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.     

Effect of Antibiotic-containing Extenders on Taylorella equigenitalis Contaminated Semen

Taylorella equigenitalis is a gram-negative coccobacillus and the causative agent of a transmissible venereal disease in horses known as contagious equine metritis. Outbreaks of contagious equine metritis have been documented in various countries since 1977, with the most recent discovery in the United States in December 2008. During disease occurrences, culturing semen samples for T equigenitalis before breeding may help to prevent transmission of this disease; however, little is known about the antimicrobial activity of equine semen extenders against the organism. The purpose of this study was to investigate the infectivity levels of T equigenitalis in three equine semen extenders inoculated with known concentrations of the organism. The semen extenders used for this study included INRA 96, E-Z Mixin BF, and VMDZ. In addition, Timentin was added to INRA 96 at three different concentrations (0.5, 1.0, and 1.5 mg/mL) to investigate possible synergistic effects of antibiotic supplementation of extenders. Results were based on the visual counting of the colonies on chocolate Eugon agar plates. Both INRA 96 (with added Timentin) and VMDZ (as supplied by the manufacturer) significantly reduced the numbers of T equigenitalis isolated from semen extenders as compared with INRA 96 (as supplied by the manufacturer) or the antibiotic free E-Z Mixin BF. Our findings indicate that INRA 96 (with added Timentin) or VMDZ may significantly decrease the growth of T equigenitalis in extended semen; however, it is also important to consider the possible effects of antibiotic supplemented extenders on sperm longevity and fertility in addition to eliminating specific pathogens in semen.     

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.     

Analysis of chromosome-sized DNA and genome typing of isolated strains ofTaylorella equigenitalis

Analysis of chromosome-sized DNA and genome typing ofTaylorella equigenitalis NCTC11184, Kentucky 188, and five strains ofT. equigenitalis isolated in Japan were carried out. The three restriction enzymes used,ApaI,NaeI andNotI, cleaved the genomic DNAs of five Japanese strains ofT. equigenitalis into relatively limited numbers of restriction fragments, which were well resolved on crossed-field gel electrophoresis (CFGE). The respective profiles after CFGE of the restriction fragments from all five strains were essentially identical to each other after digestion byApaI,NaeI orNotI. Hence it appears that these strains have a common genome type with respect to these three restriction enzymes. It was also shown that the respective profiles from these strains were essentially different from those ofT. equigenitalis NCTC11184 and those of Kentucky 188 after digestion withApaI,NaeI orNotI.Abbreviations CFGE crossed-field gel electrophoresis - FIGE field inversion gel electrophoresis - PFGE pulsed-field gel electrophoresis     

Nucleotide Sequencing and Analysis of 16S rDNA and 16S-23S rDNA Internal Spacer Region (ISR) of Taylorella equigenitalis, as an Important Pathogen for Contagious Equine Metritis (CEM)

The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184^T, Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184^T and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184^T and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNA^Ile-tRNA^Ala-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.     

A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses

: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.