奶牛乳房炎无乳链球菌Fc-Sip和金黄色葡萄球菌Fc-FnBPB-ClfA亚单位疫苗的免疫试验研究

Immunological study of Streptococcus agalactia Fc-Sip and Staphylococcus aureus Fc-Fn BPB-Clf A subunit vaccine against dairy cow mastitis

为探究由IgG FcRn介导奶牛乳房炎无乳链球菌(S.agalactiae,Sgc)和金黄色葡萄球菌(Staphylococcus aureus,Sau)保护性抗原亚单位疫苗的免疫效果,在本实验室前期研究基础上,本研究按体积11的比例将Sgc的Fc-Sip和Sau的Fc-FnBPB-ClfA两种蛋白分别与1%的盐酸左旋咪唑(1%LH)充分混合,制备两种亚单位疫苗。将即将干奶(妊娠220 d)、CMT试验为"++"以上的奶牛随机分为4组,分别将Fc-Sip+Fc-FnBPB-ClfA二联亚单位疫苗(A组)、Fc-Sip亚单位疫苗(B组)、Fc-FnBPB-ClfA亚单位疫苗(C组)通过首次肌肉免疫和二次乳头管灌注免疫方式进行免疫试验。通过免疫前后对乳样细菌分离、体细胞数测定及免疫血清抗体滴度测定,评价上述亚单位疫苗的免疫效果。结果显示,免疫前免疫组(A、B、C)奶牛的乳样细菌分离率分别为80%、90%、80%,其中Sgc检出率分别为20%、30%、10%,Sau检出率分别为30%、20%、10%;首免90 d后3个免疫组奶牛乳样细菌分离率分别降至20%、20%、40%,其中Sgc的检出率分别降至0、0、10%,Sau的检出率分别降至10%、10%、0,对照组免疫前后以上各项检测无明显变化。使用利拉伐牛奶体细胞检测仪检测免疫前后试验奶牛乳样中的体细胞数,免疫前免疫组(A、B、C)奶牛体细胞数均值分别为6.19×105个/mL、5.16×105个/mL、5.49×105个/mL,首免90 d后3个免疫组奶牛乳样中体细胞数均值分别为2.05×105个/mL、2.04×105个/mL、2.52×105个/mL,而对照组奶牛乳样体细胞数与免疫前无明显变化。检测首免后0、7 d、14 d、28 d、60 d、90 d的血清抗体滴度,结果显示,首免后7 d,免疫组(A、B、C)80%的奶牛血清抗体滴度为11024;第14 d后,免疫组(A、B、C)90%奶牛血清抗体滴度达到12048;第28 d后,免疫组(A、B、C)80%奶牛血清抗体滴度在14096~18192,比对照组平均上升3~4个抗体滴度,对照组奶牛血清抗体滴度一直保持在1256~1512。综合分析以上试验结果显示免疫A组(Fc-Sip+Fc-FnBPB-ClfA)综合免疫效果在各免疫组中最佳。本研究结果表明Fc-Sip+Fc-FnBPB-ClfA二联亚单位疫苗对无乳链球菌和金黄色葡萄球菌性奶牛乳房炎具有较好的治疗和预防作用,为奶牛隐形乳房炎的防治提供了实验依据。

To explore the immune effect of cow mastitis’Streptococcus agalactiae(Sgc)and Staphylococcus aureus(Sau)protective antigen subunit vaccine mediated by IgG Fc Rn,and to verify the important role of IgG FcRn in the local immune process of dairy cows,based on our previous research,Sgc Fc-Sip and Sau Fc-FnBPB-ClfA were mixed with 1%levamisole hydrochloride(1%LH)respectively,in a volume at ratio of 1:1 to prepare two subunit vaccines.Cows with a dry period(220 days of gestation)and a CMT test of"++"or more were randomly divided into four groups as test animals.The Fc-Sip+Fc-FnBPB-ClfA subunit vaccine(group A),The Fc-Sip subunit vaccine(group B)and Fc-FnBPB-ClfA subunit vaccine(group C)were tested by primary muscle immunization and secondary nipple immunization.The immune effects of the above subunit vaccines were evaluated by the isolation of bacteria in milk samples,the determination of somatic cell number and antibody titer of immune serum before and after immunization.The results showed that bacteria isolation rates of milk in the pre-immunization groups(A,B,C)were 80%,90%and80%,among which the Sgc detection rates were 20%,30%and 10%,and the Sau detection rates were 30%,20%and 10%;after 90 days,the bacteria isolation rates in milk of the three immunized groups decreased to 20%,20%and 40%,and the detection rate of Sgc decreased to 0,0 and 10%,the detection rate of Sau decreased to 10%,10%and 0;Detecting the number of somatic cells in milk samples from test cows with a DeLaval milk somatic cell tester before and after immunization,the average number of somatic cells in the pre-immune groups(A,B,C)were 6.19×105 cells/mL,5.16×105 cells/mL and 5.49×105 cells/mL;After 90 days,the average cell numbers of three immunization groups were 2.05×105 cells/mL,2.04×105 cells/mL and 2.52×105 cells/mL,while there’s no significant change of the number in the control group before and after immunization.The serum antibody titers of the immunized groups were measured at 0,7,14,28,60 and 90 days.On the 7 th day after the first injecting,80%of the cows in the immunized groups(A,B,C)had a serum antibody titer of 11024;On the 14 th day,90%of the cows in the immunized groups(A,B,C)had a serum antibody titer of 12048;On the 28 th day,80%of the cows in the immunized groups(A,B,C)had a serum antibody titer of14096-18192,which increased by 3-4 titers on average compared with the control group,and the serum antibody titer in the control group remained at 1256-1512.Group A showed comprehensive immune effect which was the best in three immunization groups.In summary,the Fc-Sip and Fc-FnBPB-ClfA subunit vaccines have better therapeutic and preventive effects on Streptococcus agalactiae and Staphylococcus aureus cow mastitis,providing experimental basis for the prevention and treatment of the subclinical mastitis of cows.