Preparation of antigens from midgut of Hyalomma anatolicum anatolicum and determining their immunoprotective effects

Partially fed (۴ -۵ days) H .a. anatolicum (Iranian isolate) female adult ticks were used for preparation of antigens of midgut which are, the gut supernate antigen (GS), gut membrane antigen (GM), triton extracted gut membrane antigen ( TX-GM ) and sodium dodecyl sulphate treated Triton extracted gut membrane antigen (SDS-TX-GM). The New Zealand white rabbits were immunized with GS, GM, TX- GM and SDS-TX-GM antigens. The first inoculation on day ۰ was administered after emulsification with Freund`s complete adjuvant and second inoculation was given on day ۱۴ with incomplete Freund`s adjuvant and third inoculation was given on day ۲۸ with incomplete Freund`s adjuvant. Blood drawn was from the marginal ear vein on day ۳۸. The humoral immune response was studied by Enzyme Linked Immunosorbent Assay (ELISA) and antibody titers in the sera of immunized rabbits on ۱۰ days after the last booster dose of antigen was ۱/۳۲۰۰۰. After electrophoresis of purified midgut antigens (GS, GM, TX-GM, SDS-TX-GM) ۴µg of each were applied on a ۱۰% acryl amide gel and blotted to nitrocellulose paper for reaction with its concern immune sera. By analysis of this Western blot, we got bands of ۱۱۰, ۹۵, ۸۵, ۶۶, ۵۹, ۴۹, ۴۲ Kda for GSAg, ۹۵, ۸۵, ۶۶, ۴۹, ۴۲ Kda for GM Ag, ۶۶, ۴۹ Kda for TX-GMAg and ۶۶Kda for SDS-TX-GMAg responsible for induction of resistance in the host. The prepared antigens showed engorged nymphs ranged from ۳۲-۴۱%, ۲۹-۵۶%, ۲۹-۴۹% and ۲۹-۴۷% in response to GS, GM, TX-GM and SDS-TX-GM antigens respectively. Immunodominant antigenic proteins of ۶۶ Kda were detected in the sera of rabbits immunized by all above antigens which can be candidate for single immunogen if in future studies show acceptable results. The immunized rabbits were challenged, ۱۰ days after the last booster dose of antigens, with ۷۰۰ larvae of H. a. anatolicum ticks by releasing them on one ear of rabbits and maximum protection in rabbits against H. a. anatolicum was induced with the one immunized with GSAg.

Keywords:

Hyalomma anatolicum anatolicum , Midgut , Western blot , ELISA