1株鸡源肠炎沙门氏菌的分离鉴定及致病性分析

doi:10.16431/j.cnki.1671-7236.2022.10.042

摘要/Abstract

摘要： 【目的】确定云南某蛋鸡场送检的疑似沙门氏菌病病料中的主要病原菌种类,并测定其致病性及药物敏感性。【方法】通过细菌分离培养、形态观察、生化试验、血清学鉴定及分子学方法进行细菌分离鉴定;通过对鸡的致病性试验及毒力基因检测分析分离菌的致病性;通过药物敏感性试验检测分离菌耐药情况。【结果】分离菌株在沙门氏菌显色培养基上形成紫色菌落,镜检为革兰氏阴性短杆菌,疑为沙门氏菌,命名为S2;生化试验结果显示,菌株S2赖氨酸脱羧酶、硫化氢、卫矛醇试验结果均为阳性;靛基质、尿素酶、氰化钾、山梨醇、水杨苷、β-半乳糖苷、丙二酸盐生化试验结果均为阴性,判断菌株S2为典型沙门氏菌属;血清学鉴定结果显示,菌株S2属于D群肠炎沙门氏菌,其抗原式为1,9,12:g,m;遗传进化分析结果显示,菌株S2与肠炎沙门氏菌MT621365处于同一分支,自展值为99%;致病性试验结果表明,分离菌株对雏鸡有较强的致病性,感染6 d后雏鸡全部死亡(10/10);毒力基因检测结果显示,菌株S2携带invJ、hilA、ssaB、misL、mgtC、orf319、sopB、spvA、spvB、spvC、spvR、stn和fimA毒力基因,不携带sopA、fliC毒力基因;菌株S2对多数抗菌药敏感程度很高,对阿莫西林-克拉维酸、头孢西丁、四环素等12种抗菌药均敏感,对甲氧嘧啶、复方新诺明和林可霉素耐药。【结论】本研究分离到了1株肠炎沙门氏菌,其毒力较强,可造成雏鸡较高的死亡率,对大部分常用抗菌药敏感,对甲氧嘧啶、复方新诺明和林可霉素具有耐药性。本研究结果可为沙门氏菌病的临床诊断和药物有效性评价提供一定参考依据。

关键词: 肠炎沙门氏菌; 分离鉴定; 致病性; 药物敏感性试验

Abstract: 【Objective】 The purpose of the test was to determine the main pathogenic bacteria in the suspected Salmonella disease materials sent by a laying hens farm in Yunnan,and to determine their pathogenicity and drug sensitivity.【Method】 The strains were identified by bacterial isolation and culture,morphological observation,biochemical test,serological identification and molecular methods.The pathogenicity of the isolated bacteria was analyzed through the pathogenicity test of chicken and the detection of virulence genes.The drug resistance of strains was studied by antibiotic sensitivity test.【Result】 The isolated strain formed purple colony on the Salmonella chromogenic medium,and was Gram-negative Brevibacterium and suspected to be Salmonella,named S2.The results of biochemical test showed that the results of lysine decarboxylase,hydrogen sulfide and dulcite were positive,and indol,urease,KCN,sorbitol,salicin,β-galactoside and malonate were negative,and S2 was judged to be a typical Salmonella.The results of serological identification showed that S2 belonged to Salmonella Enteritidis group D,and its antigen formula was 1,9,12:g,m.The results of genetic evolution analysis showed that S2 and Salmonella Enteritidis MT621365 were in the same branch,and the self-development value was 99%.The results of pathogenicity test showed that the isolated strain had strong pathogenicity to chickens,and all chickens died after 6 days of infection (10/10).The results of virulence gene detection showed that S2 carried invJ,hila,ssaB,misL,mgtC,orf319,sopB,spvA,spvB,spvC,spvR,stn and fimA virulence genes,but did not carried sopA and flic virulence genes.The isolated strain S2 was highly sensitive to most antibiotics,and it was sensitive to 12 antibacterial drugs including amoxicillin-clavulanic acid,cefoxitin,tetracycline and so on,but it was resistant to methoxypyrimidine,compound sulfamethoxazole and lincomycin.【Conclusion】 A strain of Salmonella Enteritidis was isolated in this study.It had strong virulence which could cause high mortality of chickens,and it was sensitive to most commonly used antibiotics,but was resistant to methoxypyrimidine,compound sulfamethoxazole and lincomycin.This study provided a certain reference basis for the clinical diagnosis of salmonellosis and the evaluation of drug effectiveness.

Key words: Salmonella Enteritidis; isolation and identification; pathogenicity; drug sensitivity test

中图分类号:

S852.61^＋2

张甜甜, 吕文亮, 林显华, 鹿瑶, 徐海燕, 谷巍, 郝木强, 盛永杰. 1株鸡源肠炎沙门氏菌的分离鉴定及致病性分析[J]. 中国畜牧兽医, 2022, 49(10): 4097-4105.

ZHANG Tiantian, LYU Wenliang, LIN Xianhua, LU Yao, XU Haiyan, GU Wei, HAO Muqiang, SHENG Yongjie. Isolation,Identification and Pathogenicity Analysis of a Strain of Salmonella Enteritidis from Chicken[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 4097-4105.

参考文献

[1] 刘洋.肠炎沙门氏菌耐药性分析及环丙沙星诱导耐药对其毒力的影响[D].烟台:鲁东大学,2021. LIU Y.Antimicrobial drug resistance and influence of ciprofloxacin-induced resistance on the virulence of Salmonella enterica serovar Enteritidis[D].Yantai:Ludong University,2021.(in Chinese)
[2] FLOCKHART L,PINTAR K,COOK A,et al.Distribution of Salmonella in humans,production animal operations and a watershed in a FoodNet Canada sentinel site[J].Zoonoses Public Health,2017,64(1):41-52.
[3] HENNESSY T W,CHENG L H,KASSENBORG H,et al.Egg consumption is the principal risk factor for sporadic Salmonella serotype Heidelberg infections:A case-control study in FoodNet sites[J].Clinical Infectious Diseases,2004,38(Suppl 3):S237.
[4] LI S,HE Y,MANN D A,et al.Global spread of Salmonella Enteritidis via centralized sourcing and international trade of poultry breeding stocks[J].Nature Communications,2021,12(1):5109.
[5] MIZUMOTO N,SASAI K,TANI H,et al.Specific adhesion and invasion of Salmonella Enteritidis in the vagina of laying hens[J].Veterinary Microbiology,2005,111(1-2):99-105.
[6] WOODWARD C,KWON Y,KUBENA L,et al.Reduction of Salmonella enterica serovar Enteritidis colonization and invasion by an alfalfa diet during molt in Leghorn hens[J].Poultry Science,2005,84(2):185.
[7] JIAN Z,LUO W,LIU G,et al.High genetic similarity of Salmonella Enteritidis as a predominant serovar by an independent survey in three large-scale chicken farms in China[J].Poultry Science,2021,100(4):100941.
[8] 杨保伟,张秀丽,曲东,等.2007－2008陕西部分零售畜禽肉沙门氏菌血清型和基因型[J].微生物学报,2010,50(5):654-660. YANG B W,ZHANG X L,QU D,et al.Serotypic and genotypic characterization of Salmonella serovars from retails meat in Shaanxi province (2007-2008)[J].Acta Microbiologica Sinica,2010,50(5):654-660.(in Chinese)
[9] TACK D M,MARDER E P,GRIFFIN P M,et al.Preliminary incidence and trends of infections with pathogens transmitted commonly through food-foodborne diseases active surveillance network,10 U.S.Sites,2015-2018[J].Morbidity and Mortality Weekly Report,2019,68(16):369-373.
[10] EUROPEAN CENTRE FOR DISEASE PREVENTION AND CONTROL,EUROPEAN FOOD SAFETY AUTHORITY.Multi-country outbreak of Salmonella Enteritidis sequence type (ST)11 infections linked to eggs and egg products-8 February 2022[J].EFSA Supporting Publications,2022,19(2):7180E
[11] 国家卫生和计划生育委员会,国家食品药品监督管理总局GB 4789.4－2016 食品安全国家标准食品微生物学检验沙门氏菌检验[S].2016. CHINA HEALTH AND FAMILY PLANNING COMMISSION,CHINA FOOD AND DRUG ADMINISTRATION.GB 4789.4－2016.National food safety standard.Food microbiological examination:Salmonella[S].2016.(in Chinese)
[12] 杨文文,李玉保,路建彪,等.山东省鸡源沙门氏菌的分离鉴定及毒力基因分析[J].中国畜牧兽医,2021,48(8):3069-3078. YANG W W,LI Y B,LU J B,et al.Isolation,identification and virulence gene analysis of Salmonella from chickens in Shandong province[J].China Animal Husbandry & Veterinary Medicine,2021,48(8):3069-3078.(in Chinese)
[13] 王学硕,赵琳娜,刘娜,等.乳粉中沙门氏菌能力验证样品研制及应用评价[J].食品安全质量检测学报,2021,12(17):6803-6809. WANG X S,ZHAO L N,LIU N,et al.Preparation and application evaluation of Salmonella proficiency test samples in milk powder[J].Journal of Food Safety and Quality,2021,12(17):6803-6809.(in Chinese)
[14] 郑林,祝令伟,郭学军,等.沙门氏菌主要流行血清型耐药性的研究进展[J].江苏农业科学,2020,48(6):8-12. ZHENG L,ZHU L W,GUO X J,et al.Research progress on drug resistance of main epidemic serotypes of Salmonella[J].Jiangsu Agricultural Sciences,2020,48(6):8-12.(in Chinese)
[15] 刘新静.河北省鸡沙门氏菌的分离鉴定及耐药性分析[D].泰安:山东农业大学,2015. LIU X J.The separation of chicken Salmonella identification and drug resistance analysis in Hebei province [D].Tai’an:Shandong Agricultural University,2015.(in Chinese)
[16] 赵建梅,李月华,张青青,等.2008—2017年我国部分地区禽源沙门氏菌流行状况及耐药分析[J].中国动物检疫,2019,36(8):27-35. ZHAO J M,LI Y H,ZHANG Q Q,et al.Analysis on the prevalence and antimicrobial resistance of poultry Salmonella in some regions of China during 2008 to 2017[J].China Animal Health Inspection,2019,36(8):27-35.(in Chinese)
[17] 王迪轩,高东阳,张天姿,等.华中地区鸡源沙门菌分离鉴定及耐药性分析[J].中国畜牧兽医,2019,46(3):931-939. WANG D X,GAO D Y,ZHANG T Z,et al.Isolation,identification and drug resistance analysis of avian Salmonella in Central China[J].China Animal Husbandry & Veterinary Medicine,2019,46(3):931-939.(in Chinese)
[18] 孔宪刚.兽医微生物学[M].北京:中国农业出版社,2013. KONG X G.Veterinary Microbiology[M].Beijing:China Agriculture Press,2013.(in Chinese)
[19] WIGLEY P,HULME S D,POWERS C,et al.Infection of the reproductive tract and eggs with Salmonella enterica serovar Pullorum in the chicken is associated with suppression of cellular immunity at sexual maturity[J].Infection & Immunity,2005,73(5):2986-2990.
[20] 刘菲菲.鸡源沙门氏菌流行株毒力基因分布与三种毒力基因PCR同步检测方法的建立[D].武汉:武汉大学,2019. LIU F F.Virulence genes distribution of Salmonella isolates recovered from chickens and establishment of a multiplex PCR assay for rapid detection of three virulence genes[D].Wuhan:Wuhan University,2019.(in Chinese)
[21] LIN H H,CHEN H L,JANAPATLA R P,et al.Hyperexpression of type Ⅲ secretion aystem of Salmonella Typhi linked to a higher cytotoxic effect to monocyte-derived macrophages by activating inflammasome[J].Microbial Pathogenesis,2020,146:104222.
[22] 聂青青,张璇,黄攀,等.牛源肺炎克雷伯菌菌毛类型鉴定及fimA、mrkA基因序列分析[J].现代畜牧兽医,2022(4):38-43. NIE Q Q,ZHANG X,HUANG P,et al.Fimbriae types identification and fimA,mrkA genes characterization of isolated bovine Klebsiella pneumonia[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2022,4:38-43.(in Chinese)
[23] 申永秀,周丽萍,王艳,等.不同来源沙门氏菌耐药性及相关性研究[J].食品安全质量检测学报,2018,9(7):1513-1517. SHEN Y X,ZHOU L P,WANG Y,et al.Antimicrobial resistance and correlation of Salmonella from different sources[J].Journal of Food Safety and Quality,2018,9(7):1513-1517.(in Chinese)
[24] KARIUKI S,REVATHI G,KARIUKI N,et al.Increasing prevalence of multidrug-resistant non-typhoidal Salmonellae,Kenya,1994-2003[J].International Journal of Antimicrobial Agents,2005,25(1):38-43.
[25] VAZ C S L,VOSS-RECH D,ALVES L,et al.Effect of time of therapy with wild-type lytic lacteriophages on the reduction of Salmonella Enteritidis in broiler chickens[J].Veterinary Microbiology,2019,240:108527.
[26] 高尚,王鹏勇,鞠辉明,等.江苏省部分地区禽源沙门菌的分离鉴定及耐药性分析[A].中国畜牧兽医学会2018年学术年会禽病学分会第十九次学术研讨会[C].2018. GAO S,WANG P Y,JU H M.et al.Isolation,identification and drug resistance analysis of avian Salmonella in some areas of Jiangsu province[A].The 19th Academic Seminar of Avian Disease Branch of 2018 Academic Annual Meeting of China Society of Animal Husbandry and Veterinary Medicine[C].2018.(in Chinese)
[27] 汤全英,崔家瑞,张梦寒,等.苏州市3起疑似肠炎沙门菌食物中毒事件的病原学及耐药分析[J].江苏预防医学,2022,33(2):192-193. TANG Q Y,CUI J R,ZHANG M H,et al.Etiology and drug resistance analysis of 3 suspected Salmonella Enteritis food poisoning cases in Suzhou city[J].Jiangsu Journal of Preventive Medicine,2022,33(2):192-193.(in Chinese)
[28] 李廷翠,严红亚,常志顺,等.云南蛋鸡源肠炎沙门氏菌的分离鉴定及生物学特性[J].微生物学通报,2021,48(1):82-92. LI T C,YAN H Y,CHANG Z S,et al.Isolation,identification and characterization of Salmonella Enteritis from Yunnan laying hens[J].Microbiology China,2021,48(1):82-92.(in Chinese)