Functional characterization of candidate antigens of Hyalomma anatolicum and evaluation of its cross-protective efficacy against Rhipicephalus microplus.

Hyalomma anatolicum and Rhipicephalus microplus seriously affect dairy animals and immunization of host is considered as a sustainable option for the management of the tick species. Identification and validation of protective molecules are the major challenges in developing a cross-protective vaccine. The subolesin (SUB), calreticulin (CRT) and cathepsin L-like cysteine proteinase (CathL) genes of H. anatolicum were cloned, sequenced and analysed for sequence homology. Both Ha-SUB and Ha-CRT genes showed very high level of homogeneity within the species (97.6-99.4% and 98.2-99.7%) and among the tick species (77.3-99.3% and 85.1-99.7%) while for Ha-CathL the homogeneity was lower among ticks (57.5-89.5%). Besides tick species, both Ha-SUB and Ha- CRT genes showed high level of homogeneity with dipterans (47.2-53.4% and 72.0-74.4%) and nematodes (64.0% by CRT). The level of expression of the conserved genes in different stages of the tick species was studied. The differences in fold change of expression (FCE) of the targeted genes in life stages of tick were not statistically significant except Ha-SUB in eggs and in frustrated females, Ha-CRT in fed male and Ha-CathL in unfed and frustrated females where highest FCE was recorded. The functional properties of the genes were studied by RNAi technology and a significant level of gene suppression (p<0.05) resulted in very low percentage of engorgement of treated ticks viz., 3.7%, 11.1% and 30.0% in Ha-SUB, Ha-CRT and Ha-CathL respectively, in comparison to control was recorded. The recombinant proteins rHa-SUB, rHa-CRT and rHa-CathL encoded by the genes were expressed in prokaryotic expression system. They were evaluated for cross-protective efficacy and found to be respectively, 65.4%, 41.3% and 30.2% protective against H. anatolicum and 54.0%, 37.6% and 22.2%, against R. microplus infestations.