Michal Pakandl

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 parasites life-cycle as it is in other apicomplexan species investigated so far.

Some properties of sialic-acid binding systems in Tritrichomonas suis and Tritrichomonas foetus.

Hemagglutination of normal and enzyme-treated red blood cells and its inhibition, in vitro adherence to porcine caecal mucus and kinetic properties of neuraminidase were carried out with Tritrichomonas suis and T. foetus. All tested strains adhered extensively to porcine caecal mucus in vitro and agglutinated human (A1, A2, B and O), rabbit, porcine and hen red blood cells. Different inhibitors were efficacious in hemagglutination activity (HA) tests using neuraminidase treated and untreated red blood cells. The Scatchard plot showed an independent type of cooperativity in porcine strain 41, while in bovine strain KVC-1, a positive type of cooperativity was observed.

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.

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

Abstract 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.

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.

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.

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.

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

Abstract 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 (FACStarplus). 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.

Migration of sporozoites and merogony of Eimeria coecicola in gut-associated lymphoid tissue.

The invasive phase ofEimeria coecicola was studied during the first 80 h postinoculation (p.i.). Using a method that synchronized the life cycle, sporozoites were observed in the duodenum and the jejunum until 32 h p.i. They were seen first in the villous epithelial cells or in host cells resembling intraepithelial lymphocytes (IEL). Later they were observed in IEL in the lamina propria. After 48 h p.i., no coccidian stage was identifiable in the mucosa of the small intestine but sporozoites appeared in the lymphoid cells of lymphatic follicles of the gut-associated lymphoid tissue (vermiform appendix, sacculus rotundus, and Peyers patches). The first merogony was observed 64 h p.i. in these lymphoid cells and in membranous epithelial cells (M-cells) but was never seen in the epithelium itself. Morphologically there were two types of meronts, depending on the host cell type, but in both cases the merozoites contained a refractile body and resembled sporozoites. The first meronts of the second generation were observed 80 h p.i. in the villous epithelial cells of the domes of the follicles of the gut-associated lymphoid tissue, where the further development of thisEimeria takes place. This pattern of invasion strongly suggests that sporozoites take an exclusively extraintestinal route to reach the target cells. Moreover, to our knowledge this is the first description of an eimerian merogony that does not take place in epithelial cells.

Blastocystis sp. from pigs: ultrastructural changes occurring during polyxenic cultivation in Iscove's modified Dulbecco's medium.

Abstract A porcine strain of Blastocystis sp. grown in LES medium was transferred to Iscoves modified Dulbeccos medium (IMDM) and encystation medium (EM). In comparison with the cells maintained in LES medium, the cells cultivated for several days in both IMDM and EM exhibited considerable differences in their morphology and ultrastructure. The central vacuole was mostly electron-opaque, usually with electron-lucent granules in its center. Mitochondrial matrix became less electron-dense and cristae were shortened and reduced in number. Two membranes of the nuclear envelope were dilated and the intramembranous space was filled with intermediately electron-dense bodies. Two or three nuclei surrounded by one joint outer membrane were often seen. In the old cultures the surface coat often disappeared and electron-dense pits on the cell surface were much more numerous than in young cultures or in the cells grown in LES medium. The possible role of the cells with peculiar ultrastructural features in the Blastocystis life cycle is discussed.