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Eimeria motelo sp. n. is described from faeces of the yellow-footed tortoise, Geochelone denticulata (L.). Oocysts are irregularly ellipsoidal or cylindrical, with slightly expressed lobed protrusions and irregularities at the poles, possibly caused by wrinkling of the oocyst wall, 17 (15-19) × 9.4 (8.5-11) µm, shape index (length/width) being 1.81 (1.45-2). The oocyst wall is smooth, single-layered, 0.5 µm thick with no micropyle. There are no polar bodies. Sporocysts are ellipsoidal, 8.9 (7.5-10) × 4.4 (4-5) µm, shape index 2.03 (1.7-2.5). A sporocyst residuum is present, composed of many granules of irregular size. The sporozoites are elongate, lying lengthwise in the sporocysts. Comparison with other species of the genus Eimeria parasitising members of family Testudinidae indicates that the presently described coccidium represents a new species. The name of Eimeria carinii Lainson, Costa & Shaw, 1990 is found to be preoccupied by a homonym, Eimeria carinii Pinto 1928 given to a coccidium from Rattus norvegicus. Therefore, it is replaced by Eimeria lainsoni nom. nov.

The genetic diversity of cryptosporidium in reptiles was analyzed using PCR-restriction fragment length polymorphism and sequence analysis of a small subunit rRNA gene. In the study samples of snakes, lizards and tortoises were analyzed and results suggest extensive genetic diversity.

Describes three species of eimerian coccidia discovered from rodents of the Ruwenzori Mountains in Uganda. Eimeria livingstonei found in Lophuromys flavopunctatus; Eimeria stanleyi found in Otomys denti; Eimeria spekei found in Otomys denti.

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.

Morphological features of the eugregarine Gregarina garnhami (Canning, 1956) parasitic in the caeca and mid-gut of the desert locust, Schistocerca gregaria, have been studied by transmission and scanning electron microscopy, with particular attention to the epimerite and the relationship between the epimerite and the host epithelium. The cytoplasmic core of the globular epimerite is overlain by a distinct cortical zone, limited on its cytoplasmic face by a membrane-like structure, with an underlying layer of mitochondria. The periphery of the cortical zone is strengthened by a mass of fine filaments, especially at its base. Fine tubular structures, apparently arising from the membrane-like structure, pass through the cortical zone and attach to the epimerite-host cell interface. The base of the cortical zone is supported by a distinct osmiophilic ring. The epimerite is separated from the rest of the gregarine body by a discontinuous septum. Maturing and mature trophozoites possess conically arranged fibrils, which arise from the epimeritic septum and continue into the protomerite region. The epimerite and associated structures are here discussed with regard to the detachment of the trophozoite from the host epithelium. In individuals already detached from the host epithelium, a central depression remained at the top of their protomerite, in the area formerly bearing the epimerite.

Cryptosporidium fragile sp. n. (Apicomplexa) is described from black-spined toads, Duttaphrynus melanostictus (Schneider) (Amphibia, Anura, Bufonidae) from the Malay Peninsula. The parasitized animals were directly imported from Malaysia and harboured C. fragile at the time of arrival. Oocysts were subspherical to elliptical with irregular contour in optical section, measuring 6.2 (5.5-7.0) x 5.5 (5.0-6.5) microm. Oocyst wall was smooth and colourless in light microscopy. The endogenous development of C. fragile in the stomach of black-spined toad was analysed in detail using light and electron microscopy. Cryptosporidian developmental stages were confined to the surface of gastric epithelial cells. In transmission experiments, C. fragile has not been infective for one fish species, four amphibian species, one species of reptile and SCID mice. Full length small subunit rRNA gene sequence was obtained. Phylogenetic reconstruction revealed distinct status of C. fragile within the clade of species with gastric localisation including Cryptosporidium muris Tyzzer, 1907, Cryptosporidium serpentis Levine, 1980 and Cryptosporidium andersoni Lindsay, Upton, Owens, Morgan, Mead et Blagburn, 2000. Described characteristics differentiate C. fragile from the currently recognized Cryptosporidium species. Our experience with the description of C. fragile has led us to revise the recommended criteria for an introduction of a new Cryptosporidium species name. C. fragile is the first species described and named from an amphibian host. Its prevalence of 83% (15/18) in black-spined toads within the 3 months after importation calls for strict quarantine measures and import regulation for lower vertebrates.

Cryptosporidium muris oocysts suspended in 200 microl of water were pipetted into plastic microcentrifuge tubes which were stored at 4 degrees C or frozen at -5 degrees C for 1, 3, 5, 7, and 10 days and at -20 degrees C for 1, 3, 5, and 8h, respectively. Other samples of C. muris oocysts suspended in water were heated in the metal block of a thermal DNA cycler. Block temperatures were set at 5 degrees C incremental temperatures from 40 to 70 degrees C. At each high temperature setting microcentrifuge tubes containing C. muris oocysts were exposed for 1 min. Both, frozen and heated oocyst suspensions as well as untreated control oocyst suspensions were then inoculated into each of four ICR mice by gastric intubation. Untreated, freeze-thawed or heated oocysts were considered infectious when oocysts of C. muris were found microscopically in the faeces of mice after inoculation. All inoculated mice that received oocysts frozen at -5 degrees C for 3, 5, 7, and 10 days and -20 degrees C for 1, 3, 5, and 8h had no oocysts in faeces. In contrast, C. muris oocysts frozen at -5 degrees C for 1 day remained infective for inoculated mice. Our results also indicated that when water containing C. muris oocysts was exposed at a temperature of 55 degrees C or higher for 1 min, the infectivity of oocysts was lost.

The enzyme-linked immunosorbent assay (ELISA) method is recommended for farm surveillance programs and may be useful for epidemiological studies in wildlife or for establishing Trichinella-free areas. In this study, our interest was to compare the specificity and the time of seroconversion of excretory-secretory (E/S) antigens prepared from Trichinella spiralis. A group of eight pigs was inoculated with 500 T. spiralis larvae per animal, and blood sampling was performed at 3 and 4-day intervals during all experiments. The numbers of muscle larvae were determined in four different muscles groups. The larvae per gram burden shows that the most heavily parasitized muscles were the diaphragm [mean = 43.7 larvae per gram (lpg)] and the tongue (mean = 16.9 lpg). Antibody responses were detected by any of eight infected pigs of T. spiralis. Using the ELISA method with E/S antigen, antibodies to T. spiralis were first found on the day 21st p.i. The initial detection of antibodies varied from 21st to 31st day p.i., and the peak was reported 42nd day p.i. Dynamic of antibodies was stable or increased slightly throughout the experimental period (60 days post-inoculation). Our results represent important data for validation of a serological test, especially if blood samples are taken during early stages of infection.

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.

This study investigated the humoral immune responses of goats experimentally infected with Fascioloides magna and Fasciola hepatica to F. magna excretory/secretory products (FmESP) or F. hepatica excretory/secretory products (FhESP), respectively. An enzyme-linked immunosorbent assay (ELISA) was used to determine serum antibody responses and for possible discrimination of F. magna and F. hepatica infections in goats. Comparison of ESPs of both flukes and evaluation of ESP antigenicity was also studied applying immunoblotting techniques. In all infected goats, antibody level was significantly increased (against negative control) since 2 weeks post infection (WPI). However, the dynamics of antibodies varied between F. magna and F. hepatica groups during the course of the infection. The cross-reaction of antibodies developed against F. magna and F. hepatica with ESP proteins was recorded by ELISA. The species-specific proteins 40, 120 kDa from FmESP and 80, 160 kDa from FhESP (with no antibody cross-reaction) were detected by two dimensional electrophoresis and immunoblot as the potential immunodiagnostic markers. Our results suggest that F. magna and F. hepatica infection could be distinguished by common immunological techniques based on species-specific antigen-antibodies interaction.

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 Sumava 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).

Herbivorous animals can play a very important role in spreading trichinellosis. In the study presented here, the susceptibility and distribution of Trichinella spiralis infection was examined in 16 goat kids. The goats were inoculated with 10,000 T. spiralis larvae isolated by artificial digestion methods. The animals were necropsied per two animals in weekly intervals, and the larval burdens in different muscle tissue and anti-Trichinella antibodies measured with the indirect enzyme-linked immunosorbent assay (ELISA) serological method using excretory-secretory (E/S) antigen for detecting anti-Trichinella antibodies were assessed during the experiment. T. spiralis larval burden was maximal at 6 weeks postinoculation (480-5,057 larvae/g according to locality), and the larvae were also found in the myocardium (0.77 larvae/g). In this paper, our next step was to compare the specificity and the time of seroconversion by means of ELISA based on E/S antigen prepared from T. spiralis. Antibody response was detected in all 16 goats. The ELISA test carried out showed the first increments in optical density 2 weeks postinfection (p.i.), reached their peak 4 weeks p.i., and remained elevated from that day until the end of the experiment (10 weeks p.i.). These results indicated that specific anti-Trichinella antibodies in goats persist for a relatively long time.

A new microsporidian Trachipleistophora extenrec n. sp. was isolated from a muscle lesion of the streaked tenrec Hemicentetes semispinosus Cuvier, 1798 (Mammalia, Tenrecidae), an insectivore endemic to Madagascar. The spores isolated from the tenrec were infectious to severe combined immunodeficient (SCID) mice by intramuscular injection. Material obtained from muscular lesions in mice was used for the parasite description. All developmental stages of the microsporidian were covered by a dense coat, which during sporogony changed into the sporophorous vesicle wall. Eight, 16, 32, or more spores were formed inside the sporophorous vesicle as the result of the division by plasmotomy and sequential fission of a multinucleate sporogonial plasmodium. Spores were ovoid, 4.7 x 2.8 microm in size, had a large posterior vacuole, and had an isofilar polar tube with 15-16 coils. Although the fine structure and the developmental pattern of the organism were in some respects similar to the genus Vavraia, molecular phylogeny based on the gene sequences of the small subunit rRNA and RNA polymerase subunit II indicated that the organism belongs to the genus Trachipleistophora. The diagnostic characters of the genera Trachipleistophora and Vavraia are discussed as well as the discrepancies between the phylogenies of these two microsporidian genera based on morphology and molecules.

The ultrastructure of merozoites, gamonts and oocysts of the neogregarine Mattesia dispora and their development in larvae of the flour moth Ephestia kuehniella were studied by electron microscopy. The apical complex of free macronuclear merozoites was very distinct in micrographs of sections, the polar rings being especially prominent. Two gamonts associated in head-to-head syzygy and the apical complexes served as the contact point during pairing. At this stage the rhoptries became reduced and the conoid widened. The gamonts had a foam-like appearance in the light microscope. Paired gamonts formed an envelope and developed into a gametocyst, within which the gamonts were separated by a distinct border. Four gametes and two residual cells developed inside the gametocyst. The gametes were covered with a single membrane. The gametes fused in pairs to form two spherical zygotes, each covered by two membranes and with one large nucleus. The external layer appeared more undulated than the inner one. A single membrane covered each residual cell. Walls were formed around both zygotes to produce two oocysts. Each mature oocyst was lemon-shaped with polar plugs and eight peripheral sporozoites, which had a pellicle similar to that of the merozoites, lay beneath the thick oocyst wall.

Three new coccidian (Apicomplexa: Eimeriidae) species are reported from the lesser seed-finch, Oryzoborus angolensis from Brazil. Sporulated oocysts of Isospora curio n. sp. are spherical to subspherical; 24.6 x 23.6 (22-26 x 22-25) microm, shape-index (SI, length/width) of 1.04 (1.00-1.15). Oocyst wall is bilayerd, approximately 1.5 microm thick, smooth and colourless. Micropyle and oocyst residuum are absent. The sporocysts are ovoid, 13.2 x 10.9 (15-17 x 10-13) microm, SI = 1.56 (1.42-1.71), with a small Stieda body and residuum composed of numerous granules scattered among the sporozoites. Sporozoites are elongated and posses a smooth surface and two distinct refractile bodies. Oocysts of Isospora braziliensis n. sp. are spherical to subspherical, 17.8 x 16.9 (16-19 x 16-18) microm, with a shape-index of 1.06 (1.00-1.12) and a smooth, single-layered wall approximately 1 microm thick. A micropyle, oocyst residuum and polar granules are absent. Sporocysts are ellipsoid and slightly asymmetric, 13.2 x 10.8 (12-14 x 9-12) microm, SI = 1.48 (1.34-1.61). Each sporocyst contains a barely visible Stieda body and a residuum composed numerous of granules. Sporozoites are elongated and each of them contains two distinct refractile bodies. Oocysts of Isospora paranaensis n. sp. are subspherical to broadly ellipsoid 24.3 x 19.8 (22-26 x 18-22) microm, SI = 1.22 (1.15-1.38) with smooth single-layered wall approximately 1.5 microm thick. A micropyle and oocyst residuum are absent, but one distinct ellipsoid polar granule (2.5-3.5 x 1.5-2.5 microm) is present. Sporocyst are ovoid, 15.7 x 10.1 (14-18 x 8-12) microm, SI = 1.46 (1.31-1.72), with distinct Stieda and sub-Stieda bodies. Each sporocyst contains a spherical sporocyst residuum, 4 microm in diameter All described isosporan species represent a possible cause of acute coccidiosis for O. angolensis in captivity.