奶牛乳腺炎的病原菌分离鉴定、毒力基因检测与基因治疗研究
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摘要
乳腺炎一直是危害奶牛业的重要疾病,但随着抗生素的长期和盲目使用,其防治难度越来越大。为了深入调查奶牛乳腺炎的病原,该研究从江苏省5个牛场采集了100份临床显性和隐性乳腺炎奶样,并进行了细菌分离与生化鉴定。结果显示在乳腺炎患牛奶样中,大肠杆菌的分离率最高(82%),其次是乳房链球菌(53%)、金黄色葡萄球菌(41%)、停乳链球菌(29%)和无乳链球菌(27%)。在临床显性乳房炎中,乳房链球菌和金黄色葡萄球菌的混合感染普遍,而且以前认为无致病性的表皮葡萄球菌和腐生葡萄球菌也占一定比例(分别为15%和10%)。选用16种针对革兰氏阳性菌的抗生素,对30株乳房链球菌、20株无乳链球菌、10株停乳链球菌和40株金黄色葡萄球菌进行了药物敏感性实验,结果显示大多数链球菌菌株对米诺环素、环丙沙星高度敏感,对临床常用的复方新诺明、苯唑青霉素等具有较高的耐药性；大多数金黄色葡萄球菌对万古霉素和壮观霉素高度敏感,对临床常用的呋喃妥因、氯霉素、米诺环素等具有较高的耐药性。
为了研究致奶牛乳腺炎大肠杆菌的毒素基因、毒力岛基因、粘附素基因、溶血素基因、外膜蛋白基因等分子流行病学,对分离自江苏5个奶牛场的123株致奶牛乳房炎大肠杆菌进行了相关毒力因子的PCR检测。结果显示从76株(61.8%)致奶牛乳房炎大肠杆菌中检测到毒素基因(LT1、ST1、ST2、Cnfl、Cnf2),其中LT1+占17.1%,ST2+24.4%,Cnf2+占20.3%,未检测到ST1、Cnf1毒素基因；从68株(55.3%)致奶牛乳房炎大肠杆菌中检测到毒力岛相关基因(irp2、Ecs3703、aer), ETT2毒力岛的检出率最高(36.6%),其次为HPI(14.6%),检测率最低的为摄铁相关基因(4.1%)；从8株(6.5%)致奶牛乳房炎大肠杆菌中检测到粘附素相关基因(clpG、pap、sfa、afaD8、afaE8、F17b、F17a),其中2株(1.6%)为F17a+,3株(2.4%)携带P菌毛,3株(2.4%)携带S菌毛,未检测到携带非菌毛粘附素、CS31A粘附素和F 17b基因；在123株致奶牛乳房炎大肠杆菌中,8株(6.5%)产生溶血素,21株(17.1%)产生血清抗性因子。
为了便于防治奶牛乳腺炎基因药物的推广应用,在前期试验基础上,用无针头注射器将表达人溶菌酶的重组质粒在干乳期注射健康奶牛乳腺或泌乳期乳腺炎患牛乳腺,根据乳汁的CMT检测结果判定防治效果。结果显示每乳区两次注射500微克重组质粒,对乳腺炎的预防效果在90%以上,对泌乳期隐性乳腺炎的治疗效果为81.8%,与乳腺基部穿刺法的防治效果相当,说明无针注射法能取代常规有针注射法用于奶牛乳腺炎的防治,具有无需保定、无交叉污染和效率高等优点。
为了进一步评价基因药物治疗奶牛乳腺炎的安全性,将15头患隐性乳腺炎的奶牛随机均分为3组,分别以400μg／乳区、600μg／乳区、800μg／乳区的剂量,通过乳房基部穿刺注射表达人溶菌酶基因的重组质粒p215C3LYZ,24 h后重复注射一次,观察奶牛的精神、食欲、心率、呼吸、体温等临床指标。结果显示基因治疗奶牛的精神、食欲、心率、呼吸、体温等临床指标在注射前后无显著差异。另选取3头患隐性乳腺炎的奶牛,通过乳房基部穿刺注射重组质粒p215C3LYZ,剂量为1600μg／头,24 h后重复注射一次,定期采集试验组奶牛的乳样、粪样、尿样以及试验牛舍的垫料、水样,用PCR检测重组质粒在试验牛体内外的转移扩散。结果发现除再次注射后24h可从奶样中检测到重组质粒外,其他样品均为阴性,表明重组质粒不能在奶牛体内长期残留,也不能在环境中扩散。于第二次注射重组质粒后20天,将试验奶牛扑杀,采集血液、心、肝、脾、肺、肾、肌肉以及乳腺等样品进行PCR检测,结果表明重组质粒在奶牛体内未发生转移。
Bovine mastitis remains to be one of the most important diseases of dairy industry. To investigate the prevalence of bovine mastitis pathogens,100 milk samples were collected from cows with clinical or sub-clinical mastitis in 5 farms in the Jiangsu Province. All the samples were submitted to bacterial isolation and biotyping. The experiments showed that Escherichia coli had a highest isolation rate (82%) in mastitis milk samples, followed by Streptococcus uberis (53%), Staphylococcus aureus (41%), Streptococcus dysgalactiae (29%) and Streptococcus agalactiae (27%). In addition, Str. uberis and S. aureus were more frequently associated with clinical mastitis than sub-clinical case, while the infection rates of other bacteria were similar. Interestingly, Staphylococcus epidermids and Staphylococcus saprophyticus, which were considered as environmental bacteria, had relatively high (15% and 10%, respectively) in the milk samples tested.
To investigate the prevalence of antibiotics-resistant mastitis-causing bacteria in Jiangsu Province,30 Streptococcus uberis isolates,20 Streptococcus agalactiae isolates,10 Streptococcus dysgalactiae isolates and 40 Staphylococcus aureus isolates were submitted to antibiotics sensitivity test. Among 16 different antibiotics tested, most of the Streptococcus were highly sensitive to minocycline and ciprofloxacin, but resistant to chemitrim and oxacillin. While most of Staphylococcus aureus were highly sensitive to vancomycin and spectinomycin, but resistant to furantoin, amphemycin and minocycline.
To investigate the prevalence of the virulent genes of mastitis-causing Escherichia coli, 123 islates from different dairy farms were submitted to PCR detection for genes coding for toxins, pathogenicity islands, adhesin, hemolysin and outer membrane protein. The results showed that 76 (61.8%) isolates were positive for toxins gene, among them ST2 was the most common gene (24.4%), followed by Cnf2 (20.3%) and LT1 (17.1%). Among 123 isolates tested,68 isolates (55.3%) were positive for PAI-associated genes, including 36.6% for ETT2,14.6% for HPI and 4.1% for aer, respectively. Adhesin-associated gene detection showed that only 8 isolates were positive, aomong which 3 isolates carried P fimbriae,3 contained S fimbriae and 2 had F17a fimbriae. None of the isolates were PCR-positive for ST1, Cnf1, afaD8, afaE8, CS31A and F17b genes. In addition, aomong 123 isolates tested, 8 isolates produced hemolysin and 21 isolates produced serum resistance factor.
To facilitate clinical application of the recombinant vector for treating bovine mastitis, 500μg of the recombinant plasmid was injected into each quarter of milk-dryng or diseased cows using a needle-free injector. After repeat injection on the following day, their milk samples were collected and somatic cells numbers were counted by CMT assay. The results showed>90% prophylactic efficacy for healthy cows and 81.8% cure rate for the diseased animals. These data suggest that the needle-free injector can replace conventional injection method for treating bovine mastitis without animal restraint and advantage of quick and massive injection.
To further evaluate the biosafety of gene therapy for treating bovine mastitis, the recombinant plasmid harboring human lysozyme cDNA was injected into 18 lactating cows with subclinical mastitis with an injection dose of 400μg,600μg or 800μg/quarter or into 6 healthy cows with an injection dose of 400μg/quarter. After repeat injection with the same daose, the experimental animals were observed for 28 days for changes in psychosis, appetite, heart rate, respiratory rate and anus temperature and therapeutic efficacy was determined based on somatic cell numbers of the milk samples. The data showed that all animals were normal in terms psychosis, appetite, heart rate, respiratory rate and anus temperature. Three additional lactating cows with subclinical mastitis, as well as two healthy cows, were injected twice with 1600μg (400μg/quarter) of the recombinant vector and their milk, feces, urine, litte and water samples were collected. On day 20 after the secondary injection, the experimental animals were sacrified and their heart, liver, spleen, lung, kidney, muscle and mammary gland samples were collected. PCR detection showed that, except for the milk samples to be positive 24 h after secondary injection, all other samples were negative. These data suggest that intramammary injection of the recombinant vector is safe for both healthy and diseased cows without in vivo transmission and enviorment contamination of the plasmid.
为了研究致奶牛乳腺炎大肠杆菌的毒素基因、毒力岛基因、粘附素基因、溶血素基因、外膜蛋白基因等分子流行病学,对分离自江苏5个奶牛场的123株致奶牛乳房炎大肠杆菌进行了相关毒力因子的PCR检测。结果显示从76株(61.8%)致奶牛乳房炎大肠杆菌中检测到毒素基因(LT1、ST1、ST2、Cnfl、Cnf2),其中LT1+占17.1%,ST2+24.4%,Cnf2+占20.3%,未检测到ST1、Cnf1毒素基因；从68株(55.3%)致奶牛乳房炎大肠杆菌中检测到毒力岛相关基因(irp2、Ecs3703、aer), ETT2毒力岛的检出率最高(36.6%),其次为HPI(14.6%),检测率最低的为摄铁相关基因(4.1%)；从8株(6.5%)致奶牛乳房炎大肠杆菌中检测到粘附素相关基因(clpG、pap、sfa、afaD8、afaE8、F17b、F17a),其中2株(1.6%)为F17a+,3株(2.4%)携带P菌毛,3株(2.4%)携带S菌毛,未检测到携带非菌毛粘附素、CS31A粘附素和F 17b基因；在123株致奶牛乳房炎大肠杆菌中,8株(6.5%)产生溶血素,21株(17.1%)产生血清抗性因子。
为了便于防治奶牛乳腺炎基因药物的推广应用,在前期试验基础上,用无针头注射器将表达人溶菌酶的重组质粒在干乳期注射健康奶牛乳腺或泌乳期乳腺炎患牛乳腺,根据乳汁的CMT检测结果判定防治效果。结果显示每乳区两次注射500微克重组质粒,对乳腺炎的预防效果在90%以上,对泌乳期隐性乳腺炎的治疗效果为81.8%,与乳腺基部穿刺法的防治效果相当,说明无针注射法能取代常规有针注射法用于奶牛乳腺炎的防治,具有无需保定、无交叉污染和效率高等优点。
为了进一步评价基因药物治疗奶牛乳腺炎的安全性,将15头患隐性乳腺炎的奶牛随机均分为3组,分别以400μg／乳区、600μg／乳区、800μg／乳区的剂量,通过乳房基部穿刺注射表达人溶菌酶基因的重组质粒p215C3LYZ,24 h后重复注射一次,观察奶牛的精神、食欲、心率、呼吸、体温等临床指标。结果显示基因治疗奶牛的精神、食欲、心率、呼吸、体温等临床指标在注射前后无显著差异。另选取3头患隐性乳腺炎的奶牛,通过乳房基部穿刺注射重组质粒p215C3LYZ,剂量为1600μg／头,24 h后重复注射一次,定期采集试验组奶牛的乳样、粪样、尿样以及试验牛舍的垫料、水样,用PCR检测重组质粒在试验牛体内外的转移扩散。结果发现除再次注射后24h可从奶样中检测到重组质粒外,其他样品均为阴性,表明重组质粒不能在奶牛体内长期残留,也不能在环境中扩散。于第二次注射重组质粒后20天,将试验奶牛扑杀,采集血液、心、肝、脾、肺、肾、肌肉以及乳腺等样品进行PCR检测,结果表明重组质粒在奶牛体内未发生转移。
Bovine mastitis remains to be one of the most important diseases of dairy industry. To investigate the prevalence of bovine mastitis pathogens,100 milk samples were collected from cows with clinical or sub-clinical mastitis in 5 farms in the Jiangsu Province. All the samples were submitted to bacterial isolation and biotyping. The experiments showed that Escherichia coli had a highest isolation rate (82%) in mastitis milk samples, followed by Streptococcus uberis (53%), Staphylococcus aureus (41%), Streptococcus dysgalactiae (29%) and Streptococcus agalactiae (27%). In addition, Str. uberis and S. aureus were more frequently associated with clinical mastitis than sub-clinical case, while the infection rates of other bacteria were similar. Interestingly, Staphylococcus epidermids and Staphylococcus saprophyticus, which were considered as environmental bacteria, had relatively high (15% and 10%, respectively) in the milk samples tested.
To investigate the prevalence of antibiotics-resistant mastitis-causing bacteria in Jiangsu Province,30 Streptococcus uberis isolates,20 Streptococcus agalactiae isolates,10 Streptococcus dysgalactiae isolates and 40 Staphylococcus aureus isolates were submitted to antibiotics sensitivity test. Among 16 different antibiotics tested, most of the Streptococcus were highly sensitive to minocycline and ciprofloxacin, but resistant to chemitrim and oxacillin. While most of Staphylococcus aureus were highly sensitive to vancomycin and spectinomycin, but resistant to furantoin, amphemycin and minocycline.
To investigate the prevalence of the virulent genes of mastitis-causing Escherichia coli, 123 islates from different dairy farms were submitted to PCR detection for genes coding for toxins, pathogenicity islands, adhesin, hemolysin and outer membrane protein. The results showed that 76 (61.8%) isolates were positive for toxins gene, among them ST2 was the most common gene (24.4%), followed by Cnf2 (20.3%) and LT1 (17.1%). Among 123 isolates tested,68 isolates (55.3%) were positive for PAI-associated genes, including 36.6% for ETT2,14.6% for HPI and 4.1% for aer, respectively. Adhesin-associated gene detection showed that only 8 isolates were positive, aomong which 3 isolates carried P fimbriae,3 contained S fimbriae and 2 had F17a fimbriae. None of the isolates were PCR-positive for ST1, Cnf1, afaD8, afaE8, CS31A and F17b genes. In addition, aomong 123 isolates tested, 8 isolates produced hemolysin and 21 isolates produced serum resistance factor.
To facilitate clinical application of the recombinant vector for treating bovine mastitis, 500μg of the recombinant plasmid was injected into each quarter of milk-dryng or diseased cows using a needle-free injector. After repeat injection on the following day, their milk samples were collected and somatic cells numbers were counted by CMT assay. The results showed>90% prophylactic efficacy for healthy cows and 81.8% cure rate for the diseased animals. These data suggest that the needle-free injector can replace conventional injection method for treating bovine mastitis without animal restraint and advantage of quick and massive injection.
To further evaluate the biosafety of gene therapy for treating bovine mastitis, the recombinant plasmid harboring human lysozyme cDNA was injected into 18 lactating cows with subclinical mastitis with an injection dose of 400μg,600μg or 800μg/quarter or into 6 healthy cows with an injection dose of 400μg/quarter. After repeat injection with the same daose, the experimental animals were observed for 28 days for changes in psychosis, appetite, heart rate, respiratory rate and anus temperature and therapeutic efficacy was determined based on somatic cell numbers of the milk samples. The data showed that all animals were normal in terms psychosis, appetite, heart rate, respiratory rate and anus temperature. Three additional lactating cows with subclinical mastitis, as well as two healthy cows, were injected twice with 1600μg (400μg/quarter) of the recombinant vector and their milk, feces, urine, litte and water samples were collected. On day 20 after the secondary injection, the experimental animals were sacrified and their heart, liver, spleen, lung, kidney, muscle and mammary gland samples were collected. PCR detection showed that, except for the milk samples to be positive 24 h after secondary injection, all other samples were negative. These data suggest that intramammary injection of the recombinant vector is safe for both healthy and diseased cows without in vivo transmission and enviorment contamination of the plasmid.
引文
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