The role of the protein with molecular weight of 22 kD in the phagocytosis of vaccine strain of Yersinia pestis EV

The comparative study of different stages of the phagocytosis of vaccine strain Y. pestis EV and its achromogenic variants (AV), has been carried out with the use of peritoneal macrophages as an in vitro experimental model. As revealed in this study, AV whose outer membrane contains no protein with a molecular weight of 22 kD exhibit lower capacity for adherence and for being ingested by phagocytes than the initial strain. The absence of this protein does not inhibit the multiplication of AV inside phagocytes, leading to incomplete phagocytosis, which is characteristic of the initial strain. The suggestion is made that the 22 kD protein may be one of the adhesion factors, necessary for ensuring the initial stages of phagocytosis.     

Immunogenicity of Yersinia pestis grown on nutrient media at 28 and 37 degrees C

The immunogenicity of Y. pestis strain EV, grown in yeast-casein medium, yeast medium with Hottinger digest and yeast medium with sunflower-seed protein at 28 degrees C and 37 degrees C, for guinea pigs and white mice has been studied. As revealed in this study, these media ensure the formation of highly immunogenic populations of Y. pestis strain EV and, therefore, can be used for growing Y. pestis vaccine strains. Considerable fluctuations in the content of such highly protective antigen as fraction 1 do not affect the immunogenicity of live cultures of Y. pestis strain EV. This is due to the leveling of differences in the content of this antigen in the process of the multiplication of these bacteria in laboratory animals.     

Identification of the autoagglutination factor of Hms- cells of the plague agent

A search for cellular components responsible for autoagglutination (AA) in broth and salt solutions of Hms- cells of the plague agent Yersinia pestis was performed. The AA- mutants were obtained using vaccine strain Y. pestis EV76 derivative containing one species-specific plasmid pYP. The mutants were shown to differ from the parent strain by the decreased surface hydrophobicity, insensitivity to plague diagnostic L-413c bacteriophage and negative haemagglutination reaction with antibodies to F1 capsular substance of the plague agent. The mutants did not differ from the parent strain by electrophoretic mobility and immunochemical activity of LPS but were characterized by the absence of a 17 kDa protein on the cell surface. The AA+ cells that lost this protein after weak alkali extraction were less hydrophobic and failed to express AA in 0.5 M ammonium sulfate. After the extraction, the cells lost the ability to neutralize L-413c and to react with the anti-F1 antibodies, while both activities as well as 17 kDa protein were detected in the extracts. Thus, the 17 kDa protein is suggested to be a hydrophobic surface antigen which acts as a receptor of the L-413c bacteriophage and represents an AA factor of Hms- cells of Y. pestis.     

Lectin of Yersinia pestis vaccine strain EV and its immunological properties

As the result of the chromatographic separation of Y. pestis EV membrane proteins, a protein fraction with hemagglutinating activity was obtained. The isolated preparation was glycoprotein with a molecular weight of 22 kD, contained 16% of carbohydrates and exhibited thermolabile properties. The determination of the carbohydrate specificity of this glycoprotein revealed that it belonged to the class of lectins. Changes in the content of 11 corticosteroids and the population composition of lymphocytes, as well as the detection of specific antibodies in the blood serum of guinea pigs immunized with lectin, were indicative of the fact that the preparation was sufficiently immunogenic and induced the activation of the processes of proliferation and activation of lymphocytes during immunogenesis. The lectin isolated from Y. pestis EV outer membrane may be regarded as an additional factor ensuring the contact of the pathogen with the cells of the body and as a promising component of combined plague vaccine.     

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.     

The mutagenic effect of polymorphonuclear leukocytes on Yersinia pestis

As the result of in vitro experiments, Y. pestis auxotrophic mutants have been obtained under the influence of polymorphonuclear lymphocytes obtained from guinea pigs, previously immunized with Y. pestis strain EV. The mutagenic effect has been found to occur at minute 45 of phagocytosis. The control treatment of the bacteria with the lysate of neutrophils, homologous serum, penicillin (antibiotic-influenced selection) has not been found to lead to the appearance of auxotrophicity. These data suggest that the polymorphonuclear leukocytes of animals, having a set of powerful cytocidal systems, play an active role in the process of the natural variability of Y. pestis.     

Development of a diagnostic test for Yersinia pestis by the polymerase chain reaction

A 501 bp caf1 gene fragment and a 443 bp of pla gene fragment carried by 100 kb (pFra) and 10 kb (pPst) species-specific extrachromosomal replicons, respectively, were used as targets to study the conditions under which DNA amplification by polymerase chain reaction (PCR) may be applied to detect and identify Yersinia pestis DNA in cell lysates of pure cultures and biological samples. The sensitivity limit of PCR with the crude cell lysates of Y. pestis EV was estimated as 10–50 cfu in reaction mixture. When target Y. pestis EV cells were mixed with fresh blood of white mice, which contained 0.4% potassium citrate, the PCR detection level varied from 400 to 100 cfu ml^-1 of blood depending on the method used for preparing the sample. In our tests PCR was effective for the detection of yersinia in the blood of white laboratory mice experimentally infected with virulent Y. pestis KM638 strain. This method can be considered convenient for routine detection and identification of Y. pestis.     

Isolation, purification and physicochemical properties of pesticin I from cells of Yersinia pestis strain EV

Pesticin I has been isolated and purified from Y. pestis strain EV. The homogeneous preparation of Pesticin has been shown to be monomer protein with a molecular weight of 65000 daltons, having three immunologically identical alpha-, beta- and gamma-forms with different isoelectric points. The amino acid composition of Pesticin I is presented. Rabbit anti-serum to the beta-form of the preparation of Pesticin has been obtained.     

The genetic control of the F1-specific components of the capsular antigen in Yersinia pestis

The preparation of Y. pestis capsular antigen F1, isolated from Y. pestis Fra-positive strain by Baker's method, has been shown to have a composition and contain three components: protein, glycoprotein and a lipid-containing component. Each of them equally reacts with antibodies to F1 and diagnostic preparations based on these antibodies. The synthesis of protein and glycoprotein is temperature-dependent and controlled by pYT (Caf). The synthesis of glycoprotein is constitutive and determined by chromosomal genes. Protein and glycoprotein have almost identical mol. wt., 18 and 17 kD respectively, but their localization is different: protein is secreted on the surface of bacterial cells and into the environment, while glycoprotein can be found inside the cells and is similar to intracellular glycoprotein of different enterobacteria, described in our earlier works and exhibiting F1 specificity. Different biological role of these F1-specific components is suggested.     

Exposure of small rodents to plague during epizootics in black-tailed prairie dogs

Plague, caused by the bacterium Yersinia pestis, causes die-offs of colonies of prairie dogs (Cynomys ludovicianus). It has been argued that other small rodents are reservoirs for plague, spreading disease during epizootics and maintaining the pathogen in the absence of prairie dogs; yet there is little empirical support for distinct enzootic and epizootic cycles. Between 2004 and 2006, we collected blood from small rodents captured in colonies in northern Colorado before, during, and for up to 2 yr after prairie dog epizootics. We screened 1,603 blood samples for antibodies to Y. pestis, using passive hemagglutination and inhibition tests, and for a subset of samples we cultured blood for the bacterium itself. Of the four species of rodents that were common in colonies, the northern grasshopper mouse (Onychomys leucogaster) was the only species with consistent evidence of plague infection during epizootics, with 11.1-23.1% of mice seropositive for antibody to Y. pestis during these events. Seropositive grasshopper mice, thirteen-lined ground squirrels (Spermophilus tridecemlineatus), and deer mice (Peromyscus maniculatus) were captured the year following epizootics. The appearance of antibodies to Y. pestis in grasshopper mice coincided with periods of high prairie dog mortality; subsequently, antibody prevalence rates declined, with no seropositive individuals captured 2 yr after epizootics. We did not detect plague in any rodents off of colonies, or on colonies prior to epizootics, and found no evidence of persistent Y. pestis infection in blood cultures. Our results suggest that grasshopper mice could be involved in epizootic spread of Y. pestis, and possibly, serve as a short-term reservoir for plague, but provide no evidence that the grasshopper mouse or any small rodent acts as a long-term, enzootic host for Y. pestis in prairie dog colonies.     

The role of IS-elements of Yersinia pestis (Lehmann, Neumann) in the emergence of calcium-independent mutations

Calcium independent mutants of two Yersinia pestis strains were studied. Insertions of IS100 element at three different sites of plasmid pCad within calcium dependence region were detected in Y. pestis EV, as well as two extensive deletions covering the whole region. It was shown that IS100 carries no HindIII sites. Novel IS element of Y. pestis designated IS101 was discovered in strain 358, in addition to IS100. It is distinguished by a slightly smaller size, HindIII site presence and high specificity of integration.     

Evaluation of outcomes of combined specific prophylaxis with the antibiotic resistant immunogenic plague pathogen strain and emergency prophylaxis with aminoglycosides in the experimental plague model in white mice]

It was shown that aminoglycosides (streptomycin, kanamycin, gentamicin, sisomicin, tobramycin, amikacin) prevented manifestation of postvaccine immunity in albino mice immunized by vaccine strain Yersinia pestis EV. Avirulent strain Y. pestis 363 Monr with chromosome resistance to aminoglycosides of the 1st, 2nd and 3rd generations provided manifestation of antiplague immunity when streptomycin, kanamycin, gentamicin and amikacin were administered for prophylaxis. ED50 achieved 1.0-1.2 x 10(3) CFU and in control group (without treatment) 9.3 x 10(2) CFU. Gentamicin and amikacin were highly effective for experimental plague prophylaxis (90-100% animal survival), but inhibited development of postinfective immunity. Protective index (PI) value was 1.1 x 10(2). It was demonstrated that combination of specific prophylaxis (Y. pestis 363 Monr) and emergency prophylaxis with aminoglycosides in albino mice infected with approximately 1000 LD50 of virulent strain Y. pestis 358 (5 hours after infection) was highly effective and provided protective effect against subsequent infection with plague pathogen. Value of PI was 1.1 x 10(5) and practically did not differ from PI (1.7 x 10(5)) in control group (intact mice, immunized with strains EV [symbol: see text] 363 Monr).     

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28 degrees C and 37 degrees C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Several proteins with molecular weights ranging from 34 kDa to 71 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could also be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discussed.     

No evidence of persistent Yersina pestis infection at prairie dog colonies in north-central Montana

Sylvatic plague is a flea-borne zoonotic disease caused by the bacterium Yersinia pestis, which can cause extensive mortality among prairie dogs (Cynomys) in western North America. It is unclear whether the plague organism persists locally among resistant host species or elsewhere following epizootics. From June to August 2002 and 2003 we collected blood and flea samples from small mammals at prairie dog colonies with a history of plague, at prairie dog colonies with no history of plague, and from off-colony sites where plague history was unknown. Blood was screened for antibody to Y. pestis by means of enzyme-linked immunosorbent assay or passive hemagglutination assay and fleas were screened for Y. pestis DNA by polymerase chain reaction. All material was negative for Y. pestis including 156 blood samples and 553 fleas from colonies with a known history of plague. This and other studies provide evidence that Y. pestis may not persist at prairie dog colonies following an epizootic.     

Yersinia pestis lacZ expresses a beta-galactosidase with low enzymatic activity

Although very little, if any, beta-galactosidase activity is detected in Yersinia pestis by a standard Miller assay, we found that Y. pestis KIM6+ cells formed blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). Searches of the Y. pestis genome databases revealed the presence of noncontiguous sequences highly homologous to Escherichia coli lacZ, lacY, and lacI. Yersinia pestis lacZ is predicted to encode a 1060 amino-acid protein with 62% identity and 72% similarity to beta-galactosidase from E. coli. A deletion in the Y. pestis lacZ gene caused the formation of white colonies on X-gal-containing plates and beta-galactosidase activity was at background levels in the KIM6+lacZ mutant, while the complemented strain expressed about 190 Miller units. The Y. pestis lacZ promoter was not regulated by isopropylthiogalactoside or glucose. Finally, uptake of lactose by Y. pestis may be impaired.     

Changes in the functional activity of neutrophils during their interaction with Yersinia pestis in vitro

The functional state and electrochemical properties of human blood neutrophil leukocytes after their in vitro interaction with Y. pestis cells, strain EV, was analyzed. A considerable decrease in the electrophoretic mobility of neutrophil leukocytes and a considerable increase in their phagocytic indices was shown. At the same time the maximum phagocytic activity in the total pool of isolated neutrophil leukocytes was registered in fractions having higher electrophoretic mobility. The dependence of electrophoretic mobility on the state of neutrophil membranes, as well as the degree of their activation, is discussed.     

Clonal structure of Yersinia pestis populations in experimental soil ecosystems

Two basic tendencies--formation of latent (uncultivable) form (LF) and hemin storage variability--has been revealed during study of clonal structure dynamics of Y. pestis populations in artificial soil ecosystems in long-term incubation conditions. Y. pestis populations disappeared within 3 - 6 months at 18 - 22 degrees C, whereas at 4 - 8 degrees C a subsequent replacement of vegetative cells on LF, which are capable to prolonged survival (up to 22 months) in soil with ability to reversion in the presence of abundance of nutrients, has been observed. Bacteria of virulent strain retained all determinants of pathogenicity when reverted to LF, whereas bacteria of avirulent strain (defective on plasmid of Ca-dependence), on the contrary, undergo further degradation that resulted in loss of a pgm locus and gradual disappearance of population. LF revertants of highly virulent strain restored properties of initial population and were highly virulent.     

Immunochemical study of heterogenous antigens Y. pestis EV similar to human red cells]

Franctions containing heterogenous antigens Y. pestis EV similar to human red cells can be obtained by the method of immunosorption of antigens by fixed antibodies on polyacrylamide gel.     

Evaluation of vaccinal immunogenesis by the peroxidase method

The possibility of evaluating functional immunomorphogenesis in the course of the vaccinal process after the injection of conjugated and live brucellosis vaccines, as well as conjugated plague antigen and Yersinia pestis strain EV, to guinea pigs has been shown by means of the direct and two-layer variants of the immunoperoxidase assay. The dynamics of the accumulation of globulin-producing cells in the immunocompetent organs and the time course of immunoglobulin titers in the peripheral blood after the injection of live and conjugated vaccines have been followed up. These data may be used for the morphological evaluation of approved preparations.     

The colonization of plants by Yersinia pestis EV in an experiment

The penetration of Y. pestis (strain EV) into the stem of Impatiens walleriana via its roots, submerged into microbial suspension, was observed under experimental conditions. This was indicative of the colonization of plants by Y. pestis, thus confirming the possibility of their preservation in plants during periods between epizootics at the territories of natural foci.