Functional analysis of the B. bovis bovipain-2 using a transfection approach

LACY P; ASCENCIO M; SCHNITTGER L; FLORIN-CHRISTENSEN M; SUAREZ CE

Conferencia; Vaccines R&D 2016; 2016

The tick-transmitted apicomplexan hemoprotozoon Babesia bovis imposes serious limitations to cattle development worldwide and improved control is urgently needed. Peptidases have been shown to play vital functional roles in apicomplexan parasites and have been proposed as vaccine candidates. Based on research in related Plasmodium parasites, we previously identified the cysteine protease bovipain-2 as a viable target for vaccine and drug development. We propose defining the phenotypic characteristics of B. bovis parasites with a KO in bovipain- 2 as a strategy aimed at defining its role in pathology and as a possible virulence factor. We first generated a stably transfection plasmid aimed at integration into the bovipain-2 locus, in a way that interrupts the integrity of the bovipain-2 gene ORF. We then electroporated B. bovis T3B parasites with the KO plasmid construct and successfully generated stably transfected fluorescent parasites expressing GFP. We verified that the parasites have an insertion in the correct bovipain-2 locus by performing integration PCR analysis. Sequencing of the PCR products confirmed the insertion of the gfp construct interrupting the bovipain-2 ORF. Importantly, Southern blot analysis using gfp and bovipain-2 probes are also consistent with a single gfp insertion disrupting the bovipain-2 locus. The data strongly suggests that bovipain-2 is not essential for the growth of B. bovis in in vitro cultures. Future work includes comparing the in vivo infective phenotype of wild type and bovipain-2 mutated parasites by inoculating bovines with both strains.