The use of monoclonal antibodies for detecting the common antigenic determinants of Brucella spp. and Yersinia enterocolitica O:9

Monoclonal antibodies (McAb) 2AH10, specifically reacting with protein preparations having mol. wt. of 18 and 38 kD and not interacting with brucellar lipopolysaccharide (LPS) and protein-polysaccharide antigen, have been obtained. As shown with the use of McAb 2AH10, Brucella spp. and Yersinia enterocolitica O:9 possess common antigenic determinants, localized not only in the area of their LPS, which is generally known, but also in the area of their outer cell-wall proteins with mol. wt. 18 and 38 kD. The sensitivity of the solid-phase enzyme immunoassay and the latex agglutination test with the use of McAb 2AH10 is essentially higher in the detection of B. abortus and B. suis, than B. melitensis and B. rangiferi, as well as Y. enterocolitica O:9. Essential differences observed in the detected concentrations of different Brucella species and Y. enterocolitica O:9 are seemingly linked with different expression of specific antigenic determinants, detected with the use of McAb 2AH10 in the corpuscular antigens under study.     

Use of the phenol fraction of Brucella for isolating a specific serum against Yersinia enterocolitica serovar O9

A phenol fraction, capable of adsorbing cross-reacting antibodies from Y. enterocolitica O9 antiserum without affecting its serological activity against specific antigens, has been obtained from a Brucella strain. The physicochemical and antigenic characteristics of the preparation are presented. It is recommended that the proposed method be tested in differentiation of intestinal yersiniosis caused by Y. enterocolitica O9 from Brucella infection. This work has resulted in obtaining highly specific antiserum to Y. enterocolitica, serovar O9.     

A real-time PCR assay for the specific identification of serotype O:9 of Yersinia enterocolitica

A real-time PCR assay was developed based on a 181-bp fragment of the recently cloned per gene, including an internal amplification control (124 bp), for the detection of Yersinia enterocolitica O:9 (Ye O:9). The validation included 48 Ye O:9, 33 Y. enterocolitica non-O:9 and 35 other closely-related bacterial strains, containing per gene homologies. The assay was specific for the Ye O:9 tested, the detection limit was 1-10 genome copies of purified DNA and amplification efficiency was between 90.5-103%, indicating a linear regression throughout the detection window.     

Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages

Abstract The survival of Yersinia enterocolitica serotype O9 within mouse peritoneal macrophages was investigated. To evaluate the role of the virulence plasmid in the resistance to intracellular killing, an isogenic pair of virulent (plasmid-bearing) and avirulent (plasmid-less) O9 strains was used. The virulent strain was able to express plasmid-encoded outer membrane proteins and to colonize the Peyer's patches of orally infected mice. When mice were infected intraperitoneally, both strains were recovered at similar rates and over the same time from the peritoneal cavity. When in vitro assays were performed, both strains showed similar resistance to intracellular killing by monolayers of resident and inflammatory peritoneal macrophages. Previous opsonization of bacteria did not modify their survival within macrophage monolayers. We concluded that serotype O9 strains display a chromosome-mediated resistance to intracellular killing by mouse peritoneal macrophages. Moreover, macrophage resistance does not seem to be of importance for virulence of serotype O9 strains in mice.     

Characterization and biological role of the O-polysaccharide gene cluster of Yersinia enterocolitica serotype O:9

Yersinia enterocolitica serotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) in Y. enterocolitica O:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer of N-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of the gnd gene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster, galF and galU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.     

Model experiments to establish behaviour of Yersinia enterocolitica O:9 strains in various types of fresh dry sausage

In model experiments different kinds of raw sausages were inoculated with liquid cultures of virulent-plasmid-carrying clinical Yersinia (Y.) enterocolitica (e.) strains of the O:9 serotype, doses being between 10⁴ and 10⁵ cfu g^-1. The sausage samples were stored at 3–5° and 13–16°C. During the first 10 d of storage the Y.e. plate count was detected with Desoxycholate-Citrate-Lactose-Sucrose Agar every day, later on in addition to it with phosphate buffer-enrichment and with enrichment according to Schiemann (1982) in intervals of several days' duration. The pH and a _w values, the contents of salt and water were detected. The multitude of complexly acting factors and substances prevents obviously the proliferation of Y.e. in fresh dry sausages. Decay dynamics of Y.e. were found to be considerably affected by storage temperature. Cold storage, basically, had a conservation effect and thus delayed the dying process of model strains. Yersinia enterocolitica -contaminated fresh dry sausage may cause potential danger to consumers, because of relatively extended survival periods of the pathogen. Therefore, manufacturers are expected to observe most stringent hygienic rules of Good Manufacturing Practice.     

Immunochemical studies on R mutants of Yersinia enterocolitica O:3.

Three mutants of Yersinia enterocolitica O:3, namely: YeO3-R1, YeO3-RfbR7 and YeO3-c-trs8-R were classified on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) profile of isolated lipopolysaccharides (LPS) as belonging to the Ra- (the first) and the Rc-type (the other two mutants). Methylation analysis, in addition to 13C and 1H NMR studies of purified core oligosaccharides revealed structures similar to those established previously for the full core of Y. enterocolitica O:3 in the case of the Ra mutant, and identical to that reported for the Rc mutant Ye75R, in the case of the two other mutants. The O-specific sugar, 6d-L-altrose, which forms a homopolymeric O-chain, was present in small amounts in all three LPS preparations, as well as in the core oligosaccha ride preparations along with the Ra and the Rc sugars, characteristic of the Y. enterocolitica O:3 core. This result is in line with genetic data, indicating that it is the inner core region which is the receptor for the O-specific chain in Y. enterocolitica O:3. This region seems likewise to be the anchoring region for the enterobacterial common antigen (ECA), as shown by SDS/PAGE/Western blot analysis with monoclonal antibodies against ECA. In addition, we also demonstrated that the Ye75R mutant Rc and its parental strain Ye75S, both were ECA-immunogenic strains. So far, ECA-immunogenic strains, i.e. those with LPS-linked ECA, were only identified in E. coli mutants of the R1, R4 and K-12 serotype.     

Yersinia enterocolitica O9 as a Possible Barrier Against Y. pestis in Natural Plague Foci in Ningxia, China

A survey of Yersinia spp, as related to plague control, was made in Haiyuan of Ganning loess plateau plague focus, Yanchi of Inner Mongolia plateau plague focus, and Yinchuan city, as a control area, in Ningxia, China. In Haiyuan, where the main plague reservoir was Mongolian ground squirrel (Citellus alaschanicus) living in the prairie, Y. enterocolitica O9 was frequently isolated from pigs, dogs, rodents living in and around houses, but only rarely from hare and Mongolian ground squirrel. In Yanchi, where the main plague reservoir was Mongolian gerbil (Meriones unguiculatus) living in the prairie and Y. pestis, which was isolated from rodents up to 1991, Y. enterocolitica O9 was sometimes isolated from pigs and rodents. In all areas, some strains of Y. enterocolitica O3 and Y. pseudotuberculosis serotypes 3 and 4b were also isolated from pigs, dogs, and from rodents. We propose that an epidemiological link exists between the prevalence of Y. pestis and Y. enterocolitica O9 in domestic and rodents living in these areas in China. The residential area in Haiyuan may be protected against Y. pestis by the domestic animals and rodents which acquired cross-protection against Y. pestis by infection with Y. enterocolitica O9, but this is not the case in the Yanchi district. Received: 14 February 2000 / Accepted: 17 July 2000     

Complete genome sequence of Yersinia enterocolitica subsp. palearctica serogroup O:3

We report here the first finished and annotated genome sequence of a representative of the most epidemiologically successful Yersinia group, Y. enterocolitica subsp. palearctica strain Y11, serotype O:3, biotype 4. This strain is a certified type strain of the German DSMZ collection (DSM no. 13030; Yersinia enterocolitica subsp. palearctica) that was isolated from the stool of a human patient (H. Neubauer, S. Aleksic, A. Hensel, E. J. Finke, and H. Meyer. Int. J. Med. Microbiol. 290:61-64, 2000).     

Structure of the O-chain of the phenol-phase soluble cellular lipopolysaccharide of Yersinia enterocolitica serotype O:9

The phenol-phase soluble cellular lipopolysaccharide isolated by the phenol/water extraction method from Yersinia enterocolitica serotype O:9 cells was shown by hydrolytic, periodate oxidation, methylation and nuclear magnetic resonance studies to be an S-type lipopolysaccharide with a linear O-antigenic polysaccharide of 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The serological cross-reactivity between Y. enterocolitica serotype O:9 and the lipopolysaccharides of Vibrio cholerae and Brucella species can now be related to the presence of N-acylated 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residues in their respective O-antigenic chains.     

The osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain's Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.     

Experimental pig yersiniosis to assess attenuation of Yersinia enterocolitica O:8 mutant strains

An experimental oral pig model was used to assess the pathogenic and immunogenic potential of Yersinia enterocolitica serotype O:8 wild-type strain 8081-L2 and its lipopolysaccharide (LPS) mutant derivatives: a spontaneous rough mutant 8081-R2, strain 8081-DeltawzzGB expressing O-antigen with uncontrolled chain lengths, and strain 8081-wbcEGB expressing semirough LPS with only one O-unit. Microbiological and immunological parameters of the infected pigs were followed from day 7 to 60 postinfection. The wild-type and all LPS mutant strains persisted in the lymphoid tissue of tonsils and small intestines, causing asymptomatic infection without any pathological changes. Although the pig is known as a reservoir of Yersiniae, a precise analysis of pathogenic and immunogenic parameters based on different in vitro tests (hematological response, killing ability of leukocytes and blood sera, antibody response, hydrogen peroxide production by macrophages, classical and alternative pathways of complement activation), revealed significant attenuation in the pathogenicity of the LPS mutant strains but not the loss of immunogenic potential. In comparison with the other strains, strain 8081-DeltawzzGB demonstrated more continuous leucocytosis with monocytosis, higher invasive potential, significant activation of hydrogen peroxide production by macrophages and an effective immunoglobulin G immune response accompanied by relevant histological immunomorphological rearrangements.     

Transmission of Yersinia enterocolitica 4/O:3 to pets via contaminated pork

AIMS: This study was conducted to investigate sources of Yersinia enterocolitica 4/O:3 infections in dogs and cats. METHODS AND RESULTS: Transmission of Y. enterocolitica 4/O:3 to pets via contaminated pork was studied using PFGE with NotI, ApaI and XhoI enzymes. A total of 132 isolates, of which 16 were from cat and dog faeces and 116 from raw pork samples, were recovered in Finland during 1998-99. Cat 1, whose diet consisted mostly of raw pig hearts and kidneys, excreted Y. enterocolitica 4/O:3 of genotype G4. This predominant genotype was also found in isolates recovered from the pig heart, liver, kidney, tongue and ear, and minced pork samples. Dog 2, which was fed raw minced pork, excreted Y. enterocolitica of genotype G13. This genotype was also identified in isolates recovered from the pig heart, kidney and tongue, and minced pork samples. CONCLUSION: These results show that raw pork can be an important source of Yersinia enterocolitica 4/O:3 infections in dogs and cats. Significance and Impact of the Study: Raw pork should not be given to pets.     

Structure of the O-specific polysaccharide of the Yersinia enterocolitica serovar O:4.32 lipopolysaccharide. Serologic relations of lipopolysaccharides of Y. enterocolitica O:4.32 and Y. intermedia O:4.33

O-Specific polysaccharide has been isolated on mild acid hydrolysis of the lipopolysaccharide from Yersinia enterocolitica O: 4.32 (strain 96) and shown to consist of yersiniose B (3,6-dideoxy-4-C-(1-hydroxyethyl)-D-xylo-hexose, YerB) acetylated at C1' and 2-acetamido-2-deoxy-D-galactose residues in a molar ratio 1:2. Acid hydrolysis, methylation and 13C NMR studies indicated the polysaccharide to be composed of trisaccharide repeating units of the following structure: (sequence; see text) The data obtained revealed structural and serological interrelation between O-antigens of Y. enterocolitica O:4.32 and Y. intermedia O:4.33.     

Studies on the interactions of immunostimulated macrophages and Yersinia enterocolitica O:8

Immunological and electron microscopy investigations of the phagocytic and killing activities of peritoneal macrophages from rats and mice against Yersinia enterocolitica serotype O:8 cells were performed. The effect of in vivo application of cytoplasmic membranes (CM) from the stable Escherichia coli WF+ L-form on macrophage activity was also studied. It was established that rat macrophages more actively phagocytosed the plasmidless pYV(-) Y. enterocolitica cells, compared to the plasmid-bearing pYV(+) Y. enterocolitica cells. The killing ability against both variants of the Y. enterocolitica strain was significantly enhanced in macrophages from CM-treated rats after 2 h, 4 h, and 24 h incubation. The CM treatment enhanced the phagocytic activity of the macrophages. The in vitro interaction of normal and immunostimulated rat macrophages with both pYV(+) and pYV(-) variants of Y. enterocolitica did not lead to any additional apoptotic and necrotic changes in macrophages compared to control macrophages, which were cultivated without Y. enterocolitica. Electron-microscopic investigation showed that mouse macrophages eliminated Y. enterocolitica pYV(+) cells in vivo after 24 h. No engulfed or digested bacterial cells were observed. Activation of cell surfaces and vacuolization of macrophage cytoplasm, both of CM-treated non-infected and infected mice, were observed. The experimental results showed that Y. enterocolitica pYV(+) cells could be eliminated by peritoneal macrophages.     

Yersinia enterocolitica arthritis in southern Sweden: a four-year follow-up study.

Thirty-eight cases of suspected yersinia arthritis occurring in southern Sweden in 1975-6 were reviewed four to five years later. In 31 cases the diagnosis was confirmed. At follow-up three of the patients had definite ankylosing spondylitis, three radiologically confirmed sacroiliitis, three extensor tenosynovitis, five isolated articular joint disease, and 10 localised arthralgias; one patient had developed seropositive rheumatoid arthritis. Only six of the 31 patients were free of joint symptoms. These results suggest that although the acute symptoms of yersinia arthritis disappear within 12 months, the long-term prognosis may be less favourable than previously thought.     

Structural identification of the lipopolysaccharide O-antigen produced by Yersinia enterocolitica serotype O:28.

The structure of the O-antigenic polysaccharide (O-PS) component of the lipopolysaccharide produced by Yersinia enterocolitica serotype O:28 has been elucidated. From chemical methods involving glycose analysis, periodate oxidation, methylation and the use of one- and two-dimensional NMR spectroscopy, the O-PS was found to be a polymer of repeating branched hexasaccharide units composed of L-rhamnose (four parts), 2-acetamido-2-deoxy-D-glucose (one part), and 2-acetamido-2-deoxy-D-galacturonic acid (one part) having the following structure: