B.  H.  Hoyer,  D.  B.  Lackman,  R.  A.  Ormsbee, 

E.  Ribi 

Cooperating  Units:       None 

Man  Years  (calendar  year  i960): 
Total:        12.5 
Professional:  k.5 
Other:         8.0 

Project  Description: 

Objectives: 

In  view  of  the  wide-spread  distribution  of  Coxiella  burnetii 
among  dairy  cattle  throughout  the  United  States,  continued  investigations 
on  this  problem  are  warranted.  The  objectives  of  these  studies  are 
fourfold:  (l)  to  evaluate  the  public  health  aspects  of  Q  fever,  particu- 
larly the  role  of  dairy  cattle  in  the  epidemiology  of  this  disease,  (2) 
to  determine  the  means  by  which  C_.  burnetii  spreads  among  livestock, 
(3)  to  reduce  the  toxicity  and  improve  the  immunogenic ity  of  Q  fever 
vaccine,  and  (U)  to  develop  a  practical  and  safe  procedure  for  immuni- 
zation of  man. 

This  project  will  be  considered  in  two  categories:  "A"  deals 
primarily  with  field  investigations  and  "B"  with  laboratory  studies. 

A.  Field  Investigations 

Methods: 

Methods  consist  of  field  studies  supported  by  laboratory  work, 
including  complement-fixation  tests,  capillary  agglutination  tests,  and 
isolation  of  strains  of  Q  fever  in  suitable  laboratory  animals.   In 
Montana  these  studies  were  restricted  chiefly  to  Gallatin  and  Ravalli 
counties,  whereas  studies  in  Idaho  were  concentrated  in  an  area  surround- 
ing Boise. 


Part  B  included:   Yes 


16 


Serial  No.  NIAID-172 
Major  findings: 

1.  During  the  past  year,  the  infection  rate  among  herds  of  dairy 
cattle  in  Montana  almost  doubled.  In  the  two-county  study  area  in 
Montana,  herd  infection  rate  increased  from  2$  in  1959  to  18$  in  i960. 
Preliminary  observations  indicate  that  many  factors  are  involved  in  dis- 
semination of  Q  fever  rickettsiae  among  livestock.  Although  the  purchase 
of  infected-herd  replacements  is  responsible  for  dissemination  of  Q  fever, 
a  significant  proportion  of  newly- infected  herds  was  closed  to  outside 
replacements.  Hence,  the  disease  might  have  been  spread  by  arthropods, 
wind,  or  mobile  fomites. 

2.  In  two  studies  designed  to  evaluate  the  role  of  dairy  cattle 
in  the  epidemiology  of  Q  fever,  similar  results  were  obtained.   In  an 
area  in  Ada  and  Canyon  counties  in  Idaho,  where  very  few  sheep  are  raised 
and  approximately  half  the  herds  of  dairy  cattle  are  infected,  27$  of  199 
persons  living  on  infected  premises  had  antibodies,  whereas  lk%>   of  260 
persons  residing  on  Q-f ever-free  premises  possessed  antibodies.  However, 
the  incidence  of  illnesses  resembling  Q  fever  among  seropositives  was 
essentially  similar  to  that  observed  among  seronegative  persons.  In  the 
study  in  Ravalli  County,  Montana,  involving  kyo   persons,  18$  of  those 
residing  on  infected  premises  possessed  antibodies,  whereas  ^$  of  those 
residing  on  Q-f ever-free  premises  were  seropositive.  Only  1  of  30  posi- 
tive individuals  had  experienced  an  illness  compatible  with  that  of  Q 
fever. 

3.  In  a  study  involving  190  human  volunteers  at  the  Montana  State 
Prison,  persons  who  failed  to  react  to  a  skin  test  of  0.02  complement- 
fixing  (CF)  units  of  antigen  were  safely  immunized  with  10  CF  units 
administered  subcutaneously.   In  a  limited  number  of  volunteers  a  dose 
of  20  CF  units  of  vaccine  resulted  in  painful  reactions  and  areas  of  in- 
duration at  the  sites  of  vaccination.  Antibody  response  was  detected 
more  readily  by  the  cepillary  agglutination  (CA)  test  than  by  the  CF  test. 
After  1,  2,  and  3  doses,  27$,  53$>  and  68$,  respectively,  developed  anti- 
bodies detectable  by  the  CA  test,  but  only  U.5$  of  these  persons  developed 
antibodies  detectable  by  the  CF  test.  Dermal  hypersensitivity  of  16.5$ 

of  these  persons  converted  from  negative  to  positive  after  vaccination. 

k.   Investigation  of  the  feasibility  of  combining  strain  19  Brucella 
abortus  vaccine  and  Q  fever  vaccine  for  immunization  of  calves  revealed 
that  the  antibody  response  in  calves  given  both  vaccines  was  similar  to 
that  obtained  in  calves  given  only  Q  fever  vaccine  or  strain  19  vaccine. 

5.  Mature  sheep  immunized  with  varying  doses  of  Q  fever  vaccine 
developed  high  levels  of  antibody  which  were  still  detectable  6  months 
after  vaccination. 


17 


Serial  NO.  NIAID-172 

Significance: 

The  failure  to  associate  clinical  illness  with  the  presence  of 
antibodies  in  persons  exposed  to  infected  dairy  cattle  conflicts  with 
observations  made  earlier  in  southern  California.  Most  of  the  strains 
of  C.  burnetii  isolated  from  dairy  cattle  in  Idaho  and  Montana  are  only 
mildly  pathogenic  for  guinea  pigs  and  this  may  explain  the  relative  in- 
ability of  these  organisms  to  cause  disease  in  man. 

The  presence  of  antibodies  in  a  large  proportion  of  rural  residents 
creates  a  diagnostic  problem.  A  clinical  diagnosis  of  Q  fever  should  be 
based  upon  the  judicious  evaluation  of  a  rise  in  antibody  titer  or  actual 
isolation  of  the  organism.  Because  of  the  known  propensity  of  C.  burnetii 
to  cause  human  disease,  the  large  reservoir  of  infection  in  the  dairy 
cattle  population  of  this  country  must  be  considered  a  potential  public 
health  problem.  The  high  incidence  of  sterile  abscesses  and  tissue  re- 
actions among  sensitized  persons  given  Q  fever  vaccine  and  the  absence 
of  such  reactions  in  nonsensitized  persons  emphasized  the  need  of  a  skin 
test  before  the  administration  of  Q  fever  vaccine. 

Proposed  course: 

In  studies  completed  to  date,  minor  illnesses  attributable  to  in- 
fection with  C.  burnetii  could  not  be  accurately  evaluated.  As  Q  fever 
continues  to  spread  through  dairy  cattle  in  Ravalli  County,  rural  popu- 
lations will  be  observed  closely  to  detect  any  minor  illnesses  attribut- 
able to  Q  fever. 

Epizootiological  studies  will  be  continued  to  elucidate  the  means 
whereby  the  organism  is  disseminated  throughout  the  livestock  population. 
Isolates  from  man  and  livestock  will  be  studied  and  characterized  in  an 
attempt  to  explain  the  relative  inability  of  these  organisms  to  cause 
human  illness. 

B.  Laboratory  studies: 

Methods: 

Organisms  grown  in  tissue  culture  or  embryonated  chicken  eggs  are 
purified  and  subjected  to  chemical  and  physical  fractionation.     Fractions 
are  then  assayed  for  complement-fixing  activity  and  immunogenic  proper- 
ties by  appropriate  serologic  and  biologic  tests. 

Major  findings: 

1.  A  method  of  purifying  rickettsiae  by  centrifuging  10  percent 
yolk-sac  suspensions  in  molar  salt  solution  in  a  continuous -flow  centri- 
fuge has  been  found  to  be  particularly  useful  for  purifying  large 
quantities  of  Q  fever,    spotted  fever,    and  typhus  rickettsiae. 

18 


Serial  No.  NIAID-172 

2.  The  density -gradient-sedimentation  technique  was  found  to  be 
useful  for  separating  cell  walls  from  intact  cells. 

3.  In  chemical  fractionation  studies,  dimethyl  sulfoxide  (EMS) 
extracts  of  phase  II  rickettsiae  were  CF  specific  hut  non immunogenic  in 
guinea  pigs,  whereas  EMS  extracts  of  phase  I  rickettsiae  were  immunogenic. 

Lauryl  sulfate  extracts  react  specifically  in  the  CF  test  and 
elicit  protective  antibodies  in  guinea  pigs.  This  chemical  agent  appears 
to  extract  phase  I  antigen  from  mixtures  of  rickettsiae  containing  both 
phase  I  and  II  components. 

h.   In  physical  fractionation  studies,  preparations  of  cell  walls 
and  protoplasm  were  prepared  from  purified  suspensions  of  C.  burnetii. 
Preliminary  studies  indicate  that  the  immunogenic  and  CF  activity  is 
situated  chiefly  in  the  cell  wall.  On  a  weight  basis,  cell  wall  prepa- 
rations showed  25-fold  greater  protection  than  did  protoplasm.  Fractions 
of  C .  burnetii  were  toxic  when  inoculated  intraderraally  into  normal 
rabbits.  The  least  dose  to  produce  a  visible  reaction  was  0.125  micro- 
grams for  whole  cells,  0.025  micrograms  for  cell  walls,  and  Uo.O  micro- 
grams for  protoplasm.  The  difference  in  dermal  activity  between  cell 
wall  and  protoplasm  is  similar  to  that  of  cellular  fractions  of  Salmon- 
ella enteritidis  and  S.  typhosa. 

5.  Comparative  studies  on  the  capillary  agglutination  test  and 
the  CF  test  on  serums  of  varied  sources  indicate  that  at  least  three 
types  of  antibodies  are  involved  in  the  immune  response  of  this  disease. 

6.  Growth  of  C.  burnetii  in  cell -free  medium  still  has  not  been 
accomplished. 

Significance: 

Studies  to  date  indicate  that  both  toxic  and  immunogenic  proper- 
ties of  the  Q  fever  rickettsiae  are  situated  in  the  cell  wall.  By  proper 
chemical  or  physical  fractionation  it  is  hoped  that  a  nontoxic,  protective 
antigen  can  be  isolated  from  this  organism.  Such  a  product  is  needed 
since  the  amount  of  vaccine  that  can  be  administered  at  the  present  time 
is  limited  by  its  toxic  properties. 

Proposed  course: 

Studies  on  the  physical  and  chemical  fractionation  of  C.  burnetii 
will  be  continued  to  obtain  fractions  which  will  be  particularly  useful 
in  immunologic  and  allergic  investigations.  Attempts  will  be  made  to 
isolate  and  identify  the  toxic  component  of  the  rickettsial  cell  wall 
since  this  has  direct  relationship  to  other  projects  dealing  with  bac- 
terial toxins. 


19 


Serial  No.  NIATD-172 

Attempts  will  be  made  to  correlate  the  virulence  of  strains  of  C. 
burnetii  with  the  chemical  composition  of  their  cell  walls.   Investigations 
on  the  relationship  of  the  phase  of  the  Q  fever  rickettsia  to  allergic 
and  protective  properties  of  various  cell  fractions  will  be  continued. 


20 


Serial  No.  NIAID-172 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Stoenner,  H.  G.  and  Lackman,  D.  B. :  The  biologic  properties  of  Coxiella 
burnetii  isolated  from  rodents  collected  in  Utah.  Am.  J.  Hyg.  71(1): 
45-51.  Jan.  I960. 

Luoto,  L. :   Report  on  the  nationwide  occurrence  of  Q  fever  infections  in 
cattle.  Pub.  Health  Rep.  75(2):  135-lto.  Feb.  i960. 

Stoenner,  H.  G.:   Q  fever.   In  A  Manual  of  Tropical  Medicine,  3rd  ed. 
W.  B.  Saunders  &  Co.,  Philadelphia,  i960,  pp.  10^-106. 

Ribi,  E.  and  Hoyer,  B.  H.:  Purification  of  Q  fever  rickettsiae  by 
density-gradient  sedimentation.  J.  Immunol.  85(3):  31^-3l8«  Sept.  i960. 

In  press: 

Pickens,  E.  G.  and  Gaon,  J.  A.:  Growth  of  Coxiella  burnetii  in  agar 
tissue  culture.  Am.  J.  Trop.  Med. 


21 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Serial  No.   HIAID-173 

Rocky  Mountain  Laboratory 
Hamilton,  Montana 


Part  A. 


Project  Title:   Diseases  having  a  reservoir  of  infection  in  the  natural 
environment  (other  than  rickettsioses) 

Principal  Investigator:   J.  F.  Bell 

Other  Investigators:     W.  L.  Jellison,  D.  B.  Lackman,  M.  T.  McKee, 

C.  R.  Owen,  and  H.  G.  Stoenner 

Cooperating  Units:       Dr.  C.  W.  Emmons,  LID,  NIAXD 

Man  Years  (calendar  year  i960): 
Total:        11.0 
Professional:   U.O 
Other:         7.0 

Project  Description: 

Objectives: 

Studies  performed  in  this  project  are  directed  toward  an  under- 
standing of  the  ecology  of  those  diseases  transmissible  to  man  from 
animals  and  the  natural  environment.  As  a  direct  continuation  of  the 
ecological  studies,  attention  is  paid  to  the  laboratory  aspects  of  the 
problems  encountered  with  special  emphasis  on  diagnosis,  treatment,  and 
control. 

Methods : 

The  methods  employed  are,  in  general,  straightforward  microbio- 
logical techniques,  with  such  variations  as  are  required  for  study  of 
specialized  problems,  rabies,  for  example. 

Major  findings: 

1.  Since  only  2  isolations  of  bat  rabies  were  made  this  year  and 
9  were  made  in  1959*  the  idea  of  an  initial  upsurge  toward  epizootic 
proportions  can  be  discounted. 

2.  Survival  of  mice  patently  infected  with  isolates  from  various 
sources  has  been  found  to  be  a  common  phenomenon  contrary  to  previous 

Part  B  included:   Yes 

22 


Serial  No.  NIAID-173 
ideas.  The  factors  responsible  are  unknown. 

3.  Continuing  presence  of  Group  A  streptococci  in  areas  of  high 
vole  populations  confirms  that  similar  earlier  observations  were  not 
isolated  ones,  but  the  epizoological  significance  remains  obscure. 

h.   In  an  initial,  long-term  experiment  designed  to  compare  the 
duration  of  protection  afforded  mice  against  P.  tularensis  when  vacci- 
nated with  ether-extracted  antigen  (LV)  and  a~~Russian  living  attenuated 
strain  (RV),  the  role  of  latent  infection  in  the  immune  response  of  the 
latter  group  could  not  be  determined.  During  the  ensuing  weeks  the  log 
protection  decreased  rapidly  or  slowly  depending  on  the  virulence  of 
challenge  material.   The  slopes  of  decreasing  protection  showed  consider- 
able similarity  between  LV  mice  challenged  with  ^25-FkJ  strain  and  RV 
mice  challenged  with  Schu.  Protection  in  KV  mice  challenged  with  l^-Fki 
remained  high  through  week  20  but  decreased  to  nearly  zero  after  52  weeks. 

5.  In  continued  comparisons  of  the  virulence  of  P.  tularense  from 
different  geographic  areas,  three  strains  from  2  patients  and  a  dead 
hare  in  Japan  were  strikingly  lower  in  virulence  for  guinea  pigs  and 
rabbits  than  3  isolates  from  hares  in  France  and  h   from  D.  variabilis 
wood  ticks  from  eastern  Montana. 

6.  Results  of  the  use  of  Ovary's  passive  cutaneous  anaphylaxis 
test  lend  weight  to  the  specific  rather  than  subspecific  differentiation 
of  P.  tularensis  and  P.  novicida.   Cross-vaccination  studies  still  leave 
a  question  as  to  their  relationship. 

7.  Isolations  of  Type  A2  influenza  virus  were  made  from  15  of  30 
throat  washings  collected  during  a  local  epidemic.   One  isolation  on 

22  January  was  earlier  than  usual  for  this  disease.  These  isolates  were 
forwarded  to  the  International  Influenza  Center  for  the  Americas. 

8.  P.  multocida  was  isolated  from  mouths  of  32  of  Vf  domestic 
cats,  one  bobcat,  and  one  of  5  dogs.  None  of  the  27  other  animals,  in- 
cluding grizzly  bears,  woodchucks,  skunks,  and  snakes,  were  positive. 

9-  The  fungal  organism  causing  haplomycosis  in  animals  has  been 
found  in  one  South  American  and  h   European  countries,  and  3  new  rodent 
hosts  were  discovered.   In  conjunction  with  Dr.  C.  W.  Emmons,  the  new 
name,  "adiaspiromycosis,"  was  applied  to  this  parasitism  because  of  the 
peculiar  lack  of  cellular  multiplication  of  organisms  in  the  animal  host. 
Massive  doses  from  cultures  given  intravenously  have  been  lethal  for 
monkeys  and  a  sheep. 

10.  Reciprocal  cross-immunity  in  guinea  pigs  and  mice  was  found 
between  Brucella  suis  and  Br.  neotomae  when  care  was  taken  to  use  only 
smooth-phase  cultures.  The  explanation  for  inconsistencies  in  earlier 
work  was  found  to  be  due  to  undetected  colonial  dissociation. 

23 


Serial  No.  NIAID-173 

11.  Leptospira  pomona  was  isolated  for  the  first  time  from  the 
spinal  fluid  of  a  bovine  animal,  a  local  dairy  cow  that  showed  signs  of 
meningitis  prior  to  death. 

12.  The  absence  of  HI  antibodies  against  swine  influenza  virus  in 
serums  from  members  of  the  Alaskan  National  Guard  confirms  epidemiologic 
evidence  that  the  1918-19  pandemic  of  influenza  failed  to  reach  some 
areas  of  Alaska.  Survivors  of  another  isolated  population  severely- 
affected  at  that  time,  still  retain  significant  levels  of  such  antibodies. 

Significance; 

Additional  laboratory  data  progressively  confirm  recent  notions 
that  rabies  virus  does  not  cause  an  invariably  fatal  disease  as  it  was 
once  thought  to  do.  The  concern  that  rabies  might  progress  to  epizootic 
proportions,  as  suggested  by  the  large  number  of  isolations  last  year, 
was  fortunately  not  justified. 

Though  human  infection  with  P.  multocida  has  not  been  observed 
locally,  isolation  from  the  mouths  of  cats  and  dogs  suggests  this  source 
as  of  potential  local  health  concern. 

A  reassessment  of  the  epidemiology  of  epidemic  nephroso-nephritis 
may  provide  a  new  viewpoint  for  a  fresh  attack  on  this  puzzling  Old  World 
entity.  Dr.  Jellison  has  made  the  novel  suggestion  that  some  2,000  cases 
of  "trench  nephritis"  in  troops  during  World  War  I  may  have  been  this 
type  of  hemorrhagic  fever. 

Studies  have  revealed  that  Br.  suis  and  Br.  neotomae  are  closely 
related  immunologically  but  are  not  completely  similar.   Br.  neotomae 
has  not  been  found  to  persist  in  tissues  of  swine.   Therefore,  if  results 
of  studies  in  mice  and  guinea  pigs  are  applicable,  this  organism  could 
conceivably  be  used  as  an  effective  vaccine  for  control  of  swine 
brucellosis. 

The  application  of  results  of  research  to  field  problems  is  one 
of  the  main  accomplishments  of  the  serology  laboratory.  The  identifi- 
cations of  infections  in  human  and  animal  populations  during  epidemiologic 
study  are  largely  the  result  of  serologic  testing.  When  it  is  possible 
to  isolate  the  causative  agent,  serology  plays  an  important  part  in  its 
final  identification  and  characterization.  While  such  work  progresses, 
improved  techniques  and  diagnostic  reagents  are  sought.  This  in  turn 
gives  rise  to  improved  prophylactic  vaccines  and  better  methods  for  their 
evaluation  and  standardization.  Results  of  serologic  testing  are  also 
utilized  for  the  identification  of  problems  in  infectious  disease  which 
may  exist  in  the  natural  environment  of  the  west. 

Pandemic  influenza  is  a  continuing  threat  to  man.  Therefore,  it 
is  important  to  detect  any  antigenic  variation  in  strains  causing  epidemics. 

2k 


Serial  No.  NIAID-173 

To  do  this  it  is  necessary  to  have  some  laboratories,  especially  ones 
located  in  isolated  areas,  which  will  forward  new  isolates  to  the  WHO 
Center.  Also  of  importance  is  the  continual  investigation  of  the  in- 
fluenza experience  of  isolated  populations  because  it  provides  information 
as  to  what  strains  should  be  included  in  vaccine.  Our  results  suggest 
that  consideration  should  be  given  to  the  inclusion  of  swine  influenza 
virus  in  vaccine  because  there  are  people  who  have  not  been  exposed  to 
this  virus  and  it  may  still  be  present  among  swine. 

Proposed  course: 

Steady  progress  in  laboratory  maintenance  of  bats  suggests  that 
experimental  infection  can  now  be  instituted  with  confidence. 

Studies  on  the  efficacy  of  vaccination  of  domesticated  beaver 
against  tularemia  are  under  way. 

Attempts  will  be  continued  to  elucidate  now  obscure  factors  in 
the  epizoology  of  Group  A  streptococci  and  "M"  organisms  (Toxoplasma 
microti)  in  Microtus  mouse  populations. 

If  facilities  become  available,  further  work  is  planned  on  differ- 
entiation of  P.  tularensis  and  P.  novicida  and  on  possible  protection  of 
swine  by  Br.  neotomae  against  Br.  suis. 

Studies  also  will  be  continued  on  virulence  and  cultural  patterns 
among  strains  of  P.  tularensis  of  diverse  origins  and  on  factors  favoring 
persistence  of  the  organism  in  natural  waters. 

Bird  tissues  collected  in  Alaska  for  test  for  arbor  viruses  will 
be  screened  in  the  serology  unit  for  viruses  of  the  psittacosis  group. 


25 


Serial  No.  NIAID-173 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 

Part  B.     Honors,  Awards,    and  Publications 

Publications  other  than  abstracts  from  this  project: 

Bell,   J.  F.   and  Moore,   G.   J.:      Rabies  virus  isolated  from  brown  fat  of 
naturally  infected  bats.     Proc.   Soc.  Exp.  Biol.  &  Med.   103(1);   lI+O-li+2. 
Jan.   I960. 

Owen,   C.   R.,  Meis,   A.,   Jackson,   J.  W.,    and  Stoenner,  H.  G.:     A  case  of 
primary  cutaneous  listeriosis.     New  Eng.  J.  Med.   262(20):    1026-1028. 
May  19,   i960. 

Emmons,  C.  W.   and  Jellison,  W.    L.:      Emmonsia  crescens  sp.  n.   and 
adiaspiromycosis  (haplomycosis)   in  mammals.     Ann.  N.Y.  Acad.   Sci.   89: 
91-101.     Aug.   27,   i960. 

Jellison,   W.   L. ,  Glesne,   L. ,    and  Peterson,   R. :     Emmonsia,    a  fungus,    and 
Besnoitia,    a  protozoan,    reported  for  South  America.     Bol.  Chilena  de 
Parasitol.  XV(3):    U6-V7.     July-Sept.  i960. 

Jellison,  W.  L.,  Vinson,   J.  W.,    and  Holager,   E.:      Haplomycosis  in  Norway. 
Acta  Pat.   et  Microbiol.  Scandinavica  ^9(4):    kd0-k8^.     i960. 

Esplin,   D.  W.,  Philip,  C.   B.,    and  Hughes,    L.  E.:      Impairment  of  muscle 
stretch  reflexes  in  tick  paralysis.     Science  132(31+32):    958-959. 
Oct.  7,   I960. 

In  press: 

Jellison,  W.  L.,  Helminen,  M.,  and  Vinson,  J.  W.:   Presence  of  a  pulmonary 
fungus  in  rodents  in  Finland.  Ann.  Med.  Exp.  et  Biol.  Fenniae  38(3): 

Jellison,  W.  L. :  Sodoku.  Rat-bite  fever  due  to  Spirillum  minus  Carter. 
Diagnostic  Procedures  and  Reagents,  Uth  ed.  American  Public  Health  Assoc, 
New  York,  N.Y. 

Larson,  C.  L. :   Tularemia.   Diagnostic  Procedures  and  Reagents,  Uth  ed. 
American  Public  Health  Assoc,  New  York,  N.Y. 


26 


Serial  No.  NIAID-171* 

Rocky  Mountain  Laboratory 
Hamilton,  Montana 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:  Transmission  of  disease  agents  by  certain  vectors 
Principal  Investigator:  C.  B.  Philip 

Other  Investigators:     J.  F.  Bell,  J.  M.  Brennan,  W.  Burgdorfer, 

C.  M.  Clifford,  W.  L.  Jellison,  G.  M.  Kohls, 

D.  B.  Lackman,  V.  F.  Newhouse 

Cooperating  Units:       Laboratory  of  Immunology,  NIH,  NAMRU  3 

Man  Years  (calendar  year  i960) : 
Total:        13.5 
Professional:   4.5 
Other:        9*0 

Project  Description: 

Objectives: 

This  group  directs  its  studies  toward  problems  of  the  intimate 
relationships  between  infectious  agents  and  their  actual  or  potential 
invertebrate  vectors.  Occasionally,  symbiotic  organisms  in  arthropods 
come  under  scrutiny.  Efforts  during  the  past  year  have  been  directed 
primarily  toward  continuation  of  experimental  tick  paralysis,  completion 
of  studies  on  transovarial  tick  transmission  of  Pasteurella  tularensis, 
intensification  of  studies  on  relationships  of  Rickettsia  rickettsii  and 
Colorado  tick  fever  virus  in  vectors,  and  a  reconsideration  of  evidence 
on  the  epidemiology  of  the  hemorrhagic  fever  known  as  epidemic  nephroso- 
nephritis.  An  intensive  attack  on  potential  disease  relationships  of 
chigger  mites  in  Panama  was  organized. 

Methods: 

Field  and  laboratory  methods  are  mainly  those  developed  at  the 
Rocky  Mountain  Laboratory,  including  more  recent  fluorescent  microscopy 
as  applied  to  vertebrate -invertebrate  cycles  of  disease  agents. 

Major  findings: 

1.  A  successful  cryostat  technique  for  the  thin-sectioning  of 
Part  B  included:  Yes 

27 


Serial  No.  NIAID-171* 

fresh-frozen  tissues  of  hard  and  soft  ticks  was  developed  in  conjunction 
with  Dr.  J.  E.  Tobie  of  the  Laboratory  of  Immunology,  NIH.  This  was  a 
necessary  antecedent  to  refined  studies  of  certain  tick-borne  agents  by 
fluorescent  microscopy.   In  comparison  with  older  techniques  this  method 
is  much  more  rapid  and  is  a  marked  improvement  for  sectioning  and  stain- 
ing both  nymphs  and  adults  of  these  ticks  in  various  states  of  engorgement. 

2.  The  incidence  of  tick  paralysis  in  man  is  not  necessarily  a 
reflection  of  the  relative  ability  of  indigenous  ticks  to  produce  the 
disease  in  hamsters.  From  2U$  to  90$  of  test  lots  of  ticks  (k&   lots) 
from  3  areas  in  Colorado  caused  experimental  tick  paralysis  though  only 
3  cases  were  reported  in  the  entire  state  up  to  1950;  whereas  only  from 
0$  to  50$  of  similar  lots  (39)  from  3  areas  in  a  single  county  (Ravalli) 
in  western  Montana  were  paralytic  though  7  local  cases  have  been  reported 
in  the  same  period.  On  the  other  hand,  75$  of  16  lots  from  adjoining 
Missoula  County  were  positive.  If  ecologic  factors  were  responsible  for 
these  differences,  they  have  so  far  eluded  solution. 

3-  Species  of  ticks  of  the  genus  Argas,  which  are  virtually  indis- 
tinguishable in  the  adult  stage,  have  been  separated  by  taxonomic  char- 
acters present  in  the  larval  stage.  This  has  resulted  in  clarification 
of  a  subspecies  which  infest  bats  in  Egypt  and  in  the  discovery  that  a 
species  found  in  cliff  swallow  nests  in  western  North  America  and  another 
from  birds  in  Chile  are  new.  Present  classification  of  other  species, 
particularly  parasites  of  migratory  birds  and  bats,  probably  requires 
clarification  as  a  prerequisite  to  investigations  of  the  disease-carrying 
potential  of  the  ticks. 

h.   A  new  funguslike  agent  which  causes  death  in  white  mice  and 
embryonated  chicken  eggs  has  been  isolated  from  Argas  found  in  nests  of 
local  cliff  swallows.  Studies  are  under  way  to  assess  its  systematic 
relationship . 

Significance: 

New  data  confirm  the  utility  of  fluorescent  microscopy  in  study 
of  arthropod-borne  agents  as  applied  particularly  to  rickettsial  agents 
(see  No.  171)-  Special  tissue  tropisms  are  now  more  readily  detectable, 
and  a  tool  is  provided  that  may  enable  estimation  of  growth  in  different 
stages  and  periods  of  a  given  cycle.  The  new  techniques  for  tick  section- 
ing will  be  applicable  to  other  fields  and  right  now  mosquitoes  are  being 
subjected  to  similar  techniques. 

Previous  advances  in  knowledge  of  the  chigger-mite  fauna  of  Central 
and  South  America  will  now  pay  dividends  in  facilitating  the  testing  of 
species  in  Panama  for  presence  of  pathogenic  agents.  This  illustrates 
the  advantages  of  fore -knowledge  of  parasitic  arthropods  in  a  given  fauna 
over  information  obtained  after  a  health  problem  has  arisen.  In  Korea, 
for  example,  a  hurried  survey  of  medically  important  parasites  lagged 

28 


Serial  No.  NIAID-17^ 

behind  the  emergency  need  for  epidemiologic  studies  of  hemorrhagic  fever 
in  military  forces. 

Several  faunal  studies  of  this  nature  on  parasites  in  various 
parts  of  the  world  were  completed  or  were  under  way  during  the  year  by 
appropriate  staff  specialists;  the  significance  of  these  studies  may  only 
become  apparent  when  some  future  need  arises. 

Proposed  course; 

As  opportunity  arises,  the  relationship  of  acarine  and  insect- 
borne  pathogens  will  continue  to  be  investigated.  Strains  of  supposed 
Rickettsia  prowazekii  reported  to  have  been  isolated  from  tick  and  domes- 
tic  animal  sources  in  Abyssinia  are  now  under  study.  Attempts  to  purify 
the  salmon -poisoning  agent  for  antigenic  purposes  were  not  fruitful  during 
the  year,  but  the  problem  will  be  attacked  by  a  new  technique.  Some 
initial  promising  fluorescent  antibody  studies  on  this  agent  will  be 
elaborated. 

The  Panama  chigger-mite  unit  may  be  expected  to  have  some  results 
from  initial  field  and  laboratory  tests. 

A  revision  of  the  world  systematics  of  the  important  tick  genus, 
Argas,  in  collaboration  with  Dr.  Harry  Hoogstraal  of  NAMRU  3  in  Cairo, 
Egypt,  should  be  well  advanced  by  the  end  of  next  year. 


29 


Serial  No.  NIAID-171* 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Brennan,  J.  M.:  Ectonyx,  a  new  Neotropical  genus  of  chiggers  (Acarina: 
Trombiculidae) .  Acarologia  Il(l):  88-91.  i960. 

Brennan,  J.  M.  and  Dal  mat,  H.  C:  Chiggers  of  Guatemala  (Acarina: 
Trombiculidae).  Ann.  Ent.  Soc.  Amer.  53(2):  183-191.  March  i960. 

Philip,  C.  B.:  New  North  American  Tabanidae.  XI.  Supplemental  notes 
pertinent  to  a  catalog  of  Nearctic  species.  Ann.  Ent.  Soc.  Amer.  53(3): 
36U-369.  May  I960. 

Brennan,  J.  M.  and  White,  J.  S.:  New  records  and  descriptions  of  chiggers 
(Acarina:  Trombiculidae)  on  bats  in  Alabama.  J.  Parasitol.  U6(3):  3^+6- 
350.  June  i960. 

Kohls,  G.  M.:   Records  and  new  synonymy  of  New  World  Haemaphysalis  ticks, 
with  descriptions  of  the  nymph  and  larva  of  H.  juxtakochi  Cooley.  J. 
Parasitol.  U6(3):  355-361.  June  i960. 

Newhouse,  V.  F.:   Birds  of  selected  irrigated  river  valleys  of  west- 
central  Idaho.  Murrelet  Ul(l):  1-6.  i960. 

Philip,  C.  B.:   Further  records  of  Neotropical  tabanidae  (Diptera)  mostly 
from  Peru.  Proc.  Calif.  Acad.  Sci.,  1+th  Series  XXXl(3):  69-102. 
July  8,  i960. 

Brennan,  J.  M.  and  Maki,  W.  A.:  An  inexpensive  and  effective  indoor 
insectary.  J.  Econ.  Ent.  53(h):   685-688.  Aug.  i960. 

Hoogstraal,  H.  and  Kohls,  G.  M.:  Observations  on  the  subgenus  Argas 
(ixodoidea,  Argasidae,  Argas) .  1.  Study  of  A.  reflexus  reflexus 
(Fabricius,  179*0,  the  European  bird  argasid.  Ann.  Ent.  Soc.  Amer.  53(5): 
611-618.  Sept.  I960. 

Kohls,  G.  M.  and  Hoogstraal,  H.:  Observations  on  the  subgenus  Argas 
(ixodoidea,  Argasidae,  Argas) .  2.  A.  cooley i,  new  species,  from  western 
North  American  birds.  Ann.  Ent.  Soc.  Amer.  53(5):  625-63I.  Sept.  i960. 

Philip,  C.  B.:  Another  hcQarc tic  species  of  Tabanidae  (Diptera).  Canad. 
Ent.  XCII(9):  697-699-  Sept.  i960. 

30 


Serial  No.  NIAID-171* 

Kohls,  G.  M.:  Ixodides.  McGraw-Hill  Encyclopedia  of  Science  and 
Technology.  McGraw-Hill  Book  Co.,  Inc.,  New  York.  i960.  Vol.  7, 
pp.   298-299. 

Kohls,   G.  M.:      Ixodes  (Endopalpiger)    zaglossi,   n.    sp.   from  the  long-beaked 
echidna  of  New  Guinea  (Acarina,    Ixodidae) .      Acarologia  2(U):    M+7-I+52. 
Oct.   i960. 

Brennan,   J.  M.:      Eight  new  species  of  Pseudoschongastia  from  Mexico  and 
Panama  with  a  revised  key  to  species  (Acarina:    Trombiculidae) .     Acarologia 
2(U):    I+8O-U92.     Oct.   i960. 

Brennan,   J.  M.   and  Jones,   E.  K. :     Chiggers  of  Trinidad,   B.W.I.,    (Acarina: 
Trombiculidae).     Acarologia  2(U) :    1+93-51+0.     Oct.   i960. 

Philip,  C.   B.:     Malaysian  Parasites.     XXXV.     Description  of  some  Tabanidae 
(Diptera)   from  the  Far  East.     Malaysian  Parasites  No.   29,  pp.   1-32.     i960. 

Philip,  C.   B.:     Malaysian  Parasites.     XXXVI.     A  summary  review  and  records 
of  Tabanidae  from  Malaya,   Borneo,    and  Thailand.     Malaysian  Parasites  No. 
29,  PP.   33-78.     I960. 

In  press: 

Philip,  C.  B.  and  Mackerras,  I.  M.:  On  Asiatic  and  related  Chrysopinae 
(Diptera:  Tabanidae).  Philippine  J.  Sci. 

Philip,  C.  B.:  Additional  records  of  Tabanidae  (Diptera)  from  the  West 
Coast  of  South  America.  Pan-Pacific  Ent. 

Fairchild,  G.  B.  and  Philip,  C.  B.:  A  revision  of  the  Neotropical  genus 
Dichelacera,  subgenus  Dichelacera,  Macquart  (Diptera,  Tabanidae).  Studia 
Entomologica,  Brazil. 

Philip,  C.  B.:  Three  new  Tabanine  flies  (Tabanidae,  Diptera)  from  India. 
Ent.  J.  India. 

Hoogstraal,  H.  and  Kohls,  G.  M.:  Observations  on  the  subgenus  Argas 
(ixodoidea,  Argasidae,  Argas) .   3-  A  biological  and  systematic  study  of 
A.  reflexus  hermanni  Audouin,  1827  (revalidated),  the  African  bird  argasid. 
Ann.  Ent.  Soc.  Amer. 

Brennan,  J.  M.  and  Jones,  E.  K.:   New  genera  and  species  of  chiggers  from 
Panama  (Acarina:  Trombiculidae).   J.  Parasitol. 

Brennan,  J.  M.  and  Jones,  E.  K.:  Chiggers  of  Peru  (Acarina:  Trombiculidae). 
Acarologia. 


31 


Serial  No.  NIAID-17^ 

Kohls,  G.  M.  and  Clifford,  CM.:  A  new  species  of  Ixodes  ( Lep_idixodes ) 
from  bats  in  Malaya,  North  Borneo,  and  the  Congo  (Acarina-Ixodidae) . 
Acarologia. 

Philip,  C.  B.  and  Burgdorfer,  W.:  Arthropod  vectors  as  reservoirs  of 
microbial  disease  agents.  Ann.  Rev.  Ent.  6:   I96I. 

Philip,  C.  B.:  Arthropod  vectors  in  relation  to  the  reservoir  mechanism 
of  microbial  agents  of  animal  diseases.  Presented  at  11th  Internat.  Ent. 
Cong.,  Vienna,  Aug.  i960.  Acta  Tropica. 

Philip,  C.  B.:  Proposal  to  validate  under  the  plenary  powers  the  specific 
name  Akamushi  (Trombidium)  Brumpt,  (Class  Acarina) .  Z.N.(S)  1+00. 
Internat.  Bull.  Zool.  Nomenclature. 

Philip,  C.  B.:  Proposal  to  validate  under  the  plenary  powers  the  specific 
name  Dermacentor  andersoni  Venus tus .   Internat.  Bull.  Zool.  Nomenclature. 

Philip,  C.  B.:  New  North  American  Tabanidae.  XIII.  Change  of  name  for 
a  well-known  species  of  Chrysops.  Ent.  News. 

Honors  and  Awards  relating  to  this  project: 

Glen  M.  Kohls 

Continued  as  an  Assistant  Editor  for  the  Journal  of  Parasitology. 

Invited  by  the  Editor  of  the  Annals  of  the  Entomological  Society  of 
America  to  prepare  a  review  of  Dr.  D.  R.  Arthur's  monograph  on  several 
genera  of  ticks  published  by  the  Cambridge  University  Press. 

Vice  President  and  member  of  the  program  committee  of  the  International 
Northwest  Conference  on  Diseases  in  Nature  Communicable  to  Man. 

Dr.  C.  B.  Philip 

Invited  to  serve  on  the  "Research  and  Engineering  Advisory  Panel  on 
Biological  and  Chemical  Defense"  under  the  office  of  the  Director  of 
Defense  Research  and  Engineering.   (May  6,  i960) 

Received  the  Outstanding  Achievement  Award  of  the  University  of 
Minnesota,  which  is  reserved  for  former  students  of  the  institution 
who  have  attained  "high  eminence  and  distinction."   (June  k,    i960) 

Invited  to  be  a  Visiting  Lecturer  in  the  program  of  the  Academic  Year 
Institute  for  High  School  Teachers  of  Science  and  Mathematics,  I96O-6I. 

Continued  as  lecturer,  CDC  Training  Course,  PES,  Atlanta,  February 
1960-61. 

32 


Serial  No.  NIAID-175 

Rocky  Mountain  Laboratory 
Hamilton,  Montana 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:  Allergy  and  immunology  of  fungal  infections  and  the 
mechanisms  of  allergic  phenomena 

Principal  Investigator:   S.  B.  Salvin 

Other  Investigators:     M.  B.  Gregg,  R.  F.  Smith 

Cooperating  Units:       None 

Man  Years  (calendar  year  i960): 
Total:        13-25 
Professional:    k.25 
Other:         9-00 

Project  Description: 

Objectives: 

Further  efforts  to  uncover  the  mechanisms  responsible  for  delayed 
allergy  have  been  stimulated  by  relationships  recently  established  between 
delayed  allergy  and  homograft  rejection  and  collagen  or  "auto-immune" 
disease.  Three  main  objectives  of  this  project  are:   (l)  to  study  the 
biologic  mechanisms  responsible  for  the  development  and  expression  of 
delayed  hypersensitivity  in  experimental  animals;   (2)  to  relate  delayed 
allergy  to  other  immunologic  phenomena  such  as  antibody  formation,  contact 
hypersensitivity,  or  tolerance;  and  (3)  to  apply  such  knowledge  of  delayed 
hypersensitivity  to  its  possible  role  in  the  pathogenesis  of  "auto-immune" 
diseases  such  as  lupus  erythematosis  and  rheumatoid  arthritis,  as  well  as 
to  certain  infectious  diseases,  such  as  rheumatic  fever,  tuberculosis,  or 
histoplasmosis. 

Methods: 

Although  biologic  and  chemical  procedures  typical  of  immunologic 
investigations  are  used,  emphasis  has  been  placed  on  some  quantitative 
immunochemical  procedures  for  analyses  and  study  of  allergy  and  antigen- 
antibody  reactions.  Highly  purified  proteins,  such  as  diphtheria  toxoid, 
egg  albumin,  and  bovine  gamma  globulin,  are  used  as  antigens,  either 
singly  or  conjugated  with  a  wide  variety  of  haptenic  groupings  and  carbo- 
hydrates. A  new  technique  involves  the  use  of  radioactive  conjugates  to 

Part  B  included:  Yes 

33 


Serial  No.  NIAID-175 

trace  the  progress  of  antigen -antibody  reactions  within  the  cell  by  auto- 
radiographic procedures.  Also,  electron  opaque  metals  conjugated  to 
conventional  proteins  are  used  to  trace  the  course  of  the  antigen  within 
the  cell. 

Major  findings: 

Previous  studies  have  shown  that  delayed  allergy  in  guinea  pigs 
is  superseded  by  circulating  antibody  and  Arthus  reactions  if  a  large 
antigenic  dose  is  given.  When  guinea  pigs  are  sensitized  with  a  small 
dose,  only  delayed  hypersensitivity  develops.  Animals  given  a  smal 1 
dose  of  antigen  develop  maximum  anamnestic  responses  when  a  second  injec- 
tion is  given  at  the  time  of  maximum  delayed  hypersensitivity.  These  and 
other  observations  strongly  indicate  that  delayed  hypersensitivity  is  an 
immature  form  of  the  immune  response. 

1.  In  studies  on  the  anamnestic  response  to  conjugated  diazotized 
proteins,  guinea  pigs  developed  delayed  hypersensitivity  to  the  protein 
moiety,  but  developed  specific  circulating  antibody  directed  toward  small 
chemical  configurations  present  only  in  the  booster  antigen. 

2.  Carbohydrates,  when  conjugated  to  proteins,  act  as  haptens  and 
can  be  used  to  detect  circulating  antibody.  Since,  however,  the  speci- 
ficity of  delayed  allergy  is  directed  toward  the  protein,  the  carbohydrate 
can  neither  induce  nor  detect  delayed  hypersensitivity. 

3.  Contact  hypersensitivity,  which  is  believed  to  be  analogous  to 
delayed  allergy,  may  be  induced  in  experimental  animals  by  repeated  appli- 
cation of  a  simple  chemical  (2,U-dinitro-l-flurobenzene,  DFB)  on  the  skin 
or  by  intradermal  injection  of  the  chemical  in  Freund' s  adjuvant.  Animals 
sensitized  with  a  simple  chemical  develop  circulating  antibody  and  Arthus 
reactions  to  a  conjugate  composed  of  hapten  and  a  protein  such  as  guinea 
pig  serum  and  react  to  a  surface  application  of  the  hapten  only.   In  con- 
trast, animals  sensitized  with  the  conjugate  (DFB  guinea  pig  serum) 
develop  Arthus  reactions  and  circulating  antibody  directed  toward  the 
hapten  and  produce  typical  delayed  allergy  to  subsequent  intradermal  in- 
jection of  the  conjugate,  but  do  not  react  to  surface  application  of  the 
simple  chemical.  This  inconsistency,  that  synthetic  conjugates  fail  to 
induce  contact  skin  hypersensitivity  to  the  hapten,  but  that  hapten  in- 
duces Arthus  reactions  to  the  conjugate,  may  be  explained  by  one  of  two 
possible  hypotheses:  (a)  When  the  host  animal  is  injected  with  simple 
chemicals,  these  substances  may  enter  intracellular  areas  inaccessible  to 
extracellular  proteins.  Subsequent  conjugation  leads  to  conjugates  which 
exist  in  intracellular  sites  inaccessible  to  conjugates  prepared  in  vitro. 
Several  workers  have  provided  evidence  that  this  phenomenon  may  be  func- 
tional,  (b)  The  protein  members  of  in  vivo  conjugates  are  unique  and 
have  not  been  duplicated  by  proteins  chosen  for  in  vitro  preparations. 
The  observation  that  intradermal  injection  of  an  in  vitro  conjugate  of 
soluble  guinea  pig  skin  protein  with  DFB  produces  contact  hypersensitivity 

3h 


Serial  No.  NIMD-175 

to  surface  application  of  the  hapten  as  well  as  delayed  and  Arthus 
reactions  to  skin  tests  with  the  conjugate  supports  this  hypothesis. 
Thus,  the  production  of  contact  hypersensitivity  is  directly  related  to 
a  particular  protein  or  proteins  in  the  host  tissue  and  the  specificity 
of  contact  hypersensitivity  is  analogous  to  that  of  delayed  reactions. 

h.   In  further  substantiating  the  role  of  delayed  allergy  in  the 
immune  process,  immunologic  studies  were  conducted  in  newborn  guinea 
pigs  because  they  may  show  immaturity  in  the  formation  of  delayed  allergy 
and  circulating  antibody.  When  these  animals  are  sensitized  within  12 
hours  after  birth,  typical  delayed  responses  cannot  be  evoked  for  about 
2  weeks.  By  this  time,  however,  circulating  antibody  has  appeared,  and 
animals  develop  typical  Arthus  reactions  when  challenged.   If  sensitiza- 
tion of  neonatal  animals  is  postponed  for  12  to  lk   days  after  birth, 
delayed  allergy  is  not  apparent,  but  circulating  antibody  appears  about 
11  days  after  sensitization.  When  the  neonatal  animal  is  sensitized 
later  than  lk   days  after  birth,  allergic  and  antibody  responses  are  simi- 
lar to  those  of  the  adult.  These  observations  suggest  either  that  delayed 
hypersensitivity  cannot  be  produced  by  the  neonatal  animal  but  conventional 
antibody  can,  or  that  the  basic  mechanism  of  delayed  hypersensitivity  is 
present  but  cannot  be  made  manifest  by  the  host  animal  because  of  some 
inherent  deficiency.  The  following  observations  suggest  that  the  latter 
hypothesis  is  true:  (a)  delayed  hypersensitivity  cannot  be  transferred 
passively  to  newborns  with  lymph-node  cells  from  highly  sensitized  adults, 
(b)  delayed  hypersensitivity,  however,  can  be  transferred  passively  to 
normal  adults  with  lymph-node  cells  from  highly  sensitized  neonatal  guinea 
pigs  which  themselves  do  not  show  delayed  hypersensitivity,  and  (c)  con- 
tact hypersensitivity  can  be  exhibited  in  neonatal  animals  although  it 
is  somewhat  slow  in  onset. 

5.  Guinea  pigs  sensitized  to  purified  ultraviolet-killed  Type  II 
poliovirus  develop  an  anamnestic  response  to  a  second  injection  of  antigen. 

6.  Through  chemical  and  physical  fractionation  procedures,  a  spe- 
cific skin-test  antigen  has  been  prepared  from  Cryptococcus  neoformans, 
a  pathogenic  fungus  frequently  isolated  from  pigeon  excreta  in  the 
vicinity  of  New  York  City.  This  antigen  produces  large  and  impressive 
delayed-type  skin  reactions  in  guinea  pigs  previously  infected  with  C. 
neoformans.  The  active  fraction  of  this  antigen  has  been  purified  and 
its  biochemical  properties  determined. 

Significance: 

To  date, experimental  data  support  the  hypothesis  that  delayed 
hypersensitivity  is  an  immature  phase  of  the  classical  immune  response. 
Studies  on  contact  sensitivity  induced  by  simple  chemicals  indicate  that 
its  specificity  and  dynamics  are  similar  to  those  of  conventional  delayed 
hypersensitivity.  The  inability  to  invoke  typical  delayed  responses  in 
a  neonatal  animal  appears  to  be  attributable  to  an  inherent  deficiency 

35 


Serial  No.  NIAID-175 

which  prevents  the  young  host  from  manifesting  an  established  hypersensi- 
tive state.  Hence,  even  in  the  young  animal,  delayed  allergy  is  an  early 
phase  in  the  antibody  response. 

In  view  of  the  prevalence  of  C.  neoformans  in  pigeon  excreta  in 
urban  areas,  large  numbers  of  people  are  exposed  to  this  fungus.  There- 
fore, cryptococcosis  in  a  mild,  unrecognized  form  may  be  much  more  preva- 
lent than  the  relatively  infrequent  occurrence  of  this  disease  would 
indicate.  The  preparation  of  a  specific  skin-test  antigen  creates  a  new 
tool  for  conducting  epidemiologic  studies  of  this  disease. 

Proposed  course: 

Studies  on  the  immune  mechanism  in  the  neonatal  animal  will  be 
continued.  The  possibility  exists  that  a  cellular  or  humoral  factor  is 
necessary  or  supplemental  for  manifestation  of  delayed  hypersensitivity 
in  an  infant  or  in  a  newborn  animal.  Experiments  are  in  progress  to  ex- 
amine this  hypothesis.  In  addition,  skin  grafts  from  adults  to  newborn 
and  vice  versa  are  being  used  to  determine  whether  maturity  of  the  skin 
is  essential  for  delayed  reactions.  Since  gamma  radiation  of  newborn 
guinea  pigs  delays  the  appearance  of  Arthus  reactions,  this  technique  is 
also  being  used  to  postpone  the  appearance  of  antibody  and  thus  aid  in 
the  determination  and  examination  of  delayed  reactions. 

Studies  with  live  bacteria  such  as  Mycobacterium  tuberculosis 
have  been  initiated  to  determine  if  the  response  relative  to  hypersensi- 
tivity and  immunity  of  the  newborn  differs  from  that  of  the  adult. 

Further  investigation  will  be  made  on  the  effect  of  age  and  the 
role  of  skin  and  other  cellular  elements  in  the  allergic  response. 

To  clarify  the  part  delayed  allergy  plays  in  antibody  formation, 
antigen  should  be  traced  through  the  cell,  and  its  particular  activity 
within  the  cell  should  be  correlated  with  the  gross  reaction  of  the  host. 
Two  techniques  will  be  used.  One  involves  the  injection  of  guinea  pigs 
with  the  electron-opaque  protein  conjugate  and  the  determination  of  the 
exact  locus  and  relationship  of  the  antigen  within  the  cell  by  examination 
of  ultrathin  sections  under  the  electron  microscope.  The  other  method 
involves  a  study  of  the  cellular  disposition  of  injected  radioactive 
proteins  and  the  position  of  antigen  within  the  cell.  Radioactive  anti- 
gens and  subsequent  antibody  combinations  are  traced  by  autoradiographic 
techniques. 

Experiments  on  sensitization  of  guinea  pigs  with  purified  polio- 
virus  will  be  continued  in  order  to  learn  whether  sensitization  with 
Types  I  or  III  will  produce  an  anamnestic  response  on  later  introduction 
of  Type  II  virus. 


36 


Serial  No.   NIA  33-175 

Standardization  of  the  skin-test  antigen  for  detecting  crypto- 
coccosis in  man  and  collaborative  epidemiologic  studies  of  this  disease 
are  contemplated. 


37 


Serial  No.  NIAID-175 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Salvin,  S.  B.:   Resistance  of  animals  and  man  to  histoplasmosis.   In 
Histoplasmosis.  Chas.  C  Thomas,  Springfield,  111.,  i960,  pp.  99-112. 

Salvin,  S.  B.  and  Smith,  R.  F.:   Specificity  of  allergic  reactions. 

I.  Delayed  versus  Arthus  hypersensitivity.  J.  Exp.  Med.  111(4):  U65-483. 
April  I960. 

Salvin,  S.  B.  and  Smith,  R.  F.:  Delayed  hypersensitivity  and  the 
anamnestic  response.  J.  Immunol.  8U(5):  UU9-U57.  May  i960. 

Salvin,  S.  B.  and  Smith,  R.  F.:   Specificity  of  allergic  reactions. 

II.  Azoproteins  in  the  anamnestic  response.  Proc.  Soc.  Exp.  Biol.  &  Med. 
10U(U);  584-590.  Aug. -Sept.  i960. 

Honors  and  Avards  relating  to  this  project: 

Dr.  S.  B.  Salvin 

Invited  to  contribute  paper  in  "Progress  in  Allergy,"  S.  Karger, 
Basel,  Switzerland,  publisher. 

Invited  to  present  series  of  lectures  and  seminars  on  allergy  and 
immunology  at  the  University  of  Montana,  Missoula,  Montana. 


38 


Serial  No.   NIAID-I76 

Rocky  Mountain  Laboratory 
Hamilton,   Montana 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:  Viruses,  virus  components,  and  virus  surfaces 

Principal  Investigator:   B.  H.  Hoyer 

Other  Investigators:     R.  K.  Gerloff,  F.  G.  Jarvis,  R.  A.  Ormsbee,  and 

D.  B.  Ritter 

Cooperating  Units: 

Man  Years  (calendar  year  i960): 
Total:         11.5 
Professional:    3*5 
Other:         8.0 

Project  Description: 

Objectives: 

Elucidation  of  the  mechanisms  involved  in  the  synthesis  of  virus 
protein  and  RNA  and  clarification  of  fundamental  relationships  between 
virus  protein  and  RNA  continue  to  be  major  objectives.  The  finding  of 
others  that  poliovirus  protein  and  RNA  are  synthesized  simultaneously  at 
similar  rates  required  us  to  make  changes  in  our  investigations.  Current 
interest  in  live  poliovirus  vaccines  has  stimulated  studies  on  the  behavior 
of  surface  proteins  of  virulent  and  avirulent  poliovirus  in  column  chroma- 
tography.  A  sensitive  precipitation  system  for  detecting  antibody  against 
poliovirus  is  being  developed. 

Methods: 

Quantitative  and  qualitative  methods  appropriate  for  the  study  of 
virus  proliferation  and  cell  metabolism  are  employed.  Cellular  and  virus 
fractions  are  separated  and  characterized  by  chemical  and  physical  means. 
Radioactive  labels,  p32  and  S^ ,  are  used  in  following  certain  biologic 
processes  and  in  studying  biophysical  properties  of  viral  components. 


Part  B  included:  Yes 

33 


Serial  No.  NIA3D-176 
Major  findings; 

op 

1.  Previous  studies  have  shown  that  ¥->   -labeled  type  3  Sauckett 
virus,  type  2  MEF-1  virus,  and  type  1  Mahoney  virus  can  be  separated  by 
column  chromatography.   In  continued  investigations,  Leon  and  Sauckett 
type  3  viruses  could  be  separated  from  each  other  by  elution  from  the 
DEAE  column  at  different  pH  (7.7  vs.  7.1)  values. 

2.  CHAT  poliovirus,  an  avirulent  type  1,  was  separated  from  the 
virulent  parent  by  elution  in  a  salt  or  pH  gradient,  but  FOX  and  Wi-1 
could  not  be  separated  from  their  virulent  counterparts.  CHAT  and  Wi-1, 
both  avirulent  variants,  could  be  separated  from  each  other  by  virtue  of 
different  elution  properties. 

3-  After  prolonged  storage,  avirulent  poliovirus  binds  more  firmly 
to  cellulose  ion  exchange  columns  than  does  its  virulent  counterpart. 
This  property  is  probably  due  to  a  more  rapid  change  in  avirulent  virus 
protein  and  indicates  a  less  stable  surface. 

h.   Because  of  the  large  ratio  (1:10  to  1:500)  between  plaque -forming 
units  (PFU)  and  physical  particles,  virus  proteins  could  not  be  character- 
ized without  knowledge  of  the  proportion  of  total  virus  that  reacts  with 
the  cell.  After  S^-7-  or  p32_iabeled  polioviruses  were  mixed  with  KB  cells 
in  a  virus-cell  ratio  of  20  to  1,  samples  were  removed  at  intervals  and 
examined  in  CsCl  or  RbCl  equilibrium-density  gradients.   Radioactivity 
and  infectivity  disappeared  at  the  same  rate  from  the  supernatant  medium, 
and  the  virus  shifted  from  the  density  zone  characteristic  of  whole  virus. 
Most  of  the  radioactivity  that  had  left  the  virus -density  zone  was  con- 
centrated in  a  well-defined  region  in  the  upper  level.  Alteration  of  the 
density  of  original  virus  particles  indicates  that  most  of  the  virus 
actually  reacts  with  susceptible  cells. 

5.  Infectious  RNA  was  prepared  from  highly  purified  poliovirus  by 
phenol  extraction.   RNA  preparations  of  fairly  uniform  physical  properties 
had  an  Sgo  w  °^  37 •   Also,  a  30$  increase  in  ultraviolet  absorption  after 
treatment  with  RNAase  indicates  an  initial  high  degree  of  polymerization. 
Both  octyl  alcohol-chloroform  and  neutral  hydroxylamine  (0.1  M)  destroyed 
more  than  99$  of  RNA  infectivity  after  10  minutes  at  room  temperature, 
but  the  molecular  properties  of  RNA,  as  determined  by  ultracentrifugation 
patterns,  were  not  affected. 

6.  In  a  preparation  of  RNA  yielding  1$  of  the  PFU  present  in  whole 
virus,  the  s35  content  was  only  0.001$  of  that  of  the  whole  virus.  Thus, 
residual  protein  cannot  account  for  more  than  0.1$  of  the  RNA  activity. 

7.  The  method  originally  used  for  preparation  of  purified  poliovirus 
was  modified  by  treating  crude  DEAE  filtrates  of  tissue-culture  fluids 
with  0.2$  DEXTRAN  500,  6.1+5$  Carbowax  6000  and  0.3  M  NaCl  (final  concen- 
tration). After  standing  for  2k   to  h&   hours  at  h     C.,  liquids  separate 


Serial  No.  NIMD-176 

into  two  phases,  the  lower  of  which  contains  all  the  virus.  From  2,700  ml. 
of  culture  fluid,  all  the  virus  will  be  present  in  the  lower  20  ml.  of 
liquid. 

8.  An  extremely  sensitive  and  specific  precipitation  test  for 
detecting  antibody  against  poliovirus  has  "been  developed.  A  serum  with 
a  neutralizing  antibody  titer  of  1:256  possesses  a  precipitation  titer  of 
l:l6,38U.  Serum  dilutions  are  mixed  with  p32-iabeled  poliovirus  and 
incubated.   Antihuman  gamma  globulin  is  added,  followed  by  further  incu- 
bation. The  precipitate  formed  is  then  removed  by  centrifugation.   If  a 
significant  amount  of  radioactivity  is  present  in  the  sediment,  precipita- 
tion of  virus  has  occurred. 

Significance: 

The  behavior  of  derived  avirulent  poliovirus  in  column  chromatog- 
raphy indicates  some  alterations  in  surface  protein  differing  from  that 
of  parent  strains.  Although  other  workers  claim  that  avirulent  poliovirus 
can  be  separated  uniformly  from  virulent  virus  because  the  avirulent 
strains  are  absorbed  more  firmly  to  cellulose  columns,  our  findings  indi- 
cate that  generalizations  regarding  the  binding  properties  and  the  elution 
profiles  of  avirulent  strains  cannot  be  made. 

In  spite  of  the  well-established  high  ratio  between  the  PFU  and 
virus  particles,  the  bulk  of  virus  in  an  inoculum  actually  binds  to  the 
cells  and  forms  material  of  lower  density.   Thus,  this  wide  ratio  is 
probably  explained  by  the  limited  ability  of  RNA  to  enter  sites  where 
virus  production  can  be  influenced.  Other  workers  have  proposed  that  RNA 
infectivity  may  be  destroyed  if  only  a  small  group  of  the  long  molecular 
chain  is  inactivated.   Our  findings  indicate  that  major  molecular  changes 
do  not  occur  when  RNA  is  inactivated  by  hydroxylamine  or  octyl  alcohol- 
chloroform.  Neither  of  these  should  harm  RNA,  and  it  is  entirely  possible 
that  infectivity  of  the  relatively  large  RNA  molecule  can  be  destroyed  by 
uncoupling  of  one  link.  If  this  is  true  and  if  the  link  is  unique,  an 
approach  to  virus  chemotherapy  is  indicated. 

The  case  for  infectivity  of  RNA  per  se  was  considerably  strengthened 
by  demonstrating  that  only  0.1$  of  the  RNA  infectivity  could  be  accounted 
for  by  residual  protein. 

Proposed  course: 

The  protein  composition  of  polioviruses  will  be  studied  by  protein 
finger  printing  of  virulent-avirulent  combinations  which  will  indicate 
possible  qualitative  differences  in  their  proteins.  Column  elution  proper- 
ties and  biophysical  tools  such  as  electrophoresis,  ultracentrifugation, 
and  equilibrium-density  centrifugation  may  indicate  differences  in  folding 
if  qualitative  differences  do  not  exist.   Our  ability  to  label  both  protein 
and  nucleic  acid  of  the  poliovirus  will  facilitate  analytical  work  in 

III 


Serial  No.  NIAID-I76 

regard  to  these  components  of  the  viruses. 

Investigations  will  be  continued  on  the  use  of  labeled  viruses  as 
fundamental  and  diagnostic  reagents.  Collaborative  studies  will  be  con- 
tinued on  the  anamnestic  response  elicited  in  guinea  pigs  against  purified 
poliovirus  proteins.   Investigations  are  currently  under  way  to  determine 
if  "hybrid"  animal  cells  can  be  produced  and  if  animal  cells  exposed  to 
bacterial  KNA  will  form  bacterial  protein  in  the  same  manner  that  these 
cells  form  virus  protein  when  exposed  to  virus  RNA. 


42 


Serial  No.  NIAID-1T6 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 

Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Perrine,  T.  D.:  Method  for  attaching  glass  water  aspirators  to  water 
lines.  J.  Chem.  Education  37:  h8l.     Sept.  i960. 

Oertli,  E.  and  Perrine,  T.  D.:  Magnetically  stirred  separatory  funnel. 
Chemie-Ingenieur-Technik  32(8):  55^-555.  i960.  Also  in  Chemie  fur  Labor 
und  Betrieb  8:  i960. 

Holland,  J.  J.,  Hoyer,  B.  H.,  McLaren,  L.  C,  and  Syverton,  J.  T.:  Enter- 
oviral  ribonucleic  acid.  I.  Recovery  from  virus  and  assimilation  by  cells. 
J.  Exp.  Med.  112(5):  821-839-  Nov.  1,  i960. 

Holland,  J.  J.,  McLaren,  L.  C,  Hoyer,  B.  H.,  and  Syverton,  J.  T.:  Enter- 
oviral  ribonucleic  acid.   II.  Biological,  physical  and  chemical  studies. 
J.  Exp.  Med.  112(5):  81+1-86^.  Nov.  1,  i960. 

Reinhard,  K.  R.  and  Gerloff,  R.  K.:   Immunity  towards  poliovirus  among 
Alaskan  natives.  II.  Am.  J.  Hyg.  72(3):  298-307.  Nov.  i960. 

Reinhard,  K.  R. ,  Gerloff,  R.  K.,  and  Philip,  R.  N.:   Immunity  towards 
poliovirus  among  Alaskan  natives.  III.  Am.  J.  Hyg.  72(3):  308-320.  Nov.1960. 

Honors  and  Awards  relating  to  this  project: 

Dr.  B.  H.  Hoyer 

Invited  to  be  Visiting  Professor  in  the  Department  of  Bacteriology  and 
Immunology,  The  Medical  School,  University  of  Minnesota,  196O-6I. 
Invitation  not  accepted. 

Invited  to  present  series  of  lectures  in  Virology  at  the  University  of 
Washington,  May  i960. 

Invited  to  participate  in  Animal  Virus  Symposium,  Berkeley,  California, 
I960. 

Appointed  Lecturer  in  Microbiology  196O-61,  Montana  State  University, 
Missoula,  Montana. 

Dr.  R.  A.  Ormsbee 

Appointed  lecturer  in  Microbiology,  Montana  State  University,  Missoula, 
Montana. 

K3 


Serial  No.  NIAID-177 

Rocky  Mountain  Laboratory 
Hamilton,  Montana 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:   Immune  prophylaxis  of  mycobacterial  infections 

Principal  Investigator:  E.  Ribi 

Other  Investigators:     W.  T.  Haskins  and  C.  L.  Larson 

Cooperating  Units: 

Man  Years  (calendar  year  i960): 
Total:         ^.75 
Professional:   0.75 
Other:         U.00 

Project  Description: 

Objectives:  ; 

The  primary  objective  of  this  program  has  been  to  investigate  the 
role  of  live  or  dead  tubercle  bacilli  or  fractions  thereof  in  the  production 
of  resistance  to  infection  with  virulent  tubercle  bacilli.  First,  it  was 
considered  essential  to  develop  a  test  based  upon  a  more  natural  challenge 
with  virulent  organisms  than  had  been  used  previously  and,  as  a  consequence, 
the  present  method  of  challenge  with  a  small  number  of  organisms  given  by 
aerosol  was  developed.  The  objectives  also  include  the  application  of 
this  method  as  a  control  procedure  in  the  production  of  vaccine  and  the 
use  of  the  method  to  determine  the  mechanism  upon  which  immunity  is  based. 

The  role  of  physical  and  chemical  components  of  tubercle  bacilli 
and  other  acid-fast  organisms  in  producing  or  eliciting  delayed  reactions 
and  circulating  antibodies  has  also  been  investigated. 

Methods: 

The  methods  used  have  been  in  large  measure  those  developed  at  the 
Rocky  Mountain  Laboratory.   Fractionation  of  organisms  is  accomplished  by 
means  of  mechanical  disruption.  Vaccines  are  evaluated  by  all  means 
available,  but  special  emphasis  is  placed  upon  aerosol  infection  of  immu- 
nized mice. 


Part  B  included:   Yes 


W 


Serial  No.  NIAID-177 
Major  findings: 

1.  Animals  (guinea  pigs  or  rabbits)  infected  with  live  cells  or 
sensitized  with  killed  whole  cells  or  cell  walls  of  various  acid-fast 
organisms  develop  specific  sensitivity  to  protoplasm  derived  from  these 
organisms. 

2.  Mice  immunized  with  live  cells  of  H37Ra  administered  either 
intravenously  or  by  aerosol  develop  significant  resistance  to  subsequent 
aerosol  infections  with  H37Rv.  Animals  immunized  with  H37R&  hy  any  other 
route  fail  to  show  similar  resistance. 

3«  Resistance  engendered  by  H37Ra  does  not  appear  to  be  induced  by 
interference,  for  administration  of  this  organism  either  2h   hours  before 
or  after  administration  of  HSTRv  does  not  influence  the  course  of  infection. 
In  these  studies  both  organisms  were  given  as  an  aerosol. 

k.   Continued  experience  demonstrated  that  C.  burnetii,  Br.  neotomae, 
or  P.  tularensis  infections  do  not  affect  the  course  of  pulmonary  tuber- 
culosis in  mice.  These  findings  establish  the  specific  nature  of  this 
test  for  studying  immunity  against  tuberculosis. 

5.  Live  or  dead  acid-fast  organisms  (M.  tuberculosis,  M.  phlei, 
M.  butyricum,  M.  smegmatis,  and  atypical  acid-fasts)  produce  lesions  in 
the  lungs  of  guinea  pigs  only  if  the  organisms  are  suspended  in  Freund's 
adjuvant  prior  to  subcutaneous  injection  into  the  animals. 

6.  Antibodies  were  not  detected  in  serums  from  persons  ill  with 
tuberculosis  when  tested  by  the  double  diffusion  technique  of  Parlett  and 
Youmans  or  by  Ovary's  method. 

7.  Previous  treatment  of  animals  with  BCG  organisms  produces 
hyperreactivity  to  subsequent  injections  of  Salmonella  toxins.  Relatively 
large  doses  of  cell  walls  are  required  to  produce  such  hyperreactivity. 

8.  Previously,  it  was  shown  that  the  production  of  isoallergic 
encephalitis  in  guinea  pigs  was  enhanced  when  0.05  mg.  of  Mycobacterium 
cell  walls  was  added  to  a  subcutaneously  administered  dose  of  brain 
antigen  mixed  with  Freund's  adjuvant.  As  much  as  5  mg.  of  protoplasm 
mixed  with  the  antigen  failed  to  do  so.  Further  studies  have  shown  that 
0.05  mg.  of  a  water-soluble  fraction  of  cell  walls  has  the  same  enhance- 
ment potential  as  that  possessed  by  an  equal  weight  of  cell  walls. 

Significance; 

Since  small  amounts  of  cell  walls  produce  hypersensitivity  but 
fail  to  produce  hyperreactivity  to  toxins,  it  would  appear  that  these 
two  phenomena  are  distinct.  So  far,  cell  walls  and  the  cord  factor  have 
been  the  only  fractions  of  tubercle  bacilli  capable  of  causing  hyper- 
reactivity in  mice. 

1*5 

2 


Serial  No.  NIAID-177 

Studies  of  hypersensitivity  show  that  specific  delayed  reactions 
are  induced  by  cell  walls  or  whole  cells  of  various  organisms  and  that 
the  delayed  reactions  may  be  specifically  elicited  by  protoplasm  of  these 
organisms.  These  studies  indicate  that  it  is  feasible  to  employ  proto- 
plasmic fractions  of  such  organisms  as  the  Battey  strain  to  determine  the 
role  of  this  and  other  atypical  acid-fast  bacilli  in  the  production  of 
nonspecific  tuberculin  reactions  in  man.  As  Palmer  and  his  group  have 
emphasized,  the  problem  of  determining  the  role  of  these  organisms  in 
producing  disease  (symptomatic  or  asymptomatic)  in  man  has  yet  to  be 
evaluated. 

Our  recent  findings  that  mice  can  be  immunized  against  infections 
with  virulent  tubercle  bacilli  when  both  organisms  are  administered  by 
aerosol  also  have  shown  that  the  test  we  have  developed  is  specific  and 
that  the  resistance  noted  is  not  based  on  interference.  To  date,  only 
live  organisms  have  been  found  to  engender  resistance,  but  the  resistance 
noted  is  of  such  character  that  it  indicates  the  usefulness  of  live 
organisms  in  the  prevention  of  tuberculosis  under  experimental  conditions. 

Proposed  course: 

It  is  hoped  that  a  study  can  be  initiated  to  determine  the  role 
of  acid-fasts  in  producing  disease  and/or  hypersensitivity  in  a  normal 
rural  population  in  the  western  United  States. 

Studies  will  be  continued  on  immunity  against  infections  with 
virulent  tubercle  bacilli.  Plans  are  being  made  to  examine  lots  of  BCG 
vaccine  produced  in  various  laboratories  to  determine  the  value  of  our 
mouse  test  as  a  control  for  manufacture  of  BCG  vaccines. 

Studies  of  antibody  production  by  various  fractions  of  tubercle 
bacilli  will  be  expanded. 

Studies  of  isoallergic  encephalitis  will  be  continued.  Our  results 
indicate  that  it  now  may  be  possible  to  study  this  phenomenon  at  a 
molecular  level. 


kS 


Serial  No.  NIAID-177 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

In  press: 

Larson,  C.  L.,  Ribi,  E.,  Wicht,  W.  C,  and  List,  R.:   Skin  reactions 
produced  in  rabbits  by  cell  walls  and  protoplasm  of  Mycobacterium 
tuberculosis  and  M.  butyricum.  The  Amer.  Rev.  of  Respiratory  Diseases. 


hi 


Serial  No.  NIAID-I78 

Rocky  Mountain  Laboratory 
Hamilton,  Montana 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:   Immune  prophylaxis  of  Sal monella  infections 

Principal  Investigator:  E.  Ribi 

Other  Investigators:     W.  T.  Haskins,  F.  G.  Jarvis,  and  K.  C.  Milner 

Cooperating  Units:       Dr.  Maurice  Landy,  NCI,  Dr.  Emanuel  Suter,  U.  of 

Florida,  and  Dr.  Erwin  Neter,  U.  of  Buffalo. 

Man  Years  (calendar  year  i960): 
Total:         5.5 
Professional:  2.5 
Other:        3^0 

Project  Description: 

Objectives: 

Since  immunogenic  properties  of  Gram-negative  bacteria  are  closely 
related  to  potent  antigens  located  in  the  cell  wall,  studies  are  directed 
toward  the  acquisition  of  a  more  precise  knowledge  of  the  chemical  struc- 
ture of  such  antigens  and  the  correlation  of  such  structure  with  biologic 
function.  Particular  attention  has  been  given  to  a  correlation  of  chemi- 
cal composition  with  biologic  activity,  particularly  that  reported  to  be 
attributable  to  the  so-called  "lipid  A"  fraction. 

Methods: 

Bacterial  fractions  isolated  by  physical  and  chemical  methods  are 
characterized  by  appropriate  biophysical,  biologic,  and  immunologic 
assays.   In  addition  to  the  use  of  electron  microscopy,  complete  chemical 
and  physical  analyses  are  made  to  verify  purities  of  fractions. 

Major  findings: 

Endotoxins,  which  are  generally  regarded  as  combinations  of  lipid, 
polysaccharide,  protein,  and  peptidelike  substances,  are  so  complex  that 
precise  relationships  between  chemical  constitution  and  biologic  function 
are  still  unknown  and  are  subject  to  controversy.  German  workers  contend 

Part  B  included:  Yes 


Serial  No.  NIAID-I78 

that  the  biologic  properties  of  these  complexes  are  attributable  to  the 
firmly  bound  lipid  content,    so-called  "lipid  A."     Isolated  lipid  A  is 
relatively  nontoxic,   but  these  investigators  believe  that  this     component 
is  toxic  when     conjugated  with  polysaccharide  of  the  original  complex. 

Last  year  it  was  demonstrated  that  most,   but  not  all,   of  the  pro- 
tein, peptide,    and  lipid  portions  could  be   removed  from  aqueous-ether 
extracts  without  altering  the  whole  array  of  in  vivo  and  in  vitro  biologic 
activities  of  the  original  complex.      In  comparison  of  a  Boivin-type  ex- 
tract with  a  19.2$  lipid  content  and  an  aqueous-ether  extract  with  a  1.256 
lipid  content,    large  but  not  significant  differences  in  biologic  properties 
were  disclosed.     Since  firmly  bound  lipid  had  been  determined  by  the 
Freeman  method  and  not  in  terms  of  lipid  A,    additional  investigations  were 
conducted  to  delineate  the  biologic  properties  of  bound  lipid. 

1.  Endotoxin  of  low  lipid  content  prepared  from  Salmonella 
enteritidis  by  the  aqueous-ether  method  has  been  treated  further  for  the 
removal  of  bound  lipid  by  nonhydrolytic  procedures.     Such  endotoxin,    con- 
taining 2$  to  3$  lipid  A,   was  as  potent  as  that  prepared  by  the  well-known 
phenol -water  or  Boivin  procedures,   which  yield  products  of  high  lipid  A 
content  (20$  to  30$).     To  verify  differences  in  lipid  content  of  aqueous- 
ether  preparations  and  other  types  of  endotoxins,    three  different  methods 
of  lipid  analysis  were  employed.     These  three  methods  were  in  accordance 

in  demonstrating  the  magnitude  of  differences  in  Freeman  lipid  (chloroform- 
soluble  material  released  by  hydrolysis  with  acetic  acid),   lipid  A 
(material  released  with  hydrochloric -acid  treatment),   and  esterified  fatty 
acid. 

2.  With  modified  methods,    endotoxins  having  a  nitrogen  content  of 
less  than  0.5$  and  a  fatty  acid  ester  content  of  1.7$  were  prepared.     The 
analytical  values  for  these  potent  endotoxins  did  not  differ  appreciably 
from  those  of  the  classic  haptenic  polysaccharide  in  respect  to  nitrogen, 
phosphorus,    aminohexose  and  fatty  acid  ester  content. 

3.  The  chemical  analyses  of  a  series  of  endotoxins  prepared  from 
different  bacterial  species  indicate  that  firmly  bound  lipid  (lipid  A) 
varies  according  to  the  strain  of  bacteria  and  to  the  extracting  agent. 

Introductory  information  for  the  next  three  findings. 

German  workers  have  contended  that  the  biologic  activity  of  lipid 
A  would  be  comparable  to  that  of  endotoxin  if  it  were  possible  to  disperse 
the  free  lipid  A  in  water  in  a  manner  similar  to  the  original  state 
whereby  it  is  bound  to  the  polysaccharide  carrier.     These  workers  have 
reported  that  lipid  A,   when  dispersed  in  detergent  or  when  coupled  to 
inert  protein,   has  l/lO  and  l/5,    respectively,   of  the  activity  of  the 
intact  endotoxin.     However,    in  making  these  comparisons,    endotoxin  and 
lipid  A  were  not  derived  from  the  same  source.      In  view  of  our  findings, 
which  were  at  variance  with  these  reports,    and  in  view  of  the  scarcity  of 


Serial  No.  NIAID-I78 

pertinent  biologic  data,    comparative  studies  on  the  biologic  effects  of 
lipid  fractions  and  the  endotoxins  from  vhich  they  were  prepared  were 
made  by  well-established  dose-response  assays.     Bioassays  were  based  on 
the  following  host  responses:      fever,    resistance  to  infection,    tumor 
damage,  primary  inflammation  of  the  skin,    and  toxic  death. 

h.   Without  exception,  preparations  of  lipid  A  had  only  a  small 
fraction  of  1$  of  the  biologic  activity  of  the  endotoxins  from  which 
they  were  derived. 

5.  Lipoidal  fractions  were  dissociated  from  endotoxins  by  nonhydro- 
lytic  procedures  without  appreciable  reduction  in  potency  of  the  endo- 
toxins. These  lipids  exhibited  biologic  effects  of  the  same  low  order  as 
lipid  A  isolated  by  acid  hydrolysis,  a  method  which  is  known  to  be 
destructive  to  the  potency  of  endotoxin. 

6.  Lipid  A  is  a  material  of  a  heterogeneous  nature.  For  example, 
hexosamine-free  lipids  have  been  prepared  which  are  of  comparable  potency 
to  lipid  A  containing  18$  to  20$  hexosamine.  The  German  workers  consider 
this  substance  to  be  an  essential  component  of  lipid  A. 

7.  In  order  to  obtain  information  on  the  effect  of  the  hydrophilic 
carrier  to  lipid  A  activity,  the  capacities  to  evoke  various  host  responses 
of  original  endotoxin  and  of  artificial  lipoprotein  prepared  from  it  were 
determined  in  parallel-dose  responses.  Although  artificial  lipoprotein 
had  a  biological  potency  at  least  100 -fold  lower  than  endotoxin,  it  was 
greater  than  that  of  lipid  A.  However,  less  severe  acid  treatment  is 
used  to  prepare  artificial  lipoprotein  than  is  used  to  release  lipid  A. 
The  less  severe  acid  treatment,  rather  than  increased  solubility  of 

lipid  A,  was  thought  to  be  responsible  for  the  increased  activity  of 
lipoprotein.  To  prove  this  interpretation,  a  kenetic  study  was  made  of 
the  rate  of  reduction  of  biologic  activity  and  the  rate  of  release  of 
firmly  bound  lipid  by  acid  hydrolysis.  Biologic  activity  of  endotoxin, 
as  measured  by  5  quantitative  biologic  assays,  disappeared  before  appre- 
ciable amounts  of  firmly  bound  lipid  were  released.  The  loss  of  biologic 
potency  cannot,  therefore,  be  explained  by  the  separation  of  the  water- 
insoluble  lipid  from  the  endotoxin  complex.  At  the  stage  where  acid 
treatment  in  the  kenetic  study  was  comparable  to  that  used  for  the  prepa- 
ration of  artificial  lipoproteins,  the  biologic  activity  of  residual 
endotoxin  was  about  equal  to  that  of  artificial  lipoprotein. 

8.  A  method  which  includes  2  cycles  of  curtain  electrophoresis 
was  developed  for  the  quantitative  separation  and  recovery  of  both  Vi 
and  0  antigens.  Because  this  method  does  not  involve  hydrolysis,  certain 
labile  acetyl  groups  are  not  affected,  and  the  recovered  antigen  is  about 
ten  times  more  active  than  preparations  heretofore  available.  The  biologic 
activity  of  Vi  antigen  was  shown  to  be  dependent  upon  the  presence  of 
these  labile  acetyl  groups. 

50 


Serial  No.  NIAID-I78 
Significance; 

The  injection  into  animals  or  man  of  minute  quantities  of  endo- 
toxins extracted  from  Gram-negative  bacteria  gives  rise  to  an  array  of 
striking  physiological  effects.  Among  those  which  have  been  the  subject 
of  extensive  study,  the  following  are  especially  noteworthy:   stimulation 
of  resistance  to  infection  with  both  homologous  and  heterologous  organ- 
isms, enhancement  of  antibody  production,  protection  against  radiation 
injury,  pyrogenicity,  and  induction  of  a  state  of  tolerance  to  endotoxins. 

Even  though  they  have  been  studied  intensively,  endotoxins  are 
sufficiently  complex  that  precise  relationships  between  chemical  consti- 
tution and  the  capacity  to  elicit  characteristic  reactions  in  mammals 
are  still  unknown  and  are  subject  to  controversy.  The  consensus  is  that 
these  complexes,  as  ordinarily  isolated,  consist  of  lipid,  polysaccharide, 
protein,  and  peptidelike  substances. 

Although  we  had  shown  that  the  major  portion  of  the  lipid  may  be 
removed  while  retaining  the  entire  array  of  biological  properties,  these 
results  are  at  variance  with  the  widely  accepted  view  that  firmly  bound 
lipid  is  the  actual  toxic  principle  of  endotoxins  and  that  such  lipids 
could  be  split  off  by  acids  (lipid  A)  and  their  toxic  activity  restored 
by  suitable  dispersion  in  water. 

A  major  contribution  from  this  laboratory  involves  the  use  of  a 
number  of  dose-related  quantitative  assays  to  determine  in  parallel  tests 
the  relative  potencies  of  endotoxins  and  the  lipid  fractions  derived  from 
them.  The  use  of  these  quantitative  measurements  revealed,  to  a  degree 
previously  not  suspected,  that  endotoxins  are  far  more  potent  than  their 
corresponding  lipids. 

The  study  of  the  kinetics  of  acid  hydrolysis  of  endotoxin  showed 
a  progressive  destruction  of  potency  which  was  virtually  complete  before 
any  separation  of  firmly  bound  lipid.  It  is  believed  that  the  small 
amount  of  activity  exerted  by  lipid  fractions  is  of  a  special  kind, 
unrelated  to  the  major  activity  of  the  complete  endotoxin.  This  con- 
clusion is  supported  by  the  finding  that  lipids  which  had  been  dissociated 
by  nonhydrolytic  means,  without  appreciable  alteration  of  the  activity  of 
endotoxin,  were  at  least  as  potent  as  lipid  A  recovered  by  acid  hydrolysis, 
the  remainder  of  which  is  inactive. 

Proposed  Course: 

Despite  considerable  progress  in  stripping  away  nonfunctional 
parts  of  endotoxic  extracts,  much  more  can  be  done  toward  reducing  com- 
plexes to  pure  active  principles.  This  aspect  of  the  work  will  be 
continued  in  an  effort  to  define  the  minimum  constitution  of  an  endotoxin 
or  of  an  0  antigen.   It  is  sufficiently  clear  that,  up  to  this  point,  all 
announcements  of  "purified"  endotoxins  have  been  premature. 

51 


Serial  No.  NIAID-178 

Studies  on  stimulation  of  specific  immunity  to  infection  will  be 
resumed,  particularly  investigation  of  leads  reported  earlier  concerning 
the  possibility  of  producing  effective  prophylactic  agents  of  modified 
toxicity.  To  implement  these  aims,  enzymatic  studies  of  the  endotoxin 
complex  will  be  added  to  our  chemical  approach  to  the  problem.  Physio- 
logical studies  of  endotoxin  shock  and  passive  protection  to  it,  assays 
for  pyrogenicity  in  rabbits,  and  perhaps  other  tests  will  be  added  to 
the  biological  work  at  this  laboratory.   It  is  expected  that  collabora- 
tive projects  with  Drs.  Suter,  Ueter,  Landy,  and  Nowotny  will  also  go 
forward  during  the  coming  year,  and  that  a  joint  project,  involving  an 
exchange  of  personnel,  will  be  organized  with  Drs.  Malmgren  and  Heden  of 
the  Karolinska  Institutet,  Stockholm,  Sweden. 


52 


Serial  No.  NIAID-I78 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Ribi,  E.,  Hoyer,  B.  H.,  Milner,  K.  C,  Perrine,  T.  D.,  Larson,  C.  L.,  and 
Goode,  G. :     Physical  and  chemical  analysis  of  endotoxin  from  Sal monella 
enteritidis.  J.  Immunol.  8^(1):  32-i+7-  Jan.  i960. 

Jarvis,  F.  G.,  Mesenko,  M.  T.,  and  Tibbs,  K.  E.:  Production  of  Vi  antigen 
on  a  chemically  defined  medium  by  a  coliform  bacterium.  J.  Bact.  80(5): 
673-676.  Nov.  i960. 

Jarvis,  F.  G.,  Mesenko,  M.  T.,  and  Kyle,  J.  E.:  Electrophoretic  purifi- 
cation of  the  Vi  antigen.  J.  Bact.  80(5):  677-682.  Nov.  i960. 

Honors  and  Awards  relating  to  this  project: 

Dr.  K.  C.  Milner 

Invited  to  spend  a  year  in  I96I-62  at  the  Karolinska  Institutet  in 
Stockholm,   Sweden. 


53 


Serial  No.  NIAID-179 

Rocky  Mountain  Laboratory 
Hamilton,  Montana 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:   Investigations  of  the  role  of  morphological  elements  of 
microorganisms  in  immunity  and  related  phenomena 

Principal  Investigator:  E.  Ribi 

Other  Investigators:     R.  L.  Anacker,  J.  E.  Coe,  C.  L.  Larson,  and 

K.  C.  Milner 

Cooperating  Units:       Dr.  J.  W.  Foster,  U.  of  Georgia;  Dr.  Philip 

Gerhardt,  U.  of  Michigan;  Dr.  Henry  Koffler, 
Dr.  John  Stasny,  and  Dr.  F.  L.  Crane,  Purdue  U. 

Man  Years  (calendar  year  i960): 
Total:         ^.0 
Professional:   1.0 
Other:         3*0 

Project  Description: 

Objectives: 

Studies  of  the  fine  structure  and  chemical  composition  of  a  variety 
of  organisms  and  the  correlation  of  such  findings  with  certain  immunologic 
and  biologic  properties  of  microbial  cells  or  fractions  thereof  comprise 
the  activities  of  this  section.   In  general,  these  projects  represent 
collaborative  efforts  with  other  investigators  who  wish  to  utilize  Dr.  Ribi's 
biophysical  methodology  to  fulfill  desired  objectives.  This  year,  studies 
have  been  concerned  chiefly  with  Brucella,  Penicillium  chrysogenum,  Bacillus 
terminalis  and  B.  subtilis. 

Methods: 

In  addition  to  the  usual  microbiologic  techniques,  special  methods, 
such  as  X-ray  diffraction,  the  pressure  cell  system  for  rupturing  bacterial 
cells,  and  linear  continuous  sucrose  or  glycerol  gradients  are  utilized. 
The  electron  microscope  is  routinely  used  to  observe  and  evaluate  induced 
morphologic  alterations.  Immunogenic  substances  are  released  from  bacterial 
cells  by  mild  chemical  treatment. 


Part  B  included:  Yes 

54 


Serial  No.  NIMD-179 
Major  findings; 

1.  Continued  studies  confirmed  initial  findings  that  immunogenic 
and  toxic  properties  of  Br.  abortus  are  located  in  the  cell  wall.  The 
limited  activity  of  protoplasm  was  thought  to  result  from  contamination 
with  cell -wall  materials.  Aqueous-ether  extracts  of  cell  walls  or  whole 
cells  possessed  far  greater  immunogenic  activity  than  did  other  fractions 
or  whole  cell  preparations.  When  extraction  is  performed  in  the  presence 
of  phosphate  buffer  (pH  7«0)>  &  more  potent  though  less  toxic  soluble 
antigen  is  released. 

2.  The  problem  of  removing  large  lipoidal  granules  from  washed 
aqueous  suspensions  of  bacterial  spores  was  solved  by  sedimenting  washed 
spore  cultures  through  a  linear  continuous  glycerin  gradient.  By  this 
technique,  dormant  spores  also  could  be  separated  from  vegetative  spores. 

3.  In  view  of  the  known  resistance  of  spores  to  heat,  drying,  and 
disinfectants,  the  fine  structure  of  the  spore  coat  was  studied  in  order 
to  explain  the  protection  afforded  the  spore  body.  When  a  purified 
fraction  of  spore  coats  was  examined  under  the  electron  microscope,  these 
coats  appeared  to  be  comprised  of  multiple  layers  or  lamellae  whose  thick- 
nesses approached  the  limit  of  resolution  of  the  electron  microscope.  The 
shape  of  fractured  edges  suggested  that  these  layers  were  composed  of 
laminar  crystals,  and  subsequent  X-ray  diffraction  patterns  confirmed  the 
character  of  the  spore  coat  as  a  perfect  crystalline  substance. 

h.   X-ray  diffraction  patterns  of  purified  cell  walls  of  Penicillium 
chrysogenum  contained  only  reflections  typical  of  chitin  and  not  of  glucan, 
a  related  polysaccharide  thought  by  collaborative  investigators  to  be 
present  in  the  cell  wall.  The  fine  structure  of  chitinous  cell  walls  was 
shown  to  be  similar  to  that  of  Histoplasma  capsulatum,  but  crystallites 
of  the  latter  were  smaller.  The  morphologic  arrangement  of  fibrils  and 
amorphous  regions  explains  such  properties  as  rigidity,  elasticity,  and 
permeability  of  the  cell  walls  of  these  fungi.  Chitin  crystallites  iso- 
lated from  fibrils  of  P.  chrysogenum  are  now  being  purified  by  Drs.  Koffler 
and  Stasny,  collaborators  on  this  project. 

5.  The  significance  of  "double  membranes"  in  osmium-stained  thin 
sections  of  cells,  nuclei,  and  other  morphologic  structures  observed  under 
the  electron  microscope  has  not  been  resolved.  This  "double  membrane" 
appears  as  2  parallel  lines  of  osmium  but  the  cellular  components  with 
which  osmium  is  associated  have  not  been  determined.  Similar  structures 
have  been  seen  when  high  speed  supernates  of  bacterial  cytoplasm  were 
desiccated  in  the  presence  of  sodium  chloride  upon  plastic  specimen- 
supporting  membranes.   It  was  determined  that  these  same  structures 
appeared  in  transparent  films  obtained  by  simply  evaporating  aqueous 
solutions  of  sodium  or  potassium  chloride  on  plastic  membranes. 


55 


Serial  No.  NIAID-179 

Significance: 

The  intricate  association  of  toxic  and  immunogenic  substances  in 
the  cell  vail  of  Br.  abortus  complicates  the  development  of  a  nontoxic 
vaccine.  However,  the  effect  of  buffer  (pH  7-°)  on  the  toxicity  of  sub- 
stances released  by  ether  extraction  suggests  that  some  selective  separa- 
tion of  toxic  and  immunogenic  fractions  is  possible.  To  date,  ether  is 
the  only  known  agent  that  will  kill  Brucella  without  destroying  its 
immunogenic  properties. 

The  ability  to  separate  various  components  of  washed  spore  cultures 
by  sedimentation  in  a  linear,  continuous  glycerin  gradient  enables  further 
definitive  characterization  of  the  coat  and  body  of  bacterial  spores. 
Additional  information  thereby  obtained  may  elucidate  the  role  of  the  spore 
coat  in  protecting  the  body  from  detrimental  effects  of  physical  and  chem- 
ical energies. 

Concurrent  with  the  use  of  thin -sectioning  technique  for  the  prepa- 
ration of  tissues  for  study  by  electron  microscopy,  numerous  reports  on 
"double  membranes"  associated  with  various  cellular  structures  have  appeared 
in  the  literature.  The  significance  of  these  structures  has  remained 
controversial.  The  results  of  present  studies,  however,  indicate  that 
these  membranes  may  be  formed  by  some  colloidal  physical  forces  which  are 
induced  during  solidification  of  inorganic  ions  on  a  surface. 

Proposed  course: 

Collaborative  studies  on  spore  coats  of  B.  subtilis  and  character- 
ization of  cellular  fractions  of  B.  abortus  and  P.  chrysogenum  will  be 
continued. 


56 


Serial  No.  NIAID-179 

PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 

Part  B.   Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Larson,  C.  L.,  Ribi,  E.,  Milner,  K.  C,  and  Lieberman,  J.  E.:  A  method 
for  titrating  endotoxic  activity  in  the  skin  of  rabbits.  J.  Exp.  Med. 
111(1):  1-20.  Jan.  i960. 

Ribi,  E.,  Brown,  W.,  and  Goode,  G.:  Preparation  of  microorganisms  for 
electron  microscopy.  J.  Bact.  79(l):  lU2-lM+.  Jan.  i960. 

Honors  and  Awards  relating  to  this  project: 

Dr.  Edgar  Ribi 

Invited  to  give  lecture  on  endotoxins  for  Department  of  Bacteriology, 
University  of  Florida,  Gainesville,  Florida. 

Participated  in  panel  discussion  of  bacterial  endotoxins  at  meeting 

of  Society  of  American  Bacteriologists,  Philadelphia,  as  representative 

of  endotoxin  group. 

Invited  to  participate  in  a  symposium  on  bacterial  endotoxins  sponsored 
by  the  Society  of  American  Bacteriologists  in  Chicago,  Illinois, 
April  1961. 


57 


Serial  No.  NIAID-180 

Rocky  Mountain  Laboratory 
Hamilton,  Montana 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:  The  encephalitides 

Principal  Investigator:  C.  M.  Eklund 

Other  Investigators:     W.  Burgdorfer,  D.  B.  Lackman,  V.  Newhouse,  and 

L.  A.  Thomas 

Cooperating  Units:       None 

Kan  Years  (calendar  year  i960): 
Total:        15.5 
Professional:   2.5 
Other:        13*0 

Project  Description: 

Objectives: 

The  objectives  of  this  program  are  to  determine  the  incidence  of 
arthropod-borne  viruses  in  the  vestern  United  States  and  to  develop 
better  methods  of  diagnosis  of  these  diseases,  a  detailed  knowledge  of 
the  ecology  and  epidemiology  of  this  group  of  diseases,  and  general  and 
specific  methods  of  control. 

Methods: 

The  methods  employed  are  in  general  those  used  in  field  studies 
of  arthropod-borne  infections  and  virological  and  serological  techniques 
suitable  for  this  type  of  study. 

Major  findings: 

1.  A  virus,  stored  since  its  original  isolation  from  Colorado 
ticks  in  1952,  was  recently  characterized  and  found  to  be  closely  related 
to  Powassan  virus  obtained  from  a  patient  in  Ontario,  Canada,  and  studied 
at  RML  last  year.  These  viruses  belong  to  the  Russian  spring-summer 
encephalitis  group,  but  they  are  not  as  closely  related  to  this  group  as 
they  are  to  each  other.  The  tick  isolate  was  neutralized  by  RSSE  immune 
serum,  but  RSSE  virus  was  not  neutralized  by  Powassan  or  tick-virus 

Part  B  included:  Yes 

58 


Serial  No.  NIAID-180 

immune  serum.  Failure  to  isolate  this  agent  in  tests  of  many  other 
field-collected  ticks  and  other  considerations  suggest  that  this  virus 
isolated  from  Colorado  ticks  was  an  accidental  infection  of  D.  andersoni. 

2.  In  contrast  to  a  very  low  incidence  of  WEE  virus  in  C.  tarsalis 
in  1959>  a  marked  increase  in  number  of  isolations  (22)  from  Idaho  and 
Oregon  was  reported  this  year.  One  strain  also  was  isolated  from  Culex 
pipiens  which,  like  a  previous  one,  shows  some  biological  differences 
from  WEE  isolated  from  C.  tarsalis.   There  were  also  k   isolations  of 
St.  Louis  virus  from  Idaho  C.  tarsalis. 

3-  Three  isolations  of  Culiseta  inornata  virus  were  made,  perhaps 
a  reflection  of  an  unexpected  increase  of  this  species  in  North  Dakota. 
Isolations  had  not  been  made  since  1952.  This  virus  belongs  to  the 
African  Bunyamwera  group  and  is  related  to  the  Cache  Valley  virus  in  Utah. 
A  colony  of  C .  inornata  has  been  established  for  transmission  studies 
with  these  strains. 

U.  A  virus  isolated  from  a  local  snowshoe  hare  has  been  identified 
as  closely  related  if  not  identical  to  so-called  California  virus.  This 
is  the  first  isolation  from  a  vertebrate,  though  antibodies  have  been 
detected  in  Bitterroot  horses.  Domestic  rabbits  have  been  refractory  to 
experimental  infection,  and  tests  of  the  susceptibility  of  native  hares 
and  rabbits  from  other  states  are  inconclusive.  The  related  trivittatus 
virus  was  not  recovered  from  mosquitoes  this  year. 

5.  Transmission  studies  showed  survival  of  the  snowshoe  hare  strain 
in  C.  tarsalis  and  A.  aegypti  for  25  days,  but  only  one  unrepeated 
passage  by  bites  of  the  latter.   D.  andersoni,  0.  parkeri  and  0.  turicata 
did  not  acquire  infection  when  fed  on  experimentally  infected  hamsters. 
This  virus  had  not  been  recovered  at  RML  in  tests  of  153>°0°  C.  tarsalis 
collected  from  various  western  states  during  the  last  10  years. 

6.  To  check  for  presence  of  possible  latent  WEE  virus  in  bloods  of 
migratory  birds  arriving  before  advent  of  the  mosquito  season,  976  serums 
of  different  species  collected  from  k   localities  in  Minnesota,  North  Dakota, 
Montana,  and  Oregon  were  tested  but  none  contained  virus.  An  additional 
lk8   serums  from  mammals  and  snakes  were  free  of  WEE  or  SLE  virus. 

7.  Of  50  garter  snakes  of  2  species  injected  intraperitoneally 
with  WEE  virus  last  fall,  virus  was  isolated  from  23  (l6  were  proved  WEE) 
after  leaving  hibernation.  Virus  circulated  in  some  snakes  up  to  70  days. 
The  complete  experimental  overwintering  cycle  was  confirmed  by  passage  of 
virus  to  other  snakes  through  the  bites  of  mosquitoes  infected  on  the 
above  snakes. 

8.  In  garter  snakes  injected  with  WEE  virus  and  held  at  k° ,  22°, 
and  31°  C,  viremia  was  observed  to  start  latest  (about  a  month)  at  the 
lowest  temperature  of  storage,  but  to  last  longest  (at  least  25  days)  at 

53 


Serial  No.  NIAID-180 

9«  Preliminary  tests  suggest  that  gopher  snakes  and  rattlesnakes 
may  not  be  as  susceptible  as  garter  snakes. 

10.  On  the  possibility  that  immunologic  tolerance  might  influence 
the  overwintering  mechanism  of  WEE  virus,  mice  in  various  states  of 
pregnancy  were  infected.  Up  to  the  10th  day  of  pregnancy,  mothers  and 
subsequent  litters  survived.  Deaths  among  mothers  infected  after  the 
10th  day  occurred  at  varying  intervals  after  inoculation;  litters  born  to 
mothers  surviving  until  parturition  occurred  died  within  2  to  3  days  after 
birth.  However,  virus  was  recovered  only  from  the  dead  mothers,  not  from 
the  litters.  Pregnancy  therefore  has  a  marked  effect  on  the  course  of  the 
disease. 

Significance; 

The  survival  of  WEE  virus  in  hibernating  garter  snakes,  the  long 
viremia,  and  demonstrated  snake-to-snake  transmission  by  the  bites  of  C. 
tarsalis  (the  important  known  natural  vector)  offer  at  least  one  explana- 
tion for  the  overwintering  of  the  virus. 

Ecologic  data  being  accumulated  on  the  natural  occurrence  of  WEE 
and  SLE  viruses  were  reviewed  in  last  year's  report  and  are  still  being 
augmented. 

Because  of  the  increasing  need  for  identification  of  various  viral 
agents  isolated  in  field  studies  or  referred  to  the  virus  unit  by  others, 
a  collection  of  37  identified  agents  is  now  available.  This  collection 
will  not  only  speed  systematic  reference  but  will  provide  data  fundamental 
to  an  understanding  of  the  relationships  of  these  so-called  ar-bo  viruses. 

The  isolation  of  California  virus  for  the  first  time  from  a  verte- 
brate should  forge  a  significant  link  in  the  epidemiologic  chain  of  the 
natural  cycle  of  this  virus  which  has  caused  some  human  infection  in 
California  and  is  probably  mosquito  borne. 

The  isolation  from  Colorado  ticks  of  a  strain  of  virus  related  to 
Powassan  virus  of  human  origin,  suggests  that  a  tick-borne  entity  with 
some  resemblance  to  the  important  Russian  spring-summer  encephalitis  of 
Eurasia  may  be  present  in  North  America. 

Proposed  course: 

Ecologic  observations  with  intensified  study  of  the  laboratory 
features  of  the  relationships  among  ar-bo  viruses  of  western  United  States 
will  be  continued.  Further  data  on  various  aspects  of  the  role  of  snakes 
are  expected  to  add  to  epidemiologic  information.  Comparative  studies 
will  be  continued  on  the  pathogenesis  of  infection,  including  duration  of 
viremia  in  mice,  chickens,  and  other  fowls.  The  susceptibility  of  verte- 
brates to  California  virus  will  be  investigated. 

60 


Serial  No.  HIAID-180 

Farther  efforts  will  be  made   to  correlate  HI,   CF,    and  neutralizing 
antibodies  and  to  determine  stability  of  neutralizing  antibodies  in  serums 
during  storage.     Additional  techniques  will  be  used  to  refine  certain 
mouse-brain  vaccines. 

The   reference  collection  of  ar-bo  viruses  will  facilitate  collab- 
orative studies  with  the  Greeley  laboratory  of  CDC  and  other  institutions 
on  the  characterization  of  unclassified  viruses. 


61 


Serial  No.  NIAID-180 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Eklund,  CM.:   Insect-borne  and  animal-borne  virus  diseases  of  man. 
Minnesota  Med.  ^3(3):  I8U-IB9,  193*  March  i960. 

Thomas,  L.  A.,  Kennedy,  R.  C,  and  Eklund,  CM.:   Isolation  of  a  virus 
closely  related  to  Powassan  virus  from  Dermacentor  andersoni  collected 
in  Cache  la  Poudre  Canyon,  Colorado.  Proc.  Soc.  Exp.  Biol.  &  Med.  10H(2): 
355-359.  June  i960. 

Thomas,  L.  A.  and  Eklund,  CM.:  Overwintering  of  Western  equine 
encephalomyelitis  virus  in  experimentally  infected  garter  snakes  and 
transmission  to  mosquitoes.  Proc.  Soc.  Exp.  Biol.  &  Med.  l£5(l):  52-55« 
Oct.  I960. 

In  press: 

Olson,  T.  A.,  Rueger,  M.  E.,  Price,  R.  D.,  Schlottman,  L.  L.,  Kennedy, 
R.  C,  and  Eklund,  CM.:  Evaluation  of  activity  of  western  equine 
encephalomyelitis  virus  in  Minnesota  by  antibody  response  of  sentinel 
pigeons.  Am.  J.  Trop.  Med.  and  Eyg. 

Honors  and  Awards  relating  to  this  project: 

Dr.  C  M.  Eklund 

Appointed  lecturer  in  Microbiology,  Montana  State  University,  Missoula, 
Montana. 

Invited  to  participate  in  a  symposium  on  the  biology  of  viruses  of  the 
tick-borne  encephalitis  complex  in  Smolenice,  Czechoslovakia. 


62 


Serial  No.  NIAID-181 

Rocky  Mountain  Laboratory 
Hamilton,   Montana 
PHS-NIH 
Individual  Project  Report 
Calendar  Year  i960 


Part  A. 


Project  Title:  Colorado  tick  fever 

Principal  Investigator:  C.  M.  Eklund 

Other  Investigators:     W.  Burgdorfer,  G.  M.  Kohls,  D.  B.  Lackman,  and 

L.  A.  Thomas 

Cooperating  Units:       None 

Man  Years  (calendar  year  i960): 
Total:        18.0 
Professional:   2.0 
Other:        16.0 

Project  Description: 

Objectives: 

The  objectives  of  this  program  are  to  develop  a  knowledge  of  the 
clinical  syndromes  which  CTF  virus  may  produce  in  human  beings  and  to 
understand  the  ecology  of  this  agent,  especially  the  role  of  tick  and 
rodent  reservoirs.   In  addition,  studies  are  under  way  to  check  possible 
interference  with  concomitant  infections  of  R.  rickettsii  in  tick  vectors. 
Development  of  a  prophylactic  vaccine  remains  a  primary  objective. 

Methods: 

The  methods  employed  do  not  differ  greatly  from  those  used  in 
laboratories  studying  field  and  laboratory  aspects  of  arthropod-borne 
viruses  except  as  they  are  adapted  to  our  local  situations. 

Major  findings: 

1.  Though  CTF  had  been  reported  from  ticks  on  Long  Island,  investi- 
gations to  date  at  RML  indicate  that  human  infection  is  limited  to  areas 
where  D.  andersoni  is  found.  Presence  of  the  virus  in  adult  tick  vectors 
depends  on  a  natural  cycle  between  the  active  immature  stages  and  their 
small  animal  hosts,  because  transovarial  maintenance  has  never  been  con- 
firmed in  exhaustive  studies. 

Part  B  included:  Yes 

63 


Serial  No.  NIAID-181 

2.  The  disease  in  human  beings  varies  from  preponderant  subclinical 
and  benign  febrile  syndromes  to  infrequent  involvement  of  the  central 
nervous  system  or  severe  bleeding.  Although  serum  samples  were  not 
especially  solicited  from  physicians,  almost  twice  as  many  isolations 
were  made  this  year  as  last  year.  Two-thirds  of  the  blood  specimens  were 
received  from  Idaho,  Oregon,  and  Wyoming. 

3.  In  tests  of  ticks  from  several  areas  in  Colorado,  infection 
rates  varied  from  zero  to  more  than  21$. 

h.   Members  of  the  Trappist  Monastery  at  Snowmass,  Colorado,  were 
vaccinated  again  without  ill  effect.  Danger  from  repeated  use  of  this 
inactivated  mouse-brain  product  appears  to  be  minimal  because  untoward 
effects  were  not  observed  in  guinea  pigs  receiving  multiple  injections. 

5.  Three  new  lots  of  vaccine  prepared  from  infected  suckling  mouse 
brains  again  provided  marked  protection,  as  shown  by  differences  in 
resistance  between  vaccinated  and  nonimmunized  control  mice. 

6.  It  has  now  been  shown  that  the  simplest  diagnostic  test  for  CTF 
is  the  complement -fixation  technique.  Antibodies  are  readily  detected  in 
serums  from  patients  convalescent  from  CTF;  they  first  appear  20  days 
after  onset  of  illness  and  may  remain  as  long  as  260  days. 

Significance: 

As  with  other  tick-borne  human  diseases,  the  number  of  annual 
cases  of  CTF  fluctuates.  Hence,  the  marked  increase  in  number  of  isola- 
tions may  not  represent  a  real  increase  in  disease.  Physicians  in  the 
Rocky  Mountain  area  have  undoubtedly  become  more  aware  of  the  prevalence 
of  this  tick-borne  disease  through  our  efforts  to  obtain  more  data  on 
severe  types  of  CTF.  This  year  one  9-year-old  Oregon  boy  had  encephalitis 
accompanied  by  increased  cell  count  in  the  spinal  fluid  and  other  aggra- 
vated symptoms. 

Since  a  high  percentage  of  ticks  in  Estes  Park  carry  CTF  virus, 
transient  visitors  are  likely  to  contract  infection  but  symptoms  do  not 
appear  until  these  persons  have  returned  home  and  it  is  probable  that 
some  cases  remain  undiagnosed. 

In  certain  areas  of  considerable  occupational  incidence,  such  as 
in  stock  handlers  in  northern  Nevada,  a  suitable  vaccine  would  be  useful. 

Proposed  course: 

As  in  the  past,  continued  efforts  will  be  made  to  prepare  a 
refined  and  potent  vaccine. 


64 


Serial  No.  NIAID-lSl 

We  are  continuing  to  accumulate  data  on  possible  interference 
between  infections  of  R.    rickettsii  and  CTF  virus  in  the  same  ticks  since 
this  is  one  possible  explanation  for  the  observed  low  incidence  of  the 
former  in  endemic  localities  of  CTF-infected  ticks.     Study  by  fluorescent 
microscopy  of  the  CTF  agent  in  vertebrate  and  acarine  hosts  is  continuing. 


G5 


Serial  No.  NIAID-l8l 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  i960 


Part  B.  Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

Burgdorfer,  W.  and  Lackman,  D.:   Identification  of  the  virus  of  Colorado 
tick  fever  in  mouse  tissues  by  means  of  fluorescent  antibodies.  J.  Bact. 
80(1):  I3I-I36.  July  i960. 

Thomas,  L.  A.  and  Eklund,  CM.:  Use  of  the  complement-fixation  test  as 
a  diagnostic  aid  in  Colorado  tick  fever.  J.  Inf.  Dis.  107(2):  235-2UO. 
Sept. -Oct.  i960. 

In  press: 

Eklund,  C.  M.,  Kohls,  G.  M.,  Jellison,  W.  L.,  Burgdorfer,  W.,  Kennedy,  R.  C, 
and  Thomas,  L.  A.:  The  clinical  and  ecological  aspects  of  Colorado  tick 
fever.  Proc.  Intern.  Cong.  Trop.  Med.  and  Malaria,  Lisbon,  Sept.  1958. 

Burgdorfer,  W.  and  Eklund,  CM.:   I.  Colorado  tick  fever  ecological 
studies  in  western  Montana.  J.  Inf.  Dis. 

Burgdorfer,  W.:  Colorado  tick  fever.  II.  The  behavior  of  Colorado  tick 
fever  virus  in  rodents.  J.  Inf.  Dis. 

Eklund,  CM.,  Kennedy,  R.  C,  and  Casey,  M. :  Colorado  tick  fever. 
Rocky  Mtn.  Med.  Jour. 


68 


LABORATORY  OF  BACTERIAL  DISEASES 


Summary. 


1 


200  -  Brucellosis 2 

201  -  Basic  Studies  on  Staphylococcus 5 

202  -  Identification  of  Pseudomonas 7 

203  -  Studies  on  Bacteria  Characterized  by  the 

Production  of  Reproductive  Filterable 
Granules ' 

204  -  Intracellular  Parasitism , H 

205  -  The  Role  of  Infection  in  the  Delayed 

Deaths  of  Mice  Following  Extensive 

Burn  Injury 13 


SUMMARY  OF  ACTIVITIES 

LABORATORY  OF  BACTERIAL  DISEASES 

December  1960 


The  research  program  of  the  Laboratory  of  Bacterial  Diseases  has 
continued  in  the  same  general  areas  as  last  year  with  the  change  of 
emphasis  noted  in  last  year's  report. 

A  greater  proportion  of  the  research  effort  has  been  directed  to 
the  studies  on  intracellular  parasitism.   These  studies  deal  with  possible 
changes  in  characteristics  of  infected  and  immune  cells  as  the  result  of 
parasitism,  and  the  effect  of  intracellular  growth  on  the  parasite.   One 
such  notable  change  of  course  is  the  production  of  specific  antibodies  by 
certain  cells  of  immune  animals.   During  the  current  year  a  considerable 
effort  has  been  directed  toward  the  study  of  antibody  production  by  cells 
in  vitro.   The  macrophage  was  selected  as  a  multipotential  cell  for  such 
study.  Macrophages  obtained  from  the  peritoneal  cavity  of  immune  guinea 
pigs  (immunized  with  egg  albumin)  have  been  found  to  release  antibody  in 
vitro  for  a  period  of  several  days.   This  provides  a  system  for  further 
study  of  the  nutritional  or  other  requirements  for  continued  antibody 
production  in  vitro,  or  even  in  serial  cultures.   Cells  derived  from 
macrophages  have  been  carried  in  serial  tissue  culture  for  several  months, 
retaining  their  phagocytic  ability.   This  promising  line  of  research  will 
be  intensively  continued  during  the  coming  year. 

Studies  on  brucellosis  are  conducted  at  a  reduced  tempo.   There  is 
continuing  need  to  collaborate  with  other  brucellosis  research  centers 
throughout  the  world  to  try  and  settle  problems  of  classification  and 
epidemiology  of  the  Brucella.   Currently  we  are  doing  some  laboratory 
testing  of  Brucellosis  Vaccine  for  Human  Use  prepared  in  Russia.   There  is 
present  interest  in  this  vaccine  by  the  World  Health  Organization  for  its 
possible  use  in  occupational  and  otherwise  continually  exposed  groups. 

Studies  on  the  Staphylococcus  are  directed  toward  determining  the 
factors  responsible  for  pathogenicity,  and  toward  development  and  standardi- 
zation of  tests  for  measuring  relative  pathogenicity  of  strains. 

Some  of  Dr.  Verder's  past  studies  on  the  genus  Pseudomonas  are  being 
prepared  for  publication. 

There  are  other  areas  within  the  province  of  this  Laboratory  which 
should  be  developed  were  space,  personnel,  and  financial  support  available. 
Among  these  are  the  diarrheal  diseases  and  the  basic  field  of  PPLO  and  L 
form  research. 

There  remains  a  continuing  need  for  certain  additional  equipment  and 
increased  monetary  support  to  pursue  the  basic  studies  mentioned. 


Serial  No.      NIA3D-200 

1.  Bacterial  Diseases 

2.  None 

3.  Bethesda,   Maryland 


PHS-NIH 

Individual   Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:   Brucellosis 

Principal  Investigator:   Norman  B.  McCullough,  M.  D. 

Other  Investigator:   None 

Cooperating  Units:   None 


Man  Years  (calendar  year  1960) 
Total:   2 
Professional:   1/2 
Other:   1-1/2 


Project  Description: 

Objectives: 

The  objectives  of  this  project  are  broad  and  studies  are 
conducted  on  all  aspects  of  brucellosis,  including  the  nutrition,  metabolism, 
and  enzymatic  make-up  of  the  organism,  mechanism  of  action  of  antibiotics  on 
the  organism,  factors  determining  virulence  of  the  organism,  the  development 
and  efficacy  of  prophylactic  immunizing  agents,  field  studies  to  determine 
the  occupational  hazard  of  the  disease,  epidemiology,  diagnosis  and  therapy 
of  the  disease  in  man. 

Clinical  studies  concern  diagnostic  criteria  for  the  disease  in 
man,  especially  in  borderline  cases  of  chronic  illness  in  which  present 
cultural  and  serological  techniques  fail  to  provide  reliable  evidence  of 
infection;  studies  of  the  pathogenesis  of  the  disease;  and  the  evaluation  of 
current  therapeutic  regimens. 


Part  B  included, 


Serial  No.      NIAID-200 


Methods  Employed: 

The  Warburg  technique  is  employed  in  enzymatic  and  metabolic 
studies;  growth  in  a  synthetic  medium  is  used  for  nutrition  experiments; 
guinea  pig  inoculation  for  virulence  and  vaccine  studies;  occupationally 
exposed  groups  for  epidemiological  studies;  human  patients  for  studies  on 
diagnosis  and  therapy. 

Major  Findings: 

The  study  of  antigenic  relationships  among  the  Brucella  has 
continued.   Br.  abortus  and  Br.  suis  constitute  a  single  antigenic  group  as 
previously  held,  whereas  Br.  melitensis  is  antigenically  more  variable.   The 
latter  species  consists  of  three  antigenic  patterns,  only  one  of  which  is 
identified  by  the  use  of  currently  prepared  mono-specific  typing  serum.   The 
recognition  of  this  antigenic  variance  allows  the  proper  classification  of 
most  of  the  so-called  "intermediate"  strains,  and  should  clarify  many  problems 
in  the  classification  and  epidemiology  of  the  Brucella. 

Significance  to  the  Program  of  the  Institute: 

In  terms  of  world-wide  significance,  brucellosis  ranks  in 
incidence  and  importance  with  malaria,  tuberculosis,  and  parasitic  diseases, 
in  relation  to  the  welfare  of  mankind.   Even  in  the  United  States,  by  reported 
incidence,  it  is  the  most  important  disease  of  animals  transmissible  to  man, 
is  the  cause  of  serious  economic  loss  to  the  livestock  industry,  is  an 
occupational  disease,  and  one  which  presents  many  problems  in  the  diagnosis 
and  therapy  of  the  infection  in  man.   The  organism  readily  lends  itself  to 
basic  studies  outlined  elsewhere  in  this  report. 

Proposed  Course  of  Project: 

Continue  studies  under  way. 

Continue  to  collaborate  with  other  Brucella  research  centers 
throughout  the  world  in  the  development  and  standardization  of  laboratory  . 
tests  for  species  identification,  and  for  diagnosis  of  Brucella  infection. 

Study  of  Brucella  bacteriophage  as  regards  basic  phenomena  of 
bacteriophagy  and  potential  use  of  phage  for  preparation  of  enzymatic  and 
subcellular  fractions  of  Brucella  for  biochemical  studies,  and  as  possible 
immunizing  agents.   Investigate  the  utility  of  specific  phages  as  an  aid  in 
identification  of  Brucella  species  and  strains. 

Continue  to  use  Brucella  as  a  basic  tool  in  the  study  of 
mechanism  of  development  of  acquired  resistance  to  antibiotics  and  in  studies 
of  intracellular  parasitism. 


Serial  No.      NIAID-200 


PHS-NIH 

Individual  Project  Report 

Calendar  Year  1960 


Part  B    Honors,  Awards,  and  Publications 

Publications  other  than  abstracts  from  this  project: 

1.  Dolan,  T.  F.,  Jr.;  McCullough,  N.  B. ;  and  Gibson,  L.  E.: 

Nocardiosis.   Report  of  Two  Cases  in  Children.  A.M. A. 
Jour,  of  Dis.  of  Child.  99:  234-237,  1960. 

2.  Dolan,  T.  F.,  Jr.;  McCullough,  N.  B. ;  and  Gibson,  L.  E.: 

Hypogammaglobulinemia.   Report  of  an  Unusual  Variation. 
Pediatrics  26:  817-821,  1960. 

Honors  and  Awards  relating  to  this  project: 

Principal  investigator  elected  to  serve  as  a  member  of  the  Executive 
Committee  and  as  General  Chairman  of  the  Annual  Brucellosis  Research 
Conference  (1960). 


Serial  No.      NIAID-201 

1.  Bacterial  Diseases 

2.  None 

3.  Bethesda,   Maryland 


PHS-NIH 
Individual   Project  Report 
Calendar  Year   1960 


Part  A. 


Project  Title:   Basic  Studies  on  Staphylococcus 
Principal  Investigator:   Elizabeth  Verder,  Ph.D. 
Other  Investigator:   None 
Cooperating  Units:   None 


Man  Years  (calendar  year  1960) 
Total:   1-1/12 
Professional:   8/12 
Other:  5/12 


Project  Description: 
Objectives: 

1.  To  develop  an  objective  means  of  measuring  virulence  of 
Staphylococci. 

2.  To  determine  the  factors  responsible  for  virulence. 

3.  To  develop  more  satisfactory  methods  for  the  identification 
of  individual  strains  used  in  measuring  virulence. 

Methods  Employed: 

The  use  of  experimental  animals  and  tissue  cultures  in  defining 
overall  virulence,  and  in  study  of  individual  fractions  or  products  of  the 
Staphylococcus. 


Part  B  not  included. 


Serial  No.      NIAID-201 


Use  of  physical  and  chemical  methods  in  fractionation  of  the 
organism  and  its  products  to  allow  assessment  of  individual  factors  in 
pathogenicity  and  in  cell  injury  in  vitro. 

Nutritional  studies  on  the  production  of  factors  concerned  in 
pathogenicity. 

Gel  diffusion  techniques  using  both  tubes  and  plates, 
agglutination,  hemagglutination  and  precipitin  tests  are  being  employed. 
An  attempt  is  being  made  to  correlate  mouse  protection  properties  of 
human  sera  with  certain  characteristics  of  jLn  vitro  reactions. 

Major  Findings: 

Numerous  serological  procedures  have  been  explored  in  an 
effort  to  develop  a  technique  for  more  satisfactory  identification  of 
individual  strains  without  immediate  success. 

Significance  to  the  Program  of  the  Institute: 

The  number  of  antigenic  components  produced  by  a  strain  of 
staphylococcus  varies  with  the  virulence.   Quantitative  estimation  of 
some  of  these  components  and  also  some  of  the  products  of  the  more 
invasive  strains  may  be  very  helpful  in  characterizing  strains. 

Proposed  Course  of  Project: 

As  described  above. 


Serial  No.     NIAID-202 

1.  Bacterial  Diseases 

2.  None 

3.  Bethesda,    Maryland 


PHS-NIH 

Individual  Project  Report 

Calendar  Year   1960 


Part  A. 

Project  Title:   Identification  of  Pseudomonas 

Principal  Investigator:   Elizabeth  Verder,  Ph.D. 

Other  Investigator:  None 

Cooperating  Units:   Dr.  Carl  Millican,  NIAMD 


Man  Years  (calendar  year  1960) 
Total:   1/12 
Professional:   1/12 
Other :   0 


Project  Description: 
Objectives: 

a)  Identification  of  strains  for  other  scientists. 

b)  Orientation  of  scientists  in  methods  used  for  study  of 
biochemical  characteristics  of  various  species  and  for  serological  typing. 

c)  Distribution  of  cultures  of  strains  of  various  species  and  of 
representative  serological  types  of  Ps.  aeruginosa  on  request  to  research 
scientists. 

d)  To  determine  whether  therapeutic  antisera  contains 
identifiable  group  specific  antibody. 


Part  B  not  included. 


Serial  No.      NIAID-202 


Methods  Employed; 

The  methods  which  we  have  employed  are  ones  developed  or 
modified  in  the  course  of  our  studies  on  the  classification  of  Pseud omonas ; 
the  studies  were  initiated  during  work  on  our  project  on  diarrheal  disease 
of  the  newborn  and  have  been  carried  on  for  eight  years.  A  proposed  scheme 
for  the  antigenic  analysis  of  strains  of  Ps.  aeruginosa  was  worked  out  and 
has  been  employed  successfully  in  identifying  strains  by  serological  typing. 

Dr.  Carl  Millican  has  succeeded  in  guiding  a  commercial 
biological  company  to  the  production  of  an  antiserum  that  has  been  used 
successfully  in  the  treatment  of  children  with  Ps.  aeruginosa  infections. 
He  has  asked  us  to  collaborate  in  studies  on  protection  of  animals  infected 
with  various  serological  types  to  determine  whether  or  not  a  group  specific 
antibody  is  responsible  for  these  protective  properties. 

Standard  antigen- antibody  techniques  will  be  employed  in  the 
study.   Gel  diffusion  studies  will  be  done  with  various  gamma  globulin  and 
serum  preparations  and  several  antigenic  fractions  of  Pseudomonas. 

Major  Findings: 

Methods  have  been  established  for  use  in  the  identification  of 
individual  strains  of  Pseudomonas  so  that  epidemiological  surveys  in 
relation  to  the  spread  of  resistant  strains  may  be  carried  out  successfully. 

Significance  to  the  Program  of  the  Institute: 

Methods  have  been  developed  for  studies  on  the  spread  of  a 
group  of  antibiotic  resistant  organisms  of  medical  importance,  particularly 
in  infections  in  individuals  with  minimal  resistance  such  as  newborns,  the 
aged,  children  with  leukemia,  individuals  with  extensive  or  deep  burns,  etc. 
With  the  increasing  importance  of  Pseudomonas  infections  the  findings  in 
these  studies  are  receiving  recognition  in  clinical  applications. 

Proposed  Course  of  Project: 

Collaborative  studies  with  Dr.  Millican  as  outlined  above. 
This  will  be  limited  because  of  time-consuming  nature  of  staphylococcus 
studies. 


Serial  No.      NIAID-203 

1.  Bacterial  Diseases 

2 .  None 

3.  Bethesda,    Maryland 


PHS-NIH 

Individual   Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:   Studies  on  Bacteria  Characterized  by  the 

Production  of  Reproductive  Filterable  Granules 


Principal  Investigator:   Elizabeth  Verder,  Ph.D. 


Other  Investigator:   Dr.  Alexis  Shelokov  was  co- investigator  on  the 
project  at  time  the  organisms  were  isolated 
and  any  reports  will  be  made  jointly. 


Cooperating  Units:   None 


Man  Years  (calendar  year  1960) 
Total:   1/12 
Professional:   1/12 
Other:  0 


Project  Description: 
Objectives: 

a)  To  learn  more  about  1)  the  classification  of  bacteria 
capable  of  producing  reproductive  filterable  granules,  2)  the  environmental 
changes  that  induce  the  formation  of  granules,  3)  the  potentialities  of 
these  incomplete  filterable  organisms  in  the  establishment  of  infectious 
processes  and  4)  the  factors  influencing  the  development  of  more  complete 
cells  from  filtered  particles. 

b)  Distribution  of  cultures  on  request  to  other  scientists. 


Part  B  not  included. 


Serial  No.      NIAID-203 


Methods  Employed: 

Through  the  use  of  special  media,  organisms  of  this  type  have 
been  isolated  from  the  peripheral  blood  of  several  patients  with  illnesses 
with  an  indefinite  diagnosis.  Methods  commonly  employed  in  the  study  of 
organisms  producing  L  bodies  and  the  pleuro- pneumonia- like  (PPLO)  group 
were  utilized. 

Major  Findings: 

More  detailed  studies  must  be  carried  out  before  significant 
findings  on  these  organisms  may  be  reported.   During  the  past  year  attempts 
to  produce  L  forms  of  several  strains  of  staphylococci  have  been  successful. 
No  detailed  physiological  studies  have  yet  been  carried  out. 

Significance  to  the  Program  of  the  Institute: 

Organisms  with  similar  characteristics  have  been  recognized 
as  important  etiological  agents  of  animal  disease  for  many  years.   Their 
presence  in  extracts  of  tissue  and  blood  serum  collected  from  infected 
embryo  or  animals  and  used  inadvertently  in  the  preparation  of  tissue 
culture  media  has  increased  interest  in  the  development  of  more  satisfactory 
methods  for  their  detection  and  identification.   Their  importance  in 
infectious  processes  in  man  has  not  been  adequately  assessed.   Since 
several  species  of  Bacterioides  are  present  in  large  numbers  in  the 
intestinal  canal  of  man  and  animals,  some  of  these  organisms  may  belong 
to  closely  related  species  possessing  filterable  granules  under  certain 
circumstances  and  be  present  in  the  peripheral  blood  frequently.   The 
importance  of  the  group  to  medical  scientists  is  emphasized  by  the  three 
day  conference  sponsored  by  the  N.  Y.  Academy  of  Sciences  held  in 
January,  1959,  for  discussion  of  various  phases  of  studies  on  this  group 
of  organisms;  Drs.  Klieneberger-Nobel  and  Edwards  of  London  and  Dr.  Freund 
of  Copenhagen  were  among  the  invited  participants. 

Proposed  Course  of  Project: 

No  additional  work  will  be  carried  on  with  these  organisms 
until  the  studies  on  the  staphylococcus  have  been  well  established. 


10 


Serial  No.      NIAID-204 

1.  Bacterial  Diseases 

2 .  None 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual   Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:   Intracellular  Parasitism 
Principal  Investigator:   Norman  B.  McCullough,  M.  D. 
Other  Investigator:   None 
Cooperating  Units:   None 


Man  Years  (calendar  year  1960) 
Total:   2 
Professional:   1/2 
Other:   1-1/2 


Project  Description: 

Objectives: 

To  study  host-parasite  relationships  in  intracellular  metabolism 
for  the  purpose  of  increasing  our  understanding  of  disease  and  immunity. 

1.  To  determine  the  effect  of  infecting  organisms  on  the 
metabolism  of  the  infected  cell. 

2.  To  determine  the  effect  of  certain  bacterial  products,  such 
as  Staphylococcus  toxin  and  bacterial  fractions,  on  cells  maintained  as 
explants  and  in  tissue  culture,  and  upon  certain  subcellular  particles. 

3.  To  determine  the  contribution  of  cells,  and  of  subcellular 
particles,  extracts,  and  enzyme  systems  to  the  nutrition  of  and  their 
effects  on  the  characteristics  of  intracellular  bacteria. 


Part  B  not  included. 

11 


Serial  No.   NIAID-204 


4.   To  study  protein  synthesis  (such  as  antibody  production) 
by  cells  from  normal  and  immunized  animals. 

Methods  Employed: 

The  study  employes  various  pathogenic  agents  such  as  Brucella, 
Staphylococcus,  Pasteurella  tularensis,  and  certain  viruses.   It  will 
entail  the  use  of  standard  tissue  culture  techniques,  chemically  defined 
media  for  bacteria,  the  Warburg  technique,  standard  chemical  procedures, 
phase  microscopy,  fluorescent  antibody  technique,  vital  dyes,  electrophoresis, 
and  chromatography. 

Major  Findings: 

Using  hemagglutination  of  tanned  red  cells  as  indicator, 
macrophages  derived  from  the  peritoneal  cavity  of  guinea  pigs  immunized 
with  egg  albumin  have  been  found  to  release  antibody  Jji  vitro  for  a  period 
of  several  days.   Production  of  antibody  appears  correlated  with  conditions 
promoting  the  best  growth  of  cells.   However,  after  a  few  days  there  is 
a  change  in  the  morphology  of  the  cells  and  antibody  production  ceases. 
Primary  stimulation  in  vitro  has  not  yielded  antibody. 

Cell  cultures  derived  from  guinea  pig  macrophages  have  now  been 
maintained  in  serial  culture  for  several  months. 

Significance  to  the  Program  of  the  Institute: 

This  study  concerns  the  basic  interactions  between  host  cell 
and  infecting  agent  and  hence  any  contribution  to  knowledge  would  be  of 
significance  to  the  entire  field  of  infectious  diseases  and  to  the  program 
of  the  Institute. 

Proposed  Course  of  Project: 

As  stated  above. 


12 


Serial  No.      NIAID-205 

1.  Bacterial  Diseases 

2 .  None 

3.  Bethesda,   Maryland 


PHS-NIH 

Individual   Project  Report 

Calendar  Year   1960 


Part  A. 


Project  Title:   The  Role  of  Infection  in  the  Delayed  Deaths  of 
Mice  Following  Extensive  Burn  Injury 


Principal  Investigator:   Elizabeth  Verder,  Ph.D. 

Other  Investigator:   None 

Cooperating  Units:   Drs.  Sanford  Rosenthal  and  Carl  Millican 


Man  Years  (calendar  year  1960) 
Total:   2/12 
Professional:   2/12 
Other :  0 


Project  Description: 
Objectives: 

a)  To  review  literature  dealing  with  increased  susceptibility  to 
infection  following  traumatic  shock,  radiation  and  extensive  thermal  injury. 

b)  To  evaluate  findings  in  extensive  studies,  carried  out  in 
1943,  on  incidence  of  bacteria  in  tissues  of  burned  mice  and  their  role  in 
infectious  processes  causing  delayed  deaths  in  mice  following  extensive 
burn  injury. 

Methods  Employed: 

Standardized  techniques  for  the  isolation  and  identification  of 
bacteria  from  infectious  processes  were  employed. 


Part  B  not  included. 

13 


Serial  No.   NIAID-205 


Major  Findings: 

Bacteria  were  isolated  from  657.  to  907.  of  the  cultures  of 
heart,  liver,  spleen  and  kidneys  of  43  mice  sacrificed  two  to  nine  days 
after  extensive  burn  injury.   It  appears  that  bacteria  of  low  virulence 
and  invasiveness  may  spread,  from  previously  localized  foci  of  latent 
infections  or  from  areas  of  colonization  on  damaged  surfaces,  and  establish 
generalized  infections. 

Significance  to  the  Program  of  the  Institute; 

Septicemias  and  other  fatal  infections  are  of  increasing 
importance  as  the  cause  of  death  following  extensive  burn  injury  in  spite 
of  advances  in  treatment. 

Proposed  Course  of  Project: 

Study  will  be  terminated  in  1960  with  the  completion  of  a  paper 
discussing  our  findings. 


m 


2303 


© 


LIBRARY 


'I'     Amazing  Rev 


Amazing  Hslp. 


http://nlhllbrary.nlh.gov 


10  Center  Drive 

Bethesda,  MD  20892-1 150 

301-496-1080 


DATE  DUE 

CAVLCO 

NIH   LIBRARY 
4   0110  0128 


NIH  LIBRARY 


3  1496  00238  2169