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1.
Four hundred and twenty intestinal content samples (not including intestinal tissues) of freshwater fishes (60 silver carps, 100 carps, 100 crucian carps, 60 catfishes and 100 zaieuws) caught from one water reservoir were examined bacteriologically for the occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (alpha, beta, epsilon and iota) for classification into toxin types and for genes encoding enterotoxin and the novel beta2 toxin for further subclassification. C. perfringens could be isolated in 75 intestinal contents samples (17.9%) from freshwater fish including: 13 silver carps, 2 carps, 12 crucian carps, 40 zaieuws, and 8 catfishes. In 75 isolates, 58 strains (77.3%) were C. perfringens toxin type C (alpha and beta toxin positive), 13 strains (17.3%) were toxin type A (alpha toxin positive) and 4 strains (5.3%) were toxin type B (alpha, beta and epsilon toxin positive). In addition, the gene encoding for beta2 toxin was found in 47 strains (62.7%) of all the isolates, seven from type A, two from type B, and 38 from type C. The gene encoding for enterotoxin was not found in any isolate. These amplified toxin gene fragment were cloned and sequenced and compared with reference strains, the identity varied from 98.15% to 99.29%. This is the first report of C. perfringens alpha, beta, epsilon, beta2 toxins in freshwater fish and of beta, epsilon toxins in fish in general, and is the first discovery that the beta2 toxin could be detected in strains of type B. The origin of this bacterium and its importance to human food poisoning in freshwater fish is discussed.  相似文献   

2.
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.  相似文献   

3.
Eleven Clostridium perfringens type C strains isolated from fatal cases of hemorrhagic enterotoxemia of Canadian calves, a piglet, and a foal were studied for the production of soluble antigens. All the isolates from calves and a foal failed to produce delta toxin, but were capable of producing large amounts of lethal beta toxin. A strain isolated from a piglet produced delta, but very little beta toxin. Other differences were relatively minor. The results indicated that young domestic animals may be susceptible to all subtypes of C. perfringens type C. A simple method of using blood agar plates coated with type A antiserum for demonstration of hemolytic patterns was found advantageous in differentiation of C. perfringens strains.  相似文献   

4.
多重PCR检测圈养牛、猪和羊源魏氏梭菌   总被引：4，自引：0，他引：4  
采用多重PCR对圈养源魏氏梭菌的α，β，ε，ι毒素基因进行了检测，结果证实该方法具有很高的特异性。通过对山东德州、枣庄、泰安、蒙阴曾经流行过魏氏梭菌病的猪场、牛场和羊场的162个粪便样品进行检测，检出率为19．1％，均为A型。应用该方法鉴别魏氏梭菌血清型快速、简便，结果对于预防治疗均具有重要的指导作用。  相似文献   

5.
Beta2 toxin, encoded by the cpb2 gene, has been implicated in the pathogenesis of porcine, equine and bovine enteritis by type A Clostridium perfringens. By incorporating primers to cpb2 into a multiplex genotyping PCR, we screened 3270 field isolates of C. perfringens. Of these, 37.2% were PCR positive for the cpb2 gene. The majority of isolates from cases of porcine enteritis were positive for cpb2 (>85%), and this was even more true for C. perfringens isolated from cases of porcine neonatal enteritis (91.8%). In contrast, isolates from normal pigs only contained cpb2 in 11.1% of cases. The correlation between enteritis in other animal species and the presence of cpb2 was not so strong. cpb2 was found in 21.4% of C. perfringens isolates from cattle with enteritis, and in 47.3% of isolates from calves with enteritis or abomastitis. The prevalence of cpb2 varied with genotype, with type A isolates being positive for this gene in 35.1% of cases. Furthermore, enterotoxigenic type D or type E strains almost always carried cpb2. We cloned a 6xHIS-tagged beta2 (HIS-beta2) and used this protein to raise antiserum against beta2. Culture supernatants from 68 cpb2-positive and 13 cpb2-negative strains were tested for the presence of beta2 by Western blotting. In cpb2-positive isolates of porcine origin, beta2 was almost always detected (96.9%). However, in cpb2-positive isolates from other animal species, only 50.0% expressed beta2 protein. The high rate of cpb2-positivity among strains from neonatal pigs with enteritis and the high correlation of genotype with phenotype, supports the contention that beta2 toxin plays a role in the pathogenesis of these infections. However, it may be important to consider the use of an additional method for the detection of beta2 toxin in non-porcine cpb2-positive isolates when making claims about the role of beta2 in enteritis in non-porcine species.  相似文献   

6.
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。  相似文献   

7.
兔的梭菌性肠炎是由产气荚膜梭菌感染引起的严重危害养兔业的一种疾病,为了更好地控制此病,本研究调查了青岛地区规模化兔场爆发此病时产气荚膜梭菌的毒素型及遗传多样性。2010年11月-2012年5月期间,采集青岛地区规模化养兔场疑似产气荚膜梭菌感染兔的肝脏进行产气荚膜梭菌的分离鉴定,采用Multiple—PCR方法对分离菌株进行毒素型分析,应用ERIC-PCR方法分析分离菌株的遗传多样性。共分离到25株产气荚膜梭菌,其中A型24株（96％）,C型1株（4％）。用ERIC—PCR方法将25株分离株分于9个聚类中,其中V型为主要流行型。结果表明：青岛地区规模化兔场中产气荚膜梭菌流行的毒素型主要为A型,且具有多种基因亚型,其中V型为主要流行型。此结果为该地区兔产气荚膜梭菌病的免疫和微生态防治提供了重要的参考依据。  相似文献   

8.
OBJECTIVE: To determine the percentage of broodmares and foals that shed Clostridium perfringens in their feces and classify the genotypes of those isolates. DESIGN: Prospective cross-sectional study. ANIMALS: 128 broodmares and their foals on 6 equine premises. PROCEDURES: Anaerobic and aerobic bacteriologic cultures were performed on feces collected 3 times from broodmares and foals. All isolates of C. perfringens were genotyped. RESULTS: Clostridium perfringens was isolated from the feces of 90% of 3-day-old foals and 64% of foals at 8 to 12 hours of age. A lower percentage of broodmares and 1- to 2-month-old foals shed C. perfringens in their feces, compared with neonatal foals. Among samples with positive results, C. perfringens type A was the most common genotype identified (85%); C. perfringens type A with the beta2 toxin gene was identified in 12% of samples, C. perfringens type A with the enterotoxin gene was identified in 2.1% of samples, and C. perfringens type C was identified in < 1% of samples. CONCLUSIONS AND CLINICAL RELEVANCE: Clostridium perfringens was identified from the feces of all but 6 foals by 3 days of age and is likely part of the normal microflora of neonatal foals. Most isolates from broodmares and foals are C. perfringens type A; thus, the clinical relevance of culture results alone is questionable. Clostridium perfringens type C, which has been associated with neonatal enterocolitis, is rarely found in the feces of horses.  相似文献   

9.
Samples of faeces were taken from 166 healthy domesticated reindeer (Rangifer tarandus tarandus) from three flocks in different reindeer husbandry districts in northern Norway and examined bacteriologically for the presence of Clostridium perfringens. The organism was isolated from 98 (59 per cent) of the reindeer. The isolates were classified into C perfringens toxin types by PCR analysis specific for the genes encoding the four major toxins (alpha, beta, epsilon and tau) and were subclassified by the detection of the genes encoding C perfringens beta2-toxin and enterotoxin. All the isolates belonged to C perfringens toxin type A. In addition, 15 of the 98 isolates were PCR-positive for the beta2-toxin gene, and two of the isolates had the the gene encoding for enterotoxin.  相似文献   

10.
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。  相似文献   

11.
Very little is known about the occurrence of Clostridium perfringens and of diseases caused by this anaerobic bacterium in marine mammals, especially those that are free-living. During a scientific expedition to the Greenland Sea (West Ice) in spring 1999, faeces samples from 70 hooded seals (Cystophora cristata) were taken to isolate C. perfiringens. Subsequently, PCR analysis of the isolates was performed with oligonucleotide primers of the genes encoding the four major lethal toxins (alpha, beta, epsilon and iota) for classification of toxin type and of the genes encoding C. perfringens beta2-toxin and enterotoxin for further subclassification. In addition, a commercial ELISA kit for detection of C. perfringens alpha, beta- and epsilon-toxin was used. C. perfingens was isolated in samples from 38 (54.3%) hooded seals. All isolates were C. perfringens toxin type A (alpha-toxin positive). This is the first report on the occurrence of C. perfringens in this arctic marine mammal species. Myositis and enterotoxemia caused by C. perfrigens were described in other marine mammals and it may be assumed that the pathogenesis of an outbreak of disease is similar to that encountered in terrestrial animals. Although there is some controversy surrounding the enteropathogenicity and virulence of alpha-toxin (concerning enterotoxemia), this study suggests that a possible outbreak of enterotoxemia caused by C. perfringens type A in hooded seals may, however, not be excluded.  相似文献   

12.
Clostridium perfringens isolated from lambs with dysentery (n=117) were analysed by a DNA amplification technique, the polymerase chain reaction (PCR), in order to determine the prevalence of the alpha-, beta-, beta 2-, epsilon-, iota- and enterotoxin genes. The most prevalent toxin type of C. perfringens found was type B, containing the alpha-, beta-, and epsilon-toxin genes, representing 46% of the cases with clostridial dysentery. C. perfringens type C containing the alpha-, and beta-toxin genes was isolated in 20% and type D, which is characterized by the alpha- and epsilon-toxin genes, was isolated in 28% of all isolates. The recently discovered, not yet assigned beta 2-toxigenic type of C. perfringens was represented in 6% of all isolates. No C. perfringens type A containing the alpha-toxin alone and no type E, which harbours the ADP-ribosylating iota-toxin, were found in the diseased animals. None of the samples contained the enterotoxin gene. Only one type of C. perfringens was found in a given herd, revealing the epidemiological use of PCR toxin gene typing of C. perfringens. The animals originated from 79 different herds with sizes ranging from 30 to 250 animals, bred in the area of northern Greece.  相似文献   

13.
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.  相似文献   

14.
OBJECTIVE: To compare the frequency of isolation, genotypes, and in vivo production of major lethal toxins of Clostridium perfringens in adult dairy cows affected with hemorrhagic bowel syndrome (HBS) versus left-displaced abomasum (LDA). DESIGN: Case-control study. ANIMALS: 10 adult dairy cattle with HBS (cases) and 10 adult dairy cattle with LDA matched with cases by herd of origin (controls). PROCEDURE: Samples of gastrointestinal contents were obtained from multiple sites during surgery or necropsy examination. Each sample underwent testing for anaerobic bacteria by use of 3 culture methods. The genotype of isolates of C. perfringens was determined via multiplex polymerase chain reaction assay. Major lethal toxins were detected by use of an ELISA. Data were analyzed with multivariable logistic regression and chi2 analysis. RESULTS: C. perfringens type A and type A with the beta2 gene (A + beta2) were the only genotypes isolated. Isolation of C. perfringens type A and type A + beta2 was 6.56 and 3.3 times as likely, respectively, to occur in samples from cattle with HBS than in cattle with LDA. Alpha toxin was detected in 7 of 36 samples from cases and in 0 of 32 samples from controls. Beta2 toxin was detected in 9 of 36 samples from cases and 0 of 36 samples from controls. CONCLUSIONS AND CLINICAL RELEVANCE: C. perfringens type A and type A + beta2 can be isolated from the gastrointestinal tract with significantly greater odds in cattle with HBS than in herdmates with LDA. Alpha and beta2 toxins were detected in samples from cows with HBS but not from cows with LDA.  相似文献   

15.
Forty-two Clostridium perfringens type A strains isolated from cases of diarrhoea in pigs were tested for their ability to sporulate and produce enterotoxin in three different sporulation media. Enterotoxin was produced by 11 of the 42 C perfringens type A isolates (26.2 per cent). Thirteen isolates (30.9 per cent) produced spores at a frequency of 10 per cent or more. Spore production was recorded in 24 (57.1 per cent) of the isolates. The titres of enterotoxin produced by the isolates ranged from 1:2 to 1:64. The enterotoxin produced was compared with that produced by a reference strain and found to be identical. Ninety-eight of 106 sow sera from four different farms were found to possess antibodies to C perfringens type A enterotoxin with titres ranging from 1:2 to 1:64. Spores of C perfringens type A were detected in pig faeces and intestinal contents in 20 of 23 cases of enteritis at levels of up to 5 x 10(6) cells/g of faeces. Smaller numbers of spores, up to 2 x 10(4)/g were present in five of 10 samples from non-diarrhoeic pigs. Enterotoxin was demonstrated by Vero cell assay in five of the 23 samples from diarrhoeic pigs but in none of the 10 samples from non-diarrhoeic animals. It was clear from these studies that C perfringens type A strains in pigs could sporulate and produce enterotoxin in vitro and in vivo and that enteritis might be associated with sporulating organisms in vivo.  相似文献   

16.
Clostridium perfringens is a cause of economically significant enteritis in livestock. Beta2 toxin, encoded by one of two cpb2 alleles, is implicated as a virulence factor in this disease. Previous studies determined that the consensus cpb2 allele is preferentially associated with C. perfringens isolated from pigs. In C. perfringens strain 13, the consensus cpb2 allele is found on the plasmid pCP13, which also carries cna, encoding a putative collagen binding protein, CpCna. This protein was shown to be a bona fide collagen adhesin, as recombinant, HIS-tagged CpCna bound collagen type I as determined by Far Western blotting. Genomic DNA from C. perfringens isolated from a variety of host species were subjected to PCR to determine the prevalence of cna in these strains and correlate its carriage with the presence and type of cpb2 allele. The cna gene was found in 55.8% of isolates from all host species (n=208) and 68.1% of porcine isolates (n=119). In cpb2+ isolates, cna was present in 69.9% of isolates from all hosts (n=153), but was found in 98.7% of porcine isolates (n=75). Furthermore in porcine isolates, the consensus cpb2 allele and cna were absolutely correlated with the presence of pcp12, a pCP13-encoded gene, and pcp12 was never found in any isolate that lacks either cpb2 allele. The finding that CpCna binds collagen and that the cna gene is associated with the consensus cpb2 allele implicates CpCna as a potential virulence factor in porcine enteritis caused by C. perfringens.  相似文献   

17.
Currently, the factors/toxins responsible for Clostridium perfringens-associated avian enteritis are not well understood. To assess whether specific C. perfringens' toxinotypes are associated with avian enteritis, the isolates of C. perfringens from 31 cases of avian necrotic or ulcerative enteritis submitted between 1997 and 2005 were selected for retrospective analysis using multiplex PCR. C. perfringens was isolated from chickens, turkeys, quail, and psittacines. The toxinotypes of isolates from diseased birds were compared against the toxinotype of 19 C. perfringens isolates from avian cases with no evidence of clostridial enteritis. All C. perfringens isolates were classified as type A regardless of species or disease history. Although many isolates (from all avian groups) had the gene encoding the C. perfirngens beta2 toxin, only 54% produced the toxin in vitro when measured using Western blot analysis. Surprisingly, a large number of healthy birds (90%) carried CPB2-producing isolates, whereas over half of the cpb2-positive isolates from diseased birds failed to produce CPB2. These data from this investigation do not suggest a causal relationship between beta2 toxin and necrotic enteritis in birds.  相似文献   

18.
A survey based on clinical, pathological and microbiological investigations was performed on 11 Brown Swiss cattle affected with depression, anorexia, agalaxia, ruminal hypomotility, abdominal pain and melaena. In eight animals, macroscopical lesions consisted in haemorrhagic enteritis in the small intestine. Seven of eight isolates from tissue samples were identified as Clostridum perfringens type A, and four were identified as C. perfringens type A with the beta2 toxin gene. Based on these observations, animals were considered affected with haemorrhagic bowel syndrome.  相似文献   

19.
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.  相似文献   

20.
Toxovars of 97 airborne C. perfringens isolates and 10 C. perfringens isolates from fecal samples of a calf stable were determined by an EIA procedure. Most airborne and fecal isolates belonged to toxovar A (88.7% and 80.0% respectively). Eight point two% of airborne C. perfringens were identified as toxovar C and 3.1% as toxovar D. Toxovar B was not found in the airborne state. Twenty% of fecal C. perfringens belonged to toxovar D. Toxovar B and C was not isolated from fecal samples. In addition, all fecal and air-borne isolates of C. perfringens toxovar D strains were analyzed in SDS-PAGE for their polypeptide pattern. All isolates from both sources exhibited the same polypeptide pattern after electrophoretic analysis in SDS-PAGE. Both results, determination of toxovars as well as polypeptide pattern analysis in SDS-PAGE, suggest that a major source of airborne C. perfringens in animal stables is animal feces.  相似文献   

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