Ribotyping and virulence markers of Yersinia pseudotuberculosis strains isolated from animals in Brazil

Ribotyping and virulence markers has been used to investigate 68 Yersinia pseudotuberculosis strains of serogroups O:1a and O:3. The strains were isolated from clinical material obtained from healthy and sick animals in the Southern region of Brazil. Ribotypes were identified by double digestion of extracted DNA with the restriction endonucleases SmaI and PstI, separation by electrophoresis and hybridization with a digoxigenin-labeled cDNA probe. The presence of the chromosomal virulence marker genes inv, irp1, irp2, psn, ybtE, ybtP-ybtQ, and ybtX-ybtS, of the IS100 insertion sequence, and of the plasmid gene lcrF was detected by polymerase chain reaction. The strains were grouped into four distinct ribotypes, all of them comprising several strains. Ribotypes 1 and 4 presented distinct profiles, with 57.3% genetic similarity, ribotypes 2 and 3 presented 52.5% genetic similarity, and genetic similarity was 45% between these two groups (1/4 and 2/3). All strains possessed the inv, irp1, and irp2 genes. Additionally, strains of serogroup O:1a carried psn, ybtE, ybtP-ybtQ, ybtX-ybtS, and IS100. As expected lcrF was only detected in strains harboring the virulence plasmid. These data demonstrate the presence of Y. pseudotuberculosis strains harboring genotypic virulence markers in the livestock from Southern Brazil and that the dissemination of these bacteria may occur between herds.     

Experimental kinetics of infection induced by Yersinia pseudotuberculosis isolated from stock animals

The course of in vivo infection of five isolates of Yersinia pseudotuberculosis was followed for three weeks in Swiss mice. The strains were isolated from diarrheic and normal feces and mesenteric lymph nodes of healthy and sick stock animals. Four strains of serogroup O:3 and one of serogroup O:1a, with and without the virulence plasmid, were inoculated intragastrically and intravenously in the mice. Groups of five animals were sacrificed at 6 h and 3, 6, 10, 15, and 21 days after inoculation, and organs and tissues were checked for possible macroscopic alterations. Development of infection was monitored at these times by performing viable bacterial counts in homogenates of selected tissues. The animals were checked daily for clinical alterations. The results of the study showed that strains with the virulence plasmid infected organs and tissues at various times and at varying intensity by both routes of infection, the strain of type O:1a being the most invasive. Moreover, clinical and pathological alterations occurred only in animals inoculated with bacteria carrying the virulence plasmid, regardless of the route of infection.     

Characteristics of Yersinia spp. isolated from wild and zoo animals

Thirteen strains of Yersinia spp. were isolated at the Rome zoo and at Castelporziano, a game preserve near Rome. The strains were tested for calcium dependency, autoagglutination, heat-stable toxin production, 50% minimum lethal dose in mice (LD50), pyrazinamidase activity and content of plasmids by electrophoresis in agarose gel. The former three tests were negative for all strains, the LD50 was always greater than or equal to 1 X 10(7.6) CFU/ml and pyrazinamidase activity was positive for all strains. Electrophoresis revealed the presence of two plasmids of 27 and 66 megadaltons (MDa) in the two strains of Y. enterocolitica of serotype 027 isolated from animals in the zoo. The two strains of the same species and serotype, isolated from wild animals harboured a 42-MDa plasmid. A small plasmid of 2 MDa was found in two strains of Y. enterocolitica of serotype 07.8 from two subsequent samples of a zoo animal.     

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.     

Virulence of Yersinia pseudotuberculosis isolated from pork and from the throats of swine

Yersinia pseudotuberculosis was isolated from retail pork and from healthy swine throats. These wild-type strains and their representative cured isogenic strains were tested for the presence of plasmids and several virulence factors, and these characteristics were compared with those of virulent strains from humans. Two pork isolates (serotype IVB) and four swine isolates (serotypes IIB, IIC, III, and IVB) harbored a 42- to 48-megadalton plasmid which had similar fragmentation patterns resulting from digestion with restriction endonuclease. These six strains were lethal for mice via oral challenge and were positive in autoagglutination and calcium dependency tests. They also invaded HeLa cells and induced cytotoxicity. Histopathological examination and indirect fluorescent-antibody staining provided definite evidence of the pathogenicity of these strains when tissue sections from orally infected mice were used. The virulence factors of wild-type pork and swine isolates with the 42- to 48-megadalton plasmid were identical to those of two human isolates (serotypes IVB and VB). Hence, these pork and swine isolates should be considered potentially pathogenic for humans. The finding suggests that retail pork and swine may play an important role in the epidemiology of human infections caused by Y. pseudotuberculosis.     

Genotyping of Yersinia pseudotuberculosis isolated in Siberia and Far East

Genotypic characteristics based three main factors of pathogenicity (presence of resident plasmids [pYV, pVM], gene of toxin-superantigen ypm and nine genes for high pathogenicity island [HPI]) of 212 strains of Y. pseudotuberculosis isolated in Siberia and Far East were studied. It was shown that strains of Y. pseudotuberculosis with one of two variants of plasmids 82:47 MDa and 47 MDa (60.8% and 31.6% respectively) are predominated. Gene ypmA was detected in 96.2% of isolated strains. Eight strains had none of the ymp gene variants. HPI were detected in 96.2% of isolated strains. Obtained characteristics of Y. pseudotuberculosis allowed to determine the dominating genogroup pWYV+, ypmA+, HPI- (95.8% of strains) that cause systemic infection.     

Virulence of Yersinia pseudotuberculosis isolated from pork and from the throats of swine.

Yersinia pseudotuberculosis was isolated from retail pork and from healthy swine throats. These wild-type strains and their representative cured isogenic strains were tested for the presence of plasmids and several virulence factors, and these characteristics were compared with those of virulent strains from humans. Two pork isolates (serotype IVB) and four swine isolates (serotypes IIB, IIC, III, and IVB) harbored a 42- to 48-megadalton plasmid which had similar fragmentation patterns resulting from digestion with restriction endonuclease. These six strains were lethal for mice via oral challenge and were positive in autoagglutination and calcium dependency tests. They also invaded HeLa cells and induced cytotoxicity. Histopathological examination and indirect fluorescent-antibody staining provided definite evidence of the pathogenicity of these strains when tissue sections from orally infected mice were used. The virulence factors of wild-type pork and swine isolates with the 42- to 48-megadalton plasmid were identical to those of two human isolates (serotypes IVB and VB). Hence, these pork and swine isolates should be considered potentially pathogenic for humans. The finding suggests that retail pork and swine may play an important role in the epidemiology of human infections caused by Y. pseudotuberculosis.     

Structure of O-specific polysaccharide isolated from the Yersinia pseudotuberculosis serotype 1A lipopolysaccharide

An O-specific polysaccharide from the lipopolysaccharide Yersinia pseudotuberculosis 1A serovar has been isolated and characterized. This compound was shown to contain residues of paratose, 6-deoxy-D-manno-heptose, D-galactose and 2-amino-2-deoxy-D-glucose in equimolar ratios. Using methylation studies, partial acid hydrolysis and 13C NMR spectroscopy, the following structure was proposed for the repeating unit of the O-specific polysaccharide: (Formula: see text).     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.     

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.     

Antitumor activity of L-asparaginase from Yersinia pseudotuberculosis

The cytotoxic activity of L-asparaginases from Yersinia pseudotuberculosis and from Erwinia carotovora were investigated in vitro using human T-lymphoblastic leukemia (Jurkat and Molt-4) and also solid tumor cell lines MCF-7 (human breast adenocarcinoma), LnCap (human prostate carcinoma), NGUK1 (rat Gasser node neurinoma). E.coli L-asparaginase produced by Medak (Germany) was used as a reference preparation. The data obtained indicate that Y. pseudotuberculosis L-asparaginase significantly inhibits growth of leukemic and solid tumor cells. Its antitumor activity is comparable to that of the reference preparation of L-asparaginase (Medak). These results suggest that the recombinant L-asparaginase can be used for the development of new preparations for the therapy of different types of tumors.     

Yersinia pseudotuberculosis nucleoside-kinase

Enzyme capable of catalyzing the phosphorylation of thymidine and uridine was isolated from Y. pseudotuberculosis cells by fractionation with the use of ammonium sulfate, ion exchange and affinity chromatography. The degree of purification of thymidine- and uridine-kinase was approximately 350 times, and at all stages of isolation the activity of both nucleoside-kinases was detected in the same peaks. The purified enzyme was capable of the phosphorylation of thymidine and uridine at temperatures of 8-10 degrees C to 50 degrees C and exhibited the maximum enzymatic activity at pH 8-8.5 and 45 degrees C in the presence of 0.5-1.0 mM MgCl2 and 2 mM ATP. The enzyme was found to have no strict substrate specificity and transferred the phosphate group from ATP to radiolabeled thymidine, uridine and desoxycytidine with different effectiveness, but did not use thymidine-monophosphate as phosphate acceptor.     

Yersinia pseudotuberculosis toxins

The review of publications about protein toxins Y. pseudotuberculosis are presented. It includes the main data obtained by domestic and foreign investigators as well as the results of our own elaboration in the study of Y. pseudotuberculosis protein toxins. The guestions of isolation, purification, characterization of physico-chemical and biological properties, the mechanism action and role of toxins on pathogenesis of infection were discussed.     

Plasmids of Yersinia pseudotuberculosis

Plasmids with the sizes of 5.7; 51; 70-77; and 120-130 kb were found in six strains among the ten strains collection of Yersinia pseudotuberculosis. The restriction endonucleases analysis. Southern-blot hybridization and physical maps construction were performed for the plasmids. The 70-77 kb plasmids were found to be analogous to the Ca2(+)-dependence plasmid pYVO19 from Yersinia pestis EV76. The difference between the plasmids of this type is in the insertions or deletions located on the similar fragments of the restriction maps. The 51 kb plasmid has no common fragments with the Ca2(+)-dependence plasmids and does not code for virulence properties of the strain harbouring it. No homology is shared by the 5.7 kb plasmid and the 10 kb plasmid from Yersinia pestis EV76. Replicon of the 5.7 kb plasmid has been used to construct the pVS11 vector plasmid.     

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.     

A new R-plasmid from Yersinia pseudotuberculosis]

Yersinia pseudotuberculosis strain 140-P isolated from soil in the Far East was found to harbour an R-plasmid different from the plasmids that had been isolated from the bacteria previously. A new R-plasmid pLD140 is conjugation proficient and codes for the cellular resistance to streptomycin, tetracycline and sulfonamides. The plasid belongs to incompatibility group IncP. Its restriction endonucleases BamHI and SalI profile is different from the ones of the plasmids belonging to the RP4 family.