Phylogenetic relationships among Eimeria spp. (Apicomplexa, Eimeriidae) infecting rabbits: evolutionary significance of biological and morphological features.

Monophyly of all 11 valid Eimeria species from rabbits (Oryctolagus cuniculus Linnaeus, 1758) was revealed based on nuclear 18S rDNA sequence data. This finding implies that these species, which vary considerably in terms of their morphology and biology, diversified on a single host or several closely related species. Phylogenetic analysis divided rabbit Eimeria species into 2 sister lineages, corresponding to the presence/absence of the oocyst residuum. Other morphological or biological traits (oocyst shape and size, presence/absence of oocyst inner structures, pathogenicity, infection site, pre-patent and patent periods, sporulation time, and number of asexual generations) do not explicitly correlate with the phylogeny of rabbit coccidia.

Selection of a precocious line of the rabbit coccidium Eimeria flavescens Marotel and Guilhon (1941) and characterisation of its endogenous cycle.

The SPF rabbits were inoculated with oocysts of Eimeria flavescens and the first newly developed oocysts were recovered. They were used for inoculation of other rabbits which consequently excreted oocysts sooner than in the previous passage. By repeated use of this method, the prepatent period was shortened after 18 passages by more than 60 h. The endogenous development of this precocious line (PL) differed from that of the original strain (OS). Compared to OS, two asexual generations, second (or third) and fourth, were absent in PL. The first merogony took place in the jejunum and ileum in OS and, in contrast, in the large intestine in PL. Like in other rabbit coccidia, two types of meronts (A and B) were seen in each generation. However, the ratio of B: A meronts in the last (fifth) asexual generation as well as ratio of microgamonts:macrogamonts differs in OS and PL.

Early events in the life cycle of the mouse coccidium Eimeria falciformis (Eimer, 1870) Schneider, 1875 in naive and immune hosts.

The initial phases of invasion of mammalian coccidia of the genus Eimeria into host tissue are still poorly known. This process, including the passage of oocysts through the intestinal lumen, excystation of sporozoites, their penetration into epithelial cells and migration to the target site was studied in both naive and immune mice infected with Eimeria falciformis. After oral infection, the intact oocysts were transported with enteral contents to the large intestine, where the excystation of sporozoites and their penetration into superficial epithelium took place. The sporozoites subsequently migrated into the epithelium of crypts, which is the specific site of asexual multiplication. The immune status of the hosts did not affect the passage of oocysts, excystation and penetration of sporozoites. However, the migration of sporozoites towards their target site (crypts) was impeded in immune mice and sporozoites tended to remain in superficial mucosa rather than migrate to the crypts.

The rabbit coccidium Eimeria flavescens Marotel and Guilhon, 1941: an electron microscopic study of its life cycle.

The endogenous cycle of Eimeria flavescens was studied in specific pathogen-free rabbits by means of histology and transmission electron microscopy. In total, five asexual generations were observed and two types of meronts and merozoites were found in each generation. Type A gave rise to a smaller number of thick polynucleate merozoites in which daughter merozoites were formed by endomerogony, while in the type B meronts slender uninucleate merozoites arose from ectomerogony. The first generation meronts were found in the crypts and proximal part of the villi of the duodenum and jejunum, whereas the three following generations developed in the superficial epithelium of the large intestine (cecum, vermiform appendix and colon). The last merogony as well as gamogony took place in crypts of the large intestine.

Electron microscopic study on the endogenous development of Eimeria mulardi, Chauve, Reynaud and Gounel, 1994: a coccidium from the mule duck.

An electron microscopic study of the endogenous development of Eimeria mulardi Chauve, Reynaud and Gounel, 1994 was carried out in mule ducks which are hybrids of the domestic duck (Anas platyrhynchos) and the muscovy duck (Cairina moschata). All of the endogenous stages were seen within the nucleus of the host cell. Merozoites arose from ectomerogony and three mutually similar merogonies were noted. The asexual stages were found in leukocyte-like cells in the lamina propria of the jejunum, ileum and caecum, while the gamonts developed in glandular epithelial cells in the same part of the intestine.

Electron microscopic study on sporocysts and sporozoites of parental strains and precocious lines of rabbit coccidia Eimeria intestinalis, E. media and E. magna.

The fine structure of sporocysts and sporozoites of parent strains and precocious lines of rabbit coccidia Eimeria intestinalis, E. magna and E. media was studied. The parent strains and precocious lines differ only in the shape and size of refractile bodies (RB). In the sporocysts of precocious lines of E. magna and E. media, one extremely large RB was seen, either inside one of the sporozoites, or free in the sporocyst. In the oocysts of the precocious strain of E. intestinalis, two sporocysts resembled those of the precocious lines of E. magna and E. media, whereas the other two sporocysts did not harbour any RB. Sporozoites of all the precocious lines contained no, or very small, RB after in vitro excystation.

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.

Life cycle of Eimeria vejdovskyi Pakandl, 1988: electron microscopy study.

The endogenous development of the rabbit coccidium Eimeria vejdovskyi was studied in SPF rabbits using light and electron microscopy. All endogenous stages were seen in the ileal epithelium. There were two types of meronts in each of five asexual generations. Type A produced fat, polynucleate merozoites while type B meronts had slender uninucleate merozoites. First- second- and third-generation meronts were in the crypts. The first meronts were at the bottom of the crypts, very often in the Paneth cells. The meronts of the fourth generation were in the middle of the villi; the fifth-generation meronts were recorded in the middle and at the top of the villi. The prepatent period was 10 days.

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

A porcine strain of Blastocystis sp. grown in LES medium was transferred to Iscove's modified Dulbecco's 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.

Eimeria magna Pérard, 1925: study of the endogenous development of parental and precocious strains.

The endogenous development of a parental strain of E. magna and its deriving precocious line was studied after inoculation of coccidia-free rabbits with oocysts or sporocysts directly into the duodenum and using electron microscopy. Four meront generations could be observed mainly in the jejunum and ileum for the parent strain. The first merogony began 24 h post-inoculation (h p.i.). and meronts were matured about 48 h p.i. The second and the third generation were complete at 66 and 84 h p.i. respectively. Thus, each generation needs approximately 16-20 h for maturation. The fourth generation appeared 102 h p.i. and the young gamonts were present 120 h p.i. As in the other rabbit Eimeria, two types of meronts were described: A and B. The morphology of all endogenous stages of the precocious line was similar but refractile bodies were absent in the first generation merozoites and the fourth generation meront was lacking.

Eimeria media Kessel 1929: comparative study of endogenous development between precocious and parental strains.

Endogenous development of a pure strain of Eimeria media and of a precocious line derived from this strain was studied in specific pathogen-free (SPF) rabbits. Endogenous development of the parental strain comprised three generations and the gamogony began 76 h post-inoculation (pi). Two types of meronts were observed in each generation. The type A meronts gave rise to large, polynucleated merozoites present in low numbers. Multiplication was carried out by endomerogony. Within type B meronts, merozoites arose from ectomerogony. These were slender and more numerous than those of type A. The sporozoite refractile body was divided and distributed into the first and second generation merozoites but not into the third. The endogenous development of the precocious line was similar to that of the parental strain except that no refractile body was observed and the last merogony was absent. Gamogony appeared 60 h pi.

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.

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.

The prevalence of intestinal protozoa in wild and domestic pigs.

We examined faecal samples from 842 domestic and 91 wild pigs in order to record the prevalence of intestinal protozoa. We used staining of faecal smears by Noller-Westphal-Gönnert method, cultivation in modified LES broth and microscopical examination of native preparations using small magnification (for detection of Balantidium coli). Moreover, the faeces from wild pigs were examined by flotation in Sheather's sugar solution for detection of coccidia. We found 8 coccidian species in the wild pigs. Except sucking piglets, high prevalence of B. coli, Entamoeba polecki, lodamoeba sp. and Tritrichomonas suis was noticed in all categories of animals. On the other hand, only sporadic occurrence of Chilomastix sp. was recorded. The intensity of infections by different protozoa was variable in all categories of animals. It is not sure whether Chilomastix sp. and Iodamoeba sp. are identical with the species known from the intestine of humans and that is why their epidemiological importance is not clear.

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

The invasive phase of Eimeria 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 Peyer's 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 this Eimeria 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.

An experimental transmission of porcine strains of Blastocystis sp. in the laboratory mice and gerbils.

Outbred laboratory mice were successfully infected with porcine strains of Blastocystis sp. using faecal stages as well as stages from fresh or passaged cultures. In contrast, inbred BALB/c mice and gerbils were only rarely infected and the intensity of their infection was mostly negligible. Blastocystis sp. was never observed inside the host cells. These results indicate a low host specificity of Blastocystis sp. as well as different sensitivity of investigated hosts to Blastocystis sp. infection.

Giardia infection in pigs: detection and in vitro isolation of trophozoites of the Giardia intestinalis group.

Giardia infections were detected in 14 out of 32 weaned, 1 to 2-month-old pigs, used in experiments with coccidia. Giardia trophozoites were present in the small intestine from the duodenum to the ileum with maximum numbers in the cranial part of the upper jejunum. They were localized mainly on the surface of intestinal crypts. No histological changes accompanied the infection and the infected animals were asymptomatic. Giardia trophozoites obtained from the intestines of necropsied pigs were isolated in axenic culture in a bile-supplemented TYI-S-33 medium. Morphological features of the trophozoites revealed by both light and scanning electron microscopy proved that the organism belongs to the Giardia intestinalis group.