Targeted disruption of the B. bovis bovipain-2 does not impair the fitness of the parasite to growth in in vitro cultures

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

Conferencia; One Health 9th Tick and Tick-borne Pathogen Conference & 1st Asia Pacific Rickettsia Conference; 2017

The tick-transmitted apicomplexan hemoprotozoon Babesia bovis imposesserious limitations to the development of cattle industries worldwide andimproved control is needed. Peptidases have been shown to play vital functionalroles in apicomplexan parasites and have been proposed as vaccine candidates.Based on research in related Plasmodiumparasites, we previously identified the cysteine protease bovipain-2 as aviable target for vaccine and drug development. We propose defining thephenotypic characteristics of B. bovisparasites with a KO in bovipain- 2 as a strategy aimed at defining its role asa possible virulence factor. We electroporated B. bovis T3B parasites with a KO plasmid construct designed todisrupt the structure of the bovipain-2 gene locus with a gfp-bsd gene, and successfully generated stably transfectedfluorescent parasites resistant to blasticidin and expressing GFP. We verifiedthat the transfected parasites have a gfp-bsdinsertion disrupting the bovipain-2 locus by performing integration PCR analysis.Sequencing of the PCR products confirmed the expected insertion of thetransfected gfp-bsd gene interruptingthe bovipain-2 ORF. Importantly, Southern blot analysis using gfp and bovipain-2probes was also consistent with a single gfp-bsd gene insertion disrupting thebovipain-2 locus in the transfected parasites. Growth curve comparisons showedthat the Bovipaine-2 KO parasites growth at a similar rate as the wild typeparasites, suggesting that bovipain-2 is not essential for the growth of B. bovis in in vitro cultures.  Futurework includes comparing the in vivoinfective phenotype of wild type and bovipain-2 mutated parasites byinoculating bovines with both strains.