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Vaccination of cattle with the *Babesia bovis* sexual-stage protein HAP2 abrogates parasite transmission by *Rhipicephalus microplus* ticks

Veterinary Science

Vaccination of cattle with the *Babesia bovis* sexual-stage protein HAP2 abrogates parasite transmission by *Rhipicephalus microplus* ticks

M. G. Silva, R. G. Bastos, et al.

This groundbreaking study explores the potential of recombinant HAP2 as a transmission-blocking vaccine against *Babesia bovis*. Vaccinated calves showcased a remarkable immune response, preventing engorged ticks from transmitting the parasite, unlike their unvaccinated counterparts. Conducted by esteemed researchers Marta G. Silva, Reginaldo G. Bastos, Jacob M. Laughery, Heba F. Alzan, Vignesh A. Rathinasamy, Brian M. Cooke, and Carlos E. Suarez, this work highlights a promising approach to combatting this significant concern in veterinary medicine.

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Playback language: English
Introduction
Bovine babesiosis, caused by the apicomplexan parasite *Babesia bovis*, is a significant economic threat to the cattle industry globally. Current control strategies, including acaricides, anti-babesial drugs, and live attenuated vaccines, have limitations such as acaricide resistance, drug costs and potential for toxicity, and live vaccine risks of pathogen contamination and reversion to virulence. The development of effective subunit vaccines remains elusive due to the complexity of the parasite-host interaction, antigenic variation, and a lack of well-defined correlates of protection. Transmission-blocking vaccines (TBVs) represent a promising alternative, targeting parasite stages in the tick vector, which are less subjected to host immune selection pressures. The HAPLESS2/GCS1 (HAP2) protein, a widely conserved gametocyte protein essential for gamete fusion in various eukaryotes, is uniquely expressed on the surface of *B. bovis* sexual-stage parasites within the tick vector. Previous in vitro studies have indicated HAP2's potential as a TBV candidate. This study aimed to evaluate rHAP2 as a TBV in a bovine/competent tick model, hypothesizing that induced antibodies against *B. bovis* HAP2 would interfere with parasite transmission by ticks.
Literature Review
The literature review highlights the limitations of current control measures for bovine babesiosis, emphasizing the need for novel strategies. Previous attempts at developing TBVs against *B. bovis*, using proteins like 6cys A and 6cys B, were unsuccessful. The conserved nature and role of HAP2 in gamete fusion across various eukaryotes, including the related *Plasmodium* parasites, were discussed, providing a strong rationale for investigating its potential as a TBV candidate. While in vitro studies suggested the potential of HAP2, this study aimed to address the gap in knowledge regarding its efficacy in vivo using a natural host-tick model.
Methodology
Three calves were vaccinated with rHAP2, and three served as controls (adjuvant only). The rHAP2 protein was expressed in *E. coli*, purified, and confirmed using SDS-PAGE, Western blot, and LC-MS/MS. Vaccination was performed subcutaneously four times at 21-day intervals. Post-vaccination, all calves were infested with *Rhipicephalus microplus* larvae and subsequently infected with virulent *B. bovis* via intravenous inoculation. Clinical parameters (rectal temperature, PCV) and parasite DNA levels (qPCR) were monitored. Engorged female ticks were collected to assess reproductive fitness (number of ticks, egg mass weight, presence of kinetes). Larvae from ticks fed on vaccinated and control calves were used in transmission-feeding experiments on naive recipient calves, monitoring clinical signs, parasite DNA, and antibody responses. Statistical analyses, including mixed models and non-parametric tests, were used to analyze the data. Indirect ELISA (iELISA) was used to measure antibody responses against rHAP2 and RAP-1 (a *B. bovis* blood-stage antigen). Quantitative PCR (qPCR) was used to detect parasite DNA in blood samples.
Key Findings
Vaccination with rHAP2 induced significant antibody responses in vaccinated calves but did not affect the clinical course of acute babesiosis after *B. bovis* challenge. However, transmission-feeding experiments revealed a complete abrogation of parasite transmission in recipient calves challenged with larvae from ticks that fed on rHAP2-vaccinated animals. No detectable *B. bovis* DNA and no seroconversion were observed in these recipient animals. In contrast, recipient calves infested with larvae from control ticks developed typical clinical signs of acute babesiosis, showed detectable *B. bovis* DNA, and seroconverted. Tick fitness analysis showed a trend towards a higher number of ticks dropping from vaccinated animals and a statistically significant increase in egg mass weight from ticks that fed on vaccinated calves suggesting that *B. bovis* infection itself impacts tick fitness.
Discussion
The findings strongly support the hypothesis that antibody responses against *B. bovis* HAP2 block parasite transmission. The lack of impact on acute babesiosis in vaccinated animals was expected, as HAP2 is a sexual-stage protein, not expressed in blood stages. The complete absence of transmission in recipient calves challenged with larvae from ticks fed on vaccinated animals demonstrates the efficacy of rHAP2 as a TBV. The absence of kinetes in ticks feeding on vaccinated animals further supports the blocking of zygote formation. This aligns with studies demonstrating HAP2's role in gamete fusion and the success of HAP2-based TBVs in other apicomplexan parasites. The observed decrease in tick fitness in the control group suggests that parasite infection may negatively impact tick reproduction.
Conclusion
This study provides strong evidence for the potential of HAP2 as a key component for a TBV against *B. bovis*. Future research should focus on evaluating the duration of protection, optimal vaccination regimens, and the combination of HAP2 with blood-stage antigens to achieve broader protection. The conserved nature of HAP2 suggests potential applications in developing TBVs against other related parasites.
Limitations
The study used a relatively small number of animals. Further studies with larger sample sizes are needed to confirm the findings and to investigate the efficacy of HAP2 vaccination against different *B. bovis* strains and other tick species. Long-term protection and the need for booster vaccinations also need further investigation.
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