商品肉鸭致病性肠炎沙门菌的分离鉴定及耐药性与致病性分析

doi:10.16431/j.cnki.1671-7236.2025.05.038

摘要/Abstract

摘要： 【目的】探究某商品肉鸭养殖场内分离到的致病性肠炎沙门菌的耐药性及毒力基因分布情况，为该菌所致疾病的防控和用药提供参考依据。【方法】采集病死肉鸭肝脏进行细菌分离，通过分离纯化、革兰染色镜检、生化试验及PCR扩增对分离菌株进行血清型鉴定。采用K-B纸片法进行药敏试验，结合菌株的耐药基因，分析其耐药情况。进行雏鸭致病性试验，监测雏鸭肛拭子排毒、致死率和体重变化，结合菌株的毒力基因，评估其致病性。【结果】分离菌株在沙门菌显色培养基上呈紫色菌落，革兰染色镜检结果为革兰阴性短杆菌。生化鉴定结果显示，分离菌株靛基质、氨基酸脱羧酶对照、尿素酶、氰化钾试验生化管和氰化钾试验对照显示阴性，赖氨酸脱羧酶显示阳性，符合沙门菌的生化特征。通过肠炎沙门菌引物PCR扩增获得大小为304 bp目的片段，与预期片段大小一致，进而鉴定分离菌株为肠炎沙门菌。药敏结果显示，分离株对氟喹诺酮类和氯霉素类药物敏感，对四环素类和β-内酰胺类药物耐药，检测出tetA和bla_TEM 2 种耐药基因。检测出11种毒力基因：bcfC、avrA、sopE、spvR、spvB、spvC、spvD、sopB、siiD、mgtC和ssaQ。雏鸭致病性结果试验显示，7日龄雏鸭攻毒致死率为90%，攻菌后2 d肛拭子开始检出沙门菌且雏鸭体重增长缓慢，剖检可见心脏、肝脏发生不同程度的纤维素渗出病变。【结论】本研究从商品肉鸭中分离鉴定出致病性肠炎沙门菌，分离菌株对肉鸭有较强致病能力，存在2种耐药基因和多种毒力基因。试验结果可为肠炎沙门菌致病机制的进一步研究和疾病防控奠定理论基础。

关键词: 商品肉鸭; 肠炎沙门菌; 分离鉴定; 致病性; 耐药性

Abstract: 【Objective】 This study was conducted to explore the distribution of drug resistance and virulence genes of pathogenic Salmonella Enteritidis isolated from a commercial meat duck farm,and provide reference for the prevention,control and medication of the disease.【Method】 The livers of dead meat ducks were collected for bacterial isolation,and the serotype of the isolated strains was identified by separation and purification,Gram staining microscopy,biochemical test and PCR amplification.The drug sensitivity test was carried out by K-B disk method,and the drug resistance of the strain was analyzed by combining the drug resistance genes.The pathogenicity test of duckling was used to monitor the virulence,mortality and body weight changes of duckling anal swabs,and to evaluate the pathogenicity of strains combined with virulence genes.【Result】 The isolated strain showed purple colonies on the Salmonella chromogenic medium,and the results of Gram staining microscopy were Gram-negative brevis.The results of biochemical identification were negative for indigo matrix,amino acid decarboxylase control,urease,potassium cyanide test biochemical tube and potassium cyanide test control,and positive for lysine decarboxylase,which was consistent with the biochemical characteristics of Salmonella.The target fragment 304 bp was further amplified by PCR primer of Salmonella Enteritidis,which was consistent with the expected fragment size,and then the strain was identified as Salmonella Enteritidis.The results of drug sensitivity showed that the isolate was sensitive to fluoroquinolones and chloramphenicols,but resistant to tetracyclines and β-lactams.Meanwhile，tetA and bla_TEM were detected as drug resistance genes.Eleven virulence genes were detected:bcfC,avrA,sopE,spvR, spvB,spvC,spvD,sopB,siiD,mgtC and ssaQ.The pathogenicity test of ducklings showed that the mortality rate of 7-day-old ducklings was 90%.Salmonella pathogen was detected by anal swab 2 days after the challenge,and the weight of ducklings increased slowly.Anatomical symptoms showed varying degrees of cellulose exudates in the heart and liver.【Conclusion】 In this study,Salmonella Enteritidis were isolated and identified from commercial meat ducks.The strain had strong pathogenic ability to meat ducks,and there were two drug resistance genes and multiple virulence genes.The experimental results could lay a theoretical foundation for further research on the pathogenesis,prevention and treatment of Salmonella Enteritidis.

Key words: commercial meat ducks; Salmonella Enteritidis; isolation and identification; pathogenicity; drug resistance

中图分类号:

S852.61

于堃, 赵杰, 马芹, 石艳红, 张筱, 刘子涵, 张新婷, 汪建华, 李玉峰. 商品肉鸭致病性肠炎沙门菌的分离鉴定及耐药性与致病性分析[J]. 中国畜牧兽医, 2025, 52(5): 2353-2363.

YU Kun, ZHAO Jie, MA Qin, SHI Yanhong, ZHANG Xiao, LIU Zihan, ZHANG Xinting, WANG Jianhua, LI Yufeng. Isolation,Identification,Drug Resistance and Pathogenicity Analysis of Salmonella Enteritidis from Commercial Meat Ducks[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 2353-2363.

参考文献

[1] 彭峻烽,曾杭,吴思凡,等.成都地区鸭源沙门氏菌的分离鉴定及其耐药特征、毒力基因分析[J].中国人兽共患病学报,2018,34(3):217-223.PENG J F,ZENG H,WU S F,et al.Prevalence,antimicrobial resistance patterns and virulence gene analysis of Salmonella spp.originated from ducks in Chengdu area,China[J].Chinese Journal of Zoonoses,2018,34(3):217-223.(in Chinese)
[2] 徐桂云,樊世杰.家禽沙门氏菌感染现状及不同国家的防治策略[J].中国家禽,2012,34(9):7-12.XU G Y,FAN S J.Poultry Salmonella infection status and the control strategy of different countries[J].China Poultry,2012,34(9):7-12.(in Chinese)
[3] 彭峻烽.肉鸭屠宰链沙门氏菌的污染情况、耐药特征、毒力基因及PFGE分型研究[D].雅安：四川农业大学,2018.PENG J F.Contamination,antimicrobial resistance patterns,virulence gene and pulsed-field gelelectrophoresis analysis of Salmonella spp.isolated from duck slaughter chain[D].Ya’an:Sichuan Agricultural University,2018.(in Chinese)
[4] 郭丰.2020—2021年吉林省腹泻病例中沙门氏菌的感染状况及耐药性分析[D].长春：吉林大学,2022.GUO F.Analysis of Salmonela infection status and drug resistancein diarrhea cases in Jilin province,2020—2021[D].Changchun:Jilin University,2022.(in Chinese)
[5] 王贤文,赵丽媛,张瑞雪,等.沙门氏菌血清分型及耐药机制研究进展[J].山东农业科学,2024,56(1):174-180.WANG X W,ZHAO L Y,ZHANG R X,et al.Research progress on serotyping and antimicrobial resistance mechanism of Salmonella[J].Shandong Agricultural Sciences,2024,56(1):174-180.(in Chinese)
[6] 杜新娜.蛋鸡沙门氏菌的分离鉴定及耐药性分析[J].当代畜牧,2023,6:7-8.DU X N.Isolation,identification and drug resistance analysis of Salmonella in laying hens[J].Contemporary Animal Husbandry,2023,6:7-8.(in Chinese)
[7] 王喜,李珂,李廷翠,等.75株蛋鸡源沙门菌的MLST分型与耐药性分析[J].畜牧兽医学报,2022,53(5):1626-1631.WANG X,LI K,LI T C,et al.MLST typing and drug resistance analysis of 75Salmonella strains isolated from laying hens[J].Acta Veterinaria et Zootechnica Sinica,2022,53(5):1626-1631.(in Chinese)
[8] 张启龙,栗云鹏,傅彩霞,等.1株信鸽源鼠伤寒沙门氏菌的分离鉴定及其耐药性和毒力分析[J].中国畜牧兽医,2021,48(1):338-347.ZHANG Q L,LI Y P,FU C X,et al.Isolation,identification,drug resistance and virulence analysis of one Salmonella Typhimurium strain from racing pigeons[J].China Animal Husbandry & Veterinary Medicine，2021,48(1):338-347.(in Chinese)
[9] 李伟.山东省及周边部分地区沙门氏菌在肉鸭养殖场中的流行特征及致病性分析[D].泰安：山东农业大学,2022.LI W.Epidemiological characteristics and pathogenicity analysis of Salmonella isolated from meat duck farms in Shandong province and surrounding areas[D].Tai’an:Shandong Agricultural University,2022.(in Chinese)
[10] 齐佳欣,邵明杰,崔平,等.马流产沙门氏菌研究进展[J].动物医学进展,2024,45(4):102-106.QI J X,SHAO M J,CUI P,et al.Progress on Salmonella abortus equi[J].Progress in Veterinary Medicine,2024,45(4):102-106.(in Chinese)
[11] 潘文章.羔羊病原性腹泻发生的原因与防治[J].四川畜牧兽医,2024,51(4):58-60.PAN W Z.Causes and prevention of pathogenic diarrhea in lambs[J].Sichuan Animal & Veterinary Sciences,2024,51(4):58-60.(in Chinese)
[12] 沈冬梅,王旭红,牧仁,等.沙门氏菌引起育肥猪死亡的案例分析[J].兽医导刊,2024,1:29-31.SHEN D M,WANG X H,MU R,et al.Case study of death caused by Salmonella in fattening pigs[J].Veterinary Orientation,2024,1:29-31.(in Chinese)
[13] LAI Y S,CHENG C C,LEE M T,et al.The prognostic value of cytokeratin and sal-like protein 4 expression in hepatocellular carcinoma and intra-hepatic cholangiocarcinoma in Taiwan[J]. International Journal of Medical Sciences,2018,15(14):1746.
[14] 陈小娇,张妹,蔡晓庆,等.市场鸡沙门氏菌分离鉴定及致病性研究[J].山东畜牧兽医,2024,45(3):9-11.CHEN X J,ZHANG M,CAI X Q,et al.Isolation,identification and pathogenicity of Salmonella in chicken[J].Shandong Journal of Animal Science and Veterinary Medicine,2024,45(3):9-11.(in Chinese)
[15] 肖胜.鸡沙门氏菌病的诊断与防治[J].今日畜牧兽医,2024,40(2):83-85.XIAO S.Diagnosis and prevention of chicken salmonellosis[J].Today Animal Husbandry and Veterinary Medicine,2024,40(2):83-85.(in Chinese)
[16] 臧金刚.鸭沙门氏菌病的诊断及中西医防治[J].养殖与饲料,2024,23(4):102-104.ZANG J G.Duck Salmonella disease diagnosis and prevention and treatment of Chinese and Western medicine[J].Animals Breeding and Feed,2024,23(4):102-104.(in Chinese)
[17] 马永生,徐永平,金礼吉,等.饲用抗生素细菌耐药性的研究进展[J].中国饲料,2005,12:5-7.MA Y S,XU Y P,JIN L J,et al.Research progress of antibiotic resistance in feeding bacteria[J]. China Feed,2005,12:5-7.(in Chinese)
[18] VO A T T,VAN DUI JKEREN E,FLUIT A C,et al.Class 1 integrons in Dutch Salmonella enterica serovar Dublin isolates from clinical cases of bovine salmonellosis[J].Veterinary Microbiology,2006,117(2-4):192-200.
[19] 孙朋浩,邱荣超,李书宁,等.广州市生鲜鸡中肠炎沙门菌的监测及耐药基因的分子鉴定[J].畜牧兽医学报,2018,49(2):388-395.SUN P H,QIU R C,LI S N,et al.Monitoring of Salmonella Enteritidis and molecular identification of antibiotic resistance gene in fresh chicken of Guangzhou city[J].Acta Veterinaria et Zootechnica Sinica,2018,49(2):388-395.(in Chinese)
[20] 刘莉,董洪燕,朱善元,等.6株鸭源沙门氏菌的分离鉴定与药敏试验[J].动物医学进展,2023,44(9):51-54.LIU L,DONG H Y,ZHU S Y,et al.Isolation,identification and drug sensitivity test of 6Salmonella isolates from ducks[J].Progress in Veterinary Medicine,2023,44(9):51-54.(in Chinese)
[21] 于堃.致病性鸭源沙门氏菌的鉴定及耐药性分析[D].沈阳：沈阳农业大学,2020.YU K.Identification and drug resistance analysis of pathogenic Salmonella from ducks[D].Shenyang:Shenyang Agricultural University,2020.(in Chinese)
[22] 刘加圣,龚建森.鸭源沙门氏菌的分离鉴定及耐药性检测[J].中国家禽,2012,34(10):54-56.LIU J S,GONG J S.Duck source separation,identification and drug resistance of Salmonella detection[J].China Poultry,2012,34(10):54-56.(in Chinese)
[23] 罗忠宝.闽北地区鸡源肠炎沙门菌的分离鉴定与致病性分析[J].福建农林大学学报(自然科学版),2023,52(6):827-836.LUO Z B.Isolation,identification and pathogenicity analysis of Salmonella Enteritidis from chickens in Northern Fujian[J].Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2023,52(6):827-836.(in Chinese)
[24] 张捷,畅晓晖,柳明,等.禽类中沙门氏菌MLST分型及毒力基因筛查[J].食品研究与开发,2021,42(13):174-179.ZHANG J,CHANG X H,LIU M,et al.MLST typing of Salmonella in poultry and screening of virulence genes[J].Food Research and Development,2021,42(13):174-179.(in Chinese)
[25] 刘理慧,储锦华,隋雨欣,等.沙门氏菌中主要毒力因子的研究进展[J].生物技术通报,2022,38(9):72-83.LIU L H,CHU J H,SUI Y X,et al.Research progress of main virulence factors in Salmonella[J].Biotechnology Bulletin,2022,38(9):72-83.(in Chinese)
[26] NIEMANN G S,BROWN R N,GUSTIN J K,et al.Discovery of novel secreted virulence factors from Salmonella enteric serovar Typhimurium by proteomic analysis of culture supernatants[J].Infectious Diseases and Therapy,2011,79(1):33-43.
[27] LOPEZ-GARRIDO J,CASADESUS J.Crosstalk between virulenceloci:Regulation of Salmonella enterica pathogenicity island 1(SPI-1) by products of the std fimbrial operon[J].PLoS One,2012,7(1):e30499.
[28] PASSARIS I,CAMBRE A,GOVERS S K,et al.Bimodal expression of the Salmonella Typhimurium spv operon[J].Genetics,2018,210(2):621-635.
[29] GEBREYES W A,THAKUR S,DORR P,et al.Occurrence of spvA virulence gene and clinical significance for multidrug-resistant Salmonella strains[J].Journal of Clinical Microbiology,2009,47(3)：777-780.
[30] SUN L Q,YANG S D,DENG Q F,et al.Salmonella effector SpvB disrupts intestinal epithelial barrier integrity for bacterial translocation[J].Frontiers in Cellular and Infection Microbiology,2020,10(10)：606541.
[31] GOPINATH A,ALLEN T A,BRIDGWATER C J,et al.The Salmonella type Ⅲ effector SpvC triggers the reverse transmigration of infected cells into the bloodstream[J].PLoS One,2019,14(12)：e0226126.
[32] CHENG R A,EADE C R,WIEDMANN M.Embracing diversity:Differences in virulence mechanisms,disease severity,and host adaptations contribute to the success of nontyphoidal Salmonella as a foodborne pathogen[J].Frontiers in Microbiology,2019,10(10):1368.
[33] CERNY O,HOLDEN D W.Salmonella SPI-2 type Ⅲ secretion system-dependent inhibition of antigen presentation and T cell function[J]. Liver International,2019,215(215)：35-39.
[34] 王路才,撒朗文朱,张焕容.禽源肠炎沙门菌的分离鉴定及耐药性与致病性分析[J].中国畜牧兽医,2023,50(4):1663-1674.WANG L C,SALANG W Z,ZHANG H R.Isolation,identification and drug resistance and pathogenicity analysis of Salmonella Enteritidis from avian[J].China Animal Husbandry & Veterinary Medicine,2023,50(4):1663-1674.(in Chinese)
[35] 薛颖,郭荣显,钱珊珊,等.沙门菌毒力岛的研究进展[J].微生物与感染,2015,10(6):381-389.XUE Y,GUO R X,QIAN S S,et al.Research progress on Salmonella pathogenicity islands[J].Journal of Microbes and Infections,2015,10(6):381-389.(in Chinese)
[36] 叶海梅,刘红,齐文岚,等.食物中毒沙门菌血清型、耐药性及毒力基因研究[J].热带医学杂志,2017,17(1):11-14.YE H M,LIU H,QI W L,et al.Detection of serotype,drug resistance and virulence genes of food poisoning Salmonella[J].Journal of Tropical Medicine,2017,17(1):11-14.(in Chinese)
[37] 赵方.江苏地区禽源沙门菌的临床检测及相关特性的分析[D].扬州：扬州大学,2016.ZHAO F.Clinic surveilling and characteristics of Salmonella isolates from avian of Jiangsu province[D].Yangzhou:Yangzhou University,2016.(in Chinese)