反刍动物源产气荚膜梭菌新疆流行株的分离鉴定及分型研究

为了弄清反刍动物源产气荚膜梭菌新疆流行株的生物学特性和分子特征,本研究从新疆不同地区规模化养殖场和屠宰场采集158份牛羊临床病料,进行产气荚膜梭菌的分离培养;通过形态学、生化特性、16S rRNA序列分析对产气荚膜梭菌分离株进行鉴定;利用多重PCR技术扩增产气荚膜梭菌α、β、ε、ι、β2基因,将测序结果与NCBI数据库比对进行新疆流行株的基因分型。结果从158份临床病料中共分离出产气荚膜梭菌67株,形态学、生化和16S rRNA序列分析鉴定结果均为产气荚膜梭菌。毒素基因检测表明,其中α基因检出率为100.00%(67/67),ε基因检出率为17.91%(12/67),β2基因检出率为37.31%(25/67),β、ι基因未检出。毒素基因分型可将产气荚膜梭菌新疆流行株分为A、D型,其中A型占82.09%(55/67),D型占17.91%(12/67),提示产气荚膜梭菌新疆流行株优势型别为A型。本研究为产气荚膜梭菌流行病学研究和防控提供科学依据。     

采用AFLP和ERIC-PCR技术研究规模化鸡场健康鸡群中产气荚膜梭菌的遗传多样性

为了调查产气荚膜梭菌在健康鸡群中的流行现状,掌握我国部分地区规模化鸡场产气荚膜梭菌的遗传多样性,分析其流行特点与地区差异的关系,利用AFLP（amplified fragment length polymorphism）和ERIC-PCR（enterobacterial repetitive intergenic consensus PCR）方法对从四川省8个市10个规模化鸡场分离得到的34株A型产气荚膜梭菌进行基因型分析。结果表明,用AFLP技术将34株菌分为12个亚型,用ERIC-PCR方法分为8个亚型。分析亚型分布情况发现：不同鸡场产气荚膜梭菌的亚型差异明显;而同一鸡场的亚型较简单,以一种亚型为主,交叉有少数其他亚型;优势基因型分别为AFLP基因Ⅷ型或ERIC-PCR基因1型。该研究结果说明：四川省规模化鸡场健康鸡群中产气荚膜梭菌的遗传多样性较低,其流行特点与地区差异相关。     

多重PCR检测产气荚膜梭菌方法的建立及其临床应用

产气荚膜梭菌是牛羊猝死和肠出血症的重要病原之一。本研究根据其常见的A、B、C、D四种菌型菌株编码α、β和ε毒素的保守序列,分别设计合成了针对这3种毒素的特异性引物,建立了多重PCR检测不同血清型产气荚膜梭菌的技术方法。通过对参考菌株的分型检测表明,该方法不仅快速、客观,而且具有良好的特异性和敏感性。对羔羊猝死症中分出的菌株进行检测,确定其为产气荚膜梭菌A型和D型混合感染;对产后母牛血痢病料中分离的菌株进行检测确定其为产气荚膜梭菌A型。     

羊源产气荚膜梭菌不同毒素型多重PCR检测方法的建立及初步应用

根据GenBank中已发布的产气荚膜梭菌α,β,ε,ι毒素基因序列,设计并合成4对特异性引物,经过优化多重PCR反应条件,建立了检测产气荚膜梭菌不同毒素型多重PCR方法。特异性试验表明,该方法对A,B,C,D,E型产气荚膜梭菌标准菌株均扩增出了相应的目的条带,而对诺维氏梭菌和腐败梭菌扩增为阴性;灵敏性试验表明,该方法对A,B,C,D,E型标准菌株基因组DNA最低检测量分别为9.0,17.8,12.2,13.8,18.5pg;重复性试验表明,该方法有很好的重复性。应用所建立的方法从21份羊临床病料中检测出9株A型和1株C型产气荚膜梭菌。本试验建立的多重PCR方法可以进行产气荚膜梭菌的快速检测及5种毒素型的鉴别。     

犬产气荚膜梭菌的分离和PCR检测方法的建立

从12头疑似产气荚膜梭菌感染的猝死警犬中分离获得了18株病原菌，经生化试验及毒素中和试验，确定为A型和C型产气荚膜梭菌。参考GenBank中登录的基因序列，设计合成了针对产气荚膜梭菌α、β、ε、ι、CPE和β2共6对分型毒素基因的引物，对以前昆明地区分离的1株产气荚膜梭菌进行了PCR扩增，结果扩增出了与预期大小相同的6个基因片段，分别为233、196、324、446、567和665 bp。建立的PCR方法能同时用于产气荚膜梭菌鉴定和毒素分型，较细菌毒素检测方法快速，与细菌分离鉴定的结果一致。     

牛出血性肠炎A型产气荚膜梭菌的分离鉴定

从新疆维吾尔自治区某牛场采集的3份疑似出血性肠炎病料中分离到8株产气荚膜梭菌,用PCR扩增保守基因16SrRNA,并进行序列测定和同源性分析,再通过多重PCR方法扩增型特异性基因进行分离菌株的分型鉴定。结果显示,所分离的8株产气荚膜梭菌之间16S rRNA基因同源型为100%,与GenBank参比序列同源性在99.8%以上,确定为产气荚膜梭菌。遗传进化分析表明,本次分离的8株产气荚膜梭菌之间拥有共同起源,但与所用的参考菌株分属不同来源。多重PCR扩增结果显示,8株菌株均为产气荚膜梭菌A型。     

产气荚膜梭菌菌落多重PCR方法的建立及初步应用

根据GenBank中已发布的产气荚膜梭菌α、β、ε、τ毒素基因序列,分别设计并合成针对4种毒素基因的特异引物,通过优化多重PCR反应条件,建立1种简单的产气荚膜梭菌定型菌落多重PCR方法。结果显示：A、B、C、D、E5型产气荚膜梭菌参考菌株均扩增出了相应的预期目的条带,而大肠杆菌、巴氏杆菌和芽孢杆菌则均未能扩增出相应条带;将单个菌落稀释100倍,仍能扩增出相应的目的片段,该方法对B型和E型参考菌株最低检测量分别为2．6×10^4cfu／mL、1．2×10^4cfu／mL。应用该多重PCR方法从106份样品中检测到30株产气荚膜梭菌且均为A型,其中病死鸡的盲肠内容物分离率为36．5％（19／52）,健康鸡群新鲜粪便样品分离率为20．4％（11／54）。本研究建立的多重PCR方法特异性强,敏感度高,重复性好,可以有效进行产气荚膜梭菌的快速检测及5种血清型的鉴别,对产气荚膜梭菌的感染及食品安全问题的研究均具有重要意义。     

甘肃省牛和羊源产气荚膜梭菌耐药性分析

旨在探究甘肃省牛和羊产气荚膜梭菌的流行情况及对常用抗菌药物的耐药性。对采集自甘肃省不同地区的229份牛、羊源肛拭子进行了产气荚膜梭菌的分离鉴定,并对分离到的产气荚膜梭菌菌株进行毒素基因检测、最小抑菌浓度(MIC)测定及四环素耐药菌株的同源性分析。结果表明,在229份样品中分离到53株A型产气荚膜梭菌,分离率达23.1%,其中36株(68%)含cpb2毒素基因;分离菌株对万古霉素、阿莫西林、美罗培南、氯霉素和头孢他啶敏感,对四环素存在13株耐药菌和8株中介菌,且四环素耐药菌株间具有较高的同源性。结果表明,产气荚膜梭菌更易存在于牛的胃肠道环境;牛源产气荚膜梭菌对四环素的耐药性强于羊源产气荚膜梭菌,且四环素耐药菌株在甘肃省不同地区牛和羊之间具有一定程度的传播。     

一例兔产气荚膜梭菌的分离与鉴定

从滨州某兔场送检的病料中分离到1株兔产气荚膜梭菌,经细菌培养、形态学检查、生化特性鉴定、毒素中和试验鉴定为A型产气荚膜梭菌,易感试验兔接种试验能够复制出典型的病例。     

动物源A型产气荚膜梭菌流行情况调查及α毒素基因分析

产气荚膜梭菌是一种重要的人畜共患病原菌,在一定条件下可引起多种严重疾病。为了调查不同动物A型产气荚膜梭菌流行情况及α毒素基因同源性,本试验从不同地区共采集病料307份,其中鸡133份(包括肉鸡112份、蛋鸡21份)、鸭65份、犬31份、猪14份、兔子20份、小鼠9份、牛粪便18份、鸵鸟粪便17份。取肠道内容物和粪便进行产气荚膜梭菌分离鉴定和毒素基因分型,并检测cpe、β2毒素基因携带率;从不同地区、不同动物源A型产气荚膜梭菌分离株挑选18株进行α毒素基因扩增,将所得基因序列进行同源性比较。结果显示,307份样品中68份(22.1%)呈产气荚膜梭菌阳性,不同动物源产气荚膜梭菌阳性率介于5.9%～44.4%;68株产气荚膜梭菌分离株α毒素基因阳性率为100%,所有分离株毒素基因分型均为A型,未检测到cpe毒素基因,β2毒素基因总阳性率为63.2%;分离株与NCBI参考菌株α毒素基因相似性介于97.8%～100%。结果表明,不同动物α毒素具有很高的同源性,本调查为研发A型产气荚膜梭菌α毒素通用疫苗提供数据支持。     

Isolation and characterization of Clostridium perfringens from apparently healthy animals of the Shandong province of China

In a pilot study the presence and frequency of Clostridium (C.) perfringens was investigated among apparently healthy farm animals in the Shandong province of China. 748 faecal samples were collected from 9 pig-, 4 sheep-, 7 cattle- and 5 rabbit farms. C. perfringens was isolated from 124 samples (16.6%). The isolates were classified into major toxin types by using PCR analysis detecting the genes encoding these toxins. All isolates were identified as C perfringens toxin type A. There are also some reports from different regions in China linking C. perfringens toxin type A strains to gastrointestinal diseases. Therefore further investigations about the epidemiologic role of C perfringens toxin type A strains in the Shandong region are necessary. Currently, cases of enterotoxemia from this region are investigated for the presence of C perfringens.     

Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens

The aim of this study was to analyse the genetic diversity among Clostridium perfringens isolates from Danish broiler chickens since both sick and presumably healthy animals were investigated. Isolates (n=279) collected from chickens from 25 farms were analysed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI. A high genetic diversity was found. Isolates with different PFGE types were toxin typed by PCR and all were found to be of type A. The results showed that healthy broiler chickens carried several different C. perfringens clones both within a flock and even within individual birds, whereas flocks suffering from necrotic enteritis (NE) or cholangio-hepatitis carried only one or two clones.     

The necrotizing enteritis by Clostridium perfringens type C in piglets: II. Molecular epidemiology study

Investigations were performed on shedding of C. perfringens in sows from four different pig farms. In two farms where no outbreaks of necrotizing enteritis had been observed, no strains of C. perfringens producing beta-toxin were detected in the faeces of sows. In contrast, C. perfringens strains producing beta-toxin were detected in sows on both farms suffering outbreaks of acute necrotizing enteritis. Strains of C. perfringens producing beta-toxin were invariably positive for the beta 2-toxin gene. However, strains carrying the beta 2-toxin gene only (i.e. negative for beta-toxin) were present in animals on all farms with roughly similar frequencies (mean 28.2% carriers). Some sows carried C. perfringens strains of both toxin genotypes simultaneously. Whereas these data further support the role of betatoxin as a cause of necrotizing enteritis, the role of beta 2-toxin in intestinal disease of piglets remains unclear. To establish the role of faecal shedding vs. environmental contamination as reservoirs of C. perfringens type C, strains were isolated from teats and feedlot trough swabs (toxin genotype beta/beta 2), as well as from fodder (genotype beta 2). However, sows carried this pathogen intermittently and in small numbers. This renders an individual, reliable diagnosis of carrier sows very difficult. Ribotyping of 34 C. perfringens isolates of different toxin genotypes showed five distinct profiles. Different toxin genotypes can belong to the same ribotype, and the same toxin genotype can be present in different ribotypes. Thus, even if a majority (79.4%) of strains investigated in a limited geographic region belonged to ribotype 1, ribotyping offered discrimination of strains beyond toxin typing.     

Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status

Due to the diminished use of growth-promoting antibiotics in the European Union, Clostridium perfringens induced necrotic enteritis and subclinical disease have become important threats to poultry health. A study was set up to genotypically and phenotypically characterise C. perfringens isolates from poultry flocks with different health status. Animals from healthy flocks were sampled by cloacal swabs, while intestinal and liver samples of animals suffering from necrotic enteritis were analysed. A total of 27 isolates was obtained from 23 broiler flocks without clinical problems and 36 isolates were obtained from 8 flocks with clinical problems. Using PFGE typing, high genetic diversity was detected between isolates from different flocks. Isolates derived from flocks where disease outbreaks occurred were clonal within each flock, but each flock harboured a different clone. All isolates were of toxin type A. Isolates from 5 out of 35 PFGE types carried the cpb2 gene, encoding the beta2 toxin, and isolates from 2 out of 35 PFGE types harboured the cpe gene, encoding the enterotoxin. In vitro alpha toxin production for all isolates was quantified by enzyme-linked immunosorbent assay. It was shown that in vitro alpha toxin production of C. perfringens isolates from diseased flocks was not higher than in vitro alpha toxin production from isolates derived from healthy flocks.     

新疆牛源大肠杆菌O157∶H7的分离鉴定及ERIC-PCR基因分型研究

为了了解牛源大肠杆菌（E.coli）O157∶H7在新疆地区的污染状况以及遗传多样性,探究不同地区分离菌株的遗传关系,为控制牛源E.coli O157∶H7的传播提供试验依据。将采集的样品在EC肉汤中进行增菌（37 ℃、180 r/min）,接着将增菌液划线接种到SMAC平板上,37 ℃培养箱中过夜培养18 h左右。挑取SMAC平板上白色或无色单菌落接种MUG培养基,37 ℃培养18 h左右,将无荧光样品接种到SMAC平板上,37 ℃培养18 h左右,隔天挑取白色或无色单菌落进行PCR鉴定,具有rfbE和fliC基因条带的即为阳性菌株。将阳性菌株进行肠杆菌基因间重复共有序列扩增（ERIC-PCR）指纹图谱聚类分析,分析菌株之间的同源性关系。ERIC-PCR结果显示,相似性100%的菌株有3组。从伊犁地区分离到的菌株差异性最大,具有6种分型;其次是乌鲁木齐,具有4种分型。菌株来源多样性最多的在D簇,由此可见通过ERIC-PCR分型,可以进行溯源观察。ERIC-PCR能够区分特定采样点或物种的分离物,它能够证明从不同来源的菌株之间,存在着某些相似的ERIC特性,并聚集在同一个簇群中。该研究中筛选出的E.coli O157∶H7菌株具有广泛的遗传多样性,该方法对于检测不同物种间的细菌差异非常敏感。由此可见ERIC-PCR可以作为E.coli O157∶H7常规监测和鉴定的一个有效的工具。     

奶牛养殖场环境大肠杆菌的ERIC-PCR分型和系统进化分群

旨在了解奶牛养殖场环境中大肠杆菌流行情况及遗传多样性，探究不同样品分离菌株的遗传关系及系统进化分群情况，收集2017—2019年新疆某大型奶牛场养殖环境中的209份样品进行大肠杆菌分离和16S rRNA鉴定，对非重复菌株进行ERIC-PCR分型和系统进化分群试验。结果显示，共分离到338株大肠杆菌，分离率为67.46%。ERIC-PCR将其分为Ⅰ~ⅩⅣ共14型。Ⅳ（196株）型为优势型；其次为Ⅰ型（59株）、Ⅴ（31株）、Ⅹ（11株）；剩余41株分布于其他10种型。除2株未分群外，其他被分为6个群，B1群（75.45%）分布最多，其次是A群（18.34%）、C群（2.96%）、D或E群（1.18%）、F群（0.30%）。综上表明，奶牛养殖场环境中大肠杆菌存在广泛的DNA多样性，且不同时间及来源菌株间存在较近的亲缘关系；系统进化分群以B1群为主。     

家兔“流行性腹胀病”的病因和防治研究初报

对近3年四川发生的兔腹胀病的流行特点、临床症状、剖解病理变化、病因和防治进行了初步的研究。结果,该兔腹胀病主要引起断奶后至3月龄左右的兔发病,发病率一般在30％～60％,死亡率达90％以上．具有一定传染性和区域流行性。病兔出现明显腹胀,胃膨胀,胃内充满食物或水样稀薄的内容物,部分胃粘膜有黑色溃疡斑点,结肠内多充满白色胶冻样半透明状物。从69例病死兔中,分离到38株魏氏梭菌（55％）、13株大肠杆菌和2株沙门氏杆菌,大多数菌株都有致病力。添加霉菌毒素吸附剂或抗球虫药物不能有效控制该病。而使用恩拉霉素和蒽诺沙星可在1周内控制该病的发生。初步的研究结果表明：该病可能是一种细菌性传染病,其病原菌有可能是以魏氏梭菌为主,还有大肠杆菌、沙门氏茵等细菌,为单一或混合感染所致。     

互助藏羊梭菌病病原体的分离鉴定与分析

青海省互助县某羊场藏系绵羊患病死亡,伴有腹泻、神经症状,为快速确诊发病原因,及时防控和治疗,采集病羊样品5份,通过镜检、细菌分离培养、分子生物学试验及致病性试验进行病原鉴定分析。结果证明此次病原菌为羊致病性D型魏氏梭菌,毒素基因分析显示该菌同时含有α和ε两种毒素基因,动物致病性试验结果表明该菌对昆明系小鼠具有较强的致病性,并从试验致死的小鼠中分离到与藏羊病原相一致的细菌。三种基因的遗传进化树显示,该菌具有较强的多样性。研究确定了本次藏羊致死的主要病原为D型魏氏梭菌,结果可为该羊场梭菌病的治疗和预防提供科学依据。     

Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence.