牛源金黄色葡萄球菌单链抗体对奶牛乳腺炎的治疗效果评价
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摘要
本文首先对患隐性乳腺炎的50头奶牛采取奶样,并对奶样进行了细菌分离鉴定及PCR检测,共检出金黄色葡萄球菌21株;随后对分离到的金黄色葡萄球菌进行了药敏试验,结果显示70%的金黄色葡萄球菌对青霉素产生了耐药性。为了研究针对金黄色葡萄球菌全菌抗原的单链抗体对奶牛乳腺炎的治疗效果,将表达单链抗体的重组质粒通过无针注射器注射到患病奶牛的乳腺(600μg/乳区),48 h后再以相同的剂量注射一次。14 d后,奶样的加利福尼亚乳腺炎试验(California Mastitis Test,CMT)结果显示,治疗奶牛乳腺炎的有效率为83.33%,其中2例完全治愈。定期采集试验奶牛的乳样,并用PCR方法进行检测,发现第二次注射后的14 d内,奶样中未检测到重组质粒,说明重组质粒不能在奶牛体内长期残留。本研究为奶牛乳腺炎治疗抗体的研发提供了理论依据。
In order to investigate antimicrobial susceptibility of the main pathogens causing bovine mastitis,21 Staphylococcus aureus( S.aureus) isolates from milk samples of 50 cows with subclinic mastitis were isolated,characterized and identified by PCR. The results showed that,in the 21 S. aureus strains,70% were resistant to penicillin. To evaluate the treatment effectiveness of the single-chain variable fragment( sc Fv) against S. aureus,600μg of the recombinant plasmids( pc DNA3. 1-sc Fvs),which expressed the sc Fv,were prepared and injected aseptically into the mammary region of cows suffering from clinic and sub-clinic mastitis. 48 h later,the same experiment was conducted again. 14 days later,the California Mastitis Test( CMT) was performed with the results that showed that the pc DNA3. 1-sc Fvs responded well to 83. 33% of the infected cows,two of which were cured. Further experiments were performed to find out whether the pc DNA3. 1-sc Fvs still existed in the milk of the cows injected with the recombinant plasmids from day 1 to day14. The PCR results showed no residue of the recombinant plasmids,implying a biosafety here. Our findings provided a scientific rationale for efficient control of bovine mastitis by applying sc Fv.
In order to investigate antimicrobial susceptibility of the main pathogens causing bovine mastitis,21 Staphylococcus aureus( S.aureus) isolates from milk samples of 50 cows with subclinic mastitis were isolated,characterized and identified by PCR. The results showed that,in the 21 S. aureus strains,70% were resistant to penicillin. To evaluate the treatment effectiveness of the single-chain variable fragment( sc Fv) against S. aureus,600μg of the recombinant plasmids( pc DNA3. 1-sc Fvs),which expressed the sc Fv,were prepared and injected aseptically into the mammary region of cows suffering from clinic and sub-clinic mastitis. 48 h later,the same experiment was conducted again. 14 days later,the California Mastitis Test( CMT) was performed with the results that showed that the pc DNA3. 1-sc Fvs responded well to 83. 33% of the infected cows,two of which were cured. Further experiments were performed to find out whether the pc DNA3. 1-sc Fvs still existed in the milk of the cows injected with the recombinant plasmids from day 1 to day14. The PCR results showed no residue of the recombinant plasmids,implying a biosafety here. Our findings provided a scientific rationale for efficient control of bovine mastitis by applying sc Fv.
引文
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[4]Middleton JR.Staphylococcus aureus antigens and challenges in vaccine development[J].Expert Review of Vaccines,2008,7(6):805-815.
[5]Kasuya K,Boyer JJ,Tan Y,et al.Passive immunotherapy for anthrax toxin mediated by an adenovirus expressing an anti-protective antigen single-chain antibody[J].Molecular Therapy 2005,11(2):237-244.
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[8]农业部.奶牛乳房炎防治技术指南(试行)(农医发[2010]29号)[Z].2010-06-07.
[9]易明梅,黄奕倩,朱建国,等.上海地区奶牛乳房炎主要病原菌的分离鉴定及耐药性分析[J].中国兽医学报,2009,29(3):360-363.
[10]刘佩红,黄忠,陆承平,等.奶牛乳腺炎病原菌的分离鉴定及耐药性分析[J].畜牧与兽医,2004,36(9):13-15.
[11]Wang M,Zhang Y,Zhu JG.Anti-Staphylococcus aureus singlechain variable region fragments provide protection against mastitis in mice[J].Applied microbiology and biotechnology 2016,100(5):2153-2162.
[12]Wolff JA,Malone RW,Williams P,et al.Direct gene transfer into mouse muscle in vivo[J].Sci,1990,247(4949):1465-1468.
[13]孙怀昌,于锋,苏建华,等.人溶菌酶基因治疗奶牛乳腺炎的初步研究[J].畜牧兽医学报,2004,35(2):227-232.
[2]马保臣,李建基,王春傲.奶牛急性乳腺炎病原菌的分离与鉴定[J].中国兽医杂志,2004,40(11):22-24.
[3]Barkema HW,Schukken YH,Zadoks RN.Invited review:the role of cow,pathogen,and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis[J].Journal of Dairy Science 2006,89(6):1877-1895.
[4]Middleton JR.Staphylococcus aureus antigens and challenges in vaccine development[J].Expert Review of Vaccines,2008,7(6):805-815.
[5]Kasuya K,Boyer JJ,Tan Y,et al.Passive immunotherapy for anthrax toxin mediated by an adenovirus expressing an anti-protective antigen single-chain antibody[J].Molecular Therapy 2005,11(2):237-244.
[6]叶应妩,王毓三,申子瑜.全国临床检验操作规程[M].3版.南京:东南大学出版社,2006:896-901.
[7]NCCLS performance standards for antimicrobial susceptibility testing:15thinformational supplement[S]NCCLS document M1002S15.Wayne,Pennsylvania:NCCLS,2005,25(1):34-74.
[8]农业部.奶牛乳房炎防治技术指南(试行)(农医发[2010]29号)[Z].2010-06-07.
[9]易明梅,黄奕倩,朱建国,等.上海地区奶牛乳房炎主要病原菌的分离鉴定及耐药性分析[J].中国兽医学报,2009,29(3):360-363.
[10]刘佩红,黄忠,陆承平,等.奶牛乳腺炎病原菌的分离鉴定及耐药性分析[J].畜牧与兽医,2004,36(9):13-15.
[11]Wang M,Zhang Y,Zhu JG.Anti-Staphylococcus aureus singlechain variable region fragments provide protection against mastitis in mice[J].Applied microbiology and biotechnology 2016,100(5):2153-2162.
[12]Wolff JA,Malone RW,Williams P,et al.Direct gene transfer into mouse muscle in vivo[J].Sci,1990,247(4949):1465-1468.
[13]孙怀昌,于锋,苏建华,等.人溶菌酶基因治疗奶牛乳腺炎的初步研究[J].畜牧兽医学报,2004,35(2):227-232.