Genetic investigation of GPI anchored Bd37 orthologs in Babesia divergens group and use of recombinant protein for ecological survey in deer.

The Glycosylphosphatidylinositol (GPI) anchored protein group has great potential as an excellent immunodiagnostic marker, because of its high expression and necessity for parasite survival. Babesia divergens /B. capreoli group includes parasites with confirmed or possible zoonotic potential to cause human babesiosis. In this study, we investigated ortholog of Bd37, a GPI-anchored major merozoite surface protein of B. divergens sensu stricto, in the Asia lineage of the B. divergens /B. capreoli group. From two genomic isolates from sporozoites/sporoblasts, three Bd37 gene variants, namely Bd37 JP-A, JP-B, and JP-C, were isolated with 62.3% -64.1% amino acid sequences identity. Discriminative blood direct PCR revealed that JP-A was exclusively encoded in all parasites infecting wild sika deer examined (n=22). While JP-B and JP-C genes were randomly detected in 12 and 11 specimens, respectively. Recombinant JP-A-based ELISA showed an overall positive rate of 13.9% in deer in Japan from north (Hokkaido) to south (Kyushu islands) (24 prefectures, n=360). This positive rate was twice as high as that examined by 18S rRNA-based PCR (6.8%). Antibodies against recombinant JP-B and JP-C were also evident in the deer. This study demonstrated that the presence of three orthologs in the Bd37 gene family in Asia lineage and identified JP- A as an informative marker for serological surveys in Japan. This is the first report that diagnostic antigen of Babesia parasite was identified by a comprehensive analysis of genetic polymorphisms from a various developmental stage in host and vector... ImportanceBabesia divergens Asia lineage in B. divergens/ B. capreoli group is a parasite closely related to zoonotic pathogen B. divergens sensu strict (EU lineage) and Babesia sp. MO1(US-lineage). Large scale serodiagnostic system for this group has not been established. As the nature of the parasites antigenic differentiation to escaping from immunological attack in the host, investigation of diagnostic markers should consider such antigenic diversity inherited (circulating) in the population. We focused on the Glycosylphosphatidylinositol (GPI) anchor protein, Bd37, a major surface protein of the EU lineage, and investigated Asia lineage infecting sika deer and taiga tick in Japan. Three Bd37 ortholog genes (JP-A, JP-B, and JP-C) were isolated from the tick and deer, though only JP-A gene was exclusively encoded in the parasites genomes (n=36). In spite of sequence polymorphism in the N-terminal region, the antibody raised against the representative recombinant antigen, rJP-A2, reacted to various JP- A proteins. rJP-A2-based ELISA system revealed a positive rate in wild sika deer was 13.9% which is two times higher than that examined by genetic examination (PCR). GPI-anchored proteins are densely expressed and required for parasite survival. We showed GPI proteins including Bd37 and its ortholog are potentially excellent immunodiagnostic markers for emerging and growing human babesiosis.