Development of a Multilocus Sequence Tool for Typing Cryptosporidium muris and Cryptosporidium andersoni

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.