首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 15 毫秒
1.
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   

2.
A wild-type isolate with similar morphological and phenotypic properties to Taylorella equigenitalis, the causative bacterial agent of contagious equine metritis (CEM), was referred for molecular identification by PCR amplification of the 16S rRNA gene. A species-specific PCR failed to yield a product compatible with that of T. equigenitalis. The direct sequencing of the universal 16S rRNA PCR amplicon suggested the presence of a Bacteroides sp., probably Bacteroides ureolyticus, with no consequent effects on the movement and transportation of the animal. Adoption of such a molecular means of identification through sequencing may aid in the identification of the atypical forms of Taylorella equigenitalis, as recently described, as well as differentiating this species from Taylorella asinigenitalis.  相似文献   

3.
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   

4.
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   

5.
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   

6.
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   

7.
: 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   

8.
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   

9.
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   

10.
Contagious equine metritis (CEM) is a highly contagious venereal disease of horses caused by Taylorella equigenitalis. During testing for semen export purposes, a stallion in Kentucky was found to be T. equigenitalis culture positive in December of 2008. This finding triggered an extensive regulatory investigation to search for additional positive horses, determine the extent of the outbreak, identify the potential source of the outbreak, and ultimately return the United States to CEM-free status. The investigation included over 1000 horses located in 48 states. Diagnostic testing found a total of 22 stallions, 1 gelding and 5 mares culture positive for T. equigenitalis. Epidemiologic analysis indicated that all of the positive horses were linked to a single common source, most likely a Fjord stallion imported into the United States in 2000. The T. equigenitalis strain subsequently spread to other stallions via undetermined indirect mechanisms at shared breeding facilities, and to mares via artificial insemination and live breeding. This CEM outbreak and investigation represent the largest ever in the United States based on the number of exposed horses tested and their geographic distribution.  相似文献   

11.
The objective of this study was to examine the degree of phenotypic and genotypic diversity between 43 French Taylorella asinigenitalis strains isolated from 22 jacks, two stallions and one mare between 1995 and 2008 by culturing genital swabs obtained during routine diagnosis for contagious equine metritis. This retrospective analysis revealed the existence of T. asinigenitalis species since 1995 and the natural colonization of a mare's genital tract in 2001. Despite the presence of 27 different patterns revealed by the combination of API ZYM, antibiogram and 16S rDNA profiles, we show that T. asinigenitalis is a highly homogeneous species. API ZYM diversity only concerns acid phosphatase and naphthol-AS-BI-phosphohydrolase activity. The majority of strains are susceptible to a wide range of antimicrobial agents but most are streptomycin-resistant (95.5%), ampicillin-resistant (88.4%), and four strains are atypical due to a high degree of resistance to at least eight antimicrobial agents. 16S rDNA sequence analysis showed only two clusters and revealed similarity of 99.3-100% between T. asinigenitalis strains. The geographic origin of the 43 isolates correlates to the two 16S rDNA clusters.  相似文献   

12.
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   

13.
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   

14.
Contagious equine metritis (CEM) has given rise to international concern since it was first recognized as a novel venereal disease of equids in 1977 and the etiologic agent was identified as a previously undescribed bacterium, Taylorella equigenitalis. Horse industry concerns over CEM centered on the ease with which this bacterium could be disseminated, the significance of T. equigenitalis as a cause of short-term infertility in the mare, and the existence of the carrier state in the stallion and the mare. The first known outbreak of CEM in the United States was in Kentucky in 1978. The economic impact on the Thoroughbred industry in the state was substantial. Before 2008, additional small-scale outbreaks occurred in Missouri in 1979, Kentucky in 1982, and Wisconsin in 2006, nearly all attributed to the importation of carrier animals. On each occasion, appropriate measures were taken to eliminate the infection, resulting in the United States regaining its CEM-free status. With the exception of the 1978 occurrence in Kentucky, none of the subsequent outbreaks significantly affected the horse industry. That changed dramatically in 2008, however, after the discovery of a Quarter horse stallion in Kentucky that cultured positive. Subsequent investigations turned up 23 carrier stallions and 5 carrier mares belonging to 11 breeds and located in 8 states. Shipment of infective semen and indirect venereal contact in stallion collection centers through the use of contaminated fomites were major factors in the spread of T. equigenitalis. Trace-back investigations of some 1,005 exposed and carrier stallions and mares in 48 states have failed to identify the origin of this latest CEM event. Neither clinical evidence of CEM nor decreased pregnancy rates were reportedly a feature in infected or exposed mares. In light of these findings, there was some question of whether or not the considerable expense incurred in investigating the latest CEM occurrence was warranted. Regaining CEM-free status for the United States will present considerable challenges.  相似文献   

15.
An enzyme-linked immunosorbent assay (ELISA) was developed for the serodiagnosis of contagious equine metritis (CEM), a sexually transmitted disease caused by Taylorella equigenitalis. Antigen preparation was simple, and antigens derived from both classical and atypical forms of T. equigenitalis enabled detection of antibody responses elicted in horses experimentally exposed to either form of the bacterium. Sera serially obtained from these horses from 0 to 63 days postexposure were tested by the traditional complement fixation test (CFT) for CEM and with the ELISA, using both antigens separately. There was close agreement between CFT and ELISA methodologies during the postexposure time period used to detect CEM serodiagnostically in regulatory animal health testing programs. Unlike the CFT, which requires an overnight incubation step, the ELISAs are more convenient and can be completed in 3 hours.  相似文献   

16.
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   

17.
The method of rapid slide agglutination and coagglutination was tested in the detection of Haemophilus equigenitalis (Taylorella equigenitalis)--the causal agent of contagious equine metritis (CEM). It was demonstrated that both methods were suitable for the serological diagnosis of the species under study. The antisera obtained from rabbits immunized with Haemophilus equigenitalis strains treated in different ways were specific, but with different antibody titres. When cross reactions with other species of microorganisms were verified, the antisera did not react with any of the strains, even after binding them to protein A of the positive strain Staphylococcus aureus--Cowan I. Coagglutination was much more rapid and pronounced than the ordinary rapid agglutination test. It was characterized by a low consumption of specific antiserum. The specific antibodies bound to staphylococci were kept at the temperature of 4 degrees C for several months without losing agglutinin activity.  相似文献   

18.
In this study 55 strains of Taylorella equigenitalis isolated from horses of four different studs in Austria, and a comparative strain from the Federal Republic of Germany were investigated by different methods. These investigations were carried out with the help of SDS-PAGE, immunoblotting, the analyses of genomes and by proof of plasmids. Furthermore, pathogenic mechanisms such as adhesion or the formation of toxins were investigated in vitro. On the basis of the results carried out by means of SDS-PAGE and immunoblotting all tested strains of Taylorella equigenitalis were alike, whereas by DNA analyses the strains could be divided into five groups. The comparative strain from the FRG, which clearly differed from the Austrian strains, formed one group all by itself. From three studs, which are related to each other because of an intensive exchange of horses, representatives (n = 53) of three DNA fingerprint groups were isolated. These three fingerprint patterns were very similar to each other, while the hybridisation patterns from the other two Austrian strains were very different. One of these strains, isolated from a diseased mare, could not be distinguished from the other strain isolated from a clinical healthy stallion from the same study by this method. Only 47.3% from the investigated strains showed attachment to HeLa cells, while cell extracts of all of them caused morphological changes of a varying degree of both Y1 and Vero cells. There were no connexions between these adhesion-cytotoxicity-properties and the DNA fingerprint groups as well as the studs, respectively. No plasmids were found in the Taylorella equigenitalis strains used in this study.  相似文献   

19.
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   

20.
The transmission of contagious equine metritis (CEM) on stud farms in Britain, Ireland and other European countries is prevented by following the recommendations in the Horserace Betting Levy Board's Code of Practice on CEM. A quantitative risk assessment was undertaken to estimate the likely impact of removing the recommendation, from the 2002 code, to culture endometrial or cervical swabs microaerophilically for the presence of Taylorella equigenitalis, the causative organism. The scientific literature was reviewed for evidence about the anatomical distribution of T. equigenitalis at different times after infection and it was found that, in chronically infected mares, the organism was detectable in the clitoral swabs of nearly 93 per cent, but in the cervical swabs of only 31 per cent. In contrast, in acutely infected mares, the organism was detectable in the clitoral swabs of nearly 69 per cent, but in the cervical swabs of 84 per cent. By using these results, a quantitative risk assessment was undertaken, assessing the likely effects of removing the recommendation that swabs from the cervix of low-risk mares should be cultured for T. equigenitalis. The results were sensitive to the prevalence of the infection, but when it was low, there appeared to be few benefits in continuing to culture cervical swabs routinely. However, such swabs are vital when the disease is suspected.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号