鸡蛋源沙门菌的分离鉴定及其耐药基因和毒力基因的检测
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摘要
沙门菌病是公共卫生学上具有重要意义的人畜共患病之一。鸡是沙门菌的最重要宿主，沙门菌可引起鸡急性或慢性疾病，感染沙门菌的鸡也可通过产出带菌的蛋导致沙门菌的垂直传播，从而形成恶性循环，当鸡群暴发沙门菌病时死亡率可达80%，对养禽业造成巨大的经济损失。人一旦摄入了含有大量沙门菌的动物性食品，可导致食源性中毒，在我国细菌性食物中毒中，有70％-80％是由沙门菌引起的，沙门菌作为禽蛋中最常见的母源或污染性细菌，已经成为重要的食源性微生物。因此，对鸡蛋中沙门菌进行检测在兽医公共卫生及食品卫生方面有着重要的意义。
1.本试验从陕西省6个不同地市的孵化场中随机采集1200份鸡蛋的蛋壳膜，利用细菌学常规分离技术、生化实验、免疫学试验及PCR方法共鉴定沙门菌60株，检出率为5.0%.
2.设计13对特异性引物对60株分离菌的耐药基因进行检测，共检测出7种耐药基因，其中flor的检出率高达100%，BlaCMY-2及tetA的检出率均高于60%。分离株药物敏感性试验显示，菌株对氨苄西林、甲氧苄啶、头孢噻吩、萘啶酸、环丙沙星、氯霉素、卡那霉素和四环素的耐药率较高，对阿莫西林、庆大霉素、诺氟沙星、妥布霉素、链霉素、氧氟沙星、头孢噻肟、头孢他啶和阿米卡星的耐药率较低，对头孢噻肟、头孢他啶和阿米卡敏感，且菌株的耐药性以三重以上耐药为主，比例高达98.4%，耐药最多的分离株可耐12种抗生素，但耐药表型与耐药基因的携带之间无显著相关性。
3.对分离株进行动物致病性试验显示，强致病性菌株有14株，占23.3%。设计5对特异性引物对沙门菌毒力岛核心蛋白基因进行检测，PCR扩增结果显示，分离株毒力基因携带情况SPI-1为60%、SPI-2为73.3%、SPI-3为100%、SPI-4为90%、SPI-5为76.7%，分析发现，毒力基因SPI-1+SPI-2组合与沙门菌致病力呈正相关，且携带SPI-1菌株的致病性略高于携带SPI-2的菌株，携带SPI-4、SPI-5毒力岛基因与菌株的致病性无显著相关性。
Salmonella infection is one of the sciences of public health significance of zoonoticdisease. Chicken is the most important host of Salmonella, any type of Salmonella can causethe chicken acute or chronic diseases. Chickens infected with Salmonella can also infect eggswhich cause vertical transmission of salmonella, thus it creates a vicious cycle. When theflock of Salmonella infection outbreaks,80%chickens will die, causing huge economiclosses to the poultry industry. Once the intake of animal foods contains a large number ofSalmonella, it can cause foodborne poisoning. Based on statistics,70％～80％of the bacterialfood poisonings were due to Salmonella in China. As the most common source of pollution ofthe eggs, salmonella has become an important foodborne micro-organism. Therefore, thedetection of Salmonella in eggs was of great significance in Food Safety and Public Health．
1.1200egg were collected in the hatchery from6different cities in Shaanxi Province,under sterile conditions, the eggshell membranes were collected and cultured, using themethod of Salmonella enrichment medium for isolating and identify Salmonella strains. Thebacteriology conventional separation techniques, biochemical tests and immunological testswere needed. The result shows that60Salmonella strains were isolated, and the rate for theisolation was5.0%.
2. According to the sequence of drug resistance genes published by GenBank, whichdesigned13pairs of the PCR primers to enlarge and detect the drug resistance genes,7kindsof drug resistance genes were detected, where the carried rate of flor were100%andBlaCMY-2were higher than60%. The drug sensitivity test shows that the60isolated strainshave high resistance rate to trimethoprim, cephalosporins, nalidixic acid, ciprofloxacin,chloramphenicol, kanamycin and tetracycline, but the resistance rate was lower toAmoxicillin, gentamicin, norfloxacin, tobramycin, streptomycin, ofloxacin, cefotaxime,ceftazidime and amikacin. The isolates that resistant to more than3kinds of antimicrobialsaccounted for98.4%. The most severe isolated strainsc can resistant to12kinds ofantimicrobials, but there was no absolute correlation between resistance phenotype and theresistance gene carried.
3. According to the animal pathogenesis test, there were14strains that have strong pathogenic among the60Salmonella strains which took a part of23.3%. According to thesequence of SPI genes published by GenBank,5pairs of the PCR primers were designed toenlarge and detect the core protein of the SPI genes.The results show that the carried rate ofthe SPI genes from the isolated strains indicated that SPI-1were60%, SPI-2were73.3%,SPI-3were100%, SPI-4were90%, and that SPI-5were76.7%, then an analysis also hasdone which knows the isolates with the genes of SPI-1and SPI-2have more intimaterelationship between the pathogenesis and the SPI carried. The pathogenic of strains whichcarried SPI-1genes was higher than strains which carried SPI-2genes, but the in carrying ofSPI-4and SPI-5there was no significant correlation between the pathogenesis and the SPIcarried.
1.本试验从陕西省6个不同地市的孵化场中随机采集1200份鸡蛋的蛋壳膜，利用细菌学常规分离技术、生化实验、免疫学试验及PCR方法共鉴定沙门菌60株，检出率为5.0%.
2.设计13对特异性引物对60株分离菌的耐药基因进行检测，共检测出7种耐药基因，其中flor的检出率高达100%，BlaCMY-2及tetA的检出率均高于60%。分离株药物敏感性试验显示，菌株对氨苄西林、甲氧苄啶、头孢噻吩、萘啶酸、环丙沙星、氯霉素、卡那霉素和四环素的耐药率较高，对阿莫西林、庆大霉素、诺氟沙星、妥布霉素、链霉素、氧氟沙星、头孢噻肟、头孢他啶和阿米卡星的耐药率较低，对头孢噻肟、头孢他啶和阿米卡敏感，且菌株的耐药性以三重以上耐药为主，比例高达98.4%，耐药最多的分离株可耐12种抗生素，但耐药表型与耐药基因的携带之间无显著相关性。
3.对分离株进行动物致病性试验显示，强致病性菌株有14株，占23.3%。设计5对特异性引物对沙门菌毒力岛核心蛋白基因进行检测，PCR扩增结果显示，分离株毒力基因携带情况SPI-1为60%、SPI-2为73.3%、SPI-3为100%、SPI-4为90%、SPI-5为76.7%，分析发现，毒力基因SPI-1+SPI-2组合与沙门菌致病力呈正相关，且携带SPI-1菌株的致病性略高于携带SPI-2的菌株，携带SPI-4、SPI-5毒力岛基因与菌株的致病性无显著相关性。
Salmonella infection is one of the sciences of public health significance of zoonoticdisease. Chicken is the most important host of Salmonella, any type of Salmonella can causethe chicken acute or chronic diseases. Chickens infected with Salmonella can also infect eggswhich cause vertical transmission of salmonella, thus it creates a vicious cycle. When theflock of Salmonella infection outbreaks,80%chickens will die, causing huge economiclosses to the poultry industry. Once the intake of animal foods contains a large number ofSalmonella, it can cause foodborne poisoning. Based on statistics,70％～80％of the bacterialfood poisonings were due to Salmonella in China. As the most common source of pollution ofthe eggs, salmonella has become an important foodborne micro-organism. Therefore, thedetection of Salmonella in eggs was of great significance in Food Safety and Public Health．
1.1200egg were collected in the hatchery from6different cities in Shaanxi Province,under sterile conditions, the eggshell membranes were collected and cultured, using themethod of Salmonella enrichment medium for isolating and identify Salmonella strains. Thebacteriology conventional separation techniques, biochemical tests and immunological testswere needed. The result shows that60Salmonella strains were isolated, and the rate for theisolation was5.0%.
2. According to the sequence of drug resistance genes published by GenBank, whichdesigned13pairs of the PCR primers to enlarge and detect the drug resistance genes,7kindsof drug resistance genes were detected, where the carried rate of flor were100%andBlaCMY-2were higher than60%. The drug sensitivity test shows that the60isolated strainshave high resistance rate to trimethoprim, cephalosporins, nalidixic acid, ciprofloxacin,chloramphenicol, kanamycin and tetracycline, but the resistance rate was lower toAmoxicillin, gentamicin, norfloxacin, tobramycin, streptomycin, ofloxacin, cefotaxime,ceftazidime and amikacin. The isolates that resistant to more than3kinds of antimicrobialsaccounted for98.4%. The most severe isolated strainsc can resistant to12kinds ofantimicrobials, but there was no absolute correlation between resistance phenotype and theresistance gene carried.
3. According to the animal pathogenesis test, there were14strains that have strong pathogenic among the60Salmonella strains which took a part of23.3%. According to thesequence of SPI genes published by GenBank,5pairs of the PCR primers were designed toenlarge and detect the core protein of the SPI genes.The results show that the carried rate ofthe SPI genes from the isolated strains indicated that SPI-1were60%, SPI-2were73.3%,SPI-3were100%, SPI-4were90%, and that SPI-5were76.7%, then an analysis also hasdone which knows the isolates with the genes of SPI-1and SPI-2have more intimaterelationship between the pathogenesis and the SPI carried. The pathogenic of strains whichcarried SPI-1genes was higher than strains which carried SPI-2genes, but the in carrying ofSPI-4and SPI-5there was no significant correlation between the pathogenesis and the SPIcarried.
引文
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