Enhanced detection of Taylorella equigenitalis by qPCR using 'Dry' swabs.

Detection of Taylorella equigenitalis (CEMO) in the horse uses genital swabs. These swabs traditionally have been put in Amies charcoal transport medium for detection by culture but are also used for PCR. We determined the suitability of swabs without transport medium (Dry swabs) for CEMO PCR compared to swabs in Amies charcoal transport medium. The experiment was a factorial design using swab type and dilution of organism in culture suspensions, done in two parts. Simulated genital swabs were prepared in the laboratory by dipping in pairs into culture suspensions containing T. equigenitalis with or without other organisms, and then inserting them into a sleeve either with or without transport medium. In study 1, the difference in Ct value for the two swab types was compared. In study 2 genital swab material was then also added to culture suspensions and the swab types again compared. The swabs were tested by a validated quantitative PCR method. The Ct value of the PCR test was used as the measure for comparison, and the effect of variables assessed with linear regression. There was an 7.7% (6.5-8.9) higher mean Ct value of TM versus Dry swabs (P<0.001) overall. The Ct difference was more marked at higher dilutions. Addition of genital swab material had no effect on the Ct value. Dry swabs perform at least as well for PCR as swabs in Amies charcoal transport medium, especially when relatively low numbers of organism are present, and are advantageous for routine sampling when culture is not being used.

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.

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.

Taylorella equigenitalis in Icelandic intact males compared with other horse breeds using natural cover.

BACKGROUND: Contagious equine metritis (CEM) is caused by Taylorella equigenitalis. It is a venereal disease that is detected in some breeds more than others and can cause temporary infertility with substantial costs for regular testing, sanitation and retesting. There was a perceived increase in T. equigenitalis-positive cases in Icelandic intact males where natural cover is common. OBJECTIVES: We aimed to investigate the prevalence of T. equigenitalis in Icelandic intact males and compare to draught horse and Haflinger intact males. We hypothesised that prevalence of T. equigenitalis is higher in Icelandic compared with draught and Haflinger intact males. STUDY DESIGN: Cross sectional. METHODS: Swabs from 76 Icelandic, 35 Haflinger, and 51 draught horse intact males were collected on 38 different farms and analysed by qPCR. Animals were further stratified into active breeding and non-breeding animals and age groups (1.5-7.0 and 8.0-26.0 years). Fisher's exact tests and mixed effect logistic regression with 'farm' as random effect were used to estimate differences in odds for T. equigenitalis-positive test results. RESULTS: The overall prevalence of T. equigenitalis in included intact males was 16.7% (27/162). The odds for T. equigenitalis-positive intact males were significantly higher in Icelandic compared with draught and Haflinger intact males (Odds ratio [OR] = 6.42, 95% confidence interval (CI) = 1.43-28.8, p = 0.02). Odds for T. equigenitalis-positive intact males were significantly lower in active breeding compared with non-breeding animals (OR = 0.09, 95% CI = 0.01-0.54, p = 0.009). Age had no significant influence on test results. MAIN LIMITATIONS: Convenience sampling with regional restrictions to Southern Germany and Austria, small sample size. CONCLUSIONS: Significantly higher odds for T. equigenitalis-positive intact males were found within Icelandic over draught and Haflinger and within non-breeding animals compared with active breeding animals. Findings suggest that non-breeding animals could be a reservoir for T. equigenitalis. Testing for CEM should therefore be routinely performed in Icelandic horses prior to breeding and investigations into epidemiology and reservoirs on affected farms should be initiated.

First report and molecular characterization of cases of natural Taylorella asinigenitalis infection in three donkey breeds in Spain.

Taylorella asinigenitalis is a non-pathogenic bacteria isolated from the genital tract of donkeys but also a cause of metritis and vaginal discharge in mares. It is closely related to Taylorella equigenitalis, the cause of Contagious Equine Metritis (CEM) in horses, and has been present in different countries in Europe since 1995. Up to date, there are no studies on the prevalence of T. asinigenitalis in the equine or asinine populations in Spain; this is the first report of the presence of T. asinigenitalis in donkeys (Equus asinus) from different breeds in three regions of Spain. A total of 106 healthy animals of three different Spanish donkey breeds: Andaluza (26), Majorera (12) and Zamorano-Leonés (68) were sampled between June and July 2017 and a real-time PCR was used to detect T. asinigenitalis in all samples. A total of 39/221 (17,65 %) samples from 22/106 (20,75 %) animals yielded a positive result and were further characterized by MLST; an allelic profile and Sequence Type (ST) could be assigned to 11 of the 39 positive samples, resulting in four novel STs and no clonal complexes within the PubMLST database. There were statistically significant differences in the percentage of positive animals by breed and sex, and also in the variability of STs between farms. Breeding management would have an influence on the percentage of positives in a farm; artificial insemination and separating jacks from jennies should be implemented. Further studies to detect and characterize T. asinigenitalis in donkeys and horses from Spain would be required to obtain a broader epidemiological picture in this country.

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.

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.

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.

Validation of an Easy Handling Sample Preparation and Triplex Real Time PCR for Rapid Detection of T. equigenitalis and Other Organisms Associated with Endometritis in Mares.

Isolation and identification of Taylorella equigenitalis, the causative agent of contagious equine metritis, by bacteriology is laborious and does not permit differentiation from the other member of the genus, Taylorella asinigenitalis. Moreover, other organisms such as Klebsiella pneumoniae and Pseudomonas aeruginosa can also cause endometritis in mares and warrant diagnostic detection. Our objectives were to develop a rapid preparation method for field swab samples and to validate this protocol using new multiplex real-time polymerase chain reaction (rtPCR) detection tools for identification of these four pathogens. The complete analytical process from sample preparation to PCR analysis was then evaluated against bacteriology, the World Organisation for Health's (OIE) gold standard method for T. equigenitalis and commonly used for the other three pathogens. The diagnostic sensitivity and specificity of this method, which used direct lysis and a multiplex rtPCR, were 100% and >92%, respectively. This study provided a simple-to-use method for prebreeding screening of mares and stallions.

Overview of spatio-temporal distribution inferred by multi-locus sequence typing of Taylorella equigenitalis isolated worldwide from 1977 to 2018 in equidae.

The accurate identification of Taylorella equigenitalis strains is essential to improve worldwide prevention and control strategies for contagious equine metritis (CEM). This study compared 367 worldwide equine strains using multilocus sequence typing according to the geographical origin, isolation year and equine breed. The strains were divided into 49 sequence types (STs), including 10 described for the first time. Three major and three minor clonal complexes (CCs), and 11 singletons, were identified. The genetic heterogeneity was low (0.13 STs/strain) despite the wide diversity of geographical origins (n = 16), isolation years (1977-2018) and equine breeds (n = 18). It was highest outside Europe and in the 1977-1997 period; current major STs and CCs already existed before 1998. Previous data associated the major CC1 with the first CEM outbreaks in 1977-1978 in the United Kingdom, Australia and the United States, and revealed its circulation in France. Our study confirms its circulation in France over a longer period of time (1992-2018) and its distribution in Spain and Germany but not throughout Europe. In addition to CC1, relationships between non-European and European countries were observed only through ST4, ST17 and ST30. Within Europe, several STs emerged with cross-border circulation, in particular ST16 and ST46 from the major complexes CC2 and CC8. These results constitute a baseline for monitoring the spread of CEM outbreaks. A retrospective analysis of a higher number of strains isolated worldwide between 1977 and the early 2000s would be helpful to obtain an exhaustive picture of the original CEM situation.

Successful Eradication of Taylorella asinigenitalis, Pseudomonas aeruginosa, and Klebsiella pneumoniae Venereal Bacterial Pathogens Using Domestic Steam Disinfection: Implications for AI Practice.

Steam disinfection has become established as a trusted method of microbial decontamination; however, there have been no reports on the use of this technology to disinfect equipment used in collection of semen in artificial insemination practice. Hence, it was the aim of this study to examine the survival of Taylorella asinigenitalis, Pseudomonas aeruginosa, and Klebsiella pneumoniae venereal bacterial pathogens using domestic steam disinfection. Sixteen bacterial pathogens from three genera Taylorella, Pseudomonas, and Klebsiella each at an inoculum density of approximately 1.5 × 107 colony-forming units were subjected to a steam disinfection cycle. No bacteria were recovered after disinfection, including following recovery and nonselective cultural enrichment techniques. In the absence of full sterilization, domestic steam disinfection of equipment offers a cheap, simple, and widely available technology for the elimination of these pathogens, thereby enhancing infection control in equine breeding.

Direct culture-independent sequence typing of Taylorella equigenitalis obtained from genital swabs and frozen semen samples from South African horses.

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.

Isolation and comparison of Arcanobacterium hippocoleae isolates from the genital tract of 15 mares.

The present study reports the isolation of A. hippocoleae from genital swabs of 15 apparently healthy mares (at least one had an abortion one month earlier) and describes the genotypic and phenotypic characterisation of these strains. The mares were of eight different breeds with a thoroughbred dominance and came from 11 breeding farms located in the French region of Brittany. 16S rRNA gene sequencing confirmed the species' identification by comparing it with reference strain A. hippocoleae CIP 106850T. Some degree of natural divergence within A. hippocoleae was observed by 16S rRNA sequencing (two 1,002-pb sequences), MALDI-TOF MS typing (two groups), a CAMP test (three different intensities of haemolysis from CAMP-positive results) and API® Coryne system (five profiles). The strains were all susceptible to the antimicrobials tested. A national prevalence survey would be required to estimate the frequency of A. hippocoleae carriage in mares and stallions and to verify the presence of A. hippocoleae outside the French region of Brittany, which is the only one found to be affected in the current study, probably because the isolates were recovered from a single field laboratory in this region.

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.