Ail Gene Research Articles

Sequence analysis of the yadA, inv, and ail genes and their expression in the main and nonmain Yersinia pestis subspecies and Yersinia pseudotuberculosis

The nucleotide sequences of the inv, yadA, and ail adhesin-invasin genes were analyzed in 24 strains of the main and nonmain Yersinia pestis subspecies, which were isolated from natural plague foci in Russia and neighbor countries, and ten Y. pseudotuberculosis strains. All of the five plague agent subspecies (main, caucasica, altaica, ulegeica, and hissarica) had the inv and yadA genes altered by insertion of the IS element and a single nucleotide deletion, respectively, as was earlier observed for the Y. pestis strains KIM and CO92. Consequently, the strains lacked functional activity of the Inv and YadA proteins. The ail gene of the main and ulegeica subspecies had a missense mutation, which replaced Val138 with Phe in the Ail protein. The strains of the caucasica subspecies had an AGT insertion in the ail gene, resulting in Ser148 insertion in the polypeptide chain. The changes in the ail sequence probably exerted no effect on ail expression, since the strains of all subspecies were resistant to blood serum complement.

A NOVEL MULTIPLEX POLYMERASE CHAIN REACTION FOR SIMULTANEOUS DETECTION OF YERSINIA ENTEROCOLITICA, STAPHYLOCOCCUS AUREUS, AEROMONAS AND SALMONELLA FROM CHICKEN MEAT AND MILK SAMPLES

ABSTRACTYersinia enterocolitica, Staphylococcus aureus, Aeromonas and Salmonella are among the most important foodborne bacterial pathogens. The majority of human infections caused by all of these organisms are associated with ingestion of undercooked and contaminated meat, dairy products and water where in the secreted bacterial toxins lead to foodborne intoxications. We, here, report a new multiplex polymerase chain reaction (mPCR) assay for the simultaneous detection of these important foodborne bacterial pathogens. The mPCR targeted Ail and virF genes of Y. enterocolitica, nuc and entB genes of S. aureus, aerA and 16S rRNA genes of Aeromonas and invA, an invasion protein A gene of Salmonella. An internal amplification control designed to check the false negative reactions in mPCR was also included. This procedure could detect initial populations of 1–100 cfu/g or /mL within 24 h in experimentally spiked food and water samples. When evaluated on a total of 104 naturally occurring food samples, the mPCR detected two samples to contain S. aureus, one was identified to contain Y. enterocolitica and four samples were identified to contain Salmonella species individually. This was compared with the standard microbiological and biochemical identification procedures.PRACTICAL APPLICATIONSAll the microorganisms selected in this study are food and waterborne and contaminate a variety of food items. Pathogenic Y. enterocolitica and Aeromonas species are able to grow and multiply and secrete toxins even at low temperatures. The high throughput and cost‐effective multiplex polymerase chain reaction method reported here could be a viable alternative for detection of pathogenic Y. enterocolitica, S. aureus, Aeromonas and Salmonella from food and environmental samples.

Variation in the Prevalence of EnteropathogenicYersiniain Slaughter Pigs from Belgium, Italy, and Spain

Tonsils of 829 fattening pigs originating from Belgium (n = 201), Italy (n = 428), and Spain (n = 200) were collected between 2005 and 2007 to study the prevalence of enteropathogenic Yersinia in slaughter pigs. Isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis was done by selective enrichment and by cold enrichment for 7 and 14 days. Pathogenic Y. enterocolitica and Y. pseudotuberculosis isolates were identified by polymerase chain reaction targeting the chromosomal genes ail and inv, respectively, as well as the plasmid-encoded virF of both species. A significantly higher (p < 0.001) prevalence of ail-positive Y. enterocolitica in Spain (93%) than in Belgium (44%) or Italy (32%) was observed. virF-positive Y. enterocolitica was present in 77% of ail-positive samples. Bioserotype 4/O:3 was the most common type in all three countries. Bioserotypes 2/O:5 and 3/O:9 were found in Italy (1%) and Belgium (9%), respectively. The prevalence of inv- and virF-positive Y. pseudotuberculosis was 2% and 1% in Belgium and Italy, respectively. Y. pseudotuberculosis was not detected in pigs from Spain. Bioserotypes 1/O:1 (20%), 1/O:2 (20%), and 2/O:3 (60%) were found in Belgium, and 1/O:1 (60%) and 2/O:3 (20%) in Italy. The most efficient method for isolation of Y. enterocolitica was combined cold enrichment for 7 and 14 days; however, the isolation method for Y. pseudotuberculosis was cold enrichment for 14 days. Fattening pigs seem to be an important reservoir of pathogenic Y. enterocolitica in Belgium, Italy, and Spain. Bioserotype 4/O:3 of Y. enterocolitica and bioserotypes 2/O:3 and 1/O:1 of Y. pseudotuberculosis have been shown to predominate.

The detection of Yersinia enterocolitica in biological samples by RT-PCR technique.

The research has been performed in order to optimize Yersinia enterocolitica diagnosis from blood samples and other biological samples from different human clinical cases. Following this objective, the general technique of collecting and processing the samples was adapted and optimized, raising the rapidity of diagnosis. There have been artificially contaminated different types of samples with Yersinia enterocolitica stems, checking the efficiency and accuracy of optimized method. In finally was developed a methodology of Yersinia identification from naturally contaminated biological samples. As a target-gene there has been used the chromosome gene ail, this being present in the pathogenic strains of Y. enterocolitica. The sample was marked at the 5’ end with a report dyeing solution and at the 3’ end with an extinction one. The reading was performed at the RT-PCR, with a work protocol consisting of 2 stages, including 45 cycles. This qualitative method demonstrated some specific results with high sensitivity, positive ail gene detection rate in Y. enterocolitica in 267 blood samples and other type of biological products, being of 82 % using PCR and 56 % using culture methods.

The detection of Yersinia enterocolitica in surface water by quantitative PCR amplification of the ail and yadA genes

Yersinia enterocolitica has been detected in surface water, and drinking untreated water is a risk factor for infection. PCR-based methods have been used to detect Y. enterocolitica in various sample types, but quantitative studies have not been conducted in water. In this study, quantitative PCR (qPCR)-based methods targeting the Yersinia virulence genes ail and yadA were used to survey the Grand River watershed in southern Ontario, Canada. Initial testing of reference strains showed that ail and yadA PCR assays were specific for pathogenic biotypes of Y. enterocolitica; however the genes were also detected in one clinical Yersinia intermedia isolate. A survey of surface water from the Grand River watershed showed that both genes were detected at five sampling locations, with the ail and yadA genes detected in 38 and 21% of samples, respectively. Both genes were detected more frequently at colder water temperatures. A screening of Yersinia strains isolated from the watershed showed that the ail gene was detected in three Y. enterocolitica 1A/O:5 isolates. Results of this study show that Yersinia virulence genes were commonly detected in a watershed used as a source of drinking water, and that the occurrence of these genes was seasonal.

Identification ofYersinia enterocoliticaat the Species and Subspecies Levels by Fourier Transform Infrared Spectroscopy

Yersinia enterocolitica and other Yersinia species, such as Y. pseudotuberculosis, Y. bercovieri, and Y. intermedia, were differentiated using Fourier transform infrared spectroscopy (FT-IR) combined with artificial neural network analysis. A set of well defined Yersinia strains from Switzerland and Germany was used to create a method for FT-IR-based differentiation of Yersinia isolates at the species level. The isolates of Y. enterocolitica were also differentiated by FT-IR into the main biotypes (biotypes 1A, 2, and 4) and serotypes (serotypes O:3, O:5, O:9, and "non-O:3, O:5, and O:9"). For external validation of the constructed methods, independently obtained isolates of different Yersinia species were used. A total of 79.9% of Y. enterocolitica sensu stricto isolates were identified correctly at the species level. The FT-IR analysis allowed the separation of all Y. bercovieri, Y. intermedia, and Y. rohdei strains from Y. enterocolitica, which could not be differentiated by the API 20E test system. The probability for correct biotype identification of Y. enterocolitica isolates was 98.3% (41 externally validated strains). For correct serotype identification, the probability was 92.5% (42 externally validated strains). In addition, the presence or absence of the ail gene, one of the main pathogenicity markers, was demonstrated using FT-IR. The probability for correct identification of isolates concerning the ail gene was 98.5% (51 externally validated strains). This indicates that it is possible to obtain information about genus, species, and in the case of Y. enterocolitica also subspecies type with a single measurement. Furthermore, this is the first example of the identification of specific pathogenicity using FT-IR.

Prevalence of EnteropathogenicYersiniain Estonian, Latvian, and Russian (Leningrad Region) Pigs

Tonsils of 457 fattening pigs from Estonia (n = 151), Latvia (n = 109), and the Leningrad Region of Russia (n = 197) were collected between 2004 and 2007 to study the prevalence of enteropathogenic Yersinia in slaughter pigs. Yersinia enterocolitica and Yersinia pseudotuberculosis were isolated by selective and cold enrichment. Pathogenic Y. enterocolitica and Y. pseudotuberculosis were identified by PCR targeting the chromosomal genes ail and inv, respectively. The presence of the virulence plasmid was confirmed by PCR targeting the virF gene of Y. enterocolitica and Y. pseudotuberculosis. The prevalence of ail-positive Y. enterocolitica was 89% in Estonia, 64% in Latvia, and 34% in Russia, with 81% of ail-positive samples being virF-positive. A statistically significant (p < 0.05) difference between Estonian and Latvian pigs and between pigs from Latvia and the Leningrad Region of Russia was observed when assuming randomized sampling. Y. enterocolitica bioserotype 4/O:3 was the only pathogenic type found. The prevalence of inv and virF-positive Y. pseudotuberculosis was higher in Russia (7%) than in Latvia (5%) and Estonia (1%), with a statistically significant difference between pigs from Estonia and the Leningrad Region of Russia when assuming both randomized sampling and clustering at farms. All Y. pseudotuberculosis isolates were bioserotype 2/O:3. A total of eight pigs (2%), one pig from Latvia and seven pigs from the Leningrad Region of Russia, carried both pathogenic Y. enterocolitica and Y. pseudotuberculosis in the tonsils. Cold enrichment was found to be a more efficient method compared to selective enrichment to isolate both species. Pigs seem to be an important reservoir of human enteropathogenic Y. enterocolitica 4/O:3 and Y. pseudotuberculosis 2/O:3 in these countries.

Detection of Yersinia enterocolitica in Alfalfa, Mung Bean, Cilantro, and Mamey Sapote (Pouteria sapota) Food Matrices Using DNA Microarray Chip Hybridization

Four different food matrices (alfalfa, cilantro, mamey sapote, and mung bean) were contaminated with three different dilutions 10(6), 10(4), and 10(3) cfu/g of Yersinia enterocolitica. DNA was isolated from each food mix and used in chromosomal amplifications. The amplified DNA was used as templates in single PCR reactions of the four genes (virF, ail, yst, and blaA) followed by mixing the four reactions for one PCR primer extension reaction. The presence and the limit of detection of four genes in four food matrices were established by microarray hybridization. Data revealed the diversity of signal intensities. Neither the microarray chip hybridization nor the single PCR amplification could detect virF's presence located on a plasmid. Ail was detected in 10(3) cfu/g, whereas blaA and yst were detected from 10(5) to 10(6) cfu/g in all food matrices. Therefore, the ail gene could be the gene of choice in identifying Y. enterocolitica in alfalfa, cilantro, mamey, and mung bean. Other genes--blaA, yst, virF--exhibited wide variability in hybridization signals, highlighting the need of a better DNA purification step prior to DNA microarray hybridization.

Clonality and Antibiotic Susceptibility ofYersinia enterocoliticaIsolated from U.S. Market Weight Hogs

Pigs are the only known animal reservoir of Yersinia enterocolitica strains pathogenic to humans. In this study 106 ail-positive pathogenic Y. enterocolitica isolates, previously recovered from 2793 swine fecal samples (3.8%) collected during National Animal Health Monitoring System's Swine 2000 study, were examined. The presence of the previously described virulence plasmid, expression of plasmid-associated virulence determinants, and serotype were correlated with genotype, expression of YopE protein, and antibiotic susceptibility. Pulsed-field gel electrophoresis using the enzyme XbaI showed that O:3 and O:5 isolates were highly clonal within a serotype regardless of geographic origin. Antimicrobial resistance profiles of 106 isolates of serotypes O:3 and O:5 were evaluated by agar disk diffusion methodology to 16 different antibiotics. All isolates were susceptible to 13 of the 16 tested antimicrobials; resistance was noted to ampicillin, cephalothin, and tetracycline. The presence of the ail gene, virulence plasmid, the expression of virulence determinants, and serotypes indicate that these isolates from U.S. swine are potentially capable of causing human foodborne illness.

THE ROLE OF PIGS AS PHARYNGEAL CARRIERS OF HUMAN PATHOGENIC YERSINIA ENTEROCOLITICA STRAINS

From March 2007 to January 2008, a total of 170 pigs at slaughter were tested for Y. enterocolitica contamination in tonsils tissue. The animals came from 125 different farms located in four regions of Northern Italy. Y. enterocolitica was isolated from 19 out of 170 (11.2%) tonsils samples. The prevalent bio-serotype (68.4%) was 4/O:3, followed by bioserotypes 1A/O:8 (15.8%), 1A/O:5 (10.5%) and 4/O:8 (5.2%). Among bio-serotype 4/O:3, several strains possessed yadA, ail and ystA virulence genes.

The Detection of Yersinia enterocolitica Strains from

Yersiniosis is an important zoonosis, causing disease to humans and animals. The available culture methods for detecting Y. enterocolitica in the samples prelevated from different categories of biological products need more time for performing and sometimes they offer satisfying results. By using methods based on DNA detection, as molecular techniques, this pathogenic agent can be detected quickly and with a higher accuracy. The purpose of this study was to establish a quicker and more veracious PCR method for the detection of this microorganism in biological product samples. There has been used a nucleotide target sequence for the PCR test, 5’ nuclease, the chromosomal gene ail, present in pathogenic strains of Y. enterocolitica. The sample was marked at 5’ end with a report dyeing substance and at 3’ end with an extinction substance. The reading was performed by RT-PCR, using a 2 stage-work protocolem consisting of 45 cycles. This qualitative PCR method demonstrated the procurement of specific results and with a high sensitivity, the detection rate of ail positive gene of Y. enterocolitica in 140 samples of biological products being of 86 % by using PCR and 37 % for culture methods.

TheYersinia pestisAil Protein Mediates Binding and Yop Delivery to Host Cells Required for Plague Virulence

Although adhesion to host cells is a critical step in the delivery of cytotoxic Yop proteins by Yersinia pestis, the mechanism has not been defined. To identify adhesins critical for Yop delivery, we initiated two transposon mutagenesis screens using the mariner transposon. To avoid redundant cell binding activities, we initiated the screen with a strain deleted for two known adhesins, pH 6 antigen and the autotransporter, YapC, as well as the Caf1 capsule, which is known to obscure some adhesins. The mutants that emerged contained insertions within the ail (attachment and invasion locus) gene of Y. pestis. A reconstructed mutant with a single deletion in the ail locus (y1324) was severely defective for delivery of Yops to HEp-2 human epithelial cells and significantly defective for delivery of Yops to THP-1 human monocytes. Specifically, the Yop delivery defect was apparent when cell rounding and translocation of an ELK-tagged YopE derivative into host cells were monitored. Although the ail mutant showed only a modest decrease in cell binding capacity in vitro, the KIM5 Deltaail mutant exhibited a >3,000-fold-increased 50% lethal dose in mice. Mice infected with the Deltaail mutant also had 1,000-fold fewer bacteria in their spleens, livers, and lungs 3 days after infection than did those infected with the parental strain, KIM5. Thus, the Ail protein is critical for both Y. pestis type III secretion in vitro and infection in mice.

Prevalence of Yersinia enterocolitica in Market Weight Hogs in the United States

Pigs are the major animal reservoir for Yersinia enterocolitica strains, which are potentially pathogenic for humans. The goals of this study were (i) to estimate the individual animal and on-farm prevalences of Y. enterocolitica in hogs based on tonsil samples collected during National Animal Health Monitoring System Swine 2002 study and (ii) to use these data with data previously published for fecal samples to determine on-farm risk factors for Y. enterocolitica. Tonsil swabs (1,218) and fecal samples (2,847) were collected on 124 farms located in the top 17 pork-producing states. Ten percent of tonsils (122 of 1,218 samples) were positive in irgasan-tiracillin-chlorate (ITC) enrichment broth by real-time PCR, but only 5.6% of samples (68 of 1,218) were positive after subculture on the more selective cefsulodin-irgasan-novobiocin (CIN) agar. For tonsils, the on-farm prevalence based on real-time PCR detection of the ail gene in ITC enrichment broth cultures was 32% (32 of 100 premises sampled); the prevalence based on subculture in CIN agar was 19.6% (20 of 102 premises). Results of bacteriological isolation and real-time PCR analysis of tonsils and feces were combined to estimate prevalence (individual animal and farm), which was subsequently correlated with 40 farm management practices. Four factors and their accompanying odds ratios (ORs) were identified in the final regression model: location in a central state (OR =0.3), vaccination for Escherichia coli (OR = 3.0), percentage of deaths due to scours (OR = 3.5), and presence of meat or bone meal in grower-finisher diet (OR = 4.1).

A real-time PCR for Detection of Pathogenic Yersinia enterocolitica in food combined with an Universal Internal Amplification Control System

A real-time PCR system with an internal amplification control was developed for detection of pathogenic Yersinia) enterocolitica in food samples. The chromosomally encoded ail gene was chosen as PCR target. Sequences of plasmid pUC19 served as target for the internal amplification control. The method was validated in combination with sample enrichment in PSB and TSB broth using different food matrices spiked with Y. enterocolitica and naturally contaminated slaughterhouse samples. The results of the real-time PCR with internal control were verified by the cultural method according to EN ISO 10273:2003. The sensitivity of the real-time PCR with internal control is about 5 genome copies per reaction. Artificial contamination of food samples resulted in a detection level of 5 cfu per 25 g Y. enterocolitica in food samples. 100% of porcine tonsils and about 22% meat from pig heads were contaminated. The screening of samples by PCR prior to cultural analysis allows focusing on positive samples in routine analysis. This could result in a higher detection rate by cultural analysis.

O:8 serotype Yersinia enterocolitica strains in China

Serotypes O:3, O:8 and O:9 Yersinia enterocolitica strains carrying virulence determinants are common pathogens causing human infections. In many years of surveillance in China for Y. enterocolitica, no pathogenic O:8 strains have been found where the isolated O:8 serotypes lacked the major virulence genes and in contrast to O:3 and O:9 strains, none of the O:8 isolates were from humans. These O:8 isolates lack ail, ystA, yadA and virF genes but possess the ystB gene and all belong to Biotype 1A. These O:8 strains did not kill mice and could protect immunized mice against challenge with a pathogenic O:8 strain. Compared to the Chinese pathogenic O:3 and O:9 strains which have similar pulsed-field gel electrophoresis patterns, the 39 Chinese O:8 animal and food isolates were different from the pathogenic O:8 reference strains. This suggests the O:8 strains lacking virulence determinants may not disseminate rapidly in humans and are maintained in animal reservoirs; and therefore exhibit higher variance and divergence from the virulent type.

Detection, isolation and enumeration of Yersinia enterocolitica from raw pork

The methods available for the isolation of Yersinia enterocolitica from foods are generally considered to be less than optimal, and methods for estimation of numbers are lacking. Such methods are needed to understand better the significance of foodborne yersiniosis and to provide data for exposure assessment. We describe a method for the detection and enumeration of Y. enterocolitica containing the pYV virulence plasmid ( YeP+) in samples from pork surfaces. The method uses a multiplex PCR targeting the ail and virF genes to detect Y. enterocolitica after incubation of surface swabs in Yersinia enrichment broth according to Ossmer. Enumeration was achieved by adapting the enrichment to a most probable number (MPN) method format. A presumptive result was available within 24 h of sample receipt, and YeP+ isolates were confirmed within four days. The presence/absence and MPN methods were evaluated in a pilot survey of 34 packs of raw pork meat purchased from retail outlets in Christchurch, New Zealand. YeP+ was detected by PCR on meat from 32% of the packs, and YeP+ isolates were obtained from 18% of the samples. YeP+ were present at numbers ranging from 0.30 to 5.42 MPN/cm 2. This improved method for the detection and enumeration of YeP+ from meat samples can be used for microbiological surveys to obtain data for assessments of consumer exposure to virulent Y. enterocolitica, and in outbreak investigations.

High Incidence ofYersinia enterocolitica(Enterobacteriaceae) in Alpine AccentorsPrunella collarisof the Tatra Mountains

Cloacal and pharyngeal swabs were sampled from 33 Alpine Accentors. A total of 32 specimens were Yersinia positive, with 73% of birds being positive for Y. enterocolitica and 51% for Yersinia spp. A comparison of host characters and environmental conditions showed these to be consistent with the different life strategies of Y. enterocolitica and other Yersinia species. Y. enterocolitica is more successful at colonizing the birds' digestive tracts — the occurrence of Y. enterocolitica was significantly higher in the cloacal than the pharyngeal swabs. The occurrence of Y. enterocolitica was high in summer, especially in the nesting period (July). In juveniles (including nestlings), there was a 100% prevalence of Y. enterocolitica, whereas only two out of nine juveniles were Yersinia spp. positive. There was no significant difference between the occurrence of Y. enterocolitica in anthropogenic and natural habitats, but the occurrence of Yersinia spp. was much greater in the former than in the latter habitats. The presence of the ail gene associated with pathogenic Y. enterocolitica strains was not confirmed in any of the samples examined.

Application of Multiplex PCR for Monitoring Colonization of Pig Tonsils by Yersinia enterocolitica, Including Biotype 1A, and Yersinia pseudotuberculosis

A multiplex PCR assay was developed for the detection and differentiation of the Yersinia enterocolitica and Yersinia pseudotuberculosis isolates in both pure bacterial cultures and pig tonsils. The assay was based on the amplification of the ail, inv, yadA, and ystB genes. The PCR products, corresponding to the ail gene and the plasmid-borne yadA gene or only one product corresponding to the ail gene, were detected in Y. enterocolitica 4 biotype isolates. All of the Y. pseudotuberculosis isolates (n = 6) tested gave a positive PCR reaction for the inv gene. For all tested Y. enterocolitica 1A biotype isolates (n = 31), one product corresponding to the ystB gene was observed. The multiplex PCR assay was used to detect Y. enterocolitica and Y. pseudotuberculosis strains in pig tonsil samples obtained from 80 slaughtered pigs from three different herds. The presence of at least one of the specific PCR amplification products of ail-, ystB-, yadA-, and inv-specific sequences was observed in 11 samples (13.75%). These results of the multiplex PCR assay were compared with the results of conventional, microbiological testing. Y. enterocolitica isolates were cultured from only 3 (3.75%) of the 80 pig tonsils examined. The multiplex PCR assay was shown to be an efficient tool for differentiation between the pYV plasmid–bearing Y. enterocolitica isolates, the plasmidless Y. enterocolitica isolates, the Y. enterocolitica biotype 1A isolates, and the Y. pseudotuberculosis isolates with and without the pYV plasmid in naturally contaminated pig tonsils. This indicates that this assay is useful to control food processing and track the source of contamination.

Yersiniosis in zoo marmosets ( Callitrix jacchuss) caused by Yersinia enterocolitica 4/O:3

The aim of this research was to describe two fatal cases of Yersinia enterocolitica bioserotype 4/O:3 infection in non-human primates and to characterise the isolates by PCR and PFGE. In July 2004, two marmosets ( Callitrix jacchuss) born in captivity in Zagreb Zoo, died following a few days of intermittent diarrhoea in intervals of 2 weeks. The pathomorphological diagnosis of the female (born in 1997) and the male (born in 1995) marmoset, was disseminated miliary necrosis of the liver. Y. enterocolitica 4/O:3 was isolated from both livers showing that monkeys are susceptible to this bioserotype. The ail gene, which is an essential chromosomal virulence factor in pathogenic Y. enterocolitica isolates, was present in the marmoset isolates. Two different PFGE patterns were obtained from the isolates of the male liver with NotI enzyme. One genotype of the male marmoset isolate was indistinguishable from the genotype of the female marmoset isolate when NotI, ApaI and XhoI enzymes were used indicating a common infection source for the marmosets. The genotypes of the marmoset isolates differed only slightly from one human (of seven Croatian isolates) and from one pig isolate (representing a common genotype found among human and porcine isolates in Germany) suggesting that raw pork fed to the marmoset could have been the infection source.

Distribution of virulence-associated genes inYersinia enterocoliticabiovar 1A correlates with clonal groups and not the source of isolation

Yersinia enterocolitica, an important food- and water-borne enteric pathogen, is represented by six biovars viz. 1A, 1B and 2-5. Some biovar 1A strains, despite lacking virulence plasmid (pYV) and chromosomal virulence genes, have been reported to cause symptoms similar to that produced by isolates belonging to known pathogenic biovars. Virulence-associated genes viz. ail, virF, inv, myfA, ystA, ystB, ystC, tccC, hreP, fepA, fepD, fes, ymoA and sat were studied in 81 clinical and nonclinical strains of Y. enterocolitica biovar 1A by PCR amplification. All strains lacked ail, virF, ystA and ystC genes. The distribution of other genes with respect to clonal groups revealed that four genes viz. ystB, hreP, myfA and sat were associated exclusively with strains belonging to clonal group A. The clonal groups A and B were differentiated previously based on rep (REP-/ERIC) - PCR genomic fingerprinting. The distribution of virulence-associated genes, however, did not differ significantly between clinical and nonclinical strains. In strains of Y. enterocolitica biovar 1A, clonal groups seem to reflect virulence potential better than the source (clinical vs. nonclinical) of isolation.