A pathological study of experimental long-standing Schistosoma bovis infection in sheep

J, Comp. Path~ 1998 Vol. l tg, 479 484

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SHORT PAPER

A Pathological Study of Experimental Long-standing S e h i s t o s o m a b o v i s Infection in Sheep M. C. Ferreras-Estrada, M.J. Garcia-Iglesias, C. P6rez-Martinez, M. Y. Manga-GonzMez*, V. Ramajo-Martin~, A. Escudero-Diez and J. F. Garcia-Marin Departamento de Patologia Anirnah Medicina Animal, Facultad de Veterinana, Universidad de Ledn, 24071 Le6n, *Departamento de Sistema.~ de Produccidny Sanidad Animal, Estacidn Agricola Experimental, Consejo Superior de Investigaciones Cientificas (CSIC), Apartado 788, 24080 Ledn and *Departamento de Patologia Animal, Instituto de Recursos Naturales y A~mbiolo~ia, Consejo Superior de Inve.~tigaeiones Oient(ficas (CSIC), C/ Cordel de Merinas 4052, 5'7001 Salamanca, Spain

Summary The pathological response of sheep to two dose levels (400 or 10000 cercariae) of Schistosoma boris was evaluated 24 weeks after infection. The results confirmed that a single low or high dose causes lesions in the liver and intestine, and that the lungs, lymph nodes, pancreas and abomasum are affected in sheep given a single high dose. In addition, the study showed that pathological changes (mainly a granulomatous inflammatory reaction) were induced not only hy eggs but also by adult worms, and that their severity was in general related to the dose of S. boris. Hoeppli reaction product, observed on the surface of adult schistosomes in some parasitic granulomas, showed no immunoreaction for IgG, IgA or lgM. 9 1998 W.B. Saunders Company Limhed

Introduction Schistosomiasis is an important parasitic disease which has adverse effects on animal health and productivity (Saad et al., 1984) and on human health (Khalil etal., 1995). Schistosoma boris Sonsino, 1876, is the main cause of ovine schistosomiasis, a disease known to occur in Spain (Sim6n and Ramajo, 1982). To gain a more complete knowledge of ovine schistosomiasis in the Iberian Peninsula, strict attention to diagnosis (based on coprology, post-mortem examination and serology) is essential, according to expert European Community advice (Hagan and Gryseels, 1994). This report describes the pathological responses of sheep to two dose levels of S. boris, the smaller of which probably resembled natural infection.

Materials and Methods Experimental Design Infective material consisted of the Salamanca strain of S. boris, isolated by Ramajo (1972) and maintained in Planorbarius metidiensis snails and sheep of the Castellana ()02 [ 9975/98/08~)479 + 06 $12.00/0

9 t998 W,B. Saundcrs Company Limited

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breed. Twelve 3-month-old Castellana sheep, free from naturally occurring helminths and kept indoors during the study, were divided into the following groups: group A, sheep infected with 400 S. bovis cercariae (n=6); group B, sheep infected with 10000 cercariae (n = 3); group C, uninfected (n = 3). The method used was the leg immersion technique (van Wyk et al., 1975). All sheep were killed 24 weeks after infection by an intravenous overdose of pentobarbital and necropsies were carried out immediately. Histopathology and Immunohistochemistry

Samples of the liver, gall bladder, small and large intestine, lymph nodes (mesenteric, mediastinal and hepatic), abomasum, pancreas, lung, testis, kidney and urinary bladder were fixed in 10% buffered formalin and embedded in paraffin wax, and sections (4g) were stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Masson-Goldner's trichrome. Egg granulomas were classified on the basis of their histopathological features, as proposed by Lindberg et al. (1993) for goats. Egg granulomas in which the mononuclear phagocyte population was dominated by giant cells (GC granulomas) or macrophages (M granulomas) were distinguished. Both types of granuloma usually contained one egg (degenerating or dead, sometimes calcified), empty egg shells and egg residues. Each type of granuloma was subdivided according to the absence ('a') or presence ('b') of fibrosis. Infiltration of granulomas with lymphocytes, plasma cells or eosinophils was recorded, as also was the occurrence of apparently intact (viable) or calcified (degenerated) eggs with little or no inflammatory reaction. The changes described above were subjectively graded as + / - - (minimal), +(slight), + +(moderate) or + + +(marked). Intravascular viable worms and the presence of a Hoeppli reaction in egg and worm granulomas were assessed. For the demonstration of immunogIobulins in Hoeppli reaction product, paraffin-wax sections were immunolabelled by the avidin-biotin complex (ABC) system (Vector Laboratories, Burlingame, CA, USA). The primary antibodies used were polyclonal swine antibodies against IgA and IgM, diluted 1 in 300 and 1 in 50 respectively (Eivai Bios Laboratories, UK), and polyclonal rabbit antibody against IgG, diluted 1 in 10000 (Vector Laboratories). Negative and positive controls for the primary and secondary antibodies were included. Results

Macroscopical lesions were mild to moderate in group A, some milky spots being found in the liver. In group B, the liver appeared firm, dark and somewhat shrunken, with many yellowish-white loci. Minute foci in the intestine and abomasum and firm dark-red foci in the lung were also observed. Adult schistosomes were found in pulmonary and hepatic vessels; thrombi were present in the hepatic veins, with associated phlebitis. The portal and the mesenteric lymph nodes were enlarged in both groups. Histopathological lesions consisted mainly of S. bovis egg- and worm-induced granulomas. Two different types of worm granuloma were observed. The first type ("primary granulomas") consisted of cell debris, degenerated worms (lacking identifiable vascular structures) and eosinophils, surrounded by macrophages, giant cells and occasional lymphocytes. Amorphous eosinophilic precipitates on the surface of some partly disintegrated worms (Hoeppli reaction) were observed (Fig. l); these precipitates gave no immunoreaction for IgG, IgM or IgA. The second type ("ageing granulomas") showed a wide area of central necrosis, sometimes with remnants of adult schistosomes, surrounded by a few macrophages, giant cells and a thick peripheral fibrous layer. In

S c h i s t o s o m a b o v i s Infection in Sheep

Fig. 1.

48 l

Ltln~ from sheep infccwd with I 0 000 S. bovi~ cercariac. Rcmnant of worms with Hoeppli reaction (arrows) and granulomatous response ("primary granuhmm" type). HE. Bar, 50 l.tm.

group A, primary granulomas were found only in the liver, but in group B they were seen in the lung and in low numbers in the liver. Numerous ageing granulomas were found only in the livers of group B animals. & boris egg granulomas were observed mainly in the liver and intestine of groups A and B (Table 1). In both infected groups the predominant finding in liver was the presence of GCb granulomas; these showed central eosinophilic necrosis only in group A (Fig. 2). The marked presence of egg granulomas throughout the small and large intestine was observed in group B animals, but in group A, such granulomas were mainly found in the jeiunum and ileum. In group B, GCa granulomas were found in the abomasum, pancreas and lung, surrounded by lymphocytes and eosinophils, and Ma granulomas and viable eggs were detected in lung and pancreas. GCb granulomas, sometimes with calcified eggs, and M granulomas, with or without fibrosis, were observed in the lymph nodes of group B sheep. No Hoeppli reaction was found on the surface of eggs in any organ examined. Both infected groups showed proliferative mesoendophlebitis in numerous portal tract veins, and occasional portal venous thrombophlebitis, caused by intravascular worms. Adult parasites were detected within the intestinal subserosal, mesenteric and pancreatic veins, without an inflammatory reaction. Apparently viable worms occurred in pulmonary arteries, with or without inflammatory lesions. Other changes observe in both inl~cted groups were hepatic periportal fibrosis, and increased numbers of intra-epithelial globule leucocytes in intrahepatic bile ducts and in the epithelium of intestinal crypts (more numerous in group B). Macrophages

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Table 1 S c h i s t o s o m e e g g - a s s o c i a t e d l e s i o n s in target o r g a n s

Site

Type of egg-associated

Egg-associated lesions in sheep of

lesion*

group A (400 cercariae) laver

Duodenum

J e j u n u m ileum

Colon

Ma Mb OC a GCb

+9 + w + + + (necrosis)

group B (10000 cercariae) m

+ +++

+1

Ma Mb GCa GCb

+++1

Ma Mb GCa GCb

+ w

+9

+ w

Ma Mb GCa GCb

+t

+++; 9 ++1 +++9 +++t w

* See Materials and Methods for abbreviations. t Some eggs calcified.

++All eggs calcified. w by lymphocytes, plasma cells and eosinophils. + + + , Marked; + + , moderate; + , slight; - , none.

of hepatic and mediastinal lymph nodes, laden with schistosomic pigment, were observed only in group B. Acinar atrophy, plasma cell infiltrates and necrosis of numerous ganglion cells adjacent to egg granulomas were also seen in the pancreas of group B animals. No schistosome-related lesions were detected in kidney, urinary bladder or testis in either infected group. Discussion In the chronic ovine schistosomiasis produced in this study, the main lesions resulted from the granulomatous inflammatory reaction to eggs, although worms were also responsible to a degree. Saad et al. (1984) found that the duodenum showed particularly severe egg-associated lesions in sheep given 4000 S. boris cercariae. Our results indicated that, after massive exposure, the main intestinal target areas were the jejunum and ileum, followed by spread of granulomatous lesions to the duodenum and colon; a dose of 400 cercariac failed to produce lesions in the duodenum. Hepatic granulomas consisting of one egg embedded within an eosinophilic core were described as "hyperergie granulomas" by Saad et al. (1984); they occurred in goats but not sheep infected with 4000 cercariae, as a result of the greater hypersensitivity of the goats. Such granulomas were also found in our group A animals, but in group B animals the granulomas also contained degenerating eggs. S. bovis worm-induced granulomas in the lungs of the more heavily infected

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Fig. 2. [,i~cr of a sheep infectedwith 400 ,S'.boeGccr~ariae. An eosinophilicnecrotic area is seen in the centre of an egg granuloma,surroundedhy a zone containingmacrophages,two conspiculusgiant cells, and a peripheral capsule of COlmCctivetissue. HI';. Bar, 50 ~tm. animals were reported for the first time in sheep; they have, however, been described in sheep and goats naturally infected with Schistosoma indicum (Singh el al., 1995). This fact, together with the presence of hepatic "ageing granulomas" - n o t reported previously in the literature in animals with high w o r m loads, m a y indicate that a strong immunological response against S. boris in unusual sites eliminated most of the adults. Hoeppli reaction product, observed on the surface of worms but not on eggs in the granulomas, has been described as an antigen antibody complex (Fransen et al., 1990). O u r results suggest that the a m o r p h o u s precipitates that form the Hoeppli reaction product do not contain IgG, IgA or IgM; IgE, which is known to be associated with protection against schistosome infection (Capron el al., 1995) was not investigated. References

Capron, A,, Riveau, G., Grzych, J. M., Boulanger, D., Capron, M. and Pierce, R. (1995). Development of a vaccine strategy against human and bovine schistosomiasis. Background and update. Proceedings of the IVth International Symposium on Sehisto.~omiasis and the IVlh ]Vhtional Meeting on Schistosomiasis, Rio de janeiro, Brazil, 29 ..Vog.,emberIo 3 December; 1993. Memorias do Instituto Oswaldo Cruz, 90, 235 240. Fransen,J., De Bont,J., Vercruysse,J., Van Aken, D., Southgate, V. R. and Rollinson, D. (1990). Pathology o[" natural injections of Schistosoma spindale Montgomery, 1906, in cattle, journal q/Comparati~,e Pathology, 103, 447 455. Hagan, P. and Gryseels, B. (1994). Schistosomiasis research and the European Community. Tropical and Geographical Medicine, 46, 259 268.

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Khalil, H. M., Sadek, M. S., E1-Baki, M. H., Khalil, N. M., Karam, M., Makled, M., Girgis, F. and Abd-el-Aziz, S. S. (1995). Detection of cellular and humoral markers (IL-2, IFN-gamma and IgE) in sera of patients with urinary schistosomiasis. Journal of the Egyptian Society of Parasitology, 25, 513 524. Lindberg, R., Monrad, J., Van Johansen, M., Christensen, N. O. and Nansen, P. (1993). Histopathology of experimental Schistosoma bovis infection in goats. Acta Veterinaria Scandinavica, 34, 183 191. Ramajo, V. (1972). Contribuci6n al estudio epizootiol6gico de la esquistosomiasis bovina (Schistosoma bovis) en la provincia de Salamanca. Anales de la Facultad de Veterinaria de Ledn, 18, 151 214. Saad, A. M., Hussein, M. F., Dargie,J. D. and Taylor, M. G. (1984). The pathogenesis of experimental Schistosoma boris infectious in Sudanese sheep and goats. Journal of Comparative Pathology, 94, 371 385. Simdn, F. and Ramajo, V. (1982). Present epizootiological state of bovine schistosomiasis in Spain. Revista Ibirica de Parasilologia, Volumen extraordinario, 423 429. Singh, S. V., Mann, H. S., Sharma, D. N., Srivastava, A. K. and Prakash, D. (1995). Pulmonary schistosomiasis in sheep and goats: incidence and pathomorphology. Indian journal of Vetehnary Pathology, 19, 38-- 39. van Wyk, J. A., Heitmann, L. P. and van Rensburg, L. J. (1975). Studies on schistosomiasis. 7. A comparison of various methods for the infestation of sheep with Schistosoma matthei. OnderstepoortJournal of VeterinaryResearch, 42, 71-74.

'[Received, August 14th, 1997] [ Accepted,June 29th, 1998 J