Whole genome sequence analysis of the 2018 Persian onager isolate suggests sublineages within the Taylorella asinigenitalis species.

In 2018, a T. asinigenitalis strain (MCE663) was isolated in a Persian onager tested for contagious equine metritis (CEM) in a United Kingdom (UK) zoo. This bacterium had never been reported in the UK and Multilocus Sequence Typing described a new atypically divergent ST (ST60). Although the causative agent of CEM is the bacterium Taylorella equigenitalis, a first natural outbreak of endometritis caused by T. asinigenitalis ST70 was reported in 2019, putting its pathogenic potential into question. In this context, we aimed to further sequence the T. asinigenitalis MCE663 genome and characterize the strain using phenotypical and genetic approaches. Results showed that it gathered all identification characteristics of T. asinigenitalis with smaller colonies and it was susceptible to all tested antibiotics. Genome-level phylogeny showed that the genome MCE663 formed a distinct phylogroup, and only shared ≈ 96.1% of average nucleotide identity (ANI) with the three published T. asinigenitalis genomes, which together shared ≈ 98.3% ANI. According to current cut-offs consensus for species and subspecies delineation (95% and 98%, respectively), our results support the first insights of a sublineage delineation within the T. asinigenitalis species.

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.

Taylorella asinigenitalis: raising awareness of its importance and presence in equine and asinine populations.

Taylorella equigenitalis has long been recognised as a causative agent of contagious equine metritis, but practitioners may be less familiar with Taylorella asinigenitalis, which has been identified more recently. Here, Abel Dorrego, Consuelo Serres and Fatima Cruz-Lopez of the Universidad Complutense de Madrid describe T asinigenitalis and report the findings of a survey they carried out in donkeys in Spain.

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.

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.

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.

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.

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.

Survival of taylorellae in the environmental amoeba Acanthamoeba castellanii.

BACKGROUND: Taylorella equigenitalis is the causative agent of contagious equine metritis, a sexually-transmitted infection of Equidae characterised in infected mares by abundant mucopurulent vaginal discharge and a variable degree of vaginitis, cervicitis or endometritis, usually resulting in temporary infertility. The second species of the Taylorella genus, Taylorella asinigenitalis, is considered non-pathogenic, although mares experimentally infected with this bacterium can develop clinical signs of endometritis. To date, little is understood about the basic molecular virulence and persistence mechanisms employed by the Taylorella species. To clarify these points, we investigated whether the host-pathogen interaction model Acanthamoeba castellanii was a suitable model for studying taylorellae. RESULTS: We herein demonstrate that both species of the Taylorella genus are internalised by a mechanism involving the phagocytic capacity of the amoeba and are able to survive for at least one week inside the amoeba. During this one-week incubation period, taylorellae concentrations remain strikingly constant and no overt toxicity to amoeba cells was observed. CONCLUSIONS: This study provides the first evidence of the capacity of taylorellae to survive in a natural environment other than the mammalian genital tract, and shows that the alternative infection model, A. castellanii, constitutes a relevant alternative system to assess host-pathogen interactions of taylorellae. The survival of taylorellae inside the potential environmental reservoir A. castellanii brings new insight, fostering a broader understanding of taylorellae biology and its potential natural ecological niche.

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.

Prevalence and persistence of Taylorella asinigenitalis in male donkeys.

This study was undertaken to investigate the prevalence of Taylorella asinigenitalis in a subset of the donkey population of Michigan and in other equids on farms on which the organism was identified. Other aims were to further characterize the carrier state in terms of persistence and preferred sites of colonization of T. asinigenitalis in the male donkey as well as determine the genotype of any isolates of the organism. Initial testing of 43 donkeys and 1 mule turned up 4 (9.3%) donkeys culture positive for T. asinigenitalis. The 4 culture-positive donkeys resided on 2 farms accommodating a collective total of 89 equids, of which 23 (25.8%) were confirmed positive for T. asinigenitalis. The positive equid population on the 2 farms comprised 14 (67%) of 21 gelded donkeys, 8 (36.4%) of 22 intact male donkeys, and 1 (25%) of 4 gelded horses. T. asinigenitalis was not isolated from 27 female donkeys, 11 female horses, 2 female mules, 1 male horse, or 1 male mule resident on these premises. Isolations of the bacterium were obtained from a number of male donkeys whenever they were sampled over a span of 33 months; preferential sites of isolation were the urethral fossa (fossa glandis), dorsal diverticulum of the urethral sinus, and terminal urethra. Isolates of T. asinigenitalis from the 23 culture-positive equids comprised 2 genotypes, one identical to the type strain isolated in California in 1997, and the other identical to 2 strains isolated from donkey jacks in Kentucky in 1998.

Comparative genomic analyses of the Taylorellae.

Contagious equine metritis (CEM) is an important venereal disease of horses that is of concern to the thoroughbred industry. Taylorella equigenitalis is a causative agent of CEM but very little is known about it or its close relative Taylorella asinigenitalis. To reveal novel information about Taylorella biology, comparative genomic analyses were undertaken. Whole genome sequencing was performed for the T. equigenitalis type strain, NCTC11184. Draft genome sequences were produced for a second T. equigenitalis strain and for a strain of T. asinigenitalis. These genome sequences were analysed and compared to each other and the recently released genome sequence of T. equigenitalis MCE9. These analyses revealed that T. equigenitalis strains appear to be very similar to each other with relatively little strain-specific DNA content. A number of genes were identified that encode putative toxins and adhesins that are possibly involved in infection. Analysis of T. asinigenitalis revealed that it has a very similar gene repertoire to that of T. equigenitalis but shares surprisingly little DNA sequence identity with it. The generation of genome sequence information greatly increases knowledge of these poorly characterised bacteria and greatly facilitates study of them.

Genomic characterization of the Taylorella genus.

The Taylorella genus comprises two species: Taylorella equigenitalis, which causes contagious equine metritis, and Taylorella asinigenitalis, a closely-related species mainly found in donkeys. We herein report on the first genome sequence of T. asinigenitalis, analyzing and comparing it with the recently-sequenced T. equigenitalis genome. The T. asinigenitalis genome contains a single circular chromosome of 1,638,559 bp with a 38.3% GC content and 1,534 coding sequences (CDS). While 212 CDSs were T. asinigenitalis-specific, 1,322 had orthologs in T. equigenitalis. Two hundred and thirty-four T. equigenitalis CDSs had no orthologs in T. asinigenitalis. Analysis of the basic nutrition metabolism of both Taylorella species showed that malate, glutamate and alpha-ketoglutarate may be their main carbon and energy sources. For both species, we identified four different secretion systems and several proteins potentially involved in binding and colonization of host cells, suggesting a strong potential for interaction with their host. T. equigenitalis seems better-equipped than T. asinigenitalis in terms of virulence since we identified numerous proteins potentially involved in pathogenicity, including hemagluttinin-related proteins, a type IV secretion system, TonB-dependent lactoferrin and transferrin receptors, and YadA and Hep_Hag domains containing proteins. This is the first molecular characterization of Taylorella genus members, and the first molecular identification of factors potentially involved in T. asinigenitalis and T. equigenitalis pathogenicity and host colonization. This study facilitates a genetic understanding of growth phenotypes, animal host preference and pathogenic capacity, paving the way for future functional investigations into this largely unknown genus.

Demonstration of the absence of intervening sequences (IVSs) within 16S rRNA genes of Taylorella equigenitalis and Taylorella asinigenitalis isolates.

A total of 57 Taylorella equigenitalis (n=22) and Taylorella asinigenitalis (n=35) isolates was shown not to carry any intervening sequences (IVSs) within 16S rRNA gene sequences. By contrast, we have already shown the genus Taylorella group to carry several kinds of IVSs within the 23S rRNA gene sequences.

Molecular identification and characterization of the intervening sequences (IVSs) within 23S ribosomal RNA (rRNA) genes of Taylorella asinigenitalis isolated in France.

In the helix 25 region, 32 French Taylorella asinigenitalis isolates carried at least one 23S rRNA gene not containing intervening sequences (IVSs). No IVSs in the region were identified in three isolates and the other remaining 29 isolates carried one or more IVSs (UCD-1(T)IVS1A, UCD-1(T)IVS1B and UK-1IVS1B) described already and two new kinds of IVS (TaIVS1C and TaIVS1D). In the helix 45 region, no T. asinigenitalis isolates not carrying any IVSs were identified. UK-1IVS2B was identified in the region from 26 isolates. Five new kinds of IVSs (TaIVS2D, E, F, G and H) occurred in the region in the 13 isolates. Distinctly different tandem repeat units (RS48 and RS32 and RS-A, -B and -C) were evident in both regions, respectively, from the French (n=32) and American (n=3) T. asinigenitalis isolates. Thus, several different kinds of tandem repeat units and their combinations in IVSs in both regions within the gene were shown in 32 French isolates.

Phenotypic and 16S ribosomal RNA gene diversity of Taylorella asinigenitalis strains isolated between 1995 and 2008.

The objective of this study was to examine the degree of phenotypic and genotypic diversity between 43 French Taylorella asinigenitalis strains isolated from 22 jacks, two stallions and one mare between 1995 and 2008 by culturing genital swabs obtained during routine diagnosis for contagious equine metritis. This retrospective analysis revealed the existence of T. asinigenitalis species since 1995 and the natural colonization of a mare's genital tract in 2001. Despite the presence of 27 different patterns revealed by the combination of API ZYM, antibiogram and 16S rDNA profiles, we show that T. asinigenitalis is a highly homogeneous species. API ZYM diversity only concerns acid phosphatase and naphthol-AS-BI-phosphohydrolase activity. The majority of strains are susceptible to a wide range of antimicrobial agents but most are streptomycin-resistant (95.5%), ampicillin-resistant (88.4%), and four strains are atypical due to a high degree of resistance to at least eight antimicrobial agents. 16S rDNA sequence analysis showed only two clusters and revealed similarity of 99.3-100% between T. asinigenitalis strains. The geographic origin of the 43 isolates correlates to the two 16S rDNA clusters.

Initial occurrence of Taylorella asinigenitalis and its detection in nurse mares, a stallion and donkeys in Kentucky.

In 1998, a newly identified bacterium Taylorella asinigenitalis was isolated from the external genitalia and reproductive tracts of nurse mares, a stallion and donkey jacks in Kentucky. An extensive regulatory effort was implemented to contain the outbreak including the tracing and testing of 232 horses and donkeys on 58 premises. T. asinigenitalis was isolated from the reproductive tract of 10 adult equids, including two donkey jacks, one Paint Quarter-horse stallion and seven draft-type breeding mares. None of the infected horses had clinical signs of reproductive tract disease. The odds of being culture positive were 20 times greater for a mare bred to a donkey than for a mare bred to a stallion. Approximately 18% of mares bred to either a carrier stallion or donkey jack were confirmed culture positive. Seventy-one percent of infected mares required more than one course of treatment to clear the organism from their reproductive tracts and one mare harbored the organism for more than 300 days.

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.

Intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella asinigenitalis UCD-1(T) strain.

PCR was performed with Taylorella asinigenitalis UCD-1(T) using two primer pairs constructed in silico for the amplification of the intervening sequences (IVSs) in the first quarter and central regions of the 23S rRNA gene. Following TA cloning and sequencing, the strain was identified to carry heterogeneous and multiple IVSs. Two similar tandem repeat units of 25 and 24 base pairs (bp) with unknown function(s) were identified within the two IVSs in the central region. Secondary structure models of IVSs, containing stem and loop structures, were demonstrated. Although 16S rRNA and 4-5S RNA species were identified in the purified RNA fraction, no 23S rRNAs were evident, resulting in the occurrence of some smaller RNA fragments from approximately 500 to 1,600 bp, in length. Thus, the 23S rRNA primary transcripts may be cleaved into some smaller fragments and IVSs. No IVS transcript was detected by northern blot hybridization analysis. The present and previous results strongly demonstrate the occurrence of heterogeneous and multiple IVSs in 23S rRNA gene sequences and 23S rRNA fragmentation, in T. asinigenitalis.