华南虎源禽致病性大肠杆菌的分离鉴定及其生物学特性研究
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摘要
【目的】对福建梅花山华南虎繁育研究所一例华南虎幼虎急性死亡病例的致病菌进行分离鉴定及生物学特性研究,为圈养华南虎疫病防控提供科学依据。【方法】无菌采取死亡幼虎的肺、肝、脾、肠道等组织病料,进行细菌分离培养,根据形态特征、培养特性、生化试验、16SrRNA PCR扩增结果进行病菌鉴定;并对该分离菌进行16S rRNA系统进化分析、禽致病性大肠杆菌PCR鉴定、药敏试验、动物致病性试验和毒力基因检测。【结果】从幼虎肝脏及肠道组织中分离得到1株大肠杆菌(E.coli),命名为FJ/Tiger2016。16SrRNA系统进化分析显示,该分离菌与大肠杆菌人源分离株(NZCP016497)、鼠源分离株(NZMBNX01000003)、鸭源分离株(EF620926)及禽源分离株(AB272358)的遗传距离较近,形成一个相对独立的小遗传分支,其中与禽源分离株(AB272358)的同源性高达99.7%。PCR鉴定及药敏试验结果表明,该分离菌为禽致病性大肠杆菌(APEC),对选用的15种抗菌药均表现敏感。动物致病性试验结果显示,经腹腔接种时该菌株对试验小鼠具有较强的致病力,小鼠攻毒后5h内全部死亡。病理剖检及组织病理切片观察结果表明,该菌株对死亡小鼠的心脏、肝脏和脾脏等多个重要组织器官均造成了较为严重的病理损伤,而经灌胃接种时试验小鼠均未出现明显的临床症状。毒力基因检测结果显示,该菌株含有10种毒力基因,暗示其可能具有很强的致病性。【结论】从死亡幼虎肝脏及肠道组织中分离到1株具有较强致病力、肠道外致病的禽致病性大肠杆菌,表明禽致病性大肠杆菌已对我国圈养华南虎造成了一定威胁,应引起重视。
【Objective】Isolation,identification and biological characteristics of the pathogenic bacteriacausing an acute death South China tiger cub in Fujian Meihuashan Institute of South China tiger breeding were studied to provide basis for prevention and control of the captive South China tiger disease.【Method】Sterile lung,liver,spleen and intestines samples of the dead tiger cub were collected for isolation and cultivation,and the isolates were identified according to morphological characteristics,culture characteristics and 16 SrRNA PCR amplification.The isolates were identified by phylogenetic analysis of 16SrRNA,avian pathogenic Escherichia coli PCR,drug sensitivity test,animal pathogenicity test and virulence gene detection.【Result】An E.coli strain,named FJ/Tiger2016,was isolated from the liver and intestinal tissues.The 16SrRNA phylogenetic analysis showed that the isolate had a close genetic distance with strains from human(NZCP016497),rat(NZMBNX01000003),duck(EF620926)and poultry(AB272358),and formed a relative independent small genetic branch far from other strains.The isolate had 99.7% homology with the strain from poultry(AB272358).The results of avian pathogenic E.coli PCR detection and drug sensitivity test showed that the isolate was an avian pathogenic E.coli,and it was highly sensitive to all of the 15 antimicrobial agents.The result of animal pathogenicity test showed that the isolate had strong pathogenicity to the experimental mice and all mice died within 5hafter intraperitoneal injection with it,and the pathological examination and histopathological observation results also showed that the isolate caused severe pathological damage to heart,liver and spleen and other important tissues and organs of the dead mice,while the mice in another group infected by intragastric administration had no clinical symptoms.The virulence gene detection showed that the isolate contained 10 of 17virulence genes,suggesting that it may have a strong pathogenicity.【Conclusion】An avian pathogenic E.coli strain with strong pathogenicity and extraintestinal pathogenic characteristics was isolated from the liver and intestine tissues of the dead South China tiger cub.The results indicate that the avian pathogenic E.coli has certain threat to captive South China tigers in China.
【Objective】Isolation,identification and biological characteristics of the pathogenic bacteriacausing an acute death South China tiger cub in Fujian Meihuashan Institute of South China tiger breeding were studied to provide basis for prevention and control of the captive South China tiger disease.【Method】Sterile lung,liver,spleen and intestines samples of the dead tiger cub were collected for isolation and cultivation,and the isolates were identified according to morphological characteristics,culture characteristics and 16 SrRNA PCR amplification.The isolates were identified by phylogenetic analysis of 16SrRNA,avian pathogenic Escherichia coli PCR,drug sensitivity test,animal pathogenicity test and virulence gene detection.【Result】An E.coli strain,named FJ/Tiger2016,was isolated from the liver and intestinal tissues.The 16SrRNA phylogenetic analysis showed that the isolate had a close genetic distance with strains from human(NZCP016497),rat(NZMBNX01000003),duck(EF620926)and poultry(AB272358),and formed a relative independent small genetic branch far from other strains.The isolate had 99.7% homology with the strain from poultry(AB272358).The results of avian pathogenic E.coli PCR detection and drug sensitivity test showed that the isolate was an avian pathogenic E.coli,and it was highly sensitive to all of the 15 antimicrobial agents.The result of animal pathogenicity test showed that the isolate had strong pathogenicity to the experimental mice and all mice died within 5hafter intraperitoneal injection with it,and the pathological examination and histopathological observation results also showed that the isolate caused severe pathological damage to heart,liver and spleen and other important tissues and organs of the dead mice,while the mice in another group infected by intragastric administration had no clinical symptoms.The virulence gene detection showed that the isolate contained 10 of 17virulence genes,suggesting that it may have a strong pathogenicity.【Conclusion】An avian pathogenic E.coli strain with strong pathogenicity and extraintestinal pathogenic characteristics was isolated from the liver and intestine tissues of the dead South China tiger cub.The results indicate that the avian pathogenic E.coli has certain threat to captive South China tigers in China.
引文
[1]谢和平,朱庆艳,陈武,等.华南虎源大肠杆菌的快速鉴定及致病特性的初步研究[J].野生动物,2012,33(3):109-112.Xie H P,Zhu Q Y,Chen W,et al.Rapid identification of an Escherichia coli isolated from South China Tiger(Panthera tigris Amoyensis)and preliminary study of pathogenic characteristics[J].Chin J Wil,2012,33(3):109-112.
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[6] Janβen T,Schwarz C,Preikschat P,et al.Virulence-associated genes in avian pathogenic Escherichia coli(APEC)isolated from internal organs of poultry having died from colibacillosis[J].Int J Med Microbiol,2001,291(5):371-378.
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[8]崔艳芳,王德丽,刘丽娜,等.一株虎源H5N1亚型禽流感病毒的分离鉴定及生物学特性研究[J].中国预防兽医学报,2015,37(12):908-911.Cui Y F,Wang D L,Liu L N,et al.Biological characteristic of an H5N1avian influenza virus isolated from tiger in Guangxi province in 2015[J].Chin J Prevent Vet Med,2015,37(12):908-911.
[9]李元果,高晓龙,桑晓宇,等.虎源伪狂犬病毒的鉴定及gB基因分析[J].中国病原生物学杂志,2014,9(12):1057-1060.Li Y G,Gao X L,Sang X Y,et al.Identification of pseudorabies virus(PrV)and analysis of the gBgene form tigers in China[J].J Path Biol,2014,9(12):1057-1060.
[10] Liu H,Yan Q,Zhao B,et al.Isolation,molecular characterization,and phylogenetic analysis of encephalomyocarditis virus from South China Tigers in China[J].Infect Genet Evol,2013,19:240-243.
[11]关乃瑜,夏爽,赵丽丽,等.断奶仔猪源小肠结肠炎耶尔森氏菌的分离鉴定及其致病性研究[J].中国预防兽医学报,2015,37(9):679-682.Guan N Y,Xia S,Zhao L L,et al.Isolation,identification,and pathogenicity of Yersinia enterocolitica from weaned piglets[J].Chin J Prevent Vet Med,2015,37(9):679-682.
[12] Hu Q H,Tu J,Han X G,et al.Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer,Escherichia coli,and Salmonella enterica simultaneously from ducks[J].J Microbiol Meth,2011,87(1):64-69.
[13] Pass M,Odedra R,Batt R.Multiplex PCR for identification of Escherichia coli virulence genes[J].J Clini Microbiol,2000,38(5):2001-2004.
[14]张铁男,李继昌,鲁成武,等.3种致泻性大肠埃希氏菌多重PCR检测方法的研究[J].中国兽医杂志,2007,43(9):17-19.Zhang T N,Li J C,Lu C W,et al.Study of a multiplex PCR assay for the detection of pathogenic genes of EPEC,ETEC and EIEC[J].Chin J Vet Med,2007,43(9):17-19.
[15] La Ragione R M,Woodward M J.Virulence factors of Escherichia coli serotypes associated with avian colisepticaemia[J].Res Vet Sci,2002,73(1):27-35.
[16]刘晓民,杨华,张润祥,等.牛源支气管败血波氏杆菌的分离与鉴定[J].中国兽医科学,2009,39(11):964-967.Liu X M,Yang H,Zhang R X,et al.Isolation and identification of Bordetella bronchiseptica from calves[J].Chin Vet Sci,2009,39(11):964-967.
[17]罗燕,王朋,赵洪明,等.林麝肺源致病性大肠杆菌分离鉴定及毒力基因PCR检测[J].中国预防兽医学报,2012,34(8):615-618.Luo Y,Wang P,Zhao H M,et al.Isolation and identification of lung pathogenic Escherichia coli from musk deer and PCR detection of the virulence genes[J].Chin J Prevent Vet Med,2012,34(8):615-618.
[18]唐婕,刘文华,王永奇.大肠杆菌引起林麝死亡的初步研究[J].特产研究,2009,31(1):23-24.Tang J,Liu W H,Wang Y Q.A preliminary study on forest musk deer death caused by Escherichia coli[J].Special Wild Economic Animal and Plant Research,2009,31(1):23-24.
[19]杨青,石有斐,陈静,等.貂源大肠杆菌的分离鉴定及耐药性研究[J].中国兽药杂志,2015,49(8):14-19.Yang Q,Shi Y F,Chen J,et al.Isolation,identification of Escherichia coli from mink and related research of some biological characteristics[J].Chin J Vet Drug,2015,49(8):14-19.
[20]钟妮娜,徐顶,胡刚,等.猕猴肠侵袭型大肠杆菌的分离鉴定及其毒力因子基因的检测[J].中国兽医科学,2010,40(11):1106-1109.Zhong N N,Xu D,Hu G,et al.Isolation and identification of enteroinvasive Escherichia coli in Macaca mulatta and detection of its virulence factor gene[J].Chin Vet Sci,2010,40(11):1106-1109.
[21] Guenther S,Grobbel M,Heidemanns K,et al.First insights into antimicrobial resistance among faecal Escherichia coli isolates from small wild mammals in rural areas[J].Sci Total Environ,2010,408(17):3519-3522.
[22] Maluta R P,Logue C M,Casas M R,et al.Overlapped sequence types(STs)and serogroups of avian pathogenic(APEC)and human extra-intestinal pathogenic(ExPEC)Escherichia coli isolated in Brazil[J].PLoS ONE,2014,9(8):e105016.
[23]宦海霞,张科.大肠杆菌致病因子研究进展[J].中国家禽,2010,32(23):45-48.Huan H X,Zhang K.Research progress on pathogenic factor of Escherichia coli[J].Chin Poultry,2010,32(23):45-48.
[24]马兴树,范翠蝶,夏玉龙.禽致病性大肠杆菌研究进展[J].中国畜牧兽医,2013,40(2):169-174.Ma X S,Fan C D,Xia Y L.Research progress on avian pathogenic Escherichia coli[J].Chin Anim Husband&Vet Med,2013,40(2):169-174.
[25]李叶芳,白灏,彭开松,等.禽致病性大肠杆菌强毒力岛摄铁功能与致病性关系的研究[J].中国兽医科学,2012,42(9):906-910.Li Y F,Bai H,Peng K S,et al.Relationship between the iron uptake function of high pathogenicity island and the pathogenicity of avian pathogenic Escherichia coli strains[J].Chin Vet Sci,2012,42(9):906-910.
[26] Gophna U,Oelschlaeger T A,Hacker J,et al.Yersinia HPI in septicemic Escherichia coli strains isolated from diverse hosts[J].FEMS Microbiol Lett,2001,196(1):57-60.
[27] Delicato E R,de Brito B G,Konopatzki A P,et al.Occurrence of the temperature-sensitive hemagglutinin among avian Escherichia coli[J].Avian Dis,2002,46(3):713-716.
[28] Moll A,Manning P A,Timmis K N.Plasmid-determined resistance to serum bactericidal activity:a major outer membrane protein,the traTgene product,is responsible for plasmid-specified serum resistance in Escherichia coli[J].Infect Immun,1980,28(2):359-367.
[29]陈文静,韩先干,何亮,等.鸭致病性大肠杆菌的分离鉴定及其生物学特性分析[J].中国动物传染病学报,2010,18(2):34-40.Chen W J,Han X G,He L,et al.Characterization of duck pathogenic Escherichia coli[J].Chin J Anim Infect Dis,2010,18(2):34-40.
[2] Kaper J B,Nataro J P,Mobley H L.Pathogenic Escherichia coli[J].Nat Rev Microbiol,2004,2(2):123-140.
[3] Ewers C,Janβen T,Wieler L.Avian pathogenic Escherichia coli(APEC)[J].Berl Münch Tierrztl Wochenschr,2003,116(9/10):381-395.
[4]白灏,冀辉,韩先干,等.禽致病性大肠杆菌江苏、安徽分离株的生物学特性分析[J].微生物学通报,2013,40(7):1315-1322.Bai H,Ji H,Han X G,et al.Characterization of avian pathogenic Escherichia coli isolated from Jiangsu and Anhui provinces[J].Microbiol China,2013,40(7):1315-1322.
[5] Tivendale K A,Logue C M,Kariyawasam S,et al.Avian-pathogenic Escherichia coli strains are similar to neonatal meningitis E.coli strains and are able to cause meningitis in the rat model of human disease[J].Infect Immun,2010,78(8):3412-3419.
[6] Janβen T,Schwarz C,Preikschat P,et al.Virulence-associated genes in avian pathogenic Escherichia coli(APEC)isolated from internal organs of poultry having died from colibacillosis[J].Int J Med Microbiol,2001,291(5):371-378.
[7]陈大庆,李林翔.华南虎患猫病毒性鼻气管炎的诊治[J].野生动物,2013,34(5):281-284.Chen D Q,Li L X.Diagnosis and treatment of feline vial rhinotracheitis in South China Tiger(Panthera tigris Amoyensi)[J].Chin J Wil,2013,34(5):281-284.
[8]崔艳芳,王德丽,刘丽娜,等.一株虎源H5N1亚型禽流感病毒的分离鉴定及生物学特性研究[J].中国预防兽医学报,2015,37(12):908-911.Cui Y F,Wang D L,Liu L N,et al.Biological characteristic of an H5N1avian influenza virus isolated from tiger in Guangxi province in 2015[J].Chin J Prevent Vet Med,2015,37(12):908-911.
[9]李元果,高晓龙,桑晓宇,等.虎源伪狂犬病毒的鉴定及gB基因分析[J].中国病原生物学杂志,2014,9(12):1057-1060.Li Y G,Gao X L,Sang X Y,et al.Identification of pseudorabies virus(PrV)and analysis of the gBgene form tigers in China[J].J Path Biol,2014,9(12):1057-1060.
[10] Liu H,Yan Q,Zhao B,et al.Isolation,molecular characterization,and phylogenetic analysis of encephalomyocarditis virus from South China Tigers in China[J].Infect Genet Evol,2013,19:240-243.
[11]关乃瑜,夏爽,赵丽丽,等.断奶仔猪源小肠结肠炎耶尔森氏菌的分离鉴定及其致病性研究[J].中国预防兽医学报,2015,37(9):679-682.Guan N Y,Xia S,Zhao L L,et al.Isolation,identification,and pathogenicity of Yersinia enterocolitica from weaned piglets[J].Chin J Prevent Vet Med,2015,37(9):679-682.
[12] Hu Q H,Tu J,Han X G,et al.Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer,Escherichia coli,and Salmonella enterica simultaneously from ducks[J].J Microbiol Meth,2011,87(1):64-69.
[13] Pass M,Odedra R,Batt R.Multiplex PCR for identification of Escherichia coli virulence genes[J].J Clini Microbiol,2000,38(5):2001-2004.
[14]张铁男,李继昌,鲁成武,等.3种致泻性大肠埃希氏菌多重PCR检测方法的研究[J].中国兽医杂志,2007,43(9):17-19.Zhang T N,Li J C,Lu C W,et al.Study of a multiplex PCR assay for the detection of pathogenic genes of EPEC,ETEC and EIEC[J].Chin J Vet Med,2007,43(9):17-19.
[15] La Ragione R M,Woodward M J.Virulence factors of Escherichia coli serotypes associated with avian colisepticaemia[J].Res Vet Sci,2002,73(1):27-35.
[16]刘晓民,杨华,张润祥,等.牛源支气管败血波氏杆菌的分离与鉴定[J].中国兽医科学,2009,39(11):964-967.Liu X M,Yang H,Zhang R X,et al.Isolation and identification of Bordetella bronchiseptica from calves[J].Chin Vet Sci,2009,39(11):964-967.
[17]罗燕,王朋,赵洪明,等.林麝肺源致病性大肠杆菌分离鉴定及毒力基因PCR检测[J].中国预防兽医学报,2012,34(8):615-618.Luo Y,Wang P,Zhao H M,et al.Isolation and identification of lung pathogenic Escherichia coli from musk deer and PCR detection of the virulence genes[J].Chin J Prevent Vet Med,2012,34(8):615-618.
[18]唐婕,刘文华,王永奇.大肠杆菌引起林麝死亡的初步研究[J].特产研究,2009,31(1):23-24.Tang J,Liu W H,Wang Y Q.A preliminary study on forest musk deer death caused by Escherichia coli[J].Special Wild Economic Animal and Plant Research,2009,31(1):23-24.
[19]杨青,石有斐,陈静,等.貂源大肠杆菌的分离鉴定及耐药性研究[J].中国兽药杂志,2015,49(8):14-19.Yang Q,Shi Y F,Chen J,et al.Isolation,identification of Escherichia coli from mink and related research of some biological characteristics[J].Chin J Vet Drug,2015,49(8):14-19.
[20]钟妮娜,徐顶,胡刚,等.猕猴肠侵袭型大肠杆菌的分离鉴定及其毒力因子基因的检测[J].中国兽医科学,2010,40(11):1106-1109.Zhong N N,Xu D,Hu G,et al.Isolation and identification of enteroinvasive Escherichia coli in Macaca mulatta and detection of its virulence factor gene[J].Chin Vet Sci,2010,40(11):1106-1109.
[21] Guenther S,Grobbel M,Heidemanns K,et al.First insights into antimicrobial resistance among faecal Escherichia coli isolates from small wild mammals in rural areas[J].Sci Total Environ,2010,408(17):3519-3522.
[22] Maluta R P,Logue C M,Casas M R,et al.Overlapped sequence types(STs)and serogroups of avian pathogenic(APEC)and human extra-intestinal pathogenic(ExPEC)Escherichia coli isolated in Brazil[J].PLoS ONE,2014,9(8):e105016.
[23]宦海霞,张科.大肠杆菌致病因子研究进展[J].中国家禽,2010,32(23):45-48.Huan H X,Zhang K.Research progress on pathogenic factor of Escherichia coli[J].Chin Poultry,2010,32(23):45-48.
[24]马兴树,范翠蝶,夏玉龙.禽致病性大肠杆菌研究进展[J].中国畜牧兽医,2013,40(2):169-174.Ma X S,Fan C D,Xia Y L.Research progress on avian pathogenic Escherichia coli[J].Chin Anim Husband&Vet Med,2013,40(2):169-174.
[25]李叶芳,白灏,彭开松,等.禽致病性大肠杆菌强毒力岛摄铁功能与致病性关系的研究[J].中国兽医科学,2012,42(9):906-910.Li Y F,Bai H,Peng K S,et al.Relationship between the iron uptake function of high pathogenicity island and the pathogenicity of avian pathogenic Escherichia coli strains[J].Chin Vet Sci,2012,42(9):906-910.
[26] Gophna U,Oelschlaeger T A,Hacker J,et al.Yersinia HPI in septicemic Escherichia coli strains isolated from diverse hosts[J].FEMS Microbiol Lett,2001,196(1):57-60.
[27] Delicato E R,de Brito B G,Konopatzki A P,et al.Occurrence of the temperature-sensitive hemagglutinin among avian Escherichia coli[J].Avian Dis,2002,46(3):713-716.
[28] Moll A,Manning P A,Timmis K N.Plasmid-determined resistance to serum bactericidal activity:a major outer membrane protein,the traTgene product,is responsible for plasmid-specified serum resistance in Escherichia coli[J].Infect Immun,1980,28(2):359-367.
[29]陈文静,韩先干,何亮,等.鸭致病性大肠杆菌的分离鉴定及其生物学特性分析[J].中国动物传染病学报,2010,18(2):34-40.Chen W J,Han X G,He L,et al.Characterization of duck pathogenic Escherichia coli[J].Chin J Anim Infect Dis,2010,18(2):34-40.