Subolesin vaccination inhibits blood feeding and reproduction of Haemaphysalis longicornis in rabbits

Abstract

Background: Ticks can transmit numerous tick-borne pathogens and cause a huge economic loss to the livestock industry. Tick vaccines can contribute to the prevention of tick-borne diseases by inhibiting tick infestation or reproduction. Subolesin is an antigenic molecule proven to be a potential tick vaccine against different tick species and even some tick-borne pathogens. However, its effectivity has not been verified in Haemaphysalis longicornis, which is a widely distributed tick species, especially in East Asian countries. Therefore, the purpose of this study was to evaluate the effectivity of subolesin vaccination against H. longicornis in a rabbit model.

Methods: Haemaphysalis longicornis (Okayama strain, female, adult, parthenogenetic strain) and Japanese white rabbits were used as the model tick and animal, respectively. The whole open reading frame of H. longicornis subolesin (HlSu) was identified and expressed as a recombinant protein using E. coli. The expression was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the immunogenicity of rHlSu against anti-H. longicornis rabbit serum was confirmed using Western blotting. After vaccination of rHlSu in rabbits, experimental infestation of H. longicornis was performed. Variables related to blood-feeding periods, pre-oviposition periods, body weight at engorgement, egg mass, egg mass to body weight ratio, and egg-hatching periods were measured to evaluate the effectiveness of subolesin vaccination.

Results: The whole open reading frame of HlSu was 540 bp, and it was expressed as a recombinant protein. Vaccination with rHlSu stimulated an immune response in rabbits. In the rHlSu-vaccinated group, body weight at engorgement, egg mass, and egg mass to body weight ratio were statistically significantly lower than those in the control group. Besides, egg-hatching periods were extended significantly. Blood-feeding periods and pre-oviposition periods were not different between the two groups. In total, the calculated vaccine efficacy was 37.4%.

Conclusions: Vaccination of rabbits with rHlSu significantly affected the blood-feeding and reproduction in H. longicornis. Combined with findings from previous studies, our findings suggest subolesin has the potential to be used as a universal tick vaccine.

Keywords: Akirin; Haemaphysalis longicornis; Subolesin; Tick; Tick-borne disease; Vaccine.

Fig. 1

The phylogenetic relationships of subolesin identified in this study. For this analysis, other subolesin sequences were obtained from the GenBank database. The tree is generated based on the Maximum-Likelihood method with 500 replications using MEGA 7.0. The sequence obtained in this study is indicated by an arrow. Bootstrap values < 70 were omitted

Fig. 2

Expression and immune response of rHlSu. a Expression and purification of recombinant Haemaphysalis longicornis subolesin (rHlSu) using an E. coli expression system. Each protein (Lanes 1–4, 6 μg; Lane 5, 2.25 μg) was applied and separated by SDS-PAGE. Lane M: protein marker; Lane 1, empty vector without IPTG induction; Lane 2, empty vector with IPTG induction; Lane 3, vector including subolesin without IPTG induction; Lane 4, vector including subolesin with IPTG induction; Lane 5, purified rHlSu. b The immune response of rHlSu was verified using Western blotting. Purified rHlSu (0.03 μg) was used for Western blotting. Serum dilutions were 1:600 for lanes anti-H. longicornis serum and Naïve serum, and 1:2000 for lane anti-rHlSu serum. Secondary antibody dilution was 1:5000 for all lanes. Anti-Hl, reaction with anti-H. longicornis serum; Anti-rHlSu, reaction with anti-rHlSu serum; Naïve, reaction with naïve serum

Fig. 3

Evaluation of antibody production after vaccination. The vaccination group and PBS group were established and tested. Antibody levels were estimated using ELISA, and the mean antibody titer is shown. Following each vaccination, there was a two-week interval before antibody levels were assessed. Statistical analysis regarding OD values between the two groups was performed using the Mann-Whitney test. a P < 0.005 to PBS group; b P < 0.005 to naïve rabbit serum

Fig. 4

Effect of rHlSu vaccination on rabbits to control Haemaphysalis longicornis infestation. The results are shown as box plots. a Blood-feeding periods. b Pre-oviposition periods. c Body weight at engorgement. d Egg mass. e Egg mass to body weight ratio. f Egg-hatching periods. Mean values are indicated with an asterisk. Statistical comparison between the two groups for each parameter was performed using Student’s t-test or Mann-Whitney test