Specific identification of pathogenic Yersinia enterocolitica by monoclonal antibodies generated against recombinant attachment invasion locus (rAil) protein

Classical pathogenic strains of Yersinia enterocolitica produce a 17 kDa outer membrane protein, Ail (attachment-invasion locus), which mediates bacterial attachment to some cultures epithelial cell lines and invasion of others. In the present study, hybridomas were developed for the production of monoclonal antibodies (MAbs) against Ail protein of Y. enterocolitica. A set of five stabilized hybridoma cell lines were generated, of which, two MAbs, YEA 302 and YEA 303, exhibited specific reaction to the native Ail protein (17 kDa) present in whole cell lysate of Y. enterocolitica strains beside having reaction with rAil. The other three MAbs, YEA 5, 17 and 32, had some cross reactions with proteins other than Ail. Two out of five MAbs were IgG1, two were IgG2b and one in IgM in nature. MAbs (YEA 302 and YEA 303) did not show any cross-reaction with whole cell lysate of Brucella abortus, Vibrio cholerae, Salmonella typhimurium and Escherichia coli and other species of Enterobacteriaceae including Y. pestis in ELISA and Western blot analysis. The presence of Ail protein among the strains recovered from pork and milk samples was evaluated by these sets of MAbs and the results were compared with the duplex PCR. Collectively, the data suggest that these MAbs may have the potential for their use in the detection of pathogenic Y. enterocolitica reliably, rapidly and at a relatively low cost.     

Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content

Aims: The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. Methods and Results: The four methods comprised of 15 isolation steps using selective enrichments (irgasan–ticarcillin–potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25°C. Salmonella–Shigella‐desoxycholate–calcium chloride agar, cefsulodin–irgasan–novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre‐enrichment step with further selective enrichment showed the highest sensitivities (55–66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. Conclusions: Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. Significance and Impact of the Study: The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.     

Identification of Heat-Stable Enterotoxin-Producing Strains of Yersinia enterocolitica and Vibrio cholerae Non-O1 by a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay

Using a mouse monoclonal antibody (MAb) 2F raised against Vibrio cholerae non-O1 heat-stable enterotoxin (NAG-ST) which also recognizes a shared epitope of Yersinia enterocolitica heat-stable enterotoxin (Y-ST), a competitive enzyme-linked immunosorbent assay (ELISA) was developed for independent detection of NAG-ST and Y-ST. There was good concordance between the Y-ST ELISA and the suckling mouse assay (SMA) for detection of Y-ST from test strains of Y. enterocolitica, and the Y-ST ELISA can effectively replace the SMA for routine detection of Y-ST. On the contrary, evaluation of the NAG-ST ELISA and the SMA using 139 strains of V. cholerae non-O1 showed discordant results and this was attributed to the presence of the suckling mice active factor(s) such as El Tor hemolysin and to the production of low amounts of NAG-ST. Concentration of culture supernatants of V. cholerae non-O1 followed by heating at 100 C was essential to obtain reproducible results by both the NAG-ST ELISA and the SMA. The ELISA developed in this study can be used for the identification of biologically active strains. While recently genetic methods such as polymerase chain reaction became available and were very reliable and simple techniques, the ELISA in this study has an advantage in detecting biologically toxic gene products of the strains. The genetic methods cannot differentiate silent STa genes which we often encounter in the case of Y. enterocolitica.     

Whole cell protein profiling reiterate phylogenetic relationships among strains of Yersinia enterocolitica biovar 1A as discerned earlier by different genotyping methods

Aim: To evaluate whole cell protein profiling vis‐à‐vis genotyping to discern phylogenetic relationships among strains of Y. enterocolitica biovar 1A. Methods and Results: Whole cell protein profiling of Y. enterocolitica biovar 1A strains was performed using SDS–PAGE. Twenty‐one distinct protein profile types were obtained among a collection of 81 strains isolated from clinical and nonclinical sources. Whole cell protein profiling exhibited discriminatory index (DI) of 0·80 and clustered the strains into two distinct clonal groups. The clinical and the aquatic serotype O:6,30–6,31 strains were clustered into two discrete subgroups. Conclusions: Whole cell protein profiling displayed sufficient diversity among strains of Y. enterocolitica biovar 1A, and the phylogenetic relationships obtained were in good agreement with those established earlier by genotyping techniques. Significance and impact of the study: Whole cell protein profiling was as discriminatory as some of the genotyping methods and has the potentiality to be used as an adjunct tool to study epidemiology of Y. enterocolitica.     

Possible use of <Emphasis Type="Italic">ail</Emphasis> and <Emphasis Type="Italic">foxA</Emphasis> polymorphisms for detecting pathogenic <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis>

Background  

Yersinia enterocolitica is an enteric pathogen that invades the intestinal mucosa and proliferates within the lymphoid follicles (Peyer's patches). The attachment invasion locus (ail) mediates invasion by Y. enterocolitica and confers an invasive phenotype upon non-invasive E. coli; ail is the primary virulence factor of Y. enterocolitica. The ferrioxamine receptor (foxA) located on the Y. enterocolitica chromosome, together with its transport protein, transports a siderophore specific for ferric ion. Currently, ail is the primary target gene for nucleic acid detection of pathogenic Y. enterocolitica.     

Molecular and biochemical characterization of urease and survival of Yersinia enterocolitica biovar 1A in acidic pH in vitro

Background  

Yersinia enterocolitica, an important food- and water-borne enteric pathogen is represented by six biovars viz. 1A, 1B, 2, 3, 4 and 5. Despite the lack of recognized virulence determinants, some biovar 1A strains have been reported to produce disease symptoms resembling that produced by known pathogenic biovars (1B, 2-5). It is therefore imperative to identify determinants that might contribute to the pathogeniCity of Y. enterocolitica biovar 1A strains. Y. enterocolitica invariably produces urease and the role of this enzyme in the virulence of biovar 1B and biovar 4 strains has been reported recently. The objective of this work was to study genetic organization of the urease (ure) gene complex of Y. enterocolitica biovar 1A, biochemical characterization of the urease, and the survival of these strains under acidic conditions in vitro.     

Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥10² CFU/ml in pure cultures and ≥10³ CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

Characterization of a Yersinia enterocolitica biotype 1A strain harbouring an ail gene

Aims: The chromosomal ail gene (attachment and invasion locus) is commonly used as target gene for the detection of pathogenic Y. enterocolitica strains in food testing. The ail PCR does not detect strains of biotype 1A (BT1A), which are regarded as non‐pathogenic because BT1A strains lack the virulence plasmid and chromosomally encoded virulence genes. In some recent reports, however, BT1A strains were discovered that harboured the ail gene. We isolated an ail‐positive strain and characterized this strain with phenotypic and genotypic methods to study its possible relation to pathogenic Y. enterocolitica strains. Methods and Results: The ail region of the BT1A strain was sequenced and compared with the corresponding region of nonpathogenic BT1A strains and pathogenic strains. Pulsed field gel electrophoresis (PFGE) analysis was applied revealing no similarity of the PFGE pattern of this strain to the patterns of pathogenic strains. Virulence‐gene‐based PCR analyses showed the strain to be positive for ystB, but negative for virulence genes ystA, virF and yadA. Whole‐cell MALDI‐TOF MS combined with a shrinkage discriminant analysis approach was applied and clearly classified the ail‐positive biotype 1A strain within the cluster of BT1A strains. Conclusions: PCR detection of ail sequences in food matrices should be followed by the isolation of the responsible strain and its characterization using phenotypic or genotypic methods. Significance and Impact of the Study: The ail gene may be present in Y. enterocolitica BT1A strains, which are commonly considered as nonpathogenic. Efficient methods such as PCR typing of other virulence genes or rapid MALDI‐TOF MS‐based bacterial profiling allow a more comprehensive assessment of the pathogenicity potential of Yersinia strains.     

Immunoassay‐based Techniques for Early and Specific Detection of Latent Postharvest Anthracnose in Mango

The postharvest anthracnose pathogen Colletotrichum gloeosporioides inciting latent or quiescent infection of mango was detected in early stages using immunoassay methods. Twenty‐five pathotypes isolated from different agroclimatic zones of Tamil Nadu, Karnataka and Pondicherry, India, revealed the variation in protein profile analysis (SDS‐PAGE). The polyclonal antibodies (PCA) were raised against the unfractioned mycelial protein (UMP) and a 40‐kDa polypeptide present in all pathotypes. Standardization of antigen and antiserum dilutions revealed that an antigen dilution of 1 : 200 (protein concentration of 20 μg/ml) and antiserum dilution of 1 : 100 (protein concentration of 40 μg/ml raised against UMP) and 1 : 200 (protein concentration of 20 μg/ml raised against 40 kDa polypeptide) was found to be optimum for the detection of anthracnose pathogen. Both antisera detected the C. gloeosporioides antigen in enzyme‐linked immunosorbent assays (ELISAs), dot immunobinding assays (DIBAs) and Western blots. The specificity in reaction was compared by isolating other Colletotrichum spp. from various hosts viz., C. lindemuthianum (beans), C. falcatum (sugarcane), C. musae (banana), C. capsici (chillies) and Botryodiplodia theobromae (mango). The antisera generated against UMP revealed the cross‐reaction with other host isolates and mango stem end rot pathogen (B. theobromae). The PCA raised against 40‐kDa polypeptide exhibited the specific reaction with C. gloeosporioides isolates in all the immunoassay techniques. By utilizing both PCA, the presence of latent infection was observed in healthy‐looking leaves, flowers and fruits in orchard conditions. The fruit tissues recorded high absorbance values followed by flowers and leaves in all the detection methods. The ELISA technique was also useful in assessing the pathogen inoculum at various biocontrol formulations sprayed mango trees under field conditions. The fluorescent pseudomonad strains mixture (KFP1 + FP7) amended with chitin sprayed at 30‐day intervals revealed the significant reduction in pathogen load than other formulations and unsprayed control.     

Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF mass spectrometry

Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level.     

virF-Positive Yersinia pseudotuberculosis and Yersinia enterocolitica Found in Migratory Birds in Sweden

During spring and autumn migrations, 468 fecal samples from 57 different species of migratory birds were collected in Sweden. In total, Yersinia spp. were isolated from 12.8% of collected samples. The most commonly found species was Yersinia enterocolitica, which was isolated from 5.6% of all collected samples, followed by Y. intermedia (3.8%), Y. frederiksenii (3.0%), Y. kristensenii (0.9%), Y. pseudotuberculosis (0.6%), and Y. rohdei (0.4%). The pathogenic, virF-positive Y. pseudotuberculosis strains were recovered from three thrushes. These strains belonged to the same bioserotype, 1/O:2, but had two different profiles as determined by pulsed-field gel electrophoresis with NotI and SpeI enzymes. In addition, 10 Y. enterocolitica strains, all from barnacle geese, belonged to bioserotype 3/O:3, which is associated with human disease. Two of the strains were pathogenic, carrying the virF gene on their plasmids. All pathogenic Y. pseudotuberculosis and Y. enterocolitica strains were recovered during the spring, and as the birds were caught during active migration they likely became infected at an earlier stage of the migration, thus potentially transporting these bacterial pathogens over long geographical distances.     

Development of a Fluorogenic 5′ Nuclease PCR Assay for Detection of the ail Gene of Pathogenic Yersinia enterocolitica

In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic Yersinia enterocolitica. The assay targets the chromosomally encoded attachment and invasion gene, ail. Three primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions of ail were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of purified Y. enterocolitica DNA. TM1 identified all 26 Y. enterocolitica strains examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of the Y. enterocolitica strains tested. None of the primer-probe sets cross-reacted with any of the 21 non-Y. enterocolitica strains examined. When the TM1 set was utilized, the fluorogenic PCR assay was able to detect ≤4 Y. enterocolitica CFU/ml in pure culture and 10 Y. enterocolitica CFU/ml independent of the presence of 10⁸ CFU of contaminating bacteria per ml. This set was also capable of detecting ≤1 CFU of Y. enterocolitica per g of ground pork or feces after a 24-h enrichment in a Yersinia selective broth.     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

Detection by using monoclonal antibodies of Yersinia enterocolitica in artificially-contaminated pork

Monoclonal antibodies against Yersinia enterocolitica were produced by fusion of NS‐1 mouse myeloma cells with spleen cells of ICR mice immunized with heat‐killed and heat‐killed plus SDS‐mercaptoethanol treated forms of Y. enterocolitica ATCC 27729 alone or mixed with Y. enterocolitica MU. The twenty‐five MAbs obtained from five fusions were divided into nine groups according to their specificities to different bacterial strains and species, as determined by dot blotting. The first five groups of MAbs were specific only to Y. enterocolitica, but did not recognize all of the isolates tested. MAbs in groups 6 and 7 reacted with all isolates of Y. enterocolitica tested but showed cross‐reaction with some Yersinia spp. and Edwardsiella tarda, especially in the case of group 7. MAbs in groups 8 and 9 reacted with all isolates of Y. enterocolitica and Yersinia spp., as well as other Gram‐negative bacteria that belong to the family Enterobacteriaceae. These MAbs recognized Y. enterocolitica antigens with apparent molecular weights ranging from 10 – 43 kDa by Western blotting, and could detect Y. enterocolitica from ～10³– 10⁵ colony forming units (CFUs) by dot blotting. The hybridoma clone YE38 was selected for detection of Y. enterocolitica in pork samples which had been artificially‐contaminated by inoculation with Y. enterocolitica ATCC 27729 at concentrations of ～10⁴– 10⁶ CFUs/g and incubation in peptone sorbitol bile broth at 4°C. Samples were collected and applied on a nitrocellulose membrane for dot blotting with trypticase soy and cefsulodin‐Irgasan‐novobiocin agars. After 48 hr of incubation, the detection limit was ～10²– 10³ CFU/g by dot blotting.     

Transport of haemin across the cytoplasmic membrane through a haemin-specific periplasmic binding-protein-dependent transport system in Yersinia enterocolitica

The Yersinia enterocolitica O:8 periplasmic binding-protein-dependent transport (PBT) system for haemin was cloned and characterized. It consisted of four proteins: the periplasmic haemin-binding protein HemT, the haemin permease protein HemU, the ATP-binding hydrophilic protein HemV and the putative haemin-degrading protein HemS. Y. enterocolitica strains mutated in hemU or hemV genes were unable to use haemin as an iron source whereas those mutated in the hemT gene were able to use haemin as an iron source. As Escherichia coli strains expressing only the haemin outer membrane receptor protein HemR from Y. enterocolitica were capable of using haemin as an iron source the existence of an E. coli K-12 haemin-specific PBT system is postulated. The first gene in the Y. enterocolitica haemin-specific PBT system encoded a protein, HemS, which is probably involved in the degradation of haemin in the cytoplasm. The presence of the hemS gene was necessary to prevent haemin toxicity in E. coli strains that accumulate large amounts of haemin in the cytoplasm. We propose a model of haemin utilization in Y. enterocolitica in which HemT, HemU and HemV proteins transport haemin into the cytoplasm where it is degraded by HemS thereby liberating the iron.     

Degradation of a Polymerase Chain Reaction (PCR) Product by Heat-Stable Deoxyribonuclease (DNase) Produced from Yersinia enterocolitica

When crude deoxyribonucleic acid (DNA) preparations by boiling were used for the polymerase chain reaction (PCR) from pathogenic and non-pathogenic Yersinia enterocolitica strains, the amplified products were degraded after their storage at 4 C. The degradation of products was prevented by the addition of ethylenediaminetetraacetate (EDTA) or treatment with proteinase K. These findings indicate that Y. enterocolitica produced heat-stable deoxyribonuclease (DNase). Proteinase K treatment would be recommended to prevent heat-stable DNase contamination in the DNA preparations for PCR from Y. enterocolitica strains.     

Isolation ofYersinia enterocolitica andYersinia enterocolitica-like organisms from raw milk in Italy

Thirty samples of raw milk, originating from individual producers in the Turin area, were examined for the presence ofYersinia enterocolitica. A cold enrichment method with phosphate-buffered saline (PBS) 1/15M, pH 7.6, and sorbitol-bile-salts broth (SB) was used. After 7, 14, or 21 days at 4°–5°C, plating was performed on selective agar media directly (MacConkey agar andSalmonella-Shigella agar) after the alkali method was used. Six strains ofY. enterocolitica (biotype 1) and 32 strainsY. enterocolitica-like (threeY. fredericksenii; nineYersinia rhamnose-, melibiose+, -methyl-d-glucoside+, raffinose+, probablyYersinia intermedia biotype rhamnose-; and 20Y. intermedia) were isolated.Yersinia strains were found in 11 samples of raw milk, andY. enterocolitica in four samples.     

Preparation and Characterization of a Monoclonal Antibody to Prunus necrotic ringspot virus

A cell line named PVRSV1D11 secreting monoclonal antibody (McAb) against the prokaryotically expressed coat protein (CP) of Prunus necrotic ringspot virus (PNRSV) was developed using hybridoma technology including animal immunization, cell fusion, cell line culture and enzyme‐linked immunosorbent assay (ELISA)‐based for screening. The specificity, titre and detection sensitivity of the McAb were determined by indirect ELISA to establish optimal conditions. The antibody reacted strongly with PNRSV and showed no cross‐reactions with the proteins of Plum pox virus, Prunus dwarf virus, Apple stem pitting virus, Apple stem grooving virus, Apple mosaic virus or Apple chlorotic leafspot virus. The ascites developed with PNRSV1D11 cell line showed high absorbance until it was diluted to over 6.6 × 10⁷ fold. The McAb belonged to IgG_2a isotype and was diluted by 1.28 × 10⁵ folds as an optimal detection concentration. The detection sensitivity of the monoclonal antibody was 11.7 ng/ml protein of PNRSV. The results indicated that the McAb against the CP of PNRSV is suitable for PNRSV detection in the plants and for monitoring the dynamics of the virus by using indirect ELISA.     

Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

Banana streak MY virus (BSMYV) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein (VAP) coded by ORF II of BSMYV was expressed in Escherichia coli, and polyclonal antibodies were raised against purified recombinant VAP (rVAP) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assays (ELISAs). In direct antigen‐coated (DAC)‐ELISA, antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction (IC‐PCR) assay was developed and compared with DAC‐ELISA. VAP antibody‐based IC‐PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV (eBSV) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV. To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC‐PCR. This VAP‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.