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1.
Profiles of the genomic DNA of 104 strains ofT. equigenitalis isolated from brood mares with contagious equine metritis in Hokkaido during the breeding seasons from 1980 to 1993, as well as those of five strains (SS28, EQ56, EQ59, EQ70 and HH139) previously isolated in Japan were examined after restriction digestion and crossed-field gel electrophoresis. These profiles were essentially identical to each other and the various isolates and strains appeared to have a common genotype, designated genotype J, with respect to two restriction enzymes,ApaI andNotI. These results suggest a common source for all these isolates obtained over the course of more than 10 years in Japan.Abbreviations CEM contagious equine metritis - CFGE crossed-field gel electrophoresis - PFGE pulsed-field gel electrophoresis  相似文献   

2.
The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184T, Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184T and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184T and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNAIle-tRNAAla-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.  相似文献   

3.
Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE profiles to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T. equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related difference in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classified into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM.  相似文献   

4.
The profiles, after digestion with ApaI or NotI and pulsed-field gel electrophoresis (PFGE), of genomic DNA from 18 strains of Taylorella equigenitalis isolated in Ireland were of three different types. The 13 strains in one of these types gave PFGE profiles identical to that of an American prototype strain, Kentucky 188, but different from strain NCTC11184T and from a strain isolated in Japan.Eight additional strains isolated in the United States gave four distinct types of genomic PFGE profiles. All four types were different from that of T. equigenitalis NCTC11184T or that of the Japanese strain. The profile of three strains of one type was identical to that of Kentucky 188.  相似文献   

5.
Eight strains of Taylorella equigenitalis were identified by a polymerase chain reaction using a primer pair specific to the 16S rDNA of T equigenitalis. These eight strains were chosen because they had previously been shown to represent eight distinct genotypes by pulsed-field gel electrophoresis analysis after separate digestion of the genomic DNA with ApaI or NotI. The eight strains could be classified into six or seven types by random amplified polymorphic DNA analysis using different kinds of primers. Amplified rDNA restriction analysis after separate digestion with five restriction enzymes, including AluI and MboI, of the 1,500 bp fragments of rDNA amplified by polymerase chain reaction did not discriminate the genomic variations among the eight strains of T equigenitalis. Thus, pulsed-field gel electrophoresis was shown to discriminate these eight organisms better than random amplified polymorphic DNA analysis, while amplified rDNA restriction analysis was found to be unsuitable for subtyping T equigenitalis.  相似文献   

6.
Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bacterial culture and polymerase chain reaction (PCR) testing. Our results indicated that IIF using polyclonal antibodies allows T. equigenitalis to be discriminated from T. asinigenitalis. This test constitutes a rapid, sensitive and specific tool for confirming presumptive colonies of T. equigenitalis.  相似文献   

7.
《Veterinary microbiology》1998,62(3):243-250
Pulsed field gel electrophoresis analysis of genomic DNA was used to investigate genetic diversity among Dichelobacter nodosus from footrot in sheep in Malaysia. Twelve Dichelobacter nodosus strains isolated from lesion materials from infected sheep were confirmed as Dichelobacter nodosus by polymerase chain reaction technique using the species-specific Dichelobacter nodosus 16S RNA sequence Ac and C as primers. Pulsed field gel electrophoresis banding profiles using restriction enzymes ApaI (5′GGGCCC3′), SfiI (5′GGCCNNNNNGGCC3′)and SmaI (′5CCCGGG3′) enabled the 12 Dichelobacter nodosus strains to be differentiated into eight different PFGE patterns and thus genome-types, with F (coefficient of similarity) values ranging from 0.17 to 1.0 (ApaI), 0.14 to 1.0 (SfiI) and 0.22 to 1.0 (SmaI). Strains with origin in different farms were shown to have different PFGE patterns (two strains, M7 and M8 were the only exception). On the basis of their PFGE, all field strains used in the study differed from the reference strains. Our data revealed that there are several clonal types of Dichelobacter nodosus isolates and indicated that there is probably more than one source of this pathogen on the farms studied. The study showed that strains of D. nodosus exhibited considerable genetic diversity using this method and that genomic analysis by pulsed field gel electrophoresis was useful in discriminating the D. nodosus strains.  相似文献   

8.
Lipopolysaccharides (LPSs) from Taylorella equigenitalis, the causative agent of contagious equine metritis, and T. asinigenitalis were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharide profiles of 11 T. equigenitalis strains were similar, but different from the profiles of 3 T. asinigenitalis strains, and the profiles of 2 T. asinigenitalis strains were similar to each other. The serological specificities of the LPSs from these 14 strains were examined by immunoblotting and enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) to the LPSs of the T. equigenitalis and T. asinigenitalis type strains and T. asinigenitalis strain 2329–98. A MAb to T. equigenitalis LPS O-polysaccharide (O-PS) (M2560) reacted with LPSs from all T. equigenitalis strains but did not react with LPSs from the 3 T. asinigenitalis strains or with 43 non-Taylorella bacteria. Three MAbs to the T. asinigenitalis type strain LPS O-PS or core epitopes (M2974, M2982, M3000) reacted with the homologous strain and T. asinigenitalis strain Bd 3751/05, but not with any of the other bacteria. Five MAbs to T. asinigenitalis 2329–98 LPS O-PS or core epitopes (M2904, M2907, M2910, M2923, M2929) reacted only with this strain. Proton nuclear magnetic resonance spectra of the O-PSs of the type strains of T. equigenitalis and T. asinigenitalis provided fingerprint identification and differentiation of these 2 organisms. The serological results were consistent with our previous finding that the O-antigen of the type strain of T. equigenitalis, being a linear polymer of disaccharide repeating [→4)-α-L-GulpNAc3NAcA-(1→4)-β-D-ManpNAc3NAcA-(1→] units, differs from that of the T. asinigenitalis O-antigen polymer that is composed of repeating [→3)-β-D-QuipNAc4NAc-(1→3)-β-D-GlcpNAmA-(1→] units. Lipopolysaccharide O-PS could be a specific marker for identification and differentiation of T. equigenitalis and T. asinigenitalis, and provide the basis for the development of specific detection assays for T. equigenitalis.  相似文献   

9.
SUMMARY Polyacrylamide gel electrophoresis and agarose gel electrophoresis were used to resolve restriction endonuclease digests of 20 Australian isolates of Leptospira interrogans cultured from urine samples of cattle with agalactia and abortion. The restriction endonuclease profiles of 19 isolates closely matched the profiles of L interrogans serovar hardjo subtype hardjobovis reference strains. The remaining isolate had a different restriction profile from subtype hardjobovis and subtype hardjoprajitno reference strains and was serologically identified as serovar pomona. Silver staining of polyacrylamide gels gave enhanced resolution of restriction fragments compared with the traditional method of ethidium bromide staining of agarose gels.  相似文献   

10.
Bacterial cell envelope preparations from eight untypable strains ofPasteurella haemolytica were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting with rabbit antisera prepared against the eight untypable strains (one untypable strain per rabbit) and with cattle antisera prepared againstP. haemolytica serotypes 1, 2, 5, 6, 9 and against one heterologous untypable strain. Numerous comigrating and shared antigens were recognized by the eight rabbit antisera and theP. haemolytica serotype cattle antisera. Comigrating antigens at 43 and 30 kilodaltons (kDa) were recognized by all eight rabbit antisera. Shared antigens, detected by all eight rabbit antisera when reacting againstP. haemolytica serotype 1, were recognized at 43, 32, 30, 20 and 15 kDa.  相似文献   

11.
Reasons for performing study: Standard bacteriological methods for identifying Taylorella equigenitalis in cervical smears are time consuming. Therefore, a more rapid real‐time PCR assay was evaluated for its suitability in screening swabs. Objective: To compare the results of a commercially available real‐time PCR assay with routine microbiological culture for the identification of T. equigenitalis, the causative organism of contagious equine metritis, in equine genital swab samples, under ‘field trial’ conditions. Materials and methods: Routine prebreeding genital swabs (n = 2072) collected from Thoroughbred mares and stallions during 2009 were examined together with stored T. equigenitalis positive material. Swabs were cultured for T. equigenitalis using standard microbiological techniques. Bacterial lysates were isolated from the swabs and examined for the presence of a 16S DNA fragment of T. equigenitalis, using a commercial multiplex real‐time PCR assay system. Results: There was complete concordance between positive and negative results obtained by the 2 methods. Real‐time PCR also detected T. equigenitalis DNA from swabs that were negative using standard microbiological culture after 6 months' storage at +4°C but from which T. equigenitalis had been isolated following collection. The sensitivities of realtime PCR and bacterial culture were both 10?3 (equivalent to 3 colony‐forming units). Conclusion and clinical relevance: Routine bacterial culture of T. equigenitalis requires an incubation period of not less than 7 days before a conclusive negative result can be obtained, whereas bacterial extraction and real‐time PCR assay can be completed in less than 6 h. The commercially‐available PCR assay tested provided a rapid and reliable method for the identification of T. equigenitalis from equine genital swabs and could be usefully employed for the screening of mares and stallions for preseason Horserace Betting Levy Board (HBLB) Code of Practice and in other situations such as for bloodstock sales screening requirements, overcoming the current delays imposed by bacterial culture requirements. Its use could be quality assured by the existing HBLB biannual testing scheme for designated laboratories.  相似文献   

12.
Mycoplasma agalactiae and Mycoplasma bovis are closely related species in phylogenetic terms. Pulsed Field Gel Electrophoresis (PFGE) of SmaI, EclXI, Bsi WI, MluI, BssHII, SalI, XhoI, NruI and ApaI digested DNAs were used to analyse and to compare restriction fragment length polymorphism between M. agalactiae and M. bovis and to estimate their genome sizes. SmaI, EclXI and Bsi WI enzymes cleaved DNAs of both microrganisms. MluI, BssHII, SalI, XhoI and NruI digested only M. agalactiae DNA whereas ApaI cut only M. bovis DNA. The total DNA length was established to be 945 ± 8.4 Kb for M. agalactiae and 961 ± 18.9 Kb for M. bovis.  相似文献   

13.
In the present study four methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains isolated from a dog (n = 3) and the anterior nares of the dog owner (n = 1) were investigated by conventional and molecular methods. The species identity of the four S. pseudintermedius strains was confirmed by conventional methods, by PCR mediated amplification of S. intermedius/S. pseudintermedius specific segments of thermonuclease encoding gene nuc and by restriction fragment length polymorphism analysis of phosphoacetyltransferase encoding gene pta. Investigation of the four S. pseudintermedius for toxinogenic potential revealed that all four strains were positive for the exfoliative toxin encoding gene siet and the leukotoxin encoding genes lukS, lukF. The oxacillin and penicillin resistance of the four S. pseudintermedius strains could be determined by cultivation of the strains on oxacillin resistant screening agar base, ChromID MRSA Agar and Brilliance MRSA Agar and by multiplex PCR detecting the resistance genes mecA and blaZ. The genetic relatedness of the strains was studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). According to PFGE all four S. pseudintermedius strains represent an identical bacterial clone indicating a cross transmission between the dog and the dog owner.  相似文献   

14.
The purpose of this study was to develop and apply a multilocus sequence typing (MLST) scheme to study the molecular epidemiology of Brachyspira hyodysenteriae, the aetiological agent of swine dysentery. Sequences of seven conserved genomic loci were examined in 111 B. hyodysenteriae strains. Fifty-eight of these previously had been analysed by multilocus enzyme electrophoresis (MLEE), and for some the results of pulsed field gel electrophoresis (PFGE), restriction endonuclease analysis (REA) and/or serotyping also were available. The discriminatory power of these methods was compared. The strains were divided into 67 sequence types (STs) and 46 amino acid types (AATs) by MLST. The Index of Association value was significantly different from zero, indication that the population was clonal. Eleven clonal complexes (Cc) comprising between 2 and 10 STs were recognised. A population snapshot based on AATs placed 77.5% of the isolates from 30 of the AATs into one major cluster. The founder type AAT9 included 13 strains from nine STs that were isolated in Australia, Sweden, Germany and Belgium, including one from a mallard. The MLST results were generally comparable to those produced by MLEE. The MLST system had a similar discriminatory power to PFGE, but was more discriminatory than REA, MLEE or serotyping. MLST data provided evidence for likely transmission of strains between farms, but also for the occurrence of temporal “micro-evolution” of strains on individual farms. Overall, the MLST system proved to be a useful new tool for investigating the molecular epidemiology and diversity of B. hyodysenteriae.  相似文献   

15.
Tuberculosis in wild seals and characterisation of the seal bacillus   总被引：3，自引：0，他引：3  
SUMMARY Tuberculosis was diagnosed in 3 otariid seals found dead on beaches at 3 locations on the south coast of Western Australian between May 1990 and March 1991. This confirms that tuberculosis is present in the 2 native seals (Neophoca cinerea and Arctocephalus forsteri) in Western Australian waters. Mycobacterium sp isolated from the lungs of 2 of the seals were studied to determine the similarity of the strains to each other, to the strains isolated during 1986 from Australian sea lions and New Zealand fur seals kept in captivity at a marine park near Perth, Western Australia, and to a strain isolated in 1988 from a seal trainer who worked with the infected captive seals for 3 years. After restriction endonuclease analysis (REA) with the endonucleases Bst Ell, Bcl I and Pvu II, one of the wild seal strains appeared to have identical DNA fragment patterns to the strains from the captive seals and the seal trainer. The other wild seal isolate had identical REA profiles using Bst EII and Bcl I, but a minor difference was detected using Pvu II. Differences in these isolates were more clearly seen in restriction fragment length polymorphisms after hybridisation with two DNA probes. The secretory protein MPB70, present in M bovis, was not detected in wild seal isolates using sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting techniques. Analysis of protein and DNA fragment profiles indicated that seal tuberculosis isolates form a unique cluster within the M tuberculosis complex.  相似文献   

16.
The effect of the presence of ruminal protozoa on the composition of fecal microbiota in cattle was investigated. Six castrated Holstein cattle (mean bodyweight 137 kg) were divided into two groups: three faunated and three unfaunated. The fecal bacterial composition of the faunated and unfaunated cattle was compared using a culture method and by terminal restriction fragment length polymorphism (T‐RFLP) analysis. Approximately 0.4 to 2.3% of the bacterial cells detected by microscopy formed colonies on medium 10. The major terminal restriction fragments were detected in the T‐RFLP profiles generated by Hha I and Msp I digestion in both the faunated and unfaunated cattle. In particular, the Bacteroides group, the Clostridium coccoides group and the Clostridium leptum subgroup might be the known bacterial groups that protozoa influence by Msp I digestion. From the dendrogram analysis by T‐RFLP patterns, the faunated and unfaunated cattle were divided into two clusters, I and II, respectively. These results suggest that absence of protozoa in the rumen changes the composition of fecal bacteria.  相似文献   

17.
The technique of RT-PCR and restriction enzyme analysis was standardized to detect and differentiate Newcastle disease viruses. Digestion of RT-PCR-amplified, F gene sequences encoding for the cleavage activation sites of fusion protein with restriction enzymes AluI, BglI, HaeIII, HinfI, HhaI, RsaI, StyI and TaqI was carried out in order to characterize Newcastle disease viruses of varying pathogenicity. Restriction enzyme digestion of the amplicons by BglI and HhaI could group eight viruses, both field isolates and known vaccine strains, into lentogenic, mesogenic and velogenic pathotypes. By employing this technique directly on a clinical sample, Newcastle disease virus of the lentogenic pathotype could be detected.  相似文献   

18.
Mycoplasma hyosynoviae strains from Denmark, Germany, Japan, Sweden, the Netherlands and the UK were examined for variations in the genomic DNA and within the 16S ribosomal RNA (rRNA) gene. Variations in the chromosomal DNA among 57 isolates recovered from the respiratory tract and joints of pigs, were investigated by analysis of amplified fragment length polymorphisms of the BglII and MfeI restriction sites and by pulsed‐field gel electrophoresis of a BssHII digest of chromosomal DNA. Both methods allowed unambiguous differentiation of the analysed strains and showed similar discriminatory potential for the differentiation of M. hyosynoviae isolates. Concordant results obtained with the two whole‐genome fingerprinting techniques evidence the considerable intraspecies genetic heterogeneity of M. hyosynoviae. Sixteen field strains of M. hyosynoviae and the type strain S16T were further examined for variation within the 16S rRNA gene. Ten field strains possessed the 16S rDNA sequences identical to the type strain, while the remaining six strains had sequences that differed by one to two nucleotides from that obtained from the type strain.  相似文献   

19.
The genetic diversity of 60 field strains of Mycoplasma mycoides ssp. mycoides, small colony type (M. mycoides SC), comprising 56 isolates from cattle in Tanzania, one from Kenya, two from Botswana and one from Portugal, as well as the type (PG1T) and vaccine (T1‐SR49) strains, was investigated. The strains were analysed for variations in the EcoRI and Csp6I restriction sites in the genomic DNA using the amplified fragment length polymorphism (AFLP) technique, and variations in the BamHI restriction sites using pulsed‐field gel electrophoresis (PFGE). Six AFLP types were detected among the analysed strains. The AFLP profiles of the type and vaccine strains were indistinguishable from each other. Indistinguishable AFLP profiles were found for 55 Tanzanian field strains, one of them isolated in 1990 and the other 54 isolated in 1998/1999, although one strain isolated in 1999 showed a different profile. Strains from different countries revealed different AFLP profiles. Six PFGE types were detected among the analysed strains, with all the 56 Tanzanian field strains displaying indistinguishable PFGE profiles. Strains from different countries revealed different PFGE profiles, and so did the type and vaccine strains. The strong genomic homogeneity among M. mycoides SC strains associated with outbreaks of contagious bovine pleuropneumonia in different regions of Tanzania suggests that the outbreaks of the disease in the 1990–99 period might have been caused by a single epidemic clone. Moreover, this study has demonstrated that AFLP and PFGE are potential tools for molecular epidemiological studies of M. mycoides SC infections.  相似文献   

20.
A rapid screening assay forCampylobacter fetus in bull semen was developed using the polymerase chain reaction (PCR) and restriction endonuclease analysis (REA) to complement isolation by culture. An oligonucleotide primer pair (C1/C2) from the hypervariable region of 16S rRNA ofC. fetus was used to amplify a 362 base pair fragment by PCR. The PCR/REA assay, which is completed in 10 hours, detected as few as threeC. fetus subsp.venerealis cells in experimentally infected raw bull semen and in semen diluted with milk or egg yolk Tris (EYT). All the strains tested, of both subspecies ofC. fetus, were amplified, as were some otherCampylobacter species. Restricting the amplified products byAluI differentiatedC. fetus from the other organisms. There was no visible product generated by PCR fromC. sputorum subsp.bubulus, a saprophytic organism found in the prepuce of bulls, or from seven other species of bacteria found in semen. A modification of the PCR assay, using another primer pair (C3/C2) and two temperature PCR cycling conditions, increased the probability of detectingC. fetus subsp.venerealis. PCR amplification followed by REA could be used to screen bovine semen rapidly forC. fetus. In most cases, sequencing of C1/C2 PCR generated products would be preferable for distinguishing between the two subspecies ofC. fetus.Abbreviations AI artificial insemination - bp base pair - Cff Campylobacter fetus subsp.fetus - Cfv Campylobacter fetus subsp.venerealis - EYT egg yolk Tris - MH Mueller-Hinton - PCR polymerase chain reaction - REA restriction endonuclease analysis - TE Tris-EDTA  相似文献   

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