Diversity of Cryptosporidium spp. infecting rodents from genus Apodemus in the Czech Republic

We investigated the species and genotypes of Cryptosporidium infecting wild rodents from the genus Apodemus in ten areas in the year 2012 in the Czech Republic. A total of 207 faecal samples, 182 samples of Apodemus flavicollis and 25 of Apodemus sylvaticus, were screened for presence of Cryptosporidium spp. using both the aniline-carbol-methyl violet staining method and molecular tools. Microscopy examination revealed the presence of Cryptosporidium spp. oocyst in 24 samples of Apodemus flavicollis and 1 sample of Apodemus sylvaticus. Genomic DNA was isolated from both microscopy positive and negative samples. Using the nested PCR amplifying gene encoding the small ribosomal subunit (SSU rRNA) 25 samples were detected positive for the presence of Cryptosporidium-specific DNA. The same results were obtained also in the nested PCR amplifying gene encoding actin. All microscopy positive samples were also PCR positive. Only 19 samples were successfully sequenced, following phylogeny analyses showed presence of two new genotypes. First genotype is phylogenetically related to Cryptosporidium ubiquitum (1 sample) and the second genotype (consisting of several subgroups) related to C. canis (18 samples). The new genotypes seem to be host specific, however this hypothesis needs to be verified using experimental infection in the future. This is the first report of these Cryptosporidium genotypes in Apodemus spp. and for the first time ever