Haemaphysalis longicornis calreticulin is not an effective molecular tool for tick bite diagnosis and disruption of tick infestations

Background: Tick calreticulin (CRT) is a calcium-binding protein secreted into the host during blood feeding. It has been used as a biomarker of tick exposure and has potential as an anti-tick vaccine, but there is no information about these uses for Haemaphysalis longicornis CRT (HlCRT). We synthesized recombinant H. longicornis CRT (rHlCRT) and evaluated its potential for tick bite diagnosis and for disrupting tick infestations. Methods: The responses of mice and rabbits exposed to H. longicornis ticks were measured with ELISA to determine the antibody level against rHlCRT. To evaluate the effects of rHlCRT-induced anti-tick immunity, engorgement weight, tick engorgement index (TEI), feeding duration, ecdysis rate, and egg weight per engorged tick were compared between ticks fed on immunized and normal mice. Results: Mean anti-tick CRT antibody levels in sera collected from mice at 1 and 15 days after primary tick exposure were not significantly different from the mean antibody levels in negative control mice that were not bitten by ticks (all P values > 0.05). No significant anti-HlCRT IgG responses developed in mice after second exposure to tick bites compared with the level of anti-HlCRT antibody response in negative control mice (all P values > 0.25). For rabbits, no significant differences in the antibody levels were observed in animals before challenge infestation and after tick exposures, and in animals after two tick exposures (all P values > 0.10). There were no significant differences in the body weight of ticks fed on immunized and normal mice (all P values > 0.15). No significant differences in TEI were observed between ticks fed on immunized mice and normal control mice (all P values > 0.50). There were no significant differences in feeding duration for female ticks, and feeding duration and ecdysis rate for nymphs in the experimental and control groups (all P values > 0.10 for feeding duration and P value = 0.19 for ecdysis rate). We did not observe a significant difference in egg weight per tick in the rHlCRT-immunized and the control groups (P = 0.88). Conclusions: HlCRT in H. longicornis tick saliva proteins appears to be nonimmunogenic to mammalian hosts like mice and rabbits. Vaccination with rHlCRT did not generate effective immunity against parthenogenetic and bisexual H. longicornis nymphs or female ticks. These results indicate that HlCRT is not a suitable molecular candidate for H. longicornis tick bite diagnosis and not effective for the disruption of tick infestations.