Dynamics and responsiveness of T-lymphocytes in secondary lymphoid organs of rabbits developing immunity to Eimeria intestinalis.

Primary infection with Eimeria intestinalis confers very effective immunity against further infections in rabbits. This study was designed to determine the onset of the immune response in primary-infected rabbits and to characterise the immune status of protected rabbits. Variations in kinetics of CD4+ and CD8+ T-cell subpopulations were followed after primary infection at the intestinal sites of penetration (duodenum) and development (ileum), in mesenteric lymph nodes (MLN) and in the spleen. The response against the parasite was measured by specific lymphocyte proliferation in the spleen and MLN and by determining specific IgG titres in serum. The mucosal immune response was strong after primary infection and was characterised by (i) transient increase in the percentages of intestinal CD4+ lymphocytes and MLN CD8+ lymphocytes 14 days PI and (ii) strong increase in the percentages of intestinal CD8+ lymphocytes from 14 days PI persisting throughout further infections. Extensive infiltration of the lamina propria with CD8+ lymphocytes was observed 14 days PI. The specific proliferative response started between 7 and 14 days PI in MLN but remained undetectable in spleens for up to 21 days, in contrast to "immunised" rabbits. The fact that systemic immune responses were low after primary infection, in contrast to indicators of mucosal immune responsiveness, suggests that protection of rabbits against E. intestinalis infection is due to an effective mucosal immune response, and that systemic responses that increase after successive infections are only reflections of repeated encounters with parasite antigens.

Tissues and cells involved in the invasion of the rabbit intestinal tract by sporozoites of Eimeria coecicola.

This study was designed to identify an extra-intestinal route of migration of Eimeria coecicola sporozoites and the types of cell harbouring the parasite during the invasion of the intestine. The presence of E. coecicola in blood, spleen and mesenteric lymph nodes of infected donor rabbits was demonstrated by immunohistology on donor organs and measurement of oocyst excretion by coccidia-free recipient rabbits injected with whole-cell suspensions prepared from donor tissues. Two types of donor lymphocyte, B (IgM+) and T (CD5+), were labelled using a two-colour immunofluorescence-labelling technique and separated with a cell-sorter (FACStar(Plus)). The presence of parasites in the sorted cells was assessed by direct examination and by using the same in vivo test after intravenous injection of IgM+ B or CD5+ T lymphocytes collected from donors at different times after inoculation. This test provided evidence that the parasites were alive and still infectious within the sorted lymphocytes. It was demonstrated that both B and T lymphocytes were infected.

Vaccination against Eimeria magna coccidiosis using spray dispersion of precocious line oocysts in the nest box.

Coccidiosis mostly affects young rabbits just after weaning (5- to 6-week-old animals). Prevention of this disease must therefore be initiated before weaning. 'Precocious lines', derived from field species display good immunogenicity, though not pathogenic when administered at the right dose. In the present work, we tested the vaccination method of the whole litter at 25 days of age by spray dispersion of oocysts of a precocious line of Eimeria magna in the nest box. Three doses were tested, d1 = 4 x 10(2) oocysts, d2 = 4 x 10(3) oocysts, d3 = 4 x 10(4) oocysts. The animals were challenged individually with 10(4) oocysts of a wild strain of E. magna at 35 days of age, 5 days after weaning. The oocyst output and weight gain were recorded after vaccination and after challenge. The vaccinated animals did not display any vaccine reaction. The dose d3 was totally effective on oocyst output and on weight gain under our experimental conditions. As our challenge conditions were severe, we think that a lower vaccine dose could be used. This method proved to be efficient, and quick and easy to use because it did not require animal manipulation.

Acquired protection of the rabbit (Oryctolagus cuniculus) against coccidiosis using a precocious line of Eimeria magna: effect of vaccine dose and age at vaccination.

Sucklings were vaccinated orally once at 25, 27 or 29 days of age with a precocious line of Eimeria magna. Each group received two doses varying from 3.5 x 10(2) to 3.5 x 10(4) oocysts. At 36 days of age, animals received a challenge inoculation with 10(4) oocysts of the wild strain of E.magna. Vaccination reduced oocyst output 10 to 1000 times after the challenge inoculation and prevented the decrease in the weight gain observed in non vaccinated challenged animals. When the vaccination was performed more than 9 days before challenge, full protection was obtained. An individual oral vaccination performed with 3500 oocysts gave total protection whatever the age at vaccination between 25 and 29 days of age.

In utero transmission of Pneumocystis carinii sp. f. oryctolagi.

Although vertical transmission of Pneumocystis in human or animal hosts has often been suspected, no evidence demonstrating this infection route has been furnished until now. This widespread parasite is constantly found in the lungs of rabbits, which spontaneously develop a benign pneumocystosis at weaning. However, the infection source, the method of entry of Pneumocystis organisms into the rabbit and when this mammal is infected, remain to be known. As a few parasites have been microscopically observed and detected by PCR in the lungs of rabbits at birth, in utero Pneumocystis infection has been hypothesized. The presence of Pneumocystis was therefore carefully assessed in 16 pregnant does, their embryos and fetuses by using several detection methods. Pneumocystis was detected by PCR in maternal blood, embryos, amniotic fluid and fetuses. The parasite was also revealed histologically and by immunofluorescence in fetal and maternal lungs and in placentas. The results suggest that vertical transmission of P. carinii sp. f. oryctolagi occurs as early as at the 10th day of pregnancy.

Eimeria coecicola Cheissin 1947: endogenous development in gut-associated lymphoid tissue.

Coccidia-free rabbits were inoculated with different doses of a pure strain of Eimeria coecicola and samples of gut were taken at 80, 96, 112, 128, 144, and 160 h postinoculation. The use of a very low infective dose (2-20 oocysts) was sufficient to study the last merogony. The number of merozoites in meronts increased when the infective dose decreased. Only the first merogony of this coccidium in lymphocytes or M-cells of gut-associated lymphoid tissue (GALT) has previously been described. Three other generations of meronts are described herein. All these endogenous stages were observed in the epithelium of the vermiform appendix, sacculus rotundus, and Peyer's patches, especially at the bases of the domes. However, in heavily infected tissues the gamonts were seen throughout the epithelium of the GALT. The third- and fourth-generation meronts were of two types. As in other eimerian species of the rabbit, type A meronts produced thick polynucleated merozoites, whereas type B meronts gave rise to large numbers of thin merozoites with one nucleus. Microgamonts were polynucleated and less numerous than macrogamonts. Type A meronts were also polynucleated and less numerous at the end of the merogony. Therefore, types A and B could correspond to a sexual phenotype differentiation occuring during the two asexual phases of multiplication.

How do sporozoites of rabbit Eimeria species reach their target cells?

In all the studies performed on the invasion of the rabbit by sporozoites of 4 Eimeria species it was shown that the sporozoites first penetrate the duodenal epithelium; and then very quickly appear within the IELs of the duodenal epithelium. A few hours later they are found in IELs of their specific site of multiplication. The relationship between an "extraintestinal" route for sporozoites and the IELs homing phenomenon is therefore an exciting hypothesis.

Eimeria magna: pathogenicity, immunogenicity and selection of a precocious line.

A precocious line (PrEmag) of Eimeria magna in rabbits was obtained by selecting for early development of oocysts. The prepatent period was shortened by 46 h. The pathogenicity of PrEmag was substantially reduced and its reproductive potential was much lower (500 times) than that of the parent strain. Rabbits given 2500 oocysts of PrEmag were almost totally protected against a challenge with the parent strain. As in other precocious lines of coccidia from the rabbit, PrEmag showed morphological anomalies of the sporulated oocysts. Each sporocyst harboured one large refractile body instead of the two smaller ones in the parent strain.

The invasion of the rabbit intestinal tract by Eimeria intestinalis sporozoites.

To follow the route of migration of Eimeria intestinalis sporozoites from the excystation stage to their development in the epithelial cells of the ileum, we inoculated sporocysts into the duodenum of coccidia-free rabbits and euthanized the animals at 10 min to 12 h post-inoculation. Excystation occurred at less than 10 min after the experimental infection. The sporozoites penetrated into the epithelium of the duodenum at as early as 10 min post-inoculation; 6 h later, the number of sporozoites had dramatically decreased in the duodenal mucosa, with a corresponding increase being noted in the ileal mucosa. These findings suggest that sporozoites invade the duodenal epithelium and migrate to the ileum by an as yet unknown nonlumenal tissue route.

Eimeria media: selection and characterization of a precocious line.

A precocious line of Eimeria media was obtained by selection for early development of oocysts in rabbits. The prepatent period was reduced from 108 to 72 h. The precocious line was less pathogenic than the original strain, and its multiplication rate was lower. Rabbits given oocytes of the precocious line were totally immune to challenge with the original strain as assessed by change in weight gain but were partially protected as assessed by oocyst output. Selection for precocious development was accompanied by morphological changes in the sporulated oocytes; each sporocyst contained only one large refractile body instead of the two smaller bodies seen in the original strain.

Eimeria sp. from the rabbit (Oryctolagus cuniculus): pathogenicity and immunogenicity of Eimeria intestinalis.

The pathogenicity and immunogenicity of Eimeria intestinalis was evaluated in SPF rabbits. The animals were given immunizing doses of 6, 6 x 10(2), 6 x 10(3), and 6 x 10(4) sporulated oocysts and were challenged with 3 x 10(3) oocysts. The criteria analysed were the daily weight gain and the oocyst output. This study showed that E. intestinalis had strong immunogenicity, as the inoculation of 6 oocysts was sufficient to minimize the clinical expression of the disease following the challenge and to reduce the oocyst output by about 60%. The immunity towards the excretion of oocysts and the illness was absolute in animals inoculated with 600 or more oocysts. Moreover, this protection seemed to be efficient at least 8 weeks after the challenge. The present results also confirm the pathogenicity of E. intestinalis, although the occurrence of diarrhoea may be irregular, and emphasize the fact that the capacity of this Eimeria for multiplication is not a criterion for clinical diagnosis of the disease.

Selection and characterization of a precocious line of Eimeria intestinalis, an intestinal rabbit coccidium.

A precocious line of Eimeria intestinalis was obtained by selection for early development of oocysts in rabbits and after six consecutive passages in animals. This line (EiP) was derived from a wild strain (EiO) isolated in 1975 from the caecal content of a rabbit with coccidiosis. The prepatent period of the EiP strain was reduced from 215 h to less than 144 h, the result being that the oocyst sporulation time was the same for both lines. The excreted and unsporulated oocysts had exactly the same shape, but microscopical examination of the sporulated oocysts showed a marked difference between EiP and EiO strains. A huge refractile globule was located in each of two sporocysts of the precocious line, whereas no refractile globule was seen in the other two. The EiP line had a reproductive potential much lower (1000 times) than that of its parent strain EiO and, as judged by the weight gain, mortality and lesions that also occurred in the jejunum and above all in the ileum, its pathogenicity was substantially reduced.