' ' Morphological study of partridge Title development in the foreign host - (Gallus gallus) Revajová, Viera, Loószová, Adrian Author(s) Maria, Zibrín, Martin, Herich, Ro Mikulas The Journal of Protozoology Resea Citation 26-32 Issue Date 2006-04 URL http://ir.obihiro.ac.jp/dspace/ha RightsNational Research Center for Prot 帯広畜産大学学術情報リポジトリ OAK:Obihiro university Archives o
J. Protozool. Res. 16, 26-32 (2006) Copyright 2006, National Research Center for Protozoan Diseases Morphological study of partridge Eimeria procera development in the foreign host - Leghorn chicks (Gallus gallus) Viera Revajová*, Adriana Loószová, Maria Goldová, Martin Zibrín, Robert Herich and Mikulas Levkut Department of Pathological Anatomy, University of Veterinary Medicine, Komenského 73, 041 81 Košice, Slovak Republic *Corresponding author: Viera Revajová, E-mail: revajova@uvm.sk ABSTRACT In order to morphological study of non-specific host immune response, cross-species experimental infection of Leghorn chicks with partridge specific coccidium, Eimeria procera (10 6 oocyst/per birds) was done. Schizonts of E. procera were histologically observed in all the parts of small intestine and caeca of chicks. Heterophilic and lymphocytic infiltration in lamina propria, and damage of villi intestine were observed. Schizonts were studied by electron microscope after 12, 30, and 60 hours post-infection (h pi). Results demonstrated immature and structurally damaged schizonts at 12 h pi, delayed maturation of first generation schizonts at 30 h pi, and phagocytosis of parasites at 60 h pi by intraepithelial lymphocytes. Key words: Eimeria procera, intraepithelial lymphocytes transmission electron microscopy, specific and non-specific host, INTRODUCTION Coccidia of the genus Eimeria are intracellular parasites, usually of the epithelial cells of the intestine of the host (Lawn and Rose, 1982). Complex life cycle of the coccidia elicits antibody and cell-mediated immune response (Lillehoj, 1998). In spite of a host and site specificity of Eimeria spp. in the digestive tract, sporozoites from various species of Eimeria could invade the intestinal mucosa of foreign hosts with tendency to limited or no further development (Norton, 1967; Doran, 1978; Long and Millard, 1979). Immunology, genetics, and nutrition of the host are believed to play a role in the innate resistance. A dominant role of the cell-mediated immunity in the host-protective response to Eimeria infection has been shown (Lillehoj and Trout, 1993). Goldová et al. (2001) reported that the infection of Leghorn chicks with specific pheasant coccidia Eimeria colchici resulted in the dispersed, impotent invasion of the caeca and small intestine of these birds. The development of schizonts in the intestinal mucosa was inhibited, and they were significantly smaller than those in natural host (Loószová et al., 2001a). The increase in the number of peripheral blood cells in general, and CD4+ cells in particular, indicate their participation in defence during the invasion and development of coccidia in a non-specific host (Loószová et al., 2001b). Goldová et al. (1996; 2000) provided ultrastructural findings of E. procera endogenous development in experimentally infected partridges (Perdix perdix) as well as the duration of merogony and gamogony. The present investigation was undertaken to obtain some knowledge of the Leghorn chicks cross-infected with partridge specific coccidia E. procera and describes some details about localisation, development, and damage in the non-specific host intestine after experimental per os inoculation of this protozoa. 26
MATERIAL AND METHODS Animals and experimental design Thirty-six white Leghorn chicks were raised in standard poultry cages with free access to non medicated food and water. At 10 days of age, the birds were divided into experimental and control groups. The experimental group were orally infected with suspension of sporulated E. procera oocysts at a dose 10 6 /per bird. A pure culture of E. procera was obtained by single oocysts isolation on agar (Tsutsumi, 1972). The control group was administrated with a "placebo" of inoculum buffer only. Six birds of each experimental and control group were sacrificed by cervical dislocation at 12, 36 and 60 h pi. At necropsy, the samples from small intestine and caeca were collected from both groups of birds for histological examination and from caeca also for transmission electron microscopy. Histological processing The caeca, duodenum, jejunum, and ileum were examined from each group autopsied at each interval. Samples were fixed in 4 % phosphate-buffered formaldehyde, paraffin-embedded, sectioned at 5 µm, and stained with haematoxylin-eosin. Light microscopy was done for evaluation of samples. Transmission electron microscopy Small pieces of caeca were fixed immediately after removing in a mixture of fixatives (2.5 % paraformaldehyde and 2 % glutaraldehyde). Osmium tetroxide 2 % solution in distilled water diluted 1:1 with 0.2 M cacodylate buffer (ph 7.2) was used as a postfixative solution. The samples were processed using conventional techniques and embedded in Durcupan AMC. Ultrathin sections were cut on an LKB Nova ultramicrotome. Sections were collected on copper grids and stained with lead citrate and uranyl acetate (Mráz and Polónyi, 1988). Samples were observed using a Tesla BS 500 at 80 kv. RESULTS Developing schizonts of Eimeria procera were observed up to the nuclei of villous epithelial cells and between them 12 h pi in all sections of the small intestine and caeca. First generation schizonts were found bellow the nuclei of villous epithelial cells, between epithelial cells, and in the lamina propria mucosae 48 h pi. Degenerated first generation schizonts were located mainly in the duodenum and caeca in cryptal epithelial cells 60 h pi. Catarrhal inflammation, desquamation of epithelial cells, rupture and shortening of villi, together with heterophilic and lymphocytic infiltration presented intestine host reaction to protozoa. At 12 h pi the first developing schizonts of E. procera were enclosed within parasitophorous vacuole in the epithelial cells of caecal crypts non-specific host - chicks (Gallus gallus) (Fig. 1). The parasitophorous vacuoles were not fully developed and schizonts appeared damaged (Fig. 2). At 36 h pi maturation of first generation schizonts was delayed. Host nucleus and nucleolus were enlarged in infected enterocytes (Fig. 3). Atypical localization of some schizonts was also observed in the connective tissue of caecal mucosa (Fig. 4). Sixty hours pi. degenerated first generation schizonts were found in caecal enterocytes (Fig. 5), and occasionally they were phagocytosed and localized in the intraepithelial lymphocytes (Fig. 6). 27
Fig. 1. Electron micrograph of caecum from chicks experimentally infected with E. procera. Young first generation schizonts (S) enclosed in the parasitophorous vacuoles (PV) could be seen in the epithelial cell, closely to the host nucleus (HN), and covered by microvilli (M) 12 h pi. Bar: 1 µm. Fig. 2. Electron micrograph of first generation schizonts (S) sometimes enclosed in parasitophorous vacuoles (PV), and in close contact with intraepithelial lymphocyte (IEL) and host nucleus (HN), after experimental infection with E. procera 12 h pi. Bar: 1 µm. 28
Fig. 3. Electron micrograph of first generation E. procera schizont (S) located in close contact to the host nucleus (HN) and nucleolus (NU) of the epithelial cell (E). A number of degenerating merozoites are visible inside of schizont up to the basal lamina (BL) at 36 h pi in chicks post-experimental infection. Bar: 1 µm. Fig. 4. Electron micrograph of degenerate schizont (S) located in the connective tissue bellow the epithelium (E) of caeca in experimentally infected chicks with E. procera at 36 h pi. Host nucleus (HN), collagen microfilaments (CMF), and bacteria (B) were noticeable. Bar: 1 µm. 29
Fig. 5. The parasitophorous vacuole (PV) of the first generation schizont within intraepithelial lymphocytes (IEL) in close contact to the epithelial cell (E) and its host nucleus (HN), 60 h pi in caeca post-infection with E. procera Bar: 2 µm. Fig. 6. Three first generation schizonts (arrows) with degenerated merozoites located in the epithelial cell of caeca 60 h pi. Bar: 2 µm. DISCUSSION Virulence of coccidia reflects a number of factors. Among these are the location and type of cell infected by various stages of organisms, the function of infected cells, and the degree of host reaction stimulated by infection (Jubb et al., 1985). Goldová et al. (1996; 2000) reported experimental infection with E. procera in specific host partridge chicks (Perdix perdix), and their findings are useful in comparing ultrastructural changes in specific and non-specific host species. The infection of specific host partridge chicks with E. procera on 12 hours pi showed the development of rounded and nucleated schizonts in the epithelial cells of caecal crypts, and enclosed within parasitophorous vacuoles, derived from the host cell by a single membrane. Further development revealed hypertrophy of the host cell nucleus (Goldová et al., 2000). 30
In the present study, using the Leghorn chicks as host species, at 12 h pi developing E. procera schizonts were enclosed within parasitophorous vacuoles in the epithelial cells of caecal crypts, nonetheless parasitophorous vacuoles were not fully developed and schizonts manifested structural damage. Maturation of the first generation schizonts was delayed having been observable only at 36 until 60 h pi in the caecal enterocytes in the present study as compared to the development of mature and oval merozoites in partridge chicks that extended to the 2 nd generation schizonts (Goldová et al., 1996). Occasionally the first generation schizonts were phagocytosed and localized within the intraepithelial lymphocytes. Atypical localization of some schizonts was also observed in the connective tissue of caecal mucosa below the intestinal epithelium. The crossing of sporozoites of the basement membrane and of their engulfment by macrophages has been reported, however Lawn and Rose (1982) observed that most sporozoites are enclosed by host cells intraepithelial lymphocyte - throughout their journey from the epithelial surface to the crypt epithelium. Lillehoj and Trout (1996) had reported that macrophages and intraepithelial lymphocytes, and mainly CD8+ cells are involved in sporozoite transportation in Eimeria acervulina. We could observe (Loószová, unpublished data) the significant increase of CD3+ positive cells in duodenum 60 h pi (P<0.01), and in caeca 48 (P<0.05), as well as 60 h pi (P<0.01) by immunohistochemistry. Flow cytometry analysis in the same experiment showed no significant increase of CD8+ cells in the peripheral blood after infection with E. procera (Loószová et al., 2005). There are also differences in size of schizonts between the non-specific (chicken) and specific host (pheasant) (Goldová et al., 2001). Schizonts found in the Leghorn chicks caeca infected with E. procera measured 5.66 x 3.43 µm at 12 h pi, and 9.77 x 7.47 µm at 36 h pi. At 60 h pi, schizonts were smaller and measured 5.75 x 3.78 µm (Loószová, unpublished data). Histological observation showed damage to villi in the form of oedematisation and rupture in some, presence of large amount of goblet cells, and desquamation of epithelial cells, shortening and thickening. Schizonts were located in the epithelial cells of villi, in the lamina propria mucosae, in the crypt epithelium and lamina of crypts. Daszak and Ball (1993) described morphological alteration of caecal epithelial cells invaded by first-generation merozoites of Eimeria tenella in vivo. In this case the infection of enterocytes resulted in loss of microvilli and extensive bulging of cytoplasm into the crypt lumen. They also observed multiple invasions of enterocytes and invasion of goblet cells, and they found merozoites within mast cells and lymphocytes in the lumen. The complete loss of microvilli on the surface of invaded cells suggests significant alteration or disruption of the cortex of the host cell by the parasites. Unlike many protozoan parasites, the primary target tissue for coccidian is the intestinal epithelium. Acquired immunity to coccidiosis may involve immune mechanisms that both reduce the number of intracellular sporozoites and inhibit the natural progression of parasites development. Understanding the immune system-parasite interactions in the gut, leading to parasite elimination, is crucial for the design of new approaches to coccidiosis control. Destruction of parasite in non-specific host and activation of immunocompetent cells could be used for promoting of poultry defence against any infectious organisms. 31
ACKNOWLEDGEMENT We are very grateful to grant agency for financial supporting. This work was supported by the VEGA Grants 1/1367/04, 1/05852/03, 1/1370/04, and 1/1363/04 from the Ministry of Education the Slovak Republic. REFERENCES Daszak, P. and Ball, S.J. 1993. Ultrastructural observation on caecal epithelial cells invaded by firstgeneration merozoites of Eimeria tenella in vivo. Ann. Trop. Med. Parasitol. 87: 359-364. Doran, D.J. 1978. The life cycle of Eimeria dispesia Tyzzer 1929 from the turkey in gallinaceous birds. J. Parasitol. 64: 882-885. Goldová, M., Letková, V., Csizsmarová, G. and Kolodzieyski, L. 1996. Ultrastructural study of developmental stages of Eimeria procera in partridges (Perdix perdix). J. Protozool. Res. 6: 52-59. Goldová, M., Letková, V. and Csizsmarová, G. 2000. Life cycle of Eimeria procera in experimentally infected grey partridges (Perdix perdix). Vet. Parasitol. 90: 255-263. Goldová, M., Revajová, V., Pistl, J., Letková, V., Levkut, M., Wagshal, E., Csizsmárová, G. and Loószová, A. 2001. Eimeria colchici and immunocompetent cells in specific and non-specific hosts. Acta Parasitol. 46: 39-44. Jubb, K.V.F., Kennedy, P.C. and Palmer, N. 1985. Pathology of domestic animals. Protozoal enteritis. Vol. 2. London, Academic Press. 188-203. Lawn, A. and Rose, M.E. 1982. Mucosal transport of Eimeria tenella in the cecum of the chicken. J. Parasitol. 68: 1117-1123. Lillehoj, H.S. 1998. Role of T lymphocytes and cytokines in coccidiosis. Int. J. Parasitol. 28: 1071-1081. Lillehoj, H.S. and Trout, J.M. 1993. Coccidia: a review of recent advances on immunity and vaccine development. Avian Pathol. 22: 3-31. Lillehoj, H.S. and Trout, J.M. 1996. Avian gut-associated lymphoid tissue and intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev. 9: 349-360. Long, P.L. and Millard, B.J. 1979. Rejection of Eimeria by foreign host. Parasitol. 78: 239-247. Loószová, A., Revajová, V., Levkut, M. and Pistl, J. 2001a. Pathogenesis of Eimeria colchici in the intestine of chickens and the related immune response. Acta Vet. Brno. 70: 191-196. Loószová, A., Revajová, V., Goldová, M., Pistl, J., Czizsmarová, G. and Letková, V. 2001b. Eimeria colchici infection in chickens: the invasion and local immune response of a non-specific host. Acta Vet. Beog. 51: 133-142. Loószová, A., Revajová, V., Levkut, M., Goldová, M. and Letková, V. 2005. Immunological changes during Eimeria procera infection in a non-specific host. Folia Veterinaria 49: 206-209. Mráz, P. and Polónyi, J. 1988. Metódy elektrónovej mikroskopie živouíšnych tkanív. Bratislava, VEDA Press. pp. 33-119. Norton, C.C. 1967. Eimeria colchici sp. as the cause of cecal coccidiosis in English covert pheasants. J. Protozool. 14: 772-781. Tsutsumi, Y. 1972. Eimeria tsunodai sp. n. (Protozoa: Eimeriidae) a caecal coccidium of Japaneses quails (Coturmix coturmix japonica). Jap. J. Vet. Sci. 34: 1-9. 32
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