Background: Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied. Methods: The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed. Results: Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro. Conclusions: These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission. Graphical

Bovine babesiosis, caused by Babesia bovis and B. bigemina, is a major tick-borne disease of cattle with global economic impact. The disease can be prevented using integrated control measures including attenuated Babesia vaccines, babesicidal drugs, and tick control approaches. Vaccination of cattle with the Rhipicephalus microplus Bm86-based recombinant vaccine reduces the fitness of R. microplus and R. annulatus, but several booster inoculations are required to maintain protection. Herein, we generated a stable transfected strain of B. bovis expressing an enhanced GFP (eGFP) and a chimeric version of Bm86 (B. bovis/Bm86/eGFP). The eGFP was expressed in the parasite cytoplasm, whereas Bm86 was displayed on the surface of merozoites. Three splenectomized calves experimentally infected with B. bovis/Bm86/eGFP showed mild signs of acute disease and developed long-lasting antibody responses to B. bovis and native Bm86. No evidence of sequestration of parasites in the cerebral capillaries was found upon postmortem analysis, confirming attenuation of the strain. This is the first report of transfected B. bovis expressing the tick antigen Bm86 on the merozoite surface that elicits an antibody response to native Bm86. These results represent a proof of concept for a novel live, attenuated, tagged dual-vaccine approach to attempt simultaneous control of babesiosis and tick infestation.

Background: The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 (Bcsbp4) gene and protein (BcSBP4) and analyzes its antigenicity in infected equids. Methods: BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified BcSBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n = 18) using an indirect ELISA (iELISA). Results: Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi-infected equids, but none out of ten negative equid control sera, gave a positive signal in an rBcSBP4 based iELISA. Conclusions: The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide.

Bovine babesiosis is caused by apicomplexan pathogens of the genus Babesia, including B. bovis. This protozoan parasite has a complex life cycle involving dynamic changes to its transcriptome during the transition between the invertebrate and vertebrate hosts. Studying the role of genes upregulated by tick stage parasites has been hindered by the lack of appropriate tools to study parasite gene products in the invertebrate host. Herein, we present tfBbo5480, a transfected B. bovis cell line, constitutively expressing enhanced green fluorescent protein (eGFP) created by a whole gene replacement transfection strategy, that was capable of completing the parasite’s entire life cycle in both the vertebrate and invertebrate hosts. tfBbo5480 was demonstrated to respond to in vitro sexual stage induction and upon acquisition by the female tick vector, Rhipicephalus microplus, the tick specific kinete stage of tfBbo5480 was detected in tick hemolymph. Larvae from tfBbo5480 exposed R. microplus female ticks successfully transmitted the transfected parasite to a naïve calf. The development of the whole gene replacement strategy will permit a deeper understanding of the biology of parasite-host-vector triad interactions and facilitate the evaluation of upregulated genes during the parasite’s journey through the tick vector leading to new intervention strategies for the control of bovine babesiosis.

Bovine babesiosis caused by the tick-borne apicomplexan parasite Babesia bovis remains a threat for cattle worldwide, and new vaccines are needed. We propose using immune-subdominant (ISD) antigens as alternative vaccine candidates. We first determined that RAP-1 NT and RRA are subdominant antigens using recombinant antigens in ELISAs against sera from B. bovis-protected cattle. Protected animals demonstrated high antibody responses against the known immunodominant rRAP-1 CT antigen, but significantly lower levels against the rRAP-1 NT and rRRA antigens. Next, a group of cattle (n = 6) was vaccinated with rRRA and rRAP-1 NT using a FliC–Emulsigen mix as the adjuvant, and there was a control group (n = 6) with the adjuvant mix alone. All but one immunized animal demonstrated elicitation of strong humoral immune responses against the two ISD antigens. Acute babesiosis occurred in both groups of cattle upon a challenge with the virulent B. bovis, but a significant delay in the average rate of decrease in hematocrit in the vaccinated group, and an early monocyte response, was found in half of the vaccinated animals. In conclusion, we confirmed the immune subdominance of rRRA and rRAP-1 NT and the ability of FliC to increase immunogenicity of ISD antigens and generate useful information toward developing future subunit vaccines against B. bovis.

The tick-transmitted disease bovine babesiosis causes significant economic losses in many countries around the world. Current control methods include modified live-attenuated vaccines that have limited efficacy. Recombinant proteins could provide effective, safe, and low-cost alternative vaccines. We compared the expression of the Babesia bovis thrombospondin-related anonymous protein (TRAP) family from parasites in bovine blood, in vitro induced sexual stages, and kinetes from tick hemolymph. Quantitative PCR showed that in blood and sexual stages, TRAP3 was highly transcribed as compared to the other TRAPs. In contrast, the TRAP1 gene was highly transcribed in kinetes as compared to the other TRAPs. Fixed immunofluorescence assays showed that TRAP2, 3, and 4 proteins were expressed by both blood and sexual stages. Conversely, TRAP1 protein, undetected on blood and induced sexual stages, was the only family member expressed by kinetes. Live IFA revealed that TRAP2, 3, and 4 proteins were expressed on the surface of both B. bovis blood and sexual stages. Modeling of B. bovis TRAP1 and TRAP4 tertiary structure demonstrated both proteins folded the metal-ion-dependent adhesion site (MIDAS) domain structure of Plasmodium TRAP. In conclusion, TRAP proteins may serve as potential vaccine targets to prevent infection of bovine and ticks with B. bovis essential for controlling the spread of bovine babesiosis.