| Bovine mastitis, one of the most significant disease of dairy herds, has huge effects on farm economics due to reduction in milk production and treatment costs. It's a multiple reasons disease, thus we must Application of a variety of measures to control bovine mastitis.As there are many kinds of Microorganism have a role in the processes of mastitis, the vaccine become a important target for control bovine mastitis.Staphylococcus aureus (S. aureus) is the main causative agent of bovine mastitis, development an efficient vaccine of S.aureus is important for control the infection,there are several vaccines used in commercially herds, but the economic benefit following such treatment has not been uniformly demonstrated. With the development of biotechnology, new type vaccine emerged one after another, researchers also used these new technology for the vaccine 'invention. Followed the knowledge improve about the S.aureus infected in mammary gland, peoples found it's the key step of S.aureus adhesion to the host celles or extracelluar matrix. The adhesion molecules yet become the potential candidate antigens. As is know about the molecules include the Fibronectin binding protein (FnBP), clumping factor A (ClfA),clumping factor B(ClfB), Collagen-binding protein Can (Can) et al.In this study, we choose prokaryotic express proteins of FnBP and ClfA, purificated for prepared the subunit vaccine. Evaluate the effectiveness on mouse model. S. aureus is a bacteria planted out of the host celles, Humoral immune palys an important role in resistancing S. aureus infection. As well know it have an important role to enhance the IgG level. That we clones the IL-6 gene of cows and expressed in prokaryotic express system. Using the IL-6 as an adjuvant for subunit vaccine. We established a mastitis mouse model before to evaluated the vaccine, take mice at 8-10days during lactating, injection different dose S. aureus or PBS into the fourth pair mammary glands,2 hours later take mother-children co-cage,24 hours apart after injection, killed mice by cervical pull, remove one side of mammary gland fixed for histopathologic evaluations, another side pulverization to detect the bacteria amount and the tumor necrosis factor (TNF-α) concentration. Through the mouse model we gained the suitable dose of S. aureus to challenge after vaccination. Take purificated proteins add adjuvant prepared subunit vaccine, and collection S. aureus lysis using Ultrasonic, add adjuvant to prepared total bacteria vaccine. Mice were vaccinated twice 14 days apart, one group cohabit to pregnancy, after parturition give S. aureus injection to challenge evaluation, another group give different adjuvant (PHA, LPS, IL-6, IFN, thymosin) with second vaccinated, haemospasia and apart serum every week, detect the IgG level in serum used the ELISA.Results, we got purificated bovine IL-6, through clone and prokaryotic expression, established a mastitis mouse model, gained the suitable dose of S. aureus was 1.0×103cfu per mouse.and the scientific criteria to evaluated the severity of mouse mastitis. Both of the two vaccine can significantly reduce the severity of mastitis in mouse model, with a light histopathologic changes, lower TNF-αlevel and a small amount of bacteria in mammary gland tissues.The total protein vaccine group shown a highest total IgG level, but the subunit vaccine group shown a highest anti-FnBP & ClfA antibody, every adjuvant can enhance the IgG level rising significantly, the thymosin was the best one between the five adjuvants. |
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