Yersinia enterocolitica 03 findings on porcine tongues in comparison with yersiniosis incidence in man in Czechoslovakia

Positive isolations of Yersinia obtained in repeated bacteriological examinations of porcine tongues at three slaughter-houses in Prague and a single examination at the slaughter-house at Kladno were compared with notified yersiniosis morbidity. The incidence of illnesses caused by Y. enterocolitica 03 does not exceed values of 4.5/100,000 and 3.5/100,000 population in the Czech and Slovak Socialist Republics, respectively, and is equal to a sixtieth part of the notified shigellosis and salmonellosis morbidity. Cultivation of 334 pooled samples consisting of 1142 porcine tongues yielded 12 strains (1.05%) of Y. enterocolitica 03, five strains (0.44%) of Y. pseudotuberculosis and 55 strains (4.82%) of other Yersinia organisms (indole-positive serotypes). Because of the low isolation rates obtained for the individual Yersinia species, Y. enterocolitica 03 in particular, the isolation efficiency of different cultivation techniques and culture media was statistically evaluated for all Yersinia organisms jointly. Primary cultivation on deoxycholate-citrate medium yielded five of the 12 Y. enterocolitica 03 strains isolated. The other Yersinia strains grew only after preliminary propagation. Yersinia pseudotuberculosis grew almost exclusively (4 out of 5 strains) on McConkey's agar.     

Invasiveness and cytotoxicity as criteria in assessing Yersinia attenuation

The electron microscopic study of cells HEp-2 and the complex microbiological and morphological study of tissues and organs of guinea pigs and mice infected with the isogenic pairs of Yersinia strains (Y. pseudotuberculosis I and Y. enterocolitica 09) differing in the presence of the calcium-dependence plasmid, as well as Y. pseudotuberculosis mutants resistant to rifampicin, nalidixic acid or crystal violet without this plasmid, have revealed that the invasiveness and cytotoxicity of the infective agents are not directly related to the presence of the above-mentioned plasmid in these bacteria. The use of the quantitative characteristics of virulence, such as penetration ability, intracellular multiplication, dissemination and the formation of degenerative changes, has made it possible to find out that the mutants of Y. pseudotuberculosis I, yielding the negative result in the keratoconjunctivitis test and resistant to the above-mentioned antimicrobial substances, can be arranged in the following order according to the degree of attenuation: rifr mutants--nalr mutants--kvlr mutants. In contrast to Y. pseudotuberculosis I, the loss of the calcium-dependence plasmid by Y. enterocolitica 09 is accompanied by an essential decrease in their invasive and cytotoxic properties, but this relationship is indirect and unstable. The proposed criteria intended for use in the evaluation of the degree of the manifestation of the invasive and cytotoxic properties of bacteria can be useful for the selection of optimally attenuated Yersinia strains showing promise as vaccine strains.     

Impact of Yersinia pseudotuberculosis on the in vitro production of cytokines by whole blood cells of donors

The impact of two plasmid (47, 82 MD), single plasmid (47 MD) and non plasmid Y. pseudotuberculosis strains, Y. enterocolitica (47 MD) as well as Y. pseudotuberculosis superantigen (YPM) on the production of interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-alpha (IFN = alpha) and tumor necrosis factor alpha (TNF-alpha) by whole blood cells obtained from donors was studied. All Y. pseudotuberculosis and Y. enterocolitica strains stimulated the production of IFN-alpha, IL-1, IL-6 and TNF-alpha by whole blood cells, but considerably less than Y. pseudotuberculosis lipopolysaccharide and YPM. These data are indicative of the pathogenetic role played by 82 MD plasmid in manifestation of Y. pseudotuberculosis immunosuppressive properties. The maximum stimulation of the production of cytokines was observed under the action of YPM, which confirmed an important role played by this superantigen in the pathogenesis of Y. pseudotuberculosis.     

Immunoelectrophoretic analysis of the antigenic composition of Yersinia

The antigenic composition of 24. Y. pseudotuberculosis newly isolated and reference strains, 7 Y. enterocolitica strains, as well as Y. pestis vaccine strain EV, has been studied by the method of immunoelectrophoresis in agar. The antigenic composition of these bacteria has been found to be complicated and to comprise not less than 8-11 antigens, and among them nonspecific protein antigens common for enterobacteria, the common generic antigen, the antigen common with Y. pestis, as well as O-antigens specific for each serovar are identified. Immunoelectrophoretic study has shown the possibility of Y. pseudotuberculosis O-antigen, serovar I, with Salmonella sera, serogroup A, and Y. enterocolitica 09 with brucellar and cholera sera.     

Distinctive electrophoretic and isoelectric focusing patterns of esterases from Yersinia enterocolitica and Yersinia pseudotuberculosis

Esterases of 53 strains of Yersinia enterocolitica sensu stricto, including five previously defined biotypes, and 30 strains of Yersinia pseudotuberculosis were analysed by horizontal polyacrylamide-agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. Esterase bands were defined by their range of activity towards several synthetic substrates, their resistance to heat and to di-isopropyl fluorophosphate. The two species were characterized by distinct electrophoretic patterns of their esterases. The apparent molecular weights of the heat-resistant esterase of Y. enterocolitica and of the major heat-resistant esterase of Y. pseudotuberculosis, as determined by polyacrylamide gradient gel electrophoresis, were estimated to be 52 000 and 250 000, respectively. On the basis of electrophoretic mobilities and isoelectric points of esterases produced by strains of Y. enterocolitica, five principal zymotypes were observed: two for strains of biotype 1, two for strains of biotypes 2 and 3, respectively, and only one for strains of both biotypes 4 and 5. The zymotypes of strains of biotypes 2, 3, 4 and 5 appeared to be more closely related to one another than to zymotypes of strains of biotype 1. Variations in number or mobility of bands observed within each biotype of Y. enterocolitica and within some serotypes of Y. pseudotuberculosis could represent an additional marker for epidemiological analysis.     

Purification of lipopolysaccharide from strains of Yersinia enterocolitica belonging to serogroups 03 and 09

Lipopolysaccharide (LPS) was purified from strains of Yersinia enterocolitica belonging to serogroups 03 and 09, by three methods, and analysed by SDS-PAGE and silver staining for carbohydrate. SDS-PAGE of LPS prepared from whole-cells by digestion with proteinase-K, produced profiles containing high molecular mass LPS and a lower molecular mass region migrating as discrete bands. LPS prepared from strains belonging to serogroup 03, using a hot-phenol procedure alone was found to contain cellular proteins, and LPS prepared from strains of serogroup 09, by this method, did not contain high molecular mass carbohydrate. A novel method of preparing LPS by digesting bacterial outer membranes with proteinase-K prior to hot-phenol extraction produced protein-free LPS from strain of Y. enterocolitica 03 and high molecular mass LPS from strains belonging to serogroup 09.     

A heat-modifiable outer membrane protein carries an antigen specific for the species Yersinia enterocolitica and Yersinia pseudotuberculosis

The outer membranes of gram-negative bacteria are considered to be of importance in host-bacteria interaction, in protective immunity, and occasionally in subclassification within a species. In this study, the outer membranes of several strains of Yersinia enterocolitica and Y. pseudotuberculosis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the appearance of the major proteins depended on the temperature at which they were solubilized in SDS. A protein was identified with the use of two-dimensional gels and preparative SDS-PAGE, which was equivalent to the "heat-modifiable protein" (protein II) of other Enterobacteriaceae species. A monoclonal antibody, 4G1, was generated against an isolated preparation of this Y. enterocolitica protein. This antibody was tested with whole cell bacterial antigens of 46 individual bacterial strains. The reactive strains included only Y. enterocolitica and Y. pseudotuberculosis. In addition, the reactivity of the 4G1 monoclonal antibody preparation could be absorbed only with Y. enterocolitica and Y. pseudotuberculosis, and not with other strains of bacteria. The reactivity of this 4G1 monoclonal antibody was also tested by the Western Blot technique. Six individual strains were tested: a Y. enterocolitica serotype 0:3, a Y. enterocolitica serotype 0:9, an Escherichia coli, a Salmonella typhimurium, a Shigella flexneri, and a Klebsiella pneumoniae. The 4G1 antibody reacted with only the proteins of the two Y. enterocolitica strains. In conclusion, the equivalent of the heat-modifiable protein was present in Y. enterocolitica and Y. pseudotuberculosis. Moreover, this protein also carried a species-specific antigenic determinant.     

The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin.

An essential virulence attribute for Yersinia enterocolitica and Yersinia pseudotuberculosis is the ability to invade the intestinal epithelium of mammals. The chromosomal invasin gene (inv) has been cloned from both of these Yersinia species, and the Y. pseudotuberculosis invasin has been well characterized (R. R. Isberg, D. L. Voorhis, and S. Falkow, Cell 50:769-778, 1987). Here we constructed TnphoA translational fusions to the Y. enterocolitica inv gene to identify, characterize, and localize the inv protein product in Escherichia coli. The cloned Y. enterocolitica inv locus encoded a unique protein of ca. 92 kilodaltons when expressed in minicells. A protein of comparable size was detected in immunoblots by using monoclonal antibodies directed against the Y. pseudotuberculosis invasin. This protein, which we also refer to as invasin, promoted both attachment to and invasion of cultured epithelial cells. These two functions were not genetically separable by insertional mutagenesis. We determined that the Y. enterocolitica invasin was localized on the outer membrane and that it was exposed on the bacterial cell surface, which may have implications for how invasin functions to mediate invasion.     

Action of sodium chloride on Yersinia pseudotuberculosis and Yersinia enterocolitica

The bacteriostatic and bactericidal action of sodium chloride on 60 Y. pseudotuberculosis strains, 75 Y. enterocolitica strains and 158 urine-fermenting strains has been studied. A new specific feature of Y. pseudotuberculosis has been revealed: high sensitivity to sodium chloride. The suitability of the sodium chloride test has been shown for the identification of Yersinia and the differentiation of Y. pseudotuberculosis and Y. enterocolitica.     

Chromosomal-encoded siderophores are required for mouse virulence of enteropathogenic Yersinia species

Abstract Yersinia enterocolitica and Y. pseudotuberculosis are enteropathogenic for humans. Essential virulence functions of these pathogens are determined by a 40-mDa plasmid. Plasmid-bearing Y. pseudotuberculosis strains and Y. enterocolitica strains of serotypes 0 : 8, 0 : 13, 0 : 20 and 0 : 40 are lethal for mice. In contrast, human pathogenic Y. enterocolitica strains of serotype 0 : 3, 0 : 9 and 0 : 5.27 are not mouse-lethal. Using a sensitive siderophore-indicator CAS-agar, we were able to detect siderophore production in all mouse-lethal Y. enterocolitica and Y. pseudotuberculosis strains mentioned above. By Tn5-transposon insertions into the chromosome of a serotype 0 : 8 strain we obtained two siderophore-deficient mutants. Introduction of the virulence plasmid did not render these mutants mouse-lethal, indicating that siderophore production is an essential virulence factor. The human nonpathogenic, aerobactin-producing strains of Y. intermedia, Y. kristensenii and Y. frederiksenii remained avirulent for mice after receiving the virulence plasmid of Y. enterocolitica . Obviously the siderophore aerobactin does not contribute to virulence in the genus Yersinia .     

Studies on the pathogenicity of Yersinia enterocolitica. III. Comparative studies between Y. enterocolitica and Y. pseudotuberculosis

Comparative studies on pathogenicity between Yersinia enterocolitica and Yersinia pseudotuberculosis were performed using experimental infection systems in vivo and in vitro. All of thestra ins of both species successfully produced experimental enterocolitis in rabbits although the severity varied with the strains challenged. The changes were characterized by granulomatous lesions with necrobiotic centers in reticuloendothelial tissues of the intestine, mesenteric lymph nodes, liver and spleen. These strains uniformly had the ability to penetrate HeLa cells and to survive or multiply within cultured rabbit peritoneal macrophages. In addition, in infections with strain TP-2 or PST-III of Y. pseudotuberculosis, catarrhal inflammation all over the small intestine and/or focal necrosis and parenchymatous degeneration in the liver were observed, along with the granulomatous lesions. These strains, at the same time, exhibited cytotoxic effects on the cultured cells. The pathogenic factors of Y. enterocolitica are discussed in comparison with those of Y. pseudotuberculosis.     

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Detection of virulence markers in clinical strains of Yersinia]

The dispersion of plasmid pYV associated virulence markers in 474 Yersinia strains isolated from people has been studied. The ability to autoagglutination, calcium dependence of growth and the specific antigens were identified in 157 strains of traditionally pathogenic Yersinia enterocolitica serovars 03, 09, Yersinia pseudotuberculosis serovar I. They were not found in 223 strains of other 12 serovars of Yersinia enterocolitica, in 40 strains of Yersinia frederiksenii, Yersinia kristensenii, Yersinia intermedia. The proportion of virulent clones in the population of Yersinia is noted to depend on the conditions of its existence in vivo or in vitro. Identification of virulence markers is acknowledged to be expedient in epidemiological and ethiological estimation of the role of isolated Yersinia strains.     

Antibodies to Yersinia enterocolitica and Proteus mirabilis in blood sera of rheumatoid arthritis patients

Data on specific antibodies to the bacterial antigens of Yersinia enterocolitica (serovars 03, 09) and Proteus mirabilis in rheumatoid arthritis (RA) patients are presented. According to the data of some researchers, these organisms play some role in the pathogenesis of RA. The highest level of IgG antibodies to Y. enterocolitica antigens of both serovars was noted. The activity of specific IgG antibodies to serovar 03 was higher than that to serovar 09. In the sera of RA patients with P. mirabilis antgens the activity of IgM antibodies was higher.     

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.     

The lytic activity of Yersinia pestis phage P 3d serovar

The lytic activity of plague phage II, serovar 3, with respect to 1,800 bacterial strains has been studied: 760 Yersinia pestis strains, 262 Y. pseudotuberculosis strains, 252 Y. enterocolitica strains, 166 Escherichia coli strains, 90 Shigella strains and 270 strains of other species. The phage has been found to lyse 81.8% of Y. pestis strains, 1 Y. pseudotuberculosis strain and 1 Y. enterocolitica strain. The representatives of other 19 bacterial species have proved to be resistant to the phage. Though having a wide range of action within Y. pestis, the phage does not lyse most of the strains of the causative agent of plague, isolated in certain natural foci. This fact offers promise for using the phage for the differentiation of Y. pestis.     

Modulation of complement activityin Vitro andin Vivo byYersinia wild and mutant strains

The ability of released proteins (Yops) and surface lipopolysaccharides (LPS) from the wild-type strain Yersinia enterocolitica 8081-L2, serotype 0:8 to influence the complement activity was determined. Yops and LPS from wild-type and mutant strains showed different ability to affect the classical pathway (CP) functional complement activity in vitro. The serum CP activity was inhibited during the infection induced with six Y. enterocolitica and three Y. pseudotuberculosis strains in rabbits. The changed complement activity might be of importance for the course of Yersinia infections.     

Evaluation of usefulness for selected virulence markers for identifying pathogenic Yersinia enterocolitica strains. IV. Genes myfA and ureC

We check by polymerase chain reaction (PCR) the presence of gene ureC and myfA, encoding subunits of urease and Myf fimbriae, among clinical and food-originated strains of Yersinia to determine their usefulness as molecular virulence markers of Y. enterocolitica. The examinations were done on 130 clinical strains of Y. enterocolitica O:3/4 isolated in Poland from humans. All strains were obtained from stool and possessed the virulence plasmid pYV. In addition 40 isogenic, plasmid-cured strains were tested. The 52 strains including Y. enterocolitica (biotype 1A, 4, 2 and 1B), Y. pseudotuberculosis, Y. intermedia, Y. frederiksenii, Y. kristensenii, E. coli, Citrobatcer, Shigella and Salmonella were used as controls. The PCR assay resulted in detection of genes: ureC and myfA in genomic DNA of all 130 tested clinical strains of Y. enterocolitica pYV+, as well as in plasmid cured strains. Furthermore, ureC was found in all tested strains of Y. enterocolitica biotype A1 and in one strain of Y. intermedia and Y. kristensenii. In contrast to ureC, myfA was detected only in strains of Y. enterocolitica considered as pathogenic. Obtained results show, gene myfA seems to be the reliable virulence marker of Y. enterocolitica, whereas ureC is not recommended for identification of pathogenic strains of this species.