Co-Immunization Efficacy of Recombinant Antigens against Rhipicephalus microplus and Hyalomma anatolicum Tick Infestations

Abstract

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 Rhipicephalus microplus BM86 and Hyalomma anatolicum subolesin (SUB) and tropomyosin (TPM) were targeted to evaluate the cross-protective potential. The sequence identities of the BM86, SUB, and TPM 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-Kluyveromyces lactis, 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 (p < 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 R. microplus and H. anatolicum and theadults of H. anatolicum, and a significant vaccine efficacy of 87.2% and 86.2% against H. anatolicum larvae and adults, respectively, and 86.7% against R. microplus was obtained. The current study provides significant support to develop a multi-antigen vaccine against cattle tick species.

Keywords: Bm86; Hyalomma anatolicum; Rhipicephalus microplus; co-vaccination; subolesin; tropomyosin.

Figure 1

Fold changes in the Bm86 gene expression at different life stages in comparison to the larvae of R. microplus IVRI-I strain [* p < 0.05; ** p < 0.0001].

Figure 2

SDS-PAGE profile of the purified rBm86 protein of R. microplus IVRI-I (A), rTPM (B), and rSUB (C) protein of H. anatolicum IVRI-II; western blot profile of three purified proteins probed with anHA-tag polyclonal antibody (D); hyper-immune sera raised in rabbits (E); three-colour protein weight marker (Puregene, USA).

Figure 3

Immuno-reactivity of the rBm86 (89 kDa), rHaTPM (36 kDa), and rHaSUB (21 kDa) proteins against sera collected from immunized calves on different days using multi-screen Western blotting apparatus.

Figure 4

rBm86-induced IgG (A), IgG1 (B), and IgG2 (C) responses in calves co-immunized with recombinant proteins. HL—H. anatolicum larvae; RL—R. microplus larvae; HA—H. anatolicum adults; Ns—not significant; ** p < 0.01; *** p < 0.001 in comparison to the previous day’s responses. Images of syringes imply days of immunization, while images of ticks indicate days of challenge.

Figure 5

rSUB-induced IgG (A), IgG1 (B), and IgG2 (C) responses in calves co-immunized with recombinant proteins. HL—H. anatolicum larvae; RL—R. microplus larvae; HA—H. anatolicum adult; ns—not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 in comparison to the previous day’s responses. Images of syringes imply days of immunization, while images of ticks indicate days of challenge.

Figure 6

rTPM-induced IgG (A), IgG1 (B), and IgG2 (C) responses in calves co-immunized with recombinant proteins. HL—H. anatolicum larvae; RL—R. microplus larvae; HA—H. anatolicum adult; ns—not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 in comparison to the previous day’s responses. Images of syringes imply days of immunization, while images of ticks indicate days of challenge.