Multilocus sequence typing and population genetic structure of Cryptosporidium cuniculus in rabbits in Heilongjiang Province, China

Multilocus sequence typing (MLST) tools have been used widely to characterize population genetic structure of some Cryptosporidium species. To understand MLST subtypes and population genetic structure of Cryptosporidium cuniculus from rabbits in Heilongjiang Province, China, 34 C. cuniculus DNA specimens were collected including VbA21 (n = 6), VbA28 (n = 2), VbA29 (n = 18) and VbA32 (n = 8). They were analyzed by nested-PCR amplification and sequencing at seven microsatellite, minisatellite and polymorphic loci including CP47, CP56, ML2, DZ-HRGP, MSC6-5, MSC6-7 and RPGR. The CP47, CP56, MSC6-5 and MSC6-7 loci were monomorphic. The remaining loci were polymorphic, with two, three and two subtypes being found at ML2, DZ-HRGP and RPGR loci, respectively. Six MLST subtypes were obtained based on sequence information of 29 DNA specimens successfully amplified at all eight loci including gp60 locus. Linkage disequilibrium (LD) analysis showed a clonal population structure of C. cuniculus in the investigated areas. STRUCTURE, neighbor-joining and network analyses indicated the presence of two distinct groups, corresponding to VbA21 subtype and VbA28, VbA29 and VbA32 subtypes. This is the first report of MLST analysis of C. cuniculus. A clonal population structure of C. cuniculus suggested the prevalence of C. cuniculus in Heilongjiang Province is not attributed to the introduction of rabbits. Thus, prevention and control strategies should be focused on making stricter measures to avoid occurrence of cross-transmission and re-infection among rabbit individuals. Based on the previous findings of VbA21 subtype only in rabbits and VbA28, VbA29 and VbA32 subtypes both in rabbits and humans, the results of subpopulation analyses might be used to assess zoonotic potential of C. cuniculus subtypes in Vb family. These data will be helpful to explore source attribution of infection/contamination of C. cuniculus and understand its transmission dynamics in humans and rabbits in the investigated areas.