Host specificity of turkey and chicken Eimeria: controlled cross-transmission studies and a phylogenetic view.

Protozoan parasites of the Eimeria genus have undergone extensive speciation and are now represented by a myriad of species that are specialised to different hosts. These species are highly host-specific and usually parasitise single host species, with only few reported exceptions. Doubts regarding the strict host specificity were frequent in the original literature describing coccidia parasitising domestic turkeys. The availability of pure characterised lines of turkey and chicken Eimeria species along with the recently developed quantitative PCR identification of these species allowed to investigate the issue of host specificity using well-controlled cross-transmission experiments. Seven species of gallinaceous birds (Gallus gallus, Meleagris gallopavo, Alectoris rufa, Perdix perdix, Phasianus colchicus, Numida meleagris and Colinus virginianus) were inoculated with six species and strains of turkey Eimeria and six species of chicken coccidia and production of oocysts was monitored. Turkey Eimeria species E. dispersa, E. innocua and E. meleagridis could complete their development in the hosts from different genera or even different families. Comparison of phylogenetic positions of these Eimeria species according to 18S rDNA and COI showed that the phylogeny cannot explain the observed patterns of host specificity. These findings suggest that the adaptation of Eimeria parasites to foreign hosts is possible and might play a significant role in the evolution and diversification of this genus.

Coccidia of turkey: from isolation, characterisation and comparison to molecular phylogeny and molecular diagnostics.

Coccidiosis is a disease caused by apicomplexan parasites of the genus Eimeria, which has a significant economic impact on poultry production. Multiple species infecting the turkey have been described; however, due to the general lack of unambiguous description, their identification and taxonomy is debatable. In this work, a systematic approach was taken to isolate, characterise and compare coccidian species in the turkey. Individual species were tracked according to their unique 18S ribosomal DNA sequence. The single-oocyst isolation technique and passaging of mixed species field isolates in selectively immunised birds enabled the derivation of pure species. Six distinct strains representing five eimerian species that infect the turkey were obtained. It appears highly probable that these species represent all species described in the past with the exception of Eimeria subrotunda. The species were analysed using both traditional methods and DNA sequencing. For each strain the oocyst morphology, prepatent period, gross pathology, pathogenicity, host specificity and endogenous cycle were studied. Antigenic similarity was investigated in multiple cross-immunity experiments. For identification and quantification of each individual species or strain, quantitative real-time PCR markers were also developed. Parallel characterisation of pure strains allowed comprehensive comparison with the original descriptions and assignment of correct species names. The species Eimeria meleagridis, Eimeria dispersa, Eimeria gallopavonis, Eimeria meleagrimitis and Eimeria innocua were identified. Comparison of our data with those of previous studies indicates that Eimeria adenoeides is most probably a synonym for either E. meleagridis or E. gallopavonis, or a description based on a mixture of these species, and thus nomen dubium. The species E. dispersa and E. innocua were also found to infect Bobwhite Quail. Phylogenetic reconstruction based on 18S rDNA and cytochrome c oxidase subunit I gene (COI) sequences showed that these two species form a distinct clade unrelated to other turkey coccidia and point to a polyphyletic origin of the species infecting the turkey.

The discovery of the two types of small subunit ribosomal RNA gene in Eimeria mitis contests the existence of E. mivati as an independent species.

Although the validity of the coccidian species, Eimeria mivati, has been questioned by many researchers for a long time there has not been any molecular analysis that would help resolve this issue. Here we report on the discovery of the two types of small ribosomal subunit (18S) gene within the Eimeria mitis genome that correspond to the known 18S sequences of E. mitis and E. mivati, and this is in conflict with the existence of E. mivati as an independent species. We have carried out five single oocyst isolations to obtain five single-oocyst-derived strains of E. mitis and these were analyzed by the sequencing of 18S and mitochondrial cytochrome c oxidase subunit I genes. The two types of 18S gene were found to be present in each strain in roughly equal ratios. This indicates that if the strains carrying only one or the other 18S type exist, they will likely cross-breed and still represent a single species. However, the more probable explanation is that all strains of E. mitis contain two types of 18S gene and that the occasional detection of only one or the other type by sequencing might be caused by insufficient sampling. This is also the first report of the two types of 18S gene in Eimeria, which has already been described in some other apicomplexan species, most notably Plasmodium. We also found that these two types of ribosomal RNA differ significantly in their secondary structure. The biological significance of the two 18S gene variants in E. mitis is not known, however, we hypothesize that these variants might be used in different stages of the parasite's life-cycle as it is in other apicomplexan species investigated so far.

Development of molecular assays for the identification of the 11 Eimeria species of the domestic rabbit (Oryctolagus cuniculus).

Coccidiosis are the major parasitic diseases in poultry and other domestic animals including the domestic rabbit (Oryctolagus cuniculus). Eleven distinct Eimeria species have been identified in this host, but no PCR-based method has been developed so far for unequivocal species differentiation. In this work, we describe the development of molecular diagnostic assays that allow for the detection and discrimination of the 11 Eimeria species that infect rabbits. We determined the nucleotide sequences of the ITS1 ribosomal DNAs and designed species-specific primers for each species. We performed specificity tests of the assays using heterologous sets of primers and DNA samples, and no cross-specific bands were observed. We obtained a detection limit varying from 500fg to 1pg, which corresponds approximately to 0.8-1.7 sporulated oocysts, respectively. The test reported here showed good reproducibility and presented a consistent sensitivity with three different brands of amplification enzymes. These novel diagnostic assays will permit population surveys to be performed with high sensitivity and specificity, thus contributing to a better understanding of the epidemiology of this important group of coccidian parasites.

Coccidia of rabbit: a review.

This article summarises the current knowledge of the rabbit coccidia and the disease they cause. Various aspects, such as life cycles, localisation in the host, pathology and pathogenicity, immunity and control, are discussed.

Dependence of the immune response to coccidiosis on the age of rabbit suckling.

To study the immune response to coccidiosis, the suckling rabbits were inoculated with 2,000 oocysts of either Eimeria intestinalis or Eimeria flavescens at 19, 22, 25, 29, and 33 days of age (DA) and in the case of E. intestinalis at 14 and 16 DA as well and sacrificed 14 days later. Another group served as an uninfected control and the rabbits were killed at the same age as their infected counterparts. Unlike the antibody response, the parameters reflecting cellular immunity (total number of leukocytes in mesenteric lymph nodes, lymphocyte proliferation upon stimulation with specific antigen and the dynamics of CD4+ and CD8+ cell proportions in the intestinal epithelium at the specific site of parasite development) were significantly changed from about 25 DA onwards. In contrast to the rabbits infected with weakly immunogenic coccidium E. flavescens, the proportions of CD4+ and CD8+ lymphocytes in intraepithelial lymphocytes from the specific site of parasite development were considerably changed after infection with highly immunogenic species E. intestinalis. As the immune system of sucklings from about 25 DA reacts to the infection, this age may be considered in terms of vaccination against coccidiosis.

The endogenous development of the rabbit coccidium Eimeria exigua Yakimoff, 1934.

The coccidium Eimeria exigua from the tame rabbit (Oryctolagus cuniculus) has been neglected so far since it was considered to be an invalid species. Indeed, little is known about this coccidium. We have studied its endogenous development in domestic rabbits by means of light and electron microscopy. The first meronts appeared 72h post-inoculation (h.p.i.), but a total of four asexual generations developed from 72 to 144h.p.i. Meronts and gamonts were localized in the small intestine and the asexual stages successively moved from the duodenum to ileum. All parasite stages were found exclusively in the epithelium of the walls and tops of the villi. Two types of meronts developing in parallel during the asexual phase were observed from at least the 2nd generation. As in other rabbit coccidia, these forms corresponded to type A, which usually develops two polynucleate merozoites where endomerogony occurs, and type B, characterized by more numerous uninucleate merozoites arising by ectomerogony. This report is the first description of the endogenous stages of E. exigua.

Immune response to rabbit coccidiosis: a comparison between infections with Eimeria flavescens and E. intestinalis.

Seven- to eight-week-old rabbits were infected with Eimeria intestinalis Cheissin, 1948, a highly immunogenic coccidium, or Eimeria flavescens Marotel et Guilhon, 1941, which is weakly immunogenic. Immune response was investigated at 7, 14 and 21 days post inoculation (DPI). The level of serum immunoglobulins, lymphocyte proliferation stimulated by parasite antigens and weight of mesenteric lymph nodes (MLN) showed similar dynamics in rabbits inoculated with both coccidia species. The amount of serum IgG and IgM, but not IgA, was increased from 14 DPI. The lymphocytes from MLN of infected animals significantly reacted to stimulation with parasite antigen 14 and 21 DPI and MLN were enlarged at 14 DPI. Thus, both parasite species elicited immune response characterized by these parameters in a similar manner despite of their different immunogenicity. The only apparent difference in the responses was in the percentage of CD8+ lymphocytes in the specific site of parasite development (the last third of the small intestine in E. intestinalis, caecum in E. flavescens), which increased in rabbits infected with E. intestinalis but not with E. flavescens. This parameter reflects the status of local immunity and hence the results suggest that the local reaction plays an important role in induction of protective immunity to coccidia in rabbits.

The reproduction of Eimeria flavescens and Eimeria intestinalis in suckling rabbits.

Suckling rabbits from six litters were orally inoculated at various ages with oocysts of Eimeria flavescens or Eimeria intestinalis. The total number of oocysts in the ceca served to estimate parasite reproduction. The rabbits at 14 and 16 days old remained uninfected, and in 19-day-old animals, low or no oocyst shedding was observed. All the older rabbits were infected and the number of oocysts in the ceca increased with the age of animals at inoculation. The dependence of oocyst production on the age of inoculated rabbits was similar in both coccidian species.

The rabbit coccidium Eimeria piriformis: Selection of a precocious line and life-cycle study.

Specific pathogen-free (SPF) rabbits were inoculated with oocysts of an original strain (OS) of Eimeria piriformis and the first newly developed oocysts recovered from the intestine were used for infection of other rabbits. The prepatent period (PP) was shortened after 12 passages from 194 to 170 hours and remained stable after 5 passages without any selection pressure. Oocysts of the precocious line (PL) exhibited peculiar morphology. Besides refractile bodies (RB) within sporozoites, one huge RB joined with a residual body was present inside each sporocyst. The parasite developed in the proximal colon and, to a lesser extent, in other parts of the large intestine. All stages were seen in the epithelium of crypts. In OS, four asexual generations preceded gamogony and, like in other rabbit coccidia, two types of meronts were observed: meronts of type A that develop into polynucleate merozoite, in which endomerogony takes place, and meronts of the type B that form uninucleate merozoites. The endogenous development of PL was identical with that of OS except for the last merogony which was absent. This resulted in earlier appearance of gamogony and shortening of PP. These observations of the life cycle of E. piriformis substantially improve on its description made 50-60 years ago.

Invasion of the intestinal tract by sporozoites of Eimeria coecicola and Eimeria intestinalis in naive and immune rabbits.

Naive and immune specific-pathogen-free rabbits were inoculated in the duodenum with sporocysts of Eimeria coecicola or Eimeria intestinalis. Samples were taken from the following tissues: duodenum (site of penetration of sporozoites), ileum (specific target site of the endogenous development of E. intestinalis), vermiform appendix (target site of E. coecicola) and two extraintestinal sites, mesenteric lymph nodes (MLNs), and spleen. The presence of sporozoites was checked by immunohistochemistry. In rabbits primary-infected with E. coecicola, large numbers of sporozoites were detected in the duodenum, extraintestinal sites, and vermiform appendix. The abundance of sporozoites in the spleen, MLN, and appendix was significantly reduced in the immune rabbits, and the migration seemed impeded. In the rabbits infected with E. intestinalis, sporozoites were absent in the spleen and MLN, indicating that the route of migration is different from that of E. coecicola. The number of sporozoites in the crypts of the ileum was markedly reduced in the immune animals.