| The mastitis model induced by Staphylococcus aureus (S.aureus) Newbould305 via the base of the mammary glands in mice was established successfully in this study. Therefore the protective effect of recombinant Staphylococcal Beta-hemolysin vaccine on immune defensive system and the inflammation-associated factors of mammary gland and systematic level by using this mastitis model were investigated. The result of this study provided a new for the biotherapy of mastitis.1. Establishment of lactation period mammary gland attacked by Staphylococcus aureus isolated from cows in mice48 mice were randomly divided into 6 groups(n=8),5.0×10~2CFU/50μL(E1),1.0×10~3CFU/50μL(E2),5.0×10~3CFU/50μL(E3).1.0×10~4CFU/50μL(E4) 1.0×10~5CFU/50μL(E5) and control group were inoculated into the base of the mammary glands 7-10 days after parturition. All the mice were sacrificed at 48h after infection. The content changes of mouse-interleukin 2(IL-2),mouse-interleukin 6(IL-6),mouse-tumor necrosis factor a(TNF-α),histopathologic observations and the amount of bacteria from mammary glands of mice. Breast tissue in the experimental groups, the number of isolated bacteria showed a downward trend after the first rise, E3 highest. Histopathologic examination of mammary gland revealed that the acinar structural of the control group were present integrity. The El and E2 groups were present primarily within the acinus serous effusion-based, histopathologic examination of the E3 group showed that numbers of polymorphonuclear neutrophils (PMN) were increased and acinus wall thickening were thickend. As the dose increase, the acinus wall of the E4 and E5 groups ruptured, and even individual samples appeared necrotic foci. In comparison with the control group, TNF-α,IL-2 and IL-6 concentration of the mammary gland tissue of the E3 group were significantly (P< 0.01) higher and overall tend showed increased at the beginning,and then decreased. The results demonstrated that 5.0×10~3CFU/50μL (E3) under the attack of doses of the Staphylococcus aureus was the same as inflammatory response and immunopathology, so choose this dose as the mice with mammary gland infection in the pathological model of modeling dose.2. The Protective Effect of recombinant Staphylococcal Beta-hemolysin vaccine on Mice with experimental Mastitis Induced by Staphylococcus aureus Infection48 mice were randomly divided into blank control group and blank control challenged group and pET-32a (+) vaccinated without challenged group and pET-32a (+) vaccinated challenged group and P-HL vaccinated without challenged group andβ-HL vaccinated challenged group (n=8), Ni-NTA Agarose was used to purify purpose fusion protein and protein content were determined with Bradfrod method, then, the purpose protein purified were aseptically made into vaccine. All the mice of all the challenged groups were inoculated into the base of the mammary glands 7-10 days after parturition with 5.0×10~3CFU/50μL of bacterial suspension. The mammary glands were harvested and ground for calculating colony for units and viewing the content changes of the inflammation-associated factors of mammary gland. Agarose immune-double diffusion test was reviewed if pET-32a (+) has an impact against recombinant Staphylococcal Beta-hemolysin.The results showed that there were expected protein bands with molecular mass of 57kD (purifiedβ-HL) and 20.5kD (purified pET-32a (+) in SDS-PAGE, and the concentration were 950μg/ml and 1.45mg/ml in differences. The blood sera of all vaccinated mice were detected specific antibodies. Moreover, the antibody titers were present to step up with increased number of times of vaccination. The concentrations of mouse-tumor necrosis factor a(TNF-α) and mouse-interleukin 2(IL-2) in mammary gland from pET-32a (+)vaccinated challenged group and P-HL vaccinated challenged group were a degree to change for comparison with the blank control challenged group (P> 0.05), however, in comparison with corresponding to control groups, there were present in statistical significance (P< 0.05). However, The content of mouse-interleukin 6(IL-6) fromβ-HL vaccinated challenged group were only present in statistical significance (P< 0.05) for comparison with the blank control challenged group. Agarose immune-double diffusion test revealed that the holes of theantibody of purifiedβ-HL fusion protein and the antibody of purified pET-2a (+) carrier protein emerge sediment lines with that of disintegration suspension of Staphylococcus aureus (S.aureus) Newbould305.Based on these results, we investigated that the purified fusion proteins had good fineness and injected vaccine to mice can produce a higher antibody titer and the pET-32a (+) carrier can increase the antigenicity of the purifiedβ-HL fusion protein. In conclusion, the preventive efficacy of the recombinantβ-HL vaccine against infection in lactating mice inoculated with Staphylococcus aureus (S.aureus) Newbould305 couldn't lessen inflammation symptom of mice, however, which could decrease degreely bacterial numbers in mammary gland.
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