Břetislav Koudela

Redescription of Neospora caninum and its differentiation from related coccidia

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Genetic diversity of Cryptosporidium spp. in captive reptiles.

ABSTRACT The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium. Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards.

Dog shedding oocysts of Neospora caninum: PCR diagnosis and molecular phylogenetic approach

Results of molecular determination of a dog isolate of Neospora caninum in the Czech Republic are presented. Colorless bisporocystic oocysts measuring 10-13 micro m x 10-11 micro m were recovered from feces and used for DNA isolation. A diagnostic PCR procedure using previously described molecular methods was performed to determine the species. The N. caninum species-specific primers based on the Nc 5 region produced a positive result, while primers specific for Hammondia heydorni rDNA internal transcribed spacer 1 (ITS1) was negative. Sequencing and phylogenetic comparison of ITS1 rDNA and the D2 domain of the large subunit rDNA (D2 LSU) determined our isolate to be N. caninum. Phylogenetic analysis of closely related genera Toxoplasma, Neospora and Hammondia based on ITS1 and D2 LSU robustly distinguished three clades: (i). Toxoplasma gondii + Hammondia hammondi, (ii). N. caninum + Neospora hughesi, and (iii). H. heydorni. Based on phylogenetic relationships we propose three acceptable suggestions to solve the problem of taxonomy of these genera.

The Phylogeny of Goussia and Choleoeimeria (Apicomplexa; Eimeriorina) and the Evolution of Excystation Structures in Coccidia

The phylogenetic relationships of Goussia janae and Choleoeimeria sp. were analyzed using the small subunit ribosomal RNA gene (SSU rDNA). This is a first attempt to study the molecular phylogeny of coccidian genera parasitizing strictly poikilotherm hosts. The biliary Eimeria-like coccidia of reptiles classified into the genus Choleoeimeria form a sister clade to the family Eimeriidae, which confirms the separate generic status of the genus Choleoeimeria. The position of Goussia is less robustly resolved, since it forms a trichotomy with the Eimeriidae and Sarcocystidae, or alternatively constitutes the earliest branch of the coccidian lineage. Morphological similarities, namely the extracytoplasmic location of the endogenous stages, and the presence of sutures in the sporocyst wall are discussed in the context of the traditional classification of eimeriids. In contrast to the morphology-based systematics, the monophyly of Goussia and Choleoeimeria is not supported by the SSU rDNA data.

SEQUENCE ANALYSIS OF RIBOSOMAL AND MITOCHONDRIAL GENES OF THE GIANT LIVER FLUKE FASCIOLOIDES MAGNA (TREMATODA: FASCIOLIDAE): INTRASPECIFIC VARIATION AND DIFFERENTIATION FROM FASCIOLA HEPATICA

Complete sequences of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna are presented. In particular, small subunit (18S) and internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene (rDNA), as well as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) of the mitochondrial DNA (mtDNA), were analyzed. The 18S and ITS sequences were compared with previously published sequences of the liver fluke Fasciola hepatica. Fixed interspecific genetic differences were determined that allow molecular differentiation of F. magna and F. hepatica using either the PCR-RFLP method or PCR amplification of species-specific DNA regions. Additionally, intraspecific sequence polymorphism of the complete cox1 and nad1 mitochondrial genes in geographically distinct F. magna populations was determined. Based on the sequence divergences, short (<500 bp) variable regions suitable for broader biogeographical studies of giant liver fluke were designed.

Coccidiosis in goats in the Czech Republic

An observational study was conducted to determine coccidial infections in goats of 13 farms in the Czech Republic. The prevalence of oocysts of Eimeria species in kids (less than 3 months old), weaned but not served goats (from 3 months to 1 year), and adult goats (1 year or more) was determined. Nine Eimeria species were identified in fecal samples by Sheathers sugar flotation technique. The overall prevalence of Eimeria oocysts in fecal specimens was 92.2%. Eimeria arloingi was the most common species with an overall prevalence of 84%, followed by E. hirci (63%) and E. ninakohlyakimovae (56%). Other species present were E. christenseni (55%), E. alijevi (36%), E. caprina (25%), E. aspheronica (12%), E. capriovina (6%) and E. jolchijevi (2%). Two or more Eimeria species were detected in 88% of the samples. The most prevalent species in kids was E. arloingi, while in weaned but not served and adult goats E. ninakohlyakimovae was the most frequently found. The number of oocysts excreted was generally lower in adult goats (2567.3+/-12678 OPG), whereas higher number oocyst per gram of feces (OPG) were found in kids (18565+/-24888 OPG). Clinical coccidiosis was detected in two farms, and E. arloingi and E. ninakohlyakimovae were implicated as its cause. Disease was observed in kids 2 to 4 weeks after weaning and watery feces with clumps of mucus, and color changes from brown to yellow or dark tarry, weight loss, and dehydration were the most conspicuous clinical signs. At necropsy, macroscopic changes included mucosal hemorrhages and whitish nodular polyps in the jejunum were found. Histopathological changes were characterized by local hypertrophy and hyperplasia of intestinal villi, villus blunting and inflammatory infiltration in the lamina propria. Numerous developmental stages of the parasites were observed in enterocytes and lacteals of intestinal villi.

Infectivity of Cryptosporidium muris isolated from cattle

The infectivity of a bovine isolate of Cryptosporidium muris for various animals was studied by transmission experiments. Neonatal BALB/c mice, adult BALB/c mice, SCID mice, common voles (Microtus arvalis), bank voles (Clethrionomys glareolus), common field mice (Apodemus sylvaticus), Mongolian gerbils (Meriones unguiculatus), desert gerbils (Gerbilus gerbilus), guinea pigs, rats, rabbits and goats were used to test the infectivity of this isolate. Among these host species, only Mongolian gerbils were susceptible to the infection and discharged C. muris oocysts in their faeces. The prepatent period for 8-week-old Mongolian gerbils was 15-19 days, the patent period varied between 18 and 36 days. More protracted chronic infections have been observed in gerbils immunosuppressed with methylprednisolone. No signs of clinical illness or macroscopic changes were seen in infected gerbils. Cryptosporidial developmental stages were detected in the stomach, histopathological changes were characterized by epithelial hyperplasia and mucosal hypertrophy without inflammatory exudate. In spite of the fact that C. muris was able to infect gerbils, we do not consider gerbils to be a true hosts for C. muris of cattle origin. Based on our results, we suggest that significant differences in host specificity of individual C. muris isolates exist, and that wild rodents are not reservoir for C. muris infection of cattle.

An Ultrastructural Comparison of the Attachment Sites Between Gregarina steini and Cryptosporidium muris

ABSTRACT. Early developmental stages of Gregarina steini Berndt, 1902 from the intestine of Tenebrio molitor larvae were studied by transmission electron microscopy. The formation and structure of the eugregarine attachment site were compared with comparable features found on the feeder organelle of Cryptosporidium muris Tyzzer, 1907, from the stomach of experimentally infected rodents. The similarity of the attachment strategy between both organisms was revealed. The membrane fusion site in G. steini, formed by the trophozoite plasma membrane, host cell plasma membrane and a membrane‐like structure limiting the cortical zone of the epimerite, resembles the Y‐shaped membrane junction between the host cell plasma membrane, the trophozoite plasma membrane and membrane surrounding the anterior vacuole in C. muris. The anterior vacuole of C. muris appears to be the precursor of the feeder organelle and its structure is very similar to the epimeritic bud and the cortical zone of G. steini trophozoites. In both investigated organisms, the apical complex disappears early during cell invasion. The possibility of the epicellular location of Cryptosporidium on the surface of host cells is discussed.

Development of a Multilocus Sequence Tool for Typing Cryptosporidium muris and Cryptosporidium andersoni

ABSTRACT Although widely used for the characterization of the transmission of intestinal Cryptosporidium spp., genotyping tools are not available for C. muris and C. andersoni, two of the most common gastric Cryptosporidium spp. infecting mammals. In this study, we screened the C. muris whole-genome sequencing data for microsatellite and minisatellite sequences. Among the 13 potential loci (6 microsatellite and 7 minisatellite loci) evaluated by PCR and DNA sequencing, 4 were eventually chosen. DNA sequence analyses of 27 C. muris and 17 C. andersoni DNA preparations showed the presence of 5 to 10 subtypes of C. muris and 1 to 4 subtypes of C. andersoni at each locus. Altogether, 11 C. muris and 7 C. andersoni multilocus sequence typing (MLST) subtypes were detected among the 16 C. muris and 12 C. andersoni specimens successfully sequenced at all four loci. In all analyses, the C. muris isolate (TS03) that originated from an East African mole rat differed significantly from other C. muris isolates, approaching the extent of genetic differences between C. muris and C. andersoni. Thus, an MLST technique was developed for the high-resolution typing of C. muris and C. andersoni. It should be useful for the characterization of the population genetics and transmission of gastric Cryptosporidium spp.

The giant liver fluke Fascioloides magna (Bassi 1875) in cervids in the Czech Republic and potential of its spreading to Germany

The giant liver fluke Fascioloides magna is an important parasite of cervids in Europe. From September 2003 to December 2005, faecal samples and livers of red deer (Cervus elaphus) and fallow deer (Dama dama) were investigated to determine the current distribution of the fluke in the Czech Republic. Faecal samples were collected from 20 different areas, and livers of hunted deer were dissected from each locality to confirm F. magna infection. The prevalence of F. magna in examined areas determined by coprological examination varied from 4% to 95%. Moreover, new foci of F. magna infection were discovered in all localities in the Šumava mountains where F. magna was observed; this has epizootiological importance due to the possibility of the spread of F. magna into the German territory (Bavaria).