山东省鸡源沙门氏菌的分离鉴定及毒力基因分析

doi:10.16431/j.cnki.1671-7236.2021.08.040

摘要/Abstract

摘要： 本研究旨在调查山东地区鸡源沙门氏菌的流行情况及毒力基因分布。在山东地区规模化养鸡场采集病料，利用四硫磺酸钠煌绿增菌液（TTB）进行沙门氏菌选择增菌，并对分离菌株进行纯化培养、革兰氏染色、生化试验、培养基菌落形态鉴定，利用沙门氏菌属及血清型特异性引物对分离菌进行PCR扩增鉴定，并利用PCR方法检测分离菌中33种毒力基因分布。结果显示，分离菌在LB固体培养基和麦康凯培养基上呈无色半透明、光滑且边缘整齐的菌落，镜检为革兰氏阴性、两端钝圆的小杆菌。生化试验、培养基菌落形态鉴定、沙门氏菌属及血清型特异性引物PCR扩增结果显示，共分离到25株鸡源沙门氏菌，其中肠炎沙门氏菌9株，鸡白痢沙门氏菌和鼠伤寒沙门氏菌各8株。毒力基因检测结果显示，33种毒力基因在25株分离菌中均有检出，其中20种毒力岛基因（invJ、virK、sopA、mogA、hilA、hisJ、ssaB、ssaQ、ssiD、misL、mgtC、orf319、siiE、siiD、bcfA、pipC、sopB、araB、spoB和avrA基因）、肠毒素基因（stn基因）及菌毛毒力基因（fimA基因）检出率均为100%；2种毒力岛基因（sipA和sodC基因）及4种毒力质粒基因（spvA、spvC、spvD和spvR基因）检出率均为96%，spoE基因检出率为68%，fliC基因检出率为36%，gipA与spvB基因检出率均为32%，sseL基因检出率为4%；25株鸡源沙门氏菌中，分别携带31种（8株）、30种（1株）、29种（15株）和24种（1株）毒力基因；毒力基因gipA和spvB只在鼠伤寒沙门氏菌中检出，且在鼠伤寒沙门氏菌中检出率为100%。本研究结果表明，山东地区鸡源沙门氏菌主要为肠炎沙门氏菌、鸡白痢沙门氏菌和鼠伤寒沙门氏菌，分离菌皆携带多种毒力基因，其中gipA和spvB毒力基因只在鼠伤寒沙门氏菌中检出，可作为鼠伤寒沙门氏菌鉴定的备选基因之一。

关键词: 鸡; 沙门氏菌; 分离; 鉴定; 毒力基因

Abstract: This experiment was aimed to investigate the prevalence and virulence genes distribution of Salmonella from chicken in Shandong province.Samples were collected from large-scale chicken farms in Shandong province, and tetrathionate broths (TTB) were used as Salmonella selective enrichment broths before bacterial isolation.The isolated strains were purified and cultured, and Gram staining, biochemical tests, colony morphology, PCR amplification with Salmonella and serotype specific primers were carried out.33 kinds virulence genes were detected by PCR to study the virulence genes distribution.The results showed that the isolated strains had grown on LB and MacConkey media with colorless, translucent, smooth and neat edged colonies, and the bacterial morphology were Gram-negative brevibacterium with blunt ends at both ends.25 Salmonella strains were isolated, including 9 Salmonella Enteritis, 8 Salmonella Pullorum strains and 8 Salmonella Typhimurium strains.The detection rates of 20 genes that virulence island (invJ, virK, sopA, mogA, hilA, hisJ, ssaB, ssaQ, ssiD, misL, mgtC, orf319, siiE, siiD, bcfA, pipC, sopB, araB, spoB and avrA genes), enterotoxin gene (stn gene) and fimbriae virulence genes (fimA gene) were 100%.The detection rates of 2 virulence island genes (sipA and sodC genes) and 4 virulence plasmid genes (spvA, spvC, spvD and spvR genes) were 96%.The detection rate of spoE gene was 68%, fliC gene was 36%, gipA and spvB genes were 32%, and sseL gene was 4%.In the 25 Salmonella strains, the numbers of carrying virulence genes were 31(8 strains), 30(1 strain), 29(15 strains) and 24(1 strain), respectively.gipA and spvB genes could be detected only in Salmonella Typhimurium, and the positive rate was 100%.The above results showed that Salmonella Enteritis, Salmonella Pullorum and Salmonella Typhimurium were the main pathogens of salmonellosis in Shandong province.All the chicken Salmonella carried many virulence genes, gipA and spvB genes detected only in Salmonella Typhimurium, it could be used as one of the candidate genes for the identification of Salmonella Typhimurium.

Key words: chicken; Salmonella; isolation; identification; virulence gene

中图分类号:

S852.61⁺2

杨文文, 李玉保, 路建彪, 司振书, 张凯悦, 管玉堂, 庞喆羽, 徐欣璐, 孟凡达, 丁宁宁. 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 Wenwen, LI Yubao, LU Jianbiao, SI Zhenshu, ZHANG Kaiyue, GUAN Yutang, PANG Zheyu, XU Xinlu, MENG Fanda, DING Ningning. Isolation,Identification and Virulence Gene Analysis of Salmonella from Chickens in Shandong Province[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 3069-3078.

参考文献

[1] PRISNYI A A,MOISEEVA A A,SKVORTSOV V N,et al.The effect of enrofloxacin on blood values of chickens in experimental salmonellosis[J].Earth and Environmental Science,2020,548(4):37-42.
[2] 刘聪,黄世猛,赵丽红,等.凝结芽孢杆菌对感染肠炎沙门氏菌蛋鸡肠道形态、抗氧化能力及沙门氏菌定植的影响[J].中国农业大学学报,2019,24(1):69-75. LIU C,HUANG S M,ZHAO L H,et al.Effects of dietary supplementation with Bacillus coagulans on the intestinal morphology,plasma anti oxidation capacity and colonization of Salmonella in the caecum of S. Enteritidis infected laying hens[J].Journal of China Agricultural University,2019,24(1):69-75.(in Chinese)
[3] 孟繁新.鸡白痢沙门氏菌病的流行病学、临床症状、诊断与防控措施[J].现代畜牧科技,2020,49(11):90-91. MENG F X.Epidemiology,clinical symptoms,diagnosis and control measures of Salmonella Pullorum disease in chicken[J].Modern Animal Husbandry Science & Technology,2020,49(11):90-91.(in Chinese)
[4] 苏兵.鸡沙门氏菌病的流行特点与防控[J].养殖与饲料,2020,19(10):88-89. SU B.Epidemic characteristics and control of chicken salmonellosis[J].Animals Breeding and Feed,2020,19(10):88-89.(in Chinese)
[5] 牛琼,杨斌,杨溢,等.禽源沙门氏菌净化检测技术研究进展[J].中国预防兽医学报,2020,42(10):1073-1078. NIU Q,YANG B,YANG Y,et al.Updated study on detection and eradication technology of avian-derived Salmonella infection[J].Chinese Journal of Preventive Veterinary Medicine,2020,42(10):1073-1078.(in Chinese)
[6] PARK C J,LI J F,ZHANG X L,et al.Diverse lineages of multidrug resistant clinical Salmonella enterica and a cryptic outbreak in New Hampshire,USA revealed from a year-long genomic surveillance[J].Infection,Genetics and Evolution,2021,87:104645.
[7] BARBOSA P M,RUVIARO A R,MARTINS I M,et al.Enzyme-assisted extraction of flavanones from citrus pomace:Obtention of natural compounds with anti-virulence and anti-adhesive effect against Salmonella enterica subsp.enterica serovar Typhimurium[J].Food Control,2021,120:107525.
[8] ZOU W,ALKHALDI S F,BRANHAM W S,et al.Microarray analysis of virulence gene profiles in Salmonella serovars from food/food animal environment[J].Journal of Infection in Developing Countries,2011,5(2):94-105.
[9] LIN H H,CHEN H L,JANAPATLA R P,et al.Hyperexpression of type Ⅲ secretion system of Salmonella Typhi linked to a higher cytotoxic effect to monocyte-derived macrophages by activating inflammasome[J].Microbial Pathogenesis,2020,146:104222.
[10] MASAYUKI N,EIKI Y,AKITOYO I,et al.Salmonella enterotoxin (stn) regulates membrane composition and integrity[J].Disease Models & Mechanisms,2012,5(4):515-521.
[11] 范忠军,许保疆,翟崇凯,等.鸡源肠炎沙门氏菌快速检测方法的建立及其分离株的耐药性分析[J].河南农业科学,2017,46(10):137-142. FAN Z J,XU B J,ZHAI C K,et al.Establishment of rapid detection method of Salmonella Enteritidis and drug resistance analysis of isolates from chickens[J].Journal of Henan Agricultural Sciences,2017,46(10):137-142.(in Chinese)
[12] 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)
[13] 朱春红,陶志云,刘宏祥,等.鸡白痢与鸡伤寒沙门菌双重PCR方法的建立和初步应用[J].中国兽医杂志,2019,55(8):74-76. ZHU C H,TAO Z Y,LIU H X,et al.Detection of Salmonella Gallinarum and Pullorum strains by double PCR[J].Chinese Journal of Veterinary Medicine,2019,55(8):74-76.(in Chinese)
[14] LI K,YE S,ALALI W Q,et al.Antimicrobial susceptibility,virulence gene and pulsed-field gel electrophoresis profiles of Salmonella enterica serovar Typhimurium recovered from retail raw chickens,China[J].Food Control,2017,72:36-42.
[15] 刘芳萍,王德宁,李昌文,等.鸡源沙门氏菌耐药性的分析及毒力基因的检测[J].中国兽医科学,2013,43(12):1236-1239. LIU F P,WANG D N,LI C W,et al.Analysis of antimicrobial resistance of Salmonella isolated from chickens and detection of virulence genes of isolates[J].Chinese Veterinary Science,2013,43(12):1236-1239.(in Chinese)
[16] 刘书宏,吴科敏,周辰瑜,等.食源性沙门氏菌分离株致病性研究和毒力相关基因检测[J].食品科学,2018,39(17):182-186. LIU S H,WU K M,ZHOU C Y,et al.Foodborn Salmonella isolates:Pathogenicity evaluation and detection of virulence genes[J].Food Science,2018,39(17):182-186.(in Chinese)
[17] 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)
[18] 宋雪,赵格,刘文化,等.不同来源沙门氏菌的毒力基因检测与耐药性分析[J].中国动物检疫,2017,34(5):40-46. SONG X,ZHANG G,LIU W H,et al.Comparative analysis of virulent genes and drug resistance of Salmonella isolated from different sources[J].China Animal Health Inspection,2017,34(5):40-46.(in Chinese)
[19] 王晶钰,董睿,王利勤,等.市售鲜鸡蛋中沙门氏菌的分离鉴定及毒力岛基因检测[J].食品科学,2012,33(16):154-158. WANG J Y,DONG R,WANG L Q,et al.Isolation,identification and pathogenicity island gene detection of Salmonella in commercial eggs[J].Food Science,2012,33(16):154-158.(in Chinese)
[20] 张芬,周秀娟,宋启发,等.宁波地区沙门氏菌耐药性监测及毒力基因筛查[J].中国食品学报,2019,19(2):216-222. ZHANG F,ZHOU X J,SONG Q F,et al.Analysis of antimicrobial susceptibility and surveillance of virulence genes in Salmonella isolates from Ningbo China[J].Journal of Chinese Institute of Food Science and Technology,2019,19(2):216-222.(in Chinese)
[21] 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.Current situation of Salmonella infection in poultry and control strategies in different countries[J].China Poultry,2012,34(9):7-12.(in Chinese)
[22] LAI J,WU C M,WU C B,et al.Serotype distribution and antibiotic resistance of Salmonella in food-producing animals in Shandong province of China,2009 and 2012[J].International Journal of Food Microbiology,2014,180:30-38.
[23] LU Y,WU C M,WU G J,et al.Prevalence of antimicrobial resistance among Salmonella isolates from chicken in China[J].Foodborne Pathogens and Disease,2011,8(1):45-53.
[24] LI R C,LAI J,WANG Y,et al.Prevalence and characterization of Salmonella species isolated from pigs,ducks and chickens in Sichuan province,China[J].International Journal of Food Microbiology,2013,163(1):14-18.
[25] 薛俊龙,田林君,张国权,等.山西省鸡沙门氏菌病流行病学调查分析[J].山西农业科学,2010,38(9):58-62. XUE J L,TIAN L J,ZHANG G Q,et al.Investigation and epidemiological survey on chicken Salmonella in Shanxi province[J].Journal of Shanxi Agricultural Sciences,2010,38(9):58-62.(in Chinese)
[26] 陈培荣,刘锦妮,何斌.皖北地区禽源性沙门氏菌分离鉴定及耐药性研究[J].湖北农业科学,2013,52(17):4161-4163. CHEN P R,LIU J N,HE B.Isolation,identification and drug resistance of fowl Salmonella in Northern Anhui[J].Hubei Agricultural Sciences,2013,52(17):4161-4163.(in Chinese)
[27] 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)
[28] 施开创,李凤梅,邹联斌,等.鸡源致病性沙门氏菌的分离鉴定及血清型和药物敏感性分析[J].中国畜牧兽医,2015,42(8):2160-2168. SHI K C,LI F M,ZOU L B,et al.Isolation,identification and analysis of serotype and antibiotic susceptibility of pathogenic Salmonella from chickens[J].China Animal Husbandry & Veterinary Medicine,2015,42(8):2160-2168.(in Chinese)
[29] 段星星,曾晓琮,周露.沙门氏菌检验及鉴定方法研究进展[J].食品安全质量检测学报,2020,11(16):5364-5370. DUAN X X,ZENG X Z,ZHOU L.Research progress on the detection and identification methods of Salmonella[J].Journal of Food Safety & Quality,2020,11(16):5364-5370.(in Chinese)
[30] 李玉燕,巩新民,祝永华,等.鸡白痢、鼠伤寒和肠炎沙门氏菌在不同鉴别培养基上的形态[J].家禽科学,2019,7(7):42-44. LI Y Y,GONG X M,ZHU Y H,et al.Morphology of Salmonella Pullorum,Typhimurium and Enteritis on different media[J].Poultry Science,2019,7(7):42-44.(in Chinese)
[31] 赵玮,郭龙宗,张孝兰,等.有效选择沙门氏菌培养基[J].家禽科学,2020,3(3):46-48. ZHAO W,GUO L Z,ZHANG X L,et al.Effective selection of Salmonella culture medium[J].Poultry Science,2020,3(3):46-48.(in Chinese)
[32] 黄秀梅,盖文燕,曲志娜,等.市售猪肉中沙门氏菌耐药基因与毒力基因筛查及PFGE分型研究[J].中国动物检疫,2018,35(11):81-86. HUANG X M,GAI W Y,QU Z N,et al.Screening of drug-resistance genes and virulence genes and PFGE typing of Salmonella from retail pork products[J].China Animal Health Inspection,2018,35(11):81-86.(in Chinese)