ANNUAL REPORT
OF
PROGRAM ACTIVITIES
NATIONAL INSTITUTES OF HEALTH
1 9 6 0
NATIONAL INSOTTE OF ALLERGY
AND INFECTIOUS DISEASES
A'MawAL •Msravn-s Of health
PUBLIC HEALTH SERVICE
U. S. DEPARTMENT 0F mXm, EDUCATION, AND
•>(■*■*■
ANNUAL REPORT OF PROGRAM ACTIVITIES
VX.^ National Institute of Allergy and Infectious Diseases
January - December, 1960
l^0
INDEX
ANNUAL REPORT
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Director, NIAID l
Extramural Programs
Intramural Programs
Laboratory of Clinical Investigation
Summary
10 (c) - Office of the Chief 5
12 (c) - Respiratory Viral Diseases 6
13 (c) - Antimicrobial Drug Therapy 1°
14 (c) - Staphylococcal Disease 13
15 (c) - Hepatitis and Mononucleosis 16
16 (c) - Aseptic Meningitis 17
17 (c) - Allergy-Immunology 19
18 (c) - Enteric Diseases 28
19 (c) - Clinical Investigations on Helminthic Diseases 31
20 (c) - Clinical Investigations on Protozoan Infections
and Diseases
37
21 (c) - Cystic Fibrosis of the Pancreas 44
22 (c) - Basic Biochemical Studies ^5
23 (c) - Systemic Fungal Disease 50
24 (c) - Sarcoidosis 54
25 (c) - Pyelonephritis 55
26 (c) - Urinary Tract Viruses 56
27 (c) - Biochemical Studies on Staphylococcal Cell Walls 59
28 (c) - Pathogenesis of Viral Infections 61
Laboratory of Cell Biology
Summary
50 - Biosynthesis in Mammalian Cell Cultures 3
51 - The Mechanism of Production of Poliovirus in Cultured
Mammalian Cells 8
52 - Effects of Animal Viruses on the Metabolism of
Mammalian Cell Cultures and the Mechanism of Viral
Replication 11
53 - Mechanism of Drug Resistance in Cultured Mammalian Cells 14
54 - Metabolic Control Mechanisms in Cultured Mammalian Cells 17
55 - Application of the Technique of Tissue Cultures to
Selected Genetic Diseases 19
Laboratory of Infectious Diseases
Summary 1
60 - Studies on Cellular and Cell-Free Staphylococcal
Penicillinase 44
61 - Antibiotic Activity of Sea Water 47
62 - The Electron Transport System in an Autotrophic
Hydrogenomad 50
62-A - Microbiological Fractionation of the Hydrogen Isotopes . 55
63 - Non-specific Immunity Associated with Serum Sidero-
philin and the Iron Metabolism of Both Pathogen and
Host 57
63-A - Chemical Aspects of the Specific Binding of Iron to
Serum Siderophilin and Egg White Conalbumin 62
63-B - Biochemistry of the Acquisition of Iron by Mammalian
Tissues as Mediated by the Iron-Binding Protein,
Siderophilin • • • 66
63-C - Development of an Economical 280mu Light Source Suit-
able for Detecting Proteins in Effluents from
Chromatographic Columns 69
64 - Detoxification of Potential Tuberculostatic, Fungi-
static, Parasiticidal and Virucidal Agent 71
64-A - Potential Fungistatic and Parasiticidal Agents 75
64-B - Isolation of Antibacterial and Antiviral Substances
from Shellfish 77
65 - Longitudinal Studies of Beta-hemolytic Streptococcal
Isolations 80
65-A - Streptococcal M Protein, Virulence, and Type-
Specific Immunity 83
66 - Epidemiologic Studies of Illnesses and Microbial
Experience of Junior Village Nursery Children 89
66-A - Epidemiologic Studies of Host Parasite Disease
Relationships 93
66-B - Epidemiologic Studies of New Vaccines and Chemo-
prophylactic Agents 95
66-C - The Etiology of Respiratory Disease on a College
Campus 97
66-D - The Role of Viruses in the Etiology of Ear Disease 99
66-E - A Study of the Influence of Influenza A and B Mineral
Oil Adjuvant Vaccines on Influenza Antibody Patterns,
8-9 Years after Vaccination 100
67- - Studies on Human Enteroviruses, Adenoviruses, and
Reoviruses 103
67-A - Studies on Viruses of the Enteric Tract of Cattle 107
67-B - Studies on the Etiology of Eosinophilic Meningitis
in French Polynes ia 110
II
68 - Studies of Tumor Viruses in Nature 114
68-A - Seroepidemiology of Virus Infections 123
69 - Study of Viruses as Causes of Respiratory Illness
in Infancy and Early Childhood 126
69 -a - Viral Pneumonia: Etiology, Therapy, and Prevention 130
69-B - Study of the Laboratory Aspects of Respiratory
Virus Illness in a Welfare Orphanage 134
70 - Laboratory Studies of Newly Recognized Viruses
Associated with Respiratory Illness 138
71 - Studies of Tumor-Producing Viruses 142
71-A - Continuing Studies of Adenoviruses and Salivary
Gland Viruses as Examples of Latent Viruses 147
71-B - Studies of Mouse Viruses with Special Reference
to their Importance as Extraneous Viruses (back-
ground noise) in Mouse Tumor Systems 150
72 - Laboratory Studies of Enteroviruses 154
72-A - Study of Viral Respiratory Disease in a Military
Population 157
72-B - Study of Respiratory Viruses in Human Volunteers ... 161
73 - Application of Fluorescent Antibody Staining
Technique to the Study of Respiratory Viruses 163
74 - Study of Bovine Respiratory Diseases 165
74-A - A Study of Squamous Cell Carcinoma (Cancer Eye)
in Catt le 170
75 - Laboratory and Epidemiologic Studies of Viruses
as Possible Etiologic Agents of Acute Leukemia
in the Pediatric Age Group 172
76-A - Ecologic Studies of Fungi Pathogenic for Man 176
76-B - In vivo Tests of Antimycotic Drugs and Anti-
biotics 180
76-C - Identification and Study of New and Unusual Fungi
from Mycoses • 183
78-A - Biochemistry and Physiology of Pathogenic Fungi
(In vitro Studies of the Action of Antifungal
Agents on Pathogenic Fungi) 186
78-B - Studies on the Physiology of Coccidioldes
immitis 188
79-A - Immunity Studies with Pathogenic Fungi 191
79-B - Antigenic Studies on Pathogenic Yeasts 193
79-C - Virulence and Pathogenic Studies with Yeasts 196
III
Laboratory of Biology of Viruses
Summary ■ • 1
80 - Basic Studies of Virus-Host Cell Relationships 3
80-A - Rabies Prophylaxis 6
81 - Mechanism of Oncogenic Activity Polyoma Viruses 8
82 - Cell Growth Inhibiting Substances 11
83 - Mutation in Animal Viruses 13
84 - Host-Parasite Relations 15
85 - Investigations of Animal Virus Reproduction 19
85-A - Animal Virus Synthesis 22
86 - Investigations of Interference Enzymatic Functioning of
Mitochondria as a Mechanism for Carcinogenesis 24
86-A - Demonstration of Glucose Metabolism and Peptide Bond
Formation by Isolated Brain and Liver Mitochondria .... 27
87 - Cytopathogenic Effect in Single Cells in Tissue Culture 29
87-A - Characterization of Mitochondria 31
88 - Biology of Mitochondria and Its Relation to Endogenous and
Viral Diseases 34
88-A - Comparison of the Properties of Crude and Crystallized
Coxsackie A-10 Virus, of Cloudman S 91, Mouse Melanoma
and of Mouse Muscle Nucleoprotein 36
89 - Kinetics and Sites of Coxsackie Virus Multiplication in
the Monkey Kidney Cell 38
89-A - Virus Structure 40
Laboratory of Tropical Virology
Middle America Research Unit
Arthropod-Borne Virus Section
Summary 1
93 - Studies on Arthropod-Borne Viruses in the Sub-tropical
Areas of the United States 6
94-A - Studies of Arthropod-Borne Viruses in Tissue Culture...
Part 1. Evaluation of Tissue Culture Systems for Use
in Viral Isolation, Identification, and in Serological
Tests for Viral Antibodies 8
94-B - Studies of Arthropod-Borne Viruses in Tissue Culture.
Part 2. Development of a Cell Culture System Utilizing
Arthropod Tissue 11
95-A - Studies on Antigen- Antibody Reactions of Arthropod-
Borne Viruses. Part 1. Kinetic Studies of the Serum
Neutralization of the Arthropod-Borne Virus 13
95-B - Studies on Antigen- Antibody Reactions of Arthropod-
Borne Viruses. Part 2. Development of a Practical and
Specific Flocculation Test for the Demonstration of
Arthropod-Borne Virus Antibodies 15
IV
95-C - Typing of Viruses by Combinations of Antiserum Pools.
Application to Typing of Arthropod-Borne Viruses .... 17
96-C - Survival Potential of the Adult of Haemagogus Equinus,
A Sylvan Vector of Yellow Fever 19
97-A - A Qualitative Evaluation of Experimentally-Induced
Eastern Equine Encephalomyelitis (EEE) Virus Infec-
tion in Horses 22
97-B - The Development of an Inactivated Vaccine Against
Eastern Equine Encephalomyelitis (EEE) Virus 24
100-A - Virological Investigation of Clinical Cases and
Epidemic Outbreaks in Panama and Other Countries of
Middle America 27
100-B - A Clinical and Virological Study of Oropharyngeal
Lesions in Panamanian Children 33
101-A - Virological Aspects of a Cooperative Investigation
on the Ecology of Arthropod-Borne Viruses 35
101-B - The Role of Chiggers and Other Acarina of the American
Tropics in the Maintenance and Transmission of Animal
Infectious Agents: 1. Viral Aspects 39
101-C - "Jungle Fever" in U.S. Military Personnel 41
102-A - Enterovirus Infections of Rural Guatemalan Children
in Relation to Nutrition 43
102-B - Laboratory Support of Phase 1, National Program for
Poliovirus Vaccine Administration in Costa Rica, 1959 45
102- C - Enterovirus Flora of Panamanian Children: A Twelve
Month Survey 48
103-A - Use of Filter Paper Discs for Virus Isolation and
Serological Testing 50
103-B - Eastern Equine Encephalomyelitis (EEE) Virus Infec-
tion in Panama 53
103-C - Encephalomyocarditis (EMC) Virus Infection. Studies
on Pathogenesis in Swine, Virus Reservoirs in Rodents
and Antibody Status of Human and Animal Populations.. 55
108 - Studies of Histoplasmosis on the Isthmus of Panama .. 59
109 - Studies of Superficial and Deep Mycoses in Panama
and Central America 63
Laboratory of Germfree Animal Research
Summary 1
110 - Pathogenesis of Amoebiasis 5
111 - The Use of Germfree Animal and Tissue for the Study
of Viruses 8
112 - Biology of Germfree Animals 10
113 - Origin of Anti-Human Blood Group B Agglutinins in
White Leghorn Chicks 14
114 - Studies on Bacterial Interactions and Host Bacterial
Relations in the Germfree Animal 16
115 - Behavior of Parasitic Protozoa in Germfree Hosts .... 18
116 - Studies on Helminthic Infections in Germfree Hosts .. 20
V
Laboratory of Parasitic Diseases
Summary *
120 - Administration and Research Planning and Coordination . 6
121-A - Host Parasite Relations in Worm Infections in Labor-
atory Animals 7
121-B - Role of Helminths in the Causation of Cancer 9
121-C - Destruction of Molluscs by Chemical Means 11
121-D - Part A. Investigation of Intermediate Hosts and
Vectors of Human Diseases Caused by Worms 14
121-E - Development of Methods for the Cultivation of
Parasitic Helminths in vitro, and the Determination
of the Nutritional Requirements of Such Organisms
in vitro 17
121-G - The Development of Helminths in Germfree Animals 21
121-1 - Pathological Physiology of Worm Infections 23
121-J - Serum Protein Studies on Germfree Animals Infected
with Various Parasites 25
121-K - Effects of Nutrition on Chemotherapy of Parasitic
Diseases 27
121-L - Effects of Improving the Nutrition of Malnourished
People Infected with Schistosoma Mansoni 30
121-M - Ammonia Metabolism and Toxicity in Relation to Liver
Disease 33
121-N - Parasitological Investigations at the Marine Biological
Laboratory, Woods Hole, Massachusetts 36
123-A - Fundamental Physiological and Biochemical Studies on
Parasites, Intermediate Hosts, and Parasitized Animals 38
123-B - Pathological Physiology and Histochemistry of Parasitic
Diseases 42
123-C - Biochemical Mechanisms of Energy Metabolism in Normal
and Parasitized Animals 45
123-D - Biology of Trypanosomes 49
127-A - Studies on Toxoplasmosis 52
127-B - Studies on Entamoeba Histolytica and Other Parasitic
Protozoa 57
127-C - Trichomoniasis 60
127-D - Biochemistry of Parasitic Protozoa 63
Laboratory of Parasite Chemotherapy
Summary 1
130 - Administration, Research Planning and Coordination .... 6
130-A - Studies in Human Malaria 10
131 - Drug Resistance in Experimental Malaria 15
131-A - The Effect of Adrenal Cortical Hormone on Chemotherapy of
Experimental Malaria 17
VI
131-B - Antimalarial Drugs and Nucleic Acid Synthesis 19
131-C - Experimental Chemotherapy of Helminthic Diseases .... 22
131-D - Effects of Nutrition onChemotherapy of Parasitic
Diseases 25
131-E - Comparison of Antimalarial Drugs Administered Singly
and in Various Combinations 28
131-F - Effect of Nutritional Status of Host on the Action
of Antimalarial Drugs 30
132 - Studies on Human Malaria 32
132-A - Chemotherapy of Intestinal Parasites 37
132-B - Physiology of Human Parasites Especially as Related
to Nucleic Acids and Drug Action 40
132-C - Virus-Parasite Association 43
132-D - Development of Human Pathogens in Immature Insects .. 47
132-E - Insect Tissue Culture 49
133 - Studies of the Biology of Exoerythrocytic Phases of
Primate Malaria 51
133-A - Chemotherapeutic Studies of the Direct Effect of Drugs
on the Exoerythrocytic Stages of Primate Malaria .... 54
133-B - Studies of Simian Malarias in Man to Determine If
Malaria Is a Zoonotic Disease in Areas in Which Man
and Monkey Are Closely Associated 57
133-C - In Vitro Studies of Malaria Parasites and Liver
Tissue 61
133-D - Immunological Studies of Malaria Parasites 66
Laboratory of Immunology
Summary 1
140 - Studies in the Transplantation of Tissues 4
141 - Studies on Antibody Production and Mechanism of Hyper-
sensitivity
142 - The Immunology of C h Mouse High Line Sarcoma 9
143 - Characterization of Substances of Bacterial Origin
Affecting Resistance to Infection 11
144 - Pathogenesis of Allergic Encephalomyelitis 14
145 - Studies of Auto-Antibodies in Human Disease States and
in Experimental Animals 19
146 - The Resistance of Fetal and Neonatal Gonads to Damage
by Maternal Immunization to Testicular Antigens 23
147 - Immunochemical and Immunogenetic Studies of the
Protein Isoantigens in Serum 25
147-A - Immunochemical Studies on Human Serum Antigens and
Antibodies 32
148 - Basic Studies on the Cellular Localization of Anti-
gens, Antibodies, and Other Substances by the
Fluorescent Antibody Technique and Fluorescence
Micros copy '. 34
150 - The Relationship of Delayed and Immediate Hypersensi-
tivity to Allergic Thyroiditis in Inbred Guinea Pigs 39
VII
152 - The Chemistry of Antibodies 42
153 - Basic Studies on the Chemical, Physical and Skin-
Reactive Properties of Extracts of House Dust 47
Rocky Mountain Laboratory
Summary 1
170 - Office of the Director 11
171 - Rickettsial Infections 12
172 - Q Fever 13
173 - Diseases Having a Reservoir of Infection in the
Natural Environment (other than rickettsioses) 22
174 - Transmission of Disease Agents by Certain Vectors .. 27
175 - Allergy and Immunology of Fungal Infections and the
Mechanisms of Allergic Phenomena 33
176 - Viruses, Virus Components, and Virus Surfaces 39
177 - Immune Prophylaxis of Mycobacterial Infections 44
178 - Immune Prophylaxis of Salmonella Infections 48
179 - Investigations of the Role of Morphological Elements
of Microorganisms in Immunity and Related Phenomena 54
180 - The Encephalitides 58
181 - Colorado Tick Fever 63
Laboratory of Bacterial Diseases
Summary 1
200 -Brucellosis 2
201 - Bas ic Studies on Staphylococcus 5
202 - Identification of Pseudomonas 7
203 - Studies on Bacteria Characterized by the Production
of Reproductive Filterable Granules 9
204 - Intracellular Parasitism 11
205 - The Role of Infection in the Delayed Deaths of Mice
Following Extensive Burn Injury 13
VIII
DIRECTOR - NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS
DISEASE
Summary 1
Annual Report of Program Activities
National Institute of Allergy and Infectious Diseases
January - December 1960
OFFICE OF THE DIRECTOR
1. The calendar year 1960 witnessed significant extension of both
intramural and extramural activities carried on or supported by the National
Institute of Allergy and Infectious Diseases. These are detailed in succeed-
ing sections .
2. Of special significance were the steps taken during the year
toward the development of International Centers for Medical Research and
Training. These are to provide tangible implementation, in addition to the
research presently supported overseas by the various Institutes, for Senator
Hill's "International Health Research Act of I960" (Public Law 86-610).
These Centers have evolved from proposals submitted originally to
this Institute for research on diseases in the tropics. This area of con-
cern has now been expanded to encompass most of the research and research
training interests supported by the National Institutes of Health. Each
Center represents a collaborative undertaking between a sponsoring medical
segment of an American university and a host institution of medical learning
overseas. Within this setting, it is expected that faculty members, post-
doctoral trainees, graduate students, and other specially qualified indivi-
duals from this country may work together with foreign scientists and their
staffs in solving problems of universal medical interest, in extending scien-
tific knowledge, and in creating and fostering international fellowship and
good will .
While the initial emphasis is on the medical and supporting sciences,
it is anticipated and hoped that these Centers may provide overseas oppor-
tunities of interest to the socio-technical and related research interests
supported by foundations or institutions outside the National Institutes of
Health.
Four Centers* have been recommended for approval. Administrative and
operational responsibilities are assigned, for the present, to the National
Institute of Allergy and Infectious Diseases. The Councils of the other
* University of California
Tulane University
Johns Hopkins University
University of Maryland
University of Malaya, at Singapore
University of Malaya, at Kuala Lumpur
Universidad del Valle, Cali, Colombia
All-India Institute of Hygiene,
Calcutta, India
Field Unit, Rawalpindi, Pakistan
Institutes and the Division of General Medical Sciences have agreed to make
available to the National Advisory Allergy and Infectious Diseases Council
funds appropriated for International health research in Fiscal Year 1960,
with the understanding that 1) regular progress and status reports will
be made to the other National Advisory Councils, and 2) that incorporation
of the lists of grants awarded in the formal minutes of the other Councils
will constitute formal endorsement of these Councils.
A standing committee has been established to advise the Director,
National Institutes of Health, with reference to the future conduct of the
Centers, and to evaluate and advise regarding applications for additional
Centers .
3. Staffing, space, and funds for the support of the National
Institute of Allergy and Infectious Diseases have been .reasonably satisfac-
tory for current needs this last year, with one exception. The death of
Dr. Jules Freund, since the last Annual Report was written, has left a
unique void in competent leadership in immunology and allergy that is
proving difficult to fill. Some eight outstanding scientists and leaders
in this field have been seriously considered, brought to the Institute to
see and to be seen, and certain of them have been invited to take over this
responsibility. This activity has become of special importance since 1955,
when the title of the Institute was amended to include the word "Allergy."
For various reasons, mainly satisfaction with their current situations, it
has not been possible to persuade any of these candidates to accept the
position as Chief of the Laboratory of Immunology.
While the existing staff is operating admirably in the discharge of
its responsibilities, the lack of expert leadership is recognized. Every
effort will be made in the forthcoming year to attract a competent individual
to this important position.
EXTRAMURAL PROGRAM
Summary.
Annual Report of Program Activities
National Institute of Allergy and Infectious Diseases
January- December, i960
Extramural Programs
In spite of the commonly held misconception that modern
antibiotics have relegated the infectious diseases to a position
of relative unimportance in human health, recent statistics from
the U. S. National Health Survey indicate that Infection and al-
lergy cause more than two-thirds of the acute illness in the
United States, and furthermore are responsible for almost one-
fifth of the chronic disease in the Nation. Fully cognizant of
these facts, the Institute has continued to build a balanced
program of research and training emphasizing the fundamental
nature of pathogenic organisms and allergens, the mechanisms by
which they reach and damage their hosts, and the means which may
be developed to combat them.
Research Grants
The distribution of research funds in support of the Institute's
objectives is shown in Tables I and II. Data regarding the review
and approval of research grants are given in Table III.
The Council continued to emphasize and support areas of special
research need. Under the Committee on Standardization of Allergens
an important program is progressing along three fronts: chemical
isolation of the allergenic fractions of ragweed pollen; immuno-
logical comparison and standardization of the active fractions; and
clinical testing to determine which fractions are important in human
hay fever. Information exchanged last April during an informal con-
ference of the scientists working on these three aspects indicated
that the substance known as trifidin apparently is the purest com-
ponent yet isolated, but that at least eight different ragweed pollen
fractions seem to be active allergens in laboratory studies.
To maintain continued stimulation of influenza research between
epidemics, the Institute established a program of "Special Grants"
which the Council authorized under the Subcommittee of Investigators
of the Committee on Influenza Research. Selected scientists work
toward specified objectives under protocols developed in collaboration
with the Subcommittee. For this purpose the Council recommended a
reserve of $175 .> 000 from each of two fiscal years, with unused funds
reverting to the regular research grant program. At the close of the
-1-
year 10 such projects for $115,713 are being supported. In February
the Institute sponsored a symposium on the 1957-5& Asian influenza
pandemic. This conference emphasized deficiencies in present know-
ledge and thus suggested areas for intensive research stimulation.
As a further service to research workers, funds were provided for the
preparation of an extensive annotated bibliography of influenza research,
which has been distributed to all medical libraries in the United States
and to the principal medical libraries of the world. In addition, the
Subcommittee of Investigators and the Committee on Influenza Research
promulgated a series of recommendations for dosage and criteria for
vaccination against influenza in the civilian population.
Inability of the antibiotics to control resistant strains of staphylo-
cocci, especially in hospital epidemics, created a critical and alarm-
ing situation which prompted Congress to Include in the 1959 appropriat-
ion for this Institute a one million dollar increase over 195^ levels
specifically for research in this area. With the impetus of these
additional funds the Institute so accelerated its grants program that
it now supports 102 staphylococcal research projects totalling $1,922,
283. More than one-third of these studies are directly related to
hospital problems, especially epidemic outbreaks in nurseries or post-
operative Infection of surgical wounds. One such project concerns the
design of hospital nurseries with relation to the spread of organisms;
another is a large-scale coordinated study by five medical school
hospitals and the National Research Council to evaluate ultraviolet
irradiation of operating rooms in the prevention of wound Infections.
Approximately one-third of the Institute's program in this area deals
with basic studies on the fundamental nature of the staphylococcus
organisms and their relationships to the host. Other grant- supported
areas of investigation include clinical aspects of staphylococcal
disease, new drugs, and the role of these organisms in food poisoning.
The Council pointed up special needs in three other areas through
partial sponsorship of research conferences, namely (l) the "Second
Conference on Medical Mycology," (2) a "Symposium on Immunochemical
Approaches to Problems in Microbiology, " and (3) a "Symposium on Air-
borne Infection." Moreover, consonant with the Institute's ongoing
interest in tropical medicine and related fields the Council supported
the "Fourth Conference on Research Needs in Tropical Medicine." This
meeting brought together a group of selected authorities to delineate
the specific aspects which should be emphasized by future research and
training in the broad fields of tropical medicine and environmental
health.
As a consequence of the Council's interest and positive action
based on a recommendation of the Virology and Rickettsiology Study
Section, an Adenovirus Committee was established early in the year
to provide needed leadership for research on the adenovirus group of
filtrable agents by providing a generally- accepted set of standards on
which identification of these viruses can be based. The most urgent of
the Committee's objectives has been the production of standardized
reagents which would permit research workers to identify adenovirus
strains. Through a technical subcommittee specifications have "been
developed for the production of type- specif ic antisera for the first
2.6 adenovirus types. Proposals have been solicited from prospective
producers and it is expected that contracts can be negotiated soon
so that the needed reagents will be available for testing and certi-
fication by the end of 1961. The Adenovirus Committee now plans to move ,
against other problems in the standardization and characterization of
this group of viruses.
Recognizing that significant research of high quality is being
conducted at many institutions outside the United States, the Council
has felt it both justified and desirable to support carefully selected
projects wherever they may be located. Thus research grants have been
awarded to scientists at foreign Institutions whenever a competent
Investigator proposed a project which for geographical or other reasons
could best be done abroad, or when the investigator was so out standing
that his contribution would have general scientific significance. On
this basis some 68 grants for $925,829 have been supported during the
year.
The current appropriation for this and several other Institutes
includes funds for the planning, negotiating, and initial support of
clinical research units spanning the interests of the several sup-
porting institutes. Such centers are being established in large medi-
cal research institutions throughout the United States. Central review
and administration of this program is the responsibility of the National
Advisory Health Council and the Division of General Medical Sciences.
In addition, $1,4-54-, 000 from the current appropriation of this Institute
has been reserved to establish the Institutional Grants Program.
To provide more expeditious payment of continuation grants when
the appropriation is delayed beyond the beginning of the fiscal year,
all research grants having anniversary dates in the first two quarters
are being forward financed to provide for renewal on December 1 or
later. This process will be completed before the end of the current
fiscal year.
In conformity with the criterion regarding scientific excellence
which the President set forth in approving the 1959 appropriation bill,
the Council has continued to give special consideration to all appli-
cations falling within the lowest ten per cent of the priority list for
each study section.
Training Grants
Following a policy initiated last year, the Institute has con-
tinued to develop its training grants program along lines designed to
emphasize disease-oriented interests in allergy and infectious disorders,
-3-
To Insure highly competent professional review of applications, two
panels of consultants were established to cover the Institute's "broad
training areas. These are the Allergy and Immunology Training Grant
Committee, and the Infectious Diseases and Tropical Medicine Training
Grant Committee.
Table IV indicates the distribution of training grant funds in
support of the Institute's objectives. Table V gives data regarding
review and approval of applications during the calendar year.
The Institute now has completed the forward financing of all
training grants so that continuation years will begin on July 1. Under
this arrangement it now is possible to assure grantees of continuation
support several months in advance of their needs, thus obviating the
uncertainty and inconvenience formerly caused by delays in the appro-
priation of funds.
Certain foreign institutions have opportunities for providing out-
standing training in special research areas to American students as
well as their own nationals. Recognizing this fact the Institute cur-
rently is supporting one training grant at a Canadian institution, with
the proviso that stipends from the grant be awarded to American citizens
only. Two other training grant applications from foreign institutions
are pending review. The Council has continued to insist that, after
the first year, at least half the funds awarded through all training
grants be devoted to stipends and other direct trainee support such as
tuition, travel expenses, and consumable supplies.
As the culmination of more than two years of planning by the
Council and the Institute's staff, a program of tropical medicine
training centers was established. Four of the six centers originally
included in this series subsequently have been broadened under auth-
ority of PI 86-610 (see below). The two remaining centers will pro-
vide opportunities for field experience in tropical medicine at cooper-
ating research establishments in various overseas locations. Support
for five years was recommended, with re- evaluation after three years.
In recognition of the high cost of this type of training only 30 percent
of the yearly budgets after the first year must be used for stipends and
direct trainee benefits.
International Centers for Medical Research and Training
Under the International Health Research Act of i960 (PL 86-6lO)
the Congress made funds available to the NIH for the establishment of
International Centers for Medical Research and Training. The Council
of this Institute accepted responsibility for the review of applications
for grants to support multidisciplinary projects under this new program,
to be financed from funds made available under PL 86-610 to the Division
of General Medical Sciences and various Institutes of NIH. Four of the
programs already activated as Tropical Medicine Training Centers (see
above) were appropriately expanded into research and training centers
encompassing the categorical Interests of the several Institutes and
the Division of General Medical Sciences. In making its recommendations
the Council had the technical advice of the Committee on International
Centers for Medical Research and Training. It is anticipated that addi-
tional universities will be considered as potential program participants
early in the coming year.
Research Fellowships
Table VI shows the distribution of awards during the year, by
areas of study and level of support. The data regarding review and
approval of "applications . are presented in Table VII. The fellowship
program of the Institute has followed the pattern set last year, with
increasing emphasis on awards at the postdoctoral and special levels
to produce increasing numbers of competent independent investigators.
Beginning July 1, i960, the funding as well as the administration of
all predoctoral awards was taken over by the Division of General Medi-
cal Sciences. The data given in Tables VI and VII for predoctoral
fellowships apply to those awarded during FY i960 and funded by this
Institute.
Three additional Fellowships Programs, designed to support faculty
positions at different levels, are being activated by the Institute and
will be in operation during the third and fourth quarters of FY 1961.
The Career Research Professor Grant Program has been established to
support individuals of demonstrated capacity to pursue with distinction
a professorial career in independent research and teaching. The Senior
Fellowship Grant Program, formerly limited to preclinical science depart-
ments and administered only by the Division of General Medical Sciences,
has been expanded to include clinical departments as well as certain
departments in university graduate schools and other appropriate insti-
tutions. These awards are for the support of individuals with at least
five years of relevant research experience beyond the doctorate who have
demonstrated high potential for a research or academic career. The
Special Fellowship Grant Program will support the promising young invest-
igator not yet eligible for an award at a higher level but who the appli-
cant institution feels would be an important adjunct to its teaching and
research staff. This program is in addition to the present Special
Fellowship awarded directly by the Public Health Service to individuals
for advanced or special training.
-5-
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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
0
31
._
0
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._
$852,681
$527,304
June
New
25
$1,791,122
15
$1,436,275
Continuations*
83
108
2,633,605
$4,424,727
83
98"
2,633,605
$4,069,880
* (includes continuation grants for which previously recommended support
was reaffirmed) .
November
New 13 $408,090** 9 $277,730**
Continuations 0 — 0
13 $408,090 ~9 $277, 730
** (includes $32,349 to forward finance one request).
l/ Amounts do not include $50,000 approved as a Chairman's grant, nor
$383,162 needed to forward finance 17 grants. Neither do they include
awards for four International Centers for Medical Research and Training
at a total of $878,342 (lst year $478,566; 2nd year $399,776).
12/15/60
-ID-
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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 inherent in the currently used
methods of direct darkfield examination, cultural procedures or animal
inoculations. The new serodiagnostic modification becomes positive with-
in three weeks after infection and, in the later stages of the disease,
continues to indicate the presence of leptospiral antigen for several
weeks after it is no longer demonstrable by laboratory animal inocul-
ations.
Expecially significant findings have been reported in the field of
staphylococcal disease and the special problems presented by antibiotic-
resistant strains of staphylococci. Recent reports by one of our grantees
at the Hospital for Joint Diseases, New York City, suggest that the
emergence of drug resistance actually represents a selection of pre-
existing resistant strains rather than the development of new ones. In
studying 1°^- staphylococcal strains isolated between 1927 snd 19^7.*
before antibiotics were widely used, this investigator found that 22
per cent belonged to the "phage type 8o/8l" which has been particularly
dangerous in hospital infections during recent years. This observation
dispels the apparently common belief that strains of "type 80/'8l" are
"new" staphylococci of recent origin. Another grantee, at the University
of Texas Southwestern Medical School, has shown that the genetic factor
responsible for resistance to streptomycin can be transferred from
resistant to drug- sensitive strains of staphylococci by the viruses
(phages) which parasitize them.
At Baylor University Medical Center a grant- supported project- has
shown that normal human white blood cells, which usually destroy
bacteria by ingesting them, actually provide protection against anti-
biotics for pathogenic staphylococci ingested by the cells. In these
studies, 10-100 fold increases in the concentration of penicillin and
other antibiotics failed to kill staphylococci which had been ingested
by the white blood cells..
-15-
Two groups have reported interesting findings on their studies
of staphylococcal infections in hospital nurseries. Working in a
hospital that permitted intermittent "rooming- in" of infants with their
mothers, investigators at the University of California at Los Angeles
found that both the infants and the air of the nursery had a very low
incidence of staphylococci while the mothers had a relatively high
incidence of bacteria which they did not transmit to their rooming- in
infants. Another research team, at the New York Hospital- Cornell
Medical Center, found that most newborn infants who are infected with
staphylococci have a relatively low index of infectivity, while a small
minority are highly infectious. Because these latter infants literally
are surrounded by clouds of bacteria they are designated "cloud babies."
Follow-up studies showed that their explosive role in hospital epidemics
continues, in the family unit, after they leave the hospital. The
explosiveness of the "cloud baby" outbreaks, the authors say, .makes it
imperative to design nursery units in such a way as to prevent airborne
dissemination of infection.
A great deal of research progress has been reported by our grantees
in the field of virology. A research team at Harvard Medical School
revealed poliovirus variants which resisted temperatures generally
believed to be rapidly destructive of all three types of virus. Whereas
previous studies have shown that poliovirus is rapidly destroyed at
temperatures slightly exceeding 60 degrees centigrade, the present
series of experiments indicated that suspensions of the virus grown in
monkey kidney cells were not completely inactivated following exposure
for one hour to temperatures of 60 degrees and 65 degrees centigrade.
In one instance infectious virus was demonstrated in Type II poliovirus
after heating at 75 degrees centigrade for one hour. Progeny of virus
surviving at this temperature showed increased thermoresistance. These
results suggested that pasteurization of milk and other food products
as now carried out cannot assure the complete inactivation of these
agents.
Grantees at the University of Pittsburgh found that previously
recognized dengue and chikungunya viruses, as well as two new dengue-
like agents, apparently have an etiologic role in a new and frequently
fatal type of human hemorrhagic fever occurring in the Philippine Is-
lands and Thailand. Aedes aegypti mosquitoes were incriminated as the
important vector in two epidemics. This work included the first reported
instance of the isolation of a dengue virus from wild-caught mosquitoes.
Among young adults at the University of Wisconsin, the measure of
acute respirator/disease which can be identified etiologically has been
raised to over 28 per cent by one of our grantees. In previous work
this investigator had identified about 20 percent of these illnesses
as specifically due to streptococci, influenza virus, adenovirus, or
bacteria other than streptococci. The present studies on 227 students
hospitalized with respiratory illness over a two year period showed
-16-
that the hemadsorption viruses are responsible for another 8 to 10 per-
cent of the cases and thus are the most frequent cause of sporadic
respiratory infection yet found in the student body. A clear definition
of the relationship between specific viruses and clinical symptomatology
in terms of age group, frequency of attack and types of population
involved will facilitate the creation of practical measures for the
prevention of respiratory disease.
Impressive evidence for the effectiveness of an attenuated virus
vaccine for measles has been recently presented in a series of reports
by 23 investigators in 11 institutions working on a coordinated program
under grant support from seven organizations, including this Institute.
The investigators agree that any proposed vaccine for measles must be
justified as both desirable and acceptable. In measles, because of the
significant mortality, frequency of bacterial complications, occasional
involvement of the central nervous system, widespread morbidity of the
uncomplicated disease, heightened virulence of outbreaks in isolated
populations and unfavorable effects on certain other pre-existing ill-
nesses, a preparation conferring immunity comparable to natural measles
seems desirable. The present experimental vaccine may be acceptable
because of the following properties delineated in the various reports:
ease of administration; lack of local reactions; absence of communicability;
high index of serological response; elimination of bacterial complications;
and demonstrated prophylactic efficacy. The work specifically supported
by this Institute and carried out by grantees at Yale University and
Western Reserve University, respectively, compared (l) the effectiveness
of the subcutaneous route of injection with others that might be involved
in natural transmission of measles and (2) the clinical, antigenic and
prophylactic effects of the vaccine in institutionalized and home-
dwelling children.
-17-
ASSOCIATE DIRECTOR IN CHARGE OF RESEARCH
INTRAMURAL RESEARCH PROGRAM
Summary
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
ANNUAL REPORT OF THE ASSOCIATE DIRECTOR IN CHARGE OF RESEARCH
INTRAMURAL RESEARCH PROGRAM
CALENDAR YEAR 1960
The year 1960 has seen both a widening of the scope of research activi-
ties and an intensification of the pursuit of promising lines of investigation
in the National Institute of Allergy and Infectious Diseases. The coming year
will see even more effective utilization of scientific resources, as new space
becomes available and programs initiated this year begin to achieve their
objectives.
Clinical investigations of the Institute have advanced in several ways.
Of great significance for present and future programs has been the increased
utilization of prisoner volunteers for clinical studies, through cooperation
with the Federal Bureau of Prisons. Volunteers have been hospitalized in the
Clinical Center, exposed to specific respiratory viruses, observed for clinical
manifestations, and examined by precise laboratory techniques for evidence of
infection. In a short time, these human volunteer studies have made available
a vastly greater amount of detailed information concerning the respiratory
syncytial virus and the Eaton agent than would have been possible by the usual
clinical and epidemiological observations in the general population.
In connection with investigations on simian malaria, prisoner inmates
have been inoculated with various strains of the parasite in the Atlanta
Penitentiary. Some of them were transferred to the Clinical Center for
precise clinical observations. This study also has permitted the extension
of clinical and laboratory observations not possible in any other way. Other
clinical studies underway for some years such as treatment of fungus infections,
bacterial complications of cystic fibrosis, and drug resistance in staphy-
lococcal infection have continued to add to specific knowledge of these
diseases.
During the year an observation of great potential importance to public
health was made by Institute malaria investigators and quickly substantiated
by two other laboratories. Two scientists working with a strain of monkey
malaria were accidentally infected, probably by a mosquito bite. This was the
first clear-cut evidence that certain strains at least, of simian malaria,
are pathogenic for man. An intensive investigation using all available
resources quickly showed that man could be repeatedly infected by this strain
through mosquito bite, that it could be transferred from man to man by blood,
and that monkeys could be infected by mosquitoes fed on human cases. These
observations formed the basis of a greatly expanded program on the pathology
and biology of malaria, including the establishment of a field party in Malaya,
the origin of the monkey strain infectious for man.
The Middle America Research Unit in Panama, established three years
ago under the sponsorship of this Institute and the Walter Reed Army Institute
of Research, has intensified its studies of the arthropod-borne viruses in
the tropics. Laboratory procedures have been developed and diagnostic
reagents prepared for working with a large proportion of the more than 125
arthropod-borne viruses. The Middle America Research Unit also has extended
its studies to include special epidemiologic observations of poliomyelitis
in Panama, and investigations of the role of mites in Central American virus
infections. In conjunction with Gorgas Memorial Laboratory workers, Institute
scientists recovered two strains ol vesicular stomatitis virus from Phlebotomus
flies. This is the first time this group of insects has been definitely
incriminated as a possible vector of this important virus.
Increasing interest in immunologic phenomena manifests itself in
nearly all aspects of Institute research activities. The development of new
immunologic techniques, such as Immunoelectrophoresis and immunofluorescence,
has stimulated new approaches to new and old questions and attracted investi-
gators into the field. The result has been a noticeable resurgence of clinical
and scientific interest in allergic diseases. The Institute initiated a new
program in clinical immunology during the year which will investigate such
autoimmune diseases as lupus erythematosus, and thyroiditis, as well as
certain clinical aspects of hypersensitivity and mechanisms of resistance.
The very important studies on respiratory infections which have been
underway for some years continue. It is now clear that the Eaton agent is,
as long suspected, an important cause of primary atypical pneumonia. Develop-
ment of more precise laboratory techniques for isolation of viruses from
animals, demonstrated that many laboratory mouse stocks are grossly contami-
nated with normally occurring latent viruses. At least five different viruses
are now known to infect apparently normal mice. This has complicated studies
on the relation of viruses to cancer, and confused much of the work in this
field conducted over the last few years. It seems unavoidable that the
development of pathogen-free animal stocks, particularly mice, will be
essential for future Investigations in cancer and in other diseases as well.
The development of some means to eliminate or control these contaminating
viruses from experimental animals poses a major and immediate problem to this
Institute and to investigators elsewhere.
The year has witnessed also the planned development of research
experience for junior investigators and of opportunities for permanent staff
members to work in research centers outside the National Institutes of Health.
The key to productive and significant research is, as has been pointed out
repeatedly, the selection of imaginative, energetic and well trained investi-
gators and the development of a stimulating environment in which these
investigators have the freedom and resources to pursue their scientific
ideas. One approach to this goal is to recruit competent interested young
men soon after completion of their doctoral training. During the last year
three such young men Joined this Institute as a part of the NIH Research
Associate Program and seven as part of the Clinical Associate Program. Six
others were recruited to specialized research activities making a total of
16 new scientific staff members at the Junior level. From these no doubt
will come a number of our future permanent staff and all will become
indoctrinated with research experience of great value to their eventual
scientific development. Thus, the intramural activities perform an Important
role for training in research methods and goals to the future benefit of
scientific endeavor, wherever these men may work- in universities, hospitals,
or in the practice of medicine. 2
The Institute has pursued vigorously a policy of encouraging work
assignments of its permanent staff in other well known research centers. One
scientist is working at the Karolinska Institutet, Stockholm, Sweden; two are
at the Pasteur Institute, Paris, France; one at the Max-Planck Institute for
Virus-forshung, Tubingen, Germany; one in collaboration with the University
of California and Institut de Recherches Medicales de la Polynesie Francaise,
Papeete, Tahiti; one at the Wallaceville Animal Research Station, Wellington,
New Zealand; and one with the Virus Laboratory, California State Health
Department. Others have spent shorter periods of time in Malaya, Africa, and
Brazil, pursuing specific research problems. A number of the staff have served
on WHO Expert Committees and on special assignments, particularly in the areas
of tropical medicine and parasitology. In this field, this Institute has
probably the largest group of investigators of any other center in this
country.
Research in tropical diseases will be expanded still further under the
provisions of P.L. 480 which permits the use of foreign currencies to support
specific intramural research projects in certain countries. Projects have
been formulated and implementation of them during the coming year will permit
the selected expansion of investigations important on a worldwide scale.
These include problems in such diseases as malaria, schistosomiasis, fungus
infections, arthropod-borne viruses, and filariasis. These international
activities emphasize the expanding scope of our research responsibilities.
The Board of Scientific Counselors met twice during the year. The
first meeting considered Institute investigations underway on respiratory
diseases of viral etiology. The second meeting was held at the South Carolina
State Hospital, Columbia, South Carolina where the Epidemiology Section of the
Laboratory of Parasite Chemotherapy is located. Institute staff members
reviewed studies on simian malaria, chemotherapy of malaria, and intestinal
parasite infections.
This report arranged by laboratory activities will summarize the
major scientific accomplishments and advances achieved during the year in
the direct intramural program.
LABORATORY OF CLINICAL INVESTIGATION
Clinical research activity has expanded during the past year largely
as a response to enlarging the professional staff and cooperation with other
Institute research units. A further period of growth will be needed to staff
the clinical service in a manner consistent with optimum research productivity.
INFECTION OF VOLUNTEERS An extensive clinical study of acute viral
WITH RESPIRATORY VIRUSES respiratory disease was begun this year in
association with staff members of the
Laboratory of Infectious Diseases. The initiation of this project required
much work but with the unstinting assistance of the Clinical Center, very
satisfactory arrangements have been made to transfer to the Clinical Center,
Federal prisoner-volunteers for study. This has been done with the permission
and considerable assistance of Mr. James Bennett, Director, and Dr. Harold
Janney, Chief Physician of the Bureau of Prisons, Department of Justice.
A team of custodial officers has also been assigned to the Clinical Center
by the Bureau of Prisons to oversee the volunteers. The volunteers have
uniformly cooperated with the program despite some extended periods of room
isolation, frequent blood-letting, and other inconveniences. Many adminis-
trative arrangements have been developed so that the program has worked
increasingly smoothly.
The results so far justify the investment in money and effort. It
has been possible to produce in human volunteers a rather uniform "cold"
with the respiratory syncytial virus. It may occur without respect to
preinfection immunity, but the subsequent rise in complement-fixing anti-
body appears to correlate with severity of illness. Forty-six men have so
far participated in this study.
Approximately 24 other volunteers have participated in studies with
para-influenza 4 virus or Eaton (primary atypical pneumonia) agent. Future
studies are planned with human influenza virus and with the recently defined
group of REO viruses.
NEW ANTI- Beginning about four years ago, the Mycology Section of
FUNGAL DRUG the Laboratory of Infectious Diseases began studies on an
antifungal drug produced by Hoffman La Roche, designated
as RO-2. This agent, an antimicrobial, was found to be the most active
material ever tested in vitro and in animals against several of the patho-
genic fungi, notably histoplasmosis and blastomycosis.
In the intervening years, 30 patients have been treated in the
Clinical Center. Extremely favorable results have been observed in several
patients severely ill with these diseases. From the standpoint of therapeutic
activity, it appears that this drug is the best available for blastomycosis
and histoplasmosis.
During the studies it was noted that the agent produced unusual hepatic
changes. It was found that the dye, bromsulphalein, normally rapidly trans-
ferred from the blood to the bowel by the liver, was retained in high concen-
tration in the blood in patients treated with RO-2. This effect appeared in
a day or two after start of treatment, before tissue changes would likely
occur, suggesting competition of the new drug for the liver excretory mechan-
ism for bromsulphalein. After treatment was stopped, dye excretion promptly
returned to normal or nearly normal . Liver biopsy has revealed changes indi-
cative of minor hepatic damage in some cases. Because of the great importance
of having a drug in addition to the relatively toxic agent, amphotericin B,
for the treatment of fungal diseases, this new agent continues under investi-
gation.
VOLUNTEERS INFECTED Following the demonstration of the infectivity of
WITH SIMIAN MALARIA Plasmodium cynomolgi bastianellii for man, several
inmates at the Federal prison in Atlanta volunteered
for exposure to this agent. The need for careful clinical characterization
of this disease in man led to the transfer of two infected volunteers to the
Clinical Center. Both men developed acute malaria which was carefully studied
throughout its course. Significant alterations in urinary steroid excretion
were detected, an unusual elevation of serum cholesterol occurred in one, and
liver lesions not previously described, resembling but not identical with exo-
erythrocytic phase parasites, were demonstrated in both patients.
PENICILLINS AND Previous work here had defined nutritional requirements
PENICILLINASE for penicillinase production by staphylococci and many
parameters of its interaction with benzyl penicillin
(penicillin G) . During the year English investigators, working with the
stripped molecule of penicillin, 6-amino-penicillanic acid, produced a new
compound largely resistant to destruction by penicillinase. Working with
this and several other penicillin derivatives, it was found that resistance
to penicillinase is greatly influenced by steric positions of ethoxy groups
on the side chain and that failure to be destroyed by penicillinase is
associated with greater penicillinase inducing-capacity . Perhaps most
significantly, this work has added to the now substantial indirect evidence
that the major reason for present resistance of staphylococci to penicillin
is the capacity of these micro-organisms to produce penicillinase upon contact
with even low doses of penicillin.
Clinical studies with the penicillinase-resistant penicillin, dimethoxy-
phenyl-penicillin, have revealed that it possesses resistance to penicillinase
in vivo, and that it is a powerful and effective anti-staphylococcal drug.
After treatment of some patients for periods of three to five months, no
penicillin-resistant staphylococci have been isolated. It has long been con-
sidered that chronic staphylococcal infection resembles tuberculosis and for
the first time it appears that an agent is available which can be employed
for extended periods of treatment without loss of effect, such as isonicotinic
acid hydrazide in tuberculosis. If long-term therapy can thus be regularly
given an enormous benefit will accrue to thousands ill with chronic staphy-
lococcal disease.
ASCITES IN MICE BY In the past year a staff member has continued
INJECTION OF ADJUVANTS to study ascites induced in mice by the in-
jection of adjuvant mixtures. This procedure
has provided a much needed laboratory method for producing antibodies of a
wide variety and a means of evaluating antigens. Recently, interest has
been focused on the pathology and abnormal physiology of the lesion. It has
been found that strain differences are associated with differences in
susceptibility to ascites. Since ascites was found associated with a local
plasma cell reaction, which in some strains of mice went on to plasma cell
tumors, many implications toward problems in neoplasia have also been raised.
These studies have become the basis for biochemical and pathologic studies
with other Institutes.
HYPOGAMMAGLOBULINEMIA Staff members have shown that patients with
hypogammaglobulinemia possess low, but definitely
measurable, levels of antibody to the enteric viruses. The implication of
this finding, in view of the normal resistance of these patients to viral
infections, is that extremely low levels of specific antibody may provide
adequate resistance against viral diseases. Subsequent, unpublished studies
have indicated that these patients will develop circulating antibodies to
Salk polio vaccine.
BENTONITE The modification of the bentonite flocculation test
FLOCCULATION TEST for the detection of gamma globulin promises to
provide the practicing physician with an accurate,
convenient laboratory aid in the diagnosis of hypogammaglobulinemia. In
contrast to the electrophoretic method, the results with the bentonite test
can be known to the physician within a few minutes of arrival of the specimen
to the laboratory. Other modifications of this technique will also give the
levels of albumin and other protein constituents of blood and other fluids
without resort to the more cumbersome method of electrophoresis.
The DNA-bentonite test for systemic lupus erythematosus has also
been developed in the past year. This test measures the antibody in lupus
serum which is directed against nuclear material. The specificity of this
test is greater than any previously described test for lupus. This test,
however, is positive primarily in those patients with active disease and
only rarely is positive when the disease is in remission. A modification of
this test, the nucleoprotein-bentonite test, has retained all the attributes
of the DNA test in regard to specificity, while achieving a much higher level
of sensitivity for cases in remission. These promise to become important
standard tests in the diagnosis of systemic lupus erythematosus.
CYSTIC FIBROSIS OF Despite the commonly accepted point of view that
THE PANCREAS antibiotics are helpful in this disease, observations
of nasopharyngeal cultures failed to reveal any
appreciable effect of antimicrobial treatment on Staphylococcus aureus.
This does not negate a possible clinical benefit but does appear to minimize
its value. It was coincidentally observed that Escherichia coli was not
present in the nasopharyngeal flora of any child over the age of eight.
ROCKY MOUNTAIN LABORATORY
At the Rocky Mountain Laboratory research has continued directed
toward both basic laboratory investigations and field studies of insect-
and animal-borne diseases. In the first category are those projects con-
cerned with the chemistry and surface properties of viruses, the highly
intriguing relations of hypersensitivity and humoral immunity, and the basic
relation of structural elements of microorganisms to the activity of the
agents, both in vivo and in vitro. Field work is the foundation of projects
related to studies of Q fever, tularemia, Colorado tick fever, and the ar?bo
viruses. In addition, the combined efforts of the staff are directed to
many other areas of research. Such projects include Q fever, tuberculosis,
influenza, poliomyelitis, and cryptococcosis.
HYPERSENSITIVITY Studies of hypersensitivity at the Rocky Mountain
Laboratory have been directed primarily toward
clarification of the relations existing between delayed hypersensitivity
and circulating antibodies and determination of the factors responsible for
induction of contact hypersensitivity. It has been demonstrated previously
that delayed hypersensitivity precedes circulating antibodies. This delayed
hypersensitivity is directed toward protein. When circulating antibody
appears, as occurs with relatively large amounts of conjugated protein, or
when booster doses are administered, the specificity of the antibody response
becomes oriented toward smaller configurations on the antigen molecule.
Studies of immunity in neonatal animals have revealed that these animals,
when injected with an antigen 12 hours after birth, develop circulating
antibodies but fail to develop delayed hypersensitivity. Additional
experiments suggest that this inability of neonatal animals to express
such reactions is due to a deficiency in a skin reactive factor rather than
an inability to respond to a primary injection of antigen.
It was shown that contact hypersensitivity is directed toward a
hapten and that simple compounds rather than protein conjugates of these
compounds produce contact hypersensitivity when administered to experimental
animals. It is considered, however, that the production of this phenomenon
is related to the specific proteins present in the host tissues because a
conjugate containing soluble guinea-pig skin proteins and a hapten produces
in guinea pigs delayed reactions and Arthus reactions to the conjugate and
contact hypersensitivity to the hapten.
POLIOVIRUS Continued studies of poliovirus have yielded considerable
fundamental data. In cooperation with the group at the
University of Minnesota, it was shown that agents such as octyl alcohol-
chloroform and neutral hydroxylamine do not materially change the physical
properties of purified infectious RNA of poliovirus but destroy over 99Z
of the infectivity of this material. The destruction of infectivity by
uncoupling of a single link in a large particle (probably an acyl link of an
amino acid with a phosphate group at the end of an RNA chain) suggests a
possible approach to virus chemotherapy. Other studies have revealed that
only 0.17« of RNA infectivity could be accounted for by residual protein,
thus strengthening the concept that RNA does indeed constitute the infectious
portion of the poliovirus moiety. By the use of chromatographic methods, it
was also demonstrated that only certain of the avirulent strains of polio-
virus can be differentiated from the virulent strains from which they are
derived. This is in direct contrast to work reported by others. In studies
of the infectivity of RNA it was found that the bulk of virus particles react
with susceptible cells and that the relatively poor correlation between virus
particles and PFU is due to the poor efficiency of RNA at entering sites
where it can influence virus production. A precipitation test for detection
of antibodies against poliovirus has been developed which is more sensitive
than the neutralization test presently employed. The antigen used is radio-
active virus.
ENDOTOXINS IN Research on endotoxins derived from Gram-negative
BACTERIAL FRACTIONS organisms has continued. As is so frequently the
case, a fresh outlook on an old problem yields
results of great value. The ideas held by Dr. Westphal, which attributed the
activity of endotoxins to the presence of a firmly bound lipid ("lipid A"),
were apparently generally accepted until presentation of the work done at RML.
The finding that lipid A was not active and that deproteinized and "delipif ied"
endotoxin was active stirred considerable controversy. In fact, the controversy
was so intense that efforts to develop a vaccine against Salmonella infections
have been diverted to settling this issue. Recent studies of the kinetics
of inactivation of toxin by hydrolysis with hot acid have given data which
should end this discussion. The old concept of "purified endotoxins" must
be abandoned since there have been no previous toxins as good as those
obtained at RML, and these are to be still further purified.
The purification of Vi antigen by curtain electrophoresis is a major
advance in the study of this most important antigen. The demonstration that
certain labile acetyl groups are responsible for the activity of Vi antigen
resulted in production of material which was ten times more active than
purified preparations prepared by mild acid hydrolysis. Emphasis should be
placed upon the new chemical, physical, and biologic methods that have been
devised to solve these problems.
TUBERCULOSIS The problem of immunity in tuberculosis has been studied
intensively. Significant findings include the fact that
mice may be satisfactorily immunized to subsequent pulmonary infection with
virulent organisms by administration of small doses of avirulent organisms
by the aerosol method. The demonstration that resistance is not due to
interference strengthens the case for the value of immunization with living
attenuated organisms for the prevention of tuberculosis. Since it has been
shown that the delayed reactions elicited by protoplasm of various acid-fast
organisms are specific in nature, it seems practicable to apply these
findings to certain diagnostic problems in man. Use of fractions of tubercle
bacilli in producing isoallergic encephalitis in guinea pigs shows that the
adjuvant effect lies in the cell walls and in a water-soluble protein prepared
from the walls. This latter finding is important since it will allow the
study of the adjuvant phenomenon from a molecular level.
Q FEVER It has been demonstrated that the number of dairy cattle
infected with Coxiella burnetii is large and is increasing,
yet it is extremely difficult to detect cases of clinical disease in man.
In Idaho and Montana we have shown that a greater number of individuals
residing on infected premises have antibodies against this organism than
do those living on noninfected premises, yet no difference can be detected
in the number of individuals who have symptoms compatible with clinical
disease. The fact that organisms isolated from cattle have been uniformly
of low virulence for experimental animals may account for the inability to
find clinical cases of disease in those exposed only to cattle.
By the use of skin tests to eliminate allergic individuals from the
study group, 190 inmates of the Montana State Prison were safely immunized
without producing such reactions as have been previously reported. It is
evident from these results, as well as from those previously obtained in
laboratory personnel, that human beings can be safely vaccinated against
Q fever if the precaution is taken to eliminate reactors by previous
administration of specific skin-test antigen.
Methods developed for growing rickettsiae on modified Zinsser tissue
cultures yielded relatively large volumes of organisms. These studies led to
others involving purification of C. burnetii by sucrose gradients and by
continuous -flow centrifugation in molar salt solution. These methods like-
wise made it possible to obtain certain chemical and physical fractions of
these organisms. It was found that dimethyl sulfoxide could extract from
Phase II C. burnetii a material which acted only as a hapten, but from
Phase I organisms the extract obtained acted as a complete antigen. Lauryl
sulfate also extracts complete antigen from Phase I organisms. Physically,
the cell walls of these organisms can be separated from the protoplasm, and
it has been noted that the cell walls are about 25 times more active in
producing immunity than is protoplasm.
These studies on Q fever are of considerable significance. The
laboratory and related studies have yielded information of both scientific
and applied interest. It is apparent now that we can safely use our present
vaccines for immunization of man and that it is feasible to produce large
numbers of organisms which can be purified and used as vaccine or manipulated
to give physical or chemical fractions which may be less toxic. The failure
to find clinical cases of Q fever in man in the face of a rising incidence of
infection in dairy cattle is highly interesting even if disappointing. The
lack of virulence of strains of C. burnetii for laboratory animals probably
is responsible for this finding.
OTHER RICKETTSIOSES Studies of rickettsiae other than C. burnetii
have been continued. By combining the methods
presently used for fluorescent microscopy, a technique for sectioning
arthropods has been developed which should be of interest to entomologists
working in the field of embryology and anatomy and to medical entomologists,
since thin sections in which the organs are not displaced can be obtained
routinely. By applying the technique to the study of ticks infected with
R. rickettsii it was found that the infection rate in local ticks varies
from 157o to 287„. Not all of the ticks found In ected by this method are
infective for laboratory animals. The value of this type of study has yet
to be fully appreciated.
The use of specific toxins has resulted in clarification of many of
the problems related to the taxonomy of rickettsiae and has proved to be
useful in ecological and epidemiological studies of this complex group of
diseases. In further studies, potent immunogenic extracts have been obtained
from certain of the rickettsiae. Their value as diagnostic and prophylactic
agents is presently under consideration.
BACTERIAL VACCINES Studies have been continued on vaccines for certain
bacterial diseases. It has been found that while
live Russian tularemia vaccine is capable of protecting mice more effectively
than does ether-extracted vaccine derived from cell walls of P. tularensis,
the protection produced by live organisms was not effective for long periods
of time. Continued studies have emphasized the value of cell walls in
producing immunity to infections with Brucella abortus in laboratory animals.
It has also been found that live cells suspended in phosphate buffer and
shaken with an excess of ether are killed but not disrupted. These cells
constitute an excellent protective antigen which is less toxic (LD50 7.5 mg.)
than aqueous ether extracts obtained by conventional methods (LD50 0-9 to
2.0 mg.).
AR-BO VIRUSES Studies of ar-bo viruses have yielded results of interest
and suggest that emphasis on field studies would greatly
increase the production of useful data. The California strain, described by
Reeves and Hammon, has been isolated from a snowshoe hare in Montana, and
serologic studies of hares obtained from Michigan indicate that the majority
possess antibodies against this virus. In California it has been demonstrated
that, although most infections in man with this agent are of the inapparent
type, some infections result in serious disease. A virus closely related to
Powassan virus was recovered from ticks from Colorado and is of importance
since viruses of this group produce serious illness in man. Studies to date
indicate that ticks probably are not the natural vector, but the relation
to Powassan virus suggests that mosquitoes would most likely be the vector
in nature. In studies of the complex relation of WEE virus with snakes and
mosquitoes it has been possible to demonstrate that the virus can be readily
overwintered in garter snakes and that mosquitoes can be infected by feeding
on such snakes. While we have not been successful in isolating virus from
snakes collected in the field, the laboratory data suggest that these or
similar animals could constitute a host suitable for overwintering of WEE
virus. In Idaho and Oregon, WEE virus was isolated with considerable
frequency during the summer season, while in North Dakota the virus did not
appear to be active. Isolations of a considerable number of strains of
trivittatus and inornata viruses were made.
Considerable research was performed to determine the level of viremia
attained in wild and domestic birds infected with ar-bo viruses. After in-
fection with WEE or St. Louis viruses, turkeys, ducks, chickens, and pheasants
display levels of viremia which should cause infection in mosquitoes feeding
on them. It is of interest, however, that in spite of considerable effort
10
we have been unable to isolate ar-bo viruses from the bloods of vertebrates.
Negative results were obtained in examination of 1,074 specimens collected
in Montana, North Dakota, Oregon, and Minnesota during the spring of 1960.
These studies fail to add weight to the contention that latent infections
of birds are a factor in overwintering or of introduction of virus into
endemic areas.
COLORADO TICK Colorado tick fever continues to be a problem in the
FEVER western United States. Without stimulation of physicians
a large number of specimens for examination were received
this year and virus was isolated from 49 of them. Our interest in the
spectrum of symptoms has continued and we still see severe cases of illness
due either to encephalitis or bleeding tendencies. It was found that the
complement-fixation reaction developed at the Rocky Mountain Laboratory is
the simplest method for diagnosis of Colorado tick fever. Vaccine has been
prepared and has been shown to be efficacious in mice. This type of vaccine
has been used repeatedly in man without ill effects, indicating that a
vaccine prepared from suckling mouse brain is harmless to man when repeated
doses are given.
Ticks collected in Estes Park, Colorado, were examined for the
presence of Colorado tick fever virus. The incidence of infection was found
to vary from 57„ to 217o. This high incidence of infection in ticks accounts
for the large number of cases of CTF reported in Colorado annually.
LABORATORY OF TROPICAL VIROLOGY
The activities of this laboratory are conducted at the Middle America
Research Unit in the Panama Canal Zone and at Bethesda. The Middle America
Research Unit is a joint research effort of the National Institute of Allergy
and Infectious Diseases which has cognizance for studies on virus diseases
and the Walter Reed Army Institute of Research which has cognizance for
studies on fungus diseases.
VIRUS ISOLATES FROM During the first 12 months of a 3-year project on
PANAMANIAN MOSQUITOES the ecology of arthropod -borne viruses in the
AND SANDFLIES tropical rain forest, conducted by the Gorgas
Memorial Laboratory with the collaboration of
MARU, major emphasis has been on virus isolation in suckling mice and hamster
kidney cell cultures. Fourteen virus strains were isolated at MARU from 412
pools and 63,000 specimens provided by GML. Virus isolation rates were for
Phlebotomus, 1:700 and for mosquitoes, 1:7000, although the rates varied
greatly with species. Of the five Phlebotomus isolates, two of broad host
range (including cell culture) and short incubation period are serologically
identical. These viruses have now been identified as the Indiana type of
vesicular stomatitis virus. The other three Phlebotomus and nine mosquito
viruses are being related to each other, to known virus groups and to human
and/or animal infection and disease.
11
EASTERN EQUINE ENCEPHALOMYELITIS The prevalence of EEE antibodies in
VIRUS INFECTION IN PANAMA horses and man in two areas of suggested
EEE virus endemicity has been determined,
allowing an evaluation of the relative usefulness of several serological
methods applicable to studies of this type. It was found that the incidence
of EEE antibodies in 460 humans tested increased with advancing age (0.87.
under 10 years with progressive increase to 97o in the 41-50 year group) .
Complement fixation results on the same sera indicated the probable presence
of other group A viruses.
Lizards of species common to this part of Panama were examined as a
possible virus reservoir. Specific EEE virus hemagglutination-inhibitors
were found in some of their sera. The occurrence of viremia and HI antibody
response following virus inoculation were experimentally confirmed by
inoculation of lizards.
ENCEPHALOMYOCARDITIS VIRUS Previously this laboratory described an
INFECTION outbreak of a fatal disease of swine caused
by the EMC virus. The outstanding lesion
in pigs dying during the outbreak was acute myocarditis. Since epidemi-
ological observations suggested that natural infection resulted from ingestion
of contaminated food, experiments were undertaken to reproduce the disease by
feeding virus to young pigs. Viremia and virus excretion from the gastro-
intestinal tract were found to occur following the administration of brain
from EMC inoculated mice. Infected pigs developed high titers of HI and
neutralizing antibody during convalescence and had myocardial fibrosis at
autopsy. Other studies included demonstration of EMC antibodies in a small
number of city rats and rats caught on the affected farm, although wild
rodents were found to be negative. Human sera were examined with interesting
differences in the results depending on the donors' age: while a substantial
proportion of the Panamanian population has been infected with EMC virus,
the antibodies were found to be more common in persons of younger age.
ENTEROVIRUS FLORA IN For a period of 12 months the enterovirus
CHILDREN OF CENTRAL AMERICA flora of infants at an outpatient clinic
in Panama City was systematically explored,
establishing a base line of enterovirus fluctuation. The majority of viruses
isolated belonged to the ECHO group, although in late 1959 and early 1960
poliovlrus type 2 had become very prevalent. This was reflected in an
uncommon occurrence of a small outbreak of paralytic disease due to type
2 poliovirus.
Other enterovirus studies have included 1) surveillance for the
presence of type 1 poliovirus in Panama in late 1960 as a check on dissemi-
nation and threatened spread of this commonly epidemic type, 2) studies on
a major epidemic of ECH0-9 virus which swept through the Republic of Panama
and the Canal Zone and 3) initiation of a collaborative project on possible
relation of enterovirus flora of Guatemalan children to their dietary status.
12
MYCOTIC DISEASES The research program on mycotic diseases has markedly
IN PANAMA increased local awareness of histoplasmosis in all of
its clinical forms, as evidenced by recognition of
three disseminated cases, two fatal and one successfully treated, within a
period of 18 months. Until then only one fatal case had been described
since Darling's original cases in 1906. Ecological and epidemiological
studies led to isolation of H^ capsulatum from eight additional soil samples,
bringing up to 16 the total number of recent isolations from Panamanian soil.
The fungus has been repeatedly recovered from the organs of trapped ground
mammals, confirming its wide dissemination in nature.
Histoplasmin skin test continues to be a major tool for the study
of epidemiology of histoplasmosis. Data on 9,200 children between six
and 19 years of age have been obtained indicating, as expected, that the
percentage of reactors increases progressively with age. The rate of
histoplasmin sensitivity varies from 13% to 587» among six-year olds and from
687. to 927» among 19 year olds, depending on location of their residence.
A survey of 631 pre-school children (six months to six years) in the Canal
Zone demonstrated an increase in hypersensitivity beginning with three years
of age. A continuing similar study of Panamanian children in a city hospital
is now in progress with information on over 800 already available.
Projects on other mycotic diseases have included diagnostic study
and therapy of moniliasis, found to be a major superficial mycosis among
both indigenous and transient population in the tropics.
ARBOR VIRUS STUDIES At Bethesda new projects involved an interesting
AT BETHESDA application of the technique of antiserum pool
combinations to typing of arthropod-borne viruses,
a wealth of data evaluating experimentally produced EEE virus infection in
horses and a promising attempt to develop an inactivated EEE virus vaccine
for human use. The infected horses yielded specific antiserum which is
being processed for prophylactic use in cases of human exposure under
laboratory or natural conditions.
Accidental labbratory infection of a staff member with an arthropod-
borne group C(Apeu) virus led to the first clinical -virological study of a
syndrome produced by this important and common group of viruses of the
western hemisphere.
LABORATORY OF BACTERIAL DISEASES
The research program of the Laboratory of Bacterial Diseases has
continued in the same general areas as last year.
INTRACELLULAR These studies deal with possible changes in characteristics
PARASITISM of infected and immune cells as the result of parasitism,
and the effect of intracellular growth on the parasite.
One such notable change of course is the production of specific antibodies
by certain cells of immune animals. Effort has been directed toward the
study of antibody production by cells in vitro and the macrophage was
13
selected as a multi potential cell for such study. Macrophages obtained from
the peritoneal cavity of guinea pigs immunized with egg albumin have been
found to release antibody in vitro for a period of several days. This provides
a system for further study of the nutritional or other requirements for con-
tinued antibody production in vitro, or even in serial cultures. Cells derived
from macrophages have been carried in serial tissue culture for several months,
retaining their phagocytic ability.
BRUCELLOSIS Studies on brucellosis are conducted at a reduced tempo.
There is continuing need to collaborate with other brucellosis
research centers throughout the world to try and settle problems of classi-
fication and epidemiology of the Brucella. Currently we are doing some
laboratory testing of brucellosis vaccine for human use prepared in Russia.
There is present interest in this vaccine by the World Health Organization
for its possible use in occupational and otherwise continually exposed groups.
Studies on the Staphylococcus are directed toward determining the
factors responsible for pathogenicity, and toward development and standard-
ization of tests for measuring relative pathogenicity of strains.
LABORATORY OF CELL BIOLOGY
The activities of the Laboratory of Cell Biology during the calendar
year 1960 have been along three major lines: (A) The continued exploration
of the metabolism of normal cultured cells, and an approach to the problem
of metabolic controls; (B) the mechanism of viral synthesis; and (C) the
study of cell cultures deriving from patients with hereditary metabolic
disease.
METABOLISM OF NORMAL A number of significant observations have been
CULTURED CELLS made with respect to the amino acid metabolism
of cell cultures. There has been no further
elucidation of the pathway of serine synthesis; but the mechanism of cystine
synthesis has been clarified, in that all the cell lines so far studied have
been shown to use the classical pathway involving the demethylation of
methionine to homocysteine, the condensation of the latter with serine to
form cystathionine, and the cleavage of the latter to cysteine and homoserine.
A dual pathway for proline synthesis has been indicated, one involving
glutamine as the source of the carbon skeleton, and the other involving
arginine by way of ornithine.
An intriguing recent observation has been the finding that a number
of factors which are rigorously required by the cells for survival and
growth can in fact be synthesized. Their nutritional requirement reflects
the fact that they are lost from the cellular pool to the medium at rates
which exceed the biosynthetic capacity of the cell; and with a sufficiently
high cell population density, when the loss to the medium per cell is
sufficiently reduced, the supposedly essential growth factors are in fact
not required for survival.
14
In these cell cultures, unlike bacteria, the biosynthesis of amino
acids is apparently not inhibited by the product of the reaction; and this
mechanism of growth control is apparently not operative. Studies are in
progress as to whether enzyme repression or feedback inhibition are
effective controls in the biosynthesis of pyrimidines. A quite different
control mechanism is perhaps indicated by the demonstration of a growth
inhibitor in the supernatant medium of heavy cultures. The chemical nature
of that inhibitor is under continuing study.
Studies on the mechanism of resistance to 2-deoxyglucose (2DG) have
shown the presence in the resistant variants of compounds which inhibit the
phosphorylation of 2DG to the metabolically active inhibitor, 2DG-phosphate.
The relationship of that inhibitor to ' the observed resistance is under
continuing study.
MECHANISMS OF A number of important new observations have been made
VIRAL SYNTHESIS with respect to the mechanisms of viral synthesis.
The puzzling wide disparity between the number of
physical particles in viral suspensions, and the number of plaque -forming
units, i.e. particles capable of initiating infection in susceptible cells,
has been partially resolved with the demonstration that after the viral
particle has been absorbed by the cell, it may undergo several alternative
fates. A large proportion are rapidly eluted into the medium, essentially
intact but no longer infectious, presumably reflecting a minor alteration
in the protein coat. Some particles remain unchanged within the cell.
Others are degraded intracellular ly, in that the nucleic acid is exposed
and becomes susceptible to intracellular ribonuclease. Only a small fraction
of the absorbed viral particles are stripped of their protein and initiate
infection.
In the case of poliovirus in the HeLa cell, although the viral protein
and RNA are synthesized concomitantly, a partial dissociation has been
achieved with appropriate inhibitors of protein synthesis, which completely
block the formation of mature virus, but not of infectious RNA. This is of
particular importance in relation to the supposedly obligatory relationship
between protein and RNA synthesis in growing cells. Of interest also is the
fact that metabolic inhibitors which effectively block the synthesis of
cellular DNA and of DNA viruses have no effect on the formation of poliovirus.
It would therefore appear that poliovirus RNA may be used directly as a
template for the formation of virus, without the necessity for intervening
DNA synthesis.
In contrast to the situation with poliovirus, in the case of vaccinia,
there was a marked lag between the formation of the viral nucleic acid (DNA)
and that of the mature virus.
CELL CULTURES FROM PATIENTS An exciting new development has been the
WITH GALACTOSEMIA successful cultivation from patients with a
hereditary metabolic disease (galactosemia)
of cells which in culture demonstrate the metabolic defect characteristic
of the disease. This suggests an entirely new experimental approach to
problems of human genetics.
15
LABORATORY OF GERMFREE ANIMAL KESEARCH
GERMFREE ANIMAL A series of observations has been made on the behavior
STUDIES of Entamoeba histolytica in the germfree host. It is
to be recalled that, in earlier studies with standardized
techniques, amoebic lesions were not produced in the germfree animal following
inoculation. In fact, the parasite failed to live in the intestine beyond
five days. Recent changes have been made in the manner of rearing and
handling the amoebae in vitro prior to inoculation which seemed to result
in more vigorous organisms. The latter have produced lesions in the absence
of bacteria, although the type and severity are still not typical of those
encountered with a bacterial associate. Thus, it would appear that the
latter is not the only determinant of the course and the pathogenesis of the
infection.
Studies have shown that the intestinal mouse parasite, Nematospiroides
dubius, does not require a flora to develop from an infective larva to the
adult form in the host. However, it apparently does require a flora or its
products, to develop from the egg to infective larva. These studies are
preparatory to those to be undertaken in an analysis of the nature of the
nutritional effects observed in certain parasitisms. One of the interesting
observations has been the finding that the sex of the host, which has been
noted by several workers to affect the outcome of the infection in con-
ventional (contaminated) animals, has not appeared to be an influence in the
germfree host. If these findings continue to hold up, a hitherto unrecognized
role (either direct or Indirect) of the flora in certain observed sex effects
may unfold.
BIOLOGY OF GERMFREE In studies on the growth and biology of germfree
GUINEA PIGS guinea pigs, a staff investigator has obtained
several advanced pregnancies in animals maintained
on irradiated diets, although no fetus was carried to term. It is to be
recalled that germfree guinea pigs have not yet been bred with success. The
importance of the intestinal flora to this species was pointed up by the
finding that conventional (contaminated) guinea pigs reproduced normally on
this same irradiated diet.
In a collaborative project with an investigator at the University of
Pennsylvania, it has also been shown that the use of large dosages of a
cathartic, or the application of tourniquet shock, increased the number of
red cells of germfree chickens coated with human B-like antigens following
monoinfection with Escherichia coli 0s6 • These studies are providing
information on the manner in which red cells of one type may acquire anti-
genic characteristics of other cell types, especially B.
GERMFREE MOUSE The germfree mouse colony has been undergoing an
COLONY intensive serologic study including an assay for the
presence of certain so-called "natural antibodies"
against a variety of bacteria. Such antibodies or antibody-like reactivities
for organisms like Staphylococcus , E. coli and Salmonella typhosa have been
found to occur in a variety of uninoculated conventional animals and are pre-
sumed to originate from encounters with the viable organisms or related
16
antigens. Animals which have lived for many generations free from contact
with live bacteria are almost the sine qua non for establishing finally the
validity of these ideas. Studies thus far, with the Communicable Disease
Center and investigators in the National Cancer Institute, have shown the
germfree animal to be singularly free from antibody-like reactivity toward
Staphylococcus and E. coli. Reactivity, however, toward £5. typhosa was
obtained in several instances, although no evidence of the presence of the
latter was found in the germfree colony. Thus, this finding strengthens
sporadic reports that non-bacterial substances (perhaps in this case dietary
components) can cause "cross" reactions with this organism.
TUMORS IN GERMFREE The germfree animal colony and a conventional colony
ANIMALS derived from the same stock now has existed for
for approximately two years. Some of our exbreeders,
in spite of the scarcity of germfree unit animal space, are one to two years
of age. Whenever a germfree or conventional animal not on an experiment dies,
especially if it is six months or more of age, it is examined thoroughly for
gross evidence of malformations or tumors. Among approximately 50 such animals
so-called spontaneous lung tumors have occurred in some of the germfree as
well as the conventional mice. While the numbers of animals are obviously
small, the incidence has been markedly higher, thus far, among the conventional
animals (those exposed to external contamination) than among the germfree.
This seems to be particularly true among animals six to twelve months of age.
LABORATORY OF PARASITE CHEMOTHERAPY
This country's commitment of 38 million dollars in Fiscal Year 1961
toward a program of world-wide malaria eradication and the long-term interest
in malaria by most of the senior staff resulted in a research effort, during
the past year, largely directed toward problems in that field. Special
emphasis was given to the study of simian malaria in man and in monkeys be-
cause malaria in simians might be a real deterrent to the eradication program.
Clinical facilities for volunteers at the Atlanta Penitentiary were enlarged
and the staff increased. A laboratory was established at Kuala Lumpur, Malaya,
in cooperation with the Malaya Institute of Medical Research and the United
States Medical Research Unit. Studies on several aspects of the simian-human-
malaria problem have been in progress there since mid-August.
As a result of the above development, it was decided to move the
Section on Cytology, now located at Memphis, to Chamblee, Georgia, early in
1961. This arrangement will bring the simian hosts closer to the human
volunteers at the penitentiary, and the insectary maintained by the Section
will be geared to accommodate the work at Chamblee and at the prison.
MALARIA - HUMAN Plasmodium falciparum (McLendon strain) : Chloroquine
(300 mg, base) and primaquine (45 mg, base) given to-
gether beginning three days after mosquito bites and weekly thereafter for a
total of eight doses, resulted in suppressive cure in 5/5 subjects. Controls
were positive 11 to 15 days after infection. After two days of parasitemia,
each control was given the above drug combination which was repeated weekly
17
for a total of three doses. Parasites were removed promptly and cure was
obtained based on no evidence of infection during 22 7 days of observation.
Primaquine, at daily doses of 0.75 mg, had some sporontocidal effect
upon Plasmodium falciparum gametocytes but none against those of P. vivax
(one case). Therapeutic doses (1.4 gm in three days) of amodiaquine had no
sporontocidal effect against gametocytes of P. falciparum (one case) . The
effect referred to is against the development of the malaria parasites in the
mosquito.
A strain of Plasmodium falciparum from Colombia, South America, was
found to be resistant to chloroquine. This finding is of utmost importance
in terms of malaria eradication.
Plasmodium vivax (Chesson strain) : A drug combination of primaquine
(45 mg) and pyrimethamine (50 mg) given weekly beginning seven days after
mosquito bite and continuing for a total of four doses, gave suppressive-
cure in 4/5 subjects; the other subject developed a patent infection 240 days
after infection. Pyrimethamine (50 mg) given alone, as above, produced
suppressive-cure in 1/4 subjects; the other three came down on days 82, 83
and 84. Five controls all came down 12 to 13 days after infection.
The Russian 8-aminoquinoline, quinocide, was compared with primaquine
and found to be distinctly inferior as a curative drug against early and
late primary attacks of Chesson vivax malaria particularly from the stand-
point of the occurrence of second and third relapses.
Another 8-aminoquinoline, Win 5037, was studied in five subjects.
Toxic effects and failure to cure made further investigation unwarranted.
Plasmodium malariae: The results of a 14-year study of the biology
of Plasmodium malariae were drawn together for publication. The highest
infectivity for mosquitoes occurred during the eighth to tenth weeks of the
primary attack. Although the infection rate of mosquitoes was ordinarily low,
the relatively long period during which mosquitoes could be infected may
explain the persistence of P. malariae in nature. The ability of the symptom-
free malarious patient to infect mosquitoes at a rate similar to that of the
symptomatic patient makes eradication difficult.
MALARIA - SIMIAN Plasmodium cynomolgi bastianellii: In early May, two
accidental sporozoite-induced infections with Plasmodium
cynomolgi bastianellii occurred at our Memphis Laboratory. This happening
was of signal importance because it showed that simian malaria, contrary to
the generally held opinion, was infectious to man. In that light, full scale
study of human infections was undertaken at our Atlanta Penitentiary installa-
tion.
Two infections were induced in inmate volunteers by inoculation of
infected blood obtained from one of the accidental sporozoite-induced in-
fections in man. Twenty inmate volunteers were infected by bites of Anopheles
quadrimaculatus or Anopheles freeborni which had fed on infected monkeys.
The prepatent period ranged from 14 to 29 days and the parasite density ranged
18
from 5 to 500/cmm. The most constant symptom was headache and the most
significant signs were fever, splenomegaly and hepatomegaly. Infections were
allowed to run their course, generally without treatment.
Anopheles freeborni were infected from two patients but attempts to
infect volunteers by their bites have yielded equivocal results. The finding
that P. £. bastianellii will grow consistently and produce clinical illness
in man suggested the possibility that malaria is a zoonotic disease, that is,
a disease which man can acquire from animals with which he is associated.
Whether or not such transfer occurs in nature is not yet determined, but
should it occur, it would be of greatest significance to the world-wide
malaria eradication program.
Plasmodium cynomolgi cynomolgi : Eleven inmate volunteers were bitten
by Anopheles freeborni infected with P. c_. cynomolgi on 8 September, and to
date (14 December) three have exhibited evidence of infection (i.e., fever).
Parasitemia has been demonstrated in only one, on the 58th day after mosquito
bites. These results show that this strain infects man far less readily than
P. £. bastianellii.
FIELD STUDIES Three staff members, Drs . Eyles, Dobrovolny, and Mr.
IN MALAYA Clinton S. Smith, were detailed to Malaya during the year
where they engaged in the study of simian and human
malaria in cooperation with the Malayan Institute for Medical Research and
the U. S. Army Medical Research Unit at Kuala Lumpur.
The epidemiology of monkey malarias is being studied and the feeding
habits of some of the Anopheles determined. By injection of uninfected
monkeys with sporozoites from natural infections, it was determined that
Anopheles hackeri is a natural vector of Plasmodium knowlesi. This is a most
important discovery, especially since the vector of this parasite has been
sought for repeatedly during the last 25 years.
Studies of malaria in aborigenes associated with monkeys have been
made. Blood passed from aborigenes to monkeys have thus far produced no
patent infection in the monkeys.
EE STAGES AND Studies were continued on the direct effect of drugs on
DRUG ACTION the exoerythrocytic stages of primate malaria. When sul-
fonamides were used with pyrimethamine to exploit the
possible synergism of the two drugs, monkeys developed parasitemia 30 to 40
days after inoculation with sporozoites even though all parasites observed
in liver biopsies were damaged. The curative efficacy of quinocide, the
Russian drug, was compared with primaquine. Even when administered at twice
the dosage used with primaquine, quinocide was less effective. Chloroquine
had no observable effect upon the liver forms of Plasmodium cynomolgi. Young
parasites appeared in the blood in large numbers on the 8th, 16th, and 24th
day indicating the existence of secondary exoerythrocytic generations.
INSECT TISSUE Blood cells from caterpillers and cells of the ovariole
CULTURE sheath of several species of moth pupae have been culti-
vated in several different media. The virus of St. Louis
encephalitis has been maintained in cultures of hemocytes from larvae of the
19
catalpa sphinx for ten days. Oocysts of Plasmodium gallinaceum attached to
the midgut of Aedes aegypti have shown growth in vitro and sporozoites have
been produced.
BIOCHEMICAL STUDIES It was shown that mosquitoes infected with malaria
have higher levels of ribonucleic acid than unin-
fected mosquitoes. Chromatographically, the acid-hydrolysate of ribonucleic
acid from a pyrimethamine -resistant strain of Plasmodium falciparum differs
from the acid-hydrolysate of ribonucleic acid from a pyrimethamine-susceptible
strain. Bephenium hydroxynaphthoate inhibited glutamic acid transaminase of
Nippostrongylus muris . Bephenium chloride and quinacrine reduced the rate
of glucose absorption by the tapeworm Hymenolepis diminuta but low concentra-
tions of dithiazanine iodide stimulated glucose absorption by this cestode.
INTESTINAL PARASITES Epidemiological studies on the inmates of a mental
institution show a high persistence of Trichuris
and hookworm for six years, with an apparent decrease in Strongyloides . To
test dithiazanine and tetrachlorethylene, alone and in combination, heavily
parasitized mental patients were given the drugs for about one year. A
large number of worms were removed but the cure rate was low and transmission
was not stopped. Bephenium hydroxynaphthoate and bephenium chloride were
used with good results against hookworm, As car is and Trichuris.
SCHISTOSOMIASIS The activity of griseofulvin observed in mice infected
with Schistosoma mansoni was not well developed in
hamsters or monkeys. A series of tetracycline analogues which show an
affinity for microfilaria did not combine with schistosomes and were without
activity. One of these analogues was significantly more active against micro-
filariae of Dirofilaria immitis than tetracycline.
In many tests, the efficacy of stibophen (Fuadin) therapy on mature
Schistosoma mansoni infections in mice was increased up to 16 times by feed-
ing a balanced semi -synthetic diet. The toxicity of the drug was not
similarly increased. The enhancement of curative action by the purified
semi -synthetic diet was thought to be due to the absence of, as yet un-
identified, inorganic salt(s) that interfere with drug activity. It was
found in mice fed on the purified semi -synthetic diet that higher blood
levels of the drug were maintained for a longer period than when the same
amount of Fuadin was injected into mice fed on the commercial pellet diet,
suggesting that the increased cure-rate was due to higher blood drug level.
Similar drug advantage was observed in mice given tartar emetic while on
the purified diet.
20
LABORATORY OF PARASITIC DISEASES
This Laboratory continues to emphasize fundamental studies on para-
sites and parasitic diseases. No important changes in the program were
instituted during the year. The program of the laboratory is well diversified
considering the size of the staff and the competencies of the various staff
members cover a large proportion of the field of parasitology.
Although the emphasis is on basic studies, this does not imply a
narrow viewpoint and the laboratory is well aware of the many practical
problems parasitic diseases create throughout the world. The laboratory is
often called upon for help and advice concerning prevention and control of
parasitic infections and so must maintain competence, and a reputation for
competence, to deal not only with basic problems of parasitism but also pro-
blems of prevention and control of parasitic diseases. Therefore, the labor-
atory continues to carry on a variety of activities which help it maintain
its international reputation and increase its capacity to cope with problems
of parasitism. Such activity also returns benefits in the form of ideas for
laboratory research and clues which may explain puzzling laboratory findings.
TOXOPLASMOSIS Studies on toxoplasmosis in New Zealand sheep have shown
that the prevalence is high. New information has been
obtained concerning the distribution of the organisms in the tissues and
their persistence there. After inoculation the distribution of the parasite
in tissues is erratic and the parasites rapidly clear from tissues other than
the muscle and placenta. Since residual infection occurs in muscle, mutton
may serve as a source of human infection. Congenital infection with Toxoplasma
is an important medical problem, therefore it is of special interest that the
sheep studies have indicated that inoculation of sheep 60 days before preg-
nancy did not result in congenital infection or abortion but inoculation at
30 days pregnancy caused abortion or foetal death with absorption. Infection
at 90 days pregnancy was less likely to be dangerous to the foetus.
The status of resistance or immunity to Toxoplasma continues to be
puzzling, since living organisms fail to protect completely animals against
challenge, especially when the challenge is great, and because low grade
parasitemia may persist for months in mice and rabbits in the presence of high
serum antibody levels. The observation that cysts of Toxoplasma probably
form in tissue cultures provides a new opportunity to study the manner of cyst
formation and the factors that lead to cyst formation.
AMOEBIASIS The work on the preservation of living Entamoeba histolytica
and other protozoa has practical significance since success
would permit retention of strains without continuous sub-culturing. This is
a relatively new field and techniques are still evolving. The work so far
has shown that this approach is feasible since four species have been frozen
and stored for periods ranging from one to four months depending on the
species involved. E. histolytica has been kept at -197° C for 24 hours,
suggesting that almost indefinite storage at this temperature may eventually
be achieved.
21
Laboratory culture of E. histolytica concinues to receive attention
since it is so important to learn more concerning its nutritional require-
ments and its pathogenicity in the absence of other organisms. It is note-
worthy that satisfactory axenic culture of this species has been achieved
for the first time. The protozoa are cultured in a complex diphasic medium
containing no cells but including chick embryo extract.
The substitution of a species of Crithidia for Trypanosoma cruzi in
cultures of E. histolytica provides a more economical and rapid way of
producing large cultures of the amoeba. Demonstration of the value of the
Coulter Counter for the enumeration of protozoa in suspension adds a valuable
tool for quantitative work and suggests this method may be applicable for
counting other organisms of similar size such as tissue culture cells.
PARASITIC INFECTIONS IN The use of germfree animals in worm-parasite
GERMFREE ANIMALS studies continues to reveal the value of this
tool and adds to our knowledge of the peculiar
nature of the germfree state. The technique seems to be particularly useful
for studying conditions that influence natural resistance and nutritional
relationships of parasite and host. For example, it was found that the
roundworm, Nematospiroides dubius , develops as well in germfree as in
conventional mice but while in conventional mice the worm recovery is much
higher from the male animals, the recovery from germfree mice is the same
for both host sexes. The cause of the difference is unknown. Also, it has
been shown that the feces of germfree mice do not support development of
N. dubius larvae and that bacteria in the feces provide important factors
for larval development. There was further evidence that the alteration in
levels of serum protein components in germfree animals is due to dietary
factors .
STERILE CULTURE Studies on the sterile culture of worms continues to
OF WORMS produce fundamental information on the nutritional re-
quirements of the parasites and brings closer the day
when we can use the axenic animals for immunologic and therapeutic studies.
Survival studies using relatively advanced larvae of Nippostrongylus muris
has produced important results. The intent has been to try, by addition of
elements to the medium, to induce the larvae to reach the adult stage.
Starting with a salt mixture, dextrose was added until the optimal level was
reached. Then casein was added and survival time rose to 11 days, but there
was not development of the larvae. Addition of a yeast extract to this
mixture not only increased survival but permitted growth to the adult stage.
Thus, a much more simple medium than used before has been evolved and the
achievement of a defined medium for culture of N. muris adults is much
closer. A similar approach is being used in attempts to culture micro-
filariae of Dirofilaria immitis.
NUTRITION AND SCHISTOSOMIASIS Although the study of the relation of
IN PUERTO RICO nutrition to schistosomiasis in Puerto
Rico is still incomplete, it appears that
enrichment of the diet does not affect the number of eggs passed in the feces.
However, it is interesting to note that the enriched diet did cause a loss of
hookworms and whipworms from the intestine. This has a bearing on the
22
problem of the existence of hookworm infection \ ithout hookworm disease. In
laboratory studies conducted in Bethesda the enhanced efficacy of stibophen
in mice receiving a semi-synthetic diet was shown to be due to the absence
from this diet of as yet unknown inorganic salts. Higher blood levels of
the drug were maintained longer when the semi-synthetic diet was used and
this may explain the greater efficacy. Demonstration of the influence of
simple salts on the efficacy of stibophen suggests that other drugs may be
similarly affected by diet. If the work with the stibophen-salt problem
progresses satisfactorily it is hoped that a test of the effect of human
diet on the action of the same drug may be tried in Puerto Rico before the
study there is concluded.
DUAL VIRUS AND HELMINTH Interaction of two pathogenic organisms in the
INFECTIONS same host has had relatively little attention
in spite of some very provocative work done in
years past. A study of simultaneous infection with encephalomyocarditis
virus and Trichinella spiralis in rats has produced striking and significant
results. While the virus alone does not injure adult white rats when given
intraperitoneally, in the presence of Trichinella spiralis infection many of
the rats are crippled and die. This potentiation of virus pathogenicity is
not due to nonspecific stress but seems to be related to the presence of
the worms on the muscles. The virus can be recovered from the muscle of T.
spiralis -infected rats but not from muscle of rats without T. spiralis.
The reason for the influence of the worm infection on the activity of the
virus is unknown. The phenomenon offers an opportunity to study some of the
fundamental factors in the pathogenesis of both the virus and the worm
parasite. It also provokes the question as to what effect this worm infection
may have on other virus infections.
AMMONIA TOXICITY The study of liver damage in relation to ammonia
IN MICE toxicity in mice has revealed that low oxygen in
breathed air greatly enhances ammonia toxicity. The
mechanism of this effect is not clear. Though hepatic coma is usually
considered to be related to ammonia toxicity none of the substances which
exacerbate hepatic coma in man increases ammonia toxicity in mice. In fact,
six of ten decrease it. Ammonia toxicity in mice was greatly reduced by
hypothermia and this suggests that the same measure may be useful in treating
hepatic coma in man. Finally, mouse liver damage was induced in eight
different ways but none caused any change in the animal's response to
intravenous ammonia. Thus, though high blood ammonia levels seem to be
related to liver damage, the causal relationships are by no means clear.
BIOCHEMICAL STUDIES Fundamental physiological studies have focused on
OF HELMINTHS the calcareous corpuscles of tapeworms and on the
phospholipids of tapeworms. The calcareous
corpuscles are amorphous but, on heating, dolomite, brucite or apatite may
be formed. Electron microscope pictures of corpuscles heated with KOH
reveal the presence of well-formed crystals. The glycerol containing
phospholipids of Taenia taeniaeformis are about half lecithid and half
cephalin. Sphingomyelin is present and more than one cephalin is known to
occur in the larvae of this tapeworm. Hexose-containing phospholipids occur
in both larvae and adults.
23
Study of the mechanism of energy metaboxism of sub-cellular elements
has dealt, among other things, with the mechanism by which mitochondria
which are depleted of high-energy phosphate intermediates are stimulated to
oxidize substrates when ATP is added. This is a complex, though fundamental,
bioenergetic system for which a better understanding is needed. Addition of
ATP not only restored succinate oxidation but also caused reduction of
intra-mitochondrial DPN. The succinate oxidation involves an energy-requiring
reaction and this energy is apparently added at one site in the respiratory
chain and used at another for reducing pyridine nucleotide.
LABORATORY OF BIOLOGY OF VIRUSES
The basic objectives of this laboratory continue to be the same as
last year. It is obvious from this annual report that four out of five units
have projects with the same general objective -- investigation of mechanisms
and localization of animal virus synthesis within the infected cell. Each of
these units is also interested in the infectious nucleic acid of viruses.
In view of the complexity of this problem and the important implications of
any information that is obtained, this "duplication" is quite justified.
Actually, it is not duplication since different approaches are used and
different virus -cell systems are studied.
The electron microscope has been installed and is now used not only
by the Biophysical Unit but also by other units of our laboratory and by
units of the Laboratory of Infectious Diseases. With studies on the structure
of viruses and a project concerned with the genetics of animal viruses added
to the biochemical and biological studies, there is now fairly complete cover-
age of the important facets of basic virus biology.
INTRACELLULAR LOCATION By use of radioautographs and staining with
OF POLIOVIRUS fluorescein tagged antiviral antibody, the intra-
cellular location of poliovirus antigen—pre-
sumably viral protein--during the cycle of virus multiplication has been
determined. Demonstrable antigen first appeared one hour after infection and
was diffusely distributed through the cytoplasm. At three hours, just before
the appearance of new virus, it was present throughout the nucleus with a
tendency to be concentrated around the periphery of the nucleolus. At five
to seven hours, particulate accumulation of antigen in the cytoplasm was noted.
Incorporation of radioactive-tagged amino acid into cell protein ceased
shortly after the start of infection, whereas incorporation of thymidine into
RNA continued until after three hours and tended to localize in the nucleoli.
MUTANTS OF Plaque type mutants of EMC virus have been found, segregated
EMC VIRUS and characterized. The stability of the mutants has been
determined and the plaque type shown to be a function of the
viral RNA. It has been shown that the difference in the size of the plaques
formed by these mutants is brought out by an inhibitor present in the agar
overlay used on the plaque plates. This inhibitor resides in the agaropectin
fraction of the agar and can be separated from the agarose fraction which
then permits both plaque type mutants to form similar sized plaques.
24
POLYOMA VIRUS By the use of a serum protection test in newborn hamsters,
evidence was found that polyoma virus transforms normal
cells to tumor cells quickly and directly without extensive virus multi-
plication being necessary. Furthermore, no evidence could be found to
suggest a lysogenic relationship of virus to tumor cell. All attempts to
show the presence of infectious or masked virus or of virus antigen in
transplantable polyoma- induced tumors have been negative. It appears that
once the virus initiates the tumor it is no longer required for tumor
growth and maintenance.
TETRACYCLINE FLUORESCENCE The discovery has been made that when the
LOCALIZED IN MITOCHONDRIA antibiotic tetracycline stains tissues in
such a way that they fluoresce under UV
light, this fluorescence is localized in the mitochondria of the cells.
This makes a convenient vital stain of these subcellular elements for
further studies. There appears to be some similar localization of the
antibiotic fluorescence in certain bacteria.
TMV MODEL A complex model of tobacco mosaic virus has been constructed
on theoretical grounds, and on checking this model against
known biochemical and biophysical properties of the virus a remarkable
consistency is found. Certain refinements of electron microscopic technics
have produced photographs of this virus which reveal previously not seen
fine structure also consistent with the theoretical model.
LABORATORY OF IMMUNOLOGY
Since the activation of the Laboratory of Immunology in 1957, the
program has been concerned, principally, with basic research. However, for
some time an important need has been felt for the initiation of clinical
studies in immunology and allergy. In September 1960 the Clinical Immunology
Section was activated and as space permits, will be expanded and will work
in close collaboration with the Laboratory of Clinical Investigation on
clinical studies involving immunological aspects of such diseases as lupus
erythematosus, nephritis, and chronic thyroiditis, in which an auto- immune
basis is suspected.
ALLERGIC THYROIDITIS Experimental allergic thyroiditis was produced
in Strain 13, inbred, histocompatible guinea
pigs by immunization with a single dose of guinea pig thyroid extract in
complete Freund's adjuvant. Thyroiditis developed as early as five days
after immunization, was present in all animals at 16 days, and by seven
weeks was consistently present and generally severe. Delayed skin test
hypersensitivity was found as early as five days after immunization in
nearly all animals, and was present in all animals with thyroiditis at
seven weeks. At seven weeks after immunization, anti-thyroid antibodies
were present, and antibody titres correlated with the presence and degree
of thyroiditis. This correlation was not found at certain other times
after immunization. The presence of delayed hypersensitivity was correlated
with experimental allergic thyroiditis, while the presence of circulating
antibody did not correlate with thyroiditis. These observations constitute
25
the earliest production of experimental allergic thyroiditis and the most
severe disease at the time intervals studied.
HOUSE DUST ALLERGENS Studies on the chemical and physical properties
of house dust extracts that are used clinically
for the diagnosis and treatment of house dust allergy have been studied to
identify the components responsible for the specific skin reactions produced
in house dust sensitive individuals. It has been found that the house dust
extracts consist of a heterogeneous mixture of acidic polysaccharides. The
heterogeneity has been demonstrated by electrophoretic and ultracentrifuge
sedimentation analysis and also by the multiplicity of cross reactions ob-
tained with antisera to the various pneumococcal polysaccharides. The chem-
ical composition of the various fractions has been shown to be roughly 5-20%
polypeptide and 80-957» polysaccharide, containing about equal amounts of
uronic acid (probably glucuronic acid), D-glucose, D-galactose, D-mannose
with lesser amounts of L-rhamnose and L-arabinose.
GENETICS OF GAMMA GLOBULIN Agar-gel immunochemical analysis of sera
from rabbit litters, with precipitating
antibodies prepared in rabbits, has shown that seven antigenic determinants
of the gamma globulins are genetically controlled by at least two gene loci
with each specificity exhibited when the appropriate allele is present. Since
the gamma globulins are soluble proteins which have properties of both an
antigen and an antibody, they should be subject to quantitative estimation
and cytological localization. This immunogenetic system, therefore, may be
uniquely suited for the study of certain basic problems in genetics, embry-
ology, immunology and protein chemistry.
In other studies, antibodies to human serum proteins were prepared in
monkeys since this animal, being a closely related species, might be more
discriminating for minor antigenic differences than a distantly related
species. Three "slow" gamma globulins were found, instead of the one usually
detected with horse or rabbit antibodies. Two of these were shown to be re-
lated to myeloma proteins. The quantitative estimation of these gamma glo-
bulins in serum should be helpful in the early diagnosis and study of diseases,
such as multiple myeloma, which involve qualitative and quantitative changes
in the gamma globulins.
MECHANISMS OF The genetically distinct guinea pigs of inbred Strains
HYPERSENSITIVITY 2 and 13 have proved to be a very important immuno-
logical tool. After studies established the fact of
skin compatibility in the two strains, experiments were conducted to transfer
cells with a measurable biological activity. Transfers of tuberculin sensi-
tivity were undertaken by the intraperitoneal injection of living lymphoid
cells from compatible donors. The almost quantitative transfers between
inbred guinea pigs were a reflection of the continued viability of the active
cells in the recipients.
Two models are being developed to study the mechanisms of immediate
and delayed hypersensitivity in the inbred guinea pigs; protracted anaphy-
lactic shock and, the massive local hemorrhagic reaction, respectively. It
has been shown that there are differences in susceptibility to hypersensitivity
26
reactions. Strain 2 guinea pigs were more resistant to death by bronchospasm
and tended toward a protracted syndrome in anaphylactic shock. Both Strain 2
and 13 guinea pigs required more mycobacteria than did random-bred Hartley
guinea pigs for inducing "delayed" sensitivity to egg albumin, using Freund's
adjuvant.
HUMAN SERUM Fractions of human serum separated by anion- exchange
AUTO-ANTIBODIES cellulose column chromatography were studied by
Immunoelectrophoresis . The conditions for elution
of eighteen immunologically distinguishable human serum proteins from the
columns were determined. Gamma globulin obtained under the appropriate
conditions by this method was found to be pure; rabbits immunized with this
fraction made antibodies to none of the other serum proteins. By the use of
anion-exchange cellulose columns, it has been found possible to separate the
7S from the 18-19S antibody activities in sera of patients with thyroiditis
and lupus erythematosus. Initial results indicate that the addition of
immunoelectrophoretic characterization of these and other sera will be
extremely helpful in our aim of characterizing the antibody activities found
in human serum.
FLUORESCENT ANTIBODY STAINING The fluorescent antibody staining of the
OF MALARIA PARASITES human malaria parasite, Plasmodium vivax,
has been recorded for the first time. A
globulin fraction of convalescent serum from a patient having a long-standing
infection with P. vivax was labeled and the fluorescent antibody applied to
thin blood films containing the parasite. The organism was visible by virtue
of its specific immuno-f luorescence. Fluorescent antibody studies were
conducted on P. cynomolgi bastianellii, the monkey malaria parasite which,
recently, has been shown transmissible to man. Considerable morphological
detail was observed at fluorescence. Preliminary studies on the serological
relationships, as based on degrees of fluorescence, indicate that P. vivax
and P. cynomolgi bastianellii parasites may have common antigens and that
the two species may be closely related.
LABORATORY OF INFECTIOUS DISEASES
In 1960 the Virus and Rickettsial and Epidemiology Sections of this
laboratory continued integrated and comprehensive efforts to define the
importance of virus infections in disease. Field investigations of human
and animal virus infections were made possible through collaboration with
a number of other organizations, including the Bureau of Medicine, USN; the
District of Columbia Children's Hospital Research Foundation; the District
of Columbia Welfare Department; the New York City Health Department; the
National Cancer Institute; the National Institute of Allergy and Infectious
Diseases; the Laboratory of Clinical Investigation, NIAID; and in Paris,
France the Laboratoire des Virus, Hopital Saint-Vincent-de-Paul; and Le
Centre Claude-Bernard de 1 'Hopital Saint Louis.
27
Natural events and opportunities afforded by our collaborators shaped
the course of most field studies. Technical breakthroughs in the laboratory
made it possible to take fuller advantage of these opportunities to study
natural disease and thus acquire not only new information about specific
virus infections, but also to move nearer our ultimate goal, namely, a clear
view of the numerous viral causes of human diseases sufficiently comprehensive
to make concerted efforts to control them appear feasible and worthwhile.
NEW CAUSES OF Pneumonia and other lower respiratory tract infections
VIRUS PNEUMONIA continue to represent major causes of death and a
large segment, presumed to be viral in origin, is
still uncontrolled. Until recently it was wholly undefined. During 1958
and 1959 our studies at Children's Hospital and Junior Village helped define
the relative importance of adenoviruses, para-influenza viruses, and
influenza viruses in causing lower respiratory illnesses of childhood. The
data suggested that as much as 40 percent of croup bronchiolitis and pneumonia
were explained by these viruses. In 1960, using more sensitive methods, we
were able to explain a much larger percentage of such illnesses, chiefly
because we were now able to assess the very significant contributions of
respiratory syncytial virus (RS) to the respiratory disease problem. Early
in the year large outbreaks of RS virus were intensively studied both at
Children's Hospital and Junior Village. Over 80 strains of RS virus were
isolated from children with pneumonia and 60 percent with bronchiolitis
yielded RS virus, whereas virus was recovered from less than one percent
of comparable control patients without respiratory illness.
Retrospective analysis of serologic surveys of respiratory illnesses
in Children's Hospital since 1957 suggested that perhaps 20 percent of all
lower respiratory illnesses observed during the last three years was due to
RS virus. Thus, considering the contributions of adenoviruses, para-
influenza viruses, influenza viruses, and "PAP" virus it now appears that
50 to 60 percent of the more severe respiratory illnesses of young children
can now be explained and, hopefully, controlled. Except for influenza virus
(which contributed probably less than 5 percent of the total), the LID
respiratory virus unit personnel played key roles in the discovery of the
first representatives of each of the other virus groups - adenovirus, para-
influenza, and RS. Delineation of still undefined viral causes of the
respiratory disease syndrome represents the major challenge to respiratory
disease investigators for 196i.
During 1960 several experimental but commercially prepared killed
vaccines containing various combinations of adenoviruses (6 types), para-
influenza viruses (3 types) , and Coxsackie B viruses (5 types) were tested
in Junior Village. The evidence suggests that while modestly antigenic, the
vaccines had insufficient potency to be regarded as satisfactory for larger
scale studies.
PRIMARY ATYPICAL The etio logic role of PAP (Eaton's virus) in primary
PNEUMONIA atypical pneumonia suggested earlier by Eaton and Liu,
was finally fully established in 1960. In cooperation
with the Bureau of Medicine, USN, the continuing "epidemic" of virus pneumonia
in Marine recruits at Parris Island was studied in several ways. Serological
28
studies showed that 51 percent of 530 pneumonia cases had antibody rises to
PAP virus; only six percent revealed contemporary infection with adeno-
viruses. Serologic studies of infection showed PAP virus to be much more
common than disease; approximately 30 recruits were infected for each case
of pneumonia, information vitally important to fuller comprehension of the
natural history of this important virus.
In 1959 treatment of Parris Island pneumonia cases with broad spectrum
antibiotics (tetracyclines) appeared to reduce the severity and the duration
of the Eaton pneumonias. In 1960 the efficacy of a new tetracycline drug,
demethylchlortetracycline, was tested in a well-controlled double blind study
including 290 pneumonia patients. The drug greatly reduced the severity and
duration of pneumonitis and fever in those shown to have serologic responses
to PAP virus. These findings, based on accurate laboratory diagnosis, fully
confirm earlier but controversial reports of the efficacy of tetracyclines
in atypical pneumonias. It also adds further support to the importance
of the Eaton virus as a cause of virus pneumonia.
An additional link in the chain of evidence establishing the PAP
virus as an important cause of pneumonia was achieved recently in collabora-
tive studies with the Laboratory of Clinical Investigation, NIAID. Volunteers
inoculated intranasally with PAP virus grown in tissue cultures reacted with
a wide gamut of respiratory signs and symptoms, including pneumonitis
characteristic of PAP.
COMMON COLDS Recent studies have served to clarify and enlarge
AND VIRUSES existing concepts of the etiology of common mild respira-
tory illnesses in adults. It is now quite clear that
instead of a few specific closely related viruses, numerous viruses belonging
to different groups each contribute in part to the syndrome called the
"common cold." Thus the newer viruses (adenoviruses, para-influenza viruses,
respiratory syncytial virus and others), together with older agents (influenza
viruses and certain bacteria), each contribute only a small proportion of the
milder respiratory ailments of adults. They contribute a larger segment of
more serious diseases, particularly in children. Very recent reports of
common cold viruses from England, together with the prior reports of agents
with somewhat similar properties in this country, served to focus our
attention on these viruses in 1960. Together with investigators elsewhere,
it was found that most, if not all, of these agents - the British HGP and
FEB, the American 2060, JH, Coe and PETT viruses which grow selectively and
rather "fussily" in human epithelial cell lines, really represent "fastidious"
enterovirus strains which have (as do almost all Coxsackie A's and some
ECHO viruses) special growth requirements. These viruses, as do a number
of still unclassified agents found in Junior Village during the past several
years, have properties very similar to the Coxsackie A viruses; indeed,
several have been shown, on the basis of serologic markers and/or by
suckling mouse pathogenicity, to be indistinguishable from Coxsackie A
viruses.
NEW SEROLOGICAL The laboratory section of the Epidemiology Section
TEST PROCEDURES concentrated on the development, application and
evaluation of i_n vitro test procedures for the identi-
fication of new viruses as well as for detecting virus infection as expressed
29
in antibody responses. Thus, using convention.! complement fixation (CF)
and newly developed hemagglutination inhibition (HI) procedures it has been
possible for our group to type thousands of virus isolates belonging to the
adenovirus, myxovirus , enterovirus, and reovirus groups. As was true during
the past several years, LID in 1960 again described and characterized more
new representatives of these viruses than all other virus laboratories in
the world combined. This was made possible during 1960 because each of
our various virus research units contributed new diagnostic techniques.
One group developed additional specific HI procedures for identifying
adenoviruses and adenovirus infections; and for reoviruses and enteroviruses
as well. Similarly, another group developed tissue culture procedures for
isolating Eaton's PAP virus, while others not only discovered several "new"
mouse viruses in tumor virus study systems, but developed serological
procedures for recognizing their presence.
SEROLOGIC REAGENTS But the availability of simplified procedures are
of very little use unless the necessary reagents
are also available. Although many virus research laboratories could do
the tests, few laboratories are able to produce the necessary reagents. The
magnitude and cost of producing and certifying them promises to continue to
exceed any possible resources available. This fact has had a very depressing
effect on research efforts aimed at the study of viruses as causes of disease,
and serves as yet another deterrent to early delineation of the common virus
diseases as public health problems. Consequently, with the help of NINDB
and Microbiological Associates, LID in 1959 and 1960 accepted responsibility
to develop and evaluate more than a hundred commercially produced virus
antigens. LID, of course, has been active in the certification of virus
prototypes and furnishes many to the Virus Registry of the American Type
Culture Collection. It is also collaborating with the Enterovirus and
Adenovirus national committees in setting up standards for large scale
production of certified antiserums for serotyping and classification of
viruses, perhaps the highest priority need of all virus laboratories con-
cerned with human infection and disease.
UNOFFICIAL WORLD REFERENCE Wholly through the operation of circum-
LABORATORY FOR VIRUSES stances, the Virus Section of LID has become
virtually the chief (in many instances only)
reference laboratory for many of the newer viruses, including adenoviruses
(about 30 human and several animal serotypes), myxoviruses (five new para-
influenzas occurring in three species), reoviruses (three serotypes In four
species) , many of the newer and some older enteroviruses (5 - 10) , salivary
gland viruses (from four species), and new mouse viruses (six), the latter
frequently found in tumor virus study systems.
Until virus reagents desperately needed for many extremely common
viruses are made available either commercially, through government agencies,
or both, LID as the sole custodian of many of these agents cannot avoid
responsibility for assisting other excellent virus laboratories to identify
their viruses, and on a pro-tem basis at least for keeping order in the
general virus field. Unfortunately it has no specific commitment to provide
such services and even worse, no specific budget to cover them, so that the
involuntary, constantly growing and unavoidable service functions must be
done at the expense of research missions.
30
However, it must be admitted that the sampler virus diagnostic tech-
niques and the availability of a complete supply of viral reagents in the
laboratory (developed out of necessity) facilitate not only epidemiologic
studies of naturally occurring virus infection but also enable it to evaluate
the significance of the data furnished by other laboratories who come for
technical assistance.
PROBLEMS OF CANCER Studies of cancer viruses can be subdivided into
VIRUSES several categories: (a) Laboratory studies of the
properties of cancer viruses and development of
laboratory tools for detecting and working with them; (b) field studies of
the behavior in nature of those tumor viruses for which suitable detection
tests are available; (c) studies of extraneous viruses ("background noise")
now preventing high caliber virologic practice in the study of animal tumor
viruses and obscuring interpretation of nearly all current observations on
them; and (d) the study of general virus experiences in relation to human
cancer - the "background noise" in the human cancer problem - which must be
done eventually if the role of viruses in human cancer is to be defined.
The approach to these various interdependent studies is based on the
following beliefs: 1) That the conventional methods of standard virology
must be applied to cancer virus research if significant progress is to be
made; 2) the study of cancer viruses obviously cannot be separated from
general virology; and 3) that the "biologic point of view" rather than
attitudes fostered by preoccupation with categorical disease, represents the
best approach to a real understanding of the natural history of cancer
viruses just as it does to other viruses.
MOUSE POLYOMA New in vitro survey tools developed during 1959 (CF, HI,
CANCER VIRUS and MAP) were evaluated and applied in 1960 in studies
of polyoma virus growth and excretion, its experimental
epidemiology, and its natural history. This interesting and versatile cancer
virus causes tumors not only in all strains of Mus mus cuius, but also in
hamsters, rats, rabbits, and guinea pigs (Stewart and Eddy). Of equal
interest is the fact that it can be studied and surveyed with the same
facility as ordinary viruses, such as influenza and polioviruses. Virus
isolation and serologic procedures, combined with epizootiologic studies
have produced the following interesting observations:
Polyoma virus was found to be widely disseminated in mouse colonies
nearly everywhere. Infection was found to be more commonly present than
absent in laboratory strains raised in experimental or commercial laboratories
and in wild strains found in city tenements. However, the basic ecology or
natural cycle appears to exist in rural areas - on farms and in feed mills
in small towns.
A full year's surveillance of Mus mus cuius infestation and polyoma
infection of crowded tenements in Harlem revealed that virus infections
persisted without exception in numerous separate foci. Three epidemiologic
factors seem most important, namely, large mouse populations capable of
furnishing adequate supplies of young susceptible mice, the extensive con-
31
tamination of the tenement environment (virus v as demonstrated in sweepings
from areas showing signs of mouse activity) , and finally the overcrowding
which insures the continuous and extensive use of communal nesting areas
(also demonstrated to be contaminated by virus) . Apartment houses having
smaller and less dense mouse infestation were generally .free of infection
and remained so during the study.
Systematic studies of polyoma in rural environments were undertaken
during the last quarter of 1960. However it appears from preliminary
data that here may be found the basic natural cycle of mouse polyoma. Mus
mus cuius infestation and polyoma infection of Mus was found to be most intense
in feed granaries on the farm and in cereal grain storage elevators in mills.
As many as 30 per cent of several hundred mice trapped in these environs
showed persistent evidence of polyoma infection, many of them apparently
excreting virus in their urine. The virus has been found on cereal grains
in the vicinity of mouse nesting areas, which appear to be very numerous in
the granaries so far examined. The actual extent of cereal grain contamina-
tion by mouse excreta containing polyoma and no doubt other microbes must
still be evaluated; however, present evidence suggests that it probably is
very extensive, if not appalling.
Since natural infection of wild mice is not limited to polyoma virus,
but includes a number of other viruses known or suspected to infect man and
domestic animals, the extension of these preliminary findings will likely
prove very interesting.
EXTRANEOUS VIRUSES IN In 1960 the "background noise" problem in cancer
CANCER VIRUS STUDIES virus research grew to almost "deafening" propor-
tions and, in the opinion of LID virologists,
constitutes the number one obstacle to intelligent and truly effective
research on cancer viruses.
Nearly every animal tumor virus system currently under study was
shown to be contaminated with extraneous agents and several viruses widely
proclaimed as "tumor" viruses turned out to be fellow traveling ordinary
viruses. To list a few examples: Friend leukemia was found contaminated
with polyoma and mouse adenovirus; Gross leukemia by polyoma, K virus and
mouse adenovirus; Schwartz leukemia with polyoma, K virus and mouse adeno-
virus; Moloney leukemia with mouse hepatitis and mouse reovirus; the polyoma
itself became contaminated with mouse adenovirus, hepatitis and salivary
gland viruses .
LID virologists showed that the "seeds" of the "background noise"
viruses are commonly present in the animals used for the induction of tumors,
and of course in the subsequent passage materials as mentioned above. The
extraneous viruses most commonly encountered in cancer systems were the
newer ones, such as polyoma, K virus, mouse reovirus and adenovirus; but
this in part may be due to newly developed easily applied survey tools for
these agents. Other viruses encountered less often (perhaps because of
comparatively less sensitive tools) were mouse hepatitis, mouse salivary
gland virus, the newly discovered "thymic agent" (TA) . Except in newborns,
most of these viruses occur subclinically and latently.
32
MEDICAL MYCOLOGY Investigations on pathogenic fungi have included broad
fields of research and although definitive goals have
been reached in most of them, all will be continued in order to further
exploit productive lines of investigation. In most cases new or additional
species of pathogenic fungi will be used in investigations, or techniques
will be altered to permit further development of experimental studies.
The antibiotic X-5079C was found to be fungistatic but not fungicidal
and its apparent low degree of jin vitro activity due to its decay in culture
medium. The yeast form of Histoplasma capsulatum is much more sensitive to
X-5079C than the mycelial form and an assay method, sensitive to 1 ug/ml
using 1U capsulatum, was developed. X-5079C has low toxicity for HeLa cells
and is active against H^ capsulatum grown in HeLa cells.
A second strain of Coccidioides immitis has been converted to serial
culture in the spherule form. Quantitative measurements show the ability of
various carbon and nitrogen sources to support growth of spherule and mycel-
ial forms of strain M-ll of C^ immitis . Only mannose is utilized as readily
as glucose by spherules. Mannose and fructose support growth of the mycelial
form as well as does glucose. A substrate which preferentially supports
growth of the spherule form was not found in this study.
Spherules were utilized to immunize mice. An increased survivor rate
in the immunized mice was noted after challenge with a lethal infecting dose
and an earlier clearance of organs (negative cultures) in the immunized mice
was observed after challenge with a sublethal dose.
CRYPTOCOCCUS By titrating and plating out organs of experimentally
NEOFORMANS infected mice, it was found that several minutes after
Cryptococcus neoformans was injected either intravenously
or intracerebrally into mice, the largest numbers of yeast cells had been
retained in the lungs. The fungus population in the lung then decreases and
2-3 days after infection multiplication in the brain is apparent. Although
the interval from infection to death of infected mice varies with the strain
of C± neoformans, the numbers of yeast cells per gram of brain tissue are
approximately the same regardless of strain.
Studies of the saprophytic occurrence in natural habitats of fungi
which cause mycoses have continued. CryptPcoccus neoformans has been iso-
lated from many additional collections of pigeon guano. When this material
is collected from old pigeon nests and from roosting sites in hay mows of
barns and upper floors of buildings, Histoplasma has never been found. There
is increasing circumstantial evidence that a presently unstudied pneumonic
form of cryptococcosis has occurred in men heavily exposed to such material
and that such epidemics have been erroneously diagnosed histoplasmosis.
EMMONS I A CRESCENS In collaboration with an investigator at the Rocky
Mountain Laboratory a new species, Emmons i a crescens,
was described. This fungus differs from the first species of Emmonsia
(E. parva) in vivo and Jji vitro at 37° C by its multinucleate condition
(instead of uninucleate), its ability to produce the in vivo form in vitro
at 37° C, and its greater size. E. parva conidia when inhaled or incubated
33
at 37° C increase in diameter from 2 - 4 u to 4J0 - 480 u. This 10 -fold
increase in volume of a single cell is very unusual in the fungi.
STAPHYLOCOCCUS STUDIES It has been established that staphylococcal
penicillinase is associated with particulate
material in the cell and thus an explanation has been given for the refrac-
toriness in preparation of this enzyme by conventional methods. New and
more potent inhibition of Staph, penicillinase have been uncovered and the
hope remains and is heightened for the ultimate finding of a chemically use-
ful inhibitor. Further, sea water has been found to possess strong inhibi-
tory activity against both penicillin-sensitive and penicillin-resistant
staphylococci (phage type 80/81).
Real progress has been reported in the understanding of iron metabolism
in the staphylococci. As a direct result of continuing work dealing with
mechanisms of the development of non-specific immunity and in particular the
function of the iron-transporting protein of plasma, siderophilin, fundamen-
tal observations on the effects of iron deficiency on the growth and metabo-
lism of S_^ aureus have been reported. Work on the biology of the staphy-
lococci so long neglected during the "antibiotic era" is cardinal to effec-
tive new therapy of staphylococcal infections.
STREPTOCOCCAL M Progress has also been made on the search for better
PROTEIN methods of isolation and purification of M protein of
streptococci. These results are of obvious importance
in the understanding of Group A streptococcal virulence. Further, highly
interesting observations have been reported dealing with the mechanism and
significance of the long-chain test for determination of anti-streptococcal
immunity .
BACTERIAL METABOLISM Real understanding of the intimate mechanisms of
energy metabolism in Hydrogenomonas in particular
and other bacteria and higher forms in general is closer as a result of work
performed in this section this year. In an enormously complicated field,
progress has occurred in the definitions of the essential reactions.
Detoxification studies on potentially useful chemotherapeutic agents
have continued and new and promising leads have been uncovered for agents
active against bacteria, fungi, parasites and, it should be added, against
cancer as well. Several of the aforementioned detoxified compounds have
passed preliminary screening processes performed by the Cancer Chemotherapy
Center.
Pinpointing of the enzymic locus of discrimination among hydrogen
isotopes by pseudomonas has been reported this year. The area lies in
formic acid metabolism.
34
LABORATORY OF CLINICAL INVESTIGATION
Summary 1
10 (c) - Office of the Chief 5
12 (c) - Respiratory Viral Diseases 6
13 (c) - Antimicrobial Drug Therapy 10
14 (c) - Staphylococcal Disease 13
15 (c) - Hepatitis and Mononucleosis 16
16 (c) - Aseptic Meningitis 17
17 (c) - Allergy- Immunology 19
18 (c) - Enteric Diseases 28
19 (c) - Clinical Investigations on Helminthic
Diseases 31
20 (c) - Clinical Investigations in Protozoan
Infections and Diseases 37
21 (c) - Cystic Fibrosis of the Pancreas 44
22 (c) - Basic Biochemical Studies 45
23 (c) - Systemic Fungal Disease 50
24 (c) - Sarcoidosis 54
25 (c) - Pyelonephritis 55
26 (c) - Urinary Tract Viruses 56
27 (c) - Biochemical Studies on Staphylococcal
Cell Walls 59
28 (c) - Pathogenesis of Viral Infections 61
Summary of Research Progress
Calendar Year 1960
Laboratory of Clinical Investigations
This year has seen some widening of the scope of our research, largely
as a response to enlarging the professional staff. We believe that a further
period of growth will be needed to staff the clinical service in a manner
consistent with optimum research productivity.
In the past the wards have operated at an occupancy rate of about 65
percent. From the service point of view, a rate of 85 percent would be most
acceptable i.e. 44 instead of 34 patients. It is estimated that the unit
could quite easily provide material for at least seven research sections (at
present there are four). We hope to have additional sections functioning as
soon as qualified investigators can be obtained. Upon the removal of the ad-
ministration offices to the new building, additional space will become avail-
able for a pediatric section, presently viewed as our most important defi-
ciency in professional staffing.
In connection with staffing, it is worth noting that it is difficult to
obtain qualified investigators to head sections. I believe the evidence will
show that civil service stipends for professional personnel are appreciably
less that those offered by the best - paying medical schools, but better than
the poorer -paying ones. Medical schools have a double standard of employment,
however, and our salaries are most competitive with the lower paying, non-
clinical appointments. I believe one can employ without great difficulty bio-
chemists, microbiologists, etc., but because of the exceedingly high returns
from the practice of medicine, the medical schools have developed private
practice and consultation arrangements for the men of their clinical faculties
which reduce the deficiency between their base pay and the return obtainable
from practice. This mechanism is not available to us.
I believe the salary issue alone would not be a crucial factor in
securing competent investigators, however, there is a feeling of reluctance
among many young investigators to accept government employment. The attitude
seems to have developed from apprehension concerning the large size of the
operation, the comparative anonymity of the individual, the division of
authority among departmental heads, the civil service organization and the
several administrative branches above the operating unit. Promotion is
usually slow and the salary increments are not large. Good men can advance
much more rapidly out of government. I don't believe the government is
adequately authorized to recognize talented young investigators who in
university life often achieve high faculty rank. It should also be recorded
that decisions concerning the salaries which can be offered are also very
slowly reached.
It is my view that the clinical sections of the National Institutes of
Health will best achieve their goals by attracting the kind of men who serve
on medical school faculties. I think we should train many who take those
positions. I believe our senior staff should also interchange with academic
medicine. Mechanisms to protect retirement and to permit such exchanges
without loss of security should be made. Such a thing as membership in the
Teachers Insurance Annuity Association as an alternative to the civil service
annuity program would be worthy of consideration.
Professional Staff
We are completing the year with 13 clinical associates and 9 senior
staff members. In July, Dr. Donald Kayhoe left the staff and in October Dr.
Howard Goodman began a joint appointment with a Section divided between this
laboratory and the Laboratory of Immunology. Mr. John Bozicevich also joined
this staff in July, by transfer from the Laboratory of Immunology. (We regret
to announce the anticipated retirement of Mr. Bozicevich early in 1961 after
30 years of service).
Research Program
Infection of volunteers with respiratory viruses. An extensive
clinical study of acute viral respiratory disease was begun this year in as-
sociation with staff members of the Laboratory of Infectious Diseases. The
initiation of this project required much work but with the unstinting assi-
stance of Dr. Clifton Himmelsbach, Associate Director, the Clinical Center,
very satisfactory arrangements have been made to transfer to the Clinical
Center, Federal prisoner-volunteers for study. This has been done with the
permission and considerable assistance of Mr. James Bennett, Director, and
Dr. Harold Janney, Chief Physician of the Bureau of Prisons, Dept. of Justice.
A team of custodial officers has also been assigned to the Clinical Center by
the Bureau of Prisons to oversee the volunteers. The volunteers have uni-
formly cooperated with the program despite some extended periods of room
isolation to prevent spread of infection, frequent blood-letting, and other
inconveniences. Many administrative arrangements have been developed so that
the program has worked increasingly smoothly.
The results so far justify the investment in money and effort. It has
been possible to produce in human volunteers a rather uniform "cold" with
respiratory syncytial virus. It may occur without respect to preinfection
immunity, but the subsequent rise in complement -fixing antibody appears to
correlate with severity of illness. Forty-six men have so far participated
in this study.
Approximately 24 other volunteers have participated in studies with
para -influenza 4 virus or Eaton (primary atypical pneumonia) virus. Future
studies are planned with human influenza virus and with the recently defined
group of REO viruses. We believe that the promise of this program is great
and that it may continue for some years.
New antifungal drug. Beginning about 4 years ago, Dr. Chester Emraone
began studies on an antifungal drug produced by Hoffman La Roche, designated
as RO-2. This agent, an antimicrobial, was found to be the most active
material Dr. Emmons had ever tested in vitro and in animals against several
of the pathogenic fungi, notably histoplasmosis and blastomycosis.
In the intervening years, the agent has been studied clinically by Dr.
Utz and his staff. Now 30 patients have been treated. Extremely favorable
results have been observed in several patients very severely ill with these
diseases. From the standpoint of therapeutic activity, it is the judgment
that this agent is the best available for blastomycosis and histoplasmosis.
Z
f>9."
During the studies it was noted that the agent produced unusual hepatic
changes. It was found that the dye, bromsulfhalein, normally rapidly trans-
ferred from the blood to the bowel by the liver, was retained in high con-
centration in the blood in patients treated with RO-2. This effect appeared
in a day or two after start of treatment, before tissue changes would likely
occur, suggesting competition of the new drug for the liver excretory
mechanism for bromsulfhalein. After treatment was stopped dye excretion
promptly returned to normal or nearly normal. Liver biopsy has revealed
changes indicative of minor hepatic damage in some cases. Because of the
great importance of having a drug in addition to the relatively toxic agent,
amphotericin B, for the treatment of fungal diseases, this new agent continues
under investigation.
Studies on volunteers infected with simian malaria. Following the
discovery by Dr. Eyeles and Dr. Coatney of the infectivity of P. cynomolgi
bastianellii for man, several inmates at the Federal prison in Atlanta
volunteered for exposure to this agent with the establishment of infection
in several. The need for careful clinical characterization of this disease
in man led to the transfer of two infected volunteers to the Clinical Center.
Both men developed acute malaria which was carefully studied throughout Its
course. All results are not yet available, but significant alterations In
urinary steroid excretion were detected, an unusual elevation of serum
cholesterol occurred in another, and liver lesions not previously described,
resembling but not identical with exoerythrocytic phase parasites, were
demonstrated in both patients. This interesting program will be continued.
Studies on penicillins and penicillinase. Previous work by Dr.
Steinman had defined nutritional requirements for penicillase production by
staphylococci and many parameters of its interaction with benzyl penicillin
(penicillin G) . During the year English investigators, working with the
stripped molecule of penicillin, 6-amino-penicillanic acid, produced a new
compound largely resistant to destruction by penicillinase. Working with
this and several other penicillin derivatives Dr .teinman has found that
resistance to penicillinase is greatly influent j t steric positions of
ethoxy groups on the side chain and that failure t be destroyed by peni-
cillinase is associated with greater penicillinase '.nducing -capacity .
Perhaps, most significantly, Dr. Steinman 's work has added to the now sub-
stantial indirect evidence, that the major reason for present resistance of
staphylococci to penicillin is the enormous capacity of these micro-organism
to produce penicillinase upon contact with. even low doses of penicillin.
Clinical studies with the penicillinase-resistant penicillin,
dimethoxy phenyl -penicillin, have revealed it to possess resistance to
penicillinase in_ vivo, and that it is a powerful and effective anti-
staphylococcal drug. After treatment of some patients for periods of 3 to 5
months no penicillin-resistant staphylococci have been isolated. It has long
been considered that chronic staphylococcal infection resembles tuberculosis
and for the first time it appears that an agent is available which can be
employed for extended periods of treatment without loss of effect such as
isonicotinic acid hydrazide in tuberculosis. If long term therapy can thu6
be regularly given an enormous benefit will accrue to thousands ill with
chronic staphylococcal disease.
Ascites induced In mice by Injection of adjuvant mixtures. In the
past year Miss Lieberman has continued to study the ascites induced in mice
by the injection of adjuvant mixtures. This discovery provided a much needed
laboratory procedure for producing antibodies of a wide variety and a means
of evaluating antigens. Recently, her interest has been focused on the
pathology and abnormal physiology of the lesion. It has been found that
strain differences are associated with differences in susceptibility to
ascites. Since ascites was found associated with a local plasma cell re-
action, which in some strains of mice went on to plasma cell tumors, many
implications toward problems in neoplasis have also been raised. These
studies have become the basis for biochemical and pathologic studies with
other Institutes.
Allergy and Immunology. Dr. Nasou and Mr. Bozicevich have shown that
patients with hypogammaglobulinemia possess low, but definitely measurable,
levels of antibody to the enteric viruses. The implication of this finding,
in view of the normal resistance of these patients to viral infections, is
that extremely low levels of specific antibody may provide adequate resis-
tance against viral diseases. Subsequent, unpublished studies have indicated
that these patients will develop circulating antibodies to Salk polio
vaccine.
The modification of the bentonite flocculation test for the detec-
tion of gamma globulin promises to provide the practicing physician with an
accurate, convenient laboratory aid in the diagnosis of hypogammaglobulinemia.
In contrast to the electrophoretic method, the results with the bentonite
test can be known to the physician within a few minutes of arrival of the
specimen to the laboratory. Other modifications of this technique will also
give the levels of albumin and other protein constituents of blood and other
fluids without resort to the more cumbersome method of electrophoresis.
The DNA-bentonite test for systemic lupus erythematosus has also been
developed in the past year. This test measures the antibody in lupus serum
which is directed against nuclear material. The specificity of this test is
greater than any previously described test for lupus. This test, however, is
positive primarily in those patients with active disease and only rarely is
positive when the disease is in remission. A modification of this test, the
nucleoprotein-bentonite test, has retained all the attributes of the DNA test
in regard to specificity, while achieving a much higher level of sensitivity £01
cases in remission. These promise to become important standard tests In the
diagnosis of systemic lupus erythematosus.
Cystic Fibrosis of the Pancreas. Despite the commonly accepted point
of view that antibiotics are helpful in this disease, observations of naso-
pharyngeal cultures failed to reveal any appreciable effect of antimicrobial
treatment on Staphylococcus aureus . This does not negate a possible clinical
benefit but does appear to minimize its value. It was coincident ally
observed that Escherichia coli was not present in the nasopharyngeal flora
of any child over the age of eight. This interesting observation will
receive further study.
Serial No. NIAID - 10 (c)
1. LCI
2. Office of the Chief
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Office of the Chief
Principal Investigator: Dr. Vernon Knight
Other Investigators: None
Man Years (calendar year 1960);
Total:
7.0
Professional:
1.0
Other:
6.5
Project Description:
Objectives:
To develop, direct and coordinate the program of the Laboratory,
as defined in the individual research projects; where necessary, to
re-direct individual projects to meet current needs and advances in
the field; to align clinical projects with patient availability; to
provide an unexcelled standard of patient care for patients utilized
in research.
Methods Employed:
Organize available staff and recruit qualified personnel, both
professional and sub-professional, to develop the program and carry-
out its aims; close liaison with area medical societies, institutions
and individuals for referral of patients whose diagnoses fall withiu
the active or proposed disease research categories; the highest
standards of patient care are maintained by selection of qualified
physicians; the development of necessary policies and continued close
and direct supervision of patient care activities; professional con-
sultant services to other institutes in the area of infectious
diseases; continued guidance of research projects undertaken by
younger investigators; maintenance of staff morale.
Part B included Yes / / No /X/
Serial No. NIAID - 12(c)
1. LCI
2. Infectious and Pediatric
Disease Services
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Respiratory Viral Diseases
Principal Investigators: Dr. John P. Utz
Dr. Vernon Knight
Other Investigators: Dr. Howard Kravetz
Dr. David Rifkind
Dr. Anderson Spickard (from 7/1/60)
Dr. Robert Carpenter (from 7/1/60)
Dr. Hugh Evans (from 7/1/60)
Clarence F. Szwed
Margret A. Huber
Cooperating Units: Dr. Robert Channock, LID, NIAID
Dr. Karl Johnson, LID, NIAID
Man Years (calender year 1960):
Total: 5.5
Professional: 3.5
Other: 2.0
Project Description:
Objectives:
1. To define clinical entities in relation to newly isolated
respiratory viruses.
2. Further to define diagnostic criteria, pathogenesis, immune
response, persistence, sites and effects on tissues of virus in certain
infections of respiratory passages and mouth.
3. To study host-parasite relationships in reference to sus-
ceptibility to chronic or recurrent respiratory diseases.
4. To improve clinical laboratory techniques in the laboratory
confirmation of respiratory viral disease diagnosis.
Serial No. NIAID - 12(c)
5. To characterize in man the clinical course and associated
virological and immunological phenomena of infection with selected
respiratory viral agents.
Methods Employed;
1. Results of clinical observations and procedures are correlated
with bacterial, mycologic and viral isolations employing both animals and
tissue culture techniques.
2. Clinical observations are made under carefully controlled
isolation following intranasal infection with certain viral agents.
Virus isolation and immunologic studies performed by recognized pro-
cedures .
Patient Material and Major Findings;
1. The Virology Unit of the Infectious Disease Service examined
a total of 1245 specimens from patients under study. Virus was isolated
in 73 of these specimens, achieving etiologic confirmation of the diag-
nosis in a total of 35 patients.
2. The status of treatment of viral pneumonias has been critically
evaluated and reported.
3. Inmates selected from volunteers from several Federal correc-
tional institutions were infected or used as controls in studies with
the following viral agents: Para- influenza 4, 12 volunteers; respira-
tory syncytial virus infection, 46 volunteers, Eaton (primary atypical
pneumonia) virus, 13 volunteers.
4. Clinical and virological infection occurred in a high pro-
portion of patients given RS virus. Infection was less frequent' with
the other agents, apparently as a result of low dosage. This matter
is receiving further study.
Significance to Microbiological Research:
Studies in the laboratory and the clinic of patients naturally
infected with respiratory viruses will hopefully provide information on
pathogenesis, distinctive clinical findings, effects of giving or with-
holding antimicrobic treatment as related to the specific, known virus
involved. This approach also provides the only opportunity of discoverir
and defining new viruses.
Studies in human volunteers may provide information on the effect
of dose, route of administration, and kind of virus on the induction of
clinical infection. They may also indicate the role of immunity and
other protective mechanisms as a defense against infection. Such studies
- 2 -
Serial No. NIAID - 12(c)
would logically precede the development of effective vaccines against
these diseases. There are approximately 100 different respiratory
viral agents whose role in human infection is not adequately defined.
Moreover, increasing evidence suggests that viruses may participate
with bacteria in the cause of infection. This may be studied by the
present methods.
Proposed Course of Project;
Studies will continue as outlined under "Objectives" in patients
referred to the Clinical Center and in other hospital or community sur-
veys when indicated. This project should be expanded because of the
great importance of careful clinical studies on patients with respira-
tory illnesses in order to link these illnesses with viral agents that
are now being isolated in a number of laboratories.
Further studies with the Eaton, RS, and Para- influenza viruses
in human volunteers are planned. Human influenza and REO virus are
also being considered for this program.
Part B included: Yes /X/ No / /
Serial No. NIAID - 12(c)
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B: Honors, Awards, and Publications
Publications other than abstracts from this project:
1. Utz, J. P.: Pneumonias, Viral. Current Therapy, 1960 ed.
2. The studies with RS virus were part of a Clinical Center Symposium,
October 20, 1960, which will be published in Annals of Internal
Medicine.
- A
Serial No. NIAID - 13 (c)
1. LCI
2. Bacteriology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Antimicrobial Drug Therapy
Principal Investigator: Dr. Vernon Knight
Other Investigators: Dr. David Rifkind
Margret Huber
Cooperating Units: None
Man Years (calendar year 1960):
Total: 2.4
Professional: 1.7
Other: 0.7
Project Description:
Objectives:
a) To evaluate an antimicrobic drug.
1. Effectiveness in treatment of penicillin-resistant
staphylococcal infections.
2. Effectiveness in treatment of other coccal infections.
3. Appropriate dosages and routes of administration.
4. Toxic and allergic side effects.
5. Effectiveness in treatment of the nasal carrier state
of staphylococci.
Methods Employed:
Hospitalized patients with staphylococcal and streptococcal
infections were evaluated by clinical and laboratory methods for
response to antimicrobics. The laboratory methods included:
a) Isolation and identification of the etiological agent.
b) Determination of the Jji vitro sensitivity of the organism
to drug.
c) Assay of serum and urine for drug concentrations during the
course of treatment.
d) Determination of the inhibitory effect of serum against the
offending microorganism.
10
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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:
la In cooperation with Dr. Howard Goodman of the National Heart
Institute and Dr. Richard Malmgren of the National Cancer Institute,
studies of lupus factor as measured by the nuclear proteins sen-
sitized and red blood cell technique and the fluorescent antibody
technique, and its clinical correlation have been conducted. This
data is currently being prepared for publication.
2. The clinical application of the procedure devised by Mr. John
Bozicevich of the Laboratory of Immunology, NIAID, the DNA-bentonite
flocculation test, has been conducted within the framework of this
laboratory. This diagnostic method for systemic lupus erythematosus
has a great specificity. Further investigations with this procedure
(the DNA-bentonite flocculation test) are underway in an attempt to
evaluate the status of the lupus factor in these patients over a
period of time.
3. Certain water precipitable proteins of the serum of patients
with collagen vascular diseases are providing interesting immunologic
phenomenon. The fractions obtained in this method of separation of
proteins are being used in the DNA-bentonite test. In doing this,
we have discovered that this test assumes even greater importance
in the diagnosis of collagen vascular diseases.
A. We are continuing clinical studies of the patient with lupus
erythematosus and agammaglobulinemia.
5. In cooperation with Dr. Samuel Baron and Dr. Eugene Barnett of
the Division of Biologic Standards, the study of agammaglobulinemia
patients and the presence of antibodies in their serum to the
enteric viruses has been conducted by use of very sensitive
techniques to detect these antibodies. It has been found that all
of these patients have significant titers. Further studies are
being conducted in this vein in the attempt to elicit antibody
formation by means of polio vaccine in those agammaglobulinemics
not showing a significant titer. These studies are now completed
and are being prepared for publication.
6. A variation of the bentonite test using nucleoprotein, instead
of DNA, has been devised by Mr. Bozicevich. This is a much more
sensitive test and is capable of detecting systemic lupus erythe-
matosus regardless of the state of activity of the disease. The
clinical evaluation of this test is almost completed.
7. A bentonite procedure for the detection of gamma globulin has
been devised by Mr. Bozicevich. This test will make an excellent
screening procedure for agammaglobulinemia since it may be done in
quantity and with accuracy at a minimun cost. Clinical evaluation
is underway.
8. Preliminary studies are underway with bentonite flocculation
procedures to detect albumin, beta-lipoprotein, and siderophilin.
9. Mr. Bozicevich has been immunizing rabbits with DNA and
nucleoprotein in an attempt to produce antinuclear antibody. To
date there is no evidence that such antibody can be formed, though
additional studies are underway.
21
Significance to Microbiological Research:
Agammaglobulinemia provides us with a biological system relatively
free of humoral defense in which we may study infectious diseases.
Systemic lupus, with its attendant hyperglobulinemia, is an ideal
disease for the study of hypersensitivity and autoimmunity.
Proposed Course of Project;
Studies will be continued in a wide variety of clinical disorders
as to the pathogen-host factors involved in infectious disease.
Study of the present methods of clinical management of agammaglo-
bulinemia patients especially as to the required frequency and
dosage of gamma globulin injection and their management with
antibiotics will be continued.
The immune mechanism of agammaglobulinemic patients will be
studied further, especially as to their response to vaccine and
other antigenic stimulation. Non-antibody factors in resistance
to disease will also be studied.
Of a special importance is the continuation of the long term
followup of patients with systemic lupus erythematosus. This group
of patients has been tediously followed in the clinic and with
frequent inpatient admission and represents an outstanding gcoup in
regard to long term followup. During the course of the coming year
it is anticipated that the relationship of the clinical course to the
level of lupus factor as detected by the bentonite flocculation test
will be a major endeavor.
The pathologic significance of the L. E. cell, especially when
found in diseases other than lupus, will be studied.
Part B Included: Yes / / No / /
22
Serial No. NIAID - 17(c)
Part B; Honors, Awards, and Publications
Publications other than abstracts from this project!
1. Antibodies to Enteroviruses in Hypogammaglobulinemic Patients.
Barnett, E. V., J. P. Nasou, J. P. Utz, and S. Baron. New
England Journal of Medicine 262:563-565, March 17, 1960.
2. Desoxyribonucleic Acid (DNA)-Bentonite Flocculation Test for
Lupus Erythematosus. Bozicevich, J., J. P. Nasou and D. E.
Kayhoe. Proceedings of the Society for Experimental Biology
and Medicine 103:636-640, 1960.
3. Clinical Evaluation of the DNA Bentonite Flocculation Test for
Systemic Lupus Erythematosus. Kayhoe, D. E., J. P. Nasou and
J. Bozicevich. New England Journal of Medicine 263:5-10,
July 7, 1960.
4. Naming the Bentonite Procedure (proposals relative to the
nomenclature of the bentonite flocculation tests). Nasou, J. P.
J. Bozicevich and D. E. Kayhoe. Journal of the American
Medical Association 174 (10) :1348, 1960.
23
Sub -project II; Ascites in Mice
Principal Investigator: Miss Rose Lieberman
Other Investigators: Mr. William Humphrey
Mr. John Douglas
Mrs. Jocelyn Blakely
Cooperating Units: None
Man Years (calendar year I960):
Total: 1.4
Professional 0.5
Other 0 . 9
Project Description:
Objectives:
1 . To determine the incidence of ascites in 12 pure inbred and 3
hybrid strains of mice employing staphylococcal -adjuvant mixtures.
2. To observe the frequency of the development of plasma cell
tumors in these various strains of mice.
3. To investigate the role of the plasma cells in the production
of specific antibody.
Methods Employed:
Ascites inducing doses of staphylococcal -adjuvant mixtures are
administered to 15 different strains of mice immunized with oval-
albumin or horse serum. The incidence of ascites, reactions to
immunizing agents, amount of ascitic fluid produced, titers of
antibody in the ascitic fluid, effect of single versus multiple
immunizing doses on titer, appearance of persistence of antibody,
and the effect of "boosters" on antibody titer are observed in
individual mice over a long period of time for each strain studied.
In collaboration with the Cancer Institute, the incidence of
plasma cell tumors appearing in the different strains of mice are
removed and transplanted into unimmunized mice for studies on
appearance and production of antibodies.
21
Major Findings!
Specific strains of mice show a higher incidence of ascites with
persistent production of large amounts of ascitic fluid. Plasma cell
tumors appear with some consistency in about 18% of the Balb/c mice
treated with the staphylococcal-adjuvant mixtures. Several of these
tumors have been successfully transplanted through 4-5 generations of
mice. Indications are that a low incidence of plasma cell tumors may
appear in other strains of mice.
Significance to Microbiological Research!
Production of ascites in mice is an excellent tool to obtain a con-
tinuous source of large amounts of potent antibody from individual
small animals over a long period of time. This method will facilitate
the study of different antibodies produced by purification and
fractionation of antigens and provide some information on the mode of
action and synthesis of antibody.
The possibility of a genetic relationship to the incidence of
ascites in mice may be ascertained.
The role of "immune" plasma cells transplanted into normal mice in
the production of antibody may be investigated.
Proposed Course of Project!
To continue as outlined under objectives.
Sub-project III; Interactions of Antibodies
Principal Investigator: Miss Rose Lieberman
Other Investigators: None
Project Description:
Objectives:
To study the effect of mixtures of antibodies in conferring
passive protection.
Methods Employed:
Mixtures of Salmonella and of Proteus immune rabbit sera are
separately investigated to determine the effect on mouse protection
of increasing, decreasing or removing specific antibody components
present in the antisera combinations.
Major Findings;
Antibodies employed in combination are either simply additive,
25
enhancing or antagonistic in conferring passive protection in mice.
Significance to Microbiological Research!
Antibodies in combination behave differently from the equivalent
amounts of the same antibodies used separately and may be more or
less effective in conferring protection.
Proposed Course of Project:
To continue as outlined under objectives.
Part B Included: Yes / / No / /
26
Serial No. NIAID - 17(c)
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B; Honors, Awards and Publications
Publications other than abstracts from this projects
1. Lieberman, R., Douglas, J. 0. A. and Mantel, N.:
Production in mice of ascitic fluid containing
antibodies induced by Staphylococcus or Salmonella-
adjuvant mixtures. J. Immunol., 84:514-529, May, 1960.
27
Serial No. NIAID - 18(c)
1. LCI
2. Bacteriology Section
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Enteric Diseases
Principal Investigator: Dr. John P. Utz
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960):
Total: 1.1
Professional: 0.1
Other: 1.0
Project Description:
Objectives:
Basic studies of the etiology, pathogenesis, and immunology of
enteric diseases which include:
1. The evaluation of antibiotic immunologic and surgical therapy
on the "carrier state" of salmonellosis.
2. Studies of cellular and humoral mechanisms in active Salmonella
infections as compared to those present in the "carrier state."
Clinical studies: To investigate the factors that produce persis-
tence of bacteria in some patients to test new antibiotics in such in-
fections .
Methods Employed:
1. Complete bacteriologic and immunologic survey of enteric
disease study patients.
(a) In vitro determinations of synergistic, or additive effects
of combinations of antibiotics on organisms isolated from
"carriers."
(b) Determination of gamma globulin, electrophoretic pattern,
and agglutinating antibodies in acute and chronic enteric
disease states.
28
Serial No. NIAID - 18(c)
(c) Cholecystectomy or abscess irradication as Indicated.
Patient Material and Major Findings:
1. Studies of the Salmonella carrier state in humans have been
continued in 2 additional patients. Gall stones removed from these
patients have been implanted, as described in last year's report, in
rabbit gallbladders. This brings to a total 6 patients who had stones
so implanted in 8 rabbits, 7 of whom became carriers. The results of
this study in abstract form were published.
2. Studies described in last year's report on the patient with
Fusobacterium septicemia were published.
Significance to Microbiological Research;
Outbreaks of salmonellosis represent an increasingly serious public
health problem. The typhoid and other Salmonella "carrier states" are
responsible for spread of the more virulent Salmonella to man, and pose
a serious problem. There is no standardized therapy for the "carrier
state."
Proposed Course of Project:
To continue as outlined under "Objectives."
Part B included Yes /X_/ No /_/
29
Serial No. NIAID - 18(c)
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
1. Tynes, B. S., and Utz, J. P.: Fusobacterium septicemia.
Am. J. Med. 29: 879-887, 1960.
30
Serial No. NIAID - 19(c)
1. Laboratory of
Clinical Investi-
gation
PHS-NIH 2. Parasitic Disease
Individual Project Report Service
Calendar Year 1960 3. Bethesda, Maryland
Part A.
Project Title: Clinical Investigations on Helminthic Diseases
Principal Investigators: Henry K. Beye, G. Robert Coatney, Leon Jacobs,
John E. Tobie, John Bozicevich
Other Investigators: Steven Schenker (since July 1959), Donald Kayhoe
(until 7/60), Sanford Kuvin (since 7/60), and
Elizabeth Guinn
Cooperating Units: Parasitic Disease Service, LCI, NIAID
Laboratory of Parasitic Diseases, NIAID
Laboratory of Parasite Chemotherapy, NIAID
Laboratory of Immunology, NIAID
Medical Division, Department of State,
Washington, D. C.
British West Indian Labour Organization
National Cancer Institute
Man Years (calendar year 1960)
Total: 3.5
Professional: 2.0
Other: 1.5
Project Description:
Objectives:
The objectives of this group of related projects which include:
(I) Filariasis, (II) Nematode Infections, (III) Trematode Infections,
(IV) Cestode Infections, are to: (1) develop improved diagnostic
techniques, (2) better understand host-parasite relationships, (3) more
accurately describe diseases associated with these parasites and (4)
elucidate the pathogenesis of the disease process.
Methods Employed:
Methods for patient procurement utilized in 1960 consist of
physician referrals, a cooperative arrangement with the Medical Division,
Department of State, whereby returning State Department employees are
screened for the presence of intestinal parasites, arrangements with the
31
British West Indian Labour Organization ut 1 11 zing British West Indian
agricultural migratory workers, when indicated and a diagnostic
serological service for indicated special tests not pei I m med elsewhere.
Two new administrative aids have come into existence this yen which
will be very helpful in future studies. The provision of funds for
aiding the transport of research candidates wi i ■ il problems,
and the use of volunteers are notable advances.
The development of a modest museum of specimens coming to the
attention of the parasitic screening laboratory Is beginning to be
fruitful in terms of investigations and training.
I. Filariasls
Patient Material and Major Findings:
Probably the two most important major findings associated wlili
this project in 1960 were (1) the demonstration of the absorption of
tetracycline by filarial worms with consequent fluorescence when exposed
to ultraviolet light and (2) the demonstration of the viability of
microfilaria of Wuchereria bancrof tl for at least 18 months when ErOEM
at -10°C. The former studies commenced in 1959 and involved the
administration of tetracycline to 2 patients exhibiting cutaneous
migrations of Loa loa . These patients, when subsequently examined under
ultraviolet light, showed fluorescence of adult filarial worms. During
the past year fluorescence has also been demonstrated to some degree In'
malaria parasites, E. histolytica and tapeworm proglottlds. Microfilaria
used in the viability studies were from British West Indian Migratory
workers, with filarial infections that were reported and studied during
1959 and discussed in last year's annual report. Specimens of heparan-
lzed blood from these individuals containing microfilaria wei ■ placid
in the refrigerator and maintained at -10°C. At approximately J oontfa
intervals, separate portions were removed and allowed Co thaw at room
temperature. As of the present date, microfilaria have retained
viability.
Attempts were made to develop a bentonite flocculation test
for use In the diagnosis of filariasls. Preliminary remiltu on the sera
obtained from immunized animals gave encouraging results. This is to be
exploited on sera obtained from infected individuals. A paper was pre-
sented at the First International Congress on Trichinosis, Warsaw, Poland
Significance to Bio-Medical Research and the Program of the Institute:
The demonstration of the phenomenon of tetracycline c< ■ i Ion
in filarial worms as well as In other parasites, has much potential
significance. There has been a great interest elicited since the
publication of the original article for using this as a diagnostic pro-
cedure. At the same time, interest has also been expressed In the
utilization of this concentration of tetracycline In the development of
more effective antlfilarial drugs. The viability studies are vary •- 1 mij> 1 <•
as performed in our laboratories and iIkt.c findings <":<y ''•' "Bt
-2 - 32
on the metabolism of these parasites. In time, such a technique wjuld
be extremely useful to medical schools providing viable microfilaria for
class demonstration and study.
Proposed Course of Project:
Additional studies will be undertaken on utilizing and under-
standing the tetracycline fluorescence phenomenon in filarial worms. In
pursuing these studies, it is hoped to utilize staff and facilities in
British Guiana where a filariasis research and control project is being
established, following the recommendations made by the senior investigator
when he served as a WHO consultant for that country in filariasis. In
addition, it is anticipated to pursue these studies by the use of PL-480
counterpart funds in various countries where filariasis is endemic. The
question of the elucidation of the pathogenesis of eye lesions in
onchocerciasis is a very important and controversial one. It is hoped
that these techniques might be applicable in this filaria infection.
II. Nematode Infections and Disease
Patient Material and Major Findings;
1. Trichostrongylus infected patients: As^ reported last year,
State Department employees continued to come to our -attention who were
infected with trichostrongylus. Species have not yet been determined
because of the difficulty of procuring adult worms. Studies conducted
this year indicate that eggs of this parasite obtained from patients
treated with tetracycline fluoresce. Attempts have been made to utilize
this phenomenon to find adult worms after treatment but so far these
attempts have been unsuccessful.
2 . Parasites among the British West Indian Migratory Workers:
This group of individuals with rather high prevalence of intestinal
nematodes is still available to us but during this past year we have
been unable to follow through with this group.
3. Visceral Larval Migrans: A very informative case referred
to the Clinical Center because of the diagnosis of eosinophilic leukemia
has been studied and proven to have visceral larval migrans. In this
case the diagnosis was established through recovering larvae of probably
Toxocara canis at liver biopsy and the adult worms were recovered from
the family dog which died and was posted. The patient has responded
well to diethy lcarbamazine . In summary, the case provides probably one
of the best documented cases of visceral larval migrans which has yet
been described. This patient was admitted to the Cancer Service and was
studied in conjunction with our laboratory. At the present time
observations are still continuing.
Significance to Bio-Medical Research and the Program of the Institute:
Probably the most significant finding in this project during 1960
was the demonstration of this case which had many characteristics of
3 -
33
eosinophilic leukemia but which responded well to diethylcarbamazine
therapy. The elucidation of the role of trichostrongylus in returning
State Department people from the Near East and further development of the
various nematode infections amongst West Indian Migratory laborers are
significant and will be pursued when time and facilities permit.
Proposed Course of the Project;
This project will be intensified, particularly in respect to
investigation of trichostrongylus and trichinosis. The further ex-
ploration of tetracycline and autof luorescence; the programming for our
activation of studies of parasitoses in 1962 in countries where counter-
part funds are available, will be pursued. The instrument section is
now building a sigmoidoscope, utilizing ultra-violet light which will be
of great help in these investigations.
III. Trematode Infections and Disease
1. Schistosomiasis and other diseases due to flukes; Little
activity has been carried on in this area during the past year, primarily
because of emphasis on simian malaria and tetracycline studies.
Dithiazanine iodide reported as being effective against Clonorchis
sinensis has been tested in this laboratory. The results of follow-up
studies are still in progress.
Significance to Bio-Medical Research and the Program of the Institute;
With the intense migration of Puerto Ricans with high infection
rates due to Schistosoma mansoni , increasing number of Americans going
to infected areas and the increasing larger areas of the world in which
infected snails are found, makes schistosomiasis one of the major world
diseases and of increasing importance to the people in the United States.
The treatment trials against Clonorchis sinensis with dithiazanine iodide
is interesting and significant in that no previous medication has been
very effective. The most universally used drug, chloroquine diphosphate,
has not been very effective in our hands as well as in other workers.
Proposed Course of the Project:
Inasmuch as facilities and staff allow, continued efforts will
be made to evaluate new drugs. The use of PL-480 funds has made it
possible to program a clinical study in schistosomiasis in Brazil with
possible activation by July 1961. This will help provide a better
understanding of the pathogenesis and the manifestations of hepatosplenic
schistosomiasis .
IV. Infections due to Cestodes
Patient Material and Major Findings:
A series of patients with Taenia saginata , mentioned in last year's
report, has now increased to approximately 26. During 1960 experimental
-4- 3H
administration has consisted of tetracycline with follow-up studies
on tetracycline fluorescence plus oral atabrine. In cases that are
unsuccessful, the treatment of atabrine transduodenal ly is employed.
Dithiazanine has been tried with inconclusive results.
Significance to Bio-Medical Research and the Program of the Institute:
Infections with these parasites are difficult to treat, and
the success of our method of using the atabrine through a duodenal
tube is a rather significant contribution.
Proposed Course of Project:
It is anticipated that a great deal of emphasis will be made in
the next year on Echinococcus disease, using patients from Alaska
or in planning Indies in Echinococcus in countries of high endemicity,
utilizing counterpart PL-480 funds.
Part B included Yes No
35
(Attachment I)
Serial No. NIAID - 19(c)
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
1. Beye, Henry K., and Gurian, Joan: The Epidemiology and Dynamics of
Transmission of Wuchereria bancrofti and Wuchereria ma lay i. Indian
Journal of Malariology, December, 1960.
2. Beye, Henry K. : International Organizations and Filarlasis Control
(Wuchereria bancrofti and Wuchereria malayi). Indian Journal of
Malariology, December, 1960.
3. Tobie, John E. and Beye, Henry K. : Fluorescence of Tetracyclines
in Filarial Worms. Proc. Soc. Exper. Biol, and Med., 104:137-140,
1960.
4. Kayhoe, Donald E. and Beye, Henry K. : The treatment of Taenia
saginata infections with quinacrine HCL (Atabrine) administered
through a duodenal tube. Presented at the American Society of
Tropical Medicine and Hygiene meeting, November 2-5, 1960.
5. Beye, Henry K. : Helminthic Diseases and Anthelminthic Drugs.
Clinical Proceedings of The Children's Hospital, In Press.
6. Beye, Henry K. : Filariasis. Cyclopedia of Medicine, Surgery and
Specialties. In Press.
36
Serial No. NIAID - 20(c)
1. Laboratory of
Clinical Investi-
PHS-NIH gation
Individual Project Report 2. Parasitic Disease
Calendar Year 1960 Service
3. Bethesda, Maryland
Part A.
Project Title: Clinical Investigations in Protozoan Infections and
Disease
Principal Investigators: Henry K. Beye, G. Robert Coatney, Leon Jacobs,
John E. Tobie and John Bozicevich
Other Investigators: Donald Kayhoe to July 1960, Morton Getz, Harvey
Elder from 7/60, Steven Schenker to 7/60,
Sanford Kuvin since 7/60, and Elizabeth Guinn
Cooperating Units: Parasitic Disease Service, LCI, NIAID
Laboratory of Parasite Chemotherapy, NIAID
Laboratory of Parasitic Diseases, NIAID
Laboratory of Immunology, NIAID
Medical Division, Department of State, Washington,
D. C.
Federal Bureau of Prisons
Man Years (calendar year 1960)
Total: 3.5
Professional: 2.0
Other: 1.5
Project Description:
Although the activities of interest on specific protozoan
infections vary from year to year, fundamental objectives and methods
remain essentially the same. Since April 1960, a great deal of the
emphasis was on the clinical investigations associated with simian
malaria. Activities in relation to amebiasis continued, but at a re-
duced activity. Little emphasis was given to trichomoniasis, toxo-
plasmosis or other protozoan diseases.
The development of a modest museum of specimens coming to the
attention of the parasitic screening laboratory is beginning to be
fruitful in terms of investigations and training.
Objectives:
(1) Improve diagnostic techniques (2) better understand host
parasite relations (3) more accurately describe disease manifestations
and host response (4) elucidate the pathogenesis of the disease process
37
in protozoan infections and (5) more accutate evaluation of various
treatment regimes.
Methods Employed:
Methods of patient procurement utilized in 1960 consisted of
physician referrals, a continuation of the cooperative arrangement
with the Medical Division, Department of State, whereby returning
State Department employees were screened for the presence of intestinal
parasites, the use of inmate volunteers, primarily through the Malaria
Project at the Federal Penitentiary in Atlanta, Georgia, a new method
started this year of the utilization of selected inmate volunteers at
the Clinical Center, and the maintenance of a research serological
diagnostic service for protozoan diseases.
Methods for investigation and study of these research candidates
remain the same, that is, close communication and liason with the basic
laboratories and workers participating in these investigations, direct
activities through our own Parasitic Disease diagnostic and investi-
gative laboratory, utilization of the Service activities provided by
the Clinical Center and finally, educational and program activities
related to patient procurement such as lectures and talks at various
universities, exhibits and discussions with organizations which might
provide a source of research candidates.
I. Blood and Tissue Protozoan Diseases
A. Simian Malaria in Man
Patient Material and Major Findings:
(1) Two blood induced infections with Plasmodium cynomolgi
bastianellii in inmate volunteers in May, (2) twenty sporozoite induced
infections with the same parasite in inmate volunteers during May and
June 1960, the infective mosquito in this instance being Anopheles
quadrimaculatus, (3) six sporozoite induced infections amongst inmate
volunteers using the same parasite but using Anopheles freeborni.
Two of these patients were transferred to the Clinical Center immediately
after being bitten while the remainder stayed for observations at the
Unit at the Atlanta Panitentiary, (4) eleven inmate volunteers bitten
by Anopheles freeborni heavily infected with Plasmodium cynomolgi. As
a result of these experiments, a new clinical entity associated with
this previously thought simian parasite has been first described.
These results can be briefly summarized as follows: Prepatent period
usually under 18 days, onset of symptoms ranging from 14 days to 2 1/2
months, extremely low parasitemias associated with rather severe
clinical manifestations in a few patients, other patients having in-
fections but with mild or asymptomatic clinical manifestations. Mani-
festations seemingly more benign in Negroes than in Caucasians. Changes
in certain laboratory tests such as increase in erythrocyte sedimentat-
ion rate, increase in cholesterol beginning early after the infection,
decrease in platelet counts and neutropenia, are associated with the
38
infection. Individuals, infected with regular cynomolgi on the other
hand, do not seem to develop the infection. A most significant finding
is the demonstration that simian malaria parasite is probably a zoonoses,
the parasite has been transmitted from monkey to man via the mosquito,
and then from man to mosquito. Suggestive evidence indicates the
transmission from man to man by the mosquito.
B. Chemotherapeutic studies using inmate volunteers at Atlanta
Penitentiary. More than a 100 volunteers participated in various
studies associated with the prophylactic and sporozonite properties of
primaquine, chloroquine and a Russian preparation, quinacide. The
results amongst these studies can be summarized as follows:
1. Suppressive cure effect of a pyrimethamine -primaquine com-
bination: Pyrimethamine 50 mgm plus primaquine 45 mgm given once a
week for four weeks, has protected five men exposed to the bites of
mosquitoes infected with Chesson strain of vivax malaria for at least
219 days of observation.
2. Suppressive cure effect of chloroquine -primaquine combination:
Ten Negro male volunteers were divided into two groups of 5 men each.
Each man in Group A received chloroquine 300 mg plus primaquine 45 mg
in a single oral dose three days prior to the date of infection, (0-3),
and on the same day of each week for a total of four doses. The sub-
jects in Group B received no drugs and served as controls.
Both groups were infected with the McLendon strain of P. falci-
parum on 1 April 1960, by the bites of ten heavily infected A. quadri-
maculatus mosquitoes. In an attempt to simulate field conditions the ten
volunteers were subjected to three subsequent bitings at irregular
intervals. At each subsequent biting time additional controls were
added.
Each of the five men in Group B developed parasitemia on the 11
to 13 experimental day. After two days of demonstrable parasites,
each was given chloroquine 300 mg plus primaquine 45 mg. This regimen
was repeated on the same day of the week for a total of three doses.
None of the five volunteers in Group A developed parasitemia
during 90 days of observation. None of the man in Group B has ex-
hibited any evidence of infection following the chloroquine-primaquine
therapy.
One control was added for each of the last 2 biting episodes.
Neither control developed malaria and the last two bitings are con-
sidered non-infecting even though sporozoites were demonstrated at
the post-prandial dissection of the mosquitoes.
3. Curative effect of CN 1115 (WIN 10448, Quinicide): To date,
8 patients have received Quinicide therapy and 10 have received Prima-
quine. Of those patients who received CN 1115, four exhibited early
primary attacks and four delayed primaries. There was a total of 6
39
recurrences in the early primary group and two in the delayed primary
group .
To date, 11 patients have received primaquine therapy and ten
have received CN 1115. The dosage of each drug was 15 mg. single dose
daily for 14 days. There have been two relapses among those who re-
ceived primaquine therapy. There have been 13 relapses among those
given CN 1115. Of these relapses, one man had four, three had two, and
three had one relapse each. Relapse infections were treated according
to the original regimen.
Significance to Bio-Medical Research and the Program of the Institute:
The significance of these investigations in terms of the world-
wide malaria eradication and in terms of more adequately understanding
the pathogenesis of malaria, including the human form, is very great.
For the first time, reservoirs of human malaria have been definitely
elucidated and perhaps for the first time, a malaria parasite with
clinical manifestations similar in monkeys and man has been found. This
will provide a very useful tool in other studies on malaria.
Proposed Course of Project:
This project in clinical investigation is one aspect of an in-
tensive program of investigating the role of simian malaria in the
epidemiology of human malarias coordinated through the Laboratory of
Parasite Chemotherapy. It is anticipated that additional work will be
done on the clinical description of the disease involving the use of
additional volunteers at the Clinical Center. Studies of cross -immunity
of the simian malarias to human malarias, particularly the regular
cynomolgi are programed. It is anticipated that some activity may be
extended to other countries through the utilization of PL-480 funds.
II. Amebiasis
A. State Department Personnel
Patient Material and Major Findings:
As indicated, patient material has consisted of the more than
6,000 State Department employees who have submitted stool specimens to
the Parasitic Disease Service for screening since the inception
of this cooperative program with the Medical Division, Department of
State. This material has been analyzed, the most interesting features
being that those individuals infected with E_. histolytica did not ex-
hibit any additional clinica 1 manifestations or did they lose more time
from work while overseas than those individuals who were not infected.
This re -emphasizes again the beginning appreciation of the benign
character of E. histolytica in normal healthy individuals. At the same
time, an occasional case was admitted to the Clinical Center with the
classical symptoms of acute amebic dysentery and one case of amebic
liver abscess from the Washington area was studied. Studies continued
40
on the development of more definitive and more perfected serological
tests for amebiasis including a bentonite flocculation test. For those
individuals who were accepted for research study, the use of the anti-
amebicide, Humatin (Puramycin) has been studied this past year. This
drug, in our present investigations, seems to be the most satisfactory
amebicide which has been our experience to study. The definitive
analysis, however, of these cases, including follow-up observations,
are still in process.
B. Immunology and Serology
Three series of rabbits were immunized to Entamoeba invadens ,
12. terrapinae and E. histolytica antigens. The antigens were grown
in vitro on the appropriate media and the animals immunized with the
supernatant fluid contining the metabolic products and the entire
organisms. At intervals, the animals were bled and complement fixation
tests were conducted with the specific and heterologous antigens. N0
cross reactions were found at any time as elicited by a positive
complement fixation test. Additional immunization was given to each
group with the specific antigen. Again, no cross reactions were en-
countered with the heterologous antigens but specific reactions were
obtained as elicited by the complement fixation test.
The purpose of the above study was to investigate the possibility
of utilizing a closely associated antigen for the complement fixation
test for the detection of E. histolytica infection. If this proves
to be successful, it would facilitate preparing antigens for the
diagnosis of amebiasis.
Significance to Bio -Medical Research and the Program of the Institute:
It is only through the understanding of the pathogenesis of E.
histolytica infections and disease that this entity will be placed in
proper prospective. Work continues on the elaboration of the patho-
genesis of IS. histolytica in man in our basic laboratories, particularly
the germ-free laboratory. The significance of our observations is rather
large in that it provides a great deal of epidemiologic data on benign
characters of this infection among foreign service personnel. The
presence of the disease, however, in occasional cases only emphasizes
the importance of understanding what factors are responsible for these
manifestations.
Proposed Course of Project:
With adequate staff and time, it is hoped that this project can,
in time, be re-oriented to concentrate on a few individual cases which
can be followed with or without treatment for long periods of time,
using in association with these studies newer techniques for the study
of the upper GI tract. This would make possible the description of the
invasiveness and the characteristics of the E. histolytica complex (E.
hartmani , small and large races of E_. histolytica) along with the
bacterial associates in the human GI tract. During this past year,
little work was done on trichomona is is, toxoplasmosis or other pro-
tozoan diseases which have received emphasis in the past. We hope to
reactivate these projects in the near future as they are of great bio-
medical significance. This is particularly true with toxoplasmosis. The
infection is wide-spread, the disease manifestations are occasionally very
severe, and the spectrum of host response and clinical manifestations
is as yet undescribed.
Part B included Yes No
m
(Attachment I)
Serial No. NIAID - 20 (c)
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
1. Beye, Henry, Brooks, Charles and Guinn, Elizabeth: Intestinal and
Blood Parasitism among British West Indian Agricultural Laborers.
Review of Protozoan and Helminthic Infestations among Migratory
Agricultural Workers. Accepted for publication by the Journal of
American Public Health Association.
2. Kayhoe, Donald E., Beye, Henry, Guinn, Elizabeth and George,
George P.: Prevalence of Intestinal Parasites Among U. S. State
Department Employees and Their Dependents. Presented at the
American Society of Tropical Medicine and Hygiene Meeting, Los
Angeles, California, November 2-5, 1960.
3. Coatney, G. Robert and Beye, Henry K. : Simian Malaria Infections
in Man. Presented at the American Public Health Association Meeting,
San Francisco, California, October 31-November 5, 1960.
4. Beye, Henry K. and Coatney, G. Robert: Preliminary Report on
Sporozoite Induced Plasmodium Infections in Man. Presented at
World Health Organization Expert Committee on Malaria.
5. Beye, Henry K., Coatney, G. Robert, Getz, Morton and Eyles, Donald:
A Vivax-Type Malaria of Simian Origin in Man. Clinical and
Parasitological Characteristics. Presented at the American Society
of Tropical Medicine and Hygiene Meeting, Los Angeles, California,
November 2-5, 1960.
6. Getz, M.E., Elder, H. A. and Todd, Chas . S., Jr.: Clinical
Manifestations of Plasmodium cynomolgi bastianellii. Presented
at the National Institute of Allergy and Infectious Diseases
and Communicable Disease Center Joint Conference, November 17-18,1960.
k3
Serial No. NIAID - 21(c)
1. LCI
2. Pediatric Service
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Project Title: Cystic Fibrosis of the Pancreas
Principal Investigator: Dr. George T. Bryan
Other Investigators: Dr. Hugh Evans (from July 1, 1960)
Dr. Philip Fireman (from July 1, 1960)
Dr. Edward Eyerman (to June 30, 1960)
Dr. Lowell Good (to June 30, 1960)
Dr. George Owen (to June 30, I960)
Cooperating Units: None
Man Years (calendar year 1960):
Total: 2.85
Professional: 2.85
1. Objectives
A. To study the microbial flora in patients with cystic fibrosis of
the pancreas .
B. To evaluate the role of staphylococcal immunology.
C. To evaluate the effect of certain antibiotic agents on the bacterial
flora of the respiratory tract.
D. To study the immunology of purified staphylococcal penicillinase.
E. To study the relationship of growth failure in these patients to
their serum growth hormone levels.
2. Patient Material
Children with proven cystic fibrosis of the pancreas who are being fol-
lowed regularly in this clinic.
3. Major findings
The major findings are limited to the study of the microbial flora in
patients with cystic fibrosis of the pancreas. These would indicate
that children with this disease have a relatively stable bacterial flora
in spite of continuous therapeutic antibiotic therapy. Investigations
are continuing in the study of microbial flora and the role of the
staphylococci in cystic fibrosis of the pancreas.
Part B included Yes f~j No /X/
W
Serial No. NIAID - 22(c)
1. LCI
2. Biochemistry and
Immunochemistry
Section
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Project Title: Basic Biochemical Studies
Principal Investigator: Dr. Harry G. Steinman
Other Investigators: Dr. Eskin Huff
Dr. Steven Schenker
Dr. Anderson Spickard
Dr. Sheldon Wolff
Cooperating Units: Hildegard Wilson, Laboratory of Nutrition and Endocrin-
ology, NIAID
Ellsworth R. Buskirk, Metabolic Diseases Branch, NIAID
Ronald H. Thompson, Metabolic Diseases Branch, NIAID
Howard Goodman, Laboratory of Immunology, NIAID
Emanuel S. Hellman, General Medicine Branch, NCI
Man Years (calendar year 1960):
Total: 4.5
Professional: 2.5
Other: 2.0
Project Description:
Objectives:
To study the biochemistry of pathogenic micro-organisms; the nature
of penicillin resistance; the mechanism of action of penicillin; the bio-
chemical abnormalities associated with specific allergic and infectious
disease processes.
Methods Employed:
1) Biochemistry of pathogenic micro-organisms. The biosynthesis
of the polysaccharides of bacterial cell wall material has been investi-
gated with emphasis on the role of certain three-carbon precursors.
2) Nature of penicillin resistance. The role of penicillinase in
the resistance of j3^_ aureus to penicillin is being investigated under a
variety of conditions.
3) Mechanism of action of penicillin. A number of new penicillins
now available are being compared in a number of biological and biochemical
systems in order to correlate chemical structure with biologic activity.
45
Serial No. NIAID - 22(c)
4) Biochemical abnormalities In allergic and infectious diseases.
The biochemical origins of a rare clinical entity known as etiocholano-
lone fever have been examined with respect to the metabolic fate of spec-
ific steroid substances. The amino acid abnormalities associated with
Familial Mediterranean Fever have been studied. The effect of chloro-
quine on producing porphyria is being investigated in both animals p.nd
patients.
Patient Material and Major Findings:
1) Biochemistry of pathopenic micro-organisms. The study on the
identification of lactaldehyde as the oxidation product of 1,2-propanediol
and the sterespecificity of the enzymatic reactions catalyzed by alcohol
dehydrogenases; which was described in a previous Project Report
(NIAID - 22(c), 1959) has been summarized in a paper entitled "The Metab-
olism of 1,2-propanediol" and which is now in press. Tt ' - publication
concludes the work on the metabolism of 1,2-propanediol and its phosphate
ester.
2) Nature of penicillin resistance. Although there is a very high
degree of coincidence between the presence of penicillinase and the peni-
cillin resistance of pathogenic J^ aureus, the direct proof that the enzyme
is responsible for the resistance has been lacking. There would appear to
be no question as to the role of penicillinase were it not for the fact
that penicillin resistance of ordinarily sensitive strains of jj^ aureus
can be readily achieved in the laboratory by a process of selection of
natural mutants. This type of natural resistance, which does not appear
to be stable, is not dependent on the presence of the destructive enzyme
penicillinase. Its true basis remains unknown. A variety of experiments
have been performed which support the thesis that penicillinase is indeed
the primary factor effecting resistance to penicillin in JL_ aureus. These
will be reported at the 5th International Congress of Biochemistry to be
held in Moscow, 1961.
3) Mechanism of action of penicillin. As a corollary of the
studies on penicillinase the activities of the reaction with several dif-
ferent penicillins had been made. It became apparent that a penicillin
which was insensitive to the action of the enzyme would be of great value
to the basic studies as well as of clinical interest. When such a peni-
cillin became available, as the result of a major breakthrough in peni-
cillin technology, our laboratory techniques were able to demonstrate
immediately the unique resistance to the destructive action by staphylo-
coccal penicillinase of the new penicillin and to indicate the potential
usefulness of the drug in therapy almost before clinical evidence of the
effectiveness in the treatment of penicillin G-resistant staphylococcal
infections was at hand. These findings were embodied in 2 papers, one
presented at a Symposium devoted to the new penicillin held in Syracuse
on September 7, 1960 and the other at the Conference on Anti-Microbial
Agents held in Washington on October 26-28, 1960. By examining a series
of penicillins in a variety of biological and biochemical reaction systems,
- 2- 1+6
Serial No. NIAID - 22(c)
it has been possible to ascertain the effect of various chemical substi-
tuents in the basic penicillin molecule on a number of specific biologic
activities. These studies will be reported at the First International
Pharmacological Meeting to be held in Stockholm, 1961.
4) Biochemical abnormalities associated with specific diseac*.
processes.
a) Etiocholanolone fever. In colloboration with Dr. H. Wilson,
NIAID, a patient with the diagnosis of etiocholanolone fever has been
studied with respect to the biochemical pathogenesis of his disease. This
disease was characterized by bouts of fever, chills, myalgia and anorexia
lasting from a day to several weeks, recurring at irregular intervals and
associated with a mild normocytic normochromic anemia and a nonspecific
granulomatous infiltration of the liver. At the time of his febrile
attacks, the patient was found to have elevated plasma levels of nonconju-
gated etiocholanolone and dehydroepiandrosterone. The i /.ness responded
dramatically to steroid therapy.
In order to elucidate the pathogenesis of this rare disease, meta-
bolic studies have been carried out to determine 1) the ability of the
patient to conjugate and excrete exogenously administered etiocholanolone
and 2) the capacity of the patient to synthesize etiocholanolone from its
precursors. Twenty five milligrams of etiocholanolone were administered
intramuscularly to the patient while his own etiocholanolone secretion
was suppressed by steroid therapy, and urine was tested for etiocholano-
lone output. Preliminary results, by the hot acid hydrolysis method,
indicate that the patient excretes 40~507o of the administered dose in
24 hours comparable with normal patients. The urine will be studied fur-
ther for total quantitative excretion of the compound as well as the amount
which is conjugated as a glucuronide and sulfate. The patient's glucuronyl
transferase system was studied by determining his ability to conjugate men-
thol and this was found to be normal. Subsequently the patient was given
doses of testosterone and hydroxy-androstenedione and his urine checked for
conversion of these substances to etiocholanolone. Initial results reveal
normal conversion. Should the patient redevelop his symptoms, it is
planned to investigate the possible beneficial role of thyroid analogues
on the course of his illness as well as to attempt to isolate a possible
thermogenic principle from the plasma.
b) Familial Mediterranean Fever. Preliminary investigations
are under way dealing with Familial Mediterranean Fever. In collaboration
with Drs. E. Buskirk and R. Thompson of NIAMD, we are studying the physio-
logic response of a patient to cold stress and also during an attack of
fever. Biochemical studies of the alpha amino nitrogen excretion and
chromatographic analyses of the amino acid content of blood and urine are
being done. Some immunologic studies in conjunction with Dr. H. Goodman,
NIAID, are planned. Some therapeutic trials are anticipated also, using
female hormones.
-3- 47
Serial No. NIAID - 22(c)
c) Porphyria. A patient with porphyria is also being studed.
Elevations in the patient's excretion of 5-hydroxyindoleacetic acid has
been noted and it is planned that this finding be looked for in other
patients and some investigation as to why this occurs will be undertaken.
Also the effect of chloroquine in this disorder is being studied by
measuring urinary porphyrins and delta aminolevulinic acid before anJ
after chloroquine therapy. The latter study and a study of the effect of
chloroquine in experimental porphyria (in rats) is being done in collabo-
ration with Dr. E. Hellman of NCI.
Significance to Biomedical Research;
The discovery of the high degree of resistance of the new penicillin,
2,6-dimethoxyphenylpenicillin, to the destructive action of staphylococcal
penicillinase supplied the scientific basis for pursuing the clinical
trials of the drug. Other laboratory data helped establ -h proper dosage
and treatment schedules. The new antibiotic shows great promise of
ameliorating the problem of hospital-borne staphylococcal infections.
However, its intrinsic chemical properties prevent it from being used
except under hospital conditions because it is not absorbed into the
blood stream well enough to be given orally, but must be injected intra-
venously (or intramuscularly). Our laboratory know-how in this field
will enable us to evaluate immediately and exploit any superior penicillin
that may appear in the future.
The studies on the biochemical observations associated with such
obscure diseases as etiocholanolone fever, Familial Mediterranean Fever,
and chloroquine-induced porphyria will be helpful in understanding the
pathogenesis of these diseases.
Proposed Course of Project;
These studies are being continued.
Part B included yes /X/ no / /
-4- 1*8
Serial No. NIAID - 22(c)
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
1. Huff, E. : The Metabolism of 1,2-Propanediol. Biochem. et Bio-
phys. Acta. In press.
2. Steinman, H.G.: Factors modifying induced formation of penicil-
linase in Staphylococcus aureus. J. Bacteriol. In press.
3. Steinman, H.G.: A biochemical comparison of 6-aminopenicillanic
acid, benzylpenicillin, and 2,6-dimethoxyphenylpenicillin. Proc.
Soc. Exptl. Biol. Med. In press.
4. Steinman, H.G.: Comparative activities of 6-aminopenicillanic
acid, benzylpenicillin, and 2,6-dimethoxyphenylpenicillin. Paper
delivered at State University of New York, Upstate Medical Center,
Syracuse, New York, September 7, 1960; to be published in
Bunn, P. A. (ed.): A Symposium on a New Synthetic Penicillin,
Syracuse University Press, 1961.
5. Steinman, H.G.: A comparison of the biochemical activities of
6-aminopenicillanic acid, benzylpenicillin, 2,6-dimethoxyphenyl-
penicillin, 2-phenoxyethylpenicillin, and phenoxymethylpenicillin.
Paper delivered before the Society for Industrial Microbiology,
Washington, D. C. , October 27, 1960; to be published in Trans-
actions of the Conference on Antimicrobial Agents, 1960.
49
Serial No. NIAID - 23(c)
1. LCI
2. Infectious Disease Service
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Systemic fungal disease
Principal Investigator: Dr. John P. Utz
Other Investigators: Dr. Vincent T. Andriole
Dr. Michael W. Brandriss
Margret A. Huber
Cooperating Units: Laboratory of Infectious Diseases
Man Years (calendar year 1960):
Total: 2.1
Professional: 2.1
Other: 0.0
Project Description:
Objectives:
1. To study diagnosis, pathogenesis and natural course of sys-
temic fungus infections.
2. To study the problem of fungus disease due to overgrowth dur-
ing antibiotic and steroid treatment.
3. To evaluate new agents in antibiotic and chemotherapeutic
treatment of systemic fungus infections.
4. To set up means of measuring blood levels of new antifungal
agents and to study acute and long term toxic effects of the drugs.
5. To study various immunologic aspects of this group of
diseases .
Methods Employed:
Patients with suspected fungal infections are studied diagnostical-
ly using the skin test, serologic, histologic, and cultural methods.
Family contacts are similarly investigated and environmental studies
undertaken, when indicated. Infecting fungi from such patients are
50
Serial No. NIAID - 23(c)
screened for sensitivity against a considerable number of cheraothera-
peutic or antibiotic agents and therapy is undertaken after such studies
and where clinically advisable.
Drugs are screened for activity against an organism isolated from
a patient by Jji vitro methods. Promising drugs are then tested in
animals for acute and long-term toxicity and for therapeutic effectiveness.
New methods are being developed for detemining serum drug levels,
rates of excretion of drug, and jji vitro screening of drugs.
Patient Material and Major Findings;
1. Studies were continued on patients with a variety of fungal
diseases. New patients with culturally proved disease this year in-
cluded cases of cryptococcosis (7), coccidioidomycosis 3), histoplas-
mosis (5), sporotrichosis (5), nocardiosis (1), blastomycosis (3),
cardidiasis (1), aspergillosis (2), and mucormycosis (1).
2. Continued observation of a total of patients treated with
amphotericin B has permitted more conclusions as to effectiveness.
Although clear-cut treatment failures have occurred in 3 cases of cryp-
tococcosis, 7 patients have "apparently recovered" (clinically well with
normal cerebrospinal fluid) from their infections 32, 41, 31, 30, 12, and
6 months after cessation of therapy. One patient died from an unrelated
disease. An additional 6 patients are improved (clinically well, but
with cerebrospinal fluid not yet normal). An analysis of the failures
of amphotericin- treated patients was completed and presented at the 2nd
Conference on Medical Mycology, New York Academy of Sciences, January 1960.
3. Eleven new patients with various culturally proved fungus
diseases, histoplasmosis (3), blastomycosis (2), coccidioidomycosis (1),
aspergillosis (1), mucormycosis (1), and sporotrichosis (3), have been
treated with the new antifungal drug R0-2-7758, making a total of 26
patients now treated with this drug. R0-2 seems effective in histo-
plasmosis, blastomycosis, aspergillosis, maduramycosis , mucormycosis,
and sporotrichosis.
A. An investigation has been undertaken of the renal toxic effects
of amphotericin. Renal plasma flow glomerular filtration rate in addi-
tion to concentrating ability in the kidney have been determined in 5
patients treated with this drug.
5. An investigation of the toxic effects of RO-2 on the liver
ha? also been undertaken. Liver biopsy and BSP determinations have
been evaluated in patients treated with this drug.
51
Serial No. NIAID - 23(c)
6. An exhibit showing the results of therapy with amphotericin
B and RO-2 was prepared and presented at the Annual Meeting of the
American Medical Association at Miami Beach in June 1960.
7. An exhibit showing the results of RO-2 was prepared and
presented at the Annual Meeting of the American Public Health Associa-
tion in San Francisco, California, in October 1960, and at the Clinical
Meeting of the American Meeical Association in Washington, D. C, in
November 1960.
8. A clinical study of Candida endocarditis was undertaken and
observations made on 6 patients with this disease.
9. A study is being made of granulomatous disease of undifferentiated
cause occurring in man.
Significance to Microbiological Research:
The group of fungus diseases under study is poorly understood;
there is no uniformly successful treatment. Apparently, certain of
them, such as histoplasmosis, are being recognized as quite prevalent; \
others such as candidiasis create problems by the very ubiquity of the
organism, which may grow more abundantly and cause systemic disease in
the presence of most antibiotics and steroids, creating serious and
extensive clinical problems. \
Proposed Course of Project;
To proceed as outlined under "Objectives" and "Methods."
Part B included: Yes /X/ No / /
52
Serial No. NIAID - 23(c)
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
1. Utz, J. P., and Andriole, V. T.: Analysis of amphotericin treat-
ment failures in systemic fungal disease. Ann. N. Y. Acad. Sci.
89: 277, 1960.
Awards
Emmons, C. W. , and Utz, J. P.: New chemotherapy cc systemic
mycosis: Experimental and clinical studies. An exhibit with
brochure. Honorable mention, Experimental Medicine & Therapeutic
Section of Scientific Exhibits at the Annual Meeting of the AMA,
Miami Beach, June 1960.
- 4 -
53.
Serial No. NIAID - 24(c)
1. LCI
2. Infectious Disease Service
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Sarcoidosis
This project has been temporarily inactivated due to
lack of principal investigator. Project to be reactivated at
a proper time in future.
5H
Serial No. NIAID - 25(c)
1. LCI
2. Infectious and Pediatric
Disease Services
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Pyelonephritis
This project has been temporarily inactivated due to
lack of investigators. Project to be reactivated at a proper
time in the future.
55
Serial No. NIAID - 26(c)
1. LCI
2. Infectious Disease Service
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Urinary Tract Viruses
Principal Investigator: Dr. John P. Utz
Other Investigators: Dr. Hugh Evans (from 7/1/60)
Clarence F. Szwed
Cooperating Units: None
Man Years (calendar year 1960):
Total: 2.8
Professional: 0.8
Other: 2.0
Project Description:
Objectives:
1. To study the urine of all patients with viral infection for
the presence of viruses and to study renal function concurrently.
2. To investigate in animals the effect on the kidneys of the
inoculation of virus recovered from human urine.
3. To attempt to produce pyelonephritis in animals by virus
inoculation alone or accompanied by secondary physiologic trauma or
bacterial infection.
Methods Employed:
Clinical studies: Patients with a variety of viral infections
will be studied intensively.
Laboratory studies: Tissue culture and animal inoculation tech-
niques will be used.
Patient Material and Major Findings:
1. Two additional viral isolates have been recovered from the
urine of patients under study here.
2. In continuing animal studies on the effects on the kidney of
viral infections, microscopic lesions, not found in controls, have
56
Serial No. NIAID - 26(c)
been observed in the proximal convoluted tubules of mice infected with
Coxsackie B-3 virus. The significance of these lesions is being studied
further with fluorescent antibody techniques.
3. A direct comparison has been made of tissue culture (cytopathic
effect), hemadsorption, and embryonated hens egg techniques in the isola-
lation of mumps virus from urine of patients. In 20 patients mumps virus
was recovered from urine by criteria of cytopathic effect in 14, by egg
inoculation in 1 and by both methods in 2. In general hemadsorption oc-
curred when cytopathic effect was seen in tissue culture but occasionally
one manifestation of infection was present without the other.
4. Because of the isolated reports of illness resembling mumps in
dogs and of viral isolations from dogs, studies were undertaken to define
the matter by inoculation of quantitative amounts of virus into puppies.
In 4 animals so inoculated no virus has been recovered subsequently and
no detectable disease has been produced. Presence or absence of antibody
response will be determined, in addition.
Significance to Microbiological Research:
The recovery of a number of viruses from the urine of patients
raises the question of the role they play in disease. The relationship
of this infection to subsequent pyelonephritis needs to be explored.
Proposed Course of Project:
As outlined in "Objectives" and "Methods."
Part B included: Yes /X/ No / /
- 2 - 57
Serial No. NIAID - 26(c)
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
1. Habel, K. and Utz, J. P.: Mumps, Ped. Clin. N. America.
7(4): 979-988, 1960.
- 3 -
58
Serial No. NIAID - 27(c)
1. LCI
2. Section on Biochemistry
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Biochemical Studies on Staphylococcal Cell Walls.
Principal Investigator: Dr. Eskin Huff
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960):
Total: 0.4
Professional: 0.4
Objectives :
The general objective of this project is an understanding of the
chemical mechanisms involved in the synthesis of staphylococcal cell
walls and the manner in which penicillin affects this synthesis. It
is hoped that a study of differences in the chemical composition and
synthesis of cell walls by penicillin sensitive and resistant staphlo-
cocci will throw some light on the mechanism of antibiotic resistance
in these organisms.
Specifically an attempt is being made to chemically characterize
the organic phosphate containing compounds occurring in the cell walls
of staphylococcal species.
Methods Employed:
A strain of Staphylococcus aureus (obtained from Miss Rose
Lieberman) has been grown in large batches on trypticase-soy-broth and
the cells broken by shaking with glass beads in a Nossal Shaker. The
cell walls have been harvested and purified by differential centri-
fugation using a Spinco preparative ultracentrifuge. Lithium bromide
solutions have been used to extract organic phosphate esters from the
cell wall material and these organic phosphate esters are being studied
at present.
59
Major Findings; Serial No. NIAID - 27(c)
No major findings can be reported at present. However, it
has been observed that staphylococcal cell walls have a high density
(specific gravity 1.5 to 1.6) as compared to most proteins (specific
gravity less than 1.4). It has also been noted that a considerable
amount of the organic phosphate containing compounds present in these
cell walls can be extracted by means of treatment with solutions of
lithium bromide.
Significance to Microbiological Research!
Badiley e_t a_l have evidence for the presence of polymers of
ribitol phosphate and glycerol phosphate in the cell walls of Bacillus
subtilis, Lactobacillus casei, and Staphylococcus aureus. These
workers have also demonstrated that In some strains of S^. aureus,
D-ala-nine and N-acetyl-glucosamine are attached to these polymers
which they call teichoic acids. However, other than the studies by
these workers little is known of the nature of the phosphate contain-
ing compounds present in staphylococci. In some of the bacteria
studied the phosphate containing compounds make up as much as 60% of
the weight of the cell wall material. In Staphylococcus aureus these
phosphate containing compounds may account for as much as 307. of the
weight of the cell walls. Although it has been demonstrated that
penicillin stops the synthesis of cell wall peptides in S. aureus with-
out an effect on the synthesis of intracellular protein (work by
Mandelstam and Rogers), it is not known what effect penicillin has on the
synthesis of the phosphate containing compounds present in these cell
walls .
A study of the biosynthesis and chemistry of the phosphate
compounds of Staphylococcal cell walls appears to be a reasonable ap-
proach to an understanding of the manner in which cell wall synthesis
is affected by penicillin.
Proposed Course of Project:
At present organic phosphate containing compounds have been
prepared from a penicillin sensitive strain of S_. aureus . A study of
the chemical nature of these compounds is planned for the immediate
future. In the future, it is also planned to study the molecular
weight, mode of attachment to cell wall, and pathway of biosynthesis
of these phosphate containing compounds. These questions are being
pursued in the penicillin sensitive strain of S_. aureus now under
study. Eventually the properties of the cell walls of these penicillin
sensitive organisms will be compared to the properties of the cell walls
of penicillin resistant organisms.
Part B included Yes f~l No /X/
60
Serial No. NIAID - 28(c)
1. LCI
2. Infectious Disease Service
PHS-NIH 3. Bethesda, Maryland
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Pathogenesis of Viral Infections
Principal Investigator: Dr. Julius A. Kasel
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960) :
Total: 1.3
Professional: 1.0
Other: 0.3
Project Description:
Objectives:
1. To investigate the prevalence of antigens separable from
infectious virus and their significance in relationship to
the pathogenesis of virus infection.
2. To study exfoliated cells in the urine, buccal mucosa and
blood cells from patients ill with viral infections for
the presence of viral antigen by fluorescent microscopy
concurrently with virus isolation procedures.
3. To determine sialic acid levels of serum and urine from
patients ill with respiratory viruses.
Methods Employed:
Clinical studies: Material from patients with viral infections
and non-viral illnesses will be utilized in the projects under study.
Laboratory studies: Tissue culture, chromatography,
fluorescent microscopy, serologic and biologic techniques will be
utilized.
Patient Material and Major Findings:
1. Recent adenovirus type 1 and 2 isolates and prototype
strains 1,2,4 and 15 were found to possess the property of removing
61
Serial No. NIAID - 28(c)
erythrocyte receptor sites possibly by enzymatic action, from human
red blood cells for three of five adenovirus hemagglutinins. The
antigen responsible for this property is separable from the infectious
virus particle and not related to the antigens causing cell detachment
and early cytopathogenicity . Studies indicated that the adenovirus
factor was a macromolecular substance, heat stable, resistant to
proteolytic and nuclease enzymes and was neutralized by homologous
but not heterologous serum.
Virus suspensions devoid of infectious virus, cell-detaching
and early cytopathic factors exhibited an "inhibiting property" for
certain adenovirus serotypes in tissue culture.
2. Viruses sharing antigenic properties of adenovirus types
9 and 15 were isolated during an investigation of an outbreak of
febrile illness among children.
In one patient convalescing from rheumatic pancarditis, the
viral infection was associated with signs and symptoms interpreted
as consistent with re-activation of the rheumatic process.
3. Significant neuraminidase activity was associated with
prototype adenovirus type 15 grown in HeLa tissue cultures. Screening
of type 15 suspensions were constantly negative for the presence of
myxovirus contaminants. The enzyme activity appeared to be
associated with the virus particle since suspensions adsorbed with
susceptible erythrocytes demonstrated a marked decrease.
Significance to Microbiological Research:
1. The presence of viral antigens other than infectious
viral particles in infectious tissue culture fluids raises the
question of their significance in disease and immunity. Their role
in viral reproduction and possible effect on the human host needs to be
defined.
2. Since prolonged excretion in the urine of mumps virus
raises the possibility of virus growth in the kidney; a study of
exfoliated cells in the urine for presence of virus needs to be
studied.
3. Determination of sialic acid levels in the blood and urine
of patients ill with viral infections may warrant this biochemical
assay as a clinical tool» The further definition of the enzyme-like
factor associated with adenoviruses needs to be further explored for
reaction products also, for consequent use as a diagnostic test.
62
Serial No. NIAID - 28(c)
Proposed Course of Project:
As outlined in "Objectives and Methods."
Part B included: Yes /J7 No AT
3
63
Serial No. NIAID - 28(c)
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B: Honors, Awards, and Publications.
Publications other than abstracts from this project:
1. Kasel, J. A., Rowe, W. P. and Nemes, J. C: Modification of
Erythrocyte Receptors by a Factor in Adenovirus Suspensions.
Virology, 10: 389-391, 1960.
2. Cramblett, H. G., Kasel, J. A., Langmack, M. and Wilken, F. D. :
Illnesses in Children Infected with an Adenovirus Antigenically
Related to Types 9 and 15. Pediatrics, 25: 822-828, 1960.
6«*
LABORATORY OF CELL BIOLOGY
Summary 1
50 - Biosynthesis in Mammalian Cell Cultures.. 3
51 - The Mechanism of Production of
Poliovirus in Cultured Mammalian Cells... 8
52 - Effects of Animal Viruses on the
Metabolism of Mammalian Cell Cultures
and the Mechanism of Viral Replication... 11
53 - Mechanism of Drug Resistance in Cultured
Mammalian Cells 14
54 - Metabolic Control Mechanisms in Cultured
Mammalian Cells 17
55 - Application of the Technique of Tissue
Culture to Selected Genetics Diseases... 19
SUMMARY
Research Projects from
The Laboratory of Cell Biology
(Serial Nos. NIAID 50-55)
The activities of the Laboratory of Cell Biology during the calendar
year 1960 have been along 3 major lines: (A) The continued exploration of
the metabolism of normal cultured cells, and an approach to the problem of
metabolic controls; (B) the mechanism of viral synthesis; and (C) the study
of cell cultures deriving from patients with hereditary metabolic disease.
A.
A number of significant observations have been made with respect to the
amino acid metabolism of cell cultures. There has been no further eluci-
dation of the pathway of serine synthesis since last year's report; but the
mechanism of cystine synthesis has been clarified, in that all the cell lines
so far studied have been shown to use the classical pathway involving the
demethylation of methionine to homocysteine, the condensation of the latter
with serine to form cystathionine, and the cleavage of the latter to
cysteine and homoserine. A dual pathway for proline synthesis has been
indicated, one involving glutamine as the source of the carbon skeleton,
and the other involving arginine by way of ornithine.
An intriguing recent observation has been the finding that a number of
factors which are rigorously required by the cells for survival and growth
can in fact be synthesized. Their nutritional requirement reflects the fact
that they are lost from the cellular pool to the medium at rates which exceed
the biosynthetic capacity of the cell; and with a sufficiently high cell
population density, when the loss to the medium per cell is sufficiently
reduced, the supposedly essential growth factors are in fact not required
for survival.
In these cell cultures, unlike bacteria, the biosynthesis of amino
acids is apparently not inhibited by the product of the reaction; and this
mechanism of growth control is apparently not operative. Studies are in
progress as to whether enzyme repression or feedback inhibition are
effective controls in the biosynthesis of pyrimidines. A quite different
control mechanism is perhaps indicated by the demonstration of a growth
inhibitor in the supernatant medium of heavy cultures. The chemical nature
of that inhibitor is under continuing study.
Studies on the mechanism of resistance to 2-deoxyglucose (2DG) have
shown the presence in the resistant variants of compounds which inhibit the
phosphorylation of 2DG to the metabolically active inhibitor, 2DG-phosphate.
The relationship of that inhibitor to the observed resistance is under
continuing study.
- 1 -
B.
A number of important new observations have been made with respect to
the mechanisms of viral synthesis. The puzzling wide disparity between the
number of physical particles in viral suspensions, and the number of plaque-
forming units, i.e. particles capable of initiating infection in susceptible
cells, has been partially resolved with the demonstration that after the
viral particle has been absorbed by the cell, it may undergo several
alternative fates. A large proportion are rapidly eluted into the medium,
essentially intact but no longer infectious, presumably reflecting a minor
alteration in the protein coat. Some particles remain unchanged within the
cell. Others are degraded intracellular ly, in that the nucleic acid is
exposed and becomes susceptible to intracellular ribonuclease. Only a
small fraction of the absorbed viral particles are stripped of their protein
and initiate infection.
In the case of poliovirus in the HeLa cell, although the viral protein
and RNA are synthesized concomitantly, a partial dissociation has been
achieved with appropriate inhibitors of protein synthesis, which completely
block the formation of mature virus, but not of infectious RNA. This is of
particular importance in relation to the supposedly obligatory relationship
between protein and RNA synthesis in growing cells. Of interest also is the
fact that matabolic inhibitors which effectively block the synthesis of
cellular DNA and of DNA viruses have no effect on the formation of polio-
virus. It would therefore appear that poliovirus RNA may be used directly
as a template for the formation of virus, without the necessity for
intervening DNA synthesis.
In contrast to the situation with poliovirus, in the case of vaccinia,
there was a marked lag between the formation of the viral nucleic acid
(DNA) and that of the mature virus.
C.
An exciting new development has been the successful cultivation from
patients with a hereditary metabolic disease (galactosemia) of cells which
in culture demonstrate the metabolic defect characteristic of the disease.
This suggests an entirely new experimental approach to problems of human
genetics.
2 -
NIAID 50
Laboratory of Cell Biology
Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Biosynthesis in Mammalian Cell Cultures
Principal Investigator: Dr. Harry Eagle
Other Investigators: Dr„ Jacob Maizel, Mr. Ralph Fleischman, Mr„ Aaron
Freeman, Miss Mina Levy and Mr. Vance I. Oyama
Cooperating Units: NIDR, Dr. Karl A. Piez
New York University, Dr. Bernard Horecker
Man Years (calendar year 1960):
Total: 7 1/2
Professional: 5 1/4
Other: 2 1/4
Project Description:
Objectives : This program involves a broad exploration of the
nutritional requirements, metabolic activities and biosynthetic pathways of
a variety of human and animal cells, under varying conditions of culture.
Methods Employed: The techniques used in the culture and chemical
fractionation of these cells, in the separation and identification of their
metabolic products, and in the use of isotopically-labeled precursors, have
been described in detail in previous publications.
Major Findings;
1. Amino acid biosynthesis and interconversion
a. Metabolic independence of serine and alanine. The so-called
"glucose family" of amino acids (alanine, serine and glycine), all of which
derive their carbon solely from glucose when cells are grown in a limiting
minimal medium, actually fall into two sharply defined groups, between which
there is little or no metabolic inter-conversion. Serine is the immediate
precursor of glycine; but there is no significant metabolic relationship
between alanine and these two amino acids. When cells are provided withc1^-
labeled serine or glycine, these two amino acids/are heavily labeled, but
' in the cell
Part B included.
- 1 -
NIAID 50
protein,
essentially none of the isotope appears in the alanine residues of the cell/
Further, when cells are grown on a medium which contains ribose and
pyruvate in lieu of glucose, the serine and glycine derive almost completely
from the ribose, while the alanine derives completely from the pyruvate.
This finding provides a direct approach to the as yet unknown mechanisms
involved in the biosynthesis of serine.
b. Cystine synthesis. It has now been clearly established that all
serially propagated human cell lines so far studied are indeed capable of
synthesizing cystine from methionine and glucose by the classical homo-
cysteine-cystathionine pathway. The capacity to synthesize cystine is
therefore not limited to liver cells, as had previously been presumed to be
the case.
c. Proline synthesis. In a minimal medium, the glutamine family
of amino acids (aspartic and glutamic acids, asparagine and proline) devolve
primarily from glutamine. In the case of proline there has however been a
consistent discrepancy, in that only half of the carbon skeleton derived
from glutamine, about 10 per cent additionally from glucose, while the rest
was unaccounted for. This has now been resolved with the finding that
slightly more than 1/3 of the proline carbon derives from arginine by way
of ornithine.
d. Negative feedback. There is now an increasing body of evidence
that the biosynthetic control mechanisms operative in bacteria, in which
the product of a given reaction serves to limit its synthesis, is not
operative in mammalian cells, at least with respect to the amino acids.
In the examples so far studied the provision of serine, cystine, and
homocystine has not served to curtail their continuing biosynthesis.
2. The anamolous requirement by human cells for metabolites which
they can synthesize. The interesting observation has been made that
cultured mammalian cells frequently require metabolites which they are
actually capable of synthesizing, and in amounts which should suffice for
growth. This has been found to be the case for cystine, homocystine,
serine, inositol, and in the case of certain cell lines, for pyruvate as
well. This paradoxical observation has now been resolved, in that the
function of the added metabolite is simply to prevent the loss of the
newly synthesized compound from the cellular pool to the medium at a rate
which exceeds the biosynthetic capacity of the cell. When the population
density is sufficiently increased, the exogenous metabolite is no longer .
required for cell survival and growth. When the task of "conditioning"
the medium is then shared by a sufficiently large number of cells, the
biosynthetic capacity of an individual cell is no longer exceeded.
3. Chemostat studies. The development of a chemostat for mammalian
cell cultures was described in last year's progress report. It has since
been learned that when the rate of growth is greatly reduced by appropriately
limiting the rate at which fresh medium is introduced Into the culture, the
factor which then limits the growth of the cells is not the depletion of
NIAID 50
medium, but instead, the elaboration of an active growth inhibitor. This
growth inhibitor is not dialyzable and is presumably associated with the
proteins of the medium. Attempts at its isolation and characterization are
now in progress.
4. Protein growth factor. Most human and animal cells in serial
culture have an absolute requirement for serum protein. That protein
however is not used to a significant degree for the synthesis of cell
protein, but instead has a dual role: it promotes the adhesion of cells
to glass surface, and provides essential and as yet undefined growth
factors. The active factor has proved to be, not the protein molecule as
such, but diffusible compounds of small molecular weight which are formed
from it on its proteolytic digestion. It is not yet clear whether those
growth factors are peptides, or compounds gf^tmntfel^aaolfce.ular- wffiighA^
initially bound to the protein and released from it as it is digested. In
either case, the dialysate of such a digested protein preparation supports
the indefinite growth of a wide variety of human cells in suspension
culture. Studies on the identification of the active compounds in such
dialysates are now in progress.
5. The utilization of inositol phosphates. Dr. S. J. Angyal of the
University of New South Wales, Australia, has prepared all the inositol
phosphate isomers. All have proved to be equally active in supporting the
growth of human cells. It is not yet clear whether the phosphates are
taken into the cell directly, or whether they must first be hydrolyzed
extracellularly.
Significance to the Program of the Institute: Studies on the metabolic
activities of human and animal cells are of obvious basic significance. For
NIAID research specifically, these studies are relevant to the problems of
antibody production, allergy, phagocytosis, inflammation, virus propagation,
and many other problems which are the concern of the microbiologist
interested in the study of human disease.
Proposed Course of Program; It is clear from the foregoing that none
of these are finished projects. The long-range program envisages a similar
broad-scale study on the metabolism of tissue explants. This will have the
multiple purpose of pin-pointing possible differences between the metabolism
of dispersed and structurally organized cells, exploring the conditions
necessary for the preservation of specialized functions ijn vitro,/ following
the chromosomal aberrations as the cells multiply under varying conditions
of growth.
- 3 -
NIAID 50
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Eagle, H„ Nutritional Requirements for Cell Growth and Poliovirus
Propagation. Perspectives in Virology 75-87. John Wiley & Sons,
Inc., N. Y. 1959.
Eagle, H. Metabolic Studies with Normal and Malignant Human Cells
in Culture. The Harvey Lectures, 1958-1959. Academic Press, Inc.,
No Y. 156-175, I960,
Eagle, H. The Sustained Growth of Human and Animal Cells in a
Protein-Free Environment. Proc. Natl. Acad. Sci. 46: 427-432,
April 1960.
Eagle, H. and Piez, K„ A. The Utilization of Proteins by Cultured
Human Cells. J. Biol. Chem. 235: 1095-1097, April 1960.
Eagle, H., Oyama, V. I. and Piez, K. A. The Reversible Binding of
Half-cystine Residues to Serum Protein, and Its Bearing on the
Cystine Requirement of Cultured Mammalian Cells. J. Biol. Chem.
235: 1719-1726, June 1960.
Eagle, H., Agranoff, B. W. and Snell, E. E. The Biosynthesis of
meso-Inositol by Cultured Mammalian Cells, and the Parabiotic Growth
of Inositol-dependent and Inositol-independent Strains. J. Biol.
Chem. 235: 1891-1893, July 1960.
Piez, K. A., Levintow, L., Oyama, V. I. and Eagle, H. Proteolysis
in Stored Serum and its Possible Significance in Cell Culture. Nature
188: 59-60, October 1960.
Darnell, J. E., Jr. and Eagle, H. The Biosynthesis of Poliovirus in
Cell Cultures. Advances in Virus Research. In press.
Cohen, E. P., Nylen, M. U. and Scott, D. B. Micros true tural Changes
Induced in Mammalian Cell Cultures by Omission and Replenishment of
a Single Essential Amino Acid. Exptl. Cell Research. In press.
Cohen, E. P. and Eagle, H. A Simplified Mammalian Cell Chemostat.
J. Exptl. Med. In press.
Levintow, L. and Eagle, H. The Biochemistry of Cultured Mammalian
Cells. The Annual Review of Biochemistry. In press.
Eagle, H., Piez, K. A. and Oyama, V. I. The Biosynthesis of Cystine
and Mammalian Cell Culture. In preparation. Q
- 1 -
NIAID 50
Honors and Awards relating to this project:
Invited Lectures
January 22-26, 1960: Perspectives in Virology. New York City.
February 9, 1960: NIH Lecture Series. Clinical Center.
March 24, 1960: Bristol Laboratories. Syracuse, New York.
March 25-26, 1960: Symposium on the Phenomena of Tumor Viruses.
N.Y.C. Sponsored by The Virology and
Rickettsiology Study Section, DRG-NIH.
June 20-22, 1960: Kingston, Rhode Island. University of Rhode Island.
Symposium: Newer Chemical and Biological Techniques
of the 1960 Medicinal Chemistry Section of the
American Chemical Society.
July 5, 1960: Seminar, Research Division of Ethicon. Somerville, N. J.
August 9, 1960: Long Island Biological Labs., Cold Spring Harbor, N. Y.
September 12, 1960: American Chemical Society, New York City.
September 28, 1960: Brandeis University seminar, Waltham, Massachusetts,
November 4, 1960: University of Delaware seminar, Wilmington, Delaware.
November 5, 1960: Eastern Psychiatric Research Assoc, symposium.
N. Y. C.
November 13-15, 1960: American Cancer Society Lecturer, Purdue
University, Lafayette, Indiana.
- 2 -
NIAID 51
Laboratory of Cell Biology
Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: The Mechanism of Production of Poliovirus in Cultured
Mammalian Cells
Principal Investigator: Dr. Leon Levintow
Other Investigators: Dr. James E. Darnell, Jr., Dr. W. K. Joklik,
Dr. Jacob V. Maizel, Mrs. Marilyn M. Thoren
and Mr. J. Leonard Hooper
Cooperating Units: None
Man Years (Calendar year 1960)
Total: 7 1/4
Professional: 2 1/4
Other: 5
Project Description:
Objectives: To define the events during the adsorption and
maturation of poliovirus in biochemical terms, and to investigate the
mode of replication of viral protein and RNA.
Methods Employed: The program is based primarily on the following
techniques: a) Infection of cells with poliovirus under controlled
conditions in a chemically defined medium; b) Purification of the polio-
virus so produced; c) Preparation of infectious RNA from infected cultures,
and from purified virus; d) Assays of the infectivity of whole virus and
infectious RNA by a plaque assay on HeLa cell monolayers.
Additional techniques include paper and column chromatography,
preparative and analytical ultrcentrifugation, and, in collaboration with
other groups, electron microscopy.
Major Findings: The events early in the infective cycle have been
studied with purified poliovirus labeled with P32. Virtually all virus
particles are capable of being adsorbed to HeLa cells; once adsorbed, a
particle has several alternative fates. It may: 1) be eluted into the
medium, essentially intact but no longer infectious; 2) be degraded intra-
cellular ly so that the P^2 label becomes acid-soluble; 3) be retained
essentially unchanged within the cell; or 4) be stripped of its protein
coat and initiate infection.
Part B included. -s
-i- 3
N1AID 51
These observations provide an explanation for the low ratio of
infectious: physical particles characteristic of poliovirus.
Following adsorption of virus to cells, there is a latent period
of 2-2 1/2 hours during which there is appreciable synthesis of viral
material. The synthesis of viral RNA and protein then begin simultaneously,
and mature virus begins to appear shortly thereafter. Unlike the situation
in most other viral systems, there is at no time a sizable pool of
unassembled viral macromolecules. The synthesis of viral protein and RNA
appears to be more closely coordinated than in any viral system heretofore
studied. This coupling of RNA and protein synthesis is not obligatory,
however; for in the presence of p-f luorophenylalanine, an inhibitor of
protein synthesis, the synthesis of viral RNA is initiated at the usual
time, but protein and mature virus are not formed. When the inhibition
of protein synthesis is reversed by the subsequent addition of phenylalanine,
the synthesis of viral protein begins, shortly followed by the appearance
of mature virus. These observations imply that the synthesis of viral RNA
is not dependent on concurrent protein synthesis.
Significance to the Program of the Institute: Virus replication
provides a useful model of a rapidly reproducing unit at the cellular and
subcellular level in which protein and nucleic acid synthesis can be
studied chemically and biologically. Such studies not only provide
insights into the mode of the replication of protein and nucleic acid
molecules, but have an obvious and important bearing on the evolution of
viral disease at both the cellular and whole animal level.
Proposed Course of the Projecti
1. The procedure for the purification of poliovirus is being
adapted to provide the larger quantities of pure virus necessary for the
physico-chemical characterization of the protein subunit.
2. Further investigation will be made of the fate of the infecting
viral particle, a problem complicated by the large number of ineffective
virus particles.
3. The reasons for the delay between adsorption and the initiation
of virus replication will be investigated.
4. Further studies will be carried out on the chemical, physical
and biological properties of the infectious RNA extracted from purified
virus.
5. Experiments on the relationships between the synthesis of viral
protein and RNA synthesis will be continued, using appropriate metabolic
antagonists.
- 2 -
NIAID 51
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Levintow, L. and Darnell, J. E., Jr. A Simplified Procedure for
Purification of Large Amounts of Poliovirus: Characterization and
Amino Acid Analysis of Type 1 Poliovirus. J. Biol. Chem. 235; 70,
1960.
Darnell, J. E., Jr. and Levintow, L. Poliovirus Protein: Source
of Amino Acids and Time Course of Synthesis. J„ Biol. Chem. 235:
74, 1960.
Darnell, J. E., Jr., Levintow, L., Thoren, M. M. and Hooper, J. L.
The Time Course of Synthesis of Poliovirus RNA. Virology. In press.
Joklik, W. K. and Darnell, J. E., Jr. The Adsorption and Early Fate
of Purified Poliovirus in HeLa Cells. Virology. In press.
Darnell, J. E., Jr. and Eagle, H. The Biosynthesis of Poliovirus in
Cell Cultures. Advances in Virus Research. In press.
- 1 -
10
NIAID 52
Laboratory of Cell Biology
Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Effects of Animal Viruses on the Metabolism of Mammalian
Cell Cultures and the Mechanism of Viral Replication
Principle Investigator: Dr. Norman P. Salzman
Other Investigators: Mr. Edwin D. Sebring, Dr. William Munyon and
Dr. Chiaki Nishimura
Cooperating Units : None
Man Years (calendar year 1960) :
Total: 3 3/4
Professional: 1
Other: 2 3/4
Project Description:
Objectives : To determine the alterations in cell metabolism
resulting from infection with poliovirus and vaccinia virus, and to study
the primary site of action of the virus; to determine the time course of
synthesis of the component parts of vaccinia virus (nucleic acid and
protein) as related to the formation of infectious virus, and to study the
effect of inhibitors on these synthetic processes.
Methods Employed: The techniques employed involve cell fractionation,
isolation of the purified component nucleic acid bases, the use of radioactive
precursors, the various techniques required for the viral infection of cells,
the plaque assay for measurement of infectious virus, and chemical methods of
virus purification.
Major Findings: The findings are in three separate areas.
1. Interrelation between cellular RNA, DNA and protein. Using a
specific inhibitor of DNA synthesis, 5-f luorodeoxyuridine, the presence of
two distinct classes of RNA and protein have been demonstrated in animal
cells. When DNA synthesis is inhibited, nuclear RNA and nuclear protein
synthesis are also inhibited; under these conditions however, cytoplasmic
protein and RNA synthesis continue at optimal rates. Because of the linkage
of nuclear RNA and protein synthesis to DNA synthesis, certain species of
protein will be synthesized only in growing cells, for under non-growing
conditions, there is no increase in DNA. This may be a fundamental mechanism
for the control of the levels of various enzymes in mammalian cells.
li
i"art: B included. - 1 -
NIAID 52
2 . Incorporation of nucleic acid analogues into pollovlrus. Our
previous studies established that the presence of 5-f luorouracil or its
deoxyriboside in the medium did not affect the de_ novo synthesis of polio-
virus. Since poliovirus synthesis occurred under conditions where no DNA
synthesis is possible, it seems likely that poliovirus RNA is used directly
as the template for the synthesis of poliovirus protein. Changes in the
virus RNA template might then be expected to produce changes in the virus
protein. Thus far an altered template has been produced by the incorporation
of 5-f luorouracil into virus RNA. The degree of incorporation of 5-fluoro-
uracil is concentration dependent. Even when 20 per cent of the uracil in
viral RNA has been replaced by 5-f luorouracil, the resultant virus is fully
infectious. Further studies on the properties of f luorouracil-containing
poliovirus are planned.
3» Vaccinia virus formation. The kinetics of formation of
vaccinia virus DNA and of infectious virus was previously determined,, The
synthesis of viral DNA was almost complete prior to the formation of any
new infectious virus. It has now been shown that the viral protein is
synthesized in the time between DNA formation and the appearance of
infectious virus.
Significance to the Program of the Institute: The effect of viruses
on animal cells, the manner in which viruses replicate, and the effect of
inhibitors on these processes is of obvious relevance to an understanding
of viral infection, and may conceivably suggest new areas of exploration
in relation to viral chemotherapy.
Proposed Course of Projects
1. The properties of a limited number of enzymes involved in
cellular metabolism will be examined to determine if they are associated
with the nucleus or the cytoplasm. The importance of these findings as a
basic mechanism in growth regulation will be evaluated.
2. By paper electrophoresis of the RNA nucleotides of purified
poliovirus, it will be determined if labeled fluorouracil is incorporated
as the nucleotide. The percent replacement of uracil by fluorouracil will
be measured by the amount of radioactive precursor incorporated and by
spectrophotometric determinations of the eluted nucleotides. The chromato-
graphic analysis of the amino acid residues of both fluorouracil-substituted
and control viral protein is planned. Other possible effects of fluoro-
uracil incorporation into poliovirus will be studied, such as altered UV
inactivation, heat inactivation, specific infectivity, and possible changes
in the one step growth curve.
3. Procedures for vaccinia virus purification from cell cultures
are being studied. The various protein components of the virus will be
purified and used to prepare specific precipitating antisera. These will be
used in studies on the kinetics of viral protein formation.
Part B included. 12
NIAID 52
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B, Honors, Awards, and Publications
Publications other than abstracts from this project:
Salzman, N. P. The Rate of Formation of Vaccinia Deoxyribose
Nucleic Acid and Vaccinia Virus. Virology, 1£: 150 (1960).
Salzman, N. P. and Sebring, E. D. The Source of Poliovirus
Ribonucleic Acid. Virology. In press.
Honors and Awards relating to this project: None.
- 1 -
13
NIAID 53
Laboratory of Cell Biology
Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Mechanism of Drug Resistance in Cultured Mammalian Cells
Principal Investigator: Dr. Stanley Barban
Other Investigators: Mr. Henry 0„ Schulze
Cooperating Units: None
Man Years (calendar year 1960)
Total: 2 3/4
Professional: 1
Other: 1 3/4
Project Description:
Objectives: To study the mechanism of resistance to a glucose
analogue, 2-deoxyglucose (2DG), by a resistant strain of human cells in
culture.
Methods Employed: By the use of cloning procedures, a strain of
HeLa cells has been isolated which can grow in the presence of high
concentrations of 2DGo Other techniques have included procedures for the
separation and identification of the carbohydrates and their metabolic
products, preparation of enzymatic extracts of drug-sensitive and drug-
resistant strains, and the use of isotopically labeled precursors. All
these procedures have been described in detail in previous publications
relating to this project.
Major Findings: A strain of HeLa cells has been developed which
can grow in the presence of a molar ratio of 2DG:glucose of 10:1, while the
parent strain is completely inhibited at equimolar concentrations of the
inhibitor :glucose. The resistant cells grow at approximately one-half the
rate of the parent strain, and the addition of pyruvate to the basal medium
is essential for its continued propagation.
2DG is rapidly metabolized by HeLa cells to its phosphorylated
intermediate, 2-deoxyglucose-6-phosphate. There is a significant difference
Part B included.
1*
NIAID 53
in the metabolism of both compounds by the sensitive and resistant strains,
in that the rate of 2DG metabolism in the resistant cells was markedly
reduced as much as 5-fold. In cell extracts there was a corresponding
marked depression of the enzyme which phosphorylates 2DG, as well as the
enzyme which phosphorylates glucose, fructose and mannose. The decreased
kinase activity for 2DG in cells resistant to this compound may well be
the primary basis of their resistance.
It has also been found that an extract of resistant cells inhibits
the enzymatic activity of a sensitive extract, suggesting that the resistant
extracts contain an active inhibitor of hexose phosphorylation.
Significance to the Program of the Institute; The mechanism of the
development of resistance to a glucose analogue in HeLa cells may serve as
a model for the development of drug resistance. This is relevant to the
general problem of chemotherapy, not only of infectious diseases, but of
other disease areas (e0g. cancer, heart disease, etc.)0
Proposed Course of Project;
1. Attempts will be made to Isolate and characterize the nature of
the metabolic inhibitor in resistant extracts.
2. The enzymatic differences between drug-resistant and -resistant
strains will be further explored.
3. The nutritional requirements of normal and resistant variants
will be explored, with particular reference to the essential role of
pyruvate in the growth of resistant cells.
4. Attempts will be made to transform 2DG-sensitive cells to drug
resistance, using the genetic material of the 2DG-resistant cells.
- 2 -
15
NIAID 53
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project: None
Honors and Awards relating to this project: Member, Society of
Biological Chemists.
16
- i -
NIAID 54
Laboratory of Cell Biology
Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Metabolic Control Mechanisms in Cultured Mammalian Cells
Principal Investigator: Dr. Jacob V. Maizel, Jr.
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960):
Total: 1
Professional: 1/2
Other: 1/2
Project Description:
Objective: To study the intracellular mechanisms for the control of
metabolic activities in mammalian cell cultures.
Methods Employed: Enzymatic assays by modifications of techniques
employed in other systems.
Major Findings:
1. Phosphatases.
In Escherichia coli alkaline phosphatase is a repressible enzyme,
the synthesis of which is affected by the level of orthophosphate in the
medium. In mammalian cells also the level of alkaline phosphatase was found
to be variable, in that cultures grown as monolayers attached to glass
regularly had at least ten times as much alkaline phosphatase activity as
suspension cultures. However, the high activity of monolayer culture was not
decreased at elevated phosphate concentrations, nor was the low level in
suspension cultures increased by growth at low phosphate concentrations.
When suspension cultures were planted as monolayers, the alkaline phosphatase
levels increased ten-fold in less than two days, independent of the
concentrations of phosphate or calcium, or population density. These findings
suggest a control mechanism unlike that of bacterial systems.
The phenomenon cannot be explained as the result of a generalized loss
of enzymes from cells in suspension, since the levels of acid phosphatase
17
NIAID 54
and aspartate transcarbamylase were the same in suspension and monolayer
cultures.
II. Control of pyrimidine synthesis.
Aspartate transcarbamylase is involved in the biosynthesis of
pyrimidines in a number of systems, and in Escherichia coli is involved in
the control of pyrimidine synthesis by exogenously supplied pyrimidine,,
The enzyme was found in extracts of HeLa cells and equally in both monolayer
and suspension cultures. Again unlike bateria, the enzyme level was
unaffected by the addition of pyrimidines to the medium.
Proposed Course of Program;
a. The question as to whether the product controls pyrimidine
biosynthesis will be approached by using appropriate radioisotope precursors
in the presence and absence of added preformed pyrimidines.
b. The enzymatic steps in pyrimidine synthesis remain to be
elucidated.
c. Factors of possible importance in the regulation of alkaline
phosphatase activity remain to be investigated.
Knowledge of the control of protein synthesis is of fundamental
significance to studies of antibody production and virus propagation.
- 2 -
ia
NIAID 55
Laboratory of Cell Biology
Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Application of the Technique of Tissue Culture to
Selected Genetics Diseases
Principal Investigator: Dr. Robert S. Krooth
Other Investigators: Miss Mary Jane Madden
Cooperating Units: NIAMD, Dr. Arnold W. Weinberg, and Dr.J„ H, Tjio
NINDB, Dr. Paul Altrocci
Man Years (calendar year 1960) :
Total: 3 3/4
Professional: 1
Other: 2 3/4
Project Description:
Studies to follow a number of genetic diseases in tissue culture
are being continued. Biopsies are performed on affected patients, and cell
lines are then developed from the excised tissue. Evidence bearing on the
persistence of the defect in cell lines from affected persons (compared with
cell lines from controls) is obtained. Ultimately, mutant cell lines from
patients with inborn metabolic errors should prove useful in the study of
genetic exchange among human cells as well as in other aspects. The system
permits one to study mutant human cells in a way which hitherto could be
applied only to mutants of bacteria and fungi. However, the number of
genetic diseases which are susceptible to this approach is at present quite
limited.
Galactosemia has been shown to persist as a defect in culture, even
after 6 months of propagation and an increase in cell number known to exceed
30 billion-fold. The defect can be shown by growth studies, the ability of
the cells to metabolize galactose-l-C^, and direct enzymatic assays. The
galactosemic cells show a pattern of galactose sensitivity similar to the'
transferaseless mutants of E. coli. Corresponding data have been obtained
for one heterozygote, and here also the defect persists.
Part B included.
- 1 -
19
NIAID 55
Wilson's disease and hypocatalaseraia are also under study by this
approach.
Our efforts to follow Gaucher's disease and certain other histiocytic
states in culture (referred to in the last report) have not yet succeeded.
Indefinite propagation of recognizably affected cells has not been achieved
although the cells will attach to glass, spread, and appear to increase in
number for a few weeks. Thereafter, though the cells can be made to survive
for months, no evidence of growth is found.
A search is being made in our laboratory, as in others, for new
chromosomal abnormalities in human disease. Cell lines have been developed
from biopsies on 5 patients with congenital malformations, in each case of
a kind not previously reported. In every instance thus far the chromosomes
have been normal in morphology and number.
- 2 -
20
NIAID 55
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Krooth, R. S., and Weinberg, A. (1960) Properties of Galactosemic
Cells in Culture. Biochem. Biophys. Research Comm., in press.
Honors and Awards relating to this project: None.
21
LABORATORY OF INFECTIOUS DISEASES
Summary 1
60 - Studies on Cellular and Cell-Free
Staphylococcal Penicillinase 44
61 - Antibiotic Activity of Sea Water 47
62 - The Electron Transport System in an
Autotrophic Hydrogenomad 50
62-A - Microbiological Fractionation of the
Hydrogen Isotopes 55
63 - Non-specific Immunity Associated with
Serum Siderophilin and the Iron
Metabolism of both Pathogen and Host 57
63-A - Chemical Aspects of the Specific Binding
of Iron to Serum Siderophilin and Egg
White Conalbumin 62
63-B - Biochemistry of the Acquisition of Iron
by Mammalian Tissues as Mediated by the
Iron-Binding Protein, Siderophilin 66
63-C - Development of an Economical 280mu
Light Source Suitable for Detecting
Proteins in Effulents from Chromatographic
Columns 69
64 - Detoxification of Potential Tuberculo-
static, Fungistatic, Parasitic idal. and
Virucidal Agent „ . 71
64-A - Potential Fungistatic and Parasiticidal
Agents 75
64-B - Isolation of Antibacterial and Antiviral
Substances from Shellfish 77
65 - Longitudinal Studies of Beta-hemo lytic
Streptococcal Isolations 80
65-A - Streptococcal M Protein, Virulence, and
Type-Specific Immunity 83
66 - Epidemiologic Studies of Illnesses and
Microbial Experience of Junior Village
Nursery Children 89
66-A - Epidemiologic Studies of Host Parasite
Disease Relationships 93
66-B - Epidemiologic Studies of New Vaccines and
Chemoprophy lactic Agents 95
66-C - The Etiology of Respiratory Disease on a
College Campus 97
66-D - The Role of Viruses in the Etiology of
Ear Disease 99
66-E - A Study of the Influence of Influenza
A and B Mineral Oil Adjuvant Vaccines
on Influenza Antibody Patterns, 8-9
Years after Vaccination 100
67 - Studies on Human Enteroviruses,
Adenoviruses, and Reoviruses 103
6 7-A - Studies on Viruses of the Enteric Tract
of Cattle 107
67-B - Studies on the Etiology of Eosinophilic
Meningitis in French Polynesia 110
68 - Studies of Tumor Viruses in Nature 114
68-A - Seroepidemiology of Virus Infections 123
69 - Study of Viruses as Causes of Respiratory
Illness in Infancy and Early Childhood... 126
69-A - Viral Pneumonia: Etiology, Therapy, and
Prevention 130
69-B - Study of the Laboratory Aspects of
Respiratory Virus Illness in a Welfare
Orphanage 134
70 - Laboratory Studies of Newly Recognized
Viruses Associated with Respiratory
Illness 138
71 - Studies of Tumor-Producing Viruses 142
71-A - Continuing Studies of Adenoviruses and
Salivary Gland Viruses as Examples of
Latent Viruses 147
71-B - Studies of Mouse Viruses with Special
Reference to their Importance as
Extraneous Viruses (background noise) in
Mouse Tumor Systems 150
72 - Laboratory Studies of Enteroviruses 154
72-A - Study of Viral Respiratory Disease in a
Military Population 157
72-B - Study of Respiratory Viruses in Human
Volunteers 161
73 - Application of Fluorescent Antibody
Staining Technique to the Study of
Respiratory Viruses 163
74 - Study of Bovine Respiratory Diseases 165
74-A - A Study of Squamous Cell Carcinoma (Can-
cer Eye) in Cattle 170
75 - Laboratory and Epidemiologic Studies
of Viruses as Possible Etiologic Agents
of Acute Leukemia in the Pediatric Age
Group 172
76-A - Ecologic Studies of Fungi Pathogenic for
Man 176
II
76-B - Iji vivo Tests of Antimycotic Drugs
and Antibiotics 180
76-C - Identification and Study of New and
Unusual Fungi from Mycoses 183
78-A - Biochemistry and Physiology of
Pathogenic Fungi. (In vitro Studies
of the Action of Antifungal Agents on
Pathogenic Fungi) 186
78-B - Studies on the Physiology of
Coccidioides immitis 188
79-A - Immunity Studies with Pathogenic Fungi. 191
79-B - Antigentic Studies on Pathogenic
Yeasts 193
79-C - Virulence and Pathogenic Studies with
Yeasts 196
III
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Yoar I960
Administrative Office - Edward F. Zadai
Introduction
The Administration of the Laboratory of Infectious Diseases
is responsible for furnishing all services, except the actual conduct
of scientific research, required for the numerous scientific investi-
gation programs of the 4 sections. In fulfilling a mission of
conducting integrated laboratory-scientific research, investigators
must be provided with individual personal attention, an earnest
interest in their work, and all the administrative support possible.
This responsibility requires continuous evaluation of activities in
all of the Laboratory of Infectiou the multiple
services needed both directly and indiroctly.
Personnel
During the p nfhs we have been fortunate in almost
ninating the problem of carrying vacant positions in our laboratoi^
We have added two professional members to our staff (Dr. Shug - from
the Veterans Administration and Dr. Huff - from rhe laboratory of
Clinical Investigation, NIAID). We have further employed two
additional Medical Biology Technicians and 2 more Laboratory Animal
Caretakers, leaving only two nonprofessional vacancies in tho entire
laboratory which we hope to fill before the completion of this
calendar year. Seven been promoted during the
year with three promotions still pending final action •
Physical Changes
This past year has seen the completion of planning and the
beginning of construction of 3 major renovations in dosigning newly
assigned laboratory space. Rooms 333 and 534 In Muilding 7 will be
converted from animal rooms into laboratories with work scheduled
for completion March 5, 1961. Also additional laboratory space
will be acquired and renovated in Room 313 for use by a collaborative
research study with rhe National Institute Ol Ni uiological Diseases
and Blindness. Lastly Rooms 204 and 206 in Building 5 have been
vacated by the Division of Biologies Standards and will be renovated
for use by our Medical and Physiological Bac feriology Section.
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
Scientific Exchange
An exchange of scientific personnel at the international level
was carried out during the year by sending one of our scientists to
Paris, France, to participate in research studies on auto-immunity in
viral infections and related virus research. In turn we received
Dr. Dietrick Falke, Senior Research Assistant in virus research
from the Hygiene Institute, Marburg, Germany. Dr. Falke is presently
spending a one year stay with us as a guest worker studying tumor-
inducing viruses.
Tra i n i nq
Courses in outside training under sponsorship of NiH were
completed by eight professional employees and it is certainly anti-
cipated that this will bring promising results in our scientific
projects.
Probl ems
The ever present scarcity of good personnel and adequate space
has certainly affected our progress. Many paths toward completion
of good scientific experiments have been narrowed or blocked due to
the lack of facilities. This Administrative Office has just completed
preliminary negotiations in purchasing two house trailers to be used
as a field laboratory. In this manner we will be able to continue
progress on a virus study that would have become useless without a
means of isolating the mice necessary for experiments.
Research Associates
In the role of a leading research facility, the Laboratory of
Infectious Diseases also conducts formal advanced training for young
scientists in certain specialties. The Research Associate program
provides exposure to techniques and methodology in microbiologic
research. In I960 there were several applicants from leading
universities and hospitals in this country for these type positions
which we were forced to reject. It would be of great value if we
could expand our facilities to accept more of these applications
because recognition of the quality of experience in our scientific
programs is a significant factor in the recruitment and retention
of staff members.
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
Cone I us ion
The Laboratory of Infectious Diseases has earned the reputation
of a progressive organization in the field of infectious disc.)
research. There have been many problems in retaining extremely
competent professional, technical, and administrative personnel. We
are fortunate in having a devoted highly productive and intelligent
senior staff made up of internationally known young workers with
the large part of their careers still before them. Results of signi-
ficant value in the conquest of disease have been achiovod, and it
is the desire of the Administrative Office to meet the challenge of
the future with earnest and sincere efforts. The Administrative
Office is fully aware of the job whicl. of us if we are
to support rather than contain our scientists. We feel very strongly
that the time is growing near will have to expand our
facilities if we wish to continue maximum progress.
Below is a highlight summarization of some of the honors
awarded LID scientists during the past year:
Dr. Chester W. Emmons
Elected President, Mycological Society of America
Elected Counc i lor-at-Large, Society of American Bacteriologists
Chairman, Second Conference on Medical Mycology, N.Y. Acad. Sc i .
Appointed Board Member of Amer. Acad, of Micro. (3 year term)
Chairman, Standards and Examination Committee, Amer. Acad. Micro.
Convenor of Mycological Symposium, III National Congress of
Microbiology, Mexico
Honorary mention for exhibit on Chemotherapy of Systemic
Mycoses, AMA meeting in Florida
Appointed to WHO Expert Advisory Panel on Parasitic Diseases
(5 year term)
Member of Committee on National Index of Fungus Cultures,
Quartermaster Research and Development, National Academy-
Research Council (3 year term)
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, N I AID
Calendar Year I960
Dr. Robert J. Huebner
Elected to National Academy of Sciences
Appointed to Enterovirus and Adenovirus Committees of NIAID, NCI
Invited to give several honorary lectures, including the
I960 Harvey Lecture
Dr. Wal I ace P. Rowe
Awarded Eli Lilly Award at the Annual Meeting of the American
Society of Bacteriologists
Appointed to Research Advisory Committee on Etiology of
Cancer of the American Cancer Society
Dr. Robert M. Chanock
Elected to Society for Clinical Investigation
Full membership to the ARD Commission of the AFEB
Dr. Leon Rosen
Appointed to the Enterovirus Committee, NCI
Received a letter from the Governor General of French Polynesia
to the Surgeon General of the United States Public Health
Service citing him for his research on viruses and
eosinophilic meningitis in French Polynesia
Dr. Arthur K. Saz
Chairman, President's Fellowship Committee, Society of American
Bacteriologists
Dr. Roy Repaske
Elected to Membership, American Society of Biological Chemists
Dr. Arthur L. Schade
Invited to following European universities to deliver lectures:
Vienna, Marburg and Geissen, Saar, Freiburg, Berne,
Zurich, Tubingen, Frankfurt, Berlin, Lund
Dr. Robert C. Woodworth
Awarded NIH Fellowship )NHI) to spend a year at Lund University,
Ma I mo, Sweden
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAIO
Calendar Year I960
Virus and Epidemiology Sections
\ .0 Introduction
In I960 the Virus and Rickettsial and Epidemiology Sections
continued integrated and comprehensive efforts to define the importance
of virus infections in disease. Field investigations of human and
animal virus infections were made possible through collaboration with
a number of other organizations, including the Bureau of Medicine,
USN; the District of Columbia Children's Hospital Research Foundation;
the District of Columbia Welfare Department; the New York City Health
Department; the National Cancer Institute; the National Institute
of Allergy and Infectious Diseases; the Laboratory of Clinical
Investigations, NIAIO; and in Paris, France the Laboratoire des
Virus, Hopital Saint-Vincent-de-Paul; and Le Centre Claude-Bernard
de I 'Hopital Saint Louis.
Natural events and opportunities afforded by our collaborators
shaped the course of most of our field studies. Technical breakthroughs
in the laboratory made it possible for us to take fuller advantage
of these opportunities to study natural disease and thus acquire
not only new information about specific virus infections, but also
to move nearer our ultimate goal, namely, a clear view of the numerous
viral causes of human diseases which is also sufficiently comprehensive
to make concerted efforts to control them appear feasible and
worthwhi le.
In the long run, a research organization stands or falls on
its puolished work, so that if we have anything deserving attention
it is in the content of our scientific reports. Nearly 50 manuscripts
were published or accepted for publication and 10 more papers are
either in the Editorial Board or about to be submitted. It is not
surprising therefore that even a summary makes for a rather long
document. However, it may help if I "highlight" certain specific
findings which while not necessarily more important have a more
immediate bearing on disease, and merely mention others.
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases
Calendar Year I960
2.0 Respiratory Virus Disease Studies
2. J New Causes of Virus Pneumonia
Pneumonia and other lower respiratory tract infections
continue to represent major causes of death and a large segment,
presumed to be viral in origin, is still uncontrolled. Until
recently it was wholly undefined. During 1958 and 1959 our studies
at Children's Hospital and Junior Village helped define the relative
importance of adenoviruses, para-inf luenza viruses, and influenza
viruses in causing lower respiratory illnesses of childhood. The
data suggested that as much as 40 per cent of croup bronchiol itis
and pneumonia were explained by these viruses. In I960, using more
sensitive methods, we were able to explain a much larger percentage
of such illnesses, chiefly because we were now able to assess the
very significant contributions of respiratory syncytial virus (RS)
to the respiratory disease problem. Early in the year large outbreaks
of RS virus were intensively studied both at Children's Hospital and
Junior Village. Over 80 strains of RS virus were isolated from
children with pneumonia and 60 per cent with bronchiolitis yielded
RS virus, whereas virus was recovered from less than one per cent
of comparable control patients without respiratory i Iness.
Retrospective analysis of serologic surveys of respiratory
illnesses in Children's Hospital since 1957 suggested that perhaps
20 per cent of all lower respiratory illnesses observed during the
last three years was due to RS virus. Thus, considering the contri-
butions of adenoviruses, para-inf I uenza viruses, influenza viruses,
and "PAP" virus it now appears that 50 to 60 per cent of the more
severe respiratory illnesses of young children can now be explained
and,hopef ul ly, controlled. Except for influenza virus (wLich
contributed probably less than 5 per cent of the total), the LID
respiratory virus unit personnel played key roles in the discovery
of the first representatives of each of the other virus groups -
adenovirus, para- influenza, and RS . Delineation of still undefined
viral causes of the respiratory disease syndrome represents the
major challenge to respiratory disease investigators for 1961.
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
A good start toward meeting this challenge has already
been made. Preliminary analysis of nearly 1800 enterovirus infections
observed in our childhood study populations showed that certain of
them such as Coxsackie B 3, poliovirus 2, and ECHO 3 virus were
responsible for febrile respiratory illnesses. From recent results
obtained by other workers, and previously by our own group, it is
likely that newly recognized but extremely common enteroviruses
closely related to the Coxsackie A virus, will be found to contribute
a significant proportion of still unexplained respiratory illnesses.
2 12 Respiratory Virus Vaccines
The fact that a very significant proportion of the more
severe respiratory illnesses occuring in childhood can now be
explained makes the development of effective preventive vaccines
against the known agents a worthwhile undertaking. The viruses
involved (see above) are antigenic and, although reinfection can
occur, evidence indicates that specific antibodies provide
significant protection against the more severe manifestations of
these viruses during subsequent infections. Unfortunately, progress
towards the development of the complex vaccines containing many
different viruses can be expected to be very slow unless more interest
and attention can be focused on the public health importance of such
efforts.
Unlike poliomyelitis and other dread and dramatic illnesses,
common respiratory disease, although responsible for much more
illness and possibly more deaths as well, do not have national
foundations devoted to their control or eradication. The relative
lack of large scale goal -oriented research activities contrasts
vividly with the dimensions and technical needs of contemporary
respiratory virus disease research; both exceeding by several
magnitudes that of poliomyelitis.
During I960 several experimental but commercial ly prepared
killed vaccines containing various combinations of adenoviruses
(6 types), para- influenza viruses (3 types), and Coxsackie B viruses
(5 types) were tested in Junior Village. The evidence suggests
that while modestly antigenic, the vaccines had insufficient
potency to be regarded as satisfactory for larger scale studies.
Additional studies of vaccines are planned for 1961, but only at
a pilot study level, since the space and personnel available to our
Virus and Epidemiological groups is scarcely sufficient to continue
our laboratory and field studies of the viruses as causes of
respiratory disease.
8
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAIO
Calendar Year I960
2.3 Pneumonia in Adults
The etiologic role of PAP (Eaton's virus) in primary atypical
pneumonia suggested earl ier by Eaton and Liu and explored by us in
1959, was finally fully established in I960. In cooperation with
the Bureau of Medicine, USN, the continuing "epidemic" of virus
pneumonia in Marine recruits at Parris Island was studied in several
ways. Serological studies showed that 51 per cent of 530 pneumonia
cases had antibody rises to PAP virus; only 6 per cent revealed
contemporary infection with adenoviruses. Serologic studies of
infection showed PAP virus to be much more common than disease;
approximately 30 recruits were infected for each case of pneumonia,'
information vital ly important to ful Ier comprehension of the natural
history of this important virus.
2.4 Treatment of Primary Atypical Pneumonia (PAP)
In 1959 treatment of Parris Island pneumonia cases with broad
spectrum antibiotics (tetracyclines) appeared to reduce the severity
and the duration of the Eaton pneumonias. In I960 the efficacy of
a new tetracycline drug, demethy Ichlortetracycl ine, was tested in
a we I I -control led double blind study including 290 pneumonia patients.
The drug greatly reduced the severity and duration of . pneumonitis
and fever in those shown to have serologic responses to PAP virus.
These findings, based on accurate laboratory diagnosis, fully confirm
earlier but controversial reports of the efficacy of tetracyclines
in atypical pneumonias. It also adds further support to the importance
of the Eaton virus as a cause of virus pneumonia.
An additional link in the chain of evidence establishing the
PAP virus as an important cause of pneumonia was achieved recently
in collaborative studies with the Laboratory of Clinical Investiga-
tions, N I A ID. Volunteers inoculated intranasal ly with PAP virus
grown in tissue cultures reacted with a wide gamut of respiratory
signs and symptoms, including pneumonitis characteristic of PAP.
8
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
2.5 Common Colds and Other Mild Respiratory Illnesses in Adults
Recent studies have served to clarify and enlarge existing
concepts of the etiology of common mild respiratory illnesses in
adults. It is now quite clear that instead of a few specific
closely related viruses, numerous viruses belonging to different
groups each contribute in part to the syndrome called the "common
cold." Thus the newer viruses (adenoviruses, para-i nf I uenza
viruses, respiratory syncytial virus and others), together with
older agents (influenza viruses and certain bacteria), each
contribute on I y a small proportion of the milder respiratory ailments
of adults. They contribute a larger segment of more serious
diseases, particularly in children. Very recent reports of common
cold viruses from England, together with the prior reports of
agents with somewhat similar properties in this country, served
to focus our attention on these viruses in I960. Together with
investigators elsewhere, it was found that most, if not all, of
these agents - the British HGP and FEB, the American 2060, JH,
Coe and PETT viruses which grow selectively and rather "fussily"
in human epithelial cell lines, really represent "fastidious"
enterovirus strains which have (as do almost all Coxsackie A's
and some ECHO viruses) special growth requirements. These viruses,
as do a number of still unclassified agents found in Junior Village
during the past several years, have properties very similar to
the Coxsackie A viruses; indeed, several have been shown, on the
basis of serologic markers and/or by suckling mouse pathogenicity,
to be indistinguishable from Coxsackie A viruses.
Enteroviruses as Specific Causes of Mild Respiratory Disease
In previous years we have reported enteroviruses in relation
to respiratory illnesses in Junior Village; JVI virus (now ECHO 20)
was one such case in point.
10
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
ECHO 28 (2060-JH Viruses )
Several years ago investigators at Great Lakes Naval
Training Station and Johns Hopkins reported viruses called
respectively "2060" and "JH" in association with mild respiratory
illness. Subsequently these agents were classified as ECHO 28
in the enterovirus family. During I960 our Virus Section developed
a complement fixation test for this virus as well as for most of
the 58 known enteroviruses. Serologic surveys of respiratory
illnesses have revealed high prevalence of antibodies, but thus
far rises in titer in relation to illness have been rare.
ECHO 3
During I960 an outbreak of ECHO 3 virus infections in
Junior Village was analyzed in relation to contemporary illness.
The study showed a temporal relation to mild respiratory illness
attended by brief febrile responses. A report is in preparation.
Coe Virus and Other New Viruses in Military Personnel
During I960, in cooperation with the Bureau of Medicine and
Surgery, USN, studies of mild respiratory illness in military
recruits were conducted at Camp Lejeune, North Carolina. Initial
findings suggested that new para-inf I uenza viruses and respiratory
syncytial virus, in addition to adenoviruses, were contributing
in part to the syndrome. Although the clinical importance of
these latter agents must still be determined, the fact that they
are encountered in adults is of considerable interest.
However, more recently (since October I960) we have observed
large outbreaks of Coe virus - over 70 strains of virus were
isolated from as many cases of mild respiratory illnesses.
Although identical serologically with the prototype Coe virus,
these new strains exhibit a hemagglutinin not previously reported.
These strains furthermore produce effects in suckling mice which
are indistinguishable from those produced by Coxsackie A viruses.
Thus one more "new" virus, at first regarded as wholly unique, is
now found with further study to belong to a well established virus
family - namely the Coxsackie viruses.
10
II
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, N I AID
Calendar Year I960
At the same time and in the same recruit population
numerous isolations of the so-called "fussy" enteroviruses also
have been recovered from persons with respiratory illness, using
the special techniques described last year by British investigators,
The role of these newer enteroviruses in causing illness are now
under study.
Future Studies of Colds due to Enteroviruses
We are making arrangements with the Bureau of Medicine
and Surgery, USN, not only to continue but to extend our studies
of the enteroviruses as causes of respiratory illnesses in the
large military population at Camp Lejeune. We hope to include
observations on the numerous dependents and on permanent cadre
personnel as well. Thus our studies of enterovirus-caused
respiratory illnesses will cover experiences in children as
well as adults, and will include all seasons of the year.
11
12
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, Nl AID
Calendar Year I960
3.0 Virus Diagnostic Reagents and Their Publ ic Health Importance
The public health importance of the more than 100 newly
recognized viruses which commonly infect man is now beginning to be
appreciated. However, only a handful of public health laboratories
are currently making systematic attempts to diagnose viral infections
and even these are limiting their surveillance to a pitifully small
group of viruses, including usually no more than 3 pol iovi ruses,
2 influenza viruses, and several viral zoonoses. This involuntary
indifference to man's largest morbidity problem is largely due to
the fact that they lack the necessary viral reagents and of course
personnel trained to use them. Realizing that control of respiratory
diseases must remain an unattainable objective unless and until
acute respiratory diseases (see 1957-58 National Health Survey) can
eventually be defined as a public health problem, we have made the
development of simple and reliable virus diagnostic procedures a
major part of our research program.
12
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAIO
Calendar Year I960
5.1 New Serological Test Procedures
The laboratory section of the Epidemiology Section therefore
concentrated on the development, application and evaluation of in
vitro test procedures for the identification of new viruses as well
as for detecting virus infection as expressed in antibody responses.
Thus, using conventional complement fixation (CF) and newly developed
hemagglutination inhibition (HI) procedures it has been possible for
our group to type thousands of virus isolates belonging to the
adenovirus, myxovirus, enterovirus, and reovirus groups. As was
true during the past several years, LID in I960 again described and
characterized more new representatives of these viruses than all
other virus laboratories in the world combined. This was made
possible during I960 because each of our various virus research units
contributed new diagnostic techniques. Dr. Rosen's group developed
additional specific HI procedures for identifying adenoviruses and
adenovirus infections; and for reovi ruses and enteroviruses as well.
Similarly, Chanock, Johnson and Cook developed tissue culture procedures
for isolating Eaton's PAP virus, while Rowe and Hartley not only
discovered several "new" mouse viruses in tumor virus study systems,
but developed serological procedures for recognizing their presence.
3.2 Serologic Reagents
But the availability of simplified procedures are of very
little use unless the necessary reagents are also available. Although
many virus research laboratories could do the tests, few laboratories
are able to produce the necessary reagents. The magnitude and cost
of producing and certifying them promises to continue to exceed any
possible resources available. This fact has had a very depressing
effect on research efforts aimed at the study of viruses as causes of
disease, and serves as yet another deterrent to early delineation
of the common virus diseases as public health problems. Consequently,
with the help of NINDB and Microbiological Associates, LID in 1959
and I960 accepted responsibility to develop and evaluate more than a
hundred commercially produced virus antigens. LID, of course, has
been active in the certification of virus prototypes and furnishes
many to the Virus Registry of the American Type Culture Collection.
We are also collaborating with the Enterovirus and Adenovirus national
committees in setting up standards for large scale production of
certified antiserums for serotyping and classification of viruses;
perhaps the highest priority need of al I virus laboratories concerned
with human infection and disease.
13
14
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
3.3 LID as the Unofficial World Reference Laboratory for Viruses
Wholly through the operation of circumstances, the Virus
Section of LID has become virtually the chief (in many instances only)
reference laboratory for many of the newer viruses, including adeno-
viruses (about 30 human and several animal serotypes), myxoviruses
{o new para-inf I uenzas occurring in 3 species), reovi ruses (3 serotypes
in 4 species), many of the newer and some older enteroviruses (5 - 10),
salivary gland viruses (from 4 species), and new mouse viruses (6),
the latter frequently found in tumor virus study systems.
Until virus reagents desperately needed for many extremely
common viruses are made available either commercially, through
government agencies, or both, LID as the sole custodian of many of
these agents cannot avoid responsibility for assisting other excellent
virus laboratories to identify their viruses, and on a pro-tern basis
at least for keeping order in the general virus field. Unfortunately
we have no specific commitment to provide such services and even
worse, no specific budget to cover them, so that our involuntary,
constantly growing and unavoidable service functions must be done
at the expense of our research missions.
However, it must be admitted that the simpler virus diagnostic
techniques and the availability of a complete supply of viral reagents
in our laboratory (developed out of necessity) facilitate not only
our own epidemiologic studies of naturally occurring virus infection
but also enable us to evaluate the significance of the data furnished
by other laboratories who come to us for tec hn Tea I assistance. As
long as LID continues its policy of working on the frontiers of virus
disease problems we have no way to avoid the responsibilities that
devolve on us as a result, and for that matter no real desire to do so.
However the dimensions of this frontier have grown geometrically in
recent years, and unless LID and other virus laboratories can grow
with it we cannot hope to continue to be effective and to play such
a decisive role in the future. There may be some justification to
the desire to see this happen, but unless other laboratories step
into the breach (it will take many years to build other groups with
the overall competence of the Virus units in LID), the effect of such
a policy will be to slow up progress not only in LID but in many other
virus laboratories concerned as we are with viruses as agents of
human disease. This may seem a presumptuous statement, but the facts
of the matter are clear and objective enough to justify it.
15
11
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
4.0 Sero-epidemioloq ic Studies of Virus Disease
Comprehensive serological studies of over 75 common viruses*
became feasible in I960 as the result of successful commercial
production of experimental lots of antigens. This was achieved
collaboratively with the NINDB Collaborative Cerebral Palsy Study
Program and Microbiological Associates, Inc. (MBA), with the Virus
Section of LID serving as project director for the production
items actually produced by M8A.
During I960 all the antigens were shown to have homologous
reactivity and many of them were evaluated in conventional complement
fixation (CF) and hemagglutination inhibition (HI) tests, and some
were employed in routine diagnostic tests in our epidemiological
studies of respiratory disease.
However before definitive sero-epidemio logic surveys of
virus infections in relation to birth defects can be monitored,
al I the antigens and test procedures must be put through "shake
down" evaluations with standard human and animal serums representing
experiences with each of the viruses, thus achieving quantitative
information on sensitivity and specificity. Fortunately many of
these standard sera are available; however, others must still be
acquired in 1 961 .
Costs and Micro-techniques.
The development of meny antigens required concentration and
other special procedures, thus increasing the estimated cost of
even routine production to as much as $10.00 per ml - a prohibitive
cost when considered in relation to the large amount of testing
which must be done in our sero-epidemiologic surveys. Consequently
we have turned the attention and the efforts of our serology teams
to the development and evaluation of micro-techniques.
''Influenza, para-influenza, poliovirus, ECHO, Coxsackie A, Coxsackie B,
adenovirus, reovirus, and other new virus groups.
I5
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
The studies have been postponed because of the delay in
obtaining the necessary precision instruments. However negotiations
with an importer to obtain them from Hungary promises to solve
the non-scientific obstacles to current progress. Preliminary
studies suggest that the Takatsy micro-technique can be applied
without modification in our Hi tests and also suggest that slight
modifications of the equipment will permit micro-CF tests as well.
Comment
The commercial production of numerous satisfactory complement-
fixing and hemaggl ut inating antigens now makes possible broad and
comprehensive sero-epidemiologic studies of virus infections which
previously could not even be contemplated. However practical
considerations, particularly their high production cost, and the
lack of standard serums for evaluation (in which there is also
a cost factor) will undoubtedly slow up transition from the stage
of possibility to that of general feasibility, - perhaps until
current concepts concerning acceptable costs for such research
activities are revised upwards.
16
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
5.0 Studies of Cancer Viruses
Our studies of cancer virus problems can be subdivided into
several categories: (a) Laboratory studies of the properties of
cancer viruses and development of laboratory tools for detecting
and working with them; (b) field studies of the behavior in nature
of those tumor viruses for which suitable detection tests are
available; (c) studies of extraneous viruses ("background noise")
now preventing high caliber virologic practice in the study of
animal tumor viruses and obscuring interpretation of nearly alt
current observations on them; and (d) the study of general virus
experiences in relation to human cancer - the"background noise"
in the human cancer problem - which we feel must be done eventually
if the role of viruses in human cancer is to be defined.
Our approach to these various interdependent studies is
based on the following beliefs: I) That the conventional methods
of standard virology must be applied to cancer virus research if
significant progress is to be made; 2) the study of cancer viruses
obviously cannot be separated from general virology; and 3) that
the "biologic point of view" rather than attitudes fostered by
preoccupation with categorical disease, represents the best approach
to a real understanding of the natural history of cancer viruses
just as it does to other viruses (see Introduction).
17
18
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
5.1 Laboratory Studies - Mouse Polyoma Cancer Virus*
New in v i tro survey tools developed during 1959 (CF, HI,
and MAP) were evaluated and applied in I960 in studies of polyoma
virus growth and excretion, its experimental epidemiology, and
its natural history. This interesting and versatile cancer virus
causes tumors not only in all strains of Mus muscul us, but also
in hamsters, rats, rabbits, and guinea pigs (Stewart and Eddy).
Of equal interest is the fact that it can be studied and surveyed
with the same facility as ordinary viruses, such as influenza
and pol ioviruses. Virus isolation and serologic procedures,
combined with ep izootiologic studies have produced the following
interesting observations:
Polyoma virus was found to be widely disseminated in mouse
colonies nearly everywhere. Infection was found to be more
commonly present than absent in laboratory strains raised in
experimental or commercial laboratories and in wild strains found
in city tenements. However the basic ecology or natural cycle
appears to exist in rural areas - on farms and in feed mills in
smal I towns.
In the laboratory the virus is maintained and disseminated
by experimentally and spontaneously infected carrier mice which
excrete virus in saliva, feces and urine, the latter appearing
to be the most important vehicle of spread. Infected infant mice
excrete so much virus (up to a million ID50's per ml of urine)
into laboratory environments that much of the data on polyoma
acquired in such environs is subject to question, particularly
if adequate controls (uninoculated mice) are not included with
every animal experiment. Polyoma virus was also shown to represent
one of the more common extraneous agents, complicating interpreta-
tion of laboratory experimentation with other mouse tumor viruses.
* Collaborators at the National Cancer Institute - Drs. L. W. Law,
C. J. Dawe, W. 6. Banfield and H. Kahler.
18
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
5.2 Natural Behavior of Polyoma
Studies of the behavior of polyoma in Mus muscul us were
also carried out in three different field environments, in densely
populated tenement areas of a large city, on farms producing
grain and livestock, and in grain mills located in small rural
towns. Each presents a different ecology but in each one focal
environmental contamination appears to be quite important to the
survival and persistence of polyoma infection in the mouse
populations observed.
Harlem Studies
A full year's surveillance of Mus muscul us infestation and
polyoma infection of crowded tenements in Harlem revealed that
virus infections persisted without exception in numerous separate
foci. Three epidemiologic factors seem most important, namely,
large mouse populations capable of furnishing adequate supplies
of young susceptible mice, the extensive contamination of the
tenement environment (virus was demonstrated in sweepings from
areas showing signs of mouse activity), and finally the over-
crowding which insures the continuous and extensive use of communal
nesting areas (also demonstrated to be contaminated by virus).
Apartment houses having smaller and less dense mouse infestation
were generally free of infection and remained so during the study.
19
20
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, N I AID
Calendar Year I960
Rural Studies
Systematic studies of polyoma in rural environments were
undertaken during the last quarter of I960. However, it appears
from preliminary data that here we may have found the basic natural
eye le of mouse polyoma. Mus musculus infestation and polyoma
infection of Mus was found to be most intense in feed granaries
on the farm and in cereal grain storage elevators in mills. As
many as 30 per cent of several hundred mice trapped in these
environs showed persistent evidence of polyoma infection, many of
them apparently excreting virus in their urine. The virus has
been found on cereal grains in the vicinity of mouse nesting
areas, which appear to be very numerous in the granaries so far
examined. The actual extent of cereal grain contamination by
mouse excreta containing polyoma and no doubt other microbes must
still be evaluated; however, present evidence suggests that it
probably is very extensive, if not appalling.
The I iterature on the ecology of Mus muscul us centers on
the infestations of rural grain storage areas - particularly in
grain "bins" and the traditional grain "ricks." Natural ecologic
arrangements such as are reported here are no doubt much older
and probably much more of a factor in the maintenance of natural
mouse agents than the conditions that exist in infested urban
areas or, for that matter, in production and experimental areas
housing laboratory mice.
Since natural infection of wild mice is not limited to
polyoma virus, but includes a number of other viruses known or
suspected to infect man and domestic animals, the extension of
these preliminary findings will likely prove very interesting.
Should our observations on Maryland farms prove not to be
unique - and there is no reason to suspect that they would be -
then extensive contamination of cereal grains with hardy viruses
such as polyoma provides a logical vehicle of infection for labora-
tory mice with polyoma and no doubt with some of the other extraneous
agents found in most production and experimental mouse colonies.
It is interesting to consider the fact that man and his domestic
animals have long been exposed to foodstuffs heavily contaminated
by mice and other rodents and that the viruses and the cancers
known to occur in these species are not remarkably different.
20
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
Comment - Cancer a Possible Zoonosis?
The fact that polyoma virus, avian sarcoma and bovine
papilloma, although well established tumor viruses, cross over
species lines - polyoma infecting rats, hamsters, guinea pigs,
and rabbits; Rous sarcoma, turkeys, ducks; and bovine
papilloma, horses - seriously challenges we think prevailing
concepts about the narrow species predilection supposedly exhibited
by cancer viruses. It is clearly necessary now to examine the
hypothesis that some cancers in man and domestic animals could
be due to viruses which like the zoonoses have their basic natural
cycle in lower commensal animals. Such studies can now be done
on some of the known animal tumor viruses, such as polyoma.
Our field projects have developed much information of
value in defining not only the natural behavior and the basic
cycle of polyoma in nature, but also the probable sources of virus
infections in both experimental and production colonies. Although
serologic studies do not support the hypothesis that polyoma virus
can infect man, widespread access of humans through mouse contam-
inated environments and contamination of food-stuffs with mouse
excreta makes consideration of possible human infection rather
more than academic. The lack of serologic correlations with
cancer in man, such as occurs in mice, may not represent con-
clusive counter-evidence, since hamsters with polyoma induced
tumors rapidly lose all evidence of infection with polyoma
(Habel), despite the fact that the tumors persist.
21
22
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
Proposed Course of the Natural History Studies
The natural history of polyoma and other animal tumor viruses
are important subjects in their own right and our studies represent
some of the first effective efforts to develop such information.
As noted above, there can be no higher order of information about
infectious agents than that derived from nature. Information
derived from carefully planned longitudinal studies of tumor viruses
are relevant to any consideration of possible human cancer viruses.
The possibility that cancer could be the result of a zoonotic
infection no longer appears so very unlikely. We plan therefore
to study possible polyoma infection in various animal species,
particularly those having repeated exposure to environments and
to materials known to contain tumor viruses, such as the polyoma
and leukemia viruses of mice, and the leukemia and sarcoma viruses
of chickens.
22
23
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, N I AID
Calendar Year I960
6.0 Extraneous Viruses as "Background Noise" in Cancer Virus Studies
In I960 the "background noise" problem in cancer virus research
grew to almost "deafening" proportions and, in the opinion of LID
virologists, constitutes the number one obstacle to intelligent and
truly effective research on cancer viruses-
Near ly every animal tumor virus system currently under study
was shown to be contaminated with extraneous agents and several
viruses widely proclaimed as "tumor" viruses turned out to be fellow
traveling ordinary viruses. To list a few examples: Friend leukemia
was found contaminated with polyoma and mouse adenovirus; Gross
leukemia by polyoma, K virus and mouse adenovirus; Schwartz leukemia
with polyoma, K virus and mouse adenovirus; Moloney leukemia with
mouse hepatitis and mouse reovirus; the polyoma virus itself became
contaminated with mouse adenovirus, hepatitis and salivary gland
vi ruses.
New "tumor viruses" grown in tissue culture systems or propagated
in mice were reported and later found to be extraneous agents. Thus,
to give one example, the tissue culture grown "VL" strain of lympho-
matosis was shown to be an almost universally prevalent adenovirus
of chickens now called "GAL" virus. LID virologists showed by
serological and virus isolation methods the lack of association
between most of these agents and the specific cancers with which
they are associated, confirming in several instances similar findings
in other laboratories.
23
24
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
6. 1 Extraneous Viruses in Tumor Virus Study Systems
LID virologists showed that the "seeds" of the "background
noise" viruses are commonly present in the animals used for the
induction of tumors, and of course in the subsequent passage materials
as mentioned above. The extraneous viruses most commonly encountered
in cancer systems were the newer ones, such as polyoma, K virus, mouse
reovirus and adenovirus; but this in part may be due to newly
developed easily applied survey tools for these agents. Other
viruses encountered less often (perhaps because of comparatively
less sensitive tools) were mouse hepatitis, mouse salivary gland
virus, the newly discovered "thymic agent" (TA) . Except in newborns,
most of these viruses occur subc I inical ly and latently. Not found
or seldom encountered in tumor systems were Theiler's virus, LCM,
PVM and mouse pox (ectromel ia) , all familiar to most virologists.
Since most of these older agents generally produce very obvious
clinical effects in mice, they are perhaps more easily eliminated
from production colonies.
All mouse colonies used for tumor virus induction were shown
to contain at least one easily demonstrable extraneous virus; but
more than one (usually three to five) was the rule. Discoveries of
new mouse viruses and the development of techniques for selecting
them enabled our scientists to survey and define many mouse production
colonies and to some extent predict some of the problems encountered
in current extensive efforts to establish cancer viruses in animal
and tissue culture systems.
2't
25
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, Nl AID
Calendar Year I960
6.2 Proposed Course of "Background Noise" Studies
The first principle of microbiology calls for "pure culture"
of microbes. The extraneous virus problem in virus study systems
must be controlled before "good" virology can be done on cancer
viruses in such systems. The extraneous agents are in most cases
slow-growing, latent, subclinical agents. While they do not seriously
handicap the study of the acutely virulent viruses such as the
encephalitis and Coxsackie viruses, they do however pose serious
problems in studies of the slow-growing and also generally latent
cancer viruses.
LID scientists visualize several steps that must be taken:
I) Further development and application of sensitive techniques for
detecting extraneous viruses; 2) Surveillance and monitoring of
existing mouse colonies and experiments for known agents, thus helping
to define current problems. This calls for the production and
evaluation of diagnostic reagents; 3) Establishment of relatively
"clean", "virus defined" colonies and animal research areas for
pilot studies aimed at the elimination of extraneous viruses from
study systems; 4) since all cancer virus passage materials passed
through experimental animals either are known or suspected to contain
extraneous viruses, they also must be "cleaned" up in order to
utilize "clean" animals in "clean" areas and finally achieve the
goal of "pure culture"; and 5) When sufficient information is available,
"c lean"animal production colonies and "clean" experimental areas must
eventually be established wherever such animals are to be used for
study of latent or tumor virus effects.
Comment . It is clear that the task described here will
appear monumental to some, insuperable to others, and to still others
too expensive. However, it is also clear that reluctance to meet
this problem squarely and failure to eventually achieve its solution
is to accept pre-Pasteurian concepts as guides for modern virology,
to waste uncounted dollars and to accept in the beginning of new
and very expensive enterprises on animal tumor viruses the probability
of f inal fai I ure.
LID scientists who have helped to develop and define this problem
recognize the importance of its solution and accept responsibility to
work towards that necessary goal. We must do so if we are to satisfy
our desire to properly study the role of latent viruses in chronic and
neoplastic diseases. It is our opinion further that the National
Institutes of Health with its large commitment to support i i rus
research cannot avoid this responsibility and that the most effective
action towards the solution of this critical problem depends on
well directed and concerted action by N I AID and NCI. 25
26
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
7.0 Studies of Common Virus Infections in Animals Likely to
be Important in Human Medicine
A. Bovine Virus Infections
During the past several years LID scientists in colla-
boration with other groups have helped to enlarge current notions
of common virus infections of dairy and beef cattle. The occurrence
of several "new" viruses, such as reoviruses types I, 2 and 3,
were reported in I960, and additional viruses, chiefly bovine
enteroviruses, are currently under study. Observations on the
properties, clinical importance and prevalence of para-inf I uenza 3
virus, infectious bovine rh i notrachei tis and bovine adenovirus
were also extended, and potencies of para-inf I uenza 3 vaccines
were explored in cattle. A clear view of the importance of these
viruses in bovine disease and their significance for human health
must await much larger and comprehensive efforts. Such an effort
however far exceeds our currently available facilities and
resources. Consequently, we plan in 1961 to continue only to a
limited degree to develop our laboratory and natural history
studies of these agents.
B. Virus Infections of House Mice (Mus musculus)
One of the more intimate of man's domestic creatures is
the house mouse. It is perhaps no accident that the known
viruses of mice closely resemble those of man; at least 13
mouse viruses have human counterparts and several are known to
be identical with human agents. Thus, mouse reoviruses
types 2 and 3 discovered during I960 in both laboratory and wild
mice, and in mouse tumor passage materials (see 6.0 Extraneous
Viruses above) are indistinguishable from the prototype human
strains. Of course LCM and Sendai viruses of mice and man were
previously shown to be identical.
26
27
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
Although many of these viruses are now important because
they produce "background noise" in cancer virus studies, they must
be regarded as important in their own right, both as possible
sources of infection to man and to other of his more desirable
domestic animals. They also serve as interesting natural models
of similar human infectious disease processes.
The contamination of cereal grains by polyoma virus have
been mentioned above (in 5.0). Since other common viruses
(such as mouse reoviruses and adenoviruses, etc.) are also
excreted in saliva, urine or feces of mice, it seems inevitable
that they 'will soon be found on grain, which of course if fed
uncooked to I ivestock.
Human infection with mouse viruses due to exposure to
cereal grains although less likely because they are almost always
cooked, could also occur through occupational and household
exposure to raw grains in the process of grinding, processing,
cooking and baking with flour. Needless to say, we plan sero-
epidemiological studies designed to answer the questions posed
here.
27
28
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, Nl AID
Calendar Year I960
Medical Mycology Section
The Mycology Section reported 8 projects for calendar year
I960. All these projects have included broad fields of research and
although definitive goals have been reached in most of them, all will
be continued in order to further exploti productive lines of investi-
gation. In most cases new or additional species of pathogenic fungi
will be used in investigations, or techniques will be altered to
permit further development of experimental studies. Results of
investigations have been published in 9 papers.
Dr. George W . Lones, investigating antibiotic X-5079C, found
that it is fungistatic but not fungicidal and that its apparent low
degree of jm vitro activity is due to its decay in culture medium.
The yeast form of Histoplasma capsulatum is much more sensitive to
X-5079C than the mycelial form and an assay method, sensitive to
I ug/ml using ]H. capsulatum, was developed. X-5079C has low
toxicity for HeLa cells and is active against H. capsulatum grown
in HeLa eel Is.
Dr. Lones has converted a second strain of Coccidioides immitis
to serial culture in the spherule form. He has made quantitative
measurements of the ability of various carbon and nitrogen sources
to support growth of spherule and mycelial forms of strain M-l I of
C. i mm i t i s . Only mannose is utilized as readily as glucose by
spherules. Mannose and fructose support growth of the mycelial
form as well as does glucose. A substrate which preferentially
supports growth of the spherule form was not found in this study.
Dr. Herbert F. Hasenclever utilized spherules to immunize
mice and found an increased survivor rate in immunized mice when
challenged with a lethal infecting dose and earlier clearance of
organs (negative cultures) in immunized mice challenged with a
sublethal dose.
Dr. Hasenclever found two antigenic groups within the species
Candida albicans. One is simi lar antigenical ly to C. tropical is and
one is antigenically indistinguishable from C. stel latoidea.
28
29 **u
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
Dr. Hasenclever found by triturating and plating out organs
of experimental ly infected mice, that several minutes after
Cryptococcus neoformans was injected either intravenously or intra-
cerebral ly into mice the largest numbers of yeast cells had been
retained in the lungs. The fungus population in the lung then
decreases and 2-3 days after infection multiplication in the brain
is apparent. Although the interval from infection to death of
infected mice varies with the strain of C. neoformans, the numbers of
yeast cells per gram of brain tissue is approximately the same
regardless of strain.
Dr. Hasenclever has found that many strains of Candida
tropical is are as pathogenic for mice as for rabbits and that
multiplication in vivo is similar to that of C. albicans. The kidneys
are the organs showing greatest tissue damage and the highest yeast
eel I population.
Dr. Chester W. Emmons and Mr. Willard Piggott, with the
assistance of Mr. William Hill, have continued studies of the
saprophytic occurrence in natural habitats of fungi which cause
mycoses. Cryptococcus neoformans has been isolated from many
additional collections of pigeon guano. When this material is
collected from old pigeon nests and from roosting sites in hay mows
of barns and upper floors of buildings, Histoplasma has never been
found. There is increasing circumstantial evidence that a presently
unstudied pneumonic form of cryptococcosis has occurred in men heavily
exposed to such material and that such material and that such epidemics
have been erroneously diagnosed histoplasmosis.
Dr. Emmons and Mr. Piggott designed a simple, convenient and
clean device for exposing mice by inhalation to dry spores of patho-
genic fungi. This has been used to stimulate natural conditions of
infection.
Continuing in vivo testing of antibiotics and of drugs prepared
by Dr. Benjamin Prescott have not yielded any chemotherapeutic agent
equal to X-5079C in the treatment of several mycoses.
23
30
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
Or. Emmons has continued to collaborate with Dr. William J.
Jellison, Dr. L ie-Kian-Joe, Dr. Charles Bridges and others, in the
study of new or unusual mycoses and the fungi which cause them.
Studies of phycomycoses have continued. With Dr. Bridges, a study
of several cases of hyphomycosis destruens in horses and of its
etiological agent has been submitted for publication. The fungus
is described under the old name, Hyphomyces destruens. It appears
to be a Phycomycete but its complete life history has not been
determined.
In collaboration with Dr. Jellison a new species, Emmons i a
crescens, was described. This fungus differs from the first species
of Emmons i a (E_. parva) in vivo and _i_n vitro at 37° C by its multi-
nucleate condition (instead of uninucleate), its ability to produce
the jm vivo form _j_n vitro at 37° C, and its greater size. £. parva
conidia when inhaled or incubated at 37° C increase in diameter
from 2 - 4 u to 400 - 480 u. This lO^-fold increase in volume of
a single cell is very unusual in the fungi.
30
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
Medical and Physiological Bacteriology Section
Dr. A. K. Saz
Introduction
Significant progress has been made during the past year on
all scientific problems under investigation by the staff of the
Medical and Physiological Bacteriology Section. All professional
investigators in the Section have published in the past calendar
year a minimum of one paper, and in most instances more, in reputable,
first-line scientific journals. The cohesiveness of the Section
has been further evidenced by the holding of regularly scheduled
informal section seminars at which the various professionals present
their current raw data for criticism. The program of the Section is
varied but the one theme common to all projects is the pursuit of
knowledge dealing with fundamental biological activity of various
bacteria. The cross-fertilization which has occurred as a result
of the many-faceted problems under investigation by the professional
personnel of the section has been of prime importance in progress
made and offers further confirmatory evidence for the validity of
the philosophy that team projects are not the sole road to scientific
knowledge.
It has been established that staphylococcal penici I I inase is
associated with particulate material in the cell and thus an explana-
ion has been given for the refractoriness in preparation of this
enzyme by conventional methods. New and more potent inhibition of
Staph, penic i I I inase have been uncovered and the hope remains and is
heightened for the ultimate finding of a chemically useful inhibitor.
Further, sea water has been found to possess strong inhibitory
activity against both penic i I I in-sensitive and penici I I in-resistant
staphylococci (phage type 80/81).
Real progress has been reported in the understanding of iron
metabolism in the staphylococci. As a direct result of continuing
work dealing with mechanisms of the development of non-specific
immunity and in particular the function of the iron-transporting
protein of plasma, siderophi I in, fundamental observations on the
effects of iron deficiency on the growth and metabolism of S_. aureus
have been reported. Work on the biology of the staphylococci so
long neglected during the "antibiotic era" is cardinal to effective
new therapy of staphylococcal infections.
31
32
PHS-NIH
Summary Statement
Labc -atory of Infectious Diseases, NIAID
Calendar Year I960
Progress has also been made on the search for better methods
of isolation and purification of M protein of streptococci. These
results are of obvious importance in the understanding of Group A
streptococcal virulence. Further, highly interesting observations
have been reported dealing with the mechanism and significance of
the long-chain test for determination of ant i -streptococcal immunity.
Real understanding of the intimate mechanisms of energy meta-
bol ism in Hvdroqenomonas in particular and other bacteria and higher
form in general is closer as a result of work performed in this section
this year. In an enormously complicated field, progress has occurred
in the definitions of the essential reactions.
Detoxification studies on potentially useful chemotherapeutic
agents have continued and new and promising leads have been uncovered
for agents active against bacteria, fungi, parasites and it should
be added, against cancer as well. Several of the aforementioned
detoxified compounds have passed preliminary screening processes
performed by the Cancer Chemotherapy Center.
Pinpointing of the enzymic locus of discrimination among
hydrogen isotopes by Pseudomonas has been reported this year. The
area lies in formic acid metabolism.
During the past year, we were unfortunate in losing the
services of Dr. Robert C. Woodworth who has left this section, but
this was compensated for by the selection of Dr. Austin Shug,
Project Associate, Enzyme Institute, University of Wisconsin, to
replace him. Dr. Shug will concern himself with studies on the
iron metabolism of staphylococci.
We were saddened by the passing of Mr. Charles Offord, Animal
Caretaker in this section. Mr. Jack Kelly has ably replaced Mr. Offord.
Dr. Eskin Huff, LCI, has been transferred to this section and
will work on the mode of action of penicillin and the biochemical
mechanisms of the development of resistance thereto. He is joined
by Miss Harriet Milner, Biologist, and will occupy the space formerly
assigned to Dr. Repaske, who in turn will have new quarters in the
space left vacant by the departure of DBS personnel from the area.
32
33
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
Authorization still has not been obtained for the hiring of
a microbial geneticist. It cannot be emphasized too strongly that
this sectionis mission in bacteriological research cannot be completely
fulfilled unless a program of genetic research is begun. This is a
highly important, vital area of contemporary microbiological research
and it is incomprehensible that an institute devoted to a study of
the microbial world is not represented in this parameter.
Studies on the Biochemi,~-y of Antibiotics .
Studies on the biochemistry of staphylococcal penicillinase
are continuing on two parameters: I) search for an effective
inhibitor of the enzyme, 2) purification of the enzyme. Results
reported last year indicated that various dipeptides, and particularly
those with D-val ine in the molecule were inhibitory. Other inhibitory
compounds have now been found. These are an interesting group of
substances prepared by condensing various amino acids, primarily of
the D-conf iguration, into peptides with substances that show per se
metal binding capacity but are not in themselves inhibitory to penicillin.
On a molar basis, these compounds are by far the most inhibitory of
any yet studied. The most active are D-dialanyl- and L-dialanyl-
benzidine, D-val y I -4-ami nob i phenyl and D-phenyl-L-alany l-aminof I uorene.
Inhibitions up to 60 per cent have also been observed with other
dipeptides, i.e., L-threony l-D-alanine, L-al lothreonyl-D-alanine
and D-a I I o i so I eucy l-D-alanine.
Various considerations had indicated that staphylococcal
penicillinase conceivably was associated with particulate matter
within the cell. Work performed this year has indicated that this
is indeed the case. It has been possible to secure 12-fold purifica-
tion of the cell-free enzyme by centrifuging in the Spinco Model L
centrifuge at 144,000 x g for periods of 5-15 minutes. All activity
is found in the pellet. It is hypothesized that the marked stimula-
tion of penicillinase activity by various alcohols (reported last year)
is due to the reversible solubilization of the particle mediated
by the alcohols. This activity brings the enzyme and the substrate
(penicillin into apposition) .
33
34
Mill
n i
Lab-.' , NIAID
A corol lary ol Hi m ■< '■ i portad >fni n1 of
.! new , h ophotom i hod of ponl' 1 1 I i
i coloi of -i I'll Indh 'ii i
of panic! I loi< i' i 'i i "i ma1 Ion ai I ilnfl from fni action of panic) I
lapti 'i df row 1 1 / U
of Di .Nil , NIAMD.
it :i i ',i - ol laboi atl rlth
. aphd in 1 1 1 .ii ion lnv< -i Igatoi i hha I
. .i mar ki /Ity fa
man If il n»1 bo1 h I pan fell i odm I ng(
pan id 1 1 In-raala1
ii f-.) Is hea
undlml ■ Uvfty It I col lab
; ion "iii ' onl Inua.
pr ItK ipal r>' I • I'
Bot'
">ra
/o
Y Q HD
Q H2O Q HD
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, Nl AID
Calendar Year I960
Fractionation of deuterium from protium apparently takes place
in the step HCOOH > CO2 + Ho' °^ +fie *nree possible enzyme systems
involved in equilibrium reactions of atomic and molecular hydrogen,
viz. hydrogen lyase, dehydrogenase and hydrogenase, only the first
one is inhibited by deuterium in concentrations equal or less than
66 per cent D2O or by completely deuterated formate. These findings
strongly suggest that at ieast part of the fractionation effect
leading to light hydrogen gas can be attributed to inhibition of
specific enzymes by deuterium.
Studies with tritium have involved the reactions between
tritiated water and organic constituents in living cells. The
Pseudomonad quickly absorbs tritiated water in various media and
splits the water molecule containing tribium. Whereas the amino
acids from bacterial protein show little or no tritium bonding, the
carbohydrate fraction including polysaccharides appears to contain
a large percentage of the bound tritium. Much of the tritium
entering the cell as tritiated water diffuses out later as carbo-
hydrate or polysaccharide. Further studies are contemplated to
identify the tritium-bound organic matter. Since the equilibrium
constant for tritium is K = Q HTO Q H2 = 5.93 + 0.08 @ 25° C
Q H20 Q HD
which is appreciably higher than deuterium, it may be expected that
the fractionation factor involving atomic and molecular compounds
of tritium will likewise be higher.
Aside from the demonstration of deuterium and tritium
fractionation, these studies have revealed the rapidity which
microorganisms absorb and split or bind water. Tracer methods using
tritiated water reveal that the Pseudomonad pumps water molecules
at the rate of 10 '3 molecules per hour at 25° C.
The implications from this study include the following:
1) The natural isotope, deuterium, appears to inhibit specific
enzymes in living cells which may affect the overall metabolism.
2) Bacteria in marine sediments may play an important role
in the concentration of deuterium in the marine environment.
36
35
PHS-NIH
Summary Statement
I -tboratory of Infectious Diseases, NIAID
Calendar Year I960
3) Bacteria may prove to be effective agents for the concentra-
tion of natural and radioactive isotopes. The former has practical
significance in the production of fusion fuel (HDO) and valuable
trace elements; the latter may be important in problems dealing
with water pollution from radioactive isotopes.
The Electron Transport System in an Autotrophic Hydroqenomonad.
Hydroqenomonas eutropha (Bovell) is an autotrophic bacterium
which is capable of obtaining all of its energy for growth, mainten-
ance, and repair from the oxidation of hydrogen gas by oxygen (ZhU + C>2
> 2H2O) . Although the oxidation of the particular substrate,
hydrogen, is unusual, the reaction is fundamentally a general one
in which hydrogen replaces the ordinary organic substrates such as
glucose, glycerol or other complex compounds. Another unique feature
of H. eutropha is its ability to synthesize al I eel I material from
carbon dioxide with energy secured from the oxidation of hydrogen.
Nitrogen is obtained from ammonium salts, sulfur from sulfate.
This organism then is a complete biological synthetic machine which
manufactures the most complicated structural proteins, enzymes,
vitamins, carbohydrates and lipids from three gases (H2, O2 and C02)
and simple inorganic compounds.
Tremendous amounts of energy are needed for these processes,
yet the mechanism of energy production during oxidative metabolism
is not known for this organism or any other aerobic organism.
It has been observed that various biological systems share
many similar biochemical reactions; this has given rise to the concept
of comparative biochemistry. Consequently it is assumed that the
mechanism of oxidative phosphorylation in H. eutropha is analogous
to that in other forms of aerobic biological life. Biochemists
working with bacterial, animal and plant material have applied
themselves to the problem of coupled oxidation and phosphorylation
for the past 35 years, but the mechanism for trapping energy in the
form of ATP during oxidations of the type mentioned has thur far
defied resolution because (I) the enzymes of the electron transport
system and the interacting energy yielding system are both associated
with particulate cellular elements, and (2) resolution of one or the
other system into a soluble state have generally resulted in loss
of both activities.
36
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAID
Calendar Year I960
Hydroqenomonas eutropha is a bacterium with unusual possibilities
for the study of oxidative phosphorylation. Present evidence
indicates that the electron transport system is soluble; consequently,
classical enzyme purification techniques should result in separation
of the individual enzymes leading to an elucidation of their function.
It seems probable that the enzymes responsible for trapping the
resultant energy in the form of ATP are also soluble and they may
be similarly studied.
Since an understanding of the electron transport system is
prerequisite to a study of the energy trapping system, we have begun
studies on the hydrogen oxidizing system. We have found that the
initial steps involve the activation of hydrogen gas and the passage
of electrons to the coenzyme diphosphopyr idine nucleotide (DPN) .
A second coenzyme requirement, riboflavin phosphate (FMN) has also
been found, and the data suggest that it functions as the cof actor
between hydrogen and DPN. Extreme caution must be exercised in
handling the enzyme because of its sensitivity to oxygen. This
has necessitated the design of several new pieces of equipment and
of techniques which minimize oxygen contamination during handling
of the enzyme. To date the enzyme has been purified three to four
fold from crude, cell-free extracts by protamine sulfate and
calcium phosphate gel treatments. Additional purification will
be necessary before investigating the next enzymic reaction, which
is probably cytochrome linked.
Preliminary studies reveal that H. eutropha contains but one
cytochrome of the "c" type; in contrast, most other aerobic organisms
contain three of four cytochromes. This fact again indicates that
this organism possesses a less complicated electron transport system
than is usual ly encountered.
37
38
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, NIAIO
Calendar Year I960
Detoxification of potential Chemotherapeutic Agents;
Further studies are in progress to increase the tolerated
dose of various chemotherapeutic agents, such as isoniazid (INH),
streptomycin (SM) and Kanamycin for tuberculosis, Neomycin for
other infections, Mystatin for fungal infections; Miracil D,
piperazine and complexes of tetracycline for parasitic infections
and certain agents in use in cancer therapy, by concomitant
administration of various metabolites. Studies on the tolerance
of mice for streptomycin and/or isoniazid have shown that a lethal
dose of either drug dissolved in glycerol formal as solvent (25-30
per cent) permitted 90-100 per cent survival in white and DBA mice.
Mixed with SM-INH, 85 per cent of the mice tolerated a lethal dose
of 10 mg SM and 4 mg INH and 80 per cent survived with 10 mg SM
and 8 mg INH. In addition, studies with steroids as adjuvants showed
that a lethal dose of 30 mg SM and 4 mg INH (5 mg is the tolerated
dose of SM) administered simultaneously with 25 mg of sodium
taurocholate permitted 55 per cent survival in two strains of mice.
With twice the amount of sodium taurocholate, 100 per cent of the
mice tested tolerated a 30 mg dose of Miracil D (6 x lethal dose).
A number of potential ly non-toxic new chemotherapeutic agents
against fungal and parasitic infections have been synthesized.
Among these, long chain thiosemicarbazones, piperazines and numerous
tetracycline complexes of atabrine, chloroquine, sulfanilic acid
and naphthoquinones have already demonstrated high in v i vo activity
and yet drew negligible toxicity for mice. Several of the long
chain thiosemicarbazones have also passed initial tests in mice
when tested by the Cancer Chemotherapy Screening Center.
Investigations of the antimicrobial activity of substances
from shellfish are being considered. Two groups of fractions were
isolated from abalone juice by passage through an an ion-exchange
(diethyl ami no-ethyl eel I ulose) column followed by elution with a
series of Tris-H3P04 buffers. Antibacterial activity of a group
of the early eluates has been reported (Proc. Soc. Exp. Biol, and
Med., in press). These fractions showed no inhibition in tissue
culture against Japanese 365 strain of influenza A virus and
polyoma virus. A grouping of later eluates including a final M-NaCI
wash from the column contained no antibacterial activity. However,
these combined eluates exhibited definite inhibition against
influenza virus and polyoma virus. When monkey kidney cells were
treated for 24 hours before virus inoculation with a fraction of
this group at a concentration of 50 mg per cent, virus multiplication
could not be demonstrated.
39
38
PHS-NIH
Summary Statement
LaLoratory of Infectious Diseases, NIAID
Calendar Year I960
Studies on the Growth and Metabolism of Staphylococci and on
the Iron Metabolism of the Host as Mediated by the Iron-binding Serum
Protein S iderophi I in.
Research on this project is oriented towards elucidation of
the following problems:
1) The role of siderophilin as a non-specific immune factor
in the control of bacterial infection through nutritional iron
restriction.
In the circulatory system, both in blood and in the lymph,
the iron availability to the bacterial cells as well as to the host
tissue cells is regulated by the protein constituent siderophilin.
Workers in this section have discovered that the growth of Staph.
albus in serum is completely inhibited by siderophilin if, as occurs
in normal and in many pathologic sera, the protein is not completely
saturated with iron. Staph, aureus . on the other hand, grows in
serum at a rate which is a function of the percentage iron saturation
of the siderophilin. For example, if the siderophilin in serum is
100 per cent saturated, Staph, aureus grows ten times faster than
when the siderophilin is only 30 per cent iron-saturated (the
percentage saturation of normal serum). Further, the lag period of
growth, under the experimental physiological conditions employed,
is extended from 8 hours to 27 hours at the lower percentage
saturation. Since, in various pathologic states, the percentage
iron-saturation levels of serum siderophilin vary in a diagnostical ly
significant manner far above and below the usual normal value to
30 per cent, the suitability of such sera as in v i vo culture media
for the growth of many pathogens and, in particular, Staph, aureus,
is a factor in the defense mechanism of the host against the establish-
ment of a given bacterial infection. A phenomenon associated with
infection and worthy of further investigation is the response of
the host to decrease the percentage iron-saturation of its siderophilin
by lowering the concentration of circulating serum iron. One
consequence of this response is to provide a more unsuitable
nutritional environment for the infecting bacterium; another
consequence is to increase the iron concentration in the reticulo-
endothelial system. The mechanism by which the response is effected
is unknown and the significance of the heightened iron concentration
in the R. E. systen likewise requires elucidation.
2) The effect of the siderophi I i n-control I eo
iron concentration on the metabol ic activities of Sj
33
40
PHS-NIH
Summary Statement
Lauoratory of Infectious Diseases, NIAID
Calendar Year I960
It has been found that the carbohydrate metabolism of cells
of Staph, aureus grown in medium restrictive as to available iron
(low percentage iron-saturation of siderophi I i n) is greatly different
from those grown in media providing 5 to 20 times as much free
iron (high percentage iron-saturation of siderophi I in) . One of
the most striking effects, among others, is the failure of "low
iron" cells to oxidize the important pivotal substrate, pyruvic
acid. The concentration of certain iron enzymes, as cytochrome
oxidase and catalase, is severely reduced in "low iron" cells.
The biochemical mechanisms by which iron controls the overall
metabolism of Staph, aureus are being investigated and the changes
in enzymatic function and localization of iron-containing components
within various cellular structures are being studied.
3) A comparison of the mechanisms by which mammalian host
tissues acquire iron from iron-si derophi I in with that operating in
the eel Is of Staph, aureus.
It has been determined that eel Is of Staph, aureus, grown
in the presence of serum, obtain their iron from iron-si derophi I in
as free ionic iron. The source of this ionic iron is the natural
dissociation of the metal protein complex under physiological
conditions of pH and bicarbonate concentration. The relationship
of pH and bicarbonate concentration changes to the dissociation
rate of the metal complex have been investigated. It has been
established that bone marrow tissue and reticulocytes take up iron
from i ron-si derophi I i n in serum, or from a bicarbonate buffer
solution of the metal complex, not as ionic iron but by some type
of direct transfer from the complex to the cells. Investigation
of the basis for this direct transfer is contemplated. Whether
other tissue ceJIs than bone marrow and reticulocytes depend for
their iron on similar direct transfer of iron from iron-si derophi I in
complex or whether they are similar to Staph, aureus eel Is in
fulfilling their iron requirements through diffusion of ionic iron
is a current problem of active interest.
Effective use of Staph, aureus and of rabbit reticulocytes have
been made as indicators of the integrity of isolated, purified
s iderophi I ins on the basis of their efficacy in the donation of iron
to the given cells. Further development of the applicability of
these agents to such purpose is required.
HO
41
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, N I AID
Calendar Year I960
Streptococcal M Protein, Virulence, and Type-specific Immunity.
This unit has been interested in the bacterial flora of the
upper respiratory tract, chiefly the gram-positive cocci, because
of their prevalence and pathogenicity. Chief among these are the
beta-hemo lytic streptococci of Group A. Despite more than 15 years
of antibiotic usage, these organisms appear to be as ubiquitous as
ever. Widespread use of antibiotics and prophylactic programs have
caused, however, decreases in severity and in non-suppurati ve
sequelae such as rheumatic fever and acute glomerulonephritis. The
continuing high incidence of Group A streptococcal infections is
probably caused by many repeat infections with the same type, due
in turn to the suppression of adequate type-specific antibody
response by early administration of antibiotics. The role of the
presumptive virulence factor, M protein, in such infections is-
moderately well-established by association, but the mechanism
of its action is obscure.
For the past several years, work in the Respiratory Bacteriology
Unit has emphasized cultural studies in field situations, balanced
by increasing basic laboratory investigations into the nature of
streptococcal virulence and of the protective type-specific anti-
bodies. It is felt that such studies may ultimately aid also in
determining the nature of virulence in, and of specific resistance
to, other gram-positive cocci and particularly the staphylococci
which, although they produce certain extracellular materials in
common with the streptococci, are less well classified serologically
or by antigenic dissection.
Cultural studies in calendar I960 have been limited to
serologic identification of beta-hemo lytic streptococci isolated
sequentially from personnel stationed in the Antarctic. The study,
done in conjunction with a group at the School of Hygiene of Johns
Hopkins University, has shown that delayed isolation of beta-
hemo lytic streptococci from frozen glycerine-broth is feasible; and
that the majority of streptococci so isolated were of Group A.
The correlation of serologic group with bacitracin sensitivity,
however, was low. The latter thus appears to be a poorer screening
procedure than indicated by previous workers. Continuing field
studies of this sort are planned, both in the Antarctic and Arctic.
In addition, the antibody responses of carriers will be studied by
new methods.
42
kl
PHS-NIH
Summary Statement
1 aboratory of Infectious Diseases, NIAID
Calendar Year I960
Basic investigations have been concerned with the development
of new methods for extracting and purifying M protein; and with
improving tests for type-specific antibody. M protein of several
types have been prepared as the acid-alcohol soluble picrates.
These preparations are simple to make, are antigenic, and cross-react
only when the organism from which they are made is known to contain
a cross-reacting "R" protein. Further purification and analysis
by antigenicity, agar gel diffusion and Immunoelectrophoresis are
in progress. The use of picrates and of conventionally-prepared
M proteins in enhancement of virulence or protection against
phagocytosis, is being studied. Fragmentation of M protein by
heat, pH, and enzymes to define the portion active in virulence,
as we I I as in other reactions, is under way.
Improved methods for the detection of type-specific antibody
are being pursued. These include the long chain test and inhibition
of specific fluorescence. The former has been increased in simplicity,
speed, sensitivity and statistical control by using chi-square
analyses of chain-length frequency distributions. The importance
of time-interval sampling, and the relation of time to antibody
and antigen concentrations used in the test, have been shown. As
a result, it is now possible to titrate readily antisera for type-
specific antibody to a degree not previously possible.
The inhibition of specific fluorescence appears to be a
sensitive screening test for type-specific antibody, but its use
in titration is hampered by subjective determination of the degree
of fluorescence as an end-point. Type-specific fluorescent antisera,
needed for this test, were readily prepared by absorption of labelled
antisera to whole cells, and did not cross-react. Such antisera
have also been used, together with group antisera, to study the
location of M protein in the cell wall. It has been shown that
type-specific antibody will block group antibody, although the
reverse is not true. These results appear to verify the superficial
location of M and the deeper position in the eel I wal I of the group
carbohydrate antigen.
43
PHS-NIH
Summary Statement
Laboratory of Infectious Diseases, N I AID
Calendar Year I960
Group A streptococci have been shown to grow well in, and to
form long chains in, f I uorescein-l abel led homologous type-specific
antiserum. Once antibody in such a system is depleted, however,
subsequently-formed portions of the cell wall are not fluorescent,
thus providing a sharp visible differentiation of old and new cell
wall. Similar studies, using ferri tin-label led antibody and
visualization under the electron microscope, are planned.
Ant i body- I abel I ing techniques are also being used in studies
on the nature of type-specific antibody. Univalent antibody has
been produced by pepsin and cysteine treatment, and is being
examined by a variety of tests.
k3
44
&d
Serial No. NIAID 60
1. Infectious Diseases
2. Medical & Physiological
Bacteriology
3. Bethesda, Maryland
PHS-NLH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Studies on Cellular and Cell-Free
Staphylococcal Penicillinase
Principal Investigator: Dr. Arthur K. Saz
Other Investigators: Nona
Cooperating Units: None
Han Years (calendar year i960)
Total: 22/12
Professional: 8/l2
Other : II4/12
Project Description:
Objectives: To purify and study penicillinase from penicillin-
resistant staphylococci. To investigate the possibility of strongly in-
hibiting cellular penicillinase.
Methods Employed: Isolation and purification of cell-free
penicillinase from staphylococcal cells by standard biochemical techniques.
Measurement of penicillinase activity manometrically and iodome trie ally.
Major Findings^ In continuation of one aspect of this problem
carried over from last year, the search for inhibition of penicillinase
activity has continued. Various compounds prepared by condensing amino
acids and metal binders have been tested for anti-penicillinase activity.
Of these, trie extremely insoluble compounds D-dialanyl benzidine and L-
dialanyl benzidine show marked inhibitory activity. Due to the very low
solubility of these compounds, an accurate inhibitory concentration has
been difficult to detemine. However, these compounds are, on a molar basis,
by far the most active of all inhibitors thus far found. Lesser activities,
but still potent, have been noted for D-valyl U-aminobiphenyl and D-phenyl-
alanyl L-aminofluorene. Inhibitions of penicillinase activity up to 60$
have been observed with L-threonyl-D-alanine, L-allothreonyl-D-alanine and
D-alloisoleucyl-D-alanine.
Efforts at purification of staphylococcal penicillinase have con-
tinued. To date, the most active preparations exhibit twel" uri-
Part B included: Yes -1- ^
Serial No. NIAID 60
fication. Various considerations indicated that the penicillinase
as it occurs intracellular ly, was not a soluble enzyme, but rather
was particulate. This has proved to be the case. Cell -free extracts
of the penicillin-resistant staphylococci prepared by sonic or Nossal dis-
integration were subjected to ultracentrifugation in the Spinco Model L
preparative centrifuge. After only five minutes centrifugation at
lUit,000 x g, all penicillinase activity appeared in a small pellet with
a concomitant h to 6-fold purification. Electron microscopic observation
of the pellet showed particulate matter, presumably though not definitively
associated with penicillinase activity. This observation conceivably
could explain the marked stimulation of penicillinase activity, as re-
ported previously, by various alcohols, and particularly n-propanol and
n-butanol. In this instance, the alcohol activity might parallel the
stimulatory effects of various detergents and solvents on mitochondria
derived from mammalian tissue. The solvents presumably render the par-
ticles more permeable to substrate. This possibility is under investi-
gation.
Significance to bio-medical research and the program of the
Institute! If a non-metabolizable non-toxic inhibitor of staphylo-
coccal penicillinase could be found it would go a long way toward solv-
ing the penicillin-resistant staphylococcus problem. Any information
concerning penicillinase is of interest to this Institute.
Proposed course of project; 1. To purify penicillinase fur-
ther and to study its properties. 2. To continue the search for an ef-
fective inhibitor. Animal studies will be used as indicated. 3. To
study the mechanism of the alcohol stimulation of penicillinase.
-2-
k5
Serial No. NIAID 60
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Saz, Arthur K. and Martinez, L. Marina: Enzymatic Basis of Resistance
to Aureomycin. III. Inhibition by Aureomycin of Protein-Stimulated
Electron Transport in Escherichia coli. J. Bact., 79, 527-531> I960*
Honors and awards relating to this project:
Chairman, President's Fellowship Committee, Socieiy of American
Bacteriologists.
Organizer and Convener of Panel on Mode of Action of Antibiotics,
Conference on Anti-Microbial Agents - Mayflower Hotel, Washington, D.C.
Oct. 26-28, I960.
Lecturer (Professor) in Botany, Howard University, Washington, D. C,
"6
Serial No. NIAID 61
1. Infectious Diseases
2. Medical & Physiological
Bacteriology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A,
Project Title: Antibiotic Activity of Sea Water
Principal Investigator: Arthur K. Saz
Other Investigators: Dr. Stanley Watson
Woods Hole Oceanographic Institution
Woods Hole, Massachusetts
Cooperating Units: Woods Hole Oceanographic Institution
Man Years (calendar year I960)
Total: 6/12
Professional: 6/12
Other:
Project Description:
Objectives: To investigate the reported antibiotic activity of
sea water. To isolate from sea water, compounds active against fecal
organisms and penicillin-resistant staphylococci.
Methods Employed: Treatment of sea water by various methods
(extraction, chromatography, electrophoresis) in order to isolate in-
hibitory factors present. Standard bacteriological techniques.
Major Findings: It has been possible to confirm a weak anti-
coliform activity of sea water. Perhaps of more interest, however, is
the unequivocal demonstration for the first time of an antistaphylococcal
activity in sea water. The reputed strong anti-coliform activity of sea
water is in large part due simply to the effect of increased salinity
in a sea water environment. Most previous reports of this activity
failed to use adequate controls as evidenced by close inspection of the
data. Indeed, on occasion, Escherichia coli, inoculated into either
filtered or raw sea water, increased in numbers during the observation
period of 72 hours. On other occasions and with different specimens of
water, a weak activity (survival of $0% after 72 hours) was evident.
Part B included: Yes
-1. »7
Serial No. NIAID 61
The problem of adequate controls for the demonstration of activity
was investigated. A small amount of organic material, proper pH,
temperature of 20-25° and the use of sea water in diluting inoculated
samples for plate count proved to be essential for demonstration of ac-
tivity. Further it was essential to run a control in each experiment
of 2.5a> buffered NaCl (the saline concentration of sea water) to eliminate
the possibility of kill due to salinity. alone.
Experiments reported hereafter were performed, because of crowded
conditions at the Woods Hole Oceanographic Institution, with a strain of
penicillin-sensitive S. aureus . However, preliminary experiments per-
formed at N.I.H. indicate that a penicillin-resistant S taphylococ cus
(80/81) is killed by sea water. In a typical experiment, 2.6 x 10^
staphylococci per ml were inoculated with raw sea water. After 2k hours,
5.7 x loVml organisms were viable and in I4.8 hours, 3.2 x 10^ organisms/
ml survived. There were only 10 viable organisms/ml after 72 hours. In
contrast, the saline control for the same periods showed 1.1 x 10 ,
0.3 x 10 , and 3.6 x lO-3 survivors/ml. Similar results were obtained us-
ing filtered sea water. By contrast, when organisms were inoculated into
autoclaved sea water, the anti-staphylococcal activity was completely
lost. In a preliminary experiment raw sea water, diluted 1:8, exhibited
as much activity as whole sea water over a 2k hour period.
It was also observed that not all samples of sea water collected
exhibited activity. To date, of three samples studied two have been
active. These results are presently being written up for publication.
Significance to bio-medical research and the program of the
Institute; The public health aspects of the mechanism of the kill-
ing of fecal organisms in sea water are obvious. An inhibitor of peni-
cillin-resistant staphylococci would be of prime importance.
Proposed course of project: A cooperative arrangement has been
worked out with Woods Hole Oceanographic Institution for continuation of
the problem both here at the NIH and it is hoped at the Oceanographic
Institution. Isolation of the substance(s) will be attempted. Since
all the waters studied in this report were surface (10 'r|) it would be of
interest to determine activity of deeper continental shelf water and
deep, open ocean water. To this end, it is hoped a stay at W.H.0.1.
this summer can be arranged so that use of the Oceanographic institu-
tion research vessels can be utilized for this purpose.
48
-2-
Serial No. NIAID 6l
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
none
Honors and awards relating to this project:
Appointed guest worker at the Oceanographic Institution,
Woods Hole, Massachusetts.
-3-
_ . , >T lTTATT, NIAID-62
Serxal No. NIAID
1. Infectious Diseases
2. Medical & Physiological
Bacteriology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A
Project Title: The electron transport system in an
autotrophic hydro genomad.
Principal Investigator: Dr. Roy Repaske
Other Investigators: None
Cooperating Units: None
Man Years (calendar year I960)
Total: 2U/12
Professional: 12/12
Other: 12/12
Project Description:
Objectives: This research has as its object the identifica-
tion of individual enzymes and intermediary cofactors which are opera-
tive in electron transport. This information may be transposable in
part to mitochondrial systems and provide important clues about the
electron transport sequence in these particulate structures. The long
range plan will include an investigation of the energy yielding reac-
tions coupled to electron transport.
Methods Employed: The methods employed will be those neces-
sary for mass cultivation of organisms and for isolation and purifica-
tion of the electron transport enzymes. Growth of large quantities of
Hydro genomonas eutropha is technically difficult because of the require-
ment for large volumes of an artificial gas mixture. Apparatus has been
designed and is being built for this purpose. The assay of the initial
enzymatic reactions in cell free preparations also requires a modified
procedure because the enzymic activity must be measured in a hydrogen
atmosphere in a spectrophotometer. Protein fractionation will be
achieved by procedures involving ammonium sulfate precipitation, gel
adsorption and elution, solvent precipitation, and chromatography.
Major Findings: It was readily apparent from the atypical
kinetics and other indirect evidence that the enzymatic reaction beingr
Part B included. Yes -1-
Serial No. NIAID"62
studied, he oxidation of hydrogen by diphosphopyridine nucleotide (DPN),
involved either a second enzyme or a cofactor. The cofactor which was
first found in a boiled cell extract has been identified as riboflavin
phosphate (FMN), but its requirement could be shown only when the enzyme
was preincubated under hydrogen and reducing agent was added to the assay
system.
Enzyme stability has been controlled by storing and handling the
enzyme under an atmosphere of hydrogen and in the presence of cationic
buffers of relatively high concentration (0.05> to 0.2 M) . The pH opti-
mum for stability is 8.0. A sharp pH optimum for enzyme activity was
found at pH 7.5.
The enzyme catalyzing the initial reaction, i.e., DPN reduction,
has been purified only three fold by calcium phosphate gel adsorption
and ammonium sulfate fractionation. Unaccountable losses of enzyme
activity during these procedures are at the moment unexplained, but
may reflect inherently important characteristics of the complex. Con-
sequently the reason for this loss of activity is under intensive in-
vestigation.
The most purified enzyme preparation has a very high affinity for
riboflavin phosphate but it has not been possible to demonstrate FMN
reduction of added exogenous FMN, indicating that the FMN is tightly
bound by the enzyme and/is not in equilibrium with the exogenous FMN
pool.
Significance to bio-medical research and the program of the In-
stitute; Hydrogenomonas species are unique organisms which obtain
energy for all of their life processes through oxidation of hydrogen
gas in the presence of oxygen. This is in contrast to animal cells
and most bacteria which oxidize organic substances for energy. Although
the initial substrates are different, there is every reason to believe
that both types of systems have much in common. Both serve the identi-
cal function of providing energy for synthesis, growth and repair, and
both utilize cytochromes and diphosphopyridine nucleotide to mediate
the transfer of electrons to oxygen. Biochemical studies in the past
have revealed that a common denominator exists between biochemical pro-
cesses of various organisms, and this well supported concept of com-
parative biochemistry is the rational basis for investigating a given
reaction in an organism most amenable to study.
Previous research on this organism was carried out at another
institution by the principal investigator. The potential advantage
of studying these reactions in H. eutropha lies in the fact that the
system in this organism is soluble and therefore it can be fraction-
ated by classical protein fractionation procedures.
-2-
51
Serial No. NIAID
-62
The electron transport system in all other organisms studied to
date has been associated with particulate elements of the cell. Ef-
forts to dissociate some of these enzymes from the particles have
met with limited success while other enzymes cannot be solubilized.
Concomitantly with the occurrence of electron transport, ATP is generated;
of the two processes, ATP generating system is the most labile during
manipulation. As a result of these characteristics, our knowledge of
electron transport and simultaneous ATP formation is sketchy and indi-
rect. It is felt that the limitations associated with other systems
may not exist in the H. eutropha system.
Proposed course of Project; More extensive purification of the
DPN reducing system is planned to eliminate interfering enzymes and
compounds and to demonstrate with reasonable assurance that only one
enzyme participates in this reaction. The remaining reactions between
reduced DPN and oxygen will be segregated so that the characteristics
and requirements of each individual reaction in the sequence will be
known. The system reconstructed with purified enzymes should have the
same characteristics as those found in the crude extract. During these
studies, the occurrence of ATP formation during electron transport will
be explored as a preliminary survey to an investigation of this system.
52
-3-
NIAID - 62
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Repaske, R. and Seward, C.: FHN as a cofactor in the enzymatic reduction
of DPN by hydrogen. Biochem. Biophys. Res. Coram., 2_, 397-UOl, I960,
Litwack, G., Repaske, R. and Myrvik, Q.: Lysozyme as related to problems
in Microbiology. Purdue University, I960, 39 p.
Honors and awards relating to this project:
Gave the following invited seminars:
"Enzymatic Reduction of DPN by Hydrogen" - Gerontology Branch, NHI-
NIH, at City Hospital, Baltimore, Maryland - Mar. 23, I960.
"Enzymatic Oxidation of Hydrogen Gas by Extracts of an Autotrophic
Bacterium" - Dept. of Bacteriology, Pennsylvania State University, State
College, Penn. - Oct. 13, I960.
"Enzymatic Reduction of DPN by Hydrogen with Extracts of Hydrogeno-
monas" - Dept. of Bacteriology, Indiana State University, Bloomington,
Indiana, Nov. 16, I960.
Panel Member in a Symposium on Lysozyme presented at Society of
American Bacteriologists national meetings in Philadelphia, May I960.
Elected to membership, American Society of Biological Chemists.
Nominated as candidpte for Secretary of the General Division of the So-
ciety of American Bacteriologists.
Invited to participate in the 2nd International Symposium on Fleming? s
Lysozyme in Milan, Italy - April 7-9, 1961.
53
_ . . .. NIAID-62
Serial No.
PHS-NIH
Individual Project Report
Calendar Tear I960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Repaske, R. and Seward, C: FMN as a cofactor in the enzymatic reduction
of DPN by hydrogen. Biochem. Biophys. Res. Comm., 2, 397-1*01, I960.
Litwack, G., Repaske, R. and Myrvik, Q.: Lysozyme as related to problems
in Microbiology. Purdue University, I960, 39 p.
Honors and awards relating to this project:
Gave the following invited seminars:
"Enzymatic Reduction of DPN by Hydrogen" - Gerontology Branch, NHT-NLH,
at City Hospital, Baltimore, Maryland - Mar. 23, I960
"Enzymatic Oxidation of Hydrogen Gas by Extracts of an Autotrophic Bac-
terium" - Dept. of Bacteriology, Pennsylvania State University, State
College, Perm. - Oct. 13, I960
"Enzymatic Reduction of DPN by Hydrogen with Extracts of Hydrogenomonas" -
Dept. of Bacteriology, Indiana State University, Bloomington, Indiana,
Nov. 16, I960.
Panel Member in ^Symposium on Lysozyme presented at Soc. of American
Bac^riSlblists1 fiatio'Ka! fie e t-^0^ Pniladelphia, May I960.
Elected to membership - American Society of Biological Chemists.
Nominated as candidate for Secretary of the General Division of the So-
ciety of American Bacteriologists.
Invited to participate in the 2nd International Symposium on Fleming's
Lysozyme in Milan, Italy - Apr. 7-9, 1961.
-u- Sij
Serial No. NIAID - 62-A
1. Infectious Diseases
2. Medical Sc Physiological
Bacteriology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Microbiological Fractionation of the Hydrogen
Isotopes
Principal Investigator: Dr. F. D. Sisler
Other Investigators: Dr. Benjamin Prescott
Cooperating Units: U. S. Geological Survey
Man Years (calendar year I960)
Total: 17/12
Professional: 9/12
Other: 8/12
Project Description:
Objectives: To determine the mechanism of hydrogen isotope
fractionation by microorganisms j the divergent metabolic pathways
of protium, deuterium and tritium; the effect of the heavy hydrogen
isotopes on the physiology of the simple biological systems; the eco-
logical and evolutionary significance of the isotope effect in the
biosphere.
Methods Employed: Pseudomonas GI4A and other bacterial cells
and cell products, from culture media containing normal and enriched
amounts of hydrogen isotopes (protium, deuterium, tritium)previously
incubated under a variety of conditions simulating various environ-
mental variables will be analyzed using methods involving the mass
spectrometer, radioactive counting equipment designed for tritium com-
pounds, paper chromatography, infra red and conventional biochemical
analytical procedures.
Major Findings: The three isotopes of hydrogen, viz. pro-
tium, deuterium and tritium are selectively fractionated by micro-
organisms during metabolic processes involving hydrogen compounds.
Samples of gas from marine sediments and from bacterial fermenta-
tion analyzed by mass spectrometer reveal a protium concentration
in excess over that expected from non-biological processes. It
would appear that in the marine environment various microorganisms
Part B not included. j* 55
Serial No. HIAID - 62-A
particularly those containing hydro genlyase and hydrogenase enzymes
play an important role in hydrogen isotope equilibria. Mechanisms
by which microorganisms may fractionate the hydrogen isotopes involve
enzyme specificity, bond cleavage rates, cell wall diffusion and the
formation of large insoluble molecules, e.g. protein-bound polysac-
charides. The latter process resembles a sequestration effect in that
the heavy isotope atoms are prevented from equilibrating with the medium
so long as the cell wall is intact. After cell death and disintegration,
predicted chemical equilibria is reestablished. Laboratory studies irvolv-
ing microbial activity on tritium compounds reveal a multi-staged process
by which protium and tritium are fractionated. It is concluded that
marine microorganisms are important geochemical agents in dynamic processes
involving hydrogen and other isotopes in the sea. The evidence accumulated
to date suggests that microbiological fractionation of the hydrogen iso-
tope is a ubiquitous phenomenon throughout the biosphere.
Significance to bio-medical research and the program of the
Ins titute : A knowledge of hydrogen metabolism of biological systems can
be considered as one of the most fundamental of metabolic processes. Hy-
drogen metabolism (hydrogen transfer, electron transfer) is synonymous
with the energetics of living processes.
With increasing use of both natural and radioactive isotopes in
medical research, it is considered of utmost importance that attention
be focused on fundamental studies which may contribute to the understanding
of the isotope effects in biological systems. This study of the hydrogen
isotopes in simple cell metabolism should, therefore, contribute basic in-
formation of value towards the solution of problems of public health and
economic importance such as implications in radioactive waste disposal in
marine environments, toxic effects of deuterium and other isotopes and
further is of fundamental importance in studying new metabolic pathways
in microbial and other systems.
Proposed course of project: To continue current research with
the following objectives in view:
1. Isolation and identification of intracellular compounds
primarily responsible for deuterium and tritium fractionation,
2. Examination of extracellular products showing heavy isotope
enrichment.
3. Evaluation of the chemical thermodynamics involved in isotope
fractionation, comparing experimentally established values with the
theoretical.
U. Further studies on transport of isotopes across cell membranes?
rate processes and steady state equilibria of all phases involved in
isotope fractionation.
-2-
Serial No. NIAID 63
1. Infectious Diseases
2. Medical & Physiological
Bacteriology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Non-specific Immunity Associated with Serum
Siderophilin and the Iron Metabolism of
both Pathogen and Host.
Principal Investigator: Dr. Arthur L. Schade
Other Investigators: Dr. Lathrop E. Roberts
Cooperating Units: None
Man Years (calendar year 1°60)
Total: 30/12
Professional: 18/12
Other: 12/12
Project Description
Objectives: To investigate the contribution of blood serum
siderophilin tc the natural resistance of the host; to investigate the
iron requirements of selected pathogens and the effect of the iron-
chelating siderophilin and conalbumin on in vivo iron accumulation,
growth, and metabolism of these pathogens; to investigate the factors
leading to changes in concentration of both serum iron and siderophilin
in circulating blood as a result of infection and other abnormal, condi-
tions, and the consequence of such changes on resistance to infection,
iron absorption by the host, and anemia.
Methods Employed: Bacterial and cultural procedures in spe-
cially deironized raedia; spectrophotometric, chromatographic, and
radioisotopic methods; metabolic and enzymatic analytical procedures;
and methods particularly suited to estimation of iron chelated
either to siderophilin or conalhumin and to their relative unsatura-
tions.
Part B included: Yes
57
Serial No. NIAID 63
Major Findings: Previous findings have established that some
bacteria vail not grow in serum unless the contained siderophilin is
saturated with iron, while the growth rate of many others in serum is
a function of the percentage iron-saturation of the siderophilin. We
have, for our more intensive research investigations, restricted our-
selves to Staphylococcus albus, whose growth in normal serum is negli-
gible in the absence of excess iron, and to Staphylococcus aureus,
whose growth rate in serum reflects the amount of bound serum iron com-
pared with its total iron-binding capacity.
A- Proceeding from our preliminary studies on the metabolism of Staph .
aureus cells grown in medium non-restrictive with respect to iron and
those grown in a medium whose concentration of free ionic iron was se-
verely limited by the presence of either siderophilin or conalbumin at
low iron saturation ($%) we have observed the following facts:
1. The rates of oxygen consumption of both high and low iron
cells in the presence of glucose are essentially the same but the
aerobic and anaerobic glycolytic rates of the low iron cells are signifi-
cantly increased over those of the high iron cells. Differences of
rates up to three-fold have been observed. High iron cells oxidize
pyruvate at a rate similar to that of glucose, while lactate is oxi-
dized at approximately twice the rate as that obtained with pyruvate.
Acetate and succinate are oxidized at rates hP% and \2% that found with
pyruvate as substrate. Low iron cells, on the other hand, despite the
similarity to high iron cells in their attack on glucose, fail to oxi-
dize pyruvate but continue to oxidize lactate, albeit at a rate 1/3 to
l/U that of the high iron cells. Low iron cells fail also to attack
either acetate or succinate,
2. The extent of oxidation of several substrates by Staph, aureus
cells cultured in media of "high" and "low" iron content was determined:
Molecules O2 Taken up per Molecule of Substrate
Theoretical Value for Observed Values*
Substrate Complete Oxidation "High" Fe Cells "Low" Fe Cells
Glucose 6 3 1
Formate 0.5 0.5 0
Acetate 2 2 0
Pyruvate 2.5 1.5 0
Lactate 3 1.5 0.5
■> Endogenous oxygen uptake subtracted.
3. The rate of anaerobic dismutation of 2 moles of pyruvate to
one mole each of lactic acid, acetic acid, and CO2 has been studied
-2-
58
Serial No. HIAID 63
manometrically and by chemical analysis. High iron cells carry out
this reaction about 3§ times as rapidly as low iron cells.
It is clear from these metabolic observations that the carbohy-
drate metabolism of Staph, aureus is profoundly affected by the avail-
ability to them of iron during their growth period. The results
likewise indicate that iron, either directly or indirectly, participates
in enzymatic activities not generally appreciated as being mediated by
iron enzjTTies, e.g. the anaerobic dismutation of pyruvate. We recall
that under physiological conditions, sera are often found in which the
availability of iron to this and other pathogens is either greatly
restricted or relatively unrestricted depending upon the percentage
saturation of the siderophilin of the sera. Many of these results
present new challenges to our understanding of the metabolism of Staph.
aureus and require further investigation to elucidate the physiological
response of this pathogen to the environmental changes that naturally
occur in serum in vivo,
B. In an effort to prepare an iron -low, satisfactory growth medium for
Staph, aureus, we devised a simple procedure for rapid and efficient
iron removal from the complex culture medium, trypticase. The procedure,
to be published, makes use of the difficultly water soluble bathophenan-
throline as iron coraplexer followed by extraction of the remaining traces
of bathophenanthrollne in the medium with organic, immiscible solvents.
The method reduces the labor of preparing an iron-low medium from a tedi-
ous, uncertain, time-consuming chore to a 3 to h hour treatment to yield
large quantities of final medium ■whose iron content is below the sensi-
tivity of the most sensitive colorimetric test available. Use of iso-
topic iron Fe£? indicates that the iron concentration is below 0.001 u-g
per ml of final medium.
C. In association with Dr. Knight of the Laboratory of Clinical Inves-
tigations we participated in an extended study of a case of Lupus Nephrosis
which, upon examination, disclosed an unusually low concentration of
siderophilin in the serum. The serum iron was extremely low because of
the severely reduced siderophilin concentration and the low percentage
iron-saturation of that present. We suggested the possibility of sider-
ophilin loss in the urine and proceeded to analyze uring samples for this
protein. At the height of the proteinuria, we found approximately 10$
of the total protein lost in a 21; hour period (ca. 20 grams) to be sider-
ophilin. Iron was also being lost through the kidney along with the sider-
ophilin. Calculation showed that the patient was losing an amount of her
iron-binding protein in 2U hours which was equivalent to that in the circu-
lation at any given moment. The details of this investigation and the ac-
companying clinical data were presented by Dr. David Rifkind, et al, at
the recent (Nov. $, i960) Regional Meeting of the American College of
Physicians.
53
Serial No. NIAU) 63
D. Our method employing Staph, aureus for the evaluation of the integri-
ty of isolated siderophilins has been improved by the use of the ultra-
filtrate of normal human serum plus 0.1$ iron-free trypticase as bacterial
growth medium. This medium better approximates whole serum employed as a
control than previously used media. Three new isolated siderophilin
preparations have now been found by this method to be equivalent in their
iron-donation capacities to the native siderophilin present in serum.
There is now considerable hematological interest in the possibility t
human tissues other than bone marrow and reticulocytes may obtain their
iron from the siderophilin-iron complex by the mechanism for which Staph.
aureus is the model.
Significance to the bio-medical research and the program of the
Institute ; The study of the naturally occurring iron-chelating serum
protein, siderophilin, contributes to our understanding of the non-
specific immunity mechanism available to humans for protection against
microbial infection. Investigation of the qualitative and quantitative
effects of siderophilin on the growth and metabolism of the pathogen
Staphylococcus aureus will provide useful information on the pathogenicity
of this bacterium especially under conditions closely approximating in
vivo conditions.
Proposed Course of Project: We shall investigate in detail the
growth, metabolism, and enzymatic capabilities of S. aureus when grown
under conditions approximating the in vivo state with especial regard
to the effect of different serum iron-saturation levels on such charac-
teristics. Special emphasis will also be placed upon the biochemical
mechanisms by which iron controls the overall metabolism of ihe orga-
nism. In these studies, changes in the enzymatic function and the lo-
calization of iron containing components within various cellular struc-
tures will be investigated. Additional biological features such as
hemolysis, effectiveness of phagocytosis, and possible differences in
response to antibiotics of high and low iron -containing cells will be
studied.
60
SERIAL No. NIAID 63
Part B. Honors Awards, and Publications
Publications other than abstracts frora this project:
Schade, Arthur L. : The microbiological activity of siderophilin.
Clinica Chimica Acta, in press.
Schade, Arthur L.: Methods applicable to the study of siderophilin.
Behringwerk Mitteilungen, in press.
Honors and awards relating to this project:
Lectures:
By invitation, visited the laboratories and gave lectures ap-
propriate to the interests of the audience on different aspects of,
"Iron metabolism of bacterial pathogens and host tissues as mediated
by siderophilin", at the following institutions:
1. Univ. of Vienna; Dept. of Physiology, Prof. Auerswald - Oct. 21 & 2lw
2. Universities of Marburg and Giessen; Medical School, Prof. Schultze.
Nov. 3 and U.
3. Univ. of the Saar; Medical School, Prof. Ruranel - Nov. h and £.
U. Univ. of Freiburg; Medical School, Prof. Heilmeyer. Nov. 7 and 8.
$. Univ. of Berne; Dept. of Organic Chemistry, Prof. Nitschmann. Nov.9 & 10,
6. Univ. of Zurich; Medical School, Dr. Hitzig. Nov. 11 and 12.
7. Univ. of Tubingen; Medical School, Prof. Bennhold. Nov. lU and l£.
8. Univ. of Frankfurt; Dept. of Pharmacology, Prof. Heinz. Nov. 17 •
9. Berliner Mikrobiologische Gesellschaft Meeting; Prof. Horing oflftie
Free University of Berlin. Nov. 22.
10. Univ. of Lund; Dept. of Clinical Chemistry (Malmfl), Prof. Laurell.
Nov. 28 and 29.
61
Serial No. NIAID 63-A
1. Infectious Diseases
PHS-NIH 2» Jfedical & Physiological
Individual Project Report , _.. . Bacteriology
Calendar Year I960 3' Bethesd^ Haiyland
Part A.
Project Title: Chemical Aspects of the Specific Binding of
Iron to Serum Siderophilin and Egg White
Conalbumin.
Principal Investigator: Dr. Robert C. Woodworth
Other Investigators: Dr. Arthur L. Schade
Cooperating Units: None
Man Years (calendar year I960)
Total: 12/12
Professional:
Other: 5/l2
Project Description:
Objectives : The goals of this project are to investigate the
chemistry of the reactive groups in the siderophilin molecule responsi-
ble for the specific binding of iron and to determine the physico-chemical
means by which the iron -siderophilin complex can be dissociated under
physiologic conditions obtaining in pathologic and normal hosts.
Methods Employed: Methods include the employment of immunological
assays for purity and for content of the iron-binding proteins, radioac-
tive tracers, spectral analyses, ion-exchange chromatography, electropho-
resis, polarography, and polarimetry.
Major Findings: The previously reported technique of determining
the iron-release rate of the iron-sidercphilin complex by me£p^lggeJJcor&9
isotope exchange has been applied to a detailed study of various/ affect-
ing the dissociation of iron from siderophilin.
Reaction: siderophilin-Fe2^ + 2Fe^9 > siderophilin-Fe2^ + 2Fe^ .
Assay: antibody + siderophilin-Fe2^9 + 56)..— .> antibody-siderophi-
lin-Fe2^ + ^ '| . Coun Lpitate for Fe™ activity.
Part B included. Yes
62
Serial No. NIAID 63-A
After requisite mathematical manipulation, the data
were plotted and consistently showed two intersecting straight
lines, rather than a single straight line. The occurrence of
the two intersecting lines may be interpreted to mean that the
two irons bound to a siderophilin molecule are attached to two
distinct sites whose properties differ in such a way that a
specific site must release its iron first in order that the
other site may release its iron. A mathematical analysis of
the system under study has been generously provided by Dr.
Clifford S. Patlak, Biometrics Branch, NIMH. By setting up
and solving the applicable differential equations, Dr. Patlak
has provided us with a method for obtaining from our data the
different dissociation rates of the two bound iron atoms.
The effect of pH on these dissociation rates has been of
considerable interest to us. Studies of the two iron-release
rates were made in whole serum at various pH's which were
established by varying the percentage composition of the carbon
dioxide-nitrogen atmosphere above the serum. It appeared that
the first dissociation rate was dependent on the square of the
hydrogen ion concentration, i£., if the hydrogen concentration
were doubled, the rate quadrupled; and that the second dissoci-
ation rate was independent of pH. A study of the effect of
buffer concentration at constant pH revealed, however, that the
first dissociation rate was dependent on the first power of the
concentration of the carbon dioxide-bicarbonate buffer system.
Since this buffer system had been used for the pH studies, it
became apparent that the rate of dissociation of the first iron
was actually dependent on the first power of the hydrogen ion
concentration and on the first power of the carbon dioxide-bi-
carbonate buffer.
In an attempt to determine whether the iron release rate is
subject to "general acid catalysis" we examined the concentration
effects of two other buffer systems, namely tris-(hydroxyraethyl)
amino methane and glycylglycinate, both in the region of pH 7*
Not only were the isotope exchange rates considerably slower in
these buffers than in the carbon dioxide-bicarbonate system, but
they were not appreciably affected by large shifts in buffer con-
centration. It would thus appear that hydrogen ion and carbon
dioxide-bicarbonate buffer are specific acid catalysts for the
dissocation of iron from iron-siderophilin.
Associated with each iron in the iron-siderophilin complex is a
bicarbonate ion. We found we could tag this bicarbonate with C11* by sup-
plying C-L'^02 as the sole source of bicarbonate when forming the complex
from iron (ferrous) and iron-unsaturated siderophilin. The C^ tag re-
mained with the siderophilin during precipitin and washing procedures.
A "double-tagging" experiment in which we followed both the rate of
63
Serial No. NIAID 63-A
dissociation of HCr-^C^" and the rate of incorporation of Fe'* showed that
the bicarbonate may be released from the complex 10 to 100 times faster
than the ir.n released. However, this experiment should be repeated tinder
conditions more favorable to accurate determination of the desired rates.
The effect of temperature on both iron dissociation rates was
studied. Both rates appeared to decrease with decreasing temperature, but
no quantitative treatment of the data has been carried out.
Significance to bio-medical research and the program of the
Institute: The shifts in bound iron levels occurring as a result of
infectious diseases, acute infections, and allergic reactions are recog-
nized. The availability of plasma iron to bacterial pathogens is a func-
tion of the siderophilin molecule's affinity for iron and of its degree
of iron-saturation. Knowledge of the chemical bases of this affinity
should elucidate the manner in which siderophilin governs the transfer of
iron to the host as well as to the pathogen.
Proposed course of project; Since Dr. Woodworth is presently
on an N.I.H. Research Fellowship with Prof. C.-E. Laurell in Malratt,
Sweden and since he may choose to seek a University professorship rather
than apply for reassociation with N.I.H. it appears that the course of
this project will be: first, to prepare in comprehensive form for pub-
lication the accumulated data thus far obtained, and, second to pursue,
as opportunity permits in his absence, the problem of the rates of as-
sociation of iron with siderophilin in serum, particularly under physio-
logical conditions, so that from the rates of dissociation and association,
the concentration of free ionic iron in serum can be approximated.
-3-
G*{
Serial No. NIAID 63-A
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Woodworth, Robert C. and Schade, Arthur L#: Immunological Precipitin
Titrations based on Radioactive Tagginf of the Iron Naturally
Chelated by the Proteins Siderophilin and Conalbumin. Accepted
for publication, Biochimica et Biophysica Acta.
Honors and Awards relating to this Project:
Dr. Robert C. Uoodworth was awarded an N.I.H. Fellowship (National
Heart Institute) to study during 1960-61 with Prof. Dr. C.-B. Laurell,
Head of the Keraiska centrallaboratoriet, Lund University, Malmfl, Sweden,
-U-
Serial No. NIAID 63-B
1 . Infectious Diseases
2. Medical & Physiological
PHS-NIH Bacteriology
Individual Project Report 3. Bethesda, Maryland
Calendar Year I960
Part A.
Project Title: Biochemistry of the acquisition of iron by
mammalian tissues as mediated by the iron-
binding protein, siderophilin
Principal Investigator: Dr. Arthur L. Schade
Other Investigators: Dr. Robert C. Woodworth
Cooperating Units: None
Man Years (calendar year I960)
Total: 17/12
Professional: 6/12
Other: ll/l2
Project Description:
Objectives: To investigate the mechanism by which mammalian
tissues acquire from serum siderophilin the iron required for normal
growth and function and to study the effect of physiological conditions
upon this exchange.
Methods Employed: Hematological procedures, radioactive
tracer techniques, manometric techniques and immunologic analyses.
Major Findings: Reticulocytes remove iron from siderophilin
up to 67 times as fast as the natural dissociation rate of iron-
siderophilin complex under physiologic conditions of pH and ionic
strength. The rate of removal of iron from siderophilin by reticulo-
cytes is independent of percentage saturation of siderophilin. Both
iron atoms are removed by reticulocytes from this chelating protein
even in the presence of excess ionic iron while iron chelated with
conalbumin or complexed with ethylenediarnine tetraacetic acid (versene)
or in an ionic form is not removed or taken up at significant rates.
The absolute rate of iron uptake by reticulocytes appears to be a func-
tion, inter alia, of the concentration of iron-siderophilin in such
manner as to suggest that the cells possess a finite number of specific
sites for accommodating the iron-siderophilin complex. The amount of
iron taken up by a given blood sample is dependent on the number of
reticulocytes present and no demonstrable iron is absorbed by the mature
red cells. Reticulocyte uptake of iron is markedly restricted by
anaerobic conditions. This fact suggests either that the iron exchange
Part B included: Yes gg
Serial No. NIAID 63-B
from siderophilin to the cell is an oxidative, energy requiring process
or that the synthesis of an iron acceptor system leading to hemoglobin
production is oxygen dependent. Attempts thus far to approximate the
iron uptake of the intact reticulocytes by a cell-free hemolysate pre-
pared from reticulocytes have not yet yielded definitive results.
A significant practical consequence of this work is our development
of a simple physiological test of the "integrity" of isolated, purified
serum siderophilins in terms of their iron-donating or iron exchange
capabilities. Progress in the study of the clinical uses of such isolated
siderophilins has been stifled by the absence of such a test. Now, the
employment of our reticulocyte iron-uptake method promises to stimulate
the production of isolated siderophilin and its application to a great
variety of experimental and clinical investigations. Our reticulocyte
and Staphylococcus aureus iron-uptake tests together have served as
guides to the American National Red Cross in its efforts to produce, on
a large scale, purified siderophilin for such investigations. New methods
of siderophilin isolation are being sought so as to preserve these phy-
siological activities intact.
Significance to bio-medical research and the program of the Insti-
tute: As a first approximation, the normal functioning of host tissues
requires iron for synthesis of their heme enzymes. Hence, the conditions
of availability of serum iron to host tissues and the mechanism of its
transfer from siderophilin are of significance to host-parasite relation-
ships obtaining in infections.
Proposed course of project; We propose to investigate with the
satisfactorily active isolated siderophilin, which heretofore has not
been available but is now at hand in small quantity, the relationships
of the absolute concentrations of iron-siderophilin to iron exchange and
metabolism by reticulocytes and Staphylococcus aureus cells. We wish
further to investigate the basis for the indicated catalysis of iron ex-
change by bone marrow and reticulocytes and to determine whether such
a mechanism applies to non -erythropoietic tissues as well.
67
-2-
Serial No. NIAID 63-B
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Avrards, and Publications
Publications other than abstracts from this project:
Honors and Awards relating to this Project:
Named consultant to the American Red Cross to gather information
for them on the serum protein fraction, siderophilin, at Wiesbaden
and Bruges internists1 and scientists1 meetings in the Spring of
I960.
Gave invited lecture on "Siderophilin, Its Characteristics and
Functions" to the research laboratory personnel of Parke, Davis & Co,
on August 3>, I960.
-3- 68
Serial No. NIAID 63-C
1. Infectious Diseases
PHS-NIH 2. Medical Sc Physiological
Individual Project Report 3. Bethesda, Maryland ^
Calendar Year i960
Part A.
Project Title: Development of an Economical 280mu. Light
Source Suitable for Detecting Proteins in
Effluents from Chromatographic Columns.
Principal Investigator: Dr. Robert C. Wood-worth
Other Investigator:
Cooperating Units: None
Man Years (calendar year i960)
Total: 2/12
Professional: l/l2
Other: 1/12
Project Description:
Objectives: We desired to develop a relatively low-cost light
source for continuous photometric monitoring of chromatographic column
effluents at X 280mu-.
Methods Employed: Spectral analyses of filters developed in the
course of the research and of the light source itself with various fil-
ters.
Major Findings: Filter combinations have been described by others
for the isolation of various regions of the ultraviolet line spectrum of
mercury. These filters provided a starting point for our investigations,
but the combination suggested for the 280mu- region provided too wide a
band-pass for our needs. This particular filter combination consists
of (1) a 2mm Corning red-purple Corex A filter No. 9863, (2) a 1M NiSOk
aqueous solution in a 1 cm light path vycor or quartz cuvette, and (3)
a 0.02^ aqueous solution of 2,7-dimethyl-3,6-diazocycloheptadiene-l,6-
iodide (hereinafter referred to as "cyanine" iodide) in a 1 cm light path
vycor or quartz cuvette. These three are placed in series between the
lamp and photocell. The "cyanine" iodide possesses an absorption minimum
at 263m[i. Dr. E. Kravitz of the National Heart Institute provided a
pure sample of phenazine-a-carboxylic acid (produced by Pseudomonas
aureofaciens). A 0.02$ aqueous solution of this substance (neutralized
with KOH to make it soluble) possesses an absorption minimum at 290n|i..
An equal -weight mixture of "cyanine" iodide and phenazine-a-oarboxylic
acid made to a total of 0.02^ in water (with KOH just s
dissolve the acid) possesses an absorption minimum at 280m(j.. T
69
Part B not included. -1-
Serial No. NIAID 63-C
solution in a 1 cm vycor or quartz cuvette, in series with a Corning red-
purple Corex A filter and 1 cm of 1M NiSOr, as described above, provides
a spectrum -ath only a single, narrow transmission band with a maximum of
280mp.. Appreciable transmission appears again only in the far red
(750mu.), a wavelength region to which commonly-used phototubes, i.e.,
No. 93S>> are completely insensitive. The filter has proved stable to
ultraviolet radiation over an in-service period of six months, i.e., the
transmission spectrum of the filter has remained unchanged.
When the output of a General Electric H3FE low-pressure mercury a
lamp is passed through this filter system and analyzed with a Beckman
HJ spectrophotometer, one finds many mercury emission lines other than
280mu.. This output spectrum is closely similar to that obtained from
the same lamp filtered by a Corning red-purple Corex A filter together
with a Baird Atomic 280mji interference filter. This latter filter system
and light source are used in a commercially available protein-monitoring
unit. A vast improvement in the mono chromic ity of filtered light from
this lamp is obtained from our filter system by increasing the "mixed-
organic" filter ($0% each of "cyanine iodide~phenazine-a-carboxylic
acid) concentration three-fold to 0.06$. If the 280. hmu. transmission is
now adjusted to lOOJo, the only other significant transmission lines
found are 2h% at 275mu and 21$ at 289*2mjx, both of which are strongly
absorbed by proteins.
The "mixed-organic" filter has the additional virtue of possessing
a variable transmission maximum between 263 and 290mu., which is dependent
on the per cent composition of the filter. Thus, it may be useful for
isolating other spectral lines than 280mu. in this region.
The shops at NIH have done preliminary work on a stable photometric-
recording system for use with this light source, but it is as yet not
ready for use.
Significance to bio-medical research and the program of the In-
stitute; The availability of an economical photometric analyzing system
for protein-containing chromatographic effluents makes possible the
release for more important work of an individual, professional or tech-
nical, from the time-consuming, routine task of reading individual frac-
tions in a manual spectrophotometer. Further, the use of a light source
which will be specifically absorbed by proteins is of paramount importance,
in order to provide adequate sensitivity for minor components and to
make possible quantitative estimations of protein content, if this should
be desired.
The 25Limu, line of mercury utilized in some commercially-available
ultraviolet monitoring devices is not suitable for use with proteins be-
cause of its demonstrated denaturing effects.
Proposed Course of Project: A more satisfactory light source might
be found, i.e., one with fewer emission bands close to the 280mn line.
Such a lamp would allow for a lower concentration of the "mixed-organic"
filter and thus permit a higher intensity of usable light. Search iov-, ^
such a light source will continue. ' '-'
Serial No. NIAID 6U
1. Infectious Diseases
2. Medical & Physiological
Bacteriology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Detoxification of Potential Tuberculostatic,
Fungistatic, Parasiticdal and Viricidal Agents.
Principal Investigator: Dr. Benjamin Prescott
Other Investigators: None
Cooperating Units: None
Man Years (calendar year I960)
Total: 13/12
Professional: 3/l2
Other: 10A2
Project Description:
Objectives: Further studies on toxicity and detoxification
of the tuberculostatic drugs streptomycin (SM), isoniazid (INH) and
mixtures of streptomycin-isoniazid (SM-INH) in two strains of mice.
In addition, to find means of reducing the toxicity of the widely used
therapeutic agent Neomycin and of several parasiticidal drugs.
Methods Employed: The effect of various chemicals on the
toxicity of INH and/or SM and Neomycin was measured by the effect on
survival time of normal mice following simultaneous subcutaneous ad-
ministration of a given adjuvant with a lethal dose of drug (both
single and multiple administrations). The in vitro bacteriostatic action
of the drug-adjuvant combination was also determined.
Major Findings:
1. With glycerol formal as solvent. Studies initiated
with glycerol formal as solvent for INH and/or SM have shown that
with a lethal 6 mg oral dose of INH in a 2$% glycerol formal solution
(in water) permitted 95% survival of a group of 60 white mice and
90^ survival in DBA mice (50). However, on repeated daily administration,
Part B included: Yes
71
Serial No. NIAID 6U
optimal results with no toxic manifestations were found in mice given
a 5tng dose (LD_, ) in 2>S% solvent.
The use of this solvent with SM has thus far shown that a single
subcutaneous administration of a lethal 15 mg dose of SM in 3S% solu-
tion permitted 100/! survival of mice. Decreasing the concentration
of adjuvant to 25% permitted 100% survival and a 1$% solution 90^.
The detoxifying action of this solvent was tested on SM-INH mixtures
in two strains of mice. 85$ of the mice tolerated a lethal mixture
of 10 mg SM and h mg INH and 80% survival with 10 mg SM and 8 mg INH.
2. With steroids; Further studies with steroids showed
that 55$ of both white and DBA mice tolerated a lethal dose of 30 mg SM
and h mg INH when administered subcutaneous ly with 25 mg of sodium tauro-
cholate. With a lethal dose of 20 mg SM and 6 mg INH or 15 mg SM and
8 mg INH, the same amount of adjuvant permitted 80 and 90% survival,
3. With L-argininyl-L-glutamate : Mice survived a lethal
15 mg dose of SM when it was administered in combination with 58 mg of
the peptide L-argininyl-L-glutamate in both single and repeated doses,
U. With Miracil D: Further studies with this drug included
the use of various steroids as possible detoxifying agents. Four
steroids (cholic acid, sodium taurocholate, sodium glycocholate and so-
dium glycotaurocholate) were tested as adjuvants with various doses of
Miracil D. Of the four steroids tested, sodium taurocholate showed good
detoxifying activity. 100$ of the mice tolerated a 30 rag dose of Miracil
D (6 x lethal dose) when it was administered simultaneously with 50 mg of
sodium taurocholate,
5. Detoxification of Neomycin: Since sodium glucuronate
and glycine proved effective in permitting mouse survival when administered
simultaneously with toxic doses of INH, SM and mixtures of SM-INH, the
detoxifying activities of these adjuvants were tested with Neomycin in two
strains of mice. When 10 mg of Neomycin per 20 g mouse (singly lethal)
was injected simultaneously with a mixture of 50 mg glycine and 50 mg
sodium glucuronate, 99% of the mice survived. Even if the Neomycin dose
was increased to 20 mg, 90% of the mice survived. In chronic toxicity
tests, 60$ of the animals tolerated 15 repeated injections of the 10 mg
dose of Neomycin if 50 mg glycine and 50 mg sodium glucuronate were in-
jected simultaneously. No interference by the detoxifying agents tested
was observed on the in vitro bacteriostatic action of Neomycin on one
strain of Staphylococcus aureus.
-2-
72
Serial No. NIAID 6U
6. YJith certain vitamins; Of 16 vitamins similarly
tested with Neomycin, nicotinic acid and calcium pantothenate exhibited
detoxifying activity. The last named in a molar ratio adjuvant and
drug gave optimal results in combination with 10 mg of Neomycin. Sur-
vival was 100£ in two strains of mice. Up to 1$ daily doses of the
Neomycin with calcium pantothenate were tolerated by 100% of the mice.
7. With amino acids: On single administration of a 1
mg dose of Neomycin per 20 gram mouse, D-glutamic, L-aspartic, and
acetyl DL-methionine permitted 70 to 86^ survival of white micej 18 other
amino acids tested showed little or no activity.
Significance to bio-medical research and the program of the
Institute: The widespread use of the chemotherapeutic agents strepto-
mycin and isoniazid in the treatment of human tuberculosis is limited
by their toxicity. Similarly use of Neomycin is also restricted.
Means of increasing the tolerance of these toxic chemotherapeutic drugs
should permit more effective therapy.
Proposed course of the Project: This study will be extended
to include in vivo tests and to a few more adjuvants that show promise.
73
-3-
Serial No. NIAID - &k
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Prescott, B., Kauffmann, G. and Stone, H. J.: Means of increasing
the tolerated dose of isoniaaid-streptomycin mixtures in mice.
II. Certain Vitamins. Antibiotics and Chemotherapy 10,
163-168, I960
Honors and Awards relating to this Project:
Invited to participate in "Symposium uber experimentelle und
klinische Pharmakologie der Antibiotika" to be held in Aachen, Germany,
May 18-19, 1961.
-k- v*
Serial No. NIAID 6U-A
1. Infectious Diseases
2. Medical & Physiological
Bacteriology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Potential Fungistatic and Parasiticidal Agents.
Principal Investigator: Benjamin Prescott
Other Investigators: None
Cooperating Units: Section on Mycology, LID-NIAID
Dr. Chester W. Emmons - Serial No. ?6~B
Section on Chemotherapy, LPC-NIAID
Dr. George Luttermoser ■ - Serial No.
Man Tears (calendar year I960)
Total : 13/12
Professional: 3/12
Other: 10/12
Project Description:
Objectives: Synthesis of new potential non-toxic cheraothera-
peutic agents against fungal and parasitic infections.
Methods Employed:
Antifungal agents: A series of 30 dithiooxamide derivatives
were synthesized by treating a solution of dithiooxamide with hydrazine
hydrate forming a dihydrazide. The resulting compound was then con-
densed with various aliphatic and aromatic aldehydes in $$% ethyl alco-
hol. The final recrystallized products were analyzed for carbon, hydro-
gen, nitrogen and melting points obtained. Toxicity studies were per-
formed in white mice. Since thiosemicarbazones have been shown to have
chemo therapeutic activity (tibione against tuberculosis and isatin thio-
semicarbazone against viruses), a series of 50 long chain thiosemicarba-
zones were synthesized by first preparing octadecylthiosemicarbazide and
condensing this compound with various aliphatic, aromatic and heterocyclic
aldehydes.
Part B not included.
75
Serial No. NIAID 6U-A
Additional derivatives of thymol, naphthoquinones and
U,U'diaminodiphenylsulfone were synthesized. In mice, toxicity of
these compounds was of a very low order. Dr. Chester W. Emmons,
LID-NIAID, will test the fungistatic activity of these compounds.
Parasiticides; A new series of piperazine derivatives
were synthesized by condensing N-aminoethyl piperazine with aldehydes,
carboxylic acids, sulfonic acids, isocyanates and isothiocyanates.
In addition, H5> derivatives of tetracycline were prepared by coupling
tetracycline with compounds like atabrine, chloroquine, piperazine
monocarboxylic acid, sulfanilic acid, hetrazan and several naphtho-
quinones.
Major Findings; Several of the tetracycline derivatives
demonstrated considerable in vitro activity against parasites and
showed negligible toxicity in mice.
Significance to bio-medical research and the program of
the Institute; Because of high tolerance and effectiveness, the
tetracycline derivatives may be useful in the chemotherapy of human
parasitic infections.
Proposed course of Project; Tests for usefulness of the
antifungal and antiparasitic compounds are to be performed in vivo
in laboratory animals (mice and dogs) to determine their effectiveness
in experimental infections. All the compounds synthesized for anti-
fungal and antiparasitic activity are also being tested for anti-tumor
activity. About UOO compounds have been sent to the Cancer Chemo-
therary screening program. Several long chain thiosemicarbazones have
passed the initial tests in mice.
76
-2-
Serial No. NIAID 6U-B
1. Infectious Diseases
PHS-NIH 2. Medical & Physiological
Individual Project Report ^ Bethesda, Ha^land10^
Calendar Year I960 J ' *
Part A.
Project Title: Isolation of Antibacterial and Antiviral
Substances from Shellfish
Principal Investigator: Benjamin Prescott
Other Investigators: None
Cooperating Units: Laboratory of Virology and Rickettsiology,
Division of Biologies Standards
Dr. Chen Pien Li - Serial No. DBS
Man Tears (calendar year I960)
Total: 16/12
Professional: 6/l2
Other: 10/12
Project Description:
Objectives: To isolate and purify potential therapeutic
agents from shellfish.
Methods Employed: Juice from fresh frozen abalone and extracts
of oysters and clams were each dialyzed against distilled water, the
residue adjusted to pH 7.8 and applied to anion-exchange (diethylaraino-
ethylcellulose)columns set up on automatic fraction collectors in the
cold (U° C). ELution of material from the column was carried out' with a
series of tris-HoPO^ buffers of varying ionic strength and pH (3.7 - 7.6)
and a final wash with M-NaCl. Eluates were consecutively collected.
Several pools were made of various fractions, dialyzed against distilled
water and lyophilized.
Major Findings: It was found that the early eluate pool frac-
tions contained antibacterial activity against both penicillin-sensitive
and —resistant strains of Staphylococcus aureus, a beta-hemolytic strain
of Streptococcus pyogenes, Salmonella typhi, and S. paratyphi A and B.
The growth of 10,000 or wore organisms per ml was inhibited by these
fractions in a concentration of 10 mg per cent. These fractions showed
no antiviral activity. The fraction isolated from the final wash of
the column with M-NaCl showed no antibacterial activity. However,
there was definite inhibitory activity against influenza A virus and poly-
oma virus in tissue culture.
Part B included: Yes
-i. 77
Serial Mo. NIAID 6U-B
S' -mif icance to bio-medical research and the program of the
Institute: The abundance and variety of shellfish is a potential
source of antibacterial and antiviral agents. Since fractions isolated
fron this source were found to possess marked antibacterial activity,
they may be valuable as therapeutic and prophylactic agents.
Proposed course of Project: Preparation of large quantities
of active fractions and chemical identification of the agent are in pro-
gress. In addition, the therapeutic efficiency of the agents is being
studied in protection experiments in mice experimentally infected with
streptococci and pneumococci.
-2-
78
Serial No. NIAID 6U-B
PHS-NIH
Individual Project Report
Calendar Year I960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Prescott, B. and Li, C. P.: Abalone juice: Fractionation and antibac-
terial spectrum. Proc. Soc. Exp. Biol. & Med. (in press)
Honors and Awards relating to this Project:
None
-3- 73
Serial No. NIAID 65
1. Infectious Diseases
2. Medical & Physiological
Bacteriology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Longitudinal Studies of Beta-hemolytic
Streptococcal Isolations.
Principal Investigator: Roger M. Cole
Other Investigators: Edythe J. Rose
Cooperating Units: Dept. of Pathobiology .
School of Hygiene and Public Health
Johns Hopkins University
(Dr. Wm. J. L. Sladen)
Bacteriology Laboratory
Johns Hopkins Hospital
(Dr. Jack Causton
Man Tears (calendar year I960):
Total: 30/12
Professional: 12/12
Other: 18/12
Project Description:
Objectives: An aspect of studies continued in this laboratory
for several years, to extend knowledge of human experience with strepto-
cocci through culture, serologic and epidemiologic methods.
Methods Employed: In general, similar to laboratory and epi-
demiologic methods described in projects for other years. In particular,
throat swabs taken routinely from personnel in isolated situations in
Antarctica were mixed in glycerine broth which was then deep-frozen and
transported to the United States. Some months later, samples were plated
from the thawed fluids and colonies of beta-hemolytic streptococci picked
for serologic identification and tested for bacitracin sensitivity at a
level said to distinguish Group A from other beta-hemolytic streptococci.
Sera from the same personnel were taken at intervals and kept frozen for
future reference. Simultaneous throat swabs were taken for eola-
tion by another group (Virus & Rickettisal Section, LID). In the past,
Part B included: Yes
_!_ 80
Serial No. NIAID 65
somewhat similar studies have been made in institutionalized children
(Jr. Village, D.C.); and studies of other methods of transport of
streptococci prior to culturing were made in collaboration with inves-
tigators in California (U. Cal. Berkeley: see publication, Part B).
Major Findings: Approximately" 2lj.O cultures of beta-hemolytic
streptococci have been received from the collaborating laboratory for
identification. Most of these represent the same persons, repeatedly
positive for streptococci of the same group or type of Group A. About
38$ of the cultures were Group A, 25$ Group B, 6% Group C, 30$ Group
G, and the small remainder failed to grow or to react serologically.
About Gh% were bacitracin-sensitive by the test used: 98$ of Group
A were sensitive as expected, but so were 57$ of Group C, U9$ of Group
G and 31$ of Group B. The test as used is obviously not an adequate
screening method for distinguishing Group A from other beta-hemolytic
streptococci.
Significance to bio-medical research and the program of the
Institute: Only in isolated or semi-isolated situations with limi-
ted possibilities for introduction of new organisms can the persistence
of throat flora be adequately examined. Whether, under such conditions of
carriage, beta-hemolytic streptococci of Group A induce antibody forma-
tion (and if so, how or why they persist) is a matter of considerable in-
terest in evaluating the various aspects of streptococcal virulence and
resistance to streptococcal infections.
Proposed Course of Project: Continue. Antibody responses will
be tested by improved methods described under another project. Addi-
tional culture and serum specimens are expected from Arctic areas within
the next year.
81
Serial No. NIAID 65
PHS-NIH
Individual Project Report
Calendar Year I960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Hollinger, N. F., Lindberg, L. H. , Russell, E. L., Sizer, H. B.,
Cole, R. M. , Brewne, A. S., and Updyke, E. L. : Transport
of streptococci on filter paper strips. Pub. Hlth. Rep.,
75: 251-259, I960.
Honors and Awards relating to the project:
None
82
Serial No. NIAID 65-A
1. Infectious Diseases
2. Medical & Physiological
Bacter iology
3. Be t he s da, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Streptococcal M protein, virulence, and
type-specific immunity.
Principal Investigator Dr. Jerome J. Hahn & Dr. Roger M. Cole
Other Investigators: None
Cooperating Units: Laboratory Histology and Pathology,
NIDR (Dr. David Scott)
(Dr. Marie Nylen) NIDR " **
Laboratory Biology of Viruses,
NIAID (Dr. C.F.T. Mat tern)
Man Years (calendar year I960):
Total : 50/12
Professional : 20/12
Other: 30 /\2
Project Description:
Objectives: In general, to study G, up A streptococcal M
protein and of antibody thereto, in order to oetter understand viru-
lence and resistance to infection among gram-positive cocci. Speci-
fically; (a) to improve methods of preparing antigenic M protein and
to determine the differences between M proteins which account for type
specificity; (b) to investigate and improve methods for detecting anti-
body to M protein; (c) to study the relation between M protein, or other
possible cellular components, to streptococcal virulence; and to deter-
mine more precisely the cell wall localization of M protein and its
significance.
Methods Employed:
(a) Neutralized acid extracts of cells of 5 strepto-
coccal types were precipitated with saturated picric acid, and the
precipitates extracted with 8o£ acid ethanol: supernates were pooled
Part B included: Yes
-1- 83
Serial No. NIAID 6g-A
and further precipitated by acetone. These washed and dried precipi-
tates were dissolved in saline and gradually neutralized, with removal
of precipitates as formed. Active material was in the supernates,
which were analyzed for M protein immunologically, for carbohydrate
by the Molisch reaction, and for nucleic acid by the orcinol method.
The immunologic tests were made by antigenicity in rabbits and by "type-
specific precipitation in liquid and in agar gel diffusion systems.
M proteins, prepared by both the conventional (acid hydrolysis-
ammonium sulfate precipitation) method and the picrate method, have
been subjected to Immunoelectrophoresis. In addition, attempts to
fragment M protein into immunologically reactive portions are being
made by differential heating with pH control and by fractional enzy-
matic degradation.
(b) Detection of type-specific antibody to M protein
has been examined by two methods: (l) the long chain reaction, in
which streptococcal chains are significantly longer in homologous anti-
serum than in heterologous or normal serum, and (2) inhibition by un-
labelled test antisera of fluorescence on addition of fluorescein-
labelled specific antiserum. Both methods have been compared with the
usual precipitin and bactericidal tests. The nature of type-specific
antibody is being examined by conversion to univalent fragments by
pepsin-cysteine or papain treatment. Such univalent antibody will be
tested by blocking of precipitation, blocking of fluorescence, precipitation,
specific fluorescence after labelling, bactericidal activity, and the long
chain reaction.
(c) Virulence of streptococcal strains is tested by intra-
peritoneal injection of culture dilutions in mice, with subsequent de-
termination of $0% lethal doses. Bactericidal and phagocytic tests in
rabbits or human whole blood with or without added specific sera, or with
mouse leukocyte suspensions, are also used to detect presence in living
cells of the presumptive virulence factor, M protein. Addition of homo-
logous and heterologous, partially purified, M proteins to phagocytic
systems, has been used to determine if the extracted proteins enhance
virulence by protection against phagocytosis.
Differential blocking of fluorescence by group and type-specific
antisera applied in different sequences, is being used to aid in localiza-
tion of M and of group carbohydrate in the streptococcal cell wall. Sec-
tions of fixed and embedded streptococci have been made and examined under
the electron microscope, as a preliminary to the use of ferritin -labelled
antibody for the electron microscopic localization of cell wall components.
8'<
Serial No. NIA3D 6g-A
TTajor Findings:
(a) M protein picrates, made as described, contain
less than 0.1$ each of carbohydrate and nucleic acid. During alka-
linization in their final preparation, picrates of Types 1 and 18
form precipitates at a lower pH (3.7) than do those of Types 2, kf
and 28 (pH U.5>). Picrates of Types 1, h, and 18 give precipitin re-
actions only with their homologous type antisera, whereas that of
Type 28 reacts also with antisera to Types 2, 13 and Uii. The possi-
bility of the presence of "R" protein, instead of, or in addition to,
M is thus raised; and preliminary studies in agar gels indicate that
at least two reactive antigens may be present in the picrate prepara-
tions. Early results from intramuscular injection of a Type 1 picrate
indicate that the material is weakly antigenic, requiring h weekly in-
jections of $ mg. each to produce antibody detectable in undiluted
serum by the long chain test.
Immunoelectrophoresis of conventionally prepared M proteins demon-
strates differences between Types 1 and 23. A single precipitin band
is obtained when the former is tested against homologous antisera,
whereas Type 23 always shows two bands or spurs which move together and
cannot be separated by variations in buffers, pH, voltage, nor time of
run.
Boiling M protein at an acid pH for between 30 and 60 minutes ap-
pears to produce a fragement which binds with but does not precipitate,
specific antibody. Similar studies, using enzymes, are in progress,
(b) The long chain test for type-specific antibody has
been statistically improved by a more rapid and simpler method, uti-
lizing the chi-square-analyzed differences in frequency distributions
of chains above and below a predetermined length, in test and normal
sera. Interval sampling has shown that time of incubation prior to
reading the test is of prime importance : chains increase in 1 ength,
and long chains increase in frequency, to a maximum with time and then
decrease. The decrease depends on antibody depletion; and maximum
length or frequency is reached sooner and is lower in low concentra-
tions of antibody than in high. As a result, time interval sampling
allows titration of antibody by determining the highest dilution which
gives a positive result according to predetermined statistical criteria.
The effect of dilution of streptococcal inoculum in test is opposite
to that of antibody dilution. Total coccal growth is unaffected by
the presence of antibody. The Size Class Frequency method of determining
a positive long chain reaction has been shown to be reproducible and more
sensitive than the previous method using mean chain lengths.
Fluorescein-labelled group-specific and type-spec ;era have
been successfully prepared. No cross reactions among antisera to several
85
Serial No. NIAID 6g-A
different tvpes have been shown by this method. Inhibition by unlabelled
antisera of specific fluorescent antisera appears to be a simple and sen-
sitive method for determining the presence of type-specific antibody. Its
use in titration is being compared with the long chain and bactericidal
tests, but appears somewhat limited by the subjective determination of the
fluorescent end point.
A method of labelling portions of the streptococcal cell wall by
growth in fluorescent antiserum with subsequent removal of excess anti-
body, has been devised. Its use in following cell wall formation and in
analysis of the mechanism of the long chain test is being investigated.
Univalent antibody, produced by pepsin-cysteine treatment of whole
antiserum, has been shown capable of blocking precipitin and fluorescent
reactions, but does not produce long chains. Additional studies of the
nature of streptococcal type-specific antibody are under way.
(c) M protein of Type 23 has been reported to enhance phago-
cytosis when added to a mouse leukocyte system containing antibody and
virulent Type 23 streptococci. This experiment was repeated and confirmed,
but similar enhancement in a Type 1 system was not found. No increase in
phagocytosis by heterologous II protein occurred. Because M, as the pre-
sumptive virulence factor, was expected to prevent phagocytosis rather
than the reverse, the possibility of a different virulence factor — at
least in Type 23 — arose. To see if removal of anti-M from a serum made
against virulent organisms would leave an "anti-virulence factor", M+
avirulent variants were derived from Types 1 and 23, and then used to
absorb sera prepared against virulent organisms. These antisera contained
" an ti -virulence factor", as shown by their mouse -protective capacities.
After absorption to the point of removal of type-specific precipitins, but
not of long chain producing antibodies, mouse protective antibody was still
present. Further absorption removed both long-chain producing and mouse
protective antibodies from both Type 1 and Type 23 antisera. The effect
appears to be a quantitative one, and this method failed to distinguish a
different virulence factor than M protein.
The suspected surface location of M protein on the streptococcal cell
wall was verified by fluorescence -inhibition experiments. Unlabelled
group-specific antiserum failed to block the fluorescence of labelled type-
specific antiserum, whereas unlabelled type-specific antiserum inhibited
the fluorescence of added group-specific antiserum. The type-specific
antigen (M) therefore appears to be superficial to the group antigen, or
a peculiar steric relationship may exist. Similar experiments, using
ferritin -labelled antibody and the electron microscope, are being initiated.
.u- 86
Serial No. NIAID 65 -A
Significance to bio-medical research and the program of
the Instioute: Group A streptococcal infections in man are common,
even today with the widespread use of antibiotics. Immunity to such
infections, shown in a limited number of instances and by use of dif-
ficult and tedious methods, has been said to be type-specific. The
use of rapid and improved methods such as we have described will
greatly facilitate verification and expansion of these findings and
allow epidemiological and clinical serologic studies on a scale not
previously possible.
The role in virulence of the M protein which stimulates such
type-specific antibodies is widely accepted but not understood. Mouse
virulence is the usual measure, but many 11+ strains freshly isolated from
man are not mouse-virulent without passage: other discrepancies occur,
and the possibility of other virulence factors requires further study.
Investigations of streptococci, which are well characterized anti-
genically, may serve as useful models for other gram-positive cocci such
as staphylococci which are at present poorly defined antigenically.
Proposed course of Project: Continue along lines indicated.
-5- 87
Serial No. NIAID «"*
PHS-NIH
Individual Project Report
Calendar Year I960
Part B Honors, Awards, and Publications'
Publications other than abstracts from this project:
Kantor, F. S., & Cole, R. M.: Preparation and antigenicity of M protein
released from Group A, Type 1 streptococcal cell walls by phage-
associated lysin. J. Exp. Med., 112: 77-96, I960
Honors and Awards relating to the project:
None
.6- 88
Serial No. NIAID 66
PHS-NIH
Individual Project Report
Calendar Tear I960
1. Infectious Diseases
2. Epidemiology
3. Bethesda, Maryland
Part A
Project Title: Epidemiologic Studies of Illnesses and Microbial
Experience of Junior Village Nursery Children
Principal Investigator: Dr. Joseph A. Bell, Dr. Albert Z. Kapikian
Other Investigators: Dr. Francis M. Mastrota and Dr. Robert J. Huebner
Cooperating Units: Virus and Rickettsial Diseases Section, LID,
(Dr. Chanock, Dr. Johnson), Epidemiology Laboratory
Unit, (Dr. Rosen), Oncolytic and Oncogenic Virus Unit,
(Dr. Rowe), Virology Section, Perinatal Research Branch,
NINDB (Dr. Sever), Parasitic Disease Service, LCI,
NIAID (Dr. Beye), Infectious Disease Service, LCI,
(Dr. Utz), Pediatrics Service, D. C. General Hospital,
(Dr. Reichelderfer)
Man Tears (Calendar year I960):
Total: 62/8
Professional: 19/8
Otheri U3/8
Project Description:
This is a long term, very intensive study of the illness and
microbial experiences of nursery children at Junior Village, a District
of Columbia Welfare Institution. It began in July 1955 and provides
research material for many projects, e.g. 66-A, 66-B and others.
(A) Basic Objectives: The general objective is to maintain under observa-
tion a population group suitable for epidemiologic study of occurrence of
infection and disease and host-parasite-disease-relationships as they occur
naturally in this group and as they can be altered by chemo prophylaxis
and new vaccines. One of the chief interim objectives is the development
of epidemiologic, clinical and laboratory tools and methods for studying
infectious diseases. A concerted effort is being made to find new micro-
biologic agents which cause disease, methods of identification of these
agents, modes of spread and methods for prevention and treatment of acute
illnesses, particularly respiratory illnesses. The study is designed to
shed light on the nature and scope of studies which should be pursued in
further search for methods of control of acute infectious diseasea
particularly, the large mass of acute respiratory diseases, including the
common cold.
Part B included /T7 Tea / / No 83
1
Serial No. NIAH) 66
Methods Employed and Patient Material:
The study children are located in Southwest Washington, D. C. The daily
population is now close to 130 white and negro babies six to I|0 months of age
who are in residence in Eisenhower Cottage and the Infirmary for domiciliary
care. The mean duration of residence is approximately 17 weeks per child.
Children with illnesses are studied either in their domicile or the infirmary
at Junior Village, or the Clinical Center at N. I.H., or at the D. c. General
Hospital, depending upon the severity of illness and study interest.
A full-time pediatrician, three nurses, and four nurses' assistants
maintain constant medical surveillance of the children, record rectal tempera-
tures twice every day, collect specimens for laboratory study and prepare
clinical records on each child each day, regardless of whether or not ill.
Both throat and anal specimens for virus study are collected on admission,
and three times weekly from every child, and otherwise when indicated. Blood
specimens are collected on admission, on discharge, six weeks after admission,
every three months during residence, and under special circumstances.
Major Findings:
Epidemiologic methods have been devised for recognition of febrile
departure from normal health and illnesses have been classified as (A) ques-
tionable fever, (B) definite fever with clinical findings (FUO) and (C) defi-
nite illnesses, i.e., fevers associated with clinical findings.
Two major epidemics of interest have occurred in the past year in Junior
Village.
In a period from April 2k to May 13, 36 or k0% of the 90 residents
developed pneumonia. In the Eisenhower Cottage where the older children
resided, 16 (2$%) of the 65 children contracted pneumonia while in the infirm-
ary where the younger children resided 20 (80$) of 2$ developed pneumonia.
Respiratory syncytial virus isolation from throat swabs was associated with the
pneumonia illness indicating an etiologic relationship. Also 91% of 80 infants
and children tested showed U-fold or greater rises to RS antigen by complement
fixation and/ or neutralization tests. A four-day incubation period of RS
pneumonia was also clearly shown. The pneumonia illness was severe with the
average of the highest fever in each patient being 103° F. (Rectal) and with the
duration of a fever of 100. 6°F. or greater being about k days.
An interesting outbreak of infectious lymphocytosis was recognized in
August I960 when k of the children were discovered to have white blood counts
over 60,000 per cu. mm with 95% lymphocytes on differential smear. This
initiated an intensive effort to study the epidemiology, the clinical coarse,
and the laboratory aspects of the disease. White blood counts were done once
weekly on all the 130 residents. Appropriate material has been studied in
laboratory animals and in tissue culture in attempts to f-j Jologic
agent. Stools have been tested for ova and parasites. Patients have been
admitted to the Clinical Center and D. C. General Hospital to study the
90
Serial No. NIAID 66
clinical findings. Thus far, no clinical finding except for the lymphocytosis
has been sigrificantly associated with the disease. Over 25 children have
contracted the lymphocytosis with no relationship seen to age, or length of
time in residence at Junior Village. An incubation period of 11-13 days is
postulated. With the paucity of literature on the subject of infectious
lymphocytosis, this study should add measurably to the definition of the
disease.
Significance to 3io-medical Research and the Program of the Institute:
This project provides a source of specimens for finding new causes of
illness, for studying their etiologic significance and for testing methods
of control. It is significant that long term Junior Village studies of host-
parasite-disease-relationships are being carried out on the bulk of diseases
which commonly cause misery and absenteeism from schools and industry. The
studies are of such a nature that they are not likely to be conducted by other
than a government group. The studies are progressing meticulously and fairly
satisfactorily to the investigators.
Proposed Course of Project:
Clinical epidemiological observations are being continued together with
collection of throat and anal specimens one to three times weekly for virus
study and collection of routine blood samples. It is planned to continue the
study for as long as it continues profitable.
91
Serial No. NIAID 66
PHS-NIH
Individual Project Report
Calendar Year I960
Part B Honors, Awards, and Publications
■** A Study of the Hemadsorption ( Para-Influenza)* And Other Viruses
in Children with and without Respiratory Disease
Albert Z. Kapikian, M.D., Robert M. Chanock, M.D.,
Joseph A. Bell, M.D., Dr. P.H., Thomas E. Reichelderfer, M.D., M.P.H.,
Robert J. Huebner, M.D.
** Published in PEDIATRICS August 1?60
92
PHS-NIH
Individual Project Report
Calendar Year I960
Serial No. NIAID 66-A
1. Infectious Diseases
2. Epidemiology
3. Bethesda, Maryland
Part A
Project Title: Epidemiologic Studies of Host Parasite Disease Relation-
ships
Principal Investigator: Dr. Joseph A. Bell
Other Investigators: Dr. Francis M. Mastrota, Dr. Albert Z. Kapikian,
Dr. Robert J. Huebner
Cooperating Units: Virus and Rickettsial Diseases Section, LID,
(Dr. Chanock, Dr. Johnson), Epidemiology Laboratory
Unit, (Dr. Rosen), Oncolytic and Oncogenic Virus
Unit, (Dr. Rowe)
Man lears (Calendar year I960):
Total: 15/8
Professional: 5/8
Other: 10/8
Project Description:
(1) Objectives: To delineate which of the many microbial agents are
causing acute illness in Junior Village nursery and describe clinical
and epidemiological nature of etiologic entities found.
Patient Material:
The Junior Village nursery studies are described in project number 66.
Epidemiologic methods are being devised to determine which of the 53 typed
viruses and the many typed bacteria, which have been isolated, were causing
illness.
The high frequency of illness occurrence (mean weekly definite ill-
ness attack rates of 25 %) and the even higher frequency of new and over-
lapping infections with potentially pathogenic microbial agents presents
an extremely complex epidemiological problem to establish specific etio-
logic relationships. One of the chief problems is serological determina-
tion of susceptibility and immunity to the many specific infections, or
illness attributable thereto; with the limited amounts of serums available.
To guide the most profitable use of these serums and for other purposes
preliminary analyses of temporal relationships between onset of specific
infection and onset of acute undifferentiated illnesses have been
Part B included / J Yes /"*~7 u0 q «
Serial No, NIAH) 66-A
completed and top priority for use of available serums can be established
on the basis of resolving the etiologic relationship of the more serious
illnesses, study of the many newly discovered viruses and study of new
vaccines. Within these limits the meticulous and time consuming analysis
of specific etiologic relationships is in progress.
Major Findings;
The preliminary analyses of temporal relations between onset of
specific infection and onset of acute undifferentiated febrile illness
using both horizontal and cross sectional controls have shown no evidence
that the following agents were causing illness in the nursery babies:
Adenovirus 2j Polio 3; Coxsackie B 5; ECHO 7, 8, 11, 12, 13, lU, 18, 19,
20, 25 and JV 5; nontypable Group A Streptococci; Hemophilus Influenza B}
Pneumonia Type 19, 23 J Staphylococci having Phage Type 81 in their anti-
genic pattern; Enteropathogenic coli of various types, and alkalescens
dispar. Infection with the following agents were significantly associated
with the occurrence of acute, febrile, undifferentiated illness; Adeno-
virus 1, 3, and 5j Influenza virus Asianj parainfluenza virus 1 and 3j
Poliovirus 2, Coxsackie virus B 3; Group A Streptococci types ky 12, 23,
and 1, 2, and 5; and Shigella Sonnei. In general, it is crudely estimated
that some h$% of the more severe illnesses of the nursery group may be
accounted for by the above agents and that measles and adenovirus infec-
tions account for the bulk of the more severe illness.
Significance to Bio-medical Research and the Program of the Institute:
Although the study is deliberately limited to a group of young
children who have a high proportion of susceptibles to various microbial
infections, it shows some of the potentialities of such infections in
older susceptible persons. This project represents a systematic approach
to determining the various etiologies of acute febrile illnesses so as
to guide research on development of methods for control.
Proposed Course of Project:
It is planned to continue the study for as long as continued collec-
tion of data and data analyses appear profitable.
sh
Serial No. NIAID 66-B
PHS-NIH
Individual Project Report
Calendar Year I960
1. Infectious Diseases
2. Epidemiology
3. Bethesda, Maryland
Part A
Project Title: Epidemiologic Studies of New Vaccines and Chemoprophy-
lactic Agents
Principal Investigator: Dr. Joseph A. Bell
Other Investigators: Dr. Francis M. Mastrota, Dr. Albert Z. Kapikian,
Dr. Robert J. Huebner
Cooperating Units: Virus and Rickettsial Diseases Section, LID,
(Dr. Chanock, Dr. Johnson) Epidemiology Laboratory
Unit, (Dr. Rosen), Oncolytic and Oncogenic Virus
Unit, (Dr. Rowe)
Man Years (Calendar Year I960)
Total: 29/8
Professional: l*/8
Other: 25/8
Project Description:
Objectives: (A) To design and have prepared monovalent and multlpolyvalent
vaccines with inactivated viruses which have shown evidence of producing
important illnesses. (B) To carry out preliminary trials of such vaccines
for determination of antigenicity and untoward reactions. (C) To observe
whether such vaccines substantially reduce the occurrence of infection
or illnesses in the Junior Village Institution.
Patient Material:
The Junior Village nursery studies are described in project numbers
66 and 66-A. The rather intense daily clinical observation permits an
evaluation of vaccine induced reactions. The vaccines which have been
used are: (1) Adenovirus types 1, 2, 3, and 5> (2) Adenovirus types 3,
h, and 7; (3) Coxsackie B virus types 1, 2, 3> h, and 5j (U) Rubeola
virus; (5) Respiratory vaccine containing 12 virus strains, namely,
monkey kidney cell grown influenza Al, A2, and B viruses; Adenovirus
types 1, 3, hy 5, 7j Parainfluenza virus types 1, 2, and 3j (6) Polio-
virus types 1, 2, 3 - three different products; (7) Parainfluenza 1 and
3 - egg grown. In addition, a daily oral dose of 300,000 units of
benzathine Penicillin has been given to a 20$ random sample
ren. The vaccines are given at time of admission, with the second
three weeks later and a third dose at three months. The vacc:.nes are
Part B included / J Yes [TJ No 95
Serial No. NIAID 66-B
given in one ml doses to preselected random samples of all children admitted
to the Junior Village nursery group. Blood sera for antibody studies is
collected on admission, at six weeks, and every three months thereafter and
at discharge.
Major Findings;
Analysis of the previously mentioned products is in progress currently.
Preliminary analysis showed the inactivated rubeola monkey kidney vaccine to
offer no protection in preventing rubeola. Serologic studies on this vaccine
are in progress.
The respiratory vaccine containing 12 virus strains did not alter the
illness pattern in those receiving it with the exception of a period in
January 1959 when Asian influenza was prevalent at Junior Village. At that
time, those children who had received the vaccine had significantly less
illness than those without the vaccine. The ability of the vaccine to
produce CF antibody to Asian influenza antigen was shown. Differences between
vaccinees and controls at other times of the year were not evident.
Significance to Bio-medical Research and the Program of the Institute;
It is obvious that the many acute febrile respiratory diseases and fevers
of undetermined origin which are a major public health problem are caused by
a great multiplicity of etiologic agents. To prevent or ameliorate these
illnesses by vaccine prophylaxis requires a multipolyvalent vaccine in as
much as no one of these known agents are producing a very large proportion
of these diseases. It seems timely to initiate controlled epidemiologic
studies in the use of multivalent vaccines and their possible enhancement or
interference with each other.
Proposed Course of Project;
It is planned to continue the study for as long as collection of data
and data analyses appear profitable.
96
Serial No. NIAID 66-C
PHS-NIH
Individual Project Report
Calendar Year I960
1. Infectious Diseases
2. Epidemiology
3. Bethesda, Maryland
Part A
Project Title: The Etiology of Respiratory Disease on a College Campu^
Principal Investigator: Dr. Albert Z. Kapikian
Other Investigators: Dr. Karl Johnson, Dr. Robert Chanock, Dr. Joseph Bell
Dr. Robert Huebner
Cooperating Units: University of Maryland Infirmary
Man Years (Calendar Year I960)
Total: 0/8
Professional: 0/8
Other: 0/8
Project Description:
Objectives : (1) To determine the role of known viruses as etiologic
agents in a young adult population; (2) To find new agents which might be
associated etiologically with respiratory disease.
Patient Material:
With the cooperation of the University of Maryland Infirmary, a study
was undertaken in October 1958 to determine the role of viruses in the
etiology of respiratory diseases in a young adult population. Patients
who appeared with complaints referable to their respiratory tract were
examined. Throat swabs and acute and convalescent bloods were also
obtained. An attempt to have a control group of nonrespiratory patients
was unsuccessful. The throat specimens were tested for viruses in appro-
priate tissue culture and the blood was tested for CF antibody against a
battery of respiratory virus antigens.
Major Findings:
The finding of a new parainfluenza virus type h was presented previously
Two hundred twenty-four patients with respiratory disease were seen--
among these, approximately 20$ had fever of 99°F. or greater. Virus
isolation attempts were markedly unsuccessful, possibly a result of
problems of storing of specimens enroute from Maryland University to N.I.B.
However, paried bloods were obtained from 129 of the total. Thirty or 2
Part B included / J Yes / x / No Q "J
Serial No. NIAID 66-C
of this latter group showed a four-fold increase tc one of the following
respiratory viral antigens by CF test: Parainfluenza 1, 3» 1»,J Adenovirus;
Influenza A and Bj Respiratory Syncytial Virus. Of the 30 showing CF antibody
rises over one-half ($2%) were to Influenza B, 17% to Respiratory Syncytial,
17% to Parainfluenza 1, and 3% to each of the other antigens. The illnesses
of the patients with the rises to each of the antigens was not associated
with any specific clinical syndrome.
Significance to Bio-medical Research and the Program of the Institute:
This project attempted to show the etiology of respiratory diseases
with respect to viruses. It is seen clearly that serologically, only 23% of
the respiratory diseases could be associated with viruses and if this had been
a year when influenza B was not prevalent, the figure might have been even
lower. The task of further attempting to find those agents which are causing
viral respiratory diseases is apparent.
Proposed Course of Project:
There are no plans to renew this study at the present time.
98
Serial No. NIAID 66-D
PHS-NIH
Individual Project Report
Calendar Year I960
1. Infectious Diseases
2. Epidemiology
3. Bethesda, Maryland
Part A
Project Title: The Role of Viruses in the Etiology of Ear Disease
Principal Investigator: Dr. Albert Z. Kapikian
Other Investigators: Dr. Joseph A. Bell, Dr. Robert J. Huebner
Cooperating Units: Washington Hospital Center, Ear, Nose and Throat
Department
Man Years (Calendar Year I960)
Total: 0/8
Professional: 0/8
Other: 0/8
Project Description:
For many years, it has been stated that viruses were responsible for
many nonbacterial ear infections such as serous otitis media. It was attempted
to study patients with serous otitis media to determine the role of viruses in
this disease.
Patient Material:
Patients at the Washington Hospital Center ENT clinic with serous
otitis media were examined. Fluids from the middle ear were tested in appro-
priate tissue culture lines. Acute and convalescent bloods were obtained.
Major Findings:
Thus far only a limited number of specimens from the ear have been
tested with no virus yet isolated. Serologic tests on the sera are in progress.
Significance to Bio-medical Research and the Program of the Institute:
The role of viruses in ear disease is uncertain. This project attempts
to clarify their role in the etiology of middle ear disease, especially serous
otitis media.
Proposed Course of Project:
It is planned to renew attempts to obtain appropriate specimens dur-
ing the year. J
Part B included / J Yes / x / No 93
Serial No. NIAID 66-E
PHS-NIH
Individual Project Report
Calendar Year I960
1. Infectious Diseases
2. Epidemiology
3. Bethesda, Maryland
Part A
Project Title: A Study of the Influence of Influenza A and B Mineral Oil
Adjuvant Vaccines on Influenza Antibody Patterns, 8-9 Years
after Vaccination
Principal Investigator: Dr. Joseph A. Bell
Other Investigator: Dr. Robert J. Huebner
Man Years (Calendar Year I960)
Total: 0/8
Professional: 0/8
Other: 0/8
Project Description:
Objectives: (1) To see if Mineral Oil Adjuvant Influenza A and B vaccines
influenced influenza antibody patterns, 8-9 years after vaccination.
Patient Material;
Approximately 100 N.I.K. personnel were given one dose of a mineral
oil adjuvant influenza vaccine between 1951 and 1952. Twenty-five hundredths
ml of a 100 CCA unit vaccine was administered. Influenza type A and B products
were assigned by a strictly random sampling process so that any follow-up of
the group would give two strictly comparable groups of near equal size, each
of which had received one dose of either type A or type B Influenza adjuvant
product 8-9 years previously. The A and B groups should have remained compara-
bly equal (within the range of sampling variation) with respect to all attri-
butes including influenza infection, disease, vaccination, antibody response,
etc., except for the influence of the different study vaccines given. The
100 N. I.H. personnel who were given 1 of the li adjuvant products and who were
all pre-bled before vaccination are in the process of being bled currently.
Hemagglutination inhibition tests are to be done to see if the adjuvant vaccines
given 8-9 years ago are still influencing the antibody pattern.
Major Findings: None
Significance to Bio-Medical Research and the Program of the Institute:
The study attempts to show the effectiveness of mineral oil adjuvant
vaccines in producing long lasting immunity. This may be of importance in
future vaccine programs.
Part B included /x~7 Yes 1 / J No *^
Serial No. NIAID 66-E
Proposed Course of Project;
No further field work is contemplated currently, but the laboratory
analysis will be carried on.
101
Serial No. NIAID 66-E
PHS-NIH
Individual Project Report
Calendar Year I960
Part B
Honors, Awards and Publications
Bell, J. A., Craighead, J.E. , and James, R.G.
Epidemiologic Observations on Two Outbreaks of Asian Influenza in a
Children's Institution
American Journal of Hygiene - Jan. 1961
Philip, R.N., Bell, J. A., Davis, D.J., Beem, M.O., Beigelman, P.M.,
Engler, J. I., Mellin, G.W., Johnson, J.H., Lerner, A.M.
Epidemiological Studies on Influenza in Familial and General Population
Groups 2. Characteristics of Occurrence
American Journal of Hygiene - Mar. 1961
Davis, D.J., Philip, R.N., Bell, J. A., Vogel, J.E., Jensen, D.V.
Epidemiological Studies on Influenza in Familial and General Population
Groups 3. Laboratory Observations
American Journal of Hygiene - Mar. 1961
Bell, J. A., Philip, R.N., Davis, D.J., Beem, M.O., Beigelman, P.M.,
Engler, J.I., Mellin, G.W., Johnson, J.H., Lerner, A.M.
Epidemiological Studies on Influenza in Familial and General Population
Groups U. Vaccine Reactions
American Journal of Hygiene - Mar. 1961
102
Part A
Serial No. NIAIO - 67
PHS-NIH
Inoivioual Project Report
Calenoar Year 1960
Project Title:
Studies on human enteroviruses, adenoviruses, and
reoviruses
Principal Investigator: Dr. Leon Rosen
Other Investigators: Dr. Joseph A. Bell, Dr. Robert J. Huebner, Dr.
Albert B. Sabin, Dr. D. Mendez-Cash ion, Mr.
Jerome Kern, and Mrs. Janet Hovis
Cooperating Units: D. C. Welfare Department; Children's Hospital
Research Foundation, Cincinnati, Ohio; School of
Medicine, University of Puerto Rico, San Juan.
Man Years: (Calendar year i960)
Total: 72/12
Professional: 32/12
Other: 40/12
Project Description:
Object i ves: To determine the role in human biology of entero-
viruses, ADENOVIRUSES, AND REOVIRUSES. To
ELUCIDATE THE NATURAL HISTORY OF THESE VIRUSES.
Methods Employed!
The principal population group under study
is the Junior Village nursery group which
has been described in detail in previous re-
PORTS of the Epidemiology Section.
Recently, a collaborative study was institu-
ted with the Department of Pediatrics of the
University of Puerto Rico. In this study,
specimens from children with a variety of
acute neurological disoroers will be stuoied
IN Bethesda.
A COLLABORATIVE STUDY WAS ALSO UNDERTAKEN
with Dr. A. B. Sabin of the Children's Hos-
pital Research Foundation of Cincinnati.
Dr. Sabin has supplied several thousand non-
polio ENTEROVIRUS ISOLATES FROM MEXICO.
Part 8 Included Yes
103
Serial No. NIAID - 67
Part A Methods Employeo (continued)
These are being studied for the production of hemagglutinins in an
attempt to obtain hemagglut i n at i ng strains of virus types which are
not now known to have this useful property.
Collaborative studies with other institutions outside of the National
Institutes of Health and with various intramural laboratories are un-
dertaken FROM TIME TO TIME AS OPPORTUNITIES ARE PRESENTED.
Major Findings: An extensive outbreak of infection with ECHO type 3
virus occurred in the Junior Village nursery population. This is the
first known outbreak of infection with this virus - the prototype
having been described from a single isolate. preliminary analysis of
data indicates that infection with this agent was associated with a
mild febrile disease.
Hemagglutinins were found for the first time for ECHO virus type 21,
COXSACKIE B VIRUS TYPES 1 AND 5t *ND A PREVIOUSLY UNRECOGNIZED ENTERO-
VIRUS (JV-9) WHICH IS CYTOPATHOGEN IC ONLY IN HUMAN TISSUE CULTURES.
Simple H-l procedures were developed for identifying enteroviruses
AND ALSO FOR PERFORMING SEROLOGIC TESTS WITH SERA FROM PERSONS SUS-
PECTED OF HAVING ENTEROVIRUS INFECTIONS.
TWO NEW ENTEROVIRUS SEROTYPES (iN ADDITION TO THE PREVIOUSLY REPORTEO
JV-1) HAVE BEEN CHARACTERIZED AND ACCEPTED FOR NUMBERING BY THE ENTERO-
VIRUS Committee. Several probably new serotypes are currently under
STUDY.
Four new adenovirus serotypes have been characterized. Simple sero-
logic PROCEDURES WERE DEVELOPED FOR IDENTIFYING ADENOVIRUSES AND FOR
performing serologic tests with sera from persons suspected of having
adenovirus infections.
Hemagglutination of rhesus erythrocytes by measles virus was demon-
strated AND A SIMPLE H-l TECHNIQUE FOR WORK WITH THIS VIRUS WAS
developed.
Significance to the Program of the Institute; The role of the numerous
enteroviruses, adenoviruses, and reoviruses in human biology is only
beginning to be studieo. data obtained thus far indicate that a num-
ber of these viruses are quite important in human pathology. further
progress in this field depenos to a great extent on the development of
simple laboratory procedures to cope with the more than 90 viruses now
known to belong to these groups.
Proposed Course of the Project; The study of these three families of
viruses will be continued both in the field, as opportunities are pre-
sented, and in the laboratory. laboratory studies will be devoted pri-
Part B Included Yes inii
Part A Proposed Course of the Project (continued)
Serial No. NIAID - 67
marily to the orderly classification of existing ano newly recognized
serotypes and to the development of simple in-vitro techniques.
Part B Included Yes
105
Serial No. NIAIO - 67
PHS-NIH
Individual Project Report
Calendar Year i960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
1. Rosen, L.: Serologic Grouping of Reoviruses by Hemagglutinati on-
Inhibition. Amer. J. Hyg., 71:2^2-2^9, 1960
2. Rosen, L., Hovis, J. F., Mastrota, F. M. , Bell, J. A., and
Huebner, R. J.: Observations on a Newly Recognized Virus (Abney)
of the Reovirus Family. Amer. J. Hyg., 71:258-265, 1960
3. Rosen, L., Hovis, J. F., Mastrota, F. M»., Bell, J. A., and
Huebner, R. J.: An Outbreak of Infection with a Type 1 Reovirus
among Children in an Institution. Amer. J. Hyg., 71 • 266-27** »
1960
h. Rosen, L.: A Hemagglut inati on-I nh ib ition Technique for Typing
Adenoviruses. Amer. J. Hyg., 71:120-128, i960
5. Rosen, L., Bell, J. A., and Huebner, R. J.: Enterovirus In-
fections of Children in a Washington, 0. C, Welfare Institution.
In "Viral Infections of Infancy and Childhood," Rose, H. M. , ed.,
Hoeber-Harper, New York, 1960, pp. 119-127
6. Rosen, L.: Hemagglutination and Complement Fixation. In "Per-
spectives in Pediatric Virology," Thirty-third Ross Conference
on Pediatric Research, Cramblett, H. G. , eo., Columbus, Ohio,
Ross Laboratories, 1959» pp« 53-56
7. Philipson, L. and Rosen, L.: Identification of a Cytopathogen ic
Agent called U-Virus Recovered from Patients with Non-
Diphtheritic Croup and from Day-Nursery Chiloren. Archiv. fur
die Gesamte Vi rusforschung, 9_;25-30, 1959
(Not included in 1959 report)
8. Rosen, L.: Hemagglutination and Hemagglutinati on-I nhibition
with Measles Virus. Accepted for publication in Virology.
Honors and Awards relating to this project:
1. Visiting lecturer -jn Epidemiology, School of Public Health,
University of California
2. Appointeo to the Enterovirus Committee of the National Cancer
Institute
106
Serial No. NIAID - 67A
1. Infectious Oiseases
PHS-NIH 2. Epidemiology
Individual Project Report 3. Bethesda, Maryland
Calendar Year i960
Part A
Project Title: Studies on viruses of the enteric tract of cattle
Principal Investigators: Dr. F. R. Abinanti and Or. Leon Rosen
Other Investigators: None
Cooperating Units: Dairy Husbandry Department, University or
Maryland; Home of Correction, Jessup, Maryland
Man Years: (Calendar year 1960)
Total: 6/12
Professional: 3/12
Other: 3/12
Project Description:
Objectives: To investigate the biologic properties and nat-
ural history of the viruses inhabiting the enteric
tract of cattle
Methods Employed: A longitudinal study of dairy cattle on
three different farms has been carried out
for approximately two years. monthly fecal
swabs and serum specimens are obtained from
each animal and these are studied in the lab-
oratory by a variety of virus isolation and
serologic procedures.
Major Findings: Reoviruses of three different types serologic-
ally INDISTINGUISHABLE FROM THE THREE TYPES
found in humans have been recovereo on numer-
ous occasions during this study. calves have
been infected with each of the three types of
human origin. no evidence of disease was noted
in either the naturally or experimentally in-
fected animals.
An apparently new hemadsorbing virus was re-
covered FROM CATTLE ON ALL THREE FARMS. THE
CHARACTER OF THE HEMADSORPTION PRODUCED BY THIS
VIRUS APPEARS TO BE DIFFERENT FROM THAT OF ANY
OF THE OTHER HEMADSORBING VIRUSES.
Part 8 Incluoed Yes
107
Serial No. NIAIO - 67A
Part A Major Findings (continued)
Several hundred isolates of enteroviruses have been recovered from
CATTLE IN THIS STUDY. The RELATIONSHIP OF THESE VIRUSES TO EACH
other and to the enteroviruses of man is currently under study.
Significance to the Program of the Institute: The recovery of vi-
ruses (such a,s reoviruses) from cattle which are ant i g en i c ally
identical with those which occur in man is of obvious interest.
Aside from the possible importance of cattle as potential sources of
infection, a number of aspects of the natural history of these agents
CAN BE CLARIFIED BY THEIR STUDY IN LOWER ANIMALS. In ADDITION, VI-
ruses recovered from cattle which are analogous, but not identical,
to those of humans also proved a mechanism for gaining useful infor-
mation about the latter agents.
Proposed Course of the Project; The animals in the longitudinal
study will be followed for about another six months. however, a
considerable amount of laboratory work will remain to be finished
after this time.
Part B Included Yes
108
Serial No. NIAID - 67A
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, ano Publications
Publications other than abstracts from this project:
Rosen, L. and Abinanti, F. R.: Natural and Experimental In-
fection of Cattle with Human Types of Reoviruses. Amer. J. Hyg.,
71:250-257,1960
103
Serial No. NIAID - 67B
1. Infectious Diseases
PHS-NIH 2. Epidemiology
Individual Project Report J>. Bethesda, Maryland
Calendar Year 1960
Part A
Project Title: Studies on the etiology of eosinophilic meningitis
in French Polynesia
Principal Investigator: Dr. Leon Rosen
Other Investigators: None
Cooperating Units: School of Medicine, University of California, Los
Angeles and Institut de Recherches Medicales oe la
Polynesie Franchise, Papeete, Tahiti
Man Years: (Calendar year i960)
Total: V12
Professional: 3/12
Other: 1/12
Project Description:
Objecti ves: To determine the etiology of eosinophilic
meningitis
Methods Employed: A comprehensive epidemiologic study was undertaken
of this unusual disease which occurred on tahiti.
Blood, spinal fluid, and stool specimens were col-
lected FOR A VARIETY OF LABORATORY STUDIES.
Major Findings: Beginning in March 1958 many hundreds of cases of an
unusual type of meningitis of unknown etiology oc-
curred on the island of Tahiti in French Polynesia.
Since the most characteristic feature of this menin-
gitis WAS A PLEOCYTOSIS CONSISTING IN LARGE PART OF
EOSINOPHILS, THE DISEASE WAS CALLED EOSINOPHILIC MEN-
INGITIS.
The MOST CHARACTERISTIC clinical FEATURES of THE DIS-
EASE WERE HEADACHE, STIFFNESS OF THE NECK AND BACK,
AND PARESTHESIAS OF DIFFERENT TYPES. APPROXIMATELY
FIVE PERCENT OF CASES HAD A FACIAL PARALYSIS OF THE
PERIPHERAL TYPE. ThE DURATION OF ILLNESS VARIED FROM
SEVERAL DAYS TO SEVERAL MONTHS AND REOCCURRENCES WERE
COMMON. No DEATHS OCCURRED.
Part B Incluoed Yes
110
Serial No. NIAID - 67B
Part A Major Finqings (continued)
More than one-half of the cases had a pleocvtosis of 500 °" more
cells per cu. mm. of cerebrospinal fluid, and in 8^ percent of all
cases eosinophils accounted for more than one-fourth of the
pleocytosis.
Examinations of spinal fluid, blood, and feces were negative for the
presence of viruses and pathogenic bacteria and fungi. no helminthic
parasites were found other than those commonly infesting the inhabit-
ants of the area. serologic examination of convalescent sera against
a number of different arthropod-borne viruses, enteroviruses, lepto-
spira, and other microbial agents failed to provide an indication of
the etiologic agent.
the disease occurred primarily in adults and affected both sexes
equally. Persons of Polynesian or part-Polynesian origin appeared to
have a higher attack rate than europeans or chinese.
There was no sharp seasonal distribution and cases occurred in every
month of the year.
The geographic distribution of cases corresponded roughly to the dis-
tribution OF THE POPULATION AND THERE WAS NO DEFINITIVE EVIDENCE OF
a higher attack rate in either rural or urban areas.
no evidence was found to suggest transmission of the disease from per-
son to person or from one geographic area to another. the aggregation
of cases by household suggested the effect of a common exposure than
person-to-person transmission.
The incubation period of the disease was estimated to be between two
and four weeks.
Although the etiology of the disease was not determined, the sum of
the clinical ano epidemiologic evidence suggested the hypothesis
that the oisease was caused by a helminthic parasite of the oceanic
BONITO OR SKIPJACK TUNA (KATSUWONUS PELAMI s) WHICH IS COMMONLY EATEN
raw in the area.
Significance to Program of the Institute: Outbreaks of eosinophilic
meningitis have occurred in new caledonia and the caroline islands,
AS WELL AS IN FRENCH POLYNESIA. It IS OBVIOUSLY OF GREAT PRACTICAL
INTEREST TO OETERMINE THE ETIOLOGY OF THIS DISEASE IN ORDER THAT AP-
PROPRIATE PREVENTIVE MEASURES CAN BE INSTITUTED. FURTHERMORE, IF. IT
IS SHOWN THAT THE OISEASE IS INDEED ACQUIRED FROM A MARINE FISH, IT
WILL BE THE FIRST INDICATION THAT A PARASITIC DISEAsE OF MAN CAN BE
ACQUIRED FROM SUCH A SOURCE.
Part B Included Yes
Ill
Serial No. NIAIO - 67B
Part A (continued)
Proposed Course of Project; Additional field studies will be under-
taken early in 1961 in an attempt to obtain further data on the
etiology of the disease - especially with regard to the hypothesis
that the disease is acquireo from eating raw fish.
Part B Included Yes
112
Serial No. NIAIO - 67B
PHS-NIH
Inoivioual Project Report
Calendar Year i960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
None
Honors and Awaros relating to this project:
1. Letter from Governor-General of French Polynesia to Sur-
geon General of the U.S. Public Health Service expressing
appreciation for the studies carried out on Tahiti by
Dr. Leon Rosen.
113
Serial No. NIAID-68
1. Infectious Oiseases
2. Virus and Rickettsial
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A
Project Title: Studies of Tumor Viruses in Nature.
Principal Investigators: Dr. Robert J. Huebner and
Dr. Wal lace P. Rowe
Other Investigators:
Dr. Janet W. Hartley, Mr. William T. Lane
and Mr. John D. Estes
Cooperating Units
New York City Health Department
Dr. David Johnson, Smithsonian Institute
Dr. E. Baker, Smithsonian Institute
Dr. B. Burmester, Regional Poultry
Research Laboratory, Agricultural
Research Service, East Lansing, Michigan
Man Years: (Calendar Year I960)
Total: 48/12
Professional : 16/12
Other: 32/12
Project Description:
Ob jecti ves : This project is concerned with elucidating natural
behavior of tumor viruses in their natural animal hosts in natural
environments. We have devoted most of our attention so far to those
viruses for which survey tools have been developed, namely, polyoma
and papi I loma.
Methods Employedit Survey tools - direct virus demonstration
and isolation techniques plus serologic procedures (CF, HI, MAP, N
tests) are evaluated and used in examining specimens collected in
field studies for evidence of contamporary and prior virus infection.
Part B incl uded :
Yes
114
Serial No. NIAID-68
PHS-NIH
Individual Project Report
Calendar Year I960
Field Studies : The natural history of polyoma virus infection
of Mus muscul us, the common house mouse, is under study in a densely
populated urban area (Harlem, N.Y.C.), and in a rural area on Maryland
farms and grain mills. The distribution of rabbit papilloma virus
(independent of tumors) in cottontails was studied in an area known
to be heavily infected (Kansas) and in a presumably tumor-free area
(Maryland) .
The natural occurrence of "VL" virus initially reported
as a tissue culture grown avian lymphomatosis virus was determined
by serologic surveys of commercial and experimental populations.
Specimens : Mice were live trapped, weighed, sexed, bled,
marked, and generally released (in same area as caught). Tissues
and urine were collected for virus isolations. Environmental
materials contaminated by mouse excreta such as trash and grain
were collected in sterile containers for virus study. Rabbits and
chickens, both with and without tumors, were bled and the sera
studied for papilloma and "VL" antibodies.
Certain Problems : Both virus demonstration and serological
techniques required extensive evaluation to determine the reliability,
accuracy and sensitivity of the available techniques. For polyoma
serology the HI test was shown to be more sensitive than the CF
test, and when sera was treated with RDE and heat, almost entirely
equivalent to the more cumbersome neutralization tests; hence it
was selected, not as the sole serologic survey instrument, but as the
test of choice.
The most severe laboratory problem that had to be solved
was the problem of spontaneous infection of laboratory mice with
laboratory strains of polyoma virus in our Bethesda laboratory;
this, plus the intrusion of other extraneous agents in the mouse
study systems (see Project NIAID-71 B) invalidated most efforts to
demonstrate virus in the MAP test, our most sensitive system. To
obtain high order information and insure bona-fide field isolations,
we eventually were required to set up field laboratories complete
with constant monitoring and adequate controls. Fortunately, we
were finally able to obtain isolations of virus which surely originated
in the field specimens.
115
Serial No. NIAID-68
PHS-NIH
Individual Project Report
Calendar Year I960
Major Findings;
Po I yoma : Polyoma was found to be widely distributed in
natural colonies of Mus muscul us, not only in Harlem but on livestock
farms and grain mills furnishing feed for livestock. As with labora-
tory mice, virus was shown to be excreted in saliva and excreta
by carrier wild mice. Not only the tenement environments and community
mouse nests in Harlem, but even the cereal grains in the feed mills
were found contaminated; presumably the farm granaries where infected
mice also abound are heavily contaminated as well. Polyoma virus
has been easily recovered from mice in all three studies; however,
the relative extent of grain and environmental area contamination
must be determined.
Polyoma, a ubiquitous and persistent natural infection
in Mus muscul us has now been studied in three separate ecologies -
laboratory breeding colonies, urban tenement houses, and in rural
agriculture establishments. The basic cycle in nature would appear
to be the latter, where extensive contamination of cereal grains
on the farm and the feed mill not only explains persistent foci of
this highly resistant virus in these areas, but also suggests that
the wide distribution (and occasional sudden outbreaks) of polyoma
virus infection in laboratory colonies are induced and maintained
by uncooked cereal grains commonly fed to such mice.
Longitudinal studies continued in Harlem tenements
suggest that large and very dense populations of mice, plus con-
taminated foci, particularly "community nests", are the chief
factors in maintaining continuous infections through several succeeding
generations of mice. Polyoma virus was isolated from debris and dust
in one Harlem kitchen cabinet near a nesting area over intervals
exceeding eight months. During the year of surveillance in New York,
negative premises tended to remain negative, and positive premises
without exception remained positive.
Despite wide distribution and high infection rates, the
role of polyoma virus in the genesis of naturally occurring cancers
in mice is still undetermined, despite the fact that field isolates
are quite as oncogenic as established strains when given to infant
laboratory mice and hamsters. Several thousand mice have now been
I ive trapped - some of them several times over periods of several
months. To date only one naturally occurring tumor has been
observed - a mammary tumor in a mouse negative for polyoma. However,
it appears that few wi Id mice I ive beyond six months of age in the
urban environment where polyoma is prevalent.
116
Serial No. NIAID-68
PHS-NIH
Individual Project Report
Calendar Year I960
Additional Findings: These studies promise to provide
interesting information on the natural histories of other latent
viruses of Mus muscul us including mouse sal ivary gland virus, K virus,
and reovi ruses. It is probable as we intensify our laboratory study
of field specimens that other latent and possibly tumor viruses may
come to I ight.
Observations on the ecology of Mus muscul us have led to
interesting findings. In the grain mill and the feed barn, just as
in the tenement house, mice tend to inhabit the upper floors presumably
because rats are more common in basements and lower floors; female
mice are more numerous than males, apparently live longer, and as
adults are more frequently positive for polyoma. The latter presumably
can be explained by greater exposure of females to infected communal
nesting areas, which in turn is due to the much greater amounts of
virus excreted by mice infected as infants (as much as 10 virus/ml
of urine may be excreted for several weeks).
Rabbit Papi I loma : Differential centrif ugation combined with
several washings to free antibody bound papilloma virus and
complement-fixing antigens in cottontail papillomas obtained from
Kansas provided more sensitive tests for live virus and CF antibody.
Complement-fixing antibody was found to be present in 50 Kansas
rabbits carrying visible papillomas; Kansas rabbits free of
papillomas were most often negative but occasionally they also were
positive in the CF test. The CF antibodies were shown to correlate
with the presence of neutralizing antibodies in the domestic rabbit
test. Maryland rabbits are reportedly free of papilloma and indeed
none were seen on some 30 rabbits live trapped in this area. To
date all Maryland rabbits have been negative in the papilloma
complement fixation test.
This test appears to be very sensitive and quite specific,
thus permitting surveys for virus prevalence independently of tumor
production. Thus far, unlike polyoma (which it resembles in many
physical and biologic traits) papilloma virus would seem to induce
tumors in the majority (If not all) Kansas cottontail rabbits which
are successful ly infected.
Studies of modes of spread - a search for attenuated or non-
tumorigenic strains appears feasible; similarly, studies of possible
antigenic relationships with papilloma agents of other species, Homo
sapiens included, are indicated.
117
Serial No. NIAID-68
PHS-NIH
Individual Project Report
Calendar Year I960
"VL" (GAL) Virus: This tissue culture grown virus was proven
during the year to be an extremely prevalent extraneous adenovirus-
like chicken virus and not the agent of avian visceral lymphomatosis
as originally reported by Burmester and Sharp I ess. This dismal
discovery was made simultaneously by Burmester, Sharp I ess in labora-
tory experiments, and by our own sero-epizootio logic studies of
the prevalence of antibodies to this agent in chicken flocks.
Virtually all commercial flocks and 90 per cent of the chickens in
such flocks revealed neutralizing antibody to the "VL" or "GAL"
virus (Gal I us adenovirus- 1 ike) . Studies of isolated outbreaks of
visceral and neural lymphomatosis revealed that while most were
associated with GAL infection, some typical leukemia outbreaks
occurred in the complete absence of evidence for GAL infection.
Surveys of Burmester* s 15 I strains of RPL-12 susceptible
birds revealed that nearly all of them had antibodies to GAL virus.
Fortunately, additional surveys have revealed a substrain of 15 I
chickens free of GAL virus, which will be used for a re-examination
of visceral lymphomatosis in a system free of an extraneous virus
possessing many of the properties of lymphomatosis itself.
Significance to the Program of the Institute: In LID our approach
to cancer research is based on a "biological" instead of the more
prevalent c I inico-patho logic point of view. We do not disdain the
use of laboratory models (we employ them as indicated); but our
major interest is conditioned by a concept which we think is funda-
mental to an intelligent and logical approach to the study of cancer
viruses. We take it as axiomatic that if a cancer is caused by a
specific virus, then as is true of all other microbial illnesses, the
microbe is the central issue and the natural behavior of such a virus
the most needed and highest order information achievable on the
subject.
This project has already developed much information of value
in defining not only the natural behavior and the basic cycle of
polyoma in nature, but also the probable sources of infection in
both experimental and production colonies.
118
Serial No. NIAID-68
PHS-NIH
Individual Project Report
Calendar Year I960
The demonstration by Eddy and Stewart of the tumorigenic
activity of polyoma in hamsters, rats, guinea pigs, and rabbits
reveals an alarming capacity for polyoma to cross species barriers.
Although serologic studies do not support definite infection of man
with this virus, widespread access of humans through mouse contaminated
environments and apparently extensive contamination of food-stuffs,
renders the question of possible human infections rather more than
academic. The lack of serologic correlation with human cancer such
as would be expected in mice may not represent conclusive countei —
evidence, since hamsters with polyoma induced tumors lose their
antibodies to polyoma rapidly despite the persistence of eventually
fata I tumors .
Proposed Course of the Project: The natural history and behavior
of polyoma and other animal tumor viruses are important subjects
in their own right and studies of them represent some of the first
extensive efforts to develop such information about latent viruses.
As noted above, there can be no higher order of information about
infectious agents and such information derived from careful
exhaustive longitudinal studies are directly relevant to the question
of human cancer viruses.
The possibility that cancer could be the result of a zoonotic
infection no longer appears so very unlikely. We plan, therefore,
to study possible polyoma infection of man and other animal species,
such as domestic animals (cattle, hogs, dogs, and cats), or other
wild rodents likely to be exposed repeatedly to infection with known
cancer viruses of mice (such as polyoma) and chickens.
113
Serial No. NIAID-68
PHS-NIH
Individual Project Report
Calendar Year I960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Huebner, R.J.: Some questions about possible approaches to
research on viruses as a cuase of cancer. Cancer Res., 20 : 669-830.
I960.
Huebner, R.J.: Viruses in search of cancer. Monograph -
PERSPECTIVES IN VIROLOGY II, I960.
Huebner, R.J.: New possibilities in virus disease control.
Medical Section Proceedings of American Life Convention, I960.
Halonen, P. and Huebner, R.J.: ECHO and poliomyelitis virus
ant i sera in guinea pigs with f I uorocarbon-treated cell culture
antigens. Proc. of Soc. for Exper. Biol, and Med., 105 ;46-49, I960.
Honors and Awards relating to this project:
Participated in Variety Children's Research Foundation Symposium,
and dedication ceremonies of new research building; talk: "Viruses
in Children, I960". Miami, Florida, January I960.
Participated in the Gustav Stern Symposium Perspectives in
Virology II. Talk: "Viruses in Search of Cancer." New York,
New York, January I960.
Participated in NCI Viruses and Cancer Panel. New York, New York,
January I960.
Invited speaker, Epidemiology for Veterinarians course, - "New
Horizons in Domestic Animal Virology." Communicable Disease Center,
Atlanta, Georgia, February I960.
Participated in International Conference on Asian Influenza.
Discussant, "Methods of Diagnosis." National Institutes of Health,
Bethesda, Maryland, February I960.
Participated in Phenomena of Tumor Viruses Symposium, New York,
New York, March I960.
120
Serial No. NIAID-68
PHS-NIH
Individual Project Report
Calendar Year I960
Invited speaker, Frederick County Tuberculosis and Public Health
Association, Frederick, Maryland. April I960.
Invited speaker, American College of Physicians - "Viruses and
Cancer." San Francisco, California, April I960.
Society of American Bacteriologists. Talk: "Viral Agents in
Relation to Tumors". Philadelphia, Pennsylvania, May I960.
Annual Health Conference, Inc. Talk: "The Growing Importance
of Virology in Public Health."" New York, New York, May I960.
Invited speaker, American College of Obstetricians and Gynecologists -
"Virus Infections in I960." Cincinnati, Ohio, April I960.
Invited speaker, The Medical Section, American Life Convention -
"New Possi bi I i ties in Virus Disease Control ." White Sulphur Springs,
West Virginia, May I960.
Invited speaker, Kansas Trudeau Society - "Viral Respiratory
Diseases." Kansas City, Kansas, September I960.
Invited speaker, The American Academy of General Practice - "What
is Non-Paralytic Polio." Kansas City, Kansas, September I960.
Participant in discussions, University of Illinois Center for
Zoonoses Research. Urbana, Illinois, September I960.
Invited speaker, NCI Staff Conference - "Background Noise in
Virus Study Systems." National Institutes of Health, October I960.
Section chairman, Southwest Section of American Association for
Cancer Research. Talk: "Viruses as a Cause of Cancer." Galveston,
Texas, October I960.
Invited speaker, Academy of Medicine of Cincinnati - "Viruses and
the Common Cold." Cincinnati, Ohio, November I960.
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Serial No. NIAID-68
PHS-NIH
Individual Project Report
Calendar Year I960
Invited speaker, The Harvey Society - "Cancer as an Infectious
Disease." New York, New York, November I960.
Invited speaker, Chicago Medical School, Abbott Laboratories
and Marquette University - "Respiratory Disease due to Viruses -
A Comprehensive View." Chicago, Illinois, November I960.
Chairman and discussant, AMA Clinical Session Symposium on
Respiratory Virus Disease. Washington, D.C., November I960.
Elected to membership, The National Academy of Sciences,
April I960.
122
Serial No. NIAIO - 68-A
1. Infectious Diseases
2. Virus & Rickettsial Oiseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Sero-ep idem io logy of virus infections.
Principal Investigator: Or. John L. Sever, Dr. Robert J. Huebner
Other Investigators:
Cooperating Units:
Anita Ley, Flora Wolman, Renee Traub,
Joan Austin
NINDB, Collaborative study on cerebral
palsy, mental retardation, and other neuro-
logical and sensory disorders of infancy
and childhood (Dr. Richard Masland).
MBA - Or. Gabriel Caste llano
Man Years (calendar year I960):
Total: 74/12
Professional: 38/12
Other: 36/12
Project Description:
Objectives: To utilize available serologic technique in an
intensive study of newly recognized viruses as to their relation to
the high incidence of acute undifferentiated respiratory diseases,
chronic diseases, birth defects, and cancer. To develop, wherever
technically possible, the refinements of serologic methods necessary
for a large scale investigation of the natural course of the disease as
caused by viral infections.
Methods Employed: Serologic techniques, including complement
fixation tests, hemagglut inat ion- inh ibit ion, hemadsorption, viral
neutralization, and tissue culture neutralization tests are now
developed for the identification of over 100 viral infections.
Part B Included:
No
123
Serial No. NIAIO - 68-A
Two major phases of this project have been pursued. First, the
commercial production and standardization of antigens and ant i sera
suitable for the performance of the serological tests. Second, the
development of an integrated laboratory facility, employing trained
technicians, capable of handling large scale testing.
Human sera for viral serologic analysis and identification of
previous viral experiences are available from studies of common acute
undifferentiated respiratory diseases, and various detailed studies
of special virus disease problems of current interest to the Laboratory
of Infectious Diseases. A large number of serial bleedings are being
obtained from mothers during the course of pregnancy and from infants
4 months after birth in the Collaborative Study on cerebral palsy,
mental retardation, and other neurological and sensory disorders of
infancy and childhood of NINOB. These sera are now becoming available
for testing.
Major Findings; Complement fixing antigens for more than 70
viruses have now been prepared by Microbiological Associates, Inc.,
in consultation with the Virology Section, LID and NINDB. The antigens
which have been prepared are titered in both laboratories and when
found acceptable are produced in quantities of 100 to 1000 ml. These
include complement-fixing antigens for: Adenoviruses (common antigen),
Coxsackie A and B viruses (25 types), Influenza A, B, C, mumps, Para-
influenza (4 types), Pol ioviruses (3 types), ECHO viruses (28 types),
measles, and Herpes Simplex and Respiratory syncytial. Viral antigens
for hemagglutination tests have also been produced for 24 adenoviruses,
4 parainfluenza viruses, respiratory syncytial virus, 3 Reoviruses,
mumps, salivary gland virus, Q Fever, Psittacosis, and other viruses
which are utilized in routine tests in our laboratories.
A large scale program for producing specific immune antisera in
human volunteers has been initiated and will be pursued during the
next calendar year. After the completion of safety tests, groups of
volunteers will be given purified viral antigens. The pedigreed stock
antisera obtained in this way will be standardized for evaluating the
potency of antigen preparations.
The application of a micro technique to both hemagglutination and
complement fixation tests will permit the use of considerably less
sera and antigen for the performance of the various tests. The
technique has now been refined and developed to the point where it may
be applied in lieu of standard techniques whenever screening information
is desired. Through the use of highly developed spiral loops, accurate
dilutions may be made rapidly in the system. The technique should be
of particular value in studying the serological background of animals
from which only a small amount of sera is available.
124
Serial No. NIAIO - 68-A
Significance to the Program of the Institute: The availability of
reliable standard and micro serological techniques for a large group of
new viruses provides an opportunity to investigate the course of human
disease caused by viruses which are either difficult to isolate or are
resistant to evaluation because the clinical effects are delayed until
a long time after infection has subsided. This is particularly true in
the case of birth defects and animal cancers. The application of this
tool for analysis should provide considerable information on the
epidemiological aspects of virus infections.
The program conducted in association with the NINDB should provide
the tools necessary to help establish the clinical importance of over
100 new viruses of man.
Proposed Course of the Project: During the year 1961, the sero-
logical program will be expanded both in terms of antigenic materials
and space for the performance of an increased testing program. The
space available has already been increased and will be enlarged to
include a well equipped laboratory and of f ice faci I it ies. The con-
tinuing process of evaluating new antigens and the volume production
of antigens will be continued. The evaluation of serial specimens
from the NINDB study will proceed in conjunction with information on
birth defects being supplied by the Collaborating Institutions.
125
Serial No. NIAID - 69
1. Infectious Diseases
2. Virus & Rickettsial Diseases
3. Bethesda, Maryland
PHS-N I H
Individual Project Report
Calendar Year I960
Part A.
Project Title: Study of viruses as causes of respiratory illness
in infancy and early childhood.
Principal Investigator: Dr. Robert M. Chanock
Other Investigators: Dr. K.M. Johnson, Dr. R.J. Huebner
Cooperating Units: Dr. R.H. Parrott, Children's Hospital
Research Foundation, Washington, D.C.
Man Years (calendar year I960):
Total: 30/12
Professional: 5/12
Other: 25/12
Project Description:
Ob jecti ves: I) To search for new agents responsible for
respiratory illness in infancy and childhood. 2) To delineate the
epidemiology of virus which have recently been associated with
respiratory illness. 3) To continue the study of certain well
established respiratory viruses, i.e., their epidemiology and
contribution to the overall respiratory disease experience during
nfancy and childhood. 4) To determine how much pediatric respiratory
llness can be associated with known viruses as a guide to future
mmunoprophy I axi s.
Methods Emp I oyed : Infants and children with respiratory illness
and suitable controls without such illness will be studied at
Children's Hospital, District of Columbia. The severe lower respiratory
syndromes will be investigated in hospitalized patients, while the
milder febrile respiratory disease syndromes will be studied among
cl in ical patients.
Part B Included: Yes
126
Serial No. NIAID - 69
The main emphasis will be on the use of various tissue culture
systems and the fluorescent antibody technique for virus isolation.
An attempt will be made to inoculate specimens from patients directly
into tissue culture without prior freezing and thawing since such
treatment appears to rapidly inactivate respiratory syncytial virus and
the current strains of influenza B. Fluorescent antibody techniques
will be applied to the search for agents which may possibly grow in
tissue culture without causing cell destructive effects.
After the various isolates are identified, their contribution
to the different respiratory disease syndromes will be estimated by
comparing the recovery rate in such groups with that observed for
healthy children free of respiratory symptoms. Children with severe
illness admitted to the hospital, as well as control subjects, will be
studied serologically for evidence of infection with 15-20 known
respiratory viruses as well as any new agent which may emerge as a
potentially important pathogen.
Major Findings;
Respiratory syncytial (RS) virus; During the past 3 years RS
infection was detected by serologic means in \\% of children with severe
lower respiratory tract illness. The agent was found to be extremely
labile and virus recovery was rarely accomplished until specimens were
immediately inoculated into tissue culture without prior freezing.
Employing this technic, 57 strains of RS virus were recovered from
children with respiratory illness from March through July, I960. Virus
was recovered most frequently from infants less than 7 months of age
who were hospitalized for pneumonia (54%) or bronchiolitis (59#). The
RS agent was isolated from 32$ of children of all ages with bronchio-
litis or pneumonia during this period. Virus was recovered significantly
less often (\%) from control subjects.
Serologic studies condirmed the virus isolation data and pro-
vided additional evidence that the RS virus is a major respiratory
pathogen of infancy and childhood. When the serologic technics were
re-examined in the light of the virus recovery data, it was found that
the CF test was only 50% efficient in detection of infection. This
suggested that 22# of the total severe pediatric respiratory illness
seen in Washington, D.C., during the past 3 years was associated with
RS infection.
Para influenza viruses; Similar to the pattern established
during the past 2 years the para influenza types I and 3 viruses were
active in infants and children of the Washington, D.C., area during
most months of the current year. These agents continued to play an
important role in all types of pediatric respiratory disease,
especially infantile croup which is the most serious respiratory
emergency of childhood. For the first time evidence was obtained which
127
Serial No. NIAIO - 69
associated type 2 (CA) virus with illness. This agent was associated
with 10 cases of croup hospitalized during November through January.
Adenoviruses; During the past 3 years 344 adenoviruses have
been recovered from 3624 patients with respiratory disease -
isolation rate = 9.5%. These viruses are currently being typed. An
analysis of their contribution to pediatric illness must await
completion of typing. It is important to assess the role of each
adenovirus type separately since the recovery rate for all adenoviruses
from control subjects is so high (.5%), and since adenoviruses not
only cause acute illness but persist for long periods in a latent form
in lymphoid tissues.
Enteroviruses; Currently under investigation is the importance
of the recently described enterovirus- 1 ike agents which grow only in
human tissue culture cells and which have very fastidious conditions
for such growth. The agents have been recovered from 6% of children
with various types of respiratory disease. The recovery rate, however,
from control subjects was the same {6%). Numerous conventional
enterovirus infections have also been observed in this study
population during the past few years. Analysis of their clinical
importance awaits specific identification of the many strains of
virus recovered.
Significance to the Program of the Institute; Respiratory
disease is the most common infectious ailment of man. Such infections
occur commonly and in their severest form during infancy and early
childhood. Detailed knowledge of the agents responsible for
respiratory disease and their natural history are a necessary prelude
to attempts at either immunoprophyl axis or chemotherapy. As has been
shown with the respiratory syncytial and para influenza viruses, the •
significance to be derived from newer respiratory agents discovered
in childhood illness is also increased because they are associated
with infections which may produce a general I y mi I der i I Iness in adults.
Proposed Course of the Project; It is planned that this study
will continue for a period of several years, since a large segment of
childhood respiratory illness remains to be elucidated. In addition,
viruses responsible for respiratory illness vary at different times
and in different localities. Therefore, surveillance should continue
in order to comprehend the larger picture.
128
Serial No. NIAID - 69
Part B. Honors, Awards, and Publ ications
Publications other than abstracts from this project:
Chanock, R.M., Bell, J. A., and Parrot, R.H.: Natural history of
para influenza infection. Monograph in: PERSPECTIVES IN
VIROLOGY II. In press, 1961.
Chanock, R.M., and Johnson, K.M.: Infectious Diseases
(respiratory viruses). In: Annual Review of Med. Pub.;
Annual Reviews, Inc. In press, 1961.
Honors and awards related to this project:
Invited to attend The Gustav Stern Symposium on Perspectives in
Virology II, and present paper entitled, "Observations
on natural history of infection with certain recently
recognized respiratory viruses." January 25, 26., I960, New York
City, N.Y.
Invited to participate at Asian Influenza Conference (International),
and present paper entitled, "Hemadsorption". February 17, 18,
19, I960, National Institutes of Health, Bethesda, Md.
Invited to participate in Armed Forces Epidemiology Board meeting,
April 4,5,6, I960, Kenwood Country Club, Bethesda, Md.
Elected Full Member, Armed Forces Epidemiology Board-Commission on
Acute Resp iratory Disease, June, I960.
Invited to participate in the American Medical Association Clinical
Sessions, and present paper on respiratory virus infections,
November 28, 29, and December I, I960, Washington, D-C.
Invited to contribute chapter on Respiratory Viruses for publication
in ANNUAL REVIEW OF MEDICINE - 1961.
123
Serial No. NIAID - 69A
1. Infectious Diseases
2. Virus & Rickettsial
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Viral pneumonia: etiology, therapy, and
prevention.
Principal Investigator: Or. Robert M. Chanock
Other Investigators: Or. Karl M. Johnson
Cooperating Units: Dr. J. Kingston, Dr. H. Bloom,
Dr. M. Mufson, and Dr. F. Gordon,
Bureau of Medicine and Naval Medical
Research Institute, U.S. Navy
Man Years (calendar year I960):
Total: 50/12
Professional: 10/12
Other: 40/12
Project Description:
Objectives: I) To define the etiology of viral pneumonia as it
occurs in persons of all ages. 2) To determine the relative importance
of various newly recognized viruses in the pneumonia syndrome in
different populations and at different times. 3) To define the natural
history of certain agents which appear to be responsible for a
significant proportion of pneumonia in the young (para influenza 3,
respiratory syncytial, adenovirus, and Eaton agent) and in the adult
(Eaton agent). 4) To continue the search for other as yet unrecognized
viral agents which cause pneumonia. 5) To evaluate the effect of
tetracycline chemotherapy in Eaton pneumonia.
Methods Employed: Current emphasis has been placed on the role of
Eaton agent in human pneumonia. The presence and quantity of antibody
for the Eaton agent was determined by the indirect fluorescent antibody
technique, employing frozen sections of infected chick embryo lung.
Epidemiologic field studies were carried out at a Marine Recruit
Training Center (Parris Island, S.C.) which has a high rate of Eaton
infection. Patients with pneumonia, febrile respiratory illness without
pneumonia, afebrile respiratory illness and comparable control subjects
free of respiratory illness were studied serologically for evidence of
Part B Included Yes 13 U
Serial No. NIAIO - 69A
infection with Eaton agent, as well as other respiratory viruses.
The effect of demethylch lortetracycl ine on the course of Eaton
pneumonia was determined in a double blind therapy study in which the
clinical observers were unaware of the patients diagnostic or therapy
status. Laboratory tests were performed without knowledge of which
patients received drug and which patients received placebo. The
patients were given a daily physical examination, temperature was
recorded four times a day, and chest X-rays were taken every third day.
Major Findings;
A. Epidemiologic findings. Over a I -year period Eaton infection
was associated with 5\% of the 530 pneumonias admitted to the Naval
hospital. Adenovirus infection contributed significantly less to the
pneumonia syndrome (6%).
Eighty-three per cent of the recruits lacked detectable antibody
for Eaton agent when they entered recruit training. Fifty-three per
cent of these sero-negat i ve men developed antibody during the 5-month
training period. For the group of incoming recruits as a whole, the
risk of infection during training was 44#. Over a 6-month period the
risk of pneumonia during training was 2% and the risk of an Eaton
positive pneumonia was I .5%. The infection to clinical pneumonia
ratio was estimated to be 30 to I .
Infection was widely disseminated through the recruit center,
and occurred in almost every platoon of recruits. Movement of
infection was slow; onsets of Eaton pneumonia occurred over a 9-week
period in certain platoons. Long incubation period and slow movement
of infection are attributes ideally suited to maintain this agent in
an ever changing recruit population. The fluorescent antibody test
proved to be twice as sensitive as the cold agglutinin technic in the
diagnosis of Eaton pneumonia.
B. Chemotherapy. In a control led double bl ind study involving
290 patients, demethy I ch lortetracyc I ine significantly reduced the
duration of fever, rales cough, malaise, and fatigue. Therapy stopped
the progression of pulmonary infiltration and accelerated its clearing.
Fever did not return when therapy was stopped. These findings strongly
suggest that the drug exerted a direct action upon the pulmonary disease
process.
Demethylch lortetracyc I ine had no apparent effect on the course of
a small mixed group of illnesses associated with other known respiratory
viruses.
131
Serial No. NIAIO - 69A
Significance to the Program of the Institute; As seen during
the past 2 years, the consequences of infection with Eaton agent appear
to be of considerable public health importance in both children and
adults. The demonstration of a marked beneficial effect of demethyl-
chlortetracycl ine on Eaton pneumonia thus constitutes a finding of
major importance in the therapy of viral pneumonia.
Proposed Course of the Project: The searcti for other as yet
unrecognized viruses which cause pneumonia will continue.
An unexpected finding in the chemotherapy study suggests that
another tetracycline sensitive agent was responsible for a significant
proportion of the pneumonias which were investigated. Efforts to
recover this agent are currently in progress.
In addition to the unexplained pneumonias of early life, future
studies will concentrate upon the heretofore completely neglected
problem of pneumonia in the aged. Emphasis will be placed in these
two areas since it is here that the problem of pneumonia mortality is
greatest.
132
Serial No. NIAID - 69A
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Awards, and Publ ications
Publications other than abstracts from this project:
Johnson, R.T., Cook, M.K., Chanock, R.M., and Buescher, E.L.:
Family outbreak of primary atypical pneumonia associated with
the Eaton agent. New England J. Med., v: 262, 817-819, I960.
Chanock, R.M., Cook, M.K., Fox, H.H., Parrott, R.H., and Huebner,
R.J.: Serologic evidence of infection with Eaton agent in
lower respiratory illness in childhood. New England J. Med.,
v:262, 648-654, I960.
Cook, M.K., Chanock, R.M., Fox, H.H., Buescher, E.L., Johnson, R.T.,
and Huebner, R.J.: Studies on the role of Eaton agent in lower
respiratory tract illness. Evidence for infection in adults.
Brit. Med. J., v: I , 905-911, I960.
Chanock, R.M., Mufson, M.A., Bloom, H.H., James, W.D., Fox, H.H.,
Kingston, J.R.: Eaton agent pneumonia. I. Ecology of infection
in a military recruit population. J. A.M. A. In press, I960.
133
Serial No. NIAIO - 69B
1. Infectious Diseases
2. Virus & Rickettsial Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Study of the laboratory aspects of respiratory
virus illness in a welfare orphanage.
Principal Investigator: Dr. Robert M. Chanock
Other Investigators: Dr. K.M. Johnson, Dr. A.Z. Kapikian,
Dr. R.J. Huebner, Dr. J. A. Bell
Cooperating Units: None
Man Years (calendar year I960):
Total: 62/12
Professional: 12/12
Other: 50/12
Project Description:
Objectives: To elucidate the natural history of certain newly
discovered respiratory viruses by means. of a longitudinal study of an
orphanage population. This study supplements our cross-sectional
studies in pediatric hospitals, thus adding additional dimensions
to our observations and increasing the scope of our comprehensive study
of respiratory infections.
Methods Employed: The population under study is an orphanage
nursery in the District of Columbia (s a Project Report NIAID-66) whose
average population is 90-100 children beJween the ages of 6 months and
3 years. The turnover of infants and children is rather rapid, and, as
a result, a great deal of respiratory illness occurs in this population
throughout the year.
Rectal temperatures are taken twice a day, and the population
is carefully observed by a full-time pediatrician who also provides
medical care. Throat and rectal swabs are collected three times a
week. Serum specimens are obtained on admission and discharge, and
at various intervals between. Throat swabs are tested in monkey kidney
tissue culture for the presence of various myxoviruses and in Hep-2
cultures for respiratory syncytial (RS) and adenoviruses. Serum
Part B Included: Yes
131
Serial No. NIAIO - 69B
specimens are tested for antibody to the various myxoviruses and
other respiratory agents by the neutralization, hemagglutination-
inhibition, or complement fixation technique. Laboratory data are
then correlated with clinical, bacteriological, and epidemiological
information in an effort to determine qualitative and quantitative
virus disease relationships. As was true in previous years witti the
adenoviruses and the newer myxoviruses (para influenzas) and certain
enteroviruses, Junior Village provided in I960 additional opportuni-
ties for defining the clinical importance of viruses in childhood
di sease.
Major Findings:
Respiratory Syncytial (RS) Virus: An abrupt outbreak of
pneumonia occurred during the last week of April and the first 2
weeks of May, I960. Thirty-six (or 40£) of the 90 infants and
children in residence at Junior Village developed pneumonia. The
usual rate of pneumonia at the nursery is I - 2 cases per month.
The widespread occurrence of RS infection in the general
Washington, D.C. community at the same time, and its association with
lower respiratory tract illness, (see Project Report NIAIO 69-B 1959)
suggested that the nursery outbreak might also be caused by RS virus.
Laboratory techniques were modified accordingly, and throat swab
specimens were tested within a few hours after collection without prior
freezing. The inoculation of fresh specimens is necessary because of
the extreme lability of RS virus. Unfortunately this type of testing
was not initiated until the last half of the outbreak. Nevertheless,
24 strains of RS virus were recovered, 18 of them from patients in
the acute phase of a pneumonia illness. An analysis of the virus
recovery and illness data strongly suggested that the RS agent was
etiological ly associated with pneumonia. Serologic tests indicated
that approximately 90£ of children in residence during the outbreak
developed a rise in antibody for RS virus.
Significance to the Program of the Institute: The information
gained during the RS virus outbreak has yielded additional evidence
that this agent is a respiratory pathogen of major importance in
infancy and early childhood. Study of the RS pneumonia outbreak
provided an unusual opportunity to observe the clinical consequences
of infection, and to determine the risk of lower respiratory tract
involvement in a young population. The finding that A0% of the
infants and children in residence developed pneumonia constitutes
high order information wnich has implications both in the natural
history of infection with this agent, and in attempts to control its
more severe effects by immunoprophyl axis.
135
Serial No. NIAID - 69B
Proposed Course of the Project: The project will continue in
an effort to gain a better understanding of the newer respiratory
viruses. A retrospective serological analysis of the pneumonia
experience at Junior Village will be undertaken with special emphasis
on the role of para influenza 3 and respiratory syncytial viruses since
these agents have been associated with the majority of much illness in
the past 2 years.
136
Serial No. NIAID - 69B
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Awards, and Publ ications
Publications other than abstracts from this project:
Chanock, R.M., Wong, O.C., Huebner, R.J., and Bell, J. A.:
Serologic response of individuals infected with para
influenza viruses. Am. J. Pub. Hlth., I960.
Johnson, K.M., Chanock, R.M., Cook, M.K., and Huebner, R.J.:
Studies of a new human hemadsorption virus. I. Isolation,
properties, and characterization. Am. J. of Hyg., v:7l,
81-92, I960.
137
Serial No. NIAID - 70
1. Infectious Diseases
2. Virus & Rickettsial Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Laboratory studies of newly recognized viruses
associated with respiratory illness.
Principal Investigator: Dr. Robert M. Chanock
Other Investigators: Dr. K.M. Johnson, Dr. A. Kisch,
Dr. F.R. Abinanti
Cooperating Units: None
Man Years (calendar year I960):
Total: 20/12
Professional: 10/12
Other: 10/12
Project Description:
Ob iecti ves: I) To investigate the biologic and antigenic
properties of the newly recognized para influenza viruses. 2) To study
the properties of Eaton agent and its growth in various types of tissue
culture. 3) To develop simple procedures for the recovery, identifi-
cation, and serologic study of Eaton agent.
Methods Employed: The standard tools of virology — neutralization,
complement-f ixation, and hemagglut ination- inh ibition are employed to
determine the antigenic specificity and existence of shared antigens
among the para influenza viruses.
The fluorescent antibody technique employing frozen sections
of chick embryo lung is used as the standard of reference in all
studies with Eaton agent. Attempts are made to cultivate the agent in
various types of tissue culture and laboratory animals. Growth of
Eaton agent is determined by titration of tissue culture fluid or
organ suspension in embryonated eggs combined with immunof luorescent
examination of embryo lung tissue. Cytopathic effects, cytochemical
changes, and inclusion bodies are searched for in the inoculated tissue
cultures. Fluids harvested from inoculated tissue cultures are tested
Part B Included: Yes
138
Serial No. NIAID - 70
for antigens which might fix complement with potent convalescent serum
from patients with Eaton pneumonia.
Major Findings:
1 . Para influenza 3. The hemadsorption type I (HA- I) virus
recovered from humans with respiratory disease and the SF-4 virus
recovered from cows with shipping fever were shown to be distinct
ant igenical I y. Previously ,these agents had been considered very
closely related or identical. Guinea pigs infected with the HA- I
or SF-4 viruses developed a hemagglutinat ion-inh ibition and neutrali-
zation antibody response which permitted differentiation of these
agents. Complement fixation tests employing viral or soluble antigens
indicated that the two viruses were sufficiently related to be
classified together as sub-types or para influenza 3. Each of 23
human isolates from various parts of the world resembled the HA- I
prototype. Each of the 7 bovine isolates from different parts of the
United States were indistinguishable from the bovine prototype SF-4
virus. These findings suggest that these viruses do not cross species
boundaries.
2. Eaton agent. The Eaton agent was shown to multiply without
cytopathic effect in chick embryo entodermal, chick kidney, Hep-2,
human kidney, and monkey kidney tissue cultures. The highest level
of replication occurred in monkey kidney cultures where infected
fluids contained up to I04 egg infectious doses of the agent. Antigen
concentration within the infected tissue culture cells was insufficient
to permit visualization by immunofluorescence. Complement fixing
antigens were not detected in fluids from infected tissue cultures.
An eclipse phase was demonstrated during replication in monkey
kidney cells. This suggests that the agent should be classified as a
virus according to a recent definition proposed by Burnet.
In a simultaneous test, monkey kidney tissue culture was found to
be as sensitive as embryonated eggs for the recovery of naturally
occurring strains of Eaton agent. Fourteen strains were recovered in
tissue culture from 17 patients with serologically positive pneumonia.
Preliminary studies indicate that the agent is inhibited in
tissue culture by 1.5 micrograms per ml. of demethylchlortetracycl ine
(Dec lomyc in) . This concentration is approximately 1/2 that found in
the blood 24 hours after oral administration of a small dose (0.5 gm)
of the drug. These findings are consistent with the therapeutic effect
which we have recently demonstrated for demethylchlortetracycl ine in
Eaton pneumonia. Preliminary studies suggest that the drug inhibits
intracellular synthesis of Eaton agent, and has no effect upon the
agent when it is in the free extracellular state.
X39
Serial No. NIAIO - 70
Significance to the Program of the Institute: Knowledge of the
antigenic properties of the para influenza viruses is required in
order to perform meaningful epidemiologic studies with these agents.
Information bearing upon the species specificity of the para influenza
viruses greatly adds to our understanding of the natural history of
these agents.
The growth of Eaton agent in various types of tissue culture
and the recovery of naturally occurring strains in monkey kidney
culture represents a significant forward step in the study of this
respiratory pathogen. This technique has made it possible to recover
strains which were suitable for administration to human volunteers,
and to study the behavior of the agent in such infected individuals.
Tissue culture studies with demethylch lortetracycl ine provide an
experimental basis for understanding the chemotherapeut ic effect of
this drug in Eaton pneumonia, and offer a tool for further analysis of
this phenomenon.
Proposed Course of the Study: Efforts will continue to maintain
surveillance of the antigenic properties of the para influenza viruses.
The search for simpler laboratory procedures for the study of
Eaton agent will continue. Various types of tissue culture will be
tested for the capacity to support multiplication of this agent, pre-
ferably with cytopathic effect. Complement fixing antigen and
hemagglutinin will be sought.
1U0
Serial No. NIAID - 70
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Chanock, R.M., Johnson, K.M., Cook, M.K., Wong, O.C., and
Vargosko, A.: The hemadsorption technique with special reference
to the problem of a naturally occurring simian para influenza
virus. Proc. of the International Conf. on Asian Influenza.
In: AM. REV. OF RESPIR. DISEASES (book). |n press, 1961.
Craighead, J., Cook, M.K., and Chanock, R.M.: Infection of hamsters
with para influenza 3 viruses. Proc. Soc. Exptl. Biol. & Med.,
v: 104, 301-304, I960.
Chanock, R.M., Fox, H.H., James, W.O., Bloom, H.H., Mufson, M.A.:
Growth of laboratory and naturally occurring strains of Eaton
agent in monkey kidney tissue culture. Proc. Soc. ExptJ . Biol.
4 Med., v:l05, 371-375, I960.
Gordon, F.B., Quan, A.L., Cook, M.K., Chanock, R.M., and Fox, H.H.:
Growth of the''Eaton agent of primary atypical pneumonia in chick
entodermal tissue culture. Proc. Soc. Exptl. Biol. & Med.,
v: 105, 375-377, I960.
1U
Serial
1 .
2.
3.
No. NIAIO - 71
Infectious Diseases
Virus & Rickettsial
Bethesda, Maryland
Indi vi
Cal
PHS
dual 1
endar
-NIH
Project Report
Year I960
Part A.
Project Title: Studies of tumor-producing viruses.
Principal Investigator: Or. Wallace P. Rowe
Other Investigators: Or. J.W. Hartley, Dr. R.J. Huebner,
Mr. L.W. Smith
Cooperating Units: Or. L.W. Law and C.J. Dawe, NCI, NIAIO
Man Years (calendar year I960):
Total: 45/12
Professional: 16/12
Other: 29/12
Project Description:
Object i ves: To characterize the viruses which produce tumors
in animals from the standpoints of their laboratory properties,
natural behavior, and modes of spread.
Methods Employed; Application of standard and newly developed
virologic procedures for detection and quantitation of animal tumor
viruses.
Major Findings:
A. Mouse polyoma virus. Work with' this agent has continued
along the I ines of the past year. The newer emphasis has been
primarily on the natural history in wild mouse populations as
described in Project No. 68.
I. The patterns of infect ion i in individual animals have
been studied in greater detail. It has been found that the virus
produces a prolonged chronic infection when inoculated into weanling
mice. The weanling mice sporadically excrete the virus in the urine.
Part B included: Yes
112
Serial No. NIAIO 71
2. Serial passage lines of the virus have been carried in
newborn mice with passages made at the peak of viral infectivity.
In this way strains have been obtained which produce extremely high
hemagglutinin titers in suckling mouse tissue, and which have altered
patterns of disease production in newborn mice, producing greater
tendencies for inducing the runting form of infection, and an increased
tendency to produce bone tumors.
3. The laboratory aspects of the epidemiological work in wild
mouse populations have shown the existence of local infections in
rural farm mice and in mice in feed mills. The virus recovered from
wild New York mice was shown to be oncogenic in baby mice and hamsters;
the oncogenic activity of rural polyoma virus strains is under study.
4. It has been found that the virus is frequently present
in transplanted tumor lines.
5. Production stocks of laboratory mice freed of infection by
rearing under conditions similar to specific pathogen-free methods.
6. Polyoma virus in baby mice was shown to be a potent
interfering agent, producing interference against the lethal effects
of vesicular stomatitis and Coxsackie A viruses.
7. The reaction of mucoprotein non-specific inhibitors with
the virus has been further characterized. The reaction with the
inhibitor was found to induce marked aggregation of the virus. The
strains which have become highly sensitive to inhibitors were shown to
have lost, to a great extent, their ability to induce tumors in mice.
B. Mouse leukemia viruses. Studies of the Gross, Schwartz, and
Moloney mouse leukemia viruses have centered primarily on reproducing
the disease in laboratory mice, in attempts to free these viruses of
contaminating mouse viruses, and on developing simpler assay pro-
cedures for the leukemia viruses.
I. Moloney and Gross leukemia viruses have been propagated
through several serial mouse passages, produced a high incidence of
leukemia with relatively short incubation periods. By passage of the
Gross virus in the presence of suitable antiserum, it has been freed
of polyoma and K viruses for long periods, which are contaminants
of the standard Gross passage A virus. This should make it possible
to attempt the development of in vitro assay procedures which will be
specific for the leukemia virus..
113
Serial No. NIAID - 71
C. Lymphomatosis virus of chickens. The sero-ep idem io logic
studies of antibodies to the cytopathic virus of Sharpless and
Burmester indicated definitely that this virus is a contaminating
agent and not the etiologic agent of visceral lymphomatosis (see
Project Report 68-A).
Significance to the Program of the Institute: The contributions
which can be made to the knowledge of the biology of tumor viruses,
such as polyoma Friend leukemia and rabbit papilloma, when studied
with standard virologic techniques is now becoming well recognized.
The advantages of rapid indirect procedures for assay of tumor
viruses (and extraneous viruses as well) as compared with the slow
unpredictable tumor response endpoints make it imperative to attempt
to develop such procedures for additional animal tumor viruses in
order for reliable progress to be made in clarifying the viral
etiology of cancer.
Proposed Course of the Project; The emphasis in the coming year
will be to develop in vitro systems for recognition of the mouse
leukemia viruses, particularly the Gross and Moloney agents, with
special reference to a propagation in tissue culture. The epidemiologic
studies of polyoma virus will be continued.
3M
Serial No. NIAIO - 71
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Awards, and Pub I ications
Publications other than abstracts from this project:
Rowe, W.P., Huebner, R.J., and Hartley, J.W.: The ecology of a
mouse tumor virus. Monograph in: PERSPECTIVES IN VIROLOGY II.
In press.
Huebner, R.J.: Viruses in search of cancer. Monograph in:
PERSPECTIVES IN VIROLOGY II. In press.
Rowe, W.P., Hartley, J.W., Estes, J.D., and Huebner, R.J.: Growth
curves of polyoma virus in mice and hamsters. National Cancer
Institute Monograph No. 4 - Symposium on Phenomena of the Tumor
Viruses, New York City, N.Y. , March 25 and 26, I960.
Rowe, W.P.: The epidemiology of mouse polyoma virus infection.
Bacteriological Reviews. In press.
Rowe, W.P., Hartley, J.W., and Huebner, R.J.: The natural history
of polyoma virus infection - a summary. In: CANADIAN CANCER
CONFERENCE. In press.
Oawe, C.J., Rowe, W.P., and Law, L.W.: Influence of age, species,
and immune factors on the response of salivary gland to polyoma
virus in tissue culture. Pathologie et Biologie. In press.
Law, L.W., Rowe, W.P., and Hartley, J.W.: Studies of mouse polyoma
virus infection. V. Relation of virus infection to lymphocytic
neoplasms of the mouse. The J. of Exp. Med., v: I I 1 , 517-523,
I960.
Honors and awards related to this project:
Eli Lilly Award - Meeting of the Society of American Bacteriologists,
May, I960, Philadelphia, Pa.
Invited to participate in Symposium On Perspectives in Virology II,
and present paper entitled "The Ecology of a Mouse Tumor Virus"
January 25 and 26, I960, New York City, N.Y.
Invited to attend Symposium On Phenomena of the Tumor Viruses, and
present paper entitled "Biologic Behavior of the Polyoma Virus
in Tissue Culture" March 26, I960, New York City, N.Y.
145
Serial No. NIAID - 71
PHS-NIH
Individual Project Report
Calendar Year I960
Appointed to Research Advisory Committee on Etiology of Cancer of
the American Cancer Society, September, I960.
1*46
Serial No. NIAIO - 7IA
1. Infectious Diseases
2. Virus 4 Rickettsial
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Continuing studies of adenoviruses and salivary
gland viruses as examples of latent viruses.
Principal Investigator: Dr. Wallace P. Rowe
Other Investigators: Dr. J.W. Hartley, Dr. R.J. Huebner
Cooperating Units: Dr. J. A. Kasel, LCI
Man Years (calendar year I960):
Total: 5/12
Professional: 1/12
Other: 4/12
Project Description:
Objectives: To characterize the salivary gland viruses and
adenoviruses, and to determine their epidemiology and clinical
importance.
Methods Employed: Serologic epidemiology and virus isolation
in tissue culture. Studies of the biology of adenoviruses and
salivary gland viruses in lower animals.
Major Findings:
Complement fixation tests with the human salivary gland virus
have been done on sera of a number of suspected or diagnosed cases
of cytomegalic inclusion disease of the newborn submitted by workers
in various parts of the world. The findings suggest that in the
newborn and young infant, the test is of I ittle or no value in
diagnosis, in that most cases have not yet responded with antibody
during the period when diagnosis is necessary.
The finding of high prevalence of salivary gland virus
infection in wild mice has been extended by testing mice from a
number of rural areas, and the same high frequency of infection has
been found. It has also been found that the infection in the wild
Part B included: Yes
147
Serial No. NIAID - 7IA
mice is of long duration, the mice excreting virus in the saliva for
as long as eight months after capture.
A new virus has been isolated from Microtus mice which appears
to represent the Microtus strain of salivary gland virus group as
detailed in Project No. NIA 10-71 B.
We have continued to work with Or. Kasel of the LCI in
characterization of the enzyme formed by certain adenovirus types
which destroys hemagglutination receptors on human erythrocytes.
It has been established that the factor is neutralized type specifi-
cally by homologous rabbit antiserum, that it is separable from the
infectious virus, and that its activity shows many characteristics of
an enzymatic reaction.
Significance to the Program of the Institute: Th i s I aboratory
is one of the very few in the world which has competence in handling
the salivary gland viruses. There is great interest in clinical
fields in the infections produced by this virus, and a corresponding
interest in laboratory diagnosis of cases. The existence of
comparable viruses in laboratory animals provides an excellent
opportunity to study model infections.
Proposed Course of the Project: The diagnostic studies and
the evaluation of the serologic tests will be continued. Epidemiologic
studies of the spread of the virus in laboratory mice will be carried
out by exposing mice through infected contacts under varying
conditions.
148
Serial No. NIAIO - 7IA
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Awards, and Publ ications
Publications other than abstracts from this project:
Hartley, J.W., and Rowe, W.P.: A new mouse virus apparently
related to the adenovirus group. Virology, v: 1 1 , 645-647, I960.
Thai hammer, 0., and Rowe, W.P.: Gibt es im Raum von Wien
cytomegal ie? Wiener klinische Wochenschrl ft. Sonderabdruck
aus 72. Nr. 36, S. 621-624, I960.
Kasel, J. A., Rowe, W.P., and Nemes, J.L.: Modification of
erythrocyte receptors by a factor in adenovirus suspensions.
Virology, v: 10, 388-391, I960.
Rowe, W.P.: Adenovirus and salivary gland virus infections in
children. In: VIRAL INFECTIONS OF INFANCY AND CHILDHOOD.
Ed. Harry M. Rose. Hoeber-Harper book, 205-214, I960.
143
Serial No. NIAIO - 7 IB
1. Infectious Diseases
2. Virus & Rickettsial
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Studies of mouse viruses with special reference
to their importance as extraneous viruses
("background noise") in mouse tumor systems.
Principal Investigator: Dr. Janet W. Hartley, Dr. Wallace P. Rowe
Other Investigators: Mr. L.W. Smith, Dr. R.J. Huebner
Cooperating Units: None
Man Years (calendar year I960):
Total: 87/12
Professional: 30/12
Other: 57/12
Project Description:
Object i ves: To characterize and develop methods for
recognizing infection and determine the epidemiological patterns of
the various mouse viruses, for the purpose of defining and eliminating
the problem of extrinsic viruses as complicating factors in studies
of mouse tumor viruses.
Methods Employed: Serologic, tissue culture, and animal
pathogenicity studies; attempts to isolate agents from laboratory and
wild mice and from mouse tumors. Studies of the epidemiology of
the agents under natural and artificial conditions.
Major Findings:
A. Mouse adenovirus. The new virus recovered from General
Purpose Swiss mice which produces myocarditis and adrenal necrosis,
has been identified as a mouse representative of the adenovirus group.
Infection with this agent is found in many laboratory mouse colonies,
but, so far, has not been encountered in wild mouse populations.
Part B Included: Yes
150
Serial No. NIAID - 7 IB
Virus is found in urine of spontaneously and artificially infected
mice for prolonged periods up to 8 months or longer, and it is con-
cluded that the chief mode of spread of this virus is by urinary
excretion.
B. Thymic agent (TA). A new virus has been recovered from
the NIH General Purpose Swiss mice and from wild house mice. This
virus induces a unique necrosis of the thymus when inoculated into
infant mice. It produces a chronic infection with prolonged
excretion of virus in saliva.
C. Reovi ruses have been found to be very common agents of
laboratory mice. Reovirus 3 has been found in the majority of mouse
colonies, and suggestive evidence of its presence in germ-free mice
has been obtained. The hepatoencephal it is virus of Stanley, which
was isolated in 1953, and considered by some workers to be a mouse
virus, has been identified as a Reo 3 strain.
D. A new virus, presumably of mouse origin, has been isolated
from Qrn ithonyssus bacoti mites which were feeding on laboratory mice.
This virus induces an encephalitis in baby mice.
E. In the course of studies of polyoma epidemiology in wild
rodent populations, a new virus isolated from the Microtus mouse.
This virus perhaps is the Microtus representative of the salivary
gland virus group, but it differs from other species salivary gland
viruses by its ability to grow in tissue cultures from many diffei —
ent species. A complement fixation test has been developed for this
agent.
F. "K" virus. The K virus of mice has been found to be
prevalent in almost all colonies tested. Suggestive evidence of its
presence in germ- free mice has been obtained. K virus has also been
isolated from wild mice, being found in saliva or urine. A complement
fixation test has been developed which currently is being analyzed.
G. Mouse hepatitis virus. Studies have been made of the mouse
hepatitis virus and its activation by Eperythrozoon coicoides as
described by Gledhill. Hepatitis virus can be activated in specific
pathogen-free mice with the same regularity as in conventional lab-
oratory mice, suggesting that the virus is transplacental I y acquired.
H. Mouse salivary gland virus has been found to be prevalent
in virtually all wild Mus musculus colonies both urban and rural. Its
natural history is currently under study.
151
Serial No. NIAIO - 7 IB
Significance to the Program of the Institute: With the con-
tinual expansion of attempts to develop indirect assay procedures
for mouse tumor viruses, it has become increasingly evident that the
viral flora of the mouse is a major stumbling block to developing
reliable study systems. In this and other laboratories, attempts
to develop such assays for tumor viruses have repeatedly foundered
because of the emergence of extraneous mouse viruses in the indicator
systems. Only by a thorough understanding of the extraneous agents
which are likely to be encountered, and the ability to recognize
them can tumor virus studies proceed intelligently with the hope of
obtaining reliable information of high order. Secondly, the increasing
evidence of the importance of the interference phenomenon in deter-
mining the outcome of viral experiments in mice makes it important
to evaluate the importance of extraneous viruses as interfering
factors in tumor igenesis studies. Thirdly, the possibility must be
considered that so-called "non-tumor viruses" may play a role in the
etiology of neoplasms; the mouse viruses provide an excellent oppor-
tunity to test this hypothesis. Fourthly, the viruses of mice have
long been recognized to be useful models of virus infections of
humans (some of them are identical or similar to human areas) and on
their own merit are deserving of study.
Proposed Course of the Project: Epidemiologic studies of the
newer mouse viruses will be continued, and additional attempts will
be made to develop and evaluate serologic tests useful for detection
of infection.
Extension of current serological surveys are planned to detei —
mine if associations exist between certain mouse tumors and past
infection with the known and newly discovered mouse viruses. Mice,
hamsters, and other laboratory species will be inoculated with sub-
lethal doses of the various mouse viruses and long-term observation
will be carried out for possible tumor induction.
Studies will be made of the influence of various mouse breeding
practices such as sterilization of food and rearing under specific
pathogen-free and germ-free conditions as they influence the
occurrence of mouse viruses in the hope that practices can be designed
to eliminate mouse viruses as an uncontrolled variable in study
systems.
152
Serial No. NIAID - 7 IB
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Awards, and Pub I ications
Publications other than abstracts from this project:
Hartley, J.W., and Rowe, W.P.: A new mouse virus apparently
related to the adenovirus group. Virology, v: I 1 , 645-647, I960.
153
Serial No. NIAID - 72
1. Infectious Diseases
2. Virus & Rickettsial Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Laboratory studies of enteroviruses.
Principal Investigator: Dr. Karl M. Johnson
Other Investigators: Dr. Robert M. Chanock, Dr. Robert J. Huebner
Cooperating Units: Dr. L. Rosen, Epidemiology Section, LID
Man Years (calendar year I960):
Total: 14/12
Professional: 4/12
Other: 10/12
Project Description:
Objectives: To develop simple reproducible methods for laboratory
manipulation of enteroviruses, particularly the large group of newly
recognized agents which appear to be potential causes of respiratory
disease. Studies are designed specifically to determine optimum methods
of cultivation of the agents in either tissue culture or small laboratory
animals. In addition, efforts are made to a) determine the optimum
manner of preparing specific antisera, b) to evaluate various serologic
procedures potentially of use in field studies, and c) since many new
viruses are fastidious in their growth requirements, to study where
indicated certain fundamental properties of these viruses.
Methods Employed: These will be described where pertinent
under consideration of results so far obtained with certain of the viruses.
Major Findings:
I . Coe virus: Newly recovered strains of Coe virus have proven
to have properties differing from those described for the original
prototype agent in several important respects. The presence of hemagg-
lutinin is demonstrable when human type 0 erythrocytes are employed
and the test performed at 4° centigrade. The reaction is temperature
dependent and can be reversed at 34° C. Elution, however, is not
enzymatic since the erythrocyte receptors are not altered following
Part B Included: No
154
Serial No. NIAID " 72
reversal of hemagglutination. The component of the virus which
participates in the hemagglutination reaction contains specific Coe
antigen as indicated by the inhibitory effect of specific antiserum.
The hemagglutinin is quite stable at 4° and at -20° C, but is fairly
rapidly inactivated at 34° C. Studies of virus antibody kinetics in
the hemagglutination inhibition (HI) test indicate that optimal virus
antibody reaction occurs following incubation at 24° for approximately
2 hours, or at 4° for 14 hours. Using the latter procedure, a rise in
HI antibody was demonstrated for 15 of 19 men from whom the virus was
recovered.
Comparative studies indicate that primary human embryonic kidney
cells (HK) are more sensitive than serially passaged human cells to
natural strains of Coe virus. In addition, HK cells produce a greater
quantity of hemagglutinin than do continuous cell lines of human origin
regardless of the nutrient medium employed. For optimum results with
Coe virus it is necessary that the culture tube be rotated rather than
incubated in the conventional stationary position.
Our strain of Coe virus, unl ike the prototype, has been success-
ful ly adapted to the suckling mouse. Typical Coxsackie-I ike hind leg
paralysis has been produced through 5 consecutive passages. This
observation is currently being exploited in attempts to prepare specific
viral antisera, as well as for experimental production of a complement
fixing antigen.
2. Newly described enterovirus- I ike agents: In January of this
year English workers described several strains of viruses with certain
properties of enteroviruses, which they had recovered from nasal
secretions of volunteers with clinical cold-like illnesses. They
reported that an unusual set of laboratory conditions was required for
a successful cultivation of these agents. In genera I,, we have
succeeded in confirming their observations and have made further progress
in efforts to adapt and manipulate these viruses in the laboratory.
Human embryonic kidney seems to be necessary for recovery of most of
these agents. Preliminary studies suggest that there is an inverse
relationship between the gestational age of the fetus from whom the
cultures are derived and the occurrence of cytopathic effects produced
by these agents. At least two of these viruses have been successfully
adapted in this laboratory to continuous type cultures of human
epithelial origin. Using these cells as a source of viral antigen,
high-titered specific immune serum has been prepared in guinea pigs
and a reproducible neutralization technique worked out. Current efforts
are being directed toward a search for complement fixing antigens and
hemagglutinins. Employing human embryonic kidney tissue cultures,
numerous new virus strains have been recovered which seem to possess
properties similar to those of the English viruses. Sorting out these
new isolates, and the establishment of new serotypes, is currently
under way.
155
Serial No. NIAIO - 72
Significance to the Program of the Institute; These studies
provide new diagnostic tools and opportunities for epidemiologic
investigation of a large group of viruses which are potentially
significant in human respiratory disease.
Proposed Course of the Project: Efforts will be made to simplify
the identification of the enteroviruses which grow predominantly in
human tissue culture cells. This includes sorting out of the available
isolates and the recognition of new serotypes. Fortunately, methods
for CF and HI identification of enteroviruses developed by the
laboratory unit of the Epidemiology Section and by the Serology Unit
of LID, promises to reduce the time and work required to compare
presumably "new" enteroviruses with the 58 serotypes already classified.
Attempts will be made to develop simplified methods of antibody assay
for use in epidemiologic studies. Until this is done, understanding of
the role these agents play in human disease will proceed at a slow
pace.
156
Serial No. NIAID - 72A
1. Infectious Diseases
2. Virus & Rickettsial Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title; Study of viral respiratory disease in a military
population.
Principal Investigator: Dr. Karl M. Johnson
Other Investigators: Dr. Robert M. Chanock, Dr. Robert J. Huebner
Cooperating Units: Dr. H.H. Bloom, Dr. M. Mufson, Naval Medical
Field Research Laboratory, Camp Lejeune,
North Carol ina
Man Years (calendar year I960):
Total: 24/12
Professional: 6/12
Other: 18/12
Project Description:
Ob ject i ves: To study on a continuing basis the role of newly
recognized viruses in adult respiratory illness. This study was
organized in order to compare viral experience of men fresh from recruit
training with that of individuals forming the stable cadre personnel.
An opportunity is also provided to assay the effectiveness of certain
viral vaccines administered to personnel during their recruit training
at another mil itary center.
Methods Employed: Clinical records and appropriate specimens are
collected routinely from individuals in various dispensaries at Camp
Lejeune, North Carolina. Such materials are also obtained from indivi-
duals hospitalized at this base, and in certain instances, from children,
wives and mothers seen in dependent outpatient clinics. Special emphasis
has been placed upon the collection of specimens from matched control
groups.
CI inical specimens are tested in monkey kidney and Hep-2 tissue
cultures. Certain other tissue cultures are employed whenever
indicated. Paired serum samples are available from well over 90# of
individuals in the study and are assayed for development of antibody
for various viral agents. Specimens for virus isolation are divided
into two portions and stored in this laboratory and the Camp Lejeune
Part B Included: Yes
157
Serial No. NIAID - 72A
laboratory respectively.
Major Findings;
1 . Adenoviruses: In February, March, and April of I960 adeno-
viruses were responsible for a major outbreak of respiratory disease in
the post recruit population (Infantry Training Regiment, ITR). Attack
rates in this unit attained a level of nearly 200 per 1000 week.
Infection and apparent illness were also disseminated into the cadre
population, although there was a clearly diminishing incidence in
correlation with length of time individuals had been in military service.
2. Myxoviruses (parainf luenzas) < During February and March of
I960 sporadic isolations of para influenza viruses were also recorded.
Although insufficient data were obtained to permit epidemiologic
association of para influenza virus infection with illness, several new
laboratory phenomena were observed which had not been previously
encountered with these viruses in the study of pediatric populations.
With only one exception, all 15 individuals from whom virus was
recovered had significant neutralizing antibody against the infecting
agents in their acute phase sera. Virus recovery proved difficult, and
many of the isolates were not recognized until after two weeks of tissue
culture incubation. Re-isolation of the agents proved exceedingly
difficult. Appropriate controls were included in each test to insure
that the isolates did not represent contamination from the laboratory
environment. Serologic confirmation of infection was obtained in only
9 of the 15 cases despite the utilization of neutralization, hemagg-
lutination-inhibition, and complement fixation techniques. The lack of
serologic response to infection was most common with para influenza I
virus. These observations suggest that the infections were mild and
that very smal I amounts of virus were excreted by the infected indivi-
duals. Information derived from this experience, however, has allowed
a better formulation of laboratory procedures designed to elucidate
the role of these agents in adult respiratory illness in the future.
3. Coe virus outbreak; During October and November of I960, a
major outbreak of respiratory disease associated with Coe virus, a
newly discovered enterovirus- 1 ike agent, was observed. Virus was
recovered significantly more often from men with respiratory disease
than from comparable control subjects free of such illness. Attack
rates have not yet been calculated, but they seem to have been
extremely high. Clinical illness consisted of mild upper respiratory
infection with or without fever. Mild pharyngitis was frequently
observed. Hospital surveillance indicated no significant increase in
the number of admissions for pneumonia, nor were there any clinical
cases of herpangina, pleurodynia, or aseptic meningitis. The occurrence
of infection in children during this time has also been documented
but the role of this virus in pediatric illness remains to be assessed.
158
Serial No. NIAID - 72A
The current outbreak represents the first documented association of
an enterovirus with a large scale occurrence of mild respiratory illness
in adults.
Significance to the Program of the Institute; This project
represents a major continuing inquiry into the nature of respiratory
disease in adult populations. It has already yielded evidence for the
association of a new enterovirus with respiratory disease, as well as
providing needed laboratory information concerning para influenza virus
infection in adults. A well documented collection of case histories,
specimens, and paired sera provides an excellent source of clinical
material which is available for the evaluation of newly discovered
agents, as we I I as those not yet known to us.
Proposed Course of the Project: Field surveillance is being
continued. Extension of the program to incorporate dependents (both
adults and children) is being undertaken. In this way a broader picture
of the natural history of newly recognized respiratory viruses can be
acqu ired.
A study designed to test the effectiveness of adenovirus vaccines
is also being organized. Since all personnel at the Infantry Training
Regiment represent immediate graduates of the recruit training base at
Parris Island, South Carolina, and since all these men are currently
being given adenovirus vaccine upon entry into the Marine Corps the
following procedures will be employed: A large number of men undei —
going training at Parris Island will be bled approximately 4 to 5 weeks
following administration of adenovirus vaccine. Recruits are routinely
bled when they enter service and this serum is available to us. These
individuals will be observed during their time at the Infantry Training
Regiment, and a certain percentage of them will also be available for
study following this period of training. In this way the immunogenic
potency of the vaccine as well as its effectiveness in preventing
illness may be assessed.
In cooperation with personnel at the hospital at Camp Lejeune a
study of the effectiveness of tetracyclines in the therapy of febrile
respiratory disease with and without pneumonia is also being organized.
Among the agents to be included in this study will be pneumonia caused
by the Eaton virus which has recently been shown to respond to chemo-
therapy.
153
Serial No. NIAID - 72A
PHS-NIH
Individual Project Report
Calendar Year I960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Bloom, H.H., Johnson, K.M., Chanock, R.M., Huebner, R.J., and
Jacobsen, R.F.: Recovery of para influenza viruses from adults.
J. of Clin. Investigation. In press.
160
Serial No. NIAID - 72B
1. Infectious Diseases
2. Virus 4 Rickettsial Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Study of respiratory viruses in human volunteers.
Principal Investigator: Dr. Robert M. Chanock, Dr. Karl M.
Johnson
Other Investigators: Dr. V.L. Knight, Dr. H.M. Kravetz,
Dr. D.E. Rifkind, Dr. J. P. Utz
Cooperating Units: Laboratory of Clinical Investigations,
NIAID
Man Years (calendar year I960):
Total: 17/12
Professional: 5/12
Other: 12/12
Project Description:
Object i ves: To determine whether newly recognized viruses of
potential respiratory pathogenicity are capable of inducing mild
illness in adults under controlled conditions. To examine the
experimental pathogenesis of such viral infection, and to provide
new information specifically designed to facilitate the study of
naturally occurring mild respiratory illness in adults.
Methods Employed: Young adult male volunteers from federal
prisons are selected on the basis of a) general good health, and b)
neutralizing antibody status for the particular virus to be studied.
Volunteers are maintained in groups of three in strict isolation on
a clinical service of the NIAID. Histories and routine physical
examinations, as well as certain basic laboratory procedures are
performed upon admission. Known amounts of test viruses are
administered intranasal ly, and daily clinical observations, as well
as appropriate specimens for laboratory analysis, are obtained.
Virus specimens are inoculated into appropriate tissue cultures and/or
animals. Serologic tests are performed on suitably collected serum
samples.
Part B Included: No
161
Serial No. NIAID - 72B
Major Findings:
Para influenza 4; In a single experiment, approximately
10,000 TCD50 of monkey kidney adapted para influenza 4 virus infected
only two of six volunteers receiving the inoculum. No clinical
illness was observed.
Respiratory Syncytial Virus: In four experiments, volunteers
were successfully infected when given a small dose (160 to 640 TCD50)
of respiratory syncytial (RS) virus. One half of the volunteers
developed typical cold-like illness without fever or pharyngitis.
The occurrence of illness correlated with the length of time virus
was shed and the onset of virus excretion was coincident with or
preceded the appearance of cl inical illness in every instance, pro-
viding evidence that the observed colds were actually produced by the
inoculated virus. Development of RS virus complement fixing antibody
correlated with the occurrence of illness. Since all volunteers in
this study possessed RS neutralizing antibody prior to challenge,
these experiments represent examples of mild illness produced by
viral reinfection. The occurrence of illness was not related to the
level of neutralizing antibody present prior to administration of
virus. Studies designed to investigate the circumstances associated
with this interesting and potentially significant finding are
currently in progress.
Significance to the Program of the Institute: This project has
resulted in the experimental demonstration of a significant new concept
concerning the pathogenesis of mild respiratory illness in adults. It
has also provided valuable information pertinent to the design and
execution of future field studies of RS virus infection.
Proposed Course of the Project; Volunteer studies involving
the Eaton agent (associated with all, or almost all, cold agglutinin
positive atypical pneumonia and a significant proportion of cold
agglutinin negative pneumonia) are in progress. This investigation
should yield important information relating to the varying clinical
consequences of infection with this agent. Future studies are planned
to evaluate certain other potential respiratory viruses, including a
para influenza virus, the family of Reoviruses, as well as certain
newly described enteroviruses.
Influenza A and B viruses grown in monkey and human tissue
cultures will be studied in volunteers as part of a search for
attenuated variants suitable for inclusion in a live virus vaccine.
162
Serial No. NIAIO - 73
1. Infectious Diseases
2. Virus A Rickettsial Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Application of fluorescent antibody staining
technique to the study of respiratory viruses.
Principal Investigator: Dr. Alexander L. Kisch
Other Investigators: Dr. Robert M. Chanock, Dr. Karl M. Johnson
Cooperating Units: None
Man Years (calendar year I960):
Total: 8/12
Professional: 6/12
Other: 2/12
Project Description:
Ob jecti ves: I) To study the sequence of intracellular events
leading to the production of viral antigen and the production of
syncytia by respiratory syncytial (RS) virus, and to differentiate
this virus from others, such as measles, also producing syncytia in
tissue culture. 2) To develop, if possible, a rapid diagnostic test
for certain viral agents causing respiratory disease in children
and adults utilizing fluorescent antibody technique. 3) To determine
the nature of the intracellular substance which is labelled by
f luoresce in-l abel led antibody using cytochemical methods.
Methods Employed: The agents under study, specifically RS virus
and Eaton agent, are grown in tissue culture using a variety of tissues.
At timed intervals, infected covers I ips are removed and stained by
several methods: fluorescent antibody, Toluidine Bl ue-Mol ybdate (for
RNA), Feulgen (for DNA), Hematoxyl in-eosin (for inclusion bodies),
Sudan Black and PAS (for definition of "golgi material"). RS virus is
compared with measles virus, which also leads fo syncytia production
in the attempt to further characterize and classify RS virus.
Further, cell-bearing secretions from the respiratory tract of
volunteers, some of whom have been infected with either RS or PAP virus,
are stained by indirect fluorescent antibody technique utilizing
Part B Included: No
163
Serial No. NIAID - 73
appropriate controls in the attempt to diagnose infection early
and specifically. If this becomes possible, it would have significant
therapeutic implications in one of the few virus diseases susceptible
to broad spectrum antibiotics.
Major Findings;
It was possible to stain cells infected with RS virus
specifically using human convalescent serum and f luorescein-label led
anti-human globulin. Fluorescent staining antigenic material is
restricted to the cytoplasm, and is first demonstrable after
approximately 12 hours. Individual infected cells show fluorescence
24 hours before the earliest characteristic CPE is recognizable by
light microscopy. No intranuclear fluorescence was observed despite
the appearance of nuclear changes occurring earlier and simultaneously
in infected cells as demonstrated by Toluidine Blue-Mol ybdate
staining.
Attempts to demonstrate specifically-staining antigen in cells
from the nasopharynx of volunteers infected with RS virus have been
unsuccessful to date.
Significance to the Program of the Institute: Th i s project has
resulted in a development in this laboratory of a technically diffi-
cult technique of wide applicability to the study of viral infection
in tissue cultures as well as in infected patients. Should it become
possible to diagnose human infections with RS virus and/or Eaton
agent by this method, this will be of value in clinical and epidemio-
logic studies.
Proposed Course of the Project: Further studies to determine
whether it is the viral or the soluble antigen of the RS virus which
is stained by immunofluorescence, will be carried out. Studies on
secretions obtained from volunteers infected with Eaton virus are
under way, and further studies on subjects infected with RS virus
will be carried out at a later date.
164
Serial No. NIAID-74
1. Infectious Diseases
2. Virus and Rickettsial
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A
Project Title: Study of Bovine Respiratory Diseases.
Principal Investigator: F. R. Abinanti
Other Investigators: R. J. Huebner (LID); Robert Byrne (University
of Maryland); Alvin Hoerlein (University of
I I I inois)
Cooperating Units: Veterinary Science Department, University of
Maryland; College of Veterinary Medicine,
University of Illinois
Man Years: (Calendar Year I960)
Total: 6/12
Professional : 3/12
Other: 3/12
Project Description:
Objectives : This is a continuation of a project in effect
during the preceeding fiscal year. Essentially these investigations
include studies of outbreaks of respiratory virus disease in cattle,
of experimental pathogenesis of the viruses injected into cattle,
and conducting sero-epi zootio logic surveys of their prevalence.
Experimental vaccines are also prepared and their antigenic potency
tested in cattle.
Specific Objectives: (I) The isolation of viral agents from
the respiratory tract of normal cattle and cattle with respiratory
disease; (2) to assess the role of myxovirus para-inf I uenza 3
virus and other newly recognized viruses in respiratory disease
of cattle; (3) to determine the relationships of the human and
bovine strains of myxovirus para-inf I uenza 3; and (4) to evaluate
antiviral vaccines.
Part B incl uded : Yes
165
Serial No. NIAIO-74
PHS-NIH
Individual Project Report
Calendar Year I960
Methods Employed: Specimens of nasal secretions were taken
from cattle and inoculated onto tissue cultures of bovine and
monkey kidney. The viruses isolated are characterized and compared
to known viral agents of man, cattle, and other animals. Serial
samples of blood are taken from normal cattle and acute and con-
valescent specimens from diseased cattle, and hemagglutination
inhibition, complement fixation, and neutralization tests are used
to obtain information concerning the prevalence, spread and natural
history of respiratory viruses. Groups of cattle are vaccinated
with experimental vaccines and potency assessed by various serologic
tests.
Major Findings: (I) Eighteen recoveries of myxovirus para-
influenza 3 virus have been made from cattle suffering from respiratory
disease and serologic evidence of a relationship of this virus to
the outbreaks of respiratory disease was found. Extensive sero-
epidemiologic studies provided evidence that infection with this
virus is widespread. In a preliminary test, young cattle developed
good levels of antibody to experimental vaccines prepared against
both human and bovine strains of para-inf I uenza 3; they appeared
to be refractory to an aerosol challenge by live virus.
(2) Several large groups of cattle were vaccinated under
field conditions with commercially produced para-inf I uenza 3 (bovine
strain) vaccines. These vaccines also produced good levels of
circulating antibody. Unfortunately, in these field trials little
or no respiratory disease occurred.
(3) Comparative studies of the human and bovine strains
of the virus showed that they can be distinguished by means of
prototype guinea pig sera in the HI test.
(4) Infectious bovine rhinotracheitis, a virus usually
recovered from cattle with respiratory disease, was recovered from
cattle involved in a large outbreak of bovine conjunctivitis -
attended by little or no respiratory disease. The virus strain
recovered produced conjunc itivtis and minimal respiratory illness
in experimentally inoculated calves.
166
Serial No. NIAID-74
PHS-NIH
Individual Project Report
Calendar Year I960
Significance to the Program of the Institute: The discovery
of viruses in domestic animals which are similar or identical to
those responsible for large amounts of human respiratory disease
is of obvious importance. Furthermore, the study of respiratory
disease in domestic animals and the efficacy of antiviral vaccines
in controlling such illnesses provide incomparable models of similar
problems in man. In addition, solutions to some of the common
viral diseases of food animals have great significance for human
health in many areas of the world where animal protein is deficient.
The prevalence of these domestic animal viruses suggests the
possibility of a widespread zoonotic potential.
Proposed Course of Project: Work on this project has been
temporarily discontinued. We do not have sufficient resources to
mount adeguate field studies, and so far we have found it impossible
to find collaborators able to conduct studies designed to answer
definitive guestions on the etiology of bovine respiratory diseases.
167
Serial No. NIAID-74
PHS-NIH
Individual Project Report
Calendar Year I960
Part B: Honors, Awards, and Publications
Publ ications other than abstracts from this project:
Abinanti, F.R., Byrne, R.J., Watson, R.L., Poelma, L.J., Lucas,
F.R., and Huebner, R.J.: Observations on infection of cattle
with myxovirus para-inf I uenza 3. Amer. J. Hyg., 71 : 52-58,
I960.
Abinanti, F.R. and Plumer, G.J.: The isolation of infectious
bovine rhinotracheitis (IBR) virus from cases of conjunctivitis
and observations on the experimental infection. A.V.M.A.
Research J. Accepted for publication.
Byrne, R.J., Abinanti, F.R., and Huebner, R.J.: Vaccination of
cattle with myxovirus para-inf I uenza 3. Cornell Vet.
Accepted for publication.
Abinanti, F.R., Hoerlein, A.B., Watson, R.L., and Huebner, R.J.:
Serological studies of myxovirus para-inf I uenza 3 in cattle
and prevalence of antibodies in bovines. J. Immunol. Accepted
for publ ication.
Book: ADVANCES IN VETERINARY SCIENCE. Chapter on "'Shipping
Fever in Cattle", by S . K. Sinha and F.R. Abinanti. 100 pages.
To be included in 1962 edition.
168
Serial No. NIAID-74
PHS-NIH
Individual Project Report
Calendar Year I960
Honors and Awards relating to this project:
Invited to participate in Annual Conference of Veterinarians.
Talk: "Respiratory Diseases of Cattle" - University of Pennsylvania,
Phi ladelphia, May, I960.
Program Chairman and participant in Maryland Veterinary Medical
Association Meeting - Ocean City, Maryland, June, I960.
Participated in Conference on Zoonoses, University of Illinois,
Urbana, September, I960.
Invited to participate in CDC Biennial Public Health Conference.
Talk: "Respiratory Diseases of Man and Animals" - Atlanta, Georgia,
September, I960.
Participated in Conference of Public Health Veterinarians,
American Public Health Association, San Francisco, California,
November, I960.
169
Serial No. NIAID- 74A
1. Infectious Diseases
2. Virus and Rickettsial
3. Bethesda, Maryland
PHS-NIH
ndividual Project Report
Calendar Year I960
Part A
Project Title: A Study of Squamous Cell Carcinoma (Cancer Eye)
in Cattle.
Principal Investigators: Dr. F. R. Abinanti, Dr. W. Gay (DRS),
Dr. M. Stanton (NCI)
Other Investigators: Dr. R. J. Huebner
Cooperating Units: Bay Manor Farms (owner Mr. Otis Smith),
Lewes, Delaware; M.D. Anderson Hospital
(Dr. Russell), Houston, Texas
Man Years: (Calendar Year I960)
Total: 9/12
Professional : 6/12
Other: 3/12
Project Description:
Ob.jecti ves : There are approximately 600 hereford cattle on
the Bay Manor Farms, Lewes, Delaware, and about 30 per cent of these
cattle have precancerous or cancerous lesions of the eyes and
orbital appendages. The principal efforts are to determine if a
virus is responsible for this condition and to study the natural
history of the disease.
Methods Employed: Monthly inspections are made of the herd
and a photographic record is being made of the progress of the
lesions. At this time eye swabs are taken and blood for possible
future serological studies. Dr. William Gay surgically removes
affected tissues at intervals which are examined histologically
by Dr. M. Stanton, and virological ly by Dr. F. R. Abinanti. Further
frozen sections will be examined by the fluorescent technique to
explore the possibility of there being a specific antigenic material
present in the cancers.
Part B Incl uded : No
170
Serial No. NIAID- 74A
PHS-NIH
Individual Project Report
Calendar Year I960
A sero-ep izootio logical study of cattle with and without
"cancer eye" has been planned in cooperation with the M.D. Anderson
Hospital "Cancer-Eye" research group under Dr. William Russell.
Several hundred sera will be collected by this group from cattle
with cancer eye and matched controls. The specimens will be tested
for antibodies vs. bovine viruses and for some of the mouse, chicken
and rabbit tumor viruses.
Major Findings: None. This is a new project.
Significance of the Program to the Institute: Provides an
opportunity to explore the possible viral etiology of squamous
cell carcinoma. The Bay Manor herd provides excellent nearby
material for epidemiological and natural history studies of the
cancer itself. Since the precancerous lesions are papillomatous,
the possible relation of the lesions and papilloma viruses is
currently under study.
Proposed Course of the Study: The continuation of this
project will depend on whether practical leads concerning causative
viruses can be found in the near future; in that event, the termina-
tion date of this project cannot at this time be predicted.
171
Serial No. NIAID- 75
1. Infectious Diseases
2. Virus and Rickettsial
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A
Project Title: Laboratory and epidemiologic studies of viruses
as possible etiologic agents of acute leukemia
in the pediatric age group.
Principal Investigator: Dr. M. Katherine Cook
Other Investigators:
Cooperating Units:
Dr. Robert J. Huebner
Mr. Horace C. Turner
Dr. Charles Chany, Laboratoire des Virus,
Hopital Saint-Vincent-de-Paul , Paris;
Dr. Jean Bernard, Hematology Service,
le centre Claude-Bernard de M Hopital
Saint Louis, Paris
Man Years: (Calendar Year I960)
Total: 14/12
Professional : 14/12
Other : None
Project Description:
Objectives: ( I ) To search for viral agents, including unknown
tumor viruses, in children with acute leukemia; (2) To study the
role of these viruses in the etiology of acute childhood leukemia
and to attempt to obtain some evidence to support or disprove the
theory of the viral etiology of this disease; (3) To determine the
effect of cortisone on the incidence of viral infections in children,
(4) To study the possibility of lysogenic systems in herpes virus
and enterovirus infections.
Part B Included: Yes
172
Serial No. NlAin- 75
PHS-NIH
Individual Project Report
Calendar Year I960
Methods Employed: The study population includes 110 children
hospitalized for acute leukemia at I 'Hopital Saint Louis, Paris;
the control populations are (I) children hospitalized for other
blood disturbances in the hematology wards with the acute leukemia
patients; and (2) children of similar age, economic background, sex,
and race hospitalized in the general pediatric wards at Hopital
Saint-Vincent-de-Paul, Paris. The children hospitalized at St. Louis
were admitted from all parts of France, and, on a consultation basis,
from all over Europe. The only treatments currently being employed
for acute leukemia at Saint Louis are X-radiation, cortisone,
bone-marrow transplants, and transfusions with whole blood and/or
packed red eel Is.
It is postulated that if the virus (or viruses) responsible
for the leukemia were maintained in a "provirus" state in the cell
of the individual that it should manifest itself by the occasional
production of mature virus and that heavy cortisone treatment may
enhance the possibility of recovering the agent, if present. It
has been possible to study approximately 70 of the leukemic children
from the day of diagnosis until their deaths.
Conventional virus isolation attempts in tissue cultures
are used on throat and anal swabs taken weekly from every child
for the duration of his hospital ization and at the time of his
visits to the out-patient clinic. Bone-marrow specimens are
obtained whenever possible. Blood specimens for serologic studies
are obtained every two weeks during the period of hospitalization
and also when the child reports to the out-patient clinic. In
addition to the standard isolation techniques, the interference
technique using para-inf luenza I (Sendai) as an indicator virus
and the indirect fluorescent antibody technique are employed to
study a number of the specimens. Serologic surveys with known
viruses, including known animal tumor viruses, are being performed
to obtain information on the incidence of past infection in the
acute leukemia group as compared to the two control groups.
Laboratory data are correlated with clinical and epidemiologic
information in an effort to determine virus-disease relationships.
Major Findings : This is a new project and present information
is limited to evaluation of the interference technique and indirect
fluorescent antibody techniques for demonstration of viruses which
do not cause cytopathogenic effects or other changes in tissue
culture, and to the incidence of known agents in the study population.
173
Serial No. NlAin- 75
PHS-NIH
Individual Project Report
Calendar Year I960
It can be stated, however, that herpes virus was extensively prevalent
in the leukemic children and that enteroviruses were conspicuously
absent as compared to the control group.
Significance to the Program of the Institute: This project, a
controlled longitudinal epidemiologic and laboratory approach to
the study of the viral etiology of acute lymphocytic leukemia in
the pediatric age group, represents a rather unique and intensive
effort to acquire a knowledge of the virologic experiences in
children with acute leukemia. Such a study is needed to establish
suitable baselines to interpret future laboratory studies of specimens
from leukemia patients. At least one full year of epidemiologic
and laboratory studies are essential to account for possible seasonal
variations in the exacerbation of viruses. It has been reported
earl ier this year that two animal tumor viruses, Rous sarcoma virus
(Prince, I960) and polyoma virus (Vogt and Dulbecco, I960) appear
to undergo something suggestive of lysogeny. In view of these
findings the potential importance of persistent and recrudescent
viruses, such as recurrent herpes and varicella, and incomplete
enteroviruses participating in a lysogenic system in the human
leukemia patient needs investi gat-ion. The effect of cortisone
on bacterial infection in man has been well documented but little
information is available on its activity in viral infection. The
extensive clinical use of this drug is sufficient justification for
a control led study to determine the effect of cortisone on viral
infections in man.
Proposed Course of Project: During the next one-two months
serologic studies with sera from the leukemia patients and the two
control groups will be completed to determine the relative prevalence
of viral infections in the leukemia group with viruses that are
known to produce proliferative growth in tissue culture, with various
animal tumor viruses and with the more common viral pathogens
as compared with those in control populations.
In collaboration with Dr. Chany, anal specimens will
continue to be collected from leukemia and control children at
I'Hopital St. Louis to determine if the rate of enterovirus isolations
in the study group continues to remain low when compared with the
control group. Serologic studies of the prevalence of the prevalence
of infection with the more common respiratory viruses in cortisone
treated and non-cortisone treated children will be completed.
174
Serial No. NIAID- 75
PHS-NIH
Individual Project Report
Calendar Year I960
Laboratory investigation of incomplete herpes virus and incomplete
herpes virus and incomplete enteroviruses will be pursued at the
Max-Planck Institute fur Virusforschung, Tubingen, Germany, where
training received in nucleic acid work, high tension electrophoresis,
ul tracentrifugat ion and other physical-biological techniques will
be applied to these problems.
PART B Honors, Awards, and Publications
Publications other than abstracts from this project:
Chany, C. and Cook, M.K. : Sur un facteur collulaire induit par
le virus entrainant la formation de syncytium en culture de tissue.
Ann. Inst. Pasteur. In press.
175
PHS - NIH Serial No. NIAID-76-A
Part A
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
A. Project Title: Ecologic studies of fungi pathogenic for man.
Principal Investigator: C. W. Emmons
Other Investigator: Willard Piggott
Cooperating Units: Individual studies within this project
have been conducted in cooperation with
Dr. Gordon Clark, Patuxent Wildlife
Refuge and with Mr. Charles Hunt, U. S.
Geologic Survey, Denver, Colorado.
Man Years (calendar year I960):
Total: 20/12
Professional: 10/12
Other: 9/12
Project Description:
Objectives:
To find new environmental habitats of fungi which cause sys-
temic mycoses; to investigate the factors which limit growth of
these fungi to special habitats; to investigate methods of elimi-
nating pathogenic fungi from specific habitats and to apply the
information so obtained to studies of the epidemiology of the sys-
temic mycoses.
Methods Employed:
Samples of soil were collected from sites suspected of being
sources of infection in individual cases of mycoses, from types of
habitats known to harbor fungi in order to extend experience in
this study and repeatedly in certain selected sites known to harbor
pathogens in intensive studies of their ecology and associated sap-
Part B included
176
Serial Mo. NIAID-76-A
rophytic microflora. The specimens were processed by conventional
methods of mouse inoculation with soil suspensions.
A laboratory device for exposure of mice by inhalation of dry
spores of fungi was designed in collaboration with Mr. Willard
Piggott and has been used to simulate natural conditions of expos-
ure in pursuing the objectives of this project.
Major Findings;
The association between Cryptococcus neoformans and pigeon
excreta has been confirmed in many additional collections of spec-
imens. Histoplasma is not found in this association. The actual
occurrence of an acute pneumonic form of cryptococcosis has not yet
been proved because there has been no opportunity to study such an
outbreak. However, circumstantial evidence for the occurrence of
this type of cryptococcosis continues to accumulate.
Significance to bio-medical research and the program of the Insti-
tute;
Environmental sources of infection urn-elated to patients or
infected animals are almost invariably associated with systemic
human mycoses. Some of the diagnoses in reported epidemics or
focal outbreaks of mycoses still remain equivocal until the type
of information sought in this project adds to our knowledge about
the epidemiology of the mycoses.
Proposed Course of Project;
This project will be continued indefinitely.
177
PHS - NIH Serial No. NIAID-76-A
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
Part B. Honors, Awards and Publications.
Publications other than abstracts from this project:
Emmons, Chester W. Prevalence of Cryptococcus neo fornans in Pigeon Habitats.
Public Health Reports, 75:362-365, Apr., I960.
Piggott, Willard R. and Ernmons, Chester W. Device for Inhalation Exposure
of Animals to Spores. Soc. Exp. Biol. & Med., 103:805-806, I960.
Honors and Awards relating to this project:
Presidential Address before the Mycological Society of America at
Stillwater, Oklahoma, August, I960, entitled: "The Jeykll-Hydes of Mycology",
Presentation of a paper before the III National Congress of Microbiol-
ogy, Mexico City, Oct. 12, I960, entitled: "Inhalation Route of Infection
in Experimental Cryptococcosis".
Presentation of a paper before the Washington Speleological Society,
Dec. 6, I960, entitled: "The Occurrence of Pathogenic Fungi in Caves".
Participation in "Fireside Conference", the American College of Chest
Physicians, Washington, D. C, Nov. 27, I960. Geographic Environment and
the Systemic Mycoses.
President, Mycological Society of America, Aug. 1959-1960.
Elected Councilor-at-Large, Society of American Bacteriologists, July
1, I960.
Chairman, Second Conference on Medical Mycology, New York Academy of
Science, Jan. 11 and 12, I960.
Appointed Board Member of the American Academy of Microbiology for a
three -year term.
Chairman, Standards and Exaioination Committee, American Academy of
Microbiology.
Convenor of Mycological Symposium, III National Congress of Microbiolo-
gy, Mexico City, I960.
178
Serial No. NIAID-76-A
Honors and Awards relating to this project (continued):
Member of Study Section on Mycology and Bacterial Diseases, NIH.
Invited lecture at Annual Meeting of the State Society of Bacteriolo-
gists, University of North Carolina, Chapel Hill, No. Carolina, Apr., I960.
179
PHS - NIH Serial No. NIAID-76-B
Part A
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
Project Title: I_n vivo tests of antimycotic drugs and antibiotics.
Principal Investigator: C. \1. Emmons
Other Investigators: Prescott, Benjamin, Piggott, Millard,
Utz, John P. and Andriole, Vincent -
MPB Serial No. 62^-Aj LCI Serial No. 23-C.
Cooperating Units: Medical Physiological Bacteriology Section
and Laboratory of Clinical Investigations.
Man Years (calendar year I960):
Total: 22/12
Professional: 8/12
Other: 12/12
Project Description:
Objectives:
To test the efficacy and safety of new drugs and antibiotics
in experimental mycoses in mice and cooperate with the Laboratory
of Clinical Investigate ons in clinical trials.
Methods Employed:
Mice are infected intravenously with a dose of fungus cells
determined in prior experimental work to be sufficient to kill un-
treated (control) mice in 1U-21 days. Treated mice are given test
drugs at doses up to tolerance and effectiveness of drug is meas-
ured by its ability to extend survival time of treated mice and to
clear the animals of infection as determined by autopsy and culture
of the surviving animals when experiment is terminated. Collabora-
tor has provided some of drugs tested and has synthesized drugs on
his initiative or of types suggested by principal investigator.
Part B included.
180
Serial No. NIAID-76-B
Major Findings;
A new antibiotic first received by the principal investigator
four years ago has now been on clinical trials for 1 1/2 years and
has been found to be superior to other antimycotic drugs in h myco-
ses.
Significance to bio-medical research and the program of the Insti-
tute;
Presently available antimycotic drugs are too toxic and too
ineffective for safe and ideal clinical use. Experimental thera-
py in animals should precede clinical trial.
Proposed Course of Project;
This project will be continued indefinitely.
181
PHS - NIH Serial No. NIAID-76-B
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
Part B. Honors, Awards, and Publications,
Publications other than abstracts from this project:
Emmons, C. W. Failure of Griseofulvin to Control Experimental Systemic
Mycoses in Mice. A.M.A. Arch. Derm. 81:700-702, May I960.
Honors and Awards relating to this project:
Presentation of a paper entitled: "A New Antimycotic Antibiotic" at
the Fifth Annual Meeting of VA-Armed Forces Coccidioidomycosis Cooperative
Study", Los Angeles, Calif., Dec. 8 - 9, I960.
Honorary Mention for an Exhibit on Chemotherapy of Systemic Mycoses
at the American Medical Association Meeting in Miami Beach, Florida, held
in June I960.
Appointed to WHO Expert Advisory Panel on Parasitic Diseases for a
term of 5 years.
Presided at a session on Antifungal Agents at the I960 Conference on
Antimicrobial Agents, sponsored by the Society for Industrial Microbiology,
Oct. 26, I960.
Appointed on Scientific Advisory Committee, Institute of Microbiology,
Rutgers - The State University, New Brunswick, N. J. for a term of three
years.
Member of Committee on National Index of Fungus Cultures, Quartermaster
Research and Development, National Academy - Research Council, Natick,
Massachusetts, for a term expiring June 1963.
Special Lecturer on Medical Mycology, George Washington University
School of Medicine, for Fiscal Year beginning Aug. I960.
182
PHS - NIH Serial No. NIAID-76-C
Part A
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
C. Project Title: Identification and study of new and unusual fungi
from mycoses.
Principal Investigator: C. W. Emmons
Other Investigators: William L. Jellison, RML, Montana, Lie-Kian-
Joe, Univ. of Indonesia, Djakarta, and
Charles Bridges, Agricultural and Mechanical
College, Texas.
Cooperating Units: Rocky Mountain Laboratory and many hospitals,
diagnostic laboratories and individual physi-
cians.
Man Years (calendar year I960):
Total: Ui/12
Professional: 6/12
Other: 7/12
Project Description:
Objectives:
Studies of new mycoses and their etiologic agents, of unusual
strains of pathogenic fungi, and of the geographic distribution of
mycoses. Support and encouragement of medical mycology in labora-
tories which lack trained mycologists.
Methods Employed:
Routine methods of fungus identification, adaptation of such
methods when necessary, tests of pathogenicity and virulence in
experimentally infected animals.
Part B included.
183
Serial No. NIAID-76-C
Major Findings:
Additional studies of ohycomycosis have been carried on. A
new phyconycosis in horses has been studied and a paper submitted
for publication.
Significance to bio-medical research and the program of the Insti-
tute;
This combines a useful diagnostic service with an important
research function because it brings under scrutiny unusual, inter-
esting and important pathogens.
Proposed Course of Project:
This project will be continued indefinitely.
18 '4
PHS - NIH Serial No. NIAID-76-C
Individual Project Report
Calendar Year I960
Part B: Honors, Awards, and Publications.
Publications other than abstracts from this project:
Joe, Lie-Kian, Eng, Njo-Injo Tjoei, Tjokronegoro, Sutomo, and Emmons,
Chester W. Phycomycosis (Mucormycosis) in Indonesia — Description of
a Case Affecting the Subcutaneous Tissue. A. J. Trop. Med. & Hyg.
9:Ui3-Hj8, Mar., I960.
Emmons, C. W. and Jellison, W. L. Emmonsia Crescens Sp. N. and Adiaspiro-
mycosis (Haplomycosis) in Mammals. N. Y. Acad. Sci. 89:91-101, Aug.
I960.
Honors and Awards relating to this project:
Invited lecture at the Agricultural and Mechanical College, College
Station, Texas, July, I960.
Presentation of a paner entitled: "Emmonsia Crescens Sp. N. and
Adiaspiromycosis (Haplomycosis) in Mammals" at the Second Conference on
Medical Mycology, New York Academy of Science, Jan., I960.
185
PHS - NIH
Individual Project Report
Calendar Year I960
Serial No. NIAID-78-A
1. Infectious Diseases
2. Medical Mycology
3. Bethesda, Maryland
A. Project Title: Biochemistry and Physiology of Pathogenic Fungi.
(In vitro studies of the acti on of Antifungal agents
on pathogenic fungi).
Principal Investigator: George W. Lones
Other Investigators: None
Cooperating Units:
None
Man Years (calendar year I960):
Total: 1 2/12
Professional: 9/12
Other: 5/12
Project Description:
Objectives:
To investigate the quantitative relationships of antifungal
drugs with respect to fungicidal and fungistatic action; to study
certain nhysical and chemical factors influencing this action; to
study the effects of these drugs on certain metabolic activities of
fungi; to study the emergence of strains resistant to these drugs;
and ultimately to understand the mechanism of action of certain of
the antifungal antibiotics.
Methods Employed:
Interest during the past year has centered on a new antibiotic,
X-5079C Conventional methods are used for determining the quanti-
tative relationships of growth of Histoplasma cansulatum in the pres-
ence of varying concentrations of the antibiotic in suitable growth
media under a variety of conditions. HeLa cell cultures are infected
with H. capsulatum in the yeast phase and the effect of the antibiot-
ic on the development and persistance of the fungus determined by
Part B not included.
186
Serial No. NIAID-78-A
microscopic examination of stained preparations and by subculture
following trypsinization.
Major Findings;
Antibiotic X-5079C is fungistatic rather than fungicidal.
Contrary to early impressions the agent is active against H. cap-
sulatun in vitro as well as in animals. The sensitivity of the
yeast phase of H. capsulatum to antibiotic X-5079C is substantial-
ly greater than that of the mycelial phase. An assay method for
antibiotic X-5079C sensitive to 1 ug/ml has been developed with a
strain of H. capsulatum as the test organism. Antibiotic X-5079C
is active against H. capsulatum in HeLa cell culture, and factors
influencing activity in this system have been investigated. It is
of low toxicity for HeLa cells.
Significance to bio-medical research and the program of the Insti-
tute:
The need for new and more effective therapeutic agents for
treatment of the systemic mycoses continues.
Antibiotic X-5079C has demonstrated impressive therapeutic
effect in experimental mycoses in animals and encouraging results
have been obtained in humans. The continued study of this and
other antifungal agents is essential.
Proposed Course of Project:
It is planned to continue these studies. The investigation
may well be extended to pathogenic fungi other than H. capsulatum.
The effect, if any, of the antibiotic on respiration and assimila-
tion will be determined. Attempts will be made to improve the
assay procedure, since a sensitive assay method is essential for the
determination of serum levels and excretion rates in man and animals.
187
PHS - NIH Serial No. NIAID-78-B
Part A
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
B. Project Title: Studies on the physiology of Coccidioides immitis,
Principal Investigator: George W. Lones
Other Investigator: None
Cooperating Units: None
Man Years (calendar year I960):
Total: 1 2/12
Professional: 3/12
Other: 11/12
Project Description:
Objectives:
The biochemistry and metabolism of this dimorphic pathogen
have been little studied. It is the purpose of this project to
obtain information on the metabolic characteristics of this
microorganism, and in particular to discover metabolic differ-
ences in the two forms.
Methods Employed:
Shake culture techniques are employed to produce the parasitic
and saprophytic morphologic modifications of C. immitis in sizeable
quantities for examination by standard chemical and biochemical
techniques.
Major Findings:
A second strain of C. immitis has been successfully converted
and maintained in the spherule form. Quantitative measurements
have been obtained of the ability of a variety of potential carbon
Part B included
188
Serial No. NIAID-78-B
and nitrogen sources to supnort the growth of the spherule phase
and the mycelial phase of strain M-ll. No substrate has been found
in this study which preferentially supports the growth of the
spherule form. Only mannose is utilized as readily as glucose by
the spherules. Mannose and fructose support growth of mycelium as
well as glucose.
Significance to bio-medical research and the program of the Insti-
tute;
Progressive coccidioidomycosis is a disease with a high mor-
tality. A better knowledge of the causative agent may favorably in-
fluence our diagnosis, prevention and treatment of the infection.
Proposed Course of Project;
Examination of the metabolism of the two morphological forms
of the fungus will be extended. The current nutritional study will
be completed.
18S
PHS - NIH
Individual Project Report
Calendar Year I960
Serial No. NIAID-78-B
1. Infectious Diseases
2. Medical Mycology
3. Bethesda, Maryland
Part B. Honors, Awards, and Publications.
Publications other than abstracts from this project:
Lones, G. W. and Peacock, Carl L. Role of Carbon Dioxide in the Dimorphism
of Coccidioides immitis. J. 3act. 79:308-309, I960.
Lones, G. W. and Peacock, Carl L. Studies of the Growth and Metabolism of
Coccidioides immitis. Annals N. Y. Acad. Sci. 89:102-108, I960.
Honors and Awards relating to this Project:
Presentation of a paper entitled: "Studies of the Growth and Metabolism
of Coccidioides immitis", Second Conference on Medical Mycology, New York
Academy of Science, January, I960.
190
PHS - NIH Serial No. HTAID-79-A
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
A. Project Title: Immunity Studies with Pathogenic Fungi.
Principal Investigator: H. F. Hasenclever
Other Investigators: None
Cooperating Units: None
Man Years (calendar year I960):
Total: S/12
Professional: 2/12
Other: 3/12
Project Description:
Objectives:
To obtain fundamental knowledge pertaining to the development
of acquired resistance in laboratory animals to coccidioidomycosis.
Methods Employed:
Immunization procedures utilizing spherule cultures of Coccid-
ioides immitis as vaccines are being studied. The vaccines are
treated with formaldehyde to destroy viability. Mice immunized with
this ^reparation are challenged wi th viable C. immitis arthrospores
or spherules.
Major Findings:
Immunized mice show an increased survivor rate when compared to
normal mice challenged with similar infecting doses of C. immitis.
In experiments where sublethal infecting doses are utilized, the im-
munized mice are culturally negative sooner than normal control mice.
Part B not included.
191
Serial No. NIAID-79-A
Significance to bio-medical research and the program of the Insti-
tute;
Me are utilizing for this investigation cultures of spherules
grown in vi tro » V/hile mycelial preparations have been investigated,
the "parasitic form" of C. irriitis, as an immunizing agent, has been
studied very little. This project has been undrr investigation for
only a short tine, but the results thus far are encouraging.
Coccidioidomycosis is a disease for which, if a satisfactory vaccine
were available, immunization would be of value and practical.
Proposed Course of Project;
This nroject is to be continued.
192
PHS - NIH Serial No. NIAID-79-B
Part A
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
B. Project Title: Antigenic Studies on Pathogenic Yeasts.
Principal Investigator: H. F. Hasenclever
Other Investigators: None
Cooperating Units: None
Man Years (calendar year I960)
Total:
9/12
Professional:
li/12
Other:
5/12
Project Description:
Objectives:
To learn more about the antigenic interrelationships of some
of the provisional pathogens with particular emphasis upon the
genus Candida.
Methods Employed:
Antisera to the desired species and strains of Candida are pre-
pared in rabbits. Homologously and heterologously adsorbed antisera
are utilized to show antigenic similarities or dissimilarities that
exist within species or between species. Tube agglutination reac-
tions have been used predominately and the applicability of comple-
ment fixation and agar gel diffusion has been tested.
Major Findings:
These studies have shown that two antigenic groups are present
in the species Candida albicans. One of these groups is antigenical-
ly similar to C. tronicalis, while the other group is identical to
C. stella toidea.
Part B included.
193
Serial No. NIAID-79-B
Significance to bio-medical research and the program of the Insti-
tute;
These studies have contributed basic and fundamental informa-
tion about the antigenic structure of these yeast-like fungi.
Proposed Course of Project;
To be continued with an effort to demonstrate qualitative anti-
genic differences between these two groups.
19'*
PHS - NIB Serial No. NIAID-79-B
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
Part B. Honors, Awards, and Publications.
Publications other than abstracts from this project!
Hasenciever, H. F. and Mitchell, William 0. The observation of Two Antigenic
Groups in Candida albicans. Bact. Proc. I960.
Honors and Awards relating to this Project;
Convenor of Session on Medical Mycology at the I960 Annual Meeting of
the Society of American Bacteriologists.
Invitational lecture entitled: "Clinical and Mycological Aspects of
Tinea pedis" before the Annual Scientific Convention of the American Podia-
try Association, Nov., I960,
195
PHS - NIH Serial No. NIAID-79-C
Part A
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
C. Project Title: Virulence and pathogenic studies with yeasts.
Principal Investigator: H. F. Hasenclever
Other Investigators: Vincent T. Andriole - LCI Serial #23-C.
Cooperating Units: Laboratory of Clinical Investigations.
Man Years (calendar year I960) :
Total: 18/12
Professional: 10/12
Other: 8/12
Project Description:
Objectives:
To learn more about the host-parasite relationship, virulence
and pathogenicity, and the in vitro multiplication rates of several
yeasts in normal and physiologically altered mice.
Methods Employed:
Virulence determinations are calculated by injecting graded
doses of yeast cells into different groups of mice. The fifty per
cent endooint is utilized and death or survival of the test animals
is the criterion employed. In vivo growth rates are obtained by
determining the viable yeast cells in the various organs, at differ-
ent stages of disease, in animals infected with a standard number of
yeasts. Similar studies have been done using mice with alloxan in-
duced diabetus mellitus.
Part B included.
196
Serial No. HIAID-79-C
Major Findings:
Studies with several strains of Cryntococcus neoformans have
shown that several minutes after injection the largest number of
yeast cells are found in lung tissue, regardless of whether the
mice were injected intravenously or intracerebrally. Soon the pop-
ulation in the lungs decreases, and by 2-3 days after injection,
multiplication in the brain is apparent. The time required to pro-
duce terminal cryptococcosis after injection in mice varies with
the strain. In moribund mice, however, the number of viable yeast
cells per 0.1 gm of brain tissue is about the same regardless of
strain.
Studies with Candida tropicalis have shown that most strains
of this yeast are quite pathogenic for mice but not for rabbits.
Multiplication in vivo is quite similar to that of Candida albicans,
i.e., the kidneys are the organs showing the greatest amount of
tissue destruction, and the highest yeast cell population.
An effective dose of alloxan monohydrate which would produce
constant glycosuria without a great number of fatalities was deter-
mined.
Lethality studies revealed that certain strains of Candida
albicans and C_. tropicalis caused death in alloxanized mice at a
faster rate than in normal control animals. This was not true for
certain strains of C_. parap silo sis and C. guilliermondii.
Plating experiments revealed that the course of C. albicans
infection in the mouse was more severe when the animal" had been
physiologically altered with alloxan monohydrate.
Significance to bio-medical research and the program of the Insti-
tute:
These investigations have contributed to knowledge about the
parasite multiplication within host tissues. We have shown that
mice are quite susceptible to infections due to £. tropicalis.
Proposed Course of Project:
To be continued with further study to be given to the effects
of physiological alteration of the host in relation to increased
or decreased susceptibility to fungus infections.
197
PHS - NIH Serial Ho. HIAID-79-C
Individual Project Report 1. Infectious Diseases
Calendar Year I960 2. Medical Mycology
3. Bethesda, Maryland
Part B. Honors, Awards and Publications.
Publications other than abstracts from this project:
Hasenclever, H. F. and Mitchell, William 0. Virulence and Growth Rates of
Crvptococcus neoformans in Mice. Annals N. Y. Acad. Sci. 89:1^6-162,
i960:
Kasenclever, H. F. and Mitchell, Uilliam 0. Pathogenicity of Candida albi-
cans and Candida tropicalis. Sabouraudia (in press).
Honors and Awards relating to this project:
Presentation of a paper entitled: "Comparative Pathogenicity of
Candida albicans and Candida tropicalis," at the Annual Meeting of the
Mycological Society of America, American Institute of Biological Sciences,
I960, Stillwater, Oklahoma.
Presentation of a paper entitled: "Virulence and Growth Rates of
Cryotococcus neoformans in Mice", at the Second Conference on Medical Mycol-
ogy, Hew York Academy of Science, Jan., I960.
198
LABORATORY OF BIOLOGY OF VIRUSES
Summary 1
80 - Basic Studies of Virus-Host Cell Rela-
t ionships 3
80-A - Rabies Prophylaxis 6
81 - Mechanism of Oncogenic Activity Polyoma
Viruses 8
82 - Cell Growth Inhibiting Substances 11
83 - Mutation in Animal Viruses 13
84 - Host-Parasite Relations 15
85 - Investigations of Animal Virus Repro-
duction 19
85-A - Animal Virus Synthesis 22
86 - Investigations of Interference Enzymatic
Functioning of Mitochondria as a
Mechanism for Carcinogenesis 24
86-A - Demonstration of Glucose Metabolism and
Peptide Bond Formation by Isolated Brain
and Liver Mitochondria 27
87 - Cytopathogenic Effect in Single Cells in
Tissue Culture 29
87-A - Characterization of Mitochondria 31
88 - Biology of Mitochondria and Its Relation
to Endogenous and Viral Diseases 34
88-A - Comparison of the Properties of Crude
and Crystallized Coxsackie A- 10 Virus,
of Cloudman S 91, Mouse Melanona and of
Mouse Muscle Nucleoprotein 36
89 - Kinetics and Sites of Coxsackie Virus
Multiplication in the Monkey Kidney Cell 38
89-A - Virus Structure .. ... 40
PHS-NIH
Summary Statement
Office of Chief
Laboratory of Biology of Viruses
Calendar Year i960
This laboratory has been anticipating a physical shift in most
of its activities to space in Building 5. Although scheduled to take
place during the past calendar year, it is now obvious that it will
not take place until well into 1961. Because of this delay, space
problems have been severe. For instance, it has been impossible to
establish our planned central tissue culture production unit although
the subprofessional responsible for this program has been on duty
since March i960. She has become well acquainted with our technics,
and equipment necessary for the operation has been collected. A
second severe limitation of space involves the newly established
Physical Biology Unit. Dr. Mattern has had to install his electron
microscope in one-half of a basement room measuring 8 x l'+ and this
has been the total working space for a whole year for him and one
professional assistant.
During this year one whole unit, The Rickettsial Biology and
Metabolism Unit involving 3 professional and 2 subprofessional staff
members, was transferred from this laboratory to the Division of
Biologies Standards. No replacement personnel, budget or space was
made available for this loss.
One new research associate joined the staff this year, making
two such now a part of this laboratory. One of these associates has
been recruited to become a part of the permanent staff at the end of
this fiscal year.
The basic objectives of this laboratory continue to be the
same as last year. It is obvious from this annual report that four
out of our five units have projects with the same general objective —
investigation of mechanisms and localization of animal virus synthesis
within the infected cell. Each of these units is also interested in
the infectious nucleic acid of viruses. In view of the complexity of
this problem and the important implications of any information that
is obtained, we feel that this "duplication" is quite Justified.
Actually, it is not duplication in so far as different approaches are
being used and different virus-cell systems studied.
The electron microscope has been installed and is now being
used not only by the Biophysical Unit but also in collaboration with
other units of our laboratory and units in the Laboratory of Infectious
Diseases. With studies on the structure of viruses and a project
Summary Statement — LBV/NIAJD (Cont.)
concerned with the genetics of animal viruses added to our biochemical
and biological studies, we feel that ve now have fairly complete cover-
age of the important facets of basic virus biology.
By the use of radioautographs and staining with fluorescein
tagged antiviral antibody, the intracellular location of poliovirus
antigen — presumably viral protein — during the cycle of virus multi-
plication has been determined. Demonstrable antigen first appeared
one hour after infection and was diffusely distributed through the
cytoplasm. At 3 hours, just before the appearance of new virus, it
was present throughout the nucleus with a tendency to be concentrated
around the periphery of the nucleolus. At 5 to 7 hours, particulate
accumulation of antigen in the cytoplasm was noted. Incorporation of
radioactive-tagged amino acid into cell protein ceased shortly after
the start of infection, whereas incorporation of cytidine into RNA
continued until after 3 hours and tended to localize in the nucleoli.
Plaque type mutants of EMC virus have been found, segregated
and characterized. The stability of the mutants has been determined
and the plaque type has been shown to be a function of the viral RNA.
It has been shown that the difference in the size of the plaques formed
by these mutants is brought out by an inhibitor present in the agar
overlay used on the plaque plates. This inhibitor resides in the
agaropectin fraction of the agar and can be separated from the agarose
fraction which then permits both plaque type mutants to form similar
sized plaques.
By the use of a serum protection test in newborn hamsters,
evidence was found that polyoma virus transforms normal cells to tumor
cells quickly and directly without extensive virus multiplication being
necessary. Furthermore, no evidence could be found to suggest a lyso-
genic relationship of virus to tumor cell. All attempts to show the
presence of infectious or masked virus or of virus antigen in trans-
plantable polyoma- induced tumors have been negative. It appears that
once the virus initiates the tumor it is no longer required for tumor
growth and maintenance.
The discovery has been made that when the antibiotic tetracycline
stains tissues in such a way that they fluoresce under UV light, this
fluorescence is localized in the mitochondria of the cells. This makes
a convenient vital stain of these subcellular elements for further
studies. There appears to be some similar localization of the anti-
biotic fluorescence in certain bacteria.
Summary Statement — LBV/NIAID (Cont.)
A complex model of tobacco mosaic virus has been constructed
on theoretical grounds, and on checking this model against known
biochemical and biophysical properties of the virus a remarkable
consistency is found. Certain refinements of electron microscopic
technics have produced photographs of this virus which reveal
previously not seen fine structure also consistent with the theoreti-
cal model.
Serial No. NIAID-80
1. Biology of Viruses
2. Viral Growth
3. Bethesda, Maryland
FHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Basic Studies of Virus-Host Cell Relationships
Principal Investigator: Dr. Karl Hahel
Other Investigators: Rosalie Silverherg
Cooperating Units: None
Man Years:
Total:
1
Professional:
5/12
Other:
7/12
Project Description:
Objectives:
To determine mechanism of and factors influencing attachment,
invasion, multiplication and release of viruses from susceptible and
resistant cells.
Methods Employed:
Various strains of tissue culture cells either suspended or on
glass are exposed to viruses and at different stages of infection the
cells are studied for viability and virus content. All quantitation is
by plaque methods.
Major Findings:
After rapid adsorption of poliovirus to HeLa cells the virus
exists in h different relationships with the cell:
(1) Virus loosely bound that will wash off,
(2) Virus firmly bound but still available to extracellular
antibody,
(3) Virus firmly bound but not available to antibody,
(h) Virus already eclipsed.
Part B included: Yes
Serial No. NIAID-80
Although all four states may exist at one time, the normal
sequence of events seems to be (l), (2) and (4). The virus in (3) may-
go to (U) and thus initiate infection but this may be a relatively
inefficient process compared to the other sequence.
Significance to Bio-medical Research and the Program of the Institute:
These findings suggest that effective initiation of the infectious
cycle can occur rapidly but that under certain circumstances virus is
firmly attached to the cell before being eclipsed. Understanding of
these events occurring at the start of infections at the cell surface
may provide a logical target for attempts at inhibition of infection
■without serious deleterious effects upon the cell.
Proposed Course of the Project:
These studies will be continued with attempts to determine
morphological and biochemical cellular components responsible for the
phenomena already demonstrated.
Serial No. NIAID-80
PHS-NIH
Individual Project Report
Calendar Year i960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Habel, Karl and Utz, John P. Mumps. Pediatric Clinics of
North America, Vol. 7, No. k, November, i960.
Honors and Awards relating to this project:
None
Serial No. NIAID-80A
1. Biology of Viruses
2. Viral Growth
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Rabies Prophylaxis
Principal Investigator: Dr. Karl Habel
Other Investigators: Rosalie Silverberg
Cooperating Units: Expert Committee on Rabies, World Health
Organization
Man Years:
Total: 1 6/12
Professional: 6/12
Other: 1
Project Description:
Objectives:
To improve methods of rabies prophylaxis in man and to reduce
non-specific reactions caused by vaccines.
Methods Employed:
This year 's activity has been chiefly aimed at developing a
tissue culture source of virus for vaccine production.
Major Findings:
A practical tissue culture system consisting of normal chicken
embryo cells in medium 199 has been infected with a fixed strain of
rabies virus and propagated through 10 serial passages. Some passage
fluids have had an infectious titer as high as 105 and with more
concentrated inocula produced a cytopathic effect which was neutral-
izable with antirabies serum.
Part B included: No
Serial No. NIAJD-80A
Significance to Bio-medical Research and the Program of the Institute:
A practical tissue culture source of inactivated rabies vaccine
would represent a tremendous improvement in the type of biological product
used for rabies prophylaxis in man.
Proposed Course of the Project:
Tissue culture as a possible source of vaccine will be further
explored.
Serial No. WIAID-81
1. Biology of Viruses
2. Viral Growth
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Mechanism of Oncogenic Activity of Polyoma Virus
Principal Investigator: Dr. Karl Habel
Other Investigators: Rosalie Silverberg and Dr. Lowell Glasgow
Cooperating Units: None
Man Years:
Total: 3 5/12
Professional: 2
Other: 1 5/12
Project Description:
Objectives:
To determine biological factors involved in the induction of
tumors by oncogenic viruses.
Methods Employed:
By studying the virus-tumor cell relationships of polyoma virus
in animal and tissue culture systems, the events occurring in newborn
versus adult mice and hamsters on injection of virus are being compared.
Attempts are being made to induce malignant characteristics in cells
in tissue culture and in adult animals by varying the physiological
state during infection with polyoma virus.
Major Findings:
Tumors produced in newborn hamsters by polyoma virus when passive
antibody is present, occur only at the site of virus inoculation, contain
no demonstrable virus and frequently do not result in an active anti-
viral antibody response.
Part B included: Yes
Serial No. NIAID-81
Tumors can be produced in adult hamsters by applying virus to
the granulation tissue resulting from scarification but not by simple
intradermal inoculation of virus.
Attempts to "transform" normal mouse and hamster embryo tissue
culture cells to tumor by establishing in them a continuing infection
with polyoma virus have been negative.
Significance to Bio-medical Research and the Program of the Institute:
Any information concerning factors responsible for oncogenic
properties of tumor viruses may well apply to the oncogenic effects of
other agents and provide leads for understanding tumor development
under natural conditions in man.
Proposed Course of the Project;
Attempts will be made to find factors that will enhance the
ability of polyoma virus to induce tumors in adult animals and to
inhibit this process in suckling animals.
Serial No. NIAID-81
PHS-NIH
Individual Project Report
Calendar Year i960
Part B Honors, Avards, and Publications
Publications other than abstracts from this project:
Habel, Karl and Silverberg, Rosalie J. Relationship of Polyoma
Virus and Tumor in Vivo. Virology 12: I+63-I+76, November i960.
Honors and Awards relating to this project:
None
10
Serial No. NIAID-82
1. Biology of Viruses
2. Viral Growth
3. Bethesda, Maryland
FHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Cell growth inhibiting substances
Principal Investigator: Dr. John W. Hornibrook
Other Investigators: None
Cooperating Units: Viral Biochemistry Unit, LBV.
Man Years:
Total: 2 7/12
Professional: 1
Other: 1 7/12
Project Description:
Objectives:
To find and investigate substances and mechanisms which prevent
the growth of cells in the adult animal.
Methods Employed:
Isolation from serum and tissues of substances which will inhibit
the growth of mammalian cells in tissue culture.
Major Findings:
An inhibitor from serum has been partially purified. It is
apparently not a carbohydrate and does not adsorb U.V. radiation at
280 or 260 mu. It is active at approximately 0.3 mg/ml.
Methods of isolating and purifying this material are being
improved and this work will continue.
Part B included: No
11
Serial No. NIAID-82
Significance to Bio-medical Research and the Program of the Institute:
Information concerning the existence and mode of action of mitotic
inhibitors in tissues or body fluids is of fundamental biological
importance and of practical significance to those engaged in tissue
culture and cancer research.
Proposed Course of the Project:
Attempts will continue to purify and identify these substances
and study their mode of action.
12
Serial No. NIAID-83
1. Biology of Viruses
2. Viral Growth
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Mutation in Animal Viruses
Principal Investigator: Dr. K. K. Takemoto
Other Investigators: Dr. Harvey Liebhaber
Cooperating Units: None
Man Years:
Total: 3 6/12
Professional: 2 l/l2
Other: 1 5/12
Project Description:
Objectives:
1. To isolate. and characterize virus mutants and to investigate
the chemistry and biology of these mutants.
2. To investigate various methods for concentration and purifi-
cation of viral mutants in order to obtain sufficient quantities of
virus for chemical as well as biological investigations.
Methods Employed:
Genetically pure lines of virus are isolated by plaque tech-
niques. Mutants forming plaques which differ from the parental type
are isolated and studied further.
Part B included: No
13
Serial No. NIAID-83
Major Findings:
1. Encephalomyocarditis (EMC) virus produces plaques which are
small and ragged with diffuse boundaries. A mutant of this virus has
been isolated which forms large plaques with sharply defined boundaries.
Both virus types are immunologically identical and do not differ in
their growth rates, thermal stability or mouse virulence. However,
the large plaque mutant differs in its hemagglutinating properties,
having a significantly higher HA/pfu ratio.
2. The basis for plaque size differences has been found to be
due to an inhibitory factor in the agar used in the overlay medium.
The primary effect of the inhibitor appears to be interference with
adsorption of virus.
3. Infectious RNA extracted from both types of virus have
yielded progeny which produce plaques identical to those from which
the RNA was obtained, proving that genetic information determining
plaque type is carried solely by the viral nucleic acid.
h. Preliminary experiments have indicated that large volumes
of EMC virus can be concentrated and purified by a simple procedure
which utilizes the partition coefficient of the virus between two
phases of aqueous solutions of high molecular weight polymers.
Significance to Bio-medical Research and the Program of the Institute:
Studies on variations and mutations of animal viruses not only
lead to further knowledge and understanding of the nature of viruses
but have practical implications in the development of vaccines in
virus diseases.
Proposed Course of the Project:
1. Utilizing the genetic markers of plaque type and hemaggluti-
nation, experiments are planned to demonstrate various types of genetic
interaction such as recombination and reactivation.
2. Procedures are being developed for large scale purification
of virus so that sufficient quantities of relatively pure virus will
be available for studies on viral nucleic acid and protein.
14
Serial No. NIAID-8^
1. Biology of Viruses
2. Virus Host Relationship
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Host-Parasite Relations
Principal Investigator: Victor H. Haas
Other Investigators: None
Cooperating Units: Miss Delta Uphoff (NCl)
Man Years:
Total: 2
Professional: 1
Other: 1
Project Description:
Objectives:
(a) To continue studies on relationships between the virus of
lymphocytic choriomeningitis (LCM) and the transmissible ascitic tumor
P288 in mice, particularly in mice immune to the virus.
(b) To study the effect of various antigens, including tumor
material and a virus strain derived from LCM-tumor passage, on immune
response to the P288 tumor.
(c) To conclude studies on LCM immunity in X-rayed mice kept
alive by marrow and spleen transplants .
Methods Employed:
A line of P288 tumor carrying LCM with it during passage was
maintained in CDBA mice, some of them previously immunized against LCM.
A strain of LCM virus derived from the tumor passage line was separated
from the tumor and maintained by serial passage in non-immune mice.
Part B included: Yes
15
Serial No. NIAID-8^
The P288 tumor was passaged in CDBA mice and from time to time in general
purpose mice, the latter being treated with amethopterin. Detection of
LCM in various passage mice was by demonstration of viremia or by immunity
tests . Bone marrow and spleen transplants were done by Miss Uphof f in
mice previously prepared by me in respect to immune status against LCM,
and irradiation was obtained from Mr. Meyer of NCI.
Major Findings:
Two years ago, I established a passage strain of LCM virus that
was carried with the ascites tumor P288 through mice immune to LCM. For
the virus to survive, it was necessary to treat the mice with amethopterin.
After nearly a year of such passage, I derived from it a strain of LCM
which survived in immune mice, when passed with the tumor P288, even
though no amethopterin was given. During the current year I have passed
this latter strain of LCM through non- immune, non-tumor bearing mice and
found that it produced a benign, transmissible ascites of a type which I
have not encountered in my ordinary passage strains of LCM. White (general
purpose) mice recovering from this ascitic infection frequently have been
resistant to P288 tumor, when the tumor was given during amethopterin
treatment, a combination generally fatal to these mice. No other instances
of tumor -immunizing capability, and none of tumor-producing capability, of
this virus strain have been detected, despite repeated efforts.
Earlier work in this laboratory demonstrated that general purpose
mice are normally refractory to the P288 tumor, but that fatal ascitic
tumors developed if they were treated with amethopterin after tumor
injection. During this year, I have found that injections of tumor given
without amethopterin treatment made the mice immune to later challenge
with the same tumor plus amethopterin. Attempts to immunize with disrupted
tumor cells (freezing-thawing) have indicated that such preparations may
have an immunizing effect but these experiments are not as yet complete.
A similar effect seemed to occur when normal tissues of the CDBA strain of
mice (the strain in which tumor P288 originated and is maintained) were
used in lieu of tumor preparations for immunizing the white mice.
Experiments on immunity to LCM in X-rayed mice kept alive by
marrow and spleen transplants have been completed. The immune status of
the X-rayed mouse before radiation and tissue transplant determined its
response to later LCM challenge . Marrow and spleen from immune donors did
not confer immunity on the recipients which had not been previously
immunized.
Significance of the Program to the Institute;
A significant part of the Institute 's program on basic research in
virology concerns the alterations produced in the virus-infected animal,
as an entity and on a cellular basis. One such alteration could be the
induction of tumor growth, another could be the induction of resistance
16
Serial No. NIAID-8U
to tumors. These experiments have yielded information on how a particular
virus — LCM — and a transplantable tumor — P288 — become inter-related during
passage together. The alteration of the general purpose mouse's response
to the P288 tumor by amethopterin provides an opportunity for studying
the immune reaction to various antigens as measured by resistance to
challenge with the tumor.
Proposed Course of the Project:
This project comes to an end this year because of my retirement.
17
Serial No. NIAID-8^
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Haas, Victor H. Serial passage of a lymphocytic tumor and
choriomeningitis virus in immune mice. Jour. Natl.
Cancer Inst. 25: 75-83 (i960).
Uphoff, Delta E. and Haas, Victor H. Immunologic response
to lymphocytic choriomeningitis virus in lethally
irradiated mice treated with bone marrow. Jour. Natl.
Cancer Inst. 25: 779-786 (i960).
Honors and Awards relating to this project:
None
18
Serial No. NIAID-85
1. Biology of Viruses
2. Viral Growth
3- Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Investigations of Animal Virus Reproduction
Principal Investigator: Dr. Hilton B. Levy
Other Investigators: Dr. Frank De Filippes
Cooperating Units: None
Man Years:
Total: 2 11/12
Professional: 11/12
Other: 2
Project Description:
Objectives:
To gain information relative to the relationship that exists
between the infected cell and the virus reproducing therein. More
particularly, to determine at a molecular level the mechanisms by which
a virus is reproduced by the infected cell. Knowledge of either the
details of altered cell metabolism or mechanisms of virus reproduction
might be useful in the development of chemotherepeutic agents to inhibit
virus growth and to aid the host. Comparison of oncogenic viruses such
as Rous Sarcoma with cytocidal viruses such as poliovirus should give
insight into growth controlling mechanisms.
Methods Employed:
The program for this year sought the intracellular localization
of the infecting virus particle during the course of the infection, and
also where the components of the new virus were made. High resolution
autoradiography and fluorescent antibody techniques were used. Nucleic
acid bases containing the weak beta particle emitter tritium were used
to study nucleic acid metabolism and to prepare labelled poliovirus.
Tritium labelled histidine was used for protein studies. Rabbit anti-
serum to highly purified poliovirus was prepared for the fluorescent
antibody work.
Part B included: Yes
13
Serial No. NIAID-85
Major Findings;
The first detected change in poliovirus infected HeLa cells occurs
about an hour after infection, when there is seen increased turnover of
RNA, particularly and almost exclusively in the nucleoli, (it is about
this time that our earlier work detected increased glycolytic energy
production.) This increased RNA turnover continues for 3 l/2 to k hours
after which it greatly decreases. At about 1 l/2 to 2 hours after
infection, there appears a virus specific antigen in the cytoplasm, even
though total cell protein metabolism, as measured by tritiated histidine,
has declined markedly. By 3 to 3 l/2 hours after infection, viral
antigen appears in the nucleus. This nuclear antigen does not occur in
the nucleolus, which latter structure has a bright thin ring of stained
antigen around it. It might be that this nuclear antigen is made at
the periphery of the nucleolus, or is made in the nucleolus but is not
susceptible to reaction with fluorescent antibody until released at its
surface. By about h to 5 hours after infection there is a decline in
the number of cells showing nuclear antigen and the appearance of
brightly staining antigen in the cytoplasm, suggesting that the nuclear
material has migrated there. Since parallel viral growth studies show
the first appearance of new virus at this time, it would suggest that
this nuclear to cytoplasmic migration is the step that forms new virus.
Whatever nucleic acid or specific protein synthesis was implicated in
the increased nucleolar RNA turnover decreases at this time. Shortly
thereafter, the increased energy production stops.
Significance to Bio-medical Research and the Program of the Institute:
Increased research in biochemistry along the lines of protein and
nucleic acid biosynthesis has resulted in the synthesis of at least two
specific proteins in cell free systems. The way seems clear to do this
with virus protein. The intracellular localization of where specific
virus protein is made indicates which subcellular components to use in
such an attempt.
Comparable studies with other cytocidal as well as with oncogenic
viruses will add to the Institute 's program directed to understanding
mechanisms by which host cells are diverted from normal cell behavior to
the production of new virus or to tumor characteristics.
Proposed Course of the Project;
Attempts will be made to utilize the information obtained this
past year to synthesize some viral components in cell free systems.
Comparison with other viruses will be made. Further studies on the
nature of the increased nucleolar activity will be made to see if viral
nucleic acid is being made.
20
Serial No. NIAID-85
PHS-NIH
Individual Project Report
Calendar Year i960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Levy, Hilton B. and Sober, Herbert. A Simple Chromatographic
Method for Preparation of Gamma Globulin. Proc. Soc.
Exptl. Biol. & Med. 103: 250-252, i960.
Levy, Hilton B. and Snellbaker, LeRoy. Phosphorus Metabolism
in Infection vith Murine Leukemia Virus. Proc. Soc.
Exptl. Biol. & Med. 103: 503-506, i960.
Levy, Hilton B. and Lynt, R. K. Heterogeneity in Cytoplasmic
RNAs of Mouse Spleen and Effect Thereon of a Leukemia
Virus. Submitted for publication.
Honors and Awards relating to this project:
The work on the Friend leukemia virus was chosen to be
published in the M. D. Anderson Hospital annual publication of
significant reports in cancer research.
21
Serial No. NIAID-85A
1. Biology of Viruses
2. Viral Growth
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Animal Virus Synthesis
Principal Investigator: Dr. Frank M. DeFilippes
Other Investigators: Dr. Hilton B. Levy
Cooperating Units: None
Man Years:
Total: 1 1/12
Professional: 1 1/12
Other: 0
Project Description:
Objectives:
1. To investigate the synthesis of an animal virus in a tissue
culture system by studying subcellular particles which are involved in
protein synthesis in many animals.
2. To increase the efficiency of infection of monkey kidney cells
with RNA extracted from purified poliovirus to a level similar to that
obtainable with whole virus.
Methods Employed:
1. HeLa cells infected with poliovirus are grown in a radioactive
medium. The cells are collected at different times and cell fractions
are isolated with particular attention being given to the ribosomal
material. The specific radioactivity of the ribosomes is followed during
the increase of intracellular virus and compared to ribosomes isolated
from uninfected cells. The ribosomal material is identified by its
spectrum and also by electron microscopy. The variation of radioactivity
of other cell fractions isolated during the purification of the ribosomes
is also under investigation.
Part B included: No
22
Serial No. NIA3D- 85A
2. Monkey kidney cell monolayers are infected vith RNA extracted
from purified poliovirus under a variety of ionic and pH conditions.
Major Findings;
1. A procedure has been worked out which consistently allows the
isolation of at least 1C$ of the ribosomal material from HeLa cells in
a relatively pure state. The ribosomal ribonucleoprotein particles
may be separated from the viral ribonucleoprotein particles by passage
through an ECTEOLA-cellulose column.
The specific activity of ribonucleoprotein particles from
infected cells is less than that of the ribosomal material of uninfected
cells 7 hours after the addition of virus under conditions of high and
low multiplicity of infection. With a high multiplicity, the cellular
protein sedimented at 15,000g for 10 minutes shows a dramatic and
continuous decline in specific activity.
2. The efficiency of infection with extracted RNA has been brought
to a level of about 0.5$ that obtainable with whole virus.
Significance to Bio-medical Research and the Program of the Institute:
The program is designed to lead to a general picture of the
synthesis of an animal virus by following events at a molecular level.
It is hoped that the key steps involved in the conversion of the cellular
machinery from normal metabolic activity to a virus producing system will
be elucidated. Interference with these key. steps may lead to new and
general methods of arresting viral disease.
Proposed Course of the Project;
Investigation of cellular fractions and especially ribosomal
material will be continued under conditions where the cells are infected
with different virus multiplicities. It is hoped that infection with
very high multiplicities may remove all remnants of normal activity and
clarify the situation with respect to virus growth. Also ribosomes from
cells actively synthesizing new protein during infection will be compared
to cells which are infected while they are in a resting state.
Infection of monkey kidney cells with RNA extracted from polio-
virus and the subsequent isolation and purification of ribosomal material
will also be attempted to study the role of the viral genetic material.
23
Serial No. NIAID-86
1. Biology of Viruses
2. Virus-Host Relationship
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Investigations of interference with enzymatic
functioning of mitochondria as a mechanism for
carcinogenesis
Principal Investigator: Dr. Marie L. Hesselbach
Other Investigators: None
Cooperating Units: Laboratory of Pathology & Histochemistry, NIAMD
Laboratory of Pathology, NCI
Man Years:
Total: 10/12
Professional: 9/12
Other: l/l2
Project Description:
Objectives:
To determine whether interference with the functioning of mito-
chondrial enzymes leads to neoplasia. Specifically, it is desired to
find a single dye which: l) is adsorbed by mitochondria; 2) effects
the functioning of mitochondrial enzymes, and 3) induces neoplasia.
Then, perhaps, a connection between these two functions of a single
agent can be demonstrated.
Methods Employed:
Conventional Warburg manometry for metabolic study of induced and
transplanted tumors, also for analysis of interaction of the dyes under
study, with mitochondrial enzymes.
Different methods of preparation of tissue for metabolic studies:
slicing, homogenization, and differential centrifugation.
Part B included: No
2k
Serial No. NIAID-86
Chemical analyses for total nitrogen, lactic acid and inorganic
phosphorus .
Histological preparation and examination of treated areas, tumors,
and organs .
A study of vehicles which would allow repeated injections of
Janus green B over a long period has been made. Non-aqueous media were
found to be necessary. Prolonged testing has made it possible to choose
the best of these .
Major Findings:
Absence of glucolysis in most of the Fast Green- and Light Green-
induced tumors and their early transplant generations was found to
correlate with the large amounts of enzymatically inert collagen present
in them. Age of tumor was found not to play a role in absence of
glucolysis. In later generations glucolysis was more commonly seen and
collagen decreased. Mitochondrial preparations which glucolyzed could
be prepared from the most metabolically active tumors.
It has been demonstrated that Fast Green and Light Green can be
added to total rat brain homogenates at concentrations which inhibit and
at other concentrations which stimulate oxygen uptake with added glucose
as substrate. The same concentrations which stimulate with glucose,
fail to do so with fructose-diphosphate . The dyes do not appear to be
uncouplers of oxidative phosphorylation.
Repeated injection of Janus Green B has induced gross changes
suggestive of tumor formation, but these have not yet been checked
histologically.
Significance to Bio-medical Research and the Program of the Institute:
Both viruses (exogenous) and altered subcellular particles
(endogenous) have been implicated as the cause of cancer. This study is
an effort to determine the relation of mitochondria to these possible
oncogenic agents.
Proposed Course of the Project:
Further studies of the Fast Green- induced tumor transplant line
will be made to see whether the homogenates acquire glucolysis "spontaneously,"
or by changes in preparative procedure, or the cofactors added. It will
also be determined which of the 3 enzymes which convert glucose to fructose-
diphosphate are destroyed by homogenization.
25
Serial No. NIAID-86
The study of the interaction of Fast Green and Light Green with
mitochondrial enzymes will be extended to include all possible enzyme
systems. The question of adsorption and physical interaction of these
dyes with the mitochondrial substance will be taken up.
Now that it is possible to give repeated doses of Janus Green B,
an experiment will be set up to see whether this dye is cancerogenic.
The treated areas, and any tumors formed, will be analyzed histologically.
Any induced tumors will be transplanted, and studied metabolically. The
interaction of Janus Green B and mitochondrial enzymes will be examined
in detail.
26
Serial No. NIA3D-86A
1. Biology of Viruses
2. Virus-Host Relationship
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Demonstration of Glucose Metabolism and Peptide
Bond Formation by Isolated Brain and Liver
Mitochondria
Principal Investigator: Dr. Marie L. Hesselbach
Other Investigators: Dr. H. G. du Buy
Cooperating Units: Analytical Chemistry Section, NIAMD
Man Years:
Total: V12
Professional: 3/12
Other: 1/12
Project Description:
Objectives:
To demonstrate that isolated mitochondria participate in protein
metabolism and that not only isolated brain mitochondria but also liver
mitochondria contain the complete enzyme systems to metabolize glucose.
Methods Employed:
Conventional Warburg manometry, chemical lactic acid determinations,
centrifugal separation of subcellular elements, biochemical and bio-
physical approach to choice of materials for a suspension medium vhich
will keep mitochondria structurally and functionally intact, biochemical
approach to determining materials to be added to isolated mitochondria
to restore in vivo enzymatic activity. For demonstration of peptide-
bond formation: one -dimensional paper chromatography.
Major Findings:
Work was continued on trying to demonstrate glucose utilization
by isolated rat liver mitochondria. Many variations in medium composition
Part B included: No
27
Serial No. NIAID-86A
were used. These involved physico-chemical agents such as methocel,
salts, chelating agents, protein derivatives, ribonucleic acid, and
phospholipid derivatives. The effects of the hormones epiniphrine and
glucagon were studied, as well as the enzymes 0!- and (3-amylase and
hexokinase. The reducing agent and cof actor, glutathione, and the
diabetogenic agent phlorizin, were also tried. Some of these substances
were used in the isolation medium, while others were added to the
reaction vessels.
At times hexokinase greatly increased glucose utilization, at
others it had little or no effect. Some of the other materials, such
as glutathione, seemed to increase glucose utilization very slightly,
but not to a significant level.
Addition to liver mitochondria of the natural "fat, " collected
from the surface of these aqueous preparations, was more successful in
increasing glucose utilization than anything except the specific enzyme,
hexokinase.
Significance to Bio-medical Research and the Program of the Institute:
The relation of viruses to mitochondria (site of virus repro-
duction, origin of viruses, site of neoplastic change) can only be fully
understood when the physical and chemical characteristics of mitochondria
become known. It would be strange if liver tissue could not metabolize
glucose, since it stores it (as glycogen), etc. It would also be
strange if this essential, energy- producing metabolism were not located
on the mitochondria in liver cells as it is in brain cells.
If isolated mitochondria can be shown to synthesize the peptide
bond, it will add significantly to our knowledge of the role of mito-
chondria in the living cell.
Proposed Course of the Project;
It is desired to obtain liver mitochondria which will readily
oxidize glucose in significant quantity, and will synthesize the peptide
bond. Further studies will be made to obtain better media and to
determine what other chemical or physical agents can be added to obtain
sustained enzyme activity of a number of complete enzyme sequences on
mitochondria in vitro.
28
Serial No. NIAID-87
1. Biology of Viruses
2. Virus-Host Relationship
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Cytopathogenic effect in single cells in tissue
culture.
Principal Investigator: JansShowacre
Other Investigators: None
Cooperating Units: None
Man Years:
Total: 6/12
Professional: V12
Other: 2/12
Project Description:
Objectives:
To study the morphologic and metabolic effects of virus infection
on individual virus infected cells in tissue culture.
Methods Employed:
Primary and established cell lines were cultured on coverslips
and then mounted in a special tissue culture flow chamber which permits
direct, phase and darkfield microscopy under conditions of cell growth.
Cells growing in the chamber were then infected with virus and changes
in morphology studied. Phase and fluorescent microscope observations
were made in rapid succession on identical living cells immediately
after mounting on regular slide mounts.
Part B included: No
29
Serial No. NIAID-87
Major Findings:
Studies with EMC, HA 1 and M 25 infected tissue cultures have
continued. Marked changes in cell morphology have not been observed
in initial stages of infection. Fluorescent antibodies against HA 1
and M 25 gave readily recognizable peripheral staining of living infected
cells in late stages of infection, similar to those reported by 0 'Dea
and Dineen with Herpes simplex. Antibody against EMC is being obtained
to determine whether increased titer and purification will improve the
efficiency of the technique in early stages of infection. Primary
cultures of embryonic and adult mouse brain have been obtained for an
in vivo study of the effects of neurotropic viruses on Nissl substance.
Significance to Bio-medical Research and the Program of the Institute;
Morphological evidence of early specific effects of virus invasion
of a cell may indicate the intracellular locus of virus activity.
Proposed Course of the Project:
Further study of morphological and physiological changes of
subcellular elements following the introduction of viruses and other
pathogenic micro-organisms.
30
Serial No. NIAID-87A
1. Biology of Viruses
2. Virus-Host Relationship
3. Bethesda, Maryland
PHS-NU
Individual Project Report
Calendar Year i960
Part A
Project Title: Characterization of mitochondria
Principal Investigator : Jane Showacre
Other Investigators: Dr. H. G. du Buy
Cooperating Units: None
Man Years:
Total: 8/12
Professional: 7/12
Other: 1/12
Project Description:
Objectives:
To study the localization of non-toxic fluorophors in living
cells.
Methods Employed:
Living, unfixed tissues vere examined by phase contrast and
fluorescence microscopy following exposure to fluorescent compounds.
Preparations were made from animal organs, a number of tissue cultures
including monkey kidney, HeLa and strain L and from cultures of micro-
organisms. The results were recorded photographically.
Major Findings:
Of the fluorescent compounds studied the most promising have been
the tetracyclines. These antibiotics, tetracycline, oxytetracycline,
and chlor tetracycline, were found to specifically combine with mito-
chondria of living cells in tissue cultures or in fresh preparations
from various organs of mice, and in bacteria such as Salmonella typhosa.
Part B included: Yes
31
Serial No. NIAID-87A
Significance to Bio-medical Research and the Program of the Institute:
Tetracyclines can now serve as an additional vital stain for the
characterization of mitochondria. As such these compounds may aid in
the determination of subcellular changes under different conditions,
e.g., viral infection. In this connection the specific localization
suggests that mitochondria are implicated in the fatty degeneration
occurring in liver following prolonged tetracycline therapy. Further,
the fluorescent properties may serve to identify the site of antibiotic
action in bacteria and elucidate differences in antibiotic effectiveness
under different environmental conditions.
Proposed Course of the Project;
Studies are continuing on factors influencing the retention of
tetracyclines by mitochondria as a preliminary to investigations of
possible change in the staining properties of mitochondria during
infection of cells with viruses, during cell division and following
cell fractionation. Bacteria under different environmental conditions
are also being examined.
32
Serial No. NIAID-87A
PHS-NIH
Individual Project Report
Calendar Year i960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
duBuy, H. G. and Showacre, J. L. Selective localization
of tetracycline in the mitochondria of living cells.
Accepted for publication in Science.
Honors and Awards relating to this project:
None
33
71 3.1
9''
Serial No. NIAID-88
1. Biology of Viruses
2. Virus -Host Relationship
3. Bethesda, Maryland
FHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Biology of mitochondria and its relation to
endogenous and viral diseases.
Principal Investigator: Dr. H. G. duBuy
Other Investigators: Dr. M. L. Hesselbach and J. L. Shovacre
Cooperating Units: Analytical Services Unit, Mr. H. G. McCann
Man Years:
Total: 1 Vi2
Professional: 10/12
Other: 6/12
Project Description:
Objectives:
Biological definition of normal versus pathogenic or virus-
altered mitochondria.
Methods Employed:
The methods encompass the applications of the cytochemical
findings reported under project No. NIAID-87A. They also include
Warburg metabolic techniques and determinations of oxidative phos-
phorylation in order to define different mitochondria metabolically.
The results are applied to in vitro cultivation of isolated mito-
chondria.
Major Findings:
A manuscript is in preparation on evidence that the ~,yme
behavior of mitochondria obtained by the sucrose gradient . .iainly
due to the unavoidable dilution of mitochondria, when this t chnique
of separation is used. The results are applied specifically to the
loss of enzymes by melanized mitochondria of the Cloudman S 91 mouse
melanoma.
Part B included: No
34
Serial No. NIA33-88
Significance to Bio-medical Research and the Program of the Institute:
The maintenance or cultivation of mitochondria in vitro when
accomplished should facilitate the investigation of many metabolic
activities of normal cells as compared to tumor cells or those infected
vith various types of viruses.
Proposed Course of the Project:
Further studies of mitochondria from different sources will be
carried out in order to learn more about the characteristics of these
elements. Additional experiments will be done to explore further the
complete enzymatic complement of mitochondria, especially as this
relates to synthetic activities. All information obtained will be
applied to continued attempts at in vitro cultivation of mitochondria.
35
Serial No. NIAID-88A
1. Biology of Viruses
2. Virus-Host Relationship
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Comparison of the properties of crude and
crystallized Coxsackie A- 10 virus, of Cloudman S 91*
mouse melanoma and of mouse muscle nucleoprotein.
Principal Investigator: Dr. H. G. duBuy
Other Investigators: H. Sasame
Cooperating Units: None
Man Years:
Total: 1 k/12
Professional: 3/12
Other: 1 1/12
Project Description:
Objectives:
To compare preparations of purified virus with those of mouse
muscle and melanoma nucleoprotein, chemically and immunologically.
Methods Employed:
For virus purification: Coxsackie A- 10 virus of suckling mouse
origin is purified and concentrated "by chemical, physical and ultra-
centrifugal means. Purified virus is analyzed for protein and nucleic
acid content.
For muscle and S 91 nucleoprotein preparation available methods
vere not applicable to the materials under study. Some steps, used
for the preparation of so-called ribosomes, followed by modifications
of existing purification procedures, have given promising results.
Part B included: No
36
Serial No. NIAID-88A
Major Findings;
Melanin granules, isolated by selective centrifugation, contained
30 to kO percent of the total ribose nucleo-protein of the melanoma cell.
This supports the view that the granules are modified mitochondria. The
antigenic activity of this material is determined by the number of
"takes" of transplanted melanoma cells in mice which have previously
been injected with immunizing doses of the melanoma nucleoprotein, as
compared with non- immunized mice.
Purified Coxsackie A- 10 virus has been introduced into normally
resistant cells by cellular uptake of glass-adsorbed virus.
Significance to Bio-medical Research and the program of the Institute:
The introduction of virus into cells which are normally not
susceptible to this virus, except in its nucleic acid stage, might
throw further light on the conditions which govern virus multiplication
in host cells.
The introduction of a self -duplicating portion of mitochondria
from cancer cells into susceptible hosts might lead to formation of
this specific neoplasm, and thus bridge the virus and the mitochondrial
theories of carcinogenesis.
Proposed Course of the Project; .
To obtain sufficient quantities of the three nucleoproteins, each
with a standard nucleic acid-protein ratio as an index of purity, to
allow quantitative antigenic studies. At this time, purified muscle
nucleoprotein to be used for control studies has not yet been obtained.
37
Serial No. NIAID-89
1. Biology of Viruses
2. Viral Growth
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Kinetics and Sites of Coxsackie Virus Multiplication
in the Monkey Kidney Cell
Principal Investigator: Dr. C. F. T. Mattern
Other Investigators: Lotta Chi
Cooperating Units: None
Man Years:
Total: 1
Professional: 1
Other: 0
Project Description:
Objectives:
To establish the intracellular site or sites of viral RNA and
protein synthesis.
Methods Employed:
Coxsackie A-9 virus is cultivated in monkey kidney cells for
various time periods. Cells are fractionated by the Dounce Citric Acid
Procedure and by conventional homogenization. Two major fractions are
now being investigated for mature virus content, namely the nuclear
fraction and the remainder of the cell, called the "cytoplasmic" fraction.
It has been previously shown by this group that cold phenol will not
extract RNA from purified Coxsackie. Also the suckling mouse inoculated
I.M. is an excellent assay system for the RNA, whereas tissue culture is
poor. These cell fractions are assayed for mature virus and extracted
with cold phenol for a virus precursor, whether free RNA or a RNP other
than mature virus.
Part B included: No
38
Serial No. NIAID-89
Major Findings:
1. Mature virus, that is virus refractory to cold phenol extraction
of its RNA, is clearly associated with the cytoplasmic fraction at all
time periods from 2 to 12 hours post- inoculation.
2. There is evidence of a cold phenol extrac table precursor. This
precursor has been extracted from whole cells and cell homogenates and
appears maximally produced by 6 hours, remaining constant in quantity
thereafter. The titer of mature virus, on the other hand appears to
continue to increase until 9-12 hours post- inoculation. Most of this
"precursor" appears associated with the "nuclear" fraction, thus show-
ing a distribution within the cell that is different from that of
"mature" virus.
Significance to Bio-medical Research and the Program of the Institute:
This study is intended to contribute to our knowledge of the
nature of the processes by which viruses multiply. It has been proposed
by others that viral RNA is synthesized in the nucleus on the basis of
indirect evidence. The studies herein described would be the first
direct evidence of viral RNA synthesis in the nucleus.
Proposed Course of the Project:
It is proposed to continue this project in order to evaluate the
significance of our findings to date, especially with respect to whether
they are representative of actual intracellular events or artifacts of
cell fractionation. Other cell fractionation techniques will be employed.
Our first "control" experiments indicated, for example, that this
distribution of "precursor" is not the result of selective absorption
of "cytoplasm precursor" by nuclei. In addition a collaborative project
with Dr. Hilton Levy is planned in which these events will be followed
by autoradiography in the Coxsackie-monkey cell system. Dr. Levy has
been utilizing a poliovirus-HeLa cell system and has made observations
which seem compatible with our interpretation of our infectivity data.
Since there is reason to believe that this "precursor" is a RN
protein, we plan to attempt to isolate and characterize this component.
33
Serial No. NIAID-89A
1. Biology of Viruses
2. Viral Growth
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year i960
Part A
Project Title: Virus Structure
Principal Investigator: Dr. C. F. T. Mattern
Other Investigators: Lotta Chi
Cooperating Units: None
Man Years:
Total: 1
Professional: 1
Other: 0
Project Description:
Objectives:
This project involves the construction of "biological and related
biochemical models of viruses by a new model approach. The inmediate
objective is to test the "reality" of the virus models by electron
microscopy and X-ray diffraction.
Methods Employed:
1. Electron Microscopy. The substructure of viruses is being
examined by several techniques: conventional shadowing, negative stain-
ing with phosphotungstic acid, and positive staining with uranium salts.
In addition, a new shadowing procedure has been developed and its
potentialities in revealing substructure are being studied.
2. X-ray diffraction. This involves an indirect approach in which
theoretical diffraction patterns may be calculated and compared with
patterns obtained from viruses by others.
Part B included: No
40
Serial No. NIAID-89A
Major Findings;
1. A new model for Tobacco Mosaic Virus has been constructed
which contains a substantial number of those structural elements known
from a wide variety of biochemical and biophysical data of others.
2. The model has predicted several gross features which differ
from currently accepted models and which should be demonstrable by
electron microscopy. Our electron micrographs taken with metal shadow-
ing and negative staining are remarkably compatible with the model.
Significance to Bio-medical Research and the Program of the Institute:
This project is an effort to elucidate the three dimensional
structure of viruses, beyond the subunit organization. The selection
of TMV for constructing a detailed model was necessary because it is
the most thoroughly studied virus and the only one about which there
is sufficient structural data to attempt to construct a detailed model.
The structure of other viruses, in particular animal viruses, will
also be investigated by this approach as additional data accumulates.
Proposed Course of the Project:
Because of the controversial nature of this project, it is
felt advisable to proceed to accumulate a substantial amount of
experimental data before challenging the currently held views on virus
structure, and in particular TMV. A purely hypothetical paper describ-
ing the model building system, without detailed reference to specific
structures, is in the process of being written.
41
LABORATORY OF TROPICAL VIROLOGY
Middle America Research Unit
Arthropod-Borne Virus Section
Summary
93 - Studies on Arthropod-Borne Viruses in the Sub-
Tropical Areas of the United States 6
94-A - Studies of Arthropod-Borne Viruses in Tissue
Culture. Part 1. Evaluation of Tissue Culture
Systems for Use of Viral Isolation, Identifica-
tion, and in Serological Tests for Viral
Antibodies 8
94-B - Studies of Arthropod-Borne Viruses in Tissue
Culture. Part 2. Development of a Cell Cul-
ture System Utilizing Arthropod Tissue 11
95-A - Studies on Antigen- Antibody Reactions of
Arthropod-Borne Viruses. Part 1. Kinetic
Studies of the Serum Neutralization of the
Arthropod-Borne Virus 13
95-B - Studies on Antigen- Antibody Reactions of
Arthropod-Borne Viruses. Part 2. Develop-
ment of a practical and Specific Flocculation
Test for the Demonstration of Arthropod -Borne
Virus Antibodies 15
95-C - Typing of Viruses by Combinations of Antiserum
Pools. Application to Typing of Arthropod-
Borne Viruses 17
96-C - Survival Potential of the Adult of Haemagogus
Equinus, A Sylvan Vector of Yellow Fever 19
97-A - A Qualitative Evaluation of Experimentally
Induced Eastern Equine Encephalomyelitis
(EEE) Virus Infection in Horses 22
97-B - The Development of an Inactivated Vaccine
Against Eastern Equine Encephalomyelitis (EEE)
Virus 24
100-A - Virological Investigation of Clinical Cases
and Epidemic Outbreaks in Panama and Other
Countries of Middle America 27
100-B - A Clinical and Virological Study of Oro-
pharyngeal Lesions in Panamanian Children 33
101-A - Virological Aspects of a Cooperative Investi-
gation on the Ecology cf Aithropod-Borne
Viruses 35
101-B - The Role of Chlggers and Other Acarlna of
the American Tropics in the Maintenance
and Transmission of Animal Infectious
Agents: 1. Viral Aspects 39
101-C - "Jungle Fever" in U.S. Military Personnel 41
102-A - Enterovirus Infections of Rural Guatemalan
Children in Relation to Nutrition 43
102-B - Laboratory Support of Phase 1, National Program
for Poliovirus Vaccine Administration in Costa
Rica, 1959 45
102-C - Enterrovirus Flora of Panamanian Children: A
Twe lve Month Survey 48
103-A - Use of Filter Paper Discs for Virus Isolation
and Serological Testing 50
103-B - Eastern Equine Encephalomyelitis (EEE) Virus
Infection in Panama 53
103-C - Encephalomyocarditis (EMC) Virus Infection.
Studies on Pathogenesis in Swine, Virus
Reservoirs in Rodents and Antibody Status of
Human and Animal Populations 55
108 - Studies of Histoplasmosis on the Isthmus of
Panama 59
109 - Studies of Superficial and Deep Mycoses in
Panama and Central America 63
II
PHS-NIH
Summary Statement
Office of Chief
Laboratory of Tropical Virology
Calendar Year i960
I. ADMINISTRATIVE ASPECTS
The second year of the Laboratory's administrative existence was event-
ful and stormy. Activities of the Arthropod Borne Virus Section (ABVS) in
Bethesda have been affected by the uncertainties in connection with Dr. William
Pond's resignation from the position of Section Head and Assistant Chief,
LTV. Difficulties were compounded by the resignation of Dr. Herbert T. Dalmat,
who temporarily was Acting Head of the Section. Mr. Clarence J. Gibbs, Jr.
then became Acting Head under difficult administrative circumstances aggra-
vated by loss of several technicians and the usual problems in communications
with Office of the Chief physically located in Panama. The Section has weath-
ered the storms, carried on the active research program and is now anticipat-
ing assignment of replacements for Drs. Pond and Dalmat.
As all other NIH components in Bethesda, the ABVS is plagued with a
shortage of space. Besides a modest area allotment in square feet, the lab-
oratory is located in a somewhat uninviting basement of Building 5. Sched-
uled expansion into several rooms finally vacated by the Division of Biologic
Standards will alleviate the work space shortage. However, such plans as
the creation of a third section of LTV to be located in Bethesda (proposed a
year ago) are hardly realistic.
The Panama Laboratory has also had space problems - but in reverse:
for the past ten months the headaches were due to planning and execution of
reconstruction to convert some of the ample space into a functional research
laboratory and supporting service areas. Although the job is not finished,
sometime early in 1 96 1 the virus research area will be more than doubled.
Especially needed are the several isolation cubicles constructed on the second
floor and provision of air-conditioned desk space for Investigators near
their own working areas.
With clarification of the MARU mission and commitment to several import-
ant projects there has been a commensurate gradual increase in personnel,
particularly at the sub-professional level.
The next calendar year will bring many more changes in the Laboratory
of Tropical Virology. At MARU there will be a new Director, a new Head of
Virus Section and a new Head of Mycology Section; in Bethesda, a new Head
of the Section and physically relocated Laboratory Chief from Panama. Un-
doubtedly, these factors will affect administration as well as the research
program of the laboratory.
I. RESEARCH HIGHLIGHTS
MARU. C. Z. - VIRUS SECTION
1 . Virus Isolates from Panamanian Mosquitos and Sandflies
During the first 12 months of a 3-year project on the ecology of
arthropod borne viruses in the tropical rain forest, which is being con-
ducted by GML with the collaboration of MARU, major emphasis has been
on virus isolation in suckling mice and hamster kidney cell cultures.
Fourteen virus strains were isolated at MARU from 412 pools and 63,000
specimens provided by GML. Virus isolation rates were for Phlebotomus
1:700 and for mosquitoes 1:7000, although the rates varied greatly with
species. Of the five Phlebotomus isolates, two of broad host range
(including cell culture) and short incubation period are serologically
identical. These viruses have now been identified as the Indiana type
of vesicular stomatitis virus. The other three phlebotomus and nine
mosquito viruses are being related to each other, to known virus groups
and to human and/or animal infection and disease.
2. Eastern Equine Encephalomyelitis Virus Infection in Panama
The prevalence of EEE antibodies in horses and man in two areas of
suggested EEE virus endemicity has been determined, allowing an evalua-
tion of the relative usefulness of several serological methods applic-
able to studies of this type. It was found that the incidence of EEE
antibodies in 460 humans tested increased with advancing age (0.8% under
10 years with progressive increase to 9% in the 41-50 year group).
Complement fixation results on the same sera indicated the probable pre-
sence of other group A viruses.
Lizards of species common to this part of Panama were examined as
a possible virus reservoir. Specific EEE virus hemagglutination-
inhibitors were found in some of their sera. The occurrence of viremla
and HI antibody response following virus inoculation were experimental ly
confirmed by inoculation of lizards.
3. Etiology of "Jungle Fever"
About 200 paired specimens were collected from military students
participating in jungle warfare training courses in the Canal Zone.
From one of the specimens obtained during an episode of fever following
known exposure to jungle environment, a virus was isolated. This agent
as yet has not been related to viruses known to be active on the Isthmus
of Panama.
4. Encephalomyocard? tis Virus Infection
Previously this laboratory described an outbreak of a fatal disease
of swine caused by the EMC virus. The outstanding lesion in pigs dying
during the outbreak was acute myocarditis. Since epidemiological obser-
vations suggested that natural infection resulted from ingestion of con-
taminated food, experiments were undertaken to reproduce the disease by
feeding virus to young pigs. Viremia and virus excretion from the gas-
trointestinal tract were found to occur following the administration of
brain from EMC inoculated mice. Infected pigs developed high titers of
HI and neutralizing antibody during convalescence and had myocardial
fibrosis at autopsy. Other studies included demonstration of EMC anti-
bodies In a small number of city rats and rats caught on the affected
farm, although wild rodents were found to be negative. Human sera were
examined with interesting differences in the results depending on the
donors* age: while a substantial proportion of the Panamanian population
has been infected with EMC virus, the antibodies were found to be more
common in persons of younger age.
5. Enterovirus Flora in Children of Central America
For a period of 12 months the enterovirus flora of Infants at an out-
patient clinic in Panama City was systematical ly explored establishing a
base line of enterovirus fluctuation. The majority of viruses isolated
belonged to the ECHO group, although in late 1959 and early I960 polio-
virus type 2 had become very prevalent. This was reflected in an un-
common occurrence of a small outbreak of paralytic disease due to type
2 pol iovirus.
Other enterovirus studies have Included 1) surveillance for the pre-
sence of type 1 pol iovirus in Panama in late I960 as a check on dissemina-
tion and threatened spread of this commonly epidemic type, 2) studies on
a major epidemic of Echo-9 virus which swept through the Republic of
Panama and the Canal Zone and 3) initiation of a collaborative project
on possible relation of enterovirus flora of Guatemalan children to
their dietary status.
6. Mite Virus Project
Ors. J. M. Brennan and C. E. Yunker of Rocky Mountain Laboratory
staff have been assigned to LTV component in Panama to conduct a two-
year study on the possible role of chiggers and other Acarina of the
American tropics in the maintenance and transmission of animal viruses.
To our knowledge this is the first serious attempt to explore this im-
portant area.
MYCOLOGY SECTION, MARU
The research program of the Section has markedly increased local
awareness of histoplasmosis in all of its clinical forms, as evidenced
by recognition of three disseminated cases (2 fatal and one success-
fully treated) within a period of 18 months (until then only one fatal
case had been described since Darling's original cases in 1906) .
Ecological and epidemiological studies led to isolation of H. capsulatum
from eight additional soil samples bringing up to sixteen the total
number of recent isolations from Panamanian soil (while only a single
positive soil sample was recorded until this Section was established).
The fungus has been repeatedly recovered from the organs of trapped
ground mammals confirming its wide dissemination in nature.
Histoplasmin skin test continues to be a major tool for the study
of epidemiology of histoplasmosis. Data on 9,200 children between 6
and 19 years of age have been obtained indicating, as expected, that
the percentage of reactors increases progressively with age. The rate
of histoplasmin sensitivity varies from 13 to 58% among six-year olds
and from 68 to 92% among 19 year olds, depending on location of their
residence. A survey of 631 pre-school children (6 months to 6 years)
in the Canal Zone demonstrated an increase in hypersensitivity beginning
with three years of age. A continuing similar study of Panamanian child-
ren in a city hospital is now in progress with information/over 800
already available.
Projects on other mycotic diseases have included diagnostic study
and therapy of moniliasis, found to be a major superficial mycosis among
both indigenous and transient population in the tropics.
LTV. BETHESDA - ABV SECTION
In spite of the difficult administrative and working conditions,
the research staff pursued the several important projects initiated
during the preceding calendar year. New projects Involved an interest-
ing application of the technique of antiserum pool combinations to typing
of arthropod borne viruses, a wealth of data evaluating experimentally
produced EEE virus infection in. horses and a promising attempt to develop
an inactivated EEE virus vaccine for human use. The Infected horses
yielded specific antiserum which is being processed for prophylactic use
in cases of human exposure under laboratory or natural conditions.
Accidental laboratory infection of a staff member with an arthropod
borne group C (Apeu) virus led to the first cl inical-virological study
of a syndrome produced by this important and common group of viruses of
the western hemisphere.
III. PERSONNEL
ABVS. Bethesda - Dr. W. L. Pond and Dr. H.T. Dalmat resigned
during second half of the year; not replaced at the end of Calendar
Year.
Virus Section, MARU:
Dr. J. E. Craighead and Dr. C. G. Dobrovolny resigned in midyear.
Dr. Craighead's position at MARU is now occupied by Or. E. A. Bruckner
transferred from Bethesda to the Canal Zone. Dr. J. V. Ordonez, staff
member of Bacteriology Section of Instituto de Nutricion de Centro
America y Panama (Guatemala) began a one year fellowship in virology
(under the sponshorship of Parke, Davis & Co.) in July i960. Drs. J. M.
Brennan and C. E. Yunker transferred from RML to LTV-MARU for a period
of 2 years to initiate a project on the role of Acarina in transmission
of infectious diseases.
Mycology Section, MARL);
No professional personnel changes. The Research and Development
Command of US Army has accepted Mrs. M. Shacklette and Mr. J. Fuentes
by transfer from NIH to US Army Caribbean payroll.
Serial No. NIAID - 93
1. Tropical Virology
2. Arthropod-borne Virus
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Studies on arthropod-borne viruses in the sub-
tropical AREAS OF THE UNITED STATES
Principal Investigator: Robert M. Pennington
Other Investigators: Clarence J. Gibbs, Jr.
William L. Pond
Cooperating Units: University of Miami School of Medicine
Southwest Blood Banks, Inc.
Man Years (calendar year 1960)
Total: 2-3/4
Professional: 1-1/4
Other: 1/2
Project Description:
Object ives:
Testing of sera from human beings residing in the Southern
United States has been in progress to establish a baseline for
the interpretation of tests used in investigations of viral diseases
in non-temperate climates. Moreover, these tests can be expected to
give an indication as to which arthropod-borne viruses are present
and which viruses are probably absent from this area.
Methods Employed:
Neutralization, hemagglutination inhibition, and complement
fixation tests of the arthropod-borne viruses are being carried
out on sera obtained from residents of subtropical areas of the
United States.
Major Findings:
Neutralization and hemagglutination inhibition tests on 125
sera from Miami residents indicate activity of St. Louis enceph-
alitis, encephalomyocardit is, and possibly Ilheus virus. There
Serial No. NIAID - 93
ARE ALSO STRONG INDICATIONS THAT ANOTHER GROUP "B" VIRUS IS ACTIVE OR HAS
BEEN ACTIVE IN
INVEST IGATED.
the Miami area. The identity of this agent is being
Significance to Program of the Institute:
Potential and actual public health importance of arthropod-borne
viruses for residents of the Southern United States should be determined
as part of investigations of tropical viral diseases.
Proposed Course of Project:
Neutralization, hemagglutination inhibition, and complement fixation
tests will be carried out on sera not already tested and the serum
collections on hand will be augmented with additional sera from other
subtropical areas of the united states.
Part B included
No
Part A
Serial No. NIAID - 94-A
1 . Trop ical Virology
2. Arthropod-borne Virus
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Project Title: Studies of arthropod-borne viruses in tissue culture.
Part 1. Evaluation of tissue culture systems for use
in viral isolation, identification, and in serological
tests for viral antibodies.
Principal Investigator: Charles R. Rosenberger
Other Investigators: Walter L. Newton
Cooperating Units: Laboratory of Germfree Animal Research,
NIAID-118
Man Years (calendar year 1960)
Total: 1-7/8
Professional: 7/8
Other:
1
Project Description:
Objectives:
Tissue culture studies are being applied to arthropod-borne
viruses to (a) develop a more efficient system for the successful
ISOLATION OF VIRUSES FROM VECTORS AND NATURALLY INFECTED HOSTS, (b)
aid in classification, identification, and characterization of these
viruses through development of new techniques utilizing cell cul-
tures, and (c) provide a source of virus material suitable for use
in vaccine development.
Methods Employed:
: studied in a
l and human
infection and
Propagation and cytopathogen ic ity of viruses af
variety of cell cultures prepared from selected anim'
tissues. Maximum sensitivity of cell lines to virus
proliferation is determined by type and degree of ci opathic changes
as well as by titers of virus obtained in cell cultui es and in mice.
Viruses are identified and classified into serological groups by
Serial No. NIAID - 94-A
neutralizing, with specific antiserum, the ability of the virus to
cause cytopathic changes, form plaques, or produce hemadsorption.
Strains of virus which show reduced pathogenicity in animals, greater
growth or cytopathogen ic ity in cell cultures, or other desired
characteristics, are selected by serial passage in cell cultures or
by selecting individual virus particles through plaque techniques.
Major Findings;
Hamster kidney cell cultures (HKTC) have been found to be partic-
ularly USEFUL IN PROPAGATING ARTHROPOD-BORNE VIRUSES. PRESENTLY, MORE
THAN 20 OF THESE VIRUSES HAVE BEEN GROWN AND OBSERVED TO PRODUCE
CYTOPATHIC CHANGES IN THIS TYPE CELL LINE.
A CONTAMINATING NON-VIRAL ORGANISM WAS ISOLATED FROM APPARENTLY
normal HKTC. This organism appears to be a bacterial L form. It
produces a "hemonuclear adsorption reaction1' in cell cultures which
results in dissolution of the cytoplasm of nucleated chick erythro-
cytes leaving nuclei adsorbed onto cells of infected cultures. the
organism will propagate in a suspension of chick erythrocytes in
balanced salt solution. the "hemonuclear adsorption*.' phenomenon has
proven to be reliable and is used routinely in our laboratory as a
test to detect the presence of this organism in cell cultures.
in collaboration with dr . walter l. newton, laboratory of germ-
free Animal Research, many cell lots have been prepared from germ-
free animals. Comparative tests, with kidney cell cultures prepared
from germfree and conventional mice, have not shown differences in
growth or cytopathogen ic ity with the following viruses: yellow fever
(French neurotropic strain), Anopheles A, Murray Valley encephalitis,
and Oriboca. Previously reported preliminary results of greater
cytopathogen ic ity with dengue type 1 virus (moch i zuk i strain) in
kidney cells prepared from germfree mice than in cells from conven-
tional mice have not been consistent on additional investigations.
Significance to the Program of the Institute:
These studies shall provide additional methods for more success-
ful isolation, identification, and classification of ARBOR viruses.
They will facilitate investigations into the mechanism of virus-cell
interrelationships.
Serial No. NIAID - 94-A
Proposed Course of the Project:
The study or ARBOR viruses in tissue culture systems will con-
tinue WITH GREATER UTILIZATION OF PLAQUE TECHNIQUES.
a study of the bunyamwera group of viruses has been initiated.
Cell line susept ib i l ity, growth characteristics, plaque production,
and antigenic relationship between the viruses of this group will be
investigated. these studies will provide definitive tests for the
identification and classification of these viruses.
Part B included - No
10
Serial No. NIAID - 94-B
1 . TROP ICAL VlROLOGr
2. Arthropod-borne Virus
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Studies of arthropod-borne viruses in tissue culture,
Part 2. Development of a cell culture system utili-
zing ARTHROPOD TISSUE.
Principal Investigator: Charles R. Rosenberger
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960)
Total:
1/8
Profess ional:
1/8
Other:
None
Project Description:
Objectives:
To develop methods of preparing cell cultures from arthropod
tissues. To provide a sensitive and more suitable cell culture
system for physiological studies of arthropod-borne virus cell-host
interact ions.
Methods Employed:
Specific tissues are dissected from arthropods and prepared as
cell cultures. the blood of the arthropod is sometimes collected
and added to the culture media. various culture media and culture
techniques are used in attempts to provide a system suitable for a
particular tissue. certain arthropods are reared under sterile
conditions to eliminate contaminating organisms when cell cultures
are prepared.
Major Findings:
Treatment of silkworm ovarian tissue with a balanced salt solu-
tion EXTRACT OF THE CROP OF THE BLUE CRAB CAUSES D I SASSOC I AT I ON OF
cells. This treatment provides a suspension of cells rather than
TISSUE FRAGMENTS OR CLUMPS OF CELLS. In CULTURE, CELLS ATTACH TO THE
GLASS SUBSTRATUM BUT FAIL TO PROLIFERATE IN WYATT'S MEDIUM WHEN HELD
AT A VARIETY OF TEMPERATURES OF INCUBATION.
11
Serial No. NIAID - 94-B
Significance to the: Program or the Institute;
Cell cultures of arthropod tissues would provide a completely
new approach for studies of viruses, host cells, and their inter-
ACTIONS. In the field of arthropod-borne virus research, insect
CELL CULTURE MAY PROVIDE A SENSITIVE AND MORE SUITABLE CULTURE
MED IUM.
Proposed Course of the Project:
Basic studies in the field of cell culture of arthropod tissues
will be continued. the possibility of maintaining tissue and cell
suspension type cultures prepared from mosquitoes reared under
sterile conditions will be investigated.
Part B included - No
12
Serial No. NIAID - 95-A
1 . Trop ical Virology
2. Arthropod-borne Virus
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Studies on antigen-antibody reactions of arthropod-
borne viruses. Part 1. Kinetic studies of the serum
neutralization of arthropod-borne viruses.
Principal Investigator: Robert M. Pennington
Other Investigators: William L. Pond
Cooperating Units: None
Man Years (calendar year 1960)
Total: 1
Professional: 1/2
Other: 1/2
Project Description:
Object i ves:
To investigate the variables of the neutralization test.
Specifically time (as an independent variable) and the relative
degree of reaction (amount of virus neutralization) due to prior
incubation of the virus-serum reactants. Such information is
expected to give insight into the kinetics of virus-antibody
combination. The rate of reactivity of the virus-antibody
systems may indicate if the instantaneous reaction of homologous
bacterial antigen-antibody systems is also applicable to homologous
virus-antibody systems.
Methods Employed:
The rapid in vitro combination of virus with homologous antibody
and the comparatively slow speed of reaction (avidity) with heter-
ologous ANTIBODY HAS BEEN SUCCESSFULLY APPLIED TO MARKEDLY INCREASE
the specificity of the arthropod-borne virus neutralization tests.
Project type serological tests, results of which are un interpretable
because of broad cross-reactive antigenic relationships, were
studied by kinetic neutralization tests.
13
Serial No. NIAID - 95-A
Major Findings;
The concept of "instantaneous" neutralization of viruses with
homologous antibody was tested using group "b" moderately reactive
sera obtained from residents of guatemala and group "b" broadly
reactive sera obtained from indigenous residents of southeast asia.
the data indicate that the neutralization of virus by homologous
antibody is an instantaneous phenomenon. the guatemalan sera, as
expected, due to their moderate cross reactivity, as demonstrated in
the conventional neutralization test, were differentiated by neutral-
ization index using the "mod i f l_edm neutralization test ( no incubation
of the virus-serum reactants) /.discussed in this reporj/. the southeast
Asian sera however, remained broadly cross reactive, although to a lesser
degree in the modified neutralization test than in the conventional
neutralization test.
Significance to Program of the Institute:
The kinetic neutralization studies have shown that the specificity
of the mouse neutralization test may be increased by eliminating the
prior incubation of virus-serum reactants. wlth cross reactivity at a
minimum, a specific virus may be identified as causing a once broadly
cross reactive serum antibody. this study demonstrates that the basic
phenomenon of instantaneous homologous antigen-antibody combinat-ion
existing in bacterial antibody systems functions in the same manner
with virus antibody systems.
Proposed Course of Project:
The instantaneous neutralization of viruses with homologous and
heterologous antibody is to be studied using sera on hand collected from
human beings residing in the miami area. these studies will be enlarged
to include additional serological tests designed to elicit serological
SURVEY SPECIFICITY. In ADDITION, ANIMAL SERA, PREPARED BY SINGLE AND DUAL
INFECTIONS, WILL BE STUDIED TO DETERMINE THE CORRELATION OF SEROLOGICAL
TEST RESULTS OBTAINED WITH THE MlAMI SERA AND THE LABORATORY ANIMAL SERA.
Part B included - No
14
Serial No. NIAID - 95-B
1 . Trop ical Virology
2. Arthropod-borne Virus
3. Bethesda, Maryland
PHS-NIH
ndividual Project Report
Calendar Year 1960
Part A
Project Title: Studies on antigen-antibody reactions of arthropod-
borne viruses. Part 2. Development of a practical
and specific flocculation test for the demonstration
of arthropod-borne virus antibodies.
Principal Investigator: Robert M. Pennington
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960)
Total: 1/2
Professional: 1/2
Other: None
Project Description:
Object i ves:
Development of a rapid flocculation test for the detection of
arthropod-borne virus antibodies and its evaluation as a worthwhile
laboratory procedure. '
Methods Employed:
Arthropod-borne viruses, contained in cell culture supernatant
fluids, are adsorbed onto clay particles in the manner previously
described by bozicevich of the laboratory of clinical investigations
for the bentonite flocculation test for trichinosis and for other
diseases. Flocculation of the viral ant igen-coated Bentonite
particles is demonstrated in the presence of the specific viral
ant ibody.
Major Findings:
The Bentonite Flocculation Test using Eastern equine encephalo-
myelitis (EEE), and St. Louis encephalitis (SLE) viruses as proto-
types AGAINST HOMOLOGOUS ANTISERA HAS GIVEN SPECIFIC HIGH TITERED
REACTIONS. NO CROSS REACTIVITY WAS DEMONSTRABLE IN FLOCCULATION
15
Serial No. NIAID - 95-B
tests with related group "a" sera or related group "b" sera. however,
antibody titers, using the same immune sera and different lots of
infective hktc fluid, have been variable. this variability is most
likely a function of virus antigen concentration and/or the amount
of adsorption of virus to the bentonite clay particles.
Significance to the Program of the Institute;
The Bentonite Flocculation Test, as applied to the arthropod-
borne VIRUSES, MAY CONSTITUTE ANOTHER POSSIBLY VALUABLE SEROLOGICAL
test to be used in conjunction with the standard complement fixation,
hemagglutination inhibition, and neutralization tests. moreover,
preliminary observations indicate that the ant i gen-benton i te combina-
tion may be of value in subsequent adsorption of selected antibody
from serum.
Proposed Course of the Project;
Evaluation of the Bentonite Flocculation Test (as applied to
arthropod-borne virus systems) in terms of (1) improved antigen
adsorption to Bentonite, (2) reproducible results, (3) stability of
the test reagents, (4) improvements in the methods for preparing test
reagents and techniques of the test.
Part B included - No
16
Serial No. NIAID - 95-C
1 . Trop ical Virology
2. Arthropod-borne Virus
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Typing of viruses by combinations of antiserum pools.
Application to typing of arthropod-borne viruses.
Principal Investigator: Clarence J. Gibbs, Jr.
Other Investigators: None
Cooperating Units: None
Man Years: (calendar year 1960)
Total: 1
Professional: 1/2
Other: 1/2
Project Description:
Object i ves:
The purpose of this investigation is to develop methods by which
individual tests of an unknown ARBOR virus against a number of typing
sera can be replaced by tests against a small number of pools of
these sera. the serum pools must yield combinations of results
specific for each type according to the distribution of the sera in
the pools.
Methods Employed:
Viruses thus far employed in these studies are prototype strains
of representative arthropod-borne viruses of serological groups A, B,
and C. These are Eastern equine encephalomyelitis, Japanese B
ENCEPHALITIS, AND APEU, CARAPARU, ORIBOCA, MARITUBA, MURUTUCU, AND
Itaqui viruses. In ADDITION, AN UNKNOWN VIRUS ISOLATED FROM A
laboratory investigator and suspected of belonging to group c, was
employed. Without exception, the antisera employed in these studies
were prepared by hyper i mmun i z at i on of rabbits with mouse adapted virus
strains. Neutralization tests were carried out by intracerebral
inoculation of 5-7 day old suckling mice. In all tests equal
volumes of inactivated undiluted serum, or sometimes sera, were mixed
with equal volumes of varying dilutions of viruses. combination serum
17
Serial No. NIAID - 95-C
POOLS WERE PREPARED BY MIXING KNOWN SPECIFIC ANTISERA ON A 1:1 RATIO
WITHOUT REGARD TO QUANTITATIVE LEVELS OF NEUTRALIZING ANTIBODY.
Combination pools of group C antisera were prepared on the basis of
serological sub-groups. controls consisted of mixtures of equal
volumes of virus with undiluted normal rabbit serum or ]0% normal
rabbit serum in borate-kcl buffer solution at ph 9.0.
Major Findings;
Prototype viruses were neutralized to a greater extent when
tested against their homologous antiserum alone or against pools
which contained such antiserum. heterologous neutralization of
prototype viruses occurred only between members of the same
serological group. there was no serological crossing over between
groups a, b, or c. an unknown viral isolate, suspected of being
apeu, a member of serological group c, when tested against combination
serum pools was neutralized by combination of group c antisera but not
by pools of groups a or b. furthermore, pools containing apeu anti-
serum showed greater degrees of neutralization than did pools that did
not contain specific apeu antisera. thus, our data show that this
method can be applied successfully in group typing of an unknown
virus if the virus does not cross react to any great extent with
heterotypic antisera and if the potency of the antisera permits
their mutual dilution when mixed together.
Significance to the Program of the Institute;
the method described in this report reduces the number of tests
that have to be done in identifying and classifying an unknown virus.
With further refinements, it should allow a central laboratory to
supply other laboratories with pools of antisera representing all
serological groups of arbor viruses made and tested in bulk. without
the necessity of having to carry stocks of specific antisera to all
types, any field laboratory can type an unknown virus as a member of
a serological group. it will facilitate handling of epidemics due to
arbor viruses as the group reactivity of several isolates should
point to where emphasis must be placed.
Proposed Course of the Project;
These studies are to be continued along the lines described and
will be broadened to include all of the known serological groups of
arthropod-borne viruses. Methods of combining specific antisera in
order to provide broadly reactive but group specific pools of
ANTISERA WILL BE DEVELOPED. Th I S TECHNIQUE WILL BE EMPLOYED AS AN
aid in classifying and further identifying unknown viruses submitted
to this Section for study.
Part B included - No
18
Serial No. NIAID - 96-C
1 . Tropical Virology
2. Arthropod-borne Virus
3. Bethesda, Maryland
PHS-MIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Survival potential of the adult of Haemagogus equinus,
a sylvan vector of yellow fever.
Principal Investigator: Paul A. Woke
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960)
Total: 1
Professional: 1
Other: 0
Project Description:
Objectives:
To improve the opportunities for more individuals in caged
populations of adults to live out their potential life spans,
thus providing healthier and longer lived experimental insects
for use in studies on the interrelationships between viruses,
vectors, and the environment, for studies on the spread of virus
diseases among vertebrate hosts in normal situations and in
studies directed toward the control of arthropod-borne virus
d iseases.
Methods Employed:
Haemagogus equinus is used as the experimental species for which
IT IS suited by reason of its importance as a vector and its adapt-
ability to laboratory experimental conditions. Populations were set
up under conditions for survival that were the best possible at the
time. The mosquitoes were closely observed throughout their lifespans
in order to learn all preventable causes of deaths. Corrections were
applied and means devised by which to reduce and/or eliminate the
current causes of mortality. The influence of parental age, age of
eggs at the time of hatching, and conditions under which the larvae
were reared were determined by trial. survival served as a criterion
of suitability. j q
Serial NIAID - 96-C
Proposed Course of the Project:
Continue to improve conditions for the maintenance of experimental
stocks of haemagogus equ inus and to apply the findings to other species
of vectors and potential vectors of arthropod-borne diseases.
Utilize the methods and findings in studies on the interrelationships
OF viruses, vectors, and the environment.
Apply the findings in studies directed toward the development of
control measures for arthropod vectors of virus diseases.
Part B included - No
20
Serial No. NIAID - 96-C
Major Findings:
Survival of Haemagogus equinus adults is greatest in those popu-
lations THAT ARISE FROM EGGS LAID BY YOUNG FEMALES FERTILIZED BY YOUNG
males, from surviving eggs of batches that have been held for periods
of time up to near the maximum periods of survival under adverse
conditions, and from larvae that developed at lower temperatures, and
is greatest in those populations of adults that are maintained in
varied rather than constant temperatures and humidities. survival is
reduced by higher temperatures, by excessive activity induced by high
intensities of white light, and by air-borne vaporized oil of exceed-
ingly low concentration.
Under present laboratory conditions the 70# survival point for
Haemagogus equ inus adults is 50 days for males and 64 days for females;
the 90# survival point is 33 days for males and 44 days for females.
The maximum life span has been increased from 11 days for males and 39
days for females to 107 days for males and 108 days for females. known
circumstances indicate that the survival potentials and potential life
spans are still above these values.
Significance to the Program of the Institute:
Haemagogus equinus adult mosquitoes can now be chosen for labora-
tory EXPERIMENTS WITH REASONABLE CERTAINTY OF SURVIVAL ACCORDING TO A
predictable distribution when maintained within a certain generally
useful set of conditions. the life expectancy is such as to permit the
completion of experiments involving all normal events; the survivorship
distribution probably comes near to the potential for the species.
Long survival is accompanied by health and vigor. Needed laboratory
investigations can now be planned intelligently in numerous areas on
the basis of the known lifespan of the species when maintained by the
methods that have been developed. thus, insects cultured according to
the methods now available are suitable for laboratory investigations of
virus-vector-environment interrelationships and of factors important
in the transmission of the virus and spread of the viral disease, and
for investigations directed toward the development of control measures
against the vector. analytical studies of the effects on survival of
specific environmental factors are now possible. information that has
been and can be derived from laboratory experimentation can be used in
field studies on the spread of arthropod-borne virus diseases and on
means by which to control the vectors. the spread of viruses and
control of vectors in the natural habitat are influenced by the envi-
ronment, and efficiency as a vector depends in part on longevity of the
vector. Methods, experience, and information which has been gained in
this study of Haemagogus equ inus will be useful in similar work with
other vectors of arthropod-borne viral diseases.
21
Serial No. NIAID - 97-A
1 . Trop ical Virology
2. Arthropod-borne Virus
3. Bethesda, Maryland
PHS-NIH
ndividual Project Report
Calendar Year 1960
Part A
Project Title: A qualitative evaluation of experimentally induced
Eastern equine encephalomyelitis (EEE) virus
INFECTION IN HORSES
Principal Investigator: Clarence J. Gibbs, Jr.
Other Investigators: William L. Pond
Robert J. Byrne (University of Maryland)
Charles R. Rosenberger
Cooperating Units: Grayson Laboratory
University of Maryland
Man Years (calendar year 1960)
Total: 3
Professional: 1-1/2
Other: 1-1/2
Project Description:
Objectives:
An immunological investigation of experimentally induced EEE
VIRUS infection in horses designed to (1) elicit the formation,
development, and persistence of viremia and hemagglut inat ion-
inhibiting (HAI ), complement fixing (CF) and neutralizing (Neut)
antibodies; (2) to establish a reservoir of standardized reference
EEE antiserum for use in immunological and serological investiga-
tions of EEE and related arthropod-borne viruses.
Methods Employed:
Each of three horses free of serologically detectable EEE anti-
bodies was pre-bled and then injected with 10,000 MOUSE intracerebral
(IC) LDc-n doses of infectious EEE virus. Following inoculation, small
volume BLEEDINGS (50 ml.) were taken on the first 6 days and large
VOLUME (500 ML.) BLEEDINGS APPROXIMATELY EVERY 5 DAYS THEREAFTER FOR
A TOTAL OF 50 DAYS. AFTER DAY 50, BLEEDINGS WERE PERFORMED AT 90
DAY INTERVALS. BLEEDINGS ON EACH OF THE FIRST 6 DAYS WERE PROCESSED
22
Serial No. NlAID - 97-A
IMMEDIATELY FOR VIREMIA STUDIES. SUBSEQUENT BLEEDINGS WERE PROCESSED
FOR SERUM 24 HOURS AFTER STORAGE ON THE CLOT AT 4-8 C. VlREMIA
DETERMINATIONS WERE PERFORMED ON A COMPARATIVE BASIS IN ONE DAY OLD
CHICKS, HAMSTER KIDNEY CELL CULTURES, AND IC IN 3 DAY OLD AND 21 DAY
OLD MICE TO ELICIT THE BEST SYSTEM TO BE USED IN FIELD ISOLATIONS OF
EEE virus. HAI, CF, and Neut tests are being carried out on all
serum samples collected.
Major Findings:
Following the exposure of horses to EEE virus, viremia is
detectable within 24 hours and persists at a significant level
for 72 hours. The 1 day old chick is the most efficient system
for the detection of eee virus in the horse blood. significant
levels of hai antibody are present on the 10th day after inoculation
(1:160, 1:80, 1:80) and are maintained at a high level 30 days post-
inoculation (1:1280, 1:640, 1:320). Complement-fixing antibodies are
detectable 10 days after inoculation (1:4, 1:256, 1:8) and reach a
maximum level between days 30 and 35 (1:1024, 1:2048, 1:1024). High
levels of cf antibodies are detectable through day 50. neutralizing
antibodies also are detectable at a significant level 10 days after
inoculation (logs protection 1.3, 2.0, 1.7), reach a high level
between the 21 and 30 days (2.5, 3.8, 3.0) and persist through
THE 204 DAY AFTER I NOCULAT I On(3 .4, 3.6, 2.7).
Significance to Program of the Institute:
This study is providing detailed serological and immunological
data of EEE virus infection in horses. The North American enceph-
ALITIDES HAVE BEEN CAPABLE OF PRODUCING EXPLOSIVE EPIDEMICS IN
ANIMALS AND HUMAN BEINGS; E.G., MASSACHUSETTS, NEW JERSEY, AND
Panama, with high mortality in animals and men infected with EEE
virus. After being properly checked for safety and sterility, the
serum obtained during this study will provide a post-exposure
prophylactic serum for inoculation of personnel exposed to the virus.
It also provides the Public Health Service with standardized reference
antiserum as part of arbor virology investigations throughout the
WORLD.
Proposed Course of Project:
It is intended that HAI, CF, and neutral i zat ing antibody persis-
tence WILL BE FOLLOWED ON A CONTINUING BASIS IN AT LEAST ONE OF THE
THREE HORSES. In ADDITION, THE CRITICAL NEED FOR STANDARDIZED
REFERENCE POLYVALENT ANTISERUM NECESSITATES INOCULATION OF EEE IMMUNE
HORSES WITH OTHER GROUP A VIRUSES IN ATTEMPTS TO BROADEN THE SPECTRUM
OF SEROLOGICAL REACTIVITY.
Part B included - No
23
Serial No. NIAID - 97-B
1 . Trop ical Virology
2. Arthropod-borne Virus
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: The development of an inactivated vaccine against
Eastern equine encephalomyelitis (EEE) virus.
Principal Investigator: Clarence J. Gibbs, Jr.
Other Investigators: Charles R. Rosenberger
Cooperating Units: None
Man Years (calendar year 1960)
Total: 1-1/2
Professional: 1
Other: 1/2
Project Description:
Object i ves:
The purpose of this study is to develop an ant igen ically potent
and safe inactivated vaccine suitable for immunizing animals and
human beings against eee virus infection.
Methods Employed:
Hamster kidney cell cultures (HKC) are inoculated with cell
culture propagated eee virus. infected cultures are maintained
in medium 199 free of serum at 35 c. until a viral cytopathic
effect on the cells of 3+ or greater (4+ = 100$ cellular destruction)
has been observed microscopically. the supernatant material,
containing virus and cellular debris, is asept1cally harvested,
pooled, and clarified by centr i fugat i on at an r.c.f. of 1 070xg . in
an International refrigerated centrifuge. The supernatant material
is separated and aliquots removed for virus infectivity titration
in hkc and suckling mice as well as for bacteriological sterility
checks on blood agar plates and in th i ogl ycollate broth. the
remainder of the supernatant material is formalinized to a final
concentration of 0.1 percent neutral formalin by volume. the
formalinized preparation is held at 37 c. for 72 hours and at 4-6 c.
for an additional 6 days prior to testing for viable virus.
2k
Serial No. NIAID - 97-B
Safety tests consist of inoculating 400 gram guinea pigs intra-
cerebrally (ic) with 0.1 ml. of undiluted vaccine. our safety
requirements also include ic inoculation of 8-10 gram mice with
0.03 ml. of vaccine undiluted and diluted 1:10, 1:100, 1:1000 with
survival of all animals as the criterion of safety.
Antigenic potency of the vaccine is determined by injecting 600
gram guinea pigs i ntradermally with two 0.1 ml. doses at 7 day
intervals. Fourteen days after the last dose of vaccine, animals
are challenged IC WITH 100-1000 LD,-0 doses (per 0.1 ML.) OF AN EEE
STRAIN DIFFERENT FROM THAT USED TO PREPARE THE VACCINE. In ORDER TO
meet the minimum requirements of the bureau of animal industry,
Department of Agriculture, at least 2/3 of the vaccinated guinea
pigs must survive the challenge. there are no prescribed minimum
requirements for use in human beings.
Pre and post-vacc inat ion bleedings are done on all test animals
to determine serologically detectable response TO THE vaccine.
Major Findings:
So FAR, TWO LOTS OF EEE-HKC VACCINE HAVE BEEN PREPARED AND
assayed. Our data show that neutral formalin, in the concentration
employed, is capable of completely inactivating detectable viable
virus without destroying the antigenicity of the product. vaccinated
animals have been able to survive ic challenge with as many as 1000
ic guinea pig ldc0 doses of virus. serological data show the vaccine
to be capable of eliciting neutralizing antibodies (log. of neut.
indices 1.5 - 2.2), but that hemagglut i nat i on- i nh i b i t i ng antibodies
are not detectable. tests to determine complement fixing antibody
response have not been done.
Significance to the Program of the Institute:
eee virus constitutes a serious veterinary and human public
health problem in many areas of the world. the virus is also
hazardous to handle in the laboratory. no licensed eee vaccine is
available for immunizing human beings at risk in the laboratory or
in an epidemic area because a safe and potent vaccine has not been
developed.
25
Serial No. NIAID - 97-B
Proposed Course of the Project:
Additional lots of vaccine will be prepared employing EEE
INFECTED HKC CULTURES AS THE SOURCE OF ANTIGEN. INASMUCH AS
FORMALIN MAY REDUCE SOME OF THE ANTIGENICITY OF THE VACCINE,
OTHER METHODS OF INACTIVATION WILL BE STUDIED; E.G., ETHYLENE
OXIDE VAPORS AND INACTIVATION BY USE OF HIGH INTENSITY LIGHT
SOURCES (PHOTO INACT I VAT ION) OTHER THAN ULTRA-VIOLET. STUDIES
TO DETERMINE THE EFFECTS OF FILTRATION ON THE VACCINE WILL BE
CARRIED OUT. ADDITIONAL SAFETY AND STERILITY STUDIES ARE PLANNED.
Part B included
No
26
offer viral diagnostic services to the community and public health agen-
cies.
Methods Employed:
Clinical case specimens are either submitted by practicing physicians
or public health officials in Panama, the Canal Zone and the neighboring
countries, or are collected by MARU medical personnel. In the case of
epidemic outbreaks already in progress (or when leads of epidemic import-
ance are uncovered in the course of testing clinical case specimens) ar-
rangements for field activities are initiated either by appropriate of-
ficials or by Director of MARU. In the Republic of Panama such arrange-
ments are made through Director of Gorgas Memorial Laboratory and in the
Canal Zone through Health Director's office. For other Middle America
countries two-way communications between MARU and the National Governments
are always through Office of the Representative, Zone 1 1 I of Pan-American
Sanitary Bureau, Pan-American Health Organization, WHO.
Laboratory procedures with specimens from either individual clinical
cases or from epidemic outbreaks are generally the same; appropriate cell
cultures (as available) and laboratory animals are inoculated for virus
isolation, while serological testing of blood specimens for the presence
of specific antibodies is performed by standard techniques.
Major Findings:
1. CI inical Cases. A variety of viral agents has been recovered
with the predominance of enteroviruses (pol iovi ruses, Coxsackie A & B,
ECHO) from throat and rectal swabs, cerebrospinal fluid and patients* sera.
Strains of myxoviruses, adenoviruses and Herpes simplex have also been
recovered. In many cases the virus isolates were related to the clinical
illness by serological tests.
2. CA Virus Infection in Adults. Croup-Associated (parainfluenza 2)
virus infections were demonstrated in two young adults. The virus was re-
covered from throat swabs taken at the time of acute illness, utilizing
human amnion cultures and the hemadsorption test; significant rises in HI
and neutralizing antibodies during convalescence were demonstrated. The
findings indicate that CA virus can cause clinical illness in adults.
3. Epidemic Influenza, 1959. Last year's report referred to etiol-
ogical studies on an epidemic of influenza in Panama due to influenza
B-virus and a major epidemic in British Guiana during the same months of
mid-1959 due to the Asian influenza virus (A2) • A report describing
these findings has been accepted for publication in the Am. ^J. Trop. Med.
& Hyg.
k. Viral CNS Disease in a Guatemala Nn-sery. Last year's report
referred to a tragic occurrence of fatal pai jlytic poliomyelitis cases
and many aseptic meningitis cases due to po o and non-pol iovi ruses in a
Guatemala City charity nursery. The laboratory findings could not be
fully reported at the time. A total of kh < iterovirus strains were 00
Serial No. NIAID-100-A
offer viral diagnostic services to the community and public health agen-
cies.
Methods Employed:
Clinical case specimens are either submitted by practicing physicians
or public health officials in Panama, the Canal Zone and the neighboring
countries, or are collected by MARU medical personnel. In the case of
epidemic outbreaks already in progress (or when leads of epidemic import-
ance are uncovered in the course of testing clinical case specimens) ar-
rangements for field activities are initiated either by appropriate of-
ficials or by Director of MARU. In the Republic of Panama such arrange-
ments are made through Director of Gorgas Memorial Laboratory and in the
Canal Zone through Health Director's office. For other Middle America
countries two-way communications between MARU and the National Governments
are always through Office of the Representative, Zone 1 1 I of Pan-American
Sanitary Bureau, Pan-American Health Organization, WHO.
Laboratory procedures with specimens from either individual clinical
cases or from epidemic outbreaks are generally the same; appropriate cell
cultures (as available) and laboratory animals are inoculated for virus
isolation, while serological testing of blood specimens for the presence
of specific antibodies is performed by standard techniques.
Major Findings;
1. CI inical Cases. A variety of viral agents has been recovered
with the predominance of enteroviruses (pol iovi ruses, Coxsackie A £■ 8,
ECHO) from throat and rectal swabs, cerebrospinal fluid and patients' sera.
Strains of myxoviruses, adenoviruses and Herpes simplex have also been
recovered. In many cases the virus isolates were related to the clinical
illness by serological tests.
2. CA Virus Infection in Adults. Croup-Associated (parainfluenza 2)
virus infections were demonstrated in two young adults. The virus was re-
covered from throat swabs taken at the time of acute illness, utilizing
human amnion cultures and the hemadsorption test; significant rises in HI
and neutralizing antibodies during convalescence were demonstrated. The
findings indicate that CA virus can cause clinical illness in adults.
3. Epidemic Influenza, 1959. Last year's report referred to etiol-
ogical studies on an epidemic of influenza in Panama due to influenza
B-virus and a major epidemic in British Guiana during the same months of
mid-1959 due to the Asian influenza virus (A2) . A report describing
these findings has been accepted for publication in the Am. ^J. Trop. Med.
£• Hyg.
k. Viral CNS Disease in a Guatemala Ni-sery. Last year's report
referred to a tragic occurrence of fatal pai jlytic poliomyelitis cases
and many aseptic meningitis cases due to po o and non-pol iovi ruses in a
Guatemala City charity nursery. The labora' >ry findings could not be
fully reported at the time. A total of kk > iterovirus strains were 00
Serial No. NIAID-100-A
isolated from 103 individuals tested during five months of the study.
Two or more specimens were available from the majority of affected in-
fants. Twenty-two attendant nursing personnel were found to be negative
for any enterovirus isolations.
From the two initial paralytic fatal cases Type 1 poliovirus strains
were isolated from several sources (in one - from the throat and rectal
swabs, cerebrospinal fluid and brain sections, and in the other from se-
veral areas of the brain). Two nonparalytic cases occurring during the
early part of the outbreak also yielded Tl poliovirus. The only other
Tl isolate was from an infant, undoubtedly suffering from non-polio viral
meningitis, who was infected with Tl after many days in a hospital ward.
No strains of poliovirus T2 were found, but 2 strains of poliovirus T3
and 3 strains of Coxsackie A were isolated from asymptomatic infants.
The remaining 3^ viruses apparently belonged to the ECHO group: 9 ECHO
types 2, 11 and 12 and 17 which were either untypable or were not typed.
Work on the remaining 8 strains, recovered during post-epidemic spread,
was abandoned.
5. Poliovirus Type 2, Panama. A small outbreak of paralytic polio-
myelitis caused by T2 virus was investigated. Of the 15 clinical cases,
11 were under 2k months of age. Type 2 viruses were recovered from 12
cases. During the epidemic (October 1959 to March I960) a survey of en-
terovirus flora in Panamanian children was conducted as part of another
study. It indicated a wide dissemination of the virus in the community.
The emergence of poliovirus T2 during the past two years as an epidemic
virus in Central America will be closely followed by MARU staff.
6. ECH0-9 Virus Epidemic, Panama. A major epidemic of ECH0-9 virus
disease swept through Panama and the Canal Zone from July to October I960.
To date isolations have been made from 20 patients. In these clinical
cases ECHO 9 virus was isolated most commonly from cerebrospinal fluid,
then throat swabs and least often from rectal swabs.
During the epidemic (but apparently after its peak) 189 Panamanian
children at the Hospital del Nino Outpatient Clinic were surveyed with
throat swabs and rectal swabs taken from all and 98 venous blood speci-
mens drawn at the same time as finger puncture blood was absorbed on
filter paper discs. The purpose was to document occurrence of ECHO 9
virus (and possibly pol iovi ruses) and to study the practical applications
of filter paper discs as a serological tool. In contrast to clinical
cases, the virus was much more frequently recovered from rectal swabs
than from throat swabs. Relevant serological results with filter paper
disc eluates are discussed under a separate project heading.
7. Measles Epidemic, Panama. In June i960 the Ministry of Health
of Panama requested MARU assistance. A staff medical officer accompanied
two Panamanian physicians in a U.S. Air Force helicopter to Las Barretas
village in the interior of Panama where a serious epidemic of measles
had occurred: between late April and early June 35 deaths were reported
in a population of 1 500. During the 2 days' stay the sick were treated
and specimens collected. <^o
Serial No. NIAID-100-A
The area is not endemic for measles and the last epidemic occurred
in 1952. Of 33 fatal cases whose age was known, 27 were seven years old
and less. Forty-seven sera were tested for complement fixing antibodies:
The majority of individuals over seven years of age with no history of a
recent characteristic rash had the CF titer of less than 1:4. Studies
by others In areas where measles is endemic revealed that CF titer per-
sists in a fashion similar to neutralizing antibody titers. By using
the neutralization test on sera from communities where measles has been
absent for known intervals, we hope to clarify the immunological status
of populations in non-endemic areas.
8. Epidemic Poliomyelitis, Nicaragua. A severe epidemic of polio-
myelitis occurred in Nicaragua in late December 1959 through March I960.
Nicaraguan public health authorities and the Pan-American Sanitary Bureau
invited the laboratory participation of MARU. Specimens for virus isola-
tion were sent to both MARU and the Lederle laboratories. The epidemic
was of special interest since Nicaragua had suffered an epidemic of un-
precedented severity due to poliovirus Type 2 in 1958 and most of the
children had received the Lederle live poliovirus vaccine. Unfortunately,
the initial campaign concentrated on manovalent T2 vaccine with much
sparser coverage by T3 and Tl vaccine strains.
Of 66 paralytic cases from whom specimens were received, 49 virus
isolates were made - 47 of Tl and 2 of T2. The findings were reported
to the Government of Nicaragua and the Pan-American Sanitary Bureau.
Representatives of both of these agencies made a report on the joint find-
ings at the Second International Conference on Live Poliovirus Vaccines
held in Washington, D. C. in June i960.
9. Community Spread of Poliovirus Type 1, Panama. Following the
isolation of poliovirus Tl from two paralytic cases during the first week
of November I960, a survey of enterovirus flora was conducted in Panama
and the Canal Zone. From children at four Canal Zone locations on the
Pacific Coast 112 rectal swab specimens were collected and 90 rectal swabs
were procured from the out-patient clinic of Hospital del Nino.
Testing of these in monkey kidney cell cultures failed to support the
suspicion of wide dissemination and spread of Tl poliovirus strains in
the community. A most striking finding was the abundance of enteroviruses
among children of the poorer families in Panama and the sparsity of isola-
tions from the Canal Zone, regardless of the community, ethnic background
or relative income.
10. Another Outbreak of Fatal Swine Disease, Panama. An epizootic
of fatal disease at a major commercial pig farm, similarly affected two
years ago, was investigated in October jointly with other agencies. The
clinical picture was different from the EMC syndrome described by our
group for the I958 outbreak (Science 131 : 498-499, i960). Fever, lethargy,
difficulty in standing and walking, coughing and nasal discharge, labored
breathing and diarrhea were now common. Miscellaneous specimens were
collected from 17 live pigs; organs from two autopsied animals. Serum,
rectal swabs and a few urine and nasal swabs were tested for virus issjte-
4 Serial No. NIAID-100-A
lation in suckling mice, monkey kidney and hamster kidney cell culture
tubes, and chick embryo cells in bottles. No virus isolations were ac-
complished in these systems. Subsequently, it was established by patho-
logical and bacteriological studies at Laboratorio Veterinario and Gorgas
Memorial Laboratory that this was an outbreak of hog cholera (swine fever)
complicated by Pasteurel la suis infection. Unfortunately, the outbreak
continued to spread involving many animals. Our study was terminated
early.
Significance to Bio-medical Research and the Program of the Institute;
Besides providing viral diagnostic services to the medical community,
this project represents in essence epidemic intelligence indispensable to
proper functioning of this field station. With but a few exceptions,
such as Bocas del Toro study, the virus projects have been an outgrowth
of careful consideration of leads discovered in the course of our willing
cooperation with the health agencies and individual physicians in Panama
and nearby countries.
Proposed Course of the Project:
Individual subprojects will be either terminated or developed into
planned major projects as indicated. Participation in work-up of epidemic
outbreaks and of promising individual cases will continue.
31
Serial No. NIAID-100-A
Serial No.
PHS-NIH
Individual Project Report
Calendar Year i960
Part B; Honors, Awards and Publications
Publications other than abstracts from this project:
1. Craighead, J.E., Shelokov, A., Vogel J.E., Peralta, P.H.
An Outbreak of Influenza B in Panama. Accepted for publication in
Amer. J. Trop. Med. S- Hyg.
2. Craighead, J. E., Shelokov, A., Peralta, P.H., Vogel, J.E.
Croup Associated Virus Infection in Adults: Report of Two Cases.
Accepted for publication in New Eng. J. Med.
3. Chi, L., Vogel, J.E., Shelokov, A., Selective Phagocytosis of
Nucleated Erythrocytes by Cytotoxic Amebae in Cell Culture. Science,
130; 1763 (Dec.) 1959 (LISTED LAST YEAR AS ACCEPTED FOR PUBLICATION)
Honors and Awards relating to this project:
None
?2
Serial No. NIAID-100-A
Serial No. NIAID-100-B
1. Tropical Virology
2. Middle America Research Unit
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year i960
Part A:
Project Title: A Clinical and Virological Study of Oropharyngeal
Lesions in Panamanian Children
Principal Investigator: Dr. J. V. Ordonez
Other Investigators: Dr. S.de Leon (H.del N.), Dr. J. A. Brody,
Dr. E. Bruckner
Cooperating Units: Hospital del Nino, Panama City
Man Years (calendar year i960)
Total: 3/12
Professional: 2/12
Other: 1/12
Project Description
Objectives:
Clinical and virological evaluation of oropharyngeal lesions
in Panamanian children.
Methods Employed:
Swabs are collected in skim milk medium from oropharyngeal
lesions in children examined in a pediatric outpatient clinic.
Each group of specimens (collected over a half-day period) is
kept frozen until inoculation into the test systems (monkey kidney,
hamster kidney and chicken fibroblast cell cultures, as well as
suckling and weanling mice).
Patient Material:
Children with observed oropharyngeal lesions attending the
outpatient clinic of Hospital del Nino in Panama.
Major Findings:
This study is still in its initial stages and consequently no
data are as yet available for presentation.
Part B Included No ~ ,,
~~ , 33
Significance to Blo-medical Research and the Program of the Institute;
Distressing and even painful lesions of the oropharynx are not un-
common among children seen in the outpatient clinics of Panama. To our
knowledge, no virological investigation regarding the etiology of such
lesions has ever been carried out in the American tropics.
Proposed Course of the Project:
Work has been initiated as proposed and will be carried out for
at least the next six months.
34
Serial No. NIAID-100-B
Serial No. NIAID-101-A
1. Tropical Virology
2. Middle America Research Unit
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year I960
Part A:
Project Title: Virological Aspects of a Cooperative Investigation
on the Ecology of Arthropod Borne Viruses.
Principal Investigator: Dr. A. Shelokov
Other Investigators GML: Dr. P. Galindo, Dr. E. de Rodaniche and
Dr. C. M. Johnson
MARU: Dr. P.H. Peralta, Dr. C.G. Dobrovolny,
D. . Longfellow, J. Vogel, Dr. E.A. Bruckner,
Dr. J.V. Ordonez
Cooperating Units: Gorgas Memorial Laboratory, Panama City
Hospital Div., Chiriqui Land Co., Almirante, R.de P.
Arthropod Borne Virus Section, LTV (Bethesda)
Man Years: (calendar year I960)
Total: 5-9/12
Professional: 3-1/12
Other: 2-8/12
Project Description
Objectives:
1) To isolate and identify arthropod-borne viruses occurring
in the tropical rain forest area near Almirante, Bocas del Toro,
Province, R.de P.; 2) To investigate the role of the virus isolates
in human and domestic animal disease; 3) To study the role of arth-
ropod vectors and animal reservoirs in the epidemiology of infec-
tions due to the arthropod borne viruses in the study area and other
parts of the Isthmus.
Methods Employed:
In accordance with the original plan for the joint project,
Gorgas Memorial Laboratory (GML) has provided MARU with arthropods
collected by standard techniques and identified as to genus and in
most cases species. MARU has also received aliquots of all human
sera jointly collected at Almirante in October i960 and from the
small mammals bled by GML collectors earlier in the year. An ex-
35
Part B included no ^ Serial No. NIAID-101-A
perimental method for the collection of bird blood by saturation of filter
paper discs has been utilized for virus isolation attempts at MARU. All
birds caught or shot by the GML team beginning October are being thus
sampled.
Standard methods have been used for: 1) isolation attempts in suck-
ling mice (SM) and hamster kidney cell cultures (HKTC) ; 2) passages of
isolates in these and other host systems for characterization and adapt-
ation; 3) desoxycholate and ether sensitivity tests; h) preparation of
viral antigens and specific antisera and 5) serological tests - comple-
ment fixation (CFT), hemagglutination (HA), hemagglutination- inhibition
(HI), and neutralization (NT). Preparation of "immune" ascitic fluid
in laboratory animals has been initiated on a small scale.
Major Findings:
1. Isolation. The results of virus isolation from mosquito and
phlebotomus pools during the first year at MARU and GML are shown by
species in the attached table. In the new year of operations a few
additional isolations have been made at MARU, including three from
Psorophora ferox and 1 from Aedes (Ochlerotatus) spp. Isolation attempts
with bird bloods eluted from discs were begun late in the year, without
positive results so far.
2. Characterization and Identification, a) Phlebotomus Isolates:
Three of these are similar in that they infect SM i.e. only, with a
somewhat irregular incubation period (3-5 days) even after repeated pass-
age; titers of SM brain pools have been low,making them difficult to
work with. A sufficiently high titer has been obtained with one of these
to permit testing by NT against antisera to the two types of Sandfly fe-
ver virus with negative results.
The other two sandfly agents appear indistinguishable in their bio-
logical characteristics and serological cross-reactivity (both CFT and
NT). BT-78 (chosen as prototype), a DCA and ether-sensitive agent, was
not neutralized in HKTC by antisera to the two types of Sandfly Fever,
Herpes-B, EMC or the New Jersey type of Vesicular Stomatitis virus (VSV).
It was neutralized by an antiserum for the Indiana type VSV, with which
It also was reactive by CF test.
b) Mosquito Isolates: The twelve mosquito isolates fall into sever-
al groups according to their behavior in mice and MKTC. The only definite
serological cross-relationship so far demonstrated (both CFT and NT) was
between the two virus strains from pools of C.vomerifer. None of the
several isolates tested has been cross-reactive with Eastern or St. Louis
encephalitis, llheus or Mayaro (known or suspected in the area) or the
viruses isolated from Panamanian mosquitoes collected in 1958-59.
3. Antibody Survey. Preliminary testing of human sera on hand from
several areas in Panama against phlebotomus virus isolate BT-78 indicated
that 20-35% of the population in certain areas possess neutralizing anti-
bodies. Convalescent sera from 27 Panama City and Canal Zone patients
2 3QSerial No. NIAID-101-A
with inf luenza-1 ike illnesses, suspected CNS disease or FUO were negative
by NT.
Among domestic animals, 4/28 pigs (from two areas) and 19/63 horses.
and mules (from 5 localities on the isthmus) were shown to have neutral-
izing antibodies, with 65% of the equines positive in one area.
Significance to Bio-medical Research and the Program of the Institute:
The stated objective of the Virus Section of MARU has been "to eval-
uate the significance of viral agents found among the inhabitants of
Middle America as related to causation of human and animal disease. Spe-
cial emphasis is placed on arthropod borne viruses, their natural reser-
voirs and vectors as well as other agents which may be of potential dan-
ger to the population of other American countries, including the United
States". The Bocas collaborative project is a major endeavor to fulfill
this mission in a most thorough, efficient and economical way by combin-
ing and supplementing the efforts and skills of Gorgas Memorial Laboratory
and Middle America Research Unit.
Proposed Course of the Project:
This field and laboratory project is now entering the second of its
three scheduled years. Isolation attempts will be continued as the GML
field team proceeds with arthropod collection. Cul icoides midges will
be collected during the second year. Bird and small animal blood and or-
gans will be obtained in increasing numbers for virus isolation and sero-
logical testing. Sentinel suckling mouse techniques are being adapted
for field use in Panama. Characterization and identification procedures
will be accelerated with the increasing availability of immune sera to
project isolates, type specific and groupings sera (obtained with the
collaboration of ABVS-LTV). Plans include the use of additional tech-
niques (such as TC plaques, agar-gel diffusion) as they become feasible
and desirable for the execution of the project.
The second and third objectives of the project will be actively pur-
sued with arthropod borne virus isolates for which the vector- reservoir
relationships and public health significance should be sought.
37
Serial No.NIAID-101-A
BOCAS DEL TORO
PROJECT
First Year's Summary
' of Mosquito and Sandfly
Col lections"
* and
Virus
solations at GML and MARU
1 Sept. 1959 - 31 Aug. I960
/Data f
rom Dr. P. G<
al indo,
GML/
Pools Inoc.
Specimens
Inoculated
Virus
Isol.
Species
GML
MARU
GML
MARU
GML
MARU
Haemagogus spp. ,
15
12
884
787
Aedes (Ochlerotatus) spp.
69
59
9,670
8,199
1
leucocelaenus clarki
4
3
168
116
leucotaeniatus
1
22
(Finlaya) spp.
1
6
(Howard ina) spp.
3
2
85
40
Psorophora albipes
13
15
1,651
2,032
1
ferox
12
8
1,401
1,079
3
2
lutzii
1
1
101
97
1
cingulata
spp.
6
4
543
420
36
38
4,932
5,142
1
1
Mansonia venezuelensis
67
67
9,849
9,291
t i ti 1 lans
20
19
2,400
2,257
arribalzagae
4
2
248
227
indubi tans
3
54
nigricans
1
34
Culex nigri palpus-^
223
139
23,296
20,058
1
1
declarator
5
4
432
410
coronator
8
5
552
598
cornlger
4
1
234
105
vomer if er
15
15
1,782
1,749
2
taeniopus
9
7
816
627
elevator
6
6
558
555
chrysonotum
5
4
418
351
spp.
9
8
704
653
Jranotaenia spp.
3
4
95
89
i> ichoprosopon spp.
7
7
609
585
Wye omyia spp.
15
14
1,531
1,513
Sabethes chloropterus
17
13
1,344
1,311
1
Sabethes spp
5
5
283
175
Anopheles neivai
3
1
68
27
Anopheles spp.
14
13
1,408
1,366
1
1
Phlebotomus spp.
18
18
2,721
3,391
2
5
Totals:
1 _ —
622
494
68,899
63,160
12
13
* BY G0RGAS MEMORIAL LABORATORY STAFF
1.
Includes:
A.
2.
Includes:
A.
P.
3.
Includes:
C.
serratus, A. angustiv? ttatus, A. hastatus, A. tormentor,
ol igopistus, A. fulvus. _Q
ferox, £. albipes and £. lutzi 1 OO
inf 1 ictus.
L Serial No. NIAID-101-A
Serial No. NIAID-101-B
1. Tropical Virology
2. Middle America Research Unit
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year 1 960
Part A:
Project Title: The Role of Chiggers and Other Acarina of the
American Tropics In the Maintenance and Trans-
mission of Animal Infectious Agents: X»Viral Aspects.
Principal Investigator: Dr. J. M. Brennan
Other Investigators: Dr. C. E. Yunker
Cooperating Units: Rocky Mountain Laboratory, NIAID
Gorgas Memorial Laboratory, R.de P.
U.S. Army Malaria Control & Survey Branch, C.Z,
Veterinary Division, Health Bureau, C.Z.
Man Years (calendar year i960):
Total: 1-2/12
Professional: 1
Other: 2/12
Project Description:
Objectives:
To explore the role of chiggers and possibly other Acarina,
such as mites and ticks, in the maintenance and transmission of
viral agents of actual or potential importance in Panama and nearby
tropical areas.
Methods Employed:
The problem will be approached by trapping, bleeding, preparing
and identifying hosts of parasitic Acarina, collecting their mites
and processing for inoculation (by the staff of Virus Section, MARU)
into laboratory animals and tissue culture. If viral agents are
isolated, they will be identified, related to possible occurrence
of viremia or the presence of antibodies in the donor host by stand-
ard virological procedures.
Part B included No 33
Major Findings (during the calendar year):
This a is new collaborative project in a field setting which has
taken several months to initiate. The many anticipated and some
unexpected problems have been largely resolved and the project should
be in full operation by the beginning of the new calendar year. In
the meantime, approximately *f00 animals, including bats, snakes, birds
and rodents, have been examined for parasitic mites.
In the course of initial exploratory studies a discovery was made
of two new genera and species of chiggers parasitic In the nasal passages
of Panamanian bats. Although intranasal chiggers have been recovered
from rodents in Africa and Malaysia, to our knowledge no chiggers util-
izing an intranasal habitat have ever been recorded for Chlroptera.
Significance to Bio-medical Research and the Program of the Institute:
The role of chiggers and other Acarina in transmission of certain
diseases is well known, (e.g., scrub typhus, rickettsialpox, Russian
spring-summer encephalitis, etc.). However, no serious attempt has
been made so far to define their role in transmission of classical and the
newly recognized arthropod borne viruses. Hence positive or negative
information will constitute a distinct contribution to our limited
understanding of the ecology, epidemiology and epizootiology of this
important group of pathogens.
Proposed Course of the Project:
The field project will continue as proposed until leads Indicate
the desirability of faunal or geographical specialization. In the event
of bona fide isolation of viral agents, emphasis will be placed on a
particular species relative to its abundance, distribution, host rela-
tionships, disease transmission potential, including field ecological
studies and laboratory colonization attempts. To determine the poten-
tial public health significance of the viral isolates attempts will be
made to relate them serologically to human and animal infection.
As opportunities arise, it is planned to explore further the role
of Acarina in maintenance and transmission of other pathogenic agents,
including rickettsiae, protozoa, bacteria and fungi.
40
Serial No. NIAID-101-B
Serial No. NIAID-101-C
1. Tropical Virology
2. Middle America Research Unit
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year i960
Part A:
Project Title: Viral "Jungle Fever" in U.S. Military Personnel
Principal Investigator: Dr. J. A. Brody
Other Investigators: D. Longfellow, Dr. J. V. Ordonez,
Dr. E. Bruckner and Dr. A. Shelokov
Cooperating Units: Lt. Col. J. E. Goldoni, CO,
Jungle Warfare Training Center (JWTC) U.S. Army
Col. H. B. Leach, MC, Chief Surgeon, USARCARIB
Man Years (calendar year I960)
Total: 8/12
Professional: 3/12
Other: 5/12
Project Description
Objectives:
1. Isolation of viral agents from the blood of men with minor
illness following known exposure to jungle environment.
2. To determine the rate of human infection with the newly
isolated as well as viruses known to be active on the Isthmus.
Methods Employed:
Blood samples are drawn from military students participating in
a 3-week course at JWTC. Specimens are secured by corpsmen from
men reporting to the aid station with other than traumatic complaints.
Specimens are tested for virus isolation in suckling mice (SM)
and hamster kidney cell cultures. Three successive cycles of classes
at JWTC were bled before and after the 3-week course. Some 200 paired
specimens were collected.
Part B included No
m
Major Findings:
Because of reticence to be bled only 18 specimens for Isolation
have been secured from the first 900 candidates. These have yielded
one definite virus isolate ( JW- 10). The agent was isolated and re-
isolated from a soldier with fever of 101 F and malaise. The virus pro-
duces illness in SM inoculated intracerebral ly in 3 days with 100% mor-
tality in 5 days. When inoculated intraper i tonea 1 ly the illness and
death are delayed 1 to 2 days. It Is pathogenic for weanling mice i.e.
but not i.p. Virus pools have been prepared and an Immune mouse serum
is available. Adequate CF and HA antigens are being worked out. Pre-
liminary results with an HA antigen at low pH (5.2 - 5.7) are promising.
To date attempts to adapt this agent to various cell culture lines have
not been successful.
Significance to Bio-medical Research and the Program of the Institute:
The likelihood that isolates from human blood are of pathogenic
significance makes information from this study important in defining
not only the local health problems but viral infections of importance
to the military training program of the area.
Proposed Course of the Project:
Although the numbers of specimens for isolation are small, each
isolate is significant. Therefore, attempts at Isolation will continue.
Paired bloods will be tested for antibody rises against known isolates
by suitable means and will also be tested against other MARU virus
isolates as indicated.
hi
Serial No. NIAID-101-C
Serial No. NIAID-102-A
1 . Tropical Vi rology
2. Middle America Research Unit
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year 1 960
Part A:
Project Title: Enterovirus Infections of Rural Guatemalan
Children in Relation to Nutrition
Principal Investigators: Dr. A. Shelokov & Or. W. Ascol i (INCAP)
Other Investigators MARU: Dr. J. V. Ordonez, Dr. J. A. Brody,
Dr. P. H. Peralta, Dr. E. Bruckner
INCAP: Miss V. Pierce, Mr. H. Bruch,
Dr. N. Scrimshaw £■ Dr. J. Gordon
Cooperating Units: Instituto de Nutricion de Centro America
y Panama (INCAP), Guatemala
Man Years (calendar year I960)
Total: 1/12
Professional: 1/12
Other: None
Project Description:
Objectives:
1. To compare the incidence and nature of viral flora among
the children in three carefully controlled villages under constant
survei 1 lance.
2. To explore, if possible, the relationship of certain entero-
viruses to the occurrence of clinical cases of diarrhea.
Methods Employed:
Each village comprises a population of approximately 1,000
with 200 children under age 5. Socio-economic, climatic, and
topographical aspects are comparable. One village serves as con-
trol, in the other all children under age 5 receive liberal diet
supplements, while in the third the diet is unchanged but sanitary,
prophylactic, and therapeutic measures to prevent and ameliorate
cases of diarrhea are enforced.
Part B included No
43
The entire study group population of each village will be surveyed
every six weeks by rectal swabs for isolation of cytopathogenic entero-
viruses. These will be identified and grouped as far as possible. Vi-
rus isolates will be related to clinical cases of diarrhea by epidem-
iological and serological techniques.
Major Findings (during the calendar year):
This is a new collaborative project between INCAP and MARL) which
has been planned during the calendar year. The INCAP group is engaged
in a 3-year epidemiological study of the relationship between infectious
diseases and nutrition in children under 5 years of age in these three
rural Guatemalan communities. This has presented an unusual opportunity
for a concurrent investigation of the viral flora during the second year
of the INCAP study. Dr. J. V. Ordonez, staff member of INCAP' s Bacter-
iology section was assigned to MARL) in August i960 on a 1-year fellow-
ship (Parke, Davis & Company) to participate in the laboratory work and
to provide liaison.
The first group of specimens should be received before the end of
the calendar year, but no findings can be listed as yet.
Significance to Bio-medical Research and the Program of the Institute:
Diarrhea in young children ranks high among serious public health
problems and causes of death in Guatemala and all other countries of
Central America. Any and all information resulting from this study will
provide much needed background. The unusual opportunity to explore the
epidemiological aspects of enterovirus infections in relation to the nu-
tritional status of controlled population groups would in itself justify
this laboratory project.
Proposed course of Project:
It is anticipated that collection of appropriate specimens from
children in the study groups and their testing will continue for one
year. Another year will be devoted to identification of virus isolates,
serological studies (if possible) and correlation of laboratory and
epidemiological information.
Serial No. NIAID-102-A
Serial No. NIAID-102-B
1. Tropical Virology
2. Middle America Research Unit
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year I960
Part A:
Project Title: Laboratory Support of Phase I, National Program for
Poliovirus Vaccine Administration in Costa Rica, 1959*
Principal Investigator: Or. A. Shelokov
Other Investigators: Dr. J. A. Brody, D. Longfellow & J. E. Vogel
Cooperating Units: Ministry of Public Health, Costa Rica
Pan-American Sanitary Burea/PAHO/WHO
San Juan de Oios Hospital, San Jose, Costa Rica
PASB-TC Lab. at Univ. del Valle, Cali, Colombia
Division of Biological Standards, NIH
Epidemiology Branch, CDC, BSS
Man Years (calendar year I960):
Total: 2/12
Professional: 1/12
Other: 1/12
Project Description:
Objectives:
1. To support surveillance of neurological illnesses during
the administration of live poliovirus vaccine in San Jose, Costa
Rica.
2. To provide virological information on a series of well
studied cases of neurological illness in a Central American country.
Methods Employed:
All cases of neurological illness during the oral vaccination
program in the capital city were investigated by Dr. Brody on duty
in Costa Rica. From almost every patient throat and rectal swabs
and paired blood samples were obtained.
Specimens for isolation were tested at MARU in monkey kidney
cell cultures and suckling mice. Duplicate specimens were tested
by Dr. H. Doany (PASB-TC Laboratory, Cali) in HeLa cells and suck-
ling mice. Serological testing of acute and convalescent blood
Part B included Yes 1 ^^
specimens was performed at the Cali laboratory, Division of Biological
Standards in Bethesda and to a limited extent at MARU.
Major Findings:
Most of the work was accomplished during the preceding calendar year.
In summary, 51 cases or siblings were examined. Six of these had clinical
poliomyelitis and in 6 more the final diagnosis of poliomyelitis could
not be ruled out. There were no cases in which the vaccine could be
clearly related to the clinical illness.
Eleven viral agents from 10 individuals were isolated by testing
8h specimens. The isolations included one strain of poliovirus Tl , two
poliovirus T2, two poliovirus T3, two Coxsackie Qk, two ECHO ]k, one
ECHO 8 and one unidentified virus. Serological studies against homologous
virus isolates supported the isolation findings. Determinations of anti-
bodies for the 3 types of poliovirus revealed occasional vaccine failures.
Among the cases of clinical poliomyelitis only 2 were seen early
enough in the course of illness to make specimens meaningful. Poliovirus
T2 was isolated from one and no virus was isolated from the other.
Neither patient had been vaccinated. Of the 6 cases in which the diag-
nosis of poliomyelitis could not be ruled out, the following poliovirus
isolations were made: T3 - 2 cases, T2 - 1 case, Tl - 1 case. Coxsackie
Qk was recovered from two cases: from one Tl poliovirus was also recovered
and the other had a serological rise to T3 poliovirus. The latter
patient had not been vaccinated, while the others had received at least
one dose of oral vaccine.
Significance to Bio-medical Research and the Program of the Institute:
This study failed to raise serious doubts as to the safety of the
Lederle oral live poliovirus vaccine. It emphasized the difficulties
in diagnosis and interpretation of laboratory results on hospitalized
patients with neurological symptoms, especially when fed live virus vac-
cine. It also revealed that in the absence of a clear cut syndrome the
virus yield in this hospitalized population was small.
Proposed Course of Project:
The project has been terminated and the findings were reported to
the Government of Costa Rica and the Pan-American Sanitary Bureau.
46
Serial No. NIAID-102-B
Serial No. NIAID-102-B
PHS-NIH
Individual Project Report
Calendar Year I960
Part B:
Publications other than abstracts from this project:
1. Quirce, J. M., Vargas Mendez, 0., Nunez J., Montoya, J. A.,
Brody J. , Henderson, 0. A., and Martins Da Silva, M. "Vaccination
with Attenuated Polioviruses in Costa Rica" in Live Poliovirus Vaccines:
Papers Presented and Discussions Held at the First International Con-
ference on Live Poliovirus Vaccines, Washington, D. C, Pan American Sani-
tary Bureau/WHO, 1959, 713 pp.
2. Quirce, J. M. , Nunez, J., Guevara, E. C, Montoya, J. A.,
Doany, H., and Shelokov A. "Vaccination with Attenuated Polioviruses in
Costa Rica. Second Progress Report, Section II: Surveillance Program"
in Live Poliovirus Vaccines: Papers Presented and Discussions Held at
the Second International Conference on Live Poliovirus Vaccines. Wash-
ington, D. C, Pan American Health Organizat ion/PASB/WHO, i960, 63*+ pp.
Honors and Awards relating to this project:
None
47
Serial No. NIAID-102-C
1. Tropical Virology
2. Middle America Research Unit
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year I960
Part A:
Project Title: Enterovirus Flora of Panamanian Children: A Twelve-
Month Survey.
Principal Investigator: Dr. J. E. Craighead
Other Investigators: Dr. A. Shelokov
Cooperating Units: Hospital del Nino
Man Years (calendar year 1980) :
Total: 6/12
Professional: 2/12
Others VI 2
Project Description:
Objectives:
This study was undertaken with the purpose of gaining informa-
tion on the enteric virus flora of infants and young children re-
siding in a tropical environment. Surveying the population at re-
gular intervals for a year should reveal fluctuations in the inci-
dence and type of virus infections and contribute to a better un-
derstanding of community outbreaks of clinical disease due to
enteroviruses.
Methods Employed:
During the 12 months of the study, 30-35 rectal swab specimens
were obtained every two weeks by the same MARU investigator from
children under the age of three years in the out patient clinic of
Hospital del Nino. Specimens were tested for virus isolation in
MKTC. Identification of cytopathogenic agents was accomplished
by neutralization tests employing antiserum for the three types of
poliovirus, five Coxsackie B types and Coxsackie Ag. No attempt
was made to identify agents not neutralized by one of these specific
antisera.
Part B included No
48
Major Findings;
A total of 805 specimens was collected during the 12 months of the
study; of these 256 were obtained in the months of January, February,
March and April of 1 960. The results during I960 followed the trends
established in 1 959- Forty six cytopathogenic agents were recovered
during the four months of i960 (if contaminated with bacteria specimens
were excluded from the final computations). Twenty per cent of rectal
swabs satisfactorily tested in i960 and 25% of specimens tested over the
12 months of the study were positive for enterovirus isolation. In
January 19°0, 19% yielded viruses, in February 20%, in March 16% and in
April 25%.
Throughout the survey surprisingly few Coxsackie 8, Coxsackie An and
poliovirus type 1 and 3 strains were isolated. The majority of agents
recovered were not identified by the techniques used (presumably they
are miscellaneous ECHO viruses). In late 1959 and the early months of
I960, a substantial proportion of children tested yielded poliovirus
Type 2. The appearance of this virus in the survey was associated with
an outbreak of paralytic poliomyelitis due to the same virus type In
Panama City. The outbreak is reported under another heading. In this
study intestinal viral infection was not associated with a recognizable
clinical syndrome. Viruses were recovered as frequently from well child-
ren as from children with diarrhea, fever and respiratory symptoms. No
significant seasonal variations in the incidence of enterovirus infection
were demonstrated.
Significance to Bio-medical Research and the Program of the Institute;
Infantile diarrhea and severe respiratory infections have been among
the most serious health problems in Central and South America. Viruses
are often blamed but while in North America and Europe enterovirus flora
has been carefully investigated, practically no information is available
on the frequency and nature of enterovirus infection in the American
tropics. The recognized role of non-polio enteroviruses in the causation
of aseptic meningitis (and occasionally muscle paresis) underscores the
need for long-range evaluation of local enterovirus flora.
Proposed Course of Project;
Specimen collections for this 12-month survey (which developed from
an earlier 6-week exploratory study in 1958) were completed in May i960.
Interesting and significant information is emerging from analysis of the
data, providing background for subsequent studies. One such investiga-
tion on the community spread of poliovirus type 1 is already in progress.
43
Serial No.NIAID-102-C
Serial No. NIAID-103-A
1. Tropical Virology
2. Middle America Research Unit
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year I960
Part A:
Project Title: Use of Filter Paper Discs for Virus Isolation
and Serological Testing.
Principal Investigator: Dr. J. A. Brody
Other Investigators: Dr. J. V. Ordonez and D. Longfellow
Cooperating Units: Staff of Gorgas Memorial Laboratory and
Hospital del Nino
Man Years (calendar year I960)
Total: 6/12
Professional: 3/12
Other: 3/12
Project Description
Objectives:
To develop a field tool for virus isolation and for sero-
logical testing.
Methods Employed:
Discs produced by Carl Schleicher and Schnel 1 Co. (cat. No.
7^+0-E) are used in all experiments. Humans are bled by finger
puncture and small animals, particularly suckling mice (SM), by
opening the chest and absorbing the blood onto the discs.
For virus isolation, Eastern equine encephalitis (EEE) virus
has served as a model. Virus is inoculated into SM which are bled
in 36 hours. Whole blood and discs are collected and treated under
varying conditions. At definite intervals the dried discs are re-
suspended in diluent and titered. Virus determinations are con-
ducted in hamster kidney cell cultures and occasionally in SM. In
addition St. Louis encephalitis (SLE), llheus and yellow fever
viruses have been studied to a limited extent.
Serological experiments have employed standard neutralization
procedures in monkey kidney cell cultures using ECHO-9 and polio-
viruses. Discs are resuspended overnight before use and compared
Part B included No 1 50
with sera obtained by venipuncture from same subjects.
Major Findings:
EEE virus, titering 10 in whole SM blood, was absorbed on discs and
left in the refrigerator and at ambient temperatures. After 12 days in the
refrigerator the titer on the disc was 10 and after 32 days 10^*5. The
environmental temperature disc titered 10^-5 at k days and the virus was
recoverable (in SM) at 8 days. Crude attempts to dehumidify the discs did
not seem to enhance preservation of the virus. Various methods of resus-
pension have not improved on the basic method of O.occ of diluent per disc
left overnight at k C. Freezing wet and dry discs did not alter titers
appreciably. SLE virus is fairly stable on dried discs. V/hen viremia ti-
tered 10°*-' in SM, the discs left at 26°C contained 10 '"' logs of virus
at 21 days and the discs left k days at ambient temperatures contained
10',-J logs of virus. Ilheus and yellow fever viruses are more fragile on
discs. Both were recoverable from discs after k days at k°, but not from
discs at environmental temperatures.
Because of the practical advantages of the disc method for isolation
of viruses from small wild mammals and birds, Gorgas Memorial Laboratory
is cooperating in a field trial. Birds shot at Bocas del Toro are being
bled by cutting the jugular vein to saturate the discs then sent to MARU
under refrigeration. Three discs are combined, resuspended and inoculated
into SM. To date no virus isolations have been made. (GML reports that
no viruses have been isolated by standard methods from sera of the same
birds).
Serological studies have been performed using finger puncture discs
and venous blood sera from the same individuals. We have established that
blood resuspended from discs can not be titered out and can not be stored
at room temperature for more than ^-h weeks.
Significance to Bio-medical Research and the Program of the Institute:
The original work by Karstad and Hanson at University of Wisconsin
showed that EEE could be recovered from discs. We are amplifying this find-
ing by exploring the stability of viruses dried on discs. The potential
value of discs as a field tool is considerable: increasing emphasis is
being placed on cycles involving nestling birds and small mammals in the
ecology of arthropod borne viruses; study of these cycles is limited be-
cause their bloods are difficult to collect; the discs may offer a partial
solution to the problem of virus isolations in the field.
The possibilities of a reliable serological screening tool which does
not require a syringe and vein are enormous. In tropical areas, where con-
version rates are high in the young children (who are difficult to bleed)
this procedure should be especially helpful.
Proposed Course of Project:
1) To continue field trials for virus isolations; 2) To explore fur-
51
2 *«*■ Serial No. NIAID-103-A
ther the use of discs as a reliable serological method for human surveys;
3) To experiment with CF and HAI antibodies on disc-eluted bloods; k) To
attempt serological procedures for arthropod borne viruses on disc-absorbed
blood specimens from wild life.
52
Serial No.NIA|D-103-A
Serial No. NIAID-103-B
1. Tropical Virology
2. Middle America Research Unit
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year i960
Part A:
Project Title: Eastern Equine Encephalomyelitis (EEE)
Virus Infection in Panama
Principal Investigator: Dr. J. E. Craighead
Other Investigators: Dr. P. H. Peralta, D. Longfellow
J. E. Vogel and Dr. A. Shelokov
Cooperating Units: Gorgas Memorial Laboratory (GML)
Laboratorio Veterinario, Panama
U.S. Army Mission to Panama
Vet. Div., WRAIR, Washington, D. C.
Man Years (calendar year I960)
Total: 9/12
Professional: 5/12
Other: VI 2
Project Description:
Objectives:
The I960 objectives were an outgrowth of our investigation of a
1958 EEE outbreak in horses: 1) To determine the prevalence of EEE
antibody in horses and man in two areas of probable EEE virus en-
demicity; 2) To evaluate the possible role of lizards as reservoirs
of EEE virus in Panama; 3) To evaluate the relative usefulness of
serological methods applicable to studies of this type.
Methods Employed:
Neutralization (NT) in mice, complement fixation (CFT) and hemag-
glut ination-inhibi tion (Hi) were used as classical techniques or
with recently described but widely accepted modifications. EEE virus
isolate (1958) from a Panamanian horse brain was used both as in-
fectious agent and antigen.
Major Findings:
1. Human Population: Four hundred sixty serum specimens col-
lected by GML and MARU in Darien and Panama provinces of the Republic
53
Part B included No 1
were surveyed for EEE antibodies by HI test. Reactive sera were tested
in mouse NT. Thirty-two sera (7-6%) were reactive by HI test at > 1:20;
12(3%) neutralized ^ 1.7 logs of virus in the adult mouse test and k
additional sera neutralized ;>, 1.7 logs of virus when tested l.P. in suck-
ling mice. The incidence of EEE antibodies in humans increased with ad-
vancing age (0.8% under 10 years with progressive increase to 9% in the
41-50 year group). The mean percentage of positive sera was 3-7%. Anti-
bodies were equally common in both sexes. Complement fixation tesir
were carried out on all k60 sera, but the results were difficult to inter-
pret, suggesting heterologous antibodies to other group A viruses.
2. The Virus Reservoir: Many lizards of several species were ob-
served on the ranches where the outbreak had occurred. Other investigat-
ors (RML) have indicated the possible role of reptiles in the ecology of
ARBOR viruses. Because of these considerations, 2^6 wild lizards caught
in the suspecL area were examined by the HI test. Specific EEE virus
hemagglutination inhibitors were found in a small proportion of these
sera.
Several experiments were carried out with lizards of one of the
three species common in Panama (Ameria festiva) confirming the occurrence
of viremia and HI antibody response following virus inoculation.
3. Evaluation of Methods: The horse serum HI results were compared
with CFT and NT results on same sera. Kaolin and acetone procedures for
removal of non-specific serum hemagglutination inhibitors were also com-
pared. It was concluded that the HI test is useful for rapid evaluation
of sera for antibodies, since HI negative sera were consistently nega-
tive by NT and CFT. Further, large numbers of specimens can be quickly
tested by surveying sera at a single dilution. However, a titer can be
considered only suggestive of specific EEE antibodies and the NT is
needed for confirmation, particularly in areas of likely activity of
other group A viruses (6 of the 13 sera tested were shown to have VEE
neutralizing antibodies by a cooperating laboratory). Our studies sug-
gest that kaolin treatment of sera is not as efficient as acetone ex-
traction for routine removal of inhibitors, but acetone may also remove
much heterologous antibody, which can be disadvantageous in some survey
situations.
Significance to Bio-medical Research and the Program of the Institute:
EEE virus continues to cause not only epizootics, but occasional
epidemics associated with human fatalities and permanent sequelae.
Studies on the ecology and epidemiology of the disease utilizing improved
sero-diagnost ic procedures should contribute to control of this public
health problem common to both temperate and tropical areas.
Proposed Course of Project:
Experimental work on this project was concluded in June I960 and the
results are being prepared for publication.
54
2 Serial No. NIAI0-103-B
Serial No.NIAID-103-C
1. Tropical Virology
2. Middle America Research Unit
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year I960
Part A:
Project Title: Encephalomyocardl tls (EMC) Virus Infection. Studies on
pathogenesis In swine, virus reservoirs In rodents and
antibody status of human and animal populations.
Principal Investigator: Dr. J. E. Craighead
Other Investigators: Ma j . T. G. Murnane, VC, USA, Dr. P. H. Peralta
Dr. A. Shelokov
Cooperating Units: Laboratorio Veterinario, R.de P.
U. S. Army Mission to Panama
Man Years (calendar year 1 960)
Total: 8/12
Professional: 4/12
Other: VI 2
Project Description:
Objectives:
1) To conclude a basic investigation on the pathogenesis of
EMC infection in swine; 2) To examine small wild rodents for EMC
antibodies as possible reservoir hosts; 3) To determine the pre-
valence of EMC infection in the Panamanian population by testing
randomly collected human sera for antibodies.
Methods Employed:
Laboratory methods developed during the preceeding calendar
year were employed in these studies. Relevant details are mentioned
under Major Findings.
.Major Findings:
1. Biology and Pathogenesis of EMC Infections. Following
the feeding of virus, 3 pigs without pre-existing antibodies devel-
oped viremia for 2-k days and excreted virus in the feces for as
long as 10 days. During the period of active infection the pigs
exhibited no evidence of illness. All 3 developed substantial in-
crease in antibody by hemagglutination inhibition (Hi) and neutral-
Part B included Yes
55
ization (NT) tests. The 2 autopsied pigs had histological evidence
of myocardial fibrosis compatible with earlier myocarditis. A fourth
pig, wi th pre-existing antibody, developed viremia transiently, excreted
virus for only two days and exhibited an antibody rise for only a brief
period. A fifth pig was fed a large amount of virus after only one mouse
passage. This pig died (in arrythmia?) 5 days later with histologic evi-
dence of acute myocarditis. The virus titer of the heart tissue was
greater than 10°» other organs yielded lesser amounts.
2. Virus Reservoirs: In an attempt to find possible virus reservoirs
In nature, wild rodents were trapped, bled and tested for EMC antibodies.
Two of 53 rats (Rattus rattus) caught in or in the vicinity of Panama
City were found to have antibodies, but all sera from 42 other small ro-
dents of several species were negative.
3. Human Serological Studies: Eighteen percent of 158 sera from 4
localities in Panama were found to contain significant levels of EMC an-
tibodies by NT in HeLa cell cultures (TC). There were interesting differ-
ences in the results with age. Thus, in the group under 10 years 23%
were positive, 10-19 years - 23%, 20-29 years - 17%, 30-49 years - 17%,
while only 7% of persons over the age of 50 possessed antibodies. These
studies were carried out employing diluted serum and varying dosages of
virus. Sera considered positive neutralized 2.5 logs or more of virus.
Many of the positive sera were evaluated in the diluted state against ap-
proximately 100 TCDcjq doses of virus. More than half of the sera tested
in this way had demonstrable antibodies at a dilution of 1:4 or greater.
A large number of NT negative and positive sera were tested by HI. Twenty-
nine of 40 NT positive sera were also positive at a dilution of 1:4 or
greater in the HI test, whereas only 4 of 36 NT negative were positive.
Many of the HI positive sera had demonstrable antibodies at high dilu-
tions. Limited studies were carried out in an attempt to compare the
results of HeLa TC and mouse NT tests. While many sera were positive by
both tests, TC positive sera frequently were negative in mouse NT. These
data suggest that a substantial proportion of the Panamanian population
has been infected with EMC virus.
The explanation for the relatively high incidence of antibodies in
the younger persons is obscure. Possible explanations are: a) the virus
has been recently introduced, b) infection occurs early in life and an-
tibodies disappear with time, c) the virus infection ultimately contri-
butes to mortality at a young age, d) the "antibodies" are actually non-
specific neutralizing substances (this is highly unlikely).
Significance to Bio-medical Research and the Program of the Institute:
This project was based on our demonstration of natural infection of
swine with EMC virus causing the most extensive outbreak of EMC infection
in man or animals in which the virus was recovered. Our subsequent stud-
ies suggest that rats may constitute a natural source of infection, while
pigs contract the disease by ingestion of rats and/or droppings. Improved
laboratory diagnostic procedures developed during this study allowed ready
demonstration of specific antibodies in many Panamanians, especially In
56
Serial No.NIAID-103-C
the younger age groups. These findings may contribute to a definition
of not only another veterinary problem, but of possibly an important
zoonosis, especially In the Caribbean area and Southern United States.
Proposed Course of the Project:
The project has been terminated and the findings are being reported
in medical and veterinary research journals.
57
Serial No. NIAID-103-C
Serial No. NIAID-103-C
PHS-NIH
Individual Project Report
Calendar Year I960
Part B: Honors, Awards and Publications
Publications other than abstracts from this project:
Murnane, T. G., Craighead, J. E., Mondragon, H., Shelokov, A,
Fatal Disease of Swine Due to Encephalomyocardi tls Virus,
Science, 131:498-*f99 (Feb.) I960 /reported as "accepted for
publication" last year/.
Honors and Awards relating to this project:
None
58
Serial No. NIAID-108
1. Tropical Virology
2. Mycology Section, MARU
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year i960
Part A;
Project Title: Studies of Histoplasmosis on the Isthmus of Panama
Principal Investigator: Capt. R. Taylor, MSC
(R&D Command, U.S. Army)
Other Investigators: Dr. F. Abildgaard, Coco Solo Hospital
Dr. C. G. Dobrovolny, MARU
Cooperating Units: Walter Reed Army Institute of Research
Gorgas Memorial Laboratory, R.de P.
Hospital del Nino, R.de P.
Gorgas and Coco Solo Hospitals, C. Z.
Ministry of Health, R.de P.
Canal Zone Health Bureau
Army, Navy and Air Force Surgeons
U.S. Army Malaria Control and Survey Branch
U.S. Army Preventive Medicine Officer
Veterinary Health Offices, C. Z.
Man Years (calendar year 1 960)
Total: 3-1/2
Professional: 3/k
Other: 2- 3 A
Project Description:
Objectives:
1. To determine the extent of histoplasmosis on the Isthmus of
Panama and the significance of this disease In the military person-
nel stationed In an area of histoplasmosis endemlclty.
2. To explore the epidemiology and ecology of HIstoplasma
capsulatum on the Isthmus of Panama and Central America.
3. To provide diagnostic aids and adequate clinical follow-up
on all recognized cases of histoplasmosis In this area.
k. To evaluate experimental therapy of histoplasmosis (In co-
operation with Gorgas Hospital).
Part B Included Yes
5S
Methods Employed;
In cooperation with military and civilian health authorities obtain
data on hypersensitivity to histoplasmin in individuals arriving for duty
on the Isthmus, as well as long time residents of the Canal Zone and the
Republic of Panama. Provide diagnostic assistance to Isthmian medical
facilities in locating and following all clinical cases of histoplasmosis.
Collect soil samples from areasof suspected endemicity as suggested
by patient interviews to demonstrate the presence of H. capsulatum and
the foci of infection. Determine the extent of naturally occurring
histoplasmosis in the wild animal population as possible reservoirs of
H. capsulatum and define the local areas of endemicity.
Major Findings:
In the first eleven months of 1 960, forty-three cases of acute his-
toplasmosis in the Canal Zone were serologically confir ^d. In addition,
one case of disseminated histoplasmosis in an 11 month old infant was
culturally proven and the child successfully treated at Coco Solo Hos-
pital. The first known case of cavitary histoplasmosis in this area oc-
curred in a North American employee in the Canal Zone, who was evacuated
to the Clinical Center NIH for therapy.
Histoplasmin skin testing was completed in the Canal Zone School
system yielding data on 9»200 children between 6 and 19 years of age.
This survey provides data on the entire school-age population permitting
comparison of hypersensitivity rates according to location and length
of residence, race, age and sex. As expected, the percentage of react-
ors increases progressively from the younger to older children. A si-
milar increase occurs in the same age group, as length of residence in-
creases. The rate of histoplasmin sensitivity in life-residents varies
from 13 to 58% among 6 year olds and 68 to 92% among 19 year olds, de-
pending on location of residence. The Central area of the Isthmus
showed the highest sensitivity rates followed by the Pacific and Atlantic
areas.
A completed survey of 631 pre-school children on the Atlantic side
of the Isthmus provides data on children from 6 months to six years of
age. The results of this study indicate a steady increase in the rate
of hypersensitivity to histoplasmin from approximately 3 years to 6
years of age when the results dovetail with the results obtained in
the school system on the Atlantic side. A similar study is currently
under way at Hospital del Nino to further augment the data on the younger
children. This survey has been in progress for 11 months, and results
on approximately 800 children are available.
Ecological and epidemiological studies resulted in the isolation
of _H. capsulatum from 8 soil samples: one was from the residence of a
clinical case of histoplasmosis; one from a U.S. Army training area;
another from the sparsely populated remote province of Darien, and four
60
2 Serial No. NIAID-108
from below bat roosts in a cave frequented by Canal Zone residents. The
remaining isolation was from a repeatedly positive tree buttress and con-
firms the ability of H. capsulatum to propagate in this site through
both rainy and dry seasons. This brings the total number of isolations
from soil up to sixteen, representing six widely separated locations on
the Isthmus of Panama.
The trapping of wild animals continued this year and H. capsulatum
v/as recovered from the livers and spleens of 5 spiney rats and 9 oppos-
sums trapped on the Atlantic side. The area of trapping is extensively
utilized by the U.S. Army as a training site and is the same area where
H. capsulatum was isolated from soil.
Significance to Bio-medical Research and the Program of the Institute:
As would be expected the research program has resulted In a greater
local awareness of histoplasmosis in all of its clinica; forms, as evi-
denced by recognition of three disseminated cases (two fatal and one
successfuly treated) within a period of eighteen months. Previously,
only one fatal case had been described since Darling's original cases
in 1906.
It is hoped that the current studies on ecology and epidemiology
will elucidate the clinical cases of this disease, frequently misdiag-
nosed as tuberculosis, carcinoma and FUO, among military and civilian
personnel and their dependents in this strategically important area.
The study offers an opportunity to study the organism in a tropical
climate where the ecology may be considerably different from the United
States.
Proposed Course of the Project;
Clinical, ecological and epidemiological investigations will be
continued utilizing methods and techniques now employed, as well as
those being developed in this laboratory and elsewhere, until either the
major objectives are achieved or the studies become obviously unproductive.
61
Serial No. NIAID-108
Serial No. NIAIO-108
1. Tropical Virology
2. Mycology Section, MARU
3. Panama, Canal Zone
PHS-NIH
Individual Project Report
Calendar Year I960
Part B: Honors, Awards, and Publications
Publications other than abstracts from this project:
Abildgaard, C. F. and Taylor R. L.
Generalized Histoplasmosis in a Panamanian Infant: Case Report
Am. Jour, of Trop. Med. & Hyg. %: 400-01, (July) i960
Taylor, R. L. and Oobrovolny, C. G.
The Distribution of Histoplasmin Sensitivity in Guatemala
Am. Jour, of Trop. Med. & Hyg. 2.: 518-22 (Sept.) i960
Honors and Awards relating to this project:
None
62
Serial No. NIAID-109
1. Tropical Virology
2. Mycology Section, MARU
3. Panama Canal Zone
PHS-NIH
Individual Project Report
Calendar Year i960
Part A:
Project Title: Studies of Superficial and Deep Mycoses In Panama
and Central America
Principal Investigator: Capt. L. Taylor, MSC, R&D Command, U.S. Army
Other Investigators: Capt. A. W. McFadden, MC, Gorgas Hospital
Cooperating Units:
Gorgas Hospital, C.Z.
U. S. Military Dispensaries, C.Z.
Veterinary Division, Health Bureau, C.Z.
Raymond Clinic, R.de P.
Santo Tomas Hospital, R.de P.
Man Years (calendar year I960)
Total: 1/2
Professional: \/k
Other: \/k
Project Description:
Objectives:
To establish: 1) the nature of common superficial mycoses In
the tropics and the efficacy of newer therapeutic regimens; 2) the
extent and types of deep mycoses and 3) to correlate the abundant
airborne mold spores to respiratory allergy.
Methods Employed:
Standard cultural diagnostic assistance has been made avail-
able to local physicians and medical facilities where patients
with mycoses are commonly seen. A survey of the airborne molds of
Panama was conducted by regularly exposing Petri dishes with medium
at six specified locations for one year. Hairbait cultures were
utilized for recovery of keratophilic fungi from soil samples which
are being processed for Histoplasma capsulatum.
Part B Included:
No
63
Major Findings:
1. Airborne fungi in Panama. A one year survey of low altitude
mold spores was completed this year. Plates were exposed twice weekly
at six locations on the Pacific side of the Isthmus. Over 3,000 colo-.
nies were identified: Hormodendrum predominated, followed by
Asper igi 1 lus and Penici 1 1 ium species. The total counts showed a seasonal
variation with a peak in July and August and a low from November to
February. Meteorological data were obtained and a correlation of total
counts with relative humidity, temperature, rainfall and wind velocity
wtl 1 be attempted.
2. Griseofulvln Therapy. Therapy of chronic derma tophytos is with
griseofulvin is being conducted by Dr. A. W. McFadden, dermatologist,
Gorgas Hospital, with MARU furnishing cultural diagnosis and follow-up.
The patients selected are primarily those with recalcitrant nail Infec-
tions. The causative agent Is determined prior to therapy and the pa-
tient is followed by a series of clinical laboratory tests and additional
cultures to determine toxicity and efficacy of the drug. Excel lent "cl I-
nical results have been obtained to date; however, no new findings on
the use of the drug are anticipated.
3. Therapy of Moniliasis. A "blind" comparative clinical evalua-
tion of Amphotericin B and Mycostatin for monilial infections is being
conducted by Dr. A. W. McFadden and the physicians at the military dis-
pensaries. All cultural material is submitted to MARU for identifica-
tion of the causative agent. Preliminary results indicate one of the
two drugs to be more effective (the code has not been broken). This
study has brought out the major importance of moniliasis in a population
living in the tropics.
k. Hair-bait Culture of Soils. Microsporum gypseum has been isol-
ated repeatedly from soil using the hair-bait culture technique. No
clinical correlation has been attempted.
5. Epidemiology of Animal and Human Dermatophytosis. The occur-
rence of dermatophytosis in animals and their role in human infection
is being studied in collaboration with the Canal Zone Veterinary Clinic.
Several isolations have been made from clinical infections in animals with
only one highly suspicious but unproved case of transmission to humans.
6. Deep Mycoses. The utilization of MARU's cultural diagnostic
facilities by Dr. C. Calero (Raymond Clinic in Panama) resulted in con-
firmation of one case of cervico-facial actinomycosis. This success
may arouse further interest in diagnosis of other mycoses previously
unrecognized in this area.
Significance to Bio-medical Research and the Program of the Institute:
As a by-product of the long-range major histoplasmosis project
there are opportunities to explore other mycological problems. While
much of this information can be classified as "geographical pathology",
some of it may be obviously important to public health authorities of
2 Q l| Serial No. NIAID-109
the area while Interesting leads of general concern may be uncovered.
Proposed Course of the Project;
The course of these studies is determined by the time and personnel
available as well as the relative importance of the findings to National
public health authorities of Panama and the U.S. military personnel and
dependents in the area.
65
Serial No. NIAID-109
LABORATORY OF GERMFREE ANIMAL RESEARCH
S ummary .
1
110 - Pathogenesis of Amoebiasis 5
111 - The Use of Germfree Animal and Tissue for
the Study of Viruses 8
112 - Biology of Germfree Animals 10
113 - Origin of Anti-Human Blood Group B
Agglutinins in White Leghorn Chicks 14
114 - Studies on Bacterial Interactions and
Host Bacteria Relations in the Germfree
Animal 16
115 - Behavior of Parasitic Protozoa in
Germfree Hosts 18
116 - Studies on Helminthic Infections in
Germfree Hosts 20
Laboratory of Germfree Animal Research
National Institute of Allergy and Infectious Diseases
Summary statement of research progress, calendar year 1960
Program developments and trends:
During the year, Laboratory plans to broaden the scope of its
research activities showed considerable and rapid progress. Authorization
to occupy completely the south end of the third floor of Building 8,
following the departure of DBS from this space, was finally clarified. The
contract for the remodeling of this area was awarded and renovations got
underway early in the Fall. It is anticipated that this space will be
ready for occupancy (according to the schedule) around March or April of
1961. Following the transfer of LGAR personnel to the south end, alterations
in the north end, converting the entire area into space for germfree animal
units and maintenance, will commence. This will be effecte-l slowly by NIH
personnel, with the view of disrupting normal activities as little as
possible. When this is accomplished, additional germfree animal units will
be obtained and put into operation.
As a part of the broadening in research scope, it was deemed advisable
to strengthen the program by the addition of pathology and virology activities,
The staffing for this has proceeded well. Of the 5 new positions authorized
by the Scientific Director up through the fiscal year ending July 1961,
personnel for 4 are already committed. The new personnel will enter on duty
in the Spring when the space is available. For the pathology group,
Dr. Edwin Lerner, Senior Surgeon, will transfer from NIAMD to our Laboratory.
Dr. Fred Gorstein, S. A. Surgeon, who has had training in pathology will
enter on duty about July 1. Technical assistance for these people has been
arranged for. The only position outstanding for which a candidate is not
yet lined up is the virologist. Efforts are currently being made to locate
one at the level of a GS-11 or 12.
The alteration of the air conditioning system for other institutes
on the first, and especially the second, floor during the last five or six
months of the year has caused some inconvenience especially with noise,
vibration and occasional shutting down of a utility. However, the research
program continued at a satisfactory pace in spite of some of these drawbacks.
As was true of last year, the program reflected an increased variety of
interests and an increase in the number of cooperative projects planned or
underway with other laboratories, and even other institutes. As examples,
we have recently undertaken on a limited scale with Dr. Rowe, LID, serologic
and tissue studies for the possible presence of viruses in our germfree
animal colony. Such data will establish baselines for future studies with
virus-free animals, or, if it turns out that way, animals containing a few
known viruses. We have, in collaboration with Dr. Landy of the NCI,
initiated studies on the origin and specificity of natural antibodies to
enteric bacteria, and similar studies in connection with Staphylococcus with
the CDC. Experiments in germfree animals would appear to provide the crucial
- 1
information in these areas. Also, such studies can provide information on
the nature of the mechanism whereby endotoxin alters resistance to infection
with gram negative pathogens. With Doctors Kelly and O'Gara, also of the
NCI, we have initiated on a small, but satisfactory, scale a study to
ascertain whether the incidence of chemically- induced lung tumors is the
same in germfree mice as in conventional mice of the same strain. In view
of the large numbers of viruses demonstrated as occurring in experimental
mice, (Huebner, Rowe) , the question arises whether the chemicals induce the
tumors by activating viruses, or some other living agents that are present
in the animal. Such studies in an organism- free host could provide rather
significant leads in the approach to understanding tumor-virus relations.
The apparent increase in the interest in germfree animals in medical
research stimulated a request by Dr. Peterson to have a film made on this
subject for use by medical schools, universities, research institutions, etc.
The Audio-Visual Aids section of the CDC came to the Laboratory and we made
a 19-minute color movie. The popularity of the subject matter is pointed
up by the fact that, recently, all 28 copies of the film which CDC had in
its library were booked up on loan for a month in advance.
Significant scientific advances:
With regard to the progress in some of the research projects, an
interesting series of observations has been made by Mr. Phillips on the
behavior of Entamoeba histolytica in the germfree host. It is to be recalled
that, in earlier studies with standardized techniques, amoebic lesions were
not produced in the germfree animal following inoculation. In fact, the
parasite failed to live in the intestine beyond 5 days. Recent changes have
been made in the manner of rearing and handling the amoebae In vitro prior
to inoculation which seemed to result in more vigorous organisms. The latter
have produced lesions in the absence of bacteria, although the type and
severity are still not typical of those encountered with a bacterial associate-
Thus, it would appear that the latter is not the only determinant of the
course and the pathogenesis of the infection.
In collaborative studies with Dr. Weinstein of LPD, we have shown
that the intestinal mouse parasite, Nematospiroides dubius, does not require
a flora to develop from an infective larva to the adult form in the host.
However, it apparently does require bacteria, or their products, to develop
from the egg to infective larva. These studies are preparatory to those to
be undertaken in an analysis of the nature of the nutritional effects
observed in certain parasitisms. One of the most interesting observations
has been our finding that the sex of the host, which has been noted by
several workers to affect the outcome of the infection in conventional
(contaminated) animals, has not appeared to be an influence in the germfree
host. If these findings continue to hold up, a hitherto unrecognized role
(either direct or indirect) of the flora in certain observed sex effects
may unfold.
In studies on the growth and biology of germfree guinea pigs,
Dr. Horton has obtained several advanced pregnancies in animals maintained
on irradiated diets, although no fetus was carried to term. It is to be
recalled that germfree guinea pigs have not been bred with any success.
The importance of the intestinal flora to this species was pointed up by
the finding that conventional (contaminated) guinea pigs reproduced normally
on this same irradiated diet.
In a collaborative project with Dr. Springer of the University of
Pennsylvania, Dr. Horton has also shown that the use of large dosages of a
cathartic, or the application of tourniquet shock, increased the number
of red cells of germfree chickens coated with human B-like antigens following
mono-infection with E. coli 086- These studies are providing information
on the manner in which red cells of one type may acquire antigenic character-
istics of other cell types, especially B.
Our germfree mouse colony has been undergoing an intensive serologic
study from several points of view. One of the most interesting has been
an assay for the presence of certain so-called "natural antibodies" against
a variety of bacteria. Such antibodies or antibody- like re? -t:ivities for
organisms like Staphylococcus, E. coli and S_. typhosa have been found to
occur in a variety of uninoculated conventional animals and are presumed
to originate from encounters with the viable organisms or related antigens.
Animals which have lived for many generations free from contact with live
bacteria are almost the sine qua non for establishing finally the validity
of these ideas. Studies thus far, with the CDC and with Dr. Landy of NCI,
have shown the germfree animal to be singularly free from antibody-like
reactivity toward Staphylococcus and E. coli, but reactivity toward S_. typhosa
was obtained in several instances. We are, of course, unable to find
evidence of the presence of the latter in the germfree colony. Thus, this
finding strengthens sporadic reports that non-bacterial substances (perhaps
in this case dietary components) can cause "cross" reactions with this
organism.
We have now had our germfree animal colony and a conventional colony
derived from the same stock for approximately two years . Some of our ex-
breeders which we try to keep, in spite of the scarcity of germfree unit
animal space, are of the order of 1-2 years of age. We have made it a
practice that whenever a germfree or conventional animal not on an experiment
dies, especially if it is 6 months or more of age, it is checked thoroughly
for gross evidence of malformations or tumors. As of now, we have informa-
tion on a total of more than 50 animals . It is of interest that we have
found so-called spontaneous lung tumors in the germfree as well as the
conventional mice. Also, while the numbers of animals are obviously
relatively small, the incidence has been markedly higher, thus far, among
the conventional animals, i.e., those exposed to external contamination,
than among the germfree. This has been particularly true among animals
6-12 months of age. These observations have been discussed with Doctors
Kelly and O'Gara of the NCI who find them of considerable interest. Such
data will continue to be collected and will serve as corollary information
along with experiments in which such tumors are induced chemically in both
germfree and contaminated animals. If this difference in incidence continues,
it could point up further the importance of microbial agents per se, or
3 -
stresses -oduced by such agents, in the induction of these tumors. While
it is, v- ourse, too soon to say much in this regard, the possibilities
suggests jy even this limited information are very intriguing.
Serial No. NIAID-110
1. Germfree Animal Research
2. Pathogenesis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Pathogenesis of amoebiasis
Principal Investigator: Bruce P. Phillips
Other Investigators: None
Cooperating Units: NIAID-127-B
Man Years (calendar year 1960):
Total: 1 3/4
Professional: 3/4
Other : 1
Project Description:
Objectives:
To study the etiology, pathogenesis, and pathology of
intestinal amoebiasis with a view toward ascertaining the role of
the associated intestinal flora.
Methods Employed:
Guinea pigs are inoculated intracecally with Entamoeba
histolytica trophozoites cultivated in vitro without bacteria.
Conventional, germfree and ex-germfree animals with various known
single or multiple bacterial infestations are used, and the disease
processes produced, if any, are compared grossly and histologically.
Modifications of techniques for cultivating and harvesting the
amoebae are also employed in an effort to obtain maximum virulence.
Major Findings:
Amoebic lesions did not occur in germfree animals which
received amoebic inocula cultivated and prepared by previously
standardized procedures, even though such inocula regularly pro-
duced amoebic ulceration in conventional hosts. Amoebic ulceration
varying in extent and severity did occur following introduction of
similar amoebic inocula into ex-germfree animals harboring each of
the following bacteria as a monocontaminant: Escherichia coli,
Aerobacter aerogenes, Streptococcus faecalis, Bacillus subtilis,
- 1 - *
Serial No. NIAID-110
Lactobacillus acidophilus, Staphylococcus aureus, Micrococcus sp.
(from conventional guinea pig) . More recently, amoebic lesions
have been produced in germfree animals following intracecal
inoculation of E. histolytica cultivated and harvested by newer
procedures developed as a part of these investigations . However,
these lesions were not typical of those observed in animals
harboring bacteria.
Significance to Bio-Medical Research and the Program of the Institute:
The wide range of host-parasite relationships which characterize
enteric amoebic infection have long been a matter of considerable
scientific interest. The demonstration of bacterial influence on
development of the disease, although as yet incomplete, may provide
at least partial explanation for the diverse manifestations of
amoebic infection which range from asymptomatic infestation to acute,
sometimes fatal ulcerative enteritis. Recent success in producing
enteric lesions in germfree animals following inoculation of very
large members of vigorous amoebae has not altered the concept that
bacterial participation is essential for the development of
symptomatic intestinal amoebiasis. It does provide, however, an
opportunity to study intestinal tissue changes resulting from the
activity of E_. histolytica, alone, disassociated from all bacterial
influence.
Proposed Course of the Project:
With reference to the recent finding that amoebic lesions can
be produced in germfree hosts, there are two areas in particular
that require clarification. First, since the lesions appear to
emanate possibly from the site of the puncture wound resulting from
injecting inoculum through the cecal wall, it may be that such trauma
is a prerequisite. Secondly, since E. histolytica has not been
cultivated axenically, our amoebic inocula contain invertebrate forms
of Trypanosoma cruzi, with which amoebae are grown ±n vitro. The
possibility exists, therefore, that the flagellates may participate
in the development of amoebic lesions in the germfree animals. Efforts
will be directed toward ascertaining whether either, or both, of
these points is a factor.
Part B Included: Yes
2 -
Serial No. NIAID-110
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this Project:
Phillips, Bruce P., and Wolfe, P. A.: Pneumonic disease in germfree
animals. J. Inf. Dis . (In press)
Honors and Awards relating to this project: None
3 -
Serial No. NIAID-111
1. Germfree Animal Research
2. Pathogenesis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: The use of germfree animals and tissues for the
study of viruses
Principal Investigator: Walter L. Newton
Other Investigators: Charles Rosenberger, Laboratory of Tropical
Virology
Wallace P. Rowe, Laboratory of Infectious
Diseases
Cooperating Units: NIAID-
Man Years (calendar year 1960) :
Total: 1
Professional: 1/4
Other: 3/4
Project Description:
Objectives:
To explore the possibility that germfree animals and tissues
might serve as particularly favorable experimental tools for certain
aspects of virus study. To ascertain whether viruses are present
in the germfree mouse colony.
Methods Employed:
Comparative studies are carried out on the relative susceptibili-
ties of germfree and conventional mice to infection with certain
viruses. Cultures prepared from germfree animal tissues are also
used. Sera are examined for evidence of antibodies to viruses.
Standard virologic techniques are employed.
Major Findings:
1. Preparations containing Dengue type I virus (Mochizuki
strain) continued to cause much more CPE in kidney tissue cultures
prepared from germfree mice than in cultures prepared from
conventional mouse kidney. However, in recent animal tests to
8
- i -
Serial No. NIAID-111
ascertain whether an increase in viral growth was associated with
the difference in CPE, no evidence of such growth was obtained.
Furthermore, essentially no CPE was obtained following inoculation
of the same material into either cell type. Efforts are being
directed toward resolving this apparent inconsistency.
2. Sera from several germfree mice 1 year or more of age have
been examined for evidence of the presence of certain mouse viruses.
Numbers are too few for negatives to be conclusive, but thus far no
positives for polyoma and mouse adenovirus have been obtained.
Evidence for the presence of Reo 3 is questionable. However, there
is good evidence that K virus may be present.
Significance to Bio-Medical Research and the Program of the Institute:
The study of viruses in animals without the poss ioility of
influence of other concomitant infections, or with known infections,
should provide special insight into virus-host relations. Also,
with the increase in the number of viruses shown to occur in the
conventional mouse, animals with a defined viral status and for
which exposure to infection can be controlled become increasingly
more valuable. This is particularly true in view of the current
interest in tumor-virus studies.
Proposed Course of the Project:
Efforts will be directed toward clarifying the variation in the
behavior of dengue virus in the germfree animal tissue culture. Also,
further serological and tissue analysis of the viral state of the
germfree mouse colony will continue.
Part B Included: No
•
2 -
Serial No. NIAID-112
1. Germfree Animal Research
2. Biology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Biology of germfree animals
Principal Investigator: Richard E. Horton
Other Investigators: Walter L. Newton
William B. DeWitt, Laboratory of Parasitic
Diseases
N. A. Jaworski and John L. S. Hickey, Division
of Research Services
Cooperating Units: DRS 6.4
Man Years (calendar year 1960) :
Total: 2%
Professional: 1%
Other: 1
Proiect Description:
To obtain descriptive bioj.ugn.cii aaca on germfree animals for
the purposes of determining in what manner they may differ from the
conventional animal, and establishing baseline values for other
studies employing these animals.
Methods Employed:
Comparative studies are made on germfree, specifically-
contaminated, and conventional animals in such areas as hematology,
blood chemistry, serology, gross and microscopic anatomy, growth
curves, longevity, etc. Standard laboratory techniques are employed.
Major Findings:
In efforts to improve germfree guinea pigs by maintaining them
on diets sterilized by irradiation instead of steam, some success
was obtained with the use of irradiated semisynthetic diet. However,
neither conventional nor germfree animals on irradiated diet grew
as well as conventionals on non-irradiated diet. It was observed
10
Serial No. NIAID-112
that the growth rate of germfree animals reared on a semisynthetic
diet sterilized by 2 million rad was greater than that of animals
reared on the same diet sterilized by 3 million rad. Irradiation
has an obvious detrimental effect on the nutrition adequacy of the
diet.
Five pregnancies have been observed in adult germfree guinea
pigs maintained on the irradiated ration. None of the females was
able to carry the fetuses to term; they usually aborted near the
5th or 6th week of gestation. In most instances, procidentia of
the uterus was a sequela that eventually led to the death of the
animal. The administration of additional amounts of Vitamins K and
E to the last two pregnant animals did not appear to alter the course
of their pregnancies. This failure to reproduce has not been
observed in the colony of conventional guinea pigs maintained on the
same sterilized diet for four generations. Histology :al examination
of several of the older germfree guinea pigs (approximately a year
old) reared on the irradiated diet has revealed fatty degeneration
of the liver.
Further study of the serum proteins in germfree and conventional
mice has provided data similar to those reported earlier with germ-
free guinea pigs: Gamma globulin levels in the germfree were the
same as those in mice from a conventional colony maintained on the
same sterilized diet, but both groups showed values lower than those
of mice from the NIH conventional colony which is maintained on a
non-sterilized diet.
Data are being accumulated on the incidence of tumors (especially
lung) in our germfree and conventional mice (the same genetic stock)
dying or sacrificed after 6 months of age. Lung tumors have been
found in the germfree mice, but there is some evidence that the
incidence is lower than in their counterparts exposed to the outside
environment. The possibility of this interesting difference will
continue to be explored:
Significance to Bio-Medical Research and the Program of the Institute;
Studies of this nature provide baseline data for current and
future experiments that utilize the germfree animal. Also, they
provide an opportunity to study the role that the "normal" flora may
play in establishing hematological and serological values and general
growth, longevity, and fecundity.
Proposed Course of the Project;
Studies will continue along essentially the same lines. Where
differences between the germfree and the conventional "contaminated"
animals are encountered, efforts will be made to establish whether
11
Serial No. NIAID-112
fundamental principles are involved, and whether a particular
difference is worth further exploitation. Also, attempts will be
made (through the use of hormones, vitamins, etc.) to determine
reasons for the germfree guinea pig's failure to reproduce
satisfactorily.
Part B Included: Yes
12
Serial No. NIAID-112
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this Project:
Horton, R. E. and Hickey, J. L. S.: Irradiated Diets for Rearing Germfree
Guinea Pigs. Proc. of Animal Care Panel. (In press)
Newton, Walter L . , Pennington, Robert M. and Lieberman, Jacob E.: Compara-
tive Hemolytic Ccnolement Activities of Germfree and Conventional
Guinea Pig Serum. Proc. Soc. for Exper . Biol, and Med., 1960, Vol. 104,
Pp. 486-488.
Baer, Paul N., and Newton, Walter L.: Studies on Periodontal Disease in
the Mouse. III. The Germ-Free Mouse and Its Conventional Control. Oral
Surg., Oral Med. and Oral Path., Vol. 13, No. 9, Pp 1134-1144, Sep. 1960.
Honors and Awards relating to this project:
Dr. Newton was invited to present a paper on the work on gamma globulin in
germfree guinea pigs at a special session of the Fifth International
Congress on Nutrition.
13
Serial No. NIAID-113
1. Germfree Animal Research
2. Biology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Origin of Anti-Human Blood Group B Agglutinins in
White Leghorn Chicks
Principal Investigator: Richard E. Horton
Other Investigators: G. F. Springer, University of Pennsylvania
Cooperating Units: None
Man Years (calendar year 1960) :
Total: 1
Professional: 1/4
Other: 3/4
Project Description:
Objectives:
To determine if the blood group B active polysaccharide of
E. coli Oq/- B:7 is absorbed onto the erythrocytes of monocontaminated
chicks harboring the organism in the intestinal tract.
Methods Employed:
Groups of germfree and conventional White Leghorn chicks are
inoculated orally with E. coli 086 B:7 at selected ages. Techniques
which increase permeability of bacterial endotoxin across the
intestinal wall are employed: the induction of chemical enteritis
by prolonged feeding of drastic cathartics; and the induction of
tourniquet shock. Blood samples are taken shortly after the period
of physical stress by cardiac puncture. Standard serological
testing procedures are used to determine the presence of group B
active polysaccharide on erythrocytes and the titer of anti B
agglutinins in blood samples.
Major Findings:
In vitro studies have shown that erythrocytes of White Leghorn
chicks are easily and irreversibly coated with blood group B active
bacterial products. We have found that erythrocytes of the germfree
14
Serial No. NIAID-113
chicks used in our studies do not contain blood group B-like antigen.
When healthy germfree chicks are infected with E. coli 086> only a
minority of the animals acquire B-like antigens on the red cells.
When a series of large doses of a cathartic (cascara sagrada) is
fed to the E. coli Ogg infected chicks, the proportion of animals
having antigen coated red cells is increased. Tests made on blcod
samples taken several hours after E. coli 0g6 infected chicks have
been subjected to tourniquet shock show that almost all chicks
possess varying amounts of antigen-coated erythrocytes. Two to three
days after application of tourniquet shock, the anti-B titer of
these animals was found to rise considerably, but there was no
demonstrable decrease in coated erythrocytes.
Significance to Bio-Medical Research and the Program of the Institute:
Studies of this nature may help explain the mec.i.mism by which
human patients with severe intestinal disorders (e.g. cancer of the
colon, incarcerated hernia, etc.) acquire a transitory B antigen on
their erythrocytes and the altered polyagglutinability of those red
cells which accompany this phenomenon.
Proposed Course of the Project:
In vivo studies will be conducted to determine extent of coating
of the erythrocytes with B antigen when germfree chicks and chicks
monocontaminated with E. coli 0g6 are given purified B-substance
parenterally . When these results have been obtained, further work
on this project will be terminated.
Part B Included: No
15
Serial No. NIAID-114
1. Germfree Animal Research
2. Biology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Studies on bacterial interactions and host
bacteria relations in the germfree animal
Principal Investigator: Norman S. Ikari
Other Investigators: Walter L. Newton
Maurice Landy, National Cancel, institute
Cooperating Units: CDC, Atlanta, Georgia
Man Years (calendar year 1960):
Total: 1 1/4
Professional: 1/2
Other: 3/4
Project Description:
Objectives:
To determine whether certain bacterial phenomena observed in
vitro, e.g. genetic recombination between two enteric strains of
bacteria, occur within the intestine of the living mammalian host.
To evaluate host responses to particular bacterial species without
interference from, and in association with, other known organisms.
Methods Employed:
In vitro studies have shown that the ability to produce colicine,
an antibiotic-like substance lethal to some enteric bacteria, can be
genetically transferred from one bacterial strain to another. In our
studies, E. coli K 12 Row (streptomycin-resistant, colicine-sensitive)
is established in the germfree mouse intestinal tract by inoculation
of the water bottles. Later, Paracolon CA62 (streptomycin-sensitive,
colicine-positive (col+)) is fed by mouth tube, and fecal pellets are
collected at intervals thereafter. Saline dilutions of these pellets
are assayed for col+ colonies by plating onto streptomycin agar plates
Sera of germfree and variously contaminated mice are compared
for levels of antibody activity towards specific organisms using
Ouchterlony and CF techniques.
16
Serial No. NIAID-114
Major Findings:
Col+, streptomycin-resistant hybrids resembling the E. coli
K 12 parent were obtained at every sampling for periods ranging from
24 hours to one month after the Paracolon feeding. Further examina-
tion of the col+ colonies confirmed the stability of this transfer
and revealed the possibility of an additional change(s) in these
hybrids not previously shown in _in vitro studies. Sharp dichotomy
was noted between germfree and conventional animals with respect to
staphylococcal antibody. No antigen-antibody lines were noted in
the germfree serum diffused against Cowan I soluble antigens, whereas
one or more lines were obtained with conventional mouse serum.
Significance to Bio-Medical Research and the Program of the Institute;
Studies on whether genetic recombination and ouar bacterial
interactions observed in the test tube occur in natural ecological
surroundings can provide information on ways in which a host may
affect these phenomena. Also, since backgrounds of "normal" antibodies
(e.g., to organisms like staphylococcus) that are often observed in
conventional animals can complicate attempts at serologic typing and
fluorescent antibody study, the potential value of the germfree
animal for such studies is well worth exploring.
Proposed Course of the Project;
Other known jin vitro genetic recombination systems will be
attempted in the germfree animal. The finding of large numbers of
very mucoid variants completely different from either parental type
or the col+ hybrids has suggested possible studies in a different
direction.
Thorough analysis of the germfree mouse for the presence of
antibody or antibody-like reactivity to a variety of organisms,
especially gram negatives, will continue.
Part B Included: No
17
2 -
Serial No. NIAID-115
1. Germfree Animal Research
2. Pathogenesis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Behavior of parasitic protozoa in germfree hosts
Principal Investigator: Walter L. Newton
Other Investigators: Bruce P. Phillips
Lucy V. Reardon, Laboratory of Parasitic
Diseases
Cooperating Units: NIAID-127-C
Man Years (calendar year 1960);
Total: 1%
Professional: ^
Other: 3/4
Project Description:
Objectives:
To determine whether the presence of bacteria affect the course
of, and response of the host to, protozoan infections other than
those caused by E. histolytica.
Methods Employed:
Germfree, monocontaminated, and conventional guinea pigs are
inoculated with axenically-reared or micro-isolated species of
Trichomonas, Trypanosoma, or Giardia. The inoculated animals are
examined for the presence and type of lesions, time of death, etc.
Major Findings:
It has been shown previously that T. vaginalis, when injected
subcutaneous ly, soon disappears in conventional guinea pigs, but
multiplies and produces a severe lesion in germfree animals. However,
when the germfree animal is orally contaminated with even a single
species of bacteria, its response is like the conventional animal. In
recent studies, the infection has been followed in the germfree animals
until subsidence -- often requiring several weeks. When such animals
are later re-exposed to the parasite, the pattern has been like that
in the conventional animal. The encounter with the infection, even
. i- 13
Serial No. NIAID-115
though it eventually disappeared (and the animal became "germfree"
again), seemed to activate a defensive response which persisted.
Techniques to inoculate germfree animals with sterile Giardia
cysts have been worked out. Preliminary attempts at infection have
not been successful, although the numbers of organisms inoculated
have been small, thus far.
Significance to Bio-Medical Research and the Program of the Institute:
Studies of this type can lead to a better understanding of the
possible effects of the "normal flora" in maintaining a host's
natural defensive mechanisms. It would appear that this system
(involving a host and an organism to which it is resistant in the
conventional but not the germfree state) provides a good tool for
such studies. Also, the role of the intestinal flor* in the patho-
genesis of a variety of protozoal diseases has yet to be established.
Proposed Course of the Project:
Additional experiments involving the "conditioning" of germfree
animals with non-living stimuli such as dead bacteria, egg albumen,
etc., prior to challenging with T. vaginalis are planned. A compara-
tive study of the tissue responses in the conditioned and unconditioned
germfree animal is planned. Attempts at establishing other parasitic
protozoa in germfree animals will continue.
Part B Included: No
«
Serial No. NIAID-116
1. Germfree Animal Research
2. Pathogenesis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Studies on helminthic infections in germfree hosts
Principal Investigator: Walter L. Newton
Other Investigators: Paul P. Weinstein, Laboratory of Parasitic
Diseases
Cooperating Units: NIAID-121-G
Man Years (calendar year 1960) :
Total: 1%
Professional: %
Other : 1
Project Description:
Objectives:
To ascertain what effect the bacteria and other organisms
normally present in the conventional animal have upon the course of
helminth infections, and upon the host's response to these parasites.
Methods Employed:
Germfree animals are fed or inoculated with sterilized eggs, or
axenically-reared infective larvae, of various parasitic helminths.
The development of these parasites is followed by established pro-
cedures, and lungs, intestine, and other appropriate tissues are
examined histologically for the study of host response. The findings
are compared with those obtained in conventional controls.
Major Findings:
Mature, fertile adult worms were obtained in germfree as well as
conventional mice, inoculated with infective larvae of Nematospiroides
dubius. This indicated that the lack of bacteria appeared to have
little or no effect on the development of the parasite from the
infective larva to the adult stage in the host.
However, development of the parasite from the egg to infective
larva in feces from germfree mice was extremely poor. Fatty degenera-
20
- i -
Serial No. NIAID-116
tion not unlike that associated with B-vitamin deficiency occurred.
If living bacteria from a conventional animal were added to the
germfree feces, development of the larvae progressed normally.
Apparently, feces without bacteria fail to provide certain essentials
for development of the larvae.
Of special interest was the apparent indication that the sex of
the host, which is a factor in parasitism in the conventional animal,
was unimportant among the germfree animals. In the conventional
animals, males had 2-3 times the worm burden at necropsy that the
females had. Among the germfree animals, however, average worm counts
were about the same in both sexes.
Significance to Bio-Medical Research and the Program of the Institute;
The fact that bacteria, per se, are apparently ™ot required for
full development of some parasites opens the way for other studies in
the areas of nutrition and parasitism, chemotherapy, in vitro culti-
vation, and physiologic studies. Also, opportunity is provided for
the study of eggs and larvae in fresh (non-sterilized) feces without
bacteria, and perhaps to ascertain factors contributed by the latter
toward their proper development. If the sex phenomenon holds up in
future tests, interesting and potentially important relationships
may be uncovered.
Proposed Course of the Project:
Of course, the validity and significance of the difference in
the host sex effect between germfree and conventional mice will be
studied. Efforts to complete the entire life cycle of the parasite
in a sterile environment will be continued. Attempts may be made to
ascertain the role of bacteria in certain parasite-diet relationships
noted in conventional mice.
Part B Included: No
21
LABORATORY OF GERMFREE ANIMAL RESEARCH
Summary 1
110 - Pathogenesis of Amoebiasis 5
111 - The Use of Germfree Animal and Tissue for
the Study of Viruses 8
112 - Biology of Germfree Animals 10
113 - Origin of Anti-Human Blood Group B
Agglutinins in White Leghorn Chicks 14
114 - Studies on Bacterial Interactions and
Host Bacteria Relations in the Germfree
Animal 16
115 - Behavior of Parasitic Protozoa in
Germfree Hosts 18
116 - Studies on Helminthic Infections in
Germfree Hosts 20
Laboratory of Germfree Animal Research
National Institute of Allergy and Infectious Diseases
Summary statement of research progress, calendar year 1960
Program developments and trends:
During the year, Laboratory plans to broaden the scope of its
research activities showed considerable and rapid progress. Authorization
to occupy completely the south end of the third floor of Building 8,
following the departure of DBS from this space, was finally clarified. The
contract for the remodeling of this area was awarded and renovations got
underway early in the Fall. It is anticipated that this space will be
ready for occupancy (according to the schedule) around March or April of
1961. Following the transfer of LGAR personnel to the south end, alterations
in the north end, converting the entire area into space for germfree animal
units and maintenance, will commence. This will be effected slowly by NIH
personnel, with the view of disrupting normal activities as little as
possible. When this is accomplished, additional germfree animal units will
be obtained and put into operation.
As a part of the broadening in research scope, it was deemed advisable
to strengthen the program by the addition of pathology and virology activities,
The staffing for this has proceeded well. Of the 5 new positions authorized
by the Scientific Director up through the fiscal year ending July 1961,
personnel for 4 are already committed. The new personnel will enter on duty
in the Spring when the space is available. For the pathology group,
Dr. Edwin Lerner, Senior Surgeon, will transfer from NIAMD to our Laboratory.
Dr. Fred Gorstein, S. A. Surgeon, who has had training in pathology will
enter on duty about July 1. Technical assistance for these people has been
arranged for. The only position outstanding for which a candidate is not
yet lined up is the virologist. Efforts are currently being made to locate
one at the level of a GS-11 or 12.
The alteration of the air conditioning system for other institutes
on the first, and especially the second, floor during the last five or six
months of the year has caused some inconvenience especially with noise,
vibration and occasional shutting down of a utility. However, the research
program continued at a satisfactory pace in spite of some of these drawbacks.
As was true of last year, the program reflected an increased variety of
interests and an increase in the number of cooperative projects planned or
underway with other laboratories, and even other institutes. As examples,
we have recently undertaken on a limited scale with Dr. Rowe, LID, serologic
and tissue studies for the possible presence of viruses in our germfree
animal colony. Such data will establish baselines for future studies with
virus-free animals, or, if it turns out that way, animals containing a few
known viruses. We have, in collaboration with Dr. Landy of the NCI,
initiated studies on the origin and specificity of natural antibodies to
enteric bacteria, and similar studies in connection with Staphylococcus with
the CDC. Experiments in germfree animals would appear to provide the crucial
- 1
information in these areas. Also, such studies can provide information on
the nature of the mechanism whereby endotoxin alters resistance to infection
with gram negative pathogens. With Doctors Kelly and O'Gara, also of the
NCI, we have initiated on a small, but satisfactory, scale a study to
ascertain whether the incidence of chemically- induced lung tumors is the
same in germfree mice as in conventional mice of the same strain. In view
of the large numbers of viruses demonstrated as occurring in experimental
mice, (Huebner, Rowe) , the question arises whether the chemicals induce the
tumors by activating viruses, or some other living agents that are present
in the animal. Such studies in an organism- free host could provide rather
significant leads in the approach to understanding tumor-virus relations.
The apparent increase in the interest in germfree animals in medical
research stimulated a request by Dr. Peterson to have a film made on this
subject for use by medical schools, universities, research institutions, etc.
The Audio-Visual Aids section of the CDC came to the Laboratory and we made
a 19-minute color movie. The popularity of the subject matter is pointed
up by the fact that, recently, all 28 copies of the film whx^h CDC had in
its library were booked up on loan for a month in advance.
Significant scientific advances:
With regard to the progress in some of the research projects, an
interesting series of observations has been made by Mr. Phillips on the
behavior of Entamoeba histolytica in the germfree host. It is to be recalled
that, in earlier studies with standardized techniques, amoebic lesions were
not produced in the germfree animal following inoculation. In fact, the
parasite failed to live in the intestine beyond 5 days. Recent changes have
been made in the manner of rearing and handling the amoebae in vitro prior
to inoculation which seemed to result in more vigorous organisms. The latter
have produced lesions in the absence of bacteria, although the type and
severity are still not typical of those encountered with a bacterial associate.
Thus, it would appear that the latter is not the only determinant of the
course and the pathogenesis of the infection.
In collaborative studies with Dr. Weinstein of LPD, we have shown
that the intestinal mouse parasite, Nematospiroides dubius, does not require
a flora to develop from an infective larva to the adult form in the host.
However, it apparently does require bacteria, or their products, to develop
from the egg to infective larva. These studies are preparatory to those to
be undertaken in an analysis of the nature of the nutritional effects
observed in certain parasitisms. One of the most interesting observations
has been our finding that the sex of the host, which has been noted by
several workers to affect the outcome of the infection in conventional
(contaminated) animals, has not appeared to be an influence in the germfree
host. If these findings continue to hold up, a hitherto unrecognized role
(either direct or indirect) of the flora in certain observed sex effects
may unfold.
In studies on the growth and biology of germfree guinea pigs,
Dr. Horton has obtained several advanced pregnancies in animals maintained
- 2
on irradiated diets, although no fetus was carried to term. It is to be
recalled that germfree guinea pigs have not been bred with any success.
The importance of the intestinal flora to this species was pointed up by
the finding that conventional (contaminated) guinea pigs reproduced normally
on this same irradiated diet.
In a collaborative project with Dr. Springer of the University of
Pennsylvania, Dr. Horton has also shown that the use of large dosages of a
cathartic, or the application of tourniquet shock, increased the number
of red cells of germfree chickens coated with human B-like antigens following
mono- infection with E. coli 086- These studies are providing information
on the manner in which red cells of one type may acquire antigenic character-
istics of other cell types, especially B.
Our germfree mouse colony has been undergoing an intensive serologic
study from several points of view. One of the most interesting has been
an assay for the presence of certain so-called "natural antibodies" against
a variety of bacteria. Such antibodies or antibody- like re£'_^ivities for
organisms like Staphylococcus, E. coli and S. typhosa have been found to
occur in a variety of uninoculated conventional animals and are presumed
to originate from encounters with the viable organisms or related antigens.
Animals which have lived for many generations free from contact with live
bacteria are almost the sine qua non for establishing finally the validity
of these ideas. Studies thus far, with the CDC and with Dr. Landy of NCI,
have shown the germfree animal to be singularly free from antibody- like
reactivity toward Staphylococcus and E. coli, but reactivity toward S_. typhosa
was obtained in several instances. We are, of course, unable to find
evidence of the presence of the latter in the germfree colony. Thus, this
finding strengthens sporadic reports that non-bacterial substances (perhaps
in this case dietary components) can cause "cross" reactions with this
organism.
We have now had our germfree animal colony and a conventional colony
derived from the same stock for approximately two years . Some of our ex-
breeders which we try to keep, in spite of the scarcity of germfree unit
animal space, are of the order of 1-2 years of age. We have made it a
practice that whenever a germfree or conventional animal not on an experiment
dies, especially if it is 6 months or more of age, it is checked thoroughly
for gross evidence of malformations or tumors . As of now, we have informa-
tion on a total of more than 50 animals . It is of interest that we have
found so-called spontaneous lung tumors in the germfree as well as the
conventional mice. Also, while the numbers of animals are obviously
relatively small, the incidence has been markedly higher, thus far, among
the conventional animals, i.e., those exposed to external contamination,
than among the germfree. This has been particularly true among animals
6-12 months of age. These observations have been discussed with Doctors
Kelly and O'Gara of the NCI who find them of considerable interest. Such
data will continue to be collected and will serve as corollary information
along with experiments in which such tumors are induced chemically in both
germfree and contaminated animals. If this difference in incidence continues,
it could point up further the importance of microbial agents per ^e, or
stresses produced by such agents, in the induction of these tumors. While
it is, of course, too soon to say much in this regard, the possibilities
suggested by even this limited information are very intriguing.
4 -
Serial No. NIAID-110
1. Germfree Animal Research
2. Pathogenesis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Pathogenesis of amoebiasis
Principal Investigator: Bruce P. Phillips
Other Investigators: None
Cooperating Units: NIAID-127-B
Man Years (calendar year 1960):
Total: 1 3/4
Professional: 3/4
Other: 1
Project Description:
Objectives:
To study the etiology, pathogenesis, and pathology of
intestinal amoebiasis with a view toward ascertaining the role of
the associated intestinal flora.
Methods Employed:
Guinea pigs are inoculated intracecally with Entamoeba
histolytica trophozoites cultivated jLn vitro without bacteria.
Conventional, germfree and ex-germfree animals with various known
single or multiple bacterial infestations are used, and the disease
processes produced, if any, are compared grossly and histologically.
Modifications of techniques for cultivating and harvesting the
amoebae are also employed in an effort to obtain maximum virulence.
Major Findings:
Amoebic lesions did not occur in germfree animals which
received amoebic inocula cultivated and prepared by previously
standardized procedures, even though such inocula regularly pro-
duced amoebic ulceration in conventional hosts . Amoebic ulceration
varying in extent and severity did occur following introduction of
similar amoebic inocula into ex-germfree animals harboring each of
the following bacteria as a monocontaminant: Escherichia coli,
Aerobacter aerogenes, Streptococcus faecalis, Bacillus subtilis,
5
Serial No. NIAID-110
Lactobacillus acidophilus, Staphylococcus aureus, Micrococcus sp.
(from conventional guinea pig). More recently, amoebic lesions
have been produced in germfree animals following intracecal
inoculation of E. histolytica cultivated and harvested by newer
procedures developed as a part of these investigations. However,
these lesions were not typical of those observed in animals
harboring bacteria.
Significance to Bio-Medical Research and the Program of the Institute:
The wide range of host-parasite relationships which characterize
enteric amoebic infection have long been a matter of considerable
scientific interest. The demonstration of bacterial influence on
development of the disease, although as yet incomplete, may provide
at least partial explanation for the diverse manifestations of
amoebic infection which range from asymptomatic infestation to acute,
sometimes fatal ulcerative enteritis. Recent success in producing
enteric lesions in germfree animals following inoculation of very
large members of vigorous amoebae has not altered the concept that
bacterial participation is essential for the development of
symptomatic intestinal amoebiasis. It does provide, however, an
opportunity to study intestinal tissue changes resulting from the
activity of E. histolytica, alone, disassociated from all bacterial
influence.
Proposed Course of the Project:
With reference to the recent finding that amoebic lesions can
be produced in germfree hosts, there are two areas in particular
that require clarification. First, since the lesions appear to
emanate possibly from the site of the puncture wound resulting from
injecting inoculum through the cecal wall, it may be that such trauma
is a prerequisite. Secondly, since E. histolytica has not been
cultivated axenically, our amoebic inocula contain invertebrate forms
of Trypanosoma cruzi, with which amoebae are grown ±n vitro. The
possibility exists, therefore, that the flagellates may participate
in the development of amoebic lesions in the germfree animals. Efforts
will be directed toward ascertaining whether either, or both, of
these points is a factor.
Part B Included: Yes
- 2
Serial No. NIAID-110
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this Project:
Phillips, Bruce P., and Wolfe, P. A.: Pneumonic disease in germfree
animals. J. Inf. Dis . (In press)
Honors and Awards relating to this project: None
Serial No. NIAID-111
1. Germfree Animal Research
2. Pathogenesis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: The use of germfree animals and tissues for the
study of viruses
Principal Investigator: Walter L. Newton
Other Investigators: Charles Rosenberger, Laboratory of Tropical
Virology
Wallace P. Rowe, Laboratory of Infectious
Diseases
Cooperating Units: NIAID-
Man Years (calendar year 1960) :
Total: 1
Professional: 1/4
Other : 3/4
Project Description:
Objectives:
To explore the possibility that germfree animals and tissues
might serve as particularly favorable experimental tools for certain
aspects of virus study. To ascertain whether viruses are present
in the germfree mouse colony.
Methods Employed:
Comparative studies are carried out on the relative susceptibili
ties of germfree and conventional mice to infection with certain
viruses. Cultures prepared from germfree animal tissues are also
used. Sera are examined for evidence of antibodies to viruses.
Standard virologic techniques are employed.
Major Findings:
1. Preparations containing Dengue type I virus (Mochizuki
strain) continued to cause much more CPE in kidney tissue cultures
prepared from germfree mice than in cultures prepared from
conventional mouse kidney. However, in recent animal tests to
8
Serial No. NIAID-111
ascertain whether an increase in viral growth was associated with
the difference in CPE, no evidence of such growth was obtained.
Furthermore, essentially no CPE was obtained following inoculation
of the same material into either cell type. Efforts are being
directed toward resolving this apparent inconsistency.
2. Sera from several germfree mice 1 year or more of age have
been examined for evidence of the presence of certain mouse viruses.
Numbers are too few for negatives to be conclusive, but thus far no
positives for polyoma and mouse adenovirus have been obtained.
Evidence for the presence of Reo 3 is questionable. However, there
is good evidence that K virus may be present.
Signif icanct: to Bio-Medical Research and the Program of the Institute:
The study of viruses in animals without the possJ-ility of
influence of other concomitant infections, or with known infections,
should provide special insight into virus-host relations. Also,
with the increase in the number of viruses shown to occur in the
conventional mouse, animals with a defined viral status and for
which exposure to infection can be controlled become increasingly
more valuable. This is particularly true in view of the current
interest in tumor-virus studies .
Proposed Course of the Project;
Efforts will be directed toward clarifying the variation in the
behavior of dengue virus in the germfree animal tissue culture. Also,
further serological and tissue analysis of the viral state of the
germfree mouse colony will continue.
Part B Included: No
Serial No. NIAID-112
1. Germfree Animal Research
2. Biology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A;
Project Title: Biology of germfree animals
Principal Investigator: Richard E. Horton
Other Investigators: Walter L. Newton
William B. DeWitt, Laboratory of Parasitic
Diseases
N. A. Jaworski and John L. S. Hickey, Division
of Research Services
Cooperating Units: DRS 6.4
Man Years (calendar year 1960) :
Total: 2\
Professional: 1%
Other : 1
Proiect Description:
To obtain descriptive bioiogicai aaca on germfree animals for
the purposes of determining in what manner they may differ from the
conventional animal, and establishing baseline values for other
studies employing these animals.
Methods Employed:
Comparative studies are made on germfree, specifically-
contaminated, and conventional animals in such areas as hematology,
blood chemistry, serology, gross and microscopic anatomy, growth
curves, longevity, etc. Standard laboratory techniques are employed.
Major Findings:
In efforts to improve germfree guinea pigs by maintaining them
on diets sterilized by irradiation instead of steam, some success
was obtained with the use of irradiated semisynthetic diet. However,
neither conventional nor germfree animals on irradiated diet grew
as well as conventionals on non- irradiated diet. It was observed
10
Serial No. NIAID-112
that the growth rate of germfree animals reared on a semisynthetic
diet sterilized by 2 million rad was greater than that of animals
reared on the same diet sterilized by 3 million rad. Irradiation
has an obvious detrimental effect on the nutrition adequacy of the
diet.
Five pregnancies have been observed in adult germfree guinea
pigs maintained on the irradiated ration. None of the females was
able to carry the fetuses to term; they usually aborted near the
5th or 6th week of gestation. In most instances, procidentia of
the uterus was a sequela that eventually led to the death of the
animal. The administration of additional amounts of Vitamins K and
E to the last two pregnant animals did not appear to alter the course
of their pregnancies . This failure to reproduce has not been
observed in the colony of conventional guinea pigs maintained on the
same sterilized diet for four generations. Histolog-! ral examination
of several of the older germfree guinea pigs (approximately a year
old) reared on the irradiated diet has revealed fatty degeneration
of the liver.
Further study of the serum proteins in germfree and conventional
mice has provided data similar to those reported earlier with germ-
free guinea pigs: Gamma globulin levels in the germfree were the
same as those in mice from a conventional colony maintained on the
same sterilized diet, but both groups showed values lower than those
of mice from the NIH conventional colony which is maintained on a
non-sterilized diet.
Data are being accumulated on the incidence of tumors (especially
lung) in our germfree and conventional mice (the same genetic stock)
dying or sacrificed after 6 months of age. Lung tumors have been
found in the germfree mice, but there is some evidence that the
incidence is lower than in their counterparts exposed to the outside
environment. The possibility of this interesting difference will
continue to be explored:
Significance to Bio-Medical Research and the Program of the Institute:
Studies of this nature provide baseline data for current and
future experiments that utilize the germfree animal. Also, they
provide an opportunity to study the role that the "normal" flora may
play in establishing hematological and serological values and general
growth, longevity, and fecundity.
Proposed Course of the Project:
Studies will continue along essentially the same lines. Where
differences between the germfree and the conventional "contaminated"
animals are encountered, efforts will be made to establish whether
11
Serial No. NIAID-112
fundamental principles are involved, and whether a particular
difference is worth further exploitation. Also, attempts will be
made (through the use of hormones, vitamins, etc.) to determine
reasons for the germfree guinea pig's failure to reproduce
satisfactorily.
Part B Included: Yes
12
3 -
Serial No. NIAID-112
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this Project:
Horton, R. E. and Hickey, J. L. S.: Irradiated Diets for Rearing Germfree
Guinea Pigs- Proc . of Animal Care Panel. (In press)
Newton, Walter L . , Pennington, Robert M. and Lieberman, Jacob E.: Compara-
tive Hemolytic Complement Activities of Germfree and Conventional
Guinea Pig Serum. Proc. Soc. for Exper . Biol, and Med., 1960, Vol. 104,
Pp. 486-488.
Baer, Paul N., and Newton, Walter L.: Studies on Periodontal Disease in
the Mouse. III. The Germ-Free Mouse and Its Conventional Control. Oral
Surg., Oral Med. and Oral Path., Vol. 13, No. 9, Pp 1134-1144, Sep. I960.
Honors and Awards relating to this project:
Dr. Newton was invited to present a paper on the work on gamma globulin in
germfree guinea pigs at a special session of the Fifth International
Congress on Nutrition.
13
Serial No. NIAID-113
1. Germfree Animal Research
2. Biology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Origin of Anti-Human Blood Group B Agglutinins in
White Leghorn Chicks
Principal Investigator: Richard E. Horton
Other Investigators: G. F. Springer, University of Pennsylvania
Cooperating Units: None
Man Years (calendar year 1960) :
Total: 1
Professional: 1/4
Other: 3/4
Project Description:
Objectives:
To determine if the blood group B active polysaccharide of
E. coli 0q£ B:7 is absorbed onto the erythrocytes of monocontaminated
chicks harboring the organism in the intestinal tract.
Methods Employed:
Groups of germfree and conventional White Leghorn chicks are
inoculated orally with E. coli 086 B:7 at selected ages. Techniques
which increase permeability of bacterial endotoxin across the
intestinal wall are employed: the induction of chemical enteritis
by prolonged feeding of drastic cathartics; and the induction of
tourniquet shock. Blood samples are taken shortly after the period
of physical stress by cardiac puncture. Standard serological
testing procedures are used to determine the presence of group B
active polysaccharide on erythrocytes and the titer of anti B
agglutinins in blood samples.
Major Findings:
In vitro studies have shown that erythrocytes of White Leghorn
chicks are easily and irreversibly coated with blood group B active
bacterial products. We have found that erythrocytes of the germfree
14
Serial No. NIAID-113
chicks used in our studies do not contain blood group B-like antigen.
When healthy germfree chicks are infected with E. coli 086> only a
minority of the animals acquire B-like antigens on the red cells.
When a series of large doses of a cathartic (cascara sagrada) is
fed to the E. coli Ogg infected chicks, the proportion of animals
having antigen coated red cells is increased. Tests made on blood
samples taken several hours after E. coli 086 infected chicks have
been subjected to tourniquet shock show that almost all chicks
possess varying amounts of antigen-coated erythrocytes. Two to three
days after application of tourniquet shock, the anti-B titer of
these animals was found to rise considerably, but there was no
demonstrable decrease in coated erythrocytes.
Significance! to Bio-Medical Research and the Program of the Institute:
Studies of this nature may help explain the mec'.rnism by which
human patients with severe intestinal disorders (e.g. cancer of the
colon, incarcerated hernia, etc.) acquire a transitory B antigen on
their erythrocytes and the altered polyagglutinability of those red
cells which accompany this phenomenon.
Proposed Course of the Project:
In vivo studies will be conducted to determine extent of coating
of the erythrocytes with B antigen when germfree chicks and chicks
monocontaminated with E. coli Ogg are given purified B-substance
parenterally . When these results have been obtained, further work
on this project will be terminated.
Part B Included: No
15
Serial No. NIAID-114
1. Gennfree Animal Research
2. Biology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Studies on bacterial interactions and host
bacteria relations in the gennfree animal
Principal Investigator: Norman S. Ikari
Other Investigators: Walter L. Newton
Maurice Landy, National Cancel Institute
Cooperating Units: CDC, Atlanta, Georgia
Man Years (calendar year 1960):
Total: 1 1/4
Professional: 1/2
Other: 3/4
Project Description:
Objectives:
To determine whether certain bacterial phenomena observed in
vitro, e.g. genetic recombination between two enteric strains of
bacteria, occur within the intestine of the living mammalian host.
To evaluate host responses to particular bacterial species without
interference from, and in association with, other known organisms.
Methods Employed:
In vitro studies have shown that the ability to produce colicine,
an antibiotic-like substance lethal to some enteric bacteria, can be
genetically transferred from one bacterial strain to another. In our
studies, E. coli K 12 Row (streptomycin-resistant, colicine-sensitive)
is established in the germfree mouse intestinal tract by inoculation
of the water bottles. Later, Paracolon CA62 (streptomycin-sensitive,
colicine-positive (col+)) is fed by mouth tube, and fecal pellets are
collected at intervals thereafter. Saline dilutions of these pellets
are assayed for col+ colonies by plating onto streptomycin agar plates,
Sera of germfree and variously contaminated mice are compared
for levels of antibody activity towards specific organisms using
Ouchterlony and CF techniques.
16
- i -
Serial No. NIAID-114
Major Findings:
Col+, streptomycin-resistant hybrids resembling the E. coli
K 12 parent were obtained at every sampling for periods ranging from
24 hours to one month after the Paracolon feeding. Further examina-
tion of the col+ colonies confirmed the stability of this transfer
and revealed the possibility of an additional change(s) in these
hybrids not previously shown in j^n vitro studies. Sharp dichotomy
was noted between germfree and conventional animals with respect to
staphylococcal antibody. No antigen-antibody lines were noted in
the germfree serum diffused against Cowan I soluble antigens, whereas
one or more lines were obtained with conventional mouse serum.
Significance to Bio-Medical Research and the Program of the Institute:
Studies on whether genetic recombination and o:.i ar bacterial
interactions observed in the test tube occur in natural ecological
surroundings can provide information on ways in which a host may
affect these phenomena. Also, since backgrounds of "normal" antibodies
(e.g., to organisms like staphylococcus) that are often observed in
conventional animals can complicate attempts at serologic typing and
fluorescent antibody study, the potential value of the germfree
animal for such studies is well worth exploring.
Proposed Course of the Project:
Other known jLn vitro genetic recombination systems will be
attempted in the germfree animal. The finding of large numbers of
very mucoid variants completely different from either parental type
or the col+ hybrids has suggested possible studies in a different
direction.
Thorough analysis of the germfree mouse for the presence of
antibody or antibody-like reactivity to a variety of organisms,
especially gram negatives, will continue.
Part B Included: No
17
Serial No. NIAID-115
1. Germfree Animal Research
2. Pathogenesis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Behavior of parasitic protozoa in germfree hosts
Principal Investigator: Walter L. Newton
Other Investigators: Bruce P. Phillips
Lucy V. Reardon, Laboratory of Parasitic
Diseases
Cooperating Units: NIAID-127-C
Man Years (calendar year 1960);
Total: Ik
Professional: %
Other: 3/4
Project Description:
Objectives:
To determine whether the presence of bacteria affect the course
of, and response of the host to, protozoan infections other than
those caused by E. histolytica.
Methods Employed:
Germfree, monocontaminated, and conventional guinea pigs are
inoculated with axenically-reared or micro- isolated species of
Trichomonas, Trypanosoma, or Giardia. The inoculated animals are
examined for the presence and type of lesions, time of death, etc.
Major Findings:
It has been shown previously that T. vaginalis, when injected
subcutaneous ly, soon disappears in conventional guinea pigs, but
multiplies and produces a severe lesion in germfree animals. However,
when the germfree animal is orally contaminated with even a single
species of bacteria, its response is like the conventional animal. In
recent studies, the infection has been followed in the germfree animals
until subsidence -- often requiring several weeks. When such animals
are later re-exposed to the parasite, the pattern has been like that
in the conventional animal. The encounter with the infection, even
1 -
13
Serial No. NIAID-115
though it eventually disappeared (and the animal became "germfree"
again), seemed to activate a defensive response which persisted.
Techniques to inoculate germfree animals with sterile Giardia
cysts have been worked out. Preliminary attempts at infection have
not been successful, although the numbers of organisms inoculated
have been small, thus far.
Significance to Bio-Medical Research and the Program of the Institute:
Studies of this type can lead to a better understanding of the
possible effects of the "normal flora" in maintaining a host's
natural defensive mechanisms. It would appear that this system
(involving a host and an organism to which it is resistant in the
conventional but not the germfree state) provides a good tool for
such studies. Also, the role of the intestinal flora in the patho-
genesis of a variety of protozoal diseases has yet to be established.
Proposed Course of the Project:
Additional experiments involving the "conditioning" of germfree
animals with non-living stimuli such as dead bacteria, egg albumen,
etc., prior to challenging with T. vaginalis are planned. A compara-
tive study of the tissue responses in the conditioned and unconditioned
germfree animal is planned. Attempts at establishing other parasitic
protozoa in germfree animals will continue.
Part B Included: No
19
2 -
Serial No. NIAID-116
1. Germfree Animal Research
2. Pathogenesis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A:
Project Title: Studies on helminthic infections in germfree hosts
Principal Investigator: Walter L. Newton
Other Investigators: Paul P. Weinstein, Laboratory of Parasitic
Diseases
Cooperating Units: NIAID-121-G
Man Years (calendar year 1960) :
Total: 1%
Professional: %
Other: 1
Project Description:
Objectives:
To ascertain what effect the bacteria and other organisms
normally present in the conventional animal have upon the course of
helminth infections, and upon the host's response to these parasites.
Methods Employed:
Germfree animals are fed or inoculated with sterilized eggs, or
axenically-reared infective larvae, of various parasitic helminths.
The development of these parasites is followed by established pro-
cedures, and lungs, intestine, and other appropriate tissues are
examined histologically for the study of host response. The findings
are compared with those obtained in conventional controls.
Major Findings:
Mature, fertile adult worms were obtained in germfree as well as
conventional mice, inoculated with infective larvae of Nematospiroides
dubius. This indicated that the lack of bacteria appeared to have
little or no effect on the development of the parasite from the
infective larva to the adult stage in the host.
However, development of the parasite from the egg to infective
larva in feces from germfree mice was extremely poor. Fatty degenera-
20
- i -
Serial No. NIAID-116
tion not unlike that associated with B-vitamin deficiency occurred.
If living bacteria from a conventional animal were added to the
germfree feces, development of the larvae progressed normally.
Apparently, feces without bacteria fail to provide certain essentials
for development of the larvae.
Of special interest was the apparent indication that the sex of
the host, which is a factor in parasitism in the conventional animal,
was unimportant among the germfree animals . In the conventional
animals, males had 2-3 times the worm burden at necropsy that the
females had. Among the germfree animals, however, average worm counts
were about the same in both sexes.
Significance to Bio-Medical Research and the Program of the Institute:
The fact that bacteria, per se, are apparently "ot required for
full development of some parasites opens the way for other studies in
the areas of nutrition and parasitism, chemotherapy, ^n vitro culti-
vation, and physiologic studies. Also, opportunity is provided for
the study of eggs and larvae in fresh (non-sterilized) feces without
bacteria, and perhaps to ascertain factors contributed by the latter
toward their proper development. If the sex phenomenon holds up in
future tests, interesting and potentially important relationships
may be uncovered.
Proposed Course of the Pro.ject:
Of course, the validity and significance of the difference in
the host sex effect between germfree and conventional mice will be
studied. Efforts to complete the entire life cycle of the parasite
in a sterile environment will be continued. Attempts may be made to
ascertain the role of bacteria in certain parasite-diet relationships
noted in conventional mice.
Part B Included: No
21
LABORATORY OF PARASITIC DISEASES
Summary 1
120 - Administration and Research Planning and
Coordination 6
121-A - Host Parasite Relations in Worm Infections
in Laboratory Animals 7
121-B - Role of Helminths in the Causation of
Cancer 9
121-C - Destruction of Molluscs by Chemical Means 11
121-D - Part A. Investigation of Intermediate
Hosts and Vectors of Human Diseases
Caused by Worms 14
121-E - Development of Methods for the Cultiva-
tion of Parasitic Helminths in vitro, and
the Determination of the Nutritional Re-
quirements of Such Organisms in vitro ... 17
121-G - The Development of Helminths in Germ-
free Animals 21
121-1 - Pathological Physiology of Worm Infec-
tions 23
121-J - Serum Protein Studies on Germfree Animals
Infected with Various Parasites 25
121-K - Effects of Nutrition on Chemotherapy of
Parasitic Diseases 27
121-L - Effects of Improving the Nutrition of
Malnourished People Infected with
Schistosoma Mansoni 30
121-M - Ammonia Metabolism and Toxicity in Rela-
tion to Liver Disease 33
121-N - Parasitological Investigations at the
Marine Biological Laboratory, Woods Hole,
Massachusetts 36
123-A - Fundamental Physiological and Biochemical
Studies on Parasites, Intermediate Hosts,
and Parasitized Animals 38
123-B - Pathological Physiology and Histochemis-
try of Parasitic Diseases 42
123-C - Biochemical Mechanisms of Energy Metabo-
lism in Normal and Parasitized Animals .. 45
123-D - Biology of Trypanosomes 49
127-A - Studies on Toxoplasmosis 52
127-B - Studies on Entamoeba Histolytica and Other
Parasitic Protozoa 57
127-C - Trichomoniasis 60
127-D - Biochemistry of Parasitic Protozoa 63
II
Laboratory of Parasitic Diseases
National Institute of Allergy and Infectious Diseases
Summary statement of research activities, calendar year 1960
Program development and trends:
Two of the regular staff members, Dr. Jacobs and Dr. Weinbach, have
been on research assignment outside the country for more than half of the year.
On the other hand, we have had two persons on assignment to the Laboratory from
other countries, Mr. Ian Sommerville from the McMaster Laboratory, Sydney,
Australia; and Dr. Iain R. Bowman recently from the University of Aberdeen,
Scotland. Mr. Sommerville has been working with Dr. Weinstein on problems re-
lated to sterile culture of worms, while Dr. Bowman has been conducting bio-
chemical studies with Dr. von Brand. In July Dr. Allen W. Cheever was assigned
to the Laboratory as a Research Associate. He has been placed in the Section
on Helminthic Diseases and is conducting research on schistosome pathology, and
on fundamental problems related to immunity and strain differences in suscepti-
bility to the liver tapeworm of small rodents, with Dr. Olivier.
The Laboratory continues to emphasize fundamental studies on parasites
and parasitic diseases. No important changes in the program were instituted
during the year. The program of the laboratory is well diversified, consider-
ing the size of the staff, and the competencies of the various staff members
cover a large proportion of the field of parasitology.
Although the emphasis in on basic studies, this does not imply a nar-
row viewpoint and the Laboratory is well aware of the many practical problems
parasitic diseases create throughout the world. The Laboratory is often called
upon for help and advice concerning prevention and control of parasitic infec-
tions and so must maintain competence, and a reputation for competence, to deal
not only with basic problems of parasitism but also problems of prevention and
control of parasitic diseases. Therefore, the Laboratory continues to carry
on a variety of activities which help it maintain its international reputation
and increase its capacity to cope with problems of parasitism. Such activity
also returns benefits in the form of ideas for laboratory research and clues
which may explain puzzling laboratory findings.
The Laboratory is prepared to carry out field studies when these are
logical and reasonable extensions or applications of laboratory work. The pro-
ject in Puerto Rico on the relation of nutrition to schistosomiasis is a case
in point. Also, during the year steps were taken to extend laboratory findings
and interests into foreign laboratories and into foreign field situations through
the use of Public Law 480 funds in various countries. A series of project pro-
posals has been made for work in Israel, Poland, Yugoslavia, India, and Brazil.
Negotiations for two PL 480 projects in Brazil are actively under way and it
is expected that both will be started in 1961.
The PL 480 projects should return substantial dividends, not only in
new data which could not be obtained in the Bethesda Laboratory, but also in-
valuable experience and sophistication for the staff members involved. The
projects are designed so as to cause minimal interference with essential lab-
oratory activities. If the Laboratory is to consider more than a very small
number of PL 480 projects it would have to add personnel to staff them and
would probably need additional funds to cover incidental expenses.
The Laboratory has continued to maintain liaison with international
agencies interested in health problems. Two of the staff (Olivier and Berry)
have been chosen to serve on the WHO Expert Panel on Parasitic Diseases. These
same staff members were loaned for short periods to WHO as consultants on
schistosomiasis research and control activities. Four staff members (Berry,
von Brand, Olivier, Weinstein) made important contributions to international
symposia or conferences dealing with research on human disease.
The development of cooperative clinical studies with the Laboratory of
Clinical Investigation has been disappointing. Relatively little significant
cooperative clinical work was done during the year. This did not result from
lack of desire on the part of either the Laboratory of Clinical Investigations
or the Laboratory of Parasitic Diseases since as excellent rapport has been
maintained, but rather because of problems inherent in procurement of useful
patients and also, to some extent, to failure to form a productive "team".
It is hoped that progress along this line can be made in 1961.
Scientific advances - made in 1960:
The Laboratory has produced a number of noteworthy advances in knowl-
edge during the year. Many of these can be classified as being additions to
"basic" knowledge but some have "practical" implications. Selection of items
for emphasis is sometimes presumptuous and always risky since the importance
of an individual item is hard to judge and often the "small" contribution may,
in the long run, turn out to be "large". Nevertheless, attention is called
to the following:
The studies on toxoplasmosis in New Zealand sheep (127-A) have shown
that the prevalence is high. Considerable new information has been obtained
concerning the distribution of the organisms in the tissues and their persis-
tence there. After inoculation the distribution of the parasite in tissues
is erratic and the parasites rapidly clear from tissues other than the muscle
and placenta. Since residual infection occurs in muscle, mutton may serve as
a source of human infection. Since congenital infection with Toxoplasma is
an important medical problem, it is of special interest that the sheep studies
have indicated that inoculation of sheep 60 days before pregnancy did not re-
sult in congenital infection or abortion but inoculation at 30 days pregnancy
caused abortion or foetal death with absorption. Infection at 90 days preg-
nancy was less likely to be dangerous to the foetus.
The status of resistance or immunity to Toxoplasma continues to be
puzzling, since living organisms fail to completely protect animals against
challenge, especially when the challenge is great, and because low grade para-
sitemia may persist for months in mice and rabbits in the presence of high ser-
um antibody levels. The observation that cysts of Toxoplasma probably form in
tissue cultures provides a new opportunity to study the manner of cyst forma-
tion and the factors that lead to cyst formation (127 -A).
The work on the preservation of living Entamoeba histolytica and other
protozoa (127-B) has practical significance since success would permit reten-
tion of strains without continuous sub-culturing. This is a relatively new
field and techniques are still evolving. The work so far has shown that this
approach is feasible since four species have been frozen and stored for periods
ranging from one to four months depending on the species involved. Entamoeba
histolytica has been kept at -197° C for 24 hours, suggesting that almost in-
definite storage at this temperature may eventually be achieved.
Laboratory culture of Entamoeba histolytica continues to receive atten-
tion since it is so important to learn more concerning its nutritional re-
quirements and its pathogenicity in the absence of other organisms. It is
noteworthy that satistactory axenic culture of this species has been achieved
for the first time (127-B). The protozoa are cultured in a complex diphasic
medium containing no cells but including chick embryo extract. This is a long
and very important step forward.
The substitution of a species of Crithidia for Trypanosoma cruzi in
cultures of E. histolytica provides a more economical and rapid way of pro-
ducing large cultures of the amoeba. Demonstration of the value of the Coulter
Counter for the enumeration of protozoa in suspension adds a valuable tool for
quantitative work and suggests this method may be applicable for counting other
organisms of similar size such as tissue culture cells (127-B).
The use of germ free animals in worm-parasite studies continues to re-
veal the value of this tool and adds to our knowledge of the peculiar nature
of the germ free state. The technique seems to be particularly useful for
studying conditions that influence natural resistance and nutritional rela-
tionships of parasite and host. For example, it was found (121-G) that the
roundworm, Nematospiroides dubius, develops as well in germ free as in con-
ventional mice but while in conventional mice the worm recovery is much higher
from the male animals, the recovery from germ free mice is the same for both
host sexes. The cause of the difference is unknown. Also, it has been shown
that the feces of germ free mice do not support development of N. dubius larvae
and that bacteria in the feces provide important factors for larval develop-
ment (121-G). There was further evidence that the alteration in levels of
serum protein components in germ free animals is due to dietary factors (121-G).
Studies on the sterile culture of worms continues to produce fundamen-
tal information on the nutritional requirements of the parasites and brings
closer the day when we can use the axenic animals for immunologic and thera-
peutic studies. Survival studies using relatively advanced larvae of Nippos-
tronqylus muris has produced important results (121-E). The intent has
been to try, by addition of elements to the medium, to induce the larvae to
reach the adult stage. Starting with a salt mixture, dextrose was added until
3 3
the optimal level was reached. Then casein was added and survival time rose
to 11 days, but there was not development of the larvae. Addition of a yeast
extract to this mixture not only increased survival but permitted growth to
the adult stage. Thus, a much more simple medium than used before has been
evolved and the achievement of a defined medium for culture of N. muris adults
is much closer. A similar approach is being used in attempts to culture mi-
crofilariae of Dirof ilaria immitis (121-E).
Although the study of the relation of nutrition to schistosomiasis in
Puerto Rico is still incomplete, it appears that enrichment of the diet does
not affect the number of eggs passed in the feces. However, it is interesting
to note that the enriched diet did cause a loss of hookworms and whipworms
from the intestine (121-L). This has a bearing on the problem of the exist-
ence of hookworm infection without hookworm disease. In laboratory studies
conducted in Bethesda the enhanced efficacy of stibophen in mice receiving
a semi-synthetic diet was shown to be due to the absence from this diet of as
yet unknown inorganic salts (121-K). Higher blood levels of the drug were
maintained longer when the semi-synthetic diet was used and this may explain
the greater efficacy. Demonstration of the influence of simple salts on the
efficacy of stibophen suggests that other drugs may be similarly affected by
diet. If the work with the stibophen-salt problem progresses satisfactorily
it is hoped that a test of the effect of human diet on the action of the same
drug may be tried in Puerto Rico before the study there is concluded.
Interaction of two pathogenic organisms in the same host has had rela-
tively little attention in spite of some very provocative work done in years
past. A study of simultaneous infection with encephalomyocarditis virus and
Trichinella spiralis in rats has produced striking and significant results
(121-A). While the virus alone does not injure adult white rats when given
intraperitoneally, in the presence of Trichinella spiralis infection many of
the rats are crippled and die. This potentiation of the virus pathogenicity
is not due to non-specific stress but seems to be related to the presence of
the worms on the muscles. The virus can be recovered from the muscle of T.
spiralis-infected rats but not from muscle of rats without T. spiralis. The
reason for the influence of the worm infection on the activity of the virus
is unknown. The phenomenon offers an opportunity to study some of the funda-
mental factors in the pathogenesis of both the virus and the worm parasite.
It also provokes the question as to what effect this worm infection may have
on other virus infections.
Continued study of the hepato-splenic syndrome in mice infected with
Schistosoma mansoni has added new evidence to show that, in mice at least,
the schistosome eggs are the prime cause of liver damage and therefore the
chief cause of fibrosis, portal hypertension, and collateral circulation which
are so often the cause of morbidity and mortality in human schistosome infec-
tion (121-1). Diet, dead worms, and toxins produced by the worms seem to be
less important in contributing to liver damage related to schistosome infec-
tion.
The study of liver damage in relation to ammonia toxicity in mice has
revealed that low oxygen in breathed air greatly enhances ammonia toxicity
(121-M). The mechanism of this effect is not clear. Though hepatic coma is
usually considered to be related to ammonia toxicity none of the substances
which exacerbate hepatic coma in man increases ammonia toxicity in mice. In
fact, 6 of 10 decrease it. Ammonia toxicity in mice was greatly reduced by
hypothermia and this suggests that the same measure may be useful in treating
hepatic coma in man. Finally, mouse liver damage was induced in eight differ-
ent ways but none caused any change in the animal* s response to intravenous
ammonia. Thus, though high blood ammonia levels seem to be related to liver
damage, the causal relationships are by no means clear.
Fundamental physiological studies have focused on the calcareous cor-
puscles of tapeworms and on the phospholipids of tapeworms (123-A). The cal-
careous corpuscles are amorphous but, on heating, dolomite, brucite or apatite
may be formed. Electron microscope pictures of corpuscles heated with KOH re-
veal the presence of well-formed crystals. The glycerol containing phospholi-
pids of Taenia taeniaef ormis are about half lecithid and half cephalin. Sphin-
gomyelin is present and more than one cephalin is known to occur in the larvae
of this tapeworm. Hexose-containing phospholipids occur in both larvae and
adults.
Study of the mechanism of energy metabolism of sub-cellular elements
has dealt, among other things, with the mechanism by which mitochondria which
are depleted of high-energy phosphate intermediates are stimulated to oxidize
substrates when ATP is added. This is a complex, though fundamental, bioener-
getic system for which a better understanding is needed. Addition of ATP not
only restored succinate oxidation but also caused reduction of intra -mitochon-
drial DPN. The succinate oxidation involves an energy-requiring reaction and
this energy is apparently added at one site in the respiratory chain and used
at another for reducing pyridine nucleotide (127-D).
Serial No. NIAID-120
1. Parasitic Diseases
2. Office of the Chief
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Administration and research planning and coordination.
Principal Investigator: Leon Jacobs
Other Investigators: Louis J. Olivier
Cooperating Units: None
Man Years (calendar year 1960):
Total: 2)4
Professional: \
Other: 2
Project Description:
This project furnishes supervisory and administrative services to
all research projects in the Laboratory, as folows:
Over-all evaluation of research plans and initiation of field
projects; integration of laboratory research activities with clinical
studies of the Clinical Center; editing scientific and technical reports
and manuscripts; preparation of reports, budget estimates, and exhibits;
supervision over personnel, travel, correspondence, and maintenance;
requisitioning and supervision of equipment and supplies; maintenance of
reference library, etc. ; and consultatory services to individuals,
academic and other organizations, and liaison activities with other
branches of the Service and the Federal Government.
Research projects are reviewed and, in consultation with Section
heads, changes in research program are considered and initiated. The
emphasis is placed on fundamental aspects of medical parasitology;
extensions of projects into the fields of clinical or practical preventive
medicine are attempted only when the laboratory work has progressed to a
point where a sound basis exists.
Serial No. NIAID-121-A
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Host parasite relations in worm infections in
laboratory animals.
Principal Investigator: Louis J. Olivier
Other Investigators: Allen W. Cheever
Cooperating Units: Laboratory of Viral Products, DBS
Man Years (calendar year 1960):
Total: 2&
Professional ]H
Other: 114
Project Description:
Objectives:
To study the characteristics of infection in laboratory animals;
to learn how resistance to infection may be enhanced and reduced; to
learn how the host defense mechanisms act to resist infections; to study
variations in resistance of host strains; to study the mechanism of
natural resistance to parasites; to study how two simultaneous infections
may affect each other.
Methods Employed:
Infect animals in such a way as to produce predictable infections;
treat with hormones and antigens and so forth; challenge with infective
worms or other agents; observe the host and parasite; and determine the
degree of resistance to infection, the host reaction, and the effect of
one agent on another.
Major Findings:
Encephalomyocarditis virus is pathogenic to very young rats, but
is not ordinarily pathogenic to older ones. However, this virus is
highly pathogenic in older rats if the latter harbor new infections with
Trichinella spiralis. The rats are crippled by the infection and many
die. Moreover, the virus is recoverable from trichinous muscle in
greater quantity than from non-trichinous muscle. The enhanced patho-
Serial No. NIAID-121-A
genicity is not due to non-specific stress since other stresses, such as
chilling, fighting, and so forth, do not give this result. This potenti-
ation of virus pathogenicity by T. spiralis is very striking, but not
well understood as yet. Schistosome infection does not potentiate the
virus.
Significance to the Program of the Institute:
Problems of resistance and immunity are basic to understanding of
host parasite relationships and pathogenicity of parasites. Natural
resistance is an elusive phenomenon, but worm infections provide a use-
ful and attractive system for its study since infective doses, antigenic
mass, and lesion size can be controlled and measured quite accurately.
Some parasites do not multiply in the host and are not attacked by
phagocytes. These facts simplify the study of the effects of hormones
and other agents on infections.
The revelation of the potentiation of EMC by T. spiralis may pro-
vide a wonderful opportunity to study the pathways of host damage by
both the virus and the worms.
Proposed Course of the Project:
To test whether mice develop acquired immunity to the liver
parasite of rats; to study paths of transfer of immunity of rats; to
study further the affect of hormones on this resistance.
To study natural resistance to Cysticercus fasciolaris in mice
and other rodents.
To follow the EMC-T. spiralis relationship and to study other
possible virus worm associations. To determine the role of the reticulo-
endothelium system of the liver in resistance through the use of reticulo-
endothelial system stimulating and depressing agents. To determine
whether immunologic tolerance to Cysticercus fasciolaris can be induced
in rodents.
Part B included No
3
Serial No. NIAID-121-B
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Role of helminths in the causation of cancer
Principal Investigators: Elmer G. Berry and Louis J. Olivier
Other Investigators: None
Cooperating Units: Pathologic Anatomy Branch, National Cancer Institute,
NCI— 525
Laboratory of Viral Products, DBS
Man Years (calendar year 1960)
Total: 1/4
Professional: 1/4
Other: 0
Project Description:
Objectives:
To test whether worm parasites can induce malignant growths in their
hosts. To test whether Schistosoma haematobium infections in hamsters
cause tumors of the digestive or urinary tract. To test whether the ma-
lignant tumors produced in rat liver by larval tapeworms (Taenia taeniae-
formis) are virus-induced. To test whether cats, the host of the adult
tapeworm, have a virus which can be related to these tumors.
Methods Employed:
Schistosoma haematobium. Hamsters are infected with S. haematobium.
The animals are allowed to live as long as possible to determine whether
the schistosome infection induces malignant growths.
Taenia taeniaeformis. Rats are infected and the liver cysts are
ground and injected into rats and other laboratory animals. Material
from cats and rats is studied by virological and immunological methods.
Major Findings:
Rat virus has been isolated from two rats having the liver sarcomas.
Serial No. NIAID-121-B
Significance to the Program of the Institute:
Cancer of the bladder appears to be much higher in Egypt and
Mozambique than it is in other areas. Although this has been attributed
to the particular strain of S. haematobium which occurs in these countries
the actual cause for these differences is not known. It is hoped that
this study might help to solve the problem.
The problem of virus etiology of cancer is at the center of
concern in the cancer field.
Proposed Course of the Project;
Termination of the S. haematobium study when the last infected
animals die.
Continued study of the rat tumor project and possible addition of
similar projects.
Part B included No
10
Serial No. NIAID-121-C
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Destruction of molluscs by chemical means.
Principal Investigator: Louis J. Olivier
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960)
Total:
1
Professional:
1/2
Other:
1/2
Project Description:
Objectives:
To discover more effective means for killing snail vectors of
schistosomiasis. To study the mode of action of chemicals used as mol-
luscicides. To develop more effective means for study of snail poisons
in the laboratory.
Methods Employed:
Snail vectors, or their eggs, are exposed to chemicals and the de-
structive efficacy of the chemicals is recorded. The optimal conditions
for use of chemicals against snails are sought. By quantitative methods
the optimal duration of exposure, concentration, temperature, etc. are
determined.
Major Findings:
Using standardized methods the sodium pentachlorophenate LD50 was
determined for eggs and adults of Australorbis qlabratus. Eggs are more
susceptible to the chemical than adults by a chemical concentration fac-
tor of about 5. Strain differences in susceptibility were not great.
Very small differences were found between young and old post-enbryonic
snails.
11
Serial No. NIAID-121-C
Following failure of NaPCP to kill snails as predicted in some
field situations it was shown that ultra-violet light distroys the com-
pound rapidly and that this could explain some of the field failures.
Quantitative laboratory data showed the half-life of NaPCP solutions in
sunlight may be less then an hour whereas the duration of an effective
field concentration probably has to be 8 or more hours.
Significance to the Program of the Institute:
For the present, at least, the best means to control schistoso-
miasis is interruption of transmission from man to man by destruction of
the snail intermediate hosts. The molluscicides now available are not
ideal. The project attempts to find better and more economical ways to
use the available molluscicides. The evidence from the work done so far
suggests the possibility that more efficient use may be made of mollusci-
cides if they are directed against the snail eggs than against the adults.
The project puts the Institute in a better position to take part in
measures directed against the schistosomes which are rapidly coming to
prominence as dangerous parasites of man.
Proposed Course of the Project:
To continue with efforts to standardize the laboratory method for
study of molluscicides with the aim of perfecting a method for highly
analytical quantitative work with snail poisons and also with the aim of
providing laboratories interested in chemical screening with a better
tool. To investigate the action of chemicals already studied upon other
species of vector snails. To try, under the same conditions, other mol-
luscicides of high promise so that there may be accurate and quantitative
data by which molluscicides can be compared. To devise a method for
screening for compounds which may be active but insoluble.
Part B included Yes
12
Serial No. NIAID-121-C
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Olivier, L. and Haskins W. T. The effects of low concentrations of sodium
pentachlorophenate on the fecundity and egg viability of Australorbis qlabratus.
Amer. Jour. Trop. Med. Hyg. 9; 199-205 (1960)
Olivier, L. Factors affecting the survival of aestivating pulmonate vectors
of schistosomiasis. Anniversary Volume for Dr. Caballero y Caballero. pp. 215-
225 (1960)
Hiatt, C. W. , Haskins, W. T. , and Olivier, L. The action of sunlight on so-
dium pentachlorophenate. Amer. Jour. Trop. Med. Hyg. 9; 527-531. (1960)
Awards: None
Honors: Dr. Louis J. Olivier served in Geneva as Consultant to the WHO Section
on Endemic Diseases from 18 September to 10 October 1960. During this time he
took part in a meeting of the WHO Expert Comittee on Bilhorziasis which met
from 26 September to 10 October 1960.
Dr. Louis J. Olivier was invited to present one of the papers in the Fourth
Conference of the Industrial Council for Tropical Health which met on July
20 - 22 in the Harvard School of Public Health.
Under the sponsorship of WHO Dr. Louis J. Olivier visited three laboratories
studying chemicals useful as animal toxins in water in order to gather infor-
mation and increase exchange of information and ideas among those interested
in the subject.
Dr. Louis J. Olivier has been chosen to serve on the WHO Expert Advisory
Panel on Parasitic Diseases.
13
Serial No. NIAID-121-D
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Investigation of intermediate hosts and vectors of
human diseases caused by worms.
Principal Investigator: Elmer G. Berry
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960):
Total: 1 3/4
Professional: 3/4
Other: 1
Project Description:
Objectives:
To investigate the distribution, life history, and ecology of
snail intermediate hosts and to develop a rational and useful system of
classification and means of identification.
Methods Employed:
Colonies of snails which serve as intermediate hosts of schisto-
somes are maintained in the laboratory where they are available for
intensive study. Laboratory-reared specimens are exposed to miracidia
to evaluate the susceptibility and to determine whether strain differences
are present.
Major Findings:
Additional specimens belonging to the genera Bulinus and Biompha-
laria were collected from the Belgian Congo, Mozambique, Kenya, and the
Transvaal. Colonies of these species are now established in the labora-
tory. Continued progress has been made toward clarification of taxonomic
problems.
n
Serial No. NIAID-121-D
Significance to the Program of the Institute;
This knowledge is essential in understanding the epidemiology of
bilharziasis and its control.
Proposed Course of the Project:
Continuance of the anatomical studies of the snails which serve
as intermediate hosts and to compile this information into a manual.
Part B included Yes
15
Serial No. NIAID-121-D
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project: None
Awards: None
Honors: Dr. Elmer G. Berry served as a Consultant and Leader of Discussion
on Snail Control by Chemical Means at the African Symposium on Bilharziasis
which met in Lourenco Marques, Mozambique, 30 March to 8 April 1960.
Dr. Elmer G. Berry served as Consultant on bilharziasis research at the Expert
Committee on Bilharziasis (Molluscicides) which met in Geneva, Switzerland
26 September to 1 October 1960.
Dr. Elmer G. Berry was appointed to serve on the World Health Organization
Expert Advisory Panel on Parasitic Diseases for a period of five years,
beginning June 1, 1960.
1G
Serial No. NIAID-121-E
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Development of methods for the cultivation of parasitic
helminths ki vitro, and the determination of the nutri-
tional requirements of such organisms in vitro.
Principal Investigator: Paul P. Weinstein
Other Investigators: I. R. Sommerville (visiting scientist)
T. K. Sawyer
Cooperating Units: None
Man Years (calendar year 1960):
Total: 4
Professional: 2 1/4
Other: 1 3/4
Project Description:
Objectives:
To develop media and physical conditions suitable for the in vitro
cultivation of helminths throughout their life cycle, and to obtain infor-
mation on their specific nutritional requirements.
Methods Employed:
Worm eggs, infective larvae, or partially matured worms are iso-
lated from charcoal cultures, rodent hosts, or mosquitoes, depending on
the helminths involved. These are inoculated into culture media to be
tested for growth -promoting properties, and are observed for development
and differentiation.
Major Findings:
1. Cultivation of trichostronqylids. Survival of fourth stage
larvae of Nippostronqylus muris was studied in salt solutions to which
nutrients were added. In a modified Krebs-Ringer salt solution, survi-
val was limited to 3 or 4 days, but the addition of dextrose increased
the survival time to 6-8 days. The optimal concentration of dextrose was
0.014 M. Change in phosphate concentration was without effect. When
IT
Serial No. NIAID-121-E
casein was added to the solution of salts and dextrose, survival time in-
creased to 11 days, but no developmental changes were observed. Replace-
ment of casein with either enzymic or acid hydrolysates of casein gave
inferior results, and acid hydrolysates appeared to be toxic. The addition
of a water-soluble extract of yeast to a solution containing casein, dex-
trose and salts not only enhanced survival but yielded adult worms. Better
survival, but no development was obtained when a mixture of L-amino acids
in the proportions in which they occur in casein was used in place of
casein.
13
Serial No. NIAID-121-E
Although N. muris does not mate when grown in a complex medium
composed of chick embryo extract, vitamin mixture and serum, it was
found that the third, fourth, and early fifth stages, if taken from
such cultures and put directly into the small intestine of rats will
mate and produce viable eggs.
The closely related nematode, Nematospiroides dubius, was placed
in the same medium which was not conducive to the mating of N. muris.
Under these conditions, N. dubius was observed to mate. This makes more
likely the possibility that a complete generation of a parasitic nematode
may be obtained in vitro.
2. Cultivation of Dirofilaria immitis microfilariae. Survival
in balanced salt solutions alone is very short. Addition of dextrose
increased survival time about fourfold. There is a direct relation
between concentrations of magnesium and dextrose and survival time.
With sodium to potassium ratios near that of mammalian blood (29:1) or
close to that of some insect fluids (5:1 or less), survival is good,
whereas at intermediate ratios (20:1, 10:1) it is relatively poor.
3. Nematode coelomocytes and vitamin B]?. This vitamin added
to a defined medium resulted in the development of a deep rose-pink pig-
ment in the coelomocytes of the dog hookworm Ancylostoma caninum.
similar to what was previously reported for N. muris. It is suspected
that this pigment represents a specific concentration of Bi2»
Significance to the Program of the Institute:
The growth jji vitro of parasitic helminths will provide an impor-
tant tool with which to study the physiology and metabolism of these
organisms, and should facilitate the isolation and preparation of meta-
bolic antigens involved in the development of functional immunity.
Proposed Course of the Project:
Work will continue on the specific nutritional requirements of
N. muris and N. dubius in vitro.
The study of the interaction of B^ and coelomocytes will be
extended to other nematodes.
Dirofilaria uniformis infections in rabbits have now been es-
tablished in the laboratory, and coupled with D. immitis from dogs,
studies will continue on the growth requirments of filariids in. vitro.
Part B included Yes
13
Serial No. NIAID-121-E
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Weinstein, Paul P.: Excretory mechanisms and excretory products of nematodes:
An appraisal. In "Host Influence on parasite physiology." Rutgers University
Press: 65-92. (1960).
Weinstein, Paul P. : The current status of the axenic cultivation of helminths.
(In press).
Honors: Dr. Weinstein was invited to participate in the Annual Conference on
Protein Metabolism entitled, "Host Influence on Parasite Physiology," sponsored
by Rutgers University, January 1960.
Dr. Weinstein was invited to present the "Annual Address" to the Annual
Meeting of the Midwestern Conference of Parasitologists held in conjunction
with the International Symposium on Growth at Purdue University to commemorate
the dedication of the Life Sciences Building, June 1960.
Dr. Weinstein was invited by the First Pan-American Congress on Biology and
Experimental Pathology to participate in a symposium entitled, "Parasitic
Biodynamics," held in Caracas, Venezuela, September 1960.
Awards: None
20
Serial No. NIAID-121-G
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: The development of helminths in germfree animals.
Principal Investigator: Paul P. Weinstein
Other Investigators: I. R. Sommerville, (visiting scientist), LPD
Thomas K. Sawyer, LPD
Cooperating Units: Laboratory of Germfree Animal Research, NIAID Serial
No. NIAID-116
Man years (calendar year 1960):
Total: Vt
Professional: \
Other: J£
Project Description:
Objectives:
To establish helminth infections in germfree animals so that prob-
lems involving host-parasite relations, nutrition, immune response, and
pathology can be studied in the absence of other complicating microorganisms.
Methods Employed:
Larvae are reared to the infective stage under axenic conditions,
and eggs and larvae are also isolated from natural sources and rendered
axenic; these are used to infect germfree animals.
Major Findings:
Axenically reared Nematospiroides dubius larvae will consistently
mature in germfree mice. Total worm recoveries from germfree animals have
been equivalent to those from conventional ones. While in conventional
animals there is a considerable higher worm recovery from male than from
female mice, in germfree mice worm recovery was essentially the same from
both sexes. Apparently, diet does not affect this difference.
N. dubius larvae which hatched from eggs passed in axenic feces did
not develop normally in such feces maintained axenically. When feces from
21
Serial No. NIAID-121-G
conventional animals containing bacteria was added to such cultures, rapid,
normal larval development to the infective stage occurred, indicating that
bacteria in feces provide important components for larval development.
Significance to the Program of the Institute.
These experiments demonstrate the feasibility of using the axenic
helminth parasite and host in studying factors which concern the host par-
asite relationship. This technique has value particularly where intesti-
nal parasites are concerned in studying conditions which influence natural
resistance, host specificity, and nutritional relationships of parasite
and host.
Proposed Course of the Pro.ject:
Further work will be done to compare the susceptibility to infection
of male and female conventional and germfree mice with a helminth parasite.
It is also planned to attempt to establish the infection on a continuing
basis in the germfree environment through successive generations of host
and parasite, and to follow the course of the infection under such condi-
tions. Nutritional factors relating to infection are to be studied. It
is also planned to use worms developing in axenic hosts as inocula for cul-
tures for in vitro study under axenic conditions.
Part B included No
22
Serial No. NIAID-121-I
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Pathological physiology of worm infections
Principal Investigators: Kenneth S Warren and Allen W. Cheever
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960):
Total: 1)4
Professional: 3/4
Other: J£
Project Description:
Objectives:
To study the dynamic relationships existing between worms and
their hosts. Specifically to study the syndrome in mice which resembles
human hepato-splenic schistosomiasis mansoni and learn its causes; to
use this syndrome as a tool for the study of liver diseases; to study
the pathogenesis of schistosome infection, using mice as tools.
Methods Employed:
Use unisexual and bisexual infections to determine the cause of
schistosome liver disease; evaluation by use of portal pressure deter-
minations, study of tissues, surgical procedures, nutritional changes,
liver function tests, and physiological studies. Study vascular damage
in liver by injection of colloidal carbon or carmine. Study importance
of eggs, dead worms, etc. as factors in schistosome liver damage.
Major Findings:
In mice hepato-splenic schistosomiasis occurs in the presence of
good nutrition and is not exacerbated by poor nutrition; unisexual in-
fections do not cause typical hepato-splenic schistosomiasis, but in
male infections there is a moderate, transient splenomegaly and an
increase in portal pressure. The production of eggs by the worms appears
23
Serial No. NIAID-121-I
to be necessary before hepato-splenic schistosomiasis develops. There is
no lung shift of worms following the use of high doses of Fuadin. There
is some evidence that the liver recovers rapidly from damage due to dead
worms.
Significance to the Program of the Institute:
Much controversy exists concerning the relative roles played by
ova, toxins, dead worms, and immunity in the production of schistosoma
liver disease. The dispute is of practical importance since those who
believe dead worms to be the chief pathogenic agent do not treat
infected patients. In general, pathogenesis of worm parasites needs more
attention. Its study should return fundamental information of broad
usefulness.
Proposed Course of the Project:
To continue to study the relations of schistosomes to their host
by studying the role of male schistosomes in the cause of liver damage;
by injecting schistosome eggs into the portal system in order to observe
the damage they cause alone; by attempting to produce immunologic tolerance;
by studying the role of immunity in host damage; by testing further the
role of dead worms in causing liver damage; and other similar approaches
to the problem.
It is also intended to study the pathogenesis of tapeworm in-
fections in rat liver.
Part B included No
24
Serial No. NIAID-121-J
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Serum protein studies on germfree animals infected with
various parasites.
Principal Investigator: William B. De Witt
Other Investigators: None
Cooperating Units: Laboratory of Germfree Animal Research, NIAID-112.
Man YearsCcalendar year 1960):
Total: 1/4
Professional: 0
Other: 1/4
Project Description:
Objectives:
To determine the effects of a germfree environment on the relative
distribution of various protein components of germfree guinea pigs, mice,
and other small laboratory animals. This work is being done to obtain
base line values in preparation for contemplated serologic studies on the
pathogenesis of parasitic infections in germfree hosts.
Methods Employed:
Serum from animals maintained in germfree tanks on a sterilized
ration is compared with that obtained from animals housed in conventional
cages and given the same ration. A third group of animals, housed in
conventional cages and fed a commercial pellet diet is also studied.
Paper electrophoretic studies are carried out according to standard
techniques.
Major Findings:
This project has been relatively inactive during the past year.
However, further confirmatory evidence was obtained, indicating that
previously observed alterations in levels of serum protein components
in the germfree mouse was due mainly to dietary factors rather than to
the germfree state.
25
Serial No. NIAID-121-J
Significance to the Program of the Institute:
Before the effects of parasitic infections on the relative distri-
bution of serum protein components of the gerrafree animal can be studied,
it is first necessary to establish baseline values in the axenically
reared animal. It is hoped that serum protein studies on animals experi-
encing for the first time the invasion and development of parasites will
shed light on the role played in the defense of the host by circulating
antibodies associated with the various globulin components.
Proposed Course of the Project:
The present study is to be extended to determine the effect of age
and various nutritional factors on the relative distribution of the serum
protein components. Later the response to infection with a variety of
parasites will be measured.
Part B included No
26
Serial No. NIAID-121-K
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Effects of nutrition on chemotherapy of parasitic diseases
Principal Investigator: William B. DeWitt
Other Investigators: None
Cooperating Units: Laboratory of Parasite Chemotherapy, NIAID-131-D.
Man Years (calendar year 1960):
Total 1 3/4
Professional: 1/2
Other 1 1/4
Project Description:
Objectives:
To determine whether the nutrition of the host can influence the
efficacy of parasite chemotherapy.
Methods Employed:
Animals maintained on defined diets either enriched or deficient
in certain components are exposed to a single species of parasite and the
developing infection is treated either in the prepatent or patent periods.
The influence of the diet on treatment is determined by (1) the reduction
of the number of parasites, (2) condition of the parasites, (3). growth
and survival of the host, and (4) pathologic conditions developing in the
host.
Major Findings:
The efficacy of stibophen (Fuadin) therapy on mature Schistosoma
mansoni infections in mice was increased up to 16 times by feeding a
balanced semi-synthetic diet. The toxicity of the drug was not similarly
increased. The enhancement of curative action by the purified semi-
synthetic diet was found to be due to the absence of as yet unidentified
inorganic salt(s) that interfere with drug activity. It was found in
mice fed on the purified semi-synthetic diet that higher blood levels of
the drug were maintained for a longer period than when the same amount
27
Serial No. NIAID-121-K
of Fuadin was injected into mice fed on the commercial pellet diet,
suggesting that the increased cure-rate was due to the higher blood
drug level.
Significance to the Program of the Institute:
Since parasitic infections occur most frequently in backward
areas where malnutrition is also prevalent, it is necessary to determine
the relation between the nutritional status of the people and the results
obtainable by chemotherapy. Failure of chemotherapy is a common experi-
ence in such areas.
Pronosed Course of the Project:
The present study is to be extended to determine the specific
nutritional factors involved in the enhancement of Fuadin efficacy in
schistosomiasis. Other drugs used for the treatment of this and other
parasitic diseases will be investigated under similar experimental
conditions.
Part B included Yes
28
Serial No. NIAID-121-K
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Luttermoser, George W., and DeWitt, William B. : Studies on interrelations
of nutrition and treatment of schistosomiasis I. Enhancement of stibophen
(Fuadin) activity against Schistosoma mansoni in mice by feeding purified
semi-synthetic diets. (In press)
Honors: None
Awards: None
25
Serial No. NIAID-121-L
1. Parasitic Diseases
raS-NIH 2. Helminthic Diseases
Individual Project Report 3# Bethesda, Maryland
Calendar Year 1960 ' ' 3
Part A.
Project Title: Effects of improving the nutrition of malnourished
people infected with Schistosoma mansoni.
Principal Investigator: William B. De Witt
Other Investigators: None.
Cooperating Units: School of Medicine, San Juan, P. R.
Rio Piedras Hospital, Rio Piedras, P.R.
Man Years (calendar year 1960):
Total: 1
Professional: 1/2
Other: 1/2
Project Description:
Objectives:
To evaluate the effects of providing malnourished people infected
with Schistosoma mansoni with an enriched diet containing an abundance
of animal protein.
Methods Employed:
Malnourished people suffering from schistosomiasis are given
high-protein, high-caloric, vitamin-enriched food over an extended
period so that the effects of the improved diet on their well-being and
on the parasitic infections may be determined. Such factors as the
following are observed before and at various intervals after the patients
are given the enriched diet: (1) Nutritional status as determined by
physical examinations and biochemical tests; (2) egg-production and via-
bility of eggs of the parasite; (3) antibody levels; (4) skin sensitivity
to schistosome antigens; (5) intestinal uptake of essential nutrients;
(6) liver function, as determined by BSP and thymol turbidity measure-
ments; and (7) liver biopsy examinations. After the effects of the
improved diet on the health of the patients have been determined, the
patients will be given a standard course of treatment so that the effects
of the improved nutrition on the efficiency of the drug can be determined.
Suitable control patients are likewise to be studied.
30
Serial No. NIAID-121-L
Major Findings;
The first part of this project has been completed and preliminary
analysis of the data indicates: (1) Malnourished people suffering from
schistosomiasis respond favorably to an enriched diet. Weight gain and
improvement in general well-being were particularly marked. (2) The
effects of the enriched diet on passage of schistosome eggs did not
appear to be significant. However, with some patients a decrease was
noted in the number of schistosome eggs passed. (3) Several patients
with concomitant hookworm and whipworm infections apparently underwent
self-cure while on the enriched diet. Similar cures were not observed
among the control subjects. (4) Biochemical studies made on patients
before they received the improved diet revealed a complete absence of
urinary vitamin C in more than half of those examined. Most of those
studied also had low serum vitamin A levels.
Significance to the Program of the Institute:
This project is a direct extension of some of our basic labora-
tory studies which indicate that the normal growth and development of
schistosomes is prevented when the experimental host is given a deficient
diet. Many of the worms remain sexually immature and are unable to pro-
duce eggs, which are important factors in the pathogenic mechanism of
schistosomiasis. No information is available concerning the interrela-
tion of diet and schistosomiasis in humans. If, as has been shown in
animal experiments, the efficacy of treatment of schistosomiasis in
humans can be greatly enhanced by providing an enriched diet, it would
be a major accomplishment. As to date, no satisfactory treatment of the
disease has been discovered.
Proposed Course of the Project:
The second phase of the project is concerned with determining the
effects of dietary improvement on the efficacy of drugs against schisto-
somiasis. The curative action of the drug (Fuadin) will be evaluated
when different regimens are employed and the occurrence and severity of
side reactions will be noted.
Part B included Yes
31
Serial No. NIAID-121-L
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project: None
Honors: Dr. William B. De Witt was made editor of Tropical Medicine
and Hygiene News.
Awards: None
32
Serial No. NIAID-121-M
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Ammonia metabolism and toxicity in relation to liver
disease.
Principal Investigator: Kenneth S Warren
Other Investigators: None
Cooperating Units: Laboratory of Clinical Investigations, NIAID
Man Years (calendar year 1960):
Total: lfc
Professional: 1
Other: J£
Project Description:
Objectives:
To study the biochemical pathway of ammonia toxicity and the
possible relation between ammonia toxicity and hepatic coma. More speci-
fically, to determine whether drugs which exacerbate hepatic coma or are
used in its treatment effect ammonia toxicity; to test whether the sus-
ceptibility of animals to ammonia toxicity is altered by different types
of liver disease.
Methods Employed:
Ammonia toxicity to mice is established by determining the intra-
venous LD50. The modification of the LD50 by drugs, anti-metabolites
and liver disease is then determined. Blood and brain ammonia concen-
trations, glutamine concentrations, and blood pH are also studied.
Major Findings:
1. Low oxygen in breathed air enhances ammonia toxicity, but
hypoglycemia and cyanide have no such effect. Uncoupling agents have a
slight effect and substances which effect the lower part of the Embden-
Myerhoff cycle and the Krebs cycle up to succinate increase ammonia
toxicity. Ammonia toxicity bears no relation to brain acetyl-choline
33
Serial No. NIAID-121-M
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Warren, K. S, Iber, F. L., Dolle, W., and Sherlock, S: The effect of
alterations in blood pH on the distribution of ammonia from blood to
cerebrospinal fluid in patients in hepatic coma. J. Lab. Clin. Med. 56: 687
Warren, K. S, and Schenker, S. : Hypoxia and amonia toxicity. Am. J. Physiol.
(Dec. 1960).
Honors: None
Awards: None
35
Serial No. NIAID-121-N
1. Parasitic Diseases
2. Helminthic Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Parasitological investigations at the Marine Biological
Laboratory, Woods Hole, Mass.
Principal Investigator: Paul P. Weinstein
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960):
Total: Y4
Professional: 54
Other: None
Project Description:
Objectives:
A. To study the ectocommensal associates of the horseshoe crab,
Limulus polyphemus. and the factors which influence the host-ectocommensal
relationship.
B. To study the little-understood life cycles of trypanorhynchid and
tetraphyllidean tapeworms.
Methods employed:
A. Various stages of Limulus were collected and the parasites were
studied in vivo and in. vitro.
B. Usual biological methods were used to study life cycles of the
tapeworms.
Major Findings:
A. A new species of oncholaimid nematode was discovered as an ecto-
commensal on the ventral surface of Limulus. Its entire life cycle has
been found to take place in this habitat, and various stages from the egg
to the adult have been studied. Information has been obtained on seasonal
changes in growth of the worm, prevalence rate on different instars of
Limulus, and the relationship of molting of Limulus to continued "infestation.
3G
Serial No. NIAID-121-N
The turbellarian ectocommensals, Bdelloura Candida. B. propinqua and
Syncoelidium pellucidum were studied "in vitro", and information was ob-
tained on feeding response to various substances. Cocoon deposition, which
ordinarily only occurs on the gills of Limulus. was obtained hi vitro, and
these cocoons were successfully embryonated and hatched.
Studies made on Limulus from the trilobite stage to the adult gave
some information on the sequence of "infestation" of this host with its
nematode and turbellarian ectocommensals, and has clarified somewhat the
"epidemiological" picture.
B. Studies done with the trypanorhynch, Lacistorhynchus tenuis indi-
cate that certain copepods will support rapid development of procerci after
exposure to coracidia. No procerci were observed following exposure of co-
pepods to the eggs of two species of Calliobothrium (tetraphyllidean), nor
were procerci of either genus found in the exposed benthic fauna.
Significance to the Program of the Institute:
A. The ectocommensals of Limulus have a strict host specificity; they
are found nowhere else in nature. Delineating the factors underlying such
a relationship should contribute toward an understanding of ectocommensalism,
which is one of the interesting categories of animal association, and is in
an evolutionary sense a forerunner to parasitism.
B. The tapeworms studies have given information on the first inter-
mediate hosts of a relatively primitive group of tapeworms, whose life cycles
are essentially unknown. The trypanorhynchid cycle may prove to be similar
to that of Diphyllobothrium latum, the fish tapeworm of man.
Proposed Course of the Project:
This project has been terminated to permit time to develop plans for
the contemplated filariasis program under PL 480.
Part B included No
37
Serial No. NIAID-123-A
1. Parasitic Diseases
2. Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Fundamental physiological and biochemical studies on
parasites, intermediate hosts, and parasitized animals.
Principal investigator: Theodor von Brand
Other investigators: Eleanor J. Tobie, Patricia A. McMahon, Iain B.
Bowman (Guest worker).
Cooperating Units: Laboratory of Histology and Pathology, NIDR.
Man Years (calendar year 1960)
Total: 4 1/2
Professional 3
Other 1 1/2
Project Description:
Objectives:
The objective is to gain knowledge on the chemical composition and
the metabolism of parasites, intermediate hosts, and parasitized animals.
Methods Employed:
Trypanosoma cruzi, T. qambiense and T. rhodesiense are cultivated
in wholly fluid media and trehalose utilization is studied by quantita-
tive methods. Insight into a major metabolic pathway of Trypanosoma cruzi
is sought by estimating, with the help of radioactive CO2, the carbon di-
oxide fixation into succinic acid. The aerobic and anaerobic metabolism
of Taenia taeniaef ormis are studied by determining quantitatively the
elimination of various metabolic end-products and the utilization of car-
bohydrate. The calcareous corpuscles of the same tapeworm are isolated
by various means, treated in a muff lefurnace, and then turned over to the
collaborating laboratory for diffraction and electronmicroscopic studies.
The phospholipids of larval and adult Taenia taeniaef ormis are fraction-
ated and studied by electrophoretic, chromatographic, and chemical
methods, stress being laid on the quantitative relationships between var-
ious components.
o
3
Serial No. NIAID-123-A
Major Findings:
The trypanosome species studied are not capable of utilizing
trehalose and the presence of this disaccharide does not render cultures
of African trypanosomes infective. The production of succinic acid by
the culture form of T. cruzi is dependent on the presence of carbon
dioxide in its surroundings. Taenia taeniaeformis produces anaerobically
much more succinic acid than it does aerobically. Stored lipids are not
utilized by this worm during short periods of aerobic or anaerobic
starvation. Upon heating the calcareous corpuscles of larval or adult
Taenia taeniaeformis to 300° C diffraction lines appear that are very
close, if not identical, to those of dolomite. Upon heating to higher
temperatures, the dolomite-like substance is decomposed to calcium oxide
and magnesium oxide. Upon treating of corpuscles with KOH or NaOH, no
dolomite is formed, but brucite and apatite are produced. Highly magni-
fied electron-microscope photographs of KOH-isolated corpuscles reveal
the presence of well formed crystals. The glycerol-containing phospho-
lipids of both larval and adult Taenia taeniaeformis are approximately
half lecithin and half cephalin. Electrophoretic studies confirmed the
presence of sphingomyelin and indicated two cephalin fractions in larval
T. taeniaeformis as well as a lecithin differing from that of the adult
tapeworm. Hexose-containing phospholipids have been found in both the
larval and the adult worms.
Significance to the Program of the Institute:
The studies on metabolism and chemical composition of parasites,
intermediate hosts, and parasitized animals are essential for an under-
standing of the pathogenesis of parasitic diseases. They also lay a
foundation for a rational approach to chemotherapy.
Proposed Course of the Project:
The studies on the metabolism of trypanosomes will be continued
with emphasis on tracer studies. The studies on the overall metabolism
of Taenia taeniaeformis will be terminated within the next few months.
A study of the influence of oxygen tension and carbon dioxide tension on
the metabolism of tapeworms will be initiated. The studies on the
calcareous corpuscles will be continued, but may be terminated some time
during the coming year. The studies on phospholipids will be continued
with emphasis on electrophoretic studies.
Part B included Yes
39
Serial No. NIAID-123-A
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
von Brand, Theodor: Recent advances in carbohydrate biochemistry of helminths.
Helminthological Abstracts 29: 97-111 (1960).
von Brand, Theodor: Der Stoffwechsel der Trypanosomen. Ergejibnisse der
Bio log ie _12: 30-46 (1960)
von Brand, Theodor: Influence of oxygen on life processes. In: Sasser, J.N.,
and Jenkins, W.R. (Editors): Nematology. The University of North Carolina
Press, pp 242-248. (1960).
von Brand, Theodor: Influence of size, motility, and age on metabolic rate.
In: Sasser, J.N., and Jenkins, W.R. (Editors): Nematology. The University
of North Carolina Press, pp. 233-241. (1960).
von Brand, Theodor: Influence of pH, ions, and osmotic pressure on life
processes. In: Sasser, J.N. and Jenkins, W.R. (Editors): Nematology.
The University of North Carolina Press, pp. 249-256. (1960).
von Brand, Theodor: Influence of temperature on life processes. In: Sasser,
J.N. and Jenkins, W.R. (Editors): Nematology. The University of North
Carolina Press, pp. 257-266. (1960).
von Brand, Theodor: Introductory remarks. In: Stauber, L.A. (Editor):
Host influence on parasite physiology. Rutgers University Press, pp. 1-3.
(1960).
von Brand, Theodor, Mercado, Teresa I., Nylen, M.U., and Scott, D.B. :
Observations on function, composition, and structure of cestode calcareous
corpuscles. Exper. Parasitol. 9: 205-214. (1960).
Thompson, M.J., Mosettig, E., and von Brand, T. : Unsaponif iable lipids of
Taenia taeniaeformis and Moniezia sp. Exper. Parasitol. 9: 127-130. (1960).
Bowman, Iain B.R., von Brand, Theodor, and Tobie, Eleanor J.: The cultivation
of trypanosomes in the presence of trehalose with observations on trehalase
in blood serum. Exper. Parasitol. (In press).
von Brand, Theodor: The metabolism of trypanosomes with special reference to
Trypanosoma cruzi. Proc. 1. Internat. Congr. Chagas Dis. (In press).
Serial No. NIAID-123-A
von Brand, Theodor: Influencia del tamano, motilidad, ayuno y edad sobre la
actividad metabolica. Biologia 28: 117-128. (1959). (Issued April 1960 and
therefore not reported as published in 1959).
Awards: None
Honors: Theodor von Brand presented the introductory remarks on the occasion
of the symposium "Host influence on parasite physiology," Rutgers University's
16th Annual Conference on Protein Metabolism, New Brunswick, January 1960.
Theodor von Brand served as coordinator of the panel "Impact of modern
instrumentation on medicine in the tropics." Fourth Conference on Research
Needs in Tropical Medicine. New Orleans, La. April 1960.
Theodor von Brand served as chairman of the"Symposium sobre biodinamia para-
sitaria". I. Congreso Panamericano de Biologia y Patologia Experimental,
Caracas, Venezuela, September 1960.
Theodor von Brand gave a series of seven lectures on parasite physiology
before the Instituto de Medicina Tropical, Universidad Central de Venezuela,
September 1960.
Theodor von Brand was nominated "Huesped de Honor" by the Medical Faculty
of the Universidad Central de Venezuela, September 1960.
11
Serial No. NIAID-123-B
1. Parasitic Diseases
2. Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Pathological physiology and histochemistry of parasitic
diseases.
Principal Investigator: Teresa I. Mercado
Other Investigators: Theodor von Brand
Cooperating Units: None
Man Years (calendar year 1960):
Total: 2)4
Professional: 1#
Other: 1
Project Description:
Objectives:
To gain a better understanding, by means of studies at the
cellular level, of the pathological physiology of parasitic diseases
as related to interaction between parasite and host.
Methods Employed:
Normal rats and rats infected with Plasmodium berqhei are fed
large amounts of hytakerol (dihydrotachysterol) for 4, 5, or 6 days.
They are then killed and various organs are fixed. Sections are
stained for calcium, primarily by the von Kossa method, and for
mucopolysaccharides by the alcian blue procedure. The slides are then
studied in respect to the possible presence of quantitative and quali-
tative calcification differences between normal and infected animals.
Major Findings:
In most organs studied, such as kidney, heart, lung, and other
essentially the same type of calcification occurred in normal and
parasitized animals; the question of possible occurrence of quantitative
differences (either in respect to the number of animals showing calci-
fications, or in respect to the degree of calcification) requires
further analysis of the prepared slides. A significant qualitative
difference between malarious and control rats was observed in stomach
calcification. The majority of infected animals showed pronounced l|2
Serial No. NIAID-123-B
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Mercado, Teresa I., and von Brand, Theodor: Histocheraical studies of
liver glycogen and lipid in some parasitic infections. J. Infect. Dis. 106:
95-105. (1960).
Honors: None.
Awards: None.
43
Serial No. NIAID-123-B
calcium deposits between the glands of the stomach mucosa, resulting
in a mosaic-like pattern. This was found only in a few non-infected
animals; when the latter showed calcification, the deposits were seen
more frequently in the muscular coat.
Significance of the Program to the Institute:
Histocheraical studies concerned with the pathological physiology
of parasitic diseases allow the study of some facets of the complicated
interaction between parasite and host that cannot be investigated by
purely chemical means. As such, they may be used to throw light on
such fundamental questions as the elusive question concerning the
possible production of specific parasite substances damaging the host,
the intimate distribution of enzyme patterns, and others; all of them
important for the advancement of modern chemotherapeutic and immuno-
logical concepts.
Proposed Course of the Project;
The calcification studies will be terminated during the coming
year. H is proposed to study by modern histochemical procedures cer-
tain enzymes, such as succinoxidase or cytochrome oxidase, in the liver
of normal and parasitized rats.
Part B included Yes
44
Serial No. NIAID-123-C
1. Parasitic Diseases
PHS-NIH 2. Physiology
Individual Project Report 3. Bethesda, Maryland
Calendar Year 1960
Part A.
Project Title: Biochemical mechanisms of energy metabolism in
normal and parasitized animals.
Principal Investigator: E. C. Weinbach
Other Investigators: None
Cooperating Units: Laboratory of Parasite Chemotherapy; Wenner-Grens
Institute Stockholm, Sweden.
Man Years (calendar year 1960)
Total: ]%
Professional: 1
Other: 1/4
Project Description:
Objectives:
The objective is to conduct fundamental studies on the mechanism
of energy metabolism in normal and parasitized animals.
Methods Employed:
Mammalian tissues are fractionated by mechanical disruption of
the cells and the subcellular elements are isolated by differential
centrifugation. Mitochondria are fragmented further by various
chemical and mechanical means to obtain membranes bearing the electron
transport system. These cellular and subcellular fractions are employed
as biological catalysts to study electron transport, oxidative phos-
phorylation, and related exergonic reactions. Intramitochondrial sub-
strates and cofactors of metabolism are determined by suitable enzymatic,
chemical, radiochemical, and spectrophotometric techniques. Manometric
procedures and oxygen electrode recordings are used to measure oxidation
rates. Radioisotope techniques are used to investigate subtle metabolic
changes. Similar studies are conducted with tissues obtained from
parasitized animals.
Major Findings:
The stability of isolated liver mitochondria is related to the
endogenous oxidative phosphorylation. Oxygen electrode recording of
45
Serial No. NIAID-123-C
the respiratory control index has revealed that liver mitochondria are
more stable when they exhibit a high endogenous metabolism.
Phosphoenolpyruvate is formed from endogenous substrates in
isolated liver mitochondria.
Work at the Wenner-Grens Institut:
Studies are conducted on the oxidation of f lavosubstrates by the
mitochondrial respiratory chain and on the regulation by f lavosubstrates
of the redox state of the intramitochondrial pyridine nucleotides.
Specifically, the present study concerns the mechanism by which the
addition of ATP to mitochondria which are depleted of their endogenous
high energy-phosphate intermediates stimulates the oxidation of a
flavosubstrate such as succinate and, simultaneously, reduce pyridine
nucleotides. In effect, this is a "reversal" of oxidative phosphory-
lation and it appears to be the most powerful tool to date for studying
the mechanism of this complex bioenergetic process.
Major Findings:
Mitochondria, depleted of endogenous high-energy phosphate,
exhibit a marked diminution in the capacity to oxidize succinate.
Addition of ATP restored the succinate oxidation and, simultaneously,
initiated reduction of intramitochondrial DPN. Since the ATP effect
was obtained in the presence of uncoupling agents, it must be exerted
at a site which is not accessible to mitochondrial ATPase. This
suggests compartmentalization in the mitochondria and preliminary
studies with P3^ supports this concept. The important fact is that the
oxidation of succinate is linked to an energy-requiring reaction, and
that this energy is invested at one site of the respiratory chain and
then utilized at another site for reducing pyridine nucleotide.
Significance to the Program of the Institute:
This investigation, because of its fundamental nature in eluci-
dating one of the vital processes in all living cells, has obvious
significance to biochemistry in general, and provides a firm basis for
our understanding of the subtle biochemical changes in energy metabolism
associated with parasitism. In addition, it provides a solid foundation
for future studies on the complex bioenergetic mechanisms of parasitic
forms.
Proposed Course of the Project:
Efforts aimed at obtaining additional information on the basic
metabolism of mitochondria, and an increased understanding of the
fundamental mechanism of oxidative phosphorylation will be continued.
Serial No. NIAID-123-C
Specifically at the Wenner-Grens Institut;
We intend to measure the requirement for high-energy phosphate
of different systems capable of succinate oxidation. Since the ATP
effect is not found with non-phosphorylating submitochondrial prepa-
rations, we plan to study the aerobic oxidation of succinate in sub-
mitochondrial fragments which have different degrees of phosphorylating
capacity. An intense effort, employing radioisotopes, will be made to
examine the compartmentalization concept of mitochondrial organization.
Part B included Yes
4?
Serial No. NIAID-123-C
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Weinbach, E.C. : Biochemical changes in mitochondria associated with age.
In: Strehler, B.L. (Editor): The Biology of Aging. A Symposium. American
Institute of Biological Sciences, Washington, D.C. Publication No. 6:
pp. 328-331. (1960).
Weinbach, E.C. : Oxidative phosphorylation with endogenous mitochondrial
substrates. Acta Chem. Scand. (In press).
Schellenberg, K.A., and Weinbach, E.C: The endogenous formation of
phosphoenolpyruvate by rate liver mitochondria. Biochem. et Biophys. Acta.
(In press).
Honors: None
Awards: None
H8
PHS-NIH
Individual Project Heport
Calendar Year 1960
Part A.
Serial No. NIAID-123-D
1. Parasitic Diseases
2. Physiology
3. Bethesda, Maryland
Project Title: Biology of trypanosomes.
Principal Investigator: Eleanor J. Tobie
Other Investigators: None
Cooperating Units: Applied Immunology Section, Laboratory of Immunology,
NIAID. Serial No. NIAID-148.
Man Years (calendar year 1960):
Total: 1 3/4
Professional: 3/4
Other: 1
Project Description:
Objectives:
To clarify the life cycle of Trypanosoma ranqeli in relation to
its vertebrate and invertebrate hosts; to study ways of maintaining and
enhancing infectivity of strains.
Methods Employed:
Young nursling rats and Rhodnius prolixus are used as hosts. A
colony of R. prolixus is maintained. One strain of T. ranqeli is
maintained in R. prolixus- and several are maintained in vitro. Rats and
R. prolixus are infected, both by artificial and natural means. Infec-
tions in rats are determined by microscopic examination of blood or by
in vitro cultivation of blood. Infections in R. prolixus are proved by
microscopic examination of fecal material and hemo lymph, feeding on
fresh rats, and by dissection, with microscopic examination of body fluids
and organs. Fresh frozen sections are made of infected R. prolixus,
stained and examined microscopically. Culture material is grown and
harvested for immunizing of rabbits.
Major Findings:
Various strains of T. ranqeli react differently in the white rat
as well as in R. prolixus. Evidence suggests that R. prolixus may not be
Serial No. NIAID-123-D
the natural invertebrate host for all strains of T. ranqeli or that
trypanosomes designated as T. ranqeli actually represent more than one
species. Passage through R. prolixus. following inoculation into the
hemocoele enhanced the ability of one strain to invade the salivary
glands, making it possible to maintain the strain by cyclical trans-
mission. The infectivity of i_n vitro strains can be extended by
repeated isolation in vitro and passage through the rat. R. prolixus
does not develope resistance to T. ranqeli when the infection does not
pass beyond the digestive tube. Artificial transfer of parasites from
fecal material to the hemocoele resulted in completion of the cycle.
Significance of the program to the Institute:
Intensive biological studies of this species may provide new and
valuable information general to trypanosomes. This is a valuable
laboratory tool.
Proposed Course of the Project:
Studies will be directed to determining the life cycle of the
parasite within the insect host by localizing the organism during its
stages of development in the various organs by means of the fluorescent
antibody technique.
Part B included Yes
r
0
Serial No. NIAID-123-D
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
von Brand, Theodor, and Tobie, Eleanor J: The mechanism of elimination of
certain strains or species of trypanosomes when mixed in experimental
infections. J. Parasitol. 46 (2): 129-136. (1960).
Tobie, Eleanor J. : Experimental transmission and biological comparison of
strains of Trypanosoma ranqeli. Exp. Parasitol. (In press).
Honors: None
Awards: None
51
Serial No. NIAID-127-A
1. Parasitic Diseases
2. Protozoal Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Studies on toxoplasmosis
Principal Investigator: Leon Jacobs
Other Investigators: Milford N. Lunde, Marjorie L. Melton,
Anastasia S. Stanley
Cooperating Units: Ophthalmology Branch, NINDB
Man Years (calendar year 1960):
Total: 5%
Professional: 3J£
Other: 2
Project Description:
Objectives:
The accumulation of data on the occurrence of toxoplasmosis as an
acute and chronic infection of man and animals; the study of the biology
of the proliferative parasite and the cyst, with the aim of evaluating
their importance in the epidemiology of toxoplasmosis and in the pro-
duction of chronic disease; the determination of the usefulness of
immunological tests in diagnosis, and the identification of antigens and
antibodies; the description of factors related to the virulence of
Toxoplasma, and of those involved in susceptibility and immunity of the
host, and in the occurrence of congenital disease; the elucidation of the
mechanism of cyst formation, relative to factors in the host; the critical
study of toxoplasmicidal agents against proliferative parasites and cysts;
the study of the role of hypersensitivity in ocular toxoplasmosis.
Methods Employed:
Standard procedures of animal inoculation plus special techniques
for injection into the anterior or posterior chambers of the eye. A
microisolation apparatus is used for handling cysts. Histochemical pro-
cedures are being initiated to complement electron-microscopic work.
52
Serial No. NIAID-127-A
Tissue cultures are used for morphological, histochemical, and
electron-microscopic studies and for attempts at growing Toxoplasma cysts
in vitro. The dye and hemagglutination tests are used for serological
studies, and the antigens are investigated by biochemical and electro-
phoretic techniques.
Major Findings:
Sheep Studies in New Zealand: The prevalence of Toxoplasma in
New Zealand sheep is very high. The distribution of parasites in the
tissues of sheep is erratic. Parasitemia was found only in the first
week of infection with two strains of Toxoplasma given by various routes.
Toxoplasma in the sheep is rapidly cleared from tissues except for muscle
and placenta. Residual infection in sheep is more likely to persist in
muscle than in brain. Mutton can therefore definitely serve as a source
of human infection.
The following statements concerning the circumstances of con-
genital transmission appear justified from data obtained, although not
yet complete: Infection 60 days prior to onset of pregnancy does not
result in congenital infection or abortion. Abortion or foetal death
and resorption occurs following infection of the ewe at 30 days
pregnancy. Infection at 90 days pregnancy, by various routes, sometimes
results in abortion or death of the lamb soon after birth. In other
cases, the lamb survives, although the foetal cotyledons are infected.
Natural immunity or active immunity produced by vaccination with live
parasites prior to mating does not protect ewes from Toxoplasma abortion
when the challenge inoculum is high. When congenital transmission does
occur, only one of twin lambs may be affected. The foetal cotyledons are
more consistently infected than are the tissues of the lambs.
Studies at N.I.H: The failure of immunization with live organisms
completely to protect animal from a challenge infection has been demon-
strated in further tests in guinea pigs.
Further studies have been conducted on parasitemia in chronically
infected mice and additional work has been done on rabbits. Both species
may exhibit low grade parasitemia for months. Since these animals have
high antibody levels, it is difficult to explain the persistent activity
of the parasite.
Various domestic animals have a heat-labile, non-specific, anti-
Toxoplasma activity in their serum. It has been found also in human
sera. The use of citrate in the collection of accessory factor sera for
the dye test diminishes or eliminates this non-specific activity.
A serological survey of cattle sera indicates that in these animals
there is considerable difference in hemagglutination and dye test titers.
53
Serial No. NIAID-127-A
Ultra violet absorption at 260 and 280 millimicrons shows a
relatively large amount of nuclear protein material in the hemagglutina-
tion antigen. Agar gel diffusion with the hemagglutination antigen and
its rabbit antiserum indicates at least two separate antigen-antibody
systems are present.
The complement-fixation test for toxoplasmosis has been set up
as an additional diagnostic procedure.
Use of pyrimethamine in mice with chronic infections has reduced
the number of cysts. This has important implications, possibly, relative
to control of toxoplasmic chorioretinitis. The same effect has been
observed with sulfadiazine.
Cysts of Toxoplasma have been found at all levels of the intes-
tinal tract and in the lung of chronically infected mice three to four
months after infection. There is evidence that cysts of Toxoplasma form
in tissue cultures. Suspensions of cultures have infected mice after
pepsin-HCl digestion, a treatment which kills proliferative forms.
Significance to the Program of the Institute:
Knowledge of the transmission of Toxoplasma in nature is requisite
as a basis for recommendations for prevention of the infection. Continued
work is necessary to explain mechanisms of transmission to herbivores
and vegetarian human beings.
The analysis of antigens derived from Toxoplasma has importance
in relation to an understanding of the course of the disease and the
action of various antibodies, as well as diagnosis. Continued work on
the hemagglutination test may establish it as the best practicable pro-
cedure for diagnosis of human toxoplasmosis.
Extensive and intensive basic study of Toxoplasma infection will
furnish explanations for clinical observations and contribute to a
general understanding of chronic infections. This is of great importance
in relation to ocular disease and may also reveal instances of chronicity
in systemic infections.
Proposed Course of the Project:
Attention will be paid to: the epidemiology of toxoplasmosis in
herbivorous animals; the circumstances of cyst-formation and rupture in
chronic infections; the relation of cysts and dormant parasites to
chronic disease; the cultivation of cysts in tissue culture; the mechanism
of proliferation of Toxoplasma in cells in tissue cultures; the relation
of antibodies to particular antigenic components and to persistence of
5h
Serial No. NIAID-127-A
chronic infections; the effect of drugs on cyst and on activity of the
parasite during chronicity; the relation of local tissue immunity to
the spread of challenge infections; and the significance of non-specific
ant i -Toxoplasma activity of serum.
Part B included Yes
55
Serial No. NIAID-127-A
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Jacobs, Leon, Remington, Jack S., and Melton, Marjorie L. The resistance of
the encysted form of Toxoplasma gondii. J. Parasitol. 46: 11-21. (1960)
Jacobs, Leon, Remington, Jack S., and Melton, Marjorie L. A survey of meat
samples from swine, cattle, and sheep for the presence of encysted toxoplasma.
J. Parasitol. 46: 23-28 (1960)
Remington, Jack S. , Jacobs, Leon, and Kaufman, Herbert. Toxoplasmosis in the
adult. New England Journal Medicine 262: 180-186, 237-241. (1960)
Frenkel, J. K., Weber, R. W., and Lunde, M. N. Acute toxoplasmosis. Effective
treatment with pyrimethamine, sulfadiazine, leucovorin calcium, and yeast.
J.A.M.A. 173: 1471-1476. (1960)
Jacobs, Leon. Ocular toxoplasmosis: Laboratory contributions to diagnosis
and chemotherapy. Reprinted from Human Toxoplasmosis Copenhagen: Munksgaard.
(1960)
Remington, Jack S., Jacobs, Leon, and Melton, Marjorie L. Chronic toxoplasma
infection in the uterus, (in press) J. Lab and Clin. Med.
Remington, Jack S., Jacobs, Leon, and Melton, Marjorie L. Congenital transmis-
sion of toxoplasmosis from mother animals with acute and chronic infections.
J. Inf. Dis. (in press)
Honors and Awards:
Dr. Leon Jacobs was awarded a Fulbright Fellowship for study of problems
in the epidemiology and pathology of toxoplasmosis in New Zealand. He departed
on this assignment May 1.
Dr. Leon Jacobs visited Brisbane, Sydney, and Melbourne from 10 to 24
October on a visiting lecturer under the Australian Fulbright Program. He
lectured in universities, hospitals and veterinary research institutions.
Dr. Leon Jacobs was awarded a Guggenheim Fellowship which will permit him
to visit a number of Asian and European parasitology laboratories on his return
trip from New Zealand.
56
Serial No. NIAID-127-B
1. Parasitic Diseases
2. Protozoal Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Studies on Entamoeba histolytica and other
parasitic protozoa.
Principal Investigator: Louis S. Diamond
Other Investigators: Harry D. Baernstein
Lucy V. Reardon
Cooperating Units: Biophysics Laboratory, Naval Institute for
Medical Research.
Man Years (calendar year 1960):
Total 2%
Professional: 1J£
Other: 1
Project Description:
Objectives:
To study metabolism of E. histolytica, methods of diagnosis,
mechanisms of pathogenesis, factors in host susceptibility, methods of
culture, immunology, and variation. To study the cultural requirements
of axenically cultivated Entamoeba spp. of lower animals in order to
extend our knowledge of the biology of these important parasites. To
devise a method for the axenic cultivation of Trichomonas tenax, the
oral trichomonad of man. To develop techniques for the preservation of
parasitic protozoa by freezing and storage at low temperatures and by
freezing and storage in the dry state.
Methods Employed:
Usual methods for axenic studies and for use of single bacterial
or protozoan associates; micro-isolation methods; immunological methods,
including adsorption and extraction. For preservation studies, protozoa
are suspended in a variety of suitable agents and cooled slowly from
ambient temperatures to -25° C, then stored at this temperature or at
temperatures of -79° C and -197° C; or the protozoa are frozen rapidly
in vacuo at a temperature of -30° C, then stored at -197° C; or protozoa
are supercooled, frozen and dried i_n vacuo.
57
Serial No. NIAID-127-B
Major Findings;
Axenic cultivation of E. histolytica and T. tenax was achieved in
an anaerobic medium supplemented with a cell free extract of chick embryo.
Neither species grew in the absence of the extract. Furthermore, growth
of E. histolytica occurred only when the medium was made up in a diphasic
form, i.e., a solid agar slant with a liquid overlay.
A substitute for T. cruzi as an associate in the monoxenic culti-
vation of E. histolytica was found in the form of a new species of
Crithidia isolated from the gut of a hemipteran. The new associate in-
creases the yield of amoebae by a factor of 4, does not require a separate
medium for maintenance of stock cultures, and is not known to be a para-
site of man.
Several batches of antigen have been prepared from monoxenic E.
histolytica - T. cruzi cultures.
Studies on the cultural requirements of E. inyadens resulted in
the following: a) development of a technique for counting the amoebae
with a Coulter Electronic counter; b) the finding that the amoebae re-
acted adversely to any change in concentrations of trypticase, yeast
extract or horse serum; c) that yeast extract could not be replaced by
Eagle's vitamin B mixture.
There has been progress toward development of a method for preser-
vation of E. histolytica and other protozoan species by freeze-drying, but
so far it has not been possible to store the freeze-dryed material success-
fully and still preserve viability.
Attempts to preserve the protozoans by freezing without drying
had been more successful. The slow freezing and subsequent storage at
-79° C of T. vaginalis, T. hominis, T. gallinae, and E. histolytica
suspended in dimethyl sulfoxide has resulted in preservation, to date,
of these protozoa for periods of 4 months, 3 months, 30 and 29 days
respectively. E. histolytica cooled to a temperature of -197° C by a
similar technique has been stored successfully at this temperature for
a period of 24 hours. This last finding is most encouraging, since
theory predicts that a cell capable of withstanding exposure to -197° C,
even for such a short time, should be capable of almost indefinite
storage at such temperatures.
Significance to the Program of the Institute:
The development of a technique for growing E. histolytica under
axenic conditions should open the way to the solution of many unsolved
problems in amoebiasis. It should be possible, for example, to determine
whether or not £. histolytica, by itself, is capable of initiating the
53
Serial No. NIAID-127-B
lesions of araoebiasis; to prove or disprove the existence of lytic
substances of amoebic origin; and to determine growth requirements in
vitro. Solution of the technical problems associated with axenic culti-
vation of the amoebae and T. tenax should find application in solving
similar problems met with in the cultivation not only of other parasites,
but mammalian cells as well.
Techniques developed for the preservation of parasitic protozoa
by freezing and freeze-drying should be applicable to preservation of
other types of cells.
Development of a technique for enumerating protozoa in culture
with the aid of the Coulter Electronic counter has extended the use of
this instrument, which was originally designed to count blood cells, and
gives indication that this instrument could be utilized profitably in
counting mammalian cells from tissue culture.
Proposed Course of Project:
Efforts will be made to define and improve media employed in
axenic cultivation of E. histolytica and T. tenax; to develop methods
for axenic cultivation of other parasitic protozoa of man; to develop
more sensitive antigens for serological diagnosis; to study factors
associated with invasiveness and pathogenicity of strains of E. histo-
lytica in experimental hosrs; to study cytochemical changes during
division of E. histolytica; to refine techniques for preservation of
protozoa by freezing and freeze-drying.
Part B included No
5S
Serial No. NIAID-127-C
1. Parasitic Diseases
2. Protozoal Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Trichomoniasis
Principal Investigator: Lucy V. Reardon
Other Investigators: Louis S. Diamond
Cooperating Units: Laboratory of Clinical Investigations, NIAID
Laboratory of Germfree Animal Research, NIAID
Laboratory of Pathology and Histochemistry, NIAMD
Clinical Investigations Branch, NIDR
Man Years (calendar year 1960):
Total: 1-3/4
Professional: 3/4
Other: 1
Project Description:
Objectives:
Continued study of the pathogenesis of Trichomonas vaginalis;
transfer of genetic characters in strains of T. vaginalis; antibody for-
mation and hypersensitivity in relation to resistance and pathogenesis;
the action of purported trichomonacides (as well as amoebacides) as
determined by in vitro tests; the possible relation of oral protozoa to
peridontal disease.
Methods Employed:
Pathogenesis/trichomonads is tested in mice by intraperitoneal
injection of concentrated organisms. Microbe-free cultures, maintained
without use of antibotics, furnish the inocula.
Pathogenesis is also studied in germfree guinea pigs. Microbe-
free material is injected subcutaneously into germfree pigs, monocon-
taminated pigs, and conventional pigs. The effect of the sera from the
above guinea pigs on washed concentrates of trichomonads is observed
microscopically.
60
Serial No. NIAID-127-C
For studies on genetic transfer, organisms of the non-virulent
R strain are associated with horaognates of the virulent C-l strain
processed to contain DNA of the strain and then injected into mice for
evidence of alteration of characters. Genetic transfer is also attempted
through efforts to produce an aureomycin resistant strain of the organism.
The haemagglutination test is used for evidence of antibody production.
Human sera and vaginal exudates are likewise tested. Evaluation of pur-
ported trichomonacides (as well as amoebacides), is made by i_n vitro
tests. Diseased and normal tissues from patients with peridontal
disease are examined for oral protozoa, both by direct examination of
the material and by cultures.
Major Findings:
Virulent and avirulent T. vaginalis were retested in mice after
having been in continuous in vitro cultivation 4 and 5 years respectively.
No change in virulence was observed.
Experimental studies of germfree guinea pigs, monocontaminated
pigs (as well as conventional pigs) showed as in previous experiments,
large, palpable swellings or abscesses occurring at the site of in-
jection in germfree pigs, less marked swelling in the monocontaminated
pigs, and none in conventional pigs. Germfree pigs, rechallenged with
trichomonads after subsidence of swellings, failed, in one experiment,
to develop new swellings. Sera from germfree or monocontaminated pigs
appeared to have a deleterious effect on the trichomonads. Sera from
the conventional pigs had no effect on the trichomonads other than to
cause agglutination.
Additional experiments on transformation of virulence by assoc-
iation of the non-virulent strain with DNA of the virulent, showed no
evidence of such transformation.
The preparation of T. vaginalis antigens was continued. Pro-
duction in guinea pigs of high titers of haemagglutinins against T.
vaginalis demonstrated the usefulness of whole organisms as a source of
antigen. Haemagglutinins against an enzyme preparation of sonicated
T. vaginalis were also produced in guinea pigs. These enzyme haemag-
glutinins tested in assay system for malic dehydorgenase showed decided
inhibitory action against this enzyme.
Humatin, (Paromomycin sulfate), failed to cause complete inhi-
tition of activity of T. vaginalis in concentrations of the drug up to
800 ug/ml of medium. E. histolytica in association with Trypanosoma
cruzi was not inhibited in concentrations of the drug up to 64 ug/ml.
However, in association with a Crithidia (an insect flagellate) as its
supporting organism, E. histolytica appeared inhibited in concentrations
of 32 and 64 ug/ml but not in lower concentrations. The Crithidia was
likewise not inhibited in the lower concentrations of the drug.
61
Serial No. NIAID-127-C
Significance to the Program of the Institute.
Such studies should give new basic information on the trichomonads
and lead to a better understanding of their pathogenecity, variation,
transmission, life history, etc.
Proposed Course of Project:
Attempts will be made to induce haemagglutinis against individual
enzymes of T. vaginalis and to develop a hemagglutination test for tri-
chomoniasis. Transformation and strain studies will be continued.
Part B included No
62
Serial No. NIAID-127-D
1. Parasitic Diseases
2. Protozoal Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Biochemistry of parasitic protozoa.
Principal Investigator: Harry D. Baernstein
Other Investigators: L. V. Reardon
L.S. Diamond
Cooperating Units: None
Man Years (calendar year 1960):
Total: 3/4
Professional: 3/4
Other: None
Project Description:
Objectives:
To study the biochemistry of the parasitic protozoa: especially
the intracellular enzymes and biochemical problems related to axenic and
monoxenic cultivation.
Methods Employed:
Cultivation of various species and evaluation of growth rates in
relation to composition of the medium by use of the Coulter Counter.
The isolation and purification of intracellular enzymes by con-
ventional precipitation methods and by electrophoresis, and spectro-
photometry methods to evaluate activities related to the concentration
of protein present. Relations of the organisms to their hosts are
studied by analysis for antienzymes and other antibodies developed in
serum against purified antigens.
Major Findings:
Studies on the enzymatic activation of acetate in T. vaginalis
showed the presence of such activation as determined by hydroxamic acid
production. However, the type of hydroxamic acid produced must await
purification of the enzyme and identification. Preliminary experiments
on acetylation of sulfanilamide were negative.
63
Serial No. NIAID-127-D
Significance to the Program of the Institute:
The elucidation of biochemical properties of these parasitic
organisms should help us understand their pathogenicity and their
susceptibility to drugs. Specific enzyme-inactivating antibodies can
be useful in the study of enzyme function and differentiation and identi-
fication of enzymes. The techniques developed for producing these anti
enzymes should be applicable in the study of enzymes of a variety of cells.
Proposed Course of Project:
Analysis of the complex components of media employed in cultiva-
tion of parasitic protozoa. Analysis of the enzyme systems present in
the organisms. Better preparations with fewer interfering enzymes
should result by application of electrophoresis. The problem of multiple
enzymes having the same substrate specificity (isoenzymes) will be
studied. The special biochemical problems associated with anaerobiasis
such as electron transport will be explored.
Part B included Yes
Oh
Serial No. NIAID-127-D
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Baernstein, H. D. : Malic dehydrogenase of Trichomonas vaginalis.
J. Parasitol. (in press).
Honors: None
Awards: None
LABORATORY OF PARASITE CHEMOTHERAPY
/-"
/
Summary 1
130 - Administration, Research Planning and
Coordination 6
130-A - Studies in Human Malaria 10
131 - Drug Resistance in Experimental
Malaria 15
131-A - The Effect of Adrenal Cortical
Hormone on Chemotherapy of
Experimental Malaria 17
131-B - Antimalarial Drugs and Nucleic Acid
Synthesis 19
131-C - Experimental Chemotherapy of
Helminthic Diseases 22
131-D - Effects of Nutrition on Chemotherapy
of Parasitic Diseases 25
131-E - Comparison of Antimalarial Drugs
Administered Singly and in Various
Combinations 28
131-F - Effect of Nutritional Status of Host
on the Action of Antimalarial Drugs.. 30
132 - Studies on Human Malaria 32
132-A - Chemotherapy of Intestinal Parasites. 37
132-B - Physiology of Human Parasites
Especially as Related to Nucleic
Acids and Drug Action 40
132-C - Virus-Parasite Association 43
132-D - Development of Human Pathogens in
Immature Insects 47
132-E - Insect Tissue Culture 49
133 - Studies of the Biology of
Exoerythrocytic Phases of Primate
Malaria 51
133-A - Chemotherapeutic Studies of the
Direct Effect of Drugs on the
Exoerythrocytic Stages of Primate
Malaria 54
133-B - Studies of Simian Malarias in Man
to Determine if Malaria is a Zoonotic
Disease in Areas in which Man and
Monkey are Closely Associated 57
133-C - In Vitro Studies of Malaria Parasites
and Liver Tissue 61
133-D - Immunological Studies of Malaria
Parasites 66
Laboratory of Parasite Chemotherapy, NIAID
Summary Statement of Research Activities, Calendar Year 1960
This Country's commitment of 38 million dollars in Fiscal Year 1961
toward a program of world-wide malaria eradication and the long-term interest
in malaria by most of the senior staff resulted in a research effort, during
the past year, largely directed toward problems in that field. Special
emphasis was given to the study of simian malaria in man and in monkeys be-
cause malaria in simians might be a real deterrent to the eradication program.
Clinical facilities at the Atlanta Penitentiary were enlarged and the staff
increased. A laboratory was established at Kuala Lumpur, Malaya in cooperation
with the Malaya Institute of Medical Research and the United States Medical
Research Unit. Studies on several aspects of the simian-human-malaria problem
have been in progress there since mid-August.
As a result of the above development, it was decided to move the Section
on Cytology, now located at Memphis, to Chamblee, Georgia, early in 1961.
Alterations necessary to accommodate this move are nearing completion. This
arrangement will bring the simian hosts closer to the human volunteers at the
penitentiary, and the insectary maintained by the Section will be geared to
accommodate the work at Chamblee and at the prison.
Dr. Jeffery of the Section on Epidemiology is spending a year in
graduate study at Yale University and therefore the Section has been without
his services since July. The program of the Laboratory was reviewed by the
Board of Scientific Councilors who visited the Laboratory at Columbia, November
9-10.
The work of the Section on Chemotherapy has been curtailed due to two
resignations, Drs . Gaudette and Schellenberg, during the past year. During
this period, Dr. Jacobs joined the staff of that Section and has initiated a
program of research aimed at an assessment of the place of nutrition in
chemotherapy.
MALARIA - HUMAN Plasmodium falciparum (McLendon strain) : Chloroquine
(300 mg, base) and primaquine (45 mg, base) given together
beginning 3 days after mosquito bites and weekly thereafter for a total of 8
doses, resulted in suppressive cure in 5/5 subjects. Controls were positive
11-15 days after infection. After 2 days of parasitemia, each control was
given the above drug combination which was repeated weekly for a total of 3
doses. Parasites were removed promptly and cure was obtained based on no
evidence of infection during 227 days of observation.
Primaquine, at daily doses of 0.75 mg, had some sporontocidal effect
upon Plasmodium falciparum gametocytes but none against those of P. vivax
(one case). Therapeutic doses (1.4 gm in 3 days) of amodiaquine had no
sporontocidal effect against gametocytes of P. falciparum (one case). The
effect referred to is against the development of the malaria parasites in the
mosquito.
A strain of Plasmodium falciparum from Colombia, South America, was
found to be resistant to chloroquine. This finding is of utmost importance
in terms of malaria eradication.
Plasmodium vivax (Chesson strain): A drug combination of primaquine
(45 mg) and pyrimethamine (50 mg) given weekly beginning 7 days after mosquito
bite and continuing for a total of 4 doses, gave suppressive-cure in 4/5
subjects; the other subject developed a patent infection 240 days after infec-
tion. Pyrimethamine (50 mg) given alone, as above, produced suppressive-cure
in 1/4 subjects; the other 3 came down on days 82, 83 and 84. Five controls
all came down 12 to 13 days after infection.
The Russian 8-aminoquinoline, qulnocide, was compared with primaquine
and found to be distinctly inferior as a curative drug against early and late
primary attacks of Chesson vivax malaria particularly from the standpoint of
the occurrence of second and third relapses.
Another 8-aminoquinoline, Win 5037, was studied in 5 subjects. Toxic
effects and failure to cure made further investigation unwarrented.
Plasmodium malar iae: The results of a 14-year study of the biology
of Plasmodium malariae were drawn together for publication. The highest
infect ivity for mosquitoes occurred during the 8th to 10th weeks of the
primary attack. Although the infection rate of mosquitoes was ordinarily low,
the relatively long period during which mosquitoes could be infected may ex-
plain the persistence of P. malariae in nature. The ability of the symptom-
free malarious patient to infect mosquitoes at a rate similar to that of the
symptomatic patient makes eradication difficult.
MALARIA - SIMIAN Plasmodium cynomolgi bastianellii: In early May, two
accidental sporozoite-induced infections with Plasmodium
cynomolgi bastianellii occurred at our Memphis Laboratory. This happening
was of signal importance because it showed that simian malaria, contrary to
the generally held opinion, was infectious to man. In that light, full scale
study of human infections was undertaken at our Atlanta Penitentiary installa-
tion.
Two infections were induced in inmate volunteers by inoculation of in-
fected blood obtained from one of the accidental sporozoite-induced infections
in man. Twenty inmate volunteers were infected by bites of Anopheles
quadrimaculatus or Anopheles freeborni which had fed on infected monkeys. The
prepatent period ranged from 14 to 29 days and the parasite density ranged from
5 to 500/cmm. The most constant symptom was headache and the most significant
signs were fever, splenomegaly and hepatomegaly. Infections were allowed to
run their course, generally without treatment.
Anopheles freeborni were infected from two patients but attempts to
infect volunteers by their bites have yielded equivocal results. The finding
that P. c. bastianellii will grow consistently and produce clinical illness in
man suggested the possibility that malaria is a zoonotic disease, that is, a
disease which man can acquire from animals with which he is associated. Whether
2
or not such transfer occurs in nature is not yet determined, but should it
occur, it would be of greatest significance to the world-wide malaria eradica-
tion program.
Plasmodium cynomolgi cynomolgi: Eleven inmate volunteers were bitten
by Anopheles freeborni infected with P. c_. cynomolgi on 8 September, and to
date (14 December) three have exhibited evidence of infection (i.e., fever).
Parasitemia has been demonstrated in only one, on the 58th day after mosquito
bites. These results show that this strain infects man far less readily than
P. c. bastianellii.
FIELD STUDIES Three staff members, Drs. Eyles, Dobrovolny, and Mr. Clinton
IN MALAYA S. Smith, were detailed to Malaya during the year where they
engaged in the study of simian and human malaria in coopera-
tion with the Malayan Institute for Medical Research and the U. S. Army Medical
Research Unit at Kuala Lumpur.
The epidemiology of monkey malarias is being studied and the feeding
habits of some of the Anopheles determined. By injection of uninfected
monkeys with sporozoites from natural infections, it was determined that
Anopheles hackeri is a natural vector of Plasmodium knowlesi. This is a most
important discovery, especially since the vector of this parasite has been
sought for repeatedly during the last 25 years.
Studies of malaria in aborigenes associated with monkeys have been
made. Blood passed from aborigenes to monkeys have thus far produced no
patent infection in the monkeys.
EE STAGES AND Studies were continued on the direct effect of drugs on the
DRUG ACTION exoerythrocytic stages of primate malaria. When sulfonamides
were used with pyrimethamine to exploit the possible syner-
gism of the two drugs, monkeys developed parasitemia 30 to 40 days after
inoculation with sporozoites even though all parasites observed in liver
biopsies were damaged. The curative efficacy of quinocide, the Russian drug,
was compared with primaquine. Even when administered at twice the dosage
used with primaquine, quinocide was less effective. Chloroquine had no
observable effect upon the liver forms of Plasmodium cynomolgi. Young para-
sites appeared in the blood in large numbers on the 8th, 16th and 24th day
indicating the existence of secondary exoerythrocytic generations.
INSECT TISSUE Blood cells from caterpillers and cells of the ovariole
CULTURE sheath of several species of moth pupae have been cultivated
in several different media. The virus of St. Louis encepha-
litis has been maintained in cultures of hemocytes from larvae of the catalpa
sphinx for 10 days. Oocysts of Plasmodium gallinaceum attached to the midgut
of Aedes aegypti have shown growth in vitro and sporozoites have been produced.
BIOCHEMICAL STUDIES It was shown that mosquitoes infected with malaria
have higher levels of ribonucleic acid than uninfected
mosquitoes. Chromatographically, the acid-hydrolysate of ribonucleic acid
from a pyrimethamine-resistant strain of Plasmodium falciparum differs from
the acid-hydrolysate of ribonucleic acid from a pyrimethamine-susceptible
strain. Bephenium hydroxynaphthoate inhibited glutamic acid transaminase of
Nippos trongy lus muris . Bephenium chloride and quinacrine reduced the rate of
glucose absorption by the tapeworm Hymenolepis diminuta but low concentrations
of dithiazanine iodide stimulated glucose absorption by this cestode.
INTESTIONAL PARASITES Epidemiological studies on the inmates of a mental
institution show a high persistence of Trichuris
and hookworm for six years, with an apparent decrease in Strongyloides . To
test dithiazanine and tetrachlorethylene, alone and in combination, heavily
parasitized mental patients were given the drugs for about one year. A large
number of worms were removed but the cure rate was low and transmission was
not stopped. Bephenium hydroxynaphthoate and bephenium chloride were used with
good results against hookworm, Ascaris and Trichuris .
SCHISTOSOMIASIS The activity of griseofulvin observed in mice infected with
Schistosoma mansoni was not well developed in hamsters or
monkeys. A series of tetracycline analogues which show an affinity for micro-
filaria did not combine with schistosomes and were without activity. One of
these analogues was significantly more active against microfilariae of Diro-
f ilaria immitis than tetracycline.
In many tests, the efficacy of stibophen (Fuadin) therapy on mature
Schistosoma mansoni infections in mice was increased up to 16 times by feeding
a balanced semi-synthetic diet. The toxicity of the drug was not similarly
increased. The enhancement of curative action by the purified semi-synthetic
diet was thought to be due to the absence of, as yet unidentified, inorganic
salt(s) that interfere with drug activity. It was found in mice fed on the
purified semi-synthetic diet that higher blood levels of the drug were main-
tained for a longer period than when the same amount of Fuadin was injected
into mice fed on the commercial pellet diet, suggesting that the increased
cure-rate was due to higher blood drug level. Similar drug advantage was
observed in mice given tartar emetic while on the purified diet.
DRUG COMBINATIONS Various combinations of primaquine, chloroquine, amodia-
quine, and pyrimethamine proved to be no more effective
than single drugs against blood induced Plasmodium berghei in mice.
NUTRITION AND MALARIA An intensive study of the influence of nutritional
deficiencies on the activity of antimalarials has been
initiated.
ADRENAL HORMONE The influence of cortisone on antimalarial activity was
AND MALARIA investigated in Plasmodium berghei and Plasmodium gallina-
ceum. The drugs used were primaquine, chloroquine, and
pyrimethamine. The only combination in which cortisone exerted an adverse
effect on antimalarial action was primaquine and Plasmodium gallinaceum.
In Plasmodium berghei, cortisone has a slight synergistic effect when given
concurrently with antimalarial drugs.
Serial No. NIAID-130
1. Parasite Chemotherapy
2. Office of the Chief
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Administration, research planning and
coordination
Principal Investigator: G. Robert Coatney
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960) :
Total: 1 3/4
Professional: 3/4
Other: 1
Project Description:
This project furnishes certain technical, supervisory, and administrative
services to all research projects in the Laboratory, as follows:
(a) Over-all planning of research and the coordination of research
activities in the various Sections of the Laboratory.
(b) Integration of Laboratory research activities with clinical studies
of the Clinical Center.
(c) Supervision over personnel, maintenance, travel, and correspondence.
(d) Requisitioning and supervision of equipment and supplies,
(e) Preparation of reports, budget estimates, and exhibits and other
public relations materials.
(f) Maintenance of reference library, reprint files, card catalogues,
and specimen collections.
(g) Consul tatory services to individuals, commercial organizations,
nongovernmental organizations, Government agencies including liaison activities
with other branches of the Service, foreign governments, and international
agencies .
Serial No. NIAID-130
(h) Reviewing, editing, and revising scientific and technical reports and
manuscripts.
Progress on research projects is reported quarterly and, when necessary,
are discontinued or revised in order to meet current objectives. During the
past year some changes have been made in the research program of the laboratory,
impetus given to others, and certain activities directed into new channels.
The discovery that simian malaria was infective to man brought about changes
in three directions: (1) Plans were laid for moving the Memphis activities to
Chamblee, Georgia in order to have a flourishing insectary and the simian hosts
close to the inmate volunteers at the Atlanta prison, (2) A laboratory was
established at Kuala Lumpur, Malaya, to carry out investigations in the field
relative to all aspects of the simian-human-malaria chain, and (3) Studies
in human volunteers at the Atlanta prison were tailored to an all-out study
of simian infections in man. Continued emphasis was placed on the fundamental
aspects of other problems in tropical parasitology and chemotherapy. These
included the culture of insect tissues, intensive and extensive study of the
development and chemotherapy of fixed tissue stages of certain mammalian
malarias, the tissue culture of mammalian malarias, the mechanism of drug
action on parasites and the inhibiting effect of antimetabolites, the effect
of nutrition on chemotherapy, drug resistance by parasites, chemotherapy of
intestinal parasites, and the carriage of viruses by parasites.
The cooperative study with Dr. John Tobie, LI (Ser. No. NIAID-148), on the
application of fluorescent antibody techniques to the staining of human Plas-
modia for mass screening with a mechanical scanner was continued.
Part B included - Yes
Serial No. NIAID-130
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Gaudette, Leo E. and Coatney, G. R. Stability of primaquine diphosphate
under various conditions. Am. J. Trop. Med. & Hyg. 9_: 532-535, 1960.
Coatney, G. R. , Elbel, R. E., and Kocharatana, P. Some blood parasites
found in birds and mammals from Loei Province, Thailand. J. Parasitol. 46:
701-702, 1960.
Coatney, G. R. and Greenberg, J. The effect of a diet deficient in
Factor 3 on the course of Plasmodium berghei infection in mice. Proc. . VI
Internatl. Congress Trop. Med. &Mal. (in press).
Coatney, G. R. and Hinman, E. H. Malaria. Cyclopedia of Medicine,
Surgery and Specialties (in press).
Honors and Awards relating to this project:
Visiting Professor, Department of Preventive Medicine and Public Health,
School of Medicine, Howard University.
Visiting Lecturer on Tropical Public Health, Harvard School of Public
Health.
Visiting Lecturer on chemotherapy, Malaria Eradication Training Center,
Kingston, Jamaica.
Consultant to Chief, Section on Malaria Eradication, Pan American
Sanitary Bureau.
Vice President, American Society of Tropical Medicine and Hygiene.
President-Elect, American Society of Tropical Medicine and Hygiene.
Vice Chairman for the meeting of the Expert Committee on Malaria held in
Geneva, Switzerland, 25-30 July 1960.
Chairman, Technical Meeting on Malaria, Geneva, Switzerland, 14-19
November 1960.
Member of the Expert Advisory Panel on Malaria of the World Health
Organization.
Serial No. NIAID-130
Consultant to the Director, Division of Malaria Eradication, World Health
Organization, Geneva, Switzerland, on the place of drugs in malaria eradication
in Africa, and in that capacity, visited Liberia, French Equitorial Africa,
Belgian Congo, Zanzibar, Tanganyika, Southern Rhodesia, and Geneva, during
March and April, 1960.
Serial No. NIAID-130-A
1. Parasite Chemotherapy
2. Office of the Chief
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Studies in human malaria
Principal Investigator: G. Robert Coatney
Other Investigators: Morton E. Getz, Henry K. Beye, Harvey Elder,
Don E. Eyles, and Martin D. Young
Cooperating Units: Department of Justice, Bureau of Prisons, and
Institute for Medical Research, Christ Hospital,
Cincinnati, Ohio
Man Years (calendar year 1960) :
Total: 3 3/4
Professional: 1 1/4
Other: 2 1/2
Project Description:
Ob jectives :
1. To evaluate new antimalarial drugs and/or drug combinations and
methods of administration applicable to mass chemotherapy as applied to
malaria eradication.
2. To study the clinical manifestations and parasitology of simian malaria
infections in man with special emphasis on Plasmodium cynomolgi bastianellii.
Methods Employed: Normal inmate volunteers at the Federal Penitentiary,
Atlanta, Georgia, are screened before they are admitted to the project.
Volunteers are under the care of the resident investigators whether handled
as outpatients or as inpatients. They take drugs as prescribed, submit to
necessary laboratory procedures and to infection by bites of infected mosquitoes.
Participation may be for 6 weeks to 2 months or up to one year depending on the
nature of the project.
Major Findings:
McLendon strain of falciparum malaria.
Chloroquine (300 mg, base) and primaquine (45 mg) given together in a
10
Serial No. NIAID-130-A
single oral dose beginning day 0-3, and weekly thereafter for a total of 8
doses, resulted in suppressive cure of 5/5 subjects. Controls were positive
11-15 days after infection. After 2 days of parasitemia, each control was
given the above drug combination as a single dose and then weekly for a total
of 3 doses. Parasites were removed promptly and cure was obtained based on no
evidence of infection following 227 days of observation.
Chesson strain of vivax malaria.
Primaquine, given 30 mg weekly (single dose) beginning day 0-1 and con-
tinuing on the same day each week and, to another group, at 3 mg daily starting
day of infection, failed to prevent or suppress infection. Treated and control
patients all exhibited patent infections 9 to 12 days after infection.
A drug combination of primaquine (45 mg) and pyrimethamine (50 mg) given
in a single dose weekly, beginning on day 0+7, and weekly thereafter for a
total of 4 doses, gave suppress ive-cure to 4/5 subjects; the other subject
developed a patent infection 240 days after infection. Pyrimethamine (50 mg)
given alone, as above, produced suppress ive-cure in 1/4 subjects; the other 3
came down on day 82, 83, and 84, respectively, after infection. The controls
(5) all came down 12 to 13 days after infection.
An 8 - amino quino line allied to primaquine and known as CN1115 and Win
10,448 in the United States and as quinocide by the Russians was purported to
produce radical cure and result in less toxicity than primaquine. The effec-
tive dosage of each drug was supposed to be the same, 15 mg daily x 14.
Primaquine compared with quinocide against sporozoite-induced
Chesson strain P. vivax; each drug at 15 mg daily x 14 after
600
mg base chloro
quine
Type of
attack
1st relapse
(Days)
2nd relapse
(Days)
3rd relapse
(Days)
Days of
observation*
PRIMAQUINE
E. P.
D. P.
1/5 (48)
1/5 (35)
1/5 (250)
281-446
401-593
QUINOCIDE
E. P.
D. P.
3/4 (7-356)
2/4 (32-140)
3/4 (21-218)
1/4 (161)
2/4 (46-68)
405-446
443-446
11
Serial No. NIAID-130-A
E. P. = early primary attack
D. P. = delayed primary
( ) = days to relapse since last Rx
* = as of 1 December 1960
The results show that primaquine is distinctly superior to the other
compound.
Reports on a new 8 - amino quino line, 6 methoxy-8(5 propylaminoamylamino)
quinoline phosphate, Win 5037, indicated that this drug was as therapeutically
effective as chloroquine and as effective as primaquine in producing cure.
At dosages of 20 mg b.i.d. for 2 days, and then 10 mg daily x 12, and at 30
mg b.i.d. x 2, and then 20 mg daily x 12, the parasites were removed and the
fever subsided almost as rapidly as following 600 mg of chloroquine, but the
entire regimen did not produce cure. Those on regimen 1 (3 pts) relapsed
after 35 to 45 days and were re-treated on two occasions; at neither time was
the blood cleared of parasites. Those on regimen 2 (2 pts.) each relapsed on
day 103 and were cured with chloroquine and primaquine. Because of toxic
effects, failure to cure, and drug resistance after the initial trial, further
investigation was not considered warranted.
Simian malaria, Plasmodium cynomolgi bastianellii.
In early May, two infections were induced in inmate volunteers by inocula-
tion of infected blood obtained from a sporozoite-induced accidental infection.
Following these infections, individuals (whites and Negroes) have been infected
by bites of infected Anopheles quadrimaculatus or Anopheles freeborni fed on
infected monkeys. In cases where typical parasites could be demonstrated, the
prepatent period ranged from 14 to 29 days. The parasite density ranged from
5 to 500/cmm. In some patients, parasitemia was demonstrated before the onset
of symptoms. The most frequent symptom was headache, followed in decreasing
frequency by anorexia, abdominal pain, joint pain, nausea, myalgia, back pain,
vomiting, chest pain, chills, and cramping. Headache was generally described
as an aching, bilateral, frontal pain, fairly persistent and often extending
to the back of the head and down the back and the lateral aspects of the neck.
There was often muscle tenderness in the head and neck. Abdominal pain was
often in the right and left lower upper quadrants. It occasionally was in-
creased on deep inspiration. The most significant findings were splenomegaly
and hepatomegaly usually associated with tenderness. In those patients ex-
hibiting a tertian fever, the pattern was initiated between the 19th and 23rd
day; there was no correlation between parasitemia and febrile response.
Observations to date would show no febrile response after the 75th day of
infection (based on 24 patients).
Anopheles freeborni were infected after feeding on two different patients
with gametocytes as evidenced by finding normal looking oocyts on the gut and
'normal' sporozoites in the salivary glands, but attempts to infect volunteers
12
Serial No. NIAID-130-A
by mosquito bite have so far yielded equivocal results. One patient became
patent after 58 days but the source of the circulating parasites is open to
question.
Plasmodium cynomolgi cynomolgi.
Eleven inmate volunteers were bitten by large numbers, up to 40, of in-
fected mosquitoes (A.f_.) on 8 September, and to date three patients have ex-
hibited evidence of infection (i.e., fever); parasitemia has been demonstrated
in only one and that on the 58th day after infection. Blood was drawn from
each man on day 8, and subsequently when there was clinical evidence of infec-
tion, and inoculated into clean monkeys at Dr. Schmidt's laboratory, Cincinnati.
So far no infections have developed in monkeys except in those inoculated with
blood from the one patient shown to have had parasites on smear. These results
show that P. £. cynomologi will produce infection in man but that it is far
less infective than P.c_. bastianellii.
Significance to Bio-medical Research and the Program of the Institute:
This country, along with other countries of the world, is committed to a
program of world-wide eradication of malaria and to that end the Congress
appropriated thirty-eight million dollars for fiscal year 1961. It was
considered by malar iological consultants that non-human reservoirs would not
be a problem in this effort, and this may still be true. However, the ease
with which P. c. bastianellii of monkeys can be transmitted to man raises an
important question about the place of simian hosts in human disease. Studies
in volunteers have a two-fold purpose in terms of malaria eradication:
(1) clinical and parasitological studies of simian malarias in man, and
(2) evaluation of drugs and drug combinations. It is expected that the know-
ledge obtained can be applied in this field.
Proposed Course of the Project: This project is being enlarged in order
to include studies on simian malarias in man along with the assessment of
methods of mass chemotherapy and the/parasite resistance in terms of malaria
eradication. stuay of
Part B included - Yes
13
Serial No. NIAID-130-A
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Eyles, Don E., Coatney, G. R. , and Getz, M. E. Vivax-type malaria
parasite of Macaques transmissible to man. Science 131 ;1812-1813, 1960.
1'*
Serial No. NIAID-131
1. Parasite Chemotherapy
2. Chemotherapy
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Drug resistance in experimental malaria
Principal Investigator: William F. Cantrell
Other Investigators: None
Man Years (calendar year 1960) :
Total: 1 1/2
Professional: 1/2
Other: 2/3
Project Description:
Objectives : To obtain strains of malaria resistant to a variety of anti-
malarial drugs and to study the genetics of drug resistance by producing
hybridization of these strains in the mosquito phase of the life cycle.
Methods Employed: Large populations of Plasmodium gallinaceum are ex-
posed to the pyrimethamine, chloroquine or primaquine by treating infected
chickens. After one to three days, 0.5 ml of blood is transferred to a new
chicken and treatment is repeated.
Major Findings : Repeated exposure of large populations of malaria para-
sites resulted in resistance only in the case of pyrimethamine. It appears
that even in very large populations parasites resistant to chloroquine or prima-
quine do not occur. An attempt was made to select a chloroquine resistant
strain from the pyrimethamine resistant strain without success.
Significance to Bio-medical Research and the Program of the Institute:
The development of resistance to chloroquine or primaquine is a greatly feared
possibility in the world-wide malaria eradication program. Fundamental know-
ledge about the process by which malaria parasites become resistant may lead
to means for preventing drug resistance or combating drug resistant strains.
Proposed Course of the Project: Other drugs will be tried in Plasmodium
gallinaceum in order to obtain genetic markers. Experiments will be made with
Plasmodium berghei in which species chloroquine-resistant strains are known.
Here the mosquito phase of the work will be more difficult and will require
special study.
15
Serial No. NIAID- 131
The possibility of using X-ray to speed up the development of resistant
strains will be explored.
The pyrimethamine resistance characteristic will be employed in studies
directed toward the prevention of the development of resistance by simultaneous
administration of drugs and other means.
Part B included - No
16
Serial No. NIAID-131-A
1. Parasite Chemotherapy
2. Chemotherapy
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: The effect of adrenal cortical hormone on
chemotherapy of experimental malaria
Principal Investigator: William F. Cantrell
Other Investigators: None
Cooperating Units : None
Man Years (calendar year 1960) :
Total: 1 1/2
Professional: 1/2
Other : 1
Project Description:
Objectives : To determine whether adrenal cortical hormones affect the
action of antimalarial drugs adversely or otherwise.
Methods Employed: Chickens infected with Plasmodium gallinaceum or mice
infected with Plasmodium berghei are treated simultaneously with cortisone and
one of the antimalarials and the effect is compared with the effect of the
antimalarial alone and cortisone alone.
Major Findings : In the case of P. gallinaceum, cortisone (or hydrocorti-
sone) alone was without effect on the course of the infection. In the case of
P. berghei, cortisone (or hydrocortisone) alone has a weak but significant
antimalarial effect. In the case of P. gallinaceum treated with primaquine,
cortisone has an adverse effect on antimalarial action. In the case of P.
gallinaceum, treated with chloroquine or with pyrimethamine, cortisone is
without effect even in high doses. In the case of P. berghei, the antimalarial
action of cortisone (or hydrocortisone) is combined with the antimalarial
action of chloroquine, primaquine or pyrimethamine giving a slightly increased
antimalarial effect.
Significance to Bio-medical Research and the Program of the Institute:
The adverse effect of cortisone on host defenses in a number of experimental
infections and in a few human diseases suggested that more information with
regard to malarial infections would be useful. The diverse responses found
with different drugs and different host-parasite systems indicates that no
direct application should be made to human malaria. On the other hand, the
17
Serial No. NIAID-L31-A
finding that there is system in which cortisone has a favorable effect on
therapy is of some value from the point of view of experimental chemotherapy
as a science. The finding that cortisone interferes with chloroquine activity
against P. gallinaceum but not with primaquine nor pyrimethamine suggests
that primaquine activity depends more on a cooperative action by the host
defense system.
Proposed Course of the Project: No further exploration of this phase of
the work is planned, but studies of other physiological factors affecting the
response of malaria to chemotherapy will be made.
Part B included - No
18
Serial No. NIAID-131-B
1. Parasite Chemotherapy
2 . Chemo ther apy
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A
Project Title: Antimalarial drugs and nucleic acid synthesis
Principal Investigator: Karl A. Schellenberg
Other Investigator: G. Robert Coatney
Cooperating Units : None
Man Years (calendar year 1960) :
Total: 1 1/3
Professional: 2/3
Other: 2/3
Project Description:
Objectives : To determine the effects of antimalarial drugs on nucleic
acid synthesis in experimental malaria.
Methods Employed: The incorporation of radioactive phosphate into ribo-
nucleic acid and deoxyribonucleic acid was measured and the influence of
several antimalarial drugs and other substances was determined in Plasmodium
gallinaceum and Plasmodium berghei.
Major Findings: Quinine, chloroquine, and quinacrine inhibited the in-
corporation of P32 into both RNA and DNA, whereas pyrimethamine and the tria-
zine metabolite of chloroguanide specifically inhibited incorporation of P32
into DNA. The inhibition by pyrimethamine was not reversed by folic acid,
folinic acid, thymidine, deoxyuridine, uracil, thymine, glycine or adenine.
Chloroguanide was active in vivo but not in vitro.
Significance to Bio-medical Research and the Program of the Institute:
The highly specific effect of pyrimethamine on deoxyribonucleic acid metabolism
occurring at concentrations which may be reached in actual therapy affords an
explanation of drug action on a more fundamental level than is available for
most drugs. This linking of drug action to synthesis of the genetic material
of a parasitic organism affords an approach to the discovery of new drugs
through advances in the highly active field of nucleic acid enzymology.
13
Serial No. NIAID-131-B
Proposed Course of the Project: Dr. Schellenberg has resigned in order
to continue advanced study in biochemistry. Continuation of this important
line of research depends on finding a suitably trained person.
Part B included - Yes
20
Serial No. NIAID-131-B
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Schellenberg, K. A. and Coatney, G. R. The Influence of antimalarial
drugs on nucleic acid synthesis in Plasmodium gallinaceum and Plasmodium
berghei. Biochem. Pharmacol, (in press).
Schellenberg, K. A. and Weinbach, E. C. The endogenous formation of
phosphoenolpyruvate by rat-liver mitochondria. Biochim. et Biophys. Acta
45:593-595, 1960.
21
Serial No. NIAID-131C
1. Parasite Chemotherapy
2. Chemotherapy
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Experimental chemotherapy of helminthic diseases
Principal Investigator: George V. Luttermoser
Other Investigators: None
Cooperating Units: Dr. B. Prescott, LID, NIAID-6lAand Dr. J. Tobie, LI,
NIAID-1U8
Man Years (calendar year I960):
Total: 1 1/2
Professional: l/2
Other* 1
Project Description:
Objectives: To discover new and more effective schistosomacides and
compounds active against other helminth parasites. To learn how the chemicals
selectively kill those parasites.
Methods Employed: Mice are infected with cercariae of Schistosoma mansoni
and treatment is begun (l) immediately (prophylaxis) or (2) after 35 days
(curative). The mice are examined $0 days or more after exposure to infection
or for the presence of dead worms. In critical evaluation of drug activity,
the liver and portal system are perfused and the live or dead worms are counted.
Live mature worms removed from treated and untreated animals are observed in
maintenance culture for activity and survival. Drugs are added to some of the
cultures. Monkeys are infected with the same parasite and dogs, with 8.
japonicum (Formosan). A test for either prophylactic or curative activity of
promising compounds was conducted in the larger animals in a similar fashion
to the mouse test except that fecal egg counts were also utilized as an indi-
cator of the status of the infection.
For studies on nematode parasites, dogs naturally infected with Diro-
filaria immitis and mice infected with pinworms were utilized. Observations
were made on the parasiticidal effects of the drugs in vivo and in vitro.
Fluorescence of the helminth was read in UV light which indicated whether the
drugs were absorbed.
22
Serial No. NIAID- 131C
Major Findings; Completion of the test for prophylactic activity of
the antibiotic S1629 (Griseofulvin or Fulvicin) indicated that oral adminis-
tration of 50 mg/kg of the drug daily starting the fourth day before exposure
to S. iransoni and continuing for 12 days brought about a reduction in the
numbers of schistosomes developing in these animals. This was not found in
similar experiments with hamsters. There was, however, an indication of weak
prophylactic activity in two monkeys started on the drug two days before
exposure and continued en a daily dose for 9 days.
Since filariids and other helminths absorb tetracycline and will
fluoresce (Tobie and Beye, I960), a study was made of the effects of tetra-
cycline and 12 analogues of tetracycline on Dirofilaria iremitis and on mouse
pinworms. A comparison was made of the longevity of the larvae and adults
of these nematodes in maintenance media with and without drugs added at final
concentrations of from 1 ug to 1 mg per ml. One of the analogues (1686-7)
killed the microf ilariae of D. immitis at in -vitro concentrations as low as
10 ^ug/ml in a period of h to 9 hours, while tetracycline itself did not do
so. The oral administration of 15 daily oral doses of 20 mg of this analogue
per kg body weight likewise caused a marked reduction in the microfilarial
blood count. Nevertheless, many of the micro- and macrofilaria which fluoresced
after in vitro exposure to other of these "tetracyclines", continued to live
for several hours afterward. In mice with pinworm inlection, the analogue
1686-7 was not more active than tetracycline itself in clearing the animals of
pinworms. However, a 2 day regimen of another of the analogues (168°) cleared
the pinworm infections from most of the mice while a similar regimen of an
active piperazine did not. Weak fluorescence was only observed in small areas
of the upper intestine and of the genital system of the pinworm whereas strong
fluorescence was observed throughout the body wall and intestine of the
filariids.
The analogues of tetracycline tested have not been found to be schisto-
somacidal nor have they been found to cause the schistosome to fluorece.
Significance to the program of the Institute; New information with
regard to a reaction between a chemical and a parasite may be a "lead" for
the development of better drugs needed for control of parasitic infections
such as schistosomiasis and filariasis. Current treatments for these
infections are inadequate.
Proposed Course of the Project; The principal investigator will be
Visiting Professor at the American University of Beirut, Lebanon, during
196l. Studies of effects of new drugs on schistosomes will be continued by
Dr. William Cantrell.
23
Serial No. NIAID-131C
PHS-NIH
Individual Project Report
Calendar Year I960
Part B Honors, Awards, and Publications
Honors and Awards relating to this project:
President of the Helminthological Society of Washington, I960.
Appointed consultant on schistosomiasis to WHO and accepted assignment
to Ministry of Health of Venezuela for the period 22 January to 18 March,
I960.
Named Visiting Research Professor in the Department of Tropical Health,
American University, Beirut, Lebanon, for the year 196l,
24
Serial No. NIAID-131-D
1. Parasite Chemotherapy
2. Chemotherapy
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A
Project Title: Effects of nutrition on chemotherapy of parasitic
diseases.
Principal Investigator: George W. Luttermoser
Other Investigators: None
Cooperating Units: Dr. W. DeWitt, LPD, NIAID-121K
Man Years (calendar year I960) :
Total: 1 1/2
Professional: l/2
Other: 1
Project Description:
Objectives: To determine whether the nutrition of the host can influence
the chemotherapy of parasitic infections.
Methods Employed : Animals maintained on defined diets either enriched or
deficient in certain components are exposed to a single species of parasite
and the developing infection is treated in either the prepatent or patent
period. The influence of the diet on treatment is determined by (l) the
reduction of the number of parasites (2) the condition of the parasites (3)
growth and survival of the host and (Li) the pathologic conditions developing
in the host.
Major Findings: In many tests, the efficacy of stibophen (Fuadin) therapy
on mature Schistosoma mansoni infections in mice was increased up to 16 times
by feeding a balanced semi-synthetic diet. The toxicity of the drug was not
similarly increased. The enhancement of curative action by the purified semi-
synthetic diet was thought to be due to the absence of, as yet unidentified,
inorganic salt(s) that interfere with drug activity. It was found in mice
fed on the purified semi-synthetic diet that higher blood levels of the drug
were maintained for a longer period than when the same amount of Fuadin was
injected into mice fed on the commercial pellet diet, suggesting that the
increased cure-rate was due to higher blood drug level. Similar drug advantage
was observed in mice given tartar emetic while on the purified diet.
25
Serial No. NIAID-131-D
Significance to the Program of the Institute : Since parasitic infections
occur most frequently in backward areas where malnutrition is also prevalent,
it is necessary to determine the relationship between the nutrition of the
people and the results obtainable by chemotherapy. Failure of chemotherapy
is a common experience in such areas. Projects for screening drugs for
schistosomacidal activity might be improved if more optimum dietary conditions
for experimental chemotherapy are found.
Proposed Course of the Project; During the next year the Principal
Investigator will be Visiting Professor at the American University, Beriut,
Syria. During his absence Dr. DeWitt (LPD) will continue the investigation
to determine the specific nutritional factors involved in the enhancement of
the efficacy of stibophen in schistosomiasis. Other drugs used for the
treatment of this and other parasitic diseases will be investigated under
similar conditions with different animals.
Part B included - Yes
2C
Serial No. NIAID-131-D
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Luttermoser, G. W. and DeWitt, W. B. Studies on interrelations of
nutrition and treatment of schistosomiasis I. Enhancement of stibophen
(Faudin) activity against Schistosoma mansoni in mice by feeding purified
semi-synthetic diets. Am. J. Trop. Med. & Hyg. (in press).
27
Serial No. NIAID-131-E
1. Parasite Chemotherapy
2 . Cheruo therapy
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A
Project Title: Comparison of antimalarial drugs administered singly and
in various combinations.
Principal Investigator: Richard L. Jacobs
Other Investigators: William F. Cantrell
Cooperating Units : None
Man Years (calendar year I960) :
Total : 1/3
Professional: l/6
Other: l/6
Project Description:
Objectives: To determine the dosage-level of chloroquine, primaquine,
amodiaquine and pyrimethamine required to give any. desired degree of
suppression of parasitemia in mice. To compare the effectiveness of various
combinations of these drugs to single-drug treatment.
Methods Employed: NIH Swiss female mice, averaging 20 gm/each,' were in-
fected by tail-vein injection of one million RBC infected with P. berghei
NYU-2 strain. Drugs were administered orally at various levels for five
consecutive days, beginning on day of infection. Blood smears were made on
four consecutive days, beginning on the third day after infection, for evalu-
ation of the effect of different treatments on the course of parasitemia.
Each trial involved five animals per group and all trials were repeated at
least one time.
Major Findings: Chloroquine, primaquine, and amodiaquine, when reduced
to base equivalents, were equally effective in suppressing parasitemia in mice
by the method described. Approximately 50 percent suppression in parasitemia
resulted from administering U0 /ig/mouse/day. Pyrimethamine is equally effective
at one-third the dosage level of the above drugs.
28
Serial No. NIAID-131-E
Various combinations of these four drugs were no more effective than
single-drug treatment. When combined, results indicated consistently that
the effect was additive, or less than additive.
Significance to Bio-medical Research and the Program of the Institute:
The practice of combining drugs has been used extensively, often with
favorable results. Reports have indicated that certain combinations of anti-
malarial drugs exhibit a synergistic effect in suppression of parasitemia
(quinine and pamaquine in chicks) . Also, clinical trials have indicated that
antimalarial combinations are more effective in preventing relapse in vivax
malaria. The results of this study indicate that combinations of the drugs
tested offer no advantage over single-drug therapy in suppressing erythro-
cytic forms of P. berghei. Suppressive levels of drugs have been established
for use in other studies.
Proposed Course of the Project; This project has been completed.
Part B included - No
23
Serial No. NIAID-131-F
1. Parasite Chemotherapy
2. Chemotherapy
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year I960
Part A
Project Title: Effect of nutritional status of host on the action of
antimalarial drugs.
Principal Investigator: Richard L. Jacobs
Other Investigators: William F. Cantrell
Cooperating Units: None
Man Years (calendar year l°6o)»
Total: 2/3
Professional: 1/3
Other : l/3
Project Description:
Objectives: To study the effect of the nutritional status of the host ani-
mal on the course of parasitemia in experimental malarial infection, both with
suppressive levels of antimalarial drugs and in the absence of drug therapy.
It is hoped that the results of this study will yield information regarding
the mode of action of antimalarial drugs.
Methods Employed: The parasite P. berghei, NYU-2 strain is being studied
in mice of altered nutritional status. Young NIH Swiss female mice are
obtained averaging nine to ten grams each and are maintained on various test
diets for approximately four weeks prior to infection. An alternate method
consists of the administration of antimetabolites prior to, or at, time of
infection. Effects are studied both in absence of and in suppressive levels
of chloroquine.
Major Findings: Preliminary results indicate that alterations in the
course of parasitemia in mice by dietary deficiencies are variable. The vari-
ability is due, primarily, to the degree of the deficiency. Also, the possi-
bility of multiple-deficiencies cannot be neglected when the semi -purified
diet is employed.
In tests employing folic acid deficient diets, the effect on the course
has been variable^ however, a striking suppression of parasitemia has
30
Serial No. NIAID-131-F
consistently been observed when aminopterin is administered.
The effect of nutritional deficiencies appears to be influenced by
suppressive drug treatment. Pyridoxine-deficient animals without drug treat-
ment showed an increased parasitemia; when a $0% suppressive dose of chloro-
quine was administered, the pyridoxine -deficiency appears to inhibit para-
sitemia.
Vitamin deficiency states that appear to favor the course of parasitemia
include: Pantothenic acid, biotin, riboflavin and choline.
Deficiencies that suppress parasitemia include: ascorbic acid and niacin.
Significance to Bio-medical Research and the Program of the Institute:
It is believed that the results of this study may give some insight into the
mode of action of antimalarial drugs.
Proposed Course of the Project » Investigations along the lines outlined
above will be continued and the use of antimetabolites will be extended.
Further work will be initiated to clarify promising leads.
Part B included - No
31
Serial No. NIAID-132
1. Parasite Chemotherapy
2. Epidemiology
3. Columbia, South Carolina
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Studies on Human Malaria
Principal Investigator: Martin D. Young
Other Investigators: None
Cooperating Units:
Man Years (calendar year l°60)j
Total : 3
Professional: l/2
Other: 2 l/2
Project Description:
Objectives:
To add to the biological knowledge of malaria by observations
and experiments on induced malaria. To determine the sporontocidal
activity of antimalarial drugs and how developed resistance influ-
ences transmission by mosquito vectors. To study the development
and inheritance of resistance to drugs by the parasites. To
determine if the development of resistance to insecticides by
mosquitoes alters their ability to transmit malaria. To serve as
probably the only source in the nation for established strains of
malaria for use on a nationwide basis for therapy of neurosyphilis,
nephrosis, Parkinsonism, and for experimental malaria studies.
Methods Employed:
Malaria is induced in neurosyphilitic patients and in volun-
teers under normal and experimental conditions. The patent infec-
tions are challenged by drugs. When resistance occurs measurements
are made of its characteristics. Mosquitoes are allowed to bite
patients with induced infections before and at intervals after
drug administration. The resulting infections in the mosquitoes
are correlated with the presence or absence of drug resistance
of the malaria infection in the human host. Insecticide -resistant
and normal mosquitoes are fed simultaneously for comparison of
ability to transmit malaria.
32
Serial No. NIAID-132
Major Findings;
Primaquine given 1.5 mg. daily had sporontocidal effect
upon Plasmodium vivax and P. falciparum but little effect upon
the asexual parasites. Daily doses of 0.75 mg. had some sporontocidal
effect upon P. falciparum but none against one P. vivax case.
Therefore, 1.5 mg. daily appears to be the lowest amount that is
reliably sporontocidal against both P. falciparum and P. vivax.
Therapeutic doses (I.I4 gm in 3 days) of amodlaquine had
no sporontocidal effect upon one P. falciparum case.
P. falciparum from South Rhodesia was susceptible to
pyrime thamine .
Preliminary tests indicate no diffprence in vectorial
ability of insecticide -susceptible and insecticide-resistant
mosquitoes.
P. vivax was maintained in a viable condition for five
years at -70° C. The adding of glycerine to blood containing
chicken and rat malaria aids in its preservation at low
temperatures.
A lU-year study of the biology of P. malariae , during
which time it had been passaged through neurosyphilitic patients
mainly by blood transfusions, revealed the following: there
was no apparent reduction in viabilityj the infection and com-
pletion of the sporogonous cycle in Anopheles aztecus; the com-
parative susceptibilities of the mosquitoes tried, in decreasing
order, were: A. freeborni, A_. punctipennis, A. aztecus, and A»
quadrimacula tu s ; no infections were found in A. crucians and A.
albimanus; the length of the sporogonous cycle in A. quadrima-
culatus ranged from 19 to 23 days when incubated at 7o° F.j
A. freeborni when paired with A. quadrimacula tus also had an
identical 23-day cycle; of 7,3~5U A. quadrimaculatus dissected,
3.1 per cent were infected; the oocysts per infected gut
averaged 3.0; only 3*9 per cent of the guts had 10 or more
oocysts; transmission of the malaria to six patients was
accomplished, three by the bites of infected mosquitoes and three
by the intravenous injection of the glands of these mosquitoes;
there was wide variation in the infectiousness of different
malarious patients to mosquitoes; mosquitoes became infected
in nearly every week of the first six months with the highest
inf ectivity occurring during the eighth to tenth weeks of the
primary attack; the asymptomatic parasitemias infected mosqui-
tces at about the same rate as the symptomatic parasitemias.
33
Serial N0. NIAID-132
an autochthonous chronic asymptomatic case of P. malariae of
at least 18 months duration infected mosquitoes; although
the infection rate of mosquitoes in ordinarily low, the relatively
long period of infection during which mosquitoes can be infected
may explain the persistence of P. malariae in certain areas;
the ability of the symptom-free malarious patient to infect
mosquitoes at a rate similar to that of the symptomatic patient
makes case finding more difficult, especially in important
areas of malaria eradication.
A strain of P. falciparum induced into neurosyphilitic
patients was resistant to normal and above normal doses of
chloroquine .
Significance to Bjo-Hedical Research and the Program of the
Institute ;
The finding of resistance of malaria parasites to
chloroquine is of importance to the malaria eradication pro-
grams in many parts of the world as drugs are being used as
supplemental measures in some programs. It is important to
determine if there is cross-resistance between the U-amino-
quinoline drugs. Additional information is needed on the
biology of the parasite especially in relationship to drugs.
The comparative ability of insecticide-susceptible and
insecticide -resistant mosquitoes to transmit drug-susceptible
and drug -resistant parasites needs to be determined, especially
to indicate whether the change in the vector or parasite has
significance in the epidemiology of malaria.
The low but persistent infectivity of P. malariae to
mosquitoes helps explain its epidemiology and especially its
persistence in nature.
Proposed Course of the Project;
To determine more fully the nature and extent of the
chloroquine resistance in human malaria, as well as cross-
resistance to other drugs such as the h-aminoquinolines. Further
investigate the ability of insecticide-susceptible and insecticide-
resistant mosquitoes to transmit drug-susceptible and drug-
resistant malaria parasites. Continue to obtain basic informa-
tion, especially epidemiological and host-parasite relation-
ships on induced human malarias, both domestic and foreign.
Important strains of malaria will be added to those now in the
local repository which apparently is the only existing one for
the preservation of malaria.
Part B included - Yes „ ° '
Serial No. NIAID-132
PHS-NIH
Individual Project Report
Calendar Year I960
Part B.
Honors, Awards, and Publications.
Publications other than abstracts from this project:
Young, M. D. The Effect of Snail Doses of Primaquine Upon
Malaria Infections. Ind. J. Mai. 13:69-7h. I960.
Young, M. D. MALARIA - A Study of Scientific Exploration
and Achievement. Bull. S. C. Acad. Sci. 21:32-37.
I960. —
Young, M. D. Malaria (Chapter in Manual of Tropical Medicine,
third edition, by Hunter, Frye, and Swartzwelder.
Saunders). I960.
Jeffery, G. M. Infectivity to Mosquitoes of Plasmodium vivax
and Pla sm odium falciparum Under Various Conditions.
Am. J. Trop. Med. and Hyg. 9:315-320. I960. May.
Jeffery, G. M. Book Review: The Ecology of Human Diseases,
by Jacques M. May. Am. J. Trop. Med. and Hyg. 9:
350-351. I960.
Young, M. D. Book Review: Parasitology (Protozoology and
Helminthology) in Relation to Clinical Medicine, by
K. D. Chatterjee. Am. J. Trop. Med. and Hyg. 9:
351.
Young, M. D. Chemo therapeutic agents and malaria eradication.
Proceedings of Sixth International Congresses on Trop.
Med. and Mai. (In press). I960.
Garnham, P. C. C, Jeffery, G. M., and Young, M. D. Preservation
of strains of malaria parasites. Proceedings of Sixth
International Congresses on Trop. Med. and Mai. (In
Press.) I960.
Jeffery, G. M. Inoculation of Human Malaria into a Simian Host,
Macaca mulatta. J. Parasit. (In Press.)
Young, M. D., and Burgess, R. W. The Infectivity to Mosquitoes
of Plasmodium Malariae. Am. J. and Hyg. (in press).
I960.
35
Serial No. NIAID-132
Honors and Awards Relating to this Project:
For Dr. Martin D. Young:
Visiting Lecturer, Department of Microbiology,
Meharry Medical School.
Visiting Lecturer and Temporary Advisor on Chemotherapy
PAHO Malaria Eradication Training Center, Kingston,
Jamaica.
Member, City Board of Health, Columbia, South Carolina.
Member, Expert Advisory Panel on Malaria, World Health
Organization.
Member, Editorial Board, American Journal of Tropical
Medicine and Hygiene.
3C
Serial No. NIAID-132-A
1. Parasite Chemotherapy
2. Epidemiology
3. Columbia, South Carolina
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Chemotherapy of intestinal parasites.
Principal Investigator: Martin D. Young
Other Investigators: Geoffrey M. Jeffery
Cooperating Units: South Carolina State Hospital
Man Years (calendar year 1960) x
Total: 2 5/6
Professional: 5/6
Other: 2
Project Description:
Objectives:
To find adequate drugs for the prophylaxis and treatment
of parasitic infections which constitute serious health problems
in certain types of mental patients as well as in more normal
populations; to determine the relationship of parasite load to
psychological or psychiatric considerations in the patients;
also to investigate factors influencing parasite spread and
persistence among patients.
Methods Employed:
Various segments of the institutional population are
examined to determine qualitatively, and quantitatively when
possible, their parasitic infections. Promising compounds in
varying dosages and regimens are tried against selected cases;
post-treatment evaluation determines the effectivity of the
drug against the various parasites. Occasionally careful pre-
and post-treatment psychological, psychiatric and physical
evaluations are done to determine the possible effect of
parasite removal. The results are based upon thousands of
patient-drug (individual regimens) trials.
37
Serial No. NIAID-132-A
Major Findings;
Epidemiological studies on a defined population show a
high persistence of Trichuris and hookworm for six years, with
an apparent decrease in Stronqyloides. The yearly studies
show fluctuations in protozoal infections indicating that
transmission is occurring.
To test the therapeutic and prophylactic effect of
dithiazanine and tetrachloroethylene alone and in combination,
a group of mental patients heavily parasitized were given drugs
at various intervals for about one year. A large number of
worms were removed but the cure rate was low. Treatments sub-
sequent to the initial one had reduced effectiveness. Trans-
mission was not stopped. These drugs do not appear to be
adequate prophylactic agents under conditions of high exposure
such as represented in this study.
Bephenium hydroxynaphthoate continued to exhibit
excellent results against hookworm and Ascaris infections
found in mental patients. A retrial of bephenium chloride
was begun. The results so far indicate that it may be more
effective in single comparative doses than the bephenium
hydroxynaphthoate. It is showing promising activity against
Trichuris. Combined with an anti-nausea drug, Marezine, the
side effects of nausea and vomiting appear to be reduced.
Significance to Bio-Medical Research and the Program of the
Institute;
Currently available drugs in the regimens tried have
not been shown to be adequate in the prevention of trans-
mission of Trichuris and hookworm parasites in areas of high
exposure risks. Better drugs, or better regimens of the
present ones, are needed. Information obtained should be of
value in mental hospitals and in many tropical areas.
Proposed Course of the Project:
The evaluation of promising compounds for better prophy-
lactic and parasiticidal agents will be continued with especial
interest on different regimens of bephenium chloride. Parallel
investigations are underway on the mode of action of drugs on
parasites. Efforts are being made to explain the failure of
effective drugs to produce radical cures, the reduced effective-
ness of drugs after the initial treatment, and similar problems.
Further investigations on the epidemiology of parasitic infections
in mental hospital populations will be continued.
Part B included: Yes. ° "
Serial No. NIAID-132-A
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Jeffery, G. M. A Three-Year Epidemiologic Study of Intestinal
Parasites in a Selected Group of Mental Patients.
Am. J. Hyg. 71:1-8. 1960.
Young, M. D., Jeffery, G. M., Morehouse, W. G., Freed, J. E., and
Johnson, R. S. The Comparative Efficacy of Bephenium
Hydroxynaphthoate and Tetrachloroethylene against Hookworm
and other Parasites of Man. Am. J. Trop. Med. & Hyg.
9:488-495.
3S
Serial No. NIAID-132-B
1. Parasite Chemotherapy
2. Epidemiology
3. Columbia, South Carolina
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Physiology of human parasites especially as
related to nucleic acids and drug action.
Principal Investigator: Kenneth 0. Phifer
Other Investigators: None
Cooperating Units: None
Man Years (calendar year I960):
Total: 1
Professional: 1
Other* 0
Project Description:
Objectives:
To determine the place of action of drugs upon malaria
and intestinal parasites of man. The study of the action of
drugs on malaria includes the mechanism and locale, the develop-
ment of resistance and a search for methods to avoid the
development of resistance or how to overcome it. Similar
studies are planned on the developmental chemotherapy of other
parasites, especially the worms of the intestinal tract.
The nucleic acids of malaria parasites are being
examined qualitatively and quantitatively in order to determine
whether drug-resistance affects the synthesis of these sub-
stances* To ascertain whether malaria-infected mosquitoes have
higher nucleic acid levels than non-infected mosquitoes. To
determine what effect anthelmintics have on the in vitro meta-
bolism and in vivo distribution of helminths.
Methods Employed:
Malaria-infected red blood cells and infected mosquitoes
are homogenized and fractionated into ribonucleic acid; this
40
Serial No. NIAID-132-B
fraction is then measured colorimetrically. Qualitative
analysis of the nucleic acid fraction is carried out through
the use of paper chromatography and paper electrophoresis.
Helminths are incubated both in the presence and in
the absence of anthelmintics and several measures of metabolism
are utilized; e.g., transaminase reaction and rate of glucose
uptake. Also noted is the effect of anthelmintics on egg pro-
duction and distribution of the worms in the intestine of the
host.
Major Findings:
It was shown that mosquitoes infected with malaria
parasites tend to have higher levels of ribonucleic acid
than do uninfected mosquitoes.
Chromatographically, the acid-hydrolysate of ribonucleic
acid from a pyrimethamine-resistant strain of Plasmodium falci-
parum appears to be different than the acid-hydrolysate of
ribonucleic acid from a pyrimethamine-susceptible strain of
this species. Preliminary experiments point toward similar findings
in the case of chloroquine resistance.
Bephenium hydroxynaphthoate inhibited glutamic acid-alanine
transaminase and glutamic acid-aspartic acid transaminase in
Nippostrongylus muris in vitro. This drug also appears to remove
more worms from the anterior part of the intestine of infected
rats than from more posterior segments of the gut.
Bephenium chloride and atabrine reduced the rate of
glucose absorption by the tapeworm, Hymenolepis diminuta, from
30 to 50 per cent. At low concentrations the cyanine dye,
dithiazanine iodide stimulated glucose absorption by this cestode.
Significance to Bio-Medical Research and the Program of the
Institute:
Since several antimalarial agents have chemical structures
similar to those of components of nucleic acids it seems advis-
able to determine whether these drugs affect the parasite's
basic nuclear compounds. It is felt that if it were demonstrated
that nucleic acids are altered by antimalarials, this clarifi-
cation of mechanism of drug action would provide a more logical
rationale for the synthesis of new drugs.
Serial No. NIAID-132-B
Resistance of parasites to drugs is a serious world
problem with respect to malaria eradication. Since resistance
has been shown to be under genetic control in many instances,
demonstration of changes in nucleic acid composition in
response to exposure to a drug should be of value in determin-
ing mechanisms of resistance. Once mechanisms of resistance
are delineated, the problems associated with combatting drug
resistance might be more readily solved.
A corollary aim of this project is the attempt to
develop a biochemical means of detection of infection of mos-
quitoes before morphological stages in the parasite's life
cycle are evident. If such a means proved reliable, some
insight could be gained as to the changes occurring and the
experimental procedures would be shortened.
Study of the effect of anthelmintics on helminth
metabolism will aid in a clearer understanding of drug action
and might be valuable in the design of new drugs.
Study of the effect of anthelmintics on the distribution
of helminths in the host intestine is important since in human
cases of helminthiasis, a few worms often remain after therapy
and the question of whether this is due to resistance to the
drug or a re-location in the gut should be answered.
Proposed Course of the Project;
Studies of nucleic acid composition of malaria parasites
from drug-resistant and drug-susceptible strains will be con-
tinued using both paper chromatography and absorption spectrum
analysis via spectrophotometry.
Infected mosquitoes will be examined for ribonucleic
acid level to determine conclusively whether increased nucleic
acid levels can be reliable indices of infection.
Studies with respect to the permeation of drugs into the
malaria parasite will be carried out, comparing the uptake of
drug in susceptible and resistant strains.
Animals infected with helminths will be drugged with
anthelmintics and the worms will be counted, position and size
of worm noted, and relative metabolic activity assayed. Both
cestodes and nematodes will be utilized.
Part B included: No ^*-
Serial No. NIAID-132-C
1. Parasite Chemotherapy
2. Epidemiology
3. Columbia, South Carolina
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Virus-Parasite Association
Principal Investigator: Geoffrey M. Jeffery
Other Investigators: None
Cooperating Units: None
Man Years (calendar year 1960):
Total: 1 1/3
Professional: l/3
Other: 1
Project Description:
Objectives:
To determine experimentally if parasites might be
associated with some of the virus diseases of man or animals
as vectors or in some other capacity. To study such relation-
ships under natural conditions and to investigate known virus
infections and known populations to determine existing re-
lationships, including the study of possible intestinal viruses
among institutionalized mental patients. In view of current
findings, further study the relationships of virus-malaria
and virus-endoparasite associations under experimental conditions.
Investigate the possibility that certain parasites may them-
selves be afflicted with harmful virus infections. Investigate
the effects of parasitic infections upon virus infections and
vice versa.
Methods Employed:
Small animals and their common parasites are used in
attempts at transmission of viruses. Viruses, such as lympho-
cytic choriomeningitis (LCM), polio, St. Louis encephalitis (SLE),
rabbit papilloma and fibroma are maintained in small animals by
serial transmission, providing infective virus material for
*»3
Serial No. NIAID-132-C
experimental work. Besides the common helminth parasites of
mice, Nippostronqylus muris, Nematospiroides dubius, Hymenolepis
nana, and others, the malaria parasites Plasmodium berqhei and
P. gallinaceum are also maintained as experimental parasites.
Viruses and parasites are brought together in various ways, such
as by simultaneous infection in animals, during cultivation of
the free-living stages, or by simultaneous exposure of the host
to infective parasite stages and virus suspensions. Tissue
culture or small animals are used for detection of virus infec-
tion carried by parasites from virus-infected animals, for
virus detection in parasites suspected of harboring viruses in
nature, and for detection of virus infection in arthropods.
Incidence of intestinal viruses and their possible association
with parasites in institutionalized mental patients is studied
with animal inoculation, tissue culture, and other means.
Major Findings;
In mice inoculated with SLE virus intraperitoneal^, the
presence of P. berqhei aided in the transport of the virus to
the central nervous system as shown by symptoms and by death
of the mice. The same occurred when LCM virus and P. berqhei
were combined. The virus appeared earlier and persisted longer
when malaria was present. SLE virus and P. gallinaceum com-
bined caused death earlier in some chicks and enhanced the
viremia in others over that found when malaria alone was
present.
Significance to Bio-Medical Research and the Program of the
Institute:
Many problems associated with the transmission of viruses
in human and animal populations remain unsolved. New viruses
in mammals and arthropods are continually being discovered.
There are suggestions in the literature and theoretical possi-
bilities that parasites may act as reservoirs and agents of
transmission for viruses of importance to man. Elucidation of
association of viruses and parasites, both experimental and
natural, will do much toward bringing about a more complete
understanding of the epidemiology of diseases produded by such
organisms, alone or in combination. The possible role of malaria
in transmitting virus from host to host either by extending the
viremia or by actually transporting the virus in the sporozoite
is of epidemiological importance.
4 H
Serial No. N1AID-132-C
Proposed Course of Projectt
The general course of the project will be changed little.
Repetition and expansion of the experiments combining nematode
parasites and viral agents in the vertebrate host, during free-
living stages, and in culture, will be carried out. An increased
amount of effort will be directed toward the association of
viruses and malaria parasites, and possibly other protozoans.
The detection of viruses in malaria parasites, especially the
sporozoites by fluorescent antibody techniques and by inoculation
will be attempted. Further refinement of viral detection methods
will be investigated and incorporated in the work when practicable.
Most of the experimental work planned is in the realm of artifi-
cial viral-parasite associations, but the possibility of extension
of the studies to naturally occurring associations will be always
in mind and such studies will be pursued as opportunities arise.
Part B. included* Yes.
45
Serial No. NIAID-132-C
PHS-NIH
Individual Project Report
Calendar Year 1960
Honors, Awards, and Publications
Honors and Awards Relating to this Project:
Member Council, South Carolina Academy of Science.
Editor, ASB Bulletin, published by the Association of
Southeastern Biologists.
Recipient of Jefferson Award of the South Carolina
Academy of Science.
46
Serial No. NIAID-132-D
1. Parasite Chemotherapy
2. Epidemiology
3. Columbia, South Carolina
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Development of human pathogens in immature
insects.
Principal Investigator: William E. Collins
Other Investigators: None
Cooperating Units: None
Man Years (calendar year I960):
Total: 1 1/2
Professional: l/2
Other : 1
Project Description:
Objectives :
To determine experimentally to what extent immature
stages of arthropods will support growth of pathogenic
organisms with particular emphasis on the viruses and
protozoans in mosquito larvae. If experimental evidence
indicates the growth or maintenance of these microorganisms
in insect larvae, investigations will be made to determine the
possible role larval infection plays in the transmission of
these infections to man.
Methods Employed:
The larvae of mosquitoes belonging to several different
genera are exposed to virus suspensions under varying con-
ditions. The quantity of virus present in larvae, pupae, and
adults subsequent to exposure is determined by small animal
inoculations. Transmission of the virus by the adult mosqui-
toes is made using small animals and blood pools.
^
Serial No. NIAID-132-D
Major Findinqst
Further studies confirmed the results of the prior year
where the virus of St. Louis encephalitis was taken up by the
larvae of Aed e s aeqypti and an increase in virus titer found
in subsequent larval and pupal mosquitoes. The larvae of Culex
quinquef asciatus exposed under similar conditions take up the
virus to a lesser extent, and the adults have not demonstrated
the virus. Transmission of the virus to baby chicks has been
confirmed using mosquitoes 9 to 26 days after adult emergence.
The rate of adult infection and virus transmission using St.
Louis encephalitis virus and Aedes aeqypti mosquitoes has been
less than one per cent under these experimental conditions.
Significance to Bio-Medical Research and the Program of the
Institute:
The presence of viruses and protozoans in mosquito
breeding pools as a result of improper sanitation procedures
or by death and decomposition of infected animals and arthro-
pods poses the possibility that these organisms may be taken
up by the mosquito larvae and subsequently transmitted by the
adults. Information on such larval infection will add to a
more complete understanding of the epidemiology of a number
of arthropod -borne diseases.
Proposed Course of the Project i
The general course of the project will be changed
little. The relative ability of mosquito larvae of different
genera to take up the virus of St. Louis encephalitis and the
effect of initial virus titer, incubation temperature and
larval instar on the rate of infection, virus titer and
adult transmission will be tested.
Part B included: No.
43
Serial No. NIAID-132-E
1. Parasite Chemotherapy
2. Epidemiology
3. Columbia, South Carolina
PHS-NIH
Individual Project Report
Calendar Year I960
Part A.
Project Title: Insect Tissue Culture
Principal Investigator: William E. Collins
Other Investigators: None
Cooperating Units: None
Man Years (calendar year I960):
Total : 1
Professional: l/2
Other: 1/2
Project Description:
Objectives:
To establish insect tissues in culture in order to study better
those factors necessary for insect growth aid metamorphosis and the effect
of microorganisms on insect tissues. To determine which of the viruses,
protozoans or helminths will persist, develop, or multiply in the different
insect tissues grown in vitro using both natural vectors and other species.
Methods Employed}
Tissues and cells from moth larvae and pupae and from mosquito
larvae, pupae and adults are removed and set up in experimental culture
media using several different culture methods. The presence of mitosis,
tissue contractility or general cell condition are used as indications of
tissue growth or maintenance. Viruses are added to aich cultures and periodic
samples collected and titrated in small animals. Protozoans are added and
microscopic examinations made to determine persistence or development of the
parasites. Guts of mosquitoes previously infected with Plasmodium sp. are
removed and set up in culture. Periodic examination and measurement of the
parasite development are made.
43
Serial No. MIA ID- 132 -E
Major Fjnding3t
Blood cells from caterpillars and cells of the ovariole sheath of
several species of moth prepupae and pupae have undergone growth in culture
in several different experimental media. The virus of St. Louis encephalitis
has been maintained in cultures of third, fourth, and fifth instar hemocytes
from the catalpa sphinx (Ceratomia catalpae) for 10 days with little drop in
titer over that which occurred during the first 2k hours. The oocysts of
Plasmodium gallinaceum attached to mosquito midguts in vitro have exhibited
growth and on several occasions, sporozoites have been produced.
Significance to Bio-Iledical Research and the Program of the Institute:
Insects serve as alternate hosts for a number of virus and
protozoan diseases of man. Insect tissue culture would offer the opportunity
to study these parasites and their growth in insect cells in vitro, and thus
quite possibly shed new light on: (l) parasite growth requirements (2)
arthropod tissue specificity and (3) reasons why many human pathogenic
viruses will multiply in the insect with no visible or apparent pathology.
These studies may yield information on those factors controlling the ability
of insect vectors to transmit virus and protozoan diseases whereas closely
related species of insects do not.
Proposed Course of the Project?
Various tissues of mosquito larvae, pupae, and adults, and moth
larvae and pupae will be established in culture and tested for their ability
to support the virus of St. Louis encephalitis. Microscopic studies will be
made in efforts to detect any cytopathology of the tissue as a result of
virus infection. In addition, fluorescent antibody studies will be made to
determine in which cells or tissues the virus is present. Guts from mosquitoes
infected with Plasmodium sp. will be set up in different culture media in an
attempt to determine those factors necessary for the development of sporozoites
in vitro. Further studies on the effect of virus on caterpillar blood cells
and ovariole tissue will be made.
Part B included: No.
50
Serial No. NIAID-133
1. Parasite Chemotherapy
2. Cytology
3. Memphis, Tennessee
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Studies of the biology of exoerythrocytic phases of
primate malaria.
Principal investigator: Don E. Eyles
Other investigators: Robert L. Ingram, Nell C. Owen, Frances E. Jones
and C. S. Smith
Cooperating Units: None
Man Years (calendar year i960):
Total 3
Professional: 1 1/2
Other: 1 1/2
Project Description:
Objectives: To extend the knowledge of the exoerythrocytic stages of pri-
mate malaria, determine the type of cells parasitized, and trace the development
from sporozoite to mature schizont. To determine if the liver is the sole
tissue parasitized. To determine the origin and nature of the late tissue
stages and to determine their relationship to relapse. To define in detail
quantitative relationships in the exoerythrocytic cycle. To study the
exoerythrocytic cycle as it is related to the immune processes of the monkey
host. To determine factors influencing the degree of exoerythrocytic
infection. To provide materials and correlative information to accompany
projects on growth of malaria parasites in vitro and the chemotherapy of
malaria.
Methods Employed: Plasiiiodium cynornolgi, which is closely related to
P. vivax of man, is maintained in Macaca mulata monkeys. Strains of P.
ponderi (also a simian vivax-type parasite) and P. inui (related to human
P. malariae) are also maintained. Anopheles mosquitoes of several species
are produced and mosquitoes infected in order to produce very large numbers
of infected mosquitoes. Salivary glands are dissected from these mosquitoes
and injected intravenously in order to produce patent liver infection.
51
Serial No. NIA3D- 133
At predetermined intervals, liver specimens are taken by laparotomy.
Histological studies are made on living and stained material. Flourescent-
antibody as an aid in locating parasites is employed, and other labelling
methods will be used as necessary.
Major Findings; Continued study was made on quantitative aspects of the
problem such as the relationship of inoculm size to the development of liver
parasites and subsequent parasitemia. Other studies were made which indicated
that secondary generations of exoerythrocytic parasites in the liver do occur.
Experiments on route of inoculation (ie. portal circulation versus femoral
vein) indicated no difference in degree of liver infection.
Work on this project was curtailed due to the urgency of work on malaria
as a zoonosis.
Significance to Bio-medical Research and the Program of the Institute;
Confirmation of the liver cycle of malaria puts work on chemotherapy on a
sound basis. Unelucidated aspects of the cycle may have significance in
chemotherapy and basic biology of the parasites. Study of simian species is
especially appropriate as the close relationship to the human species indicates
that any findings may be directly applied to human infection with few
reservations. The present project is closely correlated with chemotherapy and
tissue culture projects, and is another phase of the integrated malaria study
of the Laboratory of Parasite Chemotherapy.
Proposed Course of the Project; During the coming year special emphasis
will be placed on study of the exoerythrocytic stages of newly isolated strains
of simian malaria and on comparative biology of the parasites of different
species of primate malaria. Work is again proposed on the influence of
immunity on exoerythrocytic parasites, and the project will continue to be a
source of material for tissue culture work and for chemotherapy studies.
Part B included - Yes
52
Serial No. NIAID-133
PHS-NIH
Individual Project Report
Calendar Year i960
Part B« Honors, Awards, and Publications
Publications other than abstracts from this project:
Eyles, D. E. Anopheles freebcrni and A. quadrimaculatus as experimental
vectors of Plasmodium cynomolgi and P. inui« J. Parasit., U6:5UO. I960.
Eyles, D. E. The exoerythrocytic cycle of Plasmodium cynomolgi and
P. cynomolgi bastianellii in the rhesus monkey. Araer. J. Trop. Med. & Hyg.,
9 :5U3-555." I9oo;
Eyles, D. E. Susceptibility cf Anopheles alblmanus to primate and avian
malarias. Mosquito News, In press. I960.
Eyles, D. E. and Coleman, N. The effect cf metabolites on the antitoxo-
plasmic action of pyrimethamine and sulfadiazine. Am. J. Trop. Med. & Hyg.,
9:277-283
Eyles, D. E. The treatment of toxoplasmosis. From Human Toxoplasmosis,
Munksgaard, Copenhagen, pp. 127-1U5. I960.
Gibson, C. L. and Jumper, J. R. The prevalence of canine toxoplasmosis
in Memphis, Tennessee. J. Parasit., U6:55>9-565. I960
Note: Last three papers not directly associated with project but repre-
sent previous projects not reported this year.
53
Serial No. NIAID-133-A
1. Parasite Chemotherapy
2. Cytology
3. Memphis, Tennessee
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Chemotherapeutic studies of the direct effect of drugs
on the exoerythrocytic stages of primate malaria.
Principal investigator: Don E. Eyles
Other Investigators: Robert L. Ingram, Nell C. Owen,
Clinton S. Smith
Cooperating Units: None
Man Years (calendar year 1°60) :
Total: 2 l/2
Professional: 1 l/2
Other : 1
Project Description:
Objectives: To determine the direct effects upon exoerythrocytic parasites
of primate malarias of established antimalarial drugs. To describe morpho-
logical and histochemical changes and relate these to mode of action. To
determine dosages necessary for effect. To s tudy effect as related to time of
administration; that is, before infection and at various times during infection.
To study possible synergists. To study the reaction of the primate host to
killed or damaged parasites. In the case of incomplete effects, to determine
how parasites survive treatment and produce relapse.
To study the direct effect of various substances of known metabolic or
antimetabolic activity on primate exoerythrocytic stages. To relate findings
to physiological processes of the parasites, with the ultimate objectives of
obtaining leads toward development of more effective drugs. It is quite possi-
ble that incomplete effects, not detectable by indirect studies of relapse,
might be seen directly, and provide information useful in future work.
Methods Employed: Plasmodium cynomoigi, the vivax-type parasite of
macaques, is the principal parasite used; however, we plan to use the malar iae-
type, Plasmodium inui, as suitable techniques are developed. For chemothera-
peutic studies, infections are established in the livers of monkeys by the
intravenous inoculation of massive numbers of sporozoites. Patent infections
can ue produced consistently. Drugs or other substances are administered either
before or after infection is produced. In the event of prior drug administration,
5^
Serial No. NIAID-133-A
controls are established using identical inocula. When treatment is begun
after the infection is established, pre-treatment and post-treatment biopsies
are made and the parasites compared with those found in control animals.
The infections, both erythrocytic and exoerythrocytic, are followed after
treatment to obtain information on relapse.
Parasites affected by the drugs are studied morphologically and histo-
chemically by the application of various staining techniques. Drug levels
are obtained when necessary. A flexibility of procedure is maintained so that
the methods may be modified to apply to specific problems as they arise.
Major Findings; Further study of pyrimethamine confirmed the fact that
when administered on the sixth day of sporozoite induced infection all para-
sites seen on subsequent biopsy were severely damaged or killed, but para-
sitemia developed 30 to U0 days later indicating some parasites escaped the
action cf the drug. When sulfonamides were used with pyrimethamine to exploit
the possible synergistic interaction of the two drugs, similar results were
obtained — monkeys developed parasitemia 30 to U0 days after infection even
though all parasites seen were damaged.
Quinocide, the Russian 8-aminoquinoline drug, was compared with Prima-
quine. Even when administered at twice the dosage used with Primaquine the
drug was less effective. Treatment on the sixth day did not prevent the
appearance of large numbers of blood forms on the ninth day, but the liver
parasites after five days of treatment did appear damaged.
Untreated parasites and parasites treated with pyrimethamine were fixed in
Carnoy's and stained with toluidine blue before and after exposure of sections
to the action of ribonuclease . Preliminary conclusions are that the synthesis
of DNA is markedly inhibited by the drug.
Chloroquine had no observable effect upon the liver forms of Plasmodium
cynomolgi, normal exoerythrocytic stages being observed after treatment. Ring
stage parasites appeared in the blood in large numbers on the 8th, l6th and
about the 2Uth day after inoculation indicating the existence of secondary
exoerythrocytic stages.
Significance to Bio-medical Research and the Program of the Institute:
This project is part of an intergrated program of the Laboratory of Parasite
Chemotherapy to study this subject in all of its aspects. The methods used
allow direct evaluation of antimalarial drugs as illustrated by the tests with
Quinocide, and provide a means for studying drug action histochemically. Since
the exoerythrocytic stages are those responsible for relapse and must be elimi-
nated for radical cure, the studies have significance on the use of drugs in
malaria eradication.
55
Serial No. NIAID-133-A
Proposed Course of Project: The project was somewhat curtailed due to
the urgent investigation of malaria as a zoonosis. During the coming year
it is expected to increase the emphasis along lines proposed in the previous
year's reportj that is, studies on Plasmodium inui and studies on mode of
action using metabolites and cytochemical methods. Studies of resistant
strains are also contemplated.
Part B. included - No.
56
Serial No. NTATD-1 ^-b
1. Parasite Chemotherapy
2. Section on Cytology
3. Memphis, Tennessee
PHS-NIH
Individual Project Report
Calendar Year 1960
Part Aj.
Project title: Studies of simian malarias in man to determine
if malaria isazoonotic disease in areas in which
man and monkey are closely associated.
Principal investigators: Don E. Eyles, Charles G. Dobrovolny
Other investigators: Clinton S. Smith, local Malayan staff
Cooperating units: Institute for Medical Research
Federation of Malaya, Kuala Lumpur
U. S. Army Medical Research Unit
Kuala Lumpur, Federation of Malaya
Man Years (Calendar year 1960)
Total 4
Professional 1
Other 3
Project Description:
Objectives:
To determine the degree to which established strains of monkey
malaria, particularly Plasmodium cynomolqi. will grow in man. To isolate
new strains of monkey malaria for comparison with established strains
and for study in man. To work out the epidemiology and transmission of
monkey malarias in the field, and apply knowledge obtained to the problem
of inter-relationship between human and monkey malarias. To determine if
monkey malarias are acquired by man in the field and if they can maintain
themselves in man. To collect and correlate all information on distri-
bution and host relationships of primate malarias.
Methods employed:
Strains of monkey malaria are maintained in captive monkeys in the
laboratory. Using insectary reared mosquitoes, these strains are
57
Serial No. NIAID-133-B
passed from monkey to monkey and from monkey to man. Clinical study of
the malaria produced in man is described in a separate project at the
Atlanta Federal Penitentiary. Strains of malaria are compared before
and after passage through man and attempts are made to transmit the
infections man to man.
A field party is operating in the Federation of Malaya cooperating
with the Malayan Institute for Medical Research and the U. S. Army
Medical Research Unit. A systematic study is being made of the malarias
present in far eastern macaques and other monkeys. Isolation of strains
is being attempted from selected monkeys from a variety of species and
from several geographical areas. Studies are being made of the vectors
of monkey malaria as compared with the vectors of human malaria. The
prevalence of human malaria is studied in aborigines groups closely
associated with monkeys.
Major findings:
It was found that laboratory personnel bitten by mosquitoes infected
with Plasmodium cvnomolqi subspecies bastianellii will become infected
and clinically ill with malaria. This finding was confirmed immediately
by experimental inoculation of prison inmates. Clinical and parasitological
characteristics of the infections in man are being studied (separate project).
It was found that the malarias could be passed back to monkey easily
and identified by cross-immuno logical experiments. It was found possible
to infect mosquitoes on human infections, and one instance of man to man
passage by mosquitoes has been seen, the malaria being then reintroduced
into monkey successfully. Man to man blood passage was accomplished
readily.
In the field notable progress has been made in delineating the
prevalence of malarias of various types in monkeys. A number of strains
have been isolated or are in the process of being isolated. Other
infected animals are on hand for future isolation attempts. Many new
findings on host distribution and on geographic distribution of monkey
malarias have been made. Studies of malaria in aborigines associated with
monkeys have been made in Malaya revealing many malaria infections.
Blood passed from aborigines to monkeys have thus far produced no patent
infections in the monkeys.
Studies of the epidemiology of monkey malarias have been initiated
and the feeding habits of some of the Anopheles determined. By injection
of uninfected monkeys with sporozoites from natural infections it was
determined that Anopheles hackeri is a natural vector of Plasmodium knowlesi.
58
Serial No. NIAID-133-B
Significance to Bio-medical Research and the Program of the. Institute;
The finding that Plasmodium cynomolgj subspecies bastianellii will
grow consistently and produce clinical illness in man suggests the
possibility that malaria is a zoonotic disease; that is, a disease
which man can acquire from animals with which he is associated. Whether
or not such transfer of infection occurs in nature is not yet determined,
but should it occur it would be of greatest significance to the worldwide
malaria eradication program.
More basically, the findings of the study taken in conjunction with
previous findings indicate that there is not the strict specificity of
primate malarias previously supposed but rather that the specificity is
a matter of degree and varies within the species. The possibility of
variants with greater infectivity to man occuring naturally is a matter
of concern.
The full significance of the study cannot be completely evaluated
until study has been carried further, but the potential importance makes
it essential that the project be prosecuted vigorously.
Proposed Course of Project;
Isolation of strains from different monkeys and from different geo-
graphical areas shall be continued, with the view of studying these in
man and comparing them immunologically and morphologically. Work in Asia
at present should eventually be extended to Africa and the new world
tropics.
Studies shall be continued to determine if transmission from monkey
to man occurs in nature, and if so whether or not man is a "deadend" host
or can pass the malaria on man to man. This study requires further study
of populations associated with monkeys and the study of the epidemiology
of malaria in monkeys, a subject which has not been at all well
elucidated.
Part B included — Yes.
5S
Serial No. NIATD-]
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Eyles, D. E., Coatney, G. R. and Getz, M. E. Vivax-type malaria
parasite of macaques transmissible to man. Science, 131:1812-
1813. 1960.
Honors and awards relating to this project;
None.
60
Serial No. NTATT)-m-n
1. Parasite Chemotherapy
2. Section on Cytology
3. Memphis, Tennessee
PHS-NIH
Individual Project Report
Calendar Year 1960
Part Aj.
Project title: In vitro studies of malaria parasites and
liver tissue.
Principal investigator: Robert L. Ingram
Other investigators: John R. Jumper
Cooperating units: None
Man years (Calendar Year 1960):
Total:
2-3/4
Professional:
1
Other:
1-3/4
Project Description:
Objectives:
To develop techniques of maintaining adult liver tissue
in vitro in order to study the biology of the exoerythrocytic stages
of malarias, to test antimalarial drugs and antimetabolites on the
course of the infection of the tissue and other forms of the
organism and to investigate, if possible, certain liver metabolic
pathways and ways in which the parasite may interfere with normal
metabolism of the hepatic cells. Also, to study the degradation
of various therapeutic antimalarial agents by the liver, and the
parasites which inhabit the hepatic cells. And to trace the
cycle of the organism in the host.
Methods employed :
Routine tissue culture methods are used, but the mediums
employed, accessory factors, conditions of the cultures, methods of
preparation, etc., are devised to produce the desired effects.
61
Serial No. NIAID-133-C
These include considerations of the composition of the medium,
accessory factors such as glycolytic intermediates, hormones which
stimulate growth or otherwise affect metabolic pathways, gas
requirements for the proper oxygen tension, buffering capacity,
etc. The effect of various agents against sporozoites of the
malaria parasite were determined by bioassay using P_s_ gallinaceum
and baby chicks. Standard bacteriological procedures were used to
assess the effect of the compounds on the microflora of the
mosquito.
Histological sections were made and material inoculated into
experimental animal to determine the results of the studies.
Staining of the material with fluorescein labelled antibodies and
tagging with radioactive isotopes, using the autoradiographic
techniques for detection of the parasite were also used.
32 3 14
P ~~ and H - and C - labelled thymidine were obtained from
commercial sources. At first approximately 5/Lic of the H and C*4
compounds were injected into the body cavity of infected mosquitoes.
This produced a high mortality which did not appear to be^due
entirely to traumatization of the insect. Thereafter, p and
tritrated thymidine was added to the food of the mosquitoes. The
mosquitoes were examined later for oocysts and in some experiments
dissections were made for sporozoites. After 14 days the mosquitoes
were offered a blood meal on the experimental animal and the glands
or whole ground mosquitoes were injected into the animal. Tissue
sections were monitored and film strip radioautographs were made.
Chemical fraction of the nucleic acids, nucleotides and
purine and pyrimidine bases were carried out by methods of Schmidt
and Thannhauser, Schneider, and others. Separation and identification
of the nucleotides and bases have been attempted by ionexchange and
paper chromatography and spectrophotometry analysis.
Fetal human material was obtained from a local hospital.
'1ajor findings;
Slight improvements have been made for maintaining the liver
tissue over that reported last year. However, many additional
factors and conditions which needed to be tested have been studied
for their effect on liver maintenance. Also, greater success has
been had with outgrowth of "hepatic" cells which meet a single
criterion of hepatic cells, e.g. the selective uptake of neutral
red dye as reported in the literature. Sufficient quantities
of material have not been available to do further studies, espec-
ially of a biochemical nature, to characterize the cells.
62
Serial No. NIAID-133-C
We have continued to grow the HE stages of the parasite
whenever these are available in sufficient numbers in the livers
of Pj;. cvnomolgi bastianellii infected monkeys. Also, we have
demonstrated by tissue culture that the parasite is in the liver
four days after sporozoite inoculation. This represents a some-
what closer elucidation of the cycle of the parasite in the
animal.
Sulfapyrazine, 6-mercaptopurine, and pyrimethamine, at lOfj.
M per ml adversely affected the in. vitro survival of organized
liver tissue, especially when chick plasma was used as substrate
(in a tissue culture sense, not in a biochemical sense) for the
tissue fragments. Aminoptein at the same level showed little
deleterious effect. Human plasma as substrate greatly counter-
acted the inimical effect of the drugs on the survival of the
tissue.
Penicillin and streptomycin at 500-1000 units and micro-
gram per ml respectively showed very definite parasiticidal
activity. Sulfa drugs such as sulfathiozole, Gantricin, and
Elkosin at 500 micrograms per ml also showed some parasiticidal
activity, but less than that shown by the penicillin and
streptomycin combination. The broad spectrum antibiotic, Achromycin,
had less effect on the sporozoites than did the sulfa drugs. The
addition of 250 micrograms of "tycostatin, a fungicidal drug, to
certain of the other drugs did not increase their effect on the
survival of the sporozoites.
Achromycin in combination with Dow A, a fungicidal compound,
or '.Tycostatin eliminated or greatly inhibited the growth of
most of the microorganisms associated with the mosquito. Penicillin
and streptomycin and the sulfa drugs gave only partial control of
the micro flora. The studies indicate that in attempts to infect
organized tissue, cellular outgrowths, or cell lines, with
sporozoites, penicillin and streptomycin, the commonly used anti-
biotics to combat contamination in tissue cultures, should not
be used — first, because they will adversely affect the sporozoites
and secondly they are ineffective against the mosquito flora.
32
The mosquitoes which were given p showed fewer oocysts and
in some cases the oocysts at 14 days was the size of a normal 7
day old oocyst. To date, no sporozoites have been demonstrated in
these mosquitoes either microscopically or by the mosquitoes being
able to infect an experimental animal.
3
Mosquitoes which were given the H - thymidine showed more oocysts
than did those which received the p^2 which were allowed to feed upon
the same infected monkey. About 90 per cent of Anopheles f reeborni
63
Serial No. NIAID-133-C
3
H fed mosquitoes were positive for oocysts; many had over 100
oocysts on the gut. They also showed high sporozoite infections.
Experimental animals inoculated with gliffls from these mosquitoes
became infected. However, because of faulty film and other
technical difficulties radioautographs of the parasite has not,
as yet, been demonstrated. However, we have promising slides
in the process of being exposed.
The nucleic acid studies are in the formative stages with
the greatest effort at present being directed toward separation and
identification using commercially available known compounds.
Significance to Bio-medical Research and the Program of the
Institute:
The liver has such a varied and interesting function that any
possibility of maintaining it in vitro would be of interest,
especially, to biochemists; also, to tissue culture workers. The
results of the in vitro maintenance of the liver would be of
interest to. other workers who are making studies with malaria similar
to ours. The finding should be of interest to workers who are
studying infectious hepatitis and other liver infections, of which
there are a large number of parasitic ones.
The importance of the drugs, tested for their effect on the
survival of organized liver tissue in vitro, as antimetabolites and
antimalarial are such that any information concerning their mode
of action would be of general interest to biochemists, pharma-
cologists, 3nd malariologists. Also, any clues to substances which
might counter their actions such as competitive substrates, etc.,
would be of general interest to workers studying chemotherapeutic
agents and to biochemists. Many bio-medical scientists are attempt-
ing to isolate viruses from mosquitoes and other Diptera by means
of tissue culture. This is especially true of the encephalitides,
dengue and related viruses. The findings reported here concerning
the effect of antibiotics against the microflora of mosquitoes
should be useful to these investigators. The results should be
especially useful to those who attempt to infect tissue culture
material with sporozoites or who attempt embryonic inoculations
with sporozoites and hope to eliminate the possible concomitant
bacterial and fungal infections.
If we are successful in tracing the cycle of the malaria
parasite in the host by the radioisotope technique this will ad-
vance our knowledge of the parasite and should be a very useful
finding. It will probalby encourage others to use the method
in similar studies. Biochemical knowledge related to malaria
organisms is extremely fragmentary and it is bdieved that our
approach to the subject will produce fruitful results. Nucleic
Serial No. NIAID-133-C
acid studies are among the forefront of bio-medical studies
at the present time; this apparently indicates the sugnificance
with which biological scientists regard these compounds.
Proposed course of the project:
We intend to make use of the results reported here to make
a number of studies concerning the biology of the EE stages of
malaria if we discover ways in which to produce high infections in
the liver.
Work will be extended with the antimalarial and antimetabolite
drugs. We especially wish to screen a number of these drugs for
their antimalarial potency by means of tissue culture.
The tracer work will be oontinued unless it is found that
the organism will not incorporate some of the radioactive label;
this seems highly unlikely. We shall also look for mutagenic
effects of the isotopes on the parasite. Hopefully this method
can be used to determine the mode or site of action of some of the
drugs to be tested.
Part B. included — No.
G5
Serial No. NTATn-133-n
1. Parasite Chemotherapy
2. Section on Cytology
3. Memphis, Tennessee
PHS-NIH
Individual Project Report
Calendar Year 1960
Part kj_
Project title: Immunological studies of malaria parasites.
Principal investigator: Robert L. Ingram
Other investigators: John R. Jumper
Cooperating units: None.
Man years (Calendar Year 1960):
Total
1
Professional
1
Other
0
Project Description:
Objectives:
To develop ways of distinguishing closely related malaria organisms
to devise means of detecting the organisms in the tissue phase as soon
as possible after infecting the experimental animal so as to delineate
the cycle of the organism, and basic immunochemical studies concerning
antigen-antibody reactions of protozoa.
Method s employed:
Antigenic material was prepared from both the erythrocytic and the
sporozoite stages of the malaria parasites. White rabbits were given
a series of injections of these preparations and after a suitable
time, serums were collected. These were pooled, the gamma globulin was
fractionated and conjugated with fluorescein isothiocyanate (F.I.C.).
This preparation was used to stain the parasites which were then
examined using fluorescent microscopy equipment.
66
Serial No. NIAID-133-D
Major findings;
Blood forms of Ejl gallinaceum were stained with a serum prepared
by using sporozoites of this species as the antigenic material.
P. fallax and F_j_ lophurae also were stained with this labeled serum.
Blood forms of P_t. cvnomolqi bastianellii failed to stain with this
preparation. However, F.I.C. labeled serum from experimentally
infected convalescent monkeys was used to stain P^ cvnomolqi bastian-
ellii. P. gallinaceum, P. fallax and P^. lophurae failed to stain with
this preparation.
Significance to Biomedical Research and the Program of the Institute ;
The use of the fluorescent antibody technique has in the past few
years received much attention by bio-medical investigators. The
reported findings that malarias can be studied by this method will
find utility in a number of laboratories where malaria is being
studied. The fact that one stage of an organism was used to stain a
different stage of the same organism should facilitate the study by
this technique of many parasitic diseases in which the organism
undergoes several stages of development.
Proposed course of the study ;
As other species of malarias become available to us and experimental
infection with these become possible, we shall study immunological cross
reactions among the various species. We also intend to use this method
to help trace the cycle of the organism through the host. Attempts
may be made to partially purify the antigen or antigens involved in
the reaction.
67
Serial No. NIAID-133-D
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
Ingram, Robert L., Otken, Luther B., Jr. and Jumper, John R.
Staining of malaria parasites by the fluorescent antibody technique.
In press. Proc . Soc. Exp. Biol, and Med.
63
LABORATORY OF IMMUNOLOGY
Summary 1
140 - Studies in the Transplantation of
Tissues 4
141 - Studies on Antibody Production and
Mechanism of Hypersensitivity 7
142 - The Immunology of Coh Mouse High
Line Sarcoma * 9
143 - Characterization of Substances of
Bacterial Origin Affecting Resistance
to Infection 11
144 - Pathogenesis of Allergic Encephalo-
myelitis 14
145 - Studies of Auto-Antibodies in Human
Disease States and in Experimental
Animals 19
146 - The Resistance of Fetal and Neonatal
Gonads to Damage by Maternal
Immunization to Testicular Antigens. ... 23
147 - Immunochemical and Immunogenetic
Studies of the Protein Isoantigens in
Serum 25
147-A - Immunochemical Studies on Human Serum
Antigens and Antibodies 32
148 - Basic Studies on the Cellular
Localization of Antigens, Antibodies
and other Substances by the Fluorescent
Antibody Technique and Fluorescence
Microscopy 34
150 - The Relationship of Delayed and
Immediate Hypersensitivity to Allergic
Thyroiditis in Inbred Guinea Pigs 39
152 - The Chemistry of Antibodies 42
153 - Basic Studies on the Chemical,
Physical and Skin Reactive Properties
of Extracts of House Dust 47
LABORATORY OF IMMUNOLOGY
SUMMARY STATEMENT
CALENDAR YEAR i960
During the past year there has been a tremendous resurgent interest in
the field of immunology. This has been brought forcefully to our attention
by a variety of events which occurred during the Fall of i960. A very high
percentage of the many Research Associate applicants, which were interviewed
over a period of several weeks, chose the Laboratory of Bnmunology as being
the one in which they desired to conduct research for a two year period.
The interest of these physicians in immunology stems from the possibilities
that they envisage in the application of immunology and its newer techniques
to the study of human disease. Many of these exceptionally bright young men
have been stimulated by their professors at medical schools such as Harvard,
N.Y.U., etc. During the course of their medical studies they have had the
opportunity to carry on immunological investigations, which have made them
realize the importance of immunology as a fruitful area of research.
For several years the Laboratory of Immunology has conducted a bi-
weekly immunology seminar. In the past, this was attended by the members of
our staff and a few investigators from several other Institutes. In view of
the ever-increasing interest in immunology, and in anticipation of this, the
Laboratory scheduled a weekly seminar which is, at the time of this writing,
so well attended that it has been necessary to abandon the NIAID conference
room and to hold these lectures and discussions in the Clinical Center
Auditorium (l^t-th Floor). The investigators attending these seminars are from
all of the Institutes and represent a variety of disciplines. It is evident
that these scientists have found the need for incorporating immunology in
their studies in order that they may attain their research goals. This is
another example of the resurgence of immunological research at NIH and points
out the necessity for the development of a truly strong and outstanding
laboratory. At the present time we possess' a small nucleus of competent
immunologists and from this nucleus it will be possible to develop a labora-
tory which potentially can be a national and international leader in the
field.
During the year the demands on the scientific staff of the Laboratory
for consultation and collaboration have been increasing. Many of the demands
on their time center around the performance of the newer techniques and
methods of immunological research, such as immuno-electrophoresis, fluorescent
antibody, passive cutaneous anaphylaxis, immunization with adjuvants, etc.
This almost daily demand has been of a proportion as to interfere at certain
times with the scientists' own research program.
Since the activation of the Laboratory of Dmnunology in 1957* the pro-
gram has been concerned, principally, with basic research. However, for some
time an important need has been felt for the initiation of clinical studies in
immunology and allergy. In September i960 the Clinical Immunology Section
was activated with the appointment of Dr. Howard c. Goodman as Head of this
Section. For some years he has been engaged in clinical studies and more
recently has been concerned with the importance of auto-antibodies in the
■j vthogenesis of certain disease states. Dr. Goodman spent the year preced-
ing his appointment at the Pasteur Institute where he was engaged in studies
on serum proteins involving the use of the technique of Immunoelectrophoresis.
As space permits, the Section will be expanded with the addition of two investi-
gators at the doctoral level. Dr. Goodman's section will be working in close
collaboration with Dr. Vernon Knight, Clinical Director, NIAID, on clinical
studies involving the immunological aspects of such diseases as lupus erythem-
atosus, nephritis, chronic thyroiditis, and others in which an auto-immune
basis is suspected. In order that clinical studies are executed properly, it
has become apparent that the clinician must have intimate contact with the
basic scientist and the laboratory methods involved in immunological research.
The newly activated Clinical Immunology Section, being an integral part of
our Laboratory, affords us a unique opportunity to accomplish first-rate and
complete clinical studies.
Due to lack of space since the formation of the Laboratory, it was
necessary to place several of our permanent professional staff on contract
with universities. Two of these scientists resigned during the year and
accepted positions at institutions of medical research. The exact reasons
for their leaving is not completely understood but, in part, it was due to
better salary opportunities and our present lack of facilities. The loss of
these established scientists leaves a gap in certain areas of our research
program. Another scientist working on tissue transplantation resigned during
the year to enter training in surgery. It seems imperative that this person
e replaced in the near future in order that this important segment of immu-
njlogical research be represented in our Laboratory.
The activities of the staff have resulted in certain significant scien-
tific advances*
Aller^J-C Thyroiditis
Experimental allergic thyroiditis was produced in Strain 13, inbred,
histocompatible guinea pigs by immunization with a single dose of guinea pig
thyroid extract in complete Freund's adjuvant. Thyroiditis developed as early
as 5 days after immunization, was present in all animals at 16 days, and by
7 weeks was consistently present and generally severe. Delayed skin test
hypersensitivity was found as early as 5 days after immunization in nearly all
animals, and was present in all animals with thyroiditis at 7 weeks. Circu-
lating anti-thyroid antibody was absent at 5 days in animals with thyroiditis
and with delayed hypersensitivity. At 7 weeks after immunization, anti-thyroid
ntibodies were present, and antibody titres correlated with the presence and
.egree of thyroiditis. This correlation was not found at certain other times
after immunization. To intensify the formation of antibody without producing
delayed hypersensitivity, guinea pigs were immunized with thyroid extract in
Freund's incomplete adjuvant. These animals at 7 weeks showed no thyroiditis
and no delayed hypersensitivity, although they did develop low levels of circu-
lating antibody. The presence of delayed hypersensitivity was correlated with
experimental allergic thyroiditis, while the presence of circulating antibody
did not correlate with thyroiditis. These observations constitute the earliest
production of experimental allergic thyroiditis and the most severe disease
at the time intervals studied.
House Dust Allergens
Studies on the chemical and physical properties of house dust extracts
that are used clinically for the diagnosis and treatment of house dust allergy
have been studied to identify the components responsible for the specific skin
reactions produced in house dust sensitive individuals. It has been found that
the house dust extracts consist of a heterogeneous mixture of acidic poly-
saccharides. The heterogeneity has been demonstrated by electrophoretic and
ultracentrifuge sedimentation analysis and also by the multiplicity of cross
reactions obtained with antisera to the various pneumococcal polysaccharides.
The chemical composition of the various fractions has been shown to be roughly
5-20$ polypeptide and 80-95$ polysaccharide, containing about equal amounts
of uronic acid (probably glucuronic acid), D-glucose, D-galactose, D-mannose
with lesser amounts of L-rhamnose and L-arabinose. Starch block electrophore-
sis experiments at low pH and in the presence of 7M urea have shown that the
polypeptide components are covalently linked to the polysaccharides. A
number of different kinds of experiments have shown that the colored compo-
nents may be separated from the bulk of the material and the skin test data
suggest that the color is not associated with skin reactivity. The skin test
data also indicate that more than one allergen specificity is present in the
dust extracts. By starch block electrophoresis it has been shown that certain
isolated fractions have greater skin reactivity than the original extract;
however, it should be appreciated that the skin test assay is exceedingly
inaccurate so it is not possible to determine the degree of concentration of
activity.
Immuno chemistry and Genetics of Gamma Globulin
Agar-gel immunochemical analysis of sera from rabbit litters, with
precipitating antibodies prepared in rabbits, has shown that seven antigenic
determinants of the gamma globulins are genetically controlled by at least
two distinct gene loci with each specificity exhibited when the appropriate
allele is present. Since the gamma globulins are soluble proteins which have
properties of both an antigen and an antibody, they should be subject to
quantitative estimation and cytological localization. This immunogenetic
system, therefore, may be uniquely suited for the study of certain basic
problems in genetics, embryology, immunology and protein chemistry. Since
gamma globulin should be expected to pass through maternal- fetal barriers,
of considerable interest would be the study of gamma globulin synthesis in
fetal and neonatal rabbits uncomplicated by the problem of distinguishing
maternally derived gamma globulin. In other studies, antibodies to human
serum proteins were prepared in monkeys since this animal, being a closely re-
lated species, might be more discriminating for minor antigenic differences
than a distantly related species. Three "slow" gamma globulins were found,
instead of the one usually detected with horse or rabbit antibodies. Two of
these were shown to be related to myeloma proteins. The quantitative estima-
tion of these gamma globulins in serum should be helpful in the early diagnosis
and study of diseases, such as multiple myeloma, which involve qualitative and
■^antitative changes in the gamma globulins.
M ; hanisms of Hypersensitivity
The genetically distinct guinea pigs of inbred Strains 2 and 13 have
proved to be a very important immunological tool. After studies established
the fact of skin compatibility in the tvo strains, experiments were conducted
to transfer cells with a measurable biological activity. Transfers of tuber-
culin sensitivity were undertaken by the intraperitoneal injection of living
lymphoid cells from compatible donors. The almost quantitative transfers
between inbred guinea pigs were a reflection of the continued viability of
the active cells in the recipients. It was found that transfers could be
made soon after active sensitization of the donor; early transfers merely
required a longer waiting period for full sensitivity to appear in the re-
cipient. This contrasted with the need to test as soon as possible after
transfer in random-bred animals in face of a dwindling population of trans-
ferred lymphoid cells in a non- compatible host. Two models are being develop-
ed to study the mechanisms of immediate and delayed hypersensitivity in the
inbred guinea pigs; protracted anaphylactic shock and, the massive local
hemorrhagic reaction, respectively. It has been shown that there are differ-
ences in susceptibility to hypersensitivity reactions. Strain 2 guinea pigs
were more resistant to death by bronchospasm and tended toward a protracted
syndrome in anaphylactic shock. Both Strain 2 and 13 guinea pigs required
more mycobacteria than did random-bred Hartley guinea pigs for inducing "de-
layed" sensitivity to egg albumin, using Freund's adjuvant.
.. aracterization of Human Serum Auto-antibodies
Fractions of human serum separated by anion- exchange cellulose column
chromatography were studied by Immunoelectrophoresis . The conditions for
elution of eighteen immunologically distinguishable human serum proteins
from the columns were determined. Gamma globulin obtained under the appro-
priate conditions by this method was found to be pure; rabbits immunized with
this fraction made antibodies to none of the other serum proteins. By the
use of anion- exchange cellulose columns, it has been found possible to sep-
arate the 7S from the 18-19S antibody activities in sera of patients with
thyroiditis and lupus erythematosus. Initial results indicate that the addi-
tion of imraunoelectrophoretic characterization of these and other sera will
be extremely helpful in our aim of characterizing the antibody activities
found in human serum.
Fluorescent Antibody Staining of Malaria Parasites
The fluorescent antibody staining of the human malaria parasite,
. asmodium vivax, has been recorded for the first time. A globulin fraction
o convalescent serum from a patient having a long-standing infection with ■
P. vivax was labeled and the fluorescent antibody applied to thin blood films
containing the parasite. The organism was visible by virtue of its specific
immuno-fluorescence. If blood films can be stained in such a manner that,
essentially, only the parasites are visible, this could provide a method
whereby numerous blood films could be examined in a single day by means of
t.n automatic scanning microscope. Fluorescent antibody studies were conduct-
ed on P. cynomolgi bastianellii, the monkey malaria parasite which, recently,
has been shown to be transmissible to man. Considerable morphological detail
was observed at fluorescence. Preliminary studies on the serological relation-
ships, as based on degrees of fluorescence, indicate that P. vivax and P.
cynomolgi bastianellii parasites may have common antigens and that the two
species may be closely related. This is of importance in the program of the
world-wide eradication of malaria which has become more complex with the
possibility that monkeys may serve as reservoirs for human infection.
Submitted by:
c . ^Atf-^Q
JohnT;. Tobie, Ph.D., Acting Chief,
Laboratory of Immunology, NIAID
Form No. ORP-2 Serial No. NIAID - lto
Nov. i960 1. Laboratory of immunology
PHS-NIH 2. Natural & Acquired
Individual Project Report Resistance
Calendar Year i960 3« Bethesda, Maryland
Part A:
Project Title; Studies in the Transplantation of Tissues
Principal Investigator: Dr. Sanford H. Stone
Other Investigators; Dr. Joseph A. Bauer, Jr.
Cooperating Units; None
Man Years: Patient Days; None
Total: 1.25
Professional: 0.75
Other: 0.5
Project Description; Isologous and Homologous Lymphoid Cell Trans-
plants
Objectives:
To compare the relative efficiency of passive transfer of tuberculin
sensitivity and antibody producing activity between histocompatlble
guinea pigs with that between random-bred animals.
Methods Employed;
Inbred guinea pigs are sensitized with egg albumin and sheep erythro-
cytes in Freund's complete adjuvant (containing mycobacteria). Lym-
phoid cell transfers are made by intraperitoneal route into inbred and
random-bred recipients. Cell recipients are tested for skin- reactivity
and circulating antibody.
Major Findings;
1. The passive transfer of delayed hypersensitivity and antibody syn-
thesizing capacity appears to be more effective between inbred
guinea pigs than between random-bred animals.
2. Soni cation or freezing and thawing of lymphoid cells obliterates
their capacity for successful transfer.
Significance to the Program of the Institute;
Passive transfer with viable lymp^ i cells represents an important
Serial No. NIAID - lto
tool in the study of immune phenomena. In histocompatible animals, the
variable of transplantation immunity is eliminated, the transferred
cells thrive, and go on to immune maturity in the recipient (if the
lymphoid cells are harvested soon after sensitization of the donors,
a longer waiting period will intervene before full sensitivity is
manifested in the recipient).
Proposed Course of Project:
F, generation guinea pigs of the inbred Strain 13 - inbred Strain 2
crosses will be used to study problems of host vs. graft and graft vs.
host reactions during transfers of lymphoid cells.
Part B included: Yes X Ho
PHS-NIH
Individual Project Report
Calendar Year i960
(Attachment I)
Serial No. NIAID - lliO
Part B: Honors, Awards, and Publications
Publications:
Bauer, J. A., Jr. and Stone, s. H. : Isologous and Homologous
lymphoid Transplants I. The Transfer of Tuberculin Hypersensitivity
in Inbred Guinea Pigs. J. Immunol. 85: (in press), i960.
Form No. ORP-2 Serial No. NIAID - 1^1
Nov. i960 PHS-NIH 1. Laboratory of Immuno logy-
Individual Project Report 2. Natural and Acquired
Calendar Year i960 Resistance
3. Bethesda, Maryland
Part A:
Project Title; Studies on Antibody Production and Mechanism of Hyper-
sensitivity
Principal Investigator; Dr. Sanford H. Stone
Other Investigators; None
Cooperating Units; None
Man Years; Patient Days; None
Total; 1.0
Professional; 0.5
Other; 0.5
Project Description;
Objectives;
To study the mechanisms of "Immediate" and "delayed" hypersensitiv-
ity.
Methods Bnployed;
Inbred histocompatible guinea pigs permit experimentation on genetic
factors involved in hypersensitivity. The experimental models: random-
bred or inbred guinea pigs injected with antigen in complete (for "de-
layed" hypersensitivity studies) or incomplete (for "immediate" hyper-
sensitivity studies) Freund's adjuvants, and challenged by the subcu-
taneous route with homologous antigen. Methods of quantitation of the
manifestations of "immediate" (survival time in protracted anaphylaxis)
and "delayed" (amount of mycobacterium necessary to sensitize) become
feasible. The local massive hemorrhagic reaction described in previous
reports is mot versatile for manifestation of "delayed" hypersensitivity.
Major Findings;
If quantitative methods are to be applied to the field of hyper-
sensitivity research, it becomes important to know if all guinea pigs
react similarly to the conditions set up in an assay. It was found
that the NIH inbred strains 2 and 13 differ from the random-bred Hartley
guinea pigs in several significant areas concerned with hypersensitivity.
Strain 2 guinea pigs are relatively resistant to the lethal bronchospasa
7
Serial Wo. NIAID - lH
of anaphylactic shock, and tend toward the protracted type of syndrome
involving the gastrointestinal system. Both Strains 2 and 13 are more
difficult to sensitize for "delayed" type reactivity (local hemorrhagic
reaction ), requiring from 10 to 50 times as much mycobacteria in the
Freund adjuvant emulsion. Questions of genetic variance in such areas
as mast cell concentration, affinity of antibody for tissue, competence
of mononuclear cells to sensitize or of the skin to react, etc., come to
the fore.
Significance to the Program of the Institute:
The genetic influences on hypersensitivity and its manifestations
have been relatively unstudied. The significance of the availability of
the N.I.H. inbred strains of guinea pigs is best stated by quoting from
Dr. Freund' s summary statement of 1959: "The most important of these
(new methods involves) the introduction of genetically distinct, highly
inbred guinea pigs into Immunologic research work. The initiation of such
studies is of particular importance in the field of delayed type of hyper-
sensitivity. The guinea pig is unique in its suitability. . .high degrees
of local and systemic reactions can be produced."
Proposed Course of Project:
The mechanisms of protracted anaphylaxis and the local hemorrhagic
reaction will be investigated using random-bred and inbred guinea pigs.
Part B included: Yes No
Form No. ORP-2 Serial No. NIAID - 1^+2
Nov. i960 PHS-NIH 1. Laboratory of Immunology
Individual Project Report 2. Applied Immunology
Calendar Year i960 3. Paris, France
Part A:
Project Title: The Immunology of Cob. Mouse High Line Sarcoma
Principal Investigator; Dr. Philip R. B. McMaster
Other Investigators; None
Cooperating Units;
Dr. Pierre Grabar, Laboratorie de Chimle Microbienne, mstitut
Pasteur, Paris, France
Dr. Georges Barsky, The Hospice Paul Brousse, Paris, France
Man Years; Patient Days; None
Total; O.J*
Professional; 0.2
Other: 0.2
Project Description:
Objectives:
To attempt to find an antigen in the C3I1 mouse high line sarcoma,
developed by Earle and others at the NIH, which is not present in the
normal mouse tissue, as well as to determine the most effective method
of immunization to prevent the growth of subsequently inoculated tumor.
Methods Employed:
To compare by immunoelectrophoretic and other methods, the nature
and number of antigens in normal mouse tissue and the above named
sarcoma, as well as to try various methods of immunization to determine
which is the more effective in the prevention of the growth of sub-
sequently injected tumor.
Major Findings:
Study in its initial stages. No significant findings to date.
Significance to the Program of the Institute:
The continued search for antigens in tumors not present in normal
tissue may yield results which would seem to offer diagnostic and
Serial HO. NIAID - lk2
therapeutic possibilities.
Proposed Course of Project;
To be continued along the lines outlined above.
Part B included : Yes Ho X
10
Form No. ORP-2 Serial No. NIAID - 1^3
Nov. i960 PHS-NIH 1* laboratory of Immunology
Individual Project Report 2. Iramunochemistry Section
Calendar Year i960 3« New York City, New York
Part A:
Project Title; Characterization of Substances of Bacterial Origin
Affecting Resistance to Infection
Principal Investigator: Dr. Curtis A. Williams, Jr.
Other Investigators; None
Cooperating Units; Dr. Rene J. Dubos, Rockefeller Institute, N.Y., N.Y.
Man Years; Patient Days; None
Total: 1.0
Professional: 0.5
Other: 0.5
Project Description;
Objectives:
1. Further examination of biologically active materials extracted from
tubercle bacilli ^ Their isolation and composition.
2. To distinguish between the mechanisms of specific and non-specific
acquired resistance to infectious diseases.
Methods Employed;
1. Animal protection tests are performed in albino mice. They are
studied by bacterial enumeration from infected organs and by survival
time*
2. Biologically active substances from tubercle bacilli are compared
with bacterial endotoxins from gram negative organisms, which have
similar chemical composition and most of the same activities.
3. Clearance of microorganisms from the blood stream of mice.
k. Chromatography, zone electrophoresis, ultracentrifugation, standard
immunochemical tests.
Major Findings
1. There are many variables to be carefully controlled in the prepara-
tion of active substances from tubercle bacilli. Solvent ratios,
11
Serial No. NIAID - 1*»3
water content of organic solvents, and temperature of Importance In
terms of recoverable activity in products.
2. Strains of mice differ in their response to these products. C57/6
mice and Rockefeller Swiss behaved roughly as predicted from their
respective responses to BCG.
3. Studies with Serratia plymuthica have demonstrated that the use of so-
called "non-pathogens" to infectious disease may be very fruitful. The
usual serological reactions and Immunological responses are operative.
The cause of death is apparently toxemia. Survival 2b hours usually
leads to complete recovery and a high level of immune resistance.
k. The blood clearance mechanism has been examined with reference to
Immune and non-immune resistance, to strain of mouse, to physiologic
and genetic variants among bacteria. It has become clear that bacterial
variation plays a more significant role in the function of this mecha<-
nism in resistance than many host treatments or the strain of host.
Significance to the Program of the Institute;
Relating recognized resistance mechanisms in experimental animals to
the outcome of infectious diseases is of obvious importance. The study
of bacterial variation with respect to these mechanisms, however, could
lead to an eventual understanding of the terms pathogenicity, virulence,
susceptibility, and resistance.
Proposed Course of Project;
Terminated as NIAID Project June 31, i960, with the resignation of
principle investigator.
Part B included: Yes X No
12
(Attachment i)
Serial No. NIAID - 1^3
PHS-NIH
Individual Project Report
Calendar year i960
Part B: Honors, Awards, and Publications
Publications:
Williams, jr., Curtis A.: Immunoelectrophoresis. Scientific
American. 130:lU0, i960.
13
Form No. ORP-2 Serial No. NIAID - ikk
Nov. i960 PHS-NIH 1. Laboratory of Immunology
Individual Project Report 2. Clinical Immunology
Calendar Year i960 3- Bethesda, Maryland
Part A:
Project Title; Pathogenesis of Allergic Encephalomyelitis
Principal Investigator; Dr. Philip Y. Paterson
Other Investigators;
Miss Jennifer Bell and Mr. S. Martin Harwin, medical students at
N.Y-U. School of Medicine and Mr. N. C. Didakow, laboratory
technician.
Cooperating Units; None
Man Years; Patient Years; None
Total; 2
Professional; 0.5
Other; 1.5
Project Description;
The allergic encephalomyelitis (AE) induced in rats and other
animals by injection of normal nervous tissue combined with Freund
type adjuvants is under study at New York University College of
Medicine under contract with the National Institute of Allergy and
Infectious Diseases (Contrac-; Serial #SA-^3«PH 3027).
Project Subtitle I: Transfer of Allergic Encephalomyelitis (AE) by
means of Lymph Node Cells
Objectives;
To transfer AE from diseased donors to normal recipients using
lymph node cells.
Methods Employed;
Adult donor rats are sensitized to nervous tissue by injection of
guinea pig spinal cord emulsified in Freund' s complete adjuvant (oil,
emulsifier and killed mycobacteria) . At varying times thereafter,
the lymph node cells (LNC) from these donors are collected and used
for 2 different types of transfer:
(a) homologous type of transfer — the LNC are injected intra-
cerebrally into rats of a genetically different strain.
Serial No. NIAID - 1^
(b) isologous type of transfer— the LNC are injected intra-
venously into rats of the same genetic strain.
Major Findings;
1. Homologous type transfers - AE lesions may be identified in
approximately 10$ of recipients if the donor cells are injected
via the intracerebral route. Transfer of cells by other routes
(viz., intravenous, intraperitoneal) has not resulted in transfer.
2. Isologous type transfers - AE lesions may only rarely (less than
5$) be found in recipients.
Significance to the Program of the Institute;
These means of transferring AE by LNC, in addition to the technique
described in Annual Report for 1959> provides further support for the
cellular nature of the disease and offers a means for ultimately
defining the morphogenesis of the AE lesion in precise terms. The
information obtained should prove helpful in transfer studies of
analogous auto-immune disease states, e.g., thyroiditis, aspermato-
genesis.
Proposed Course of Project;
Contract terminated July 1, i960.
Project Subtitle II; Role of Freund's adjuvant components in induction
of Allergic Encephalomyelitis (AE).
Objectives;
To define which component (s) of Freund's adjuvant is required for
rapid and regular induction of AE in rats and guinea pigs.
Methods Employed;
Groups of rats and guinea pigs are given a single intracutaneous
injection of guinea pig or rat spinal cord alone or combined with
Freund's adjuvant components in varying combinations. Occurrence of
AE is determined by presence or absence of AE lesions in brains and
spinal cords of animals about 3 weeks post-sensitization.
Major Findings;
AE may be regularly induced in the rats and infrequently induced in
the guinea pig by injection of nervous tissue combined with Freund's
incomplete adjuvant, i.e., paraffin oil, emulsifying agent but no
killed mycobacteria. The paraffin oil is the important component of
2
15
Serial No. NIAID - 1U4
the adjuvant for rapid induction of the disease in the rat. In
contrast, both paraffin oil and killed mycobacteria are essential for
rapid and regular induction of disease in guinea pigs. An occasional
rat may exhibit lesions of AE if injected with nervous tissue alone;
such is not the case in the guinea pig. In both the rat and the guinea
pig, heterologous nervous tissue is more active as sensitizing antigen
than is homologous nervous tissue.
Significance to the Program of the Institute:
In addition to providing a less complex technique for induction of
the disease at least in the rat, these findings provide additional
clues to understanding the mode of action of Freund type adjuvants and
role of cellular and humoral factors in development of experimental
auto-immune disease. This work underscores the long appreciated poten-
tial hazard of using any vaccine consisting of nervous tissue for
immuni zation of man.
Proposed Course of Project:
Contract terminated July 1, i960.
Project Subtitle III: Antibodies against autologous brain in rats with
Allergic Encephalomyelitis (AE) .
Objectives:
To determine whether rats sensitized to homologous or heterologous
nervous tissue-adjuvant produce antibodies specifically directed
against their own (autologous) brain.
Methods Employed:
Rats are sensitized to nervous tissue by a single injection of rat
or guinea pig spinal cord in adjuvant. About 3 weeks later the rats
are bled, sacrificed and their sera tested (by complement fixation) for
antibodies using both autologous and homologous brain extracts as the
antigens. Appropriate controls are included in every test.
Major Findings:
More than half of the rats tested produce antibodies against their
own (autologous) nervous tissue. The antibody appears organ-specific;
it does not react with kidney tissue and does not appear in response
to sensitization with kidney-adjuvant.
Significance to the Program of the Institute:
These results add weight to the presumed auto-immune theory of AE
and analogous forms of experimental tissue damage.
18
3
Proposed Course of project;
Contract terminated July 1, i960.
Serial No. NIAID - Ikk
Part B included: Yea X No
17
(Attachment I)
Serial No. NIAID -Ihk
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B: Honors, Awards, and Publications
Publications ;
Paterson, P. Y. : Transfer of Allergic Encephalomyelitis in Rats
by Means of Lymph Node Cells. J. Exper. Med. 111:119, i960.
Bell, J., and Paterson, P. Y- : Rapid Induction of Allergic
Encephalomyelitis in Rats Without the Use of Mycobacteria.
Science. 131:lWt8, i960.
Harwin, S.M., Paterson, P. Y., and Didakow, N.C: Antibodies
Against Autologous Brain in Rats With Allergic Encephalomyelitis.
Nature (in press).
18
Form No. ORP-2 Serial No. NIAID - 1^5
Nov. i960 PHS-NIH 1. Laboratory of immunology
Individual Project Report 2. Clinical Immunology
Calendar Year i960 3. Paris, France and
Betheada, Maryland
Part A:
Project Title; Studies of Auto-Antibodies in Human Disease States
and in Experimental Animals.
Principal Investigator: Dr. Howard C Goodman
Other Investigators; None
Cooperating Units;
Dr. John L. Fahey, GM., NCI, and Dr. Richard A. Malmgren, PA., NCI.
Dr. James H. Baxter, LC., NOT,.
Dr. Leonard Laster, A&R., NIAMD.
Man Years; Patient Days; 60
Total: 2.0
Professional: 1.0
Other: 1.0
Project Description;
Objectives;
1. To detect the factors in human sera vhich react with human tissue
antigens .
2. To characterize these factors, i.e. show whether they are actually
gamma-globulins and whether the reactions with tissue antigens
behave like typical antigen-antibody reactions.
3. To attempt to produce similar auto-antibodies in experimental
animals.
k. To determine whether animals with experimentally produced auto-
antibodies develop tissue damage.
Methods Employed;
The tannic acid hemagglutination test of Boyden, gel-diffusion
antigen-antibody precipitation tests (Ouchterlony and Oudln),
Immunoelectrophoresis (Grabar and Williams), and DEAE cellulose
column chromatography (Sober and Peterson) techniques are used to
19
Serial No. NIAJD - l*+5
detect and characterize the serum factors which react with tissue
antigens .
To produce auto-antibodies in animals experimentally, rabbits,
guinea pigs and rats are Immunized with tissue extracts incorporated
in Freund's adjuvant.
Patient Material;
Patients with lupus erythematosus and idiopathic nephrotic syndrome
are admitted to the clinical center for these studies. Kidney biopsies
are performed and the clinical response to steroid administration is
studied; lipid metabolism studies on patients with the nephrotic syn-
drome have been performed in conjunction with Dr. James Baxter, NEE.
Major Findings:
During the past year at the Pasteur Institute with Professor Pierre
Grabar, the technique of imaiunoelectrophoresis was used to analyze the
fractions obtained from normal human serum after DEAE cellulose column
chromatography. The information gained about conditions for elution
of some 20 of the immunologically distinguishable serum proteins from
the cellulose columns, particularly the gamma, beta-2 A, and beta-2 M
(the macrogammaglobulins), will be immensely useful this year when we
attempt to see into which of these classes of "immune»globulins" auto-
antibodies fall. A specific example of what will be a general usefulness
of Immunoelectrophoresis in characterization of serum proteins in other
diseases (not necessarily caused by immune reactions) is the demonstra-
tion with Dr. Leonard Laster (NIAMD) and Dr. Donald Frederickson (NET)
of the absence of beta lipoproteins from the serum of their patient,
thus confirming the diagnosis of "a-betalipoproteinenda", a newly
described disease syndrome.
Attempts to detect auto-antibodies in patients with nephritis and
nephrosis and to produce auto-antibodies to kidney tissue and renal
disease in animals have not been successful so far, although these
studies continue. Auto-antibodies are detectable in patients with
lupus erythematosus, chronic thyroiditis, and a few patients with
scleroderma and rheumatoid arthritis. In patients with thyroiditis,
the serum factors which react with thyroglobulin have been shown to be
gamma-globulins and to belong to both the 7S (mol. wt. about 160,000)
and IBs (mol. wt. about 1,000,000) gamma-globulins. In the sera of
patients with lupus erythematosus, auto-antibodies reacting with com-
ponents of cell nuclei have been studied. The L. E. cell factor has
been shown to be in the TS gamma-globulins. Some of the other anti-
nucleus factors were found to be 7S gamma-globulins, and were demon-
strated for the first time to be among the l8s gamma globulins.
Rabbits injected with calf thymus and human liver nucleoprotein
extracts have been shown to develop antibodies not only to the injected
20
2
Serial No. NIAID - 1^5
nuclear material, but also auto-antibodies to their ovn cell nuclei.
So far no associated tissue damage has been detected.
Significance to the Program of the Institute:
Obese studies, as veil as those to be developed in our ovn Labora-
tory of Immunology and vith the cooperating units listed, represent
the approach of the section on clinical immunology to the study of the
role of immune mechanisms in human disease states. The question of
the importance of auto-antibodies in the pathogenesis of a great number
of diseases deserves critical study. The finding that anti-nucleus
auto-antibodies in lupus erythematosus can be macro-globulins raises
nev questions about the reasons for the production of high molecular
weight antibodies. The production of anti-cell nucleus auto-antibodies
in experimental animals should enable us to determine whether these
antibodies can produce a disease state in these animals, and thus help
decide whether the auto-antibodies are important in the pathogenesis of
the human disease states where they are found.
Proposed Course of Project;
Attempts to detect auto-antibodies in different disease states vill
continue (e.g. ulcerative colitis with Dr. Laster). The auto-anti-
bodies detected will be characterized by immunoelectrophoresis and
cellulose column chromatography. A logical extension of our studies
on serum antibodies in disease states will be to apply, in conjunction
with Dr. Wilton E. Vannier, the technique of DEAE cellulose column
chromatography and immunoelectrophoresis to the characterization of re-
agins in serum of patients with clinical allergy.
Animal studies on the production of and characterization of auto-
antibodies and production of tissue damage will continue, with emphasis
on the antibodies to components of cell nuclei to thyroid tissue and to
kidneys. In patients and in experimental animaiw where tissue damage
is associated with auto-antibodies, attempts will be made to determine
whether tissue damage is caused by hypersensitivity of the delayed type
or due to circulating antibodies. Cell and serum transfer experiments
can help decide this question, and we are in a position to do these
studies using the histocompatlble, pure strains of guinea pigs develop-
ed at NIH.
Part B included: Yes X No
21
PHS-NIH
Individual Project Report
Calendar Year i960
(Attachment i)
Serial No. NIAID - 1^5
Part B: Honors, Awards, and Publications
Publications;
Baxter, James H., Goodman, Howard C. and Havel, Richard J. Serum
Lipid and Lipoprotein Alterations in Nephrosis. J. Clin. Inv.
39:^55, I960.
Fahey, John L-, Dutcher, Tftomas E. and Goodman, Howard C. Cell
Nuclei and Gamma Macroglobulins . Royal Soc. Med. Vol. 53 No. 8, i960
Fahey, John L. and Goodman, Howard C. Characterization of Anti-
HSiyroglobulin Factors in Human Serum. J. Clin. Inv. 39:1259, i960.
Goodman, Howard C., Fahey, John L. and Malmgren, Richard A. Serum
Factors in Lupus Erythematosus and Other Diseases Reacting With
Cell Nuclei and Nucleoprotein Extracts: Electrophoretic, Ultra-
centrifugal and Chromatographic Studies. J. Clin. Inv. 39:1595,
i960.
22
Form No. ORP-2 Serial No. NIAID - 1^6
Nov. i960 PHS-NIH 1. Laboratory of Immunology
Individual Project Report 2. Applied Immunology
Calendar Year i960 3« Bethesda, Maryland
Part A:
Project Title: The Resistance of Fetal and Neonatal Gonads to
Damage by Maternal Immunization to Testicular
Antigens .
Principal Investigator; Dr. Philip R. B. McMaster
Other Investigators: None
Cooperating Units: None
Man Years: Patient Days: None
Total: 0.1*
Professional: 0.2
Other: 0.2
Project Description:
Objectives:
To demonstrate that maternal Immunization to testicular antigens Is
innocuous to the development of the offspring in contrast to the report-
ed results of maternal immunization to lense and brain, and thereby
lend support to the hypothesis that delayed hypersensitivity is a re-
quisite for the development of allergic aspermatogenesis, and also to
provide a control model for the study of vhite cell transmission across
the placenta.
Methods Employed:
Adult female outbred Hartley strain guinea pigs were immunized with
testicular extract in complete adjuvant in two groups, one shortly
before mating and the other shortly after mating. These animals were
later tested for delayed hypersensitivity and serum antibodies to
testicular extract, whereas the offspring were examined for the same
serum antibodies as well as for histologic and physiologic abnormalities
of the reproductive system.
Major Findings:
All the actively Immunized animals developed circulating antibody
and delayed hypersensitivity to testicular extract. All the off-
spring had circulating antibody in their serum, but nevertheless their
reproductive organs were normal physiologically and histologically.
23
Serial NO. NIA3D - lM?
Significance to the Program of the Institute;
!Riis study extends the previous work of Dr. Freund, who
demonstrated a correlation between aspermatogenesis and delayed hyper-
sensitivity to testicular antigens, and a lack of such a correlation
between the disease and serum antibody in adult animals. Ifcese
experiments on the development of offspring of immunized females show
that circulating anti-testicular antibody does not cause detectable
damage even when present early in life during the development of
barrier membranes and organ differentiation. This study also indicates
a lack of a transfer of prolonged nature across the placental membrane
of outbred animals, even when a high degree of delayed hypersensitivity
is present in the maternal parent.
Proposed Course of Project:
Hiis study may serve as a model control for the study of white cell
transfer across the placenta, by comparing the above with a similar
study in inbred animals, in which transferred white cells would survive.
Part B included : Yes Ho
2H
Form No. ORP-2 Serial No. NIAID - lVf
Nov. I960 PHS-NIH 1« Laboratory of Immunology
Individual Project Report 2. immunochemistry Section
Calendar Year i960 3« Bethesda, Maryland
Part A:
Project Title; Immunochemical and Immunogenetic Studies of the Protein
Isoantigens in Serum
Principal Investigator; Dr. Sheldon Dray
Other Investigators; Miss Glendowlyn 0. Young
Cooperating Unit;
Project Subtitle I - Drs. Charles W. Mcpherson and Donald Bailey,
Animal Production Section, Serial No.
Project Subtitle V - Dr. T. N. Harris, Children's Hospital of
Philadelphia, Philadelphia, Pennsylvania
Man Years; Patient Days; None
Total: 3.0
Professional: 1.5
Other: 1.5
Project Description;
Project Subtitle I: Genetic Control of Serum Isoantigens
Objectives;
To establish the genetic basis for the presence of serum isoantigens
and to develop lines of animals, (rabbits) with genetically defined iso-
antigens .
Methods Employed;
1. Qualitative tests using primarily agar-gel methods for the identifica-
tion of isoantigens in families of rabbits and in various rabbit
populations .
2. Selective breeding of rabbits to test genetic hypotheses and estab-
lish genetically defined lines.
Major Findings;
The genetic control for two gamma-globulin allotypes have been estab-
lished. A genetic theory for five additional gamma globulin allotypes is
25
Serial NO. NIAID - 3A7
"being tested utilizing the serum bank of rabbit families available from
earlier studies.
Significance to the Program of the Institute;
To know the genetic mechanism for the control of isoantigens is
essential for the usefulness of these isoantigens in studies of basic
immunology and hypersensitivity. The molecules having antigenic sites
under the genetic control postulated are soluble proteins, namely, gamma
globulins. Some of these molecules may also react as antibodies. As
antigens, precipitable by antibodies, they should be subject to quanti-
tative estimation. Moreover, these molecules may be expected to pass
through maternal- fetal barriers. Thus, this immunochemical genetic
system may be uniquely suited for studies of some basic problems in
Immunology, hypersensitivity, embryology, and protein chemistry.
Proposed Course of Project:
1. To establish genetically defined lines of rabbit gamma-globulin
types so that they may be available for other studies.
2. To study the genetic control of new gamma globulin isoantigens or
other isoantigens as they are discovered.
Project Subtitle II; Chemical Studies of Serum Isoantigens
Objectives;
1. To identify the antigens in serum which induce isoantibodies.
2. To study the chemical nature of these isoantigens.
Methods Employed;
1. Immunochemical analysis by diffusion in agar-gel; simple diffusion
in plates, and immunoelectrophoresis .
2. Passive cutaneous anaphylaxis, tanned-cell hemagglutination and
other Immunological tests for antibody.
3. Cellulose ion-exchange chromatography and other protein fractiona-
tion and purification methods.
k. Enzyme (papain) digestion of isoantigens to produce smaller frag-
ments for study.
Major Findings;
Investigations have shown that components of sera from individual
rabbits are antigenic in certain other rabbits and that isoprecipitins
can be produced which react with serum antigens having electrophorettah
2
Serial Ko. NIAID - IkJ
mobilities corresponding to alpha-, beta-, and gamma-globulins.
Significance to the Program of the Institute;
1* Differences in serum proteins between individuals of the same species
may be of genetic and clinical significance similar to that of the
blood groups. The serum isoantigens may be implicated in some of the
unexplained transfusion reactions in man.
2. Serum isoantigens may be useful in the study of "immune tolerance"
and perhaps serve as a somewhat analagous experimental model for the
study of supposed tolerance to Rh antigen*
3* Serum protein isoantigens may be particularly suitable for study of
some basic problems in immunology concerning the nature of antigen
and antibody sites, antigenicity of antibodies, chemical structure
of gamma-globulin, the antigen-antibody reaction, etc.
Proposed Course of Project;
1. Chemical characterization of the gamma-globulin isoantigens in
rabbits. Development of methods of assay for each of the iso-
antigens.
2. Studies on the chemical structure of gamma-globulin by papain diges-
tion studies and quantitative assay of antigenic sites.
3* Search for additional gamma-globulin isoantigens and additional
alpha- and beta-globulin isoantigens.
k. Specificity of rabbit isoantigens and antigenicity in other species.
5. Isoantigens in other species, particularly mice and man.,
6. isoantigens In other tissues.
7. Antibody production in rabbits of different gamma-globulin types.
Project Subtitle III; Application of serum isoantlgen-isoantlbody
systems to study of "Immune Tolerance."
Objectives;
To investigate the question as to whether the isoantigens would be
useful in the study of so-called "immune tolerance."
27
Serial No. NIAID - 1^7
Methods Employed;
1. Selective breeding of rabbits on the basis of known genetics of
gamma-globulin isoantigens*
2. Immunochemical methods to evaluate production of isoantibodies and
presence of isoantigens.
2. Quantitative assay of isoantigens by agar-gel methods and labeling
with tracers*
Major Findings;
un
Results of preliminary experiments suggest that "immunologic Respon-
siveness" to gamma-globulin isoantigens may be induced in rabbit off-
spring in the following ways. The offspring may be exposed to the
"foreign" isoantigen naturally during pregnancy as a result of the
"foreign" isoantigen of the mother passing through the maternal- fetal
barrier. The offspring may also be exposed artificially by trans-
fusion of the mother with the "foreign" isoantigen during the last week
of pregnancy.
Significance to the Program of the Institute;
"Immunologic unresponsiveness" has been one of the most interesting
problems in hypersensitivity during the last few years as a result of
new developments in our knowledge of tissue grafting. Should the iso-
antigen- isoantibody system be effective in the induction of "tolerance,"
this system may very well be most suited for a study of some of the basic
mechanisms involved in "tolerance." The isoantigen-antibody system has
the advantage that one may work with soluble proteins (rather than tissue)
and with the precipitating antibodies (rather than tissue rejection) .
Proposed Course of Project;
Discontinued at present because of lack of sufficient animals and
facilities.
Project Subtitle IV; Synthesis of Gamma-Globulins in The Fetus and
Newborn
Objectives;
To determine when and where gamma-globulins are first produced by the
fetal or neonatal rabbit and the rate at which the gamma-globulin
synthesized by the offspring appears in the serum.
Methods Employed;
1. Selective breeding of rabbits on the basis of the known genetics of
28
Serial Ho. NIAID - 1^7
gamma-globulin antigens.
2. Immunochemical methods to assay quantity of gamma-globulin iso-
antigen appearing in the serum.
3* Fluorescent antibody to localize time and site of appearance in
tissues.
Major Findings:
Preliminary findings have shown that the six-week neonatal rabbit
lacks some of the isoantlgens it later produces.
Significance to the Program of the Institute:
1. The isoantigen-isoantibody system in rabbits provides the best means
for study of gamma-globulin formation in the newborn animals since
the gamma- globulin produced by the offspring can be clearly dis-
tinguished from maternal gamma-globulins which pass the placental
barriers .
2. This study may lead into an experimental system analagous to the
Bh system*
3. Serve as a pilot study for the question of transfusion reactions to
plasma.
Proposed Course of Project;
1. Study the production of gamma globulin in offspring of mothers
Immunized to the gamma-globulin produced by the fetus in analogy to
the Bh system.
2. Use of isoantlgens as cell markers in connection with studies of skin
homograft rejection in cooperation with Dr. Joseph Bauer, Peter Bent
Brigham Hospital, Boston, Massachusetts.
3. One of the interesting by-products would be the possibility for the
study of the rate of gamma-globulin synthesis per cell.
Project Subtitle V; Use of Isoantigens as Cell Markers in Cell-Transfer
Studies.
Objectives:
To determine whether donor white cells from an immunized rabbit
produce antibody in an irradiated recipient.
29
Serial No. NIAID - IkJ
vfethods Employed;
1. Use of gamma- globulin typed rabbits as donors and recipients.
2. Fluorescent antibody to identify the source of gamma-globulin anti-
body found in the recipient.
Major Findings; None
Significance to the Program of the Institute;
The isoantigen-isoantibody system provides the means to definitively
determine the above objective, and would demonstrate the applicability
of this system to other problems in immunology involving cell transfer.
Proposed Course of Project;
Evaluate the question as to whether one or two gamma-globulin iso-
antigens may be produced in the same cell. This is a question of con-
siderable theoretical interest with regard to theories of antibody produc-
tion.
Part B included: Yes X No
30
PHS-NIH
Individual Project Report
Calendar Year i960
(Attachment i)
Serial No. NIAID - Hf7
Part B: Honors, Awards, and Publications
Publications;
Dray, Sheldon and Young, Glendovlyn, 0.: Genetic Control of Two
Gamma-Globulin Isoantlgenic Sites in Domestic Rabbits. Science,
131:738-739, i960.
31
Form No. ORP-2 Serial No. NIAID - 1^7-A
Nov. i960 PHS-NIH 1. Laboratory of Immunology
Individual Project Report 2. Immuno chemistry Section
Calendar Year i960 3. Bethesda, Maryland
Part A:
Project Title: Immunochemical Studies on Human Serum Antigens and
Antibodies
Principal Investigator; Dr. Sheldon Dray
Other Investigators: None
Man Years; Patient Years: None
Total: 2.0
Professional: 0.5
Other: 1.5
Project Description:
Objectives:
1. Identification, fractionation, purification, and chemical properties
of antigens in human serum.
2. Significance of serum antigens and antibodies in disease.
Methods Employed:
1. Immunochemical analysis by diffusion in agar-gel; simple diffusion
in tubes, double diffusion in plates, and Immunoelectrophoresis.
2. Cellulose ion-exchange chromatography, zone electrophoresis, and
other methods of protein fractionation and purification.
3> Use of primates for immunization with human serum antigens.
Major Findings:
1. Primates were selected for immunization with human serum antigens
with the idea that a closely related species to man might yield
antibody to components of human serum not readily obtainable in
other ways. Immunization of Rhesus monkeys with various antigens
from human serum has yielded precipitating antibody to many compo-
nents of human serum (albumin, alpha-, beta-, and gamma-globulins).
Precipitating antibodies were found which were specific for three
normal gamma-globulins of relatively slow electrophoretic moMllty
instead of the one usually found with rabbit or horse antisevi.
Furthermore, the immunochemical relationship between the thj«?e
normal 7S gamma-globulin antigen and two myeloma gamma-globi . .ins
32
Serial No. HIAID - lVf-A
were investigated and it was found that one of the myeloma gamma-
globulins corresponded to one of the normal gamma-globulins while a
second myeloma gamma-globulin corresponded to the second myeloma
gamma-globulin. It was further found that these globulins have
antigenic determinants in common.
2. Nine chimpanzees have been immunized with components of human serum.
The results thus far have revealed two antisera to human alpha-
globulin and several antisera to human gamma-globulin.
Significance to the Program of the Institute;
The availability of precipitating antibodies specific for three
normal human gamma-globulins should facilitate many studies of consider-
able interest concerning these gamma-globulins, such as: quantitative
estimation in serum and other body fluids; fractionation and purifica-
tion; chemical structure, particularly in the analysis of fragments re-
sulting from enzyme digestion; antibody properties in infectious dis-
eases and diseases of supposed Immunologic etiology; cytological locali-
zation by fluorescent antibody; and possible genetic differences. Of
immediate clinical interest, the quantitative estimation of these gamma-
globulins in serum should be useful for early diagnosis and study of
diseases which involve qualitative and quantitative changes in the
gamma-globulins, such as in multiple myeloma.
Proposed Course of the Project:
1. Continue the immunization of primates with various fractions of
human serum and to study the antigens which then may be detected.
2. Survey 30 multiple myeloma sera (in collaboration with Dr. John
Fahey, Metabolism Branch, NCI) with respect to their reactions with
monkey antisera.
3. immunization of monkeys with myeloma proteins, rheumatoid factor,
and other proteins of pathological origin.
k. In cooperation with Dr. Edward C. Franklin (New York University
College of Medicine) to investigate the antigenic determinants of
papain fragments of human gamma-globulins.
Part B included Yes No X
33
Form No. ORP-2 Serial No. NIAID - lh8
Nov. i960 PHS-NIH 1. Laboratory of Immunology
Individual Project Report 2. Applied Immunology
Calendar Year i960 3« Bethesda, Maryland
Part A:
Project Title; Basic Studies on the Cellular Localization of Antigens,
Antibodies and other Substances by the Fluorescent
Antibody Technique and Fluorescence Microscopy.
Principal Investigator: Dr. John E. Tobie
Other Investigators: None
Cooperating Units:
Project Subtitle II - Dr. G. Robert Coatney, LPC, Serial NO. NIAID-
130-B
Project Subtitle III - Mrs. Eleanor J. Tobie, LPD, Serial No. NIAID-
123-D
Man Years: Patient pays: None
Total: 3*0
Professional: 1.0
Other: 2.0
Project Description:
Project Subtitle I: Antibody Formation in Neonatal Rabbits
Objectives:
To perform studies on the types of cells involved in antibody forma-
tion and to determine the capacity of neonatal animqlg to synthesize
antibody.
Methods Employed;
Neonatal and adult rabbits were given a single injection of bovine
serum albumen, crystalline egg albumin or killed typhoid organisms,
combined vith hydrocarbon adjuvants, into the footpad. The npitngin
were bled frequently and tested by hemagglutination or agglutination
reactions in an effort to determine the earliest time at which
circulating antibodies first appear. In certain animals the regional
popliteal lymph node was removed and the node fluid tested for
presence of antibody. Fluorescent antibody methods will be employed
to determine the lymph node cells involved in antibody synthesis.
3k
Serial No. NIAID - lUQ
Major Findings;
Using the antibody responses after a single injection of bovine
serum albumen as an example , neonatal animals (1-3 days old when inject-
ed) were very slow in producing antibody. None of the rabbits produced
detectable antibody earlier than 3-4 weeks while in individual animals
it took as long as 100 days. Certain animals failed completely to re-
spond to a single injection when followed for as long as 1*00 days.
One animal which became positive at 102 days continued to produce anti-
bodies through k&l days, at which time the animal was sacrificed. In
contrast to the antibody response in neonatals, antibody could be
detected consistently in adult rabbits 7-9 days after a single injection.
In several animals no circulating antibody could be detected in spite
of the presence of antibody in lymph node fluid.
Significance to the Program of the Institute:
A study of the nature of the antibody response in neonatal animals
may lead to a better understanding of the mechanisms involved in
"immunological immaturity" in the infant. The influence of age upon
antibody formation is an important factor in the capacity of infants
to be actively immunized.
Proposed Course of Project:
Fluorescent antibody studies will be conducted on lymph nodes from
the neonatal rabbit because the slow antibody formation in the neonatal,
as compared with adults, seems to offer a better opportunity to study
the cellular aspect of antibody synthesis.
Project Subtitle II; Fluorescent Antibody Studies on Malaria
Parasites
Objectives:
To specifically stain human malaria parasites by the fluorescent
antibody technique in such a manner that essentially only the malaria
parasites are visible on a blood film.
To feed such blood films through an automatic scanning microscope
for the rapid detection of parasites in large-scale surveys.
Methods Employed:
Globulin fractions of sera were prepared from inmate volunteers who
had long-standing infections with Plasmodium vivax. The globulin was
conjugated with fluorescein isothiocyanate and applied to thin blood
films which had been dehemoglobinized and the films examined at fluores-
cence microscopy.
35
Serial No. NIAID - lW?
Globulin from a patient infected with the vivax-like malaria
parasite (P. cynomolgi bastianellii) of rhesus monkeys was labeled
and applied to individual blood films containing either parasites of
human origin or those of monkey origin. The cross reactions between
these two malaria species were determined by the fluorescent light
emitted.
Major Findings;
The first recorded instance of the fluorescent antibody staining of
human malaria parasites, Plasmodium vivax, and that of the monkey
parasite, P. cynomolgi bastianellii, has been accomplished. Prelim-
inary studies on the serological relationships of these Plasmodia, as
based on the degree of fluorescence, indicate that the two species may
be closely related. The separate application of P. vivax labeled anti-
body to individual blood films containing the parasites of rodent
malaria, P. berghei, or human malaria, p. vivax, resulted in the latter
fluorescing approximately 3 times as bright suggesting that these two
species probably are not as closely related.
Significance to the Program of the Institute;
The fluorescent antibody studies on malaria could lead to a method
whereby hundreds of blood films could be scanned in a single day. A
study of the serological relationships between malaria Plasmodia of
human and animal origin are of great importance in connection with the
program of the worldwide eradication of malaria which has become more
complex with the possibility that monkeys may serve as reservoirs for
human infection.
Proposed Course of Project;
To continue the studies on the serological relationships, explore
methods of producing high-titered antisera in rabbits by sporozoite
immunization and investigate the application of fluorescent stained
malaria films to rapid scanning in an automatic microscope.
Project Subtitle III; localization of Trypanosoma rangeli in the
Insect Host
Objectives;
To determine the organ localization of Trypanosoma rangeli in the
arthropod host and to study the developmental relationships between
the parasite and the insect vector.
Methods Employed;
The insect vector, Rhodnius prolixus, has been experimentally
has been experimentally infected, either artificially, by injection
of trypanosomes into the hemocoel or naturally, by allowing the on
3
Serial No. NIA3D - 1^8
insect to take "blood meals on an infected animal. Fresh-frozen sections
of the whole insect were prepared and the sections conventionally
stained. Fluorescent antibody methods also will be employed for
discrete localization in the arthropod host.
Major Findings:
The trypanosomes have been localized in the salivary glands of the
insect after artificial infection. After certain intervals, the
developmental stages of the trypanosome also were found in the intes-
tinal tract of the arthropod.
Significance to the Program of the Institute;
The successful localization of Trypanosoma rangeli in fresh-frozen
sections of whole insects opens up the possibility of the precise
localization of other infectious agents in arthropod hosts. Detailed
studies on the developmental relationships between the parasite and the
insect vector are now within the realm of possibility.
Proposed Course of Project:
Fluorescent antibody studies have been initiated in an effort to
elucidate the mechanisms involved in the insect transmission of this
trypanosome.
Part B included: Yes X No
37
PHS-NIH
Individual Project Report
Calendar Year i960
(Attachment I)
Serial NO. NIAID - Ihd
Part Bt Honors, Awards, and Publications
Publications;
Milch, Robert A., Tobie, John E. and Robinson, Robert A.:
A Microscopic Study of Tetracycline Localization in Skeletal
Neoplasms. Jour. Histochem. Cytochem., In press.
Tobie, John E. and Beye, Henry K. : Fluorescence of Tetra-
cyclines in Filarial Worms. Proc. Soc. Exp. Biol. Med.,
10U:137-l»»O, i960.
Tobie, John E., Burgdorfer, Willy and Larson, Carl L.: Frozen
Sections of Arthropods for Histological Studies and Fluorescent
Antibody Investigations. Experimental Parasitology, In press.
Tobie, John E. and McCullough, Norman B.: Serological Evidence
of Leptospira pomona Infections in Meat Inspectors. Jour. Am.
Vet. Med. Assoc. In press.
38
Form NO. ORP-2 Serial NO. NIAID - 150
Nov. i960 PHS-NIH 1. Laboratory of immunology
Individual Project Report 2. Applied Immunology
Calendar Year i960 3. Bethesda, Maryland
Part At
Project Title; The Relationship of Delayed and Immediate Hypersensi-
tivity to Allergic Thyroiditis in Inbred Guinea Pigs.
Principal Investigator; Dr. Philip R. B. McMaster
Other Investigators; None
Cooperating Units; Dr. Edwin M. Lerner, LPH, Serial NO. NIAMD • k
Man Years; Patient Days; None
Total: 1.2
Professional: 0.6
Other: 0.6
Project Description:
Objectives:
To determine the relative importance of delayed and Immediate hyper-
sensitivity to thyroid extract in the etiology and pathogenesis of
allergic thyroiditis.
Methods Employed:
Two groups of strain 13 histocompatible guinea pigs, one Immunized
with the aid of complete adjuvant, the other with the aid of incomplete
adjuvant, were compared with respect to the development of circulating
antibody, delayed hypersensitivity to thyroid extract, and thyroiditis.
Major Findings;
A close correlation between the appearance of delayed hypersensi-
tivity to thyroid extract and allergic thyroiditis has been found. The
discovery that the disease could appear as early as five days after
the start of immunization allowed the investigators to show an inverse
relation between serum antibody and the disease could exist as well as
a direct correlation between the two, depending on the interval of time
after immunization. This suggests the possibility of a similar relation-
ship between delayed hypersensitivity to the thyroid and Hashimoto's
disease.
33
Serial HO. NIAID - 150
Significance To The Program of The Institute;
As a result of these studies, a close correlation has been demon-
strated "between delayed hypersensitivity to the thyroid and experimental
allergic thyroiditis. This, therefore, provides experimental evidence
for the role of delayed hypersensitivity in the pathogenesis of experi-
mental allergic thyroiditis and suggests the possibility of a similar
role in the development of allergic thyroiditis in humans.
Proposed Course of Project;
The long term course of this disease In guinea pigs is now being
followed in preparation for a study of the effects of desensitization
to an auto-antigen upon an auto-allergic disease, for which this
particular condition is most admirably suited.
Part B included; Yes X Bo
40
PHS-NIH
Individual Project Report
Calendar Year i960
(Attachment I)
Serial no. NIAID - 150
Part Bt Honors, Awards, and Publications
Publications:
McMaster, Philip R.B.: Autoantibodies and Autosensitivity, in
Fundamentals of Modern Allergy, Edited by Samuel J. Prigal, 51-69*
i960.
McMaster, Philip R.B.: Decreased Aqueous Outflow in Rabbits With
Hereditary Bupthalmia. A.M. A. Arch. Ophth. 6Jm 388-391, i960.
McMaster, Philip R.B., Lerner, Edwin M., and Exum, Eurmal D.: The
Relationship of Delayed Hypersensitivity and Circulating Antibody
to Experimental Allergic Thyroiditis in Inbred Guinea Pigs. J. Exp.
Med. (in Press)
u
Form No. ORP-2 Serial No. NIAID - 152
Nov. i960 PHS-NIH 1. Laboratory of Immunology
Individual Project Report 2. Applied immunology
Calendar Year i960 3. Pasadena, California and
Bethesda, Maryland
Part A:
Project Title; The Chemistry of Antibodies
Principal Investigator; Dr. Wilton B. Vannier
Other Investigators: None
Cooperating Units:
Project Subtitle I - Dr. Ernest M. Heimlich of the Department of
Pediatrics of the University of California at Los Angeles
Project Subtitle II - Dr. James Miller of the Department of Infec-
tious Diseases of the University of California at Los Angeles
Project Subtitle III - Dr. Anil Saha of the California Institute of
Technology and Dr. Jean-Marie Dubert of the Pasteur Institute in
Paris
Man Years: Patient Days : None
Total: 1.16
Professional: 0.66
Other: 0.5
Project Description:
Project Subtitle I: The Chemistry of the Skin Sensitizing Antibodies
of Atopic Human Allergy
Objectives:
To study the sedimentation properties of the skin sensitizing anti-
bodies of the sera of individuals with atopic allergies.
Methods Employed:
Sera from allergic individuals were fractionated by sedimentation
in an angle rotor in the preparative ultracentrifuge, by sedimenta-
tion in the Waugh partition cell in the analytical ultracentrifuge
and by sedimentation in salt density gradients in a swinging bucket
rotor in the preparative ultracentrifuge. The fractions obtained
have been characterized by analytical ultracentrifugatlon, electro-
phoresis and passive transfer skin reaction.
42
Serial No. NIAID - 152
Major Findings:
As previously reported, our data show that the skin sensitizing
activity cannot be exclusively associated with the S19 component and
that probably the S19 component is not active at all. Our data and more
recent experiments carried out by Dr. Alec Sehon, Dr. Julius Gordon and
Dr. Ladislas Gyenes at Montreal are all consistent with the hypothesis
that the skin sensitizing activity is associated with a serum component
or components of intermediate sedimentation coefficient, i. e. between
7S and 18s. The salt density gradient studies have shown that the
sensitizing antibody appears to be associated with serum proteins of
hydrated density of about I.36 rather than being present in the lipo-
protein fractions of density 1.2 or lower. During the course of the
salt density gradient studies, it has been observed that there is a
considerable loss in antibody activity associated with the fairly high
salt concentrations used.
Significance to the Program of the Institute;
The physical properties of the skin sensitizing antibody of atopic
allergy are of fundamental importance in understanding allergic re-
actions. The skin sensitizing antibody is qualitatively different from
most ordinary precipitating antibody in its electrcphoretic and sedimen-
tation properties and in that it is retained locally when injected intra-
dermally rather than diffusing away. The process of antibody fixation
to tissues is an important problem in all allergic reactions and any
differences that can be demonstrated in the chemical or physical prop-
erties between ordinary precipitating and skin sensitizing antibody may
be important in this process.
Proposed Course of Project;
A more detailed study of the salt inactivation of the skin sensitiz-
ing antibody will be carried out.
Project Subtitle II; The Chemistry of the Antibodies of Syphilis.
Objectives:
To study the physical properties of some of the antibodies associated
with syphilitic infections and to study a macroglobulin antibody system
in which a specific purification of antibody may possibly be achieved.
Methods Employed;
Sera from individuals with various stages of syphilitic infection are
being fractionated by starch block electrophoresis and ultracentrifuga-
tion methods and the fractions tested for VDRL flocculation antibody and
for TPI antibody.
1*3
Serial No. NIAID - 152
Major Findings :
The work has not progressed far enough for any definite conclusions
to be reached.
Significance to the Program of the Institute;
There is confusion in the literature with regards to the inter-
relationship between the antibodies in syphilis as measured by the
various tests available. Studies, such as this, involving the prepara-
tion of purified antibody fractions may help to clarify the problem.
The study of the chemical and physical properties of specifically
purified macroglobulin antibody may yield basic information regarding
the structure of these antibodies and their role in immune processes.
Proposed Course of Project:
The studies will be continued as indicated. In the future further
fractionation will be attempted using ion exchange chromatography.
Project Subtitle III: The Fractionation and Specific Purification of
Antibody
Objectives:
1. To explore possible specific methods of antibody purification.
2. To achieve the chromatographic fractionation of specifically
purified antibody and study changes in the distribution of specific
bonding affinities of antibodies during the course of immunization.
Methods Employed:
Specific antigen-antibody aggregates are dissociated at low pH and
the antibody recovered. Cellulose coupled with p-arsanilic acid through
tyramine is being used as a solid adsorbent to purify and fractionate
antibody directed against the p-arsanilic acid hapten.
Major Findings:
1. Purified antibody fractions have been obtained by the low pH
dissociation of BSA antiBSA precipitates and the elution of anti-
body from the cellulose-hapten adsorbent with simple hapten (sodium
p-arsanilate) .
2. Cellulose-tyramine-p-arsanilic acid, cellulose-histamine-p-arsanilic
acid and cellulose-lysine-p-arsanilic acid adsorbents have been pre-
pared and it has been found that all three will adsorb antibody from
sera obtained by immunization with p-arsanilic acid coupled hemo-
cyanin.
3
Serial No. NIAID - 152
Significance to the Program of the Institute;
The general problem of specific purification of antibody is one that
is of basic importance for chemical work in immunology. The presence
of large amounts of nonspecific protein with antibody complicates or
prevents a study of the chemical reactions of antibodies, the structure
of antibodies or the physical properties of antibodies. A study of the
heterogeneity of antibodies and especially the influence of degree or
route of immunization on the distribution of antibodies found will pro-
vide additional basic information about the nature of the immune process.
Proposed Course of Project;
The yield, purity and immunochemical characteristics of antibody re-
moved by the specific adsorbents and purified by dissociation of specific
precipitates will be further investigated. The preparation of cold and
C^ labeled haptens for equilibrium dialysis studies with antihapten
antibody fractions will be continued.
Part B included : yes /x/ no / /
KS
(Attachment I)
Serial No. NIATD - 152
PHS-NIH
Individual Project Report
Calendar Year i960
Part B: Honors, Awards, and Publications
Publications :
Heimlich, Ernest W., Vannier, Wilton E. and Campbell, Dan H.:
Sedimentation Studies of Skin Sensitizing Antibody. J. Allergy
31:361+ (i960).
46
Form No. ORP-2 Serial No. NIAJD - 153
Nov. i960 PHS-NIH 1. Laboratory of Immunology
Individual Project Report 2. Applied Immunology
Calendar Year i960 3. Pasadena, California
and Bethesda, Maryland
Part A:
Project Title; Basic Studies on the Chemical, Physical and Skin
Reactive Properties of Extracts of House Dust
Principal Investigator: Dr. Wilton E. Vannier
Other Investigators: None
Cooperating Units:
Dr. Ernest M. Heimlich of the Department of Pediatrics of the
University of California at Los Angeles and Dr. A. M. Targov, a
practicing allergist in Los Angeles have tested various house dust
fractions for skin reactivity in specifically sensitive individuals.
Man Years; Patient Days: None
Total: O.83
Professional: O.33
Other: O.50
Project Description:
Objectives:
1. To study the chemical and physical properties of the materials used
clinically for the diagnosis and treatment of house dust allergy.
2. To try to identify the components responsible for the specific skin
reactivity.
Methods Employed:
1. Fractions of aqueous house dust extracts have been prepared by
starch block electrophoresis and evaluated for activity by direct
skin test and analyzed chemically.
2. Studies are in progress to determine the effect of chemical or
enzymatic modification of the dust fractions on the specific skin
reactivity.
Major Findings:
1. Previously it vas shown that the dust allergen fractions consist
47
Serial No. NIAID - 153
of a heterogeneous mixture of acidic polysaccharides containing 5-20$
polypeptide. Starch block electrophoresis studies at low pH and in
7M urea have indicated that the polypeptide and polysaccharide com-
ponents are linked through covalent bonds rather than being present
as separate components or linked "by ionic bonds or multiple hydrogen
bonds.
2. Preliminary experiments involving the chemical modification and
enzyme treatment of active dust fractions have been carried out.
Partial methyl esteriflcation of the free carboxyl groups of the
polypeptide portion did not affect the activity; however, the ex-
tent of conversion to ester was rather low and it will be necessary
to test materials in which a more complete methyl esteriflcation
has been achieved in order to be sure that the carboxyl groups are
not involved in determining the allergen activity. Treatment of
active dust allergen fractions with pepsin has had no influence on
their skin reactivity.
Significance to the Program of the Institute;
1. About one- third of all allergic individuals give a positive skin
reaction to house dust extracts. These extracts have been used
for many years in the diagnosis and treatment of house dust allergy.
It is of importance to investigate the chemical nature of these
materials and to find out what chemical groups are involved in the
skin reactions.
2. A rational standardization of the potency of house dust allergen
extracts might well be based on a knowledge of the chemical nature
of the active materials.
Proposed Course of Project:
The study of chemically modified and enzyme treated du3t fractions
will be continued.
Part B included: Yes/X"/ No/ — f
48
(Attachment I)
PHS-NIH Serial No. NIAID - 153
Individual Project Report ' ~~
Calendar Year i960
Part B; Honors, Awards, and Publications
Publications:
Vannier, Wilton E. and Campbell, Dan H. : A Starch Block
Electrophoresis Study of Aqueous House Dust Extracts.
J. Allergy, in press.
1*9
ROCKY MOUNTAIN LABORATORY
Summary p i
170 - Office of the Director 11
171 - Rickettsial Infections 12
172 - Q Fever 13
173 - Diseases Having a Reservoir of
Infection in the Natural Environment
(other than rickettsioses) 22
174 - Transmission of Disease Agents by
Certain Vectors 27
175 - Allergy and Immunology of Fungal
Infections and the Mechanisms of
Allergic Phenomena 33
176 - Viruses, Virus Components, and Virus
Surfaces 39
177 - Immune Prophylaxis of Mycobacterial
Infections 44
178 - Immune Prophylaxis of Salmonella
Infections 48
179 - Investigations of the Role of
Morphological Elements of Micro-
organisms in Immunity and Related
Phenomena 54
180 - The Encephalitides 58
181 - Colorado Tick Fever 63
PHS-NIH
Summary Statement
Rocky Mountain Laboratory
Hamilton, Montana
Calendar Year i960
The accompanying annual report summarizes, as well as such a report
can, the activities of the staff of the Rocky Mountain Laboratory for the year
i960. We have presented our projects in the same manner as we have in previous
years. This is necessary because of the frequent cooperation of workers in
various sections in the solution of mutual problems and because of the types
of problems in which we are engaged.
Our research has continued to be directed toward both basic laboratory
investigations and field studies of insect- and animal-borne diseases. In the
first category are those projects concerned with the chemistry and surface
properties of viruses, the highly intriguing relations of hypersensitivity and
humoral immunity, and the basic relation of structural elements of microorgan-
isms to the activity of the agents, both in vivo and in vitro. Field work is
the foundation of projects related to studies of Q fever, tularemia, Colorado
tick fever, and the ar-bo viruses. In addition, the combined efforts of the
staff are directed to many other areas of research. Such projects include Q
fever, tuberculosis, influenza, poliomyelitis, and cryptococcosis.
We are also engaged in studies of a cooperative nature with members of
the staff of the National Institutes of Health, Communicable Disease Center,
University of Minnesota, University of Georgia, Purdue University, University
of Michigan, New York University, Buffalo University, and other institutions.
The cooperators have sought our aid because of special methods, techniques, or
ideas which have been developed at RML. As a result of our past accomplishments,
63 visitors have spent some time here during the last year. I believe this
reflects in some measure the solid contributions the staff has made in both the
recent and distant past, for these visitors represent scientists interested not
only in entomological and biological problems but also those interested in
chemical, physical, and immunological problems related to many fields of
microbiology.
Hypersensitivity. Studies of hypersensitivity have been directed
primarily toward clarification of the relations existing between delayed hyper-
sensitivity and circulating antibodies and determination of the factors respon-
sible for induction of contact hypersensitivity. It has been demonstrated
previously that delayed hypersensitivity precedes circulating antibodies. This
delayed hypersensitivity is directed toward protein. When circulating antibody
appears, as occurs with relatively large amounts of conjugated protein, or when
booster doses are administered, the specificity of the antibody response becomes
oriented toward smaller configurations on the antigen molecule. Studies of
immunity in neonatal animals have revealed that these animals, when injected
with an antigen 12 hours after birth, develop circulating antibodies but fail
to develop delayed hypersensitivity. Additional experiments suggest that this
inability of neonatal animals to express such reactions is due to a deficiency
in a skin reactive factor rather than an inability to respond to a primary
injection of antigen.
It was shown that contact hypersensitivity is directed toward a hapten
and that simple compounds rather than protein conjugates of these compounds
produce contact hypersensitivity when administered to experimental animals.
It is considered, however, that the production of this phenomenon is related
to the specific proteins present in the host tissues because a conjugate con-
taining soluble guinea-pig skin proteins and a hapten produces in guinea pigs
delayed reactions and Arthus reactions to the conjugate and contact hyper-
sensitivity to the hapten.
Poliovirus. Continued studies of poliovirus have yielded considerable
fundamental data. In cooperation with the group at the University of Minnesota,
it was shown that agents such as octyl alcohol-chloroform and neutral hydroxyl-
amine do not materially change the physical properties of purified infectious
RNA of poliovirus but destroy over 99$ of "the infectivity of this material.
The destruction of infectivity by uncoupling of a single link in a large
particle (probably an acyl link of an amino acid with a phosphate group at the
end of an RNA chain) suggests a possible approach to virus chemotherapy. Other
studies have revealed that only 0.1$ of RNA infectivity could be accounted for
by residual protein, thus strengthening the concept that RNA does indeed con-
stitute the infectious portion of the poliovirus moiety. By the use of chromo-
tographic methods, it was also demonstrated that only certain of the avirulent
strains of poliovirus can be differentiated from the virulent strains from which
they are derived. This is in direct contrast to work reported by others. In
studies of the infectivity of RNA it was found that the bulk of virus particles
react with susceptible cells and that the relatively poor correlation between
virus particles and PFU is due to the poor efficiency of RNA at entering sites
where it can influence virus production. A precipitation test for detection
of antibodies against poliovirus has been developed which is more sensitive
than the neutralization test presently employed. The antigen used is radio-
active virus.
Endotoxins in bacterial fractions. Research on endotoxins derived from
Gram-negative organisms has continued to occupy much of our attention. As is
so frequently the case, a fresh outlook on an old problem yields results of
great value. The ideas held by Dr. Westphal, which attributed the activity of
endotoxins to the presence of a firmly bound lipid ("lipid A"), were apparently
generally accepted until presentation of the work done at RML. The* finding
that lipid A was not active and that deproteinized and "delipified" endotoxin
was active stirred considerable controversy. In fact, the controversy was so
intense that our efforts to develop a vaccine against Salmonella infections
have been diverted to settling this issue. Recent studies of the kinetics of
inactivation of toxin by hydrolysis with hot acid have given data which should
end this discussion. The old concept of "purified endotoxins" must be abandoned
since there have been no previous toxins as good as those obtained at RML, and
we feel these are to be still further purified. Many of our studies would have
been difficult to pursue without the active participation of Dr. Landy and
others, especially certain commercial concerns which, because of interest in
our results, provided us with mass cultures of organisms grown in commercial lots.
The purification of Vi antigen by curtain electrophoresis is a major
advance in the study of this most important antigen. The demonstration that
certain labile acetyl groups are responsible for the activity of Vi antigen
resulted in production of material which was ten times more active than purified
preparations prepared by mild acid hydrolysis. Emphasis should be placed upon
the new chemical, physical, and biologic methods that have been devised to solve
these problems.
Many of these methods and ideas have been used in studies of other
problems raised here and by visiting scientists. Contributions made to the
study of immunology of brucellosis and of Q fever will be discussed elsewhere.
The fine structure of bacterial spores has been studied in conjunction with
Dr. Gerhardt. These studies show that the coat is made up of two-dimensional
crystals. Results of a study with the group from Purdue University revealed
that the fine structure of the walls of Penicillium chrysogenum contained much
chitin and very little glucan in contrast to the previous finding of this group
of workers. These studies substantiate the results with Histoplasma capsulatum
previously reported from EML.
While these results may be considered to be of only theoretical interest,
many questions have been solved by methods applied here. It is believed that
these studies are of fundamental importance and will serve as guidelines in
future studies of the relation of morphological elements to various in vitro
and in vivo activities of microorganisms.
Tuberculosis. The problem of immunity in tuberculosis has been studied
intensively. Significant findings include the fact that mice may be satisfac-
torily immunized to subsequent pulmonary infection with virulent organisms by
administration of small doses of avirulent organisms by the aerosol method.
The demonstration that resistance is not due to interference strengthens the
case for the value of immunization with living attenuated organisms for the
prevention of tuberculosis. Since it has been shown that the delayed reactions
elicited by protoplasm of various acid-fast organisms are specific in nature,
it seems practicable to apply these findings to certain diagnostic problems in
man. Use of fractions of tubercle bacilli in producing isoallergic encephalitis
in guinea pigs shows that the adjuvant effect lies in the cell walls and in a
water-soluble protein prepared from the walls. This latter finding is important
since it will allow us to study the adjuvant phenomenon from a molecular level.
Q fever. Our studies on Q fever in many ways have been most productive
and in other ways most frustrating. It has been demonstrated that the number
of dairy cattle infected with Coxiella burnetii is large and is increasing, yet
it is extremely difficult to detect cases of clinical disease in man. In Idaho
and Montana we have shown that a greater number of individuals residing on in-
fected premises have antibodies against this organism than do those living on
noninfected premises, yet no difference can be detected in the number of indi-
viduals who have symptoms compatible with clinical disease. The fact that
organisms isolated from cattle have been uniformly of low virulence for experi-
mental animals may account for our inability to find clinical cases of disease
in those exposed only to cattle.
More satisfying experiences were noted with other phases of the studies.
By the use of skin tests to eliminate allergic individuals from the study group,
190 inmates of the Montana State Prison were safely immunized without producing
such reactions as have been previously reported. It is evident from these
results, as well as from those previously obtained in laboratory personnel,
that human beings can be safely vaccinated against Q fever if the precaution
is taken to eliminate reactors by previous administration of specific skin-test
antigen.
Methods developed for growing rickettsiae on modified Zinsser tissue
cultures yielded relatively large volumes of organisms. These studies led to
others involving purification of C. burnetii by sucrose gradients and by con-
tinuous-flow centrifugation in molar salt solution. These methods likewise
made it possible to obtain certain chemical and physical fractions of these
organisms. It was found that dimethyl sulfoxide could extract from Phase II
C . burnetii a material which acted only as a hapten, but from Phase I organisms
the extract obtained acted as a complete antigen. Lauryl sulfate also extracts
complete antigen from Phase I organisms. Physically, the cell walls of these
organisms can be separated from the protoplasm, and it has been noted that the
cell walls are about 25 times more active in producing immunity than is
protoplasm.
In addition, considerable effort was expended in evaluating Q fever
vaccines and in assembling data which might be used for promulgating minimum
standards for production of Q fever vaccines.
These studies on Q fever are of considerable significance. The labora-
tory and related studies have yielded information of both scientific and applied
interest. It is apparent now that we can safely use our present vaccines for
immunization of man and that it is feasible to produce large numbers of organ-
isms which can be purified and used as vaccine or manipulated to give physical
or chemical fractions which may be less toxic. The failure to find clinical
cases of Q fever in man in the face of a rising incidence of infection in dairy
cattle is highly interesting even if disappointing. The lack of virulence of
strains of C. burnetii for laboratory animals probably is responsible for this
finding.
Other rickettsioses. Studies of rickettsiae other than C_. burnetii have
been continued. By combining the methods presently used for fluorescent micro-
scopy, we have developed with Dr. Tobie a technique for sectioning arthropods
which should be of interest to entomologists working in the field of embryology
and anatomy and to medical entomologists, since thin sections in which the
organs are not displaced can be obtained routinely. By applying the technique
to the study of ticks infected with R. rickettsii it was found that the infec-
tion rate in local ticks varies from 15$ to 28$. Not all of the ticks found
infected by this method are infective for laboratory animals. The value of
this type of study has yet to be fully appreciated.
By application of methods developed at RML we have been able to produce
a vaccine for immunization against Rocky Mountain spotted fever which is 10 to
100 times more potent than those presently manufactured on a commercial basis.
The use of specific toxins has resulted in clarification of many of the prob-
lems related to the taxonomy of rickettsiae and has proved to he useful in
ecological and epidemiological studies of this complex group of diseases. In
further studies, potent immunogenic extracts have been obtained from certain
of the rickettsiae. Their value as diagnostic and prophylactic agents is
presently under consideration.
Tick paralysis. Studies of the incidence of ticks capable of producing
tick paralysis in hamsters have shown that 2^$ to 90$ of lots from Colorado
induce disease; from 0$ to 50$ of those from the Bitterroot Valley; and 75$
from adjoining Missoula County. The incidence of ticks with ability to produce
disease does not appear to be related to the number of cases of tick paralysis
reported in man.
Pasteurella novicida. Considerable debate has been caused by our de-
scription of P. novicida as a new species. Application of the Ovary reaction
(passive cutaneous anaphylaxis) has given weight to our description.
Bacterial vaccines. Studies have been continued on vaccines for certain
bacterial diseases. It has been found that while live Russian tularemia
vaccine is capable of protecting mice more effectively than does ether-extracted
vaccine derived from cell walls of P. tularensis, the protection produced by
live organisms was not effective for long periods of time. Continued studies,
in conjunction with Dr. Foster, have emphasized the value of cell walls in
producing immunity to infections with Brucella abortus in laboratory animals.
It has also been found that live cells suspended in phosphate buffer and shaken
with an excess of ether are killed but not disrupted. These cells constitute
an excellent protective antigen which is less toxic (ID50 7*5 rog») than aqueous
ether extracts obtained by conventional methods (LD50 0.9 to 2.0 mg.). It is
planned that Dr. Foster will continue these studies in Georgia.
Investigations of other bacterial, fungal, and viral infections common
to man and animals have been continued. P. multocida is found as a contaminant
of the oral cavity of many species of animals, and this situation is a potential
source of human infection. Leptospira pomona has been shown to be present in
cattle in western United States, and the organism was obtained from cerebro-
spinal fluid of cattle for the first time. The organism causing haplomycosis
in animals was demonstrated in tissues of animals from a number of different
countries and, in conjunction with Dr. Emmons, a new name, "adiaspiromycosis,"
was applied to this type of parasitism. It has been found that massive doses
of this organism are lethal for monkeys and sheep. The possible relation of
this agent to hemorrhagic fever has been suggested.
Rabies in bats. The study of rabies in bats has been continued and,
although the number of infected bats has not been high, it continues as a
problem. Progress has been made in maintaining bat colonies in the laboratory,
and it is now practicable to commence laboratory investigations of pathogenesis
and other aspects of rabies infections in bats.
Cryptococcosis. An antigen isolated from Cryptococcus neoformans is
now being tested in man to determine the incidence of cryptococcosis in New York,
an area in vhich pigeons have been shown to be heavily infected with this
fungus.
Ar-bo viruses. Studies of ar-bo viruses have yielded results of inter-
est and suggest that emphasis on field studies would greatly increase the pro-
duction of useful data. The California strain, described by Reeves and Hammon,
has been isolated from a snowshoe hare in Montana, and serologic studies of
hares obtained from Michigan indicate that the majority possess antibodies
against this virus. In California it has been demonstrated that, although most
infections in man with this agent are of the inapparent type, some infections
result in serious disease. A virus closely related to Powassan virus was re-
covered from ticks from Colorado and is of importance since viruses of this
group produce serious illness in man. Studies to date indicate that ticks
probably are not the natural vector, but the relation to Powassan virus sug-
gests that mosquitoes would most likely be the vector in nature. In studies
of the complex relation of WEE virus with snakes and mosquitoes it has been
possible to demonstrate that the virus can be readily overwintered in garter
snakes and that mosquitoes can be infected by feeding on such snakes. While
we have not been successful in isolating virus from snakes collected in the
field, the laboratory data suggest that these or similar animals would consti-
tute a host suitable for overwintering of WEE virus. In Idaho and Oregon,
WEE virus was isolated with considerable frequency during the summer season,
while in North Dakota the virus did not appear to be active. Isolations of a
considerable number of strains of trivittatus and inornata viruses were made.
Considerable research was performed to determine the level of viremia
attained in wild and domestic birds infected with ar-bo viruses. After infec-
tion with WEE or St. Louis viruses, turkeys, ducks, chickens, and pheasants
display levels of viremia which should cause infection in mosquitoes feeding
on them. It is of interest, however, that in spite of considerable effort we
have been unable to isolate ar-bo viruses from the bloods of vertebrates.
Negative results were obtained in examination of 1,07^+ specimens collected in
Montana, North Dakota, Oregon, and Minnesota during the spring of i960. These
studies fail to add weight to the contention that latent infections of birds
are a factor in overwintering or of introduction of virus into endemic areas.
Colorado tick fever. Colorado tick fever continues to be a problem in
the western United States. Without stimulation of physicians we still received
a large number of specimens for examination this year and isolated virus from
U9 of them. Our interest in the spectrum of symptoms has continued and we
still see severe cases of illness due either to encephalitis or bleeding tend-
encies. It was found that the complement-fixation reaction developed at the
Rocky Mountain Laboratory is the simplest method for diagnosis of Colorado
tick fever. Vaccine has been prepared and has been shown to be efficacious
in mice. This type of vaccine has been used repeatedly in man without ill
effects, indicating that a vaccine prepared from suckling mouse brain is harm-
less to man when repeated doses are given.
Ticks collected in Estes Park, Colorado, were examined for the presence
of Colorado tick fever virus. The incidence of infection was found to vary
from 5$ to 21$. This high incidence of infection in ticks accounts for the
large number of cases of CTF reported in Colorado annually.
Publications. At the time this report was written we had published k$
papers and had 27 accepted for publication during the calendar year i960. This
compares favorably with those published in previous years, and the diversity
of subjects dealt with reflects the varying interests and capabilities of the
members of the staff of the Rocky Mountain Laboratory.
Comment. Although the RML has developed a broad research program and
a staff capable of dealing adequately with the laboratory problems assigned to
it and those arising from public health needs, consideration should be given
to certain physical needs of the plant. These needs include a building suitable
for housing experimental animals and facilities for housing the library and the
administrative staff. These are matters that call for additions to our present
facilities or radical changes. The availability of funds to modernize certain
of our laboratories would result not only in some consolidation of space, but
also would lead to better safeguards for the health of our staff.
Difficulties were experienced again this year in operating the Rocky
Mountain Laboratory on a budgetary apportionment of 80$ for personal services
and 20$ for other expenses. Even though some of our allotted positions have
not been filled, 20$ of our budget has not been adequate to provide essential
materials and equipment. In view of the proposed reactivation of the pathology
unit and approved addition of personnel in the areas of epidemiology and immu-
nology, a material increase in the amount allotted for other expenses, in
addition to that allotted for staff increases, is essential for continued pro-
ductive research and for the morale of our present staff.
The quality and quantity of our field work is a specific weakness which
we hope to correct by the addition of a qualified epidemiologist to our staff.
The application of some of our basic findings to limited human experience would
establish whether or not the products developed in the laboratory might be use-
ful for control or diagnosis of disease. The recent study of Q fever vaccine
in humans, previously skin tested, to detect and eliminate reactors shows that
vaccine can be used with impunity if proper precautions are taken. Similar
studies with typhoid fever vaccine (based on observations by Ribi and Milner)
should surely be attempted on the above basis and followed by challenge studies
in cooperation with Woodward' s group. Another vaccine that should be studied
in man is prepared from B. pertussis and described by Ribi and Munoz. In
addition, purified products of C. burnetii should be examined, and the value
of various fractions of acid-fast organisms as diagnostic agents should be
investigated. Studies of this nature are essential in order to prove the value
of basic research and to shorten the temporal gap which exists between discovery
in the laboratory and application to man. This is a legitimate field for en-
deavor in this institute and in no way interferes with the programs of CDC.
It should be emphasized that the scientific stature of the RML has not
suffered from the partial shift in the research aims of the group. Exceptional
interest and competence have continued to be manifested by those members of
the staff who have followed their studies of insect- and animal-borne diseases
through field and laboratory investigations. The recent assignment of
Drs. Brennan and Yunker to MARU to study the role of mites in the transmission
of disease is a move in the direction of establishing the necessary type of
entomological field investigation.
Increases in our staff during the past few years have allowed us to
develop programs of significance in areas new to us. These areas, specifically
viral proliferation, allergy, and the relation of morphological elements of
microorganisms to their immunologic activity, have been highly productive and
even have created international interest. It should be emphasized that, in
spite of the relative isolation of this institution, new ideas, new methods,
and important scientific results are forthcoming. As a matter of fact, it is
my opinion that much of our productiveness may be a result of the relative
remoteness of the laboratory.
Carl L. Larson, M.D.
Director
December 1, i960
ROCKY MOUNTAIN LABORATORY, HAMILTON, MONTANA
ANNUAL REPORT, i960
NLAED-I70 OFFICE OF THE DIRECTOR
C. L. Larson, Director
C. B. Philip, Acting Director
H. G. Stoenner, Assistant to the Director
NIAID-171 RICKETTSIAL INFECTIONS
Principal investigator: C. B. Philip
Others: E. J. Bell, W. Burgdorfer, C. M. Clifford,
G. M. Kohls, D. B. Lackman, R. A. Ormsbee,
H. G. Stoenner
NIAID-I72 Q FEVER
Principal investigators: L. Luoto, H. G. Stoenner
Others: B. H. Hoyer, D. B. Lackman, R. A. Ormsbee,
E. Ribi
NIAID-I73 DISEASES HAVING A RESERVOIR OF INFECTION IN THE NATURAL
ENVIRONMENT (OTHER THAN RICKETTSIOSSS")
Principal investigator: J. F. Bell
Others: W. L. Jellison, D. B. Lackman, M. T. McKee,
C. R. Owen, H. G. Stoenner
NIAID-17U TRANSMISSION OF DISEASE AGENTS BY CERTAIN VECTORS
Principal investigator: C. B. Philip
Others: J. F. Bell, J. M. Brennan, W. Burgdorfer,
C. M. Clifford, W. L. Jellison, G. M. Kohls,
D. B. Lackman, V. F. Newhouse
NIAID-175 ALLERGY AND IMMUNOLOGY OF FUNGAL INFECTIONS AND THE
MECHANISMS OF ALLERGIC PHENOMENA
Principal investigator: S. B. Salvin
Others: M. B. Gregg, R. p. Smith
NIAID-I76 VIRUSES, VIRUS COMPONENTS, AND VIRUS SURFACES
Principal investigator: B. H. Hoyer
Others: R. K. Gerloff, F. G. Jarvis*, R. A. Ormsbee,
D. B. Ritter
NIAID-I77 IMMUNE PROPHYLAXIS OF MYCOBACTERIAL INFECTIONS
Principal investigator: E. Ribi
Others: W. T. Haskins, C. L. Larson
NIAID-I78 IMMUNE PROPHYLAXIS OF bAITDNELLA INFECTIONS
Principal investigator: E. Ribi
Others: W. T. Haskins, F. G. Jarvis*, K. C. Milner
NIAID-179 INVESTIGATIONS OF THE ROLE OF MORPHOLOGICAL ELEMENTS
OF MICROORGANISMS IN IMMUNITY AND RELATED PHENOMENA
Principal investigator: E. Ribi
Others: R. L. Anacker, J. E. Coe, C.
K. C. Milner
L. Larson,
NIAID-180 THE ENCEPHALITIDES
Principal investigator: C. M. Eklund
Others: W. Burgdorfer, D. B. Lackman, V. F. Newhouse,
L. A. Thomas
NIAID-181 COLORADO TICK FEVER
Principal investigator: C. M. Eklund
Others: W. Burgdorfer, G. M. Kohls, D. B. Lackman,
L. A. Thomas
*Resigned July 29, i960
10
Serial No. NIAID-170
PHS-NIH
Individual Project Report
Calendar Year i960
Rocky Mountain Laboratory
Hamilton, Montana
Part A.
Project Title: Office of the Director
Principal Investigator: C. L. Larson, Director
Other Investigators:
Cooperating Units:
C. B. Philip, Acting Director
H. G. Stoenner, Assistant to the Director
None
Man Years (calendar year i960):
Total: 31.5
Professional: 1.5
Other: 30.0
Project Description:
The over-all direction and supervision of the scientific research
program of the Rocky Mountain Laboratory; the direction of the adminis-
trative aspects of the Laboratory including all fiscal, supply, house-
keeping, and maintenance activities; and the providing of scientific
services as required by the research staff, including laboratory animal
care and breeding, sterile glassware and media preparation, special shop
services, library, graphic arts, etc.
Part B included: No
11
Serial No. NIAID-171
Rocky Mountain Laboratory
Hamilton, Montana
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Rickettsial Infections
Principal Investigator: C. B. Philip
Other Investigators: E. J. Bell, W. Burgdorfer, C. M. Clifford,
G. M. Kohls, D. B. Lackman, R. A. Ormsbee,
H. G. Stoenner
Cooperating Units: None
Man Years (calendar year i960):
Total: 11.0
Professional: 3«0
Other: 8.0
Project Description:
Objectives:
This project is concerned with elucidation of problems created by
rickettsial agents (exclusive of Coxiella burnetii) causing human and
animal diseases. In the main, attention has continued to be directed
toward the spotted fever group of diseases, their relationships, and
classification.
Methods:
The customary, standardized procedures for study of rickettsial
agents have been followed with particular attention to methods of purifi-
cation, cultivation, and use of specific rickettsial toxins in study of
relationships of the spotted fever group.
Major findings:
1. The value of the mouse protection test against specific toxin
has been demonstrated for measuring immunogenic activity of spotted fever
vaccines and for correlation with other methods of potency assay.
Par: I included: Yes
12
Serial No. NIAID-171
2. By improved standardized procedures, yolk-sac vaccines prepared
at RML were 10- to 100-fold more potent than commercial products. It was
a surprise to find that some vaccines prepared from infected ticks by the
old methods and stored at 5 C. for from 10 to 15 years exhibited immuno-
genic potency values equivalent to those of the commercial vaccines.
3. Factors affecting optimum toxin production from R. conorii and
R. rickettsii grown in fertile hen's eggs have been defined. Temperature
of incubation (33*5° C.) before and after death of the embryos is im-
portant, and yolk sacs should be harvested from 36 to k& hours after
death. However, a better yield is obtained from eggs injected with
rickettsias of Kenya and Siberian tick typhus and South African tick-bite
fever if eggs are held U8 to 60 hours after death. Snyder's solution,
which inhibits growth of organisms, is contraindicated for use as a
rickettsial seed diluent.
k. An agent consistently nonpathogenic for guinea pigs and mice
was isolated from local ticks. The identity and relationships of this
isolate, which resembles rickettsia, are under investigation. The
relationship of an atypical rickettsial isolate from a patient, who died
of an undiagnosed disease, is also being studied.
5. Dimethyl sulfoxide will extract a complement-fixing antigen
from purified R. rickettsii but not from R. prowazekii.
6. The fluorescent antibody technique was found to provide a re-
liable specific diagnostic tool for detection of R. rickettsii in
sections of infected ticks which had been held for 3 days at 37° C.
Conditions were adequately controlled by comparison with preparations of
uninfected ticks, by inhibition with unlabeled immune serums, or by use
of heterologous conjugates. Furthermore, untreated samples of fluorescent-
positive ticks, experimentally infected, caused infection in guinea pigs.
As demonstrated by this technique, natural infection rates among 2^5
ticks from k localities in the Bitterroot Valley varied from 15-5$ to
28$. However, only a small percentage of ticks representative of the
group yielded strains when tested in eggs or guinea pigs.
7. In a study of transovarial transmission, 100$ of 585 eggs and
205 larvae from 5 female ticks were fluorescent positive.
8. Sexual transmission of R. rickettsii from infected male ticks
to the progeny of noninfected females appears to be through the agency of
"contaminated" fluid surrounding the spermophores received by the female,
rather than through the medium of rickettsia-bearing sperm.
Significance:
Continued refinement of methods of preparation of potent ricket-
tsial vaccines, particularly in areas of purified antigens and
13
Serial No. NIAID-171
standardized potency assays, should benefit commercial producers. The
more precise characterization of different isolates of R. rickettsii and
relatives within the group should provide tools for improved investi-
gation of other rickettsial entities; for example, the rather bewildering
variation observed in the tsutsugamushi (scrub typhus) agents. By these
methods additional knowledge of variations in the properties of strains
from various geographic locations, both here and abroad, should become
available .
Likewise, techniques developed for study of R. rickettsii in D.
andersoni by fluorescent microscopy should be adaptable to investigations
of other rickettsia-vector relationships. A better insight into vector-
parasite relationships, for example the difference in ability of acarine
vectors and insect vectors to pass rickettsiae through their eggs, should
also be facilitated. This new technique revealed that natural infection
in ticks from the west side of the Bitterroot Valley was about double the
highest infection rate observed following inoculation of ticks into
guinea pigs during a 5-year study in this area in the 1950' s. However,
it should be noted that a considerable proportion of fluorescent-positive
ticks failed to cause infection when untreated samples were injected into
embryonated eggs and guinea pigs. This raises questions of quantitative
or qualitative factors involved.
The occurrence of fluorescing R. rickettsii in 100$ of progeny of
5 female ticks is in contrast to former reports of only incomplete trans-
ovarial passage.
Proposed course;
Completion of spotted fever assay studies is expected. Lacunae in
our knowledge of the relationships of the spotted fever group will
continue to be filled by results of studies of rickettsial toxins.
Initial observations by fluorescent microscopy on development of R.
rickettsii in ticks will be elaborated.
11
Serial No. NIAID-171
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Bell, E. J. and Stoenner, H. G.: Immunologic relationships among the
spotted fever group of rickettsias determined by toxin neutralization
tests in mice with convalescent animal serums. J. Immunol. 8^(2):
171-182. Feb. i960.
Philip, C. B.: Rickettsial diseases. In A Manual of Tropical Medicine,
3rd ed. W. B. Saunders & Co., Philadelphia, i960, pp. 6I-69, 82-IO3.
Burgdorfer, W. and Lackman, D.: Identification of Rickettsia rickettsii
in the wood tick, Dermacentor andersoni, by means of fluorescent antibody.
J. Inf. Dis. 107(2): 241-2M+. Sept. -Oct. i960.
Philip, C. B.: Microtatobiotes, Rickettsiae and Tick-borne Diseases.
McGraw-Hill Encyclopedia of Science and Technology. McGraw-Hill Book
Co., Inc., New York. i960. Vol. 8, pp. I1O3-U0U; Vol. 11, pp. 567-570;
Vol. 13, pp. 63O-63I.
In press:
Kohls, G. M.: Rocky Mountain spotted fever. History of Medical Depart-
ment of Army in World War II.
Philip, C. B.: Scrub typhus. History of Medical Department of Army in
World War II.
Tobie, E. G. and Burgdorfer, W.: Cryostat technique for sectioning
arthropods. Exp. Parasitol.
Honors and Awards relating to this project:
Dr. D. B. Lackman
Appointed lecturer in Microbiology, University of Montana, Missoula,
Montana.
Dr. H. G. Stoenner
Invited to rewrite chapter on Rocky Mountain spotted fever for Tice's
Practice of Medicine with the collaboration of Dr. C. M. Eklund and
Glen M. Kohls.
15
Serial No. NIAID-172
Rocky Mountain Laboratory
Hamilton, Montana
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Q fever
Principal Investigators: L. Luoto, H. G. Stoenner
Other Investigators: B. H. Hoyer, D. B. Lackman, R. A. Ormsbee,
E. Ribi
Cooperating Units: None
Man Years (calendar year i960):
Total: 12.5
Professional: k.5
Other: 8.0
Project Description:
Objectives:
In view of the wide-spread distribution of Coxiella burnetii
among dairy cattle throughout the United States, continued investigations
on this problem are warranted. The objectives of these studies are
fourfold: (l) to evaluate the public health aspects of Q fever, particu-
larly the role of dairy cattle in the epidemiology of this disease, (2)
to determine the means by which C_. burnetii spreads among livestock,
(3) to reduce the toxicity and improve the immunogenic ity of Q fever
vaccine, and (U) to develop a practical and safe procedure for immuni-
zation of man.
This project will be considered in two categories: "A" deals
primarily with field investigations and "B" with laboratory studies.
A. Field Investigations
Methods:
Methods consist of field studies supported by laboratory work,
including complement-fixation tests, capillary agglutination tests, and
isolation of strains of Q fever in suitable laboratory animals. In
Montana these studies were restricted chiefly to Gallatin and Ravalli
counties, whereas studies in Idaho were concentrated in an area surround-
ing Boise.
Part B included: Yes
16
Serial No. NIAID-172
Major findings:
1. During the past year, the infection rate among herds of dairy
cattle in Montana almost doubled. In the two-county study area in
Montana, herd infection rate increased from 2$ in 1959 to 18$ in i960.
Preliminary observations indicate that many factors are involved in dis-
semination of Q fever rickettsiae among livestock. Although the purchase
of infected-herd replacements is responsible for dissemination of Q fever,
a significant proportion of newly- infected herds was closed to outside
replacements. Hence, the disease might have been spread by arthropods,
wind, or mobile fomites.
2. In two studies designed to evaluate the role of dairy cattle
in the epidemiology of Q fever, similar results were obtained. In an
area in Ada and Canyon counties in Idaho, where very few sheep are raised
and approximately half the herds of dairy cattle are infected, 27$ of 199
persons living on infected premises had antibodies, whereas lk%> of 260
persons residing on Q-f ever-free premises possessed antibodies. However,
the incidence of illnesses resembling Q fever among seropositives was
essentially similar to that observed among seronegative persons. In the
study in Ravalli County, Montana, involving kyo persons, 18$ of those
residing on infected premises possessed antibodies, whereas ^$ of those
residing on Q-f ever-free premises were seropositive. Only 1 of 30 posi-
tive individuals had experienced an illness compatible with that of Q
fever.
3. In a study involving 190 human volunteers at the Montana State
Prison, persons who failed to react to a skin test of 0.02 complement-
fixing (CF) units of antigen were safely immunized with 10 CF units
administered subcutaneously. In a limited number of volunteers a dose
of 20 CF units of vaccine resulted in painful reactions and areas of in-
duration at the sites of vaccination. Antibody response was detected
more readily by the cepillary agglutination (CA) test than by the CF test.
After 1, 2, and 3 doses, 27$, 53$> and 68$, respectively, developed anti-
bodies detectable by the CA test, but only U.5$ of these persons developed
antibodies detectable by the CF test. Dermal hypersensitivity of 16.5$
of these persons converted from negative to positive after vaccination.
k. Investigation of the feasibility of combining strain 19 Brucella
abortus vaccine and Q fever vaccine for immunization of calves revealed
that the antibody response in calves given both vaccines was similar to
that obtained in calves given only Q fever vaccine or strain 19 vaccine.
5. Mature sheep immunized with varying doses of Q fever vaccine
developed high levels of antibody which were still detectable 6 months
after vaccination.
17
Serial NO. NIAID-172
Significance:
The failure to associate clinical illness with the presence of
antibodies in persons exposed to infected dairy cattle conflicts with
observations made earlier in southern California. Most of the strains
of C. burnetii isolated from dairy cattle in Idaho and Montana are only
mildly pathogenic for guinea pigs and this may explain the relative in-
ability of these organisms to cause disease in man.
The presence of antibodies in a large proportion of rural residents
creates a diagnostic problem. A clinical diagnosis of Q fever should be
based upon the judicious evaluation of a rise in antibody titer or actual
isolation of the organism. Because of the known propensity of C. burnetii
to cause human disease, the large reservoir of infection in the dairy
cattle population of this country must be considered a potential public
health problem. The high incidence of sterile abscesses and tissue re-
actions among sensitized persons given Q fever vaccine and the absence
of such reactions in nonsensitized persons emphasized the need of a skin
test before the administration of Q fever vaccine.
Proposed course:
In studies completed to date, minor illnesses attributable to in-
fection with C. burnetii could not be accurately evaluated. As Q fever
continues to spread through dairy cattle in Ravalli County, rural popu-
lations will be observed closely to detect any minor illnesses attribut-
able to Q fever.
Epizootiological studies will be continued to elucidate the means
whereby the organism is disseminated throughout the livestock population.
Isolates from man and livestock will be studied and characterized in an
attempt to explain the relative inability of these organisms to cause
human illness.
B. Laboratory studies:
Methods:
Organisms grown in tissue culture or embryonated chicken eggs are
purified and subjected to chemical and physical fractionation. Fractions
are then assayed for complement-fixing activity and immunogenic proper-
ties by appropriate serologic and biologic tests.
Major findings:
1. A method of purifying rickettsiae by centrifuging 10 percent
yolk-sac suspensions in molar salt solution in a continuous -flow centri-
fuge has been found to be particularly useful for purifying large
quantities of Q fever, spotted fever, and typhus rickettsiae.
18
Serial No. NIAID-172
2. The density -gradient-sedimentation technique was found to be
useful for separating cell walls from intact cells.
3. In chemical fractionation studies, dimethyl sulfoxide (EMS)
extracts of phase II rickettsiae were CF specific hut non immunogenic in
guinea pigs, whereas EMS extracts of phase I rickettsiae were immunogenic.
Lauryl sulfate extracts react specifically in the CF test and
elicit protective antibodies in guinea pigs. This chemical agent appears
to extract phase I antigen from mixtures of rickettsiae containing both
phase I and II components.
h. In physical fractionation studies, preparations of cell walls
and protoplasm were prepared from purified suspensions of C. burnetii.
Preliminary studies indicate that the immunogenic and CF activity is
situated chiefly in the cell wall. On a weight basis, cell wall prepa-
rations showed 25-fold greater protection than did protoplasm. Fractions
of C . burnetii were toxic when inoculated intraderraally into normal
rabbits. The least dose to produce a visible reaction was 0.125 micro-
grams for whole cells, 0.025 micrograms for cell walls, and Uo.O micro-
grams for protoplasm. The difference in dermal activity between cell
wall and protoplasm is similar to that of cellular fractions of Salmon-
ella enteritidis and S. typhosa.
5. Comparative studies on the capillary agglutination test and
the CF test on serums of varied sources indicate that at least three
types of antibodies are involved in the immune response of this disease.
6. Growth of C. burnetii in cell -free medium still has not been
accomplished.
Significance:
Studies to date indicate that both toxic and immunogenic proper-
ties of the Q fever rickettsiae are situated in the cell wall. By proper
chemical or physical fractionation it is hoped that a nontoxic, protective
antigen can be isolated from this organism. Such a product is needed
since the amount of vaccine that can be administered at the present time
is limited by its toxic properties.
Proposed course:
Studies on the physical and chemical fractionation of C. burnetii
will be continued to obtain fractions which will be particularly useful
in immunologic and allergic investigations. Attempts will be made to
isolate and identify the toxic component of the rickettsial cell wall
since this has direct relationship to other projects dealing with bac-
terial toxins.
19
Serial No. NIATD-172
Attempts will be made to correlate the virulence of strains of C.
burnetii with the chemical composition of their cell walls. Investigations
on the relationship of the phase of the Q fever rickettsia to allergic
and protective properties of various cell fractions will be continued.
20
Serial No. NIAID-172
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Stoenner, H. G. and Lackman, D. B. : The biologic properties of Coxiella
burnetii isolated from rodents collected in Utah. Am. J. Hyg. 71(1):
45-51. Jan. I960.
Luoto, L. : Report on the nationwide occurrence of Q fever infections in
cattle. Pub. Health Rep. 75(2): 135-lto. Feb. i960.
Stoenner, H. G.: Q fever. In A Manual of Tropical Medicine, 3rd ed.
W. B. Saunders & Co., Philadelphia, i960, pp. 10^-106.
Ribi, E. and Hoyer, B. H.: Purification of Q fever rickettsiae by
density-gradient sedimentation. J. Immunol. 85(3): 31^-3l8« Sept. i960.
In press:
Pickens, E. G. and Gaon, J. A.: Growth of Coxiella burnetii in agar
tissue culture. Am. J. Trop. Med.
21
PHS-NIH
Individual Project Report
Calendar Year i960
Serial No. HIAID-173
Rocky Mountain Laboratory
Hamilton, Montana
Part A.
Project Title: Diseases having a reservoir of infection in the natural
environment (other than rickettsioses)
Principal Investigator: J. F. Bell
Other Investigators: W. L. Jellison, D. B. Lackman, M. T. McKee,
C. R. Owen, and H. G. Stoenner
Cooperating Units: Dr. C. W. Emmons, LID, NIAXD
Man Years (calendar year i960):
Total: 11.0
Professional: U.O
Other: 7.0
Project Description:
Objectives:
Studies performed in this project are directed toward an under-
standing of the ecology of those diseases transmissible to man from
animals and the natural environment. As a direct continuation of the
ecological studies, attention is paid to the laboratory aspects of the
problems encountered with special emphasis on diagnosis, treatment, and
control.
Methods :
The methods employed are, in general, straightforward microbio-
logical techniques, with such variations as are required for study of
specialized problems, rabies, for example.
Major findings:
1. Since only 2 isolations of bat rabies were made this year and
9 were made in 1959* the idea of an initial upsurge toward epizootic
proportions can be discounted.
2. Survival of mice patently infected with isolates from various
sources has been found to be a common phenomenon contrary to previous
Part B included: Yes
22
Serial No. NIAID-173
ideas. The factors responsible are unknown.
3. Continuing presence of Group A streptococci in areas of high
vole populations confirms that similar earlier observations were not
isolated ones, but the epizoological significance remains obscure.
h. In an initial, long-term experiment designed to compare the
duration of protection afforded mice against P. tularensis when vacci-
nated with ether-extracted antigen (LV) and a~~Russian living attenuated
strain (RV), the role of latent infection in the immune response of the
latter group could not be determined. During the ensuing weeks the log
protection decreased rapidly or slowly depending on the virulence of
challenge material. The slopes of decreasing protection showed consider-
able similarity between LV mice challenged with ^25-FkJ strain and RV
mice challenged with Schu. Protection in KV mice challenged with l^-Fki
remained high through week 20 but decreased to nearly zero after 52 weeks.
5. In continued comparisons of the virulence of P. tularense from
different geographic areas, three strains from 2 patients and a dead
hare in Japan were strikingly lower in virulence for guinea pigs and
rabbits than 3 isolates from hares in France and h from D. variabilis
wood ticks from eastern Montana.
6. Results of the use of Ovary's passive cutaneous anaphylaxis
test lend weight to the specific rather than subspecific differentiation
of P. tularensis and P. novicida. Cross-vaccination studies still leave
a question as to their relationship.
7. Isolations of Type A2 influenza virus were made from 15 of 30
throat washings collected during a local epidemic. One isolation on
22 January was earlier than usual for this disease. These isolates were
forwarded to the International Influenza Center for the Americas.
8. P. multocida was isolated from mouths of 32 of Vf domestic
cats, one bobcat, and one of 5 dogs. None of the 27 other animals, in-
cluding grizzly bears, woodchucks, skunks, and snakes, were positive.
9- The fungal organism causing haplomycosis in animals has been
found in one South American and h European countries, and 3 new rodent
hosts were discovered. In conjunction with Dr. C. W. Emmons, the new
name, "adiaspiromycosis," was applied to this parasitism because of the
peculiar lack of cellular multiplication of organisms in the animal host.
Massive doses from cultures given intravenously have been lethal for
monkeys and a sheep.
10. Reciprocal cross-immunity in guinea pigs and mice was found
between Brucella suis and Br. neotomae when care was taken to use only
smooth-phase cultures. The explanation for inconsistencies in earlier
work was found to be due to undetected colonial dissociation.
23
Serial No. NIAID-173
11. Leptospira pomona was isolated for the first time from the
spinal fluid of a bovine animal, a local dairy cow that showed signs of
meningitis prior to death.
12. The absence of HI antibodies against swine influenza virus in
serums from members of the Alaskan National Guard confirms epidemiologic
evidence that the 1918-19 pandemic of influenza failed to reach some
areas of Alaska. Survivors of another isolated population severely-
affected at that time, still retain significant levels of such antibodies.
Significance;
Additional laboratory data progressively confirm recent notions
that rabies virus does not cause an invariably fatal disease as it was
once thought to do. The concern that rabies might progress to epizootic
proportions, as suggested by the large number of isolations last year,
was fortunately not justified.
Though human infection with P. multocida has not been observed
locally, isolation from the mouths of cats and dogs suggests this source
as of potential local health concern.
A reassessment of the epidemiology of epidemic nephroso-nephritis
may provide a new viewpoint for a fresh attack on this puzzling Old World
entity. Dr. Jellison has made the novel suggestion that some 2,000 cases
of "trench nephritis" in troops during World War I may have been this
type of hemorrhagic fever.
Studies have revealed that Br. suis and Br. neotomae are closely
related immunologically but are not completely similar. Br. neotomae
has not been found to persist in tissues of swine. Therefore, if results
of studies in mice and guinea pigs are applicable, this organism could
conceivably be used as an effective vaccine for control of swine
brucellosis.
The application of results of research to field problems is one
of the main accomplishments of the serology laboratory. The identifi-
cations of infections in human and animal populations during epidemiologic
study are largely the result of serologic testing. When it is possible
to isolate the causative agent, serology plays an important part in its
final identification and characterization. While such work progresses,
improved techniques and diagnostic reagents are sought. This in turn
gives rise to improved prophylactic vaccines and better methods for their
evaluation and standardization. Results of serologic testing are also
utilized for the identification of problems in infectious disease which
may exist in the natural environment of the west.
Pandemic influenza is a continuing threat to man. Therefore, it
is important to detect any antigenic variation in strains causing epidemics.
2k
Serial No. NIAID-173
To do this it is necessary to have some laboratories, especially ones
located in isolated areas, which will forward new isolates to the WHO
Center. Also of importance is the continual investigation of the in-
fluenza experience of isolated populations because it provides information
as to what strains should be included in vaccine. Our results suggest
that consideration should be given to the inclusion of swine influenza
virus in vaccine because there are people who have not been exposed to
this virus and it may still be present among swine.
Proposed course:
Steady progress in laboratory maintenance of bats suggests that
experimental infection can now be instituted with confidence.
Studies on the efficacy of vaccination of domesticated beaver
against tularemia are under way.
Attempts will be continued to elucidate now obscure factors in
the epizoology of Group A streptococci and "M" organisms (Toxoplasma
microti) in Microtus mouse populations.
If facilities become available, further work is planned on differ-
entiation of P. tularensis and P. novicida and on possible protection of
swine by Br. neotomae against Br. suis.
Studies also will be continued on virulence and cultural patterns
among strains of P. tularensis of diverse origins and on factors favoring
persistence of the organism in natural waters.
Bird tissues collected in Alaska for test for arbor viruses will
be screened in the serology unit for viruses of the psittacosis group.
25
Serial No. NIAID-173
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Bell, J. F. and Moore, G. J.: Rabies virus isolated from brown fat of
naturally infected bats. Proc. Soc. Exp. Biol. & Med. 103(1); lI+O-li+2.
Jan. I960.
Owen, C. R., Meis, A., Jackson, J. W., and Stoenner, H. G.: A case of
primary cutaneous listeriosis. New Eng. J. Med. 262(20): 1026-1028.
May 19, i960.
Emmons, C. W. and Jellison, W. L.: Emmonsia crescens sp. n. and
adiaspiromycosis (haplomycosis) in mammals. Ann. N.Y. Acad. Sci. 89:
91-101. Aug. 27, i960.
Jellison, W. L. , Glesne, L. , and Peterson, R. : Emmonsia, a fungus, and
Besnoitia, a protozoan, reported for South America. Bol. Chilena de
Parasitol. XV(3): U6-V7. July-Sept. i960.
Jellison, W. L., Vinson, J. W., and Holager, E.: Haplomycosis in Norway.
Acta Pat. et Microbiol. Scandinavica ^9(4): kd0-k8^. i960.
Esplin, D. W., Philip, C. B., and Hughes, L. E.: Impairment of muscle
stretch reflexes in tick paralysis. Science 132(31+32): 958-959.
Oct. 7, I960.
In press:
Jellison, W. L., Helminen, M., and Vinson, J. W.: Presence of a pulmonary
fungus in rodents in Finland. Ann. Med. Exp. et Biol. Fenniae 38(3):
Jellison, W. L. : Sodoku. Rat-bite fever due to Spirillum minus Carter.
Diagnostic Procedures and Reagents, Uth ed. American Public Health Assoc,
New York, N.Y.
Larson, C. L. : Tularemia. Diagnostic Procedures and Reagents, Uth ed.
American Public Health Assoc, New York, N.Y.
26
Serial No. NIAID-171*
Rocky Mountain Laboratory
Hamilton, Montana
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Transmission of disease agents by certain vectors
Principal Investigator: C. B. Philip
Other Investigators: J. F. Bell, J. M. Brennan, W. Burgdorfer,
C. M. Clifford, W. L. Jellison, G. M. Kohls,
D. B. Lackman, V. F. Newhouse
Cooperating Units: Laboratory of Immunology, NIH, NAMRU 3
Man Years (calendar year i960) :
Total: 13.5
Professional: 4.5
Other: 9*0
Project Description:
Objectives:
This group directs its studies toward problems of the intimate
relationships between infectious agents and their actual or potential
invertebrate vectors. Occasionally, symbiotic organisms in arthropods
come under scrutiny. Efforts during the past year have been directed
primarily toward continuation of experimental tick paralysis, completion
of studies on transovarial tick transmission of Pasteurella tularensis,
intensification of studies on relationships of Rickettsia rickettsii and
Colorado tick fever virus in vectors, and a reconsideration of evidence
on the epidemiology of the hemorrhagic fever known as epidemic nephroso-
nephritis. An intensive attack on potential disease relationships of
chigger mites in Panama was organized.
Methods:
Field and laboratory methods are mainly those developed at the
Rocky Mountain Laboratory, including more recent fluorescent microscopy
as applied to vertebrate -invertebrate cycles of disease agents.
Major findings:
1. A successful cryostat technique for the thin-sectioning of
Part B included: Yes
27
Serial No. NIAID-171*
fresh-frozen tissues of hard and soft ticks was developed in conjunction
with Dr. J. E. Tobie of the Laboratory of Immunology, NIH. This was a
necessary antecedent to refined studies of certain tick-borne agents by
fluorescent microscopy. In comparison with older techniques this method
is much more rapid and is a marked improvement for sectioning and stain-
ing both nymphs and adults of these ticks in various states of engorgement.
2. The incidence of tick paralysis in man is not necessarily a
reflection of the relative ability of indigenous ticks to produce the
disease in hamsters. From 2U$ to 90$ of test lots of ticks (k& lots)
from 3 areas in Colorado caused experimental tick paralysis though only
3 cases were reported in the entire state up to 1950; whereas only from
0$ to 50$ of similar lots (39) from 3 areas in a single county (Ravalli)
in western Montana were paralytic though 7 local cases have been reported
in the same period. On the other hand, 75$ of 16 lots from adjoining
Missoula County were positive. If ecologic factors were responsible for
these differences, they have so far eluded solution.
3- Species of ticks of the genus Argas, which are virtually indis-
tinguishable in the adult stage, have been separated by taxonomic char-
acters present in the larval stage. This has resulted in clarification
of a subspecies which infest bats in Egypt and in the discovery that a
species found in cliff swallow nests in western North America and another
from birds in Chile are new. Present classification of other species,
particularly parasites of migratory birds and bats, probably requires
clarification as a prerequisite to investigations of the disease-carrying
potential of the ticks.
h. A new funguslike agent which causes death in white mice and
embryonated chicken eggs has been isolated from Argas found in nests of
local cliff swallows. Studies are under way to assess its systematic
relationship .
Significance:
New data confirm the utility of fluorescent microscopy in study
of arthropod-borne agents as applied particularly to rickettsial agents
(see No. 171)- Special tissue tropisms are now more readily detectable,
and a tool is provided that may enable estimation of growth in different
stages and periods of a given cycle. The new techniques for tick section-
ing will be applicable to other fields and right now mosquitoes are being
subjected to similar techniques.
Previous advances in knowledge of the chigger-mite fauna of Central
and South America will now pay dividends in facilitating the testing of
species in Panama for presence of pathogenic agents. This illustrates
the advantages of fore -knowledge of parasitic arthropods in a given fauna
over information obtained after a health problem has arisen. In Korea,
for example, a hurried survey of medically important parasites lagged
28
Serial No. NIAID-17^
behind the emergency need for epidemiologic studies of hemorrhagic fever
in military forces.
Several faunal studies of this nature on parasites in various
parts of the world were completed or were under way during the year by
appropriate staff specialists; the significance of these studies may only
become apparent when some future need arises.
Proposed course;
As opportunity arises, the relationship of acarine and insect-
borne pathogens will continue to be investigated. Strains of supposed
Rickettsia prowazekii reported to have been isolated from tick and domes-
tic animal sources in Abyssinia are now under study. Attempts to purify
the salmon -poisoning agent for antigenic purposes were not fruitful during
the year, but the problem will be attacked by a new technique. Some
initial promising fluorescent antibody studies on this agent will be
elaborated.
The Panama chigger-mite unit may be expected to have some results
from initial field and laboratory tests.
A revision of the world systematics of the important tick genus,
Argas, in collaboration with Dr. Harry Hoogstraal of NAMRU 3 in Cairo,
Egypt, should be well advanced by the end of next year.
29
Serial No. NIAID-171*
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Brennan, J. M.: Ectonyx, a new Neotropical genus of chiggers (Acarina:
Trombiculidae) . Acarologia Il(l): 88-91. i960.
Brennan, J. M. and Dal mat, H. C: Chiggers of Guatemala (Acarina:
Trombiculidae). Ann. Ent. Soc. Amer. 53(2): 183-191. March i960.
Philip, C. B.: New North American Tabanidae. XI. Supplemental notes
pertinent to a catalog of Nearctic species. Ann. Ent. Soc. Amer. 53(3):
36U-369. May I960.
Brennan, J. M. and White, J. S.: New records and descriptions of chiggers
(Acarina: Trombiculidae) on bats in Alabama. J. Parasitol. U6(3): 3^+6-
350. June i960.
Kohls, G. M.: Records and new synonymy of New World Haemaphysalis ticks,
with descriptions of the nymph and larva of H. juxtakochi Cooley. J.
Parasitol. U6(3): 355-361. June i960.
Newhouse, V. F.: Birds of selected irrigated river valleys of west-
central Idaho. Murrelet Ul(l): 1-6. i960.
Philip, C. B.: Further records of Neotropical tabanidae (Diptera) mostly
from Peru. Proc. Calif. Acad. Sci., 1+th Series XXXl(3): 69-102.
July 8, i960.
Brennan, J. M. and Maki, W. A.: An inexpensive and effective indoor
insectary. J. Econ. Ent. 53(h): 685-688. Aug. i960.
Hoogstraal, H. and Kohls, G. M.: Observations on the subgenus Argas
(ixodoidea, Argasidae, Argas) . 1. Study of A. reflexus reflexus
(Fabricius, 179*0, the European bird argasid. Ann. Ent. Soc. Amer. 53(5):
611-618. Sept. I960.
Kohls, G. M. and Hoogstraal, H.: Observations on the subgenus Argas
(ixodoidea, Argasidae, Argas) . 2. A. cooley i, new species, from western
North American birds. Ann. Ent. Soc. Amer. 53(5): 625-63I. Sept. i960.
Philip, C. B.: Another hcQarc tic species of Tabanidae (Diptera). Canad.
Ent. XCII(9): 697-699- Sept. i960.
30
Serial No. NIAID-171*
Kohls, G. M.: Ixodides. McGraw-Hill Encyclopedia of Science and
Technology. McGraw-Hill Book Co., Inc., New York. i960. Vol. 7,
pp. 298-299.
Kohls, G. M.: Ixodes (Endopalpiger) zaglossi, n. sp. from the long-beaked
echidna of New Guinea (Acarina, Ixodidae) . Acarologia 2(U): M+7-I+52.
Oct. i960.
Brennan, J. M.: Eight new species of Pseudoschongastia from Mexico and
Panama with a revised key to species (Acarina: Trombiculidae) . Acarologia
2(U): I+8O-U92. Oct. i960.
Brennan, J. M. and Jones, E. K. : Chiggers of Trinidad, B.W.I., (Acarina:
Trombiculidae). Acarologia 2(U) : 1+93-51+0. Oct. i960.
Philip, C. B.: Malaysian Parasites. XXXV. Description of some Tabanidae
(Diptera) from the Far East. Malaysian Parasites No. 29, pp. 1-32. i960.
Philip, C. B.: Malaysian Parasites. XXXVI. A summary review and records
of Tabanidae from Malaya, Borneo, and Thailand. Malaysian Parasites No.
29, PP. 33-78. I960.
In press:
Philip, C. B. and Mackerras, I. M.: On Asiatic and related Chrysopinae
(Diptera: Tabanidae). Philippine J. Sci.
Philip, C. B.: Additional records of Tabanidae (Diptera) from the West
Coast of South America. Pan-Pacific Ent.
Fairchild, G. B. and Philip, C. B.: A revision of the Neotropical genus
Dichelacera, subgenus Dichelacera, Macquart (Diptera, Tabanidae). Studia
Entomologica, Brazil.
Philip, C. B.: Three new Tabanine flies (Tabanidae, Diptera) from India.
Ent. J. India.
Hoogstraal, H. and Kohls, G. M.: Observations on the subgenus Argas
(ixodoidea, Argasidae, Argas) . 3- A biological and systematic study of
A. reflexus hermanni Audouin, 1827 (revalidated), the African bird argasid.
Ann. Ent. Soc. Amer.
Brennan, J. M. and Jones, E. K.: New genera and species of chiggers from
Panama (Acarina: Trombiculidae). J. Parasitol.
Brennan, J. M. and Jones, E. K.: Chiggers of Peru (Acarina: Trombiculidae).
Acarologia.
31
Serial No. NIAID-17^
Kohls, G. M. and Clifford, CM.: A new species of Ixodes ( Lep_idixodes )
from bats in Malaya, North Borneo, and the Congo (Acarina-Ixodidae) .
Acarologia.
Philip, C. B. and Burgdorfer, W.: Arthropod vectors as reservoirs of
microbial disease agents. Ann. Rev. Ent. 6: I96I.
Philip, C. B.: Arthropod vectors in relation to the reservoir mechanism
of microbial agents of animal diseases. Presented at 11th Internat. Ent.
Cong., Vienna, Aug. i960. Acta Tropica.
Philip, C. B.: Proposal to validate under the plenary powers the specific
name Akamushi (Trombidium) Brumpt, (Class Acarina) . Z.N.(S) 1+00.
Internat. Bull. Zool. Nomenclature.
Philip, C. B.: Proposal to validate under the plenary powers the specific
name Dermacentor andersoni Venus tus . Internat. Bull. Zool. Nomenclature.
Philip, C. B.: New North American Tabanidae. XIII. Change of name for
a well-known species of Chrysops. Ent. News.
Honors and Awards relating to this project:
Glen M. Kohls
Continued as an Assistant Editor for the Journal of Parasitology.
Invited by the Editor of the Annals of the Entomological Society of
America to prepare a review of Dr. D. R. Arthur's monograph on several
genera of ticks published by the Cambridge University Press.
Vice President and member of the program committee of the International
Northwest Conference on Diseases in Nature Communicable to Man.
Dr. C. B. Philip
Invited to serve on the "Research and Engineering Advisory Panel on
Biological and Chemical Defense" under the office of the Director of
Defense Research and Engineering. (May 6, i960)
Received the Outstanding Achievement Award of the University of
Minnesota, which is reserved for former students of the institution
who have attained "high eminence and distinction." (June k, i960)
Invited to be a Visiting Lecturer in the program of the Academic Year
Institute for High School Teachers of Science and Mathematics, I96O-6I.
Continued as lecturer, CDC Training Course, PES, Atlanta, February
1960-61.
32
Serial No. NIAID-175
Rocky Mountain Laboratory
Hamilton, Montana
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Allergy and immunology of fungal infections and the
mechanisms of allergic phenomena
Principal Investigator: S. B. Salvin
Other Investigators: M. B. Gregg, R. F. Smith
Cooperating Units: None
Man Years (calendar year i960):
Total: 13-25
Professional: k.25
Other: 9-00
Project Description:
Objectives:
Further efforts to uncover the mechanisms responsible for delayed
allergy have been stimulated by relationships recently established between
delayed allergy and homograft rejection and collagen or "auto-immune"
disease. Three main objectives of this project are: (l) to study the
biologic mechanisms responsible for the development and expression of
delayed hypersensitivity in experimental animals; (2) to relate delayed
allergy to other immunologic phenomena such as antibody formation, contact
hypersensitivity, or tolerance; and (3) to apply such knowledge of delayed
hypersensitivity to its possible role in the pathogenesis of "auto-immune"
diseases such as lupus erythematosis and rheumatoid arthritis, as well as
to certain infectious diseases, such as rheumatic fever, tuberculosis, or
histoplasmosis.
Methods:
Although biologic and chemical procedures typical of immunologic
investigations are used, emphasis has been placed on some quantitative
immunochemical procedures for analyses and study of allergy and antigen-
antibody reactions. Highly purified proteins, such as diphtheria toxoid,
egg albumin, and bovine gamma globulin, are used as antigens, either
singly or conjugated with a wide variety of haptenic groupings and carbo-
hydrates. A new technique involves the use of radioactive conjugates to
Part B included: Yes
33
Serial No. NIAID-175
trace the progress of antigen -antibody reactions within the cell by auto-
radiographic procedures. Also, electron opaque metals conjugated to
conventional proteins are used to trace the course of the antigen within
the cell.
Major findings:
Previous studies have shown that delayed allergy in guinea pigs
is superseded by circulating antibody and Arthus reactions if a large
antigenic dose is given. When guinea pigs are sensitized with a small
dose, only delayed hypersensitivity develops. Animals given a smal 1
dose of antigen develop maximum anamnestic responses when a second injec-
tion is given at the time of maximum delayed hypersensitivity. These and
other observations strongly indicate that delayed hypersensitivity is an
immature form of the immune response.
1. In studies on the anamnestic response to conjugated diazotized
proteins, guinea pigs developed delayed hypersensitivity to the protein
moiety, but developed specific circulating antibody directed toward small
chemical configurations present only in the booster antigen.
2. Carbohydrates, when conjugated to proteins, act as haptens and
can be used to detect circulating antibody. Since, however, the speci-
ficity of delayed allergy is directed toward the protein, the carbohydrate
can neither induce nor detect delayed hypersensitivity.
3. Contact hypersensitivity, which is believed to be analogous to
delayed allergy, may be induced in experimental animals by repeated appli-
cation of a simple chemical (2,U-dinitro-l-flurobenzene, DFB) on the skin
or by intradermal injection of the chemical in Freund' s adjuvant. Animals
sensitized with a simple chemical develop circulating antibody and Arthus
reactions to a conjugate composed of hapten and a protein such as guinea
pig serum and react to a surface application of the hapten only. In con-
trast, animals sensitized with the conjugate (DFB guinea pig serum)
develop Arthus reactions and circulating antibody directed toward the
hapten and produce typical delayed allergy to subsequent intradermal in-
jection of the conjugate, but do not react to surface application of the
simple chemical. This inconsistency, that synthetic conjugates fail to
induce contact skin hypersensitivity to the hapten, but that hapten in-
duces Arthus reactions to the conjugate, may be explained by one of two
possible hypotheses: (a) When the host animal is injected with simple
chemicals, these substances may enter intracellular areas inaccessible to
extracellular proteins. Subsequent conjugation leads to conjugates which
exist in intracellular sites inaccessible to conjugates prepared in vitro.
Several workers have provided evidence that this phenomenon may be func-
tional, (b) The protein members of in vivo conjugates are unique and
have not been duplicated by proteins chosen for in vitro preparations.
The observation that intradermal injection of an in vitro conjugate of
soluble guinea pig skin protein with DFB produces contact hypersensitivity
3h
Serial No. NIMD-175
to surface application of the hapten as well as delayed and Arthus
reactions to skin tests with the conjugate supports this hypothesis.
Thus, the production of contact hypersensitivity is directly related to
a particular protein or proteins in the host tissue and the specificity
of contact hypersensitivity is analogous to that of delayed reactions.
h. In further substantiating the role of delayed allergy in the
immune process, immunologic studies were conducted in newborn guinea
pigs because they may show immaturity in the formation of delayed allergy
and circulating antibody. When these animals are sensitized within 12
hours after birth, typical delayed responses cannot be evoked for about
2 weeks. By this time, however, circulating antibody has appeared, and
animals develop typical Arthus reactions when challenged. If sensitiza-
tion of neonatal animals is postponed for 12 to lk days after birth,
delayed allergy is not apparent, but circulating antibody appears about
11 days after sensitization. When the neonatal animal is sensitized
later than lk days after birth, allergic and antibody responses are simi-
lar to those of the adult. These observations suggest either that delayed
hypersensitivity cannot be produced by the neonatal animal but conventional
antibody can, or that the basic mechanism of delayed hypersensitivity is
present but cannot be made manifest by the host animal because of some
inherent deficiency. The following observations suggest that the latter
hypothesis is true: (a) delayed hypersensitivity cannot be transferred
passively to newborns with lymph-node cells from highly sensitized adults,
(b) delayed hypersensitivity, however, can be transferred passively to
normal adults with lymph-node cells from highly sensitized neonatal guinea
pigs which themselves do not show delayed hypersensitivity, and (c) con-
tact hypersensitivity can be exhibited in neonatal animals although it
is somewhat slow in onset.
5. Guinea pigs sensitized to purified ultraviolet-killed Type II
poliovirus develop an anamnestic response to a second injection of antigen.
6. Through chemical and physical fractionation procedures, a spe-
cific skin-test antigen has been prepared from Cryptococcus neoformans,
a pathogenic fungus frequently isolated from pigeon excreta in the
vicinity of New York City. This antigen produces large and impressive
delayed-type skin reactions in guinea pigs previously infected with C.
neoformans. The active fraction of this antigen has been purified and
its biochemical properties determined.
Significance:
To date, experimental data support the hypothesis that delayed
hypersensitivity is an immature phase of the classical immune response.
Studies on contact sensitivity induced by simple chemicals indicate that
its specificity and dynamics are similar to those of conventional delayed
hypersensitivity. The inability to invoke typical delayed responses in
a neonatal animal appears to be attributable to an inherent deficiency
35
Serial No. NIAID-175
which prevents the young host from manifesting an established hypersensi-
tive state. Hence, even in the young animal, delayed allergy is an early
phase in the antibody response.
In view of the prevalence of C. neoformans in pigeon excreta in
urban areas, large numbers of people are exposed to this fungus. There-
fore, cryptococcosis in a mild, unrecognized form may be much more preva-
lent than the relatively infrequent occurrence of this disease would
indicate. The preparation of a specific skin-test antigen creates a new
tool for conducting epidemiologic studies of this disease.
Proposed course:
Studies on the immune mechanism in the neonatal animal will be
continued. The possibility exists that a cellular or humoral factor is
necessary or supplemental for manifestation of delayed hypersensitivity
in an infant or in a newborn animal. Experiments are in progress to ex-
amine this hypothesis. In addition, skin grafts from adults to newborn
and vice versa are being used to determine whether maturity of the skin
is essential for delayed reactions. Since gamma radiation of newborn
guinea pigs delays the appearance of Arthus reactions, this technique is
also being used to postpone the appearance of antibody and thus aid in
the determination and examination of delayed reactions.
Studies with live bacteria such as Mycobacterium tuberculosis
have been initiated to determine if the response relative to hypersensi-
tivity and immunity of the newborn differs from that of the adult.
Further investigation will be made on the effect of age and the
role of skin and other cellular elements in the allergic response.
To clarify the part delayed allergy plays in antibody formation,
antigen should be traced through the cell, and its particular activity
within the cell should be correlated with the gross reaction of the host.
Two techniques will be used. One involves the injection of guinea pigs
with the electron-opaque protein conjugate and the determination of the
exact locus and relationship of the antigen within the cell by examination
of ultrathin sections under the electron microscope. The other method
involves a study of the cellular disposition of injected radioactive
proteins and the position of antigen within the cell. Radioactive anti-
gens and subsequent antibody combinations are traced by autoradiographic
techniques.
Experiments on sensitization of guinea pigs with purified polio-
virus will be continued in order to learn whether sensitization with
Types I or III will produce an anamnestic response on later introduction
of Type II virus.
36
Serial No. NIA 33-175
Standardization of the skin-test antigen for detecting crypto-
coccosis in man and collaborative epidemiologic studies of this disease
are contemplated.
37
Serial No. NIAID-175
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Salvin, S. B.: Resistance of animals and man to histoplasmosis. In
Histoplasmosis. Chas. C Thomas, Springfield, 111., i960, pp. 99-112.
Salvin, S. B. and Smith, R. F.: Specificity of allergic reactions.
I. Delayed versus Arthus hypersensitivity. J. Exp. Med. 111(4): U65-483.
April I960.
Salvin, S. B. and Smith, R. F.: Delayed hypersensitivity and the
anamnestic response. J. Immunol. 8U(5): UU9-U57. May i960.
Salvin, S. B. and Smith, R. F.: Specificity of allergic reactions.
II. Azoproteins in the anamnestic response. Proc. Soc. Exp. Biol. & Med.
10U(U); 584-590. Aug. -Sept. i960.
Honors and Avards relating to this project:
Dr. S. B. Salvin
Invited to contribute paper in "Progress in Allergy," S. Karger,
Basel, Switzerland, publisher.
Invited to present series of lectures and seminars on allergy and
immunology at the University of Montana, Missoula, Montana.
38
Serial No. NIAID-I76
Rocky Mountain Laboratory
Hamilton, Montana
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Viruses, virus components, and virus surfaces
Principal Investigator: B. H. Hoyer
Other Investigators: R. K. Gerloff, F. G. Jarvis, R. A. Ormsbee, and
D. B. Ritter
Cooperating Units:
Man Years (calendar year i960):
Total: 11.5
Professional: 3*5
Other: 8.0
Project Description:
Objectives:
Elucidation of the mechanisms involved in the synthesis of virus
protein and RNA and clarification of fundamental relationships between
virus protein and RNA continue to be major objectives. The finding of
others that poliovirus protein and RNA are synthesized simultaneously at
similar rates required us to make changes in our investigations. Current
interest in live poliovirus vaccines has stimulated studies on the behavior
of surface proteins of virulent and avirulent poliovirus in column chroma-
tography. A sensitive precipitation system for detecting antibody against
poliovirus is being developed.
Methods:
Quantitative and qualitative methods appropriate for the study of
virus proliferation and cell metabolism are employed. Cellular and virus
fractions are separated and characterized by chemical and physical means.
Radioactive labels, p32 and S^ , are used in following certain biologic
processes and in studying biophysical properties of viral components.
Part B included: Yes
33
Serial No. NIA3D-176
Major findings;
op
1. Previous studies have shown that ¥-> -labeled type 3 Sauckett
virus, type 2 MEF-1 virus, and type 1 Mahoney virus can be separated by
column chromatography. In continued investigations, Leon and Sauckett
type 3 viruses could be separated from each other by elution from the
DEAE column at different pH (7.7 vs. 7.1) values.
2. CHAT poliovirus, an avirulent type 1, was separated from the
virulent parent by elution in a salt or pH gradient, but FOX and Wi-1
could not be separated from their virulent counterparts. CHAT and Wi-1,
both avirulent variants, could be separated from each other by virtue of
different elution properties.
3- After prolonged storage, avirulent poliovirus binds more firmly
to cellulose ion exchange columns than does its virulent counterpart.
This property is probably due to a more rapid change in avirulent virus
protein and indicates a less stable surface.
h. Because of the large ratio (1:10 to 1:500) between plaque -forming
units (PFU) and physical particles, virus proteins could not be character-
ized without knowledge of the proportion of total virus that reacts with
the cell. After S^-7- or p32_iabeled polioviruses were mixed with KB cells
in a virus-cell ratio of 20 to 1, samples were removed at intervals and
examined in CsCl or RbCl equilibrium-density gradients. Radioactivity
and infectivity disappeared at the same rate from the supernatant medium,
and the virus shifted from the density zone characteristic of whole virus.
Most of the radioactivity that had left the virus -density zone was con-
centrated in a well-defined region in the upper level. Alteration of the
density of original virus particles indicates that most of the virus
actually reacts with susceptible cells.
5. Infectious RNA was prepared from highly purified poliovirus by
phenol extraction. RNA preparations of fairly uniform physical properties
had an Sgo w °^ 37 • Also, a 30$ increase in ultraviolet absorption after
treatment with RNAase indicates an initial high degree of polymerization.
Both octyl alcohol-chloroform and neutral hydroxylamine (0.1 M) destroyed
more than 99$ of RNA infectivity after 10 minutes at room temperature,
but the molecular properties of RNA, as determined by ultracentrifugation
patterns, were not affected.
6. In a preparation of RNA yielding 1$ of the PFU present in whole
virus, the s35 content was only 0.001$ of that of the whole virus. Thus,
residual protein cannot account for more than 0.1$ of the RNA activity.
7. The method originally used for preparation of purified poliovirus
was modified by treating crude DEAE filtrates of tissue-culture fluids
with 0.2$ DEXTRAN 500, 6.1+5$ Carbowax 6000 and 0.3 M NaCl (final concen-
tration). After standing for 2k to h& hours at h C., liquids separate
Serial No. NIMD-176
into two phases, the lower of which contains all the virus. From 2,700 ml.
of culture fluid, all the virus will be present in the lower 20 ml. of
liquid.
8. An extremely sensitive and specific precipitation test for
detecting antibody against poliovirus has "been developed. A serum with
a neutralizing antibody titer of 1:256 possesses a precipitation titer of
l:l6,38U. Serum dilutions are mixed with p32-iabeled poliovirus and
incubated. Antihuman gamma globulin is added, followed by further incu-
bation. The precipitate formed is then removed by centrifugation. If a
significant amount of radioactivity is present in the sediment, precipita-
tion of virus has occurred.
Significance:
The behavior of derived avirulent poliovirus in column chromatog-
raphy indicates some alterations in surface protein differing from that
of parent strains. Although other workers claim that avirulent poliovirus
can be separated uniformly from virulent virus because the avirulent
strains are absorbed more firmly to cellulose columns, our findings indi-
cate that generalizations regarding the binding properties and the elution
profiles of avirulent strains cannot be made.
In spite of the well-established high ratio between the PFU and
virus particles, the bulk of virus in an inoculum actually binds to the
cells and forms material of lower density. Thus, this wide ratio is
probably explained by the limited ability of RNA to enter sites where
virus production can be influenced. Other workers have proposed that RNA
infectivity may be destroyed if only a small group of the long molecular
chain is inactivated. Our findings indicate that major molecular changes
do not occur when RNA is inactivated by hydroxylamine or octyl alcohol-
chloroform. Neither of these should harm RNA, and it is entirely possible
that infectivity of the relatively large RNA molecule can be destroyed by
uncoupling of one link. If this is true and if the link is unique, an
approach to virus chemotherapy is indicated.
The case for infectivity of RNA per se was considerably strengthened
by demonstrating that only 0.1$ of the RNA infectivity could be accounted
for by residual protein.
Proposed course:
The protein composition of polioviruses will be studied by protein
finger printing of virulent-avirulent combinations which will indicate
possible qualitative differences in their proteins. Column elution proper-
ties and biophysical tools such as electrophoresis, ultracentrifugation,
and equilibrium-density centrifugation may indicate differences in folding
if qualitative differences do not exist. Our ability to label both protein
and nucleic acid of the poliovirus will facilitate analytical work in
III
Serial No. NIAID-I76
regard to these components of the viruses.
Investigations will be continued on the use of labeled viruses as
fundamental and diagnostic reagents. Collaborative studies will be con-
tinued on the anamnestic response elicited in guinea pigs against purified
poliovirus proteins. Investigations are currently under way to determine
if "hybrid" animal cells can be produced and if animal cells exposed to
bacterial KNA will form bacterial protein in the same manner that these
cells form virus protein when exposed to virus RNA.
42
Serial No. NIAID-1T6
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Perrine, T. D.: Method for attaching glass water aspirators to water
lines. J. Chem. Education 37: h8l. Sept. i960.
Oertli, E. and Perrine, T. D.: Magnetically stirred separatory funnel.
Chemie-Ingenieur-Technik 32(8): 55^-555. i960. Also in Chemie fur Labor
und Betrieb 8: i960.
Holland, J. J., Hoyer, B. H., McLaren, L. C, and Syverton, J. T.: Enter-
oviral ribonucleic acid. I. Recovery from virus and assimilation by cells.
J. Exp. Med. 112(5): 821-839- Nov. 1, i960.
Holland, J. J., McLaren, L. C, Hoyer, B. H., and Syverton, J. T.: Enter-
oviral ribonucleic acid. II. Biological, physical and chemical studies.
J. Exp. Med. 112(5): 81+1-86^. Nov. 1, i960.
Reinhard, K. R. and Gerloff, R. K.: Immunity towards poliovirus among
Alaskan natives. II. Am. J. Hyg. 72(3): 298-307. Nov. i960.
Reinhard, K. R. , Gerloff, R. K., and Philip, R. N.: Immunity towards
poliovirus among Alaskan natives. III. Am. J. Hyg. 72(3): 308-320. Nov.1960.
Honors and Awards relating to this project:
Dr. B. H. Hoyer
Invited to be Visiting Professor in the Department of Bacteriology and
Immunology, The Medical School, University of Minnesota, 196O-6I.
Invitation not accepted.
Invited to present series of lectures in Virology at the University of
Washington, May i960.
Invited to participate in Animal Virus Symposium, Berkeley, California,
I960.
Appointed Lecturer in Microbiology 196O-61, Montana State University,
Missoula, Montana.
Dr. R. A. Ormsbee
Appointed lecturer in Microbiology, Montana State University, Missoula,
Montana.
K3
Serial No. NIAID-177
Rocky Mountain Laboratory
Hamilton, Montana
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Immune prophylaxis of mycobacterial infections
Principal Investigator: E. Ribi
Other Investigators: W. T. Haskins and C. L. Larson
Cooperating Units:
Man Years (calendar year i960):
Total: ^.75
Professional: 0.75
Other: U.00
Project Description:
Objectives: ;
The primary objective of this program has been to investigate the
role of live or dead tubercle bacilli or fractions thereof in the production
of resistance to infection with virulent tubercle bacilli. First, it was
considered essential to develop a test based upon a more natural challenge
with virulent organisms than had been used previously and, as a consequence,
the present method of challenge with a small number of organisms given by
aerosol was developed. The objectives also include the application of
this method as a control procedure in the production of vaccine and the
use of the method to determine the mechanism upon which immunity is based.
The role of physical and chemical components of tubercle bacilli
and other acid-fast organisms in producing or eliciting delayed reactions
and circulating antibodies has also been investigated.
Methods:
The methods used have been in large measure those developed at the
Rocky Mountain Laboratory. Fractionation of organisms is accomplished by
means of mechanical disruption. Vaccines are evaluated by all means
available, but special emphasis is placed upon aerosol infection of immu-
nized mice.
Part B included: Yes
W
Serial No. NIAID-177
Major findings:
1. Animals (guinea pigs or rabbits) infected with live cells or
sensitized with killed whole cells or cell walls of various acid-fast
organisms develop specific sensitivity to protoplasm derived from these
organisms.
2. Mice immunized with live cells of H37Ra administered either
intravenously or by aerosol develop significant resistance to subsequent
aerosol infections with H37Rv. Animals immunized with H37R& hy any other
route fail to show similar resistance.
3« Resistance engendered by H37Ra does not appear to be induced by
interference, for administration of this organism either 2h hours before
or after administration of HSTRv does not influence the course of infection.
In these studies both organisms were given as an aerosol.
k. Continued experience demonstrated that C. burnetii, Br. neotomae,
or P. tularensis infections do not affect the course of pulmonary tuber-
culosis in mice. These findings establish the specific nature of this
test for studying immunity against tuberculosis.
5. Live or dead acid-fast organisms (M. tuberculosis, M. phlei,
M. butyricum, M. smegmatis, and atypical acid-fasts) produce lesions in
the lungs of guinea pigs only if the organisms are suspended in Freund's
adjuvant prior to subcutaneous injection into the animals.
6. Antibodies were not detected in serums from persons ill with
tuberculosis when tested by the double diffusion technique of Parlett and
Youmans or by Ovary's method.
7. Previous treatment of animals with BCG organisms produces
hyperreactivity to subsequent injections of Salmonella toxins. Relatively
large doses of cell walls are required to produce such hyperreactivity.
8. Previously, it was shown that the production of isoallergic
encephalitis in guinea pigs was enhanced when 0.05 mg. of Mycobacterium
cell walls was added to a subcutaneously administered dose of brain
antigen mixed with Freund's adjuvant. As much as 5 mg. of protoplasm
mixed with the antigen failed to do so. Further studies have shown that
0.05 mg. of a water-soluble fraction of cell walls has the same enhance-
ment potential as that possessed by an equal weight of cell walls.
Significance;
Since small amounts of cell walls produce hypersensitivity but
fail to produce hyperreactivity to toxins, it would appear that these
two phenomena are distinct. So far, cell walls and the cord factor have
been the only fractions of tubercle bacilli capable of causing hyper-
reactivity in mice.
1*5
2
Serial No. NIAID-177
Studies of hypersensitivity show that specific delayed reactions
are induced by cell walls or whole cells of various organisms and that
the delayed reactions may be specifically elicited by protoplasm of these
organisms. These studies indicate that it is feasible to employ proto-
plasmic fractions of such organisms as the Battey strain to determine the
role of this and other atypical acid-fast bacilli in the production of
nonspecific tuberculin reactions in man. As Palmer and his group have
emphasized, the problem of determining the role of these organisms in
producing disease (symptomatic or asymptomatic) in man has yet to be
evaluated.
Our recent findings that mice can be immunized against infections
with virulent tubercle bacilli when both organisms are administered by
aerosol also have shown that the test we have developed is specific and
that the resistance noted is not based on interference. To date, only
live organisms have been found to engender resistance, but the resistance
noted is of such character that it indicates the usefulness of live
organisms in the prevention of tuberculosis under experimental conditions.
Proposed course:
It is hoped that a study can be initiated to determine the role
of acid-fasts in producing disease and/or hypersensitivity in a normal
rural population in the western United States.
Studies will be continued on immunity against infections with
virulent tubercle bacilli. Plans are being made to examine lots of BCG
vaccine produced in various laboratories to determine the value of our
mouse test as a control for manufacture of BCG vaccines.
Studies of antibody production by various fractions of tubercle
bacilli will be expanded.
Studies of isoallergic encephalitis will be continued. Our results
indicate that it now may be possible to study this phenomenon at a
molecular level.
kS
Serial No. NIAID-177
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
In press:
Larson, C. L., Ribi, E., Wicht, W. C, and List, R.: Skin reactions
produced in rabbits by cell walls and protoplasm of Mycobacterium
tuberculosis and M. butyricum. The Amer. Rev. of Respiratory Diseases.
hi
Serial No. NIAID-I78
Rocky Mountain Laboratory
Hamilton, Montana
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Immune prophylaxis of Sal monella infections
Principal Investigator: E. Ribi
Other Investigators: W. T. Haskins, F. G. Jarvis, and K. C. Milner
Cooperating Units: Dr. Maurice Landy, NCI, Dr. Emanuel Suter, U. of
Florida, and Dr. Erwin Neter, U. of Buffalo.
Man Years (calendar year i960):
Total: 5.5
Professional: 2.5
Other: 3^0
Project Description:
Objectives:
Since immunogenic properties of Gram-negative bacteria are closely
related to potent antigens located in the cell wall, studies are directed
toward the acquisition of a more precise knowledge of the chemical struc-
ture of such antigens and the correlation of such structure with biologic
function. Particular attention has been given to a correlation of chemi-
cal composition with biologic activity, particularly that reported to be
attributable to the so-called "lipid A" fraction.
Methods:
Bacterial fractions isolated by physical and chemical methods are
characterized by appropriate biophysical, biologic, and immunologic
assays. In addition to the use of electron microscopy, complete chemical
and physical analyses are made to verify purities of fractions.
Major findings:
Endotoxins, which are generally regarded as combinations of lipid,
polysaccharide, protein, and peptidelike substances, are so complex that
precise relationships between chemical constitution and biologic function
are still unknown and are subject to controversy. German workers contend
Part B included: Yes
Serial No. NIAID-I78
that the biologic properties of these complexes are attributable to the
firmly bound lipid content, so-called "lipid A." Isolated lipid A is
relatively nontoxic, but these investigators believe that this component
is toxic when conjugated with polysaccharide of the original complex.
Last year it was demonstrated that most, but not all, of the pro-
tein, peptide, and lipid portions could be removed from aqueous-ether
extracts without altering the whole array of in vivo and in vitro biologic
activities of the original complex. In comparison of a Boivin-type ex-
tract with a 19.2$ lipid content and an aqueous-ether extract with a 1.256
lipid content, large but not significant differences in biologic properties
were disclosed. Since firmly bound lipid had been determined by the
Freeman method and not in terms of lipid A, additional investigations were
conducted to delineate the biologic properties of bound lipid.
1. Endotoxin of low lipid content prepared from Salmonella
enteritidis by the aqueous-ether method has been treated further for the
removal of bound lipid by nonhydrolytic procedures. Such endotoxin, con-
taining 2$ to 3$ lipid A, was as potent as that prepared by the well-known
phenol -water or Boivin procedures, which yield products of high lipid A
content (20$ to 30$). To verify differences in lipid content of aqueous-
ether preparations and other types of endotoxins, three different methods
of lipid analysis were employed. These three methods were in accordance
in demonstrating the magnitude of differences in Freeman lipid (chloroform-
soluble material released by hydrolysis with acetic acid), lipid A
(material released with hydrochloric -acid treatment), and esterified fatty
acid.
2. With modified methods, endotoxins having a nitrogen content of
less than 0.5$ and a fatty acid ester content of 1.7$ were prepared. The
analytical values for these potent endotoxins did not differ appreciably
from those of the classic haptenic polysaccharide in respect to nitrogen,
phosphorus, aminohexose and fatty acid ester content.
3. The chemical analyses of a series of endotoxins prepared from
different bacterial species indicate that firmly bound lipid (lipid A)
varies according to the strain of bacteria and to the extracting agent.
Introductory information for the next three findings.
German workers have contended that the biologic activity of lipid
A would be comparable to that of endotoxin if it were possible to disperse
the free lipid A in water in a manner similar to the original state
whereby it is bound to the polysaccharide carrier. These workers have
reported that lipid A, when dispersed in detergent or when coupled to
inert protein, has l/lO and l/5, respectively, of the activity of the
intact endotoxin. However, in making these comparisons, endotoxin and
lipid A were not derived from the same source. In view of our findings,
which were at variance with these reports, and in view of the scarcity of
Serial No. NIAID-I78
pertinent biologic data, comparative studies on the biologic effects of
lipid fractions and the endotoxins from vhich they were prepared were
made by well-established dose-response assays. Bioassays were based on
the following host responses: fever, resistance to infection, tumor
damage, primary inflammation of the skin, and toxic death.
h. Without exception, preparations of lipid A had only a small
fraction of 1$ of the biologic activity of the endotoxins from which
they were derived.
5. Lipoidal fractions were dissociated from endotoxins by nonhydro-
lytic procedures without appreciable reduction in potency of the endo-
toxins. These lipids exhibited biologic effects of the same low order as
lipid A isolated by acid hydrolysis, a method which is known to be
destructive to the potency of endotoxin.
6. Lipid A is a material of a heterogeneous nature. For example,
hexosamine-free lipids have been prepared which are of comparable potency
to lipid A containing 18$ to 20$ hexosamine. The German workers consider
this substance to be an essential component of lipid A.
7. In order to obtain information on the effect of the hydrophilic
carrier to lipid A activity, the capacities to evoke various host responses
of original endotoxin and of artificial lipoprotein prepared from it were
determined in parallel-dose responses. Although artificial lipoprotein
had a biological potency at least 100 -fold lower than endotoxin, it was
greater than that of lipid A. However, less severe acid treatment is
used to prepare artificial lipoprotein than is used to release lipid A.
The less severe acid treatment, rather than increased solubility of
lipid A, was thought to be responsible for the increased activity of
lipoprotein. To prove this interpretation, a kenetic study was made of
the rate of reduction of biologic activity and the rate of release of
firmly bound lipid by acid hydrolysis. Biologic activity of endotoxin,
as measured by 5 quantitative biologic assays, disappeared before appre-
ciable amounts of firmly bound lipid were released. The loss of biologic
potency cannot, therefore, be explained by the separation of the water-
insoluble lipid from the endotoxin complex. At the stage where acid
treatment in the kenetic study was comparable to that used for the prepa-
ration of artificial lipoproteins, the biologic activity of residual
endotoxin was about equal to that of artificial lipoprotein.
8. A method which includes 2 cycles of curtain electrophoresis
was developed for the quantitative separation and recovery of both Vi
and 0 antigens. Because this method does not involve hydrolysis, certain
labile acetyl groups are not affected, and the recovered antigen is about
ten times more active than preparations heretofore available. The biologic
activity of Vi antigen was shown to be dependent upon the presence of
these labile acetyl groups.
50
Serial No. NIAID-I78
Significance;
The injection into animals or man of minute quantities of endo-
toxins extracted from Gram-negative bacteria gives rise to an array of
striking physiological effects. Among those which have been the subject
of extensive study, the following are especially noteworthy: stimulation
of resistance to infection with both homologous and heterologous organ-
isms, enhancement of antibody production, protection against radiation
injury, pyrogenicity, and induction of a state of tolerance to endotoxins.
Even though they have been studied intensively, endotoxins are
sufficiently complex that precise relationships between chemical consti-
tution and the capacity to elicit characteristic reactions in mammals
are still unknown and are subject to controversy. The consensus is that
these complexes, as ordinarily isolated, consist of lipid, polysaccharide,
protein, and peptidelike substances.
Although we had shown that the major portion of the lipid may be
removed while retaining the entire array of biological properties, these
results are at variance with the widely accepted view that firmly bound
lipid is the actual toxic principle of endotoxins and that such lipids
could be split off by acids (lipid A) and their toxic activity restored
by suitable dispersion in water.
A major contribution from this laboratory involves the use of a
number of dose-related quantitative assays to determine in parallel tests
the relative potencies of endotoxins and the lipid fractions derived from
them. The use of these quantitative measurements revealed, to a degree
previously not suspected, that endotoxins are far more potent than their
corresponding lipids.
The study of the kinetics of acid hydrolysis of endotoxin showed
a progressive destruction of potency which was virtually complete before
any separation of firmly bound lipid. It is believed that the small
amount of activity exerted by lipid fractions is of a special kind,
unrelated to the major activity of the complete endotoxin. This con-
clusion is supported by the finding that lipids which had been dissociated
by nonhydrolytic means, without appreciable alteration of the activity of
endotoxin, were at least as potent as lipid A recovered by acid hydrolysis,
the remainder of which is inactive.
Proposed Course:
Despite considerable progress in stripping away nonfunctional
parts of endotoxic extracts, much more can be done toward reducing com-
plexes to pure active principles. This aspect of the work will be
continued in an effort to define the minimum constitution of an endotoxin
or of an 0 antigen. It is sufficiently clear that, up to this point, all
announcements of "purified" endotoxins have been premature.
51
Serial No. NIAID-178
Studies on stimulation of specific immunity to infection will be
resumed, particularly investigation of leads reported earlier concerning
the possibility of producing effective prophylactic agents of modified
toxicity. To implement these aims, enzymatic studies of the endotoxin
complex will be added to our chemical approach to the problem. Physio-
logical studies of endotoxin shock and passive protection to it, assays
for pyrogenicity in rabbits, and perhaps other tests will be added to
the biological work at this laboratory. It is expected that collabora-
tive projects with Drs. Suter, Ueter, Landy, and Nowotny will also go
forward during the coming year, and that a joint project, involving an
exchange of personnel, will be organized with Drs. Malmgren and Heden of
the Karolinska Institutet, Stockholm, Sweden.
52
Serial No. NIAID-I78
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Ribi, E., Hoyer, B. H., Milner, K. C, Perrine, T. D., Larson, C. L., and
Goode, G. : Physical and chemical analysis of endotoxin from Sal monella
enteritidis. J. Immunol. 8^(1): 32-i+7- Jan. i960.
Jarvis, F. G., Mesenko, M. T., and Tibbs, K. E.: Production of Vi antigen
on a chemically defined medium by a coliform bacterium. J. Bact. 80(5):
673-676. Nov. i960.
Jarvis, F. G., Mesenko, M. T., and Kyle, J. E.: Electrophoretic purifi-
cation of the Vi antigen. J. Bact. 80(5): 677-682. Nov. i960.
Honors and Awards relating to this project:
Dr. K. C. Milner
Invited to spend a year in I96I-62 at the Karolinska Institutet in
Stockholm, Sweden.
53
Serial No. NIAID-179
Rocky Mountain Laboratory
Hamilton, Montana
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Investigations of the role of morphological elements of
microorganisms in immunity and related phenomena
Principal Investigator: E. Ribi
Other Investigators: R. L. Anacker, J. E. Coe, C. L. Larson, and
K. C. Milner
Cooperating Units: Dr. J. W. Foster, U. of Georgia; Dr. Philip
Gerhardt, U. of Michigan; Dr. Henry Koffler,
Dr. John Stasny, and Dr. F. L. Crane, Purdue U.
Man Years (calendar year i960):
Total: ^.0
Professional: 1.0
Other: 3*0
Project Description:
Objectives:
Studies of the fine structure and chemical composition of a variety
of organisms and the correlation of such findings with certain immunologic
and biologic properties of microbial cells or fractions thereof comprise
the activities of this section. In general, these projects represent
collaborative efforts with other investigators who wish to utilize Dr. Ribi's
biophysical methodology to fulfill desired objectives. This year, studies
have been concerned chiefly with Brucella, Penicillium chrysogenum, Bacillus
terminalis and B. subtilis.
Methods:
In addition to the usual microbiologic techniques, special methods,
such as X-ray diffraction, the pressure cell system for rupturing bacterial
cells, and linear continuous sucrose or glycerol gradients are utilized.
The electron microscope is routinely used to observe and evaluate induced
morphologic alterations. Immunogenic substances are released from bacterial
cells by mild chemical treatment.
Part B included: Yes
54
Serial No. NIMD-179
Major findings;
1. Continued studies confirmed initial findings that immunogenic
and toxic properties of Br. abortus are located in the cell wall. The
limited activity of protoplasm was thought to result from contamination
with cell -wall materials. Aqueous-ether extracts of cell walls or whole
cells possessed far greater immunogenic activity than did other fractions
or whole cell preparations. When extraction is performed in the presence
of phosphate buffer (pH 7«0)> & more potent though less toxic soluble
antigen is released.
2. The problem of removing large lipoidal granules from washed
aqueous suspensions of bacterial spores was solved by sedimenting washed
spore cultures through a linear continuous glycerin gradient. By this
technique, dormant spores also could be separated from vegetative spores.
3. In view of the known resistance of spores to heat, drying, and
disinfectants, the fine structure of the spore coat was studied in order
to explain the protection afforded the spore body. When a purified
fraction of spore coats was examined under the electron microscope, these
coats appeared to be comprised of multiple layers or lamellae whose thick-
nesses approached the limit of resolution of the electron microscope. The
shape of fractured edges suggested that these layers were composed of
laminar crystals, and subsequent X-ray diffraction patterns confirmed the
character of the spore coat as a perfect crystalline substance.
h. X-ray diffraction patterns of purified cell walls of Penicillium
chrysogenum contained only reflections typical of chitin and not of glucan,
a related polysaccharide thought by collaborative investigators to be
present in the cell wall. The fine structure of chitinous cell walls was
shown to be similar to that of Histoplasma capsulatum, but crystallites
of the latter were smaller. The morphologic arrangement of fibrils and
amorphous regions explains such properties as rigidity, elasticity, and
permeability of the cell walls of these fungi. Chitin crystallites iso-
lated from fibrils of P. chrysogenum are now being purified by Drs. Koffler
and Stasny, collaborators on this project.
5. The significance of "double membranes" in osmium-stained thin
sections of cells, nuclei, and other morphologic structures observed under
the electron microscope has not been resolved. This "double membrane"
appears as 2 parallel lines of osmium but the cellular components with
which osmium is associated have not been determined. Similar structures
have been seen when high speed supernates of bacterial cytoplasm were
desiccated in the presence of sodium chloride upon plastic specimen-
supporting membranes. It was determined that these same structures
appeared in transparent films obtained by simply evaporating aqueous
solutions of sodium or potassium chloride on plastic membranes.
55
Serial No. NIAID-179
Significance:
The intricate association of toxic and immunogenic substances in
the cell vail of Br. abortus complicates the development of a nontoxic
vaccine. However, the effect of buffer (pH 7-°) on the toxicity of sub-
stances released by ether extraction suggests that some selective separa-
tion of toxic and immunogenic fractions is possible. To date, ether is
the only known agent that will kill Brucella without destroying its
immunogenic properties.
The ability to separate various components of washed spore cultures
by sedimentation in a linear, continuous glycerin gradient enables further
definitive characterization of the coat and body of bacterial spores.
Additional information thereby obtained may elucidate the role of the spore
coat in protecting the body from detrimental effects of physical and chem-
ical energies.
Concurrent with the use of thin -sectioning technique for the prepa-
ration of tissues for study by electron microscopy, numerous reports on
"double membranes" associated with various cellular structures have appeared
in the literature. The significance of these structures has remained
controversial. The results of present studies, however, indicate that
these membranes may be formed by some colloidal physical forces which are
induced during solidification of inorganic ions on a surface.
Proposed course:
Collaborative studies on spore coats of B. subtilis and character-
ization of cellular fractions of B. abortus and P. chrysogenum will be
continued.
56
Serial No. NIAID-179
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Larson, C. L., Ribi, E., Milner, K. C, and Lieberman, J. E.: A method
for titrating endotoxic activity in the skin of rabbits. J. Exp. Med.
111(1): 1-20. Jan. i960.
Ribi, E., Brown, W., and Goode, G.: Preparation of microorganisms for
electron microscopy. J. Bact. 79(l): lU2-lM+. Jan. i960.
Honors and Awards relating to this project:
Dr. Edgar Ribi
Invited to give lecture on endotoxins for Department of Bacteriology,
University of Florida, Gainesville, Florida.
Participated in panel discussion of bacterial endotoxins at meeting
of Society of American Bacteriologists, Philadelphia, as representative
of endotoxin group.
Invited to participate in a symposium on bacterial endotoxins sponsored
by the Society of American Bacteriologists in Chicago, Illinois,
April 1961.
57
Serial No. NIAID-180
Rocky Mountain Laboratory
Hamilton, Montana
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: The encephalitides
Principal Investigator: C. M. Eklund
Other Investigators: W. Burgdorfer, D. B. Lackman, V. Newhouse, and
L. A. Thomas
Cooperating Units: None
Kan Years (calendar year i960):
Total: 15.5
Professional: 2.5
Other: 13*0
Project Description:
Objectives:
The objectives of this program are to determine the incidence of
arthropod-borne viruses in the vestern United States and to develop
better methods of diagnosis of these diseases, a detailed knowledge of
the ecology and epidemiology of this group of diseases, and general and
specific methods of control.
Methods:
The methods employed are in general those used in field studies
of arthropod-borne infections and virological and serological techniques
suitable for this type of study.
Major findings:
1. A virus, stored since its original isolation from Colorado
ticks in 1952, was recently characterized and found to be closely related
to Powassan virus obtained from a patient in Ontario, Canada, and studied
at RML last year. These viruses belong to the Russian spring-summer
encephalitis group, but they are not as closely related to this group as
they are to each other. The tick isolate was neutralized by RSSE immune
serum, but RSSE virus was not neutralized by Powassan or tick-virus
Part B included: Yes
58
Serial No. NIAID-180
immune serum. Failure to isolate this agent in tests of many other
field-collected ticks and other considerations suggest that this virus
isolated from Colorado ticks was an accidental infection of D. andersoni.
2. In contrast to a very low incidence of WEE virus in C. tarsalis
in 1959> a marked increase in number of isolations (22) from Idaho and
Oregon was reported this year. One strain also was isolated from Culex
pipiens which, like a previous one, shows some biological differences
from WEE isolated from C. tarsalis. There were also k isolations of
St. Louis virus from Idaho C. tarsalis.
3- Three isolations of Culiseta inornata virus were made, perhaps
a reflection of an unexpected increase of this species in North Dakota.
Isolations had not been made since 1952. This virus belongs to the
African Bunyamwera group and is related to the Cache Valley virus in Utah.
A colony of C . inornata has been established for transmission studies
with these strains.
U. A virus isolated from a local snowshoe hare has been identified
as closely related if not identical to so-called California virus. This
is the first isolation from a vertebrate, though antibodies have been
detected in Bitterroot horses. Domestic rabbits have been refractory to
experimental infection, and tests of the susceptibility of native hares
and rabbits from other states are inconclusive. The related trivittatus
virus was not recovered from mosquitoes this year.
5. Transmission studies showed survival of the snowshoe hare strain
in C. tarsalis and A. aegypti for 25 days, but only one unrepeated
passage by bites of the latter. D. andersoni, 0. parkeri and 0. turicata
did not acquire infection when fed on experimentally infected hamsters.
This virus had not been recovered at RML in tests of 153>°0° C. tarsalis
collected from various western states during the last 10 years.
6. To check for presence of possible latent WEE virus in bloods of
migratory birds arriving before advent of the mosquito season, 976 serums
of different species collected from k localities in Minnesota, North Dakota,
Montana, and Oregon were tested but none contained virus. An additional
lk8 serums from mammals and snakes were free of WEE or SLE virus.
7. Of 50 garter snakes of 2 species injected intraperitoneally
with WEE virus last fall, virus was isolated from 23 (l6 were proved WEE)
after leaving hibernation. Virus circulated in some snakes up to 70 days.
The complete experimental overwintering cycle was confirmed by passage of
virus to other snakes through the bites of mosquitoes infected on the
above snakes.
8. In garter snakes injected with WEE virus and held at k° , 22°,
and 31° C, viremia was observed to start latest (about a month) at the
lowest temperature of storage, but to last longest (at least 25 days) at
53
Serial No. NIAID-180
9« Preliminary tests suggest that gopher snakes and rattlesnakes
may not be as susceptible as garter snakes.
10. On the possibility that immunologic tolerance might influence
the overwintering mechanism of WEE virus, mice in various states of
pregnancy were infected. Up to the 10th day of pregnancy, mothers and
subsequent litters survived. Deaths among mothers infected after the
10th day occurred at varying intervals after inoculation; litters born to
mothers surviving until parturition occurred died within 2 to 3 days after
birth. However, virus was recovered only from the dead mothers, not from
the litters. Pregnancy therefore has a marked effect on the course of the
disease.
Significance;
The survival of WEE virus in hibernating garter snakes, the long
viremia, and demonstrated snake-to-snake transmission by the bites of C.
tarsalis (the important known natural vector) offer at least one explana-
tion for the overwintering of the virus.
Ecologic data being accumulated on the natural occurrence of WEE
and SLE viruses were reviewed in last year's report and are still being
augmented.
Because of the increasing need for identification of various viral
agents isolated in field studies or referred to the virus unit by others,
a collection of 37 identified agents is now available. This collection
will not only speed systematic reference but will provide data fundamental
to an understanding of the relationships of these so-called ar-bo viruses.
The isolation of California virus for the first time from a verte-
brate should forge a significant link in the epidemiologic chain of the
natural cycle of this virus which has caused some human infection in
California and is probably mosquito borne.
The isolation from Colorado ticks of a strain of virus related to
Powassan virus of human origin, suggests that a tick-borne entity with
some resemblance to the important Russian spring-summer encephalitis of
Eurasia may be present in North America.
Proposed course:
Ecologic observations with intensified study of the laboratory
features of the relationships among ar-bo viruses of western United States
will be continued. Further data on various aspects of the role of snakes
are expected to add to epidemiologic information. Comparative studies
will be continued on the pathogenesis of infection, including duration of
viremia in mice, chickens, and other fowls. The susceptibility of verte-
brates to California virus will be investigated.
60
Serial No. HIAID-180
Farther efforts will be made to correlate HI, CF, and neutralizing
antibodies and to determine stability of neutralizing antibodies in serums
during storage. Additional techniques will be used to refine certain
mouse-brain vaccines.
The reference collection of ar-bo viruses will facilitate collab-
orative studies with the Greeley laboratory of CDC and other institutions
on the characterization of unclassified viruses.
61
Serial No. NIAID-180
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Eklund, CM.: Insect-borne and animal-borne virus diseases of man.
Minnesota Med. ^3(3): I8U-IB9, 193* March i960.
Thomas, L. A., Kennedy, R. C, and Eklund, CM.: Isolation of a virus
closely related to Powassan virus from Dermacentor andersoni collected
in Cache la Poudre Canyon, Colorado. Proc. Soc. Exp. Biol. & Med. 10H(2):
355-359. June i960.
Thomas, L. A. and Eklund, CM.: Overwintering of Western equine
encephalomyelitis virus in experimentally infected garter snakes and
transmission to mosquitoes. Proc. Soc. Exp. Biol. & Med. l£5(l): 52-55«
Oct. I960.
In press:
Olson, T. A., Rueger, M. E., Price, R. D., Schlottman, L. L., Kennedy,
R. C, and Eklund, CM.: Evaluation of activity of western equine
encephalomyelitis virus in Minnesota by antibody response of sentinel
pigeons. Am. J. Trop. Med. and Eyg.
Honors and Awards relating to this project:
Dr. C M. Eklund
Appointed lecturer in Microbiology, Montana State University, Missoula,
Montana.
Invited to participate in a symposium on the biology of viruses of the
tick-borne encephalitis complex in Smolenice, Czechoslovakia.
62
Serial No. NIAID-181
Rocky Mountain Laboratory
Hamilton, Montana
PHS-NIH
Individual Project Report
Calendar Year i960
Part A.
Project Title: Colorado tick fever
Principal Investigator: C. M. Eklund
Other Investigators: W. Burgdorfer, G. M. Kohls, D. B. Lackman, and
L. A. Thomas
Cooperating Units: None
Man Years (calendar year i960):
Total: 18.0
Professional: 2.0
Other: 16.0
Project Description:
Objectives:
The objectives of this program are to develop a knowledge of the
clinical syndromes which CTF virus may produce in human beings and to
understand the ecology of this agent, especially the role of tick and
rodent reservoirs. In addition, studies are under way to check possible
interference with concomitant infections of R. rickettsii in tick vectors.
Development of a prophylactic vaccine remains a primary objective.
Methods:
The methods employed do not differ greatly from those used in
laboratories studying field and laboratory aspects of arthropod-borne
viruses except as they are adapted to our local situations.
Major findings:
1. Though CTF had been reported from ticks on Long Island, investi-
gations to date at RML indicate that human infection is limited to areas
where D. andersoni is found. Presence of the virus in adult tick vectors
depends on a natural cycle between the active immature stages and their
small animal hosts, because transovarial maintenance has never been con-
firmed in exhaustive studies.
Part B included: Yes
63
Serial No. NIAID-181
2. The disease in human beings varies from preponderant subclinical
and benign febrile syndromes to infrequent involvement of the central
nervous system or severe bleeding. Although serum samples were not
especially solicited from physicians, almost twice as many isolations
were made this year as last year. Two-thirds of the blood specimens were
received from Idaho, Oregon, and Wyoming.
3. In tests of ticks from several areas in Colorado, infection
rates varied from zero to more than 21$.
h. Members of the Trappist Monastery at Snowmass, Colorado, were
vaccinated again without ill effect. Danger from repeated use of this
inactivated mouse-brain product appears to be minimal because untoward
effects were not observed in guinea pigs receiving multiple injections.
5. Three new lots of vaccine prepared from infected suckling mouse
brains again provided marked protection, as shown by differences in
resistance between vaccinated and nonimmunized control mice.
6. It has now been shown that the simplest diagnostic test for CTF
is the complement -fixation technique. Antibodies are readily detected in
serums from patients convalescent from CTF; they first appear 20 days
after onset of illness and may remain as long as 260 days.
Significance:
As with other tick-borne human diseases, the number of annual
cases of CTF fluctuates. Hence, the marked increase in number of isola-
tions may not represent a real increase in disease. Physicians in the
Rocky Mountain area have undoubtedly become more aware of the prevalence
of this tick-borne disease through our efforts to obtain more data on
severe types of CTF. This year one 9-year-old Oregon boy had encephalitis
accompanied by increased cell count in the spinal fluid and other aggra-
vated symptoms.
Since a high percentage of ticks in Estes Park carry CTF virus,
transient visitors are likely to contract infection but symptoms do not
appear until these persons have returned home and it is probable that
some cases remain undiagnosed.
In certain areas of considerable occupational incidence, such as
in stock handlers in northern Nevada, a suitable vaccine would be useful.
Proposed course:
As in the past, continued efforts will be made to prepare a
refined and potent vaccine.
64
Serial No. NIAID-lSl
We are continuing to accumulate data on possible interference
between infections of R. rickettsii and CTF virus in the same ticks since
this is one possible explanation for the observed low incidence of the
former in endemic localities of CTF-infected ticks. Study by fluorescent
microscopy of the CTF agent in vertebrate and acarine hosts is continuing.
G5
Serial No. NIAID-l8l
PHS-NIH
Individual Project Report
Calendar Year i960
Part B. Honors, Awards, and Publications
Publications other than abstracts from this project:
Burgdorfer, W. and Lackman, D.: Identification of the virus of Colorado
tick fever in mouse tissues by means of fluorescent antibodies. J. Bact.
80(1): I3I-I36. July i960.
Thomas, L. A. and Eklund, CM.: Use of the complement-fixation test as
a diagnostic aid in Colorado tick fever. J. Inf. Dis. 107(2): 235-2UO.
Sept. -Oct. i960.
In press:
Eklund, C. M., Kohls, G. M., Jellison, W. L., Burgdorfer, W., Kennedy, R. C,
and Thomas, L. A.: The clinical and ecological aspects of Colorado tick
fever. Proc. Intern. Cong. Trop. Med. and Malaria, Lisbon, Sept. 1958.
Burgdorfer, W. and Eklund, CM.: I. Colorado tick fever ecological
studies in western Montana. J. Inf. Dis.
Burgdorfer, W.: Colorado tick fever. II. The behavior of Colorado tick
fever virus in rodents. J. Inf. Dis.
Eklund, CM., Kennedy, R. C, and Casey, M. : Colorado tick fever.
Rocky Mtn. Med. Jour.
68
LABORATORY OF BACTERIAL DISEASES
Summary.
1
200 - Brucellosis 2
201 - Basic Studies on Staphylococcus 5
202 - Identification of Pseudomonas 7
203 - Studies on Bacteria Characterized by the
Production of Reproductive Filterable
Granules '
204 - Intracellular Parasitism , H
205 - The Role of Infection in the Delayed
Deaths of Mice Following Extensive
Burn Injury 13
SUMMARY OF ACTIVITIES
LABORATORY OF BACTERIAL DISEASES
December 1960
The research program of the Laboratory of Bacterial Diseases has
continued in the same general areas as last year with the change of
emphasis noted in last year's report.
A greater proportion of the research effort has been directed to
the studies on intracellular parasitism. These studies deal with possible
changes in characteristics of infected and immune cells as the result of
parasitism, and the effect of intracellular growth on the parasite. One
such notable change of course is the production of specific antibodies by
certain cells of immune animals. During the current year a considerable
effort has been directed toward the study of antibody production by cells
in vitro. The macrophage was selected as a multipotential cell for such
study. Macrophages obtained from the peritoneal cavity of immune guinea
pigs (immunized with egg albumin) have been found to release antibody in
vitro for a period of several days. This provides a system for further
study of the nutritional or other requirements for continued antibody
production in vitro, or even in serial cultures. Cells derived from
macrophages have been carried in serial tissue culture for several months,
retaining their phagocytic ability. This promising line of research will
be intensively continued during the coming year.
Studies on brucellosis are conducted at a reduced tempo. There is
continuing need to collaborate with other brucellosis research centers
throughout the world to try and settle problems of classification and
epidemiology of the Brucella. Currently we are doing some laboratory
testing of Brucellosis Vaccine for Human Use prepared in Russia. There is
present interest in this vaccine by the World Health Organization for its
possible use in occupational and otherwise continually exposed groups.
Studies on the Staphylococcus are directed toward determining the
factors responsible for pathogenicity, and toward development and standardi-
zation of tests for measuring relative pathogenicity of strains.
Some of Dr. Verder's past studies on the genus Pseudomonas are being
prepared for publication.
There are other areas within the province of this Laboratory which
should be developed were space, personnel, and financial support available.
Among these are the diarrheal diseases and the basic field of PPLO and L
form research.
There remains a continuing need for certain additional equipment and
increased monetary support to pursue the basic studies mentioned.
Serial No. NIA3D-200
1. Bacterial Diseases
2. None
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Brucellosis
Principal Investigator: Norman B. McCullough, M. D.
Other Investigator: None
Cooperating Units: None
Man Years (calendar year 1960)
Total: 2
Professional: 1/2
Other: 1-1/2
Project Description:
Objectives:
The objectives of this project are broad and studies are
conducted on all aspects of brucellosis, including the nutrition, metabolism,
and enzymatic make-up of the organism, mechanism of action of antibiotics on
the organism, factors determining virulence of the organism, the development
and efficacy of prophylactic immunizing agents, field studies to determine
the occupational hazard of the disease, epidemiology, diagnosis and therapy
of the disease in man.
Clinical studies concern diagnostic criteria for the disease in
man, especially in borderline cases of chronic illness in which present
cultural and serological techniques fail to provide reliable evidence of
infection; studies of the pathogenesis of the disease; and the evaluation of
current therapeutic regimens.
Part B included,
Serial No. NIAID-200
Methods Employed:
The Warburg technique is employed in enzymatic and metabolic
studies; growth in a synthetic medium is used for nutrition experiments;
guinea pig inoculation for virulence and vaccine studies; occupationally
exposed groups for epidemiological studies; human patients for studies on
diagnosis and therapy.
Major Findings:
The study of antigenic relationships among the Brucella has
continued. Br. abortus and Br. suis constitute a single antigenic group as
previously held, whereas Br. melitensis is antigenically more variable. The
latter species consists of three antigenic patterns, only one of which is
identified by the use of currently prepared mono-specific typing serum. The
recognition of this antigenic variance allows the proper classification of
most of the so-called "intermediate" strains, and should clarify many problems
in the classification and epidemiology of the Brucella.
Significance to the Program of the Institute:
In terms of world-wide significance, brucellosis ranks in
incidence and importance with malaria, tuberculosis, and parasitic diseases,
in relation to the welfare of mankind. Even in the United States, by reported
incidence, it is the most important disease of animals transmissible to man,
is the cause of serious economic loss to the livestock industry, is an
occupational disease, and one which presents many problems in the diagnosis
and therapy of the infection in man. The organism readily lends itself to
basic studies outlined elsewhere in this report.
Proposed Course of Project:
Continue studies under way.
Continue to collaborate with other Brucella research centers
throughout the world in the development and standardization of laboratory .
tests for species identification, and for diagnosis of Brucella infection.
Study of Brucella bacteriophage as regards basic phenomena of
bacteriophagy and potential use of phage for preparation of enzymatic and
subcellular fractions of Brucella for biochemical studies, and as possible
immunizing agents. Investigate the utility of specific phages as an aid in
identification of Brucella species and strains.
Continue to use Brucella as a basic tool in the study of
mechanism of development of acquired resistance to antibiotics and in studies
of intracellular parasitism.
Serial No. NIAID-200
PHS-NIH
Individual Project Report
Calendar Year 1960
Part B Honors, Awards, and Publications
Publications other than abstracts from this project:
1. Dolan, T. F., Jr.; McCullough, N. B. ; and Gibson, L. E.:
Nocardiosis. Report of Two Cases in Children. A.M. A.
Jour, of Dis. of Child. 99: 234-237, 1960.
2. Dolan, T. F., Jr.; McCullough, N. B. ; and Gibson, L. E.:
Hypogammaglobulinemia. Report of an Unusual Variation.
Pediatrics 26: 817-821, 1960.
Honors and Awards relating to this project:
Principal investigator elected to serve as a member of the Executive
Committee and as General Chairman of the Annual Brucellosis Research
Conference (1960).
Serial No. NIAID-201
1. Bacterial Diseases
2. None
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Basic Studies on Staphylococcus
Principal Investigator: Elizabeth Verder, Ph.D.
Other Investigator: None
Cooperating Units: None
Man Years (calendar year 1960)
Total: 1-1/12
Professional: 8/12
Other: 5/12
Project Description:
Objectives:
1. To develop an objective means of measuring virulence of
Staphylococci.
2. To determine the factors responsible for virulence.
3. To develop more satisfactory methods for the identification
of individual strains used in measuring virulence.
Methods Employed:
The use of experimental animals and tissue cultures in defining
overall virulence, and in study of individual fractions or products of the
Staphylococcus.
Part B not included.
Serial No. NIAID-201
Use of physical and chemical methods in fractionation of the
organism and its products to allow assessment of individual factors in
pathogenicity and in cell injury in vitro.
Nutritional studies on the production of factors concerned in
pathogenicity.
Gel diffusion techniques using both tubes and plates,
agglutination, hemagglutination and precipitin tests are being employed.
An attempt is being made to correlate mouse protection properties of
human sera with certain characteristics of jLn vitro reactions.
Major Findings:
Numerous serological procedures have been explored in an
effort to develop a technique for more satisfactory identification of
individual strains without immediate success.
Significance to the Program of the Institute:
The number of antigenic components produced by a strain of
staphylococcus varies with the virulence. Quantitative estimation of
some of these components and also some of the products of the more
invasive strains may be very helpful in characterizing strains.
Proposed Course of Project:
As described above.
Serial No. NIAID-202
1. Bacterial Diseases
2. None
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Identification of Pseudomonas
Principal Investigator: Elizabeth Verder, Ph.D.
Other Investigator: None
Cooperating Units: Dr. Carl Millican, NIAMD
Man Years (calendar year 1960)
Total: 1/12
Professional: 1/12
Other : 0
Project Description:
Objectives:
a) Identification of strains for other scientists.
b) Orientation of scientists in methods used for study of
biochemical characteristics of various species and for serological typing.
c) Distribution of cultures of strains of various species and of
representative serological types of Ps. aeruginosa on request to research
scientists.
d) To determine whether therapeutic antisera contains
identifiable group specific antibody.
Part B not included.
Serial No. NIAID-202
Methods Employed;
The methods which we have employed are ones developed or
modified in the course of our studies on the classification of Pseud omonas ;
the studies were initiated during work on our project on diarrheal disease
of the newborn and have been carried on for eight years. A proposed scheme
for the antigenic analysis of strains of Ps. aeruginosa was worked out and
has been employed successfully in identifying strains by serological typing.
Dr. Carl Millican has succeeded in guiding a commercial
biological company to the production of an antiserum that has been used
successfully in the treatment of children with Ps. aeruginosa infections.
He has asked us to collaborate in studies on protection of animals infected
with various serological types to determine whether or not a group specific
antibody is responsible for these protective properties.
Standard antigen- antibody techniques will be employed in the
study. Gel diffusion studies will be done with various gamma globulin and
serum preparations and several antigenic fractions of Pseudomonas.
Major Findings:
Methods have been established for use in the identification of
individual strains of Pseudomonas so that epidemiological surveys in
relation to the spread of resistant strains may be carried out successfully.
Significance to the Program of the Institute:
Methods have been developed for studies on the spread of a
group of antibiotic resistant organisms of medical importance, particularly
in infections in individuals with minimal resistance such as newborns, the
aged, children with leukemia, individuals with extensive or deep burns, etc.
With the increasing importance of Pseudomonas infections the findings in
these studies are receiving recognition in clinical applications.
Proposed Course of Project:
Collaborative studies with Dr. Millican as outlined above.
This will be limited because of time-consuming nature of staphylococcus
studies.
Serial No. NIAID-203
1. Bacterial Diseases
2 . None
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Studies on Bacteria Characterized by the
Production of Reproductive Filterable Granules
Principal Investigator: Elizabeth Verder, Ph.D.
Other Investigator: Dr. Alexis Shelokov was co- investigator on the
project at time the organisms were isolated
and any reports will be made jointly.
Cooperating Units: None
Man Years (calendar year 1960)
Total: 1/12
Professional: 1/12
Other: 0
Project Description:
Objectives:
a) To learn more about 1) the classification of bacteria
capable of producing reproductive filterable granules, 2) the environmental
changes that induce the formation of granules, 3) the potentialities of
these incomplete filterable organisms in the establishment of infectious
processes and 4) the factors influencing the development of more complete
cells from filtered particles.
b) Distribution of cultures on request to other scientists.
Part B not included.
Serial No. NIAID-203
Methods Employed:
Through the use of special media, organisms of this type have
been isolated from the peripheral blood of several patients with illnesses
with an indefinite diagnosis. Methods commonly employed in the study of
organisms producing L bodies and the pleuro- pneumonia- like (PPLO) group
were utilized.
Major Findings:
More detailed studies must be carried out before significant
findings on these organisms may be reported. During the past year attempts
to produce L forms of several strains of staphylococci have been successful.
No detailed physiological studies have yet been carried out.
Significance to the Program of the Institute:
Organisms with similar characteristics have been recognized
as important etiological agents of animal disease for many years. Their
presence in extracts of tissue and blood serum collected from infected
embryo or animals and used inadvertently in the preparation of tissue
culture media has increased interest in the development of more satisfactory
methods for their detection and identification. Their importance in
infectious processes in man has not been adequately assessed. Since
several species of Bacterioides are present in large numbers in the
intestinal canal of man and animals, some of these organisms may belong
to closely related species possessing filterable granules under certain
circumstances and be present in the peripheral blood frequently. The
importance of the group to medical scientists is emphasized by the three
day conference sponsored by the N. Y. Academy of Sciences held in
January, 1959, for discussion of various phases of studies on this group
of organisms; Drs. Klieneberger-Nobel and Edwards of London and Dr. Freund
of Copenhagen were among the invited participants.
Proposed Course of Project:
No additional work will be carried on with these organisms
until the studies on the staphylococcus have been well established.
10
Serial No. NIAID-204
1. Bacterial Diseases
2 . None
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: Intracellular Parasitism
Principal Investigator: Norman B. McCullough, M. D.
Other Investigator: None
Cooperating Units: None
Man Years (calendar year 1960)
Total: 2
Professional: 1/2
Other: 1-1/2
Project Description:
Objectives:
To study host-parasite relationships in intracellular metabolism
for the purpose of increasing our understanding of disease and immunity.
1. To determine the effect of infecting organisms on the
metabolism of the infected cell.
2. To determine the effect of certain bacterial products, such
as Staphylococcus toxin and bacterial fractions, on cells maintained as
explants and in tissue culture, and upon certain subcellular particles.
3. To determine the contribution of cells, and of subcellular
particles, extracts, and enzyme systems to the nutrition of and their
effects on the characteristics of intracellular bacteria.
Part B not included.
11
Serial No. NIAID-204
4. To study protein synthesis (such as antibody production)
by cells from normal and immunized animals.
Methods Employed:
The study employes various pathogenic agents such as Brucella,
Staphylococcus, Pasteurella tularensis, and certain viruses. It will
entail the use of standard tissue culture techniques, chemically defined
media for bacteria, the Warburg technique, standard chemical procedures,
phase microscopy, fluorescent antibody technique, vital dyes, electrophoresis,
and chromatography.
Major Findings:
Using hemagglutination of tanned red cells as indicator,
macrophages derived from the peritoneal cavity of guinea pigs immunized
with egg albumin have been found to release antibody Jji vitro for a period
of several days. Production of antibody appears correlated with conditions
promoting the best growth of cells. However, after a few days there is
a change in the morphology of the cells and antibody production ceases.
Primary stimulation in vitro has not yielded antibody.
Cell cultures derived from guinea pig macrophages have now been
maintained in serial culture for several months.
Significance to the Program of the Institute:
This study concerns the basic interactions between host cell
and infecting agent and hence any contribution to knowledge would be of
significance to the entire field of infectious diseases and to the program
of the Institute.
Proposed Course of Project:
As stated above.
12
Serial No. NIAID-205
1. Bacterial Diseases
2 . None
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
Calendar Year 1960
Part A.
Project Title: The Role of Infection in the Delayed Deaths of
Mice Following Extensive Burn Injury
Principal Investigator: Elizabeth Verder, Ph.D.
Other Investigator: None
Cooperating Units: Drs. Sanford Rosenthal and Carl Millican
Man Years (calendar year 1960)
Total: 2/12
Professional: 2/12
Other : 0
Project Description:
Objectives:
a) To review literature dealing with increased susceptibility to
infection following traumatic shock, radiation and extensive thermal injury.
b) To evaluate findings in extensive studies, carried out in
1943, on incidence of bacteria in tissues of burned mice and their role in
infectious processes causing delayed deaths in mice following extensive
burn injury.
Methods Employed:
Standardized techniques for the isolation and identification of
bacteria from infectious processes were employed.
Part B not included.
13
Serial No. NIAID-205
Major Findings:
Bacteria were isolated from 657. to 907. of the cultures of
heart, liver, spleen and kidneys of 43 mice sacrificed two to nine days
after extensive burn injury. It appears that bacteria of low virulence
and invasiveness may spread, from previously localized foci of latent
infections or from areas of colonization on damaged surfaces, and establish
generalized infections.
Significance to the Program of the Institute;
Septicemias and other fatal infections are of increasing
importance as the cause of death following extensive burn injury in spite
of advances in treatment.
Proposed Course of Project:
Study will be terminated in 1960 with the completion of a paper
discussing our findings.
m
2303
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