Targeting Novel LPXTG Surface Proteins with Monoclonal Antibodies for Immunomagnetic Separation of Listeria monocytogenes.

The Gram-positive bacterium Listeria monocytogenes causes a significantly high percentage of fatalities among human foodborne illnesses. Surface proteins, specifically expressed from a wide range of L. monocytogenes serotypes under selective enrichment culture conditions, can serve as targets for the isolation of this pathogen using antibody-based methods to facilitate molecular detection. In this study, monoclonal antibodies (MAbs), previously raised against the L. monocytogenes LPXTG surface proteins LMOf2365_0639 and LMOf2365_0148, were investigated for their ability to isolate L. monocytogenes from bacterial samples with immunomagnetic separation (IMS). Only 1 out of 35 MAbs against LMOf2365_0639, M3644, was capable of capturing L. monocytogenes. Among all the 24 MAbs examined against LMOf2365_0148, 4 MAbs, M3686, M3697, M3699, and M3700, were capable of capturing L. monocytogenes cells specifically from abbreviated primary selective enrichment cultures in either Palcam or LEB/UVM1 media or from mixed samples containing target and nontarget bacteria. MAb M3686 showed a unique specificity with the capability to capture strains of seven L. monocytogenes serotypes (1/2a, 1/2b, 1/2c, 3a, 4a, 4b, and 4d). These promising MAbs were subsequently characterized by quantitative measurements of antigen-binding affinity using surface plasmon resonance analysis and epitope mapping using overlapping recombinant polypeptides. The usefulness of these MAbs to LMOf2365_0148 in bacterial capture was consistent with their high affinities with KD constants in the nanomolar range and can be explored further for the development of an automated IMS method suitable for routine isolation of L. monocytogenes from food and environmental samples.

Assessment of Listeria monocytogenes Surface Proteins Identified from Proteomics Analysis for Use as Diagnostic Biomarkers.

The Gram-positive bacterium Listeria monocytogenes is an important pathogen that causes a foodborne illness with a high percentage of fatalities. Surface proteins, specifically expressed from a wide range of L. monocytogenes serotypes under selective enrichment culture conditions, can serve as targets for the detection and isolation of this pathogen using antibody-based methods. Among a number of surface proteins identified by mass spectrometry in a previous proteomic study, six candidates (annotated as LMOf2365_0148, LMOf2365_0312, LMOf2365_0546, LMOf2365_1883, LMOf2365_2111, and LMOf2365_2742) were selected here for investigating their expression in the bacterial cells cultured in vitro by raising rabbit polyclonal antibodies (PAbs) against the recombinant form of each candidate. These protein candidates contained regions conserved among various L. monocytogenes isolates but variable in other Listeria species. LMOf2365_0148, an uncharacterized protein with a LPXTG motif accountable for covalent linkage to the cell wall peptidoglycan, exhibited a strong reaction signal from anti-LMOf2365_0148 PAb binding to the cell surface, as detected by immunofluorescence microscopy. Further study, through the generation of a panel of mouse monoclonal antibodies (MAbs) to the recombinant LMOf2365_0148, showed that one of the MAbs, M3686, reacted to bacterial isolates belonging to all three lineages of L. monocytogenes under Health Canada's standard enrichment culture conditions (MFHPB-07 and MFHPB-30). These results demonstrated the potential of using LMOf2365_0148 as a surface biomarker, in conjunction with specific MAbs developed here, for the isolation and detection of L. monocytogenes from foods and food processing environments. IMPORTANCE Strains of Listeria monocytogenes are differentiated serologically into at least 13 serotypes and grouped phylogenetically into 4 distinct lineages (I, II, III, and IV). No single monoclonal antibody (MAb) reported to date is capable of binding to the surface of L. monocytogenes strains representing all the serotypes. This study assessed the expression of six surface proteins selected from a previous proteomic study and demonstrated that surface protein LMOf2365_0148 has the greatest potential as a surface biomarker. A panel of 24 MAbs to LMOf2365_0148 were assessed extensively, revealing that one of the MAbs, M3686, reacted to a wide range of L. monocytogenes isolates (lineage I, II, and III isolates) grown under standard enrichment culture conditions and thus led to the conclusion that LMOf2365_0148 is a useful novel surface biomarker for identifying, detecting, and isolating the pathogen from food and environmental samples.

Evaluation of the Use of Sea Water as a Diluent for an Accelerated Hydrogen Peroxide Disinfectant for Inactivation of Avian Influenza Virus: A Surrogate for Infectious Salmon Anemia Virus.

Introduction: Use of sea water as a diluent for disinfectants has been of practical interest for control of aquaculture disease outbreaks in sea where fresh water is limited. This study evaluated the use of natural sea water (NSW), artificial sea water (ASW), or standard hard water (SHW) as a diluent for preparation of accelerated hydrogen peroxide (AHP) solutions against an avian influenza virus, a surrogate for the infectious salmon anemia virus. Methods: AHP solutions containing 0.18%, 0.35%, or 0.44% (w/w) of hydrogen peroxide (H2O2), corresponding to 1/40, 1/20, and 1/16 dilutions of the disinfectant concentrate, were evaluated at -20°C, 4°C, and 21°C. Results: When NSW was used as the diluent, a 0.35% H2O2 concentration was required to inactivate ∼6 log10 virus at 21°C in a 5-min contact time. When temperature dropped to 4°C, 0.44% H2O2 in NSW was required to obtain a similar inactivation within a 5-min contact time. At -20°C, supplemented with antifreeze agents, the 0.44% H2O2 in NSW solutions produced complete inactivation of 5.4 log10 virus within a 10-min contact time. In comparison, lower H2O2 concentrations and/or shorter contact times were needed to inactivate equal amounts of the virus at the same temperature when using SHW or ASW as a diluent to prepare disinfection solutions. Conclusion: The results suggested that NSW could be used as a diluent in disinfection solutions for virus inactivation as long as disinfectant concentrations and/or contact times are properly increased.

Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies.

Protein antigens expressed on the surface of all strains of Listeria monocytogenes and absent from nonpathogenic Listeria spp. are presumably useful targets for pathogen identification, detection, and isolation using specific antibodies (Abs). To seek such surface proteins expressed in various strains of L. monocytogenes for diagnostic applications, we focused on a set of surface proteins known to be involved or putatively involved in L. monocytogenes virulence and identified Listeria adhesion protein B (LapB) as a candidate based on the bioinformatics analysis of whole-genome sequences showing that the gene coding for LapB was present in L. monocytogenes strains and absent from strains of other Listeria spp. Immunofluorescence microscopy (IFM), performed with rabbit polyclonal antibodies against the recombinant LapB protein (rLapB) of L. monocytogenes serotype 4b strain L10521, confirmed expression of LapB on the surface. A panel of 48 mouse monoclonal antibodies (MAbs) to rLaB was generated, and 7 of them bound strongly to the surface of L. monocytogenes cells as demonstrated using IFM. Further characterization of these 7 anti-LapB MAbs, using an enzyme-linked immunosorbent assay (ELISA), revealed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted strongly with 46 (86.8%) of 53 strains representing 10 of the 12 serotypes tested (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4b, 4d, and 4e). These results indicate that LapB, together with companion anti-LapB MAbs, can be targeted as a biomarker for the detection and isolation of various L. monocytogenes strains from contaminated foods. IMPORTANCE: Strains of L. monocytogenes are traditionally grouped into serotypes. Identification of a surface protein expressed in all or the majority of at least 12 serotypes would aid in the development of surface-binding monoclonal antibodies (MAbs) for detection and isolation of L. monocytogenes from foods. Bioinformatics analysis revealed that the gene coding for Listeria adhesion protein B (LapB), a surface protein involved in L. monocytogenes virulence, was present in L. monocytogenes strains and absent from other Listeria spp. Polyclonal antibodies against recombinant LapB (rLapB) detected the exposed epitopes on the surface of L. monocytogenes Production and extensive assessment of 48 MAbs to rLapB showed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) detected the expression of LapB in a wide range of L. monocytogenes isolates representing 10 of 12 serotypes tested, suggesting that LapB, together with specific MAbs, can be targeted as a biomarker for pathogen detection and isolation.

Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools.

UNLABELLED: The Gram-positive bacterium Listeria monocytogenes causes a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range of L. monocytogenes serotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of the L. monocytogenes serotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains of L. monocytogenes which are variable among other Listeria species. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46 L. monocytogenes lineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17 L. monocytogenes lineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some other Listeria species grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker of L. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples. IMPORTANCE: L. monocytogenes is traditionally divided into at least 12 serotypes. Currently, there are no monoclonal antibodies (MAbs) available that are capable of binding to the surface of L. monocytogenes strains representing all 12 serotypes. Such antibodies would be useful and are needed for the development of methods to detect and isolate L. monocytogenes from food samples. In our study, we aimed to identify surface proteins that possess regions of well-conserved amino acid sequences among various serotypes and then to employ them as antigen targets (biomarkers) for the development of MAbs. Through bioinformatics and protein expression analysis, we identified one of the four putative surface protein candidates, LMOf2365_0639, encoded by the genome of the L. monocytogenes serotype 4b strain F2365, as a useful surface biomarker. Extensive assessment of 35 MAbs raised against LMOf2365_0639 in our study revealed three MAbs (M3643, M3644, and M3651) that recognized a wide range of L. monocytogenes isolates.

Campylobacter species in animal, food, and environmental sources, and relevant testing programs in Canada.

Campylobacter species, particularly thermophilic campylobacters, have emerged as a leading cause of human foodborne gastroenteritis worldwide, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari responsible for the majority of human infections. Although most cases of campylobacteriosis are self-limiting, campylobacteriosis represents a significant public health burden. Human illness caused by infection with campylobacters has been reported across Canada since the early 1970s. Many studies have shown that dietary sources, including food, particularly raw poultry and other meat products, raw milk, and contaminated water, have contributed to outbreaks of campylobacteriosis in Canada. Campylobacter spp. have also been detected in a wide range of animal and environmental sources, including water, in Canada. The purpose of this article is to review (i) the prevalence of Campylobacter spp. in animals, food, and the environment, and (ii) the relevant testing programs in Canada with a focus on the potential links between campylobacters and human health in Canada.

Enhanced inactivation of avian influenza virus at -20°C by disinfectants supplemented with calcium chloride or other antifreeze agents.

Avian influenza outbreaks have occurred during winter months, and effective disinfection of poultry premises at freezing temperatures is needed. The commercial disinfectants Virkon and Accel, supplemented with an antifreeze agent [propylene glycol (PG), methanol (MeOH), or calcium chloride (CaCl2)], were evaluated for their effectiveness in killing avian influenza virus (AIV) at -20°C or 21°C. An AIV suspension was applied to stainless steel disks, air-dried, and covered with a disinfectant or antifreeze agent for 5 to 30 min. Virkon (2%) and Accel (6.25%) with 30% PG, 20% MeOH, or 20% CaCl2 inactivated 6 log10 AIV within 5 min at -20°C and 21°C. At these temperatures PG and MeOH alone did not kill AIV, but the 20% CaCl2 solution alone inactivated 5 log10 AIV within 10 min. The results suggested that CaCl2 is potentially useful to enhance the effectiveness of disinfection of poultry facilities after outbreaks of AIV infection in warm and cold seasons.

Les poussées de cas d'influenza aviaire se sont produites durant les mois d'hiver, et une désinfection efficace des sites d'élevage à des températures de congélation est nécessaire. Les désinfectants commerciaux Virkon et Accel, auxquels on ajouta un antigel [propylène glycol (PG), méthanol (MeOH), ou chlorure de calcium (CaCl2)], furent évalués pour leur efficacité à tuer le virus de l'influenza aviaire (VIA) à −20 °C ou 21 °C. Une suspension de VIA fut appliquée à des disques d'acier inoxydable, séchés à l'air, et recouverts avec un désinfectant ou d'antigel pour une durée de 5 à 30 minutes. Le Virkon (2 %) et l'Accel (6,25 %) avec 30 % PG, 20 MeOH, ou 20 % CaCl2 ont inactivé 6 log10 de VIA en-dedans de 5 min à −20 °C et 21 °C. À ces températures le PG et le MeOH seuls n'ont pas réussi à tuer le VIA, mais la solution de CaCl2 à 20 % seule a inactivé 5 log10 de VIA en-dedans de 10 min. Les résultats suggèrent que le CaCl2 est potentiellement utile pour augmenter l'efficacité de la désinfection des exploitations avicoles suite à des poussées de cas d'infection par le VIA en saison chaude ou froide.(Traduit par Docteur Serge Messier).

Monoclonal Antibodies to Lipopolysaccharide O Antigens of Enterohemorrhagic Escherichia coli Strains in Serogroups O26, O45, O103, O111, O121, and O145.

Non-O157 enterohemorrhagic Escherichia coli in priority serogroups O26, O45, O103, O111, O121, and O145 are increasingly recognized as important human pathogens. In the present study, a panel of monoclonal antibodies (MAbs) to the lipopolysaccharide O antigens of E. coli in serogroups O26, O45, O103, O111, O121, and O145 was produced. The specificity was evaluated by examining the reactivity of the MAbs with 50 E. coli strains and 42 non-E. coli bacteria, and several MAbs highly specific for E. coli strains in each of the six non-O157 priority serogroups were identified. The use of these highly specific MAbs may be of considerable value for determining whether an E. coli isolate belongs to one of the six priority non-O157 serogroups, for developing specific detection assays for these organisms, and for characterizing the lipopolysaccharide O antigens of isolates in these serogroups.

Comparison of an antigen-capture enzyme-linked immunosorbent assay with bacterial culture for detection of Salmonella in poultry-hatchery environmental samples.

An antigen-capture, monoclonal-antibody-based enzyme-linked immunoassay (ELISA) that detects a broad range of Salmonella serovars in various serogroups was developed and compared with standard culture procedures for detection of Salmonella in 1055 field samples collected from poultry-hatchery environments. The diagnostic sensitivity of the ELISA relative to culture was 99.9% and the diagnostic specificity 99.6%. The extensive culture procedure included nonselective enrichment (NSE) as well as primary selective enrichment (PSE) and delayed secondary enrichment (DSE) with Hajna tetrathionate (TT) and Rappaport-Vassiliadis (RV) selective-enrichment broths. Significantly more Salmonella-positive samples were detected by ELISA and culture at the DSE stage than at the NSE and PSE stages (P < 0.05). Significantly more RV than TT broths were positive for Salmonella by culture and ELISA by the DSE stage (P < 0.05). This ELISA procedure could be a reliable screening test for the detection of Salmonella in hatchery samples.

Une épreuve immuno-enzymatique de capture d'antigène utilisant un anticorps monoclonal détectant un grand nombre de sérovars de Salmonella dans différents sérogroupes a été développée et comparée aux procédures standards de culture pour la détection de Salmonella dans 1055 échantillons prélevés de l'environnement de couvoirs de poulet. La sensibilité diagnostique de l'ELISA comparativement à la culture était de 99,9% et la spécificité diagnostique 99,6%. La procédure de culture incluait un enrichissement non-sélectif (NSE) ainsi qu'un enrichissement primaire sélectif (PSE) et un enrichissement secondaire retardé (DSE) avec les bouillons d'enrichissement sélectifs Hajna tétrathionate (TT) et Rappaport-Vassiliadis (RV). Un nombre significativement plus élevé d'échantillons positifs pour Salmonella fut détecté par ELISA et culture au stade DSE qu'aux stades NSE et PSE (P < 0,05). Un nombre significativement plus grand de bouillons RV que de bouillons TT était positif pour Salmonella par culture et ELISA au stade DES (P < 0,05). Cette procédure ELISA pourrait être une épreuve de tamisage rapide et fiable pour la détection de Salmonella dans les échantillons provenant de couvoirs.(Traduit par Docteur Serge Messier).

Influence of temperature and organic load on chemical disinfection of Geobacillus steareothermophilus spores, a surrogate for Bacillus anthracis.

This study evaluated the influence of temperature and organic load on the effectiveness of domestic bleach (DB), Surface Decontamination Foam (SDF), and Virkon in inactivating Geobacillus stearothermophilus spores, which are a surrogate for Bacillus anthracis spores. The spores were suspended in light or heavy organic preparations and the suspension was applied to stainless steel carrier disks. The dried spore inoculum was covered with the disinfectants and the disks were then incubated at various temperatures. At -20°C, the 3 disinfectants caused less than a 2.0 log10 reduction of spores in both organic preparations during a 24-h test period. At 4°C, the DB caused a 4.4 log10 reduction of spores in light organic preparations within 2 h, which was about 3 log10 higher than what was achieved with SDF or Virkon. In heavy organic preparations, after 24 h at 4°C the SDF had reduced the spore count by 4.5 log10, which was about 2 log10 higher than for DB or Virkon. In general, the disinfectants were most effective at 23°C but a 24-h contact time was required for SDF and Virkon to reduce spore counts in both organic preparations by at least 5.5 log10. Comparable disinfecting activity with DB only occurred with the light organic load. In summary, at temperatures as low as 4°C, DB was the most effective disinfectant, inactivating spores within 2 h on surfaces with a light organic load, whereas SDF produced the greatest reduction of spores within 24 h on surfaces with a heavy organic load.

Cette étude a permis d'évaluer l'influence de la température et de la charge organique sur l'efficacité de javellisant domestique (DB), de mousse de décontamination de surface (SDF) et de Virkon pour inactiver les spores de Geobacillus stearothermophilus, un substitut pour les spores de Bacillus anthracis. Les spores ont été suspendues dans des préparations organiques légères ou denses et la suspension étaient appliquées sur des disques d'acier inoxydable. L'inoculum séché de spores était recouvert avec les désinfectants et les disques étaient ensuite incubés à différentes températures. À −20 °C les trois désinfectants ont entrainé une réduction de moins de 2 log10 du nombre de spores dans les deux préparations organiques durant une période d'essai de 24 h. À 4 °C, le DB a causé, en dedans de 2 h, une réduction de 4,4 log10 de la quantité de spores dans les préparations organiques légères, à peu près 3 log10 plus élevé que ce qui a été atteint par la SDF ou le Virkon. Dans les préparations organiques denses, après 24 h à 4 °C la SDF avait réduit le dénombrement de spores par 4,5 log10, ce qui était à peu près 2 log10 plus élevé que ce qui a été obtenu avec le DB ou le Virkon. En général, les désinfectants étaient les plus efficaces à 23 °C mais un temps de contact de 24 h était requis pour la SDF et le Virkon pour réduire le nombre de spores dans les deux préparations organiques par au moins 5,5 log10. Une activité désinfectante comparable avec le DB n'a été observée qu'avec une charge organique légère. En résumé, à des températures aussi basse que 4 °C, le DB était le désinfectant le plus efficace inactivant les spores en moins de 2 heures sur des surfaces avec des charges organiques légères, alors que la SDF a causé la plus grande réduction de spores en-dedans de 24 h sur des surfaces avec une charge organique élevée.(Traduit par Docteur Serge Messier).

Development of an antigen-capture monoclonal antibody-based enzyme-linked immunosorbent assay and comparison with culture for detection of Salmonella enterica serovar Enteritidis in poultry hatchery environmental samples.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed for use as a presumptive screening test for detection of Salmonella enterica serovar Enteritidis and other group D Salmonella in poultry hatchery environments. A mixture of 2 monoclonal antibodies that recognize different forms of the lipopolysaccharide O-antigen was used for specific detection of group D Salmonella. The performance of the ELISA was evaluated in comparison to standard Salmonella culture procedures. Culture for each sample included nonselective enrichment with buffered peptone water and primary selective enrichment and delayed secondary enrichment with both tetrathionate and Rappaport-Vassiliadis broths. One thousand fifty-seven samples were collected from poultry hatcheries over a 5-year period (received in 85 submissions), and S. Enteritidis was recovered from 106 (10%) of them. The diagnostic sensitivity and specificity of the ELISA relative to culture were 97.2% and 99.6%, respectively, on a sample basis and were both 100% on a submission basis. Delayed secondary enrichment increased the number of S. Enteritidis culture and ELISA-positive samples as compared to nonselective enrichment and primary selective enrichment by 25%. A significantly higher (P < 0.05) number of S. Enteritidis culture- and ELISA-positive results were obtained from Rappaport-Vassiliadis broth than from tetrathionate broth or buffered peptone water cultures. The results indicate that this ELISA procedure may be useful for screening poultry hatchery environmental samples for the presence of S. Enteritidis.

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.

The structure of the polysaccharide of the lipopolysaccharide produced by Taylorella equigenitalis type strain (ATCC 35865).

Taylorella equigenitalis is a Gram-negative bacterium that causes venereally transmitted contagious equine metritis (CEM), and its identification and differentiation from other bacteria and Taylorella species is an important requirement for the control of CEM infection. Based on the results of NMR and MS analysis, the antigenic O-polysaccharide (O-PS) component of the lipopolysaccharide (LPS) produced by the type strain T. equigenitalis (ATCC 35865) was found to be a linear polymer composed of a repeating disaccharide unit, containing partially amidated 2,3-diacetamido-2,3-dideoxy-alpha-L-guluronic and 2,3-diacetamido-2,3-dideoxy-beta-D-mannuronic acids, terminated with a 4-O-methylated non-reducing Gulp-NAc3NAcA residue, and has the structure [structure: see text]. The O-PS of the type strain T. equigenitalis LPS provides a specific antigenic marker for the discrimination of the pathogen from the related type strain of T. asinigenitalis sp. nov, a phenotypically indistinguishable non-pathogenic bacterium having a serologically and structurally unrelated LPS O-antigen. The analysis of a structurally unusual core oligosaccharide of the LPS is also reported.

Structure of the O-polysaccharide of the lipopolysaccharide produced by Taylorella asinigenitalis type strain (ATCC 700933).

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-beta-D-quinovose (bacillosamine) and 2-acetamidino-2-deoxy-beta-D-glucuronic acid, and has the structure [-->3)-beta-D-QuipNAc4NAc-(1-->3)-beta-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.

Detection of multiple antibiotic-resistant Salmonella enterica serovar Typhimurium DT104 by phage replication-competitive enzyme-linked immunosorbent assay.

A phage replication-competitive enzyme-linked immunosorbent assay (PR-cELISA) was developed for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104. In the PR-cELISA procedure, a phage, BP1, was inoculated into a log-phase bacterial culture at a ratio of 1:100. After a 3-h incubation of the mixture, BP1 replication was measured by cELISA based on the competitive binding between BP1 and biotinylated BP1 to Salmonella Typhimurium smooth lipopolysaccharide. Among the 84 Salmonella strains and 9 non-Salmonella strains that were tested by PR-cELISA, BP1 detected 39 of 40 Salmonella Typhimurium strains, 2 of 10 Salmonella non-Typhimurium somatic group B strains, and 5 of 18 Salmonella somatic group D1 strains. With the addition of chloramphenicol to the culture medium, PR-cELISA detected all 27 multiple antibiotic-resistant Salmonella Typhimurium DT104 and none of the other Salmonella strains or non-Salmonella strains tested. The results demonstrated that PR-cELISA has potential applications for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104.