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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.

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.

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.

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.

The bacterium, Taylorella equigenitalis , is responsible for the disease contagious equine metritis (CEM), a highly contagious venereal disease of horses. There have been substantial economic losses reported in various equine industries across the world as a result of CEM. So far, there had been no reported cases of T. equigenitalis in the inland area of South Korea. This study was performed to determine the prevalence and the genotype of T. equigenitalis in the inland area of South Korea. In this study, 1 of 38 Thoroughbred horses was found positive for T. equigenitalis using bacterial culture. Multilocus sequence typing (MLST) and construction of a neighbor-joining tree based on the Taylorella spp. MLST database ( http://pubmlst.org/taylorella ) indicated that the inland South Korean T. equigenitalis strain in this study showed a distinct genotype and no epidemiologic relationship with other regional strains suggesting that the inland South Korean T. equigenitalis strain is unique. In order to prevent serious repercussions to the South Korean equine industry, a full epidemiologic investigation and comprehensive treatment regimen are needed.

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.

International audience; 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.

International audience; 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.

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.

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.

Equine population in Croatia has never been systematically tested for the presence of Taylorella equigenitalis, so the epidemiological situation is completely unknown. Both mares and stallions are tested for contagious equine metritis (CEM), but not often, usually only if they were bred abroad, and the owner of the animal they are being bred to requires they are tested. Testing is not mandatory even for licensed stallions. Out of these sporadic testings, no culture of T. equigenitalis has ever been obtained, but no molecular methods were used before 2014. During 2014, 12 animals (10 stallions and two mares) were tested, both by culture and polymerase chain reaction (PCR). All culture plates were either negative or got contaminated before the end of incubation period. Samples from three animals tested positive by PCR. Sequencing of PCR products revealed that one animal (a stallion) was positive for T. equigenitalis, and two animals were positive for T. asinigenitalis (one stallion and one mare). We report the first evidence of T. equigenitalis and T. asinigenitalis natural infections in horses in Croatia. Detecting T. equigenitalis by testing only 12 animals, suggests that incidence of infection in Croatia might be quite high. Furthermore, by detecting T. asinigenitalis in two out of 12 animals we can assume that its prevalence in equine population might also be high. Therefore, if animals are tested only by culture, in every case of positive result isolate has to be identified to the species level. This finding also suggests that equine population in Croatia, especially animals used for reproduction, should be tested for T. equigenitalis, and confirms findings of other authors, that testing only by culture is not reliable, and therefore, animals should be additionally tested with molecular methods.

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.

On PCR amplification of the intervening sequences (IVSs) in the central (helix 45) region within 23S rRNA gene sequences with T. equigenitalis (n = 34), as well as T. asinigenitalis (n = 35) and Bordetella (n = 11) isolates by using the primer pair of f-/r-23STis2, approximately 0.8 kb of the amplicons were generated, sequenced and analyzed. One IVS of approximately 70 bp in length was identified in all the Taylorella organisms but not Bordetella. PCR amplification was further developed for the convenient and rapid molecular detection of T. equigenitalis organisms with the IVS in the helix 45 region within the 23S rRNA genes as target by using the primer pairs (f-IVSde/r-23de). Thus, these results clearly demonstrated that PCR amplification with the primer pair (f-IVSde/r-23de) can be reliable in order to differentiate the T. equigenitalis isolates from both the T. asinigenitalis and Bordetella organisms. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The 3,339 base pair (bp) sequences encoding a putative open reading frame (ORF), non-coding promoter and leader regions (approximately 320 bp), full-length 16S ribosomal RNA (rRNA) gene (approximate 1,540 bp) and part of the 16S-23S rDNA internal spacer region (ISR) were determined from genome DNA libraries of the Taylorella asinigenitalis (UK-1) isolate. The non-coding promoter and leader regions included antiterminators (boxB, boxA and boxC) immediately upstream of the 16S rRNA gene sequence. An approximately 680 bp region upstream of the non-coding promoter region appears to contain a putative ORF with high sequence similarity to GTP cyclohydrolase I. In addition, a typical order of intercistronic tRNA genes with the 48 nucleotide spacer of 5'-16S rDNA-tRNA(Ile)-tRNA(Ala)-23S rDNA-3' was demonstrated in a part of the 16S-23S rDNA ISR. The antiterminators of boxB and boxA were also identified in the ISR.A phylogenetic analysis based on the 16S rRNA gene sequence information clearly demonstrated that the five T. asinigenitalis isolates formed a cluster together with the three T. equigenitalis strains, more similar to Pelistega europaea than the other beta-Proteobacteria from the 13 species of 11 genera.

An approximately 4.2 kbp region encoding 23S and 5S rRNA genes was identified when recombinant plasmid DNAs from two genomic DNA libraries and an inverse PCR product of Taylorella asinigenitalis UK-1 isolate were analyzed. Full-length genes of 23S rRNA (3,225 bp) and 5S rRNA (117 bp) of T. asinigenitalis are described. The present sequence analysis identified a non-coding hypothetically intrinsic transcription terminator region downstream of the 5S rRNA gene. The sequence, however, downstream of the 5S rRNA gene did not show any distal tRNA genes. Surprisingly, an intervening sequence (IVS) of 270 bp in length, including two specific tandem repeat units of 80 bp and one partial unit of 48 bp with unknown functions was identified in the first quarter of the 23S rRNA gene sequence. A second IVS of 70 bp in length was also identified in the central region of the 23S rRNA gene. In addition, by using PCR and sequencing procedures, two T. asinigenitalis isolates, UK-1 and UK-2, carried multiple IVSs in the first quarter and central regions. Moreover, the 23S rRNA fragmentation occurred in the UK-1 isolate. A phylogenetic analysis was first carried out based on the 23S rRNA sequence data from T. asinigenitalis UK-1 and 13 other beta-Proteobacteria. This is the first report of IVSs in the 23S rRNA gene from the beta-Proteobacteria.

Contagious equine metritis is a bacterial infectious disease of horses caused by Taylorella equigenitalis, a Gram-negative eubacterium. The disease has been described in several continents, including Europe, North America and Asia. A novel molecular method was developed to detect clustered regularly interspaced short palindromic repeats (CRISPRs), which were separated by non-repetitive unique spacer regions (NRUSRs) of similar length, in the Taylorella equigenitalis EQ59 strain using a primer pair, f-/r-TeCRISPR-ladder, by PCR amplification. In total, 31 Taylorella isolates (17 T. equigenitalis and 14 Taylorella asinigenitalis) were examined. The T. equigenitalis isolates came from thoroughbred and cold-blooded horses from nine countries during 1980–1996, whilst the T. asinigenitalis isolates all originated from donkey jacks in France and the USA during 1997–2006. PAGE fractionated all of the 13 CRISPRs separated by 12 NRUSRs in T. equigenitalis EQ59. Permutation examples of CRISPRs, which were separated by NRUSRs for small-sized ladders, consisting of two doublet bands were shown. Putative CRISPRs separated by NRUSRs were amplified with 14/17 (82.4 %) geographically disparate T. equigenitalis isolates using the newly designed primer pair. Approximately 82.4 % of the T. equigenitalis isolates had CRISPRs separated by NRUSRs. The CRISPR locus was also found in the French T. asinigenitalis strain MCE3. Putative CRISPRs separated by NRUSRs were detected similarly in 4/14 (28.6 %) T. asinigenitalis isolates. Overall, a more detailed understanding of the molecular biology of CRISPRs within Taylorella organisms may help elucidate the pathogenic virulence and transmission mechanisms associated with this important equine pathogen.

A polyphasic taxonomic study of 14 isolates recovered from various human and veterinary clinical samples was performed. Phenotypically these isolates shared several characteristics with members of the Alcaligenaceae and related genera. Random amplified polymorphic DNA fingerprinting and whole-cell protein analysis suggested the presence of multiple genomic groups, which was confirmed by DNA–DNA hybridization experiments. 16S rRNA gene sequence analysis indicated that these isolates were related to the genera Pelistega, Taylorella, Oligella, Pigmentiphaga, Alcaligenes, Kerstersia, Achromobacter and Bordetella and belonged to the family Alcaligenaceae. Based on the results of the present study the organisms were classified in a novel genus, Advenella gen. nov. This genus comprises one named species, Advenella incenata sp. nov. (type strain LMG 22250T=CCUG 45225T) and five currently unnamed genomic species. The DNA G+C content of members of the novel genus Advenella is between 54·0 and 57·7 mol%.

In the present review article, recent molecular advances relating to studies with Taylorella equigenitalis, as well as the recently described second species of the genus Taylorella, namely Taylorella asinigenitalis, have been described. Molecular genotyping of T. equigenitalis strains by pulsed-field gel electrophoresis (PFGE) after digestion with the suitable restriction enzyme(s) enabled the effective discrimination of strains, thus allowing the examination of the scientific mechanism(s) for its occurrence and transmission of contagious equine metritis (CEM). Alternatively, polymerase chain reaction (PCR) amplification and nucleotide sequencing of the 16S ribosomal DNA sequence and/or the other species specific sequence(s) as targets were confirmed to be effective for identification of T. equigenitalis. These new analytical methods at the genomic DNA level also enabled the discrimination of the newly discovered donkey-related T. asinigenitalis from T. equigenitalis, and moreover, the performance of phylogenetic analysis of genus Taylorella organisms with other closely related genera. Furthermore, detailed analysis of the genes responsible for CEM within the T. equigenitalis genome would be useful to help elucidate the pathogenic virulence and transmission mechanisms associated with the important equine pathogen associated with CEM.

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.

A gram-negative, oxidase-positive, rod-shaped bacterium isolated from the heart of a cotton-topped tamarin was characterized by 16S rDNA sequence analysis, SDS-PAGE of whole-cell proteins, fatty acid analysis and biochemical tests. Outer-membrane proteins, iron-regulated outer-membrane proteins, lipopolysaccharides and siderophore production were studied. On the basis of the results, the organism belongs to the beta-Proteobacteria where it forms a separate line of descent, for which a novel genus and species are proposed, Brackiella oedipodis (LMG 19451T = DSM 13743T = NCIMB 13739T). Nearest phylogenetic neighbours of the new genus are Taylorella, Pelistega, Bordetella, Alcaligenes and Achromobacter.

Background: A novel Gram-negative, non-haemolytic, non-motile, rod-shaped bacterium was discovered in the lungs of a dead parakeet (Melopsittacus undulatus) that was kept in captivity in a petshop in Basel, Switzerland. The organism is described with a chemotaxonomic profile and the nearly complete genome sequence obtained through the assembly of short sequence reads. Results: Genome sequence analysis and characterization of respiratory quinones, fatty acids, polar lipids, and biochemical phenotype is presented here. Comparison of gene sequences revealed that the most similar species is Pelistega europaea, with BLAST identities of only 93% to the 16S rDNA gene, 76% identity to the rpoB gene, and a similar GC content (~43%) as the organism isolated from the parakeet, DSM 24701 (40%). The closest full genome sequences are those of Bordetella spp. and Taylorella spp. High-throughput sequencing reads from the Illumina-Solexa platform were assembled with the Edena de novo assembler to form 195 contigs comprising the ~2 Mb genome. Genome annotation with RAST, construction of phylogenetic trees with the 16S rDNA (rrs) gene sequence and the rpoB gene, and phylogenetic placement using other highly conserved marker genes with ML Tree all suggest that the bacterial species belongs to the Alcaligenaceae family. Analysis of samples from cages with healthy parakeets suggested that the newly discovered bacterial species is not widespread in parakeet living quarters. Conclusions: Classification of this organism in the current taxonomy system requires the formation of a new genus and species. We designate the new genus Basilea and the new species psittacipulmonis. The type strain of Basilea psittacipulmonis is DSM 24701 (= CIP 110308 T, 16S rDNA gene sequence Genbank accession number JX412111 and GI 406042063). © 2014 Whiteson et al.; licensee BioMed Central Ltd.

Objective—To characterize clinical, serologic, bacteriologic, cytologic, and pathologic endometrial responses of mares to 2 donkey-origin atypical bacterial isolates resembling Taylorella equigenitalis. Design—Prospective in vivo study. Animals—10 healthy mares. Procedure—Mares in estrus (2/group) were inoculated by intrauterine infusion with 2 isolates of classic T equigenitalis or 2 isolates of atypical Taylorella sp or were sham-inoculated. Bacteriologic, serologic, clinical, uterine, cytologic, and pathologic endometrial responses were assessed 4, 11, 21, 35, and 63 days after inoculation and on day 111 in mares with positive culture results on day 63. Results—One atypical isolate failed to cause infection. The second atypical isolate and both classic T equigenitalis isolates induced similar transient metritis and cervicitis. Both classic isolates and 1 atypical isolate induced anti-T equigenitalis complement-fixing antibodies detectable at day 11. Classic isolates and an atypical isolate provoked intense neutrophilic endometritis followed by a resolving, subacute, neutrophilic-mononuclear endometrial response. The atypical isolate and classic isolates were recovered from the uterus, clitoral fossa, or clitoral sinus of one or both exposed mares for as long as 111 days. Conclusions and Clinical Relevance—Atypical Taylorella sp infections should be considered as a differential diagnosis of equine infertility in US-origin mares, even those not exposed to stallions from countries where contagious equine metritis occurs. The origins and prevalence of atypical Taylorella sp infection in US horses and donkeys are undetermined. (J Am Vet Med Assoc 2000;216:1945–1948)

International audience; 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.ResultsWe 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.ConclusionsThis 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.

Contagious equine metritis (CEM) is a sexually transmissible disease in mares. Although the disease is commonly diagnosed by culturing the causative bacterium Taylorella equigenitalis (T. equigenitalis) . false negative results do occur. A recently developed Polymerase Chain Reaction (PCR) assay, however, appeared to be much more sensitive, with initial results indicating an unexpected high incidence of the agent in selected horses. In this study, samples from 107 randomly selected mares with no clinical signs of CEM submitted for conventional culture were all negative for T. equigenitalis . but in the PCR-assay 54 (49%) were positive for Taylorella -DNA. Positives in the PCR-assay were found in all breeds tested, even in horses imported from the isolated population in Iceland. These findings suggest that T. equigenitalis was present long before it was first isolated in 1977, The high incidence of Taylorella in horse populations without apparent clinical signs of CEM, the occurrence of incidental clinical case and the known variability between strains, all indicate that Taylorella is endemic in the horse population. In order to explore whether the organism is present in species other than the horse, we also used the PCR-assay on clinically health donkeys (n = 14), zebras (n = 15), Przewalski horses (n = 2) and cows (n = 21). All the animals showed negative results except one of the Przewalski horses, and one cow that was repeatedly found to give positive reaction. We also found that the fertility of 7 stallions with cultures positive for Taylorella (6 used in an AI-program and 1 by natural breeding) was not affected, as shown by the normal range of foaling rates in mares inseminated or bred by these stallions. The overall results may be interpreted to mean that Taylorella is of limited significance in horse breeding.

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.

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.

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.

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.

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 ( Ta IVS1C and Ta IVS1D). 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 ( Ta IVS2D, 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.

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.

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.

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.

Taylorella asinigenitalis sp. nov is a nonpathogenic gram-negative bacterium recently isolated from the genital tract of male donkeys. The bacterium is phenotypically indistinguishable from Taylorella equigenitalis, a pathogen that is the cause of contagious equine metritis, a highly communicable venereal disease of horses. The structural analysis of the lipopolysaccharide produced by T. asinigenitalis sp. nov (ATCC 700933) demonstrated that its O-polysaccharide (O-PS) component is a linear unbranched polymer of repeating disaccharide units composed of 1,3-linked pyranosyl residues of 2,4-diacetamido-2,4-dideoxy-β-d-quinovose (bacillosamine) and 2-acetamidino-2-deoxy-β-d-glucuronic acid, and has the structure [→3)-β-d-QuipNAc4NAc-(1→3)-β-d-GlcpNAmA-(1→]n. The chemical structure and serological characteristics of the T. asinigenitalis O-PS are distinct from those of the O-PS of the T. equigenitalis type strain, thus providing a cell-surface target macromolecule that can be used to distinguish pathogenic from nonpathogenic Taylorella sp. clinical isolates.

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-1 T (ATCC 700933 T ). The T. asinigenitalis strain had a low MIC of gentamicin (MIC ≤ 1 μg/ml) but a high MIC of streptomycin (MIC > 16 μg/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.

Twelve chemolithotrophic strains were isolated from temperate orchard soil on reduced sulfur compounds as energy and electron sources and characterized on the basis of their physiological properties and ability to oxidize various reduced sulfur compounds. The new isolates could oxidize tetrathionate as well as thiosulfate, and oxidation of the latter involved conversion of thiosulfate to tetrathionate followed by its accumulation and eventual oxidation to sulfate, manifested in the production of acid. The mesophilic, neutrophilic, Gram-negative and coccoid bacteria had a respiratory metabolism. Physiologically and biochemically, all the strains were more or less similar, differing only in their growth rates and ability to utilize a few carbon compounds as single heterotrophic substrates. 16S rRNA gene sequence analysis was performed with five representative strains, which revealed a high degree of similarity (⩾99 %) among them and placed the cluster in the ‘Betaproteobacteria’. The strains showed low levels (93·5–95·3 %) of 16S rRNA gene sequence similarity toPigmentiphaga kullae,Achromobacter xylosoxidans,Pelistega europaeaand species belonging to the generaAlcaligenes,TaylorellaandBordetella. The taxonomic coherence of the new isolates was confirmed by DNA–DNA hybridization. On the basis of their uniformly low 16S rRNA gene sequence similarities to species of all the closest genera, unique fatty acid profile, distinct G+C content (54–55·2 mol%) and phenotypic characteristics that include efficient chemolithotrophic utilization of tetrathionate, the organisms were classified in a new genus,Tetrathiobactergen. nov. In the absence of any significant discriminatory phenotypic or genotypic characteristics, all the new isolates are considered to constitute a single species, for which the nameTetrathiobacter kashmirensissp. nov. (type strain WT001T=LMG 22695T=MTCC 7002T) is proposed.

The chemolithoautotrophic betaproteobacterium Tetrathiobacter kashmirensis belongs to the family Alcaligenaceae and is phylogenetically closely related to pathogens such as Taylorella and Bordetella species. While a complete inorganic sulfur oxidation gene cluster, soxCDYZAXWB , is present in its genome, pathogenicity islands or genes associated with virulence, disease, cellular invasion, and/or intracellular resistance are completely absent.

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).

This study elucidates the genomic basis of the evolution of pathogens alongside free-living organisms within the family Alcaligenaceae of Betaproteobacteria. Towards that end, the complete genome sequence of the sulfur-chemolithoautotroph Tetrathiobacter kashmirensis WT001(T) was determined and compared with the soil isolate Achromobacter xylosoxidans A8 and the two pathogens Bordetella bronchiseptica RB50 and Taylorella equigenitalis MCE9. All analyses comprehensively indicated that the RB50 and MCE9 genomes were almost the subsets of A8 and WT001(T), respectively. In the immediate evolutionary past Achromobacter and Bordetella shared a common ancestor, which was distinct from the other contemporary stock that gave rise to Tetrathiobacter and Taylorella. The Achromobacter-Bordetella precursor, after diverging from the family ancestor, evolved through extensive genome inflation, subsequent to which the two genera separated via differential gene losses and acquisitions. Tetrathiobacter, meanwhile, retained the core characteristics of the family ancestor, and Taylorella underwent massive genome degeneration to reach an evolutionary dead-end. Interestingly, the WT001(T) genome, despite its conserved architecture, had only 85% coding density, besides which 578 out of its 4452 protein-coding sequences were found to be pseudogenized. Translational impairment of several DNA repair-recombination genes in the first place seemed to have ushered the rampant and indiscriminate frame-shift mutations across the WT001(T) genome. Presumably, this strain has just come out of a recent evolutionary bottleneck, representing a unique transition state where genome self-degeneration has started comprehensively but selective host-confinement has not yet set in. In the light of this evolutionary link, host-adaptation of Taylorella clearly appears to be the aftereffect of genome implosion in another member of the same bottleneck. Remarkably again, potent virulence factors were found widespread in Alcaligenaceae, corroborating which hemolytic and mammalian cell-adhering abilities were discovered in WT001(T). So, while WT001(T) relatives/derivatives in nature could be going the Taylorella way, the lineage as such was well-prepared for imminent host-confinement.

areas of the adrenal glands. Tissues were negative for equine herpesviruses 1 and 4 and equine arteritis virus. A gram-negative motile bacterium was isolated in pure culture from a range of tissues which was identified as Salmonella abortus equi (4,12:-:e, n, x). Isolates of the bacterium were sensitive to a broad range of antibiotics. Remaining in-foal mares were treated with trimethoprim and gentamicin. After initiation of antibiotic treatment only two further abortions occurred, on November 10th and February 10th. The source of S. abortus equi was not identified. However, since recipient mares came from a range of farms, one or more mares could have been a carrier that recommenced shedding Salmonella resulting from the intercurrent stress associated with relocation. There is a need for greater awareness of S. abortus equi as a potential cause of widespread abortion in the mare and of the importance of breeding farms having in place appropriate biosecurity and preventive measures including vaccination, to minimize the risk of future occurrences of abortion due to this pathogen.

Publisher Summary The genus Haemophilus consists of small, nonmotile, facultatively anaerobic, and gram-negative pleomorphic rods or coccobacilli, which require one or both of the two defined growth factors commonly supplied as hemin and nicotinamide adenine dinucleotide. Any organism corresponding to that description is assigned to this genus. Organisms corresponding to the traditional description of Haemophilus occur in many species, most commonly as apparent commensals on mucous membranes—especially of the upper respiratory and lower genital tracts. Some have a potential for pathogenicity but few are consistently pathogenic. Haemophilus parasuis, Haemophilus gallinarum, Haemophilus haemoglobinophilus, Haemophilus paracuniculus, and Haemophilus aphrophilus have been encountered in animals. Haemophilus parasuis is a common V factor-requiring commensal of the porcine upper respiratory tract. Haemophilus gallinarum is the agent of infectious coryza, which is an upper respiratory infection of chickens. Haemophilus haemoglobinophilus—also known as Haemophilus canis—is a fairly common commensal parasite of the lower canine genital tract, particularly of males. Taylorella equigenitalis causes an acute, self-limiting, suppurative metritis in mares that leaves no permanent effects on the breeding efficiency of recovered animals. The disease is sexually transmitted, but no clinical signs develop in stallions. Both sexes remain asymptomatic carriers for extended periods of time. The chapter also illustrates the pathogenicity, isolation, identification, and cultural characteristics of Haemophilus and Taylorella.

The passive hemagglutination (PHA) test was improved to enable the detection of antibodies to Taylorella (Haemophilus) equigenitalis in the sera of mares. Horse red blood cells (RBC) fixed with glutaraldehyde were compared with similarly treated RBC of a cow, pig and sheep for the PHA test. The horse RBC were superior to those of the other animals tested in detecting mares affected with contagious equine metritis (CEM). A PHA test using these cells as indicator and an antigen prepared from T. equigenitalis by sonication following treatment with hyaluronidase was the most satisfactory in terms of sensitivity and specificity. None of the 156 serum samples from clinically healthy mares without a history of contact with T. equigenitalis-infected stallions or mares showed PHA titers greater than 1:32 and only a few samples (7.1%) showed PHA titers of 1:32. Four of the 50 serum samples from mares affected with CEM showed PHA titers of 1:32, while most of the samples (92.0%) showed PHA titers greater than 1:32. The glutaraldehyde-fixed horse RBC sensitized with the antigen had the advantage of being reproducible for at least 7 months when preserved at 4 degrees C.