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ON HOST-PARASITE INTERACTIONS !

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ELEANOR G. VIERECK

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ARCTIC AEROMEDICAL LABORATORY
FORT WAINWRIGHT
ALASKA

- 1963

PROCEEDINGS

SYMPOSIA ON ARCTIC BIOLOGY AND MEDICINE

III. INFLUENCE OF COLD ON HOST-PARASITE INTERACTIONS

Symposium held August 28, 29, 30, 1962 at the

Arctic Aeromedical Laboratory

Fort Wainwright, Alaska

Symposium Organizer and Chairman

L. Joe Berry
Department of Biology, Bryn Mawr College

Editor

Eleanor G. Viereck
Research Physiologist, Arctic Aeromedical Laboratory

Symposium held under the auspices of the
Geophysical Institute
University of Alaska
College, Alaska ■;{]/,'

1963 ■■■ .*-A\

TABLE OF CONTENTS

Part I.

1. Introduction to the Symposium, Influence of

Cold on Host- Parasite Interactions, L. Joe Berry 1

2. Difficulties in Epidemiological Studies of the

Relationship of Cold to Human Infections,

Robert I. McClaughry 11

3. Aspects of Arctic Epidemiology, Frank L.Babbott, Jr. ... 25

4. The Ecology of Enteroviruses in Alaska,

Karl R. Reinhard 47

5. Opening Remarks on Problems of Immunization

in Stressed Animals, Dan H.Campbell 81

6. Environmental Extremes and Endocrine Relationships

in Antibody Formation, Ignatius L. Trapani 89

7. Qualitative and Quantitative Aspects of the

Immune Response under Conditions of Cold

Exposure, William T. Northey 109

8. Influence of Hypothermia on the Action of

Bacterial Toxins, G. Tunevall and T. Lindner 135

Part II.

9. Effect of Low Ambient Temperatures on Specific

and Nonspecific Resistance, Fred Miya, Stanley

Marcus, and LeGrande J. Phelps 155

10. Virulence as a Factor in Host Response to

Bacterial Infection at Low Environmental

Temperature, Joseph J. Previte and L.Joe Berry .... 215

11. Endogeneous and Experimental Peritonitis and

Bacteraemia in Hypothermic Mice, G. Tunevall

and T. Lindner 239

12. The Role of Low Environmental Temperatures

in Predisposing Mice to Sectondary Bacterial

Infection, Gennaro J. Miraglia and L. Joe Berry .... 271

Part III.

13. Cold and Colds, Sir Christopher Andrewes 301

14. Effect of Environmental Temperature on

Viral Infection, Duard L. Walker, M. D 319

15. The Influence of Cold on Virus Infectivity,

Dr. T. G. Metcalf 343

16. Suppressive Effect of Low Environmental

Temperature on Viral Infection in Bats,
• S. Edward Sulkin and Rae Allen 369

17. Microbiological Aspects of Hibernation in

Ground Squirrels, J. Schmidt 399

18. Cold Therapy in Bacteremic Shock, Emil Blair 419

19. Summary of the Symposium, Walter J. Nungester 447

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DISCUSSANTS

HORACE F. DRURY, Arctic Aeromedical Laboratory,
Fort Wainwright, Alaska.

BOB HUNTLEY, Arctic Health Research Center, Anchorage,

Alaska.

R. B. MITCHELL, School of Aerospace Medicine, Brooks
Air Force Base, Texas.

JOHN A. MONCREIF, Brooke Army Medical Center,
Fort Sam Houston, Texas.

CHARLES T. MARROW, Fairbanks Medical and Surgical
Clinic, Fairbanks, Alaska.

This publication has been released to the Office of
Technical Services, U. S. Department of Commerce,
Washington, 25, D. C. , for sale to the general public.

INTRODUCTION TO THE SYMPOSIUM
INFLUENCE OF COLD ON HOST- PARASITE INTERACTIONS

L. Joe Berry

Department of Biology

Bryn Mawr College

Bryn Mawr, Pennsylvania

The purpose of the Symposium is familiar to all of you, but permit
me the privilege of stating it in my own way. It is our task in the
two and one- half days ahead to look critically at an area that is part
science, part superstition, and part unknown, with the view of deter-
mining how much time, effort, and money should be spent in its
study. We must ask now and throughout the proceedings whether
cold is an important factor in modifying or altering host- parasite
interaction. We have been brought here by a branch of the Armed
Forces of the United States because the medical authorities of that
branch charged with the responsibilities of safe- guarding the health
of military and civilian personnel, who are, for the sake of the
security of our country, forced to live in hostile environments, need
to have an answer. They arelookingtous for help, and if it is with-
in our wisdom to provide, I know we will. There is before us, there-
fore, a very real and practical problem which is our primary con-
cern; but there is also the challenge of pure science. Medical and
biological science from its earliest history has been preoccupied
and inquisitive about the role the environment plays in the behavior
and response of living organisms. I do not know to what extent the
phases of the moon exert an influence at the time of planting or
cultivating, or harvesting the yield and value of crops, but I have
heard in my youth from many firm and dogmatic assertions by
practicing farmers that they do. I also recall with some nostalgia
my mother, my great aunts, and indeed, our faithful country doctor
warning against wet feet, cold on the back of the neck, night aii;
cooling off too fast from a good sweat and the dire consequences
(all of an infectious nature) that would otherwise result. I remember
with affection my mother's wish to protect me from the hostile

BERRY

rigors of a winter in the far north of Texas, Each October I went
into long white underwear only to emerge in late March or early
April when the air warmea to a comparatively safe 80° F to 90° F.
Many of these notions are firmly implanted in the minds of mil-
lions of people more from reiteration than by demonstration, to
borrow a phrase from a friend (Schneider, 1946). But as is often
the case, where conviction is firm, there is often more than a
superstitious quicksand to give it support. We can now begin by
examining some of the bases for this support, and, I hope, finding
what there is of merit, v/hat there is that is formless and amor-
phous, and where, if anywhere, new work is needed to give form
to our knowledge.

In undertaking the responsibility of organizing this Symposium,
it seemed to me that attention should first be directed to an anal-
ysis of past experience of Armed Forces in an arctic environment.
During World War II and certainly in Korea, large bodies of men
were forced to live and fight at cruelly low temperatures. This
was particularly true on the Russian front (for which no direct
knowledge is readily accessible), but knowledge is available to an
adequate degree around the Chosin reservoir in Korea. Casual-
ties from frostbite were acknowledged, but the extent to which
trained and seasoned troops were made more susceptible to mi-
crobic disease may never be known. It is to obtain as much infor-
mation as possible on these points that Dr. McClaughry was asked
to contribute his paper. This was an unusually difficult and com-
plex assignment, and we are grateful to him for his willingness
to undertake it.

The epidemiology of the Arctic poses problems distinct from
those normally encountered. This fact has been recognized by
qualified investigators, and hence the presence of Dr. Babbott and
Dr. Reinhard. These men have each spent extended periods of
time studying the unusual facets that make arctic epidemiology
so challenging. Their experiences, it must be borne in mind, deal
primarily with indigenous populations living under conditions po-
tentially unrelated to those that might arise in a military emer-
gency. Perhaps in the discussion it will be possible to obtain
estimates of what might be expected under other circumstances
and whether there are areas where further work is needed. It is

INTRODUCTION

self-evident that knowledge gained from studies of native popula-
tions selected genetically through the centuries for life in the
Arctic might conceivably have little applicability to the behavior
of a population of greater size and concentration suddenly moved
into this environment.

Cold is a stress. How much it results in a unique response by
the body and the degree to which it resembles other stresses must
be left to the physiologists for elucidation. They have already
established that acclimatization to cold entails greater heat pro-
duction through an elevation in metabolic rate as judged by in-
creased oxygen consumption. This is believed to be mediated through
the endocrines in a highly complex, and as yet incompletely under-
stood manner (Hart, 19 58). These facts have pertinence for anyone
interested in host- parasite interactions because of the well docu-
mented contributions host metabolism makes to determining health
or illness from an infectious agent. Dubos' book, The Biochemical
Determinants of Microbial Disease, (19 54), is a milestone, and
since its appearance, numerous publications have expanded the
literature.

Mammals exposed to cold can be expected to maintain normo-
thermia unless there is excessive heat loss. Hypothermia is probably
the consequence of too little heat prcxiuction and too great heat
loss. The investigator must constantly be preparecTtodisoriminate,
therefore, between effects in normothermic cold, exposed animals
and those found in hypothermic ccld exposed animals. The literature
contains all too many examples of work in which these distinctions
were not made.

The major question confronting us is: Does cold exposure in-
fluence detrimentally man's ability to combat infectious diseases?
This question raises many more. What diseases? What men? How
much cold ana for how long?It is obviously difficult to answer these
questions, particularly with man as the experimental subject, except
for the most unusual circumstances such as those Sir Christopher
Andrewes has used so imaginatively in his studies of the common
cold. To a highly limited extent, it might be possible to utilize human
volunteers for experimental infection with a few Diseases against
which highly therapeutic agents are available. So far as I know,

BERRY

nothing of the kind has been attempted. Should it be done ? Well
might we provide the answer.

The uncertainties, the difficulties, and indeed the impossibilities
of scientific experimentation with human beings in most research
with infectious diseases forces theuseof laboratory animals. There
is ample precedence in the area of cold since a century ago when
Pasteur reported his classical work with anthrax in chickens. The
birds were resistant until chilled by a cold rain (Pasteur et al.,
1878). In the intervening years the literature is adequate to prove
that Pasteur's observations were not isolated findings. Other animals
may or may not respond in modified form to infectious challenge,
depending upon a number of variables, but there are sufficient ex-
amples to leave no room to doubt that host- parasite interaction is a
plastic phenomenon under the pressure of certain environmental
changes.

Confrontment with the established fact still leaves an enormous
area for elaboration. When animals are known to become more
susceptible to infectionj there is always the question of why. It is
axiomatic by now to offer certain tentative explanations. It can be
said, for example, that the defenses of the host have been weakened.
These include the humoral defense, the cellular defense, a weaken-
ing of the barriers to invasion, plus various combinations of them
all. Even if there is evidence for change in any one of the defenses,
it is still necessary to seek out the basis for the change. It is also
possible that virulence ofthe pathogen is enhanced. Without attempt-
ing to define virulence, agreement hopefully is assumed that it
comprises many facets, any one of which may become primary in a
particular situation. Again, it is desirable to understand the nature
of the virulence change whenever it plays a role. There are, in
addition, somewhat more elusive concepts that can be reasonably
offered to explain an elevation in host susceptibility. The in vivo
environment on which pathogen proliferation depends, be it intra-
cellular, extracellular, or both, can conceivably be enriched. There
is less direct evidence for this than one would wish, especially for
the bacteria, but virus multiplication is known to be iritimately
linked with the metabolic vigor of the parasitized cell. By contrast,
the host might be put in the position of heightened sensitivity to the
toxic manifestations of the disease state.

INTRODUCTION

The success of any Symposium usually hinges on the participation
of a few key individuals. One such person, in my judgment, whose
early commitment as a participant undoubtedly influenced the accept-
ance of others, is Dr. Dan Campbell, He will lead the session this
afternoon on the influence of cold on the immune response. Rather
than give the impression of attempting to anticipate his remarks
(which I would never presume to do), I am going to comment only
briefly on this topic, Immunologists have refined their techniques
to such a degree that they are the envy of many research biologists.
Indeed, their methods have been applied wherever possible to prob-
lems fundamentally alien to those classically within the scope of
their specialty. Immunology is quantitative biology at its best, and
may possibly be considered the parent of molecular biology. To de-
termine with a high degree of precision the level of response to
antigenic stimulation does not necessarily make evident, however,
the reason why one group of animals reacts differently from another.
We can expect to learn more about these phenomena from Drs. I, L,
Trapani and William Northey, who have been concentrating in this
area of work for some years. It is a great pleasure to welcome not
only them, but also one of two members of the group who make this
Symposium international. Dr. Gosta Tunevall will speakfirst on the
effects of hypothermia on the response of mice to bacterial toxins.
This is one of four papers (two from Dr. Tunevall) dealing specifi-
cally with animals at reduced body temperature as it influences
host- parasite interaction. The first is concerned with the important
problem of how hypothermia alters the physiological or pharma-
cological properties of a soluble toxin. These bacterial products
must exert their primary action at the enzymic level. If hypothermia
reduces metabolic rate, as it must since the velocity of all chemical
reactions is lowered by a drop in temperature, then there should
be a less acute action of toxin in animals at reduced body tempera-
ture. As logical as this may seem, there are also other alternative
effects that would make this untrue. Hypothermia, as it becomes
more and more severe, affects different organs such that their
activities do not change in parallel (Adolph, 1959), Physiological dis-
tortion occurs, compared to the integrated normal state, such that
some functions cease at a temperature where others continue.
Breathing may stop at about 15° C, but the animal can survive and
recover if artificial respiration is administered. Cessation of heart
beat then becomes a cause of death in animals so maintained and

BERRY

further cooled. These facts have relevance only to serve as warn-
ings against any attempt to generalize about the hypothermic in-
fluence on host- parasite interaction. The specific body temperature
and its duration must be specified in reporting results, and con-
clusions can never be presumed generally applicable unless data are
available to prove it.

The two sessions tomorrow will attempt to answer the question:
Does cold predispose experimental animals (and man) to infectious
disease, and if so, what diseases and under what conditions? We are
unusually fortunate in having two of the world's authorities on in-
fectious diseases to serve as moderators, I refer, of course, to
Dr. Walter Nungester and to Sir Christopher Andrewes. Dr. Nun-
gester has specialized primarily as a bacteriologist and Sir Chris-
topher as a virologist. Their names must certainly be included in
the special list of experts whose willingness to participate con-
tributed so much in attracting to the Symposium such an illustrious
group of participants. We are delighted to welcome and express our
deepest gratitude to Drs, Miya,Previte,andMiragliafor the reports
they are to give on work with bacterial diseases. We are equally
fortunate in having such distinguished virologists interested in this
area of research. I refer, of course, to the Drs. Walker, Metcalf and
Sulkin, and to perhaps the most versatile of us all, Dr. Marcus, who
is at home with both bacterial and viral diseases as well as with
immunology.

A warning has been sounded against the pitfalls of generalizing
from experiments with hypothermic animals unless sufficient know-
ledge justifies it. A similar warning must be issued in regard to
cold exposure and change in susceptibility to infection. Differences
in interpretation of results, differences in experimental design, and
differences in findings are to be not only anticipated but desired. It
is in this way that breadth of understanding is best acquired. But
let us reflect a moment on some of the obvious difficulties in this
type of work. At whattemperature are the animals exposed? "Cold",
according to the literature, can be anything from 15° C to 20° C
down to refrigerator temperatures, about 5° C, or deap freeze,
-10° C to -20° C, or it can be an arctic blizzard of -45° C. How
long are the animals exposed? This can range all the way from
continuous exposure, beginning with challenge, to an infinite varia-

INTRODUCTION

tion of intermittent exposures before or after infection. Are the
animals cold acclimatized? One must ask how acclimatization is
determined. Does it merely mean exposure for some time prior to
infection, or is there some measurement employed that proves the
animals are cold tolerant? Of greatest importance, in my judgment,
are detailed descriptions of experimental conditions of the exposure
to cold. Are the animals housed separately or in groups? Is bedding
material provided? If they are singly housed, how much space is
available? What is the velocity of air movement around the animals?
Is relative humidity controlled, and if so, at what saturation? Are
food and water continuously available?! confess as much ignorance
about these matters as most anyone, yet a few preliminary experi-
ments have clearly indicated that these factors cannot be ignored.
Another consideration is illumination. Continuous dark, continuous
light, or intermittent and erratic light are all to be avoided. Physi-
ologists concerned with biological clocks or with periodicity in
higher organisms have established beyond reasonable doubt that
carbohydrate reserves, body temperature, endocrine secretion,
eosinophile counts, and many others are all subject to highly sig-
nificant variations from one time of day to another (Halberg, 1960).
Some of these changes may be correlated with light, while others
may show free- running periods not directly associated withdetect-
able environmental phenomena. Space in which the animal is housed,
cubic volume as well as area, may contribute in unsuspected degree
to certain responses under investigation. The exigencies of space
travel is making this an important object of study. Research in in-
fectious diseases may be able to add something of significance to
this work.

As moderator for our final session, it is a particular pleasure
for me to welcome a very close friend of many years. We took
several graduate courses together, our doctoral research was
carried out in laboratories opening into the same basement corridor,
we shared our frustrations and successes with one another, and we
walked across the platform the same hot June night in Texas more
years ago than either of us cares to admit to receive our Ph. D.
degrees. Dr. R, B. Mitchell has beenthe author of numerous scien-
tific papers, and is now continuing his service to science through
primarily administrative channels as Chief of the Department of
Medical Sciences, School of Aerospace Medicine.

BERRY

The final session contains the only paper on hibernation that is
to be presented. Several participants suggested that someone con-
cerned with this subject be invited, but most investigators are
interested in the physiology of the hibernating animal. Little atten-
tion has been paid, however, to the fascinating possibilities such
animals provide for the study of host-parasite interactions, Mr,
Schmidtj who spent several years at the Arctic Laboratory, is a
pioneer in this work and will, I am sure, be welcomed back to Alaska
by his many friends.

The program is tobe concluded by two men, Drs. Emil Blair and
Colonel John Moncrief, who are really on the front line of the fight
for knowledge about cold and infectious diseases. They have success-
fully used hypothermia as a therapeutic tool primarily against
Gram negative infections in human beings. They are also applying
their clinical experience to the design of animal experiments capable
of elucidating some of the mechanisms involved inpatient recovery.
The significance of this work is evident, and we will be pleased to
hear of the progress made in this applied field.

Before proceeding with the program, I want on behalf of us all to
express our appreciation to the Arctic Laboratory for making this
Symposium possible; to Mr. Robert Becker for the detailed arrange-
ments he has made for our comfort and relaxation; to Mr, Alfred
George of the University of Alaska for his travel arrangements;
and to Dr. Eleanor Viereck for her editorial assistance, we voice
our thanks. We are grateful to Major Sproul, Commandant of the
Laboratory, for the hospitable accommodations he has provided for
these sessions.

As you are aware, Colonel John D, Fulton is not here. This
Symposium is his dream, and I hope it proves to be all he expected.
He asked me to undertake its organization and has given at all times
his fullest cooperation. If thereareany omissions or deficiencies in
the organization of the scientific program, they are mine and not
his.

INTRODUCTION
LITERATURE CITED

1. Adolf, E. F. 1959. Zones and stages of hypothermia. Ann. N. Y.

Acad. Sci. 80: 288-290.

2. Dubos, R. J. 1954. Biochemical determinants of microbial

disease. Harvard Univ. Press. Cambridge. 152 p.

3. Halberg, F. 1960. Temporal coordination of physiologic function.

Cold Spring Harbor Symp. Quant. Biol. 25: 289-310.

4. Hart, J. S. 1958. Metabolic alterations during chronic exposure

to cold. Fed. Proc. 17: 1045.

5. Pasteur, L., J, F. Joubert, and C. Chamber land. 1878. La theorie

des germes et ses applications a la medicine et a la chirurgie.
Bull, Acad, de Med., Paris, 2nd Series. 7: 432-447.

6. Schneider, H. A, 1946. Nutrition and resistance to infection: the

strategic situation. Vitamins and Hormones 4: 35-70.

DIFFICULTIES IN EPIDEMIOLOGICAL STUDIES
OF THE RELATIONSHIP OF COLD TO HUMAN
INFECTIONS

Robert I, McClaughry

Department of Medicine and Surgery

Veterans Administration

Washington 25, D. C.

ABSTRACT

This report deals with the difficulties experienced in an attempt to use Veterans
Administration hospital records to determine the influence of cold on infectious dis-
ease. Various means were explored to find if exposure to cold of the members of the
Armed Forces of the United States engaged in the Korean War influenced the occur-
rence and course of infectious diseases.

Scientific activity has risen exponentially in recent years. One
result of this phenomenon has been the accumulation of a very large
body of data. Dissemination of this information poses a major prob-
lem, which has been met in part by more scientific meetings and
journals. Strange as it seems at times to a scientist trying to find
time to get into his laboratory, data has been obtained faster than
outlets for its reporting have developed. In absolute terms, there
is no dearth of material to be discussed in scientific circles. These
events have made the ever difficult and unpopular task of presenting
negative results even more problematical. After all too lightly
accepting exactly this assignment at this symposium, the full weight
of what I had undertaken descended upon me. Justifying a negative
report to this illustrious group posed a problem of no mean propor-
tions,

I request your indulgence, therefore, for a brief description of
the reasons for my appearance here today. It all dates back to the
early summer of 19 59, whenthe Chairman of the Division of Medical
Sciences of the National Academy of Sciences - National Research

11

MC CLAUGHRY

Council asked me to direct a survey of the medical research pro-
gram of the Veterans Administration. In short order, I became
acquainted with a strong opinion held by the then Administrator of
Veterans Affairs, Mr. Sumner G. Whittier. He indicated that on the
one hand there were the many Veterans Administration medical
records, and on the other hand, there was the booming technology
of electronic data processing. Now, to continue the paraphrase, if
only a marriage of the two could be effected, surely answers to
most important medical problems would be found.

The committee of medical scientists involved in the NRG study
reached more circumspect conclusions from evaluating all the
evidence available to them. Indeed, the final statement in the section
on the use of computers in their report bears quotation in this con-
text. "It is also to be noted that only very limited use can be ex-
pected of the medical records now in existence for retrospective
studies, and that the collection of data by designing experiments for
the use of computers should be the rule,"

Despite this forewarning, which should certainly have been ade-
quate, I fell into the trap of attempting a retrospective record study.
In my defense, I can only say that the possibility of association of
exposure to cold during the Korean campaign withdifferences in the
incidence or course of infections seemed straightforward enough to
merit a try by this method. I was also somewhat influenced by the
consideration that such environmental influences on human infection
have received relatively little attention, despite their importance.

The burden of my message is really very simple. It was com-
pletely impossible to establish from the records any group who
were known to have been exposed to cold, or conversely to find a
group similar except for such exposure. This information was
simply not recorded. Furthermore, inference of the probability of
of exposure to cold by identifying the military organization to which
an individual belonged proved fruitless.

Since I am nearly as sensitive as was the fabled jackass which
was struck a hard blow on the head with a singletree to get its
attention, I wistfully gave up the record study at this point. Optimist
that I am, though, I must add that the prospect appears better for

12

DIFFICULTIES OF EPIDEMIOLOGICAL STUDIES

the success of a pre-planned epidemiological study of this question.
From the perusal of medical records, it appears that a military
population on maneuvers or in combat in a cold climate could be used
for such investigations. It would be necessary to obtain definite in-
formation about the kind and degree of environmental exposure.
Documentation of the diagnosis and treatment of various infections
is now fairly standardized.

It is enough to indicate that I failed in the first approach to find a
relation between exposure to cold and human infection, and that the
information which was available may favor a different approach. To
extend myself further would jeopardizemy inherently unstable posi-
tion by presuming to tell someone how his research should be done.
For the problem passes from the range of studies possible with
Veterans Administration r ecords intothe interest of those engaged in
field studies.

Now, having spoken for about five of the forty minutes that the
program allocated to this topic, I shall make my only significant
contribution to this symposium by sitting down and making more
time available for discussion of the substantive papers.

13

MC CLAUGHRY
DISCUSSION

MONCRIEF: I want to echo Dr. McClaughry's comments about
retrospective studies, particularly clinical situations. They really
have very little value because of the absence of the data that
you are looking for in the clinical records. We have, I feel,
probably one of the most exhaustive sources on clinical ma-
terial and data collections anywhere in the country because we
have a relatively small unit and a large number of personnel.
Although a patient has been in the hospital two months and may
have a clinical summary the size of a small telephone book,
it still does not have the data in it we want in the future, and
retrospective study is only valuable if the data that is contained
in your records is positive; then it may be of some value. If
it is negative, it may be that the doctor who made up the sum-
mary just left it out, or he may have observed it but did not
put it down in the records, so the only thing you can say with
retrospective study is whether something occurred. You can-
not say it did not occur, nor can you say what you think occurred,

MARCUS: I think since Dr. McClaughry is dealing with nega-
tive results in terms of effect of exposure to cold on infectious
disease, that we might ask him to comment about the subject
of medical- surgical use of hypothermia which has, at least
as near as I can make out from a very superficial inspection
of surgical literature, also given negative results with regard
to infections. Of course, there are circumstances involved here
which, again, are mitigating in terms of preventing infection,
but I still think it deserves some comment as an aspect of the
negative results that you surveyed.

McCLAUGHRY: I think we can have fun with the reports of
Dr. Blair and Col, Moncrief. In a pre-planned study, it is pos-
sible to measure and record the amount of cold exposure, along
with body temperatures, and then follow the incidence and course
of infectious diseases. Also, the bacterial and viral flora of
persons exposed to cold can be studied.

14

DIFFICULTIES OF EPIDEMIOLOGICAL STUDIES

MITCHELL: Dr. McClaughry, do you believe that the data
are really not available, or that they have been in a language
that is not translatable to your computer language? You are
talking about a reporting situation in which the gaps may be
far in excess of the words that you need to translate. A mar-
riage between the reporting of physicians and the computer re-
quires a single common language which permits little or no
deviation.

McCLAUGHRY: The data definitely are not available, since
they were not recorded. Of course, the question of translation
is also a stickler. Much of the present medical data would be
classified by the computer people as "soft garbage", and little
would be considered hard data. In recordings of blood pres-
sure, for example, Dickinson Richards studied Bellevue Hos-
pital records. No correlation was observed between the values
found on the clinical charts, which were random measurements,
and those measured under well controlled conditions.

Similarly, in a study done in Julius Comroe's laboratory^,
an automatic blood pressure recording device gave entirely
different blood pressure levels than those measured in the phy-
sician's office. There were patients who had been considered
non- progressive hypertensive patients who, with the automatic
recorders, were found to be normotensive.

BLAIR: I am particularly intrigued with the statement of the
negative results of hypothermia, and I stress "hypothermia",
not "cold exposure". These are two entirely different matters,
of course, but we will have to decide with regard to infections
whether or not hypothermia has a specific effect upon the or-
ganism involved or on the host himself, if he has any resistance
at all,

MARCUS: I questioned some of our thoracic surgeons and
found there were four in Salt Lake who engaged in the use of

1 Richards, D, W., Jr. Personal communication.

2 Hinmun, A. T., B. T. Engel, and A. F. Bickford. 1962. Am. Heart. J. 63: 663.

15

MC CLAUGHRY

hypothermia. They used both the external blanket procedure,
and they used extra-corporeal cooling of the blood via the heart-
lung machine, and they also told me about the experiences of
colleagues, Dr. Lewis from Chicago, and Dr. Duke and Dr. Swan
of Colorado, They told me that invariably what these individuals
in their own experiences encounter are physiological problems
in their patients; problems involving blood clotting dyscrasia,
cardiac arrhythmia, and so on.

They lower the temperature of their patients down to as low
as 27° C using external blankets for six to thirteen minutes,
and use multiple exposures of this t3TDe in carrying out their
surgical procedures. Invariably, they were surprised when I
asked them about infectious disease, and the four I spoke to
all said, "Well, of course these patients are all covered by post-
operative antibiotic treatment," but in no case were they con-
cerned about postoperative infectious disease, I think you should
know about that.

BLAIR: I am very much involved, in addition to my inter-
est in bacteremic problems, in surgery. The fact does remain
that we understand and know really very little about what hjqjo-
thermia does under these circumstances of altering the envi-
ronment of the bacteria. The University of Minnesota group
has been interested; Fisher at Pittsburgh has studied the ef-
fects of cold upon so-called host mechanisms. Whatever mecha-
nisms are concerned, we know that bacteria are cleared very
readily after injecting and introducing a tremendous number
of bacteria into the blood stream. Now, what this really means
from the standpoint of the added insult and stress of surgery,
anaesthesia and what-not, I don't know. However, purely from
the standpoint of the rather emperical criterion of the progress
of patients postoperatively, the incidence of infections post-
operatively after the use of hypothermia is related only to the
surgery. They have been traced without difficulty to errors and
faults with reference to techniques in surgery, which all sur-
geons should know a lot better about, of course. Infections have
also been traced to the machinery inusing extra- corporeal systems
of cooling.

16

DIFFICULTIES OF EPIDEMIOLOGICAL STUDIES

Granted, these are purely a matter of observation. And then
with surgeons in our position, one of the most important tools
is the ability to look at things from the standpoint of their clinical
judgments. At the University of Maryland, Division of Neuro-
surgery, in the past year almost two hundred patients have been
cooled. They are cooling as many patients as we are in cardio-
vascular surgery, and appear to be our staunchest competitor;
the instance of infections is no greater. This is not good scien-
tific evidence, but insofar as taking care of patients is con-
cerned, it does appear that cooling for periods up to about two
or three hours to levels of approximately 20° C to 30 C does
not increase the incidence of infection following surgery.

MONCRIEF: How about the incidence of infection following
profound hypothermia of 10° C?

BLAIR: The unfortunate fact does remain that the incidence
of infection following the use of profound hypothermia which re-
quires a mechanical set-up to cool a patient down this low, does
result in a higher incidence of infection than if the patient were
simply cooled. Whether this is related to the fact that the pa-
tients are cooled to the very profound and potentially lethal
levels per se, or whether it is due to the fact that it takes a
lot of equipment, we really don't know.

In fact, it does remain that no matter how carefully and how
rigidly Lister's ideas are employed today, after an ordinary
elective surgical case, if you culture very carefully the sur-
geon's hands, the instruments, and the nearby drapes, you will
always find bacteria in staggering and distressing amounts.
But these patients do not develop postoperative infections.

The question remains relatively unanswered, and the only
thing I might say about this is that the patients are kept at these
profound levels for a very, very brief period of time. In many
instances, it does hot exceed more than one hour; occasionally
two hours. At the University of Maryland -- I guess we were
a young, immature group trying to learn — this is the excuse
we give for the fact that we had cooled some patients down to
levels of 5° C for periods of about two hours. These were children.

17

MC CLAUGHRY

They all survived because they were children. Their natural
homeostasis was very kind to us.

BERRY: How much adrenal response is there in these patients?

BLAIR: The adrenal response is reduced. Again, this is the
difference between induced hypothermia and cold exposure. This
is related, of course, to the type of anesthesia used. These people,
you know, are all anesthetized. If a barbiturate anesthesia is
used, the response of the adrenals is reduced tremenduously.
Ether anesthesia invokes an increased response for a period
of time, but when the patient and the animal are cooled, then
the circulating cortico steroid and catecholamines are very
much lowered. Hypothermia allays stress response,

BERRY: In other words, hypothermia is almost anti-stress?

BLAIR: That is correct, as opposed to cold exposure,

BERRY: I think these are very important distinctions that we
should keep in mind,

MITCHELL: Dr, Blair, where are your prime heat sinks
for retaining heat calories in these bodies once you start cooling?
You take them down with cooling to 20° C and you maintain this
for quite a time. You are now ready to work and you have some
tissues there that are rather tremendous heat sinks. They re-
main warmer, let's say, and then if you move past that to these
colder temperatures, do you overcome the ability of that tis-
sue to resist, or do you make it more susceptible, and wherein
do you get these infectious processes originally? I am trying
to figure out how you approach this,

BLAIR: Are you referring to the instances of cooling either
animals or patients down to profound levels in the absence of
infections to begin with? Is this what you mean?

MITCHELL: Yes.

BLAIR: First of all, with regard to heat production during

18

DIFFICULTIES OF EPIDEMIOLOGICAL STUDIES

the induction of cooling, the fact is, of course, that the human
being, himself being a heat exchanger and a very inefficient
one in many respects, produces a series of problems outside
of the infection which have resulted in very serious consequences,
sometimes death, and this is because the specific heat of the
various tissues are so different. When I say artificial cool-
ing, I am referring to hypoxia in the systems. When a patient
is cooled down, he winds up with some very serious gradients
in various tissues, the most serious of which is skeletal muscle.
The skeletal muscle is not cooled down very much. It is kept
quite warm, as a matter of fact. The liver is cooled down quite
considerably; also the brain. The net result is that when the
cooling process is stopped and the patient is presumably sta-
bilized at a particular level of hypothermia, and this is usually
guaged by the esophageal temperature, we begin working on
the patient. Of course, a lot of other things are going on, par-
ticularly, I think, in the skeletal muscle, which when we start
to rewarm the patient, results in a metabolic situation which
has made us very, very unhappy; and this is the metabolic
acidosis.

Now, I am off the subject that you had raised. Dr. Mitchell.
With regard to infections per se, I am not aware of any. They
probably have developed and I can only reiterate the attempts
of culturing the equipment and, of course, the patient's blood
stream, but we have not found any bacteremia. The bacteria,
if present, would become overwhelming. This is a matter which
would require serious investigation, but then, I personally am
not terribly concerned about it because of the time factor. I
don't think much is going to happen in one or two hours. These
patients are cooled to 10° C in about twenty minutes. They are
warmed in about forty-five minutes, but time factors are very
short, and I can assure you that the deep level cooling main-
tained is very brief. Attempts to assay host mechanisms have
demonstrated that the period of cooling and rewarming has not
produced any longstanding effect on the host mechanisms in handling
bacterial infections.

CAMPBELL: The anesthetic must play quite a role here,
as in allergic reactions which do not occur during anesthesia.

19

MC CLAUGHRY

BLAIR: Yes, anesthesia, of course, is a two-edged sword.
We are not dealing with hypothermia per se in its native true
state by any manner of means. We are dealing with modified
hypothermia or, if you will, modified anesthesia, and this is
because in reference to anesthesia, it is necessary to anes-
thetize both humans and experimental subject to a rather deep
level. We call this surgical anesthesia, and this is the level
primarily at which the reflex mechanisms responsible for main-
taining homeothermia are depressed. They have to be depressed.
The only instance of which I am aware and in which hypothermia
is produced in the absence of anesthesia is in patients who have
been treated with hypothermia for bacteremic shock and other
problems of that nature, and again I will touch upon that later.
These people, of course, are not anesthetized.

MARCUS: Do you treat them with morphine or anything to
allay their pain?

BLAIR: Our only experience in this regard, Dr. Marcus, has
been with individuals whom we have considered to be refractory
to the standard therapy. In the judgment of the physician, these
are individuals who have become refractory to very intensive
therapy in bacteremic shock; these people are usually comatose.
Their reflexes are markedly depressed, and these people are
cold. It has been very unusual to observe shivering. As a matter
of fact, we use shivering as an index as to when to rewarm the
patient. It is a sign that the patient is getting better.

McCLAUGHRY: Dr. Campbell may have indicated one of the
very important possibilities here; namely that combining anes-
thesia with hypothermia may make it possible to sort out some
of the factors in pathogenesis of some infectious diseases. In
particular, it may provide a means of studying host responses
to stress, which are really protective mechanisms, but which
may become deranged.

BLAIR: These studies are under way, I might add. Through
a contact with the United States Army, the University of Mary-
land has recently established a clinical shock unit which is de-
voted to treatment of shock, including bacteremic shock, and one

20

DIFFICULTIES OF EPIDEMIOLOGICAL STUDIES

of the purposes is to investigate these properties we are talking
about and about which we really know so very little, particularly
in the human.

McCLAUGHRY: It occurred to me, also, that your descrip-
tion of the situation of the patient with bacteremic shock also
bears on this question of stress, because bacteremic shock
is one of the things which suppresses those functions. In one
sense, you have the anesthesia, anti- shock, and the anti- stress
state established by physiological and pathological mechanisms.

BLAIR: That is quite right. It is the pathologic state, the
comatose state, essentially. The only thir^ I am interested in,
of course, is controlling the reflex mechanism; that is, shivering.
In elective situations, we use anesthesia to depress this, and,
of course, anesthesia is a tremendous poison. It poisons a lot
of things besides the skeletal muscles.

SCHMIDT: I would like to make reference to some work we
did here in Alaska. Our original aim was to determine the in-
cidence of respiratory disease here at Ladd Air Force Base.
We had about two thousand subjects. It occurred to us that it
might be possible to investigate the influence of cold exposure
on the incidence of infection. Accordingly, in taking the his-
tories on these men, we started by asking them if they worked
outside or inside. This approach proved to be of little value,
because even though their duty stations might have been out-
side, the men were clothed to the extent that they could not be
considered to be cold exposed in the sense of being chilled.
We eventually grouped the men according to their squadron ac-
tivities, thinking that cooks and bakers, for instance, would be
less likely to encounter cold exposure than would field main-
tenance crews. About one third of the 1,985 men studied were
in squadrons which we considered would have a higher exposure
index. We were unable to demonstrate any significant differ-
ence in the incidence of upper respiratory infection attributable
to cold exposure, I think we ran up against a stone wall in trying
to determine, with any degree of certainty, whether these men
were actually cold exposed.

21

MC CLAUGHRY

BABBOTT: In an AFED report of two or three years ago
on winter maneuvers in arctic areas, they reported a great
many more cases of heat exhaustion than they did frostbite;
so even under field conditions it is very difficult to evaluate
cold exposure.

ANDRE WES: Williams and Lidwell carried out some experi-
ments on post office workers in Britain, and in comparing those
people who were delivering mail out of doors with those who
were working indoors, the incidence of respiratory infection was
rather greater in those who were working indoors, but of course
you can't draw very many conclusions from that because they
were so much more exposed to other people, and the outdoor
people had very little such contact.

MIRAGLIA: There are several reports in the literature that
indicate that people that work indoors and go into cold rooms —
butchers, for example — do have a higher incidence of sinusitis
and middle ear infections, Taylor and Watrous » made studies
of this using very small groups of individuals, and there are some
examples in Spanish literature which have approximately the
same data, but as some of us have already indicated, there are
other bits in the literature that seem to contradict this, or at
least I found evidence to the contrary,

BLAIR: The matter of respiratory infections, of course, has
risen in regard to hypothermia per se in patients and there
has been an occasional report of patients who have been cooled
who developed pneumonia, but these have been incredibly rare,
and I, in the years that I have had the privilege of working in
this particular area, have never seen respiratory infection in
a patient who has been cooled. As a matter of fact, we have
cooled patients who have had pneumonitis of one kind or an-
other and have observed it clearing up during the process of
cooling. Of course, these people are receiving antibiosis in
treatment, but the hj^othermia is not retarding the clearance

3 Taylor, H. M., and L. Y. Dyrenforth. 1938. JAMA HI: 1744-1747.

4 Watrous, R. M. 1947. Brit. J. Indust. M. 4: 111-125.

22

DIFFICULTIES OF EPIDEMIOLOGICAL STUDIES

of the respiratory infection, in any event.

MITCHELL: You are speaking about hjrpothermia, and the
other people have been talking about cold exposure, and it turns
out that people exposed to cold up here may be more likely to
suffer from heat exhaustion than from cold, and I wonder, maybe,
if we should instrument a few people and put them out in the
cold.

DRURY: Captain Veghte has considerable data on body tem-
peratures during cold exposure.

MITCHELL: I imagine that there is more stress in thinking
about having to go on one of those maneuvers than there is in
the experience.

MONCRIEF: In reference to Dr. Marcus's question to Dr.
Blair about incidence of infection with induced hypothermia.
Dr. Blair mentioned the fact that it is the individuals who have
the hypothermia induced by the extra- corporeal circuit that
have the increased incidence, if there is any. I think infection
is directly proportional to the number of couplings and number
of instruments that you have the patient hooked up to; and this,
I think, is very well pointed out in our normothermic patients
when we put on an extra- corporeal circuit such as extra- cor-
poreal hemodialysis for renal insufficiency,

REINHARD: I am impressed by the fact that the conversa-
tion of the last hour has moved us into an area that looks some-
what like a bucket of worms, and I wonder whether or not we
are trying to sum up before we have ever heard what each person
has to contribute.

23

ASPECTS OF ARCTIC EPIDEMIOLOGY
Frank L. Babbott, Jr.

University of Pennsylvania

School of Medicine
Philadelphia 4, Pennsylvania

ABSTRACT

Few human pathogens found in arctic populations are localized to this geographic
region, although some parasites may have a restricted distribution coinciding with
that of their intermediate hosts. The variety of infectious agents is more limited
than in temperate or tropical climates. With respect to spread, short chain person-
to-person transmission appears more important than dissemination by vector or
vehicle. The age distribution of cases and the clinical response of a particular eth-
nic group to a given disease can usually be explained on the basis of past exposure.
Under conditions of normal arctic living, there is little evidence that low environ-
mental temperatures directly affect the ability of the human host to react to anti-
genic stimuli. Rather, in the Eskimo village, cold is important because of its in-
direct effect on the way people live.

Epidemiology has been defined as a study of the distribution and
determinants of mass disease, or disease as it affects populations.
Consequently, our focus is not primarily on the cellular manifesta-
tions, nor even on diagnosis and treatment of the individual patient.
And yet, we must understand pathogenesis, as well as the clinical
picture, ifwearetounravelthedeterminantsof the disease as it be-
haves in populations.

Of course, cold, as a physical agent, can and does directly affect
individuals and groups of people. During World War II, cold alone, or
cold combined with wet, resulted in the hospitalization of 91,000 U. S.
Army personnelinallpartsof the world. Seventy- one thousand cases
of cold injury occurred in the European Theatre, mostly during the
winter of 1944-1945 (Whayne, 19 58). At one time in the winter of 1943,
frostbite injuries in United States heavy-bomber crews accounted
for more casualties thanallother causes combined. Gunners in B-17
and B-24 aircraft were especially at risk as they maneuvered ma-
chine guns through open"waist ports" while flying at 25,000 to
35,000 feet in temperatures ranging between 25° F and 45° F below

25

BABBOTT

zero (Davis, 1943). As you realize, the resultingdisability was often
permanent, with loss of fingers, toes, or even a whole foot. Less
severe cases required hospitalization ranging from a few days to
many months. So there can be no doubt about the importance of
direct injury by short-term exposure to cold, especially in military
populations.

But a long-term direct effect of environmental temperature on
evolving races of man has also been postulated. Howells points out
that ethnic groups arising in the warmest and coldest climates seem
to have developedaphysiqueandbody mass which permits the maxi-
mal dissipation or conservationof heat(Howells,1960). He contrasts
the large surface area of the Sudanese tribesman with the squat,
compact build of the pure-blooded Eskimo, and discusses the ad-
vantages of each with respect to the physiologic demands of his
immediate surroundings. Thus, cold seems to have a direct effect
not only on men as individuals, but even on the development of the
race itself.

However, the purpose of this symposium is not to discuss cold
injury per se, but rather to explore some of the more subtle in-
fluences of cold on infectious agents and host responses. In the
laboratory, as we will be hearing, it is possible to raise animals, in-
fect them and measure their reactions, all under hypothermic con-
ditions. Likewise, we may propagate the agent at any point on the
temperature scale compatible with its survival. Yet Dr. McClaughry
has just pointed out the difficulties encountered when we try to
undertake similar observations on human populations living in a
setting rampant with uncontrolled and even unrecognized variables.

Part of the trouble is that under natural conditions, both men and
infectious agents do their best to avoid the less than optimal environ-
ment imposed deliberately in the laboratory. We can set up an ex-
perimental hypothermic stress and force mice to a compensatory
physiologic adaptation, one manifestation of which maybe a reduced
capacity to resist infection. But when it gets cold at Wainwright or
Fort Yukon, the inhabitants merely put on an extra parka or add
more fuel to the stove. Only occasionally is a hunter caught on an
ice floe or an airman down on the tundra in circumstances where
he experiences the same sort of stress required of the laboratory

ARCTIC EPIDEMIOLOGY

mice. Likewise, bacteria and viruses grow best within a rather
narrow temperature range, although low ambient temperatures may
promote survival. If they are human pathogens, they usually prefer
an environment close to 37° C. Fortunately for these organisms,
they are seldom forced to adapt to lower temperatures, because
their human culture medium does everything possible to maintain
thermal homeostasis, regardless of external conditions.

Between 1954 and 1957, I was part of a group under the sponsor-
ship of the Armed Forces Epidemiological Board looking into the
transmission of shigella and salmonella infections in various parts
of the Arctic. Frequently when hearing of our studies, people would
ask how these bacteria withstand such an inhospitable climate. Our
answer was that shigella and salmonella grow and multiply at 37° C,
and people who inhabit the Arctic maintain the same body tempera-
ture as that of their distant relatives in better known parts of the
world.

This does not mean that there are not both obvious and unrecog-
nized physiologic responses to cold which may well have a bearing
on infectious illness, I am thinking of such things as blood flow and
secretory activity of the upper respiratory tract and certain endo-
crine responses. However, those of us more familiar with field than
experimental laboratory studies have hesitated to attribute such
unique features as we see to agents evolved under hypothermic con-
ditions or to hosts which have been forced to endure physiologic
stress from cold. Rather, if pressed, we are apt to point to such
secondary effects of cold as overcrowding within dwellings or per-
mafrost which hinders sanitation. Hopefully, following this con-
ference, our horizons will have been broadened to include more
subtle influences which we were previously unable or reluctant to
recognize.

AGENTS OF DISEASE IN ARCTIC POPULATIONS

It is convenient, and I believe justifiable, to think of mass disease

27

B ABBOTT

as having three components: the agent, the host population, and the
environment. In other words, multiple factors inevitably enter into
causation. The task of the epidemiologist is to determine the nature
and relative importance of these factors, so that we may know where
to apply control measures most effectively.

I would like to talk briefly and in very general terms about some
aspects of these three components of mass disease as they relate to
the Arctic, and we might begin with a consideration of infectious
agents. The first point to be made is that very few human pathogens
are strictly localized to arctic areas,Ofcourse,northern populations
have been studied less intensively than residents of other regions,
and yet I doubt if a totally new bacterium or virus having man as its
primary host will be isolated from these people. The same cannot
be said with equal confidence for parasites, however, because their
life cycles sometimes include intermediate hosts unique to northern
latitudes. The second point I'd like to make is that the variety of
infectious agents thus far encountered in the Arctic is limited, at
least by temperate and tropical standards. This is hardly unexpected,
since the same observation applies to arctic flora and fauna gener-
ally (Polunin, 1955; Bliss, 1962).

During our intestinal disease studies in Alaska, Greenland, and
Lapland, some 4,200 people were examined bacteriologically. Only
four types of shigella and five types of salmonella were identified,
and all were familiar pathogens. The greatest variety came from a
couple of Alaskan villagepopulations which included only 325 people.
Among nearly 2,000 well Greenlanders surveyed, the sole pathogenic
bacteria found were Sh. sonnei in three carriers and S. paratyhi B
harbored by four others (Gordon, 19 59). Except for one trematode,
Cryptocotyle lingua, the parasites identified in stools of 660
West Greenlanders were well recognized inhabitants of the human
gastrointestinal tract. These included E, coli, Endolimax nana,
Gardia lambia, Chilomastix, and, surprisingly enough. Entamoeba
histolytica in some 16 per cent (Babbott, 1961).

Hildes and his colleagues (Hildes, 19 58, 19 59) have carried out
serologic surveys in the Canadian Arctic, and Dr. Reinhard will
speak shortly about similar work here in Alaska. The agents they
identified - poliomyelitis, Coxsackievirus, psittacosis, ECHOvirus,

28

ARCTIC EPIDEMIOLOGY

herpes simplex, influenza and adeno-viruses, to name several- were
the same as one might find in many other parts of the world.

But if we are dealing with familiar agents of disease, we are also
dealing with agents which may be responsible for widespread sick-
ness and death. The impact of imported illness among Indians and
Eskimos is a sad story, and one we hardly have time to document
this morning. Suffice it to say that in the 19th century, waves of
smallpox, typhoid fever, pneumonia, meningitis and measles swept
through susceptible arctic populations, and tuberculosis has been
endemic at a high level for decades. In fact, it is only within very
recent years that accidents have replaced tuberculosis as the pri-
mary killer. Currently, accidents accountfor approximately a quar-
ter of total deaths in the Eskimos of Alaska and Greenland (Alaska
Health Dept., 1962; The State of Health in Greenland, 19 59).

As a measure of life threatening forces, you might be interested
in a few comparative statistics. In 19 59, the mortality rate for
Alaskan natives was 9.7 per 1000 population and for Greenlanders,
10.3 per 1000 (Alaska Health Dept., 1962; The State of Health in
Greenland, 19 59). This is close to the 9.3 per 1000 experienced by
the white population of continental United States, or "the lower 48",
as Alaskans call it. The birth rate for Alaskan natives that same
year was 47.7 per 1000, and for Greenlanders, 51 per 1000; more
than twice as high as the birth rate among whites in continental
United States (23.1 per 1000). Taken together, the current birth and
death rates mean that close to 40 people are being added to each
1000 in the population each year, thus giving Greenland and Alaska
one of the highest rates of natural increase in the world. If this
annual four per cent increase is maintained, the population will
double in less than eighteen years. Considering limited arctic re-
sources, and especially the limited locally available food supplies,
it is obvious that the growing population will be more and more de-
pendent upon goods and services brought in from outside.

We will hear much pertinent information about the influence of
environment on disease agents in the coming sessions. Under natural
conditions in the Arctic, a human pathogen, to survive, must either
complete its life cycle within the body of man or some other warm
blooded animal, or the agent must be able to withstand exposure to

29

BABBOTT

cold. Included among sensitive pathogens not having an animal
reservoir are the gonococcus, pneumococcus, and many respiratory
viruses. Trichinella and echinococcus typify parasites having an
intermediate host, as does rabies, an importantdisease in the arctic
setting.

Dr. Reinhard will have considerable to say about the survival of
viruses, but as examples of nonviral agents resistant to environ-
mental stress, we usually think of spore- formers or encysted proto-
zoa. Among the spore- formers, Clostridium botulinum, type E,
presents an important health problem for Eskimos living along the
Bering Sea and northern Labrador, Spores deposited in ocean silt
may contaminate marine mammals and subsequently propagate in
improperly stored meat, such as seal flippers. Between 1945 and
1960, 44 human cases and 23 deaths were reported (Dolman, 1960).
However, studies have shown a rapid die- off of cysts of End amoeba
histolytica at temperatures below freezing (Chang, 19 54), Although
we don't usually regard enteric bacteria as particularly hardy or-
ganisms, investigators from the U, S, Army Environmental Health
Laboratory conducted some interesting experiments in Fort Church-
ill, Canada, using fecal samples seeded with S. typhi, S, paratyphi B,
and Sh. sonnei (Human Wastes, 19 54), These were placed at tundra
sites, and recultured periodically from December to July. Sh. sonnei
could not be recovered after 17 days, but S. typhi was grown out after
45 days and S. paratyphi B after 135 days. The recovery period for
these salmonella organisms was even longer in feces from carriers.
They had survived, but not multiplied.

It might be appropriate here to mention modes of transmission
under arctic conditions. Summed up briefly, it appears that short
chain, person- to- person dissemination is more important than
complex, vulnerable spread involving vehicles, vectors, or extra-
human reservoirs. Certainly the latter exists, as exemplified by
prevalent diseases such as rabies, trichinosis (Thorborg, 1948;
Connell, 1948; Bradly, 19 50),echinococcosis(Rausch, 19 54),diphylo-
bothriasis, and tularemia (Philip, 1962). However, where bacterial
intestinal infections would commonly be transmitted through food
and water in other parts of the world, these illnesses in the Arctic
present an epidemiologic picture much more consistant with con-
tact spread (Gordon, 19 59; Fournelle, 1959; Gordon, 1961). The same

30

ARCTIC EPIDEMIOLOGY

goes for the enteroviruses which Dr. Reinhard will discuss shortly.
As a group, respiratory afflictions, both viral and bacterial, are
very important, and of course are spread by contact. In terms of
mortality for Alaskan natives in 1960, influenza and pneumonia
ranked second only to accidents. Tuberculosis and bronchitis were
also among the first 10 causes of death (Alaska Health Dept.j 1961).

Thus, we may conclude that while the variety of infectious agents
is limited, the organisms are, in general, very similar to human
pathogens isolated elsewhere. With regard to transmission, short
chain person- to- person spread appears at present to be more im-
portant than disseminationby vector or vehicle.

Because muchdetailed attention will be given toagents of disease,
I would now like to mention a few factors involving the host.

SOME HOST FACTORS IN DISEASE OF ARCTIC POPULATIONS

Disease in any population may be described in terms of certain
host characteristics. One of these is the race or ethnic background
of the people under study. At least until very recently, tuberculosis
in Eskimos has behaved rather differently than itdoes in Caucasians.
Prevalence was high, and as of 19 57, 30 per cent of adults living in
the Yukon- Kuskokwim delta region of Alaska had X-ray evidence of
past or current pulmonary involvement (Comstock, 19 59). The
disease ran a much more acute, often fulminating course, with many
extrapulmonary lesions (Schaefer, 19 59), It is interesting to note
that Negros in continental United States used to exhibit a similar
response to tuberculosis (Pinner, 19 32), as did Senegalese troops
during World War I, living in the same military environment as
their French cohorts (Borrel, 1920).

Of course it is often difficult to differentiate between innate host
factors of resistance and environment as it influences exposure,
particularly when we study human populations. However, Lurie has
shown significant family variations in tuberculosis susceptibility

31

BABBOTT

among rabbits receiving an identical dose of the bacillus (Lurie,
1941), It does not seem unreasonable to postulate a similar diversity
of nonspecific resistance for the human animal, even if it is more
difficult to demonstrate. In terms of species survival, it is to the
advantage of both host and parasite that they live in relative sym-
biosis. The long-term process of natural selection tends to evolve
such a relationship. But regardless of whether one puts emphasis
on host or environmental factors in explaining observed differences
in disease behavior; it is important to consider and specify all
possible attributes of the population under study, including ethnic
background.

Age is a second host characteristic, and we often find an unex-
pected age distribution of cases, especially when infectious illness
appears in isolated arctic communities. For example, measles was
imported by a Danish sailor to the vacinity of Juiianehaab, Green-
land, in 19 51. (Christensen, 19 53). An attack rate of 99 per cent re-
sulted, not just among children, but throughout the general popula-
tion. Only the very elderly were spared in appreciable numbers,
indicating that no measles had occurred in this community in more
than sixty years. Likewise, it has been possible to reconstruct the
past history of mumps, poliomyelitis and influenza here in Alaska
by relating antibody titers to the host characteristic of age (Philip,
19 59; Reinhard, 1960; Philip, 1962).

Of course, a very important component of host resistance is the
presence of specific antibody, which presupposes prior contact
with the antigen. I will speak shortly about the arctic environment
as it promotes or inhibits such contact. But let me return for a
moment to a point we touched on earlier; that is, under conditions
of normal arctic living, we lack epidemiologic evidence to show
that low environmental temperatures directly alter the man's ability
to respond to an antigenic stimulus. On the contrary, the Alaska
Department of Health and Welfare does not differentiate between
the Indians of Southeastern Alaska and the Eskimos of Northern
xA.laska when planning an immunization program, nor do they expect
resulting levels of immunity to differ from those seen in continental
United States (Pauls, 1962). Likewise, serologic studies following
natural infection fail to demonstrate deviation from an expected
distribution of antibody titers (Philip, 1959; 1962; Reinhard, I960).

32

ARCTIC EPIDEMIOLOGY

The reason for this uniformity in host response under natural
conditions appears to be that residents of Barrow or Thule or
arctic Lapland so modify their microclimate that they seldom suffer
physiologic stress from cold, although they are susceptible to some
of its secondary effects.

In the time remaining, I would like to mention briefly certain
features of the arctic environment.

ENVIRONMENTAL FACTORS IN DISEASES OF
ARCTIC POPULATIONS

The geographer defines the Arctic as that portion of the northern
hemisphere lying within the Arctic Circle, which falls at 66^ 30'
north latitude. This marks the southernboundary of territory where
at least one day each summer the sun never dips below the horizon,
and one day each winter it never appears. The characteristic types
of terrain are tundra, usually bordering the coast, taiga or inland
forested regions, and glacial topography typified by the Greenland
ice-cap and parts of the Canadian archipelago.

Theclimatologistdefines the Arctic as that territory lying within
the 50° F isotherm, an imaginary line whichbounds an area where
the average temperature during the warmest month of the year does
not exceed 50° F. To the agronomist, concerned with crop potentialj
the number of frost free days a year is much more important than
minimal winter temperatures.

Permafrost is one physical feature of the arctic environment
which influences distribution of disease. Obviously when the earth
is permanently frozen, subsurface excreta disposal is difficult or
impossible, and water must be procured from easily polluted surface
sources. A more indirect, but significant affect of permafrost is the
restriction it places on agriculture. People inhabiting such an area
must rely upon a hunting or fishing economy with its attendent lower
standard of living, unless, of course, military establishments or

33

BABBOTT
industry, such as mining, are available to provide employment.

There is no doubt that cold influences disease behavior in arctic
populations, but as I indicated, we have little evidence that under
natural conditions, cold of itself directly affects agent pathogenicity
or host resistance. Rather, it is important because of the way it
modifies the social environment or the way people live. For example,
houses must be heated ten or eleven months of the year. Fuel is
expensive in terms of either the money or the effort required to
procure it. To conserve heat, dwellings are small with consequent
overcrowding, and thus conditions are ideal for transmission of
respiratory illness.

Studies in other areas have shown that the incidence of enteric
disease is influenced more by the availability of water in adequate
quantities than by its purity (HoUister, 19 55; Schliessman, 19 58).
Because of the cold, people in arctic regions depend for many months
each year on melted ice or snow for their water supply, and this a-
gain requires the expenditure of precious fuel. It is not surprising,
then, that the level of sanitation is low, and fecal- oral spread of
intestinal organisms is easily accomplished. Yet with respect to
bacterial enteric illness , low environmental temperatures also serve
a useful purpose in that bacterial pathogens are immobilized and
killed off in large numbers, and spread by flies is greatly minimized.
Cold appears to promote survival, if not multiplication, of viral
agents, as Dr. Reinhard will point out.

Another indirect effect of cold on arctic disease has been the
limitations it has imposed upon travel until relatively recently.
Half a century ago, the inhospitable environment served to protect
people from imported pathogens. Now, there are few, if any, com-
munities which experience prolonged isolation. Fortunately, im-
proved communications brings not only more conventional patterns
of disease, but also preventative and therapeutic services as well.
Therefore, it is highly unlikely that we will see epidemics of the
magnitude or severity which characterized the Eskimos' early
contact with the outside world. As I have mentioned, death rates
among Alaskan natives currently approximate those of their fellow
citizens elsewhere. Our problem in the future is not to lower death

34

ARCTIC EPIDEMIOLOGY

rates, but rather to raise living standards, and this task is not made
easier by the rapid population increase.

In closing, I would like to touch upon a few features of the biologic
environment. If this meeting had been called in June, we would have
been very much aware ofthe high mosquito density which character-
izes an arctic summer. Surprisingly enough, mosquitos have yet to
be implicated as disease vectors in this part ofthe world, though
the possibility requires further study, with particular reference to
the ARBO viruses. As for animal reservoirs, Hildes and his group
at the University of Manitoba, conducting serologic surveys in the
Eastern Canadian Arctic,foundl5per centof 241 Eskimos possessed
complement fixing antibodies to psittacosis (Hildes, 19 58) . Although
cross reactions with other viruses, especially trachoma and in-
clusion conjunctivitis, must be considered, it is very possible these
people contacted psittacosis as a result of eating raw birds. I have
already referred to trichinosis, echinococcosis and rabiesj all of
which have mammalian reservoirs. Within recent years, brucellosis
has been discovered in both men and reindeer of the Soviet and
American Arctic (Cherchenko, 1961; Edwards, 19 59; Huntley, 1962).

Finally, to illustrate the importance of understanding arctic
ecology, let me mention a Strontium-90 survey conducted here in
Alaska with the help of Colonel Fulton (Schulert, 1962). Because
of permafrost, radioactive fallout from Soviet nuclear testing ac-
cumulates on thesurfaceof the soil where mosses, lichens and other
low vegetation grow. These plants provide forage for caribou. A
study of caribou antlers in Alaska revealed a concentration of Sr-90
more than ten times that of pooled deer antlers in California. Eski-
mos in certain areas eat large quantities of caribou, and urine
assays in these communities showed that new bone was being laid
down with about 12 mmc of Sr-90 per gram of calcium, which is four
times the average U. S. concentration. This single problem required
the interrelating of knowledge concerning meteorology, geology,
botany, anthropology, animal ecology, and radiobiology.

35

BABBOTT
SUMMARY AND CONCLUSIONS

In the short time available, it has been possible to touch upon
only afewfeatures which characterize the behavior of human disease
in the Arctic. In briefest summary, let me recapitulate:

1) Few human pathogens found in arctic populations are localized
to this geographic region, although some parasites may have a
restricted distribution coinciding with that of their intermediate
hosts.

2) The variety of infectious agents is more limited than in tropi-
cal or temperate climates,

3) With respect to spread, short chain person-to-perscn trans-
mission appears at this time more important thandissemination by
vector or vehicle.

4) The age distribution of cases and the clinical response of a
particular ethnic group to a given disease can usually be explained
on the basis of bast exposure.

5) Under conditions of normal arctic living, there is little evi-
dence that low environmental temperatures directly affect either
agent virulence or specific host responses. Rather, in arctic popu-
lations cold is primarily important because of its indirect effect on
the way people live.

I donotbelievethereis such an entity as "arctic medicine", if by
that we mean a unique set of pathologic conditions restricted to this
geographic region. However, the ecology of the Far North is ':'is-
tinctive, and may alter the epidemiology of certain diseases. If an
illness is to be effectively prevented or controlled, it is important
that we understand not only its laboratory and clinical character-
istics, but also its behavior in populations.

36

ARCTIC EPIDEMIOLOGY
LITERATURE CITED

1. Alaska Department of Health and Welfare. 1962. Bureau of Vital

Statistics. Personal Communication.

2. Babbot, F. L., Jr., W. W. Frye.and J. E. Gordon. 1961. Intestinal

parasites of man in arctic Greenland. Am. J. Tropical Med.
Hyg. 10; 185-190.

3. Bliss, L. C. 1962. Adaptations of arctic and alpine plants to en-

vironmental conditions. Arctic 15: 117-144.

4. Borrel, A. 1920. Pneumonie et tuberculose chez les troupes

noires. Ann. Inst. Pasteur 34: 10 5-148.

5. Bradly, P. J., and R.Rausch. 19 50. A preliminary note on trichi-

nosis investigations in Alaska, Arctic 3: 10 5-107.

6. Chang, S. L. 1954. Thesurvivalof cysts of Endamoeba histolytica

in human feces under low temperature conditions. Annual
Reportj Commission on Environmental Hygiene, Armed Forces
Epidemiological Board 19 53-19 54.

7. Cherchenko, I. I. 1961. Brucellosis in arctic regions. I. On

brucellosis in reindeer. Zh.Mikrobiol. 32: 135-159. J. Micro-
bial, Epidemiol. Immunobiol. 32: 554-559.

8. Cherchenko, I. I. 1961. Brucellosis in arctic regions. II. On

epidemiologic characteristics of a focus of brucellosis in
reindeer. Zh. Mikrobiol. 32: 118-123.

9. Cherchenko, I. I., and N. I. Samsonova. 1961. Brucella infection

in far northern regions. III. Clinical manifestations of "rein-
deer" brucellosis in man. Zh. Mikrobiol. 32: 51-56.

37

BABBOTT

10. Christensen, P. E,, H.Schmidt, H. O. Bang, V. Andersen, B.

Jordal, and O. Jensen. 1953. An epidemic of measels in
Southern Greenland, 1951. Measles in virgin soil. II. The epi-
demic proper. Acta Med. Scand. 144: 430-449.

11. Comstock, G. W., and M.E. Porter. 19 59. Tuberculin sensitivity

and tuberculosis among natives of the lower Yukon. Pub. Health
Rep. 74: 612-634.

12. Connell, F. H. 1948, Trichinosis in the arctic- a review. Arctic

2: 98-107.

13. Davis, L., J.E. Scarff, and M.Dickenson. 1943. High altitude frost-

bite: preliminary report. Surg., Gynec. and Obst. 77: 561-575.

14. Dolman, C. E. 1960. Type E Botulism: A hazard of the North.

Arctic 13: 230-256.

15. Edwards, S. 1959. Brucella suis in the Arctic. Alaska Med.

1: 41-44.

16. Fournelle, H. J., V. Rader, and C. Allen. 1959. Seasonal study of

enteric infections in Alaskan Eskimos. Pub. Health Rep.
74: 55-59.

17. Gordon, J. E.,and F. L. Babbott, Jr. 19 59. Acute intestinal in-

fection in Alaska. Pub. Health Rep. 74: 49-54.

18. Gordon, J. E., and F. L. Babbott, Jr. 1959. Acute intestinal in-

fection in the Arctic. Am. J. Pub. Health 49:1441-1453.

19. Gordon, J. E., E. A. Freundt, E.W.Brown, Jr., and F. L. Bab-

bot, Jr. 1961. Endemic and epidemic diarrheal disease in arctic
Greenland. Am. J. Med. Sci. 242: 374-390.

20. Hildes, J. A., J. C. Wilt, and W. Stackiw. 1959. Neutralizing viral

antibodies in Eastern Arctic Eskimos. Can. J. Pub. Health
50: 148-151.

38

ARCTIC EPIDEMIOLOGY

21. Hildes, J. A., J. C. Wilt, and F.J, Stanfield. 1958. Antibodies to

adenovirus and psittacosis in Eastern Arctic Eskimos. Can, J,
Pub, Health 49: 230-231.

22. HoUister, A. C, M. D.Beck, A, M.Gittelsohn, and E, C, Hemp-

hill, 1955, Influence of water availability on shigella prevalence
in children of farm labor families. Am. J. Pub. Health
45: 354-362,

23. Howells, W, W, 1960, The distribution of man, Sci, Am. 203:

112-127,

24. Human Wastes in Arctic and Sub- arctic. Final Report. 1954,

Army Environmental Health Laboratory of the Army Medical
Service, U. S, Army Chemical Center, Maryland.

25. Huntley, B. E., J, E, Maynard, and R, N, Philip. 1962. Prelimi-

nary studies of brucellosis in Alaska. In press.

26. Lurie, M. B. 1941, Heredity, constitution and tuberculosis: an

experimental study. Supplement to Am. Rev. Tuberc. Vol. 64.

27. Pauls, F, P, 1962, Chief of Laboratories- Southcentral Regional

Laboratory, Anchorage, Alaska. Personal Communication.

28. Philip, R, N., B, Huntley, D. B. Lackman, and G. W. Comstock.

1962, Serologic and skin test evidence of tularemia infection
among Alaska Eskimos, Indians and Aleuts. J. Infect. Dis.
110: 220-230.

29. Philip, R. N,, and D. B. Lackman. 1962. Observations on the

present distribution of Influenza A/swine antibodies among
Alaskan natives relative to the occurrence of influenza in
1918-1919, Am. J. Hyg. 75: 322-334.

30. Philip, R. N,, K. R, Reinhard, andD,B, Lackman. 1959. Obser-

vations on a mumps epidemic in a "virgin" population. Am. J,
Hyg, 69: 91-111,

39

BABBOTT

31. Philip, R. N., W. T. Weeks, K.R. Reinhard, D. B. Lackman, and

C. French. 1959. Observations on Asian influenza on two Ala-
skan islands. Pub. Health Rep. 74: 737-745.

32. Pinner, M., and J. A. Kasper. 1932. Pathological peculiarities

of tuberculosis in the American Negro. Am. Rev. Tuberc.
26: 463-491.

33. Polunin, N. 19 55. Aspects of arctic botany. Am, Sci. 43:

307-322.

34. Reinhard, K. R., and R. K. Gerloff. 1960. Immunity towards

poliovirus among Alaskan natives. II. A serologic survey of
47 native communities of western and northern Alaska, Am.
J. Hyg. 72: 298-307.

35. Reinhard, K. R,, R. K. Gerloff, and R, N. Philip. 1960. Immunity

towards poliovirus among Alaskan natives. III. A study of
naturally and artificially acquired antibodies against polio-
virus among residents of two Bering Sea communities. Am.
J. Hyg. 72: 308-320.

36. Rausch, R. 1954. Studies on the helminth fauna of Alaska, XXIV,

Echinococcus sibiricensis N. sp., from St. Lawrence Island,
J. Parasitcl. 40: 659-662.

37. Schaefer, O. 1959. Medical observations and problems in the

Canadian Arctic. Canad. M. A. J. 81: 248-253.

38. Schliessman, D. J., F. O, Atchley, M. J. Wilcomb, and S, F,

Welch, 19 58. Relation of environmental factors to the occur-
rence of enteric diseases in areas of eastern Kentucky. Public
Health Monograph No. 54. (PHS Pub. No. 591).

39. Schulert, A. R. 1962. Strontium-90 in Alaska. Science 136:

146-148.

40

ARCTIC EPIDEMIOLOGY

40. State of Health in Greenland, The. 1959. Annual Report from

the Medical Officer in Greenland. Godthaab, Greenland.
(The State of Health in Greenland).

41. Thorborg, N. B., S. Tulinius, and H. Roth. 1948. Trichinosis

in Greenland. Acta Path, et Microbiol. Scand. 25: 778-794,

42. Whayne, T. F., and M. E. DeBakey. 19 58. Cold Injury, Ground

Type. Office of the Surgeon General, Department of the
Army, Washington, D. C.

DISCUSSION

CAMPBELL; When you study these native populations, in
Anaktuvuk Pass and Point Barrow, for example, don't you have
to consider the possibility of malnutrition? It seems to me I
have heard in the past that vitamins may have an influence on
viruses.

BABBOTT: I think that is very important. It is a nonspecific
host characteristic which I neglected to bring in, but it is im-
portant. The reason I didn't bring it in was that it has been
very difficult to do detailed and sound studies on nutrition of
these people, particularly in recent years when their diet has
been undergoing such rapid change. Dr. Scott, of the Arctic
Health Research Center, I know, has worked on certain tj^Des
of anemias and on vitamin levels. I was not familiar with a
very extensive literature on this, and so I did not include it;
but it is important.

CAMPBELL: Aren't Eskimos calcium deficient?

BABBOTT : Not that I know of. Do you know anything about
that. Dr. Reinhard?

REINHARD: Not in general, but there may be specific people

41

BABBOTT

whose diet has become so perverted that they might be cal-
cium deficient. This matter of diet is a vexing socio-economic
situation here in the northland. For instance, at Anaktuvuk Pass
the diet may be highly adequate in years when the caribou are
coming through en masse, and in other years, when the cari-
bou hit another way through the mountains, the people's diet
may be down to a bare minimum level.

So dietary adequacy fluctuates in these people from time
to time. Now, in certain areas where the game animals num-
bered in the millions in the past have been depleted — such
as the Kuskoquim area — the people just don't get as many
fish, caribou, whales or seals as they did years ago. There
the former diet has given way to one supplemented to a large
extent by flour and sugar, which isn't especially nutritious.
But it is a problem that needs study. It hasn't been studied very
well, and I'm glad you brought it up. I might mention one more
thing. One of the medical cliches of the past held that Eskimos
were hypersusceptible to tuberculosis, but it has been demon-
strated by vigorous case findings, hospitalization, and ambulent
chemotherapy programs in the past eight years, that the tuber-
culosis epidemic could be abated rapidly, and I would like to
propose that the Eskimos are considerably more resistant to
tuberculosis than is generally conceded, or they might have been
dead long ago — wiped out as a race. We have to differentiate
between medically underprivileged people and hypersusceptible
people. They are not the same.

MITCHELL: Are we talking really and truly about tuberculosis,
or are we talking about X-ray evidence of a lung infection which
might be or might have been tuberculosis, or a fungus, or some-
thing other than, say, the mycobacterium?

REINHARD: We are talking about clinically proven cases
of tuberculosis, the statistics of hospital admission, the de-
crease in positive culture results in various laboratories, the
decrease in the malignancy of the cases entering hospitals,
and the decrease in death rate. These are all rather positive
changes that have occurred very rapidly in the last ten years.
In other words, there is proof there. It is not a supposition.

42

ARCTIC EPIDEMIOLOGY

MITCHELL: The avitaminoses that you are speaking of; would
you say that is something that we brought to Alaska? If we hadn't
come into Alaska and depleted the food supply, the natives of
this area would not be living on flour and sugar.

REINHARD: I think it would be well to get rid of the con-
cept that the white man has consistently done wrong to the Es-
kimo. After all, the inequities caused by interactions of col-
liding cultures are part of the normal history of the world, and
we have to recognize that they will occur. To go back to the
original question, I don't know of any real data on the general
occurrence of specific avitaminoses among Alaskan natives.

NUNGESTER: Let's go back just a moment to avitaminosis.
Is there any evidence of scurvy in the Eskimo?

REINHARD: I have heard that traditionally, Eskimos were
supposed to have no caries, no scurvy, and no body odor.

SCHMIDT: Dr. Babbott, you mentioned that the Russian atomic
tests have caused a great deal of Sr^^ fallout in various parts
of Alaska. I was under the impression that there were other
countries also testing. Did fallout from these tests not reach
Alaska?

BABBOTT: This particular study was done after the first
series of Russian tests. I am sure they are not the only ones.

MITCHELL: Dr. Babbott, we could show that people in Florida
are taller than the people in Alaska, maybe on the average, and
perhaps attribute this to atomic detonation. When you make
studies of antlers from animals here in the Arctic, do you find
an increased amount of Sr^^ ^^ those antlers compared with
the antlers of animals gathered, say, in California, and sent
to Smithsonian prior to the detonation?

BABBOTT: I think a baseline study would be very valuable.

MITCHELL: It certainly would be indicated, because I have
been plagued with information of this kind.

43

BABBOTT

BLAIR: I was most interested in the observations, Dr. Rein-
hard, of the Eskimos' response to treatment of tuberculosis.
There is so much doubt about the change in the organisms with
respect to the chemotherapy, the rate of mutation of the dif-
ferent strains and the effect of the various agents they use.
Do you have the same problem?

REINHARD: There is a paper in the 1962 AAAS Alaska Science
Conference by Shepard of the ADH^ laboratories which gives
a resumd of the decline in positive cultures over the last eight
to ten years, and then in surge, in 1962. At the time she postu-
lated that this was due to an occurrence of resisting types. How-
ever, there are many other factors that were not dealt with in
the paper, or brought under control, such as the possibility
that the ADH might have been going through another surge of
case findings. Furthermore, the laboratory hasn't been running
routine tests for resistance. So far, we don't know whether there
is an increase of the resistant types. I don't know whether the
Anchorage laboratory^ may have better reuslts than this. 1 wish
we could call up Frank Pauls right now.

BERRY: We do have a man here, Dr. Huntley, from the An-
chorage Laboratory. Do you have any information on this?

HUNTLEY: No, however, I know that the Alaska Department
of Health Laboratory is now doing an antibiotic sensitivity study
on positive TB cultures which has resulted in a tremendous
increase in the number of cultures requested from field nurses.
The study has not been under way long enough (probably 6 to
8 months), therefore, and no data are available on possible re-
sistant strains.

McCLAUGHRY: I'd like to make a comment on Dr. Babbott's
remark that there is no such thing as arctic medicine. This has
been accepted as a concept by the National Research Council
Committee on Tropical Medicines. In attempting to sharpen

1 Alaska Department of Health.

2 Laboratory, Southcentral Region, Alaska Department of Health.

44

ARCTIC EPIDEMIOLOGY

their definition of what they were concerned with in tropical
medicine, they have also come to recognize the phenomenon of
medical underprivilege.

ANDREWES: The thing that struck me about Dr. Babbott's
paper was the very small amount of evidence there was that
cold played any part in the story except indirectly. The effect
of cold on the habits and crowding of the people is obviously one
consequence, but you get crowding in underprivileged people all
over the world, including tropical areas, with the same results.
The other thing which so obviously effects the issue is the lack
of past experience to particular pathogens causing these out-
breaks. Now, particularly in relation to common cold research,
we thought it was something of great importance to conduct planned
studies on the behavior of isolated communities to see what
happened to them when they were isolated and when they made
contact with civilization again; and apparently we have missed
the bus, because there don't seem to be any isolated commu-
nities, any more. Even though they have a permanent station on
the South Pole, I doubt if we are ever going to be able to get
the kind of information we hoped to get.

REINHARD: I don't think the cause is entirely lost. There
are some semi-isolated communities right here in Alaska which
would provide beautiful study opportunities to a person if he
were willing to sit down in the community and test for every
virus that came through, but it would be a running fight all the
way. You would have to take what the Lord sent you and analyze
it without a hope for control on introduction of viruses.

45

THE ECOLOGY OF ENTEROVIRUSES IN ALASK.-!

Karl R, Reinhard

Division of Research Grants

National Institutes of Health

Bethesda 14, Maryland

.VBSTHACT

The paper considers the following factors; effects of various physical and chemical
environments on enteroviruses, the mechanism of enteroviral infections, the carrier
state in convalescent and immune individuals, and northern ethnic and social pat-
terns. The background information on the natural history of the enteroviruses is
piecemeal. Facts may be drawn from diverse works such as the virology of water
and sewage treatment, interferon and the inapparent persistence of viruses in hosts,
epidemiological episodes of enterovirus infections, the effects of cold climates on
community anfi household hygiene and sanitation, serological and cultural studies
of enterovirus ecology, and morbidity and mortality statistics on northern popula-
tions. The author attempts to discern, through this piecemeal evidence, the major
determinants of enterovirus ecology in northern areas.

The principal objective of this discussion will be the develop-
ment of concepts about the natural history of enteroviral infections
within the context of the general theme which deals with the in-
fluence of cold on host-parasite relationships. To explore ade-
quately the relationship between arctic peoples and their environ-
ments and the enteroviruses, we must borrow from such diverse
fields as virology, sanitary engineering, meteorology, anthropologyj
archaeology, epidemiology, and the more general aspects of natural
sciences. This brief discussion does not allow an exhaustive treat-
ment of all of these, but if some of the larger issues are clarified,
the author's hopes will be fulfilled.

The large Enterovirus group is comprised of particulate agents
which are commonlyrecoverablefrom the human and animal gastro-
intestinal tract. These viruses have a ribonucleic acid core; are
about 28 millimicrons m diameter; are pathogenic for primates,
suckling mice or certain t3qDes of mammalian cell tissue culture;
and are stabilized by cations against thermal inactivation (Com-
mittee on Enteroviruses, 1962). In this group are the polioviruses,

47

REINHARD

Coxsackie A and B viruses, the ECHO (Enteric Cytopathic Human
Origin) viruses, the enteric viruses of animal origin, and the REO
(Respiro- enteric) viruses. Why are the enteroviruses important?
This is well demonstrated by the kinds of diseases they cause:
poliomyelitis, encephalitis, aseptic meningitis, herpangina, pleuro-
dynia, pericarditis, myocarditis, exanthematous fevers, gastro-
enteritis, and upper respiratory disease. In addition, the entero-
viruses cause a variety of systemic diseases which cannot be
distinguished as specific syndromes, and which are usually diagnosed
clinically as fevers of undetermined etiology (FUE). These agents
are responsible for a major proportion of illnesses in children and
infants, and are therefore significant in that respect alone. The
enteroviruses are ubiquitous, and are frequently recovered from
the upper respiratory tract or feces of people who have no overt
disease. The following t3rpes of human immunological enteroviruses
are recognized: poliovirus, three types; Coxsackie A, twenty- three
types; Coxsackie B, six types; ECHO, twenty- six types; and REO,
three types (Rosen, 1960; Committee onEnteroviruses, 1962), There
are, in addition, a number of types of animal origin, particularly
bovine and porcine strains. Of the pathogenetic and infectious
characteristics, more will be related later.

All of our information on viruses emphasizes that these are
obligate parasites; physiologically and metabolically incomplete
organisms that must utilize other living animal or plant cells in
order to persist and propagate. Undoubtedly, virologists will e-
ventually produce non- cellular media composed of essential en-
zymes and metabolites for cultivating viruses. In the natural
realm, however, viruses grow only in living organisms. Their
existence outside of the living host is a passive one. For this rea-
son, considerations of virus ecology are primarily considerations
of host ecology plus physical environmental factors favoring passive
persistence or dissemination of the virus between the propagation
periods in the host. Accordingly, the effects of cold on virus ecology
consist of its effects on availability and suitability of hosts for the
viruses, and its effects on persistence of viruses in the physical
environment outside the host.

From the epidemiological standpoint, there is little reason to
believe that direct effects of cold on the human host have significant

48

ENTEROVIRUSES IN ALASKA

relation to virus ecology. The arctic resident is not a hypothermic
individual. Man lives successfully in the Arctic only because he
maintains a subtropical micro- climate within his clothing and a
temperate climate within his house. The previous speakers have
emphasized the fact that the effects of cold environments are in-
direct; that is, they have a bearing on hygiene, sanitation, social,
and individual activity patterns which affect the passage of viruses
from host to host. I do not wish to minimize the message of the
later discussions of effects of hypothermia on infection. These ex-
periments are important medically from the therapeutic standpoint,
and may also lead to basic information on the metabolic and physio-
logical aspects of cellular and systemic resistance to infection; but
their relation to the natural history of humandisease in arctic areas
is difficult to discern. Exceptions to this statement may be furnished
by the occasional excessive exposure of people to cold by accident
or improvidence, or by the excessive exposure of the upper res-
piratory tract to very cold air from extreme arctic conditions or
overexertion.

The cold climates operate in two, apparently paradoxical manners
on the ecology of the human hosts for viruses. First of all, the
aboriginal population has been forced to settle indiscrete, relatively
small, often widely- separated groups, or, in the past, to live a
migratory life to exploit the ecology of the basic food animals. Only
in certain areas, such as the fish-rich river- valleys of the past,
did boreal population groups cluster closely. The bionomics of food
resources, therefore, led to isolated human communities, often with
discontinuous communication in the colder seasons. This tended to
reduce the speed of dissemination of acute infectious disease be-
tween communities. When isolation was enforced by armed guards
along the trail, as is reputed to have occurred in Northwestern
Alaska during the 1918-19 Influenza Pandemic, villages could escape
epidemic disease. On the other hand, a cold environment causes
close congested living conditions within communities and families.
Consequently, a highly infectious epidemic disease spreads rapidly
through a village once it is established.

These diverse effects of cold climates on human ecology and com-
munication led to another paradox; i.e., the season in which arctic
villagers were more subject to inclementweather was also the time

49

REINHARD

when they were more free of acute infectious disease. Elder resi-
dents of St. Lawrence Island have recounted to the author how, in
the "old days", from fall to spring they could expose themselves to
chill and fatigue, yet never have a "cold", pneumonia, or other acute
infectious disease. However, the first boat of spring arriving from
the mainland would bring with it as invisible cargo acute infectious
disease; particularly upper respiratory disease. Thereafter, sick-
ness would be common on the island until freezup, when cessation
of traffic from the mainland occurred. Similar experiences were
common throughout the Arctic in years past. Many are the accounts
of introduction of disease into arctic villages through the advent of
people from areas with more concentrated population. Repeatedly,
epidemic diseases such as smallpox, measles, influenza, and whoop-
ing cough decimated the population in individual villages, affecting
young and old alike.

The general epidemiological patterns of the past are not generally
applicable to the Arctic today; particularly not in the Western Amer-
ican Arctic. Most villages are in relatively close communication with
urban areas because of a well-developed air transport system.
There is extensive human traffic throughout the year, and now,
therefore, very few villages experience the traditional freedom
from acute infectious disease during the colder seasons. Recent
epidemiological studies of St. Lawrence Island residents have shown
the year-round occurrence of acute infectious disease (Reinhard,
1956). However, this epidemiological shift has not been recognized
widely. Perhaps it has been poorly documented. Therefore, in the
concepts of the large group of medical and public health profession-
als in the more populated southerly areas, the Arctic still is the
place where people are exposed only sporadically to infectious dis-
ease and are hypersusceptible toitwhenitis introduced. The native
arctic population is presumed to be immunologically underdeveloped.

Adherence to these obsolete concepts caused considerable alarm
among those concerned with native health when a severe epidemic of
poliomyelitis occurred in Anchorage and Fairbanks, Alaska in 1953-
54. Previous severe epidemics in Greenland (Fog-Foulsen, 1955)
and the eastern Canadian Arctic (Peart, 1949 ; Adamson, et al., 1949;
Johnsen and Wood, 19 54) had caused great morbidity and mortality
among the native people in those areas. It was feared that similar

50

ENTEROVIRUSES IN ALASKA

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Figure 1. The prevalence of antibodies against the three types of poliovirus in
more than 700 native Alaskan males representing 47 villages in 11 geographic localities.

51

REINHARD

disaster would be caused by extension of the Anchorage epidemic
into the native villages of Alaska. However, only one satellite epi-
demic occurred; the unusual monotypic (Type 3) epidemic on St.
Paul Island (Eklund, 1956), Sporadic cases due to various polio-
virus types occurred in various native vallages, indicating that the
polioviruses were active in those areas. But other than the one
cited, no recognized epidemics of poliomyelitis occurred in native
villages. This intriguing fact led to a series of statistical (Reinhard
and Gibson, 1960) and sero- epidemiological (Reinhard and Gerloff,
1960; Reinhard, Gerloff, and Philip, 1960) studies to determine the
immunological status of Alaskan natives with reference to polio-
myelitis. The data are summarized by a table and two graphs from
a publication currently in press.

Table 1 shows clearly that between 19 50 and 19 54, Alaskan natives
experienced a much lower morbidity rate for poliomyelitis than non-
native Alaskans, The disparity was especially great in comparable
groups under 15 years of age.

Morbidity Rate per 100,000 per annum

Age

! Group

Under

55

years

5

to

9

years

10

to

m

years

15

to

19

years

20

to

24

years

25

to

29

years

30

to

34

years

35

to

39

years

UO

to

44

years

45

to

49

years

50

to

54

years

Native*

Non-Native

29

208

30

221

38

228

71

86

25

66

72

69

72

61

38

49

29

35

0

28

0

13

0

0

55 years and over

Table I. Comparative poliomyelitis morbidity in Alaskan natives ami non- natives,
by five-year age groups, based on reports to the Alaska Department of Health, 1950
to 1954. *Ad justed to standard age group proportion.

Figure 1 shows graphically the prevalence of antibodies against
the three types of poliovirus in more than 700 native Alaskan males
representing 47 villages in 11 geographic localities. The overall
prevalences of antibodies against polioviruses were: Type 1,87 per

52

ENTEROVIRUSES IN ALASKA

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Figure 2. The results of a serological cross-sectional study of the population
of St. Lawrence Islanf,

53

REINHARD

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ENTEROVIRUSES IN ALASKA

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55

REINHARD

cent, Type 2,92 per cent, and Type 3, 62 per cent. Except for cen-
tral Alaska (Fort Yukon) and St. Paul Island, the prevalencies of
T3T)es 1 and 2 were similar throughout. Antibodies against Type 3,
which is generally the rarer of the three known poliovirus type^,
tended to decrease with increasing geographic latitude and conse-
quent decreasing population density.

Figure 2 summarizes the results of a serological cross- sectional
study of the population of St, Lawrence Island. Noticeable in this
study were (1) the high prevalence of Type 2 antibodies in the chil-
dren, (2) the apparent reciprocal relationship between decline in
prevalence of Type 2 antibody and increase in Types 1 and 3, and
(3) the uniformly high prevalence of antibodies to all three types in
the advanced age groups. The results indicated that polioviruses
have been endemic, Type 2 more so than Types 1 and 3, and that this
situation may have existed for many years.

The epidemiological experiences and serological studies showed,
therefore, that Alaskan natives were generally highly immune to
polioviruses. Indeed, Alaskan natives appeared to be much more
experienced with polioviruses than the urban, non- native Alaskan
population. One might speculate, with good reason, that the urban
Alaskan epidemics of 19 53-54 and earlier years might have been de-
rived from endemic foci in the villages. A relatively higher immunity
among natives as compared with non- natives was also found by
Adamson and associates in a study of a poliomyelitis epidemic in
Whitehorse, Y.T. (19 54). Sero- epidemiological studies by Hildes,
Wilt and Stackiw (19 59) indicate current development of immunity
against polioviruses among eastern Canadian natives.

The poliovirus studies stiumlated cultural work on the ecology
of enteroviruses generally (Reinhard, 1961). Table II presents the
results of virological culture of series of stool samples from the
residents of native villages. The samplings for these virological
surveys were taken opportunistically, as facilitated by field work
for the study of other problems. Yet these random surveys yielded
a large number of enteroviral isolates. The Napaskiak-Oscarville
series are particularly significant because of the large variety of
types isolated, the wintertime epidemic of infection, and the long
persistence of these agents in so small a population group. The
samples from Ft. Yukon in September 19 58, and St. Lawrence Island

56

ENTEROVIRUSES IN ALASIC'\

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57

REINHARD

in March 19 59, yielded highly significant results in that epidemic
of Type 3 poliovirus infection were found to occur in individuals who
had received the full course of formalin- inactivated (Salk) vaccine.
None of the individuals were ill when sampled.

Table III, derived from a virological study of pediatric disease
in Anchorage shows that enteroviruses were found in every season
in this urban area in association with overt disease. Because of
overburdened laboratory facilities, virological surveys of healthy
urban residents were not conducted.

Quite unfortunately, time, facilities, and tenure of research
operations did not allow these exploratory studies to mature into
more rigorous studies of the natural history of enteroviral in-
fections in the Arctic. It would have been intriguing and profitable
to monitor selected representative villages closely by virological
and epidemiological means in order to detect the influx, spread,
and persistence of various types of enteroviruses in different geo-
graphic areas, thereby enabling one to determine the kind of clinical
diseases which may have been associated with them. Persistence
of virus in various phases of the physical environment could have
been studied both naturally and experimentally. However, incomplete
as the exploratory studies were, they did demonstrate that entero-
viruses, in surprisingly large variety and concentration, share the
arctic environment with man..,or vice versa.'

Confronted with the certainty of the presence and prevalence of
enteroviruses in Alaska, one is liable to follow the arctic epidemio-
logical cliche and presume that enteroviruses have been introduced
into Alaska perhaps early in this century or even more recently,
and have become established and entrenched in the native population
before the present days of epidemiological surveillance. However,
I would like to present the thesis that enteroviruses might have
accompanied man in his migrations into Alaska from the Asian
continent milleniums ago. Of course, this thesis is beyond proof,
and it is really presented here mostly to combat the arctic epi-
demiological cliche. There are features of the enteroviruses, how-
ever, which suggest strongly that they have been constant com-
panions of the human race since time immemorial.

58

ENTEROVIRUSES IN ALASKA

Parasitological philosophy has developed the concept that the
more successful and probably more ancient host- parasite relation-
ships are those in which the host rarely becomes seriously diseased
by the presence of the parasite, and the parasite is able to persist
in or on the hostdespite tissue or humoral reaction. As a group, the
enteroviruses qualify eminently as successful parasites. These are
the general characteristics of enteroviral infections:

(1) The incidence of infection in an exposed population group is
very high,

(2) Infections are frequently asymptomatic, usually benign or
transitory, rarely debilitating or fatal to the host.

(3) The host frequently becomes a carrier, disseminating virus
for a long period of time, particularly in the feces.

(4) Serologically immune hosts can become reinfected enterically
and disseminate virus,

(5) The various types of viruses are antigenically dissimilar to
the extent that heterologous immunization of the host is slight or
absent, but biological interference between virus types does occur.

In addition, enteroviruses are known to be highly persistent in nat-
ural environment, particularly when mixed with organic matter
stabilized by cations and in a menstruum of low biological activity
such as mightbe caused by low ambient temperature. We shall speak
of each of these points in turn.

There is ample evidence to show that enteroviruses, in general,
are spread rapidly. Eklund and Larson (1959), in their study of the
January 19 54 epidemic of poliomyelitis on St. Paul Island, showed
that the infection had pervaded the community to a major extent with-
in 18 days. Their thorough study led them to believe that the virus
may have been spread, to a large extent, from oropharyngeal secre-
tions, either by droplets disseminated by coughing and sneezing, or
by saliva exchange in the use of common utensils. Bhatt, Brooks,
and Fox (1955), in their detailed viro- epidemiological surveillance
of poliomyelitis infections in Louisiana, concluded that polioviruses

59

REINHARD

spread with such facility that the household becomes the epidemio-
logical unit; that is, if one member of a household is known to have
poliovirus infection, then all members have probably become in-
fected. Banker and Melnick (19 51) firstrecovered Coxsackieviruses
from North Alaskan regions, and Paul et al. (19 51) found a high
prevalence of antibodies against the isolate in sera from residents
of the community yielding the virus, Rosen and associates (19 58a;
19 58b), in their detailed virological study of enteroviruses in a
public child-care hospital, showed that most of the children in the
institution became infected within four weeks after the natural intro-
duction of an enterovirus. These are but few of the many descriptions
in the literature of explosive epidemics of enterovirus infection.
In the Northland, the natural close association of all individuals
within a small community facilitates the rapid dissemination of
highly infectious agents.

Although enteroviruses are highly infectious, the likelihood that
infection will result in severe disease is relatively small. In the
St. Paul Epidemic cited previously, 322 Aleuts were involved and
the epidemiological evidence indicated no previous experience with
Type 3 poliovirus. Of these, less than 2 per cent had severe patho-
logical involvement, and 0.9 per cent died from central paralysis.
Eleven (3.4 per cent) had symptoms of benign (aseptic) meningitis,
and 4,6 per cent had minor, transitory indisposition. The entero-
virus ecological surveys of native villages previously cited, in which
large proportions of the individuals sampled yielded virus, had no
relation to epidemiological episodes of recognizable disease. The
studies of child welfare institutions (Rosen et al., 1958a; 19 58b)
demonstrated high incidenceof enterovirus infection yielded no clear
picture of association of these viruses with specific disease. Some
studies have shown enteroviruses other than polioviruses to be the
cause of epidemics of distressing diseases such as pleurodynia and
herpangina (Huebner et al,, 19 51; Huebner et al., 19 53). The general
situation, however, is that a large proportion of people who become
infected with enteroviruses do not have overt symptoms of disease.
This led in the early years of enterovirus discovery to the term
"viruses in search of disease" or "orphan" viruses (Symposium,
1957), These cliches have now passed out of common usage, but the
fact remains that enteroviruses are capable of utilizing human
populations as habitation without production of a high incidence of

60

ENTEROVIRUSES IN ALASKA

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61

REINHARD

of serious disease. Even the poliovirus, the most dreaded of the
enteroviruses, causes extensive pathology or death in only a very
small proportion of their hosts. This fact aids in the persistence of
enteroviruses; but it also makes their true bionomic status a diffi-
cult study because of the crj^Jtic nature of their activity.

It is characteristic of enterovirus infection that the virus may be
present in large quantities in oropharyngeal secretions as well as
feces and intestinal secretions. Lymphoid tissue and mucosal epi-
thelium may be the site of propagation of virus. Usually, oropharjoi-
geal samples become negative for virus soon after the acute stage
of the disease, A notable exception to this is the case of Coxsackie
A- 21 infection, in which virus has been found in pharnygeal swab
specimens as long as 40 days after onset (Johnson et al., 1962).
In most enterovirus infections the virus may be shed in feces for
several weeks. Table IV shows duration of enteroviral excretion as
demonstrated by a number of workers. Rather consistently, entero-
viruses have been demonstrated inthefecesof a significant propor-
tion of individuals a month to six weeks after onset of enteroviral
infection (Rosen et al., 1958a; 1958b; Johnson et al,, 1962; Huebner
et al., 19 50). This means that the viruses may persist for a month
in people whose serum antibodies have reached immunologically
effective levels. The true duration of gastrointestinal infection is
not known, for recent studies have shown that treatment of feces
with freon, which dissociates antigen- antibody complex, may ex-
tend greatly the period in which virus can be detected in the healthy
or convalescent carrier (Howe, 1962), This prolonged carrier period
makes the isolation of villages in cold climates less effective in the
prevention of the introduction of enteroviruses, and also helps to
maintain the viruses within village and local area populations.

Since the advent of the vaccines, both the formalin- inactivated
and attenuated types against poliomyelitis, it has become quite
apparent that circulating antibodies againstpoliovirusesarenosure
indication of permanent, solid immunity. Serologically- immune in-
dividuals can become reinfected with polio viruses homologous to
the serum antibodies and can excrete virus (Horstmannet al., 1957;
Fox et al., 19 58; Gelfandetal., 1960). These reinfections are usually
limited to the enteric tract and apparently are pathologically be-
nign. However, virulent virus may be excreted for extended periods,

62

ENTEROVIRUSES IN ALASKA

leading to infection of susceptible individuals in the environment.
Our own data, previously cited, on the recovery of Type 3 polio-
virus from previously- immunized children in Ft. Yukon and St.
Lawrence Island, comprise a modest corroboration of this infectious
potential of enteroviruses. Recent work indicates that other entero-
viruses may have this same infectious potential (Henigstet al., 1961).
In view of the prolonged carrier state and potential for enteric in-
fection of serologically- immune individuals, it is not surprising
that enteroviruses can persist in small semi- isolated groups of
arctic residents. The data graphed on Figure 2 can be interpreted
to indicate that endemic poliovirus infection had persisted among
the St. Lawrence Islanders for many years, despite the group's
relatively small current size of 600 individuals and less in previous
decades.

Although enterovirus groups may have some basic antigenic simi-
larity (Halonen et al., 19 59; Melnick, 19 55; Wenner et al., 1956), they
are more dissimilar in antigenic composition in that an antibody
stimulated by infection with one kind of enterovirus may not protect
against infection by other types (Rosen et al., 1958a; 19 58b). The
earliest analyses of enteroviral antigenicity were made of polio-
viruses. Here it was found that Type 2 poliovirus shares antigens
with both Types 1 and 3, and Type 2 antibodies may protect against
infection by Types 1 and 3. But Types 1 and 3 have little antigenic
similarity and antibodies against them rarely cross-protect (Ham-
mon and Ludwig, 19 57; Wilt et al., 19 58; Faro, 19 59). This situation
is even more diversified among the large Coxsackie and ECHO
virus groups. Consequently, serial infections by different entero-
viruses occur. However, simultaneous, dual, or multiple infection
is rare. This is due to the mechanism of non-specific biological
interference mediated, according to recent discovery, by the host-
produced Interferon (Wagner, 1960; Baron and Isaacs, 1961), Thus,
one virus infection may produce a transitory non-specific reaction
of the host which renders the latter insusceptible for a period of
time to other viral infections. These phenomena, the diversity of
antigenicity and biological interference, in combination, tend to ex-
tend the period of time in which an introduced heterologous group
of enteroviruses remain active in a given population group, since
they limit superinfection but allow serial infectionby different tj^Des.

63

REINHARD

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64

ENTEROVIRUSES IN ALASKA

Considering the foregoing facts, it is not surprising that the
few exploratory studies conducted thus far have shown that entero-
viruses are and have been endemic in arctic areas. However, the
situation is not uniform throughout the Arctic; for example, severe
poliomyelitis epidemics have occurred among natives in the eastern
Canadian Arctic, in Greenland, and on St. Paul Island, yet entero-
virus infections have occurred endemically and almost cryptically
in most areas of the western American Arctic. The comparative
isolation of communities in these diverse areas, due to differing
transport and economic factors, may have much to do with the
disparity of their epidemiological experience. It would be highly
desirable to determine how long specific enteroviruses could remain
endemic in single isolated villages. At present, our experimental
approaches and methods may not be equal to the task. We do not
understand sufficiently the role of Interferon in cryptic infection.
We have inadequate information on the pathogenetic mechanism of
the carrier state in convalescents and reinfection of serologically-
immune hosts. Virological cultural methods are not adequate to
recover cryptic or sparse viral flora with qualitative or quanti-
tative reliability.

We will turn from the indirect effects of cold climates on entero-
viral ecology which are mediated by bionomics of the host, and
consider the direct effects on the persistence of viruses in the
physical environment. Salient in this respect are the studies of a
number of workers who have been concerned with the presence of
enteroviruses in sewage and in contaminated water supplies, Clarke
and associates (19 56, 19 59; Taft Report) have shown that Coxsackie
viruses survive long in pure waters with low biotic content and
activity. In temperate waters with high biotic activity and little
or no pollution, the survival of Coxsackie viruses was short. With
increasing organic pollution and consequent decrease in aerobic
biotic activity, the longevity of Coxsackie virus increased greatly.
Chang (cited by Clarke) found that Coxsackie virus stored in 10 per
cent sewage in water at 10° C survived for 440 days. Table V pre-
sents some of the data from several publications dealing with viabil-
ity of enteroviruses in water and sewage, and which serve to illus-
trate the foregoing statements. Experimentally, low ambient tem-
peratures were found to extend the survival of enteroviruses in
natural waters and sewage. The data in Table VI, extracted from

65

REINHARD

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66

ENTEROVIRUSES IN ALASKA

publications of the Robert A. Taft Sanitary Ei^ineering Center,
illustrate this conserving effect of low temperatures on entero-
virus viability. They showthatviability is enhanced further by gross
organic pollution. Type 1 poliovirus and ECHO Type 7 persist two to
three times longer at 4° C than at 28° C in relatively unpolluted
water, but in sewage poliovirus persisted seven times and ECHO- 7
four and a half times longer at the lower temperature. The pre-
serving effect of cold storage was not nearly so marked with Cox-
sackie A-9 and ECHC)-12 as with the firsttwo agents.

A few laboratory studies have given some information on persis-
tence of viability of enterovirus cultures at various temperatures. In
one investigation of viability of viruses in tissue culture held at
37° C,thefollowinghalf-lifeswereobserved:Poliovirus-l,47 hours;
ECHO-1, 24 hours; ECHO-4,18 hours;ECHO-6,40 hours;ECHO-9,19
hours; and ECHO-20,2,5hours(Lehmann-GrubeandSyverton, 19 59),
In another study, ECHO- 20, which was quite short- lived at 37° C, re-
mained fully viable for a year when stored at -20° C (Rosen et al,,
19 58a), Data kindly provided by Dr, H.G. Cramblett (1962) showed
that certain enteroviruses would survive six to eleven times longer
at 20° C, and twenty to sixty times longer at 5° C than they did at
37° C. The presence of cells and organic debris enhance viability.
Although freezing is a common means of long-term preservation of
viruses in laboratory procedures, little is known of a definitive na-
ure of the effects of successive freezing and thawing, Generally,it is
considered deleterious to virus viability but definitive information on
this point gained in controlled experiments would be desirable.

In general, the experimental evidence shows that low ambient
temperatures, high organic content, and low biological activity favor
the long-term persistence of enteroviruses in aquatic menstruum.
These favorable conditions could be provided by the haphazard human
waste disposal methods of many small arctic communities.

Limited experiments have shown that soil absorbed large quanti-
ties of poliovirus; it remained viable for three weeks in natural
soil and for six weeks in previously sterilized soil (Murphy et al.,
19 58). These experiments were carried out at 30° C, but one could
expect much longer survival of enteroviruses in soils at lower en-
vironmental temperatures. The information available is sketchy,

67

REINHARD

and much more extensive experiments should be conducted in which
the wide variance in soil character and chemical composition are
considered.

On the basis of preceding information, we can reasonable expect
that the physical environment in arctic communities should be cap-
able of maintaining viral contamination for extended periods of time.
As a further complication, the low ambient temperatures of most of
the seasons would tend to hinder means of eliminating pollution with
viruses, for it is known that the virucidal activity of halogens is
much reduced at low temperatures (Clarke and Kabler, 19 54; Clarke
et al,, 1956). Clearly, enterovirus activity in arctic communities
presents challenging issues to the microbiologist, epidemiologist,
and sanitary engineer. Imaginative and thorough research would be
required to clarify these issues.

The scope and depth of virological and immunological research
in recent years have forced a revision of many a time- honored epi-
demiological or bionomic concept. The revisions have occasionally
been drastic. In accordance with progress, it would be well to re-
examine critically the current concepts of arctic epidemiology and
change them to conform with the facts of natural history as they
are discerned by more subtle and penetrating scientific approach
and methodology. We are, however, confronted with two conditions
that are conducive to investigational inadequacy. First is the rela-
tively undeveloped condition of arctic biomedical research in that it
has been accustomed to dull, often obsolescent tools, and has
been carried out in piecemeal, often superficial fashion. Second
is the condition of the arctic human community; it is in a state of
rapid change socially, culturally, demographically, and econom-
ically. The scientific approaches must be equal to the task of dis-
cerning the forces which produce the changes as well as recording
the changes quantitatively and qualitatively.

The Arctic offers fabulous opportunities for imaginative, tech-
nologically-solid epidemiological research. The unique facilities of
the Arctic are the small villages with well defined, fairly stable
populations. These villages can serve as convenient, easily com-
prehended population study groups. The semi- isolation offers ex-
cellent opportunity for the controlled study of natural introduction,

68

ENTEROVIRUSES IN ALASKA

pathogenesis, persistence, and disappearance of infectious agents.
The people of these villages are pleasant, cooperative and highly
reliable when their individuality and dignity are respected. They
prize the opportunity to participate intelligently.

In like manner, the individuality of the microbial agent must be
respected. The natural history of enteroviruses is not equivalent to
that of Br. abortus. Each virus group, each bacterial species, as
well as each environment must be approached in a manner free of
preconception in order to derive the utmost in objective information.
There is no doubt that dedicated, persistent application of progres-
sive approaches and methodology to theproblems of arctic diseases
will uncover a large fund of information which would not only contri-
bute in application to the health of arctic residents, but which would
also yield a greater fundamental understanding of the natural history
of diseases.

LITERATURE CITED

1. Adamson, J. D., Malcolm R. Bow, and E. H. Lossing. 1954.

Poliomyelitis in the Yukon. Canad. J. Pub. Health 45: 337-344.

2. Adamson, J. D., J. P. Moody, A. F. W. Peart, R. A. Smillie,

J. C. Wilt, and J. W. Wood. 1949. Poliomyelitis in the Arctic.
Canad. Med. Assoc. J. 61: 339-348.

3. Banker, D. D., and J. L. Melnick. 19 51. Isolation of Coxsackie

virus (C virus) from North Alaskan Eskimos. Am. J. Hyg.
53: 383-390.

4. Baron, S., and A. Isaacs. Interferon and natural recovery from

virus diseases. New Scientist 11: 81-82.

69

REINHARD

5. Bhatt, P. N,, M. Brooks, and J. P. Fox, 1955. Extent of infection

with poliomyelitis virus in household associates of clinical
cases as determined serologically and by virus isolation using
tissue culture methods. Am. J. Hyg. 61: 287-301,

6. Clarke, N, A., G. Berg, P. W. Kabler, and S. L. Chang. Human

enteric viruses in water: source, survival and removability.
Mimeo reporl^Robt, A. Taft. San. Eng. Ctr., USPHS, Cinn.,
Ohio.

7. Clarke, N. A., and S. L. Chang. 1959. Enteric viruses in water,

J. Am. Water Works Assn. 51.

8. Clarke, N. A.,and P. W. Kabler. 19 54. The inactivation of purified

Coxsackie virus in water by chlorine. Am. J, Hyg. 59: 119-127.

9. Clarke, N. A., R. E, Stevenson, and P, W. Kabler. 1956. Survival

of Coxsackie virus in water and sewage. J, Am. Water Works
Assn. 48.

10. Clarke, N. A., R. E. Stevenson, and P. W. Kabler. 1956. The in-

activation of purified Type 3 adenovirus in water by chlorine.
Am. J. Hyg. 64: 314-319.

11. Committee on Enteroviruses , National Cancer Institute,NIH.1962.

Classification of human enteroviruses. Virology 16: 501-504.

12. Eklund, Carl M,, and Carl L. Larson. 1956. Outbreak of Type 3

poliomyelitis on St. Paul Island, Alaska. Am. J. Hyg. 63:
115-126.

13. Faro, S. N. 1959. Recurrentparalytic poliomyelitis. New England

J. Med. 260: 1177-1179.

14. Fog-Poulsen, M. 1955. Poliomyelitis in Greenland. Danish Med.

Bull. (Kbh.) 2: 241-246.

70

ENTEROVIRUSES IN ALASKA

15. Fox, J. P., H. M.Gelfanci,D, R.LeBlanc, and D. F. Rowan. 1958.

The influence of natural and artificially induced immunity on
alimentary infections with poliovirus. Am. J, Pub. Health 48:
1181-1192.

16. Gelfand, H. M., D. R. LeBlanc, J. P. Fox, and D, P. Conwell.

1957. Studies on development of natural immunity to poliomye-
litis in Louisiana, part II. Am, J. Hyg. 65: 367-385.

17. Gelfand, H. M.,D. R.LeBlanc, Louis Potash, D. I. Clemmer, and

J.P. Fox. 1960. The spreadof living attenuated strains of polio-
viruses in two communities in southern Louisiana. J. Pub,
Health 50: 767-778.

18. Halonen, P., L. Rosen, andR, J, Huebner.1959. Homologous and

heterologous complement fixing antibody in persons infected
with ECHO, Coxsackie and poliomyelitis viruses. Proc, Soc.
Exp, Biol. Med. 101: 236-241.

19. Hammon, W. McD., and E. H. Ludwig. 19 57. Possible protective

effect of previous Type 2 infection against paralytic poliomye-
litis due to Type I virus. Am. J. Hyg. 66: 274-280.

20. Henigst, W. W., H. M. Gelfand, D. LeBlanc, and J, P. Fox, 1961.

ECHO virus Type 7 infections in a continuously observed popu-
lation group in southern Louisiana, Am. J, Trop. Med. 10:
759-766,

21. Hildes, J. A., J, C. Wilt, and W. Stackiw. 1959. Neutralizing

viral antibodies in Eastern Arctic Eskimos. Canad, J, Pub.
Health 50: 148-151.

22. Horstmann, Dorothy M., J, R, Paul, J. L. Melnick, and J. V.

Deutsch. 1957. Infection induced by oral administration of
attenuated poliovirus to persons possessing homotypic anti-
body. J. Exp, Med. 106: 159-177.

23. Howe, Howard A. 1962. Detection of poliovirus in feces of

chimpanzees during late convalescence by means of fr eon 113.
Proc. Soc, Exp. Biol, Med. 110:110-113.

71

REINHARD

24. Huebner, R, J., C. Armstrong, E, A, Beeman, and R, M. Cole.

1950. Studies of Coxsackie viruses. JAMA 144: 609-612.

25. Huebner, R. J,, R. M. Cole, E, A. Beeman, J. A. Bell, and

J. H, Peers. 1951. Herpangina- etiological studies of a spe-
cific infectious disease. JAMA 145: 628-633.

26. Huebner, R. J,, J. A. Risser, J. A. Bell, E. A. Beeman, P. M.

Beigelman, and J. C. Strong. 1953. Epidemic pleurodynia in
Texas. New England J. Med. 248: 267-274.

27. Johnsen, H. V., and J. W, Wood. 19 54. An outbreak of poliomye-

litis at Maguse River, N.W.T. Canad. J. Pub. Health 45: 16-17.

28. Johnson, Karl M., Henry H, Bloom, Maurice A. Mufson, and

Robert M. Chanock. 1962. Acute respiratory disease associated
with Coxsackie A-21 virus infection. I. Incidence in military
personnel: observations in a recruit population, JAMA 179:
112-119.

29. Lehman-Grube,F., and J.T. Syverton. 19 59. Thermal stability of

ECHO viruses in cell culture medium. Am. J. Hyg. 69:161-165.

30. Melnick, J. L. 1955. Antigenic crossing within poliovirus types.

Proc. Soc. Exp. Biol. Med. 89: 131-133.

31. Murphy, Wm. H., Jr., O. R. Eylar,E.L. Schmidt, and J. T. Sy-

verton. 1958. Absorption and translocation of mammalian vi-
ruses by plants. Virology 6: 612-622.

32. Paul, J. R., J. T, Riordan, and L. M. Kraft. 1951. Serological

epidemiology: antibody patterns in North Alaskan Eskimos. J.
Immunol. 66: 695-713.

33. Peart, A. F. W. 1949. An outbreak of poliomyelitis in Canadian

Eskimos in wintertime. Canad. J. Pub. Health 40: 405-417.

72

ENTEROVIRUSES IN ALASKA

34. Reinhard, Karl R. 1956. Demographic and preliminary epidemi-

ological studies of the people of St. Lawrence Island, Alaska.
Proc.Med. and Piiblic Health Sect., 7th Alaska Science Confer-
ence,Juneau. (Not printed in toto. Copies may be requested.)

35. Reinhard, Karl R. Notes on the ecology of enteroviruses in west-

ern arctic and subarctic regions of North America. XIII Nor-
thern Conf. on Pathol. Microbiol., Turku, Finland, and 12th
Alaska Sci. Conf., 1961. To be published in JAMA.

36. Reinhard, Karl R., and R.K.Gerloff. 1960. Immunity toward polio-

virus among Alaska natives .II. A serologic survey of 47 native
communities of western and northern Alaska. Am. J. Hyg. 72:
298-307.

37. Reinhard, Karl R., R. K.Gerloff, and Robert N. Philip.1960. Im-

munity toward poliovirus amor^ Alaska natives.III. A study of
naturally and artificially acquired antibodies against poliovirus
among residents of two Bering Sea communities. Am. J, Hyg.
72: 308-320.

38. Reinhard, Karl R., and Harry V. Gibson. 1960. Immunity toward

poliovirus among Alaska natives. I. Comparative reported in-
cidence of clinical poliomyelitis in Alaska natives and non-
native residents. Am. J. Hyg. 72: 289-297.

39. Rosen, Leon. 1960. Serologic grouping of reoviruses by hemag-

glutination-inhibition. Am. J. Hyg. 71: 242-249.

40. Rosen, Leon, James H, Johnson, Robert J. Huebner,and Joseph

A. Bell. 1958a. Observations on a newly recognized ECHO virus
and a description of an outbreak in a nursery.Am.J. Hyg. 67:
300-310.

41. Rosen, Leon, Joseph A. Bell, and Robert J. Huebner. 19 58b. A

longitudinal study of enter oviral infections in young children,
Proc. of 6th IntLCong. on Tropical Med. and Malaria. V: 5-13.

42. Symposium. 1957. Viruses in search of disease. Ann. N.Y. Acad.

Sci. 67: 209-446.

73

REINHARD

43. Wagner, R. R,, and A. H, Levy, 1960. Biochemical aspects of

microbial pathogenicity. Ann. N. Y. Acad. Sci. 88: 1021-1318.

44. Wenner, H. A,,P. Kamitsuka,andM.Lenahan.l956, A compara-

tive study of TypeIIpoliomyelitisviruses.il. Antigenic differ-
ences relating to 18 Type 11 strains, J. Immunol, 77: 220-231,

45. Wilt, J. C, W. Stackiw, J. A. Hildes, E. Taylor, and A. J. W.

Alcock. 1958. The immune status of poliomyelitis patients,
Canad. Med. Assoc. J, 78: 32-34,

DISCUSSION

MARCUS: I have great fear of the Jovian wrath that falls
on people who ask questions in the field of enterovirus and polio,
and I have to confess, in asking this question, I am very ig-
norant on the whole subject. I am a little confused on a point
here, though. You insinuated, I think. Dr. Reinhard, that a person
who has had an enteroviral disease can recover from it and still
excrete this virus as a carrier?

REINHARD: For a period of several months. We don't know
actually how long,

MARCUS: This is an active infection, but it is somewhat
similar in nature to what is done in immunizing with oral virus
against an enterovirus. If nobody is looking, 1 will say it, polio-
virus, I read in Readers Digest and Time that when you immunize
by this means, you destroy the excretion of virus, so you don't
pollute the water supply which comes from the sewage that you
don't get back from the tap,

REINHARD: I wonder how well some of these concepts will
stand the test of time?

MARCUS: I see. In other words, you feel that perhaps some of

74

ENTEROVIRUSES IN ALASKA

the statements that are made are not based on thoroughly tested
data?

SULKIN: Not exactly. Actually, while there might be a per-
sistence of the attenuated strain, the concept is that the very
presence of the attenuated strain would prevent wild t5T)e virus
from invading the host. So, you are dealing with two different
agents; one interfering with the invasion of the host by the other.

REINHARD: You are speaking about biological interference?

SULKIN: Yes, this is precisely the mechanism which, it is
hoped, will operate as a result of oral immunization,

MARCUS: And when the excretion of the avirulent strain
ceases, you would say, then, the individual is susceptible once
again?

WALKER: He is considerably more resistant to infection,
even in the gut.

MARCUS: Is this true of the other enteroviruses?

WALKER: They can be reinfected with other antigenic strains,
but they must be more resistant.

MITCHELL: If you had virus particles of the so-called attenu-
ating type and the so-called wild type, equal in number, and
you placed them before the susceptible cells, which would the
cell choose? Would it choose the attenuated, or is the attenuated
virus more aggressive than the wild type?

SULKIN: The point is, by introducing attenuated virus, that
attenuated virus will replicate in the most desirable part of
the body for that organism, which happens to be the alimentary
canal, so that you no longer have equal numbers.

MITCHELL: It is already there and usually has the advan-
tage of an increased population. A good big man can always whip
a small man.

75

REINHARD

SULKIN: There are two factors to consider. One is the repli-
cation of attenuated virus, and the other is that it is conferring
local immunity so that small doses of a wild type virus cannot
gain a foothold in the host.

REINHARD: I'd like to propose an additional thought. You
remember that in the St. Lawrence study the graphs of Types
1, 2, and 3 poliovirus antibodies indicated interference between
the three types in the chronology of initial infection and immuno-
genesis. (See Figure 2 in manuscript.) Secondarily, there was
a fifteen to twenty year periodicity between resurgence in pre-
valence of antibodies, correlated with increase in titers. I wish
we had more extensive data to test the validity of these phe-
nomena. I am wondering, however, whether these graphs do
not give a true picture of the natural decline of antibodies in
an endemically exposed population to the point where reinfection
and reinforcement of antibodies takes place.

METCALF: Keeping in mind the so-called doctrine of original
antigenic sin^ and the experiences gained with influenza viruses,
is there anything in this data which could or might be interpreted
as heterological boostering of one type by another? Does that
happen in the polio group?

REINHARD: Not in type-specific antibodies. Does anybody
else have any information?

SULKIN: Heterotypic antibody responses have been observed
in vaccinated individuals as well as following natural infection
with poliovirus. Some time ago Sabin^ described the transitory
appearance of Type II neutralizing antibody in patients infected
with Type I poliovirus and suggested that these two types shared
a common antigen.

REINHARD: This would be due to actually shared antigens.

1 Davenport, F. M., A. V. Hennessy, and T. Francis, Jr. 1953. J. Exp. Med. 98: 641-656.

2 J. Exp. Med. 96: 99-106. 1952.

76

ENTEROVIRUSES IN ALASKA

SULKIN: Yes. In the report*^ describing the immunologic
classification of polioviruses into three distinct types it was
indicated that they did not share a common antigen. However,
other studies, such as that just referred to by Sabin, would
indicate that common antigens do exist. A soluble complement-
fixing antigen crosses in the GF test with heterotypic polio-
myelitis antibodies.

MET CALF: There are serum neutralization tests. I wonder
if there is any danger of a crossing here which you might miss
interpreting?

REINHARD: They were remarkably specific; however, in a
population cross section study like this, in the adult group,
where antibodies against all three types are present, it would
be difficult to unscramble the Type antibody reactions. But
note, however, the early prevalence of Type 2 reactions and
the later rise of Types 1 and 3 antibodies. The peak prevalence
of Type 3 antibody was hinted around twenty some years of age.
Type 1 was most prevalent at age twenty or so, as I remem-
ber. Of course, the peak prevelance of Type 2 antibodies was
in children.

SULKIN: Isn't this quite similar to the situation reported
several years ago by Hektoen and Boor when they studied simul-
taneous multiple immunization of rabbits with a variety of anti-
gens? I recall there was a remarkable response to a blunder-
buss vaccine containing as many as 35 antigens, although some
"crowding out" effect was observed, that is, failure of antibody
to develop to one or more antigens included in the vaccine. This
may account, at least in part, for the observation to which you
refer,

REINHARD: Yes.

MARROW: I should like to make one observation as a physician;

3 National Foundation Committee. 1951. Am. J. Hyg. 54: 191-204.

4 J. Infect. Dis. 48: 588-594. 1931.

77

REINHARD

the emphasis has been upon natives as contrasted to Caucasian
groups, but in a similar cultural environment in isolated geo-
logical survey parties in military and other construction groups,
there is the same phenomenon. I have been clinically associated
with medicine for fifteen years in this area. The fact is that
when, on the rare occasion, the Caucasian lives in the same
socio-economic status as the native, the pattern is very similar
to that you describe.

REINHARD: Yes, right. And if you compare, say, the Alaska
natives with the residents of Cairo, the situation is quite similar.

CAMPBELL: I want to bring up one question relative to the
persistence of virus in relation to organic composition or the
amount of organic material. I was just wondering whether this
is actually a persistence or perhaps a growing; is it static or
dynamic ?

REINHARD: The organism is static. These were viability
tests.

CAMPBELL: There is something in the high organic environ-
ment that stabilizes it.

REINHARD: Evidently, We could look back, for instance, to
the protective action of colloids. In organic contamination by
sewage, you probably have a few cations. Another factor, too.
Dr. Campbell, is the fact that particularly in sewage, where there
is an excessive amount of organic content, the aerobic biological
activity is decreased. This is evidently a conditioning factor,
for where there is a great amount of aerobic biologic activity,
the virus just does not last very long, but when the environ-
ment becomes anaerobic or abiotic, then the virus may last
a long while. For instance, in distilled water, the virus via-
bility is greatly increased. Does that answer your question?

CAMPBELL: Not entirely, no. You really have to do an ex-
periment, of course, and have those cells present.

REINHARD: The people at Sanitary Engineering Center were

78

ENTEROVIRUSES IN ALASKA

dealing with water in which there were actual active organisms,
and they were measuring organic content, biological activity,
and virus viability very closely.

WALKER: This stabilizing effect of proteins and certain ions
is a fairly well known one that is used and taken advantage of
regularly in the laboratory for stabilization of these and other
viruses, and is used to prolong storage of viruses,

NUNGESTER: And bacteria.

REINHARD: Yes.

BERRY: Thank you. Dr. Reinhard. I suppose as moderator
of this session, it is up to me to summarize everything that
has been said this morning. I would not attempt it, really, but
I would like to say that on the basis of this opening session,
it seems rather clear that there is no evidence whatsoever of
any significance that would indicate that arctic environment
is particularly predisposing to human diseases of an infectious
nature. There are certain unique situations that ari se as a re-
sult of the environment that alter housing and in certain groups
of people, the natives particularly, the diet. Their prior ex-
perience with various microbic agents, and actually these would
apply not only in the Arctic, but probably in all other environ-
ments and in all other parts of the earth, shows rather clearly
that in the human being, cold may or may not be stressful. In
fact, the measure of man's adaptation to the arctic environ-
ment is his ability to live with minimum stress, and I assume
that with minimum stress there is a minimum change and response
to bacterial and viral disease.

79

OPENING REMARKS ON PROBLEMS OF IMMUNIZATION IN
STRESSED ANIMALS

Dan H. Campbell

Department of Chemistry

California Institute of Technology

Pasadena, California

First I will orient you with our own interest which began about
when Dr. Larry Irving was settir^ up a laboratory at Pt. Barrow.
We agreed that something should be done on immunological re-
search and biochemical problems. From this first investigation it
became obvious that many problems were apparent and should be
investigated.

One of the first things that interested me was the arctic ground
squirrel whose body temperature goes down to near freezing when
it hibernates in the winter. If it gets a little colder, the animal
wakes up and shivers and then goes back to sleep. This is a rather
fantastic situation, but it might be representative of the extremes
between hypothermic and normal conditions. When the squirrel is
active, he makes up for the time he sleeps in various ways, so that
his metabolism is probably a little abnormal both in the summer
and in the winter. We first began to study the blood patterns in
normal and immunized animals to see if they would produce anti-
bodies. The idea then was to go through the gamut of tests. Most
of these have not yet been completed.

We studied antibody formation, and to some extent, persistence
or fate of antigens and antibodies. Most of this work has never been
published. One of the first interesting problems which impressed
me was that in the winter time, the squirrel's blood didn't clot.
At first this was a nuisance, because we wanted the blood to clot
for serum studies. This was significant in that as the body tempera-
ture decreased, the clotting time increased, and in the hibernating
animal, there was practically no clot formation. Back at the

CAMPBELL

California Institute of Technology, Dr. Irving and I continued this
work using rabbits. Dr. Trapani and Dr. Sutherland joined our team
there.

At the Galifcrnia Institute, we found that when we kept the rab-
bits at about -20° C, their body temperature didn't go down, ant
therefore, one couldn't call them hypothermic animals. In order
to keep rabbits alive in a "naked" state, their hair must be re-
moved slowly. In our experiment, a strip the width of a safety
razor blade was removed about every three or four days over a
period of weeks until the rabbit was shaved. If the rabbit was
previously conditioned to -20° C for about two weeks, he would
survive and live happily at -20° C, while an unconditioned shaved
rabbit would die within 24 to 28 hours. We studied these conditioned
rabbits for antibody formation, half-life of antigen, and particularly
the half- life of antibodies.

While in the Arctic, we also carried out a study of blood types,
including Rh, in several Eskimo villages. Following thiSj some
preliminary stuaies were made on lemmings. They turned out to be
very poor antibody formers, which indicates for the first time that
the metabolic state of the animal plays a very important role in
immune mechanisms. In the cold animal stored in a cold box, anti-
body disappeared very rapialy, which is probablj^ due to the rapid
protein turnover. In a normal rabbit, this process was about 1/3 as
fast.

If we put antigen in the arctic ground squirrels about the be-
ginning of hibernation, it would be there at the end of hibernation
just before they became active, and the same thing was true with
passively transferred antibody. If either antigen or antibiXiy was
injected during the summertime when they were active, it wruL^
disappear very rapidly. Rabbits were active in the coi<j ana their
metabolism was high; under these conditions the protein turned
over at a very fast rate. When antibody was injected, it rapidly
disappeared. Antigen behaves in somewhat the same way, although
little study has been made on this aspect as yet.

These experiments brought to mind some survival studies in
Sonoma Pass that I heard about in which there occurrev^ a very

82

IMMUNIZATION OF STRESSED ANIMALS

0 norma
serums

Midwest

2 serums I serum

Human serums from
Ladd Field

Figure 1. Electropheretic patterns of serums from active (normal) and hibernating
Arctic squirrels.

Normol

Hibernoting squirrels

Figure 2. Electropheretic patterns of human serums obtained during late winter from
Chicago area and Fairbanks area.

83

CAMPBELL

high incidence of respiratory diseases among the Marines in spite
of immunization. We suspect that lack of resistance was due to the
rapid metabolic turnover of Ab protein.

Shown in Figure 1 is a regular elect rophoretic pattern of awake
and hibernating squirrels. The hibernating squirrels show a higher
amount of protein than the awake squirrels which may reflect a
loss of blood volume during hibernation. Note the fast- moving com-
ponent which developed in a lot of the hibernating squirrels. The
fibrinogen, if it is there, would probably be in the beta component.
It is very difficult to interpret these data because of the extreme
abnormality of the physiological state of the animal.

Figure 2 shows a comparison of serums taken in January from
healthy volunteers at Ladd Field in Alaska. A peculiar thing was
that the beta anomaly, usually occurring inthe descending boundary
of practically all normal human serums in the temperate zones,
was absent. I'm not sure that this is significant, but this beta
boundary disturbance is in some way associated with lipoproteins.
In the summer time most serums showed this component. In the
first studies, it was concluded that serums from these men did have
a higher clotting time in winter than in the summer. I never saw
the actual data which should be more carefully studied.

Figure 3 presents data on the retention of antigen in the livers
of normal and immunized rabbits. I have included it to show that
antigen in the normal rabbit will persist for a long period of time
in the liver, and can be detected up to 350 days after the last
injection, thus making it a baseline. Recent work by Jerislav
indicates that the antigen persists a lot longer in hibernating
squirrels.

Experiments designed to determine if high altitude and low
temperature had any effect on experimental asthma in guinea pigs
were conducted by Dr. Heimlich and Dr. Trapani at the White
Mountain laboratory. The results indicated that if guinea pigs were
sensitized at room temperature and sea level, they were quite
resistant to challenge. Subsequent studies suggested that this re-
sistance was apparently a stress phenomenon, and if the guinea
pigs were allowed to adapt for a few weeks, they reacted like

84

IMMUNIZATION OF STRESSED ANIMALS

100 I

0.1

• OU

'A. *

"♦----<

"~-6^

-->^^

L_^^ \ L

10 20 40 70

70 90 110 150 190 230 270 310 350
DAYS AFTER LAST INJECTION

Figure 3. Semilog plot of retention in perfused liver tissue of single injection of 50 mg
of S"^^SA (A); single injection of 50 mgS'^^KLH (B) ; 9 injections of 10 mg each of S^^bsA
(C); 9 injections of 10 mg each of S"^^KLH (D). Each point represents average value for
3-5 rabbits. Center of circles indicates mean of distribution, indicated by the arrows.
(From Garvey and Campbell, Jour. Exp. Med., 10 5, 361. 1957.)

animals at a higher temperature and at sea level. Apparently
the answer involved hypersecretion of corticosteroids.

There are many practical problems dealing with the relation
of cold stress of adaptation to immune mechanisms. Many factors
other than metabolism must be involved. For example, skin tests
may depend to some extent on the state of peripheral capillary cir-
culation, and immune responses in general will depend upon the pre-
vious history of the subject. In finishing, I wish to emphasize the
importance of Dr. Viereck's statement; namely, that the physio-
logical state of an animal must be determined and not assumed.
Furthermore, there is a great difference between stress, adaptation,
and normal states.

85

CAMPBELL
DISCUSSION

SULKIN: In the antigen decay experiment with the Arctic
ground squiin^elj I wonder if you have attempted such an ex-
periment at a time when the animal does not ordinarily go in-
to hibernation? In other words, have you measured antigen at
another season of the year, such as the summer time?

CAMPBELL: Yes, even while they are active both antigen
and antibodies disappear very rapidly in the summer time. It
is a problem because squirrels do become active a while be-
fore they come out of their bui'rows, so actually you have to
dig them out. We did have an artificial setup at Point Barrow,
finally. It took us about two years to learn how to get them to
hibernate. The first two years they all died; the next year some-
body got some ambition and hauled down a few tons of sand from
the Mead River where these squirrels live. The sand was put
in a wire enclosure so the squirrels couldn't get out, and we
could go out to the pen and dig them up. They become active
for a couple of weeks before they come out of hibernation,

MONCRIEF: Does the inability to form antibody also imply
an increased destruction of antigen?

CAMPBELL: I can't answer that because they go hand in hand.
Now, in the production of antibody, antigen is destroyed. This
is a fact. Now, whether antigen has to be destroyed, I don't know.
Supposing it was not destroyed. If it is not broken down, it is
not antigenic. We think that antigen is broken down into par-
ticles about the size of templates, and that antibody formation
is just modified biosynthesis of gamma globulin by the RNA,
because it is always associated with the soluble RNA; we know
the soluble RNA does turn over during protein synthesis, and
the more protein being synthesized, and the more active the
cell is, the greater the rate cf this turnover. Well, when it
breaks down and turns over, then this template, or some of
it, may be lost. This is a reflection of the rapid protein syn-
thesis, and some of these fragments are always being secreted

86

IMMUNIZATION OF STRESSED ANIMALS

by the cell. There is a possibility, if antigen acted as a tem-
plate, that it may momentarily be associated with the antibody,
but actually would immediately disassociate, or disassociate
soon after it got out of the cell, probably inside it. There is no
question but that antigen breakdown and loss is associated with
antibody formation.

MONCRIEF: Not necessarily the other way around?

CAMPBELL: No.

MONCRIEF: Do you know anything about the diet of the hiber-
nating animal with respect to protein, carbohydrate, and fat
composition that these people on Ladd Field worked with when
they measured clotting time?

CAMPBELL: The hibernating animal, of course, isn't eating.

MONCRIEF: Prior to his going into hibernation.

CAMPBELL: He is in pretty good shape under natural con-
ditions. I don't know what all they do eat, besides berries and
roots, and so on.

MONCRIEF: The only reason I ask, is that a very peculiar
observation came up about a year ago; Walter Blum was put-
ting patients on starvation diets. These patients were placed
on a completely carbohydrate-free diet, nothing but fat and pro-
tein; he drew blood samples from these patients and placed them
in the freezing portion of the ice box, and a few weeks later
when I happened to be visiting him, he took samples out to show
them to me. He pulled about twenty samples of blood out of
the refrigerator, six of which were from patients on this diet.
The other fourteen were frozen solid, but the six on this diet
were still completely liquid. He later analyzed these for every-
thing he could consider possible and found nothing to be ab-
normal in the blood except an elevation of the non- ester if ied
fatty acids. Even serum osmolarity was the same.

CAMPBELL: I forgot to mention, these sera that don't clot,

87

CAMPBELL

and even the rabbit sera that clot very poorly, are very low in
complement. Complement has always been associated with clot-
ting, I don't know just what the connection is, but the French
used to consider prothrombin. Well, that turned out to be not
true, but these hibernating squirrels have practically no com-
plement, and in rabbits' sera that have been stored for a while,
the complement goes down; it goes along with the blood clotting.
It would be interesting to study the complement titer which might
go down very rapidly in some of these patients where the blood
clotting goes down after hypothermia.

BLAIR: Yes, this occurs only at fairly deep levels of hypo-
thermia, and I think it is important to bring this out. It has been
traced to reduction of platelets, and this is probably due to trap-
ping in the capillaries. The periods of hypothermia at this level
are so short that it is quite unlikely that anjrthing happens to
the fundamental mechanisms that involve the clotting. Whether
there is any actual alteration in the protein response is probably
unlikely for these short periods.

CAMPBELL: But even if the complement was reduced for a
short period, it might play a role.

TRAPANI: Are any of these serum changes detectable before
hibernation, or just following hibernation?

CAMPBELL: This is the problem, of course, and, let's see,
maybe Dr. Tunevall could tell us about this work in Sweden on
the polypeptide from the brown fat. I think they have been work-
ing on it in the porcupine. This is the problem that really in-
trigues me. The brown fat evolves during hibernation; and even
the shaved rabbits will begin to show a little brown fat. This
has always been associated with hibernation. If you could iso-
late a polypeptide, it would be the perfect anesthetic. This was
realized, I think, quite a few years ago.

88

ENVIRONMENTAL EXTREMES AND ENDOCRINE
RELATIONSHIPS IN ANTIBODY FORMATION^

Ignatius L. Trapani

Department of Experimental Immunology

National Jewish Hospital

Denver 6, Colorado

ABSTRACT

This paper concerns antibody production and decay in animals exposed to environ-
mental extremes of low temperature (-15° C) or high altitude (12,500 ft. and 14,500 ft.),
or in which an imbalance in endocrine activity has been produced. It soon became
apparent from our earlier studies that it was not possible to investigate the effect
of environmental stress without implicating physiological alterations which might
occur and thus influence the synthesis and metabolism of antibody. Our studies were
extended to include animals which were in endocrine imbalance in an attempt to iso-
late one of several physiological factors which might be altered under conditions
of stress. The study of these physiological factors, their inter- relationships, and
their influence on antibody synthesis and decay is expressed in the term immuno-
physiology. The study of adrenalectomized, thyroxin- treated, and surgically thyroid-
ectomized animals helps to explain the immune response of cold- exposed animals
which exhibit an increased thyroid activity. The immune response can be thought
of as being composed of two processes; antibody production and antibody decay, oc-
curing simultaneously, but not necessarily at the same rate. For example, a net
increase in circulating antibody might arise from (a) an unchanged production asso-
ciated with a decreased rate of decay; (b) an increased production associated with
a decreased rate of decay; or (c) a decreased production associated with a marked
decrease in decay rate.

The discussion I wish to present concerns antibody production
and decay in animals exposed to environmental extremes of low
temperature (-15° C) or high altitude (12,500 ft. and 14,150 ft.), or
in which an imbalance in endocrine activity has been produced
(Trapani, 19 57, 1960, 1961; Trapani and Campbell, 1959; Trapani,
Lein, and Campbell, 1959a and 1959b; Trapani and Jordan, 1962).

1 These studies were aided by Contract Nonr 3545(00) (NR 102-573) between the Office
of Naval Research, Department of the Navy, and the National Jewish Hospital at Denver.

89

TRAPANI

The experimental animal was the rabbit; the antigen- antibody
system used was bovine serum albumin (BSA) and anti-BSA pre-
cipitins. The low temperature studies were done in an especially
constructed cold box having a capacity of 10 rabbits. Adequate pro-
vision was made for lighting and fresh air without drafts, and the
animals were kept on wire mesh in individual cages. Water was
changed four times a day because of freezing and standard food was
provided ad libitum. Animals exposed to high altitude were main-
tained at the Barcroft Laboratory (altitude =12,500 ft.) of the White
Mountain High Altitude Station in California, or, in more recent
studies, at the Summit Laboratory (altitude =14,150 ft.) of the Inter-
University High Altitude Laboratory, Mount Evans, Colorado.

Much of the work discussed here was done in collaboration with
Professor Dan H, Campbell at the California Institute of Technology.

It became apparent from our earlier studies on the immune re-
sponse that it was not possible to investigate the effect of environ-
mental stress without implicating physiological alterations which
might occur and thus influence the synthesis and metabolism of
antibody. Our initial studies on the effect of low environmental
temperature and high altitude were extended to include experiments
on animals which were in endocrine imbalance in an attempt to
isolate one of several physiological factors which might be altered
under conditions of stress. The study of these physiological factors,
their inter- relationships, and their influence on antibody synthesis
and decay is expressed in the term immunophysiology.

The purpose of my discussion is not necessarily oriented toward
the elucidation of all of theproblemsrelatingto antibody formation,
but rather to emphasize some of its complexities and some of the
secondary factors which influence the immune response. The topic
of interest, for the moment, is not concerned with speculations re-
lating to cellular mechanisms per se which maybe responsible for,
or may participate in, the synthesis of a particular protein by
antibody forming cells. Rather, I will attempt to elucidate the role
of certain physiological factors which influence the basic synthetic
mechanisms involved in our test system.

Buried in the literature of the exploits of people exposed, either

90

ANTroODY FORMATION

by design or accident, to environmental extremes of low tempera-
ture or high altitude are accounts of changes in resistance to in-
fection and disease. However, little quantitative experimental work
on the immune response has been done under controlled environ-
mental conditions. Some of our first studies were those utilizing
rabbits acclimatized to an environmental temperature of -15° C,
or else exposed to an altitude of 12,500 ft. at the Bar croft Laboratory
of the White Mountain High Altitude Station in California.

A first approach to the investigation of the immune response was
the study of protein turnover, by passive immunization techniques.
In this procedure, homologous gamma globulin containing specific
antibody is injected intravenously into recipient animals. The
animals are bled periodically, and the concentration of serum anti-
body estimated by quantitative micro- precipitin analysis (Lanni,
Dillon, and Beard, 1950). The results are plotted semi- logarith-
mically as the percentage of the injected dose remaining in the
circulation versus time. The slope of the linear portion of the curve
(assumed to be steady state loss of antibody) is calculated by the
method of least squares, extrapolated back to zero time, and the
half-life of the injected antibody calculated from that point.

The half-life of passively administered homologous antibody for
controls, cold- exposed, and high- altitude adapted animals was:
4.7 ± 0.2, 3.4 ± 0.2, and 4.5 ± 0.2 days, respectively. The cold ex-
posed animals have a significantly increased rate of antibody turn-
over, while those at high altitude do not.

Rabbits exposed to -15° C for 10 weeks and clipped of hair during
the latter half of that period were actively immunized by the sub-
cutaneous injection of BSA (10 mg per kg body weight) in Freund's
adjuvant. Levels of circulating antibody were followed for a period
of 52 weeks (Fig. 1). Rabbits maintained at room temperature
and treated in the same manner were used as controls.

The level of circulating antibody in the cold exposed group in-
creased at a slower rate than in the control group and approached
control levels at approximately 14 weeks. There was then a decline
in both groups, but at different rates, throughout the remainder of
the experimental period, so that by the end of 52 weeks the level

91

TRAPANI

O CONTROLS

• GOLD-EXPOSED

0 4 8 12 16 20 24 28 32 36 40 44 48 52

TIME (WEEKS)

Figure 1. Circulating antibody levels in rabbits immunized with BSA plus Freund's
adjuvent (10 mg BSA per kg body wt) at time zero. The cold exposed group, kept at an
environmental temperature of -15° C, was progressively clipped during the first 8 weeks
and immunized after a 10-week exposure,

of circulating antibody in the cold exposed group was approximately
50 per cent of that in the control group.

If these data alone are considered, it would appear that the cold
exposed animal does not synthesize antibody as well as the non-
exposed animal. However, it must be remembered that curves of
this type represent not only antibody production, but also antibody
decay. Thus, part of the difference maybe attributed to an increase
in protein degradation, as inferred from passive antibody decay
studies.

Since cold exposed animals have been shown to have increased
thyroid activity, it seemed important to investigate the activity of
the thyroid gland on the immune response. Studies were based on
the a priori assumption that hj^Der activity of the thyroid contributed
to the immune response of cold exposed animals. The interesting
and complicating fact is that eventhough the assumption may be va-
lid, the experimental observations did not answer completely our
questions.

92

ANTIBODY FORMATION

100

o

UJ

z
<

>■
a
o
ffl

2 -

to.5 = 4.7 DAYS
to.5-3.8 DAYS

O = THYROIDECTOMIZED

• = CONTROLS

A •= THYROXIN-TREATED

4 6 8 10

TIME (DAYS)

12

Figure 2. Passive antibody decay in surgically thyroidectomized, thyroxin-treated
and untreated control rabbits.

93

TRAPANI

A study was made of passive and active immunization in rabbits
which were either surgically thyroidectomized or treated with thy-
roxin. The half- life of passively administered antibody in thyroidec-
tomized, thyroxin- treated, and controls (Fig. 2) was: 8.9* 0.4, 3.8 ±
0.2, and 4.7 ± 0.2 days, respectively.

Similarly prepared groups of rabbits were actively immunized
with BSA (10 mg per kg body weight) in Freund's adjuvant. Figure
3 displays the data obtained from the experiment. The circulating
antibody level in the thyroidectomized group rises gradually and ap-
proximates the control level at about 14 weeks after immunization.
The thyroxin- treated group, on the other hand, not only had an in-
itially higher level of circulating antibody, but also displayed
measurably increased amounts of antibody 3 days after immuniza-
tion. This early response was not present in either of the other
groups. After the initial high level of circulating antibody in the
thyroxin- treated group, there was a decline to levels below those of
controls, and then a second increase. Thus, there is a similarity
between hyperthyroid and cold exposed animals in terms of an in-
creased rate of proteinturnover. However, the net immune response
after active immunization shows little similarity between hyper-
thyroid and cold exposed rabbits.

Since experiments concerned with thyroid activity did not clarify
the results obtained with cold exposed animals, we next investigated
the role of the adrenal gland on the immune response. One reason
for this approach was the observationthathigh- altitude acclimatized
rabbits showed an increased immune response, and animals under
these conditions exhibit an increased adrenal activity.

Figure 4 shows the results of active immunization of rabbits ac-
climatized to 14,150 ft. at the Summit Laboratory of the Inter-
University High Altitude Laboratory at Mount Evans, Colorado,
Immunization was accomplished with a single intravenous injection
of 10 mg BSA per kg body weight. The animals were bled periodi-
cally for the next 5 weeks and then returned to Denver (altitude =
5,280 ft.) where further samples were taken. After circulating anti-
body had reached low levels, the animals were given a secondary
intravenous challenge with 10 mg BSA per kg body weight. Sixteen
weeks later, a third immunization dose was given in the same

94

ANTffiODY FORMATION

1000
700
500

300

T i \ \ r

1 r

^

3

100

(E

111

70

-J

50

b

V

z

30

>-

o

o

a

Z

10

<'

Oi

/

4.

5

• -CONTROLS

O - THYROIDECTOMIZeO

A -THYROXIN-TREATEO

5 6 7 8

TIME (WEEKS)

10

12 13

Figure 3. Circulating antibody levels in surgically thyroidectomized, thyroxin-
treated, and untreated control rabbits immunized with BSA plus Freund's adjuvant
(10 mg BSA per kg body wt) at time zero.

manner. Maximum utilization of the experiment could be had in this
way, and information relative to the time course of re-adaption to
the lower altitude obtained.

After primary injection, the animals at high altitude had levels
of circulating antibody approximately 60 per cent higher than con-
trols, and were significantly different. After the secondary challenge,
the group previously exposed to high altitude had levels of circulating
antibody approximately 35 per centhigherthan controls, even though
they had been residing at the lower altitude for about six weeks. A
third immunization, 22 weeks after descent to lower altitude, showed
no difference between the two groups. In this experiment, the time
course of response to the antigenic stimulus was similar for both
groups. The maximums reached, however , by the high- altitude group
were greater both while at the mountain and 6 weeks after returning
to the lower altitude.

95

TRAPANI

100

?

K 10

o
o

OD

<

• CONTROLS

O ADRENALECTOMIZED -

0 2 4 6 8 10

TIME (DAYS)

12 14

Figure 4. Passive antibixiy decay in bilaterally adrenalectomized and control rab-
bits.

96

30

1 "O

«» 7
^ 5

3

ANTIBODY FORMATION

T 1 — I r

T 1 r^

u;jO onp n c>-

• CONTROLS

O AORENALECTOMIZED

J- L

K) 12

14

16 18 20 22 24 26

TIME (WEEKS)

Figure 5. Circulating antibody levels in bilaterally acirenalectomized and control
rabbits immunized with BSA and Freund's adjuvent (10 mg BSA per kg body \\t) at
time zero.

1000
700

I I I I I I 1 I I I I I I I I I 1 I I I I I I
• CONTROL
2|Vv o HIGH-ALTITUDE 3

^^

I I '

30 d.

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
TIME (WEEKS)

-^1

14.150 ft.

5,280 ft.

Figure 6. Circulating antibixly levels in rabbits adapted to high altitude and im-
munized with BSA (10 mg per kg Ixjdy \i.t) intravenously. 1 indicates the primary in-
jection, and 2 and 3 indicate the second and third challenge, respectively.

97

TRAPANI

The next point of information I wish to present is that of the im-
mune response of adrenalectomized animals. Even though there is
an abundance of literature dealing with the effect of the adrenal
steroids on various aspects of the immune response, little has been
done utilizing extirpation experiments. If the reaction of the animal,
deprived of its endogenous source of hormone, can be established,
then the activity of various available steroid preparations might be
more accurately assessed.

Rabbits were bilaterally adrenalectomized ina one-stage opera-
tion (Zak, Good, and Good, 19 57) using a ventral mid- line approach.
They were maintained on 1 per cent saline for drinking water with
free access to food, and allowed to recover for 3 to 4 weeks before
being used in the experiments. Passive decay of antibody (Fig. 5) in
adrenalectomized rabbits was not significantly different from con-
trol animals. Active immunization (Fig, 6) shows an apparent net
decrease in the immune response of adrenalectomized rabbits.

It has been reported that adrenal steroids depress the immune
response. The data presented here, however, indicate that adrenal-
ectomy results in a decreased immune response. These apparently
contradictory results present a paradox, and point to the complexity
of experiments of this kind.

DISCUSSION

The immune response can be thought of as composed of two
processes (antibody production and antibody dec ay) occurring simul-
taneously, but not necessarily at the same rate. It is possible to
measure the decay rate of passively administered homologous anti-
body; however, what is measured in the actively immunized animal
is the net result of antibody production and decay. It is conceivable,
therefore, that an increase in antibody production might be offset
by an increase in antibody decay so that the net level of circulating
antibody measured is apparently unchanged.

98

ANTIBODY FORMATION

a

S

o

UJ

Oi

«s

X

t— 1

UJ

x

1

o

Q

Oi.

J

>-

o
u

s

u

to

^

u

z

>,

o c

x

D. 3

4-1

>^ O

u

•X. m >^

01

^ Q

a

»-( C

UJ >. >-. O —
2 S 2: 02 w

3 0 0 UJ

CO H H H >-

UJ

0 U U 0^ S

UJ 2:

CL, UJ UJ 0

a HH

X Q J >-l H

3 X

UJ H-l < < U

H o

1 0 Z Z UJ

s^ss?

^S

0 JE Q Q pC
U P < < H

99

TRAPANI

INCREASED / /

NET response/ ^/CONTROL

7^

/^ o,^^DECREASED

^^X NET RESPONSE

ANTIBODY DECAY

Figure 8. lielationship of antibody production and antibody decay to the net immune
response.

Some of the complexities of the physiological relationships which
might pertain to the immune response of animals exposed to environ-
mental extremes or endocrine imbalance can be derived from Figure
7. For example, cold exposed animals exhibit a decreased immune
response, as measured by circulating precipitating antibody. This
may be the result of a) an unchanged rate of production associated
with the increased rate of decay, b) a decreased rate of production
associated with an increased rate of decay, or c) an increased rate
of production which is not of sufficient magnitude to offset the in-
creased rate of decay.

Figures presents a theoretical relationship between antibody pro-
duction and decay. If production and decay are "balanced" against
each other, we then arrive at the line labeled "control". If we now
manipulate the animals' physiology so that antibody production or
decay is altered, it is possible to arrive at a net response which
is either increased or decreased. If production and decay are altered
to the same degree and in the same direction, the net response could
still fall on the control line, and the observation would be no net
change in response. As a consequence, the actual alteration would
be obscured in this test system, and other avenues would have to be
explored to arrive at a more definitive answer.

100

ANTIBODY FORMATION

The persistance of antigen (Garvey and Campbell, 1956) and its
degradation in the host animal may also be altered under conditions
of environmental extremes or endocrine imbalance. Recent studies
of antigen disappearance in hibernating ground squirrels (Jaroslow
and Smith, 1961) have shown that there was no detectable disappear-
ance of antigen during 14 days of hibernation. After arousal, however,
the induction period for antibody formation was shorter than in non-
hibernating animals. It appears that this response is a reflection of
the physiological state of the animal.

Even though certain physiological changes might be implicated
a priori, it is difficult to attribute the net immune response to only
one factor. It appears essentialto consider the immunophysiological
inter- relationships and the functioningofthe animal as an integrated
unit in host- parasite interactions.

SUMMARY

Deductions made for host-environmental interactions can often be
derived by more than one pathway involving secondary physiological
factors which may affect resistance to disease, antibody formation,
and antibody decay. The immune response of animals exposed to
environmental extremes or endocrine imbalance must be interpreted
in the light of available knowledge concerning the functioning of the
animal as an integrated unit.

101

TRAPANI
LITERATURE CITED

1. Jaroslow, B. N,, and D. E. Smith, 1961. Antigen disappearance in

hibernating ground squirrels. Science 134: 734-735.

2. Lanni, F,, M. L. Dillon, and J. W, Beard, 1950, Determination of

small quantities of nitrogen in serological precipitates and
other biological fluids. Proc. Soc. Exp. Biol, Med. 74: 4-7,

3. Trapani, I. L. 1957, Antibody decay in cold exposed rabbits,

(Abstr.) Federation Proc. 16: 436.

4. Trapani, I, L,, and D. H. Campbell. 1959. Passive antibody decay

in rabbits under cold or altitude stress. J, Appl, Physiol. 14:
424-426.

5. Trapani, I. L., A. Lein, and D. H. Campbell. 1959. The effect of

thyroidectomy and thyroxin treatment on the immune response
of rabbits. (Abstr.) Federation Proc, 18: 161.

6. Trapani, I, L,, A. Lein, and D. H.Campbell. 19 59. Passive anti-

body decay in thyroidectomized rabbits. Nature 183: 982-983.

7. Trapani, I. L. 1960. Cold exposure and the immune response. In

International Symposium on Cold Acclimatization, Buenos
Aires, Argentina, August, 1959, Federation Proc. 19:Suppl.
5, 109-114.

8. Trapani, I. L. 1961. The immune response in adrenalectomized

rabbits. (Abstr.) Federation Proc. 20: 23.

9. Trapani, I. L., and R.T. Jordan. 1962. Antibody formation in rab-

bits adapted to high altitude. (Abstr.) Federation Proc. 21: 25.

10. Zak, S.J.,R.H. Good, and R.A.Good. 1957. A technique for one-

stage bilateral adrenalectomy in the rabbit. Nature 179: 100-102.

102

ANTIBODY FORMATION
DISCUSSION

BERRY: I am very much interested in this report. Dr. Tra-
pani, and would like to point out some of the effects of exposure
to a simulated altitude higher than yours; that is, 20,000 feet.
One of the things that we have noticed is that mice show an in-
crease in urinary nitrogen excretion. By inference, this would
suggest that protein catabolism is increased. If one attains
an elevation in nitrogen excretion, this says that the animal
is breaking protein down fast. We find it in mice that have been
exposed to simulated 20,000 feet for as long as a month, and
these animals are about as fully acclimated, as judged by their
general metabolism responses as any animals that we have
studied. We have kept them for as long as three months, and
we can find no difference in animals kept for three months at
simulated 20,000 feet than those kept for three or four weeks.
We can also get this elevated urinary nitrogen excretion in
animals that have been exposed for one day at 1000 feet; and I
don't know what this means. This is a very bewildering thing
to us,

I wonder if in any of these rabbits urinary nitrogen excretion
was determined?

TRAPANI: No. I would suspect that almost any stress im-
posed on the animal would charge its urinary nitrogen excretion.
If an animal is put in a cage which is wired with electrical cur-
rent which goes on and off at intervals, I imagine this sort of
a stress might influence nitrogen excretion. Experiments that
you just mentioned are somewhat similar to those done by Mefford
and Hale^ in which the metabolic interrelationships of cold,
heat, and altitude were studied. One of their points, of course,
is that there is an increase in nitrogen excretion.

BERRY: What produces the increase in nitrogen excretion.

1 Am. J. Physiol. 193: 443. 1958.

103

TRAPANI

metabolically?

TRAPANI: Off hand, I don't know.

CAMPBELL: Doesn't the salt balance play an important part?

WALKER: Do these animals lose weight, or eat more, Dr.
Berry?

BERRY: They do not lose weight. They are in pretty good
metabolic balance as judged by weight.

WALKER: They take in more protein to keep that balance?

BERRY: Yes, but these are complicated relationships,

TRAPANI: The main purpose here, for me, was to point out
how complex this really is. These experiments are really not
sufficient yet to make a complete story by any means, and the
next step planned is to study the immune response of adrenal-
ectomized animals for a considerable period, to elucidate the
relationship between the thyroid and the adrenal. Despite all
of your excellent experiments utilizing the cold and high alti-
tude chambers, I personally like mountain top laboratories better,
since I feel that chambers do offer certain disadvantages.

MONCRIEF: Do your rabbits that you shave still shiver?

TRAPANI: I never saw them shiver.

MONCRIEF: Are they acclimatized to the cold?

TRAPANI: They seem to be. Their body temperature was
not much different from room temperature controls,

MONCRIEF: What evidence do you have of increased metabolism
of rabbits?

TRAPANI: The metabolic rate, based on oxygen consumption
measurements done by open circuit metabolimetry was approxi-

104

ANTIBODY FORMATION

mately twice that of room temperature controls. Serum pro-
tein concentration also increases. This might be an interesting
point also to mention. Animals at high altitude have an increased
serum protein concentration and a decreased plasma volume.
Animals in the cold also have an increased serum protein con-
centration, and an increased plasma volume. Their plasma volume
was measured by Evans blue disappearance and also from the
passive antibody decay studies. In the latter case, the amount
of antibody injected and the initial antibody concentration in
the serum are used to calculate "plasma volume", or perhaps
we should call it "antibody space", as compared to Evans blue
space,

TUNEVALL: Was the decreased plasma volume at high al-
titude simply a result of an adaptive polycythemia?

TRAPANI: Yes. From the information just given, it was cal-
culated that animals at high altitude had a total mass of cir-
culating proteins the same as controls, while animals in cold
had an increased mass of circulating protein. More specifi-
cally, if one makes the proper corrections, the increase in
the immune response of animals at high altitude is greater
than that which might be expected from a decreased plasma
volume.

PREVITE: Have you ever done any experiments in a shorter
period of time? For example, have you ever measured anti-
body titers in response to BSA prior to one week, or isn't that
a sufficient amount of time to get a response?

TRAPANI: It's very difficult to detect antibodies before seven
days or so.

PREVITE: Have you ever previously immunized your rab-
bits, waited a sufficient amount of time, and then put them at
-15° C?

TRAPANI: Not in the cold. We did one experiment with rab-
bits at high altitude simular to what you are thinking about.
The first year we took animals up to White Mountain in Cali-

10 5

TRAPANI

fornia. Some were immunized in Pasadena before going up,
some were immunized when they first got there, some were
immunized after seven days, some after a month, and some
were just brought up there for a month, then immunized and
returned to Pasadena. The only group which seemed to give
a fairly clear picture so that we could say, "Well, let's study
this one because it's a little simpler," was the group which
was brought to a high altitude, adapted for thirty days and then
immunized, and the immune response studied while the ani-
mals were kept at high altitude. The variation of response in
the other groups was too great to merit further study at the
time.

PREVITE: I am wondering what would be the antibody re-
sponse to cold stress within the first few days if one used a
rabbit with a known titer ? Would you expect antibody titers to
decrease?

TRAPANI: By inference, I would say antibody levels may
go down; these are given levels of circulating antibody. When
the animal is put into the cold, the metabolic rate is increased
to overcome the heat load; antibody levels might then decrease
as a consequence of an increased turnover rate.

PREVITE: It seems that most of us agree that normally we
are warmly clothed when we are outdoors in cold weather. May-
be one is only accidentally going to be stressed by the cold en-
vironment. Perhaps it is most important, in analysis of the
effects of cold exposure on infectious diseases, to determine
the response to this chance exposure.

TRAPANI: I would like to make one more comment that Dr.
Berry brought to mind. I think the antigen must be considered
not only in terms of its persistance, but also in regard to its
chemical nature. The system I used was a soluble one of BSA
and anti-BSA. If one uses a viral or bacterial system, these
different kinds of antigen might be handled differently by the
animals under these situations. Dr. Berry has shown that in
animals subjected to high altitudes, resistance to bacterial in-
fection is decreased, whereas resistance to viral infection is

106

ANTIBODY FORMATION

increased. At present, I am doing an experiment in collaboration
with Dr. M, L. Cohn in which Guinea pigs kept at -4° C at the
Denver altitude or on top of Mount Evans, are infected with
Mycobacterium tuberculosis. We just finished infecting our ani-
mals by inhalation techniques last week, and whether or not the
results will come out similar to yours won't be known for another
month or so.

107

QUALITATIVE AND QUANTITATIVE ASPECTS OF THE IMMUNE
RESPONSE UNDER CONDITIONS OF COLD EXPOSURE

William T. Northey

Department of Microbiology

Arizona State University

Tempe, Arizona

ABSTRACT

Rabbits have been exposed to lowered environmental temperatures and immunized
with various protein antigens. In certain experiments, the immunization and bleeding
schedules have also been changed. Serum samples from these animals, as well as
a "control" group receiving an identical immunization schedule, have been analyzed
by a number of immunological and immunochemical techniques. To detect any pos-
sible qualitative differences in the serum samples as a result of cold exposure, a
number of analyses have been conducted. Among the techniques used are; Ouchter-
lony gel diffusion, immunoelectrophoresisi and starch gel electrophoreses. Quanti-
tative differences in the serum proteins have been studied by a determination of
A/G ratios and by a comparison of the serum globulins by paper electrophoresis.
Precipitin titrations on rabbit sera have been conducted using the ring test, and total
antibody nitrogen has been measured by the quantitative precipitin technique.

It may seem unusual to many in this group that Arizona State
University, which is located in the heart of the Sonoran desert,
would be an institution in which studies concerned with "cold stress"
are conducted. However, forthepasttwo years, including the months
of June, July and August when the outside temperature exceeds
37.7*"* C almost daily, we have, through the marvels of modern day
refrigeration, continued our studies into the immunological aspects
of this fascinating and challenging problem.

Many observations concerning the influence of environmental
temperature have been made on experimental animals and human
beings as well. But the seasonal incidence of many diseases has
never been explained, although it has been suggested by some in-
vestigators that environmental temperatures may influence the
frequency with which a disease may occur, as well as the severity

109

NORTHEY

of the infection (Moragues and Pinkerton, 1944; Jungeblut et al. ,
1942). In numerous studies on the influence of environmental temper-
ature on both human and experimental infection, conflicting results
have been reported, even when the same infectious agents and the
same animals were used. In the majority of these studies, no attempt
was made to measure the anittoody response quantitatively of animals
under "cold stress", A notable exception to this has been work of
Campbell, Trapani and Sutherland (Campbell, 19 51;Sutherland etal.,
19 58) whose studies on the sera of cold exposed rats and rabbits has
provided valuable information concerning passive antibody decay,
changes in the serum proteins, and alterations in the antibody level
and blood chemistry. I attempted to measere qualitatively and quanti-
tatively the immune response of rabbits exposed to lowered environ-
mental temperatures.To achieve a highdegree of sensitivity, antigen-
antibody systems of known high reactivitywere used, and the results
of the immunization of both "normal" and "cold exposed" rabbits
studied. Through the use of strongly reacting protein antigen- anti-
body systems, a qualitative measure of the antibody response was
made throughtheuse of such techniques as Ouchterlony gel diffusion,
immune- electrophoresis, starch gel electrophoresis, and paper
electrophoresis. Quantitative determinations included titrations of
antibody levels by the conventional "ring" precipitin technique, and
by the much more precise method of micro-quantitative precipitin
analysis. An attempt was made to obtain quantitative results of gel
diffusion studies by measuring the intensity and homogeneity of the
antigen- antibody reaction in terms of the amount of precipitate
formed. The objective was to deter mine (1) the sensitivity of the im-
mune mechanism of animals during cold exposure versus the "nor-
mal", that is, the multiplicity of the antibody response, and (2) the
"tjTDe" of antibodyproduced during cold exposure, that is, the affinity
or avidity for its specific antigen of the antibody produced by the
"cold exposed" versus "normal" animal. The specific objective of the
qualitative studies was to determinethenumber of multiple antigen-
antibody systems which can be observed in each of the groups when
identical immunization schedules are followed. Immunization with
the same multiple antigen system will provide information regarding
the sensitivity and selectivity of the immune mechanism under con-
ditions of the cold exposure. By studying both the qualitative and
quantitative aspects of the problem, one should be able to draw cer-
tain conclusions regarding the response of the "cold exposed" ani-

110

IMMUNE RESPONSE IN COLD EXPOSURE

mals in terms of (1) the degree of response, for example, the total
amount of antibody produced, (2) the rate of appearance of antibody,
(3) the "type" of antibody produced, and (4) the influence of the period
of immunization and/or cold exposure on the response of animals to
major and minor antigens when immunized with a multiple antigen
system.

This information may serve toelucidatetheroleof the "specific"
factor of immunity, that is, circulating antibody during periods of
cold exposure, and at the same time serve to clarify the response of
the immune mechanism to immunization with multiple antigens both
during and prior to cold exposure.

In all of these studies, the rabbit was the experimental animal of
choice. The animals were housed in a walk- in cold room maintained
at a temperature of 4^C. Prior to the beginning of the immunization
schedules, the rabbits were allowed to "adapt" for a period of one
week, after which they were clipped until fur remained only on the
head and the extremities. New fur was periodically clipped. The
animals were given food ad libitum, and water was changed fre-
quently. Open wire cages were used to allow free movement of air.
Only one rabbit was housed in each cage to prevent huddling. In most
of the studies, rabbits weighing approximately 2.5 to 3.0 kg were
immunized with three weekly intra- muscular injections of equal
parts of antigen and Freund's adjuvant (Freund, 1947).

Blood samples were obtained via cardiac puncture, the serum
separated and merthiolate added in a 1:10,000 final concentration.
All of the rabbits used in these studies were carefully selected in
order to control genetic variations. Litters of six or more were
raised for this investigation, and each litter was divided into equal
study groups of experimental and control animals. The serum
samples were then subjected to a variety of immuno- chemical
analyses which were designed to detect any difference in the serum
proteins of the "cold exposed" versus the "non-cold exposed"
rabbits.

The immune response of an animal to the administration of an
infective agent is best measured in terms of the antibody formed and
directed against the microorganism and its antigenic spectrum.

Ill

NORTHEY

While the original aim of this investigation was to determine whether
or not differences exist in response to antigenic stimulus in "cold
stressed" animals as opposed to "normal" animals following im-
munization with various selected microorganisms, preliminary ex-
periments indicated that these systems were not sensitive enough,
nor did they possess a high degree of specificity. Early experiments
were designed to measure qualitative and quantitative differences
in response to the injection of various selected microorganisms
such as Salmonella typhimurium and Diplococcus pneumoniae.
Analysis of the sera collected from animals challenged in this man-
ner proved to be difficult in that as the gel diffusion technique best
serves as a measure of the "soluble" antigen content of a microbial
suspension, the degree of sensitivity which could be attained using
microbial anti- microbial systems was inadequate to detect differ-
ences in the "cold stressed" and control animals. Because of the
high degree of reactivity inherent to antigen- antibody systems in-
volving complex soluble protein antigens, a number of systems in-
volving these antigens were selected for investigation. As a result,
the original experimental protocols were modified to include a num-
ber of new systems which are of sufficient complexity and sensi-
tivity for a study of this t3q3e. These new studies included such pro-
tein antigens as human serum, egg albumin (Ea), bovine serum
albumin (BSA), and whole bovine serum. These protein antigens, al-
though not implicated in the usual "infective process", serve as a
reliable index of the degree of sensitivity and selectivity by the
antibody forming mechanisms.

METHODS

Gel Diffusion

One of the most sensitive methods of measuring the antibody
response is through the use of the gel diffusion technique devised
by Ouchterlony (1949). In this investigation a 1 per cent solution of
lonagar Number 2 (Consolidated Laboratories, Chicago Heights)
was used to prepare the gel. The pattern was cut in the agar using
a Fineberg Agar Gel Cutter Number 1802, manufactured by the

112

IMMUNE RESPONSE IN COLD EXPOSURE

Undilute
Antigen

1-

I - 2

Rabbit
anti- serum

1 - 4

Figure 1. CXichterlony gel diffusion analysis of bovine serum-- rabit antibovine
serum.

Shannon Scientific Company of London, and distributed in this
country by Consolidated Laboratories. This pattern consists of a
center well surrounded by four wells equidistant from it. These
four wells were used for the various antigen dilutions. In order to
approximate more closely the region of optimal proportions, four
samples of antigen were tested in each case; that is, undilute, 1:2,
1:4, and 1:8. Plates were read at appropriate intervals in order to
determine the time of precipitation of the first reacting components,
and the results of each of the readings were recorded on master
sheets designed for this purpose. Readings in all cases were taken
from the antigen- antibody concentrations which gave the most defin-
itive results. A sample plate is shown in Figure 1.

For the data obtained to be best evaluated, it became necessary
to adopt a more accurate method of measurementbased on statistics.
The method of choice was the Mann- Whitney U test. This nonpara-
metric test is a useful alternative to the parametric "t" test when
the measurement in the research is weaker than interval scaling.

Aladjem et al,(1959) have reported that the time lapse preceding

113

NORTHEY

Sample

N

R-Cold

R-Normal

u*

1

9

85.500

85.500

U0.500

2

9

86.500

84.500

39.500

3

9

66.000

105.000

21.000

»♦

9

68.500

102.500

23.500

5

9

65.500

105.500

20.500

6

9

70.500

100.500

25.500

7

9

63.000

108.000

18.000

Table I. Qualitative evaluation of precipitates in gel diffusion of sera from "normal'
and "cold exposed" rabbits. *U value of Mann- Whitney U test at 0.0 5 significance level =
17. Antigen- whole bovine serum.

Sample

N

R-Cold

R-Normal

u*

1

9

85.500

85.500

U0.500

2

9

83.500

87.500

38.500

3

9

80.000

91.000

35.000

4

9

82.500

88.500

37.500

5

9

68.500

102.500

23.500

6

9

69.500

101.500

24.500

7

9

85.000

85.500

40.500

Table II. Quantitative evaluation of precipitates in gel diffusion of sera from "normal'
and "cold exposed" rabbits. •U value of Mann- Whitney Utest at 0.05 significance level =
17. Antigen- whole bovine serum.

114

IMMUNE RESPONSE IN COLD EXPOSURE

visable precipitation in double diffusion tests is dependent upon
three factors: (1) the diffusion co-effecient of each of the reactants,
(2) their absolute concentrations, and (3) the speed with which the
antigen- antibody front can form insoluble complexes.

Assuming that each of the rabbit antibody preparations possesses
an equal diffusion co- efficient, and using the same antigen prepara-
tion for both systems, the time of appearance of each of the pre-
cipitating bands on the Ouchterlony gel diffusion plate should depend
upon the concentration of the antibody and/or the affinity or avidity
of the antibody for its homologous antigen.

In our evaluation of these data, three methods of measurement
were used. In order to analyze the data, (1) the total number of pre-
cipitating antigen- antibody complexes was measured at each reading;
(2) in an attempt to evaluate the results quantitatively, each of the
precipitates was visually estimated and ranked with respect to the
total amount and homogeneity of precipitate; and (3) the time of
appearance of each of the precipitating bands was recorded. Pre-
cipitates were graded one through four based upon assigned criteria,
with the sharpest and most well-defined bands being assigned the
highest number. Quantitative evaluations were based on the number
of bands of precipitate times the intensity of each precipitate; for
example, a gel diffusion plate having four sharp, heavy bands of
precipitate and one faint, poorly-defined precipitate was assigned a
total value of 17 (4x4 + 1x1). All of the data, both qualitative and
quantitative, were subjected to statistical evaluation by the Mann-
Whitney U test. The results of one of these studies are given in
Tables I and II,

From these tables it may be seen that a statistical evaluation of
both the qualitative data based on the number of precipitating bands
in the Ouchterlony gel diffusion plates and the quantitative data
based on the number of bands times the intensity of the precipitate
failed to reach a level of significance in the Mann- Whitney U test.
The U value at the 0.0 5 significance level is equal to 17. In study
groups in which the larger of two independent samples is smaller
than 9, tables for U values are not available and the P value is cal-
culated directly (Table III). It may be noted in Table III that differ-
ences which are significant at the 0.0 5 level are obtained in sample 2

115

NORTHEY

Sample N R-Cold R- Normal U P

13 0 0

2 3 6 U 0 .050

3 3 12 11 2 .200
»+ 3 11 12 «♦ .500

5 3 15 12 1 .100

6 3 20 18 1 .100

7 3 15 16 U .500

Table III. Qiualitative evaluation of precipitates in gel diffusion of sera from "nor-
mal" and "cold exposed" rabbits. Antigen-egg albumin (Ea).

of the egg albumin anti-egg albumin system. However, a significant
level was not attained at any other sample in this experiment. An
occasional significant value was also found in other experiments,
but these findings were not consistentwith most of the data. To date,
over 500 serum samples have been evaluated in this manner. The
same method has also been applied to measurements of the time
of precipitation. The results of one series of measurements at
closely spaced time intervals is given in Table IV.

"Ring" Precipitin Titrations

The "interfacial technique" has been used extensively as a
measure of the antibody "titer" of immune sera. In these studies,
serum samples from both study groups were titrated for antibody
content using different antigens and varyingbleeding schedules. The
result of one of these tests using egg albumin as antigen is shown
in Table V.

The antibody titrations by the precipitin "ring" technique failed
to reveal any consistent trend toward higher antibody levels in either
of the study groups. However, when the less complex antigens, egg
albumin (Ea) (Table V) and bovine serum albumin (BSA) were used,
the antibody levels were somewhat higher in the "cold exposed"
group, whereas the animals maintained at room temperatures and

116

IMMUNE RESPONSE IN COLD EXPOSURE

Qualitative Evaluation

Sample

Time

N

R-Cold

R- Normal

U*

2-1

8 hr.

9

85.500

85.500

40.500

2-2

12 hr.

9

88.500

82.500

37.500

2-3

20 hr.

9

81.000

90.000

36.000

2-4

28 hr.

9

87.500

83.500

38.500

2-5

36 hr.

9

86.500

84.500

39.500

Quantitative Evaluation

Sample

Time

N

R-Cold

R-Normal

U

2-1

8 hr.

9

85.500

85.500

40.500

2-2

12 hr.

9

84.000

87,000

39.000

2-3

20 hr.

9

72.500

98.500

27.500

2-4

28 hr.

9

84.000

87.000

39.000

2-5

36 hr.

9

91.500

79.500

34.500

Table IV. Statistical evaluation of gel diffusion precipitates based on time ol pre-
cipitation. *U value of Mann- Whitney U test at 0.05 significance level=17. Antigen-
whole bovine serum.

Number of Days

following Mean Antibody Titer Mean Antibody Titer*
Initial Injection Cold exposed Non-Cold exposed

1-2000

1-1600

10

1-3000

1-3000

17

1-6000

1-4000

24

30

1-8000
1-10,000

1-5000
1-7000

Table V. Mean precipitation titers of "normal"and "cold exposed" rabbits immunized
with egg albumin (Ea). *Mean titer calculated on the basis of antibody titers obtained
from precipitin titrations of 10 samples of rabbit antiserum.

117

NORTHEY

using the human serum antigen attained a slightly higher level of
antibody. Of interest too is that in the group in which daily blood
samples were taken, the additional "stress" created by cold ex-
posure did not significantly alter a steady rise in the antibody
level (Table VI and Table VII).

Number of Days

Following Mean Antibody Titer Mean Antibody Titer*

Initial Injection Cold exposed Non-Cold exposed

3 1-2500 1-3000

6 1-5000 1-8000

9 1-5000 1-10,000

12 1-5000 1-10,000

18 1-2U00 1-10,000

2U 1-6500 1-10,000

H8 1-7000 1-10,000

Table VI. Mean precipitin tilers of "normal" antl "cold exposed" rabbits immunized
with human serum, 'Mean titer calculated on the basis of antibody titers obtained from
precipitin titrations of 6 samples of rabbit antiserum.

Starch Gel Electrophoresis

The technique of electrophoresis in starch gel offers a method
of increased sensitivity over that of paper electrophoresis, and
under proper conditions, it is possibletoresolveas many as fifteen
components in human serum (Moretti et al., 1959). This method was
utilized for a comparison of the sera from "normal" and "cold ex-
posed" animals in order to detect the possible presence of any
"abnormal proteins" which might be formed as a result of cold
exposure. Conversely, the failure of the "cold exposed" animal to
form any of the "normal" serum sub- fractions could also be recog-
nized. Simultaneous analysis of sera from both "cold exposed" and
"normal" rabbits immunized with antigens of varying complexity
provides a convenient method for these evaluations.

Samples were analyzed by starch gel electrophoresis in an appa-

118

IMMUNE RESPONSE IN COLD EXPOSURE

Number of Days

following Mean Antibody Titer Mean Antibody Titer*

Initial Injection Cold exposed Non Cold exposed

1 -

2 .

3 .

if .

5 1-50 1-20

6 ** 1-60 1-20

7 1-60 1-20

8 1-100 1-60

9 1-60 1-60

10 1-100 1-60

11 1-100 1-60

12 1-250 1-60

13 ** 1-500 1-300
l** 1-300 1-500

15 1-1500 1-1000

16 1-1500 1-1000

17 1-1500 1-1500

18 1-3000 1-3000

19 1-6000 l-UOOO

20 1-6000 1-4500

21 1-7000 1-5000

22 1-7000 1-6000

23 1-8000 1-7000

24 1-8000 1-7500

25 1-10,000 1-8000

Table VII. Mean precipitin titers of "normal" and "cold exposed" rabbits immunized

with bovine serum albumin (BSA). ♦Mean titer calculated on the basis of antibody titers
obtained from precipitin titrations of nine samples of rabbit antiserum. **Dates of
immunization.

119

NORTHEY

Figure 2. Starch gel electrophoresis of sera from "cold exposed" (bottom of picture)
and "non-cold exposed" (top of picture) rabbits.

ratus of our own design which utilized a borate buffer having an
ionic strength of 0.01 in the gel and an ionic strength of 0.0 3 in the
borate bridge. In all cases, samples were analyzed simultaneously
using a plastic trench which accommodated two samples at once.
A piece of filter paper was saturated with the serum and placed in a
slit in the gel. Samples representing both the "cold stressed"
samples and "normal" samples were run side by side at a current
of 250 volts 5 ma per sample for a five hour period. At the end of
the migration period, the strips were cut and stained with amido
(black) Schwartz. Permanent records were maintained by sketching
the pattern on graph paper and by photographs. A representative
starch gel strip is shown in Figure 2.

Thus far, a total of more than 300 serum samples from "cold ex-
posed" and non-cold exposed animals have been analyzed by starch
gel electrophoresis, and a critical evaluation of the data has failed
to reveal any striking differences. The presence of any "abnormal
proteins" in the sera of cold exposed animals was not detected.

However, a serum fraction, which has tentatively been identified
as one corresponding to the B lipoprotein of paper electrophoresis
(the alpha- 2 lipoprotein of immuno- electrophoresis), has been
found to be weak or absent in a number of serum samples from

120

IMMUNE RESPONSE IN COLD EXPOSURE

"cold exposed" animals when compared to the sera from animals
maintained at normal environmental temperatures. This fraction
has been reported to contain 55 to 66 per cent of the total conjugated
lipid in human serum(UrialandGrabar,1956). A second observation
of interest, although not necessarily significant, is that the serum
fractions observed on starch gel and stained by Amido (black)
Schwartz stain consistently appeared to be more distinct and well
defined in the sera of the "cold exposed" animals.

Immune- electrophoresis

Because of the high degree of resolution possible by Immuno-
electrophoresis, this technique has proved to be invaluable in the
identification of complex antigenic mixtures from a variety of
sources (Growle, 1961). We felt that this method would prove to be
useful in determining the sensitivity and/or selectivity of the im-
mune mechanism because the sera of animals immunized during
periods of cold exposure could be compared with those of animals
maintained at "normal" temperature and immunized in an identical
manner. The ability of the antibody forming mechanism to respond
to a heterogeneous spectrum of antigens present in varying con-
centrations should provide a sensitive and reliable index of the
qualitative immune response. Sera from "cold exposed" and "nor-
mal" rabbits were analyzed simultaneously by immune- electro-
phoresis, and the results were compared. Serum samples from
each of the groups were allowed to migrate in the gel under the
influence of an electric current. Following the migration period,
the antigen was applied to a center trough and allowed to diffuse
into the gel and react simultaneously with each of the separated
antiserum preparations. The technique used is shown in Figure 3.

To date, no significant differences have been observed in the
qualitative response of "cold exposed" and "normal" animals as
measured by analysis of their sera by immune- electrophoresis.
However, application of this technique has been restricted to only
forty serum samples due to insufficient time. This phase of the
investigation is currently being intensified, and final judgment con-
cerning the applicability of this technique to these studies must
await completion of additional experiments.

121

NORTHEY

Figure 3. Immuno-electrophoretic analysis of rabbit anti-human serum antibody in
"cold exposed" (top of picture) and "non-cold exposed" (bottom of picture) rabbits.
1. Barbitol buffer strength ionic strength 0.375. 2. Stain- thiazine red R.

Quantitative Precipitin Analysis

A limited number of serum samples have been analyzed by the
micro-quantitative precipitin method of Lanni (Lanni et aU, 1950).
The results of this analyses are given in Table VIII,

Cold Exposed
Non-Cold Exposed

Antibody Protein
mg/ml
0.U28 + 0.15H8*

0.3716 + 0.0767

Table VIII. ^Quantitative precipitation analyses of sera from "cold exposed" and "non-
cold exposed" rabbits.

It may be noted from the table that the cold exposed rabbits
gave slightly higher levels than the non-cold exposed group. This
finding is in accord with that of Trapani and Campbell who noted
somewhat higher levels in the plasma proteins of cold exposed
(1° C to -15° C) rabbits (Sutherland et al., 1958).

122

IMMUNE RESPONSE IN COLD EXPOSURE

We are currently intensifying this phase of the investigation
in an attempt to obtain a much larger sample in order to reach a
definitive conclusion. More animals are necessary to lower the
standard error to an acceptable level, although population variation
here is small, and consequently, this initial estimate of serum
protein levels is statistically quite accurate.

Paper Electrophoresis

To date, a total of more than 350 serum samples have been
analyzed by paper electrophoresis. In addition, approximately
75 serum samples have been analyzed for glyco- protein content.
The results of these evaluations are not available at this time,
and are currently being statistically analyzed using the Fisher "t"
test by the General Electric computer center at Arizona State
University,

DISCUSSION

An evaluation of the data presented here concerning the effect
of "cold exposure" on the immune response of rabbits indicates
that any "differences" attributable to "cold" per se from both the
qualitative and quantitative aspects of immunity are of insufficient
magnitude to draw any definitive conclusions.

A fundamental consideration in any investigation of this type
concerns what may be construed as "stress" conditions. Again
citing the work of Sutherland, Trapani, and Campbell (1958) the
rectal temperature of rabbits maintained at 4° C was not signifi-
cantly different from that of animals maintained at a room tempera-
ture of 18° C. In the studies reported here, however, a temperature
of 4° C was considered to be a condition of "stress" for rabbits
shaved of the bulk of their pelage. This assumption was sub-
stantiated by the fact that the animals tended to huddle, their skin
was cold to the touch, some shivering was observed, and a sig-

123

NORTHEY

nificantly large number of animals succumbed from non-specific
causes during the course of the various studies in the cold exposed
group compared to deaths in those maintained at room temperature.

The quantitative evaluation of sera from each of the groups has
provided some interesting information regarding the immune re-
sponse under conditions of cold exposure. Antigens of varying com-
plexity were used, and the response of the animals measured. The
comparatively less complex antigens, egg albumin (Ea) and bovine
serum albumin (BSA), provide a sharp contrast to the very complex
human serum and bovine serum in terms of multiplicity of antigens
to which the antibody forming cells must respond. The failure to
find a sig-nificant qualitative difference in the experimental and
"control" groups indicates that cold exposure does not appreciably
affect the total response of an animal to multiple antigen systems.
The level of the response, as well as the time required for antibody
response, did not differ significantly in any of the gel diffusion
studies. In spite of the failure to attain statistical significance in
these studies, it must be remembered that these data do not provide
a final critical measure of antibody response during hypothermia,
Campbell et al. (19 51) and Sutherland et al. (19 58) have measured
the rates of antibody decay during both active immunization and
passively administered antibody. These authors report an increased
rate in decay of both actively formed and passively administered
antibody in cold exposed rabbits. Extrapolation of these findings to
the data presented here and increased rate of "decay" may provide
a more sensitive measure of the actual difference, and result in
the weighing in favor of an increase in antibody production in the
"cold exposed" animals. Such an extrapolation is, of course, im-
possible on the basis of our present knowledge of the mechanisms
of protein metabolism and protein turnover in the immunized
animal.

It should be pointed out that I am well aware of the inherent
dangers in lending too much credence to the results of an evaluation
by gel diffusion. Numerous investigators have demonstrated that
this procedure is dependent upon a variety of physico-chemical
factors, and that this technique, regardless of how critically
applied, is subject to error (Glenn, 1959; Jennings et al., 1962).

124

IMMUNE RESPONSE IN COLD EXPOSURE

Antibody titrations by the "ring" precipitin method are at best
only a crude approximation of the antibody concentration in a serum
sample. Because dilutions of antigen rather than antibody are used
as a measure of "antibody level", the validity of such titrations has
been widely criticized (Roffel, 1961). In spite of this fact, this tech-
nique has received wide application, and in this investigation, these
titrations have provided a simple and convenient method for approxi-
mating antibody levels at various dates during the bleeding schedule,
and will be supplemented by the vastly more sensitive micro-
quantitative precipitin analyses when time permits.

Baker and Sellers (1960) have observed in "cold exposed" rats
the presence of a plasma protein component which they report has
a similar electrophoretic mobility to the "transferrin" component
of human serum. This component, also known as siderophilin, serves
to bind iron in the circulating blood and migrates as a B globulin.

While we did not find the presence of a similar "abnormal" pro-
tein in the sera of "cold exposed" rabbits, this does not preclude
the possibility of the existance of such proteins. Through the use
of different stains such as those for haptoglobins or another immuno-
chemical technique, like the more definitive disc electrophoresis,
similar abnormal proteins may be recognized.

The "loss" of the B lipoprotein fraction from the sera of a
number of "cold exposed" animals as measured by starch gel
electrophoresis cannot readily be explained, andmaybedue merely
to an alteration in the rate of migration. But this observation
provides a fertile field for speculation. Masoro (1960) has reported
that the cold acclimated rat has an increased capacity to oxidize
long- chain fatty acids. A similar increased capacity on the part of
the cold exposed rabbit would partially account for a lower level of
slow migrating B lipoprotein in this animal. Bertke (unpublished
data) and others have reported a decrease in lipid content of the
adrenals during stress. Such a decrease could presumably be re-
flected in a decreased level of circulating lipoprotein. A final
speculation concerning a decreased B lipoprotein content of cold
exposed rabbits concerns the role of heparin during periods of
stress and/or shock. This compound, because of its strong polar
properties, is capable of liberating the combined lipid from lipo-

125

NORTHEY

protein complexes. An increase in heparin would presumably cause
a corresponding decrease in the level of circulating lipoprotein. An
increase in heparin could presumably also play a role in the in-
creased clotting time in cold exposed rabbits reported by Campbell
and Sutherland (19 51). It must be pointed out, however, that at the
present time, we have no evidence for an increase in heparin in the
circulating blood during periods of "cold stress".

While the consistent "tendency" of the sera of "cold exposed"
rabbits to form sharper and clearer zones on starch gel provides
a poor indication of any "differences" that might exist, this might
be due to a greater degree of homogeneity of each of the different
protein components measured by electrophoresis, which would re-
sult in a more nearly equal rate of migration. This homogeneity
would then be reflected as less "trailing" in the stained starch
gel pattern.

The value of immune- electrophoretic analyses of sera from cold
exposed and normal rabbits to the present investigation must await
further application of this technique. The limitations when this tech-
nique is applied must be recognized. Further experimentation may
also be necessary to determine optimal buffer pH values, ionic
strength, and so forth, all of which are critical in this procedure.
In spite of the apparent limitations of this procedure, however, it
offers one of the most sensitive measures of qualitative antibody
response that is presently available.

Future studies which are planned will include analysis of sera
from rabbits exposed to more severe environmental conditions
(-15 C), and determinations of possible alterations in lipoproteins,
haptoglobins, and other serum sub- fractions. Quantitative measure-
ments of the 7 S and 19 S macroglobulins will be made by density
gradient zone ultracentrifugation and chromatography on cellulose
ion- exchange columns. It is anticipated that these and future in-
vestigations will provide information useful in determining the role
of antibody in host- parasite interactions under conditions of
hypothermia.

126

IMMUNE RESPONSE IN COLD EXPOSURE
SUMMARY

Immuno- chemical studies were conducted on serafrom rabbits to
investigate the qualitative and quantitative aspects of the immune re-
sponse under conditionsof "hypothermia". Rabbits have been subjec-
ted to lowered environmental temperature (4° C) ,shaved of their pel-
age, and immunized with various protein antigens. Serum samples
obtained from varying periods during each of the studies via cardiac
puncture have been qualitatively assayed for antibody response by
Ouchterlony gel diffusion, immuno- electrophoresis, starch gel elec-
trophoresis, and paper electrophoresis. Quantitative analyses have
included "ring" precipitin titrations, quantitative gel diffusion anal-
ysis, and micro- quantitative precipitin analysis. In all of the studies
the response of the "cold exposed" animals have been compared
with the "non-cold exposed" animals treated in an identical manner,
except for maintenance at the lower environmental temperature.

LITERATURE CITED

1, Aladjem,F,, R. W. Jaross, R. L. Paldino, and J. A. Lackner.
1959. The antigen- antibody reaction. Ill, Theoretical consider-
ations concerning the formation, location, and curvature of the
antigen- antibody precipitation zone in agar diffusion plates,
and a method for the determination of diffusion coefficients of
antigens and antibodies. J. Immunol. 83: 221-231.

2. Baker, D. G., and E. A. Sellers. 1960. Unpublished, Intermediary

metabolism: Discussion. Symposium on cold acclimation.
Federation Proc. 19: Supplement No. 5, p. 133.

3, Bertke, Eldridge, (Unpublished data) Arizona State University.

127

NORTHEY

4. Campbell, Dan H. 1951. Immunochemical studies of arctic ani-

mals. A research report covering the period 1948- 19 50 for the
Office of Naval Research.

5. Crowle, Alfred J. 1961. Immunodiffusion. Academic Press,

New York.

6. Freund, Jules. 1947. Some aspects of active immunization.

Ann. Rev. Microbiol. 1: 291.

7. Glenn, William G. 1959, Some considerations in agar column

diffusion analyses. J. Immunol. 82: 120-124.

8. Jennings, Robert K., and Morris A. Kaplan. 1962. Implications of

qualitative comparative serology. Ann. Allergy 20: 15-28.

9. Jungeblut, C. W., M. Sanders, and R. R. Feiner. 1942. J. Exp.

Med. 75: 611-629.

10. Lanni, F., M. L. Dillon, and J. W. Beard. 19 50. Determinations of

small quantities of nitrogen in serological precipitates and
other biological materials. Proc, Soc. Exp, Biol. Med. 74: 4,

11. Moragues, V., and H. Pinkerton. 1944. J. Exp. Med. 79: 41-43,

12. Masoro, E. J. 1960. Alterations in hepatic lipid metabolism in-

duced by acclimation to low environmental temperatures.
Federation Proc. 19: Supplement 5, 115-119,

13. Moretti, J., G. Boussier, M, Hugou, andL, Hartmann, 1959, Bull,

Soc. Chim. Biol, 41: 79-87.

14. Ouchterlony, O. 1949. Antigen- antibody reactions in gels. Acta

Path. Microbiol. Scand. 26: 507-515,

15. Raff el, Sidney. 1961. Immunity. Appleton- Century Crafts, Inc.

New York, 1953, p. 155,

128

IMMUNE RESPONSE IN COLD EXPOSURE

16. Sutherland, G. Bonar, and Dan H. Campbell. 1956. Cold adapted

animals. I, Changes in blood clotting and electrophoretic
properties of rabbit plasma. Proc, Soc. Exp. Biol. Med.
91: 64-67.

17. Sutherland, G. Bonar, Ignatius L.Trapani,andDanH. Campbell.

1958. Cold adapted animals. II, Changes in the circulating
plasma proteins and formed elements of rabbit blood under
various degrees of cold stress. J. App. Physiol. 12: 367-372.

18. Trapani, Ignatius L,, and Dan H. Campbell. 19 59. Passive

antibody decay in rabbits under cold or altitude stress. J.
App. Physiol. 14: 424-426.

19. Trapani, Ignatius L. 1960. Cold exposure and the immune

response. Symposium on cold acclimation. Federation Proc.
19: Supplement No. 5, 109-114.

20. Uriel, J., and P. Grabar. 1956. Bull. Soc. Chim. Biol. 38:

1253-1269,

DISCUSSION

BLAIR: Is cold responsible for the changes that have been
observed both with regard to the antibody turnover rates, and
also with regard to the immune- electrophoretic studies, or is
cold simply a stimulus? In other words, could not the same
situation be produced in the laboratory with stimuli other than
temperature stimuli, but resulting in experimental preparations
metabolically, at least, similar to that induced by cold? In other
words, is not cold just a non-specific stimulus, or is cold really
responsible for these changes?

TRAPANI: Are you asking about the effect of temperature
at the cellular level?

129

NORTHEY

CAMPBELL: He is talking about hypothermia versus stress.
You can have stress without hypothermia. You need to really
lower the body temperature, which we haven't done. It has been
done in the case of the hibernating squirrels. I think you would
have to use an animal of this sort. Of course, this is an adap-
tation, and you have two problems. It seems to me one is a stress
problem and the other is not.

BLAIR: That is right. What I am trying to clarify in my own
mind is that the cold exposure is simply one of numerous stimuli
that could be used to produce the same situation.

TRAPANI: I think this is true. What we are really dealing
with in an animal that is cold exposed are the secondary factors
which affect its response.

BLAIR: Could we call this the affect of cold on immune re-
sponses, really?

TRAPANI: If you put it in a cold box or out in the snow, a
cold stress is imposed, and the index used for measuring the
response is attributable to that stress, either directly or in-
directly.

BLAIR: Suppose you produce a stress situation through another
stimulus?

PREVITE: These responses to various types of stimuli, for
example cold, heat, or sound, are not always the same.

MITCHELL: I wonder if we have a veterinarian around here
who can tell us whether this is a stress or not. I want to know
whether we are really and truly talking about a stress to these
animals, or whether we are trying to talk about what would be
a stress to a man if we did it to him?

REINHARD: Well, I think if the stress investigators con-
ducted baseline experiments to determine the characteristics
of the animal, that all animals would have similarities in their
responses to stress. There is only one area in which man is

130

IMMUNE RESPONSE IN COLD EXPOSURE

different, and that is in the neurological and psychological re-
sponse. When you talk about man, you have to introduce those
factors, also.

BERRY: You are not excluding the psychological factor in
animals, are you?

REINHARD: No, but they are quantitatively, and sometimes
qualitatively different.

VIERECK: I completely agree with these comments by Dr.
Reinhard. One type of baseline experiment would be to expose
the animal to the cold temperature and measure food intake
over a period of days. I think it is accepted that if the animals
consume considerably more food, their metabolism has been
higher and they have been under a sort of stress. Then this
brings to mind another question. Some animals have been treated
in this way, and they do indeed eat more food at certain low
temperatures; thus they are eating more protein, and if an ani-
mal is consuming more protein for a period of time, will this,
all by itself, affect the serum protein electrophoresis patterns
regardless of cold exposure? In other words, could cold act
via food intake? The protein turnover might be higher whether
the stress were cold or whether it were something else.

SULKIN: This is precisely why I asked Dr. Campbell earlier
whether he did the experiments with the arctic ground squirrels
just at the time that they were going into hibernation. A bat just
going into hibernation is not a stressed animal. However, if
you are dealing with the same bat species in the cold room in
the summertime, he is then in a state of hypothermia, and is
in a stressed condition. While I don't intend to talk about electro-
phoretic changes, we have data showir^ that there is a differ-
ence in the analysis made in hypothermic bats and in hibernating
bats, so it is important to emphasize the animal host and the
season of the year.

TRAPANI: Is it a qualitative or quantitative difference? Do
you pick up different components or the same components in
different amounts?

131

NORTHEY
SULKIN: I am not sure.

CAMPBELL: I suspect you might get a difference in the ratio
or distribution in some proteins that might be there in minor
amounts. Most of it is the ratio. Instead of being five per cent
or ten per cent of the total proteins, it may go up to twelve
or fifteen per cent. Now, under stress conditions, I cannot see
that anything can happen within an hour or two or even with-
in a few hours, other than changes in fluid balance perhaps ;
then you get changes in protein like the hibernating squirrel.
It looks like its got a high protein concentration, which it has,
but the total amount of protein may be actually the same as a
normal animal. I don't know what the blood volume was because
I didn't measure that.

PREVITE: I agree with what Dr. Sulkin stated a few minutes
ago. There is a tremendous difference between hibernation and
induced hypothermia. Dr. Lyman at Harvard has just emphasized
this in a review on hibernation.^ In answer to Dr. Mitchell's
question about stress, Dr. Cardy,^ of the University of Penn-
sylvania, noted that whether or not an animal is stressed de-
pends upon the host and the conditions of the experiment.

MITCHELL: And the interpretation of the investigator in whether
or not he is going to measure it by lowered temperature or the
presence of this or the presence of that.

REINHARD: To reply to Dr. Mitchell, there is one more im-
portant factor. I wish more people would do comparative work
using various species, because no one animal is exactly rele-
vant to man, and they do differ in basic physiology, especially
the gastroenterological portion of it.

McCLAUGHRY: I think it might be well to keep in mind here
the distinction between the physiological function of protein
metabolism as it might be reflected by the antigen- antibody

1 Lyman, C. P. 1961. Circulation XXIV.

2 Hardy, J, D. 1961. Physiol. Rev.

132

IMMUNE RESPONSE IN COLD EXPOSURE

response, and the function of the antibody as a protein com-
ponent of the serum. These two things may not be exactly the
same, and I think that there has been in the past an assumption
that we are measuring these two things simultaneously in some
of the studies reported. It occurred to me that it might be possible
to distinguish these two separate physiological functions in some
way.

NORTHEY: In terms of the comments about protein metabolism,
one can't say an animal eats more and therefore produces more
antibody. The antigenisticity of the substance under investi-
gation makes a difference in probably a multitude of other fac-
tors.

CAMPBELL: That is why I was bringing up this last point;
the persistence in antigens.

133

INFLUENCE OF HYPOTHERMIA ON THE ACTION OF
BACTERIAL TOXINS

G. Tunevall and T. Lindner

Central Bacteriology Laboratory

Box 177

Stockholm 1, Sweden

ABSTRACT

In hypothermic mice with a body temperature of 22° C to 23° C, given about one
DLy)o °^ tetanal toxin, the survival was significantly longer than in normothermic ones
given the preparatory (Hibernal- Nembutal) treatment but not chilled (54 versus 29.5
hours at 48 hours of hypothermia). This result must be cautiously evaluated. Though
hypothermia was not established until three hours after toxin injection, absorption
from the subcutaneous site may be slower in hypothermic animals. Further, narcotic
pretreatment drugs, though given also to the controls, may be more slowly eliminated
by hypothermic mice, resulting in a milder and protracted course of the toxic manifes-
tations. An attempt was done to find out if hypothermia prolonged the time during which
toxin could be neutralized by antitoxin. Antitoxin after 15 minutes resulted in survival
of all mice. At a toxin- antitoxin interval of four hours no animal was saved, but sur-
vival was prolonged and more so in hypothermic mice than was corresponded by the
length of hypothermia (80 versus 43 hours at 4 hours of hypothermia). At an interval
of 10 hours, survival was shorter in both groups, and the difference between hypothermic
and normothermic mice equalled the duration of hypothermia (50 versus 39 hours at
10 hours of hypothermia). These observations must be corroborated by further experi-
ments before they can be safely evaluated. The effects of staphylococcal toxin are less
likely to be attenuated by the premedication pertaining to our procedure for inducing
hypothermia. On the contrary, a synergism was observed between the narcotic drugs
and this toxin, as amounts less than one conventional DL.qq were sufficient to kill the
mice. Also with this toxin, however, the survival was longer in hypothermic mice
(3.5 versus 2 hours).

Induced hypothermia has been employed clinically in a variety of
conditions, among others in intoxications. As seems often to be the
case in connection with hypothermia, animal experimentation has
been scarce and tended to lag behind the applications in human
beings,

135

TUNEVALL AND LINDNER

The effect of tetanus toxin on mice subjected to low environ-
mental temperature was studied by Ipsen (1951). The survival after
large toxin doses was prolonged in chilled animals, whereas sub-
lethal doses caused more deaths in chilled mice than in those kept
at normal room temperature. Increased susceptibility to endo-
toxins from Gram- negative bacteria of mice held at 5° C and 15 C,
when compared to animals at room temperature was found by
Previte and Berry (in press). In hypothermia induced so as to avoid
stress reactions, no effect on the hematologic or histologic mani-
festations of staphylococcal exotoxin in rabbits was found by
Cole (1960).

EXPERIMENTAL

Material and Methods

Albino mice weighing 20 to 40 gm were numbered serially. The
allotment of animals to different experimental groups was done by
a random method according to the tables of Fisher and Yates (1953).

For inducing hypothermia the following procedure was used: A
subcutaneous injection of 32 mcg/g body weight of Chi orpromazine-
HCl is followed after 30 min. by an intraperitoneal injection of half
this amount of ethyl- (1- methyl-butyl) -malonyl- carbamide- Na. The
mice are then, in their narcotized state, fixed onto suitably formed
lead plates with adhesive tape and immersed in a supine position
into a 21° C water bath with only the head and part of the thorax
above the water. The rectal temperature will be stabilized within
one hour between 21.5° C and 23.5 C, and the temperature in
lower esophagus will be stabilized about 0.5° C higher. Oxygen and
carbogen are administrated continuously to the chamber formed by
the water bath and its fairly tightly closed cover.

Rewarming starts with slowly raising water temperature to 33° C
over a 4 to 5 hour period. When they begin to show activity, the
mice are freed from the lead plates, dried, transferred to their

136

HYPOTHERMIA AND BACTERIAL TOXINS

original containers, and placed into an air incubator generally at
35° C for the first 2 hours, then at 31° C for 24 hours. Humidity is
kept around 50 per cent, and oxygen administration is continued.
After this period, the mice can be kept under normal conditions.

Normothermic controls are given the same premedication, where-
by rectal temperature decreases by 2° C to 5° C, but are immedi-
ately transferred to the air incubator, and from that point treated
as above.

Tetanus and staphylococcal toxins and antitoxins were obtained
from the Swedish State Bacteriological Laboratory. In preliminary
experiments on normal mice of our breed, their MLDj^q neutral-
izing doses were determined. The routes of administration of these
products will be given for each experiment.

RESULTS

In a first experiment presented in Fig. 1, twenty mice were given
one MLD^QQ of tetanus toxin subcutaneously. Two hours later hypo-
thermia was induced in ten mice which were then kept in this state
for about 48 hours. The other ten mice received pretreatment only.
Tetanic manifestations were only slight in the hypothermic animals
in comparison with those of the controls, but at rewarming, after
48 hours, paroxysms grew more frequent and intense. One normo-
thermic mouse was lost because it was severely bitten, and two
hypothermic ones were lost from drowning. Therefore, recording
of survival times could be done only in nine normothermic mice
and in eight hypothermic mice, as reported in Fig. 1.

The average survival within the hypothermic group was sig-
nificantly longer than among normothermic controls, as is visible
from Fig. 2. In addition, it can be mentioned that two mice only
died during the period of hypothermia, two during the first stage
of rewarming in the water bath, oneduringthe stay in air incubator
at 35° C, and the remaining three 5 or 6 hours after the change of

137

TUNEVALL AND LINDNER

NORMOTHERMIC

MEAN
1

1

1

^^^^

^^^^•

•

^^^^^

•

HYPOTHERMIC

1

MEAN

^

^H

^^^H

=

^^^H

"^^^H

^^^^^^H

~^1HHH

. .

,

.

6 12 18 24 30 36 U2 48 54 60 66

■^^'^"^ survival time. hours

Figure 1. Survival times in mice given one MLD^qq of tetanus toxin. White parts of
columns mark period of hypothermia. Number of mice: Normothermic 9, hypothermic 8.

Number

Survival

Group

of
mice

in hours

Normothermic

9

29.5 + 1.7

Hypothermic

8

SU.O + 3.5

Diff.

24.5 6.2 < 0.001

Formulae used: s = -h^^/^Cx-x) ■»■ X(y-y) » smd

"^ n + n - 2

X y

t = D /"ley"
sVx + ^

Figure 2. Survival times in hours (M * e(M) ) of mice after subcutaneous injection
of equal doses of tetanus toxin. Start of hypothermia 2 hours, rewarming 48 hours aftef
the injection.

138

HYPOTHERMIA AND BACTERIAL TOXINS

temperature to 31° C."

The above results could at least partially be due to a slower
fixation of the toxin to susceptible cells in hypothermic animals.
Therefore, new experiments were set up in order to find out if
hypothermia prolonged the time during which the toxin remained
free to be neutralized by antitoxin.

5 mic»
3 ••

6 mic»

^antitoxin af l»r 15 min.
■■^IH^HHB .'MEAN

Smict

b mice

S mice

"f onti toxin after 4 hours

MEAN

f antitoxin after 4 hours
MEAN

'\ antitoxin after 10 hours
-^^mm ! MEAN

■7

- survlv ed

n fil 24 36 46 60 72 84

antitoxin of terio hours

TOXIN

96 108 hours

survival time

Figure 3. Survival times in mice given one MLD^JQ of tetanus toxin and, after Inter-
vals of 15 minutes, 4 hours and 10 hours, one neutralizing dose of antitoxin. White parts
of columns mark period of hypothermia, induced two hours after the toxin injection
and maintained for two or eight hours.

The results of one experiment are reported in Fig. 3. In all,
35 mice were given one MLD^qq subcutaneously. They were allotted
to three groups containing 11, 12, and 12 animals respectively. In
the first group, one neutralizing dose of antitoxin was given, also
subcutaneously, 15 minutes after the toxin injection. About 1-1/2

139

TUNEVALL AND LINDNER

hours later, hypothermia was induced in six mice and maintained
for 2 hours in three animals and for 8 hours in the other three.
Five mice were kept nor mother mic. In this group all eleven mice
survived, but it is not included in the table.

In the second group six mice were made hypothermic 1-1/2 to 2
hours after the toxin injection, and the other six kept normothermic.
All animals got antitoxin 4 hours after the toxin, and then the re-
warming of the hypothermic animals was immediately begun. In this
group no animal was saved, as seen from Figure 3, but the survival
was prolonged in comparison with the first experiment; especially
so in hypothermic animals. The difference effected by the hypo-
thermia was much larger than corresponded to the length of the
hypothermic period itself (80 versus 43 hours at two hours of
hj^othermia).

The third group received antitoxin 10 hours after the toxin,
and the rewarming of hypothermic mice was also begun after
10 hours. In other respects this group was treated as the second.
The survival was still prolonged, but the difference between
hjTDOthermic mice and controls was only equal to the length of
hypothermia (50 versus 39 hours at 8 hours of h3T)othermia).
This experiment is also presented in Fig. 4, which states the
significance of the observations.

As will be discussed later on, the effect of tetanus toxin may
be influenced by irrelevant factors. The next part of the study
was therefore performed with staphylococcal toxin which was,
as well as antitoxin, always administered intravenously in order
to avoid differences in absorption from local sites of injection.
Preliminary experiments had shown that a dose of staphylococcal
toxin large enough to kill all mice in the groups resulted in very
short survival, generally ranging between 1 and 4 hours. It was
further found that about 0.8 MLD,„„ was enough to kill mice
which had received the pretreatment whether they were made
hypothermic or not.

In the first series of experiments, the influence of hypothermia
on the survival times after lethal doses of the toxin was in-
vestigated. Groups of normothermic mice which received the

140

HYPOTHERMIA AND BACTERIAL TOXINS

Number of group

1.

2.

3.

^,

Toxin-antitoxin

interval, hours

1+

10

State

Normo-

Hypo-

Normo-

Hypo-

thermic

thermic

thermic

thermic

Number of

mice

6

5

5

5

Survival time

52

98

60

58

in hours

i+5

77

34

57

42

58

38

41

34

76

31

74

i+9

89

34

22

36

M + e(M)

^+3 + 2.9

80 + 6.8

39 + 5.3

50 + 8.8

Comparison 1.-2.: D = 37. t = 4.8 P< 0.001

2.-4.: D = 30. t = 2.7 0.05>P>0.02

Figure 4. Survival times in hours of mice given one neutralizing dose of antitoxin
serum 4 versus 10 hours after the tetanus toxin injection. Start of hypothermia 1-2
hours after the toxin injection, warming up immediately after the injection of anti-
toxin. (5 normothermic mice, 3 hypothermic for 3 hours, and 3 hypothermic for 9
hours, all receiving antitoxin after 15 min., survived).

pretreatment only were run. The toxin injection was always made
after the start of hypothermia. The experiments differed slightly
as to the period of h3T30thermia preceding the toxin injection
and the dose of toxin, but hypothermia was always maintained
to the end of the experiments. The results are given in Fig. 5.

Thus, in all groups, the average survival was longer in hypo-
thermic mice. The differences within the groups were significant,
and if all the material is taken together, they were even more
significant. It was next attempted to study the influence of hypo-
thermia on the time during which staphylococcal toxin could
be neutralized by antitoxin to a degree sufficient to save the
mice or at least prolong their survival in a manner similar to

141

TUNEVALL AND LINDNER

Group

1

•

2

•

3

•

>4

•

State

N

H

N

H

N

H

N

H

Survival,
minutes

105
60

100
30

100

100
75
90
80
80

225
160
250
150
150
195
2U0
230
200
230

130
160
120

180
150
215
210
210
220
230
265
230

80
190
100
150
120

200
190
2U0
230
180

110

110

lUO

50

70

3U5
370
100
195
380

M
e(M)

82
+ 7.3

203
+ 12.0

137
±12.0

212
±10.8

128
±19.3

208
±11.6

96
+16.0

278
±55.7

Diff.

121

75

80

182

t-value
Degrees
of freedom

8.58
18

3.6U
10

3.55
8

3.1U
8

P

< 0.001

0.01-0.001

0.01-0.001

0.02-0.01

Total N: 102 ± 7.6
Total H: 220 ± 11.5

Diff. 118. t = 8.09. dF 50. P< 0.001

Figure 5. Survival times in normothermic versus hypothermic mice given one
MLE>ioo of staphylococcal toxin. Hypothermia induced about 5 hours before the toxin
injection. (N ■ normothermic. H ■ hypothermic).

the work on tetanus toxin. In order to establish suitable experi-
mental conditions, a number of neutralization tests were first
set up in normothermic mice given only the pretreatment. The
results are reported in Fig. 6 and Fig. 7.

As the number of mice was small and as the results emanate
from several experiments, no statistical treatment has been done,
but generally, there was a good correlation between the length
of the toxin- antitoxin interval and the survival times. Essentially,
the experiments differed in one respect only; in one group, the anti-
toxin dose was increased from one to four neutralizing units, and
the results of this experiment are reported in Fig. 6 and Fig. 7.

142

HYPOTHERMIA AND BACTERIAL TOXINS

Survival

61- 76- 91- 121- 151- 181- 241-

minutes = 60 75 90 120 150 180 240 480

00

Interval
minutes

1

5/7

2

4

1

2

/5

3-5

1

1

1

2

1

1

/I

6-15

2

1

3

1

1

1

16-25

1

2

/I

1/3

26-45

/I

46-60

/I

Figure 6. Survival times of 51 pretreated but normothermic mice after Intravenous
Injection of one lethal dose of staphylocoocal toxin, followed by one or (bottom right In
the columns) four neutralizing units of antitoxin after different Intervals.

Common to all groups was the observation that increasing
toxin- antitoxin interval diminished the ability of antitoxin to prolong
the survival. The larger dose of antitoxin gave more absolute
survivals and at longer toxin- antitoxin intervals increased sur-
vival times.

As to the influence of hypothermia on the time relationships
described in the Figures 6 and 7, the work in this area is in
its beginning stages. Only one of the experiments has contained
five animals made hypothermic about five hours prior to the
injection of toxin. They all represent a toxin- antitoxin interval
of two minutes. The average survival time of these mice was
284 minutes, and the result is marked as a single point in the
figure. This is situated well above the curve for normothermic
mice given the same antitoxin amount, one neutralizing dose,
but there is no statistical difference between this average and
that of the normothermic mice in the same experiment (t = 1.7 ;
dF 8; P > 0.1),

143

TUNEVALL AND LINDNER

Survival time, minutes

o o o

3 4 5 7 10 12 15 20 30

Toxin-antitoxin interval, minutes

Figure 7. Average survival times of groups of normothermic mice given one
MLD^OO °^ staphylococcal toxin and, after different intervals, one or four (broken
line) neutralizing doses of homologous antitoxin. Same average for a group of five hypo-
thermic mice is marked as a single point.

DISCUSSION

It should first be pointed out that our experiments involve a
procedure for attaining hj^jothermia by the elimination of the
normal thermoregulation; thus, hypothermia is reached without
stress reactions.

144

HYPOTHERMIA AND BACTERIAL TOXINS

The first experiment indicated a significantly prolonged survival
time and milder tetanic manifestations in hypothermic mice. These
mice, however, got more pronounced tetanus and died at or very
soon after rewarming. To this result, which may mean simply that
a postponement of the toxic effect has come to an end, may be con-
tributed the trauma induced by the rewarming process itself. The
possibility also remains that warming up effects a compensatory
overnormal metabolism, thus giving the toxin an especially good
access to susceptible cells.

If the protective effect of hypothermia is due to a retarded
fixation of the toxin to its receptor cells, hypothermia should pro-
long the period during which the toxin may be neutralized by anti-
toxin before its entry into these cells. Too few experiments have
been made to allow a safe verification of this assumption, but it is
interesting to note that with a toxin- antitoxin interval of four hours,
a two hour period of hypothermia increased the average survival
to not less than 37 hours. Further experiments are planned to in-
vestigate whether this increase may be converted to a lasting sur-
vival if an adequate toxin- antitoxin interval is chosen.

The evaluation of these results with tetanus toxin must be made
with great caution. The injection of toxin was made subcutaneously
and though hypothermia did not start until about 2 hours after the
injection, part of the toxin may have been more slowly absorbed in
hypothermic animals. Furthermore, narcotics, as a rule, have an
attenuating effect on tetanic manifestations. There are good reasons
to believe that the drugs given as pretreatment are more slowly
eliminated in hypothermic animals, and this may account for at
least part of the protective effect of hypothermia. Therefore,
staphylococcal toxin was chosen for the subsequent tests. In this
case the situation is apparently reversed, in that the effect of this
toxin seems to be enhanced by the narcotics used for pretreatment.

Hypothermia was also found to prolong the survival time after
the injection of staphylococcal toxin. On the basis of the same con-
siderations as for tetanus toxin, a series of neutralization tests have
been begun which have already indicated a fairly good correlation
between the time allowed to pass between the injections of toxin and
antitoxin, and the ability of antitoxin to prolong the survival time in

145

TUNEVALL AND LINDNER

normothermic mice. The ability of hypothermia to alter this relation
is not yet verified, but according to a first experiment, it seems
probable that it will be.

SUMMARY

In hypothermic mice with a body temperature of 22° C to 23° C
given lethal doses of tetanus toxin, the survival time was sig-
nificantly longer than in normothermic controls given a preparatory
(Hibernal- Nembutal) treatment but not chilled. Thetetanus was also
less pronounced in hypothermia, but increased at the rewarming
procedure. During or soon after rewarming, the animals died.

The ability of antitoxin to prolong the survival in normothermic
mice varied with the interval between the injection of toxin and
antitoxin administration. When hypothermia was maintained during
part of this interval, the survival was significantly more prolonged,
and much more than corresponded to the length of the hypothermic
period.

For several reasons, these results must be cautiously evaluated,
but they suggest that the fixation of toxin to susceptible cells is
retarded in hypothermia.

In another series of experiments with staphylococcal toxin,
similar results were obtained. Hypothermia prolonged survival and
the neutralizing effect of antitoxin diminished when the toxin- anti-
toxin interval increased. That hypothermia may prolong the period
during which neutralization is possible is not yet known, but a first
experiment of a series to be continued in the future points in
this direction.

146

HYPOTHERMIA AND BACTERIAL TOXINS
LITERATURE CITED

1. Cole, W, R. I960. Studies in hypothermia and staphylococcus

toxin shock. Dissertation Abstr. 20: 4373.

2. Fisher, Ronald A., and Frank Yates. 1953. Statistical tables

for biological, agricultural, and medical research. Hafner
Publ. Co., Inc.

3. Ipsen, J, 1951. The effect of environmental temperature on

the reaction of mice to tetanus toxin. J. Immunol, 66: 687.

4. Lindner, T., and G. Tunevall. 1958. Hypothermia and infection.

I. Influence of hypothermia on antibody formation in mice
in the secondary response to typhoid- H- antigen. Scand.
J. Clin. Lab. Invest. 10: 142.

5. Previte, J. J., and L. J. Berry, In press. The effect of en-

vironmental temperature on the host-parasite relationship
in mice. J. Infect. Dis.

DISCUSSION

SULKIN: I think it might be of interest to recall some ex-
periments that were reported in the early twenties by Bronfen-
brenner and Weiss^ in which it was shown that ether anesthesia,
alone and in combination with specific antitoxin, decreased mor-
tality in experimental botulism in mice. On the basis of these
early studies, I became interested in the effect of anesthesia on
experimental viral infections^ and found that many animals

1 J. Exp. Med. 1924. 39: 517-532.

2 SulJdn et al. 1946. J. Exp. Med. 84: 277-292.

147

TUNEVALL AND LINDNER

would survive infection with Western equine virus if they were
kept under anesthesia for prolonged periods of time, and further-
more, by using hyper-immune serum additional animals would
survive.3

CAMPBELL: What is your prolonged period of time?

SULKIN: Three 4- hour periods of diethyl ether anesthesia
were used. A similar underlying mechanism may be involved,
since during anesthesia the temperature falls as a result of
diminished muscular activity and increased heat loss.

BLAIR: I think this is a very interesting piece of work, and
while the experimental model, Dr. Tunevall, is quite differ-
ent than the one which I will discuss the day after tomorrow,
I believe that the philosophies are probably going to be quite
similar. There are two probable foci of activity, one which
you have emphasized; that dealing with the toxin- antitoxin ac-
tivity, that is, the activity of the organism depending on the
organism itself. And of course, the other site of action is the
host itself; that is, the overall physiological integrity of the
host as a result of the induced infection. I think that the point
that you made is that hypothermia seemed to postpone the ac-
tivities. I have found this to be very much the same situation,
usir^ gram negative coliform bacillus in my own studies. I hate
to give away all my thunder, but I think it is apropos since your
work does dovetail so well with it. It is simply that the hypo-
thermia doesn't really alter these things to a tremendous ex-
tent permanently. It is particularly striking that upon rewarming
there was a very high death rate. This re- emphasizes the mat-
ter somewhat and the picture of death and prevalent death is
identical upon rewarming.

BLAIR: The point is, that we have to consider very care-
fully the level of hypothermia that we are talking about, and
using in the experimental model, particularly with relation to
overall physiological changes as presumably benefits the un-

3 Sulkin et al. 1945. Proc. Soc. Exp. Biol. Med. 60: 163-165.

148

HYPOTHERMIA AND BACTERIAL TOXINS

fortunate situation which it creates, and then, of course, the
level of hypothermia which may have a more direct effect upon
the organisms in question,

PREVITE: I am curious, Dr. Tunevall, as to why you didn't
make your nor mother mic mice swim for two hours in a water
bath at thirty- seven degrees just to make the two groups com-
parable. You tied the mice down and made them swim in cold
water, but not warm water.

TUNEVALL: I don't know how to get them to stay in the water
bath.

MIRAGLIA: If you place the animals in the containers that
have very smooth vertical sides, there is no difficulty in keepir^
the animal swimming continuously for over an hour,

MONCRIEF: At Denver, they tried that and they just stif-
fened their tails up and stood on them, or else balanced their
chins and their tails on both sides of the container.

BLAIR: Apparently I should harbor resentment. Dr. Tune-
vall, in your initial observation that the cart came before the
horse with regard to using hypothermia. I have been "guilty".
I refuse to take the stand or stand court trial on this, but hypo-
thermia probably has been exercised rather liberally with patients
before there has been, shall we say, adequate research or ex-
perimental investigation. I do wish to state, though, that there
has been a tremendous amount of research in hypothermia for
many years before this modern, so-called era of clinical ap-
plication. But it is true, however, that there are many facets
that we know very little about, particularly with regard to in-
fections, on the hazards of hypothermia. Part of this has been
demonstrated by virtue of the fact that there has been a good
deal of difference as reported on results and literature on ex-
perimentally induced infections using pheumococcus, for example.
The animals were cooled — I believe these were mice — to
very profound levels of 20° C. While there was one report of
some improvement of survival, actually there were other re-
ports which indicated a higher death rate. I think that Dr. Eisman

149

TUNEVALL AND LINDNER

did this. I was in Colorado last March and talked with one of
his young gentlemen. They have since repeated this work using
more moderate levels of cooling — 30° C — and the opposite
result was obtained. There was a much more significant rate
of survival at the more moderate level of cooling, and this ob-
viously related to the host problem that I mentioned.

TUNEVALL: My characterization of the situation relates to
the early 1950's when hypothermia was already being used ther-
apeutically in several clinical conditions. I don't think they knew
very much what they were doing then,

BLAIR: I discussed it a few moments ago. They used arti-
ficial hibernation, and I hate those words,

CAMPBELL: Do you think new immunomechanisms, antibodies,
play any part in it?

TUNEVALL: In this connection, no, I don't think so,

CAMPBELL: But there aren't any so-called natural antibodies
on the antigens that you were testing?

BERRY: In that connection, at the meetings in Montreal it
was said by one of the speakers on a symposium that all ani-
mals have a very high immunity, not a natural immunity. It
is acquired through contact with staphylococci and this is the
reason why any immunization against staphylococci is so un-
successful. They are already maximumally immunized. This
is a concept that never occurred to me, but maybe it has some
validity, at least in regard to the staphylococcin antibodies
not normally present as a result of the contact.

TUNEVALL: I think that the only way to bring active anti-
body formation into this picture wouldbe to arrange for a secondary
response.

4 Dr. David Rogers, VanderbuiU University.

150

HYPOTHERMIA AND BACTERIAL TOXINS

CAMPBELL: I was thinking of the situation in which the
antibodies are already there and they are less operative. I do
not know the mechanism on neutralization, but you do have stress
conditions in which you can reduce shock, particularly in mice,
by injecting cortico steroid. If you inject it ten or fifteen min-
utes before you inject antigen, you can reduce the hypersensitivity
of the reaction.

BERRY: Dr. Previte did some experiments with staphylococci.

PREVITE: Yes, that was part of a study on cold exposure.
However, since the experiments were of short duration, and
since rectal temperatures of staphylococcus toxin injected ani-
mals were not measured, and hypothermia was not induced, the
results are probably not applicable to Dr. Tunevall's findings.

BERRY: No, they are not directly so, but there was no effect
of cold exposure under your conditions?

PREVITE: No, because the number of animals used was not
large enough. However, the results did indicate that a signifi-
cant effect would have been demonstrable with a larger number
of mice.

MITCHELL: Saint Patrick did such a good job over in Ireland,
but would the same mechanisms that you are talking about rela-
tive to h5T3othermia, and with your so-called antitoxin, work
also for venoms of snakes?

BERRY: You are asking Dr. Tunevall?

MITCHELL: Yes.

ANDRE WES: He didn't know about Saint Patrick.

MITCHELL: Saint Patrick did a good job of cleaning up Ire-
land of snakes for the poor. Actually, what I am talking about
is whether or not the mechanism you have described, say, for
staph- toxin, and for one of your other toxins, works equally
well for venoms of what we call our rattle snake, or in our

151

TUNEVALL AND LINDNER

country, the coral snake, or perhaps the cobra; now, whether
or not these could be employed, because we do have situations
in which this occurs, becomes a very important facet of a military
operation.

TUNEVALL: I can only guess in that connection, and guesses
are not sure.

BERRY: You have not used the intravenous route of admini-
stration of toxins and antitoxins?

TUNEVALL: Yes, I have injected by the intravenous route,

PREVITE: There have been some reports in which rabbits
rendered hypothermic and infected with staphylococcus mani-
fested prolonged survival compared to homeothermic infected
controls. Staphylococci were injected into the bones of the rab-
bit.^

BLAIR: I think an important matter here is that there have
been various types of experimental models also in administration
as well as type of organism used, but the important thing to
me is the fact that while survival was prolonged, all of the ani-
mals succumbed. There have been no experiments with per-
manent survival, and that is a very important matter because,
as I will discuss again in detail the day after tomorrow, I think
that in the viewpoints concerning the role of "therapeutic" hypo-
thermia, we are going to need some very definite clarification.
Obviously, the situation which does not produce full, long-term
survivals can hardly be considered efficacious.

PREVITE: Yes, but it might be used as an adjunct with some-
thing else.

WALKER: Or it may provide you with time to do something
like administer antitoxin.

5 Grechishkin, D. K, 1956, Eksperim. Khirurgia.

152

HYPOTHERMIA AND BACTERIAL TOXINS

BLAIR: Yes.

CAMPBELL: Concerning the basic immune mechanism, this
occurred to me. I wonder if anyone has studied the effect of
hypothermia on the threshhold reaction to histamine acetycho-
line, or so-called slow reacting substance. That would be a
fairly interesting problem,

NORTHEY: Well, I'd just like to add to that a little. We have
done some preliminary studies in which we sensitized cold
exposed and control Guinea pigs to egg albumin and later on
moved the uterine horn and/or a strip of smooth muscle from
the intestine. With both the egg albumin antigen and histamine
we stimulated these tissues in the cold exposed and non-cold
exposed Guinea pigs. The responses were measured on a physio-
graph. In these preliminary experiments which were made with
ten or twelve animals per study group, we were able to see
no significant differences in the responses of the cold exposed
animals from those in the controls.

BLAIR: I can't remember the details, but the Schwartzman
phenomenon was studied. I don't know if this falls strictly in-
to the category that you mentioned, but the cutaneous mani-
festations are delayed, and if they do appear, they are consider-
ably less,

TRAPANI: Is that because skin temperature is different from
core temperature?

BLAIR: No, this is a stabilized state and during stabilized
hypothermia, the gradient between the skin and the core is very
much the same in the hypothermia as it is without the hypo-
thermia, so the skin temperature, of course, is lower, and this
might be part of that. The blood flow to the skin has been mea-
sured in hypothermia and during this stabilized state. It is re-
duced, but not considerably. It is markedly reduced, of course,
during the period of cooling, but in the so-called steady state,
the blood flow to the skin is fairly substantial.

TRAPANI: But you can still have an actual temperature effect,

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TUNEVALL AND LINDNER
per se, on the cellular mechanism?

BLAIR: Oh, yes. Of course, the skin and temperature, per
se, is lower; however, it gradually begins to rise in the room
temperature environment.

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