| الملخص: | The immunoprophylactic management of ticks is the most effective option to control tick infestations and counter spread the acaricide resistance problem worldwide. Several researchers reported an inconsistent efficacy of the single antigen-based immunization of hosts against different tick species. In the present study, to develop a multi-target immunization protocol, proteins from <i>Rhipicephalus microplus</i> BM86 and <i>Hyalomma anatolicum</i> subolesin (SUB) and tropomyosin (TPM) were targeted to evaluate the cross-protective potential. The sequence identities of the <i>BM86</i>, <i>SUB</i>, and <i>TPM</i> coding genes amongst Indian tick isolates of targeted species were 95.6–99.8%, 98.7–99.6%, and 98.9–99.9%, respectively, while at the predicted amino acid level, the identities were 93.2 to 99.5, 97.6 to 99.4, and 98.2 to 99.3%. The targeted genes were expressed in the eukaryotic expression system, pKLAC2-<i>Kluyveromyces lactis</i>, and 100 µg each of purified recombinant protein (Bm86-89 kDa, SUB-21 kDa, and TPM-36 kDa) mixed with adjuvant was injected individually through the intramuscular route at different sites of the body on days 0, 30, and 60 to immunize cross-bred cattle. Post-immunization, a statistically significant (<i>p</i> < 0.001) antibody response (IgG, IgG1, and IgG2) in comparison to the control, starting from 15 to 140 days, against each antigen was recorded. Following multi-antigen immunization, the animals were challenged twice with the larvae of <i>R. microplus</i> and <i>H. anatolicum</i> and theadults of <i>H. anatolicum</i>, and a significant vaccine efficacy of 87.2% and 86.2% against <i>H. anatolicum</i> larvae and adults, respectively, and 86.7% against <i>R. microplus</i> was obtained. The current study provides significant support to develop a multi-antigen vaccine against cattle tick species.
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