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

ANNUAL REPORT 

OF 

PROGRAM ACTIVITIES 



NATIONAL INSTITUTES OF HEALTH 
1 9 6 

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^ 



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 subco m mittee 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 
tr ain ing 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 a dmin istered 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- 



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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- 



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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 




31 


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$852,681 


$527,304 


June 












New 


25 


$1,791,122 




15 


$1,436,275 


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83 
108 


2,633,605 

$4,424,727 




83 
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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 — 



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- 



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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 i nh erent 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 



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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 : 

l a 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 . 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 ''•' " B t 

-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. N 
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 a ureus . 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 c c 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 withc 1 ^- 

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 ^ tmnt fel^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 P 32 . 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 Project i 

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 (e g. cancer, heart disease, etc.) 

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. 



I 5 



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 + H o' °^ +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 H 2 = 5.93 + 0.08 @ 25° C 

Q H 2 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 C0 2 ) 
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 e el 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 6 

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^riSlblists 1 fiatio'Ka! fie e t- ^ ^ Pn iladelphia, 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 t o 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 

Acetate 2 2 

Pyruvate 2.5 1.5 

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, aureu s, 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 approx im ates 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^ l gg e JJcor&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-Fe 2 ^ + 2Fe^ . 

Assay: antibody + siderophilin-Fe2^9 + 56)..— .> antibody-siderophi- 
lin-Fe 2 ^ + ^ '| . 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 C 11 * 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 internists 1 and scientists 1 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. B ethesda, 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. YJi th 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 ^ B ethesda, Ha^land 10 ^ 
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 6 5 



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 th e 
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 
T T ajor 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, k f 
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, includi n g the 
common cold. 

Part B included /T7 Tea / / No 83 

1 



Serial No. NIAH) 66 

Met hods 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 u 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, h y 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 * N D 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 Employedi t 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 be J ween 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 I0 4 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 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 fornan s 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 - 
su latu n 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. capsulatu m 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 t he progra m 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 bi o -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 - 
i oides 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 Cryntococ cus 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 Inst i- 
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 
Crvptoco ccus 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 Ca ndida 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: 

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: V 12 

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: V 12 

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 '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: 

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: 

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 reaction 1 ' 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_ed m 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: 

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) LD c - 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,- 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 ldc 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. 

Meth ods 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 (dur i ng 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 o f 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. 

Ma jor 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 a t 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: 

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 w ere 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. dubiu s 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: 
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 
ra S-NIH 2. Helminthic Diseases 

Individual Project Report 3# B ethesda, 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: Y 4 
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 P 3 ^ 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 







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. stua y 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.