The nutria (Myocastor coypus) is a semiaquatic rodent that originally inhabited South America. However, the animals have spread to different continents as alien species, and their numbers are quickly increasing, especially in North America, Europe, and Eastern Asia including Japan. Although nutrias have been suggested to serve as reservoirs for pathogens, including parasites, there have been few reports on this subject. In the present study, we surveyed the gastrointestinal parasites in nutrias living in Japan to better understand their prevalence in nutrias. We collected 72 samples of intestinal contents or feces from nutrias in Osaka and Okayama Prefectures. We found that 49 (68.1 %) samples were positive for Eimeria parasites, and two types of oocysts were identified: ellipsoidal (Type A) and subspherical (Type B) oocysts. In addition, Strongyloides myopotami was detected in 44 samples, and Capillaria spp. and Fasciola spp. were detected in one and three samples, respectively. Based on the morphologies of the detected Eimeria oocysts, Types A and B were identified to be E. coypi and E. fluviatilis, respectively. Phylogenetic analyses after PCR and sequencing targeting the cytochrome c oxidase subunit I gene placed the sequences of E. fluviatilis (Type B) as a cluster between the sequences of Eimeria derived from rodents. The sequences of the three subgenotypes of E. coypi (Type A) were included in the cluster containing the sequences of Eimeria spp. from rodents of multiple species, which is referred to as the Apionodes supercluster, and is separate from other clades. It has been suggested that Eimeria spp. from rodents in this cluster can quickly adapt to infect different hosts. Although further analyses are needed to construct more detailed phylogenetic trees, our results revealed the genetical positions of Eimeria spp. in nutrias. In addition, our results may be helpful when considering host specificity as well as host switching by the pathogen.
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.
One of the most important routes of transmission for Toxoplasma gondii infection is the ingestion of foods contaminated with cat feces containing sporulated oocysts. The diagnosis of T. gondii infection by fecal microscopy is complicated, as other similar coccidian oocysts are often present in the same fecal specimen. This study aimed to identify T. gondii oocysts in cat feces using a novel PCR technique. Feline fecal specimens (n = 254) were screened for coccidian oocysts by light microscopy using the Sheather's flotation method. PCR analysis performed on the same specimens targeted a 529 bp repeat element and internal transcribed spacer-1 (ITS-1) regions were used to confirm the presence of Toxoplasma oocysts. By light microscopy, 49/254 (19.3%) of specimens contained coccidian oocysts. PCR analysis demonstrated 2/254 (0.8%) and 17/254 (6.7%) positive results using Tox and ITS-1 primers, respectively. However, coccidian oocysts were not identified on microscopic examination of specimens that were PCR-positive by Tox primers. Coccidian oocysts were identified on microscopic examination of 6/17 (35.3%) of the PCR-positive fecal specimens using ITS-1 primers. The BLAST results of 16 ITS-1 sequences were identified as T. gondii (n = 12; 4.7%) and Hammondia hammondi (n = 4; 1.6%). There was slight agreement between the 529 bp and ITS-1 PCR results (κ = 0.148). This is the first report of the detection of Toxoplasma oocysts using PCR analysis on feline fecal specimens from Southern Thailand. The ITS-1 region has potential as an alternative marker to identify T. gondii oocysts in feline fecal specimens.