南京地区奶牛隐性乳房炎流行调查与病原分析及PCR检测
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摘要
奶牛乳房炎是导致奶牛业损失惨重的一种疾病，其一般可分为临床型乳房炎和隐性乳房炎。隐性乳房炎无明显症状，必须借助一定的检测方法才能诊断。该病可在牛群中广泛存在，国外隐性乳房炎的发病率为20％～50％，国内的发病率更高，在60％～80％间。隐性乳房炎可导致奶牛产奶量显著下降，乳房炎导致损失的70％可归咎于隐性乳房炎。因此，奶牛隐性乳房炎的防治具有重要的意义。本试验研究了奶牛隐性乳房炎的发病规律、病原菌分析及快速检测方法，主要研究结果如下：
1 通过对南京某奶牛场一年DHI的统计，分析了胎次、泌乳期和季节对体细胞数(SCC)和奶牛隐性乳房炎发病率的影响。结果表明：奶牛胎次对SCC和隐性乳房炎发病率影响明显，胎次增多，体细胞数和发病率都升高；泌乳期和季节对体细胞数与隐性乳房炎发病率影响不大
2 应用上海乳房炎检测法(SMT)和SCC检测了南京某奶牛场88头奶牛。以SCC作为隐性乳房炎判定基准，计算SMT检测结果与不同SCC诊断阈值检测结果的相符率。结果表明，以300000个／ml为SCC诊断阈值，SMT检测结果与SCC诊断结果的相符率最高，达46.88％。
3 从南京地区三个奶牛场采集104份隐性乳房炎乳样。经分离纯化，鉴定出93株细菌，葡萄球菌、链球菌、肠杆菌是主要致病菌。选取8株肠杆菌、10株链球菌和16株葡萄球菌进行了药敏试验、溶血性和细胞毒性试验。结果表明，三种主要细菌对多种抗生素产生了耐药性；除葡萄球菌溶血能力较强外，另两种细菌溶血能力弱或不溶血；所试验的菌株中，只有一株肠杆菌和两株葡萄球菌对Vero细胞有毒性外，9株细菌上清对Hep-2细胞有毒性。
4 建立了检测奶牛乳房炎致病菌金黄色葡萄球菌和无乳链球菌的双重PCR法。优化了双重PCR的反应条件，分析了PCR的特异性和灵敏性。利用水煮法制备模板，12株金黄色葡萄球菌临床分离株和10株无乳链球菌临床分离株，双重PCR均可扩增出目的条带。应用双重PCR和常规细菌鉴定法检测了54份隐性乳房炎奶样，双重PCR
摘要
检浏出金黄色葡萄球菌感染奶样12份，无乳链球菌感染扔样14份;常规细菌鉴定法
检浏出金黄色葡萄球菌感染奶样13份，多乳链球菌感染奶样17份.
Bovine mastitis led large loss to the dairy industry. Generally, bovine mastitis was classified into clinical mastitis and subclinical mastitis. Subclinical mastitis showed little symptom and was diagnosticated by certain detection assay. The incidence of subclinical mastitis is 20% to 50% in developed countries, while 50% to 80% in our country. Subclinical mastitis resulted in marked reduction in milk production, and 70% of loss resulted from mastitis can be due to subclinical mastitis. So, study of preventive and diagnostic measures to control subclinical mastitis was of highly significance. This study was based on epidemiology of subclinical mastitis, pathogens and rapid detection. The main results were followed:
1 With analysis of the data on DHI of a dairy farm in a year, the effect of age, seasons and lactation stage on SCC in raw milk and the incidence of subclinical mastitis was evaluated. The results showed that age might effect obviously on SCC and the incidence of subclinical mastitis. With age increasing, SCC and the incidence of subclinical mastitis rised. While lactation stage and seasons had little effect on SCC and the incidence of subclinical mastitis.
2 SCC in raw milk was regarded as the diagnostic criteria to bovine subclinical mastitis. 88 dairy cows were detected intramammary infection (IMI) by SMT and SCC. The results of detecting by SMT were contrasted to the results of detecting by different threshold of SCC, and the concilient rate of SMT to SCC was calculated. When 300,000 cells/mL as diagnostic threshold of SCC, the concilient rate was the highest, to 46.88%.
3 104 milk samples were collected from dairy cows suffering from subclinical mastitis on three dairy farms in Nanjing. 93 strains of bacteria were isolated and identificated. The pathogenic bacteria were mainly cassified to Staphylococcus, Streptococcus and coliform. 8 strains of coliform, 10 strains of Streptococcus and 16 strains of Staphylococcus were analyzed with the antimicrobial disk susceptibility test, hemolysis test and cytotoxicity test. The results indicated that the main pathogens were
resistant to several drug. Most Staphylococcus were shown to be strongly haemolylic activity, but the other bacteria were shown to be slightly or little haemolylic activity. One strain of coliform and two strains of Staphylococcus were shown to be cytotoxic to Vero cell, and 9 strains wee shown to be cytotoxic to Hep-2 cell.
4 Multiplex polymerase chain reaction assay was developed for detection of Staphylococcus aureus and Streptococcus agalactiae causes of bovine mastitis. The PCR was optimized and the specificities and sensitivities of the PCR were analyzed. DNA extracted by boiling, 12 strains of Sta. aureus and 10 strains of Str. agalactiae were detected by the PCR. Expecting fragments were amplified from all strains. 54 milk samples from cows suffering subclinical mastitis were detected by the PCR and conventional bacterial identification. Staphylococcus aureus and Str. agalactiae were detected by the PCR from 12 milk samples and 14milk samples, respectively. While Sta. aureus was isolated from 13 milk samples and Streptococcus agalactiae from 17.
1 通过对南京某奶牛场一年DHI的统计，分析了胎次、泌乳期和季节对体细胞数(SCC)和奶牛隐性乳房炎发病率的影响。结果表明：奶牛胎次对SCC和隐性乳房炎发病率影响明显，胎次增多，体细胞数和发病率都升高；泌乳期和季节对体细胞数与隐性乳房炎发病率影响不大
2 应用上海乳房炎检测法(SMT)和SCC检测了南京某奶牛场88头奶牛。以SCC作为隐性乳房炎判定基准，计算SMT检测结果与不同SCC诊断阈值检测结果的相符率。结果表明，以300000个／ml为SCC诊断阈值，SMT检测结果与SCC诊断结果的相符率最高，达46.88％。
3 从南京地区三个奶牛场采集104份隐性乳房炎乳样。经分离纯化，鉴定出93株细菌，葡萄球菌、链球菌、肠杆菌是主要致病菌。选取8株肠杆菌、10株链球菌和16株葡萄球菌进行了药敏试验、溶血性和细胞毒性试验。结果表明，三种主要细菌对多种抗生素产生了耐药性；除葡萄球菌溶血能力较强外，另两种细菌溶血能力弱或不溶血；所试验的菌株中，只有一株肠杆菌和两株葡萄球菌对Vero细胞有毒性外，9株细菌上清对Hep-2细胞有毒性。
4 建立了检测奶牛乳房炎致病菌金黄色葡萄球菌和无乳链球菌的双重PCR法。优化了双重PCR的反应条件，分析了PCR的特异性和灵敏性。利用水煮法制备模板，12株金黄色葡萄球菌临床分离株和10株无乳链球菌临床分离株，双重PCR均可扩增出目的条带。应用双重PCR和常规细菌鉴定法检测了54份隐性乳房炎奶样，双重PCR
摘要
检浏出金黄色葡萄球菌感染奶样12份，无乳链球菌感染扔样14份;常规细菌鉴定法
检浏出金黄色葡萄球菌感染奶样13份，多乳链球菌感染奶样17份.
Bovine mastitis led large loss to the dairy industry. Generally, bovine mastitis was classified into clinical mastitis and subclinical mastitis. Subclinical mastitis showed little symptom and was diagnosticated by certain detection assay. The incidence of subclinical mastitis is 20% to 50% in developed countries, while 50% to 80% in our country. Subclinical mastitis resulted in marked reduction in milk production, and 70% of loss resulted from mastitis can be due to subclinical mastitis. So, study of preventive and diagnostic measures to control subclinical mastitis was of highly significance. This study was based on epidemiology of subclinical mastitis, pathogens and rapid detection. The main results were followed:
1 With analysis of the data on DHI of a dairy farm in a year, the effect of age, seasons and lactation stage on SCC in raw milk and the incidence of subclinical mastitis was evaluated. The results showed that age might effect obviously on SCC and the incidence of subclinical mastitis. With age increasing, SCC and the incidence of subclinical mastitis rised. While lactation stage and seasons had little effect on SCC and the incidence of subclinical mastitis.
2 SCC in raw milk was regarded as the diagnostic criteria to bovine subclinical mastitis. 88 dairy cows were detected intramammary infection (IMI) by SMT and SCC. The results of detecting by SMT were contrasted to the results of detecting by different threshold of SCC, and the concilient rate of SMT to SCC was calculated. When 300,000 cells/mL as diagnostic threshold of SCC, the concilient rate was the highest, to 46.88%.
3 104 milk samples were collected from dairy cows suffering from subclinical mastitis on three dairy farms in Nanjing. 93 strains of bacteria were isolated and identificated. The pathogenic bacteria were mainly cassified to Staphylococcus, Streptococcus and coliform. 8 strains of coliform, 10 strains of Streptococcus and 16 strains of Staphylococcus were analyzed with the antimicrobial disk susceptibility test, hemolysis test and cytotoxicity test. The results indicated that the main pathogens were
resistant to several drug. Most Staphylococcus were shown to be strongly haemolylic activity, but the other bacteria were shown to be slightly or little haemolylic activity. One strain of coliform and two strains of Staphylococcus were shown to be cytotoxic to Vero cell, and 9 strains wee shown to be cytotoxic to Hep-2 cell.
4 Multiplex polymerase chain reaction assay was developed for detection of Staphylococcus aureus and Streptococcus agalactiae causes of bovine mastitis. The PCR was optimized and the specificities and sensitivities of the PCR were analyzed. DNA extracted by boiling, 12 strains of Sta. aureus and 10 strains of Str. agalactiae were detected by the PCR. Expecting fragments were amplified from all strains. 54 milk samples from cows suffering subclinical mastitis were detected by the PCR and conventional bacterial identification. Staphylococcus aureus and Str. agalactiae were detected by the PCR from 12 milk samples and 14milk samples, respectively. While Sta. aureus was isolated from 13 milk samples and Streptococcus agalactiae from 17.
引文
[1] 方远穆．反刍动物乳房炎菌苗的研究进展[J]．中兽医医药杂志，1992(3)：20～22
[2] 甘肃农业大学主编．兽医产科学口[M]．北京：中国农业出版社，2000，353～367
[3] 胡松华．谈奶牛乳腺的免疫机能[J]．中国奶牛，1997(5)：53～55
[4] 南京农业大学主编．家畜生理学[M]．北京：中国农业出版社，2000．
[5] 王树迎．王政富．动物组织学与胚胎学[M]．北京：中国农业科技出版社，2000．
[6] 伍义行，黄利权．奶牛乳房炎防治的免疫学和药理学机制[J]．乳业科学与技术，2001，97，37～43．
[7] William C．Rebhun著；赵德明，沈建中主译．奶牛疾病学[M]．北京：农业出版社，1992，27～322
[8] Colditz IG, Watson DL. The immunophysiologlcal basis for vaccinating ruminants against mastitis[J]. Australian Veterinary Journal. 1985, 62(5): 145～53
[9] Donovan GA, Risco CA, Shearer JK. Assessment of the mammary system[J]. Veterinary Clinics of North America - Food Animal Practice, 1992 8(2): 361～372,
[10] Fetherston CM, Lee CS, Hartmann PE. Mammary gland defense: the role of colostrum, milk and involution secretion[J]. Advances in Nutritional Research, 2001 10: 167～198
[11] Harmon RJ. Physiology of mastiffs and factors affecting somatic cell counts[J]. Journal of Dairy Science, 1994, 77(7): 2103～2112
[12] Kelleher SL, Lonnerdal B. Immunological activities associated with milk[J]. Advances in Nutritional Research, 2001 10: 39～65
[13] Kirk J. J. Somatic cells in milk: Current. Concept. Compend[J]. Contin. Educ. Pract. Vet., 1984, 6: 237～243
[14] Lascelles AK. The immune system on the ruminant mammary gland and its role in the control of mastitis[J]. Journal of Dairy Science, 1979 62(1): 154～167,
[15] Michel G, Schulz J. Tissue regeneration processes in the bovine mammary gland[J]. Tierarztliche Praxis, 1996, 24(3): 223～227
[16] Nickerson SC, Pankey JW. Cytologic observation of the bovine teat end [J]. Am J Vet Res, 1983, 44: 1433～1441
[17] Nickerson SC. Resistance mechanisms of the bovine udder: New implications for mastitis control at the teat end[J]. J. Am. Vet. Med. Assoc., 1987,191: 1484～1488
[18] Nickerson SC, Pankey JW. Cytologic observations of the bovine teat end[J]. Am. J. Vet. Res., 1983, 44: 1433～1441
[19] Nickerson SC. Immune mechanisms of bovine udder: An overview. J. Am. Vet. Med. Assoc., 1985,187: 41～45
[20] Paape MJ, Sharer-Weaver K., Capuco AV. Immune surveillance of mammary tissue by phagocytic cells[J].Advances in Experimental Medicine and Biology. 2000, 480: 259～277
[21] Paape M., Mehrzad J., Zhao X, et al. Defense of the bovine mammary gland by polymorphonuclear neutrophil leukocytes[J]. J. of Mammary Gland Biology and Neoplasia, 2002, 7(2): 109～121
[22] Riollet C., Rainard P., Poutrel B. Cells and cytokines in inflammatory secretions of bovine mammary gland[Y]. Advances in Experimental Medicine & Biology. 2000, 480: 247～258
[23] Senft B., Neudecker J. Defense Mechanisms of the bovine mammary gland[J]. Tierarztliche Praxis. 1991, 19(4): 357～363
[24] Sordillo LM, Streicher KL. Mammary gland immunity and mastitis susceptibility[J]. Journal of Mammary Gland Biology and Neoplasia. 2002, 7(2): 135～146
[25] Sordillo LM, Shafer-Weaver K., Derosa D. Immunobiology of the mammary gland[J]. Journal of Dairy Science, 1997, 80(8): 1851～1865
[2] 甘肃农业大学主编．兽医产科学口[M]．北京：中国农业出版社，2000，353～367
[3] 胡松华．谈奶牛乳腺的免疫机能[J]．中国奶牛，1997(5)：53～55
[4] 南京农业大学主编．家畜生理学[M]．北京：中国农业出版社，2000．
[5] 王树迎．王政富．动物组织学与胚胎学[M]．北京：中国农业科技出版社，2000．
[6] 伍义行，黄利权．奶牛乳房炎防治的免疫学和药理学机制[J]．乳业科学与技术，2001，97，37～43．
[7] William C．Rebhun著；赵德明，沈建中主译．奶牛疾病学[M]．北京：农业出版社，1992，27～322
[8] Colditz IG, Watson DL. The immunophysiologlcal basis for vaccinating ruminants against mastitis[J]. Australian Veterinary Journal. 1985, 62(5): 145～53
[9] Donovan GA, Risco CA, Shearer JK. Assessment of the mammary system[J]. Veterinary Clinics of North America - Food Animal Practice, 1992 8(2): 361～372,
[10] Fetherston CM, Lee CS, Hartmann PE. Mammary gland defense: the role of colostrum, milk and involution secretion[J]. Advances in Nutritional Research, 2001 10: 167～198
[11] Harmon RJ. Physiology of mastiffs and factors affecting somatic cell counts[J]. Journal of Dairy Science, 1994, 77(7): 2103～2112
[12] Kelleher SL, Lonnerdal B. Immunological activities associated with milk[J]. Advances in Nutritional Research, 2001 10: 39～65
[13] Kirk J. J. Somatic cells in milk: Current. Concept. Compend[J]. Contin. Educ. Pract. Vet., 1984, 6: 237～243
[14] Lascelles AK. The immune system on the ruminant mammary gland and its role in the control of mastitis[J]. Journal of Dairy Science, 1979 62(1): 154～167,
[15] Michel G, Schulz J. Tissue regeneration processes in the bovine mammary gland[J]. Tierarztliche Praxis, 1996, 24(3): 223～227
[16] Nickerson SC, Pankey JW. Cytologic observation of the bovine teat end [J]. Am J Vet Res, 1983, 44: 1433～1441
[17] Nickerson SC. Resistance mechanisms of the bovine udder: New implications for mastitis control at the teat end[J]. J. Am. Vet. Med. Assoc., 1987,191: 1484～1488
[18] Nickerson SC, Pankey JW. Cytologic observations of the bovine teat end[J]. Am. J. Vet. Res., 1983, 44: 1433～1441
[19] Nickerson SC. Immune mechanisms of bovine udder: An overview. J. Am. Vet. Med. Assoc., 1985,187: 41～45
[20] Paape MJ, Sharer-Weaver K., Capuco AV. Immune surveillance of mammary tissue by phagocytic cells[J].Advances in Experimental Medicine and Biology. 2000, 480: 259～277
[21] Paape M., Mehrzad J., Zhao X, et al. Defense of the bovine mammary gland by polymorphonuclear neutrophil leukocytes[J]. J. of Mammary Gland Biology and Neoplasia, 2002, 7(2): 109～121
[22] Riollet C., Rainard P., Poutrel B. Cells and cytokines in inflammatory secretions of bovine mammary gland[Y]. Advances in Experimental Medicine & Biology. 2000, 480: 247～258
[23] Senft B., Neudecker J. Defense Mechanisms of the bovine mammary gland[J]. Tierarztliche Praxis. 1991, 19(4): 357～363
[24] Sordillo LM, Streicher KL. Mammary gland immunity and mastitis susceptibility[J]. Journal of Mammary Gland Biology and Neoplasia. 2002, 7(2): 135～146
[25] Sordillo LM, Shafer-Weaver K., Derosa D. Immunobiology of the mammary gland[J]. Journal of Dairy Science, 1997, 80(8): 1851～1865