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Investigations into the Role of Pharmacologically Active Amines in Anaphylaxis in Calves
INVESTIGATIONS INTO THE ROLE OF PHARMACOLOGICALLY ACTIVE AMINES IN ANAPHYLAXIS IN CALVES By C.
WRA Y
*
AND
J.
R.
THOMLINSON
Department of Veterinary Pathology, University of Liverpool
SUMMARY
No release of histamine could be detected in blood plasma from calves subjected to anaphylactic shock. Symptoms and lesions resembling those of anaphylactic shock and endotoxin shock developed when calves were injected with histamine and with 5-hydroxytryptamine but dopamine elicited only mild and transient symptoms. Similar symptoms and lesions were observed following the use of the endogenous releasing agents, Compound 48/80 and reserpine. Mepyramine, cyproheptadine and diethylcarbamazine citrate failed to protect calves against anaphylactic shock. It is suggested that synergism between several pharmacologically-active substances released in low concentration may be responsible for the symptoms of acute anaphylactic shock in calves. INTRODUCTION
Anaphylactic reactions have been suspected to be associated with a number of animal diseases, including fog fever in cattle (Gibbons, 1962) and bowel oedema in pigs (Thomlinson & Buxton, 1963) . In calves, Wray & Thomlinson (1969, 1972a) observed that gastrointestinal lesions produced during protracted anaphylactic shock resembled those seen in cases of coli bacillosis. The release of pharmacologically active agents in the intact animal during anaphylaxis was first demonstrated by Dragstedt & Gebauer-Fuelnegg (1932). Subsequently, histamine has been shown to be liberated during anaphylaxis in guineapigs (Code, 1939) and in pigs (Thomlinson, 1963). Different mediators are involved in other animal species. For instance, in rabbits, 5-hydroxytryptamine (5-HT) has been shown to be the principal amine in anaphylactic reactions (Waalkes et at., 1957) . More than one mediator may be released during anaphylaxis and, in addition to histamine, bradykinin and slow reacting substance of anaphylaxis (SRS-A) have been demonstrated during anaphylaxis in the guineapig (Brocklehurst & Lahiri, 1962 ) . In calves, Lewis & Eyre (1972 ) considered that histamine and 5-HT were important but other evidence (Aitken & Sanford, 1972) suggests that SRS-A and kinins may be the major mediators; The experiments described in this paper formed part of an investigation into the role of anaphylactic reactions in the pathogenesis of colibacillosis. Attempts .Present address; M.A.F.F., Central Veterinary Laboratory, New Haw, Weybridge, Surrey.
ANAPHYLAXIS IN CALVES
were made to demonstrate a release of histamine during anaphylactic shock, and the effects of pharmacologically active amines, endogenous releasing agents and antagonistic drugs were studied. MATERIALS AND METHODS
Experimental calves Calves 7-14 days old and 30- 40 kg body weight were obtained from a dealer and fed on proprietary calf starter gruel. Eleven calves were actively sensitized by three intravenous injections of I g egg albumin on alternate days and challenged 12 days after the first injection with I g egg albumin (Wray & Thomlinson, 19 69) . Assay of histamine in blood plasma during anaphylactic shock Blood samples collected from four actively sensitized calves before challenge, one and fifteen minutes after challenge, were assayed for histamine. Estimation of histamine. Bioassay for histamine was made on guineapig ileum preparations in a tissue bath (Shultz, 1910 ; Dale, 1913). Histamine was extracted from blood samples by the method of Hinshaw et al. (1960) except that acetone was removed from the extract by passing nitrogen through at 37 °C. Intravenous injection of pharmacologically active substances One per cent aqueous solutions of these substances were injected intravenously as follows. The doses were based on the results of earlier experiments (unpublished ) in pigs and were selected with a view to producing clinical signs and lesions. Histamine. Two calves at dosage rates of 2 mg and I mg/kg bodyweight respectively. 5-HT. Three calves at dosage rates of5 mg, 3 mg and 2·5 mg/kg bodyweight respectively. Dopamine. One calf at a dosage rate of 2·5 mg/kg bodyweight. Four days later a further dose of I mg/kg was injected. Injection of releasing agents The following substances were injected intravenously as histamine and 5-HT releasing agents respectively: Compound 48/80. A I per aqueous cent solution was injected into a calf at a dosage rate of 1·5 mg/kg. Reserpine. A I per cent aqueous suspension prepared by the addition of a few drops of Teepol as a dispersing agent was injected into two calves at dosage rates of 2·5 and 5"0 mg/kg respectively. The calf which received the larger dose was given a similar dose four days later. Antagonistic drugs The following antagonists of histamine, 5-HT and SRS-A, respectively, were
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administered to actively sensitized calves as follows: Mepyramine maleate. A S per cent aqueous solution was injected intravenously into two calves S min before challenge at a dosage rate of 2 mg/kg bodyweight. Twelve days later, one of the calves was given the same dose of mepyramine maleate 30 min before a subsequent challenge. Cyproheptadine. A I per cent aqueous solution was injected intravenously into two calves at a dosage rate of I ' S mg/kg body weight I sand S min respectively before challenge. Another calf was similarly injected at a dosage rate of 3 mg/kg one hour before challenge. Diethylcarbamazine citrate. Preliminary experiments showed that 2'S ml of 40 per cent diethylcarbamazine citrate, when given intravenously, caused acute dyspnoea and collapse. For this reason, one of the sensitized calves was given 3'S ml diethylcarbamazine citrate by the intraperitoneal route 10 min before challenge. Another was given two intramuscular injections of2'S ml on each of the two days prior to challenge and 2'S ml intraperitoneally on the day of challenge.
Haematological methods The standard procedure, using improved Neubauer counting chambers, was employed for total erythrocyte and leucocyte counts. For differential counts, smears were stained by Leishman's method. Observations were made on the calves given S-HT, reserpine and compound 48/80 immediately before and S min afteJ the injection, and then at intervals of I S min for two hours. Pathological methods Calves given S-HT, histamine, dopamine and compound 48/80 were killed two hours after the injection. Of the calves given reserpine, the one that received the smaller dose was killed 30 hours after the injection and the other was killed 10 hours after the second injection. Portions of abomasum, small and large intestine, lung, thymus, liver, kidney, spleen, adrenal and mesenteric lymph node were fixed in 4 per cent formaldehyde in saline. Paraffin- wax seCtions 4 fl-m in thickness were stained with haematoxylin and eosin, and with toluidine blue to demonstrate mast cells. RESULTS
Assay qf histamine in blood plasma during anaphylactic shock No increase of histamine could be detected after challenge. Readings from samples taken before a nd after challenge ranged from o'oS- o' I 3 fl-g/ml. The effect of pharmacologically active substances Histamine Clinical Signs. Both calves collapsed immediately after the Injection, and showed profuse salivation and lacrimation. Coughing and sneezing were frequent. The calves strained continually and passed copious amounts offaeces. Five minutes after the injection dyspnoea occurred and, after 10 min progressed to
PLATE I
Fig.
Fig.
I.
2.
5-HT adm inistration. Congestion and oedema of the lungs. HE x
12 0.
R eserpin e administration. Erosion of the abomasal mucosa. HE x
1 20.
Wray and Tomlinson , S r. vet.
J.
( 1974),
I30,
5
ANAPHYLAXIS IN CALVES
hyperpnoea which lasted for 25 min. Post-mortem findings. Marked emphysema was present in the lungs of both calves. Oedema was present in the abomasal submucosa and the mesenteric lymph nodes. In the calf that received the larger dose, the small intestine mucosa was hyperaemic. Histological findings. Bronchiolar constriction and areas of haemorrhage were present in the lungs. In the small intestine, the lacteals were dilated and large numbers of eosinophils were found in the lamina propria.
S-HT • Clinical signs. The calf which received 3 mg/kg showed incoordination within 30 seconds after the injection and collapsed showing marked dyspnoea. This calf died 5 min later when tympany of the rumen was apparent. The other two calves extended their heads and necks and showed marked dyspnoea and profuse salivation. The dyspnoea lasted 15 min and then developed into hyperpnoea which lasted until the calves were killed for autopsy. During this period both calves passed copious amounts of fluid faeces. Post-mortem findings. In all three calves the lungs were congested, oedema was particularly marked in the subpleural and interlobular connective tissue and petechial haemorrhages were numerous. The cut surfaces oozed blood and froth, and copious amounts of froth were present in the tracheae. The gastrointestinal lesions varied in severity between the individual calves and were most marked in the calf that received the largest dose. In this calf, the abomasal mucosa was congested and showed scattered petechiae and erosions. In the calf that received the smallest dose, the abomasal lesions were similar except that no erosions occurred. In all three calves the mesenteric blood and lymphatic vessels were engorged and the associated lymph nodes were enlarged and oedematous. However, hyperaemia of the small intestine mucosa was observed only in the two calves that received the largest and smallest doses. Histological findings. Bronchiolar constriction, widespread emphysema and oedema occurred in the lungs of all the calves (Fig. I) . Large numbers of eosinophils, lymphocytes, plasma cells and neutrophils were found in the lamina propria of the abomasum and small intestine. The villi were congested and the lac teals were dilated. Haematological findings. These were studied in the calf which received the smallest dose. The total leucocyte count decreased from 8050 to 6250 cells/mm 3 during the 15 min after 5-HT was administered. During the same period, the neutrophil count decreased from 3220 to 1250 cells/mm 3• Other cell types were unaffected.
Dopamine Clinical signs. Initially the calf showed incoordination and a slight increase in respiratory rate. The respiratory rate returned to normal within 10 min although tympany of the rumen was observed. When the larger dose was inj ected similar symptoms were observed and there was no increase in severity. Post-mortem findings. No gross abnormalities were observed.
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Histological findings. The only abnormal finding was the presence of large numbers of eosinophils in the lamina propria of the small intestine. The ejJect if releasing agents Compound 48/80 Clinical findings. Five min after administration of the drug the calf became restless and hyperexcitable. Muscular tremors developed. The calf collapsed soon after the onset of these symptoms and showed marked dyspnoea, abdominal respiration and profuse salivation. After 25 min the calf was able to stand, although muscular tremors were still present, and it appeared to be normal after go min. Post-mortem findings . The small intestine was blanched and the mesenteric lymph nodes were oedematous. The lungs and other organs were normal in appearance. Histological findings. Oedema was present in the abomasal submucosa. Large numbers of eosinophils, lymphocytes, plasma cells and neutrophils were present in the lamina propria of the gastrointestinal tract and mast cells were degranulated. Eosinophils were present in the medullary cords of the lymph nodes. The lungs showed bronchiolar constriction. Haematological findings. The only alteration in the blood picture was a gradual increase of the neutrophil count from 1260 to a peak of2741 cells/mm 3 during the 15 min after the administration of Compound 48/80. Reserpine Clinical signs. The calf which received the larger dose showed incoordination and stood with its head lowered for 10 min after the injection. One hour later, the calf showed signs of abdominal discomfort, passed large amounts of fluid faeces and refused its food. Mter nine hours muscular tremors and champing of the jaws were observed. Twenty-four hours after injection, the calf appeared dull, and was unable to stand. Muscular tremors were still occurring and respiratory movements were short and rapid and these symptoms persisted for 24 hours. When a similar dose was given to the same calf four days later, muscular tremors and froth from the nostrils were observed 5 min after the injection. The tremors ceased within an hour although the calf showed signs of abdominal discomfort. The calf which received the smaller dose showed no immediate symptoms. Twelve hours after the injection, the calf was unable to stand, and muscular tremors and nystagmus with protrusion of the nictitating membrane developed. The calf salivated profusely and continually licked its lips until it was killed for au topsy. Post-mortem findings. In both calves, the mucosa of the small intestine was hyperaemic. The mesenteric lymph nodes were enlarged and oedematous, and the associated blood and lymphatic vessels were engorged. In the calf which received the larger dose, oedema was present in the submucosa of the abomasum and numerous small white plaques were found in the mucosa. No gross lesions were found in the lungs in either of the calves.
ANAPHYLAXIS IN CALVES
Histological findings. The white plaques in the abomasal mucosa of the calf which had the larger dose of reserpine proved to be erosions which extended deep in the glandular zone (Fig. 2). In both of the calves, aggregations of lymphocytes and neutrophils were present in the a bomasal mucosa and the submucosa was oedematous. In the small intestine, the lacteals were dilated and the blood vessels of the villi congested. The Peyer's patches were cleared of cells and lymphocytolysis was observed . Areas of pulmonary congestion were found a nd the bronchioles were constricted. Haematologicalfindings. In the calf tha t received the smaller dose of reserpine, the n eutrophil count decreased from I 1,5 I 5 to 7650 cells/mm 3 and the lymph- . ocyte count from 4506 to 2600 cells/mm 3 5 min after the injection. Similarly, in the calf that received the larger dose, the neutrophil count decreased from 6820 to 3847 cells/mm 3 and the lymphocyte count from 3850 to 263 2 cells/mm 3 • The leucocyte counts in both calves returned to their normal values an hour after the injection of reserpine. No alterations occurred in the erthrocyte count. Antagonistic drugs Mepyramine maleate One of the calves challenged with egg albumin 5 min after the inj ection of mepyramine maleate d eveloped mild incoordination and hyperpnoea that lasted for 15 min. Twelve days later the same calf was given mepyramine maleate 30 min before a subsequent challenge when it collapsed showing severe a naphylactic shock (Wray & Thomlinson, 1969). Another calf, given mepyramine maleate 5 min before challenge, also collapsed and showed severe anaphylactic shock. Cyproheptadine The calf which received the larger dose of cyproheptadine developed incoordination and hyperaesthesia after inj ection of the drug. No clinical signs were o bserved in the other two calves which received smaller doses of cyproheptadine. ''''hen the calves were challenged, they all showed severe anaphylactic shock. Diethylcarbamazine citrate When calves treated with this drug were challenged, they showed severe anaphylactic shock. DISCUSSION
Our ina bility to d etect a ny release of histamine confirms the experience of Code & Hester (1939) . The histamine levels showed close agreement with those observed by Nilsson (1963) in normal adult cattle. However, Eyre ( I97Ia) has shown that histamine is released in vitro from lung preparations from sensitized calves, but the concentration of histamine was lower than in other species such as the guineapig, rabbit and man. H e also suggested that there a ppeared to be a direct relationship between the age of the animal and lung histamine concentrations. Similarly, \\Tray & Thomlinson (1969) suggested that the severity of
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anaphylaxis in young calves may depend on the amount of pharmacodynamic agents available for release from the tissues. The present experiments were carried out in relatively young animals and it is possible that histamine may have been released in too low a concentration to be detected in the intact animal. Lewis & Eyre (1972) detected histamine at a concentration of 0'5 Il-g in an anaesthetized calf after the administration of Compound 48/80. Dopamine is known to be present in significant amounts in calves (Holtz, 1959) and is located in mast cells (Coupland & Heath, 1961 ). Eyre (197Ib) has recorded the release of dopamine from sensitized calf lung. In our experiments, however, dopamine produced only mild and transient symptoms. With histamine and 5-HT the symptoms and lesions closely resembled those of acute anaphylactic shock (Wray & Thomlinson, 1969) and endotoxin shock (Wray & Thomlinson, 1972b). Similar symptoms were observed by Aitken & Sanford (1972). These a uthors observed only minimal pulmonary oedema after the administration of 5HT. In our experiments, pulmonary oedema was marked, but larger doses of 5-HT were used. Similarly the doses of histamine were above those regarded as toxic by Desliens (1958) and Aitken & Sanford (1972) but none of the calves died. However, Desliens (1958) noted individu al variation in the susceptibility of cattle to histamine. Relatively mild symptoms developed following the · use of the endogenous releasing agents, compound 48/80 and reserpine. The effects of these drugs would be limited by the amount of the respective amines available for release. However, the effects of Compound 48/80 and reserpine might depend on the size of the dose used. Lewis & Eyre (1972) noted marked systemic toxic effects with Compound 48/80 which did not seem to be entirely due to the release of histamine or 5-HT, although the release of these amines probably played a major part in its toxic action. Erosions were observed in the abomasal mucosa of the calf which received the higher dose of reserpine, but not with Compound 48/80. Erosions were also observed in the abomasum of the calf which received the larger dose of 5-HT. Mota et al. (1956) showed that Compound 48/80, when used in rats, released histamine from mast cells but failed to release the relatively large amounts of histamine present in the fundus of the stomach. According to Espamer (1954), 5-HT is associated with the enterochromaffin system and it is possible that the erosions may have resulted from the release of S-HT in the abomasal mucosa itself. In the authors' experience, abomasal ulceration is a not uncommon sequel to coli bacillosis and it is interesting to speculate on the possible role of S-HT in the formation of these ulcers. On the other hand, Muggenberg et ai. (1966) concluded that gastric ulcers which developed in pigs following the administration of reserpine may have been associated with increased free acid and pepsin activity. Thus it is possible that ulceration may occur as a direct result of the action of reserpine. None of the antagonist drugs used in the present experiments afforded any protection against anaphylactic shock. Aitken & Sanford (1969) suggested that bradykinin and/or SRS-A may be responsible for the acute symptoms because sodium meclofenamate protected calves against anaphylactic shock. Eyre (197IC), however, observed that in in vitro experiments sodium meclofenamate
ANAPHYLAXIS IN CALVES
473
was not entirely specific in its actions against bradykinin. Although diethylcarbamazine citrate has been shown to inhibit the release of SRS-A in the rat (Orange, Valentine & Austen, 1968) it did not protect the calves in our experiments. Wells & Eyre (1972) have shown that this substance significantly inhibits cutaneous anaphylaxis in the calf and when given simultaneously with disodium cromoglycate the inhibitory effect was augmented. There is evidence (Fischel, Keller & O'Bryan, 1968) that, in mice, histamine and 5-HT are synergistic and when these substances are administered together, relatively small doses are capable of producing acute symptoms. Eyre (1971C) concluded that the in vitro reaction in the bovine pulmonary vein is associated with a complex interaction of histamine, 5-HT, SRS-A and possibly other substances. It is likely that the simultaneous release of several of these su bstances in small amounts would give rise to symptoms of acute anaphylactic shock and the results of the present experiments lend support to this view. A C KNOWLEDGEMENTS
We wish to acknowledge the financial support of the Agricultural Research Council. We should also like to thank Merck, Sharp and Dohme Ltd. for the gift of cyproheptadine. We are grateful to Mrs J. J. Jones and Mrs M. W. Harling for technical assistance and to Mr G. Weston for the photomicrographs. REFERENCES
AiTKEN, M. M. & SANFORD, j. (1969). Nature, Lond. 223, 314 AITKEN, M. M. & SANFORD,j. (1972) . J. compo Path. 82, 257 BROCKLEHURST, W. E. & LAHIRI, S. C. (1962). J. Physiol. Lond. 160, 15. CODE, C. F . ( 1939). Am. J. Physiol. 127, 78. CODE, C. F. & HESTER, H . R. (1939) . Am. J. Physiol. 127, 71. COUPLAND, R. E. & HEATH, I. D. (1961). J . Endocr. 22, 71. DALE, H . H. (1913). J. Pharmac. expo Ther.4, 16 7 DESLIENS, L. ( 1958). Presse Med. 66, 562. DRAGSTEDT, C. A. & GEBAUER-FuELNEGG, E . (1932). Am. J. Physiol. 102,5 12 . EDINGTON, N. (1968). J. Pharm. Pharmac. 20, 577· ESPAMER, V. (1954). Pharmac. Rev. 6, 425. EYRE, P. (197w). Archs into Pharmacodyn. Ther. 192, 347· EYRE, P. ( 197Ib). Br. J. Pharmac. 42,423. EYRE, P. (1971C). Br. J. Pharmac. 43, 302 FISHEL, C. W., KELLER, K. F. & O'BRYAN, B. S. (1968) . J. Immun. 101,679· GI BBONS, W. j . (1962). Mod. vet. Pract. 43, 34. HINSHAW, L. B., VICK, j . A. , CARLSON, C. H. &. FAN, Y. L. (1960). Proc. Soc. expo B iol. Med. 104, 379· HOLTZ, P. (1959). Pharmac. R ev. II, 326. LEWIS, A. J. & EYRE, P. (1972). Can. J. Physiol. Pharmacol. 50, 545· MOTA, L., BERALDO, W. T., FERRI, A. G. & j UNQ.UERIA, L. C. U. (1956). Ciba Foundation Symposium on H istamine, p. 47. London: Churchill. MUGGENBERG, B. A., KOWALCZYK, T., HOCKSTRA, W . G . & GRUMMER, R . H. ( 1966 ). Am. J. Vet. Res. 27, 1663. NILSSON, S. A. (1963). Acta vet. scand. 4, SuppJ. I. ORANGE, R. P ., VALENTINE, M. D. & AUSTEN, K . F. (1968) . J. expo Med. 12 7,7 67. SCHULTZ, W. H. (1910). J. Pharmac. exp o Ther. 1, 549. THOMLINSON, j. R. (1963)' Vet. R ec. 75, 12 46 .
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THOMLINSON,j. R . & BUXTON, A. (1963) . I mmunology, 6, 126. ' '\TAALKES, T. P., WEISSBACH, H., BOZICEVICH, j . & UDENFRIEND, I I
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S. (1957).
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P. W. & EYRE, P. (1972). Can. J. Physiol. Pharmacol. 50, 255. C. & THOMLINSON, j. R. (1969). J. Path. ga, 61. C. & THOMLINSON,j. R. ( 1972a). R es. vet. Sci. 13, 563. C. & THOMLINSON, j . R . ( 1972b ). R es. vet Sci. 13, 546. (Accepted for publication
I I
September 1973 )
Recherches concernant Ie role des arnines phannacologiquell1.ent actives au cours de l'anaphylaxie (Wray et Thonillnson) ResUll1.e. On ne put detecter aucune liberation d 'histamine dans Ie plasma sanguin des vaches soumises au choc anaphylactique. Des symptomes et des lesions ressemblant a ceux du choc anaphylactique et du choc endotoxinique se developperent lorsque I'on injecta aux vaches de I'histamine et d e la 5-hydroxytryptamine mais la dopamine provoquait d es signes moderes et transitoires. On constata des symptomes et lesions a nalogues a la suite de I'emploi d'agents de liberation endogene, Compose 48/80 et la reserpine. La mepyramine, la cyproheptadine et la diethylcarbazamine ne reussirent pas a proteger les vaches contre Ie choc anaphylactique. II est suggere, que la synergie qui existe entre les differentes substances pharmacologiquement actives liberees a basse concentration peut etre responsable des symptomes d e choc anaphylactique aigu chez les vaches.
Untersuchung iiber die Rolle, die pharll1.akologisch aktive Amine iII1. anaphylaktischen Schock spielen (Wray und Thonillnson) Zusall1.ll1.enfassung. 1m Blutplasma von Kalbern, die einen anaphylaktischen Schock erlitten hatten, konnte kein Histamin gefunden werden. Wenn K a lber Injektionen von Histamin erhielten, entwickelten sich Symptome und Manifestationen, die einem anaphylaktischen Schock und einem Endotoxinschock ahnelten, aber Injektionen mit 5-Hydroxytryptamin verursachten nur leichte und vortibergehende Symptome. Ahnliche Symptome und Manifestation en wurden beobachtet nach Verwendung von endogen entspannenden Mitteln, Compound 40/80 und Reserpin. Mepyramin, Cyproheptadin und Diathylcarbamazinzitrat schtitzten die Kiilber nicht gegen anaphylaktischen Schock. Man ist der Ansicht, das eine Synergismus zwischen verschiedenen pharmakologisch aktiven Substanzen. die in schwacher Konzentration produziert werden, besteht und moglicherweise d as Auftreten von Symptomen akuten anaphylaktischen Schocks bei Kiilbern verursacht.
Investigaclon de la {unclon de las aII1.inas {annacologicall1.ente activas en la anafilaxis (Wray y Thomlinson) ResUll1.en. No se pudo descubrir la emision de histamina en el plasma sanguineo de terneros que se sometieron a un shock analfilactico. Empezaron a mostrar sintomas y lesiones parecidas a las de shock anafilactico y shock endotoxico cuando se inyecto a los terneros con histamina y con 5-hydroxitriptamina, pero la dopamina solamente produjo sintomas leves y transitorios. Se observaron sintomas y lesiones similares tras el empleo de agentes endogenos de emision, Compuesto 48/80 y reserpina. La mepiraIl;1ina, cifroheptadina y el citrato de dietilcarbamacina no protegieron a los terneros contra el shock anafiloctico. Se sugiere que el sinergismo entre varias substancias farmacologicamente activas emitidas en concentraciones bajas pudieran ser responsables por los sintomas de shock anafilactico agudo en los tern eros.
WRA Y
*
AND
J.
R.
THOMLINSON
Department of Veterinary Pathology, University of Liverpool
SUMMARY
No release of histamine could be detected in blood plasma from calves subjected to anaphylactic shock. Symptoms and lesions resembling those of anaphylactic shock and endotoxin shock developed when calves were injected with histamine and with 5-hydroxytryptamine but dopamine elicited only mild and transient symptoms. Similar symptoms and lesions were observed following the use of the endogenous releasing agents, Compound 48/80 and reserpine. Mepyramine, cyproheptadine and diethylcarbamazine citrate failed to protect calves against anaphylactic shock. It is suggested that synergism between several pharmacologically-active substances released in low concentration may be responsible for the symptoms of acute anaphylactic shock in calves. INTRODUCTION
Anaphylactic reactions have been suspected to be associated with a number of animal diseases, including fog fever in cattle (Gibbons, 1962) and bowel oedema in pigs (Thomlinson & Buxton, 1963) . In calves, Wray & Thomlinson (1969, 1972a) observed that gastrointestinal lesions produced during protracted anaphylactic shock resembled those seen in cases of coli bacillosis. The release of pharmacologically active agents in the intact animal during anaphylaxis was first demonstrated by Dragstedt & Gebauer-Fuelnegg (1932). Subsequently, histamine has been shown to be liberated during anaphylaxis in guineapigs (Code, 1939) and in pigs (Thomlinson, 1963). Different mediators are involved in other animal species. For instance, in rabbits, 5-hydroxytryptamine (5-HT) has been shown to be the principal amine in anaphylactic reactions (Waalkes et at., 1957) . More than one mediator may be released during anaphylaxis and, in addition to histamine, bradykinin and slow reacting substance of anaphylaxis (SRS-A) have been demonstrated during anaphylaxis in the guineapig (Brocklehurst & Lahiri, 1962 ) . In calves, Lewis & Eyre (1972 ) considered that histamine and 5-HT were important but other evidence (Aitken & Sanford, 1972) suggests that SRS-A and kinins may be the major mediators; The experiments described in this paper formed part of an investigation into the role of anaphylactic reactions in the pathogenesis of colibacillosis. Attempts .Present address; M.A.F.F., Central Veterinary Laboratory, New Haw, Weybridge, Surrey.
ANAPHYLAXIS IN CALVES
were made to demonstrate a release of histamine during anaphylactic shock, and the effects of pharmacologically active amines, endogenous releasing agents and antagonistic drugs were studied. MATERIALS AND METHODS
Experimental calves Calves 7-14 days old and 30- 40 kg body weight were obtained from a dealer and fed on proprietary calf starter gruel. Eleven calves were actively sensitized by three intravenous injections of I g egg albumin on alternate days and challenged 12 days after the first injection with I g egg albumin (Wray & Thomlinson, 19 69) . Assay of histamine in blood plasma during anaphylactic shock Blood samples collected from four actively sensitized calves before challenge, one and fifteen minutes after challenge, were assayed for histamine. Estimation of histamine. Bioassay for histamine was made on guineapig ileum preparations in a tissue bath (Shultz, 1910 ; Dale, 1913). Histamine was extracted from blood samples by the method of Hinshaw et al. (1960) except that acetone was removed from the extract by passing nitrogen through at 37 °C. Intravenous injection of pharmacologically active substances One per cent aqueous solutions of these substances were injected intravenously as follows. The doses were based on the results of earlier experiments (unpublished ) in pigs and were selected with a view to producing clinical signs and lesions. Histamine. Two calves at dosage rates of 2 mg and I mg/kg bodyweight respectively. 5-HT. Three calves at dosage rates of5 mg, 3 mg and 2·5 mg/kg bodyweight respectively. Dopamine. One calf at a dosage rate of 2·5 mg/kg bodyweight. Four days later a further dose of I mg/kg was injected. Injection of releasing agents The following substances were injected intravenously as histamine and 5-HT releasing agents respectively: Compound 48/80. A I per aqueous cent solution was injected into a calf at a dosage rate of 1·5 mg/kg. Reserpine. A I per cent aqueous suspension prepared by the addition of a few drops of Teepol as a dispersing agent was injected into two calves at dosage rates of 2·5 and 5"0 mg/kg respectively. The calf which received the larger dose was given a similar dose four days later. Antagonistic drugs The following antagonists of histamine, 5-HT and SRS-A, respectively, were
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administered to actively sensitized calves as follows: Mepyramine maleate. A S per cent aqueous solution was injected intravenously into two calves S min before challenge at a dosage rate of 2 mg/kg bodyweight. Twelve days later, one of the calves was given the same dose of mepyramine maleate 30 min before a subsequent challenge. Cyproheptadine. A I per cent aqueous solution was injected intravenously into two calves at a dosage rate of I ' S mg/kg body weight I sand S min respectively before challenge. Another calf was similarly injected at a dosage rate of 3 mg/kg one hour before challenge. Diethylcarbamazine citrate. Preliminary experiments showed that 2'S ml of 40 per cent diethylcarbamazine citrate, when given intravenously, caused acute dyspnoea and collapse. For this reason, one of the sensitized calves was given 3'S ml diethylcarbamazine citrate by the intraperitoneal route 10 min before challenge. Another was given two intramuscular injections of2'S ml on each of the two days prior to challenge and 2'S ml intraperitoneally on the day of challenge.
Haematological methods The standard procedure, using improved Neubauer counting chambers, was employed for total erythrocyte and leucocyte counts. For differential counts, smears were stained by Leishman's method. Observations were made on the calves given S-HT, reserpine and compound 48/80 immediately before and S min afteJ the injection, and then at intervals of I S min for two hours. Pathological methods Calves given S-HT, histamine, dopamine and compound 48/80 were killed two hours after the injection. Of the calves given reserpine, the one that received the smaller dose was killed 30 hours after the injection and the other was killed 10 hours after the second injection. Portions of abomasum, small and large intestine, lung, thymus, liver, kidney, spleen, adrenal and mesenteric lymph node were fixed in 4 per cent formaldehyde in saline. Paraffin- wax seCtions 4 fl-m in thickness were stained with haematoxylin and eosin, and with toluidine blue to demonstrate mast cells. RESULTS
Assay qf histamine in blood plasma during anaphylactic shock No increase of histamine could be detected after challenge. Readings from samples taken before a nd after challenge ranged from o'oS- o' I 3 fl-g/ml. The effect of pharmacologically active substances Histamine Clinical Signs. Both calves collapsed immediately after the Injection, and showed profuse salivation and lacrimation. Coughing and sneezing were frequent. The calves strained continually and passed copious amounts offaeces. Five minutes after the injection dyspnoea occurred and, after 10 min progressed to
PLATE I
Fig.
Fig.
I.
2.
5-HT adm inistration. Congestion and oedema of the lungs. HE x
12 0.
R eserpin e administration. Erosion of the abomasal mucosa. HE x
1 20.
Wray and Tomlinson , S r. vet.
J.
( 1974),
I30,
5
ANAPHYLAXIS IN CALVES
hyperpnoea which lasted for 25 min. Post-mortem findings. Marked emphysema was present in the lungs of both calves. Oedema was present in the abomasal submucosa and the mesenteric lymph nodes. In the calf that received the larger dose, the small intestine mucosa was hyperaemic. Histological findings. Bronchiolar constriction and areas of haemorrhage were present in the lungs. In the small intestine, the lacteals were dilated and large numbers of eosinophils were found in the lamina propria.
S-HT • Clinical signs. The calf which received 3 mg/kg showed incoordination within 30 seconds after the injection and collapsed showing marked dyspnoea. This calf died 5 min later when tympany of the rumen was apparent. The other two calves extended their heads and necks and showed marked dyspnoea and profuse salivation. The dyspnoea lasted 15 min and then developed into hyperpnoea which lasted until the calves were killed for autopsy. During this period both calves passed copious amounts of fluid faeces. Post-mortem findings. In all three calves the lungs were congested, oedema was particularly marked in the subpleural and interlobular connective tissue and petechial haemorrhages were numerous. The cut surfaces oozed blood and froth, and copious amounts of froth were present in the tracheae. The gastrointestinal lesions varied in severity between the individual calves and were most marked in the calf that received the largest dose. In this calf, the abomasal mucosa was congested and showed scattered petechiae and erosions. In the calf that received the smallest dose, the abomasal lesions were similar except that no erosions occurred. In all three calves the mesenteric blood and lymphatic vessels were engorged and the associated lymph nodes were enlarged and oedematous. However, hyperaemia of the small intestine mucosa was observed only in the two calves that received the largest and smallest doses. Histological findings. Bronchiolar constriction, widespread emphysema and oedema occurred in the lungs of all the calves (Fig. I) . Large numbers of eosinophils, lymphocytes, plasma cells and neutrophils were found in the lamina propria of the abomasum and small intestine. The villi were congested and the lac teals were dilated. Haematological findings. These were studied in the calf which received the smallest dose. The total leucocyte count decreased from 8050 to 6250 cells/mm 3 during the 15 min after 5-HT was administered. During the same period, the neutrophil count decreased from 3220 to 1250 cells/mm 3• Other cell types were unaffected.
Dopamine Clinical signs. Initially the calf showed incoordination and a slight increase in respiratory rate. The respiratory rate returned to normal within 10 min although tympany of the rumen was observed. When the larger dose was inj ected similar symptoms were observed and there was no increase in severity. Post-mortem findings. No gross abnormalities were observed.
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Histological findings. The only abnormal finding was the presence of large numbers of eosinophils in the lamina propria of the small intestine. The ejJect if releasing agents Compound 48/80 Clinical findings. Five min after administration of the drug the calf became restless and hyperexcitable. Muscular tremors developed. The calf collapsed soon after the onset of these symptoms and showed marked dyspnoea, abdominal respiration and profuse salivation. After 25 min the calf was able to stand, although muscular tremors were still present, and it appeared to be normal after go min. Post-mortem findings . The small intestine was blanched and the mesenteric lymph nodes were oedematous. The lungs and other organs were normal in appearance. Histological findings. Oedema was present in the abomasal submucosa. Large numbers of eosinophils, lymphocytes, plasma cells and neutrophils were present in the lamina propria of the gastrointestinal tract and mast cells were degranulated. Eosinophils were present in the medullary cords of the lymph nodes. The lungs showed bronchiolar constriction. Haematological findings. The only alteration in the blood picture was a gradual increase of the neutrophil count from 1260 to a peak of2741 cells/mm 3 during the 15 min after the administration of Compound 48/80. Reserpine Clinical signs. The calf which received the larger dose showed incoordination and stood with its head lowered for 10 min after the injection. One hour later, the calf showed signs of abdominal discomfort, passed large amounts of fluid faeces and refused its food. Mter nine hours muscular tremors and champing of the jaws were observed. Twenty-four hours after injection, the calf appeared dull, and was unable to stand. Muscular tremors were still occurring and respiratory movements were short and rapid and these symptoms persisted for 24 hours. When a similar dose was given to the same calf four days later, muscular tremors and froth from the nostrils were observed 5 min after the injection. The tremors ceased within an hour although the calf showed signs of abdominal discomfort. The calf which received the smaller dose showed no immediate symptoms. Twelve hours after the injection, the calf was unable to stand, and muscular tremors and nystagmus with protrusion of the nictitating membrane developed. The calf salivated profusely and continually licked its lips until it was killed for au topsy. Post-mortem findings. In both calves, the mucosa of the small intestine was hyperaemic. The mesenteric lymph nodes were enlarged and oedematous, and the associated blood and lymphatic vessels were engorged. In the calf which received the larger dose, oedema was present in the submucosa of the abomasum and numerous small white plaques were found in the mucosa. No gross lesions were found in the lungs in either of the calves.
ANAPHYLAXIS IN CALVES
Histological findings. The white plaques in the abomasal mucosa of the calf which had the larger dose of reserpine proved to be erosions which extended deep in the glandular zone (Fig. 2). In both of the calves, aggregations of lymphocytes and neutrophils were present in the a bomasal mucosa and the submucosa was oedematous. In the small intestine, the lacteals were dilated and the blood vessels of the villi congested. The Peyer's patches were cleared of cells and lymphocytolysis was observed . Areas of pulmonary congestion were found a nd the bronchioles were constricted. Haematologicalfindings. In the calf tha t received the smaller dose of reserpine, the n eutrophil count decreased from I 1,5 I 5 to 7650 cells/mm 3 and the lymph- . ocyte count from 4506 to 2600 cells/mm 3 5 min after the injection. Similarly, in the calf that received the larger dose, the neutrophil count decreased from 6820 to 3847 cells/mm 3 and the lymphocyte count from 3850 to 263 2 cells/mm 3 • The leucocyte counts in both calves returned to their normal values an hour after the injection of reserpine. No alterations occurred in the erthrocyte count. Antagonistic drugs Mepyramine maleate One of the calves challenged with egg albumin 5 min after the inj ection of mepyramine maleate d eveloped mild incoordination and hyperpnoea that lasted for 15 min. Twelve days later the same calf was given mepyramine maleate 30 min before a subsequent challenge when it collapsed showing severe a naphylactic shock (Wray & Thomlinson, 1969). Another calf, given mepyramine maleate 5 min before challenge, also collapsed and showed severe anaphylactic shock. Cyproheptadine The calf which received the larger dose of cyproheptadine developed incoordination and hyperaesthesia after inj ection of the drug. No clinical signs were o bserved in the other two calves which received smaller doses of cyproheptadine. ''''hen the calves were challenged, they all showed severe anaphylactic shock. Diethylcarbamazine citrate When calves treated with this drug were challenged, they showed severe anaphylactic shock. DISCUSSION
Our ina bility to d etect a ny release of histamine confirms the experience of Code & Hester (1939) . The histamine levels showed close agreement with those observed by Nilsson (1963) in normal adult cattle. However, Eyre ( I97Ia) has shown that histamine is released in vitro from lung preparations from sensitized calves, but the concentration of histamine was lower than in other species such as the guineapig, rabbit and man. H e also suggested that there a ppeared to be a direct relationship between the age of the animal and lung histamine concentrations. Similarly, \\Tray & Thomlinson (1969) suggested that the severity of
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anaphylaxis in young calves may depend on the amount of pharmacodynamic agents available for release from the tissues. The present experiments were carried out in relatively young animals and it is possible that histamine may have been released in too low a concentration to be detected in the intact animal. Lewis & Eyre (1972) detected histamine at a concentration of 0'5 Il-g in an anaesthetized calf after the administration of Compound 48/80. Dopamine is known to be present in significant amounts in calves (Holtz, 1959) and is located in mast cells (Coupland & Heath, 1961 ). Eyre (197Ib) has recorded the release of dopamine from sensitized calf lung. In our experiments, however, dopamine produced only mild and transient symptoms. With histamine and 5-HT the symptoms and lesions closely resembled those of acute anaphylactic shock (Wray & Thomlinson, 1969) and endotoxin shock (Wray & Thomlinson, 1972b). Similar symptoms were observed by Aitken & Sanford (1972). These a uthors observed only minimal pulmonary oedema after the administration of 5HT. In our experiments, pulmonary oedema was marked, but larger doses of 5-HT were used. Similarly the doses of histamine were above those regarded as toxic by Desliens (1958) and Aitken & Sanford (1972) but none of the calves died. However, Desliens (1958) noted individu al variation in the susceptibility of cattle to histamine. Relatively mild symptoms developed following the · use of the endogenous releasing agents, compound 48/80 and reserpine. The effects of these drugs would be limited by the amount of the respective amines available for release. However, the effects of Compound 48/80 and reserpine might depend on the size of the dose used. Lewis & Eyre (1972) noted marked systemic toxic effects with Compound 48/80 which did not seem to be entirely due to the release of histamine or 5-HT, although the release of these amines probably played a major part in its toxic action. Erosions were observed in the abomasal mucosa of the calf which received the higher dose of reserpine, but not with Compound 48/80. Erosions were also observed in the abomasum of the calf which received the larger dose of 5-HT. Mota et al. (1956) showed that Compound 48/80, when used in rats, released histamine from mast cells but failed to release the relatively large amounts of histamine present in the fundus of the stomach. According to Espamer (1954), 5-HT is associated with the enterochromaffin system and it is possible that the erosions may have resulted from the release of S-HT in the abomasal mucosa itself. In the authors' experience, abomasal ulceration is a not uncommon sequel to coli bacillosis and it is interesting to speculate on the possible role of S-HT in the formation of these ulcers. On the other hand, Muggenberg et ai. (1966) concluded that gastric ulcers which developed in pigs following the administration of reserpine may have been associated with increased free acid and pepsin activity. Thus it is possible that ulceration may occur as a direct result of the action of reserpine. None of the antagonist drugs used in the present experiments afforded any protection against anaphylactic shock. Aitken & Sanford (1969) suggested that bradykinin and/or SRS-A may be responsible for the acute symptoms because sodium meclofenamate protected calves against anaphylactic shock. Eyre (197IC), however, observed that in in vitro experiments sodium meclofenamate
ANAPHYLAXIS IN CALVES
473
was not entirely specific in its actions against bradykinin. Although diethylcarbamazine citrate has been shown to inhibit the release of SRS-A in the rat (Orange, Valentine & Austen, 1968) it did not protect the calves in our experiments. Wells & Eyre (1972) have shown that this substance significantly inhibits cutaneous anaphylaxis in the calf and when given simultaneously with disodium cromoglycate the inhibitory effect was augmented. There is evidence (Fischel, Keller & O'Bryan, 1968) that, in mice, histamine and 5-HT are synergistic and when these substances are administered together, relatively small doses are capable of producing acute symptoms. Eyre (1971C) concluded that the in vitro reaction in the bovine pulmonary vein is associated with a complex interaction of histamine, 5-HT, SRS-A and possibly other substances. It is likely that the simultaneous release of several of these su bstances in small amounts would give rise to symptoms of acute anaphylactic shock and the results of the present experiments lend support to this view. A C KNOWLEDGEMENTS
We wish to acknowledge the financial support of the Agricultural Research Council. We should also like to thank Merck, Sharp and Dohme Ltd. for the gift of cyproheptadine. We are grateful to Mrs J. J. Jones and Mrs M. W. Harling for technical assistance and to Mr G. Weston for the photomicrographs. REFERENCES
AiTKEN, M. M. & SANFORD, j. (1969). Nature, Lond. 223, 314 AITKEN, M. M. & SANFORD,j. (1972) . J. compo Path. 82, 257 BROCKLEHURST, W. E. & LAHIRI, S. C. (1962). J. Physiol. Lond. 160, 15. CODE, C. F . ( 1939). Am. J. Physiol. 127, 78. CODE, C. F. & HESTER, H . R. (1939) . Am. J. Physiol. 127, 71. COUPLAND, R. E. & HEATH, I. D. (1961). J . Endocr. 22, 71. DALE, H . H. (1913). J. Pharmac. expo Ther.4, 16 7 DESLIENS, L. ( 1958). Presse Med. 66, 562. DRAGSTEDT, C. A. & GEBAUER-FuELNEGG, E . (1932). Am. J. Physiol. 102,5 12 . EDINGTON, N. (1968). J. Pharm. Pharmac. 20, 577· ESPAMER, V. (1954). Pharmac. Rev. 6, 425. EYRE, P. (197w). Archs into Pharmacodyn. Ther. 192, 347· EYRE, P. ( 197Ib). Br. J. Pharmac. 42,423. EYRE, P. (1971C). Br. J. Pharmac. 43, 302 FISHEL, C. W., KELLER, K. F. & O'BRYAN, B. S. (1968) . J. Immun. 101,679· GI BBONS, W. j . (1962). Mod. vet. Pract. 43, 34. HINSHAW, L. B., VICK, j . A. , CARLSON, C. H. &. FAN, Y. L. (1960). Proc. Soc. expo B iol. Med. 104, 379· HOLTZ, P. (1959). Pharmac. R ev. II, 326. LEWIS, A. J. & EYRE, P. (1972). Can. J. Physiol. Pharmacol. 50, 545· MOTA, L., BERALDO, W. T., FERRI, A. G. & j UNQ.UERIA, L. C. U. (1956). Ciba Foundation Symposium on H istamine, p. 47. London: Churchill. MUGGENBERG, B. A., KOWALCZYK, T., HOCKSTRA, W . G . & GRUMMER, R . H. ( 1966 ). Am. J. Vet. Res. 27, 1663. NILSSON, S. A. (1963). Acta vet. scand. 4, SuppJ. I. ORANGE, R. P ., VALENTINE, M. D. & AUSTEN, K . F. (1968) . J. expo Med. 12 7,7 67. SCHULTZ, W. H. (1910). J. Pharmac. exp o Ther. 1, 549. THOMLINSON, j. R. (1963)' Vet. R ec. 75, 12 46 .
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I I
September 1973 )
Recherches concernant Ie role des arnines phannacologiquell1.ent actives au cours de l'anaphylaxie (Wray et Thonillnson) ResUll1.e. On ne put detecter aucune liberation d 'histamine dans Ie plasma sanguin des vaches soumises au choc anaphylactique. Des symptomes et des lesions ressemblant a ceux du choc anaphylactique et du choc endotoxinique se developperent lorsque I'on injecta aux vaches de I'histamine et d e la 5-hydroxytryptamine mais la dopamine provoquait d es signes moderes et transitoires. On constata des symptomes et lesions a nalogues a la suite de I'emploi d'agents de liberation endogene, Compose 48/80 et la reserpine. La mepyramine, la cyproheptadine et la diethylcarbazamine ne reussirent pas a proteger les vaches contre Ie choc anaphylactique. II est suggere, que la synergie qui existe entre les differentes substances pharmacologiquement actives liberees a basse concentration peut etre responsable des symptomes d e choc anaphylactique aigu chez les vaches.
Untersuchung iiber die Rolle, die pharll1.akologisch aktive Amine iII1. anaphylaktischen Schock spielen (Wray und Thonillnson) Zusall1.ll1.enfassung. 1m Blutplasma von Kalbern, die einen anaphylaktischen Schock erlitten hatten, konnte kein Histamin gefunden werden. Wenn K a lber Injektionen von Histamin erhielten, entwickelten sich Symptome und Manifestationen, die einem anaphylaktischen Schock und einem Endotoxinschock ahnelten, aber Injektionen mit 5-Hydroxytryptamin verursachten nur leichte und vortibergehende Symptome. Ahnliche Symptome und Manifestation en wurden beobachtet nach Verwendung von endogen entspannenden Mitteln, Compound 40/80 und Reserpin. Mepyramin, Cyproheptadin und Diathylcarbamazinzitrat schtitzten die Kiilber nicht gegen anaphylaktischen Schock. Man ist der Ansicht, das eine Synergismus zwischen verschiedenen pharmakologisch aktiven Substanzen. die in schwacher Konzentration produziert werden, besteht und moglicherweise d as Auftreten von Symptomen akuten anaphylaktischen Schocks bei Kiilbern verursacht.
Investigaclon de la {unclon de las aII1.inas {annacologicall1.ente activas en la anafilaxis (Wray y Thomlinson) ResUll1.en. No se pudo descubrir la emision de histamina en el plasma sanguineo de terneros que se sometieron a un shock analfilactico. Empezaron a mostrar sintomas y lesiones parecidas a las de shock anafilactico y shock endotoxico cuando se inyecto a los terneros con histamina y con 5-hydroxitriptamina, pero la dopamina solamente produjo sintomas leves y transitorios. Se observaron sintomas y lesiones similares tras el empleo de agentes endogenos de emision, Compuesto 48/80 y reserpina. La mepiraIl;1ina, cifroheptadina y el citrato de dietilcarbamacina no protegieron a los terneros contra el shock anafiloctico. Se sugiere que el sinergismo entre varias substancias farmacologicamente activas emitidas en concentraciones bajas pudieran ser responsables por los sintomas de shock anafilactico agudo en los tern eros.