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1.
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   

2.
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   

3.
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   

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.
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   

6.
Contagious equine metritis (CEM), a contagious venereal disease of horses, invaded Japan in 1980 and spread in the Thoroughbred population of the Hidaka-Iburi district of Hokkaido. To eradicate CEM, we ran a program aimed at detecting Taylorella equigenitalis, the causal agent, in carrier horses by using the PCR test, followed by culling or treatment. In 2001, the first year of the program, 12,356 Thoroughbred racing stallions and mares were tested and 11 carriers were found. Four, two, one, and one carrier mares were detected in 2002, 2003, 2004, and 2005, respectively, by application of the program at the same scale as in 2001. No PCR-positive horses were found from 2006 to 2010. These results strongly suggest that CEM was eradicated from Japan by 2010.  相似文献   

7.
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   

8.
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   

9.
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   

10.
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   

11.
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   

12.
Contagious equine metritis (CEM) was reproduced in 3 of 4 donkey mares with an Irish streptomycin-resistant strain of Haemophilus equigenitalis isolated from an experimental case of the disease in a pony mare. Although some variability in clinical response occurred, there was no evidence that semen enhanced the clinical severity of the infection. Variable amounts of vaginal discharge and associated inflammatory changes of the vagina and/or cervix, similar to those seen in the horse, were observed. All the affected donkeys made spontaneous clinical recoveries and so far as could be detected, subsequent persistence of H. equigenitalis in the genital tract was of limited duration. Recovery of the bacterium was not associated with oestrus and there was no evidence that it persisted in the clitoral area after it could no longer be cultured from the anterior genital tract. Cytological examination of smears of intra-uterine or cervical swabs was of diagnostic value only during the clinical phase of the infection. Serological responses demonstrated in 3 of the 4 donkey mares by the agglutination, complement-fixation and passive haemagglutination tests, were of low magnitude and short duration. The diagnostic value of the agglutination and complement-fixation tests was limited by the presence of low levels of non-specific reactivity and pronounced anti-complementary reactivity, respectively, in many of the donkey sera. The passive haemagglutination test proved superior for demonstrating elevation in antibody and for confirming infection. The overall results indicate that the donkey has the potential to act as a source of CEM infection and under certain circumstances, could have a role to play in the epidemiology of this disease.  相似文献   

13.
The passive hemagglutination (PHA) test was improved to enable the detection of antibodies to Taylorella (Haemophilus) equigenitalis in the sera of mares. Horse red blood cells (RBC) fixed with glutaraldehyde were compared with similarly treated RBC of a cow, pig and sheep for the PHA test. The horse RBC were superior to those of the other animals tested in detecting mares affected with contagious equine metritis (CEM). A PHA test using these cells as indicator and an antigen prepared from T. equigenitalis by sonication following treatment with hyaluronidase was the most satisfactory in terms of sensitivity and specificity. None of the 156 serum samples from clinically healthy mares without a history of contact with T. equigenitalis-infected stallions or mares showed PHA titers greater than 1:32 and only a few samples (7.1%) showed PHA titers of 1:32. Four of the 50 serum samples from mares affected with CEM showed PHA titers of 1:32, while most of the samples (92.0%) showed PHA titers greater than 1:32. The glutaraldehyde-fixed horse RBC sensitized with the antigen had the advantage of being reproducible for at least 7 months when preserved at 4 degrees C.  相似文献   

14.
A quantitative real-time polymerase chain reaction method (qPCR) was developed and tested for the detection of Taylorella equigenitalis. It was shown to have an analytical sensitivity of 5 colony-forming units (CFU) of T. equigenitalis when applied to the testing of culture swabs that mimicked field samples, and a high analytical specificity in not reacting to 8 other commensal bacterial species associated with horses. As designed, it could also differentiate specifically between T. equigenitalis and T. asinigenitalis. The qPCR was compared to standard culture in a study that included 45 swab samples from 6 horses (1 stallion, 5 mares) naturally infected with T. equigenitalis in Canada, 39 swab samples from 5 naturally infected stallions in Germany, and 311 swab samples from 87 culture negative horses in Canada. When the comparison was conducted on an individual sample swab basis, the qPCR had a statistical sensitivity and specificity of 100% and 96.4%, respectively, and 100% and 99.1% when the comparison was conducted on a sample set basis. A comparison was also made on 203 sample swabs from the 5 German stallions taken over a span of 4 to 9 mo following antibiotic treatment. The qPCR was found to be highly sensitive and at least as good as culture in detecting the presence of T. equigenitalis in post-treatment samples. The work demonstrates that the qPCR assay described here can potentially be used to detect the presence of T. equigenitalis directly from submitted sample swabs taken from infected horses and also for determining T. equigenitalis freedom following treatment.  相似文献   

15.
Mycoplasma equigenitalium and M. subdolum have been implicated in genital disorders and infertility of horses. The reported cytopathic effects of M. equigenitalium observed in vitro underscore its potential pathogenic role in reproductive dysfunction in mares. This study was initiated to determine the prevalence of mycoplasmas in the genital tract of stallions in relationship to age, clinical signs, geographic location and semen quality. For this purpose the mycoplasma flora of the genital tract of 116 stallions of the Noric breed was determined by isolation and colony immunoblotting and by polymerase chain reaction (PCR) assays. Of 438 swabs from the genital tract, pre-ejaculatory fluid and semen samples, 352 (80%) samples were positive by PCR and 125 (29%) were positive by culture. Mycoplasmas were isolated predominantly from the fossa glandis and urethra and less frequently from the penis shaft and from semen. M. equigenitalium (89 isolates) and M. subdolum (70 isolates) were the predominant species identified. M. equirhinis and M. felis were detected in 27 and 8 samples, respectively. Comparison of these isolations with clinical signs, semen quality, and age of the stallions revealed no significant correlation. However, geographical location of the stallion significantly correlated with mycoplasma detection. These results suggest that mycoplasmas are present as commensals in the genital tract of stallions. Thus, clinically healthy stallions may present a permanent reservoir for infection of mares via venereal transmission.  相似文献   

16.
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   

17.
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   

18.
In six healthy mares and 24 mares showing reproductive disorders swab samples were taken from the fossa clitoridis to isolate Taylorella equigenitalis, and from the uterus to isolate mycoplasmas, ureaplasmas and other aerobic bacteria. Swab samples were also taken from the uterus for Chlamydia antigen ELISA and Chlamydia PCR studies. The uterus of 27 mares was examined cytologically, and biopsy samples were taken from the endometrium for histological examinations and for immunohistochemical examinations aimed at the detection of chlamydiae. T. equigenitalis, mycoplasmas, ureaplasmas and chlamydiae could not be detected from any of the mares examined. Aerobic facultative pathogenic bacteria were isolated from mares with endometritis in four cases. In 18 out of 22 mares with endometritis (82%) no infective agents could be demonstrated. Further studies are needed to elucidate the relative importance of non-infectious causes of endometritis and of anaerobic bacteria often detectable in the uterus in the aetiology of the reproductive disorders observed.  相似文献   

19.
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   

20.
Contagious equine metritis (CEM) is a highly contagious bacterial venereal disease of horses caused by Taylorella equigenitalis. CEM-PCR is a semi-nested PCR method for detecting this bacterium. Although this technique is regarded as a sensitive diagnostic method for CEM, there are risks of it generating false positive and false negative results. In this study, we constructed a recombinant plasmid (CEM-POS) as reaction control to assure adequate PCR reaction and prevent false positive results caused by contamination of the reaction control in routine CEM-PCR examinations. CEM-POS was constructed by insertion of rpoB fragments from Rhodococcus equi into CEM-1P, which is a recombinant plasmid that includes a T. equigenitalis-specific sequence region. In CEM-PCR, the size of the PCR product from CEM-POS was clearly different from the true positive PCR product. In addition, CEM-POS retained high stability under convenient storage conditions of 4 degrees C. These results suggest CEM-POS to be a useful tool as a reaction control in routine CEM-PCR examinations.  相似文献   

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