PATHOGENESIS OF Eimeria colchici IN THE INTESTINE OF CHICKENS AND THE RELATED IMMUNE RESPONSE

Loószová A. , V. Revajová, M. Levkut, J . Pist l: Pathogenesis of Eimeria colchici in the Intestine of Chickens and the Related Immune Response. Acta Vet. Brno 2001, 70: 191-196. To study the immune response in poultry, a non-specific host for pheasant Eimeria colchici coccidium, ten-day-old chickens were each orally inoculated with 106 oocysts. For determination and comparison of coccidia invasion and development, histological examinations in poultry and pheasant chickens were made. In histological sections of four intestinal regions (duodenum, jejunum, ileum and caeca), first generation schizonts were found in epithelial cells, 12, 36 and 60 h post-infection (p.i.). Invasion of chicken intestine was non-specifically localized in the duodenum and caecum. By 60 h p.i. the numbers of schizonts of E. colchici in chicken caecum were significantly lower compared to the numbers of schizonts in pheasant caecum (p < 0.005). We suppose that the size of the parasite was smaller in the fowl chickens as a result of a slower development of the first generation schizonts of E. colchici. In order to estimate the immunological response after administration, non-specific coccidia to the chickens, subpopulations CD3+, CD4+, CD8+, and BU1b+ in the peripheral blood and spleen were measured by flow cytometry. The indirect immunofluorescence method, mouse anti-chicken monoclonal antibodies, and goat anti-mouse secondary antibody were used. A significant (p < 0.05) increase of leukocytes and CD4 positive T cells was observed at 60 h post infection in chickens. We found that increase of CD4+ cells in a studied non-specific host infected with pheasant coccidia is similar to increase of these cells in host specific coccidia infection. It indicates participation of cellular immune response and one of the defence mechanisms during the invasion and development of coccidia in a non-specific host. Eimeria colchici, immunity, T-cell subsets, flow cytometry, intestine, experimental infection Marked host and characteristic colonisation site specificity defines the development of Eimeria species in the digestive tract. It is rare for this protozoan parasite to occur naturally in more than one host (Aly 1993). Doran (1978), however, was able to produce an experimental, patent infection in Leghorn chickens, the chuckar partridge, ring-necked pheasant and bobwhite quail using a turkey coccidium, Eimeria dispersa. The prepatent period was 6 h shorter in the quail and partridge than in chicks and pheasants. Furthermore, the oocyst shedding in chicks and pheasants was much lower than in partridge and quail. The only similarity found among the four hosts was the size and the nature of the second and third generation schizonts. Norton (1976) noted that E. colchici from a pheasant produced an infection in turkeys when a large number of oocyts was given to them. Infection with Eimeria induces an immune reaction in the host, including both humoral and cell-mediated responses that lead to acquired protection. Although it is generally accepted that acquired immunity is parasite-host species-specific, Augustine and Danforth (1990) showed that chickens repeatedly inoculated with E. adenoeides develop a measure of immunity that protected them at least partially from a subsequent moderate challenge with E. tenella. Reciprocal infection studies, in which turkeys were immunised with E. tenella or E. acervulina failed to show protection against challenge with E. adenoeides (Augustine ACTA VET. BRNO 2001, 70: 191–196 Address for correspondence: MVDr. V. Revajová, PhD. Dept. of Pathology University of Veterinary Medicine Komenského 73, 041 81 Ko‰ice, Slovakia Phone: +421 95 Fax: E-mail:revajova@uvm.sk http://www.vfu.cz/acta-vet/actavet.htm and Danforth 1995). Mechanisms preventing the intracellular development of Eimeria in the non-specific hosts are not fully understood. One of the mechanisms likely to be involved is the immune system of the host. A dominant role of the cell-mediated immunity in the hostprotective response to Eimeria infection has been shown (Wakelin and Rose 1990; Rose and Hesketh 1982; Lil lehoj and Trout 1993). To study the infection of E. colchici in a non-specific host we investigated whether E. colchici could successfully colonize the intestine of the chicken host. To quantify the effects of a reciprocal “cross infection”, we measured the elicited longitudinal changes in the blood and spleen lymphocyte subsets during infection. Materials and Methods Experimental design Thirty-six coccidia-free (1-day-old) White Leghorn chicks were raised in standard poultry cages and given nonmedicated feed and water ad libitum. At 10 d of age, the birds were distributed randomly into treatment and control groups (n = 18 in each). A pure culture of pheasant coccidium, Eimeria colchici, was obtained by isolation of a single oocyst on agar and infecting pheasant chicks and the oocysts collected from faeces were sporulated in 2% potassium dichromate (Goldová et al. 1998). Chicks of the treatment group were orally inoculated with 106 oocysts per bird, while the control group was sham-infected with inoculum buffer only. Six chicks of both groups were killed at 12, 36 and 60 h post infection and blood samples, and tissues were collected from spleen and intestine. Thirty two-week-old coccidia-free pheasant chicks were infected with a pure suspension of sporulated E. colchici oocysts (5 000 oocysts/per chicks). Histological examinat ion Intestinal samples were fixed in 10% neutral formalin and subjected to routine paraffin processing. From the sample blocks, 5 μm thick histological sections were cut and stained with haematoxylin-eosin. The sizes and numbers of schizonts in epithelial cells of the small intestine were measured after calibration of eyepiece micrometer in 50 microscopic fields. Counting of leukocytes and isolat ion of lymphocytes Blood samples for flow cytometry examinations and counting of leukocytes were collected from all animals by cardiac puncture into EDTA containing tubes. Lymphocytes were separated by Ficoll-Hypaque gradient sedimentation (Boyum 1974). Leukocytes were counted by routine laboratory method using Fried Lukáãová solution (475 μl solution plus 25 μl blood) (Fr ied and Janto‰oviã 1961). The spleen cells for flow cytometry were harvested by teasing and passing the splenic tissue through a 70 μm mesh screen (Hel ler 1987). Antibodies Primary antibodies: The primary mouse anti-chicken monoclonal antibodies (MoAbs) used are summarized in Table 1. They were obtained from Scandic (Czech Republic) and are produced by Veterinary Medical Research and Development Inc. (USA). The BU1b antibody was a generous gift of Prof. Heller. Secondary antibody: FITC conjugated goat anti-mouse IgG (Dakopatts, Germany). Flow cytometry analysis After the Ficoll-Hypaque separation, lymphocytes were twice washed with phosphate buffer saline (PBS) and once in a tissue cultured medium (TCM – RPMI 1640 supplemented by 2 % of foetal calf serum). Fifty μl of cellular suspension (1 x 106 lymphocytes in TCM) and 50 μl of specific or control MoAbs were mixed and incubated at 4 °C for 30 min. After incubation the cells were washed twice in the TCM, the resulting pellets were re-suspended and mixed with 25 μl of secondary antibody and incubated in the dark as described above. After staining the cells were washed twice in the TCM and once in PBS, then fixed in 1 % paraformaldehyde in PBS. Prepared samples 192 Specificity MoAbs Isotype Diluted CD3 RTMCA1378 mouse IgG1 1:25 CD4 SRTMCA1473 mouse IgG1 1:25 CD8 SRTMCA1377 mouse IgG1 1:25 BU-1b 8370-01 mouse IgG1 1:25 Table 1 Primary monoclonal antibodies