山东省鸡源沙门氏菌的分离鉴定及毒力基因分析

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。

Isolation,Identification and Virulence Gene Analysis of Salmonella from Chickens in Shandong Province

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.