[Mechanisms of formation of post-vaccinal immune response in children immunized with APDT and ADT-M preparations].

AIM: Study the mechanisms of formation of cell and humoral immunity against pertussis, diphtheria and tetanus in children immunized with immunobiological preparations (APDT vaccine and ADT anatoxin). MATERIALS AND METHODS: 30 practically healthy children (6 - 9 years of age) immunized with APDT and ADT-M preparations had TLR2, TLR4 expression determined in mononuclear cells (MNC). Vaccine preparations (APDT, ADT-M, AD-M, AT) and Corynebacterium diphtheriae gravis tox+, C. diphtheriae mitis tox- and Bordetella pertussis 345 were used as ligands. Cytokine production was determined in EIA. Content of anti-diphtheria, anti-tetanus and anti-pertussis antibodies--by PHA reaction and EIA. RESULTS: During stimulation with vaccines and B. pertussis 345 strain MNC were characterized by an increase (p < 0.05) of expression level of TLR2 and TLR4 and did not respond to stimulation with C. diphtheriae gravis tox+ and C. diphtheriae mitis tox- strains. Similar results were obtained during study of cytokine production (TNFalpha, IL-1, IL-6). A direct correlation between levels of antitoxic antibodies against diphtheria and tetanus (R = 0.486), antibacterial antibodies against pertussis and diphtheria was detected (R = 0.529). CONCLUSION: Analysis of cytokine production profile and determination of surface TLR expression can be used during evaluation of functional status of innate immunity cells and intensity of post-vaccinal immunity.

[The mode of spot test of plague and pseudotuberculosis agents mix cultures].

It is supposed to implement the polymerase chain reaction with mix of two pairs of "chromosome" primers "vlm12for'/"IS216rev" and "JS for"/JSrev" species-specific for Y. pestis and Y. psdtbc correspondingly in spot test of plague and pseudotuberculosis. The additional immunodiagnostics is applied to find defective and full-fledged on plague bacteria F1-antigen synthesis in the volume agglomeration reaction and paragglutination with diagnosticums of plasmid-dependent F1 antigen and chromosome FV determined antigen. This mode is characterized by more effectiveness and lesser labor intensiveness. It accelerates the detection of the mix of two agents in bacterial inoculations, organs bioassays and other materials. Also this mode facilitates the analysis of mix cultures of agents of plague and pseudotuberculosis, obtained in "mixed" nidi, the component identification, differentiation and isolation of pure growths of both Yersinia.

[New nutrient medium for the cultivation and isolation of the plague microbe ChDS-37 as an element of the mobilization reserve of specialized antiepidemic teams of the Russian Inspectorate for the Protection of Consumer Rights and Human Welfare].

A new nutrient medium has been designed to culture and isolate the plague microbe ChDS-37 on the basis of the pancreatic digest of baker's yeast. The results of laboratory tests of the designed medium, by using 10 plague microbe strains and those of approval during the tactical and special training of a specialized antiepidemic team (SAET), suggest that the medium has some advantage over reference media and creates prerequisites for being incorporated into the mobilization reserve of a SAET.

[Comparison of efficacy of tests for differentiation of typical and atypical strains of Yersinia pestis and Yersinia pseudotuberculosis].

AIM: To characterize species specificity of officially recommended tests for differentiation of Yersiniapestis and Yersinia pseudotuberculosis and propose additional tests allowing for more accurate identification. MATERIALS AND METHODS: Natural, laboratory and typical strains oftwo Yersinia species were studied using microbiological, molecular and biochemical methods. For PCR species-specific primers complementary to certain fragments of chromosomal DNA of each species as well as to several plasmid genes of Y. pestis were used. RESULTS: It was shown that such attributes of Y. pestis as form of colonies, fermentation ofrhamnose, melibiose and urea, susceptibility to diagnostic phages, nutritional requirements could be lost in pestis bacterial species or detected in pseudotuberculosis species. Such attribute as mobility as well as positive result of CoA-reaction on fraction V antigen are more reliable. CONCLUSION: Guaranteed differentiation of typical and changed according to differential tests strains is provided only by PCR-analysis with primers vlml2for/ISrev216 and JS respectively, which are homologous to certain chromosome fragments of one of two Yersinia species.

[Search for primers on the basis of Yersinia pestis chromosomal DNA for effective PCR identification of typical and atypical plague pathogen strains].

The authors present the results of a comparative appraisal of a set of species-specific primers in the polymerase chain reaction (PCR) identification of typical and atypical plague pathogen strains. A hundred and seventy-three strains with a species specificity of Y. pestis and 67 heterologous strains were used to appraise the well-known Y. pestis chromosomal DNA-based primers: "3a", "yp2769ms06", "vlml2for/ISrev216", "vlm33/ISfor1754", as well as "JS" specific to Y. pseudotuberculosis". In some plague bacterial strains, the DNA sequences complementary to the primers "3a" and "yp2769ms06" were not found. All conceivable plague pathogen strains were detectable only with the primers "vlml2for/ISrev216" and "vlm33/ISfor1754". The primers "JS" recognized only the strains of pseudotuberculosis causative agent. The DNA of other microorganisms failed to react with none species of primers. For the effective detection and differentiation of typical and atypical Y. pestis strains, the authors propose PCR using a few pairs of primers with the merits of the primers of the group "vlm" and 'JS".

[Approaches to species-specific typing of double mixed cultures comprising pseudotuberculosis bacteria and atypical plague bacillus strains].

The species relevance of atypical Yersinia strains was determined by various microbiological, immunological, and genetic (including polymerase chain reaction) tests. These strains were shown to represent mixed cultures of Y. pseudotuberculosis serovariant O1b and Y. pestis var antiqua. Identification-resistant cells with atypical properties and plasmid segregation were found in the populations of Y. pestis strains. Analysis of different diagnostic tests revealed the most reliable ones selected for the identification of atypical Y. pestis strains with unstable genome.

[Methods of diagnostics and differentiation of the plague agent: intraspecies differentiation of Yersinia pestis. Part II].

State Research Institute for Plague Control, Rostov-on-Don In the second part of the review aspects of intraspecies differentiation of the plague agent are discussed. Special emphasis is placed on the necessity of more precise definition of taxonomic position of plague agent isolates considering genotypical characteristics, data on their selective virulence, and evolutionary origin of the genus Yersinia.

[Pathogenicity of Corynebacterium diphtheriae circulating in Rostov-on-Don City and Rostov region during interepidemic period].

Main pathogenic characteristics (toxin production, tox-gene detection, adhesiveness) of 59 strains of C. diphtheriae circulating in Rostov-on-Don city and Rostov region in 2004-2005 were studied. Study of toxigenicity of 15 tox+ C.di phtheriae strains showed full coincidence of Elek immunoprecipitation test and polymerase chain reaction (PCR). Presence of part of A-fragment of tox-gene was detected in 5 (11.4%) of 44 C. diphtheriae strains that were negative in Elek test. Hemagglutinating activity of toxin producing strains was intermediate (40%) or high (60%). Among non-toxigenic strains those with intermediate adhesiveness were predominated (45,5%), the intermediate or high adhesiveness was detected in strains positive in PCR. Obtained characteristics of C. diphtheriae can be useful for surveillance for diphtheria infection during interepidemic period.

[Methods of diagnosis and differentiation of plague pathogen: approaches to detection of atypical strains of Yersinia pestis by molecular biology. Part I].

A review of data on the modern methods of detection of typical and atypical strains of the plague agent Y. pestis is given. The history of the development of the molecular-biological tests for the differentiation of Y. pestis from the related microorganisms is presented. The problems facing investigators during the development of these tests are discussed.

[Primary characterization of the properties and diagnostic value of the antigenic complex "fraction V" in a plague microbe].

The experimentally obtained antigenic complex isolated by extraction in the gradient surfactants from live and acetone-dried bacteria of the capsule-free vaccine strain EV76 of a plague microbe that had lost its ability to synthesize the diagnostic species-specific capsular antigen F1 was investigated. The antigenic complex fraction V (FV) was obtained after the fifth stage of extraction at a concentration of 1.28% of surfactants and after additional purification. The thermostable FV was found to consist mainly of protein. The protein having a molecular mass of about 43 kD predominates in the fraction. The latter is nontoxic for albino mice and antigenic. It forms a precipitate with commercial antiplague serum antibodies. FV antigenic sensitization of tanned sheep red blood cells gave rise to a diagnostic agent that specifically reacted with an antiplague serum rather than with heterologous sera against enterobacteria. The sera immunized with FV specifically reacted in the JDJFR with all the strains of the pathogen of plague irrespective of the temperature of their cultivation, including "fraction-free", which did not interact with a diagnosticum on F1. The animal sera immunized with capsule-free plague microbial strain reacted only with a FV-erythrocytic diagnosticum and they did not interact with F1 antigen-sensitized red blood cells. The erythrocytic FV diagnosticum was tested in ABNR with 130 typical and atypical plague microbial strains and with 133 strains of heterologous bacteria of different species of the family Enterobacteriaceae. The FV diagnosticum identified all the variants of a plague microbe, while the F1 diagnosticum revealed only its capsular variants. Among the heterologous bacteria, some strains of the closely related pathogen of pseudotuberculosis in those who were in the R form, rather than S form, positively reacted. The use of FV identified 2 groups of hybridomas obtained after immunization of albino mice with the capsule-free variant of a plague microbe. Some hybridomas reacted only with plague bacteria while others did with two above pathogens. The authors substantiate the expediency of using FV, its components, and obtained monoclonal plague pathogen antibodies to improve antiplague diagnosticums with an activity spectrum that exceeds that of the existing commercial F1 antigen-based diagnosticums. They also discuss the lines of further studies.