The Pathogenic Role Of Staphylococcal Superantigen Toxins In The Mouse Mammary

Cow mastitis is a serious disease of domestic and foreign dairy cattle breeding industry,Staphylococcus aureus(S.aureus)is one of the main pathogenic bacteria causing dairy cow mastitis,epidemiological findings show that,the superantigenic toxin produced of Staphylococcus aureus may is an important pathogenic factor of mastitis in dairy cows.In this study,we will take toxic shock syndrome toxin-1 as the research object,and the following studies were carried out:(1)In this study,we successfully expressed and purified the GST-rTSST-1 protein by using the gene engineering strain which constructed in previous studies,the rTSST-1 superantigenic toxin protein was obtained by enzyme digestion,affinity chromatography,then tested by SDS-PAGE electrophoresis,a single clear band was obtained at the corresponding molecular weight(22 kDa)of rTSST-1.We got a high-purity-target protein successfully.(2)In order to investigate the direct pathogenic effect of Staphylococcus aureus superantigenic toxin rTSST-1 on mammary gland,in this study,we divided the experimental female mice into 5 groups,rTSST-1 was injected into mice mammary respectively through teat canal,and the doses were 5 ?g,15 ?g,30 ?g,and injected with the same amount of sterile physiological saline and natural non injection as negative control,we necropsied the mice at 24 h,48 h and 72 h after infected,gross pathogenic changes were observed.The results showed that there were no inflammatory changes in the sterile physiological saline group and the natural non injection group at each phase.And 24 h after infected,in the groups injected with 5 ?g,the lesions were not obvious,the mammary is in state of health,and in the groups injected with 15?g and 30 ?g,the lesions were obvious,the parts of mammary became swelling.And 48 h,72 h after infected,the clinical changes of three dose test group were obvious,the mammary became yellow red or purple red and atrophy,and the clinical changes increase with the dose of infection.We necropsied the mice at 24 h after infected,and made pathological section of mammary.The results showed that,there were complete morphology of acini and no inflammatory cell infiltration was observed in the sterile physiological saline group and the natural non injection group.The group injected 5?g was complete,however,it was more swollen than that in control group,and the mammary acini was filled with leach liquor,at the same time,a small amount of neutrophil infiltrated;the gland bubble shape edges of the group injected 15 ?g and 30 ?g were more serious,and the neutrophil infiltration number were more,as a result,the whole breast isfilled with inflammatory cells.In summary,the TSST-1 can alone induce mammary gland inflammation,with the mammary appear pathological changes of the gross and tissues.(3)In order to investigate the pathogenic effect of Staphylococcus aureus without toxin gene on mammary gland,in this study,we divided the experimental female mice into 3 groups,S.aureus R8(102CFU),S.aureus R8(103CFU)and the same amount of sterile physiological saline(as negative control)were injected into mice mammary respectively through teat canal,we necropsied the mice at 24 h,48 h and 72 h after infected,gross pathogenic changes were observed.The results showed that the 24 h after infected,the clinical changes of two dose test group were obvious,and 48 h,72 h after infected,there were purulent foci and caseous necrosis in the mammary,and the high dose group had a severe lesion.(4)In order to investigate the pathogenic effect of superantigenic toxin TSST-1 and Staphylococcus aureus simultaneously on mammary gland,in this study,we divided the experimental female mice into 4 groups,rTSST-1(15 ?g),rTSST-1(15 ?g)+ S.aureus R8(102CFU),S.aureus R8(102CFU)and the same amount of sterile physiological saline(as negative control)were injected into mice mammary respectively through teat canal,we necropsied the mice at 24 h,48 h and 72 h after infected,gross pathogenic changes were observed.The results showed that clinical pathological changes of the rTSST-1 group is the lightest and that of the S.aureus R8 group is the most serious,the rTSST-1 + S.aureus R8 group is more serious than rTSST-1 group but is significantly lighter than S.aureus R8 group.We necropsied the mice at 24 h after infected,and made pathological section of mammary.The results showed that,there were complete morphology of acini and no inflammatory cell infiltration was observed in the sterile physiological saline group and the natural non injection group.The histopathological changes of the rTSST-1 group were similar with the rTSST-1 + S.aureus R8 group at 24 h,showed that the gland bubble shape edge was incomplete and a large number of neutrophil infiltrated in the gland bubble.Compared with the two groups,the morphological edge damage of the gland bubbles was more serious whereas the neutrophil infiltration numbers were fewer in S.aureus R8 group.Additionally,we detected the bacteria counts in the mammary gland of the group of rTSST-1+ S.aureus and S.aureus R8,at 24 h,48 h and 72 h after infection.The results showed that the bacteria counts of S.aureus R8 group is significantly higher than that of the rTSST-1 +S.aureus R8 group in these three periods.In addition,we necropsied the mice at 12 h and 24 h after infected,the contents of TNF-?,IL-1? and IL-18 in inflammatory cytokines were detected.The results showed that the levels of three kinds of inflammatory cytokines in the experimental group were higher than those in control group at 12 h after infection,there was no significant difference among the experimental groups,but the levels of rTSST-1 group and rTSST-1 +S.aureus group were higher than those of Staphylococcus aureus group.At 24 h after challenge,there were no significant differences in the levels of the three inflammatory factors in the experimental group compared with the control group.In summary,when we infected the mammary gland by TSST-1 mixed with Staphylococcus,the degree of inflammation in the breast is markedly reduced.The results indicate that the increased neutrophil infiltration which might be caused by the superantigenic activity of TSST-1 better removed infected bacteria in mammary tissues,and then reduce the inflammatory lesions of the mammary.