Current Topics in Microbiology and Immunology / Ergebnisse der Mikrobiologie und Immunitatsforschung

210 Pages·1973·23.228 MB·English

by  R. R. Avtalion, A. Wojdani, Z. Malik (auth.), W. Arber, R. Haas, W. Henle, P. H. Hofschneider, N. K. Jerne, P. Koldovský, H. Koprowski, O. Maaløe, R. Rott, H. G. Schweiger, M. Sela, L. Syruček, P. K. Vogt, E. Wecker (eds.)

Current Topics in Microbiology
and Immunology
Ergebnisse der Mikrobiologie und Immunitatsforschung
61
Edited by
W. Arber, Basle . R. Haas, Freiburg . W. Henle, Philadelphia· P. H Hofschneider,
Martinsried· N.K.Jerne,Basle. P. Koldovskj, Philadelphia· H.Koprowski, Philadelphia
O. Maaioe, Copenhagen· R. Rott, Giejen . H. G. Schweiger, Wilhelmshaven . 1M. Sela,
Rehovot . L. Syrucek, Prague· P. K. Vogt, Seattle· E. Wecker, Wiirzburg
With 24 Figures
Springer-Verlag Berlin· Heidelberg. New York 1973
ISBN-13: 978-3-642-65533-3 e-ISBN-13: 978-3-642-65531-9
DOl: 10.1007/978-3-642-65531-9
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Softcover reprint of the hardcover I st edition 1973
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Universitiitsdruckerei H. StUrtz AG, Wiirzburg
Table of Contents
AVTALION, R. A., WOJDANI, A., MALIK, Z., SHAHRABANI, R., and Du
CZYMINER, M., Influence of Environmental Temperature on the
Immune Response in Fish. With 14 Figures .......... , 1
Du PASQUIER, L., Ontogeny of the Immune Response in Cold-Blooded
Vertebrates 37
BAK, A. L., DNA Base Composition in Mycoplasma, Bacteria and Yeast.
With 10 Figures . . . . . . . . . . . . . . . . . . . . . . . 89
WITZ, 1. P., The Biological Significance of Tumor-Bound Immuno-
globulins . . . . . . . . . . . . . . . . . . . . . . . . 151
Author Index 173
Subject Index 193
Influence ofEnvttonmental Temperature
on the Immune Response in Fish
1
R. R. AVTALION, A. WOJDANI, Z. MALIK, R. SHAHRABANI and M. DUCZYMINER
With 14 Figures
Table of Contents
I. Introduction . . . . . . . 2
II. Immune Resistance of Fish to Pathogens . . . . . . . . . . . .. 2
A. Production of Agglutinins and Protective and Neutralizing Antibodies in Fish 2
B. Influence of the Seasonal Temperature on Fish Pathology ....... 5
C. Resistance of Experimentally Inoculated, Non-Immunized and Actively
Immunized Fish, with Pathogenic and Non-Pathogenic Bacteria, at Various
Temperatures . . . . . . . . . . . . . . . . . . . . . . . . . .. 6
III. Depressive Effect of Ambient Temperature on the Immune Response in Poikilo-
thermic Animals . . . . . . . . . . . . . . . . . . . . . . . . . .. 9
A. Evidence for Partial or Complete Inhibition of the Immune Mechanism at
Low Temperatures. . . . . . . . . . . . . . . . . . . . . . . .. 9
B. Evidence for Preexisting "Natural" Immunity in Fish. . . . . . . . . 11
C. Temperature Effect on Antibody Production in Carp and Frogs Immunized
against a Non-Microbial Antigen; Bovine Serum Albumin (BSA) 12
1. Primary and Secondary Response in Carp Immunized with Various
Physical Forms of BSA . . . . . . . . . . . . . . 12
2. Temperature Effect on Production of Antibodies to BSA. . . 13
IV. Mechanism of Infection of Fish at Low Temperatures . . . . . . . 14
A. Bisset's Observations and Hypothesis - Discussion of this in Light of the
Recent Findings in Carp Immunized against BSA at Various Temperatures 14
B. Action of the Adrenal Cortical Hormones on Antibody Production at Low
Temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
C. Temperature Effect in Carp Immunized with Pathogenic and Non-Patho-
genic Bacterial Antigens . . . . . . . . . . . . . . . . . . . . . . 18
D. Temperature Effect in Frogs Immunized against BSA and Other Antigens 20
E. Establishment of Immunological Tolerance at Low Temperature . . . . . 22
F. Discussion of the Infection Mechanism at Low Temperature. . . . . . . 23
V. Temperature Effect Mechanism and Approaches to the General Mechanism of
Antibody Formation . . . . . . . . . . . . . . . . . . . . 24
A. Chronological Determination of the Temperature-Sensitive Stage 25
B. Conclusions . . . . . 27
VI. Methods of Investigation 28
References . . . . . 32
Rapaport Laboratory for Microbiology, Department of Life Sciences, Bar-Ilan Uni
versity, Ramat-Gan, Israel.
1 This work was supported by grant No. 697 from the Oceanographic and Limnological
Research Company, Haifa, Israel, and by grants 161-83-09-3 and 162-83-01-2 from Bar
Ilan University, Ramat-Gan, Israel.
2 R. R. AVTALION et al.:
I. Introduction
At the end of the last century and the beginning of this century, the prob
lems of immunity in lower vertebrates and the influence of environmental
temperature attracted attention for the first time (ERNST, 1890; WIDAL and
SICARD, 1897; METCHNIKOFF, 1901). However, relatively little work has been
done on this subject until recently. The early investigators were chiefly in
terested in the immuno-pathological problems. They immunized various
species of lower vertebrates essentially with bacterial vaccines; agglutinating,
neutralizing and protective antibodies were detected in their blood. The in
fluence of environmental temperature on the immune response was investigated,
since this subject represented great economical and theoretical importance.
Epizootic diseases were observed to occur in relation to the cold season of the
year, when the decrease or spontaneous increase of water temperature occurred
(SCHAPERCLAUS, 1965; BESSE et al., 1965; KLONTZ et al., 1965 WOOD,1966).
The immunological deficiency of fish, caused by their natural or experimental
stay in cold water, is now evident for both humoral and cellular immunity. In
this review we will focus on two points: We shall attempt (1) to explain the
mechanism by which the environmental temperature influences the immune
resistance of fish to pathogens, (2) to determine the chronological location of
this temperature-sensitive stage in the process of antibody formation, and to
make some approaches to the general antibody formation mechanism. We
conceive that some various complex phenomena which follow the immune
response in mammals could easily be studied in a lower vertebrate which is
more primitive, but still presents the immune phenomenon we are interested
in investigating in its less complex primitive development. The temperature
effect could be used as a switch-on-off to separate between some stages in the
immune process which normally overlap, and to permit an easy approach to
some aspects of the mechanism of antibody formation. Therefore, we suggest
that lower vertebrates, and especially fish, could be used as laboratory animals
for the study of the general mechanism of immunity.
II. Immune Resistance of Fish to Pathogens
A. Production of Agglutinins and Protective and Neutralizing
Antibodies in Fish
The prophylactic aspect of immunity was the chief interest of the original
investigators in fish pathology. They tried to avoid disease in fish by immuniz
ing them against pathogenic bacteria and viruses. The bacterial agent of
furunculosis in salmon, Aeromonas salmonicida, and the agent of infectious
dropsy in carp, Aeromonas punctata, were used for vaccine preparations. These
bacteria were the principal known pathogenic agents responsible for epizootic
diseases which affect large fish populations and cause heavy economic losses in
various parts of the world.
BABES and RIEGLER (1903), BERGMAN (1911) and AESER (1925) were among
the first to find specific immune agglutinins in fish. Their findings were confir-
Influence of Environmental Temperature on the Immune Response in Fish 3
med by many other investigators in various species of fish; e.g., NYBELIN
(1935 and 1968), PLISZKA (1939a, b), SNIESZKO and FRIDDLE (1949), SNIESZKO
(1953), KRANTZ et al. (1963, 1964), SUMMERFELT (1966) and EVELYN (1971).
Fish antibodies have been found to possess opsonizing, neutralizing, and pro
tective activities. KRANTZ et al. (1963) demonstrated a specific protection of
salmon to the LD50 of Aeromonas salmonicida after immunizing the fish with
killed bacterial vaccine. The protective antibodies resulting from this vaccina
tion were still present in the sera of vaccinated fish more than 24 months after
immunization. In the same manner, the transfer of sera from specifically im
munized trout (rainbow trout) to juvenile coho salmon trout (Oncorhynchus
kisutch) was found to confer passive immunity against the furunculus agent
Aeromonas salmonicida (SPENCE et al., 1965). Infectious dropsy in carp was
prevented by their vaccination against Aeromonas punctata and Pseudomonas
fluorescent. GONCHAROV and MIKRIAKOV (1968) found that 82 % of the fish
in the vaccinated group were specifically resistant to these pathogenic bacteria,
and only 16 to 20 % in the non-vaccinated group were resistant. Similar results
had previously been reported by SCHAPERCLAUS (1965), using a polyvalent
vaccine, and by SUMMERFELT (1966), in golden chiner, using formalin-killed
vaccine of Aeromonas liquefaciens. POST (1966) succeeded in immunizing the
rainbow trout to crude protein extracts from Aeromonas hydrophyla. The main
difficulties in such immunization is the practical application, which is not at all
economical. For this reason oral vaccination, which is relatively easy, seems to
be economically suitable for the immunization of fish on a large scale. This
method was studied by many investigators, in fish and other animals. In
some cases it was found to confer protective immunization; e.g., oral
immunization is currently used against killed Pasteurella multocida (HEDD
LESTON and REBERS, 1964) and beta-propiolactone-treated Newcastle virus in
chickens and turkeys (STONE et al., 1969). Many attempts were made to im
munize fish by oral introduction of antigens, and the results on the efficiency
of this method are still variable.
Evidence for establishment of acquired immunity following oral administra
tion of antigens was reported by some investigators. DUFF (1942) immunized
trout (Salmo clarki) which were kept at temperatures of 7 to 8.60 C, with a
chloroform-killed virulent culture of Aeromonas salmonicida, which was added
to the food in the span of 40-70 days. The trout were then moved to 19° C.
(At this temperature the trout were usually found to be more susceptible to
the furunculosis disease.) Duff found that the fish so immunized were more
resistant when exposed to external contaminations or injected with virulent
cultures. Ross and KLONTZ (1965) reported that oral administration of phenol
killed redmouth-agent vaccine to rainbow trout gave protection against the
LD50 of this pathogen. KLONTZ (1966) reported that lymphoid cells from
orally immunized brook trout were found to react specifically, by immuno
fluorescent method, with the orally administered antigen (sonicate of Aero
monas salmonicida).
Generally, a certain degree of protection, especially to natural infection,
4 R. R. AVTALION et al.:
was conferred to the fish by oral immunization. Large-scale experiments were
done on coho salmon (Oncorhynchus kisutch) orally immunized with Aero
monas salmonicida antigen preparation. The antigen was added to the food,
and fish were exposed to natural outbreaks of the disease which occurred in
May and June. The mortality in the immunized group was found to be signifi
cantly lower than in the non-immunized group (KLONTZ, 1967; 1968). Immunity
of short duration was established after oral immunization of rainbow trout
with an alum-precipitated endotoxin of an unnamed agent of redmouth
disease. The fish were then exposed to a natural June outbreak of the disease
and the number of their losses was very low in comparison to non-immunized
trout (Ross et al., 1966). POST (1966\ reported that prolonged oral antigenic
stimulation with heat-killed culture of Aeromonas hydrophyla conferred im
munity on rainbow trout. Detectable levels of antibodies and a certain resist
ance to the LD50 of live bacteria were obtained. This immunity, while signifi
cantly efficient, was relatively low in comparison to parenterally immunized
groups. Reduction of the number of losses due to natural infection (from
48-8 %) in juvenile coho salmon was obtained by FUJIHARA (1969) following
oral immunization of this fish against heat killed Myxobacterium (Chondro
coccus columnaris) culture.
Although the above-mentioned experiments were successful in part, other
investigators fail to confer immunity on fish by this method. KRANTZ (1964)
reported that feeding of chloroform-killed or living bacteria (Aeromonas sal
monicida) did not cause any antibody stimulation in brown trout, in comparison
to parenterally immunized and non-immunized control groups. Similar results
were obtained by SPENCE et al. (1965). They did not succeed in immunizing
the coho salmon to Aeromonas salmonicida by oral introduction of the antigen.
However, control groups which were passively and actively immunized by
parenteral injections displayed good immunity. KLONTZ (1969) reported that
commercial vaccine preparations for oral immunization, while giving good
results on experimental laboratory scales, when tested on a large scale in
different parts of the U.S.A. gave inconclusive results. SNIESZKO (1970), who
reviewed this subject, concluded that oral immunization is significantly better
than no immunization at all. We think the partial failure of oral immunization
could be explained in some cases on the basis of the findings of ADAM (1966)
which demonstrated the existence of a peritrophic membrane in the intestine
of the hag fish fMyxine glutinosa); this is assumed to act as a barrier for foreign
substances such as toxins and antigens produced by microorganisms and thus
prevent the entrance of external antigenical stimuli. However, intestinalleuco
cytes were reported to play an important role in the process of intestinal
digestion in fish (AVETIKYAN, 1968; GUELIN and LABLIN, 1964), and in oysters
they were found able to pass through the intestinal wall to the lumen when
containing phagocytized bacteria (FENG, 1966). SNIESZKO (1970) claimed the
possibility that bacterial antigens could be transported by leucocytes to the
antibody producing tissues, and thus stimulate them to produce circulating
antibodies.
Influence of Environmental Temperature on the Immune Response in Fish 5
B. Influence of the Seasonal Temperature on Fish Pathology
Infection and immunity in lower vertebrates were suggested to be in
relation to seasonal fluctuations in ambient temperature.
Epizootic diseases affecting large fish populations were reported to be
plainly related to the seasonal changes in water temperature, low river levels
and oxygen depletion. Under such conditions saprophytic organisms, ordinarily
common in the slime of fish, and polluted water, could eventually cause heavy
mortality as primary or secondary infective agents. MACKIE et al. (1935) reported
that the activity and spread of furunculosis disease in salmon and trout were
increased at water temperatures of 15 -18° C, whereas, the disease was relatively
inhibited when the water temperatures were 5° C and 21 ° C. This problem was
reviewed by MEYER (1970). He reported that greatest incidence of parasitic
and bacterial diseases in fishes occurred at the beginning of spring in the U.S.A.
April proved to be the most troublesome period of the year. Aeromonas in
fections, while most acute in April, were most prevalent during the summer.
The autumn period was found to be the quietest season. Similar observations
were reported by European investigators, who considered mainly bacterial
infections. BESSE et al. (1965) in France reported that the acute form (hydrops)
of infectious dropsy in carp was observed in the spring, whereas the chronic
form (ulcerous) of the disease occurred in summer. OJALA (1968) in Finland
reported that infections in the northern pike (Esox lucius) occurred in summer,
and Goncharov, in the USSR, found that the incidence of outbreaks of rubella
and Aeromonas infections increased when the temperature reached 28-30° C.
SCHAPERCLAUS in Germany (1965) reported that in the spring of 1964 there
were very low losses in fish due to diseases. He suggested that this popUlation
of fish had a hot summer in 1963, so the fish were healthy enough to survive
the winter and spring diseases.
In contrast to bacterial and parasitic infections (with the exception of
Ichthyophthirius infections, MEYER, 1970), epizootic diseases of viral etiology
and affecting hatchery-reared salmonids occurred especially at low tempera
tures (KLONTZ et al., 1965). The Sacramento River chinook disease breaks out
when the temperature of water is between 7.2 and 12.3° C; the diseases subside
spontaneously at the higher temperature and do not occur at the lower. The
sockeye disease also breaks out in similar conditions, while the infectious pan
creatic necrosis appears throughout the higher temperature range of 8.8-18° C.
However, only the younger and smaller fish were found to be susceptible to
these viral diseases. The older and larger fish were rarely found infected
(PARISOT et al., 1965). The immunological competence situation and seasonal
stressing-factors were assumed to provide the essential factors predisposing to
disease. In the case of epizootic viral diseases it was assumed that fish less than
three or four inches long were not immunologically competent in temperatures
of about 10° C (KLONTZ et aI., 1965). BISSET (1947a) suggested that the
defensive activity of the host increases at high temperature, but at the same
time the offensive power of the microbe also increases. This idea was devel9ped
6 R. R. AVTALION et al.:
by SNIESZKO 1954; he hypothesized that the heavy mortalities which occurred
in warm water ponds were due to differences between metabolic rates of
pathogens and their hosts, since the host could not develop immunity at low
temperatures. Physiological conditions, genetic resistance, physical stresses,
sex and ages of fish were also considered as predisposing factors (SNIESZKO,
1958). However, MEYER (1970) suggested that environmental stresses provide
the major impetus for the development of certain diseases in fishes under pond
conditions rather than their specific immune deficiency related to fluctuations
in water temperature. (This questinn will be discussed further below.)
C. Resistance of Experimentally Inoculated, Non-Immunized and
Actively Immunized Fish, with Pathogenic and Non-Pathogenic Bacteria,
at Various Temperatures
Active immunization was reported to confer specific protection on lower
vertebrates. BISSET (1947c) pointed out that active immunization of frogs at
20° C confers considerable protection against a lethal dose of virulent bacteria,
and no protection was afforded by immunization at 8° C. Saprophytic bacteria
injected into fish gave highest mortality when the fish were kept at 23 ° C,
while in those fish kept at 10° C no mortality occurred. Protection against the
LD50 of Aeromonas salmonicida was obtained in brook trout and brown trout
injected with a killed bacteria and adjuvant and kept at 11 ° C (KRANTZ et al.
1963)·
Similar experiments were carried out in our laboratory on carp. The in
fluence of the environmental temperature on pathogenic effects of a pseudo
monas bacterium was investigated. We isolated this bacterium from skin
pustules of carp, and according to its bacteriological and biochemical charac
teristics it was found to belong to the Aeromonas punctata-liquefaciens group
(MALIK, 1970). This bacterium was found to be pathogenic to carp, provoking
lethality and hemorrhagic and necrotic internal and external lesions. We
isolated the same bacterium from the slime of other fish. The pathogenicity
of this bacterium was found to be influenced by water temperature, crowding
of fish and limitation of their movements, and by specific preimmunization.
A formalin-treated vaccine was prepared from fresh cultures of these bac
teria, and a group of 30 carp was immunized by intraperitoneal injections of
0.5 ml of a saline suspension containing 109 bacteria. The fish were kept for
8 days at 25° C (optimal temperature) and then moved, before appearance of
the first circulating antibodies, to low temperature (12° C) for 12 days. (The
first circulating antibodies were found to appear between the 9th and 10th days
after the first antigenic stimulation.) Rising titers of antibody of 1 : 32 to 1: 512,
detected by agglutination, were developed at this low temperature. The fish
were then removed to a high temperature (250 C) after being inoculated intra
muscularly with a dose of 5 X 109 living bacteria. Another group of 10 carp not
previously immunized underwent the same treatment. The results, as sum
marized in Table 1, showed that the active immunization developed at low
Influence of Environmental Temperature on the Immune Response in Fish 7
Table 1. Resistance of immunized and non-immunized carp inoculated with lethal doses
of Aeromonas punctata. The numbers indicate quantities of fish which died or showed
inflammation or necrosis in the area of injection
Immunized fish Non-immunized fish
days a days a
(total of 30) (total of 10)
6 10 14 14
Mortality o 6
Inflammation or necrosis in
the area of injection o o o o 3
Refractory 30 29 29 29
a Days after inoculation of 5 X 109 living bacteria.
Table 2. Temperature effect on development of infection in non-immunized fish. The
numbers represent the quantities of dead fish out of groups of ten fish kept at different
temperatures. The fish were inoculated with a suspension of living Pseudomonas punctata
containing 109 bacteria
Days after inoculation
2 3 7
Water temperature
Group 1 25° C 9 10
Group 2 16° C 0 5 6 10
Group 3 12° C 0 0 1
Group 4 12° C for 48 h then transferred to 25° C 10
temperatures gave protection to fish inoculated with lethal doses of pathogenic
bacteria.
The environmental temperature effect on the pathogenicity of this bac
terium was investigated in four other groups of 10 carp each. The carp were
inoculated intraperitoneally with 0.5 ml of a suspension of Pseudomonas punc
tata containing 109 living bacteria. They were kept at the various tempera
tures; 25, 16 and 120 C, in small cages (30 X 20 X 40 cm) which were submerged
in asbestos thermoregulated tanks of 750 liters equipped with a continuous
flow of water and air. The results presented in Table 2 show that all the fish
in the group kept at 250 died after one week, whereas very low mortality of
1 in 10 occurred at low temperature (120 C). However, the fish that were kept
at low temperature for 48 hours and then transferred to high temperature
(250 C) died during the first 24 hours. These results corroborate the above
mentioned statement that the mortality of fish occurs in nature essentially
when the water temperature undergoes a spontaneous increase after the cold
season. We suggest that in both natural and experimental cases the develop
ment of immunity and the bacterial proliferation competed. Therefore, when