Genetical survey of novel type of Cryptosporidium andersoni in cattle in Japan.

Previously, we reported that an isolate of novel type of Cryptosporidium andersoni detected in cattle in Japan contained Type A (identical to C. andersoni reported previously) and Type B (having a thymine nucleotide insertion unlike the Type A) genotypes in the 18S rRNA gene. Here, we conducted an extensive investigation of Cryptosporidium infections in adult cattle in Japan from 2004 to 2007. Consequently, Cryptosporidium sp. were detected in 12 of the 205 cattle examined (5.9%), and partial sequences of the Cryptosporidium oocyst wall protein (COWP) gene in all isolates were identical to those of the previously reported data for C. andersoni whereas two signals were observed in the sequence of the partial 18S rRNA gene in all the isolates. In transmission studies using five of the isolates, they all infected SCID mice. Modified multiplex PCR using DNA of a single oocyst isolated from the infected SCID mice revealed that the partial sequences in the 18S rRNA gene of 40-80% of 10 isolates were identical to the Type A genotype of C. andersoni and those of other samples were identical to the Type B genotype. These results suggested that the C. andersoni novel type is widespread in cattle throughout Japan, and have multiple copies (Types A and B) in the 18S rRNA gene.

Detection of a mixed infection of a novel Cryptosporidium andersoni and its subgenotype in Japanese cattle.

Cryptosporidium oocysts were detected in the feces of cattle in Saga, Japan. Isolates were morphologically large. We attempted to identify the species or genotypes of the isolates by analyzing the partial sequences of the 18S rRNA and Cryptosporidium oocyst wall protein (COWP) genes, and measuring the infectivity in mice. The isolates showed 100% homology with Cryptosporidium andersoni in the COWP gene sequence and it could be transmitted to mice, but in the 18S rRNA gene, there was an additional signal in the ABI sequence chromatogram. To examine the additional signal, we analyzed both the 18S rRNA and the COWP gene sequences of a single oocyst passaged from mice using a modified multiplex PCR that was able to amplify both genes. As a result, it was revealed that two distinct genotypes (Types A and B) of a novel C. andersoni type existed in the 18S rRNA gene, whereas the COWP gene sequences of both oocysts were identical to C. andersoni. Although the sequence of the 18S rRNA gene of Type A was identical to that of C. andersoni, that of Type B had a thymine insertion and was not identical to any sequence registered with GenBank. Here we report that this is a new type of C. andersoni.

Infectivity of a novel type of Cryptosporidium andersoni to laboratory mice.

Previously, we reported 'a novel type' of Cryptosporidium andersoni detected from cattle in Japan, and showed that the isolate was infective to mice. In the present study, we examined the patterns of oocyst shedding in both immunocompromised and immunocompetent mice, as well as pathological lesions in the infected mice. After oral inoculation with 1 x 10(6) oocysts, all five severe combined immunodeficiency (SCID) mice began to shed endogenously produced oocysts on day 6 post-inoculation (p.i.). The number of oocysts per day (OPD) reached 1 x 10(6) on day 17 p.i., and an OPD level of 1 x 10(6) to 10(7) was maintained until 91 days p.i. when the mice were sacrificed. In the five immunocompetent mice inoculated with 1 x 10(6) oocysts, the pre-patent and patent periods were 6 and 19 days, respectively, and the maximal OPD level was 1.5 x 10(5) on average. On histological examinations of infected SCID mice, a large number of parasites were present on the surface of the gastric glands of the stomach, but not in other organs examined. In conclusion, the novel type of C. andersoni, which genetically coincides with C. andersoni reported in other countries, is infective to mice, but susceptibility was lower than that of Cryptosporidium muris infecting rodents from the perspective of infectivity to immunocompetent mice.