云南地区牛源无乳链球菌分离鉴定及毒力基因和耐药性的检测

doi:10.16431/j.cnki.1671-7236.2018.11.034

摘要/Abstract

摘要：

试验旨在研究无乳链球菌的生物学特性，为防治无乳链球菌引起的奶牛乳房炎提供理论依据。根据细菌分子生物学分离鉴定无乳链球菌，参考GenBank登录的牛源无乳链球菌16S rRNA、菌属特异性cfb （CAMP因子）、毒力基因和耐药基因序列，运用Oligo 6.0和Primer Premier 5.0软件设计14对引物，建立PCR快速检测方法，并进行20种常见抗生药物的耐药试验。结果显示，试验成功鉴定出17株牛源无乳链球菌，毒力基因与NCBI上已报道的无乳链球菌相应序列具有高度同源性，均≥99%；共检测到6种耐药基因；分离菌株对青霉素、红霉素、林可霉素、克林霉素、万古霉素、氨苄西林、新生霉素、磺胺异噁唑的耐药率均较高，耐药率依次为100.0%、94.1%、94.1%、94.1%、94.1%、82.3%、82.3%和47.1%，对青霉素严重耐药；而对氨基糖苷类、四环素类、头孢菌素类、喹诺酮类耐药率均较低，耐药率分别为15.7%、14.7%、7.7%和3.9%。本研究结果表明，建立的PCR快速检测方法灵敏可靠，云南地区无乳链球菌已对部分β-内酰胺类、大环内酯类、磺胺类等抗生素出现多重耐药性。

关键词: 奶牛乳房炎; 无乳链球菌; 毒力基因; 耐药性

Abstract:

This experiment was aimed to study the biological characteristics of Streptococcus agalactiae (S.agalactiae)and provide a theoretical basis for the prevention and treatment of cow mastitis caused by S.agalactiae.S.agalactiae was separated and identified based on bacterial molecular biology;according to GenBank-registered S.agalactiae 16S rRNA,genus-specific cfb (CAMP factor),virulence gene and drug resistance gene sequences,14 pairs of primers were designed using Oligo 6.0 and Primer Premier 5.0 softwares,then,a rapid PCR detection method was established;The resistance tests of 20 common antibiotic drugs was carried out.The results showed that 17 strains of S.agalactiae were successfully identified,and the virulence genes were highly homologous to the corresponding sequences of S.agalactiae reported on NCBI,which were ≥ 99%;A total of 6 drug resistance genes were detected;The resistance rates of the isolates to penicillin,erythromycin,lincomycin,clindamycin,vancomycin,ampicillin,novobiocin and sulfisoxazole were higher,and the drug resistance rates were 100%,94.1%,94.1%,94.1%,94.1%,82.3%,82.3% and 47.1%,respectively,severely resistant to penicillin;And the resistance rates to aminoglycosides,tetracyclines,cephalosporins and quinolones were low,which were 15.7%,14.7%,7.7% and 3.9%,respectively.This study results indicated that the established PCR rapid detection method was sensitive and reliable,and there was a multi-drug resistance of some β-lactams,macrolides,sulfonamides and other antibiotics in Yunnan.

Key words: dairy cow mastitis; Streptococcus agalactiae; virulence gene; drug resistance

中图分类号:

S852.61^＋1

祝宇, 路璐, 颜兴琼, 龚蕾, 孙卫星, 曲伟杰. 云南地区牛源无乳链球菌分离鉴定及毒力基因和耐药性的检测[J]. 《中国畜牧兽医》, 2018, 45(11): 3261-3270.

ZHU Yu, LU Lu, YAN Xingqiong, GONG Lei, SUN Weixing, QU Weijie. Isolation, Identification and Detection of Virulence Genes and Drug Resistance of Streptococcus agalactiae from Cattle in Yunnan[J]. , 2018, 45(11): 3261-3270.

参考文献

[1] DE VLIEGHER S,FOX L K,PIEPERS S,et al.Invited review:Mastitis in dairy heifers:Nature of the disease,potential impact prevention,and control[J].Journal of Dairy Science,2012,95(3):1025-1040.
[2] 王丹,杨峰,李新圃,等.奶牛乳房炎病原菌诊断技术研究进展[J].中国畜牧兽医,2017,44(6):1811-1817. WANG D,YANG F,LI X P,et al.Research progress on diagnosis technology of pathogenic bacteria of dairy cow mastitis[J].China Animal Husbandry & Veterinary Medicine,2017,44(4):1811-1817.(in Chinese)
[3] 周熊艳,王松明,顾招兵,等.云南大额牛瘤胃微生物多样性分析[J].中国畜牧兽医,2016,43(5):1162-1168. ZHOU X Y,WANG S M,GU Z B,et al.Diversity analysis of rumen microbes in Yunnan gayals (Bos frontalis)[J].China Animal Husbandry & Veterinary Medicine,2016,43(5):1162-1168.(in Chinese)
[4] MAHMMOD Y S,KLAAS I C,KATHOLM J,et al.Molecular epidemiology and strain-specific characteristics of Streptococcus agalactiae at the herd and cow level[J].Journal of Dairy Science,2015,98(10):6913-6924.
[5] 闫常平,张序,关萍,等.奶牛乳房炎的防治进展[J].中国奶牛,2004,3:47-48. YAN C P,ZHANG X,GUAN P,et al.Progress in the prevention and treatment of mastitis in cows[J].Chinese Dairy Cattle,2004,3:47-48.(in Chinese)
[6] 米耀荣,郭生泉,张勇,等.奶牛乳房炎的危害及其防治措施[J].畜牧与饲料科学.2009,30(10):163-164. MI Y R,GUO S Q,ZHANG Y,et al.Dairy cow mastitis harm and its control measures[J].Livestock and Feed Science,2009,30(10):163-164.(in Chinese)
[7] GOMES F,HENRIQYES M.Control of bovine mastitis:Old and recent therapeutic approaches[J].Current Microbiology,2016,72,377-382.
[8] 吴彩霞.奶牛隐性乳房炎的防治[J].甘肃农业,2011,2:81-82. WU C X.Prevention of cows with hidden mastitis[J].Gansu Agriculture,2011,2:81-82.(in Chinese)
[9] 方维焕,张晓峰.乳铁素参与乳房链球菌对乳腺上皮细胞的粘附[J].中国兽医学报,2003,1:35-37. FANG W H,ZHANG X F.Lactoferrin participates in the adhesion of Streptococcus uberis to mammary epithelial cells[J].Chinese Journal of Eterinary Science,2003,1:35-37.(in Chinese)
[10] 车车.无乳链球菌、停乳链球菌、乳房链球菌GapC蛋白的表达与免疫原性研究[D].大庆:黑龙江八一农垦大学,2009. CHE C.Expression and immunogenicity of GapC protein of S.agalactiae,S.dysgalactiae,S.uberis[D].Daqing:Heilongjiang Bayi Agricultural University,2009.(in Chinese)
[11] DURHAM D L,MATTINGLY S J,DORAN T I,et al.Correlation between the production of extracellular substances by type Ⅲ group B streptococcal strains and virulence in a mouse model[J].Infection & Immunity,1981,34(2):448-454.
[12] YEUNG M K,MATTINGLY S J.Biosynthetic capacity for type-specific antigen synthesis determines the virulence of serotypeⅢ strains of group B strepto-cocci[J].Infection & Immunity,1984,44(2):217-221.
[13] MING C,RUI W,LUO F G,et al.Streptococcus agalactiae isolates of serotypesⅠa,Ⅲ and Ⅴ from human and cow are able to infect tilapia[J].Veterinary Microbiology,2015,180(1-2):129-135.
[14] 吕贺,段晓宇,周金玉,等.奶牛乳脂合成及其影响因素[J].中国畜牧兽医,2018,45(1):93-99. LV H,DUAN X Y,ZHOU J Y,et al.Milk fat synthesis and its influencing factors in dairy cows[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):93-99.(in Chinese)
[15] LINDAHL G,STALHAMMAR-CARLEMALM M,ARESCHOUG T.Surface proteins of Streptococcus agalactiae and related proteins in other bacterial pathogens[J].Clinical Microbiology Review,2005,18(1):102-127.
[16] ALMEIDA R A,OLIVER S P.Antiphagocytic effect of the capsule of Streptococcus uberis[J].Zentralbl Veterinarmed B,1993,40(9-10):707-714.
[17] 娄文琦,李源韬,常瑶,等.北京地区荷斯坦奶牛皮肤褶皱厚度与泌乳性能关系的研究[J].中国畜牧兽医,2018,45(2):375-383. LOU W Q,LI Y X,CHANG Y,et al.Study on the relationship between skin fold thickness and lactation performance of Holstein cows in Beijing[J].China Animal Husbandry & Veterinary Medicine,2018,45(2):375-383.(in Chinese)
[18] 朱战波,王红,刘宇,等.奶牛乳房炎链球菌分离株的鉴定及药敏试验[J].现代畜牧兽医,2007,12:8-10. ZHU Z B,WANG H,LIU Y,et al.Identification and drug susceptibility testing of dairy cows with Streptococcus isolates[J].Modern Animal Husbandry and Veterinary Medicine,2007,12:8-10.(in Chinese)
[19] 白龙,郝永清,范利霞,等.奶牛乳腺炎无乳链球菌的分离鉴定[J].畜牧与饲料科学,2010,31(1):96-97. BAI L,HAO Y Q,FAN L X,et al.Isolation and identification of Streptococcus agalactiae from mastitis in dairy cow[J].Animal Husbandry and Feed Science,2010,31(1):96-97.(in Chinese)
[20] 李宏胜,郁杰,罗金印,等.牛源性无乳链球菌血清型分布及抗生素耐药性研究[J].中国畜牧兽医,2012,39(1):164-167. LI H S,YU J,LUO J Y,et al.Study on serotype distribution and antibiotic resistance of Streptococcus agalactiae isolated from cattle[J].China Animal Husbandry & Veterinary Medicine,2012,39(1):164-167.(in Chinese)
[21] 杜琳,周雪,赵红梅,等.华北地区牛源无乳链球菌的分离鉴定及生物学特性[J].微生物学通报,2016,43(3):567-574. DU L,ZHOU X,ZHAO H M,et al.Isolation identification and biological characteristics of Streptococcus agalactiae from cattle origin in North China[J].Microbiology China,2016,43(3):567-574.(in Chinese)
[22] 庞茂达,孙利厂,何涛,等.江苏部分地区牛源无乳链球菌流行病学研究[J].扬州大学学报(农业与生命科学版),2017,38(4):25-29. PANG M D,SUN L C,HE T,et al.Epidemiological study on Streptococcus agalactiae from cattle in some areas of Jiangsu province[J].Journal of Yangzhou University (Agricultural and Life Science Edition),2017,38(4):25-29.(in Chinese)
[23] JROGENSEN H J,NORDSTAGA A B,SVILAND S,et al.Streptococcus agalactiae in the environment of bovine dairy herds——Rewriting the textbooks?[J].Veterinary Microbiology,2016,184:64-72.
[24] 申阿东,王咏红,黄醒华.B族链球菌普通和选择性培养基的比较[J].中华医学检验杂志,1999,22(6):376-377. SHEN A D,WANG Y H,HUANG X H.Comparison of common and selective media of group B Streptococcus[J].Chinese Journal of Medical Laboratory Medicine,1999,22(6):376-377.(in Chinese)
[25] 李霞,高谦.16S rRNA基因序列分析在临床微生物学中的应用[J].微生物与感染,2006,1(3):184-186. LI X,GAO Q.Application of 16S rRNA gene sequence analysis in clinical microbiology[J].Microorganisms and Infection,2006,1(3):184-186.(in Chinese)
[26] 罗晟远.奶牛乳房炎无乳链球菌的分离鉴定及药敏试验[J].当代畜牧兽医,2016,8:124-125. LUO S Y.Isolation,identification and drug susceptibility testing of milk cow Streptococcus agalactiae[J].Contemporary Livestock Science and Veterinary Medicine,2016,8:124-125.(in Chinese)
[27] PODBIELSKI A,BLANKENSTEIN O,LVTTICKEN R.Molecular characterization of the cfb gene encoding group B streptococcal CAMP-factor[J].Medical Microbiology & Immunology,1994,183(5):239-256.
[28] TAKAISI-KIKUNI N B,JRENS D,WECKE J,et al.Immunochemical localization of CAMP factor (protrin B) in Streptococcus agalactiate[J].Microbios,1997,89(360/361):171-185.
[29] MARY E H,DARIN Q,CHIA J H,et al.CAMP factor is not essential for systemi virulebce of group B Streptococcus[J].Microbial Pathogenesis,2008,44(1):84-88.
[30] 张莉莉,杨峰,王益民,等.我国西北地区奶牛源乳房炎无乳链球菌的青霉素耐药特性研究[J].黑龙江畜牧兽医,2018,4:105-106. ZHANG L L,YANG F,WANG Y M,et al.Study on penicillin resistance characteristics of Streptococcus agalactiae in dairy cattle derived from Northwestern China[J].Heilongjiang Animal Science and Veterinary Medicine,2018,4:105-106.(in Chinese)
[31] 龚团莲.内蒙古通辽市某奶牛场乳房炎无乳链球菌的分离、鉴定及药敏试验[J].畜牧与饲料科学,2016,37(2):23-25. GONG T L.Antimicrobial susceptibility of Streptococcus agalactiae isolated from dairy cows with clinical mastitis in Tongliao city of Inner Mongolia[J].Animal Husbandry & Feed Science,2016,37(2):23-25.(in Chinese)
[32] DECHEVA A,ZLATKOV V,PANDEV K,et al.Screning study on pregnant women and neonatal infection with Streptococcus agalactiae (group B streptococci)[J].Akusherstvo Ⅰ Ginekologiia,2014,52(7):4-7.