北美水貂源沙门氏菌的分离鉴定及耐药性检测

为调查秦皇岛地区北美水貂沙门氏菌的检出率及耐药情况,对65份水貂样品中的细菌进行分离纯化,采用细菌的鉴定培养、革兰氏染色、生化鉴定和16S rDNA鉴定分离菌,采用小鼠致病性试验研究分离菌株的致病性,采用KB纸片法检测分离菌的耐药性。结果显示,经鉴定共计分离出9株沙门氏菌,分离率13.85%(9/65);9株沙门氏菌均可致小鼠发病死亡,死亡率20%—100%;9株沙门氏菌分别对5—11种抗生素药物耐药,氟苯尼考、替米考星、磺胺间甲氧嘧啶、磺胺二甲氧嘧啶和复方新诺明的耐药率高达100%,土霉素敏感率最高,为88.89%(8/9)。上述研究结果表明,秦皇岛地区从水貂分离到的沙门氏菌均具有一定的致病力和多重耐药性,为该地区水貂沙门氏菌病的防治提供参考。     

沙门菌19个毒力岛标志性基因的PCR检测

建立沙门菌19个毒力岛标志性基因检测方法,了解沙门菌中毒力岛的分布情况。通过比对验证挑选出45个毒力基因作为19个毒力岛标志性基因,建立PCR检测方法,并对伤寒、肠炎、鼠伤寒及贝坎道夫沙门菌进行分布情况研究。19个毒力岛上45个标志性基因分别在伤寒、肠炎、鼠伤寒及贝坎道夫沙门菌得到验证,PCR扩增产物与预期扩增产物一致,电泳条带典型,无非特异扩增条带及杂带出现。45个标志性基因在伤寒、肠炎、鼠伤寒及贝坎道夫沙门菌检出率分别为75.56%、75.56%、66.67%和42.22%。本研究建立的45个毒力岛标志性基因检测方法具有结果准确、简单快速、费用低廉等优点,适用于沙门菌毒力岛研究及不同来源的沙门菌致病性风险评估。     

4株鸽源沙门氏菌致病性观察

为了解分离自四川地区的鸽源沙门氏菌的致病性,本研究先将分离的4株沙门氏菌,腹腔注射SPF昆明鼠,测定其半数致死量(LD50),再依据小鼠攻毒试验结果,选取致病性较强的纽波特沙门氏菌菌株,通过灌胃方式接种信鸽来观察对信鸽的致病性.结果显示:甲型副伤寒沙门氏菌、纽波特沙门氏菌、伯里沙门氏菌、基桑加尼沙门氏菌的LD50分别为3.37×10^6、2.49×10^7、5.46×10^7、3.58×107 CFU;感染鼠的肝脏、脾脏、肾脏及小肠有明显的炎性病变;纽波特沙门氏菌可造成感染鸽的肝脏、肺脏、肾脏和胰腺纤维素样变性及炎性细胞浸润等病变,回肠、空肠、盲肠的肠绒毛结构不完整、绒毛上皮脱落,绒毛及肌层有不同程度的炎性细胞浸润.以上结果表明,4株分离的沙门氏菌对试验鼠有一定的致病性,纽波特沙门氏菌可造成信鸽气囊等组织器官损伤,继而影响其飞行能力.本研究为鸽源沙门氏菌尤其是纽波特沙门氏菌的致病性研究提供一定的理论参考,具有公共卫生学意义.     

Detection of Resistance and Resistance Genes of Salmonella from Swine in Xinjiang

The objective of this study was to investigate the resistance of Salmonella and prevalence of resistance genes isolated from a pig farm in Xinjiang, and their coexistence with the major β-lactamases, 16S rRNA methylation enzyme genes and PMQR. The minimum inhibitory concentrations of Salmonella isolated from the pig farms were determined by agar dilution method, PCR was used to detect bla_TEM, bla_CMY-2, bla_CTX-M, bla_LAP-1, bla_KPC, bla_OXA and bla_SHV genes, 16S rRNA methylation enzyme genes armA and rmtB, and PMQR including qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB and aac(6')-Ib genes. The positive strains were performed by using DNA sequencing to determine the purpose of the belt. The result showed that resistant rate of Salmonella isolates from swine were highest to ciprofloxacin and apramycin sulfate (77.9%, 102/131). Resistant mainly based on 8 kinds of drug resistance (29.0%, 38/131), 11 kinds of resistance genes were detection, the coexistence of different genotypes had 24 types. The main types were bla_TEM+bla_OXA+qnrS+aac(6')-Ib-cr+oqxA+oqxB (27.6%, 32/116), bla_TEM+bla_OXA+qnrS+oqxA+oqxB (24.1%, 28/116) and bla_TEM+qnrS (18.0%, 21/116). The Salmonella isolated from the farm had phenomenon seriously resistance, it coexisted with the main β-lactamase, 16S rRNA methylation enzyme genes and PMQR factors. The result suggested that it should strengthen monitoring to the β-lactamase enzymes, 16S rRNA methylation enzyme genes and PMQR factors.     

新疆猪源沙门氏菌耐药性及耐药基因检测

为了解新疆某规模化养殖场猪源沙门氏菌对临床上常用抗菌药物的耐药情况,以及β-内酰胺酶、16S rRNA甲基化酶和质粒介导的喹诺酮耐药（plasmid mediated quinolone resistance,PMQR）基因的流行情况,本试验通过琼脂稀释法对分离的菌株进行最小抑菌浓度测定,PCR方法进行β-内酰胺酶bla_TEM、bla_CMY-2、bla_CTX-M、bla_LAP-1、bla_KPC、bla_OXA和bla_SHV基因,16S rRNA甲基化酶基因armA和rmtB及PMQR类基因qnrA、qnrB、qnrC、qnrD、qnrS、qepA、oqxA、oqxB和aac（6'）-Ib-cr的检测,确定阳性菌株,分析其携带的基因型与耐药表型之间的关系。分离的猪源沙门氏菌对环丙沙星和安普霉素耐药率最高,均为77.9%（102/131）,耐药菌主要以8耐为主（29.0%,38/131）。从被检基因中检测出11种耐药基因,不同基因共存有24种类型,主要以bla_TEM+bla_OXA+qnrS+aac（6'）-Ib-cr+oqxA+oqxB（27.6%,32/116）;bla_TEM+bla_OXA+qnrS+oqxA+oqxB（24.1%,28/116）;bla_TEM+qnrS（18.0%,21/116）形式共存。该养殖场分离的沙门氏菌耐药现象严重,分离耐药菌株存在β-内酰胺酶、16S rRNA甲基化酶和PMQR类基因共存现象,提示应加强对β-内酰胺酶、16S rRNA甲基化酶和PMQR类因子的监控。     

贵州矮马鼠伤寒沙门氏菌的分离鉴定及其致病力研究

为了探究贵州矮马的腹泻病原,试验以腹泻粪样为材料进行病原菌的分离鉴定。经SS和XLD培养基筛选,得到1株菌株S6,经革兰氏染色、生理生化检验以及16S rRNA基因序列比对分析,S6菌株可在SS和XLD琼脂培养基表面生长,革兰氏染色呈阴性;生理生化检验结果与沙门氏菌的生化特性相符;16S rRNA基因序列与已知的鼠伤寒沙门氏菌ATCC 13311（登录号：NR_119108）的亲缘关系最近,序列同源性为99％。形态学和分子系统学分析表明,该菌株为鼠伤寒沙门氏菌。采用PCR方法从S6菌株基因组中得到侵袭蛋白A（invasion protein A,invA）基因,与已知基因序列有6~8 bp不一致,编码的氨基酸发生了一个替换。通过改良寇氏法测定S6菌株对小鼠的半数致死量（LD₅₀）为4.71×10² CFU,属于强致病力菌株。推测S6 invA基因的变异可能与菌株的毒力强弱有关。贵州矮马群体中沙门氏菌的检出率为57％。本研究提示贵州矮马的腹泻病原之一可能是强毒力的鼠伤寒沙门氏菌。     

Isolation,Identification and Pathogenicity of Salmonella typhimurium from Guizhou Pony

To investigate the reason of the diarrhea in Guizhou pony,we used the feces of pony as experimental material to isolate and detect pathogenic bacteria.S6 strain was isolated from SS and XLD medium,and identified using Gram staining,biochemical tests and molecular phylogeny methods.The results showed that S6 strain could growth on SS and XLD medium,and the Gram staining was negative.Biochemical test suggested that its phenotype features were accordance with Salmonella.The 16S rRNA gene sequence of S6 strain was determined in a nucleotide sequence identity of 99% with Salmonella typhimurium ATCC 13311 (NR_119108).Based on the morphological and molecular phylogenetic results,the strain was identified as Salmonella typhimurium S6 strain.It was virulent to mice with the median lethal dose (LD₅₀) of 4.71×10² CFU.Then,we amplified the invasion protein A (invA) gene by PCR method.The invA gene isolated from S6 strain contained 6 to 8 bp different from the known gene,which resulted in only one amino acid substitution.The mutant sites of invA gene might attribute to the pathogenicity of S6 strain.The detection rate of Salmonella was 57% in Guizhou pony population.It was inferred that the diarrhea in Guizhou pony might be caused by virulent Salmonella typhimurium.     

沙门菌Ⅲ型分泌系统研究进展

沙门菌是一种重要的人兽共患肠道病原菌,在引起人类食物中毒的微生物中常列榜首。沙门菌的致病性与其Ⅲ型分泌系统(T3SS)密切相关。沙门菌能够利用T3SS选择性分泌效应蛋白至感染细胞的胞质中,在较短的时间内通过调节宿主细胞的功能,完成细菌的感染和胞内定殖过程,从而逃避机体的清除作用。沙门菌T3SS的作用机制研究对于沙门菌病的防治具有重要意义,论文主要对沙门菌T3SS的结构、组装及作用机理做一综述,以期为沙门菌病的防控提供参考。     

鸡白痢、鸡伤寒沙门菌分子分型方法的验证

为验证鸡白痢、鸡伤寒沙门菌分子分型方法的准确性,本研究以3株鸡白痢、鸡伤寒沙门菌国际参考菌株和306株国内分离株为研究对象,采用5种已知的分子分型方法开展验证性实验.结果发现,基于特定基因可变区的2种分型方法具有良好的特异性,其检测结果与生化分型结果一致,而3种基于特定基因单核苷酸多态性的分型方法则出现假阳性或假阴性的...     

林芝地区一例鸡沙门氏菌的分离与鉴定

2009年5月,林芝地区某个体养鸡户的鸡发病死亡。通过剖检病死鸡、病原菌的分离培养、生化试验、致病性试验和血清学试验等,鉴定出病料中含有鸡沙门氏菌,结合临床表现诊断为鸡白痢。     

屠宰猪肠道沙门氏菌的分离鉴定、耐药性分析及致病性试验

为了解广西南宁市猪源沙门氏菌的污染状况、耐药状况及致病力情况,在南宁市某生猪屠宰场随机直接从131头屠宰猪的肠道采集样品,采用鉴别培养基分离,生化鉴定的方法对样品中的沙门氏菌进行分离鉴定,并采用标准K-B纸片法对分离菌株进行25种抗生素敏感试验,最后对分离株进行小白鼠致病性试验。结果从131份屠宰猪的肠道中共分离到沙门氏菌45株,检出率为34.35%;其中鼠伤寒沙门氏菌14株,甲型副伤寒杆菌2株,肠炎沙门氏菌3株。45株分离菌株全部耐药,耐药率高达100%,其中44株为多重耐药菌株,占97.78%。45株沙门氏菌中有40株对小白鼠具有致病性,致病率达88.89%。这表明南宁市的屠宰猪存在一定程度的沙门氏菌污染,并且分离菌株存在较严重的耐药现象以及具有较强的致病性。应采取有效措施控制沙门氏菌在猪群中的污染和限制抗生素在养猪过程中的使用并严格遵守休药期,以减少细菌耐药性的产生,保障猪肉及猪肉制品的食品安全。     

植物乳酸杆菌对感染肠炎沙门氏菌蛋鸡生产性能、蛋品质及血浆生化指标的影响

为研究植物乳酸杆菌对感染肠炎沙门氏菌蛋鸡生产性能、蛋品质及血浆生化指标的影响,本试验选用400只沙门氏菌阴性蛋鸡,随机分成4个处理组,每处理组5个重复,每个重复20只试鸡。试验第1周,对照组（T1、T3组）饲喂基础日粮,试验组（T2、T4组）在基础日粮添加2×10⁸ CFU/g植物乳酸杆菌。饲喂1周后,T3、T4组试鸡连续2 d灌服1 mL肠炎沙门氏菌悬浮液（1.0×10⁸ CFU/mL）,T1、T2组试验鸡口服等量无菌PBS溶液。结果显示：①蛋鸡感染肠炎沙门氏菌后可极显著降低产蛋率（P_SE=0.02）及平均日采食量（P_SE=0.01）。添加植物乳酸杆菌可显著提高产蛋率（P_LP=0.02）,显著降低料蛋比（P_LP=0.04）。②蛋鸡感染肠炎沙门氏菌后,添加植物乳酸杆菌可显著增加蛋壳厚度（P_LP=0.01）和蛋壳强度（P_LP=0.02）,显著降低蛋黄颜色（P_LP=0.02）。③蛋鸡感染肠炎沙门氏菌后可极显著提高蛋鸡血浆中碱性磷酸酶（ALP）含量（P_SE<0.01）,植物乳酸杆菌干预可显著提高谷草转氨酶（AST）（P_LP=0.02）、钙（P_LP=0.04）和磷（P_LP=0.04）的含量。综上所述,植物乳酸杆菌可显著改善蛋鸡生产性能和蛋品质,维持蛋鸡生理生化指标稳定,同时可缓解沙门氏菌带来的病理损伤,保障蛋鸡机体健康。     

腹泻仔猪肠道致病性大肠埃希氏菌和沙门氏菌的分离鉴定与耐药性研究

为探明仔猪细菌性腹泻肠道致病性大肠埃希氏菌和沙门氏菌流行的血清型、耐药表型及耐药基因型,本试验采集了贵州省5个地（州）市7个规模化养猪场的128份腹泻仔猪肠道样本,并对采集的样本进行了大肠埃希氏菌和沙门氏菌分离与鉴定,通过动物试验鉴定菌株的致病性,利用血清学方法鉴定其血清型,并通过药敏纸片法对主要致病菌进行耐药性研究,采用PCR技术检测各致病菌株耐药相关基因,分析细菌耐药表型和耐药基因型相关性。结果显示,本研究共分离鉴定到78株致病性大肠埃希氏菌与21株沙门氏菌,致病性大肠埃希氏菌血清型以O138、O87为主,沙门氏菌血清型以鼠伤寒沙门氏菌、肠炎沙门氏菌居多;药敏试验结果表明,本试验分离到的78株致病性大肠埃希氏菌对β-内酰胺类药物耐药率达80%以上,对其他种类的抗菌药耐药率均超过40%,分离鉴定的21株沙门氏菌对氨基糖苷类药物耐药率达50%以上,对其他种类的抗菌药耐药率均达20%以上;本试验分离鉴定的致病性大肠埃希氏菌共检出12种耐药相关基因,沙门氏菌共检出10种耐药相关基因,两种细菌耐药基因型与耐药表型符合率均达60%以上,且均为多重耐药。本研究为仔猪腹泻的综合防控提供了理论依据。     

Isolation,Identification and Drug Resistance Analysis of Interstinal Pathogenic Escherichia coli and Salmonella Isolated from Diarrhea Piglets

In order to find out the serotype, resistant phenotype and genotype of Escherichia coli (E. coli) and Salmonella in piglets, this study collected 128 samples of diarrhea piglets from seven large-scale pig farms in five cities in Guizhou province, and the E. coli and Salmonella were isolated and identified. The pathogenicity of the strain was identified by animal test. The drug resistance of the main pathogen was tested by drug susceptibility paper. The resistance gene of each pathogen was detected by PCR. The drug resistance and genotype correlation of the bacterial were analyzed. The results showed that 78 strains of pathogenic E. coli and 21 strains of Salmonella were isolated and identified in this study. The serotypes of pathogenic E. coli were predominantly O138 and O87. Salmonella Typhimurium and Salmonella Enteritidis were predominant serotypes. The susceptibility test showed that the resistant strains of 78 strains of E. coli were more than 80% resistant to β-lactams and more than 40% for other antibacterials. The resistance rate of 21 strains of Salmonella to aminoglycosides was more than 50% and more than 20% to other types of antibacterials; 12 and 10 kinds of drug resistance-related genes of E. coli and Salmonella were detected, respectively; The coincidence rate of resistant genotype and phenotype of two kinds of bacteria were above 60%, and both were multiple drug resistance. This study provided a theoretical basis for comprehensive prevention and control of piglets diarrhea.     

饲料中沙门氏菌的检测

本实验通过常规分离培养鉴定技术,对某饲料厂随机抽取的30份鱼粉样品进行了沙门氏菌的分离鉴定;结果从这些份样品中分离到四株沙门氏菌菌株,分别为鼠伤寒沙门氏菌2株,鸭沙门氏菌1株,肠炎沙门氏菌1株,阳性率为13.3%。     

表达绿色荧光蛋白的重组减毒鼠伤寒沙门氏菌的构建与鉴定

应用DNA重组技术构建了表达绿色荧光蛋白（GFP）的重组减毒鼠伤寒沙门氏菌。在体外稳定试验中，重组菌经LB固体及液体培养基连续传15代后，单个菌落和菌液均呈绿色；在体内稳定试验中，经ICR小鼠连续传代10次，从肝脏、脾脏中分离的细菌经LB固体培养仍为绿色，证明构建的重组减毒鼠伤寒沙门氏菌是稳定的。SDS-PAGE结果显示，鼠伤寒沙门氏菌表达的GFP分子量约为27kD，这与预计的分子量大小一致。用免疫剂量的重组细菌接种BALB/C小鼠后，观察1个月未见有异常现象，同时剖栓后也未见脏吕有眼观病变。表达GFP的重组鼠伤寒沙门氏菌的构建为减毒沙门氏菌作为活载体的基因工程疫苗的研制提供一个优良模型，为研究沙门氏菌疫苗和沙门氏菌疾病的致病机理的提供了一个有力的工具，同时在相关疾病的免疫防制基础研究中亦有重要应用价值。     

华中地区鸡源沙门菌分离鉴定及耐药性分析

为了探明华中地区种鸡场沙门菌(Salmonella)的优势血清型和耐药情况,本研究从湖北、河南、湖南等省市22个规模化鸡场采集病鸡、死胚及弱雏组织样品3 724份,通过分离培养、生化试验、PCR鉴定及血清型试验确定分离菌种属及其血清型,并采用Kirby-Bauer法对分离菌株进行了耐药性分析。结果显示,本试验从3 724份病料中共分离鉴定出124株沙门菌,其中79株为D群肠炎沙门菌(63.71%,79/124),34株为D群鸡白痢沙门菌(27.42%,34/124),8株为B群鼠伤寒沙门菌(6.45%,8/124),有3株沙门菌未能确定血清型。O抗原鉴定79株肠炎沙门菌和34株鸡白痢沙门菌为O9,8株鼠伤寒沙门菌为O4。H抗原鉴定79株肠炎沙门菌为Hg,m,8株鼠伤寒沙门菌为Hi。药敏试验结果显示,124株分离菌株对萘啶酸、氨苄青霉素、四环素和多西环素耐药率分别为95.97%(119/124)、91.94%(114/124)、57.26%(71/124)和70.16%(87/124);对复方新诺明和红霉素耐药率分别为25.81%(32/124)和12.10%(15/124);对氯霉素、庆大霉素、头孢噻肟和卡那霉素耐药率分别为6.45%(8/124)、1.61%(2/124)、1.61%(2/124)和0.81%(1/124);对左氧氟沙星、阿米卡星和多黏菌素B完全敏感。99.19%(123/124)的分离株至少对一种药物耐药,87.10%(108/124)的分离株表现多重耐药。本研究为华中地区养鸡场沙门菌的诊断及防控提供了数据支撑。     

乌鲁木齐地区不同健康状态和饲养环境中宠物源沙门菌耐药特征分析

为探究不同健康状态和不同饲养环境下沙门菌对临床常用抗菌药物的耐药情况及耐药基因携带情况,采用琼脂稀释法对健康与患病、家养与流浪宠物粪样中分离出的99株沙门菌进行临床常用12种抗菌药物的耐药性检测;用PCR方法对获得的耐药菌株进行相关耐药基因的检测。结果:分离出的宠物源沙门菌对所有被检的抗菌药物耐药率均未超过10.0%,对头孢噻呋、安普霉素、阿米卡星、卡那霉素和硫酸庆大霉素100%敏感;患病动物源沙门菌对被检抗菌药物的耐药率高于健康动物源沙门菌,家养动物源沙门菌对被检抗菌药物的耐药率高于流浪动物源沙门菌(P<0.05)。耐药基因检测结果显示:floR的检出率为3.0%;ant(3″)-Ia、tetA、tetB检出率均为2.0%;blaTEM、oqxA、aac(6′)-Ib-cr基因检出率均为1.0%,未检出其他被检基因。结果表明,新疆乌鲁木齐市不同健康状态和不同饲养环境下宠物源沙门菌对被检抗菌药物的耐药率及耐药基因携带率低,仅有2株沙门菌存在ant(3″)-Ia+tetA+tetB 3种耐药基因共存情况。根据试验结果发现检出的耐药表型与耐药基因之间存在相关性,可根据药敏试验结果合理选用抗菌药物进行临床细菌病的治疗。     

猪霍乱沙门菌胞外产物的核酸酶活性及其对巨噬细胞胞外诱捕网形成的影响

旨在探究猪霍乱沙门菌胞外产物的核酸酶活性及其对巨噬细胞胞外诱捕网形成的影响。采用平板培养法、琼脂糖凝胶电泳法和琼脂扩散法观察猪霍乱沙门菌胞外产物降解λDNA的活性，同时，分析不同温度、pH和金属离子对胞外产物核酸酶活性的影响以及胞外产物对巨噬细胞胞外诱捕网形成的影响。猪霍乱沙门菌胞外产物具有降解λDNA的DNA酶活性，高温和酸性条件抑制DNA酶的活性，活性最适温度和pH分别为42℃和9.0，且胞外产物的核酸酶可被60℃以上的温度灭活。添加Na⁺、K⁺、Ca²⁺和Ni²⁺对胞外产物切割λDNA的活性没有影响；5.00和10.00 mmol·L^-1的Ba²⁺、Mg²⁺和Mn²⁺可提高胞外产物的DNA酶活性；0.01~1.00 mmol·L^-1的Zn²⁺、Cu²⁺和Fe³⁺对胞外产物的DNA酶活性具有促进作用，但Co²⁺则表现出抑制作用。荧光显微镜和定量分析均显示，猪霍乱沙门菌不能刺激巨噬细胞释放胞外诱捕网，而猪霍乱沙门菌胞外产物可显著降解白色念珠菌刺激巨噬细胞形成的胞外诱捕网。本研究证实了猪霍乱沙门菌胞外产物的DNA酶活性，为进一步探究胞外产物在猪霍乱沙门菌感染与宿主免疫互作中的作用提供参考。     

Effect of organic acids in drinking water during the last 2 weeks prior to slaughter on Salmonella shedding by slaughter pigs and contamination of carcasses

In this study, we investigated the effect of adding organic acids to the drinking water of finishing pigs 2 weeks prior to slaughter on the shedding and prevalence rate of Salmonella at slaughter. Approximately 600 animals from four Belgian pig herds infected with Salmonella were included. At two herds, the study was conducted twice. Before the start of the study, overshoes were taken at the different herds. Two weeks prior to the expected slaughter date, the pigs were randomly divided into two groups (treatment and control group) each containing on average 50 animals within each herd. The treatment group received from this day onwards acidified drinking water (pH = 3.6-4.0), the control group received non-treated water (pH = 7.8-8.5). All other housing, feeding and management factors were identical in both groups. At the slaughterhouse, 10 pigs of each group (20 pigs for each group of study group 6) were randomly selected and sampled (blood, contents of ileum and rectum, mesenteric lymph nodes and carcass swabs). All samples were immediately transported to the laboratory and submitted to Salmonella isolation. Salmonella was isolated out of 11.9% (66/554) of the samples taken at the slaughterhouse, with the highest frequency found in the content of the ileum (18.7%), followed by 17.8% in the lymph nodes, 7.2% in the content of the rectum and 3.6% in the carcass swabs. The results did not reveal a significant difference between the treatment and control groups for the different slaughterhouse samples. The study documented that the investigated control strategy namely, the strategic application of organic acids during the last 2 weeks prior to slaughter was insufficient to decrease Salmonella shedding and contamination shortly before and during slaughter.