A novel promising diagnostic candidate selected by screening the transcriptome of Babesia gibsoni (Wuhan isolate) asexual stages in infected beagles

Abstract

Background: Babesia gibsoni is one of the causative agents of canine babesiosis worldwide. Some dogs infected with B. gibsoni show severe clinical signs with progressive anemia, hemoglobinuria and splenomegaly. However, most infected dogs present a state of chronic infection and thereby may be a persistent pathogen carrier, increasing the risk of pathogen spreading. To date, little is known about this pathogen, with genomic and transcriptomic data in particular generally unavailable. This lack of knowledge extensively limits the development of effective diagnostic strategies and vaccines. Methods: High-throughput RNA sequencing of total RNA of B. gibsoni asexual stages collected from infected beagles was performed. The unigenes were annotated in seven databases. The genes were sorted according to their fragments per kilobase per million (FPKM) value, which was used as an indicator for expression level. The gene with the highest FPKM value was cloned from the genome of B. gibsoni and further tested for immunogenicity, cellular localization and efficacy as a potential diagnostic candidate for detecting B. gibsoni in sera collected from beagles. Results: A total of 62,580,653 clean reads were screened from the 64,336,475 raw reads, and the corresponding 70,134 transcripts and 36,587 unigenes were obtained. The gene with the highest FPKM value was screened from the unigenes; its full length was 1276 bp, and it was named BgP30. The BgP30 gene comprised three exons and two introns, with a 786-bp open reading frame, and encoded 261 amino acids with a predicted molecular weight of 30 kDa. The cellular localization assay confirmed the existence of P30 protein in B. gibsoni parasites. Moreover, P30 was detected in the serum of experimentally B. gibsoni-infected beagles, from 15 days up to 422 days post-infection, suggesting its usefulness as a diagnostic candidate for both acute and chronic infections. Conclusions: We sequenced the transcriptome of B. gibsoni asexual stages for the first time. The BgP30 gene was highly expressed in the transcriptome screening experiments, with further studies demonstrating that it could induce immune response in B. gibsoni-infected dogs. These results lead us to suggest that bgP30 may be a good diagnostic candidate marker to detect both acute and chronic B. gibsoni infections. Graphical Abstract: [Figure not available: see fulltext.]