Coprodiagnosis of Hammondia heydorni in dogs by PCR based amplification of ITS 1 rRNA: differentiation from morphologically indistinguishable oocysts of Neospora caninum.

Hammondia heydorni is thought to be a non-pathogenic coccidian parasite of dogs that is closely related to Neospora caninum, an important parasite of cattle and dogs. Oocysts of these two species are morphologically indistinguishable from each other. A population of 2240 dogs in the Czech Republic was screened for the presence of H. heydorni/N. caninum oocysts and five (0.22%), represented by five of 3135 faecal samples (0.16%), were positive. The internal transcribed spacer 1 region of the rRNA gene (ITS1) from two isolates were cloned and the DNA sequences were identical with those of the ITS1 of H. heydorni. Based on the rRNA sequences available for H. heydorni and related coccidia, the primer pair JS4-JS5 was designed to amplify the 3' end of the small subunit (SSU) rRNA gene and ITS1 of H. heydorni. When tested on DNA extracted from a variety of parasites, the primers amplified a specific 267 bp fragment in our isolates only. The presence of DNA equivalent to 10 oocysts was sufficient for the amplification of the ITS1. We present a PCR-based diagnostic method as the only fast and reliable method for the diagnosis of H. heydorni in dogs.

Caryospora matatu n. sp., a new coccidian parasite (Apicomplexa: Eimeriidae) from the horned bush-viper, Atheris ceratophorus Werner, 1895 from Tanzania.

Caryospora matatu n. sp. is the first species of coccidia reported from the a horned bush viper, Atheris ceratophorus endemic to Tanzania. Oocysts are spherical or slightly subspherical, 19.8 (16-23) microns, a micropyle and an oocyst residuum are absent, a single polar granule is present. An oocyst wall is bilayered, approximately 1.5 microns thick, brownish and distinctly pitted. Sporocysts are ellipsoidal, 15.6 (12.5-17.0) x 10.1 (8-12) microns, tightly fitting an internal oocyst diameter, Stieda and substieda bodies are present. An experimental inoculation of SCID mice did not confirm a facultatively heteroxenous life cycle in the studied coccidium. Morphology and evolutionary history of Caryospora species from viperids is is reviewed and discussed.

Eimeria hajeki n. sp. (Apicomplexa: Eimeriidae), a new coccidian parasite of the pygmy chameleon, Rampholeon temporalis (Matschie, 1892) (Reptilia: Chamaeleonidae) from Usambara Mountains, Tanzania.

Fecal samples from 10 pygmy chameleons, Rampholeon temporalis (Matschie, 1892), an endemic species of the Usambara Mountains in northeastern Tanzania, were examined for coccidian parasites. Two (20%) chameleons were found to be passing oocysts of Eimerio Schneider. Comparison with other species of Eimeria indicates that the coccidian found represents a new species. Sporulated oocysts of Eimeria hajeki n. sp. are oval, 30.2 (29-31) by 23.5 (22-25) microm, with a shape index (length/width) of 1.3 (1.2-1.4) and a 2-microm-thick rough, bilayered wall. Micropyle and polar granule are absent. Sporocysts are oval to rhomboidal, 10.8 (9-11.5) by 8.8 (7.5-10) microm, with a shape index of 1.2 (1.15-1.3) and a wall composed of 2 valves joined by a suture.

Phylogenetic position of a renal coccidium of the European green frogs, 'Isospora' lieberkuehni Labbé, 1894 (Apicomplexa: Sarcocystidae) and its taxonomic implications.

'Isospora' lieberkuehni, an unusual isosporoid renal coccidium that parasitizes the European water frog was isolated from the edible frog, Rana kl. esculenta, in the Czech Republic. Sequencing of the small-subunit (SSU) rRNA gene showed that it belongs to the family Sarcocystidae, being closely related to a clade comprising members of the subfamily Toxoplasmatinae. The position within Sarcocystidae correlates with the mode of excystation via collapsible plates as postulated by previous authors. Phylogenetic, morphological and biological differences between 'Isospora' lieberkuehni and the other Stiedabody-lacking members of the genus Isospora justify separation of this coccidium on a generic level. Hyaloklossia Labbé, 1896 is the oldest available synonym and is herein re-erected. The original definition of the genus Hyaloklossia is emended based on recent observations.

Multiple origin of the dihomoxenous life cycle in sarcosporidia.

Although their ssrRNA gene sequences are closely related, the lizard sarcosporidia (Apicomplexa, Sarcocystidae) Sarcocystis lacertae and Sarcocystis gallotiae posses heteroxenous and dihomoxenous life cycles, respectively. When aligned with available sarcosporidian ssrRNA genes, both species constitute a monophyletic clade that is only distantly related with sarcosporidia that have a viperid snake as their definitive host (Sarcocystis sp., Sarcocystis atheridis). To test the phyletic status of the dihomoxenous life style, Sarcocystis rodentifelis and Sarcocystis muris, two dihomoxenous parasites of mammals were included into this study. All studied species group together with former Frenkelia spp., Sarcocystis neurona and related marsupial and bird sarcosporidia in a monophyletic clade. However, the available dataset supports independent appearance of the dihomoxenous life cycle at least twice during the evolution of the Sarcocystidae.

Caryospora varaniornati sp. n. (Apicomplexa: Eimeriidae) in the Nile monitor, Varanus (Polydaedalus) niloticus species complex.

Parasitological examination of two ornate Nile monitors Varanus ornatus (Daudin, 1803) imported from Benin revealed the presence of a new species of Caryospora. Oocysts of Caryospora varaniornati sp. n. are spherical to slightly subspherical, 12.0 (11-12.5) x 11.5 (11-12) microm, without amicropyle and oocyst residuum, and occasionally possessing one small polar granule. Sporocysts are broadly ellipsoidal, 8.8 (8.5-9.5) x 6.7 (6.5-7) microm; a lentil-like Stieda body is present, ca. 0.5 x 1 microm; substieda body not visible. Experimental infection of a closely related host, Varanus niloticus (L.), did not lead to the oocyst excretion despite the fact that one of the experimentally inoculated monitors was immunosuppressed by dexamethasone. Histological examination did not reveal stages of coccidian development. Therefore, it is possible that C. varaniornati is strictly host specific.

Eimeria telekii n.sp. (Apicomplexa: Coccidia) from Lemniscomys striatus (Rodentia: Muridae): morphology, pathology and phylogeny.

Using a combination of morphological, life-cycle and molecular data, we describe a new apicomplexan parasite Eimeria telekii n.sp. from a striped grass mouse Lemniscomys striatus captured in Kenya. Oocysts are oval to spherical or ellipsoidal, 20.4 x 15.7 (15.5-25.0 x 12.0-20.0) microm with a colourless, smooth and bilayered wall. Sporocysts are ellipsoidal, 11.2 x 7.8 (10.0-12.0 x 7.0-9.0) microm with a small Stieda body and granular sporocyst residuum and contain 2 elongated, banana-shaped sporozoites with a single refractile body. Life-cycle, pathogenicity and host specificity of this parasite were studied in laboratory-bred Lemniscomys barbarus and BALB/c mice. Two asexual stages and the sexual phase took place within the enterocytes of the caecum and colon of L. barbarus but not in inoculated BALB/c mice. An infectious dose of 5000 oocysts caused severe clinical illness and mortality in 2/2 (100%) L. barbarus. Phylogenetic analysis of the small subunit rRNA gene of E. telekii and members of the genera Eimeria, Cyclospora and Isospora placed E. telekii within the eimerian rodent clade.

Sarcocystis stenodactylicolubris n. sp., a new sarcosporidian coccidium with a snake-gecko heteroxenous life cycle.

Oocysts/sporocysts of Sarcocystis sp. measuring 9.7 (9-10) x 7.6 (7-8) microns were found in the intestinal contents of the Dahl's whip snake Coluber najadum. Of wide spectrum of experimentally inoculated hosts, only species of the family Gekkonidae--Ptyodactylus guttatus and Stenodactylus grandiceps--were found to be susceptible intermediate hosts. Transparent, barely visible sarcocysts found in tail, limbs and tongue striated muscles of the geckoes were 175-200 microns x 35-50 microns in size at 78 DPI. Ultrastructurally, the primary cyst wall was characteristic by spine-like villar protrusions up to 800 nm long, 200-250 nm in diameter at their base, tapering to thinner apex. Protrusions appear typically lobular or irregular in the cross-sections. Back-transmission from P. guttatus to Coluber rogersi leaded to oocysts/sporocysts excretion since 38 days post infection. Based on sarcocyst morphology and experimental data, Sarcocystis stenodactylicolubris is apparently a new species. Based on obtained and already published results, Sarcosporidia parasitising colubrid snakes as definitive hosts are suggested to be family specific on the level of their intermediate host.

SCID mice as a tool for evaluation of heteroxenous life cycle pattern of Caryospora (Apicomplexa, Eimeriidae) species.

Adult severe combined immunodeficient (SCID) mice were inoculated with oocysts of 13 different Caryospora (Protozoa, Apicomplexa) species isolated from the faeces of 10 reptilian and three raptorial bird hosts in attempt to test heteroxenous life cycle pattern. Only three reptilian isolates originated from viperid snakes, namely from Calloselasma rhodostoma, Atheris nitschei and Vipera ursinii induced lethal dermal caryosporosis in SCID mice. Neither clinical signs nor developmental stages were observed in mice infected with further nine caryosporan isolates originated from other reptilian and raptorial bird hosts. Results of this study confirmed that SCID mice represent a useful tool for evaluation of heteroxenous life cycle pattern of caryosporan coccidia and that only the Caryospora species from viperid and crotalid snakes produce dermal caryosporosis in mice

Isospora lutrae n. sp. (Apicomplexa: Eimeriidae), a new coccidium from the European otter Lutra lutra (L.) (Carnivora: Mustelidae) from Spain.

Parasitological examination of European otter originating from Extremadura, Spain revealed the presence of a new isosporan species. Oöcysts of Isospora lutrae n. sp. are spherical to subspherical, 31.2 (27.5-32) x 29.6 (28-31) microm and have a smooth wall c. 1 microm thick. Sporocysts are ellipsoidal, 18.2 (17-19) x 14.4 (14-16) microm and lack Stieda and substieda bodies. A spherical sporocyst residuum is present, consisting of granules scattered among the sporozoites. Sporozoites are spindle-shaped, 12.4 x 2.5 microm and have anterior and posterior refractile bodies. Based on its unique morphologic structure and host, I. lutrae is considered to be new.

Six new species of coccidia (Apicomplexa: Eimeriidae) from East African chameleons (Sauria: Chamaeleonidae).

Coprological examination of 83 East African chameleon specimens revealed 32.5% prevalence of coccidian parasites. Six species are described as new: Eimeria tilburyi n. sp. from Chamaeleo jacksonii has cylindrical oocysts, 28.9 (26-33) x 16.0 (14-18) microm and occasionally a small polar granule. Sporocysts are oval to ellipsoidal, 10.6 (9-12) x 7.2 (6-8) microm, without Stieda and substieda bodies; endogenous stages were found in the gall bladder. Oocysts of Eimeria largeni n. sp. from Chamaeleo gracilis are broadly cylindrical, 31.2 (29.5-34) x 19.3 (18.5-20) microm, with 1-3 polar granules. Sporocysts are oval, 10.2 (10-11) x 7.6 (7-8.5) microm, without Stieda and substieda bodies. Eimeria bohemii n. sp. from Chamaeleo melleri has cylindrical oocysts, 25.0 (24-26) x 14.0 (13-15) microm, without a polar granule. Sporocysts are broadly oval, 9.4 (9-10) x 6.5 (6-7) microm, without Stieda and substieda bodies. Isospora wildi n. sp. from Chamaeleo dilepis has subspherical to broadly oval oocysts, 25 (22-28) x 21.4 (18-24) microm, with a smooth wall 1 microm thick. Sporocysts are broadly oval to ellipsoidal, 12.3 (12-13) x 9.7 (9-10) microm, with Stieda and substieda bodies. Oocysts of Isospora necasi n. sp. from C. melleri are subspherical to broadly oval, 26.6 (21-30) x 24.3 (20-27) microm, with a velvetlike wall 2 microm thick. Sporocysts are broadly ellipsoidal, 12.8 (12-14) x 9.8 (9-10) microm, with slightly pointed end and with Stieda and substieda bodies. Oocysts of Isospora munriyu n. sp. from C. jacksonii are spherical to subspherical, 23.6 (21.5-25) x 21.9 (21-23) microm, with a finely granulated wall 1.5 microm thick. Sporocysts are broadly ellipsoidal, 12.4 (12-13) X 8.7 (8-10) microm, with Stieda and substieda bodies.

Sarcocystis atheridis sp. nov., a new sarcosporidian coccidium from Nitsche's bush viper, Atheris nitschei Tornier, 1902, from Uganda.

Transmission experiments were performed to elucidate the life cycle of a Sarcocystis sp. found in a Nitsche's bush viper, Atheris nitschei nitschei (Serpentes: Viperidae), from Uganda. Sporocysts measuring 10.4 (10.0-11.0) x 8.0 (7.0-8.5) microm were given to laboratory mice (Crl: CD1), laboratory rats (Wistar H), and Barbary striped mice, Lemniscomys barbarus. Sarcocysts developed in the skeletal muscles of laboratory mice and L. barbarus. No sarcocyst was observed in laboratory rats. Merogony was observed in the liver of L. barbarus at 7 and 12 days postinfection. Mature sarcocysts in mice reached a length of 30 mm and did not exceed 0.9 mm in diameter at 121 DPI. The primary sarcocyst wall was 0.6-0.8 microm thick and displayed small osmiophilic knob-like protrusions that were up to 150 nm long and 90 nm wide. Two types of asexual multiplication, endodyogony and endopolygony, were found within sarcocysts. Our results indicate that the newly found Sarcocystis represents a new species.

Phylogenetic analysis of Sarcocystis spp. of mammals and reptiles supports the coevolution of Sarcocystis spp. with their final hosts.

Sequences of the small subunit rRNA genes were obtained for two coccidians, Sarcocystis dispersa and an unnamed Sarcocystis sp. which parasitise the European barn owl and an African viperid snake as their final host, respectively, and share mouse as their intermediate host. Phylogenetic analysis of the sequence data showed that Sarcocystis sp. from the viperid snake is most closely related to another Sarcocystis sp. isolated from an American crotalid snake, while S. dispersa grouped with other bird-transmitted species. The available dataset failed to resolve the evolutionary relationships among four major branches into which all Sarcocystidae and Isospora spp. were split. However, within these branches, the phylogenetic relationships of the majority of analysed members of the genus Sarcocystis reflected coevolution with their final, rather than intermediate hosts.