Homologues of insecticidal toxin complex genes in Yersinia enterocolitica biotype 1A and their contribution to virulence

Yersinia enterocolitica is an enteric pathogen that consists of six biotypes: 1A, 1B, 2, 3, 4, and 5. Strains of the latter five biotypes can carry a virulence plasmid, known as pYV, and several well-characterized chromosomally encoded virulence determinants. Y. enterocolitica strains of biotype 1A lack the virulence-associated markers of pYV-bearing strains and were once considered to be avirulent. There is growing epidemiological, clinical, and experimental evidence, however, to suggest that some biotype 1A strains are virulent and can cause gastrointestinal disease. To identify potential virulence genes of pathogenic strains of Y. enterocolitica biotype 1A, we used genomic subtractive hybridization to determine genetic differences between two biotype 1A strains: an environmental isolate, Y. enterocolitica IP2222, and a clinical isolate, Y. enterocolitica T83. Among the Y. enterocolitica T83-specific genes we identified were three, tcbA, tcaC, and tccC, that showed homology to the insecticidal toxin complex (TC) genes first discovered in Photorhabdus luminescens. The Y. enterocolitica T83 TC gene homologues were expressed by Y. enterocolitica T83 and were significantly more prevalent among clinical biotype 1A strains than other Yersinia isolates. Inactivation of the TC genes in Y. enterocolitica T83 resulted in mutants which were attenuated in the ability to colonize the gastrointestinal tracts of perorally infected mice. These results indicate that products of the TC gene complex contribute to the virulence of some strains of Y. enterocolitica biotype 1A, possibly by facilitating their persistence in vivo.     

Virulence and phenotypic characterization of Yersinia enterocolitica isolated from humans in the United States

Yersinia enterocolitica was recently reclassified into Yersinia enterocolitica sensu stricto and three additional species. With this new classification, it was of interest to reexamine pathogenicity previously ascribed to Y. enterocolitica. All available clinical isolates of Y. enterocolitica sent to the Centers for Disease Control from 1970 through 1980 were selected for characterization and comparison. One-hundred such strains had been submitted, from 21 states. Most (85%) were biotype 1, and O:8 was the most common of the 24 serotypes encountered. All strains were examined by several virulence assays. Two strains caused conjunctivitis in guinea pigs, 7 were lethal for mice, 54 invaded HEp2 cells, 18 produced a heat-stable enterotoxin, 9 were calcium dependent, 20 autoagglutinated, and 34 had a distinctive colonial morphology at 37 degrees C. Ten isolates of each of the new species that had previously been grouped with Y. enterocolitica (Y. kristensenii, Y. intermedia, and Y. frederiksenii) were characterized and were generally negative in all assays. This study points out pathogenicity differences among Yersinia species, confirms the complex nature of virulence in Y. enterocolitica, and confirms that no single current assay correlates with virulence in Y. enterocolitica.     

Yersinia pseudotuberculosis infection: study of an epizootic in squirrel monkeys.

An epizootic of an acutely fatal enteric disease in a colony of squirrel monkeys (Saimiri sciureus) was attributed to infection by Yersinia pseudotuberculosis serotype III. Of a total adult population of 96 animals at risk, there were six fatal cases of yersiniosis. Serological evaluation of the colony just after the outbreak ended revealed that 22 of 60 monkeys tested (37%) had significant antibody to Y. pseudotuberculosis (microagglutination titer of greater than or equal to 1:80) but did not have clinical disease. The outstanding pathological lesions noted in dying monkeys were acute, purulent, necrotic and focal enteritis primarily affecting the jejunum and ileum and focal hepatic necrosis and abscessation. Y. pseudotuberculosis was isolated from the organs of two of the dying monkeys. Using cold enrichment techniques, Yersinia was also isolated from the feces of two apparently healthy monkeys (both seropositive), from the spleen of a monkey dying of other causes, and from the colon contents of a stillborn squirrel monkey baby. All isolates had the same biotype and serotype. An episode of abortions was associated both temporally and spatially with the fatal cases of yersiniosis, and Y. pseudotuberculosis was cultured from the uterus of two of the dying monkeys, suggesting that yersinia infection may be associated with abortion, as well as with enteric infection, in these animals.     

Effects of iron and desferrioxamine on infections with Yersinia enterocolitica.

The effects of iron-dextran and the iron chelator desferrioxamine B mesylate (Desferal) on the course and outcome of experimental yersiniosis were investigated. Yersinia enterocolitica strains representing the three leading serogroups pathogenic for humans, O3, O8 and O9, were studied. In mice, iron-dextran reduced the median lethal dose of intraperitoneally administered Y. enterocolitica O3 and O9 ca. 10-fold, whereas Desferal reduced this value more than 100,000-fold. Experiments in which Y. enterocolitica was given orally to mice and intraconjunctivally to guinea pigs confirmed that Desferal markedly increased the susceptibility of animals to yersiniosis. Although serogroup O8 yersiniae were inherently more virulent for laboratory animals, they were less affected by Desferal than were O3 or O9 strains. In vitro experiments indicated that Desferal promoted growth of Y. enterocolitica under iron-limiting conditions and suggested that the enhanced virulence of O8 yersiniae may be due to their comparatively low requirement for iron. The adverse effect of Desferal on the course of experimental infection with Y. enterocolitica may partly explain the heightened susceptibility of iron-overloaded patients to systemic yersiniosis.     

Pathogenicity of Yersinia enterocolitica serotype O3 biotype 3 strains

It is known that Yersinia enterocolitica infection in Japan is caused mainly by serotype O3 biotype 4 strains. Recently, however, a number of serotype O3 strains which were classified biotype 3 and which ferment lactose and xylose, instead of sorbose, and give a negative Voges-Proskauer reaction have been isolated from both humans and animals. In this study, comparisons of four properties were made among isolates of Y. enterocolitica serotype O3 biotype 3 from humans, pigs, dogs, cats, and rats and the laboratory stock strains of Y. enterocolitica biotype 4. All strains were tested for the presence of plasmids, calcium-dependent growth at 37 degrees C, autoagglutination activity at 37 degrees C, and recovery of the organisms from the stools of intravenously challenged mice. Biotypes 3 and 4 were positive for these four properties. Plasmid digestion with restriction endonucleases showed the same digestion patterns in both biotypes. These results suggest that Y. enterocolitica serotype O3 biotype 3 strains are pathogenic, as are biotype 4 strains.     

Yersinia enterocolitica septicaemia from transfusion of red cell concentrate stored for 16 days.

Two cases of transfusion transmitted Yersinia enterocolitica biotype 3, serotype 09 infection occurred in south east Scotland within four months of each other. In one case, a 79 year old man died the day after receiving a unit of red cell concentrate that had been stored for 29 days after donation. In the second case a 78 year old man died three days after transfusion of a unit of red cell concentrate that had been collected 16 days before transfusion. The donors of both units had no symptoms attributed to gastrointestinal infection. Early outdating of blood for transfusion after three weeks of storage is unlikely to eradicate Y enterocolitica associated fatalities from blood transfusion, and alternative methods should be considered.     

Detection of pathogenic Yersinia enterocolitica by polymerase chain reaction and digoxigenin-labeled polynucleotide probes.

Yersinia enterocolitica is widespread in nature, but only a few bioserotypes are involved in human infections. Pigs are considered to be the major reservoirs of pathogenic strains. It is essential to have an accurate and rapid method for the detection of pathogenic yersiniae. To achieve this objective, 19-base synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) to detect the ail gene (which is conserved only in pathogenic strains) in strains of Y. enterocolitica and related species originating from pigs or pork products. Digoxigenin-labeled probes derived from the ail, inv, and yst genes were also evaluated on these strains. The PCR amplified a 273-bp fragment of the ail gene involved in eukaryotic cell invasion and serum resistance. The PCR detected template DNA only in strains of Y. enterocolitica traditionally classified as human pathogens but not in biotype 1A strains and related species. Other members of the family Enterobacteriaceae were also negative for the target gene. The digoxigenin-labeled ail probe gave identical results to the PCR. By use of this nonisotopic method, inv-homologous DNA was detected only among yersiniae, except for Y. ruckeri. Although all pathogenic serotypes of Y. enterocolitica were positive for the heat-stable enterotoxin yst gene, two strains of biotype 1A, one Y. intermedia strain, and six other species of the Enterobacteriaceae were also positive. Our results support the notion that pigs constitute an important reservoir of pathogenic Y. enterocolitica and that the inv-homologous sequence is Yersinia specific.     

Characterization of the Interaction between Yersinia enterocolitica Biotype 1A and Phagocytes and Epithelial Cells In Vitro

Yersinia enterocolitica strains of biotype 1A are increasingly being recognized as etiological agents of gastroenteritis. However, the mechanisms by which these bacteria cause disease differ from those of highly invasive, virulence plasmid-bearing Y. enterocolitica strains and are poorly understood. We have investigated several biotype 1A strains of diverse origin for their ability to resist killing by professional phagocytes. All strains were rapidly killed by polymorphonuclear leukocytes but persisted within macrophages (activated with gamma interferon) to a significantly greater extent (survival = 40.5% +/- 17.4%) than did Escherichia coli HB101 (9.3% +/- 0.7%; P = 0.0001). Strains isolated from symptomatic patients were significantly more resistant to killing by macrophages (survival = 48.9% +/- 19.5%) than were strains obtained from food or the environment (survival = 32.1% +/- 10.3%; P = 0.04). Some strains which had been ingested by macrophages or HEp-2 epithelial cells showed a tendency to reemerge into the tissue culture medium over a period lasting several hours. This phenomenon, which we termed "escape," was observed in 14 of 15 strains of clinical origin but in only 3 of 12 nonclinical isolates (P = 0.001). The capacity of bacteria to escape from cells was not directly related to their invasive ability. To determine if escape was due to host cell lysis, we used a variety of techniques, including lactate dehydrogenase release, trypan blue exclusion, and examination of infected cells by light and electron microscopy, to measure cell viability and lysis. These studies established that biotype 1A Y. enterocolitica strains were able to escape from macrophages or epithelial cells without causing detectable cytolysis, suggesting that escape was achieved by a process resembling exocytosis. The observations that biotype 1A Y. enterocolitica strains of clinical origin are significantly more resistant to killing by macrophages and significantly more likely to escape from host cells than are strains of nonclinical origin suggest that these properties may account for the virulence of these bacteria.     

Characteristics of Yersinia enterocolitica biotype 1A strains isolated from patients and asymptomatic carriers

Yersinia enterocolitica biotype 1A strains are frequently isolated from the environment, foods, and animals, and also from humans with yersiniosis. There are controversial reports on the pathogenicity of biotype 1A strains. In this study, 811 fecal samples from asymptomatic humans from Switzerland were studied for the presence of Y. enterocolitica. Nine (1.1 %) of the 811 samples were positive for Y. enterocolitica 1A. These strains were compared with 12 Y. enterocolitica 1A strains from Swiss patients with diarrhea isolated in the same year. Almost all (20/21) Y. enterocolitica 1A strains carried the ystB gene, seven strains carried the hreP gene, and none carried the ail, ystA, myfA, yadA, or virF genes. Most (17/21) Y. enterocolitica 1A strains belonged to two major clusters, A and B, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Strains of cluster B were only isolated from humans with diarrhea; however, ystB and hreP genes were detected in strains from both clinical and non-clinical samples and from strains of clusters A and B. Using ribotyping, six restriction patterns among biotype 1A strains were obtained with HindIII enzyme. The most common ribotype (RT I) was found in strains isolated from humans with and without diarrhea. All biotype 1A strains had a unique NotI profile by pulsed-field gel electrophoresis (PFGE), showing a very high genetic diversity. In this study, Y. enterocolitica 1A strains from clinical and non-clinical samples could not be clearly differentiated from each other. More research is needed in order to prove that biotype 1A strains are a primary cause for human yersiniosis and not only a secondary finding.     

Plasmids in Yersinia enterocolitica serotypes O:3 and O:9: correlation with epithelial cell adherence in vitro.

Human isolates of Yersinia enterocolitica serotypes O:3 (biotype 4) and O:9 (biotype 3) harbored plasmids sized approximately 47 and 44 megadaltons, respectively. No such plasmids were found in "apathogenic" strains of Y. enterocolitica belonging to biotype 1. There was a positive correlation among the presence of plasmid, autoagglutination, and adherence to and toxicity for HEp-2 cell cultures; all of these properties were lost by culturing at 37 degrees C in the absence of calcium. Strains of Y. enterocolitica O:3 and O:9 cured of the plasmids showed increased invasiveness in the HEp-2 cell culture model, but no invasiveness in guinea pig eye. It is suggested that the plasmids of Y. enterocolitica primarily determine epithelial cell adherence, but may also be associated with other pathogenic properties.     

Evaluation of DNA colony hybridization and other techniques for detection of virulence in Yersinia species.

The virulence of yersiniae varies according to (i) species and biotype and (ii) possession of a 67- to 72-kilobase virulence plasmid. Y. pestis, Y. pseudotuberculosis, and biotypes 1B, 2, 3, 4, and 5 of Y. enterocolitica are inherently virulent but express full virulence only when in possession of a virulence plasmid. Other Yersinia species and biotypes 1A and 3B of Y. enterocolitica are seldom implicated in disease. In this study, we prepared DNA probes from eight nonoverlapping regions of the virulence plasmid of a strain of Y. enterocolitica and from the inv and ail chromosomal loci responsible for the invasive capacity of Y. enterocolitica and Y. pseudotuberculosis. The probes were used in colony hybridization experiments to investigate 156 yersiniae of various species and biotypes and of differing virulence. Probes prepared from the inv gene of Y. pseudotuberculosis hybridized with Y. pseudotuberculosis and Y. pestis only, whereas an analogous probe prepared from Y. enterocolitica hybridized with all species and biotypes of yersiniae (but not with other bacteria) regardless of virulence or potential virulence. Probes prepared from the ail region of Y. enterocolitica reacted almost exclusively with Y. enterocolitica strains of pathogenic biotypes. Probes prepared from the virulence plasmid of a serogroup O:8, biotype 1B isolate of Y. enterocolitica identified virulent yersiniae in all species with a high degree of sensitivity and specificity. These probes did not react with yersiniae of avirulent biotypes or species. Of the other assays of virulence evaluated (calcium dependence, binding of crystal violet, and pyrazinamidase activity), binding of crystal violet provided a simple means for identifying plasmid-bearing strains.     

New selective agar medium for isolation of virulent Yersinia enterocolitica.

A selective agar medium for isolation of virulent Yersinia enterocolitica (VYE agar) was developed for the rapid and accurate isolation of virulent Y. enterocolitica from environmental samples highly contaminated with environmental Yersinia organisms, as well as for isolation from clinical specimens. VYE agar provided a quantitative recovery of 51 different strains of virulent Y. enterocolitica at 32 degrees C after incubation for 24 h. The cefsulodin, irgasan, josamycin, and oleandomycin content of the medium resulted in a high selectivity, and the mannitol and esculin content provided some differentiation. The greatest advantage of VYE agar is that virulent Y. enterocolitica, which forms red colonies, is easily differentiated from most environmental Yersinia organisms and other gram-negative bacteria, which form dark colonies with a dark peripheral zone as a result of esculin hydrolysis. Use of VYE agar led to a high recovery of Y. enterocolitica biotype 3B serotype O:3 strains from experimentally inoculated meat samples, compared with use of CIN agar. Biotype 2 serotypes O:5,27 and O:9 and biotype 1 esculin-negative serotypes O:4,32, O:8, O:13a,13b, O:18, O:20, and O:21 (American types) were readily differentiated from other environmental organisms able to grow on VYE agar. Epidemiological studies on Y. enterocolitica should be greatly facilitated by the use of this selective agar medium.     

Current evidence for human yersiniosis in Ireland

Yersiniosis associated with abdominal pain was commonly reported in Ireland in the 1980s. However, the Health Protection Surveillance Centre (HPSC) currently records only three to seven notified cases of yersiniosis per year. The most common cause of yersiniosis worldwide is Yersinia enterocolitica, and the leading source for this organism is consumption of pork-based food products. In contrast to the apparent current scarcity of yersiniosis cases in humans in Ireland, pathogenic Y. enterocolitica are detectable in a high percentages of pigs. To establish whether the small number of notifications of human disease was an underestimate due to lack of specific selective culture for Yersinia, we carried out a prospective culture study of faecal samples from outpatients with diarrhoea, with additional culture of throat swabs, appendix swabs and screening of human sewage. Pathogenic Yersinia strains were not isolated from 1,189 faeces samples, nor from 297 throat swabs, or 23 appendix swabs. This suggested that current low notification rates in Ireland are not due to the lack of specific Yersinia culture procedures. Molecular screening detected a wider variety of Y. enterocolitica-specific targets in pig slurry than in human sewage. A serological survey for antibodies against Yersinia YOP (Yersinia Outer Proteins) proteins in Irish blood donors found antibodies in 25?%, with an age-related trend to increased seropositivity, compatible with the hypothesis that yersiniosis may have been more prevalent in Ireland in the recent past.     

Low occurrence of pathogenic Yersinia enterocolitica in clinical,food, and environmental samples: a methodological problem

While Yersinia enterocolitica is an important pathogen, which can cause yersiniosis in humans and animals, its epidemiology remains obscure. The pig is the major reservoir of pathogenic Y. enterocolitica of bioserotype 4/O:3, the most common type found in humans. Y. enterocolitica is thought to be a significant food-borne pathogen, although pathogenic isolates have seldom been recovered from foods. The low isolation rate of this pathogenic bacterium in natural samples, including clinical, food, and environmental samples, may be due to the limited sensitivity of culture methods. During the last decade, numerous DNA-based methods, such as PCR and colony hybridization assays, have been designed to detect pathogenic Y. enterocolitica in natural samples more rapidly and with better sensitivity than can be achieved by culture methods. In addition, the occurrence of pathogenic Y. enterocolitica in natural samples is clearly higher with PCR than with culture methods. The methods available for detection of pathogenic Y. enterocolitica in natural samples are reviewed in this article.     

Resistance of chemokine receptor 6-deficient mice to Yersinia enterocolitica infection: evidence of defective M-cell formation in vivo

M cells, specialized cells within Peyer's patches (PPs), are reduced in number in chemokine receptor 6 (CCR6)-deficient mice. The pathogenic microorganism Yersinia enterocolitica exploits M cells for the purpose of mucosal tissue invasion exclusively through PPs. The aim of this study was to evaluate the course of yersiniosis in CCR6-deficient mice and to investigate whether these mice might be used as an in vivo model to determine M-cell function. After oral challenge with Y. enterocolitica, control mice suffered from lethal septic infection whereas CCR6-deficient mice showed very limited symptoms of infection. Immunohistochemical analysis demonstrated PP invasion by Y. enterocolitica in control mice whereas no bacteria could be found in CCR6-deficient mice. In addition, a significant induction of proinflammatory cytokines could be found in control mice whereas proinflammatory cytokine levels in CCR6-deficient mice remained unchanged. In contrast, intraperitoneal infection resulted in severe systemic yersiniosis in both mouse groups. Abrogated oral Y. enterocolitica infection in CCR6-deficient mice demonstrates the importance of CCR6 expression in the physiological and pathological immune responses generated within PPs by influencing M-cell differentiation, underscoring the important role of M cells in the process of microbial uptake. CCR6-deficient mice may therefore represent a suitable model for the study of M-cell function in vivo.     

Construction and characterization of a Yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant.

The high-temperature requirement (HtrA) family of stress response proteins are induced by different environmental stress conditions in a variety of bacteria and have been shown to contribute to the pathogenicity of some of these species. In this study, the htrA gene from Yersinia enterocolitica O:8 was amplified, cloned, and sequenced. Analysis of the deduced amino acid sequence predicted that the putative HtrA homolog contains a serine protease active site and a catalytic triad characteristic of trypsin-like serine proteases, structural features characteristic of previously described HtrA proteins. In order to evaluate the biological functions of Y. enterocolitica HtrA, an isogenic mutant was constructed by a reverse-genetics PCR-based approach. Characterization of the mutant provided evidence supporting a stress response function for the Y. enterocolitica htrA gene product. In contrast to the parent strain, the mutant showed increased sensitivity to killing by H2O2, O2- and temperature stress (50 degrees C). The mutant was avirulent in the murine yersiniosis injection model and offered partial protection to mice challenged with the parent strain. Further studies with the Y. enterocolitica htrA mutant should increase our knowledge of the host-pathogen interactions which occur during Yersinia infections.     

Effect of low- and high-virulence Yersinia enterocolitica strains on the inflammatory response of human umbilical vein endothelial cells

Pathogenic strains of Yersinia spp. inject a set of Yop effector proteins into eukaryotic cells by using a plasmid-encoded type III secretion system. In this study, we analyzed the inflammatory response of human umbilical vein endothelial cells (HUVECs) after infection with different Yersinia enterocolitica strains. We found that both expression of intercellular adhesion molecule 1 and release of the cytokines interleukin-6 (IL-6) and IL-8 by HUVECs are downregulated in a YopP-dependent way, demonstrating that YopP plays a major role in the inflammatory response of these cells. Infection of HUVECs with several low-virulence (biotype 2, 3, and 4) and high-virulence (biotype 1B) Y. enterocolitica strains showed that biotype 1B isolates are more efficient in inhibiting the inflammatory response than low-virulence Y. enterocolitica strains and that this effect depends on the time of contact. We extended the results of Ruckdeschel et al. and found that on the basis of the presence or absence of arginine-143 of YopP (K. Ruckdeschel, K. Richter, O. Mannel, and J. Heesemann, Infect. Immun. 69:7652-7662, 2001) all the Y. enterocolitica strains used fell into two groups, which correlate with the low- and high-virulence phenotypes. In addition, we found that high-virulence strains inject more YopP into the cytosol of eukaryotic target cells than do low-virulence strains.     

Evaluation of rapid SYS system as screen for Yersinia enterocolitica in the United States.

Clinical isolates (n = 150) from stool specimens were selected for evaluation of the Rapid SYS system (Analytab Products, Plainview, N.Y.) as a screening test for Shigella spp., Yersinia enterocolitica, and Salmonella spp. The Gram-Negative Identification Card (Vitek Systems, Inc., Hazelwood, Mo.) was used for identification. Although acceptable performance of the Rapid SYS system was described, the interpretative criteria provided by the vendor for previous studies led to inappropriate screening for Y. enterocolitica, particularly biotype 1. When corrected screening criteria were used for the present study, the sensitivity for the detection of 76 enteric pathogens was 98.7%. Of the 76 pathogens, 1 of 21 Shigella spp. was not detected. However, specificity was only 16.6% when 72 selected nonpathogens frequently encountered in stools were eliminated. Although the Rapid SYS system can identify Shigella spp., Y. enterocolitica, and Salmonella spp., only phenylalanine deaminase-producing and cytochrome oxidase-producing organisms can be eliminated from additional testing. Therefore, the Rapid SYS system cannot be used as a three-pathogen screen in the United States or in other geographic locales where Y. enterocolitica biotype 1 may be encountered.     

Development of a highly specific assay for rapid identification of pathogenic strains of Yersinia enterocolitica based on PCR amplification of the Yersinia heat-stable enterotoxin gene (yst).

The chromosomal gene yst, which encodes a heat-stable enterotoxin of Yersinia enterocolitica, is a useful diagnostic marker because it occurs only in invasive strains of this species. A homologous gene also occurs in some strains of Yersinia kristensenii. Sequence analysis of the yst genes from two different strains of Y. enterocolitica and from Y. kristensenii revealed a substantial number of mismatches at the 3' ends of the yst genes of the so-called American and European biotypes of Y. enterocolitica. Moreover, several mismatches and a deletion of 5 codons were found in the yst of Y. kristensenii. These findings were used to develop a PCR-based assay for yst of Y. enterocolitica which yielded a detectable product in as little as 50 min. The assay was 100% specific in terms of its ability to identify potentially pathogenic strains of Y. enterocolitica regardless of biotype or serotype. The PCR yielded an amplicon that was visible on agarose gel electrophoresis from as few as 100 CFU, or 10 CFU when the PCR was combined with dot blot hybridization with a digoxigenin-labeled oligonucleotide probe that corresponded to an internal sequence of yst. These results establish the value of the yst gene as a target for the identification of pathogenic bioserotypes of Y. enterocolitica and the usefulness of PCR for this purpose.     

The pathogenesis of Yersinia enterocolitica infection in gnotobiotic piglets

Yersinia enterocolitica is an important cause of enteritis and mesenteric adenitis in many countries. However the pathogenesis of the disease caused by this organism has not been fully elucidated. Most isolates from clinical material possess two independent properties associated with virulence whose relative contribution to the development of disease is not known. These are the ability to penetrate the intestinal wall, which is thought to be controlled by a plasmid gene, and the production of heat-stable enterotoxin, which is controlled by a chromosomal gene. In this study, we infected neonatal gnotobiotic piglets with strains of Y. enterocolitica expressing these two properties in various combinations. The suitability of the piglet model was shown in experiments in which piglets fed virulent Y. enterocolitica serogroup O3 developed a clinical illness related to the size of the inoculum, which was accompanied by intestinal lesions similar to those reported in naturally and experimentally infected people and animals. The results confirmed the key role of a 47 X 10(6)-mol. wt plasmid in the pathogenicity of Y. enterocolitica, but suggested that penetration of the intestinal wall may be governed by chromosomal rather than plasmid-borne genes. No role for enterotoxin in the pathogenesis of yersiniosis was shown, although there was evidence that enterotoxin may promote intra-intestinal proliferation of Y. enterocolitica, thus favouring increased shedding of bacteria and encouraging their spread between hosts.