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1.
PCR detection of Clostridium perfringens producing different toxins in faeces of goats 总被引:4,自引:0,他引:4
A polymerase chain reaction (PCR) was used to identify the genes encoding the major toxins of Clostridium perfringens in faeces of goats. When pure cultures of Cl. perfringens types A, B, C, D and E were used as templates in the PCR, amplicons were observed on the agarose gel as bands at approximately the 247 (alpha primers), 1025 (beta primers), 403 (epsilon primers) and 298 (iota primers) bp level of the DNA marker. When used to identify different types of Cl. perfringens in samples artificially spiked with these micro-organisms, the PCR detected as few as 1–1·5×102 cfu g−1 of the five types of Cl. perfringens tested. The PCR technique allowed the identification and typing of Cl. perfringens strains in faeces of goats, without recourse to other techniques such as the mouse neutralization test. 相似文献
2.
A. L. Warren F. A. Uzal L. L. Blackall W. R. Kelly 《Letters in applied microbiology》1999,29(1):15-19
A polymerase chain reaction (PCR) was used to identify the genes encoding the alpha, epsilon and beta toxins of Clostridium perfringens in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep. When pure cultures of Cl. perfringens types B and D were used as control templates in the PCR, products of the following sizes were observed on the agarose gel: 247 bp (alpha primers), 1025 bp (beta primers) and 403 bp (epsilon primers). When used to identify Cl. perfringens type D in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep, the PCR technique resulted in the detection of this micro-organism in 11 out of 13 samples known to be infected with Cl. perfringens. No false positive results were obtained when 13 culturally negative samples were analysed by the PCR technique. 相似文献
3.
Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens 总被引:6,自引:0,他引:6
A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 102 bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans.
The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples. 相似文献
The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples. 相似文献
4.
A polymerase chain reaction (PCR) assay based on the 16S rRNA gene and an improved DNA extraction procedure were developed for the direct detection and differentiation of Campylobacter upsaliensis and C. helveticus in seeded human faeces. The PCR assay was compared with culture detection by a membrane filter (MF) technique and on selective agar (SA) containing 8 mg l−1 cefoperazone. Both MF culture and the PCR assay detected 105 colony-forming units (cfu) g−1 faeces. Selective agar culture of some strains could detect as few as 103 cfu g−1 faeces. However, some strains were susceptible to cefoperazone and either failed to grow or were detected only with reduced sensitivity in the presence of the antibiotic. Detection by MF and SA both required 48–96 h incubation in a microaerobic atmosphere and did not specifically identify the isolate. By contrast, the PCR assay could be completed within 8 h and accurately identified the two phenotypically similar species, C. upsaliensis and C. helveticus. 相似文献
5.
E. Aranda M.M. Rodríguez M.A. Asensio & J.J. Córdoba 《Letters in applied microbiology》1997,25(3):186-190
A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2·1–8·1 cfu ml−1 ) for types A and B, but rather low (104 cfu ml−1 ) for types E and F. However, after enrichment at 37°C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g−1 of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods. 相似文献
6.
Microbiological composition of raw milk from selected farms in the Camembert region of Normandy 总被引:3,自引:1,他引:2
Raw milk from 27 farms was sampled over 6 months for listerias, salmonellas, Yersinia enterocolitica and campylobacters. Total bacterial counts and somatic cell counts were measured. Lactococci, lactobacilli, dextran-producing leuconostocs, Brevibacterium linens , yeasts and moulds, Staphylococcus aureus and other Micrococcaceae, Pseudomonas , coliforms, Escherichia coli , enterococci, Clostridium perfringens and spores of anaerobic lactate-fermenting bacteria were also counted. Pseudomonas (2000 cfu ml−1 ), lactococci (760 cfu ml−1 ) and Micrococcaceae (720 cfu ml−1 ) were the most numerous groups. Lactic acid bacteria were detected in all samples. Coliforms were present in most samples, but 84% of samples had counts <100 cfu ml−1 . Staphylococcus aureus was detected in 62% of milks, the average count was 410 cfu ml−1 . About 80% of supplies had ≤10 E. coli cfu ml−1 and all samples had 1 Cl. perfringens cfu ml−1 . Two of the tested milks were positive for salmonellas (2·9%), four were positive for Listeria monocytogenes (5·8%), 25 for Yersinia enterocolitica (36%) and one for campylobacters (1·4%). 相似文献
7.
Equine anterior enteritis is an acute syndrome with unknown aetiology, although salmonellosis and infection with Clostridium perfringens have both been suggested as potential causes. The main aim of this preliminary study was to compare the prevalence of toxigenic types of C. perfringens in clinically healthy horses and in horses with anterior enteritis. From horses admitted with colic at Phillip Leverhulme Large Animal Hospital in 1995-1996, samples of gastric reflux, small intestinal contents and faeces were taken for isolation of C. perfringens. Five of those horses were admitted as anterior enteritis cases, of which C. perfringens was isolated in pure culture in all five horses. Two of the anterior enteritis cases from which viable bacterial counts had been performed revealed 10(6) CFU/g faeces C. perfringens. Samples of gastric reflux and small intestinal contents submitted from one of these horses revealed 10(4) CFU/mL and 10(5) CFU/mL respectively. The number of C. perfringens observed in the gastric reflux was considered significant as the total volume removed was 12 L. The counts observed in faeces taken from horses admitted with anterior enteritis, were significantly higher than the <10(2) CFU/g faeces observed in faeces from healthy horses and horse presenting with colic and with other diagnoses. The major toxigenic types of C. perfringens in both healthy and diseased horses are being investigated using the polymerase chain reaction (PCR) to amplify target DNA sequences of the toxin genes. Primers have been designed from the published DNA sequences of the enterotoxin, alpha, beta, epsilon and iota toxin genes. PCR products obtained from NCTC strains of C. perfringens have been cloned and the sequenced, to verify that the amplicon sequence is correct. Initial typing suggests that C. perfringens type A is the predominant toxin type isolated from healthy horses and horses with colic with other diagnoses.C. perfringens strains isolated from horses with anterior enteritis are of type D. 相似文献
8.
The polymerase chain reaction (PCR) has the potential to detect low levels of the human pathogen Escherichia coli O157 : H7 in bovine faeces. To improve the utility of PCR for this application, several methods for preparing template DNA from bovine faeces, both directly and after non-selective enrichment, were tested. These were boiling, enzyme treatment, enzyme treatment plus phenol-chloroform extraction, and enzyme treatment plus phenol-chloroform extraction plus Geneclean® purification. Of these, the boiling method was the most consistent and had a sensitivity of approximately 3 cfu g−1 faeces, with an assay time of less than 32 h. The boiling method was also combined with immunomagnetic separation (IMS) to detect E. coli O157 : H7 in less than 8 h, but with a sensitivity of approximately 103 cfu g−1 faeces. These methods can be used to prepare template for PCR screening of bovine faeces using any appropriate PCR primers. 相似文献
9.
Differences in production of several extracellular virulence factors in clinical and food Aeromonas spp. strains 总被引:2,自引:1,他引:1
Carmen Pin M. Luisa Marín Dolores Selgas M. Luisa García J. Tormo Carmen Casas 《Journal of applied microbiology》1995,78(2):175-179
C. PIN, M.L. MARÍN, D. SELGAS, M.L. GARCÍA, J. TORMO AND C. CASAS. 1995. Production of several extracellular virulence factors (lipase, protease and haemolysin) was compared in 15 Aeromonas spp. isolated from faeces of patients with Aeromonas -associated gastroenteritis and 81 strains isolated from food. Strains from food did not show differences in production of these factors when compared with strains isolated from faeces. However, if strains were considered in relation to autoagglutination (AA) character, the AA+ differed from AA- strains in lipase and protease production. Supernatant fluids of AA+ food and human strains showed 2·5-fold more protease production than that observed in AA- strains. These two characteristics of certain Aeromonas strains could be related with the more virulent capacity. 相似文献
10.
In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth-enhancing antibiotics 总被引:2,自引:0,他引:2
Minimal inhibitory concentration (MIC) determinations were carried out with seven growth-enhancing antibiotics against 95 Clostridium perfringens field isolates obtained during 1991 and 1992 from poultry, pigs and calves. All were resistant to 64 μg ml−1 of the bambermycin antibiotic, flavomycin (flavophospholipol) and susceptible to avoparcin (MIC90 0.25 μg ml−1 ), avilamycin (MIC90 0.5 μg ml−1 ) and salinomycin (MIC90 ≤ 0.12 μg ml−1 ). Acquired resistance against bacitracin was detected in some isolates from poultry and bovines and resistance to tylosin and virginiamycin in some strains from all species investigated. Overall, the prevalence of resistance was comparable to the low levels recorded in 1979 in Cl. perfringens isolates from the same animal host species. 相似文献
11.
The seasonal variation of thermophilic campylobacters in beef cattle, dairy cattle and calves 总被引:5,自引:2,他引:3
K.N. Stanley J.S. Wallace J.E. Currie P.J. Diggle & K. Jones 《Journal of applied microbiology》1998,85(3):472-480
The epidemiology of clinical cases of campylobacter in temperate climates shows a striking seasonality. In the search for a seasonal environmental reservoir changes in the carriage rate and population size of campylobacters in bovine hosts with time have been measured. Most probable number (MPN) methodology was used to enumerate thermophilic campylobacters in samples taken from the small intestines of beef cattle at slaughter and the fresh faeces of four dairy herds and new-born calves. Statistical analyses revealed significant evidence for seasonal periodicity in the data from dairy herds ( P = 0·044). Not only was there a departure from constancy within a 12-month interval but these data revealed a true seasonality, that is, the same periodicity in numbers from one year to the next. Each herd had two peaks per year, in approximately spring and autumn. Peaks coincided in herds on neighbouring farms but those on farms in the north preceded those on farms in the south by 2 and 1 months, respectively ( P = 0·0057). Intestinal carriage by beef cattle at slaughter was 89·4% ( n = 360) with an average MPN campylobacters per gram fresh weight (MPN gfw−1 ) of 6·1 × 102 . Average MPN gfw−1 in faeces from the dairy herds and calves were 69·9 ( S.D. 3) and 3·3 × 104 ( S.D. 1·7 × 102 ). There was no evidence of seasonal periodicity in the size of the campylobacter population in beef cattle at slaughter. Calves were campylobacter free at birth but became colonized within a few days. 相似文献
12.
The use of PCR to monitor the population abundance of six human intestinal bacterial species in an in vitro semicontinuous culture system 总被引:2,自引:0,他引:2
Rong-Fu Wang Wei-Wen Cao Warren L. Campbell Latrina Hairston Wirt Franklin Carl E. Cerniglia 《FEMS microbiology letters》1994,124(2):229-237
Abstract Six PCR primer sets complementary to the 16S rDNAs (rRNA genes) were developed and shown to be specific for the following anaerobic bacteria: Clostridium clostridiiforme, C. perfringens, C. leptum, Bacteroides vulgatus, B. distasonis , and B. thetaiotaomicron , respectively. These primers were used for PCR to detect and monitor the bacteria in a semicontinuous culture system designed to mimic intestinal microflora in the human gastrointestinal tract. Except for C. perfringens , the five species of Bacteroides and Clostridia present in the in vitro culture system were detected by the PCR, and the titers varied from 10−2 to 10−6 dilutions. The role of azo dye reduction by these bacterial species in the system was examined and discussed. 相似文献
13.
Polymerase chain reaction (PCR) tests were established for detection of Serpulina hyodysenteriae , the agent of swine dysentery, and S. pilosicoli , the agent of intestinal spirochaetosis. Both reactions were specific when tested with DNA from 107 strains of various intestinal spirochaetes. For diagnostic use, faeces were plated to selective medium, and diatomaceous earth extraction used to obtain DNA prior to PCR. This procedure detected 103 –104 cells of either organism seeded into 0·2 g of faeces. When applied to 63 samples from pigs of eight piggeries naturally infected with either S. hyodysenteriae or S. pilosicoli , both PCRs were specific, more rapid, and detected more positive samples than did routine faecal culture and isolation. 相似文献
14.
A study of isolation procedures for multiple infections of Salmonella and Arizona in a wild marsupial, the quokka (Setonix brachyurus) 总被引:2,自引:2,他引:0
R. P. Hart J. B. Iveson † S. D. Bradshaw T. P. Speed † 《Journal of applied microbiology》1982,53(3):395-406
Rectal swabs and faeces were used in the regular sampling for salmonellas and Arizonas from a heavily-infected population of a marsupial, the quokka ( Setonix brachyurus ). The media used were strontium selenite A and strontium chloride B enrichment broths, with subculture onto modified bismuth sulphite agar and deoxycholate citrate agar. A study of sampling, enrichment, sub-culture and colony selection procedures produced an optimal scheme giving high yields but consistent with reasonable economy of time and materials. A three-swab sample was taken and inoculated into the two enrichment media, and with each enrichment subjected to three subcultures. The absolute efficiency of this procedure was greater than 80% (and confirmed by a serological method), compared with only 67% for a single swab in a single enrichment. Recovery of some serotypes depended on the media used; e.g. Arizonas could not be recovered satisfactorily from strontium chloride B enrichment. Faeces samples were found to be greatly superior to rectal swabs for detecting salmonellas and arizonas but they were less convenient in field studies. In a comparison of rectal swabs and faeces samples where the actual concentration of salmonellas was known, it was found that the efficiency of rectal swabs approached 100% if there were more than 103 salmonellas/g faeces, but this declined to approximately 50% if there were 102 -103 salmonellas/g faeces and only 25% if there were less than 102 salmonellas/g faeces. A new statistical procedure was introduced for comparing the number of isolations from two methods, and this should be of use in similar methodological studies. 相似文献
15.
Y.-J. Ahn C.-O. Lee J.-H. Kweon J.-W. Ahn & J.-H. Park 《Journal of applied microbiology》1998,84(3):439-443
The growth-inhibitory activity of Galla Rhois-derived materials towards 17 intestinal bacteria was evaluated using an impregnated paper disc method. The biologically active components of Galla Rhois were characterized as the tannins methyl gallate (MG) and gallic acid (GA) by spectral analysis. The growth responses varied with bacterial strain tested. In the test using 10 mg disc−1 , MG and GA produced a clear inhibitory effect on harmful bacteria such as Clostridium perfringens , Cl. paraputrificum , Eubacterium limosum , Bacteroides fragilis , Staphylococcus aureus and Escherichia coli . Methyl gallate showed no growth-inhibitory activity towards Bifidobacterium adolescentis or B. longum whereas the growth of B. bifidum , B. breve , B. infantis , B. animalis , B. thermophilum , Lactobacillus acidophilus , Lact. plantarum and Streptococcus faecalis was slightly affected. However, GA did not adversely affect the growth of the bifidobacteria and lactobacilli. At 5 mg disc−1 , MG significantly inhibited the growth of Cl. perfringens and Cl. paraputrificum but did not affect the growth of the bifidobacteria and lactobacilli. At 1 mg disc−1 , MG greatly inhibited the growth of Cl. perfringens alone. These results may be an indication of at least one of the pharmacological actions of Galla Rhois. 相似文献
16.
Aims: To determine the occurrence and proportion of Escherichia coli O157:H7 in faeces, skin swabs and carcasses before and after washing, from sheep and goats in Ethiopia.
Method and Results: Individual samples were enriched in modified tryptic soy broth with novobiocin, concentrated using immunomagnetic separation (IMS) and plated onto cefixime-tellurite containing sorbitol MacConkey agar. Presumptive colonies were confirmed by biochemical tests and subjected to latex agglutination tests. A PCR was performed on isolates for the detection of stx1 , stx 2 and eae genes. Escherichia coli O157:H7 was isolated from faeces (4·7%), skin swabs (8·7%) and carcasses before washing (8·1%) and after washing (8·7%) and on water samples (4·2%). The proportion of carcasses contaminated with E. coli O157:H7 was strongly associated with those recovered from faecal and skin samples. Both stx 1 and stx 2 genes were identified from one E. coli O157:H7 isolate from a goat carcass.
Conclusions: Even though the numbers of samples examined in this study were limited to one abattoir, sheep and goats can be potential sources of E. coli O157:H7 for human infection in the country. Control measures to reduce the public health risks arising from E. coli O157:H7 in reservoir animals need to be addressed at abattoir levels by reducing skin and faecal sources and carcass contaminations at different stages of slaughter operations.
Significance and Impact of the Study: Escherichia coli O157:H7 was detected from carcasses before and after washing during slaughtering operations, and one O157 isolate was positive for verotoxins. 相似文献
Method and Results: Individual samples were enriched in modified tryptic soy broth with novobiocin, concentrated using immunomagnetic separation (IMS) and plated onto cefixime-tellurite containing sorbitol MacConkey agar. Presumptive colonies were confirmed by biochemical tests and subjected to latex agglutination tests. A PCR was performed on isolates for the detection of stx
Conclusions: Even though the numbers of samples examined in this study were limited to one abattoir, sheep and goats can be potential sources of E. coli O157:H7 for human infection in the country. Control measures to reduce the public health risks arising from E. coli O157:H7 in reservoir animals need to be addressed at abattoir levels by reducing skin and faecal sources and carcass contaminations at different stages of slaughter operations.
Significance and Impact of the Study: Escherichia coli O157:H7 was detected from carcasses before and after washing during slaughtering operations, and one O157 isolate was positive for verotoxins. 相似文献
17.
G.L. Ercolani 《Journal of applied microbiology》1997,82(1):137-140
Clostridial spores were found in numbers from less than 1 to over 50 colony-forming units cm-2 on mature leaves of 19 species of horticultural plants under commercial cultivation in five localities in Apulia (SE Italy). Of 1828 clostridial isolates, 87% were identified phenotypically and ascribed to Clostridium pasteurianum, Cl. sporogenes, Cl. butyricum, Cl. roseum, Cl. perfringens, Cl. felsineum and Cl. acetobutylicum , in decreasing order of frequency. When spore suspensions of Cl. pasteurianum, Cl. perfringens, Cl. roseum and Cl. sporogenes were inoculated onto the leaves of basil, leaf-beet, lettuce, rocket-salad, spinach and tomato in the greenhouse, spore counts at first invariably declined shortly after inoculation, then rose again significantly for Cl. pasteurianum and Cl. perfringens on basil and for Cl. sporogenes on tomato in summer. 相似文献
18.
Gari was examined for its post-processing microbial content. Aerobic mesophilic bacteria and fungi were isolated from all samples. The total viable bacterial counts ranged from 2.0 × 102 to 8.0 × 104 cfu/g. Fungal counts ranged from 1.0 × 102 to 1.5 × 104 cfu/g. The total viable counts of fresh samples were much lower than those of market and packaged samples. Bacillus, Micrococcus and Proteus spp. were the bacteria isolated, Aspergillus niger, Aspergillus flavus and Penicillium spp. the fungi. Food borne parasites and pathogens such as Staph. aureus and Clostridium perfringens were not found. The gari samples were quite stable, having a shelf life of 3–6 months. The water activities of the samples ranged from 0.52 to 0.68. Based on the microbial counts of the samples, the critical upper limit for the safety of gari was set at 104 cfu/g dry sample. 相似文献
19.
Miserez Frey Buogo Capaul Tontis Burnens & Nicolet 《Letters in applied microbiology》1998,26(5):382-386
Clostridium perfringens isolated from sheep and goat with enterotoxaemia at necropsy and from healthy animals at slaughter were typed using specific PCR assays for the detection of the α-, β- and ε-toxin genes. Clostridium perfringens isolated from all 52 animals with pathological signs of enterotoxaemia showed the presence of the α- and ε-toxin genes but were devoid of the β-toxin gene. These strains could therefore be identified as type D, characteristic for clostridial enterotoxaemia of sheep, lambs and goats. In contrast, Cl. perfringens isolated from 11 of 13 healthy animals only contained the α-toxin gene which is typical for type A. Two of the healthy animals contained Cl. perfringens with the α- and ε-toxin genes. However, when several individual Cl. perfringens colonies were analysed from each of these two animals, only a small percentage was found to contain the ε-toxin gene, whereas the majority of the colonies were of type A with the α-toxin gene only. This is in contrast to the findings from the diseased animals which contained practically only type D Cl. perfringens . The β-toxin gene was not found in any Cl. perfringens isolate from goat and sheep. Comparison of the PCR data with results obtained by the classical biological toxin assay using the mouse model showed a good correlation. 相似文献
20.
A 24 h screen to detect viable salmonellas in faeces was developed by studying growth dynamics of salmonellas and competing flora in combinations of enrichment media and artificially-inoculated pig faeces. Muller-Kauffmann tetrathionate (MK) broth, incubated overnight at 42°C, maintained the lowest ratio of salmonella: competing flora and identified all inoculated samples. A 4 h postenrichment in M broth plus novobiocin reduced the number of false-positive results in subsequent ELISAs. Adjusting the negative cut-off values and incubation time of the chromogenic substrate from that recommended in the ELISA instructions reduced the rate of false-positive results further and allowed the detection of 103 salmonellas per ml in the presence of up to 107 ml−1 aerobic-competing cells. Suspension of faeces diluted 1 in 2 and 1 in 5, rather than 1 in 10 in MK broth did not necessitate further adjustments to the ELISA baseline values. The proposed screen protocol is an overnight incubation of faeces suspended 1 in 10 in MK broth, a 1 in 100 subculture into M broth plus 10 μg ml−1 novobiocin (MbN) for 4 h, steam inactivation of MbN cultures and testing by ELISA, and can detect three salmonella cells per g faeces. 相似文献