Genetic investigation of GPI anchored Bd37 orthologs inBabesia divergensgroup and use of recombinant protein for ecological survey in deer
Authors: Zamoto-Niikura Aya , Hagiwara Katsuro , Imaoka Koichi , Morikawa Shigeru 
Published: March 2024

AbstractThe 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. capreoligroup 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 ofB. divergenssensu stricto, in the Asia lineage of theB. divergens/B. capreoligroup. From two genomic isolates from sporozoites/sporoblasts, threeBd37gene variants, namelyBd37 JP-A,JP-B,andJP-C,were isolated with 62.3% −64.1% amino acid sequences identity. Discriminative blood direct PCR revealed thatJP-Awas exclusively encoded in all parasites infecting wild sika deer examined (n=22). WhileJP-BandJP-Cgenes 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 by18S 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 ofBabesiaparasite was identified by a comprehensive analysis of genetic polymorphisms from a various developmental stage in host and vector…ImportanceBabesia divergensAsia lineage inB. divergens/B. capreoligroup is a parasite closely related to zoonotic pathogenB. divergenssensu strict (EU lineage) andBabesiasp. MO1(US-lineage). Large scale serodiagnostic system for this group has not been established. As the nature of the parasite’s 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 onlyJP-Agene was exclusively encoded in the parasite’s 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.