PCR slippage across the ML-2 microsatellite of the cryptosporidium MIC1 locus enables development of a PCR assay capable of distinguishing the zoonotic cryptosporidium parvum from other human infectious cryptosporidium species

Abstract

Cryptosporidium are ubiquitous and significant enteropathogens of all classes of vertebrates and a major cause of human morbidity and mortality worldwide. Of the 24 recognized species, the zoonotic Cryptosporidium parvum and the host- specific Cryptosporidium hominis cause the majority of cases of human cryptospo- ridiosis. Here, we report on structural and transcriptional variability between C. parvum and C. hominis at the MIC1 locus, which encodes a microneme local- ized thrombospondin-like domain containing protein previously demonstrated to be critical for host cell infection by C. parvum. We demonstrate, using reverse transcription quantitative PCR with the aid of genomic data from the EuPathDB site, that the transcribed product in C . hominis is both truncated and significantly down-regulated in the sporozoite. We hypothesize that CpMIC1 may be a genetic factor involved in facilitating the wider host range of C. parvum in comparison with the specific host range of C. hominis. Furthermore, we show that the pres- ence of a microsatellite (ML-2) within the C. parvum MIC-1 locus enables the development of a PCR marker that can rapidly distinguish the zoonotic C. parvum from C. hominis and other significant human infectious Cryptospori- dium species due to reproducible PCR slippage across the ML-2 microsatellite. Additionally, we demonstrate that this locus is tightly linked to the GP60 locus, a locus commonly used in the genetic characterization of C. parvum and C. homin- is isolates. This marker should provide a robust and additional tool to aid in the rapid identification of C. parvum from other Cryptosporidium species.