ABSTRACT
We report herein the use of crude extracts obtained from samples of Taylorella equigenitalis-infected horses for the purpose of multi-locus sequence typing (MLST). Samples (n = 36) were collected from horses in South Africa from 1996 to 2017: 34 from genital swabs (stored at -20°C for 2-3 y) and 2 from cryopreserved raw semen aliquots (stored at -70°C for 18 y) prior to assay. The MLST assay showed a single sequence type (ST), designated ST4, that supported a point introduction and thus a common source for the South African outbreak of contagious equine metritis.
Subject(s)
Gram-Negative Bacterial Infections/veterinary , Horse Diseases/diagnosis , Multilocus Sequence Typing/veterinary , Reproductive Tract Infections/veterinary , Semen/microbiology , Taylorella equigenitalis/isolation & purification , Animals , DNA, Bacterial/analysis , Female , Gram-Negative Bacterial Infections/diagnosis , Gram-Negative Bacterial Infections/microbiology , Horse Diseases/microbiology , Horses , Male , Reproductive Tract Infections/microbiology , South AfricaABSTRACT
Misidentification between Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and Taylorella asinigenitalis is observed by the gold standard culture method. The performance of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for Taylorella species identification was evaluated using 85â¯T. equigenitalis and 28â¯T. asinigenitalis strains selected on the basis of multilocus sequence typing data. Seven of the T. equigenitalis and 9 of the T. asinigenitalis strains were used to generate in-house reference spectra to expand the existing commercial Bruker database. Two bacterial incubation times and 3 different sample preparation procedures were compared. Overall, we demonstrated the usefulness of MALDI-TOF MS as a differential diagnostic tool for CEM; however, commercial spectra databases should be expanded with T. asinigenitalis reference spectra to achieve the expected performance. Moreover, direct spotting of 48-h colonies was not only the most efficient protocol but also the easiest to implement in a clinical setting.
Subject(s)
Gram-Negative Bacterial Infections/veterinary , Horse Diseases/microbiology , Taylorella equigenitalis/classification , Taylorella equigenitalis/isolation & purification , Taylorella/classification , Taylorella/isolation & purification , Animals , Databases, Factual , Equidae , Female , Gram-Negative Bacterial Infections/diagnosis , Gram-Negative Bacterial Infections/microbiology , Horse Diseases/diagnosis , Horses , Male , Multilocus Sequence Typing , Phylogeny , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
BACKGROUND: Sets of genital swabs are routinely taken from horses to screen for the presence of Taylorella equigenitalis, the cause of contagious equine metritis. Typically, two to four different sites are swabbed at a time and tested by culture or PCR. OBJECTIVES: This study explored the feasibility of pooling these swabs for a single PCR test per animal instead of testing each swab individually. STUDY DESIGN: In vitro. METHODS: PCR signal strengths (Ct values) from 149 historical PCR positive genital swabs, together with historical data on the number of swabs in a set expected to be positive, were used to assess the suitability of pooling for screening horses for T. equigenitalis infection in the population at large. Twenty-four sets of four equine genital swabs were tested. The sets were prepared in the laboratory using one or more swabs positive for T. equigenitalis from naturally infected cases. Positive and negative swabs were selected to reflect a typical range of PCR Ct values expected in field cases of T. equigenitalis infection. These pools were tested by an established PCR to assess the impact and suitability of a PCR test on pooled swabs compared to individual swab testing, by comparing the Ct values. RESULTS: Pooling one positive swab with three negative swabs produced a small drop in Ct value but all pools were still clearly positive. MAIN LIMITATIONS: Large numbers of field positive horses are not available, but the proof of concept approach with laboratory prepared pools shows the method is applicable to field cases. CONCLUSIONS: It was concluded that pooling of swabs would confer no appreciable drop in the ability to detect a positive animal compared to individual swab testing; pooling is therefore a suitable alternative to individual swab testing with reduced costs. The Summary is available in Spanish - see Supporting Information.
Subject(s)
Genitalia, Female/microbiology , Genitalia, Male/microbiology , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/diagnosis , Polymerase Chain Reaction/veterinary , Taylorella equigenitalis/isolation & purification , Animals , Female , Gram-Negative Bacterial Infections/diagnosis , Gram-Negative Bacterial Infections/microbiology , Horse Diseases/microbiology , Horses , Male , Polymerase Chain Reaction/methods , Specimen HandlingABSTRACT
The performance of culture and PCR methods routinely used to diagnose contagious equine metritis (CEM) was evaluated and compared by two interlaboratory trials involving a total of 24 European laboratories, including 22 National Reference Laboratories for CEM. Samples were swab specimens artificially contaminated with bacteria present in the genital tract of Equidae, some with and some without Taylorella equigenitalis, the causative agent of CEM, and T asinigenitalis, responsible for possible misidentification as T equigenitalis Throughout both interlaboratory trials, PCR performed better in terms of specificity and sensitivity than the culture method, supporting the assertion that PCR should be accepted for CEM diagnosis. However, the culture performance during the second interlaboratory trial was better than during the first one, suggesting that the expertise of participants improved. This reveals the advantage of regular interlaboratory trials to constantly improve the expertise of laboratories. It also shows the need to develop new culture media that are more selective and/or better geared to the metabolism of T equigenitalis in order to improve the bacteriological diagnosis of CEM.
Subject(s)
Culture Techniques/veterinary , Endometritis/veterinary , Horse Diseases/diagnosis , Laboratories/organization & administration , Polymerase Chain Reaction/veterinary , Animals , Endometritis/diagnosis , Europe , Female , Horse Diseases/microbiology , Horses , Sensitivity and Specificity , Taylorella equigenitalis/isolation & purificationABSTRACT
REASONS FOR PERFORMING STUDY: The response to the first outbreak of contagious equine metritis in South Africa included pioneering a web-based platform to coordinate key aspects of a national, real-time polymerase chain reaction (qPCR)-based stallion screening programme to determine the distribution and prevalence of Taylorella equigenitalis in stallions and exposed mares. OBJECTIVES: To define the hypothesised pre-existing status of T. equigenitalis in the South African equine population and progression of the epidemiological investigation via the implementation of a molecular diagnostic-based surveillance programme. STUDY DESIGN: Retrospective case series. METHODS: Screening for T. equigenitalis was via a qPCR assay on genital swabs obtained from predilection sites in stallions and mares with subsequent confirmation using bacterial culture according to prescribed methods. RESULTS: The initial outbreak investigation identified 4 horses including the index stallion and mare. Traceback of in-contact horses identified 26 horses, including a subpopulation focus at the South African Lipizzaner Centre where 24/33 resident stallions tested positive for T. equigenitalis on qPCR. The national screening programme identified an additional 9 stallions. A total of 39 horses (36 stallions and 3 mares) tested positive for T. equigenitalis by qPCR and T. equigenitalis was isolated from 23 of these stallions and 2 of these mares. In addition to the index property, an artificial breeding centre where the index case was first identified, an additional 12 properties with infected horses were identified in 3/9 provinces. Horses on 11 of these 12 properties were directly linked to the index property. Two incidents of T. equigenitalis transmission associated with artificial insemination were recorded. CONCLUSIONS: T. equigenitalis was present in a subpopulation focus within the South African horse population prior to the outbreak identification in April 2011. Horizontal fomite-associated spread was the most probable route of transmission between stallions. The targeted surveillance of stallions and exposed mares using a qPCR-based screening programme expedited investigation of the distribution and prevalence of T. equigenitalis infection in South African horses. The application of qPCR provided a sensitive and practical screening test for identification of T. equigenitalis-positive animals as part of an emergency response to the first identified cases of T. equigenitalis infection in South African horses.
Subject(s)
Gram-Negative Bacterial Infections/veterinary , Horse Diseases/microbiology , Polymerase Chain Reaction/veterinary , Sexually Transmitted Diseases, Bacterial/veterinary , Taylorella equigenitalis/isolation & purification , Animals , Female , Gram-Negative Bacterial Infections/epidemiology , Gram-Negative Bacterial Infections/microbiology , Horse Diseases/epidemiology , Horses , Male , Sexually Transmitted Diseases, Bacterial/epidemiology , Sexually Transmitted Diseases, Bacterial/microbiology , South Africa/epidemiologySubject(s)
Endometritis/veterinary , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/microbiology , Taylorella equigenitalis/isolation & purification , Animals , Breeding , Endometritis/diagnosis , Endometritis/epidemiology , Female , France/epidemiology , Gram-Negative Bacterial Infections/diagnosis , Gram-Negative Bacterial Infections/epidemiology , Horse Diseases/diagnosis , Horse Diseases/epidemiology , Horses , Male , Retrospective StudiesABSTRACT
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.
Une épreuve quantitative de réaction en chaîne par la polymérase en temps réel (qPCR) a été développée et testée pour la détection de Taylorella equigenitalis. L'épreuve a démontré une sensibilité analytique de 5 UFC de T. equigenitalis à partir de prélèvements de culture qui imitent les échantillons de terrain, et une spécificité analytique élevée puisqu'elle n'a pas réagi à 8 autres espèces bactériennes commensales associées aux chevaux. D'ailleurs, sa conception peut aussi faire la différenciation entre T. equigenitalis et T. asinigenitalis. La qPCR a été comparée à la méthode de culture classique de routine dans une étude qui a inclus 45 prélèvements provenant de 6 chevaux du Canada (1 étalon, 5 juments), 39 prélèvements de 5 étalons d'Allemagne, tous infectés naturellement avec T. equigenitalis, ainsi que 311 prélèvements provenant de 87 chevaux du Canada diagnostiqués négatifs par la méthode de détection par culture. Lorsque la comparaison a été faite pour les prélèvements individuels, la qPCR a démonstré une sensibilité et spécificité statistique de 100 % et 96,4 %, respectivement, et de 100 % et 99,1 %, pour la comparaison basée sur les séries de prélèvements. De plus, une comparaison a été faite à partir de 203 prélèvements de 5 étalons d'Allemagne ayant été pris dans un intervalle de 4 à 9 mois après un traitement aux antibiotiques. La qPCR s'est avérée hautement sensible et tout au moins aussi bonne que la méthode par culture pour la détection de T. equigenitalis dans les prélèvements post-traitement. Ce projet démontré que la qPCR, décrite ici, peut être utilisée pour la détection de la présence de T. equigenitalis directement des prélèvements de chevaux infectés ainsi que pour la confirmation de l'absence de T. equigenitalis après traitement.(Traduit par Ms. Émilie Falardeau).
Subject(s)
Bacteriological Techniques/veterinary , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/microbiology , Taylorella equigenitalis/isolation & purification , Animals , Canada/epidemiology , Female , Germany/epidemiology , Gram-Negative Bacterial Infections/diagnosis , Gram-Negative Bacterial Infections/microbiology , Horse Diseases/diagnosis , Horses , Male , Sensitivity and Specificity , Sexually Transmitted Diseases, Bacterial/diagnosis , Sexually Transmitted Diseases, Bacterial/microbiology , Sexually Transmitted Diseases, Bacterial/veterinaryABSTRACT
Contagious equine metritis is a bacterial infectious disease of horses caused by Taylorella equigenitalis, a Gram-negative eubacterium. The disease has been described in several continents, including Europe, North America and Asia. A novel molecular method was developed to detect clustered regularly interspaced short palindromic repeats (CRISPRs), which were separated by non-repetitive unique spacer regions (NRUSRs) of similar length, in the Taylorella equigenitalis EQ59 strain using a primer pair, f-/r-TeCRISPR-ladder, by PCR amplification. In total, 31 Taylorella isolates (17 T. equigenitalis and 14 Taylorella asinigenitalis) were examined. The T. equigenitalis isolates came from thoroughbred and cold-blooded horses from nine countries during 1980-1996, whilst the T. asinigenitalis isolates all originated from donkey jacks in France and the USA during 1997-2006. PAGE fractionated all of the 13 CRISPRs separated by 12 NRUSRs in T. equigenitalis EQ59. Permutation examples of CRISPRs, which were separated by NRUSRs for small-sized ladders, consisting of two doublet bands were shown. Putative CRISPRs separated by NRUSRs were amplified with 14/17 (82.4 %) geographically disparate T. equigenitalis isolates using the newly designed primer pair. Approximately 82.4 % of the T. equigenitalis isolates had CRISPRs separated by NRUSRs. The CRISPR locus was also found in the French T. asinigenitalis strain MCE3. Putative CRISPRs separated by NRUSRs were detected similarly in 4/14 (28.6 %) T. asinigenitalis isolates. Overall, a more detailed understanding of the molecular biology of CRISPRs within Taylorella organisms may help elucidate the pathogenic virulence and transmission mechanisms associated with this important equine pathogen.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , DNA, Intergenic , Gram-Negative Bacterial Infections/diagnosis , Horse Diseases/diagnosis , Taylorella equigenitalis/genetics , Animals , Base Sequence , DNA Primers/genetics , DNA, Intergenic/genetics , Female , Gram-Negative Bacterial Infections/microbiology , Horse Diseases/microbiology , Horses , Male , Polymerase Chain Reaction , Sequence Analysis, DNA , Taylorella equigenitalis/isolation & purificationABSTRACT
We describe here the development of a multilocus sequence typing (MLST) scheme for Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and Taylorella asinigenitalis, a nonpathogenic bacterium. MLST was performed on a set of 163 strains collected in several countries over 35 years (1977-2012). The MLST data were analyzed using START2, MEGA 5.05 and eBURST, and can be accessed at http://pubmlst.org/taylorella/. Our results revealed a clonal population with 39 sequence types (ST) and no common ST between the two Taylorella species. The eBURST analysis grouped the 27 T. equigenitalis STs into four clonal complexes (CC1-4) and five unlinked STs. The 12 T. asinigenitalis STs were grouped into three clonal complexes (CC5-7) and five unlinked STs, among which CC1 (68.1% of the 113 T. equigenitalis) and CC5 (58.0% of the 50 T. asinigenitalis) were dominants. The CC1, still in circulation in France, contains isolates from the first CEM outbreaks that simultaneously emerged in several countries in the late 1970s. The emergence in different countries (e.g. France, Japan, and United Arab Emirates) of STs without any genetic relationship to CC1 suggests the existence of a natural worldwide reservoir that remains to be identified. T. asinigenitalis appears to behave same way since the American, Swedish and French isolates have unrelated STs. This first Taylorella sp. MLST is a powerful tool for further epidemiological investigations and population biology studies of the Taylorella genus.
Subject(s)
Bacterial Typing Techniques/methods , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/microbiology , Multilocus Sequence Typing/veterinary , Taylorella/classification , Taylorella/genetics , Animals , Female , Genes, Bacterial/genetics , Genetic Variation , Gram-Negative Bacterial Infections/microbiology , Horses/genetics , Male , Molecular Typing , Phylogeny , Taylorella/isolation & purification , Taylorella equigenitalis/classification , Taylorella equigenitalis/genetics , Taylorella equigenitalis/isolation & purificationABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPRs), of approximately 10,000 base pairs (bp) in length, were shown to occur in the Japanese Taylorella equigenitalis strain, EQ59. The locus was composed of the putative CRISPRs-associated with 5 (cas5), RAMP csd1, csd2, recB, cas1, a leader region, 13 CRISPR consensus sequence repeats (each 32 bp; 5'-TCAGCCACGTTCGCGTGGCTGTGTGTTTAAAG-3'). These were in turn separated by 12 non repetitive unique spacer regions of similar length. In addition, a leader region, a transposase/IS protein, a leader region, and cas3 were also seen. All seven putative open reading frames carry their ribosome binding sites. Promoter consensus sequences at the -35 and -10 regions and putative intrinsic ρ-independent transcription terminator regions also occurred. A possible long overlap of 170 bp in length occurred between the recB and cas1 loci. Positive reverse transcription PCR signals of cas5, RAMP csd1, csd2-recB/cas1, and cas3 were generated. A putative secondary structure of the CRISPR consensus repeats was constructed. Following this, CRISPR results of the T. equigenitalis EQ59 isolate were subsequently compared with those from the Taylorella asinigenitalis MCE3 isolate.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Taylorella equigenitalis/genetics , Base Sequence , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Expression Profiling , Japan , Molecular Sequence Data , Nucleic Acid Conformation , Open Reading Frames , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Homology , Taylorella equigenitalis/isolation & purification , Transcription Termination, GeneticABSTRACT
OBJECTIVE: To determine the impact of antimicrobial-containing semen extender on the growth of Taylorella equigenitalis in semen culture-positive for contagious equine metritis (CEM) and the development of CEM after artificial insemination with CEM-positive semen extended with antimicrobial-containing semen extender. DESIGN: Prospective study. ANIMALS: 21 mature mares free of CEM, 1 mature stallion experimentally infected with CEM, and semen from a stallion naturally infected with CEM. PROCEDURES: CEM-positive semen was incubated in semen extender with and without antimicrobials (amikacin [final concentration, 1 g/L] and penicillin G potassium [0.63 g/L]) followed by determination of the number of colony-forming units of T equigenitalis. Mares were inseminated with raw, extended, or cryopreserved semen culture-positive for T equigenitalis and observed for clinical signs of CEM. Samples for bacterial culture were obtained from the uterus, clitoral sinuses, and clitoral fossa of mares 7, 14, and 21 days after artificial insemination. RESULTS: Antimicrobial-containing semen extender significantly reduced the number of colony-forming units of T equigenitalis in CEM-positive semen. Artificial insemination with raw CEM-positive semen resulted in clinical signs of CEM, whereas artificial insemination with extended or cryopreserved CEM-positive semen did not result in clinical signs of CEM. CONCLUSIONS AND CLINICAL RELEVANCE: Antimicrobial-containing semen extender significantly reduced the risk of dissemination of CEM. The inclusion of amikacin (1 g/L) and penicillin G potassium (0.63 g/L) in extended semen reduced the transmission of CEM from stallions to mares during artificial insemination, which may result in altered dissemination of the disease.
Subject(s)
Anti-Bacterial Agents/pharmacology , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/prevention & control , Insemination, Artificial/veterinary , Semen Preservation/veterinary , Taylorella equigenitalis/isolation & purification , Animals , DNA, Bacterial , Female , Genitalia, Female/microbiology , Gram-Negative Bacterial Infections/prevention & control , Gram-Negative Bacterial Infections/transmission , Horses , Insemination, Artificial/adverse effects , Male , Semen Preservation/methodsSubject(s)
Disease Notification , Endometritis/veterinary , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/diagnosis , Taylorella equigenitalis , Animals , Endometritis/diagnosis , Female , Gram-Negative Bacterial Infections/diagnosis , Horses , Taylorella equigenitalis/isolation & purificationABSTRACT
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.
Subject(s)
Disease Outbreaks/veterinary , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/epidemiology , Horse Diseases/microbiology , Sexually Transmitted Diseases/veterinary , Taylorella equigenitalis/isolation & purification , Animals , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Female , Fluorescent Antibody Technique/veterinary , Gram-Negative Bacterial Infections/epidemiology , Gram-Negative Bacterial Infections/microbiology , Gram-Negative Bacterial Infections/transmission , Horse Diseases/diagnosis , Horse Diseases/transmission , Horses , Latex Fixation Tests/veterinary , Male , Polymerase Chain Reaction/veterinary , Pregnancy , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , Sexually Transmitted Diseases/epidemiology , Sexually Transmitted Diseases/microbiology , Sexually Transmitted Diseases/transmission , Taylorella equigenitalis/genetics , United States/epidemiologyABSTRACT
On PCR amplification of the intervening sequences (IVSs) in the central (helix 45) region within 23S rRNA gene sequences with T. equigenitalis (n = 34), as well as T. asinigenitalis (n = 35) and Bordetella (n = 11) isolates by using the primer pair of f-/r-23STis2, approximately 0.8 kb of the amplicons were generated, sequenced and analyzed. One IVS of approximately 70 bp in length was identified in all the Taylorella organisms but not Bordetella. PCR amplification was further developed for the convenient and rapid molecular detection of T. equigenitalis organisms with the IVS in the helix 45 region within the 23S rRNA genes as target by using the primer pairs (f-IVSde/r-23de). Thus, these results clearly demonstrated that PCR amplification with the primer pair (f-IVSde/r-23de) can be reliable in order to differentiate the T. equigenitalis isolates from both the T. asinigenitalis and Bordetella organisms.
Subject(s)
Bacteriological Techniques/methods , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/diagnosis , Horse Diseases/microbiology , Polymerase Chain Reaction/methods , Taylorella equigenitalis/isolation & purification , Animals , DNA Primers/genetics , Gram-Negative Bacterial Infections/diagnosis , Gram-Negative Bacterial Infections/microbiology , Horses , RNA, Ribosomal, 23S/genetics , Sequence Analysis, DNASubject(s)
Endometritis/veterinary , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/microbiology , Taylorella equigenitalis/isolation & purification , Animals , Endometritis/microbiology , Endometritis/physiopathology , Female , Gram-Negative Bacterial Infections/microbiology , Gram-Negative Bacterial Infections/physiopathology , Horse Diseases/physiopathology , Horses , MaleABSTRACT
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.
Subject(s)
Carrier State/veterinary , Endometritis/microbiology , Endometritis/veterinary , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/microbiology , Taylorella equigenitalis/isolation & purification , Animals , Carrier State/epidemiology , Carrier State/microbiology , Carrier State/transmission , Disease Outbreaks/economics , Disease Outbreaks/prevention & control , Disease Outbreaks/veterinary , Endometritis/epidemiology , Female , Gram-Negative Bacterial Infections/epidemiology , Gram-Negative Bacterial Infections/microbiology , Gram-Negative Bacterial Infections/transmission , Horse Diseases/epidemiology , Horse Diseases/transmission , Horses , Male , United States/epidemiologyABSTRACT
Taylorella equigenitalis is the etiologic agent of contagious equine metritis (CEM), a venereal disease of horses. A total of 82 strains of T. equigenitalis isolated in the United States were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of genomic DNA with restriction enzyme ApaI. Twenty-eight of those strains isolated from horses in the 2009 U.S. outbreak (CEM09) were further analyzed with NotI and NaeI enzymes. When ApaI alone was used for analysis, the 82 isolates clustered into 15 different genotypes that clearly defined groups of horses with known epidemiological connections. The PFGE profiles of the CEM09 isolates were indistinguishable after digestion with ApaI, NotI, and NaeI and did not match those of isolates from previous U.S. outbreaks in 1978 and 2006 or of any other isolate from the National Veterinary Services Laboratories (NVSL) culture library. Coupled with the fact that the CEM09 isolates are epidemiologically related, these results suggest a common source for the outbreak not linked to previous occurrences of CEM in the United States.
Subject(s)
Bacterial Typing Techniques , Electrophoresis, Gel, Pulsed-Field , Gram-Negative Bacterial Infections/veterinary , Horse Diseases/epidemiology , Horse Diseases/microbiology , Taylorella equigenitalis/classification , Taylorella equigenitalis/genetics , Animals , Cluster Analysis , DNA Restriction Enzymes/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Genotype , Gram-Negative Bacterial Infections/epidemiology , Gram-Negative Bacterial Infections/microbiology , Horses , Molecular Epidemiology , Taylorella equigenitalis/isolation & purification , United States/epidemiologyABSTRACT
Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bacterial culture and polymerase chain reaction (PCR) testing. Our results indicated that IIF using polyclonal antibodies allows T. equigenitalis to be discriminated from T. asinigenitalis. This test constitutes a rapid, sensitive and specific tool for confirming presumptive colonies of T. equigenitalis.
Subject(s)
Gram-Negative Bacterial Infections/veterinary , Horse Diseases/microbiology , Taylorella equigenitalis/isolation & purification , Animals , Antibodies, Bacterial/analysis , Antigens, Bacterial/analysis , Fluorescent Antibody Technique, Indirect , Gram-Negative Bacterial Infections/diagnosis , Horse Diseases/diagnosis , Horses , Rabbits/immunology , Taylorella equigenitalis/immunologySubject(s)
Equidae , Genital Diseases, Male/veterinary , Gram-Negative Bacterial Infections/veterinary , Taylorella equigenitalis/isolation & purification , Animals , Genital Diseases, Male/epidemiology , Genital Diseases, Male/microbiology , Gram-Negative Bacterial Infections/epidemiology , Gram-Negative Bacterial Infections/microbiology , Italy/epidemiology , MaleABSTRACT
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 degrees C but from which T. equigenitalis had been isolated following collection. The sensitivities of real-time 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.
