Core genome multilocus sequence typing analysis of Finnish Taylorella equigenitalis isolates.

In Finland, Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), was first detected in 1992. The aim of this study was to genotype Finnish T. equigenitalis isolates to investigate the epidemiology of the infection in the Finnish horse population. A total of 34 T. equigenitalis isolates from 24 horses obtained during 1992-2021 were subjected to whole genome sequencing (WGS) and subsequent local ad hoc core genome multi-locus sequence typing (cgMLST) targeting 1259 loci. Classical MLST profiles were extracted from the whole-genome sequence data. Three novel MLST types, ST81, ST82 and ST83, and four previously described sequence types, ST16, ST17, ST50 and ST63 were detected among the isolates. cgMLST minimum spanning tree analysis using 12 allele difference as threshold, resulted in five clusters and three singletons. cgMLST clusters were congruent with the MLST-defined groups, except for the ST83 isolates which were divided into two clusters. However, the high discriminatory power cgMLST allowed differentiation between isolates of the same MLST type as each isolate had a unique core genome ST. Our study suggests that cgMLST has the prospective for being a standardised typing method for T. equigenitalis in the future, and further contributes to worldwide phylogenetic and spatio-temporal analyses needed to better understand the epidemiology of the bacterium.

Surveillance of Contagious Equine Metritis: Results of the First 5-Year Period of French Proficiency Tests for Taylorella equigenitalis Detection by Real-Time PCR.

Contagious equine metritis (CEM) detection by PCR is recognized by the European Union according to Commission Implementing Regulation (EU) No 846/2014, and real-time PCR is now recommended by the World Organisation for Animal Health Terrestrial Manual at the same level as the culture method. The present study highlights the creation of an efficient network of approved laboratories in France in 2017 for CEM detection by real-time PCR. The network currently consists of 20 laboratories. A first proficiency test (PT) was organized by the national reference laboratory for CEM in 2017 to evaluate the performance of the early network, followed by annual proficiency tests organized for ongoing periodic assessment of network performance. Results of the 5 PTs organized from 2017 to 2021 are presented, during which 5 real-time PCRs and 3 DNA extraction methods were used. Overall, 99.20% of the qualitative data corresponded to expected results and the R-squared of global DNA amplification calculated for each PT varied from 0.728 to 0.899. DNA extraction is also an important step in the analytical process, and results were more favorable with direct lysis compared to column extraction. Focusing on the most commonly used PCR (PCR 1: 86.4% of results) showed lowest cycle threshold values with direct lysis compared to column and magnetic bead extractions, and with magnetic bead extraction compared to column extraction, but neither of these differences were statistically significant.

Screening for Taylorella equigenitalis in Equine Semen: An Exploratory Study.

The study examined and compared the sensitivity of culture and a quantitative PCR assay for screening equine semen for the presence of Taylorella equigenitalis (CEMO). Chilled semen samples, both raw and treated with extender, from two stallions were spiked with the organism at seven or 23 days postejaculation and prepared in serial dilutions. Culture of the 7-day raw semen readily detected CEMO at all dilutions, but extended semen yielded counts that were two log cycles lower at equivalent dilutions, with the organism being nearly undetectable at the maximal dilutions. By contrast, PCR sensitivity was not affected by extender, but for 7-day-old raw semen, PCR detection declined abruptly three log dilutions earlier than detection by culture. The more aged 23-day-old semen proved less satisfactory for spiking, with detection of CEMO by culture failing in three of the four samples due to overgrowth with commensal organisms. However, PCR performance was similar in both the 23- and 7-day spiking series. The detection limit by PCR is estimated at between 104 and 105 cfu/mL. Typical CEMO concentrations in the semen of colonized stallions are not widely reported but where natural semen contamination has been investigated, the organism was present at this order of magnitude. The reliability of detecting CEMO infection using semen samples by either method is discussed.

Comparison of five basal compositions of selective chocolate agar media for isolation of Taylorella equigenitalis.

The gold standard method to isolate and identify Taylorella equigenitalis, the contagious agent of equine metritis, is the culture method according to the World Organisation for Animal Health Terrestrial Manual. No selective T. equigenitalis chocolate agar medium has been developed since the 1980s and the existing media show limited performances due to the fastidious nature of T. equigenitalis and the presence of interfering bacteria in the genital tract of equines. Here, the growth rates of 6 T. equigenitalis strains and 7 non-T. equigenitalis strains were compared on Timoney's selective medium formulated with 5 different basal agars (Columbia, Eugon, Blood, Mueller-Hinton and Tryptose Blood) provided by 2 to 4 suppliers per basal agar. The impact of glucose and/or Vitox supplementation was also investigated. Overall, the performance of selective T. equigenitalis media could be improved by substituting Eugon or Columbia agar with Blood, Mueller-Hinton or Tryptose Blood agar. It is nevertheless essential to validate the basal agar/supplier pair using a panel of T. equigenitalis strains. Furthermore, our findings confirm the need to supplement the selective media with a mixture of amino acids, nucleotides, and organic, mineral and vitamin compounds, translated here by Vitox supplementation.

Comparison of seven nucleic acid amplification tests for detection of Taylorella equigenitalis.

Taylorella equigenitalis causes contagious equine metritis. Here we compared seven nucleic acid amplification tests for T. equigenitalis to select a rapid and reliable diagnostic method. The 95% detection limits of each assay varied greatly: real-time PCR had the lowest detection limit (0.77 fg/reaction); those of some of the conventional PCRs (cPCRs) were >100 fg/reaction. In experimentally infected samples, real-time PCR and semi-nested PCR showed the highest positive numbers (33 out of 42 samples), but two of the cPCRs detected only 2 and 7 positive results. Our results indicate that the use of sensitive molecular assays is important for the efficient detection of T. equigenitalis in clinical samples.

Development and Application of a Multiplex Real-Time Polymerase Chain Reaction Assay for the Simultaneous Detection of Bacterial Aetiologic Agents Associated With Equine Venereal Diseases.

Venereal diseases caused by bacteria are important to the equine industry due to economic losses caused by decline of conception rate in breeding horses. Therefore, identification of infected animals as well as the implementation of appropriate managerial procedures based on accurate diagnosis is critical. In this study, two types of multiplex real-time polymerase chain reaction with high sensitivity and specificity were developed for the simultaneous detection and differentiation of five commonly associated bacterial pathogens of venereal diseases in horses, consisting of Taylorella equigenitalis, Taylorella asinigenitalis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Streptococcus zooepidemicus. The assay was applied to samples collected as part of the surveillance of T.equigenitalis infection in South Korea. Swab samples collected from horses in 2015 were tested. T. equigenitalis and K. pneumoniae was detected in 21 (21.0%) and two (2.0%) samples, respectively. No samples were positive for T. asinigenitalis, P. aeruginosa, and S. zooepidemicus. Application of this assay to an existing surveillance program has allowed for an enhanced surveillance for a wider range of venereal diseases of equine to be implemented in South Korea.

Preservation of viable Taylorella equigenitalis in different commercially available transport systems.

The cultural diagnosis of the causal agent of contagious equine metritis (Taylorella equigenitalis) using transport swabs is challenging. Swabs must be placed in Amies charcoal medium, refrigerated during transport, and plated out at the laboratory no later than 48 h after sampling. In this study, the viability of T. equigenitalis strain CIP 79.7T in 11 commercial swab transport systems was initially compared at 1 day and 2 days of storage at ambient (20 ± 3 °C) or refrigerated (5 ± 3 °C) temperature. The four best swab transport systems, systems B, E, F (used as the reference) and K, were then compared at 0, 2, 3, 4, 7 and 10 days at refrigerated temperatures. Statistically significant differences were observed after 10 days only for system K compared to the reference, with approximately 95% viable T. equigenitalis recovered in system K compared to approximately 77% in system F. System K is thus promising for preservation and transport of viable T. equigenitalis for culture.

A new strain of Taylorella asinigenitalis shows differing pathogenicity in mares and Jenny donkeys.

BACKGROUND: Three horse mares inadvertently inseminated with semen from a Tayorella asinigenitalis-positive Jack donkey developed severe, purulent endometritis whereas two Jenny donkeys mated naturally to the same Jack donkey did not develop clinical signs of infection. OBJECTIVES: To isolate and identify the causative agent. STUDY DESIGN: Case report. METHODS: Endometrial swabs from the infected mares were cultured on selective and non-selective media under aerobic and microaerophilic conditions. Isolates were subjected to Gram staining, oxidase and catalase tests, the Monotayl Latex Agglutination test and PCR to test for both T. equigenitalis and T. asinigenitalis. In vitro antimicrobial susceptibility testing was performed and the bacterial isolate was genotyped using MLST. RESULTS: A new sequence type of T. asinigenitalis was confirmed. MAIN LIMITATIONS: A limited numbers of mares and donkeys are described. CONCLUSIONS: This strain of T. asinigenitalis causes a severe venereal infection in mares but not in Jenny donkeys.

Comparative Semen Microbiota Composition of a Stallion in a Taylorella equigenitalis Carrier and Non-Carrier State.

Contagious equine metritis is receiving renewed attention due to the continuous detection of carriers in apparent agent-free farms. Interactions of Taylorella with the seminal microflora may be the plausible cause behind these spontaneous changes of the carrier state. Accordingly, the aim of this study was to compare the differences in the seminal microbiome composition of one stallion in the contagious equine metritis carrier state and non-carrier state. Samples were cryopreserved after their extraction. Cell disruption was performed by high-speed homogenization in grinding media. Bacterial families were identified via V3 amplification of the 16S rRNA gene and Ion Torrent sequencing. Only bacterial families with relative abundance above 5% were taken into consideration. The positive sample contained a strong dominance of Corynebacteriaceae (37.75%) and Peptoniphilaceae (28.56%). In the negative sample, the Porphyromonadaceae (20.51%), Bacteroidaceae (19.25%) and Peptoniphilaceae (18.57%) families prevailed. In conclusion, the microbiome seminal composition varies when an individual carries Taylorella from when it is free of it. The wider differences were found in the Corynebacteriaceae, Porphyromonadaceae and Bacteroidaceae families. Due to the limitations of a single-case analysis, further studies are needed for a better understanding of the stallion seminal microflora interactions.

Ten years of Taylorella equigenitalis ring trial results comparing culture and polymerase chain reaction.

Taylorella equigenitalis, the cause of contagious equine metritis (CEM), can be detected by culture but in recent years polymerase chain reaction (PCR) has also been used. In 2008, the World Organisation for Animal Health (OIE) Reference Laboratory for CEM in the United Kingdom set up a ring trial for laboratories to assess their ability to identify T. equigenitalis in laboratory-prepared samples because the identification of T. equigenitalis in the laboratory was recognised to be difficult. Freeze-dried culture suspensions in various combinations of any of T. equigenitalis, Taylorella asinigenitalis, other typical equine contaminant organisms, or no organism were used. All laboratories provided culture results and some also gave PCR results. The results reported here cover the ten years since inception and look at the ability to identify T. equigenitalis under ideal laboratory conditions, a necessity to be able to detect its presence in equine genital samples. The detection rate was very high by both methods. The accuracy was not significantly different between the culture and PCR methods for pure T. equigenitalis samples. For T. equigenitalis mixed with contaminants, culture missed about 2% (p = 0.02) compared with PCR, which was over 99% accurate. Difficulty in differentiating T. asinigenitalis from T. equigenitalis was apparent in a number of laboratories for both culture and PCR in 2008 but was less evident in 2016. It was also noted that culture results from laboratories that also tested by PCR had around 4% higher detection rates (p < 0.05) of T. equigenitalis than those that only used culture.

La détection de Taylorella equigenitalis, l'agent causal de la métrite contagieuse équine, peut être réalisée par culture, mais aussi, depuis quelques années, par amplification en chaîne par polymérase (PCR). En 2008, le Laboratoire de référence de l'Organisation mondiale de la santé animale (OIE) pour cette maladie au Royaume-Uni a conçu une série d'essais comparatifs inter-laboratoires visant à évaluer la capacité des laboratoires à identifier des échantillons préparés de T. equigenitalis, compte tenu de la difficulté avérée de cette identification au laboratoire. Les essais ont porté sur des cultures lyophilisées en suspension contenant, dans diverses combinaisons, T. equigenitalis, Taylorella asinigenitalis, d'autres micro-organismes pathogènes des équidés, ou aucun agent pathogène. Tous les laboratoires participants ont communiqué les résultats des mises en culture et certains ont également transmis les résultats obtenus par PCR. Les résultats rapportés par les auteurs couvrent les dix années écoulées depuis le lancement des essais et visent à déterminer la capacité à identifier T. equigenitalis dans des conditions de laboratoire idéales, exigence essentielle pour pouvoir détecter la présence de cette bactérie à partir de prélèvements génitaux d'équidés. Le taux de détection s'est révélé très élevé pour chacune des deux méthodes. Il n'a pas été observé de variation significative entre l'exactitude de la mise en culture et celle de la PCR lorsque les prélèvements ne contenaient que T. equigenitalis. S'agissant de suspensions où T. equigenitalis était mélangée à d'autres agents pathogènes, les résultats font état d'environ 2 % (p = 0,02) d'échecs de l'identification par culture, tandis que l'exactitude de la PCR était de 99 %. En 2008, plusieurs laboratoires ont manifestement eu des difficultés à différencier T. asinigenitalis de T. equigenitalis aussi bien en culture que par PCR, mais cette difficulté était moins perceptible en 2016. Il a également été constaté que les identifications après culture effectuées par les laboratoires qui testaient aussi par PCR se traduisaient par un taux de détection de T. equigenitalis supérieur d'environ 4 % (p < 0,05) par rapport aux laboratoires qui ne pratiquaient que la mise en culture.

Taylorella equigenitalis, patógeno causante de la metritis contagiosa equina, puede ser detectado por cultivo, pero en los últimos años también se viene utilizando la técnica de reacción en cadena de la polimerasa (PCR). En 2008, ante la sabida dificultad que presenta la identificación en laboratorio de T. equigenitalis, el Laboratorio de Referencia de la Organización Mundial de Sanidad Animal (OIE), para esta enfermedad sito en el Reino Unido, puso en marcha una serie de pruebas de competencia para evaluar la aptitud de diferentes laboratorios para detectar la presencia de T. equigenitalis en muestras preparadas en laboratorio, empleando al efecto suspensiones de cultivo liofilizado con diversas combinaciones en las que estaban presentes T. equigenitalis, Taylorella asinigenitalis, otros patógenos equinos típicos o ningún microorganismo en absoluto. Todos los laboratorios comunicaron los resultados de las técnicas de cultivo y algunos de ellos también proporcionaron los resultados obtenidos por PCR. Los resultados aquí expuestos cubren los diez años transcurridos desde el inicio de las pruebas y dan cuenta de la capacidad para identificar a T. equigenitalis en condiciones de laboratorio idóneas, elemento imprescindible para poder detectar su presencia en muestras genitales equinas. Con ambos métodos se obtenía una tasa de detección muy elevada, sin que hubiera una diferencia de exactitud significativa entre el cultivo y la PCR en el caso de muestras puras de T. equigenitalis. Cuando este microorganismo estaba mezclado con contaminantes, «escapaban¼ al método de cultivo alrededor de un 2% (p = 0,02) de las muestras, frente a la exactitud del 99% que deparaban las técnicas de PCR. Los resultados obtenidos por una serie de laboratorios en 2008 ponían de manifiesto una evidente dificultad para distinguir entre T. asinigenitalis y T. equigenitalis, ya fuera por cultivo o por PCR, dificultad que resultaba menos obvia en 2016. También se observó que las tasas de detección en cultivo de T. equigenitalis obtenidas por laboratorios que también analizaban las muestras por PCR eran alrededor de un 4% superiores (p < 0,05) a las de laboratorios que empleaban únicamente el método de cultivo.

Acute Endometritis due to Taylorella equigenitalis Transmission by Insemination of Cryopreserved Stallion Semen.

Taylorella equigenitalis can be transmitted during artificial insemination. This report describes clinical T. equigenitalis transmission by cryopreserved stallion semen. T. equigenitalis isolates from a mare's vaginal discharge and semen from the same batch of the cryopreserved semen used for the insemination gave identical API ZYM, antibiotic susceptibility, and multilocus sequence typing results (ST-46); furthermore, the multilocus sequence typing lineage ST-46 is known to circulate in the country of semen collection. These results support the need for strict contagious equine metritis screening of processed semen before use for artificial insemination.

Evaluation of MALDI-TOF MS and an expanded custom reference spectra database for the identification and differentiation of Taylorella equigenitalis and Taylorella asinigenitalis.

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 85 T. equigenitalis and 28 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.

Towards European harmonisation of contagious equine metritis diagnosis through interlaboratory trials.

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.

Genotyping of German and Austrian Taylorella equigenitalis isolates using repetitive extragenic palindromic (REP) PCR and pulsed-field gel electrophoresis (PFGE).

A total of 124 Taylorella (T.) equigenitalis and five T. asinigenitalis field isolates collected between 2002 and 2014 were available for genotyping using REP- (repetitive extragenic palindromic) PCR and PFGE (pulsed-field gel electrophoresis). The study comprised 79 T. equigenitalis field isolates originating from ten defined breeds of German horses and revealed a spectrum of five REP (rep-E1-E4, rep-E3a) and 15 PFGE (TE-A1-A9, TE-B1-B3, TE-C, TE-E1, and TE-E2) genotypes. T. equigenitalis field isolates (n=40) obtained from Austrian Lipizzaner horses were differentiated into three REP (rep-E1, rep-E3a, and rep-E4) and three PFGE genotypes (TE-A2, TE-A5, and TE-D); those isolated from four Austrian Trotters belonged to the REP/PFGE genotype rep-E2/TE-A1. Interestingly, a T. equigenitalis isolate recovered from a Holsteiner stallion living in South Africa revealed the REP/PFGE genotype rep-E1/TE-A5 which was otherwise exclusively present in the majority of Austrian Lipizzaner horses in our study. The type strain included in this study revealed the genotype REP/PFGE rep-E1/TE-F. Six strains of T. asinigenitalis including the type strain were separated into three REP (rep-A1-A3) and six PFGE genotypes (TA-A1, TA-A2, TA-A3, TA-B, TA-C, TA-D). Overall, the generated REP and PFGE genotypes showed a good correlation, whereas REP-PCR proved to be a suitable method for molecular epidemiological screening of T. equigenitalis and T. asinigenitalis isolates that should be differentiated in detail by genotyping using PFGE.

Polymerase chain reaction-based national surveillance programme to determine the distribution and prevalence of Taylorella equigenitalis in South African horses.

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.

Contagious equine metritis in Portugal: A retrospective report of the first outbreak in the country and recent contagious equine metritis test results.

Contagious equine metritis (CEM), a highly contagious bacterial venereal infection of equids, caused by Taylorella equigenitalis, is of major international concern, causing short-term infertility in mares. Portugal has a long tradition of horse breeding and exportation and until recently was considered CEM-free. However, in 2008, T. equigenitalis was isolated at our laboratory from a recently imported stallion and 2 mares from the same stud. Following this first reported outbreak, the Portuguese Veterinary Authority (DGVA) performed mandatory testing on all remaining equines at the stud (n=30), resulting in a further 4 positive animals. All positive animals were treated and subsequently tested negative for T. equigenitalis. Since this outbreak, over 2000 genital swabs from Portuguese horses have been tested at our laboratory, with no further positive animals identified. The available data suggests that this CEM outbreak was an isolated event and we have no further evidence of CEM cases in Portugal, however, an extended and wider epidemiological study would be needed to better evaluate the incidence of the disease in Portuguese horses.

Development of loop-mediated isothermal amplification methods for detecting Taylorella equigenitalis and Taylorella asinigenitalis.

Taylorella equigenitalis is a causative bacterium of contagious equine metritis (CEM), and Taylorella asinigenitalis is species belonging to genus Taylorella. The authors developed two loop-mediated isothermal amplification (LAMP) methods, Te-LAMP and Ta-LAMP, for detecting T. equigenitalis and T. asinigenitalis, respectively. Using experimentally spiked samples, Te-LAMP was as sensitive as a published semi-nested PCR method, and Ta-LAMP was more sensitive than conventional PCR. Multiplex LAMP worked well without nonspecific reactions, and the analytical sensitivities of multiplex LAMP in the spiked samples were almost equivalent to those of Te-LAMP and Ta-LAMP. Therefore, the LAMP methods are considered useful tools to detect T. equigenitalis and/or T. asinigenitalis, and preventive measures will be rapidly implemented if the occurrence of CEM is confirmed by the LAMP methods.

The host model Galleria mellonella is resistant to taylorellae infection.

The genus Taylorella is composed of two species: (i) Taylorella equigenitalis, the causative agent of CEM, a venereally transmitted infection of Equidae and (ii) Taylorella asinigenitalis, a closely related species considered to be nonpathogenic, although experimental infection of mares with this bacterium resulted in clinical signs of vaginitis, cervicitis or endometritis. Currently, there is a need for an alternative host model to further study the taylorellae species. In this context, we explored Galleria mellonella larvae as potential alternative model hosts for taylorellae. Our results showed that infection of G. mellonella larvae with a high concentration of taylorellae did not induce overt G. mellonella mortality and that taylorellae were not able to proliferate within G. mellonella. In conclusion, G. mellonella larvae are resistant to taylorellae infection and therefore do not constitute a relevant alternative system for studying the virulence of taylorellae species. Significance and impact of the study: To date, the pathogenicity and host colonization capacity of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM) and T. asinigenitalis, the second species within the Taylorella genus, remain largely unknown. In this study, we evaluated the relevance of Galleria mellonella as an infection model for taylorellae; we showed that G. mellonella are resistant to taylorellae infection and therefore do not constitute a suitable host model for taylorellae.

Contagious equine metritis: artificial reproduction changes the epidemiologic paradigm.

Recent CEM outbreak reports reflect a novel epidemiologic manifestation with a markedly different risk association for transmission via artificial reproduction and subsequent to inadvertent importation of unapparent carrier stallions. Artificial breeding has an increased association with horizontal or fomite-associated transmission. Reported risk factors include inadequate biosecurity protocols at centralised breeding facilities associated with stallion management and methods of semen collection, processing and transport. Detection of carriers is based on traditional bacteriology from genital swabs and despite limitations inherent to Taylorella equigenitalis is currently the gold standard applied in all international trade and movement protocols. These limitations are reported to be overcome by PCR assays improving diagnostic sensitivity and specificity, practicality, turn-around times, through-put and cost efficacy. Molecular methods have increased understanding of the Taylorelleae, facilitate epidemiologic surveillance and outbreak control strategies. Validation and international regulatory acceptance of a robust PCR-based assay and the undefined risks in association with cryopreserved semen and embryos are future areas warranting further investigation.