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.

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.

Genomic diversity of Taylorella equigenitalis introduced into the United States from 1978 to 2012.

Contagious equine metritis is a disease of worldwide concern in equids. The United States is considered to be free of the disease although sporadic outbreaks have occurred over the last few decades that were thought to be associated with the importation of horses. The objective of this study was to create finished, reference quality genomes that characterize the diversity of Taylorella equigenitalis isolates introduced into the USA, and identify their differences. Five isolates of T. equigenitalis associated with introductions into the USA from unique sources were sequenced using both short and long read chemistries allowing for complete assembly and annotation. These sequences were compared to previously published genomes as well as the short read sequences of the 200 isolates in the National Veterinary Services Laboratories' diagnostic repository to identify unique regions and genes, potential virulence factors, and characterize diversity. The 5 genomes varied in size by up to 100,000 base pairs, but averaged 1.68 megabases. The majority of that diversity in size can be explained by repeat regions and 4 main regions of difference, which ranged in size from 15,000 to 45,000 base pairs. The first region of difference contained mostly hypothetical proteins, the second contained the CRISPR, the third contained primarily hemagglutinin proteins, and the fourth contained primarily segments of a type IV secretion system. As expected and previously reported, little evidence of recombination was found within these genomes. Several additional areas of interest were also observed including a mechanism for streptomycin resistance and other virulence factors. A SNP distance comparison of the T. equigenitalis isolates and Mycobacterium tuberculosis complex (MTBC) showed that relatively, T. equigenitalis was a more diverse species than the entirety of MTBC.

Development of a single multi-locus sequence typing scheme for Taylorella equigenitalis and Taylorella asinigenitalis.

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.

Genome sequence of Taylorella equigenitalis MCE9, the causative agent of contagious equine metritis.

Taylorella equigenitalis is the causative agent of contagious equine metritis (CEM), a sexually transmitted infection of horses. We herein report the genome sequence of T. equigenitalis strain MCE9, isolated in 2005 from the urethral fossa of a 4-year-old stallion in France.

Pulsed-field gel electrophoresis genotyping of Taylorella equigenitalis isolates collected in the United States from 1978 to 2010.

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.

Identification and differentiation of Taylorella equigenitalis and Taylorella asinigenitalis by lipopolysaccharide O-antigen serology using monoclonal antibodies.

Lipopolysaccharides (LPSs) from Taylorella equigenitalis, the causative agent of contagious equine metritis, and T. asinigenitalis were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharide profiles of 11 T. equigenitalis strains were similar, but different from the profiles of 3 T. asinigenitalis strains, and the profiles of 2 T. asinigenitalis strains were similar to each other. The serological specificities of the LPSs from these 14 strains were examined by immunoblotting and enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) to the LPSs of the T. equigenitalis and T. asinigenitalis type strains and T. asinigenitalis strain 2329-98. A MAb to T. equigenitalis LPS O-polysaccharide (O-PS) (M2560) reacted with LPSs from all T. equigenitalis strains but did not react with LPSs from the 3 T. asinigenitalis strains or with 43 non-Taylorella bacteria. Three MAbs to the T. asinigenitalis type strain LPS O-PS or core epitopes (M2974, M2982, M3000) reacted with the homologous strain and T. asinigenitalis strain Bd 3751/05, but not with any of the other bacteria. Five MAbs to T. asinigenitalis 2329-98 LPS O-PS or core epitopes (M2904, M2907, M2910, M2923, M2929) reacted only with this strain. Proton nuclear magnetic resonance spectra of the O-PSs of the type strains of T. equigenitalis and T. asinigenitalis provided fingerprint identification and differentiation of these 2 organisms. The serological results were consistent with our previous finding that the O-antigen of the type strain of T. equigenitalis, being a linear polymer of disaccharide repeating [-->4)-alpha-L-GulpNAc3NAcA-(1-->4)-beta-D-ManpNAc3NAcA-(1-->] units, differs from that of the T. asinigenitalis O-antigen polymer that is composed of repeating [-->3)-beta-D-QuipNAc4NAc-(1-->3)-beta-D-GlcpNAmA-(1-->] units. Lipopolysaccharide O-PS could be a specific marker for identification and differentiation of T. equigenitalis and T. asinigenitalis, and provide the basis for the development of specific detection assays for T. equigenitalis.

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

A discriminatory real time PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and the related species T. asinigenitalis was developed for the direct examination of genital swabs. The 112bp amplicons produced from the two species were discriminated from each other using TaqMan probes labelled with different fluorophores. The TaqMan PCR was shown to be specific for the 16S ribosomal DNA of the two species of taylorella and did not cross-hybridise with the 16S ribosomal DNA of other bacteria tested. Direct amplification from genital swabs was shown to be equally sensitive to that of culture methods. Prevalence in a sample set from The Netherlands was shown to be equivalent to that demonstrated by culture. A companion real time PCR that amplified a fragment of the 16S rDNA gene of equine commensal bacteria was developed to ensure bacterial DNA was extracted from swab material supplied for testing. The use of a rapid and reliable real time PCR for the organism causing CEM should aid the control of this disease.

Isolation and identification of Taylorella asinigenitalis from the genital tract of a stallion, first case of a natural infection.

Contagious equine metritis (CEM), caused by Taylorella equigenitalis, is a widely known highly contagious genital equine disease that is transmitted venereally. A new bacterium, Taylorella asinigenitalis resembling T. equigenitalis was recently isolated from three American donkey jacks, at routine testing for CEM. The purpose of this study was to identify and characterize a strain of Taylorella sp. from the genital tract of a stallion. Swab samples for culture of T. equigenitalis were taken from urethral fossa, urethra and penile sheath of a 3-year-old stallion of the Ardennes breed when it was routinely tested for CEM. A small Gram-negative rod was isolated, but the colony appearance, the slow growth rate and the results in the API ZYM test differed slightly from those of T. equigenitalis. Sequencing of the 16S rRNA gene was therefore performed and phylogenetic analysis demonstrated that the sequence of the strain Bd 3751/05 represents T. asinigenitalis and that the strain is identical with the Californian asinine strain UCD-1T (ATCC 700933T). The T. asinigenitalis strain had a low MIC of gentamicin (MIC16 microg/ml). Taylorella asinigenitalis has thus for the first time been isolated from the genital tract of a stallion with a natural infection. To determine the pathogenicity of T. asinigenitalis it will be important to conduct further experimental studies. Sequence analysis of 16S rRNA genes was shown to be a reliable tool for differentiation of T. asinigenitalis from T. equigenitalis as well as for identification of these species.

Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis.

BACKGROUND: At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences. RESULTS: Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences. CONCLUSION: High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.

Demonstration of heterogeneous genotypes of Taylorella equigenitalis isolated from horses in six European countries by pulsed-field gel electrophoresis.

Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE profiles to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related difference in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classified into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM.

Comparison of the value of pulsed-field gel electrophoresis, random amplified polymorphic DNA and amplified rDNA restriction analysis for subtyping Taylorella equigenitalis.

Eight strains of Taylorella equigenitalis were identified by a polymerase chain reaction using a primer pair specific to the 16S rDNA of T equigenitalis. These eight strains were chosen because they had previously been shown to represent eight distinct genotypes by pulsed-field gel electrophoresis analysis after separate digestion of the genomic DNA with ApaI or NotI. The eight strains could be classified into six or seven types by random amplified polymorphic DNA analysis using different kinds of primers. Amplified rDNA restriction analysis after separate digestion with five restriction enzymes, including AluI and MboI, of the 1,500 bp fragments of rDNA amplified by polymerase chain reaction did not discriminate the genomic variations among the eight strains of T equigenitalis. Thus, pulsed-field gel electrophoresis was shown to discriminate these eight organisms better than random amplified polymorphic DNA analysis, while amplified rDNA restriction analysis was found to be unsuitable for subtyping T equigenitalis.

Taylorella asinigenitalis sp. nov., a bacterium isolated from the genital tract of male donkeys (Equus asinus).

Three bacterial isolates that were phenotypically indistinguishable from Taylorella equigenitalis were obtained from the urethral fossae of three male donkeys (Equus asinus), one located in the state of California and the other two in the state of Kentucky, USA. Based on results of pulsed-field gel electrophoresis, the isolate from California differed from the two Kentucky isolates, which were the same. Mares bred artificially (California) or naturally (Kentucky) did not show signs of disease, even though infection with the organism was established in those bred naturally. Mares and, uncharacteristically, all three jacks produced antibodies that reacted in the complement fixation test utilized to identify mares recently infected with T. equigenitalis. Sequence analysis of DNA encoding the 16S rRNA revealed that the gene sequences of these isolates were virtually identical to each other (>99.8% similarity), but different (97.6% similarity) from those of several confirmed isolates of T. equigenitalis. The 16S rDNA sequences of the latter were 100% identical. DNA-DNA hybridization studies revealed a mean hybridization level of 89% between the donkey isolate from California and the donkey isolate from Kentucky. On the other hand, the mean DNA-DNA hybridization level from the donkey isolates with DNA from a strain of T. equigenitalis was 23%. The DNA G+C composition was 37.8 mol% for the two donkey isolates, as well as the strain of T. equigenitalis used in the hybridization studies. These data support our opinion that micro-organisms isolated from the male donkeys are different from T. equigenitalis and it is proposed that they be considered a new species within the genus Taylorella and named Taylorella asinigenitalis sp. nov. The type strain is strain UCD-1T (= ATCC 700933T = LMG 19572T).