Investigation into limiting dilution and tick transmissibility phenotypes associated with attenuation of the S24 vaccine strain.

BACKGROUND: Babesia bovis is the causal agent of Asiatic redwater, transmitted by the pandemic tick Rhipicephalus (Boophilus) microplus. Disease control may target the tick vector using acaricides or anti-tick vaccines, or the parasite using chemoprophylaxis or anti-parasite vaccines. Current anti-parasite vaccines comprise live blood vaccines using attenuated B. bovis strains. Attenuation is attained by rapid passage that may result in different phenotypes such as reduced virulence, non-transmissibility by the tick vector, inability to sequester in the host (lack of limiting dilution) and limited genetic diversity. Attenuation and phenotypes may be linked to selection of subpopulations during rapid passage. The South African B. bovis S24 vaccine strain comprise a subpopulation that present low virulence, non-transmissibility, lack of limiting dilution phenotype and the presence of a single A558 Bv80 allele. The S24 strain could be co-transmitted with a field strain (05-100) suggesting sexual recombination. The present study investigated the change in phenotype for the S24 vaccine strain during rapid passage and co-transmission. METHODS: Vaccine phenotype change during passage as well as co-transmissibility was monitored using Bv80 allele specific PCR, limiting dilution and Illumina-based genome sequencing. RESULTS: The S24 population could not be rescued from the S16 passage as previously attained suggesting that selection of the S24 vaccine strain was a serendipitous and stochastic event. Passage from S16 to S24 also resulted in loss of the limiting dilution phenotype. Genome sequencing indicated sexual recombination during co-transmission with the 05-100 field strain. Analysis of the recombinant strain indicate that VESA1, smORF and SBP2 family members are present and may be responsible for the limiting dilution phenotypes, while various regions may also be responsible for the tick transmission phenotype. CONCLUSIONS: The molecular basis for tick transmission and limiting dilution phenotypes may be defined in future using selection based on these traits in combination with sexual recombination.