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Logo of cjvetresCVMACanadian Journal of Veterinary ResearchSee also Canadian Journal of Comparative MedicineJournal Web siteHow to Submit
Can J Vet Res. Jan 2010; 74(1): 18–24.
PMCID: PMC2801306

Language: English | French

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

Abstract

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)-α-L-GulpNAc3NAcA-(1→4)-β-D-ManpNAc3NAcA-(1→] units, differs from that of the T. asinigenitalis O-antigen polymer that is composed of repeating [→3)-β-D-QuipNAc4NAc-(1→3)-β-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.

Résumé

Les lipopolysaccharides (LPSs) provenant de Taylorella equigenitalis, l’agent étiologique de la métrite contagieuse équine, et T. asinigenitalis ont été comparés par électrophorèse sur gel de polyacrylamide avec du sulfate de dodécyl sodique (SDS-PAGE). Les profils de LPSs de 11 souches de T. equigenitalis étaitent similaires, mais différaient des profils de 3 souches de T. asinigenitalis, et les profils de 2 souches de T. asinigenitalis étaient similaires entre eux. Les spécificités sérologiques des LPSs de ces 14 souches ont été examinées par immunobuvardage et immunoessais avec des anticorps monoclonaux (Mabs) contre les LPSs des souches types de T. equigenitalis et T. asinigenitalis et la souche 2329–98 de T. asinigenitalis. Un MAb contre le polysaccharide O (O-PS) du LPS de M. equigenitalis (M2560) a réagit avec les LPSs de toutes les souches de T. equigenitalis mais n’a pas réagit avec les LPSs des 3 souches de T. asinigenitalis ou des 43 bactéries différentes de Taylorella. Trois MAbs dirigés contre le O-PS LPS ou les épitopes du core de la souche type de T. asinigenitalis (M2974, M2982, M3000) ont réagit avec la souche homologue et avec la souche Bd 3751/05 de T. asinigenitalis, mais avec aucune autre bactérie. Cinq MAbs dirigés contre le O-PS du LPS ou les épitopes du core de T. asinigenitalis 2329–98 (M2904, M2907, M2910, M2923, M2929) n’ont réagit qu’avec cette souche. Le spectre des O-PSs des souches types de T. equigenitalis et T. asinigenitalis obtenu par résonnance magnétique nucléaire protonique a fourni des empreintes pour l’identification et la différenciation de ces 2 organismes. Les résultats sérologiques étaient compatibles avec nos résultats précédents qui indiquaient que l’antigène O de la souche type de T. equigenitalis, qui est un polymère linéaire d’unités répétées du disaccharide [4)-α -L-GulpNAc3NAcA-(14)- β-D-ManpNAc3NAcA-(1], diffère du polymère de l’antigène O de T. asinigenitalis qui est composé d’unités répétées de [3)- β-D-QuipNAc4NAc-(13)- β-D-GlcpNAmA-(1]. Le O-PS du LPS pourrait être un marqueur spécifique pour l’identification et la différenciation de T. equigenitalis et T. asinigenitalis, et il fournit les éléments de base pour le développement d’épreuves spécifiques de détection de T. equigenitalis.

(Traduit par Docteur Serge Messier)

Introduction

The gram-negative bacterium Taylorella equigenitalis is the causative agent of contagious equine metritis (CEM), a highly contagious venereal disease of members of the horse family (1). This disease was first described in Thoroughbred horses in England (2) and Ireland (3) in 1977 and has since been diagnosed in many countries worldwide (4). Contagious equine metritis is the focus of considerable international concern to the horse industry because of its potential to cause short-term infertility in broodmares, and the ease with which the carrier state can be established in both mares and stallions. Although endemic in various parts of the world, Canada and several other countries are recognized as being free of CEM. In countries free of this disease, stringent CEM regulatory diagnostic testing programs have been implemented for imported horses.

Isolation and identification of T. equigenitalis is the confirmatory test for diagnosis of CEM (5). However, the fastidious nature and relatively slow growth of this organism makes isolation difficult. Furthermore, this organism is non-reactive in many traditional metabolic and physiological tests, and identification of an isolate as T. equigenitalis is made on a limited number of positive characteristics. In addition to culture, an agglutination test that is based on polyclonal antibodies is commonly used for T. equigenitalis antigenic identification in routine testing laboratories. A complement fixation test has been used successfully as an adjunct to culture for T. equigenitalis in screening mares after being bred with a carrier stallion (6). Polymerase chain reaction (PCR) assays have also been developed for detection of this organism (79). Phylogenetically T. equigenitalis is closely related to Bordetella bronchiseptica, Alcaligenes xylosoxidans, and Oligella urethralis (10,11).

Diagnosis of CEM can be complicated by infection with T. asinigenitalis. T. asinigenitalis was first isolated from the genital tract of 3 male donkeys in the USA in 1997 (12,13), and has since been isolated in Sweden (14) and France (9). T. asinigenitalis is phenotypically very similar to T. equigenitalis, and T. asinigenitalis cells cross react with polyclonal anti-T. equigenitalis serum (5). T. asinigenitalis differs from T. equigenitalis in 16S ribosomal DNA sequence, growth rate, and disease production. T. asinigenitalis does not cause apparent disease in susceptible mares, whereas T. equigenitalis causes endometritis, cervicitis, and vaginitis.

Lipopolysaccharide (LPS), a characteristic component of gram-negative bacteria, is structurally and antigenically diverse, and may provide a basis for improved identification of T. equigenitalis and T. asinigenitalis and for differentiation of these 2 organisms. The LPS molecule generally comprises 3 regions: lipid A, core oligosaccharide, and O-polysaccharide (O-PS) (15). The O-PS has been found to be highly pleomorphic in the majority of bacterial species studied, and in general even related species have few or no O-PS types in common (16). Diversity has also been demonstrated in the LPS core region of various bacteria such as Escherichia coli and Salmonella (17).

Chemical analyses of O-PS reveal that assigned serological antigenic factors can be related to oligosaccharide structural epitopes contained within the O-PS linear structures and, in particular, to epitopes located in the non-reducing terminal oligosaccharide regions of the O-PS (1820) thus providing a molecular level understanding of antibody binding specificities. Recent work on the structural analysis of the lipopolysaccharide O-PSs produced by the type strains of T. equigenitalis (21) and T. asinigenitalis (22) showed that they had virtually identical core oligosaccharide regions. They did, however, have respective O-PS components of linear unbranched chains of repeating disaccharide units composed of chemically and structurally unrelated glycose residues. Thus, the respective LPSs provide target macromolecules for the development of both chemical and serological methods for the differentiation of the 2 Taylorella species.

In the present study, monoclonal antibodies (MAbs) were produced to the LPSs from 3 T. equigenitalis and T. asinigenitalis strains. The serological specificities of T. equigenitalis and T. asinigenitalis LPS were evaluated by examining the reactivity of the MAbs with various T. equigenitalis and T. asinigenitalis strains and with non-Taylorella bacteria. Monoclonal antibodies specific for T. equigenitalis were identified and these reagents may be potentially useful for the development of improved diagnostic tests for CEM.

Materials and methods

Bacterial strains and growth conditions

The T. equigenitalis and T. asinigenitalis strains used in this study are listed in Table I. All strains were grown on modified Eugon chocolate agar containing 5 mg/L amphotericin B (MECA + A) at 35°C in 7.5% CO2 for 48 h. Cells were harvested, suspended in 0.01 M phosphate buffered saline, pH 7.2 (PBS) to a concentration of approximately 1 × 1010 colony forming units (CFUs)/mL and stored at −20°C.

Table I
Taylorella equigenitalis and Taylorella asinigenitalis strains used to study the serological specificities of their lipopolysaccharides

Forty-three non-Taylorella bacteria were used in this study. Thirty-three of these organisms were described previously (23), and the other 10 were Achromobacter xylosoxidans Animal Diseases Research Institute Culture Collection (ADRI) 1916, Bordetella bronchiseptica American Type Culture Collection (ATCC) 10580, Mannnheimia haemolytica ADRI 1909, Moraxella bovis ADRI 1917, M. bovis ADRI 1918, Oligella urethralis ATCC 17960, Salmonella Pullorum National Veterinary Services Laboratory (NVSL) 11, S. Pullorum NVSL 77, Str. zooepidemicus ADRI 1919, and S. zooepidemicus ADRI 1920. O. urethralis was grown on MECA + A at 37°C for 48 h. B. bronchiseptica and Alcaligenes faecalis were grown on MECA + A at 37°C in 5.0% CO2 for 24 h. Arcobacter spp., Campylobacter spp., and Helicobacter spp. were grown on Mueller Hinton (MH) agar containing 10% sheep blood at 37°C under microaerophilic conditions (A. butzleri aerobically) for 48 to 72 h. The other bacteria were grown on trypticase or MH agar containing 10% sheep blood at 37°C aerobically for 16–18 h. Cells were harvested, suspended in 0.01M Tris buffer pH 7.5 to a concentration of approximately 1 × 1010 CFUs/mL and stored at −20°C.

SDS-PAGE and silver staining

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining were used to examine the LPSs of the T. equigenitalis and T. asinigenitalis strains and other selected bacteria. SDS-PAGE was conducted as described previously (24). Briefly, proteinase K (pK) cell digests were prepared by the procedure of Hitchcock and Brown (25). Components in the pK digests were separated by electrophoresis using a discontinuous buffer system with a 6% stacking gel and 12% separating gel. Electrophoresis was conducted with 20 mA constant current per gel at 15°C for approximately 3.5 h. Bands were detected by periodate oxidation-silver staining (26).

Monoclonal antibodies

Monoclonal antibodies to the LPSs of selected T. equigenitalis and T. asinigenitalis strains were produced using methods previously described (23). Briefly, BALB/c and ND4 mice were immunized on days 0, 14, 28, and 44 with formalin-killed cells of T. equigenitalis ATCC 35865, T. asinigenitalis 2329–98 or T. asinigenitalis ATCC 700933. Spleens were harvested on day 48. Hybridomas were produced by fusion of spleen cells with Sp 2/0-Ag-14 myeloma cells. Hybridoma tissue culture fluids were screened for specific antibody using an indirect enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Approximately 30 hybridomas were selected for each of the 3 Taylorella strains and cloned twice, and the isotype of the antibody produced by these hybridomas was determined. All experiments involving animals were approved by the local Animal Care Committee under the guidelines of the Canadian Council on Animal Care.

Indirect ELISA

An indirect ELISA with a pK digest of T. equigenitalis ATCC 35865, T. asinigenitalis 2329–98 or T. asinigenitalis ATCC 700933 cells as antigen was used for initial screening of hybridoma tissue culture fluids. Proteinase K digests were prepared by adding 1 volume of pK (Sigma P4914, 2.5 mg/mL in PBS) to 5 volumes of cells, heating at 60°C for 60 min and then heating at 100°C for 30 min. Microplate (Nunc 475094) wells were passively coated overnight with pK digests diluted approximately 1 in 100 in 0.06 M carbonate buffer pH 9.6. The antigen-coated plates were washed with 0.01 M phosphate buffer pH 7.2 containing 0.15 M NaCl and 0.05% Tween 20 (PBST), hybridoma tissue culture fluid was added, and the plates incubated for 2 h. The plates were washed with PBST, horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA) diluted 1 in 10 000 with PBST added, and the plates incubated for 1 h. The plates were washed again, 3,3′,5,5′-tetra-methylbenzidine/hydrogen peroxide substrate (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland, USA) was added, and the plates shaken for 10 min. The optical density (OD) was determined at 620 nm using a microplate reader (Titertek Multiskan MCC/340, Labsystems, Needham Heights, Massachusetts, USA).

A similar indirect ELISA procedure with pK digests of Taylorella and non-Taylorella bacteria as antigen and the anti-LPS MAbs in tissue culture fluid from 2X cloned hybridomas was used to determine the specificity of T. equigenitalis and T. asinigenitalis LPSs. An ELISA OD 620 ≥ 0.10 was interpreted as a positive result.

Immunoblotting

Immunoblotting with MAbs was used to analyze the LPSs of selected T. equigenitalis and T. asinigenitalis strains. The procedure used was essentially as described by Brooks et al (24). T. equigenitalis ATCC 35865, T. asinigenitalis 2329–98, and T. asinigenitalis ATCC 700933 cells were digested with pK. Lipopolysaccharides in the pK digests were separated by SDS-PAGE, and the separated components transferred electrophoretically from gels to nitrocellulose membranes. The membranes were washed for 1 min with 0.02 M Tris buffered saline pH 7.4 (TBS). The unbound sites on the membranes were blocked by incubation for 1 h at 35°C with TBS containing 0.05% Tween 20 (TBST). After blocking, the membranes were incubated for 16 h at 20°C with hybridoma tissue culture fluid diluted 1 in 2 with TBST. The membranes were washed with TBST and incubated for 2 h with alkaline phosphatase-conjugated goat anti-mouse IgG (Zymed Laboratories, supplied by Mandel, Guelph, Ontario) diluted 1 in 1000 with TBST. The membranes were washed again and incubated for 10 min with 5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazolium chloride substrate (Kirkegaard and Perry Laboratories).

Extraction of LPS, preparation of O-polysaccharides, and nuclear magnetic resonance (NMR) spectroscopy

Fermenter grown cells of the type strains of T. equigenitalis (ATCC 35865) and T. asinigenitalis (ATCC 700933) were extracted by a modified aqueous phenol procedure (27) and the LPSs were obtained (ca 6% yield) by a 3-fold ultracentrifugation (105 000 × g, 4°C, 8 h) of the concentrated dialyzed extract and the final precipitated gels were lyophilized from their water solution.

The LPS preparations (100 mg) were subjected to hydrolysis with 1.5% (v/v) acetic acid (50 mL, 100°C, 2 h) and following removal of precipitated lipid A by low speed centrifugation, the concentrated aqueous solution was subjected to Sephadex G-50 column chromatography (2 × 50 cm) in 0.05 M pyridinium acetate buffer (pH 5.2) and the high molecular mass eluting O-PS fraction (Kav 0.01–0.12) was collected and lyophilized.

1H-NMR spectra of O-PS exchanged and dissolved in 99% D2O at 25°C were recorded using a Varian Inova 500 MHz under standard conditions as previously described (28).

Results

SDS-PAGE analysis of LPS from T. equigenitalis and T. asinigenitalis

A silver-stained gel of pK-treated cell lysates of T. equigenitalis, T. asinigenitalis and other selected bacteria is shown in Figure 1. SDS-PAGE of S. Typhimurium LPS, included as a control and for comparison (Figure 1, lanes 1 and 9), produced a ladder-like pattern of bands typical of smooth-type LPS. The fastest migrating band was core oligosaccharide (no O-PS) and each successive band was core oligosaccharide plus increasing numbers of repeating O-PS units. A ladder-like pattern of bands was also seen with LPS from T. asinigenitalis strains 2329–98, ATCC 700933 and Bd 3751/05 (lanes 3, 4, 5, respectively). The profiles of T. asinigenitalis strains ATCC 700933 and Bd 3751/05 were virtually identical but differed from that of T. asinigenitalis strain 2329–98. High molecular mass LPS was detected in 2 areas of the gel but no ladder-like pattern of bands was observed with T. equigenitalis ATCC 35865 (lane 2), and a similar profile was observed with the other 10 T. equigenitalis strains (results not shown). High molecular mass LPS but no ladder-like pattern of bands was also observed with Bordetella bronchiseptica ATCC 10580 (lane 6). Low molecular mass (LMM) core oligosaccharide was observed in the LPS profiles of all 4 Taylorella strains (lanes 2–5), B. bronchiseptica (lane 6), O. urethralis (lane 7), and A. faecalis (lane 8).

Figure 1
Periodate oxidized and silver stained SDS-PAGE profiles of purified lipopolysaccharides from Salmonella Typhimurium (lanes 1 and 9), and pK digests of T. equigenitalis ATCC 35865 (lane 2), T. asinigenitalis 2329–98 (lane 3), T. asinigenitalis ...

Analysis of T. equigenitalis and T. asinigenitalis LPS by immunoblotting with MAbs

Monoclonal antibodies were produced to the LPS from 3 Taylorella strains. The reactivity of the LPS from these 3 strains was examined by immunoblotting with MAbs to the homologous LPS.

Thirty-two MAbs were produced to T. equigenitalis ATCC 35865 LPS. Two different patterns of reactivity were seen on immunoblots with these MAbs. The patterns observed with MAbs M2560 and M2565 were selected as representative (Figure 2, lanes 1 and 2, respectively). MAb M2560 reacted with O-PS whereas MAb M2565 reacted with core oligosaccharide. The isotype of MAb M2560 is IgG1 and the isotype of MAb M2565 is IgG3.

Figure 2
Immunoblots of pK digests of T. equigenitalis ATCC 35865 (lanes 1 and 2), T. asinigenitalis 2329–98 (lanes 3–7) and T. asinigenitalis ATCC 700933 (lanes 8–11) with monoclonal antibodies M2560 (lane 1), M2565 (lane 2), M2907 (lane ...

Twenty-nine MAbs were produced to T. asinigenitalis strain 2329–98 LPS. Five immunoblotting patterns were observed and are represented by the reactions with MAbs M2907, M2929, M2904, M2910, and M2923 (Figure 2, lanes 3, 4, 5, 6, and 7, respectively). Monoclonal antibodies M2907 and M2929 both reacted with O-PS and both reacted with LPS components with relatively high numbers of O-PS units. Monoclonal antibody M2929, but not M2907, also reacted with components with lower numbers of O-PS units. Monoclonal antibodies M2904, M2910, and M2923 reacted with core oligosaccharide. The isotype of MAb 2907 is IgG3, the isotype of MAbs M2929, M2904, and M2910 is IgG1, and the isotype of MAb M2923 is IgG2a.

Thirty-four MAbs were produced to T. asinigenitalis ATCC 700933 LPS. Four immunoblot patterns were obtained, as represented by the reaction with M2974, M3000, M2975, and M2982 (Figure 2, lanes 8, 9, 10, and 11, respectively). Monoclonal antibodies M2974, M3000, and M2975 reacted with O-PS. Monoclonal antibody M2975 reacted with LPS with the lower numbers of O-PS units, compared to MAbs M2974 and M3000. Monoclonal antibody M2982 reacted with both HMM and LMM LPS components. The isotype of MAb M2974 is IgG3 and the isotype of MAb M2982 is IgG1. Monoclonal antibodies M3000 and M2975 are IgA.

Specificity of T. equigenitalis and T. asinigenitalis LPS determined by ELISA with MAbs

The ELISA reactivity of LPS from T. equigenitalis, T. asinigenitalis, and 43 non-Taylorella bacteria with selected MAbs is shown in Table II. Monoclonal antibody M2560 reacted with all 11 strains of T. equigenitalis, but did not react with the 3 T. asinigenitalis strains or with the 43 non-Taylorella bacteria tested. Thus at least one epitope of T. equigenitalis O-PS appears to be highly specific for this organism. In contrast, MAb M2565 reacted with all 14 Taylorella strains and with 29 of the 43 non-Taylorella bacteria, indicating that an epitope of T. equigenitalis LPS core oligosaccharide is shared by various bacteria.

Table II
Reactivity of proteinase K digested cell lysates of Taylorella equigenitalis and T. asinigenitalis strains and non-Taylorella bacteria with selected monoclonal antibodies by ELISA

Monoclonal antibodies M2907, M2929, M2904, M2910, and M2923 reacted only with T. asinigenitalis strain 2329–98 and not with the other bacteria tested. Thus, several O-PS and core oligosaccharide epitopes of T. asinigenitalis 2329–98 LPS appear to be essentially unique to this organism.

Monoclonal antibodies M2974, M3000, and M2982 reacted only with T. asinigenitalis strains ATCC 700933 and Bd 3751/05, and therefore the LPS of these 2 strains appears to be very similar and highly specific to these 2 organisms. Monoclonal antibody M2975 reacted with T. asinigenitalis ATCC 700933 and Bd 3751/05, but also cross reacted with Mannheimia haemolytica ADRI 1909 (OD 0.26). It is of note that the conserved oligosaccharide (29) of the LPSs produced by O-serotypes of M. haemolytica in common with Taylorella cores express epitopes involving D-glycero-D-mannoheptose residues that may account for observed serological cross-reactions.

NMR spectroscopy

The proton NMR spectra of the O-PS preparations made in this study (Figure 3) were consistent with the resonance assignments previously determined from the use of 2D heteronuclear NMR experiments (21,22). The characteristic chemical shifts of the anomeric proton signals (4.5–6.0 ppm region), the unique methyl resonances of the 6-deoxyhexose residue (~1.17 ppm of D-QuiNAc4NAc in the T. asinigenitalis O-PS), and the complex of glycosyl ring region protons provide a simple fingerprint for the identification and differentiation of the respective antigenic O-PSs of T. equigenitalis and T. asinigenitalis type strains.

Figure 3
1H-NMR spectra of the O-PS preparations from the LPS of (A) T. equigenitalis (ATCC 35865) and (B) T. asinigenitalis (ATCC 700933).

Discussion

In the present study, analysis by SDS-PAGE and by ELISA with anti-LPS MAbs demonstrated that the LPSs of all 11 T. equigenitalis strains examined were virtually identical, but differed from the LPSs of 3 T. asinigenitalis strains. In addition, the LPS of T. asinigenitalis strains ATCC 700933 and Bd 3751/05 (California and Swedish isolates, respectively) were indistinguishable from each other, but distinct from the LPS of T. asinigenitalis 2329–98 (Kentucky isolate). These results show that selected anti-LPS MAbs can be employed for identification of T. equigenitalis, for differentiation of T. equigenitalis from T. asinigenitalis, and for distinction between strains of T. asinigenitalis.

In a previous study, Gradinaru et al (30) characterized LPS and protein components of 9 T. equigenitalis strains by SDS-PAGE. The pattern of LPS components detected after silver staining was similar for all strains examined, with a major component of molecular mass of approximately 22 kDa. In contrast, in the present study both HMM and LMM components were detected in the LPS profile of the T. equigenitalis type strain and the other 10 T. equigenitalis strains examined by SDS-PAGE and silver staining and by immunoblotting with MAb M2560 and other anti-T. equigenitalis O-PS MAbs. Gardinaru et al (30) also produced MAbs that were specific to T. equigenitalis but these MAbs reacted with epitopes of protein components.

Nuclear magnetic resonance spectral and chemical analysis confirmed that the LPS O-PSs from the type strains of T. equigenitalis (ATCC 35865) and T. asinigenitalis (ATCC 700933) were substantially structurally different. The T. equigenitalis O-PS antigen was found to be a polymer of partially amidated disaccharide units composed of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA) and 2,3-diacetamido-2,3-dideoxy-L-guluronic acid (D-GulNAcA3NAcA) having the structure [→4)-α-L-GulpNAc3NAcA-(1→4)-β-D-ManpNAc3NAc-(1→]n in which the terminal nonreducing α-L-GulpNAc3NAcA residue was methylated at its O-4 position. The T. asinigenitalis O-PS was determined to be a polymer of a disaccharide repeating unit composed of 2,4-diacetamido-2,4-dideoxy-D-quinovose (D-QuiNAc4NAc, 2,4-diacetamido-2,4,6-trideoxy-D-glucose) and 2-acetidimidoylamino-2-deoxy-D-glucuronic acid (D-GlcNAmA) residues having the structure [→3)-β-D-QuipNAc4NAc-(1→3)-β-D-GlcpNAmA-(1→]n. (21,22). The significant structural differences between the LPS O-PS of T. equigenitalis and T. asinigenitalis provide a basis for serological differentiation of these 2 organisms. T. equigenitalis and T. asinigenitalis can be identified by reference to their respective analyzed and reported (21,22) 2-D 1H-13C HSQC correlation spectra, or more simply, from their 1-D proton NMR spectra (Figure 3) using readily extracted LPS or derived O-PS, or by the possible use of magic angle spinning NMR spectroscopy requiring only a suspended sample of whole bacterial cells collected from a single plate colony (31). The major differences in the two structures are revealed in the glycose anomeric proton signals (5.03 and 5.05 ppm) and (4.64 and 4.51 ppm) in the respective T. equigenitalis and T. asinigenitalis spectra and the unique C-6 methyl signal (1.17 ppm) of the D-QuipNAc4NAc residue in the T. asinigenitalis O-antigen.

Since T. equigenitalis and T. asinigenitalis LPSs are structurally distinct from each other, it may also be possible to identify T. equigenitalis and T. asinigenitalis using PCR assays targeting regions within the genes involved in LPS biosynthesis. In various gram-negative bacteria, many of the enzymes involved in O-PS biosynthesis are encoded in an rfb gene cluster, and PCR assays targeting regions within rfb gene clusters have been used for identification of O serogroups of Salmonella (32), Escherichia coli (33), Shigella (34) and other bacteria. Similarily, sequences in the genes involved in the biosynthesis of the LPS O-PS and the core oligosaccharide may provide useful probes for identification and differentiation of T. equigenitalis and T. asinigenitalis.

In summary, based on serological and structural evidence, the LPS O-PS can be used as a specific marker for identification and differentiation of T. equigenitalis and T. asinigenitalis, and thus provides the basis for the development of specific detection assays for T. equigenitalis.

Acknowledgments

The authors gratefully acknowledge the expert contributions of the CFIA Ottawa Laboratory (Fallowfield) Monoclonal Antibody Unit and animal care staff in production of the monoclonal antibodies, and Perry Flemming at the NRC for the large-scale production of bacterial cell mass required for chemical structural analysis of LPS. We also thank V. Båverud for providing T. asinigenitalis strain Bd 3751/05 and J. Devenish for providing the other Taylorella strains.

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