Transfected Babesia bovis expressing the anti-tick Bm86 antigen as a vaccine to limit tick infestation and protect against virulent challenge
Authors: Knowles Donald , Leszkowicz Mazuz Monica 
Published: January 2022

Bovine babesiosis, caused by the apicomplexan parasites Babesiabovisand B. bigemina, is a major tick borne disease of cattle with significant economic importance globally. The vectors of Babesia parasites are R. (Boophilus) annulatusand R. microplus. In Israel these parasites are transmitted manly by R. annulatus. The main goal of the proposal was developing and testing a novel B. bovisvaccine based on stably transfected attenuated B. bovisexpressing the anti-tick Bm86 antigen. This required generating a transfected- attenuated B. bovisparasite containing a bidirectional promoter expressing both, the gfp- bsd selectable marker and the tick vaccine antigen Bm86. The vaccine was tested for its ability to elicit protective immune responses against T. annulatusticks. Efficient control of babesiosis is based on a complex scheme of integrated management, including preventive immunization, anti-babesial chemotherapy and control of tick populations. Live vaccines based on attenuated parasites are the most effective measure to control babesiosis, and are currently used in several countries, including Israel. Live attenuated parasites lead to a chronic infection and development of strong and long term immunity in vaccinated cattle. Still, live vaccines have several limitations, including the difficulty to distinguish among vaccinated and naturally infected cattle and potential for sporadic outbreaks in vaccinated animals. Tick limitation is essential to control babesiosis but the main measure to reduce tick infestation is traditionally approached using acaricides, which is limited by environmental concerns and the development of resistance by the ticks. Alternative tick-control measures including the use of anti-tick vaccines are emerging, and at least partial protective immunity has been achieved against tick vectors by vaccination with recombinant protective tick antigens (ie: Bm86). In addition, the Babesia vaccine development toolbox has been recently expanded with the development of transfection technology in Babesia parasites. In this approved proposal we successfully developed a Babesia live attenuated transfected vaccine, which is able to express a B. bovisMSA-1 signal-Bm86 chimera and eGFP genes under the control of the B. bovisef- 1 and actin promoters respectively. Genetic analysis demonstrated specific stable integration of the transfected genes in the expected ef-1 locus, and immunofluorescence analysis confirmed expression of Bm86 in the surface of transfected parasites. When applied to splenectomized calves, the transfected parasites were able to cause persistent B. bovisinfection with production of antibodies reactive with Bm86 for at least six months. In addition, partial protection against ticks was also observed upon challenging the vaccinated animals with R. annulatuslarvae. However, when used on intact calves, the vaccine failed to elicit detectable immune responses against Bm86, and we are still in the process of interpreting the data and make necessary changes in our experimental approaches. Overall, the results obtained here represent a step forward towards the development of integrated vaccines against both ticks and tick –borne pathogens, using the Babesia attenuated parasites as a platform to the delivery of exogenous protective antigens