Bioinformatic analysis of the glutathione S-transferase gene family in Sarcoptes scabiei and protective immunity of Delta-class SsGSTd3 and SsGSTd5 in rabbits.

Glutathione S-transferases (GSTs) are key detoxification enzymes and immune antigens in parasites. Sarcoptes scabiei has 14 GSTs in four subclasses (Delta, Mu, Kappa, Zeta), tandemly arranged in four chromosomes. Bioinformatic analysis revealed molecular weights of 22.28-43.73 kDa, isoelectric points of 4.90-8.70, and inter-subclass sequence similarity of 6.6%-21.03%. Transcriptomics and RT-qPCR showed consistent high expression of SsGSTd3, SsGSTd5, SsGSTm3, and SsGSTm4 across different host sources and stages. Since Delta GSTs are unique to S. scabiei, we expressed recombinant SsGSTd3 and SsGSTd5, and evaluated their immunoprotective effect. Western blotting confirmed the immunoreactivity of both proteins to rabbit serum; immunofluorescence showed that the SsGSTd3 localized mainly to inner organs, whereas SsGSTd5 was widely distributed within mites and possibly secreted to the host-parasite interface. Our vaccination trial demonstrated that the rSsGSTd3, rSsGSTd5, and cocktail groups showed significantly lower lesion scores and mite counts than control groups, including Phosphate-Buffered Saline (PBS) as a negative control, Quil A as an adjuvant control, and pET-32a as an empty vector control. The mean lesion scores in three experimental groups were 2.15 ± 1.24, 1.30 ± 0.49 and 1.46 ± 0.68, respectively, whereas in three control groups, the scores were 3.77 ± 0.47, 3.08 ± 0.64 and 3.54 ± 0.52, respectively. Compared to PBS (15360 ± 4102 mites), the rSsGSTd3, rSsGSTd5, and cocktail groups had 4129 ± 1348, 2831 ± 647, and 3128 ± 1060 mites, with reduction rates of 73.12%, 81.57%, and 79.63%, respectively. All recombinant proteins induced high specific IgG titers but no significant changes in IFN-γ, IL-2, IL-4, or IL-13. Overall, SsGSTd5 is the most promising vaccine candidate (lowest lesion score and highest mite reduction), with protection likely mediated by humoral immunity.