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1.
A peroxidase-antiperoxidase (PAP) technique was used to diagnose bacillary haemoglobinuria in formalin-fixed, paraffin-embedded liver tissues of cattle. The PAP method revealed Clostridium haemolyticum in the zone of liver necrosis characteristic of the disease and also in culture smears of this microorganism, but C. novyi type B, C. chauvoei, C. septicum and C. perfringens types B and C remained unstained by the PAP reaction. The PAP technique performed provides a specific, simple and rapid method to diagnose bacillary haemoglobinuria.  相似文献   

2.
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   

3.
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   

4.
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   

5.
We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens.  相似文献   

6.
二重PCR方法鉴别气肿疽梭菌和腐败梭菌   总被引：1，自引：1，他引：0  
用包含16S～23S rDNA间隔区和23S rDNA的部分序列作为气肿疽梭菌特异性标志,以α毒素部分序列作为腐败梭菌特异性标志,建立了鉴别气肿疽梭菌和腐败梭菌的二重PCR方法。结果显示：气肿疽梭菌C54-1株扩增出大小为509 bp的条带,腐败梭菌C55-1株、C55-16株均扩增出大小为148 bp的条带,均与预期吻合;而产气荚膜梭菌C57-1株、C59-37株,肉毒梭菌C62-4株,诺维梭菌C61-4株均未扩增出任何条带。扩增产物的测序结果进一步证实了本方法的特异性。菌株的生物学特性试验结果也符合相应气肿疽梭菌和腐败梭菌的特点。本研究所建立的二重PCR方法可用于气肿疽梭菌和腐败梭菌的快速鉴定。  相似文献   

7.
Characterization of flagellin from Clostridium chauvoei.   总被引：1，自引：0，他引：1  
Differential centrifugation and cesium chloride-equilibrium centrifugation were used to purify the flagella from the strain Okinawa of the formalin-fixed Clostridium chauvoei. SDS-PAGE profile of purified flagella showed that a major protein band with a molecular mass of 46 kDa, corresponding to the flagellin monomer, and at least two minor protein bands with molecular masses of approximately 73 and 100 kDa were found. The amino acid composition of C. chauvoei flagellin was similar to the flagellin of Salmonella typhimurium and Bacillus subtilis. In addition, C. chauvoei flagellin monomer shared limited sequence homology with the N-terminal amino acid sequence reported for other bacterial flagellins. N-terminal sequences of two minor bands corresponded to the flagellin monomer, indicating that higher molecular mass bands were polymeric forms of the flagellin monomer.  相似文献   

8.
Clostridium chauvoei causes blackleg, which is difficult to distinguish from the causative clostridia of malignant edema. Therefore, a single-step PCR system was developed for specific detection of C. chauvoei DNA using primers derived from the 16S-23S rDNA spacer region and partial 23S rDNA sequences. The specificity of the single-step PCR system was demonstrated by testing 37 strains of clostridia and 3 strains of other genera. A 509 bp PCR product, which is a C. choauvoei-specific PCR product, could be amplified from all of the C. chauvoei strains tested, but not from the other strains. Moreover, this single-step PCR system specifically detected C. chauvoei DNA in samples of muscle from mice 24 hr after inoculation with 100 spores of C. chauvoei, and in clinical materials from a cow affected with blackleg. These results suggest that our single-step PCR system may be useful for direct detection of C. chauvoei in culture and in clinical materials from animals affected with blackleg.  相似文献   

9.
Clostridium chauvoei is a causative agent of blackleg and the major protective antigen of the organism is the flagellar protein. Using an Escherichia coli expression library of the C. chauvoei Okinawa strain, we isolated the fliC gene encoding the flagellin protein. DNA sequence analysis revealed an open reading frame of 413 amino acid residues with a calculated molecular mass of 43819Da. Comparison of the sequence with those of flagellins from other bacteria showed considerable homology in the N-terminal and C-terminal domains. The glutathione-S-transferase (GST)-flagellin fusion protein and the purified FliC protein after removing the GST part with thrombin reacted with both polyclonal antisera and the non-protective monoclonal antibody (Mab), Mo-114. However, the protective Mab, Mo-41, which may recognize its conformational epitope, failed to react with both the GST-flagellin fusion protein and the purified FliC. Furthermore, the GST-flagellin fusion protein and the purified FliC induced very little protective immunity in mice. These results suggested that a conformation-dependent epitope play an important role in the development of immunity against blackleg.  相似文献   

10.
The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents against a total of 33 isolates of Clostridium perfringens, Clostridium septicum and Clostridium sordellii from cattle affected with malignant edema in Japan was determined. The low MIC activities of benzylpenicillin confirm the place of benzylpenicillin as the antibiotics of choice for treatment of malignant edema. Five (22%) of 23 C. septicum strains, five (71%) of seven C. perfringens strains and all strains of C. sordellii showed resistance to oxytetracycline. These oxytetracycline-resistant strains carried tetracycline-resistance genes [tetA(P), tetA408(P), tetB(P) and tetM]. The sequences of the tetracycline-resistance genes of some C. septicum strains were completely or nearly completely identical to those of strains belonging to other clostridiual species. This is the first report of resistance of C. septicum to tetracycline.  相似文献   

11.
In a bacteriological study of 173 cases of clostridial myositis in cattle, Cl chauvoei either alone or with Cl septicum was demonstrated in 97 (56 %)min a further 62 (36%) cases Cl novyi was demonstrated either alone or with Cl septicum. On 11 (6%) occasions Cl septicum only was recovered, and on another three 1-7%) Cl sordelli only. The gross pathology of the lesions was correlated with the bacteriological findings, and prophylaxis in relation to the infections identified is discussed.  相似文献   

12.
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   

13.
Over 3 years, the immunogenic responses of various batches of multi-component clostridial vaccines in sheep, rabbits and guinea pigs were compared. Fully susceptible healthy sheep were found to be more suitable than rabbits or guinea pigs for testing the potency of multi-component clostridial vaccines containing Clostridium novyi type B, C. perfringens type D, C. septicum and C. tetani, and recommendations are made that sheep are the preferred species for testing the potency of clostridial vaccines.  相似文献   

14.
Two polymerase chain reaction (PCR)-based procedures for typing Clostridium, perfringens, which affects most domestic animals, were compared and evaluated for efficiency as substitute to the guinea-pig intradermal test routinely used in our laboratory, namely a multiplex PCR and a protocol based on the individual amplification of gene sequences specific for each toxin. Reference isolates of C. perfringens types A, B, C and D as well as cultures from clinical specimens were tested. The sensitivity and specificity of the PCR was confirmed on reference isolates. There was similarity in results on 43 of the 46 samples typed by all 3 methods. Clear results were obtained by PCR on 5 clinical samples that showed either equivocal or weak skin reactions in guinea-pigs. The multiplex PCR protocol, in combination with the evaluation of bacterial growth, is a better alternative to in vivo toxin typing, since C. perfringens can only be incriminated as cause of a disease when it is present in large numbers in the intestine.  相似文献   

15.
In a Croatian outdoor pig breeding unit 32 sows (died between days 2 and 14 post partum) were subjected to gross pathological and further laboratory investigations. Necropsy findings revealed tympany and purple discoloration of the skin, the surface of the livers was dark and had honeycomb appearance with gas bubble infiltrations, congested lungs, hemorrhages, serosanguinous exsudates in body cavities and the stomachs were full. Gram-stains of smears revealed large numbers of Gram-positive rods. Anaerobic cultures yielded high numbers of Clostridium (C.) novyi and fluorescent antibody test (FAT) confirmed this diagnosis. Enzyme immunoassay and toxin testing by neutralisation in Chinese hamster ovary cells revealed toxin B. Based upon the clinical symptoms, gross-pathological signs, bacteriological findings and toxin testing we concluded that C. novyi caused sow mortality. Suboptimal outdoor environment and high outdoor infectious pressure might have contributed the C. novyi caused losses in this unit.  相似文献   

16.
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   

17.
The effect of cobra venom factor (CoVF) treatment was examined to clarify the mechanism of resistance of mice to Clostridium chauvoei infection. In CoVF-treated mice inoculated with spores of C. chauvoei, no death occurred and the organisms in the infected muscle progressively decreased, similar to that of non-treated control mice. These results indicated that C3 did not play a significant role in the resistance of mice against C. chauvoei infection.  相似文献   

18.
Using ammonium sulphate fractionation, the Clostridium chauvoei hemolysin was purified by cation exchange chromatography and sephacryl S-100 gel filtration. The molecular mass of the hemolysin, determined by SDS-PAGE was found to be approximately 27kDa. The activity of the hemolysin was determined in erythrocytes of various animals, with sensitivities observed in the order of cow, sheep, chicken, rabbit, rat, mouse, dog and horse. Temperature affected the sensitivity of erythrocytes to C. chauvoei hemolysin. These results may reflect distinct characteristics of the hemolytic activity of C. chauvoei hemolysin and that the hemolysin may be pore-forming.  相似文献   

19.
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   

20.
ABSTRACT: Clostridium chauvoei is the causative agent of blackleg, a wide spread serious infection of cattle and sheep with high mortality. In this study we have analyzed the sialidase activity of the NanA protein of C. chauvoei and cloned the sialidase gene nanA. Sialidase is encoded as a precursor protein of 722 amino acids with a 26 amino acid signal peptide. The mature sialidase has a calculated molecular mass of 81 kDa and contains the carbohydrate binding module 32 (CBM32, or F5/8 type C domain), the sialic acid binding module CBM40 and the enzymatically active sialidase domain found in all pro- and eukaryotic sialidases. Sialidase activity does not require the CBM32 domain. The NanA protein is secreted by C. chauvoei as a dimer. The nanA gene was found to be conserved and sialidase activity was found in C. chauvoei strains isolated over a period of 50 years from various geographical locations. Antiserum directed against a recombinant 40 kDa peptide containing CBM40 and part of the enzymatically active domain of NanA neutralized the secreted sialidase activity of all C. chauvoei strains tested.  相似文献   

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