| Mastitis is one of the largest production concerns in the dairy industry worldwide. Staphylococcus aureus (S.aureus) is the most common etiologic agent of mastitis in bovines and a major cause of economic losses in the dairy industry. The occurrence of bovine mastitis has serious consequences for public health. The effects of antibiotic therapy has becoming more and more worse because S.aureus has the capacity of adherence and anchorage to mammary gland epithelium and the Characteristic of easy to produce antibiotic-resistant strains. Thus, a vaccine to protect against infection would therefore be beneficial. Antimicrobial susceptibility of S.aureus isolated from bovine mastitis varies widely by region. Therefore, 60 strains S.aureus were isolated from cases of mastitis in cow herds of Inner Mongolia of China by using microbiological method. Susceptibility to ampicillin, penicillin G, amoxicillin, piperacillin , cephalexin, cephazolin, cefotaxime, ceftazidime, cefoxitin, SMZ-TMP, gentamycin, kanamycin, norfloxacin, ciprofloxacin, ofloxacin, furaxone, torlamician, roxithromycin ,clindamycin, vancomycin, determined by the K-B agar method. Antibiotic susceptibility testing showed over 90% isolates were resistant to penicillin G. Also, high level of resistance to SMZ-TMP(57.20%), furaxone(58.47%), roxithromycin(58.90%), vancomycin(53.81%) and ampicillin(47.88%) was observed among S.aureus strains.In this study, Fn-binding region D of FnBPs and fibrinogen-binding region A of ClfA were detected by PCR from clinical strains isolated from China. The results showed 59 tested strains carried at least one fnb gene and all of 60 strains tested carried region A of clfa.80% isolated strains carried FnBPA-D and FnBPB-D.A large number of research shows that recognizing and specifically anchoring of S.aureus adhesins to tissues and definite molecule of cell is one of the key steps in the initiation of an infection. Blocking the primary stages of S.aureus infection, specifically the bacterial adhesin to cell and the colonization of the mucosal surface, may be the most effective strategy for preventing infections. Clumping factor A (ClfA), fibronectin binding protein A (FnBPA) and fibronectin binding protein B(FnBPB) of S.aureus are important proteins involved in the pathogenesis of staphylococcal bovine mastitis which promote binding of fibrinogen and fibrin to the bacterial cell surface. ClfA contains region A that binds fibrinogen and is a target for vaccine development. Fibronectin binding proteins FnBPA and FnBPB contains the Fn-binding domain D which promote binding of fibrin.Numerous studies have underlined the role of ClfA, FnBPA and FnBPB in the staphylococcal infectious process, but there isn't the report on immunization effect of different immunization strategy. Thus, the region A of ClfA (ClfA-A) and region D of FnBPA(FnBPA-D) and FnBPB(FnBPB-D) of S.aureus were successfully expressed in expression vector pET-32a and detect the anti adhesiveness, specific antibody and opsonophagocytic of antiserum of the recombinant fusion protein in the present study.The results showed that sera from recombinant adhesin-immunized rabbit were more efficient than sera from PBS immunized rabbit at inhibiting bacterial adherence and producing of specific antibody (p < 0.01). Study proved the mixture of three kinds of recomimant proteins has an advantage in assey of antiadhesion effects. Moreover, opsonophagocytic test result showed that whole blood of immuned rabbit possessed effect of eliminating S.aureus, and opsonization of the mixture of three kinds of vaccinated rabbit blood was significantly higher than the other group immuned with single antigen.It is the first time to application ClfA-A, FnBPA-Dand FnBPB-D genes of S.aureus to construct subunit vaccine,and the systematic immune animal experiments were carried out to examine the effection of different immunization strategy in this paper.Thus, the study will provide new breakthrough ideas for cow mastitis S.aureus genetic engineering vaccine research. |
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