苏中地区规模化牧场奶牛乳腺炎病原菌鉴定及药物敏感性分析
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摘要
从江苏省南通市大生源牧场、盐城市泰来神牧场及连云港市三元双宝牧场采集分析了12个月的奶牛乳样,通过分析3个牧场奶牛乳腺炎发病率的季节变化发现,冬季发病率普遍低于夏秋季。以2017年7月份南通市大生源牧场321头奶牛的乳样为例,采用奶牛生产性能测定(DHI)随机筛选出20头患有临床型乳腺炎的奶牛乳样,经过细菌分离纯化培养后采用PCR技术鉴定致病菌种类。结果显示:这20份乳样中共分离鉴定出枯草芽孢杆菌、地衣芽孢杆菌、梭状芽孢杆菌、苏云金杆菌、微杆菌、金黄色葡萄球菌、格氏乳球菌、分支杆菌、大肠杆菌、气单胞菌、枸橼酸杆菌、不动杆菌、肺炎克雷伯杆菌共13种细菌,其中以金黄色葡萄球菌、大肠杆菌、枸橼酸杆菌、不动杆菌和肺炎克雷伯杆菌感染为主;此外,奶牛大都感染了3种及以上致病菌,说明奶牛乳腺炎以多种致病菌混合感染为主。选用6种抗生素药物组合对混合乳样致病菌进行药敏试验,结果显示氨苄西林钠、青霉素钾、硫酸链霉素共同处理组体外抑菌效果最好,为苏中地区大生源牧场奶牛乳腺炎防治提供一定的理论基础。
Milk samples from Dashengyuan Dairy Cattle Farm in Nantong City,Tailaishen Dairy Cattle Farm in Yancheng City and Sanyuanshuangbao Dairy Cattle Farm in Lianyungang City were collected for 12 months. Seasonal changes in incidence of diseases in the cows were analyzed,and bovine mastitis in winter was found to be of generally lower incidence than in summer and autumn. Milk samples of 321 cows in Dashengyuan Dairy Cattle Farm in July,2017; and 20 dairy cows with clinical mastitis were randomly selected those cows by DHI.Bacteria were isolated and cultured in the samples and pathogenic bacteria were identified by PCR. The results showed that the 20 milk samples contained 13 bacteria: Bacillus subtilis,Bacillus licheniformis,Clostridium prazmowski,Bacillus thuringiensis,Micro bacterium,Staphylococcus aureus,Lactococcus garvieae,Mycobacterium,Escherichia coli,Aeromonas,Citrobacter,Acinetobacter and Klebsiella pneumoniae. Among them,Staphylococcus aureus,E. coli,Citrobacter,Acinetobacter and Klebsiella pneumoniae were the major pathogenic bacteria. In addition,most cows were infected with three or more pathogenic bacteria at the same time,indicating that mastitis in cow was mainly caused by mixed infections of multiple pathogenic bacteria. Then,six kinds of antibiotics were used in combination to test drug susceptibility of mixed milk pathogenic bacteria,which showed that the combination of ampicillin sodium,penicillin potassium and streptomycin sulfate had the best antibacterial effect in vitro. The present study provided a theoretical basis for prevention and treatment of mastitis in Dashengyuan Dairy Cattle Farm.
Milk samples from Dashengyuan Dairy Cattle Farm in Nantong City,Tailaishen Dairy Cattle Farm in Yancheng City and Sanyuanshuangbao Dairy Cattle Farm in Lianyungang City were collected for 12 months. Seasonal changes in incidence of diseases in the cows were analyzed,and bovine mastitis in winter was found to be of generally lower incidence than in summer and autumn. Milk samples of 321 cows in Dashengyuan Dairy Cattle Farm in July,2017; and 20 dairy cows with clinical mastitis were randomly selected those cows by DHI.Bacteria were isolated and cultured in the samples and pathogenic bacteria were identified by PCR. The results showed that the 20 milk samples contained 13 bacteria: Bacillus subtilis,Bacillus licheniformis,Clostridium prazmowski,Bacillus thuringiensis,Micro bacterium,Staphylococcus aureus,Lactococcus garvieae,Mycobacterium,Escherichia coli,Aeromonas,Citrobacter,Acinetobacter and Klebsiella pneumoniae. Among them,Staphylococcus aureus,E. coli,Citrobacter,Acinetobacter and Klebsiella pneumoniae were the major pathogenic bacteria. In addition,most cows were infected with three or more pathogenic bacteria at the same time,indicating that mastitis in cow was mainly caused by mixed infections of multiple pathogenic bacteria. Then,six kinds of antibiotics were used in combination to test drug susceptibility of mixed milk pathogenic bacteria,which showed that the combination of ampicillin sodium,penicillin potassium and streptomycin sulfate had the best antibacterial effect in vitro. The present study provided a theoretical basis for prevention and treatment of mastitis in Dashengyuan Dairy Cattle Farm.
引文
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[22]智宇.奶牛乳腺炎乳汁中几种细胞因子与体细胞凋亡相关性的研究[D].呼和浩特:内蒙古农业大学,2014.
[23]Mark T,Fikse W F,Emanuelson U,et al.Short communication:effect of phantom parent grouping and properties of deregression for a low heritability trait[J].J Dairy Sci,2002,85(9):2393-2395.
[24]方敏,秦宜德,董琼珠.影响乳成分含量的因素[J].安徽医药,2004(2):130-132.
[25]赵春平,马云,曾全录,等.利用DHI对两个奶牛场奶牛生产性能的综合分析[J].中国牛业科学,2008(5):9-11.
[26]华实,齐新林,谭世新.DHI是提升奶牛场管理水平的核心[J].中国奶牛,2011(5):56-59.
[27]Middleton J R,Fox L K,Pighetti G,et al.Laboratory Handbook on Bovine Mastitis[M].New Prague:National Mastitis Council,2017:375-386.
[28]Koskinen M T,Wellenberg G J,Sampimon O C,et al.Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria[J].J Dairy Sci,2010,93(12):5707-5715.
[29]李波,周虚.奶牛乳房炎的预防及治疗的研究进展[J].中国畜牧兽医,2006(10):41-43.
[2]Mir A Q,Bansal B K,Gupta D K.Subclinical mastitis in machine milked dairy farms in Punjab:prevalence,distribution of bacteria and current antibiogram[J].Vet World,2014,7(5):291-294.
[3]Raszek M M,Guan Le L,Plastow G S.Use of genomic tools to improve cattle health in the context of infectious diseases[J].Front Genet,2016,7:30.
[4]Huang J M,Wang X G,Jiang Q,et al.Identification of CD14 transcript in blood polymorphonuclear neutrophil leukocytes and functional variation in Holsteins[J].Genet Mol Res,2016,15(2):1-10.
[5]Hertl J A,Leach J D,Bar D,et al.Effects of clinical mastitis caused by gram-positive and gram-negative bacteria and other organisms on the probability of conception in New York State Holstein dairy cows[J].J Dairy Sci,2010,93(4):1551-1560.
[6]Fernandes J B,Zanardo L G,Galvao N N,et al.Escherichia coli from clinical mastitis:serotypes and virulence factors[J].J Vet Diagn Invest,2011,23(6):1146-1152.
[7]胡洪杰,侯世忠,柴士名,等.应用奶牛DHI测定数据分析做好奶牛精细化管理[J].山东畜牧兽医,2013,34(8):69-72.
[8]Varela-Ortiz D F,Barboza-Corona J E,Gonzalez-Marrero J,et al.Antibiotic susceptibility of Staphylococcus aureus isolated from subclinical bovine mastitis cases and in vitro efficacy of bacteriophage[J].Vet Res Commun,2018,42(3):243-250.
[9]Contreras G A,Rodríguez J M.Mastitis:comparative etiology and epidemiology[J].J Mammary Gland Biol Neoplasia,2011,16(4):339-356.
[10]Duse A,Waller K P,Emanuelson U,et al.Risk factors for antimicrobial resistance in fecal Escherichia coli from preweaned dairy calves[J].J Dairy Sci,2015,98(1):500-516.
[11]Oliver S P,Murinda S E.Antimicrobial resistance of mastitis pathogens[J].Vet Clin North Am Food Anim Pract,2012,28(2):165-185.
[12]Paterna A,Contreras A,Gomez-Martín A,et al.The diagnosis of mastitis and contagious agalactia in dairy goats[J].Small Ruminant Res,2014,121(1):36-41.
[13]Gopinath S M,Suneetha T B,Mruganka V D,et al.Evaluation of antibacterial activity of Tabernaemontana divaricata (L.) leaves against the causative organisms of bovine mastitis[J].Int J Res Phytochem Pharmacol,2011,1(4):211-213.
[14]Basdew I H,Laing M D.Mini-review:biological control of bovine mastitis using bacteriophage therapy[M].Singapore:World Scientific,2011:386-393.
[15]Qiao J,Kwok L,Zhang J,et al.Reduction of Lactobacillus in the milks of cows with subclinical mastitis[J].Benef Microbes,2015,6(4):485-490.
[16]Iwano H,Inoue Y,Takasago T,et al.Bacteriophage ΦSA012 has a broad host range against Staphylococcus aureus and effective lytic capacity in a mouse mastitis model[J].Biology(Basel),2018,7(1):8.
[17]王庆容,李顺,宋培勇,等.2种改进的未知细菌DNA提取方法[J].安徽农业科学,2009,37(12):5381-5382.
[18]Lee J L,Levin R E.Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction[J].J Microbiol Methods,2006,67(3):456-462.
[19]Ward W R,Hughes J W,Faull W B,et al.Observational study of temperature,moisture,p H and bacteria in straw bedding,and faecal consistency,cleanliness and mastitis in cows in four dairy herds[J].Vet Rec,2002,151(7):199-206.
[20]陈福音,高艳霞,李建国.奶牛热应激的危害及营养调控措施[J].中国畜牧杂志,2012,48(8):37-41.
[21]王宗尉,魏萍,敖长林,等.猪舍小气候因素与环境菌量多元线性回归分析[J].现代畜牧兽医,2009(5):59-62.
[22]智宇.奶牛乳腺炎乳汁中几种细胞因子与体细胞凋亡相关性的研究[D].呼和浩特:内蒙古农业大学,2014.
[23]Mark T,Fikse W F,Emanuelson U,et al.Short communication:effect of phantom parent grouping and properties of deregression for a low heritability trait[J].J Dairy Sci,2002,85(9):2393-2395.
[24]方敏,秦宜德,董琼珠.影响乳成分含量的因素[J].安徽医药,2004(2):130-132.
[25]赵春平,马云,曾全录,等.利用DHI对两个奶牛场奶牛生产性能的综合分析[J].中国牛业科学,2008(5):9-11.
[26]华实,齐新林,谭世新.DHI是提升奶牛场管理水平的核心[J].中国奶牛,2011(5):56-59.
[27]Middleton J R,Fox L K,Pighetti G,et al.Laboratory Handbook on Bovine Mastitis[M].New Prague:National Mastitis Council,2017:375-386.
[28]Koskinen M T,Wellenberg G J,Sampimon O C,et al.Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria[J].J Dairy Sci,2010,93(12):5707-5715.
[29]李波,周虚.奶牛乳房炎的预防及治疗的研究进展[J].中国畜牧兽医,2006(10):41-43.