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1.
In the present review article, recent molecular advances relating to studies with Taylorella equigenitalis, as well as the recently described second species of the genus Taylorella, namely Taylorella asinigenitalis, have been described. Molecular genotyping of T. equigenitalis strains by pulsed-field gel electrophoresis (PFGE) after digestion with the suitable restriction enzyme(s) enabled the effective discrimination of strains, thus allowing the examination of the scientific mechanism(s) for its occurrence and transmission of contagious equine metritis (CEM). Alternatively, polymerase chain reaction (PCR) amplification and nucleotide sequencing of the 16S ribosomal DNA sequence and/or the other species specific sequence(s) as targets were confirmed to be effective for identification of T. equigenitalis. These new analytical methods at the genomic DNA level also enabled the discrimination of the newly discovered donkey-related T. asinigenitalis from T. equigenitalis, and moreover, the performance of phylogenetic analysis of genus Taylorella organisms with other closely related genera. Furthermore, detailed analysis of the genes responsible for CEM within the T. equigenitalis genome would be useful to help elucidate the pathogenic virulence and transmission mechanisms associated with the important equine pathogen associated with CEM.  相似文献   

2.
Contagious equine metritis (CEM), caused by Taylorella equigenitalis, is a widely known highly contagious genital equine disease that is transmitted venereally. A new bacterium, Taylorella asinigenitalis resembling T. equigenitalis was recently isolated from three American donkey jacks, at routine testing for CEM. The purpose of this study was to identify and characterize a strain of Taylorella sp. from the genital tract of a stallion. Swab samples for culture of T. equigenitalis were taken from urethral fossa, urethra and penile sheath of a 3-year-old stallion of the Ardennes breed when it was routinely tested for CEM. A small Gram-negative rod was isolated, but the colony appearance, the slow growth rate and the results in the API ZYM test differed slightly from those of T. equigenitalis. Sequencing of the 16S rRNA gene was therefore performed and phylogenetic analysis demonstrated that the sequence of the strain Bd 3751/05 represents T. asinigenitalis and that the strain is identical with the Californian asinine strain UCD-1T (ATCC 700933T). The T. asinigenitalis strain had a low MIC of gentamicin (MIC16 microg/ml). Taylorella asinigenitalis has thus for the first time been isolated from the genital tract of a stallion with a natural infection. To determine the pathogenicity of T. asinigenitalis it will be important to conduct further experimental studies. Sequence analysis of 16S rRNA genes was shown to be a reliable tool for differentiation of T. asinigenitalis from T. equigenitalis as well as for identification of these species.  相似文献   

3.
A total of 57 Taylorella equigenitalis (n=22) and Taylorella asinigenitalis (n=35) isolates was shown not to carry any intervening sequences (IVSs) within 16S rRNA gene sequences. By contrast, we have already shown the genus Taylorella group to carry several kinds of IVSs within the 23S rRNA gene sequences.  相似文献   

4.
: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.  相似文献   

5.
A direct-PCR assay was developed for the rapid detection of Taylorella equigenitalis, a Gram-negative bacterium responsible for contagious equine metritis (CEM) in Equidae. The bacteria may be detected in equine genital swabs without need for a preliminary step of DNA extraction or bacterial isolation. Specificity was determined with 125 isolates of T. equigenitalis, 24 isolates of Taylorella asinigenitalis, five commensal bacteria of the genital tract and a facultative intracellular pathogen of foals found in large concentration in soil. Our PCR is specific and amplified a 413-bp 16S ribosomal DNA product only in all T. equigenitalis.  相似文献   

6.
A 'culture-LightCycler PCR' assay has been developed for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM) in horses. The primers and hybridisation probes were derived from the 16S rDNA sequence. Their specificity was determined in two closely related organisms and six commensal bacteria of the genital tract. The assay was specific for T. equigenitalis and discriminates T. asinigenitalis isolates. It also avoids misidentifications of morphologically and phenotypically similar organisms. The sensitivity was evaluated in comparison to a standard bacteriological culture method. It detected T. equigenitalis in 10 of 52 samples that had not been identified bacteriologically. The results indicated that the assay had a greater sensitivity. This is the first real-time PCR for the detection of T. equigenitalis and avoids PCR carry-over contamination. The 'culture-LightCycler PCR' assay is specific, sensitive and reproducible, and can be used effectively for the detection of T. equigenitalis infections.  相似文献   

7.
Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is used for bacterial identification by analyzing the spectra of isolates and comparing them against a database of reference spectra; it is known for its rapidity and accuracy. Although MALDI-TOF MS is used for identification of bacterial isolates from animals, not all animal pathogens are identified correctly. In this study, we used a commercial MALDI-TOF MS identification system to examine 3724 bacterial isolates from horses and their environments. Isolates that could not be identified with MALDI-TOF MS were identified by 16S rRNA gene sequence taxonomic analysis. MALDI-TOF MS could identify 86.2% of the isolates from horses to the species level, showing that this method could be successfully applied for bacterial identification in horses. However, some species known to be equine pathogenic agents including Taylorella equigenitalis and Rhodococcus equi were difficult to identify with MALDI-TOF MS, which might be the result of an inadequate reference database. Some Prevotella, Staphylococcus, and Streptococcus isolates, which could not be identified with either MALDI-TOF MS or 16S rRNA gene sequencing analysis, formed clusters in the 16S rRNA phylogenic tree, and might be unknown species isolated from horses. Adding the spectra of isolates identified in this study to an in-house database might make MALDI-TOF MS a more useful tool for identifying equine isolates.  相似文献   

8.
It is difficult to distinguish isolates of Taylorella equigenitalis, the cause of contagious equine metritis, from a T. equigenitalis-like organism isolated from asymptomatic donkeys and horses. Although T. equigenitalis is responsible for a severe, contagious disease of the reproductive tract of equids, the T. equigenitalis-like organism, although contagious, does not appear to produce disease. Because of the economic consequences of correctly distinguishing isolates of these 2 microorganisms, a polymerase chain reaction (PCR)-based assay was developed that will distinguish isolates of T. equigenitalis from the T. equigenitalis-like microorganism. The primers used in the PCR assay were designed to amplify unique regions of the gene encoding the 16S ribosomal RNA.  相似文献   

9.
Contagious equine metritis (CEM) is an important venereal disease of horses that is of concern to the thoroughbred industry. Taylorella equigenitalis is a causative agent of CEM but very little is known about it or its close relative Taylorella asinigenitalis. To reveal novel information about Taylorella biology, comparative genomic analyses were undertaken. Whole genome sequencing was performed for the T. equigenitalis type strain, NCTC11184. Draft genome sequences were produced for a second T. equigenitalis strain and for a strain of T. asinigenitalis. These genome sequences were analysed and compared to each other and the recently released genome sequence of T. equigenitalis MCE9. These analyses revealed that T. equigenitalis strains appear to be very similar to each other with relatively little strain-specific DNA content. A number of genes were identified that encode putative toxins and adhesins that are possibly involved in infection. Analysis of T. asinigenitalis revealed that it has a very similar gene repertoire to that of T. equigenitalis but shares surprisingly little DNA sequence identity with it. The generation of genome sequence information greatly increases knowledge of these poorly characterised bacteria and greatly facilitates study of them.  相似文献   

10.
REASONS FOR PERFORMING STUDY: The prevalence of Taylorella equigenitalis infection in Slovenia is unknown and methods used to refine identification in these stallions are required. HYPOTHESIS: In diagnosis of T. equigenitalis, polymerase chain reaction (PCR) would have advantages over culture methods, especially in cases where small numbers of causal agent or intensive contamination of genital swabs are involved. METHODS: Culture method and PCR were used to examine a total of 980 genital swabs from the urethra and fossa urethralis of 245 stallions for the presence of the contagious equine metritis organism. RESULTS: Among 245 examined stallions, 225 (91.8%) were negative to T. equigenitalis by both methods. From the swabs of 17 stallions (6.9%) T. equigenitalis was isolated at first and/or second sampling. Swabs of 3 (13%) stallions were PCR positive but the isolation of T. equigenitalis failed. The rate of T. equigenitalis detection was higher with PCR than with the classic bacteriological examination. CONCLUSIONS AND POTENTIAL RELEVANCE: PCR protocol used in this study provided a specific, sensitive, and simple tool for rapid detection of T. equigenitalis. PCR is especially valuable in cases of intensive bacterial and fungal contamination of swabs where the isolation of T. equigenitalis usually fails.  相似文献   

11.
A discriminatory real time PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and the related species T. asinigenitalis was developed for the direct examination of genital swabs. The 112bp amplicons produced from the two species were discriminated from each other using TaqMan probes labelled with different fluorophores. The TaqMan PCR was shown to be specific for the 16S ribosomal DNA of the two species of taylorella and did not cross-hybridise with the 16S ribosomal DNA of other bacteria tested. Direct amplification from genital swabs was shown to be equally sensitive to that of culture methods. Prevalence in a sample set from The Netherlands was shown to be equivalent to that demonstrated by culture. A companion real time PCR that amplified a fragment of the 16S rDNA gene of equine commensal bacteria was developed to ensure bacterial DNA was extracted from swab material supplied for testing. The use of a rapid and reliable real time PCR for the organism causing CEM should aid the control of this disease.  相似文献   

12.
This study evaluated 16S rRNA gene sequence analysis methods as tools for identification of 22 phenotypically difficult to identify veterinary clinical bacterial isolates in a veterinary diagnostic laboratory. The study compared 16S rRNA gene sequencing and conventional phenotypic identification methods. Using 16S rRNA full-gene sequencing, 95% (21/22) of the isolates were identified to the genus level and 86% (19/22) to the species level. The conventional or commercially available manual identification phenotypic characterization methods presumptively identified 91% (20/22) of the isolates to the genus level and 1 isolate to the species level. However, only 55% (12/22) or 4.5% (1/22) of the phenotypic identifications were correct at the genus or species level when they were compared with the 16S rRNA full-gene sequencing. This study also compared 16S rRNA full-gene and partial-gene sequencing. The results demonstrated that the best 16S rRNA gene-sequencing approach is full-gene sequencing because it gives the most precise species identification. Sequencing of the variable regions 1, 2, and 3 of the 16S rRNA gene could be used for tentative identification because the ability of this sequencing to identify bacteria to the genus level is similar to that of the 16S rRNA full-gene sequencing. This method identified only 14% (3/22) isolates differently to the species level compared with the 16S rRNA full gene sequence. Sequencing of the variable regions 7, 8, and 9 is not recommended because it gives more ambiguous identifications. The cost of a 16S RNA full-gene-sequencing analysis was Can 160 dollars and Can 60 dollars for a partial 16S rRNA gene sequence, i.e., sequencing of variable regions 1, 2, and 3 or variable regions 7, 8 and 9.  相似文献   

13.
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   

14.
Isolation and identification of Taylorella equigenitalis, the causative agent of contagious equine metritis, by bacteriology is laborious and does not permit differentiation from the other member of the genus, Taylorella asinigenitalis. Moreover, other organisms such as Klebsiella pneumoniae and Pseudomonas aeruginosa can also cause endometritis in mares and warrant diagnostic detection. Our objectives were to develop a rapid preparation method for field swab samples and to validate this protocol using new multiplex real-time polymerase chain reaction (rtPCR) detection tools for identification of these four pathogens. The complete analytical process from sample preparation to PCR analysis was then evaluated against bacteriology, the World Organisation for Health’s (OIE) gold standard method for T. equigenitalis and commonly used for the other three pathogens. The diagnostic sensitivity and specificity of this method, which used direct lysis and a multiplex rtPCR, were 100% and >92%, respectively. This study provided a simple-to-use method for prebreeding screening of mares and stallions.  相似文献   

15.
The effectiveness of the polymerase chain reaction (PCR) as a field application test for the eradication of contagious equine metritis (CEM) was evaluated. Seven-thousands five-hundred and thirty-four genital swabs were collected from 4,026 Thoroughbred broodmares and stallions in Japan to test "high risk" horses as well as for general surveillance testing from 1998 to 2001. Bacterial isolation as well as PCR testing of original specimens and cultured specimens was performed for detection of Taylorella equigenitalis from genital swabs. As a result, T. equigenitalis was detected in 12 mares and 1 stallion by PCR, although the bacteria were isolated from only 2 of the PCR-positive mares. CEM-infected and carrier horses were treated by a combination of chemotherapy and surgery. Subsequent follow-up testing over a 3-year period did not detect T. equigenitalis. It was demonstrated that PCR testing was more sensitive than isolation as a method for the detection of T. equigenitalis from genital swabs of horses in the field. It was therefore suggested that a combination of PCR testing and treatment were useful measures in the eradication of CEM from Japan.  相似文献   

16.
OBJECTIVE: To characterize clinical, serologic, bacteriologic, cytologic, and pathologic endometrial responses of mares to 2 donkey-origin atypical bacterial isolates resembling Taylorella equigenitalis. DESIGN: Prospective in vivo study. ANIMALS: 10 healthy mares. PROCEDURE: Mares in estrus (2/group) were inoculated by intrauterine infusion with 2 isolates of classic T equigenitalis or 2 isolates of atypical Taylorella sp or were sham-inoculated. Bacteriologic, serologic, clinical, uterine, cytologic, and pathologic endometrial responses were assessed 4, 11, 21, 35, and 63 days after inoculation and on day 111 in mares with positive culture results on day 63. RESULTS: One atypical isolate failed to cause infection. The second atypical isolate and both classic T equigenitalis isolates induced similar transient metritis and cervicitis. Both classic isolates and 1 atypical isolate induced anti-T equigenitalis complement-fixing antibodies detectable at day 11. Classic isolates and an atypical isolate provoked intense neutrophilic endometritis followed by a resolving, subacute, neutrophilic-mononuclear endometrial response. The atypical isolate and classic isolates were recovered from the uterus, clitoral fossa, or clitoral sinus of one or both exposed mares for as long as 111 days. CONCLUSIONS AND CLINICAL RELEVANCE: Atypical Taylorella sp infections should be considered as a differential diagnosis of equine infertility in US-origin mares, even those not exposed to stallions from countries where contagious equine metritis occurs. The origins and prevalence of atypical Taylorella sp infection in US horses and donkeys are undetermined.  相似文献   

17.
Bovine keratoconjunctivitis (BKC), colloquially referred to as 'pinkeye', is a disease affecting cattle worldwide; it costs cattle producers millions of dollars in economic loss annually. While Moraxella spp. are the primary etiologic agent of pinkeye, surveys of flora from the conjunctivae of livestock from around the world have indicated that a variety of bacterial commensals occupy this niche. We used molecular biology-based methods to determine the composition of bacterial flora in the conjunctivae of normal dairy and beef cattle from Maryland (n=113), and beef cattle with clinical BKC from Louisiana (n=42). Three regimens were used: 16S rRNA PCR and DGGE analysis of amplicons; 16S rRNA PCR and cloning of amplicons into Escherichia coli followed by screening and sequencing of clones harboring inserts; and culture of bacteria on chromogenic agar followed by 16S rRNA PCR and sequencing. Most taxa were comprised of saprophytes found in the environment, such as Bacillus, Pantoea, E. coli, and Exiguobacterium. Moraxella spp. were infrequently observed. Some species, such as Propionibacterium acnes, represent taxa not previously associated with the conjunctivae. Bacillus pumilus and Bacillus licheniformis isolates from the conjunctivae of Maryland cattle were genetically distinct from isolates previously implicated in septic infections in cattle at the same location. We conclude that employing 16S rRNA-based methods for bacterial identification can be useful in defining the flora present in the conjunctivae of normal cattle, and those with BKC.  相似文献   

18.
In order to establish a rapid diagnostic method for contagious equine metritis (CEM), we developed and evaluated a polymerase chain reaction (PCR) test. Species-specific PCR primer sets were derived from the DNA sequence of a cloned DNA fragment of Taylorella equigenitalis that did not hybridize with the genome of a taxomonically related species, Oligella urethralis. Single step PCR with primer set P1-N2 and two-step semi-nested PCR with primer sets P1-N2 and P2-N2 detected as low as 100 and 10 CFU of the bacteria, respectively. Single-step PCR detected T. equigenitalis from genital swabs of experimentally infected mares with sensitivity comparable to that of bacterial isolation. Furthermore, two-step PCR was more sensitive than the culture method. Upon examination of field samples, 12 out of 3,123 samples were positive by single-step PCR while only 2 were positive by bacterial culture. The 12 PCR-positive samples originated from 5 mares, of which 3 animals were considered to be carriers based on previous bacteriologic and serologic diagnoses for CEM. The PCR test described in this study would provide a specific and highly sensitive tool for the rapid diagnosis of CEM.  相似文献   

19.
多杀性巴氏杆菌是一种常见病原菌，其血清型众多（16种血清型），在毒力及流行病学等方面的生物学特性差异很大。分子生物学鉴定和特性分析方法能直接揭示其基因型上的差异，具有高灵敏度和可靠性。本文综述了多杀性巴氏杆菌的PCR、菌落杂交和16S rRNA基因测序等鉴定方法以及限制性酶切分析、核糖体分型、脉冲场凝胶电泳、PCR指纹等特性分析方法。  相似文献   

20.
Because the first strains of Taylorella equigenitalis isolated in the Netherlands autoagglutinated, identification was difficult. The source of carbon dioxide to create a carbon dioxide atmosphere for incubation influenced the emulsifiability of these strains. Strains were emulsifiable when cultivated in a carbon dioxide incubator (7 per cent carbon dioxide in air), but were autoagglutinable when cultivated in a candle jar, or in a jar with a carbon dioxide system or anaerobic system without the palladium catalyst. When strains autoagglutinated, they were identified by the indirect fluorescent antibody test. Although strains of other bacterial species, in particular Pasteurella haemolytica, also showed fluorescence, which was partly caused by autofluorescence, the indirect fluorescent antibody test appeared to be a reliable additional test for identifying T equigenitalis.  相似文献   

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