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1.
The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.  相似文献   

2.
This study was undertaken to determine the in vitro susceptibility of Clostridium perfringens, isolated from poultry to antimicrobials used in poultry production. The minimal inhibitory concentration (MIC) of eight antimicrobials, including the ionophoric coccidiostat narasin, was determined for 102 C. perfringens isolates, 58 from Sweden, 24 from Norway and 20 from Denmark. Susceptibility to each antimicrobial compound was determined by broth microdilution. The isolates were obtained from broilers (89), laying hens (9) and turkeys (4), affected by necrotic enteritis (NE) or by C. perfringens associated hepatitis (CPH), and from healthy broilers. All strains, regardless of origin, proved inherently susceptible to ampicillin, narasin, avilamycin, erythromycin and vancomycin. A low frequency of resistance to virginiamycin and bacitracin was also found. Resistance to tetracycline was found in strains isolated in all three countries; Sweden (76%), Denmark (10%) and Norway (29%). In 80% of the tetracycline-resistant isolates, the two resistance genes tetA(P) and tetB(P) were amplified by PCR whereas in 20% only the tetA(P) gene was detected. No tetM gene amplicon was obtained from any of the tetracycline-resistant isolates. The uniform susceptibility to narasin revealed in this study shows that the substance can still be used to control clostridiosis. In this study, C. perfringens also showed a low degree of resistance to most other antimicrobials tested. Despite the small amounts of tetracycline used in poultry, a considerable degree of resistance to tetracycline was found in C. perfringens isolates from Swedish broilers.  相似文献   

3.
Clostridium perfringens is a well documented cause of a mild self-limiting diarrhea and a potentially fatal acute hemorrhagic diarrheal syndrome in the dog. A recent study documented that 21% of canine C. perfringens isolates had MIC's indicative of resistance to tetracycline, an antimicrobial commonly recommended for treatment of C. perfringens-associated diarrhea. The objective of the present study was to further evaluate the antimicrobial susceptibility profiles of these isolates by determining the prevalence of specific resistance genes, their expression, and ability for transference between bacteria. One hundred and twenty-four canine C. perfringens isolates from 124 dogs were evaluated. Minimum inhibitory concentrations of tetracycline, erythromycin, tylosin, and metronidazole were determined using the CLSI Reference Agar Dilution Method. All isolates were screened for three tetracycline resistance genes: tetA(P), tetB(P) and tetM, and two macrolide resistance genes: ermB and ermQ, via PCR using primer sequences previously described. Ninety-six percent (119/124) of the isolates were positive for the tetA(P) gene, and 41% (51/124) were positive for both the tetA(P) and tetB(P) genes. No isolates were positive for the tetB(P) gene alone. Highly susceptible isolates (MIC< or = 4 microg/ml) were significantly more likely to lack the tetB(P) gene. One isolate (0.8%) was positive for the ermB gene, and one isolate was positive for the ermQ gene. The tetM gene was not found in any of the isolates tested. Two out of 15 tested isolates (13%) demonstrated transfer of tetracycline resistance via bacterial conjugation. Tetracycline should be avoided for the treatment of C. perfringens-associated diarrhea in dogs because of the relatively high prevalence of in vitro resistance, and the potential for conjugative transfer of antimicrobial resistance.  相似文献   

4.
Genetic variation and cross-reactivity of Clostridium septicum alpha-toxin   总被引：1，自引：0，他引：1  
Clostridium septicum alpha-toxin genes were sequenced with the polymerase chain reaction (PCR) products amplified from DNAs of 25 C. septicum strains, and were classified into 10 patterns. Alpha-toxins were purified from the culture supernatant of four C. septicum strains (strains No. 44, Kagoshima 8, Mie and Tokachi) which were specially chosen from patterns of the deduced amino acid sequences. The molecular weights of the alpha-toxins were not different according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. However, the isoelectric points between the alpha-toxins of No. 44 and Tokachi strains differed markedly. Cross-neutralization tests were performed with purified alpha-toxins and antitoxins in mice and in Vero cells. Each antitoxin showed roughly the same titers against the four alpha-toxins in mice and completely identical titers against these in Vero cells. Calves immunized with toxoid prepared from the culture supernatant of No.44 strain were challenged by exposure to spores of Mie strain. The toxoid conferred protection against the challenge in calves. From these results, although genetic variation has been observed within the C. septicum alpha-toxin gene, C. septicum strains toxoid of strain No.44 induces protective immunity against exposure to C. septicum that produce other subtypes of alpha-toxin containing several different amino acid residues.  相似文献   

5.
Amplification of the 16S-23S rDNA spacer region by polymerase chain reaction (PCR) was used for the rapid detection of Clostridium chauvoei and C septicum. To assess its specificity, PCR was performed with total DNA from 42 strains of clostridia and three strains of other genera. PCR products specific to C chauvoei or to C septicum were generated from homologous cultures only. Clostridium chauvoer-specific or C septicum-specific amplicons were also generated from tissues of cows experimentally infected with C chauvoei or C septicum and in DNA samples from cows clinically diagnosed as having blackleg or malignant oedema. These results suggest that a species-specific PCR may be useful for the rapid and direct detection of C chauvoei and C septicum in clinical specimens.  相似文献   

6.
The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia.  相似文献   

7.
Five strains of Clostridium septicum were used to prepare bacterins, bacterin-toxoids, toxoid, and combinations of bacterins or bacterin-toxoids. These preparations were tested for immunogenicity in guinea pigs vaccinated subcutaneously with 1.0 ml of product. Usually, a second vaccination was given 21 to 24 days later. The immunity of groups of vaccinated guinea pigs was challenged with as many as 22 strains of C septicum. When challenge exposed with homologous strains at 21 to 24 days after one vaccination or 10 t0 18 days after a second vaccination, 60% to 100% of the guinea pigs in each group survived. Demonstrable cross-protection among strains of C septicum varied from none to 100% protection in vaccinated guinea pigs. A combination of bacterin-toxoid prepared from four selected strains protected 70% to 100% of the vaccinated guinea pigs challenge exposed with 21 strains. Duration-of-immunity studies demonstrated a twofold to fourfold decrease in protection when the vaccination-to-challenge interval was extended an additional 3 weeks. Strains of C septicum do not have an effective common immunogen and the stimulated immunity appears to be of short duration. Antitoxin was demonstrated to be less important than other factors in protecting against C septicum infection.  相似文献   

8.
分别采用ELISA、多重PCR方法对从一个犊牛舍分离到的魏氏梭菌进行型别研究,结果显示:ELISA检测结果为88%的菌种属于A型,3.4%为C型,8.6%是D型;而多重PCR结果均为A型。  相似文献   

9.
Faecal samples from adult horses and from foals with diarrhoea or with normal faeces were evaluated for the presence of Clostridium difficile, C. difficile toxins, C. perfringens enterotoxin (CPE) and C. perfringens spore counts. Clostridium difficile was isolated from 7/55 horses (12.7%) and 11/31 foals (35.5%) with colitis, but from 1/255 normal adults (0.4%) and 0/47 normal foals (P<0.001). Clostridium difficile toxins A and/or B were detected in 12/55 diarrhoeic adults (21.8%) and 5/30 diarrhoeic foals (16.7%) but in only 1/83 adults (1.2%) and 0/21 foals with normal faeces (P<0.001 and P<0.05, respectively). Clostridium perfringens enterotoxin was detected in 9/47 diarrhoeic adults (19%) and 8/28 diarrhoeic foals (28.6%), but was not detected in 47 adult horses (P<0.002) or 4 foals (P = 0.22) with normal faeces. The positive predictive value of isolation of C. perfringens with respect to the presence of CPE was only 60% in adult horses and 64% in foals. There was no association between total C. perfringens spore count and CPE in the faeces. The overall mortality rate from colitis was 22% for adult horses and 18% for foals. Clostridium difficile toxin-positive adult horses with colitis were less likely to survive than C. difficile-negative horses with colitis (P = 0.03). This study provides further evidence that C. difficile and enterotoxigenic C. perfringens are associated with equine enterocolitis.  相似文献   

10.
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.  相似文献   

11.
By the use of the electrophoretic casein precipitating inhibition test (CPI-test) the serological relationship between proteolytic enzymes produced by different species within the genera Clostridium and Bacillus has been tested. The proteases produced by Clostridium botulinum types A, B, C, D and F cross-reacted with each other. Clostridium botulinum strain 84 was inhibited by antiproteases produced against Clostridium sporogenes, Clostridium botulinum types C and F (protease F I and F II), but not by antiproteases against Clostridium botulinum types B and F (protease II), Clostridium bifermentans and Clostridium perfringens. The protease of the newly described Clostridium botulinum strain 89 (type G) was inhibited by Clostridium sporogenes antiprotease, but not by any of the other antiproteases. It is not possible to differentiate between Clostridium botulinum, Clostridium sporogenes and Clostridium perfringens by use of serological differentiation of their proteolytic enzymes. The protease of Clostridium bifermentans is not serologically related to any of the species tested in this investigation. Proteases produced by different Bacilli were not inhibited by antiproteases from Clostridium botulinum types B, C and F, Clostridium sporogenes, Clostridium bifermentans, and the two strains of Clostridium perfringens tested. This investigation indicates a serological relationship between proteases from different Clostridium species, but not a serological relationship between proteases produced by the Clostridium species and Bacillus species tested.  相似文献   

12.
建立了多重PCR检测产气荚膜梭菌α、β、ε和ι毒素基因的方法。该方法具有良好的特异性和敏感性,只有产气荚膜梭菌呈现阳性,被检验的其他梭菌以及大肠杆菌、葡萄球菌均为阴性;将肉汤菌液样品10倍系列稀释后进行检测,检测灵敏度达到1.2×104CFU/mL。收集40份牛粪便样品,进行PCR检测,32份样品中成功扩增出589 bp的α毒素基因片段,阳性率为80%。结果显示,建立的多重PCR检测方法可取代血清中和试验,用于产气荚膜梭菌分型,同时表明A型产气荚膜梭菌在当地奶牛场中较为普遍。  相似文献   

13.
Malignant oedema is a fatal disease of several animal species, produced by one or more members of the Clostridium genus. We report here a case of malignant oedema in a 1-year-old Friesian sheep after a blood sample was collected from the jugular vein. Clostridium septicum and Clostridium sordellii were isolated from the lesions and also demonstrated by a fluorescent antibody test. This report stresses the need for maintaining a clean environment for animals and for strict hygienic measures during procedures that generate wounds, together with immunity acquired by proper vaccination, for prevention of malignant oedema.  相似文献   

14.
Necrotising enteritis had been the cause of death of 4.9 per cent in 5,177 nursed piglets, which was established by pathological examination. The number of piglets, in that context, which had come from industrialised sow breeding units was equivalent to 92 per cent. The nursed piglet held the third position, next to smaller ruminants (19.4 per cent) and fowl (6.0 per cent), with regard to the occurrence of Clostridium perfringens enterotoxemia or necrotising enteritis in 112,218 animals which were pathologically examined after death. Necrotising enteritis so far has been rare in the GDR. No regional accumulation has been observed. Several outbreaks on industrialised sow breeding units actually remained stationary. The occurrence of the disease may be favoured by a number of factors which are conducive to accumulation of Clostridium perfringens Type C in a given stock. Group keeping of pregnant sows, simultaneous farrowing of larger groups of sows, group treatment of nursed piglets, using neomycin, chloramphenicol, oxytetracycline, and other antibiotics to which Clostridium perfringens is primarily resistant or has acquired resistance in the course of time are some of those contributive factors. Transmission of Clostridium perfringens Type C through feedstuff is possible, though it would lead to a real outbreak only by high intensity of the contamination, and it played a minor role in proliferation of the disease. 3479 Clostridium perfringens strains were isolated from 9,481 animals, both clinically intact and after death, with 30 species being included. Type classification revealed 2454 strains of Type A (70 per cent), 204 of Type D (5.88 per cent), 164 of Type C (four per cent), and 48 of Type B (1.34 per cent). There were 688 atoxic strains (17 per cent). Swine is the major carrier of Clostridium perfringens Type C, with 87 per cent of all Clostridium perfringens Type C strains having been isolated from swine. Swine was followed by fowl (four per cent), sheep (four per cent), cattle, rabbit, and dog (1.27 per cent each). Clostridium perfringens Type C was obtained from the faeces of clinically intact sows in seven instances, including two cases with sows (0.46 per cent) from farms with no previous record of necrotising enteritis.  相似文献   

15.
Nineteen Clostridium perfringens Type C strains and ten foreign control strains of subtypes C1, C3, and C4 were tested for their toxin formation and spore resistance to heat. The 19 Type C strains had been isolated from unweaned piglets in the context of necrotising enteritis outbreaks in the GDR. The Clostridium perfringens Type C strains formed beta-toxin, but they failed to form epsilon-toxin or gamma-toxin, alpha-toxin was successfully recorded from 15 of the 19 strains tested from unweaned piglets. The minor-lethal toxin fractions were also tested, with delta-toxin being recorded from all strains, non-alpha-delta-theta-toxin from six, theta-toxin from five, and K-toxin from one. Tests for delta-toxin, lambda-toxin, and mu-toxin were negative. The Clostridium perfringens Type C strains isolated in the GDR from unweaned piglets with necrotising enteritis were, basically, identical with those described in Denmark by von Hogh (1967) with regard to toxin formation. Clostridium perfringens strains cultured in broilers with necrotising enteritis were characterised by regular toxin production in the context of alpha, theta, delta as well as non-alpha-delta-theta. They failed to form beta, epsilon, gamma and lambda, while mu-toxin was formed by them quite irregularly. They, consequently, are Type A strains. Resistance to chloramphenicol and/or oxytetracycline was exhibited by 78.5 per cent of 237 tested Clostridium perfringens strains which had been isolated from unweaned piglets and broilers with necrotosing enteritis. Multiple resistance was recorded from 33.9 per cent. All strains were susceptible to penicillin, nitrofurantoin, and erythromycin.  相似文献   

16.
In cattle, sheep, and other ruminants, clostridial myonecrosis (gas gangrene) is mostly caused by Clostridium chauvoei, C septicum, C novyi and C sordellii. A polymerase chain reaction (PCR) system using common primers designed from multiple alignment of the 16S rRNA and 23S rRNA genes of Clostridium species was developed to identify pathogenic clostridia. The PCR was performed with total DNA from 26 strains which included seven different Clostridia species. These bacteria were differentiated at species level by the different PCR product patterns. To characterise the 16S-23S rDNA spacer regions of these clostridia further, most PCR products of these bacteria were sequenced. The smallest PCR products of each bacterium represented the fundamental 16S-23S rDNA spacer region; larger PCR products of each bacterium were caused by insertion sequences, i.e. tRNA gene sequences. The authors' observations indicate that the PCR patterns of the 16S-23S rDNA spacer regions have the potential to be used as an identification marker of pathogenic clostridia in gas gangrene.  相似文献   

17.
In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.  相似文献   

18.
In this prospective study, feces of dogs with diarrhea were compared with feces of normal dogs for the presence of Clostridium difficile, C difficile toxins A and B, C perfringens, and C perfingens enterotoxin (CPE). C difficile toxins A, B, or both were present in feces of 18 of 87 (21%) dogs with diarrhea and 4 of 55 (7%) normal dogs (P = 0.03), whereas CPE was present in the feces of 24 of 87 (28%) dogs with diarrhea and 3 of 55 (5%) normal dogs (P = 0.01). C difficile was isolated from 2 of 87 (2%) dogs with diarrhea but was not isolated from the feces of 55 normal dogs, possibly because of poor survival of the organism in fecal samples. C perfringens was isolated from the feces of 23 of 24 (96%) CPE-positive dogs with diarrhea, 52 of 63 (83%) CPE-negative dogs with diarrhea, and 39 of 55 (71%) CPE-negative dogs with normal feces. No correlation was found between C perfringens spore number and the presence of CPE.  相似文献   

19.
就腐败梭菌?毒素的理化性质、生物学特性、结构与功能、致病机制、免疫原性和应用等方面国内外的研究进展进行了论述,以期为腐败梭菌病的防治以及病原学和其他相关分子生物学的研究提供借鉴。  相似文献   

20.
The objectives of this study were to examine the potential roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs by comparison of isolation, determination of toxin status via enzyme-linked immunosorbent assay (ELISA), and application of multiplex polymerase chain reaction (PCR). These techniques were used to evaluate fecal specimens in 132 healthy and diarrheic dogs. These dogs were prospectively evaluated by grouping them into the following 3 categories: hospitalized dogs with diarrhea (n = 32), hospitalized dogs without diarrhea (n = 42), and apparently healthy outpatient dogs without diarrhea (n = 58). All fecal specimens were cultured using selective media for C difficile, Salmonella spp., and Campylobacter spp. and selective media after heat shock for C perfringens. No significant difference was found in the isolation of C perfringens or C difficile among the 3 groups. A significant association was found between the presence of diarrhea and detection of C perfringens enterotoxin (CPE) or toxin A via ELISA for both C perfringens and C difficile, respectively. PCR performed on C difficile isolates for toxin A and toxin B genes revealed no significant differences among the 3 groups, but diarrheic dogs were significantly more likely to be positive for the enterotoxin gene of C perfringens. Based on the results of this study, the use of ELISA for detection of CPE in feces combined with the detection of enterotoxigenic fecal isolates obtained via heat shock provides the strongest evidence for the presence of C perfringens-associated diarrhea.  相似文献   

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