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In vivo and in vitro anaphylactic type reactions in fish
DEVELOPMENTAL AND COMPARATIVE IM/~UNOLOGY, Vol. 4, pp. 55-64, 1980. 0145-305X/80/01055-I052.00/0 Printed in the USA. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.
IN VIVO AND IN VITRO ANAPHYLACTIC TYPE REACTIONS IN FISH Beverly A. Goven, Donald L. Dawe and John B. Gratzek, Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602
ABSTRACT.
Anaphylactic type reactions occurred in channel catfish (Ictalurus punctatus Rafinesque) and goldfish (Carassius auratus L.) following immunization and challenge with several protein antigens. The reactions were specific f o r the sensitizing antigen and could be passively transferred to non-sensitized recipients with serum from sensitized f i s h . In channel catfish the reaction consisted of d i s o r i e n t a t i o n , vertical swimming, increased opercular movement and gasping. In the goldfish the reaction consisted of clamping of the fins except the pectorals, loss of equilibrium, and increased defecation. Some deaths occurred with the catfish. The effects of various concentrations of histamine on the rhythmicity and tonus of goldfish and catfish i n t e s t i n a l smooth muscle were also measured in v i t r o . Isolated i n t e s t i n a l sections were maintained in a Schultz-Dale chamber which was connected to a physiograph. Serum d i l u t i o n s of histamine, ranging from I00 g to I000 g were tested. A dose response was observed.
INTRODUCTION Generalized immediate h y p e r s e n s i t i v i t y reactions have been well described in higher vertebrates; however, the existence of this type of reaction in lower vertebrates has been reported, but requires v e r i f i c a t i o n ( I - 5 ) . To date, the only modern report of induction of anaphylaxis in teleosts is that of Dryer and King, in which they described behavioral patterns symptomatic of anaphylaxis a f t e r appropriate antigenic s e n s i t i z a t i o n ( I ) . Following the intraperitoneal injections of a shocking dose of horse serum or egg albumin, these workers were able to produce anaphylactic s e n s i t i z a t i o n in four species of f i s h . The ensuing reactions were manifested by increased movements of the g i l l c l e f t s , fanning and folding of the f i n s , and loss of equilibrium. These reactions were specific for the sensitizing agents. Several other workers have since f a i l e d to produce anaphylaxis in teleosts and thereby verify these findings (2,4). An unsuccessfu attempt to produce anaphylaxis in the elasmobranch Raja clavata L. has also been reported (6).
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During the routine immunization of channel c a t f i s h , I c t a l u r u s punctatus Rafinesque, abnormal behavior and death were noted when i n t r a p e r i t o n e a l booster i n j e c t i o n s were administered. The reaction began approximately 15 minutes following the booster dose and lasted about 60 minutes. No abnormal behavior was noted in saline injected controls. Due to the nature of the reaction, anaphylactic s e n s i t i z a t i o n was suspected as the cause of the observed response. Further studies were i n i t i a t e d to determine i f the response was indeed an immediate h y p e r s e n s i t i v i t y , Type I reaction (7). In mammalian systems, histamine, serotonin, kinins and slow reacting substance (SRS-A) have been implicated as mediators of anaphylaxis. Variations e x i s t s among species as to which of the four substances assume importance. However, in the appropriate host, each of these can cause contraction of smooth muscle (8,9,10). In the t e l e o s t system, histamine has been shown to cause reactions in vivo s i m i l a r to those evidenced in anaphylactic-type reactions ( I ) . In v i t r o studies indicated that histamine, in concentrations which usually cause contraction of mammalian i n t e s t i n e , stimulated t e l e o s t stomach and intestine (I, II). In contrast, in the elasmobranchs, histamine has been shown to be i n a c t i v e in both in vivo and in v i t r o studies (6,11). Since we had observed differences in the gross sign of anaphylactic reactions in the c a t f i s h and g o l d f i s h , we proposed that there were d i f f e r e n t target organs in these f i s h . This part of the study was undertaken to determine i f isolated gut segments from c a t f i s h and goldfish would respond d i f f e r e n t l y to stimulation by histamine in v i t r o .
MATERIALS AND METHODS Experimental animals: Channel c a t f i s h , I c t a l u r u s punctatus, Rafinesque weighing approximately 8g, were maintained in 38 l i t e r glass aquaria. Common g o l d f i s h , Carassius auratus, L. weighing approximately 25g, were maintained in 76 l i t e r glass aquaria. All aquaria were equipped with undergravel f i l t e r s . Aeration was provided by a central a i r supply attached to the a i r l i f t system of the gravel f i l t e r . Water temperatures ranged from 23-25 C. Antigen preparations and administration f o r in vivo study: I . Solubilized Protozoa (SP), the c i l i a t e , Tetrahymena p y r i f o r m i s , was maintained in culture (12). Organisms were t h r i c e washed with saline and concentrated by c e n t r i f u g a t i o n . The concentrated organisms were then sonicated f o r 3 minutes at power s e t t i n g 38 on a s o n i f i e r cell d i s r u p t o r 125 (Branson Sonic Power Company). Protein concentrations were determined by the Lowry method (13). 2. Soluble Proteins. A stock solution of bovine serum albumin (BSA) was prepared by d i s s o l v i n g lOOmg of c r y s t a l l i n e BSA in lOOml of O.Olm phos-
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phate buffered s a l i n e (PBS). Working c o n c e n t r a t i o n s were prepared by d i l u t i o n of the stock s o l u t i o n w i t h PBS. V e r s a t o l , a serum reference c o n t r o l prepared from human sera, was rec o n s t i t u t e d and d i l u t e d w i t h s t e r i l e d i s t r i l l e d water to the desired concentrations. (General D i a g n o s t i c s , D i v i s i o n of Warner & Lambert, Morris P l a i n s , New Jersey 07950). Channel c a t f i s h received i n t r a p e r i t o n e a l i n j e c t i o n s of lOpg of SP. Control f i s h received 0. I ml s a l i n e . F i f t e e n days l a t e r the s e n s i t i z e d f i s h and c o n t r o l f i s h were i n j e c t e d w i t h e i t h e r lO~g of SP or BSA. G o l d f i s h received i n t r a p e r i t o n e a l i n j e c t i o n s of 36~g of V e r s a t o l . Control f i s h received 0. I ml s a l i n e . Two weeks a f t e r s e n s i t i z a t i o n , f i s h were i n j e c t e d w i t h e i t h e r 36~g of homologous Versatol or 36ug of BSA. Saline i n j e c t e d c o n t r o l s were d i v i d e d i n t o 2 groups, one challenged w i t h BSA and one challenged with Versatol. T r a n s f e r experiments: Channel c a t f i s h were s e n s i t i z e d w i t h lO~g V e r s a t o l . F i f t e e n days l a t e r these f i s h were bled by severance of the caudal peduncles and the blood c o l lected in c a p i l l a r y tubes. The serum was harvested and 0 . I ml t r a n s f e r r e d to u n s e n s i t i z e d f i s h by i n t r a p e r i t o n e a l i n j e c t i o n . Serum from u n s e n s i t i z e d f i s h also was t r a n s f e r r e d . Twenty-four hours l a t e r both groups of f i s h were i n j e c t e d w i t h IU~g of V e r s a t o l . As a c o n t r o l , f i s h p r e v i o u s l y s e n s i t i z e d w i t h Versatol were challenged with BSA. This experiment was repeated using l a r g e r (25g) c a t f i s h . S i m i l a r serum t r a n s f e r studies were also conducted. I n t e s t i n e preparation f o r in v i t r o study: The i n t e s t i n e s of g o l d f i s h and c a t f i s h were removed and flushed w i t h phosphate buffered s a l i n e (pH 7.2) to e l i m i n a t e the contents. Isolated sections of approximately 3cm lengths were cut and placed in Clark-FrogRinger's s o l u t i o n at 37C (14). A section was then prepared and secured in a Schultz-Dale chamber according to the method of Weir (15). The chamber was placed in a water bath to maintain the temperature of 37C. Oxygen was bubbled i n t o the chamber. The r e a c t i o n s of the i s o l a t e d i n t e s t i n a l sections were measured and recorded on a physiograph f o u r (E. & M. Instrument Co. I n c . , Houston, TX.) connected to the chamber through a Myograph A (Narco Bio-System I n c . , Houston, TX.) Baseline rhythm and tonus were recorded f o r f i v e minutes, a f t e r which various c o n c e n t r a t i o n s o f histamine diphosphate (Mann Research Laboratories D i v i s i o n of Becton-Dickinson, NY) ranging from lO0~g were added. Between a d d i t i o n s of histamine, the chamber was drained and prewarmed Clark-FrogRinger's s o l u t i o n was added. The section was then allowed to r e t u r n to basel i n e tonus before the a d d i t i o n of a s e r i a l histamine d i l u t i o n . The i n t e s t i n a l r e a c t i o n s were monitered f o r f i v e minutes during each drug a d d i t i o n .
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RESULTS
The sensitized catfish receiving homologous challenge showed an abnormal behavior pattern consisting of d i s o r i e n t a t i o n , v e r t i c a l swimming and "labored" breathing evidenced by gasping and increased opercular movement. Severe respiratory distress was followed by death in one-third of the fish injected. Those f i s h receiving a heterologous antigen and the unsensitized controls which received either of the antigens did not show any abnormal behavior, other than i n i t i a l alarm reactions which quickly subsided. (Table I ) . In goldfish, anaphylactic-type behavior was i n i t i a t e d approximately I0 minutes following homologous challenge with 36~g of Versatol. Heterologous challenge and saline control groups did not manifest "shock" behavior. The behavioral pettern in goldfish was somewhat d i f f e r e n t than that observed in channel catfish. On administration of the "shocking" dose, sensitized goldf i s h became disoriented, often bumping into the sides of the tank. Balance was affected in that the f i s h assumed e i t h e r head-up or down posture. All the fins except the pectorals were drawn t i g h t to the body, possibly contributing to the loss of balance (Figure I ) . The sensitized f i s h also showed increased defecation. Fish were unable to maintain balance and dropped to the bottom of the aquaria where they remained for up to four hours (Table I ) . These results indicate that the reaction is specific for the s e n s i t i z i n g antigen. Classically, Type I h y p e r s e n s i t i v i t i e s can be transferred from sensitized donor to non-sensitized recipient with serum. Anaphylactic-type behavior was observed only in the c a t f i s h that received serum from sensitized donors by homologous challenge. Serum transfer studies using Versatol and BSA as antigens were also conducted with goldfish. The results were similar to those observed in c a t f i s h , in that anaphylactic-type reactions were manifested in those f i s h receiving sensitized serum and homologous antigen (Table I ) . The results of the in v i t r o study indicated that both goldfish and c a t f i s h intestines were sensitive-~to histamine; however, a difference in s e n s i t i v i t y between the two was evidenced. In goldfish, lO0~g appeared to be near a threshold dose required for a response. Doses higher all gave a response, the only difference being the degree and i n i t i a t i o n of response. At all doses the response occurred within a minute a f t e r the addition of histamine. The i n i t i a l response appeared to be a contraction followed by relaxation of the smooth muscle indicated by the decrease in tonus. At all doses, the muscle returned to the baseline rhythmic contraction within 5 minutes (Figure 2). The i n t e s t i n e of the catfish was not as sensitive to the action of histamine. At the lO0~g dose there was l i t t l e i n t e r r u p t i o n of the baseline tonus and rhythmicity. The intestines were affected by higher doses, but not to the degree as the goldfish i n t e s t i n e s . The response in the catfish also appeared to be a relaxation of the smooth muscle as indicated by a decrease in tonus. The i n i t i a t i o n of response was longer than that of the goldfish (Figure 3). In both f i s h , the response f i r s t appeared as a contraction followed by a rapid loss in the tonus to relaxation.
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TABLE I Reactions of Channel Catfish, I c t a l u r u s punctatus and Goldfish, Carassius auratus to Various Antigenic Challenges
CATFISH
Treatment SPa
Challenge
GOLDFISH
Reaction
SP
IH (6/6) e
BSA
NR (0/4)
SP Serumd
SP
IH (4/4)
BSAb
BSA
IH (4/4)
V
NR (0/6)
BSA Serumd
BSA
IH (5/6)
Vc
V
IN (6/6)
BSA
NR (0/6)
V Serumd
V
IH (6/6)
Saline
SP
NR ( 0 / I 0 )
BSA
NR (0/5)
V
NR (0/5)
Treatment V
Challenge
Reaction
V
IH (13/15)
BSA
NR ( I / I 0 )
V Serumd
V
IH (4/5)
Saline
BSA
NR (0/5)
V
NR (0/5)
a S o l u b i l i z e d Protozoa (SP) b
Bovine Serum Albumin (BSA)
c Versatol (V)- General Diagnostics, D i v i s i o n of Warner and Lambert, Morris Plains, N.J. 07950 U.S.A. d 0. I ml of serum from sensitized f i s h injected IP in r e c i p i e n t f i s h 24 hr. before challenge. e IH - immediate h y p e r s e n s i t i v i t y type reaction. NR- no reaction. Numerator ind~atesnumber of f i s h showing reactions, denominator is the number of f i s h per group.
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Figure i. Top: Normal goldfish with full display of fins and tail and horizontal swimming attitude° Bottom: Goldfish previously sensitized with Versatol and then challenged. Note the clamping of all of the fins except the pectoral fins and the head-down swimming attitude.
1L1221
N
Figure 2° In vitro response of goldsfish gut to histamine° ao Baseline response of gut in Clark-Frog-Ringer's Solution bo -f. Respnse of dose to histamine. = Introduction of histamine. i block = 6 seconds°
HTf7 i ,! iit!-
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~ ~ ~ ~ : ~~L~-;"~'~r
~-~4-~-Ill~Gdimi
.
t::t~.~
-, .
.'
.--~_-~
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', '/t¢<-I ~
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E.. M_,F-I:ItL+!I£-IT::: -~.! !1 ".'lt~ ! t IT£+~ TtT:~i --'
t',i :~-rH-N_+~4T-£----:
~MH.
,
I ! '. I 1-L:441
,[ .......... ~ . + + T f ~ - : - I ~
Iiiii~,']-rq.I
~-t2-~_122+21ii_121ut!211.t:
I
i
I
,
I
: b
,
,
.
:--+
I-i-~
.-
'
PF~-H iF'r ~ ~;2-H~TTftH~!::::
~
.......
+
-
-
,
:;::::1:2:-:.:
•
--
: ::t! :.2~
Figure 3. In vitro response of catfish gut to histamine° a. Baseline response of gut in Clark-Frog-Ringer's Solution. b. -e. Response of dose to histamine° 1 block = 6 seconds.
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DISCUSSION Type I h y p e r s e n s i t i v i t y reactions are characterized by antigen specif i c i t y and passive transfer of the reaction with sensitized serum. Both of these t r a i t s were evidenced in the induction of anaphylaxis in f i s h . The'~hock" reaction was only observed in fish challenged with the homologous s e n s i t i z i n g antigen. Fish receiving a heterologous antigen or those receiving antigen for the f i r s t time did not manifest the shock reaction. Shock behavior was also manifested following transfer of sensitized serum and subsequent challenge with homologous antigen. Both characteristics of Type I h y p e r s e n s i t i v i t y were evidenced in these experiments, indicating that the observed reactions could be c l a s s i f i e d as anaphylactic. From the observed results in these studies, we feel that immediate type h y p e r s e n s i t i v i t y reactions were occurring in the f i s h . The reactions were specific for the sensitizing antigen and could be transferred with serum. This suggests that a c i r c u l a t i n g serum factor is involved with the reaction. Flectcher and Baldo (16) have reported an immediate skin reaction in marine f l a t f i s h and have suggested that the reaction may be mediated via C-Reactive P r o t e i n - l i k e protein in plaice serum. In mammals, anaphylaxis is mediated by vasoactive amines liberated by the degranulating of mast cells a f t e r reaction of the homocytotropic antibody with antigen. Mast-like cells have been described in f i s h ; however, the function has not yet been discerned (17). Clearly, the mechanism of immediate h y p e r s e n s i t i v i t y in teleosts, whether a non specific antibody independent mechanism of mast cells a c t i v a t i o n or tissue f i x i n g a n t i bodies, has yet to be delineated. Although both types of f i s h manifested a behavioral change following administration of the sensitizing antigen, d i f f e r e n t patterns of reactions were observed between the catfish and goldfish. In c a t f i s h the reaction was evidenced by "labored" breathing, indicating involvement of the respiratory system. The reaction in goldfish was manifested by defecation implicating smooth muscle involvement. The appearance of clamped fins is speculated to be a voluntary contraction of the skeletal muscle in response to the contraction of the smooth muscle. These differences suggest the p o s s i b i l i t y that d i f f e r e n t target organs are affected with perhaps d i f f e r e n t pharmacological mediators produced between species. This precedent is observed among the mamalian species (10,19). In dogs, for example, the early stage of anaphylaxis is often manifested by diarrhea and defecation, due to the action of vasoactive amines on the i n t e s t i n a l smooth muscle; whereas in guinea pigs the reactions presents as contraction of the smooth muscle of the bronchioles with dyspnea (9,10). The v a r i a b i l i t y in response is due not only to the target organ affected, but also to the action of the prominent mediator released during the reaction. Of the mediators, histamine, serotonin, kinins, and slow reacting substance (SRS-A), variation exists among species as to which of these is the principal effector. Histamine is regarded as the prime mediator in mammals with the exception of the bovine, in which SRS-A and kinins are the main mediators (I0).
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I
Histamine is regarded as a smooth muscle contractor; however, the effects also vary among species. Dreyer ( I I ) showed that Img of histamine caused contractions in the small i n t e s t i n e of the minnow, Fundalus h e t e r o c l i t u s , while the results of t h i s study showed a relaxation of i n t e s t i n a l smooth muscle in both the catfish and goldfish when exposed to a Img dose of histamine. These differences as well as the difference of s e n s i t i v i t y between the catfish and goldfish i n t e s t i n e , may be due to a species v a r i a t i o n . Histamine a c t i v i t y measured in v i t r o , using the Schultz-Dale method, caused guinea pig uterus to contract on exposure IOng, while rat uterus relaxed when exposed to a similar dose (8). Guinea pig ileum contracted and then repaidly relaxed when exposed to the drug. These differences coupled with the fact that certain histamine induced changes ( i . e . , gastric acid secretion) are not blocked by classical antihistamines ( i . e . , benadryl, pyribenzamine), have led workers to postulate the existence of two types of histamine receptors HI and H2 (19, 20). I t is now known that histamine stimulates both HI and H2 receptors to cause vasodilation and vasoconstriction (20). The relaxant effect of histamine on contracted rat uterus has been found to be mediated via H2 receptors. Dreyer and King ( I ) observed that f i s h administered histamine showed reactions s i m i l a r to the anaphylactic-type shock induced by sensitizing antigens. The injections of classical antihistamines, such as benadryl and pyribenzamine before and subsequent to shocking procedures had l i t t l e effect on the duration and s e n s i t i v i t y of e i t h e r histamine or antigen-produced shock reactions ( I ) . The observations of Dreyer and King in addition to the relaxation effect reported in t h i s study lead to the hypothesis that receptors of histamine in the i n t e s t i n e of f i s h are similar to the mammalian systems. I t appears possible that H2 l i k e receptors for histamine are located in the i n t e s t i n e of f i s h . Studies on the blocking of HI receptors with classical antihistamines and of H2 receptors with pharamacological agents such as cimetidine and burimanide, are required to further delineate the nature of histime receptors in f i s h intestine.
This study was supported by funds from the Georgia Veterinary Medical Experiment Station. We wish to thank Dr. N. Booth and Dr. D. Coulter of the Department of Physiology and Pharmacology for t h e i r help during t h i s study.
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REFERENCES I.
Dreyer, N.B. and King, J.W. 277, 1948.
2.
Good, R.A. and Papermaster, B.W. Ontogeny and Phylogeny of Adaptive Immunity. Adv. Immunol. 4 I , 1964.
.
4.
Anaphylaxis in the Fish.
J. Immunol. 60,
Clem, L.W. and Leslie, G.A. Phylogeny of Immunolobulin Structure and Function. In: Immunology and Development. M. Adinolfi (ed). Spastics Society Medical Publications, London. 62, 1969. Harris, J.E. The Apparent I n a b i l i t y of Cyprinid Fish to Produce Skin Sensitizing Antibody. J. Fish Biol. 5, 535, 1973.
.
Gershwin, L.J. The Phylogentic Development of Anaphylactic A c t i v i t y and Homocytotropic Antibody. De__v. & Comp. Immunol. 2, 595, 1978.
.
Spoujitch, V. Spuzic, I . , and Bata, A. Les Reactions Anaphylactiques Chez Les Animaux Poikilothermes, Acta Allergologica 25, 259, 1970.
.
Coombs, R.R.A. and Gell, P.G.H. (ed.) Clinical Aspects of Immunology. Blackwell S c i e n t i f i c Publications, Oxford, 575, 1968.
.
Levy, D.A. 1974. Histamine and Serotonin. In: Mediators of Inflammation. G. Weissmann Ed. Plenum Press. New York Chapter 5.
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Smith, H.A., Jones, T.C. and Hunt, R.D. 1972. Lea and Febiger. Phila.
Veterinary Pathology.
I0.
Tizard, I.R. 1977. An Introduction to Veterinary Immunology. Saunders Co., Phila.
W.B.
II.
Dreyer, N.B. 1948. Histamine and Posterior P i t u i t a r y Actions on Elasmobranch and Teleost Smooth Muscle. Arch. Int. Pharamacodyn. 82:440-443.
12.
Klaas, J. Two Gastric Mucin Cultivation Media and Chemically Defined Maintenance Medium for Balantidium Col. J. Parasit. 60, 907, 1974.
13.
Lowry, O.H., Rosenbrough, N.G., Farr, A.L., and Randall, R.J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem. 193, 265.
14.
Staff Department Pharmacology, University of Edinburgh. 1968. Pharmacological Experiments on Isolated Preparations. E. and S. Livingstone Ltd. Edinburgh and London: 120-121.
15.
Weir, D.M. 1978. Handbook of Experimental Immunology. S c i e n t i f i c Publications. London.
16.
Fletcher, T.C. and Baldo, B.A. Immediate Hypersensitivity Responses in Flatfish. Science 185, 360, 1974.
17.
E l l i s , A.E.
3rd Edition Blackwe
The Leucocytes of Fish: A review. J. Fish Biol. II 453, 1977.
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18.
Smith, H.A., Jones, T.C. and Hunt, R.D. Veterinary Pathology. Febiger. Phila. 1972.
Lea and
19.
Ash, A.S.F., and Schild, H.O. 1966. Receptors Mediating Some Actions of Histamine. Br. J. Pharmacol and Chemother. 27:427-439.
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Eyre, P., and Wells, P.W. 1973. Histamine Hp - Receptors Modulate Systemic Anaphylaxis: A Dual Cardiovascular Action of~Histamine in Claves. Br. J. Pharmcol. 49:364-367.
IN VIVO AND IN VITRO ANAPHYLACTIC TYPE REACTIONS IN FISH Beverly A. Goven, Donald L. Dawe and John B. Gratzek, Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602
ABSTRACT.
Anaphylactic type reactions occurred in channel catfish (Ictalurus punctatus Rafinesque) and goldfish (Carassius auratus L.) following immunization and challenge with several protein antigens. The reactions were specific f o r the sensitizing antigen and could be passively transferred to non-sensitized recipients with serum from sensitized f i s h . In channel catfish the reaction consisted of d i s o r i e n t a t i o n , vertical swimming, increased opercular movement and gasping. In the goldfish the reaction consisted of clamping of the fins except the pectorals, loss of equilibrium, and increased defecation. Some deaths occurred with the catfish. The effects of various concentrations of histamine on the rhythmicity and tonus of goldfish and catfish i n t e s t i n a l smooth muscle were also measured in v i t r o . Isolated i n t e s t i n a l sections were maintained in a Schultz-Dale chamber which was connected to a physiograph. Serum d i l u t i o n s of histamine, ranging from I00 g to I000 g were tested. A dose response was observed.
INTRODUCTION Generalized immediate h y p e r s e n s i t i v i t y reactions have been well described in higher vertebrates; however, the existence of this type of reaction in lower vertebrates has been reported, but requires v e r i f i c a t i o n ( I - 5 ) . To date, the only modern report of induction of anaphylaxis in teleosts is that of Dryer and King, in which they described behavioral patterns symptomatic of anaphylaxis a f t e r appropriate antigenic s e n s i t i z a t i o n ( I ) . Following the intraperitoneal injections of a shocking dose of horse serum or egg albumin, these workers were able to produce anaphylactic s e n s i t i z a t i o n in four species of f i s h . The ensuing reactions were manifested by increased movements of the g i l l c l e f t s , fanning and folding of the f i n s , and loss of equilibrium. These reactions were specific for the sensitizing agents. Several other workers have since f a i l e d to produce anaphylaxis in teleosts and thereby verify these findings (2,4). An unsuccessfu attempt to produce anaphylaxis in the elasmobranch Raja clavata L. has also been reported (6).
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During the routine immunization of channel c a t f i s h , I c t a l u r u s punctatus Rafinesque, abnormal behavior and death were noted when i n t r a p e r i t o n e a l booster i n j e c t i o n s were administered. The reaction began approximately 15 minutes following the booster dose and lasted about 60 minutes. No abnormal behavior was noted in saline injected controls. Due to the nature of the reaction, anaphylactic s e n s i t i z a t i o n was suspected as the cause of the observed response. Further studies were i n i t i a t e d to determine i f the response was indeed an immediate h y p e r s e n s i t i v i t y , Type I reaction (7). In mammalian systems, histamine, serotonin, kinins and slow reacting substance (SRS-A) have been implicated as mediators of anaphylaxis. Variations e x i s t s among species as to which of the four substances assume importance. However, in the appropriate host, each of these can cause contraction of smooth muscle (8,9,10). In the t e l e o s t system, histamine has been shown to cause reactions in vivo s i m i l a r to those evidenced in anaphylactic-type reactions ( I ) . In v i t r o studies indicated that histamine, in concentrations which usually cause contraction of mammalian i n t e s t i n e , stimulated t e l e o s t stomach and intestine (I, II). In contrast, in the elasmobranchs, histamine has been shown to be i n a c t i v e in both in vivo and in v i t r o studies (6,11). Since we had observed differences in the gross sign of anaphylactic reactions in the c a t f i s h and g o l d f i s h , we proposed that there were d i f f e r e n t target organs in these f i s h . This part of the study was undertaken to determine i f isolated gut segments from c a t f i s h and goldfish would respond d i f f e r e n t l y to stimulation by histamine in v i t r o .
MATERIALS AND METHODS Experimental animals: Channel c a t f i s h , I c t a l u r u s punctatus, Rafinesque weighing approximately 8g, were maintained in 38 l i t e r glass aquaria. Common g o l d f i s h , Carassius auratus, L. weighing approximately 25g, were maintained in 76 l i t e r glass aquaria. All aquaria were equipped with undergravel f i l t e r s . Aeration was provided by a central a i r supply attached to the a i r l i f t system of the gravel f i l t e r . Water temperatures ranged from 23-25 C. Antigen preparations and administration f o r in vivo study: I . Solubilized Protozoa (SP), the c i l i a t e , Tetrahymena p y r i f o r m i s , was maintained in culture (12). Organisms were t h r i c e washed with saline and concentrated by c e n t r i f u g a t i o n . The concentrated organisms were then sonicated f o r 3 minutes at power s e t t i n g 38 on a s o n i f i e r cell d i s r u p t o r 125 (Branson Sonic Power Company). Protein concentrations were determined by the Lowry method (13). 2. Soluble Proteins. A stock solution of bovine serum albumin (BSA) was prepared by d i s s o l v i n g lOOmg of c r y s t a l l i n e BSA in lOOml of O.Olm phos-
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phate buffered s a l i n e (PBS). Working c o n c e n t r a t i o n s were prepared by d i l u t i o n of the stock s o l u t i o n w i t h PBS. V e r s a t o l , a serum reference c o n t r o l prepared from human sera, was rec o n s t i t u t e d and d i l u t e d w i t h s t e r i l e d i s t r i l l e d water to the desired concentrations. (General D i a g n o s t i c s , D i v i s i o n of Warner & Lambert, Morris P l a i n s , New Jersey 07950). Channel c a t f i s h received i n t r a p e r i t o n e a l i n j e c t i o n s of lOpg of SP. Control f i s h received 0. I ml s a l i n e . F i f t e e n days l a t e r the s e n s i t i z e d f i s h and c o n t r o l f i s h were i n j e c t e d w i t h e i t h e r lO~g of SP or BSA. G o l d f i s h received i n t r a p e r i t o n e a l i n j e c t i o n s of 36~g of V e r s a t o l . Control f i s h received 0. I ml s a l i n e . Two weeks a f t e r s e n s i t i z a t i o n , f i s h were i n j e c t e d w i t h e i t h e r 36~g of homologous Versatol or 36ug of BSA. Saline i n j e c t e d c o n t r o l s were d i v i d e d i n t o 2 groups, one challenged w i t h BSA and one challenged with Versatol. T r a n s f e r experiments: Channel c a t f i s h were s e n s i t i z e d w i t h lO~g V e r s a t o l . F i f t e e n days l a t e r these f i s h were bled by severance of the caudal peduncles and the blood c o l lected in c a p i l l a r y tubes. The serum was harvested and 0 . I ml t r a n s f e r r e d to u n s e n s i t i z e d f i s h by i n t r a p e r i t o n e a l i n j e c t i o n . Serum from u n s e n s i t i z e d f i s h also was t r a n s f e r r e d . Twenty-four hours l a t e r both groups of f i s h were i n j e c t e d w i t h IU~g of V e r s a t o l . As a c o n t r o l , f i s h p r e v i o u s l y s e n s i t i z e d w i t h Versatol were challenged with BSA. This experiment was repeated using l a r g e r (25g) c a t f i s h . S i m i l a r serum t r a n s f e r studies were also conducted. I n t e s t i n e preparation f o r in v i t r o study: The i n t e s t i n e s of g o l d f i s h and c a t f i s h were removed and flushed w i t h phosphate buffered s a l i n e (pH 7.2) to e l i m i n a t e the contents. Isolated sections of approximately 3cm lengths were cut and placed in Clark-FrogRinger's s o l u t i o n at 37C (14). A section was then prepared and secured in a Schultz-Dale chamber according to the method of Weir (15). The chamber was placed in a water bath to maintain the temperature of 37C. Oxygen was bubbled i n t o the chamber. The r e a c t i o n s of the i s o l a t e d i n t e s t i n a l sections were measured and recorded on a physiograph f o u r (E. & M. Instrument Co. I n c . , Houston, TX.) connected to the chamber through a Myograph A (Narco Bio-System I n c . , Houston, TX.) Baseline rhythm and tonus were recorded f o r f i v e minutes, a f t e r which various c o n c e n t r a t i o n s o f histamine diphosphate (Mann Research Laboratories D i v i s i o n of Becton-Dickinson, NY) ranging from lO0~g were added. Between a d d i t i o n s of histamine, the chamber was drained and prewarmed Clark-FrogRinger's s o l u t i o n was added. The section was then allowed to r e t u r n to basel i n e tonus before the a d d i t i o n of a s e r i a l histamine d i l u t i o n . The i n t e s t i n a l r e a c t i o n s were monitered f o r f i v e minutes during each drug a d d i t i o n .
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RESULTS
The sensitized catfish receiving homologous challenge showed an abnormal behavior pattern consisting of d i s o r i e n t a t i o n , v e r t i c a l swimming and "labored" breathing evidenced by gasping and increased opercular movement. Severe respiratory distress was followed by death in one-third of the fish injected. Those f i s h receiving a heterologous antigen and the unsensitized controls which received either of the antigens did not show any abnormal behavior, other than i n i t i a l alarm reactions which quickly subsided. (Table I ) . In goldfish, anaphylactic-type behavior was i n i t i a t e d approximately I0 minutes following homologous challenge with 36~g of Versatol. Heterologous challenge and saline control groups did not manifest "shock" behavior. The behavioral pettern in goldfish was somewhat d i f f e r e n t than that observed in channel catfish. On administration of the "shocking" dose, sensitized goldf i s h became disoriented, often bumping into the sides of the tank. Balance was affected in that the f i s h assumed e i t h e r head-up or down posture. All the fins except the pectorals were drawn t i g h t to the body, possibly contributing to the loss of balance (Figure I ) . The sensitized f i s h also showed increased defecation. Fish were unable to maintain balance and dropped to the bottom of the aquaria where they remained for up to four hours (Table I ) . These results indicate that the reaction is specific for the s e n s i t i z i n g antigen. Classically, Type I h y p e r s e n s i t i v i t i e s can be transferred from sensitized donor to non-sensitized recipient with serum. Anaphylactic-type behavior was observed only in the c a t f i s h that received serum from sensitized donors by homologous challenge. Serum transfer studies using Versatol and BSA as antigens were also conducted with goldfish. The results were similar to those observed in c a t f i s h , in that anaphylactic-type reactions were manifested in those f i s h receiving sensitized serum and homologous antigen (Table I ) . The results of the in v i t r o study indicated that both goldfish and c a t f i s h intestines were sensitive-~to histamine; however, a difference in s e n s i t i v i t y between the two was evidenced. In goldfish, lO0~g appeared to be near a threshold dose required for a response. Doses higher all gave a response, the only difference being the degree and i n i t i a t i o n of response. At all doses the response occurred within a minute a f t e r the addition of histamine. The i n i t i a l response appeared to be a contraction followed by relaxation of the smooth muscle indicated by the decrease in tonus. At all doses, the muscle returned to the baseline rhythmic contraction within 5 minutes (Figure 2). The i n t e s t i n e of the catfish was not as sensitive to the action of histamine. At the lO0~g dose there was l i t t l e i n t e r r u p t i o n of the baseline tonus and rhythmicity. The intestines were affected by higher doses, but not to the degree as the goldfish i n t e s t i n e s . The response in the catfish also appeared to be a relaxation of the smooth muscle as indicated by a decrease in tonus. The i n i t i a t i o n of response was longer than that of the goldfish (Figure 3). In both f i s h , the response f i r s t appeared as a contraction followed by a rapid loss in the tonus to relaxation.
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TABLE I Reactions of Channel Catfish, I c t a l u r u s punctatus and Goldfish, Carassius auratus to Various Antigenic Challenges
CATFISH
Treatment SPa
Challenge
GOLDFISH
Reaction
SP
IH (6/6) e
BSA
NR (0/4)
SP Serumd
SP
IH (4/4)
BSAb
BSA
IH (4/4)
V
NR (0/6)
BSA Serumd
BSA
IH (5/6)
Vc
V
IN (6/6)
BSA
NR (0/6)
V Serumd
V
IH (6/6)
Saline
SP
NR ( 0 / I 0 )
BSA
NR (0/5)
V
NR (0/5)
Treatment V
Challenge
Reaction
V
IH (13/15)
BSA
NR ( I / I 0 )
V Serumd
V
IH (4/5)
Saline
BSA
NR (0/5)
V
NR (0/5)
a S o l u b i l i z e d Protozoa (SP) b
Bovine Serum Albumin (BSA)
c Versatol (V)- General Diagnostics, D i v i s i o n of Warner and Lambert, Morris Plains, N.J. 07950 U.S.A. d 0. I ml of serum from sensitized f i s h injected IP in r e c i p i e n t f i s h 24 hr. before challenge. e IH - immediate h y p e r s e n s i t i v i t y type reaction. NR- no reaction. Numerator ind~atesnumber of f i s h showing reactions, denominator is the number of f i s h per group.
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Figure i. Top: Normal goldfish with full display of fins and tail and horizontal swimming attitude° Bottom: Goldfish previously sensitized with Versatol and then challenged. Note the clamping of all of the fins except the pectoral fins and the head-down swimming attitude.
1L1221
N
Figure 2° In vitro response of goldsfish gut to histamine° ao Baseline response of gut in Clark-Frog-Ringer's Solution bo -f. Respnse of dose to histamine. = Introduction of histamine. i block = 6 seconds°
HTf7 i ,! iit!-
ti++i+i : " ' = I ~+~t ~ ?lli!!!!!
-+itlit
~z~¢;
, UIITT': I t l [ I ! . ~ L ] : ~ q ~ - p [ f r ~ ] 1~ t l ] : ~TI-{1 t ~ . . + t~ t i-rl--rrH- +; 2 2 ; / p _ r ~ . - d P-fI [,,]-i L'. : i t % '.'. '. ', 'I
~ ~ ~ ~ : ~~L~-;"~'~r
~-~4-~-Ill~Gdimi
.
t::t~.~
-, .
.'
.--~_-~
:~".i:',!=
:, 2 :'..'~- : : . " ~
', '/t¢<-I ~
"1'~O~ . . . .
1"-
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t',i :~-rH-N_+~4T-£----:
~MH.
,
I ! '. I 1-L:441
,[ .......... ~ . + + T f ~ - : - I ~
Iiiii~,']-rq.I
~-t2-~_122+21ii_121ut!211.t:
I
i
I
,
I
: b
,
,
.
:--+
I-i-~
.-
'
PF~-H iF'r ~ ~;2-H~TTftH~!::::
~
.......
+
-
-
,
:;::::1:2:-:.:
•
--
: ::t! :.2~
Figure 3. In vitro response of catfish gut to histamine° a. Baseline response of gut in Clark-Frog-Ringer's Solution. b. -e. Response of dose to histamine° 1 block = 6 seconds.
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DISCUSSION Type I h y p e r s e n s i t i v i t y reactions are characterized by antigen specif i c i t y and passive transfer of the reaction with sensitized serum. Both of these t r a i t s were evidenced in the induction of anaphylaxis in f i s h . The'~hock" reaction was only observed in fish challenged with the homologous s e n s i t i z i n g antigen. Fish receiving a heterologous antigen or those receiving antigen for the f i r s t time did not manifest the shock reaction. Shock behavior was also manifested following transfer of sensitized serum and subsequent challenge with homologous antigen. Both characteristics of Type I h y p e r s e n s i t i v i t y were evidenced in these experiments, indicating that the observed reactions could be c l a s s i f i e d as anaphylactic. From the observed results in these studies, we feel that immediate type h y p e r s e n s i t i v i t y reactions were occurring in the f i s h . The reactions were specific for the sensitizing antigen and could be transferred with serum. This suggests that a c i r c u l a t i n g serum factor is involved with the reaction. Flectcher and Baldo (16) have reported an immediate skin reaction in marine f l a t f i s h and have suggested that the reaction may be mediated via C-Reactive P r o t e i n - l i k e protein in plaice serum. In mammals, anaphylaxis is mediated by vasoactive amines liberated by the degranulating of mast cells a f t e r reaction of the homocytotropic antibody with antigen. Mast-like cells have been described in f i s h ; however, the function has not yet been discerned (17). Clearly, the mechanism of immediate h y p e r s e n s i t i v i t y in teleosts, whether a non specific antibody independent mechanism of mast cells a c t i v a t i o n or tissue f i x i n g a n t i bodies, has yet to be delineated. Although both types of f i s h manifested a behavioral change following administration of the sensitizing antigen, d i f f e r e n t patterns of reactions were observed between the catfish and goldfish. In c a t f i s h the reaction was evidenced by "labored" breathing, indicating involvement of the respiratory system. The reaction in goldfish was manifested by defecation implicating smooth muscle involvement. The appearance of clamped fins is speculated to be a voluntary contraction of the skeletal muscle in response to the contraction of the smooth muscle. These differences suggest the p o s s i b i l i t y that d i f f e r e n t target organs are affected with perhaps d i f f e r e n t pharmacological mediators produced between species. This precedent is observed among the mamalian species (10,19). In dogs, for example, the early stage of anaphylaxis is often manifested by diarrhea and defecation, due to the action of vasoactive amines on the i n t e s t i n a l smooth muscle; whereas in guinea pigs the reactions presents as contraction of the smooth muscle of the bronchioles with dyspnea (9,10). The v a r i a b i l i t y in response is due not only to the target organ affected, but also to the action of the prominent mediator released during the reaction. Of the mediators, histamine, serotonin, kinins, and slow reacting substance (SRS-A), variation exists among species as to which of these is the principal effector. Histamine is regarded as the prime mediator in mammals with the exception of the bovine, in which SRS-A and kinins are the main mediators (I0).
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Histamine is regarded as a smooth muscle contractor; however, the effects also vary among species. Dreyer ( I I ) showed that Img of histamine caused contractions in the small i n t e s t i n e of the minnow, Fundalus h e t e r o c l i t u s , while the results of t h i s study showed a relaxation of i n t e s t i n a l smooth muscle in both the catfish and goldfish when exposed to a Img dose of histamine. These differences as well as the difference of s e n s i t i v i t y between the catfish and goldfish i n t e s t i n e , may be due to a species v a r i a t i o n . Histamine a c t i v i t y measured in v i t r o , using the Schultz-Dale method, caused guinea pig uterus to contract on exposure IOng, while rat uterus relaxed when exposed to a similar dose (8). Guinea pig ileum contracted and then repaidly relaxed when exposed to the drug. These differences coupled with the fact that certain histamine induced changes ( i . e . , gastric acid secretion) are not blocked by classical antihistamines ( i . e . , benadryl, pyribenzamine), have led workers to postulate the existence of two types of histamine receptors HI and H2 (19, 20). I t is now known that histamine stimulates both HI and H2 receptors to cause vasodilation and vasoconstriction (20). The relaxant effect of histamine on contracted rat uterus has been found to be mediated via H2 receptors. Dreyer and King ( I ) observed that f i s h administered histamine showed reactions s i m i l a r to the anaphylactic-type shock induced by sensitizing antigens. The injections of classical antihistamines, such as benadryl and pyribenzamine before and subsequent to shocking procedures had l i t t l e effect on the duration and s e n s i t i v i t y of e i t h e r histamine or antigen-produced shock reactions ( I ) . The observations of Dreyer and King in addition to the relaxation effect reported in t h i s study lead to the hypothesis that receptors of histamine in the i n t e s t i n e of f i s h are similar to the mammalian systems. I t appears possible that H2 l i k e receptors for histamine are located in the i n t e s t i n e of f i s h . Studies on the blocking of HI receptors with classical antihistamines and of H2 receptors with pharamacological agents such as cimetidine and burimanide, are required to further delineate the nature of histime receptors in f i s h intestine.
This study was supported by funds from the Georgia Veterinary Medical Experiment Station. We wish to thank Dr. N. Booth and Dr. D. Coulter of the Department of Physiology and Pharmacology for t h e i r help during t h i s study.
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REFERENCES I.
Dreyer, N.B. and King, J.W. 277, 1948.
2.
Good, R.A. and Papermaster, B.W. Ontogeny and Phylogeny of Adaptive Immunity. Adv. Immunol. 4 I , 1964.
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4.
Anaphylaxis in the Fish.
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Clem, L.W. and Leslie, G.A. Phylogeny of Immunolobulin Structure and Function. In: Immunology and Development. M. Adinolfi (ed). Spastics Society Medical Publications, London. 62, 1969. Harris, J.E. The Apparent I n a b i l i t y of Cyprinid Fish to Produce Skin Sensitizing Antibody. J. Fish Biol. 5, 535, 1973.
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Gershwin, L.J. The Phylogentic Development of Anaphylactic A c t i v i t y and Homocytotropic Antibody. De__v. & Comp. Immunol. 2, 595, 1978.
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Spoujitch, V. Spuzic, I . , and Bata, A. Les Reactions Anaphylactiques Chez Les Animaux Poikilothermes, Acta Allergologica 25, 259, 1970.
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Coombs, R.R.A. and Gell, P.G.H. (ed.) Clinical Aspects of Immunology. Blackwell S c i e n t i f i c Publications, Oxford, 575, 1968.
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Levy, D.A. 1974. Histamine and Serotonin. In: Mediators of Inflammation. G. Weissmann Ed. Plenum Press. New York Chapter 5.
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Smith, H.A., Jones, T.C. and Hunt, R.D. 1972. Lea and Febiger. Phila.
Veterinary Pathology.
I0.
Tizard, I.R. 1977. An Introduction to Veterinary Immunology. Saunders Co., Phila.
W.B.
II.
Dreyer, N.B. 1948. Histamine and Posterior P i t u i t a r y Actions on Elasmobranch and Teleost Smooth Muscle. Arch. Int. Pharamacodyn. 82:440-443.
12.
Klaas, J. Two Gastric Mucin Cultivation Media and Chemically Defined Maintenance Medium for Balantidium Col. J. Parasit. 60, 907, 1974.
13.
Lowry, O.H., Rosenbrough, N.G., Farr, A.L., and Randall, R.J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem. 193, 265.
14.
Staff Department Pharmacology, University of Edinburgh. 1968. Pharmacological Experiments on Isolated Preparations. E. and S. Livingstone Ltd. Edinburgh and London: 120-121.
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Weir, D.M. 1978. Handbook of Experimental Immunology. S c i e n t i f i c Publications. London.
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Fletcher, T.C. and Baldo, B.A. Immediate Hypersensitivity Responses in Flatfish. Science 185, 360, 1974.
17.
E l l i s , A.E.
3rd Edition Blackwe
The Leucocytes of Fish: A review. J. Fish Biol. II 453, 1977.
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18.
Smith, H.A., Jones, T.C. and Hunt, R.D. Veterinary Pathology. Febiger. Phila. 1972.
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19.
Ash, A.S.F., and Schild, H.O. 1966. Receptors Mediating Some Actions of Histamine. Br. J. Pharmacol and Chemother. 27:427-439.
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Eyre, P., and Wells, P.W. 1973. Histamine Hp - Receptors Modulate Systemic Anaphylaxis: A Dual Cardiovascular Action of~Histamine in Claves. Br. J. Pharmcol. 49:364-367.