新疆部分地区牛源STEC的调查研究
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摘要
为了研究新疆部分地区牛源STEC在牛粪便、胴体、饲料和饮水中的存在情况,揭示其在牛场各个环节中的分布规律及致病的可能性。本试验于2015年9月至2017年1月期间,分别从牛场、活畜交易市场和牛屠宰加工厂,无菌采集牛肛拭子、粪便、胴体拭子、饲料和饮水样本,EC肉汤增菌后,用常规方法结合PCR(16S rRNA)技术进行大肠杆菌的分离鉴定,再用PCR检测大肠杆菌分离株的Stx1、Stx2基因;用腹腔注射EC肉汤增菌原液的方法进行动物攻毒试验,确定分离菌株的致病性。结果显示,从9个牛场、1个活畜交易市场和1个屠宰加工厂中共采集到1 453份样本,其中STEC阳性样本为217份(14.9%,217/1453);新疆伊犁、博乐、石河子、昌吉、五家渠及乌鲁木齐的STEC样本阳性率分别为9.9%,19.9%,4.0%,26.2%,43.0%,7.5%;春季(3～5月)、夏季(6～8月)、秋季(9～11月)和冬季(12～次年2月)的STEC样本阳性率分别为27.3%(147/538),0.8%(2/247),13.0%(49/376)和6.5%(19/292);肛拭子、胴体表面拭子、粪便、饲料和饮水样本STEC阳性率分别为19.4%(190/978),8.3%(4/48),6.7%(16/239),1.1%(1/94)和6.4%(6/94);共分离到468株STEC菌株,其中132株携带Stx1,122株携带Stx2,214株同时携带Stx1和Stx2;攻毒试验表明同时携带Stx1、Stx2基因的菌株对小白鼠有致病性。结果表明,牛源STEC普遍存在于所检测的牛场、活畜交易市场和屠宰加工厂,肛拭子样本阳性率最高,而牛胴体被污染的情况较严重;春季是STEC的排菌高峰期;分离到的牛源STEC菌株同时携带Stx1和Stx2的菌株最多,而单独携带Stx1或Stx2的菌株分别约为25%;在制定防控牛源STEC的措施时,要尽量避免牛粪便对胴体的污染,春季更要注意牛粪便的无害化处理,对小白鼠攻毒试验结果发现,分离菌株对人有潜在致病性。
To investigate the distribution of Shiga toxin producing Escherichia coli(STEC) in feces,carcasses and its rearing environments of cattle in some areas of Xinjiang.From September 2015 to January 2017,cattle anus swabs,fecal samples,carcass swabs,feed and water samples were collected from cattle farms,live animal trading market and cattle slaughtering house respectively.After EC broth enrichment,Stx1 and Stx2 genes were detected by PCR,then STEC were isolated and identified from PCR positive samples by conventional methods,and mice were challenged by intraperitoneal injection of the bacteria.A total of 9 cattle farms,a live animal trading market and a slaughter house were surveyed.Of 1453 samples,266 were identified as STEC positive.The samples' STEC positive rates of Yili,Bole,Shihezi,Changji,Wujiaqu and Urumqi in Xinjiang were 9.9%,19.9%,4%,26.2%,43% and 7.5% respectively;samples of STEC positive rates in spring(March-May),summer(June-August),autumn(September-November)and winter(Decembernext year's February)were 27.3%(147/538),0.8%(2/247),13%(49/376)and 6.5%(19/292)respectively,spring was the highest.The positive rate of STEC of cattle anus swabs,carcass swabs,fecal samples,feed and water samples were 19.4%(190/978),8.3%(4/48),6.7%(16/239),1.1%(1/94)and 6.4%(6/94)respectively and the anus swab samples was the highest.There are 468 STEC(32.2%,468/1453)strains were isolated from PCR positive samples,among them,132 were Stx1 positive,122 were Stx2 positive,and 214 were both Stx1 and Stx2 positive;the challenge experiment showed that the strains carrying both the Stx1 and Stx2 genes were pathogenic to mice.In this study,STEC were isolated from different cattle farms,live animal trading market and cattle slaughter house in some areas of Xinjiang,suggesting that there is a potential threat of STEC in the region,we should strengthen the detection and control of bovine origin STEC,thus ensuring the quality of animal products and the safety of environmental;considering the capability of STEC killing the mouse,there is a potential threatening to people.
To investigate the distribution of Shiga toxin producing Escherichia coli(STEC) in feces,carcasses and its rearing environments of cattle in some areas of Xinjiang.From September 2015 to January 2017,cattle anus swabs,fecal samples,carcass swabs,feed and water samples were collected from cattle farms,live animal trading market and cattle slaughtering house respectively.After EC broth enrichment,Stx1 and Stx2 genes were detected by PCR,then STEC were isolated and identified from PCR positive samples by conventional methods,and mice were challenged by intraperitoneal injection of the bacteria.A total of 9 cattle farms,a live animal trading market and a slaughter house were surveyed.Of 1453 samples,266 were identified as STEC positive.The samples' STEC positive rates of Yili,Bole,Shihezi,Changji,Wujiaqu and Urumqi in Xinjiang were 9.9%,19.9%,4%,26.2%,43% and 7.5% respectively;samples of STEC positive rates in spring(March-May),summer(June-August),autumn(September-November)and winter(Decembernext year's February)were 27.3%(147/538),0.8%(2/247),13%(49/376)and 6.5%(19/292)respectively,spring was the highest.The positive rate of STEC of cattle anus swabs,carcass swabs,fecal samples,feed and water samples were 19.4%(190/978),8.3%(4/48),6.7%(16/239),1.1%(1/94)and 6.4%(6/94)respectively and the anus swab samples was the highest.There are 468 STEC(32.2%,468/1453)strains were isolated from PCR positive samples,among them,132 were Stx1 positive,122 were Stx2 positive,and 214 were both Stx1 and Stx2 positive;the challenge experiment showed that the strains carrying both the Stx1 and Stx2 genes were pathogenic to mice.In this study,STEC were isolated from different cattle farms,live animal trading market and cattle slaughter house in some areas of Xinjiang,suggesting that there is a potential threat of STEC in the region,we should strengthen the detection and control of bovine origin STEC,thus ensuring the quality of animal products and the safety of environmental;considering the capability of STEC killing the mouse,there is a potential threatening to people.
引文
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[12] 潘玉辉,王艳,李玉,等.几种主要食源性致病菌 PCR 快速检测方法的建立与应用//[C]中国转化医学和整合医学研讨会,2015.
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[15] 姬小薇.肠出血性大肠杆菌EHECO157:H7的分子进化研究[D].重庆:第三军医大学,2010.
[16] 孔海深.致泻大肠埃希菌的分子分型和流行病学研究[D].杭州:浙江大学,2011.
[17] HANCOCK D D,BESSER T E,RICE D H,et al.A longitudinal study of Escherichia coli O157 in fourteen cattle herds[J].Epidemiol Infect,1997,118(2):193-195.
[18] TAHAMTAN Y,HAYATI M,NAMAVARI M.Prevalence and distribution of the stx1,stx2 genes in Shiga toxin producing E.coli (STEC) isolates from cattle[J].Iran J Microbiol,2010,2(1):8-13.
[19] GARCíAALJARO C,MUNIESA M,JOFRE J,et al.Prevalence of the stx2 gene in coliform populations from aquatic environments[J].Appl Environ Microbiol,2004,70(6):3535.
[20] BONARDI S,MAGGI E,PIZZING,et al.Faecal carriage of Verocytotoxin-producing Escherichia coli O157 and carcass contamination in cattle at slaughter in northern Italy[J].Int J Food Microbiol,2001,66(1/2):47-53.
[21] 毕旺来,谈笑,陈程,等.武汉市部分菜场肉类食品中产志贺毒素大肠杆菌污染状况分析[J].中国食品卫生杂志,2014,26(1):67-70.
[22] HUANG W C,HSU B M,KAO P M,et al.Seasonal distribution and prevalence of diarrheagenic Escherichia coli in different aquatic environments in Taiwan[J].Ecotoxicol Environ Saf,2016,124:37-41.
[23] 刘飞,宋定州,李键,等.产志贺毒素大肠杆菌的流行病学及致病因子的研究进展[J].中国畜牧兽医,2014,41(1):187-191.
[24] 刘飞,宋定州,汤承,等.川西北牦牛产志贺毒素大肠杆菌的流行病学调查[J].西南民族大学学报(自然科学版),2014,40(5):652-658.
[25] 张桃,苏战强,夏利宁,等.牛源大肠杆菌O157∶H7分离与鉴定及其毒力基因检测[J].中国人兽共患病学报,2015,31(12):1136-1141.
[2] JANEZIC K J,FERRY B,HENDRICKSE W,et al.Phenotypic and genotypic characterization of Escherichia coli isolated from untreated surface waters[J].Open Microbiol J,2013,7(1):9-19.
[3] ETCHEVERRíA A I,PADOLA N L.Shiga toxin-producing Escherichia coli:factors involved in virulence and cattle colonization[J].Virulence,2013,4(5):366-372.
[4] WELLS J G,DAVIS B R,WACHSMUTH I K,et al.Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype[J].J Clin Microbiol,1983,18(3):512.
[5] WATANABE Y,OZASA K,MERMINJ H,et al.Factory outbreak of Escherichia coli O157:H7 infection in Japan[J].Emerg Infect Dis,1999,5(3):424-428.
[6] 李洪卫,景怀琦,逄波,等.徐州市 2000 年肠出血性大肠埃希菌 O157∶H7 感染性腹泻的调查[J].中华流行病学杂志,2002,23(2):119-122.
[7] 吴瑶瑶.江苏某地区部分牛场产志贺毒素大肠杆菌的流行病学调查及其遗传相关性分析[D].江苏扬州:扬州大学,2014.
[8] MAINIL J G,DAUBEG.Verotoxigenic Escherichia coli from animals,humans and foods:who?s who?[J].J Appl Microbiol,2010,98(6):1332-1344.
[9] 白向宁,王红,赵爱兰,等.食源性产志贺毒素大肠杆菌的分离及菌株特征分析[J].中国食品卫生杂志,2014,26(4):312-317.
[10] MATHUSA E C,CHEN Y H,ENACHE E,et al.Non-O157 Shiga toxin-producing Escherichia coli in foods[J].J Food Prot,2010,73(9):1721.
[11] FARROKH C,JORDAN K,AUVRAYF,et al.Review of Shiga-toxin-producing Escherichia coli (STEC)and their significance in dairy production[J].Int J Food Microbiol,2013,162(2):190-212.
[12] 潘玉辉,王艳,李玉,等.几种主要食源性致病菌 PCR 快速检测方法的建立与应用//[C]中国转化医学和整合医学研讨会,2015.
[13] MOMTAZ H,SAFARPOOR D F,RAHIMIE,et al.Incidence of Shiga toxin-producing Escherichia coli serogroups in ruminant?s meat[J].Meat Sci,2013,95(2):381-388.
[14] BOSILEVAC J M,GUERINI M N,KALCHAYANAND N,et al.Prevalence and characterization of salmonellae in commercial ground beef in the United States[J].Appl Environ Microbiol,2009,75(7):1892-1900.
[15] 姬小薇.肠出血性大肠杆菌EHECO157:H7的分子进化研究[D].重庆:第三军医大学,2010.
[16] 孔海深.致泻大肠埃希菌的分子分型和流行病学研究[D].杭州:浙江大学,2011.
[17] HANCOCK D D,BESSER T E,RICE D H,et al.A longitudinal study of Escherichia coli O157 in fourteen cattle herds[J].Epidemiol Infect,1997,118(2):193-195.
[18] TAHAMTAN Y,HAYATI M,NAMAVARI M.Prevalence and distribution of the stx1,stx2 genes in Shiga toxin producing E.coli (STEC) isolates from cattle[J].Iran J Microbiol,2010,2(1):8-13.
[19] GARCíAALJARO C,MUNIESA M,JOFRE J,et al.Prevalence of the stx2 gene in coliform populations from aquatic environments[J].Appl Environ Microbiol,2004,70(6):3535.
[20] BONARDI S,MAGGI E,PIZZING,et al.Faecal carriage of Verocytotoxin-producing Escherichia coli O157 and carcass contamination in cattle at slaughter in northern Italy[J].Int J Food Microbiol,2001,66(1/2):47-53.
[21] 毕旺来,谈笑,陈程,等.武汉市部分菜场肉类食品中产志贺毒素大肠杆菌污染状况分析[J].中国食品卫生杂志,2014,26(1):67-70.
[22] HUANG W C,HSU B M,KAO P M,et al.Seasonal distribution and prevalence of diarrheagenic Escherichia coli in different aquatic environments in Taiwan[J].Ecotoxicol Environ Saf,2016,124:37-41.
[23] 刘飞,宋定州,李键,等.产志贺毒素大肠杆菌的流行病学及致病因子的研究进展[J].中国畜牧兽医,2014,41(1):187-191.
[24] 刘飞,宋定州,汤承,等.川西北牦牛产志贺毒素大肠杆菌的流行病学调查[J].西南民族大学学报(自然科学版),2014,40(5):652-658.
[25] 张桃,苏战强,夏利宁,等.牛源大肠杆菌O157∶H7分离与鉴定及其毒力基因检测[J].中国人兽共患病学报,2015,31(12):1136-1141.