Evaluation of the field application of PCR in the eradication of contagious equine metritis from Japan

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.     

Identification of Taylorella equigenitalis responsible for contagious equine metritis in equine genital swabs by direct polymerase chain reaction

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.     

Development of a real time PCR for the detection of Taylorella equigenitalis directly from genital swabs and discrimination from Taylorella asinigenitalis

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.     

Diagnostic and epidemiologic analysis of the 2008-2010 investigation of a multi-year outbreak of contagious equine metritis in the United States

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.     

Evaluation of a newly developed real-time PCR for the detection of Taylorella equigenitalis and discrimination from T. asinigenitalis

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.     

Quantitative assessment of the risks of reducing the routine swabbing requirements for the detection of Taylorella equigenitalis

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.     

A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses

: 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.     

Contagious equine metritis eradicated from Japan

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.     

Construction of a recombinant plasmid as reaction control in routine PCR for detection of contagious equine metritis (CEM-PCR)

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.     

Passive hemagglutination test for detection of antibodies against Taylorella (Haemophilus) equigenitalis in sera of mares

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.     

Comparison of uterine protein content and distribution of bacteria in the reproductive tract of mares after intrauterine inoculation of Haemophilus equigenitalis or Pseudomonas aeruginosa

Two groups of 3 mares were inoculated with Haemophilus equigenitalis or Pseudomonas aeruginosa on the 1st day of estrus. Uterine flushing samples were recovered on day 3 of estrus and day 8 after ovulation for each cycle. Mares were killed 22, 25, and 30 days after inoculation with P aeruginosa and 45, 46, and 49 days after inoculation with H equigenitalis. Pseudomonas aeruginosa was recovered from the uterus of 2 mares 48 hours after inoculation. Although the initial flushing sample of 1 of these 2 mares had an increased total protein concentration, there appeared to be little difference between protein concentrations of other uterine flushing samples. Haemophilus equigenitalis was recovered from the uterus of each of the 3 mares at postmortem. One mare had a slight, purulent discharge from the vulva. Total protein values were not increased in flushing samples from this mare after inoculation with H equigenitalis. Total protein values decreased in the last flushing sample of each of the 2 remaining mares. Swabbing the uterus was more effective than was homogenizing the uterine mucosa in isolating H equigenitalis.     

Prevalence of Taylorella equigenitalis infection in stallions in Slovenia: bacteriology compared with PCR examination

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.     

Study of the role of Chlamydia,Mycoplasma, Ureaplasma and other microaerophilic and aerobic bacteria in uterine infections of mares with reproductive disorders

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.     

Clinical, bacteriologic, serologic, and pathologic features of infections with atypical Taylorella equigenitalis in mares

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.     

Horse species symposium: contagious equine metritis: an insidious threat to the horse breeding industry in the United States

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.     

Multiplex PCR for the detection of Brucella ovis, Actinobacillus seminis and Histophilus somni in ram semen

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the rapid detection of Brucella ovis, Actinobacillus seminis, Histophilus somni in fresh ram semen samples. DESIGN: The multiplex assay was based on the single PCR assays published for the detection of A seminis and B ovis, and the forward primer published for the detection of H somni; an alternative reverse primer for H somni was designed in this study. PROCEDURE: Culture and PCR of 295 fresh semen samples were carried out. RESULTS: The multiplex PCR was far more successful in the detection of H somni (45/295) than culture (23/295). A seminis was also detected in more semen samples by multiplex PCR (29/295) than culture (13/295) and B ovis was detected in three samples using both PCR and culture. No amplifications were detected with DNA from a range of bacterial isolates including species associated with epididymitis in rams. CONCLUSION: This PCR could be used as a complementary test, or alternative to culture of ram semen and other biological samples for the detection B ovis, H somni and A seminis.     

Serological and bacteriological survey of three horse studs for contagious equine metritis

A bacteriological and serological survey for evidence of contagious equine metritis (CEM) was made during the 1980 breeding season on 3 horse studs in South Australia with a history of previous infection. Swabs from the clitoral sinus and the cervix were cultured for Haemophilus equigenitalis and serum was screened for antibody using the complement fixation test (CFT) and the enzyme-linked immunosorbent assay (ELISA). The specificity of both tests was greater than 0.99 but the ELISA was more sensitive in detecting antibody in infected mares. On the evidence presented it was concluded that H. equigenitalis is no longer present in the horse studs investigated.     

Development and Comparison of Sampling Techniques for a Broad Range,Semiquantitative Polymerase Chain Reaction Assay for Detection of Bacterial DNA in the Equine Uterus

Microbial culture from a double-guarded culture swab is commonly used to diagnose infectious endometritis. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR) assay to detect a broad range of bacteria from equine uterine samples. Twenty-seven mares with a clinical history of endometritis had a double-guarded culture swab collected for analysis by qPCR and microbial cultures. An additional 12 mares had a uterine biopsy sample collected for qPCR analysis, microbial culture, and histopathology. Subsequently, a double-guarded culture swab for microbial culture and a cytology brush sample were also collected. The qPCR assay detected bacterial DNA in nine of 27 mares from a double-guarded swab and six of 12 mares from an endometrial biopsy. Positive microbial growth was detected in nine of 27 mares and four of 12 mares from a double-guarded culture swab. Bacterial DNA was detected in two of 27 mares and two of 12 mares without subsequent microbial growth. The simple presence of an organism's DNA allows for detection by nonculture-based systems, both live and dead organisms can be identified. In conclusion, the qPCR assay was determined to be a sensitive diagnostic technique for identifying pathogens associated with infectious endometritis. The primary application of the qPCR assay is detection of potential pathogenic bacteria in the uterus of a mare suspected of having infectious endometritis when a traditional microbial culture is negative. Further work is warranted to determine if mares positive for bacterial DNA and negative for microbial culture are affected clinically.     

Recent advances in molecular epidemiology and detection of Taylorella equigenitalis associated with contagious equine metritis (CEM)

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.     

Detection of bacterial DNA in synovial fluid from horses with infectious synovitis

Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient. Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis. However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet. The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious synovitis. Synovial fluid samples from 6 horses with culture confirmed synovial infection were used for broad range 16S rRNA gene PCR. Synovial aspirates of 2 healthy horses were used as negative controls. Following extraction and purification of synovial fluid DNA, all samples were processed by touchdown PCR. Amplicons were detected by reverse line blot hybridisation and visualised with chemiluminescence. Pathogen-specific detection of 16S rRNA gene sequences was successful in all 6 synovial fluid samples. No bacterial DNA was detected in the aspirates from the negative control horses using touchdown PCR followed by 25 additional cycles of amplification. The identity of the pathogens was confirmed by DNA sequencing of the amplicons. It can be concluded that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation is a promising technique for detection of bacterial DNA in synovial fluid samples. Further research should aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but having negative culture results. When the 16S PCR proves to be reliable and more sensitive than standard culturing techniques, it may become a powerful tool in the diagnosis of synovial infection.