[CONSTRUCTION OF A TEST-SYSTEM WITH NANOPARTICLES OF COLLOID SILVER FOR DETECTION OF PSEUDOTUBERCULOSIS AND INTESTINAL YERSINIOSIS FOR CAUSATIVE AGENTS IN DOT-IMMUNOASSAY].

AIM: Construction of an immunologic test-system for detection of causative agents of enteropathogenic Yersinia (Yersinia pseudotuberculosis and Y. enterocolitica) by dot-immunoassay. MATERIALS AND METHODS: Nenoparticles of colloid silver sized 5-9 nm were used as a marker of specific antibodies. IgG fraction was isolated from commercial antisera to Y. pseudotuberculosis (Ο:1) and Y. enterocolitica (Ο:3 and Ο:9). Testing of the obtained test-system was carried out on 20 strains of Y.pseudotuberculosis and Y. enterocolitica (10 of each species). RESULTS: Dot-analysis had a specific character and detected enteropathogenic Yersinia at a level of 5-105 - 8.106 CFU/ml (100 - 1000 CFU in sample). Wherein cross-reaction with heterologic studied microorganisms - Escherichia coli, Salmonella typhimurium, Shigella flexneri, Yersina pestis EV - as not observed. A possibility of simultaneous detection and serotyping of Y. enterocolitica is shown, that is necessary for confirmation of their epidemic significance. CONCLUSION: The developed test-systems allow to study micro volumes of the samples under study (1 µ1), are express (1.5 - 2h), highly sensitive and specific, technically simple and do not require the use of high-cost equipment, special training of the staff, may be successfully used in practical healthcar in laboratories with varying equiptment levels.

[Molecular-biological characteristic of Yersinia enterocolitica circulating in various regions of Russian Federation].

AIM: Complex characteristic by phenotype signs and main virulence genes of Yersinia enterocolitica strains circulating in various regions of Russian Federation. MATERIALS AND METHODS: 46 strains of Y. enterocolitica of 2 - 4 biotypes and 401 strains of Y. enterocolitica IA biotype isolated in 15 administrative territories of Russian Federation (Siberian, Far Eastern, Northwestern, Urals Federal Districts) from infected people, rodents, agricultural animals, birds, the environment were studied. Phagotyping was performed in the reference laboratory of the Pasteur Institute (Paris). All the Y. enterocolitica cultures were studied for the presence of ail, ystB and ystA genes by PCR method. Presence of virulence plasmid pYVwas determined by gel electrophoresis by T. Kieser method. RESULTS: 447 strains of Y. enterocolitica biotype 1A and 2 - 4 were studied. Most of the strains belonged to serotypes O:3; O:9; O: 5; O: 6,30; O:6,31; O:7,8. Phagotyping was performed for part of the strains. Phagotypes Xz and Xo were determined in biotype 1A strains. 2 - 4 biotype strains circulating in Siberia and the Far East were characterized by phagotype VIII, X3 that are present in other countries, and phagotype Xz that is spread only in Russia. Phagotypes IXa, IXb, II that are characteristic for strains from Canada, South Africa, Japan were not detected in Russian Federation. All the strains of 2 - 4 biotypes had ail and ystA genes. Most of the recently isolated strains had pYV. The only pathogenicity factor detected in 81.3% of biotype 1A strains including 14 strains from patients was ystB gene. These infections were accompanied by an expressed clinical symptomatology of enteritis and enterocolitis. CONCLUSION: Isolation of 1A biotype strains from patients necessitates execution of diagnostic studies of intestinal yersiniosis in patients with diagnosis "acute intestinal infection of undetermined etiology".

[Molecular genetic monitoring of Yersinia pseudotuberculosis using PCR O-genotyping].

Irkutsk PCR O-genotyping of 117 Yersinia pseudotuberculosis serotype O:1-O:4 strains isolated in Siberia and Far East was performed. These methods allowed both O-genotype and its variants (a, b, c) to be detected. It was demonstrated that three Y. pseudotuberculosis O-genotypes were circulated in Siberia (O:1a, O:1b and 0:3) and six genotypes (O:1a, O:1b, O:1c, O:2a, O:3 and O:4b) were circulated at Far East. Genotype O:1b dominates at both regions (87.8%). PCR-algorithm for identification of Y. pseudotuberculosis O-genotypes having epidemic significance was developed. The identification of the etiological agent O-genotype without bacteriological isolation of the stimulus is possible by PCR analysis of the clinical material.

[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.

[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.

[The population variability of Yersinia pestis in soil samples from the natural focus of plague].

Three Y. pestis strains were found to exist in the experimental soil ecosystem at a temperature of 4 degrees - 8 degrees C for a longer period (10 months, the term of observation) than at room temperature (3.5 months). Y. pestis population structure was characterized by relative stability in strains of the subspecies altaica and heterogeneity in the strain of the main subspecies, manifested by the loss of the pgm locus by vegetative cells and the preservation of pgm+ variants in the latent (uncultivable) form (LF). In the populations of all strains uniformity in calcium dependence, the tendency towards a decrease in the synthesis of factor 1, nutritional requirements in amino acids was observed. An important factor of the preservation of Y. pestis in the soil was LF formation. At room temperature this process quickly resulted in the death of the population. At 4 degrees - 8 degrees C A. pestis altaica avirulent strain could be inoculated onto solid nutrient media for a two-fold longer period (for 4 month) than the strain with selective virulence and for 5.5 months longer than Y. pestis pestis highly virulent strain.

[Ecological regularities of the existence of pathogenic Yersinia in soil ecosystems].

In this review the data on the ecology of pathogenic Yersinia in soil ecosystems, based on prolonged observations, were analyzed and summarized. In contrast to saprophytic species, ubiquitously spread in nature, pathogenic representatives of the genus Yersinia occurred only in the soil of natural foci and of these, Y. pestis were found only in the soil of burrows of the main carriers. The complex of abiotic and biotic factors (temperature, humidity, chemical composition, interactions in biocenosis) which determined the possibility of the existence of Yersinia in the soil environment and the preservation of their pathogenic properties was considered. Special attention was paid to their geno-phenotypic variability as the main factor of the adaptation of the causative agents of plague, pseudotuberculosis and intestinal yersiniosis in the environment.

[Evaluation of the polymerase chain reaction effectiveness in the investigation of out-breaks of pseudotuberculosis]. / Otsenka éffektivnosti polimeraznoi tsepnoi reaktsii pri rassledovanii vspyshek psevdotuberkuleza.

Data on the investigation of pseudotuberculosis epidemic outbreaks with the use of the polymerase chain reaction (PCR), are presented. Specific fragments of Yersinia pseudotuberculosis DNA were detected in 81.25% of patients, in 46.83% of cases confirmed by the isolation of Y. pseudotuberculosis cultures. The study of washings from vegetables and equipment in vegetable stores and kitchens yielded positive results in PCR in 8.52% and the survey of rodents--in 3.85% of cases. In the course of the bacteriological study of these specimens 6 Y. pseudotuberculosis cultures were isolated: 3--from vegetable products, 1--from a Norway rat and 2--from house mice. The coincidence of the data obtained by bacteriological study and PCR showed that the latter method gave objective results, while being capable of ensuring rather rapid analysis. PCR should be regarded as a signal test for the bacteriological search of the definite infective agent in the material under study.

[Impact of Yersinia pseudotuberculosis on the in vitro production of cytokines by whole blood cells of donors]. / Vliianie Yersinia pseudotuberculosis na produktsiiu tsitokinov kletkami tsel'noi krovi donorov in vitro.

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.

[Mode of sample preparation for pseudotuberculosis laboratory diagnosis by polymer chain reaction method]. / Sposob podgotovki prob dlia laboratornoi diagnostiki psevdotuberkuleza metodom polimeraznoi tsepnoi reaktsii.

A mode of feces sample preparation was developed for polymerase chain reaction (PCR) assay. It was based on alkaline treatment of the material. This treatment killed the most part of indigenous microflora, whereas Yersinia survived, because it was relatively resistant to alkaline. The mode was tested using human feces artificially contaminated with Yersinia pseudotuberculosis. Positive responses in samples containing 10(3)-10(8) microbial cells per ml were obtained by PCR assay with Yersi and Yers2, Invl and Inv2, YP3 and YP4 primers. Diagnostic efficiency of PCR for patients, small mammals, and washings from environmental objects was 4.75, 1.66, and 2.12 times higher than diagnostic efficiency of bacteriological analysis of these samples, respectively. Positive results in PCR were obtained at the day of the material collection and treatment, whereas Y. pseudotuberculosis was isolated only after 8-20 days. Positive samples in PCR and in bacteriological analysis were found to coincide. A brief scheme of the Y. pseudotuberculosis laboratory diagnosis is suggested. According to this scheme, target-oriented bacteriological assay is performed only in those samples, in which preliminary PCR assay after 1-3 days of incubation gave positive results of Y. pseudotuberculosis DNA detection.

[THe characteristics of the epidemic process of pseudotuberculosis infection in the southern and northwestern foci of Irkutsk Province]. / Osobennosti épidemicheskogo protsessa psevdotuberkuleznoi infektsii v iuzhnom i severo-zapadnom ochagakh Irkutskoi oblasti.

A long-term study of pseudotuberculosis epidemiology has revealed two independent foci in the south and northwest of Irkutsk Province, which differ in the level and pattern of morbidity, seasonal features, patients' age distribution, severity of a clinical course. Each focus is characterized by the clonal structure of the pseudotuberculosis bacillus, which is persistent in terms of the plasmid profile. Strains with the two plasmids 47 mD (pYV) and 82 mD (pVM) circulate in the southern focus, while those with one plasmid 47 mD (pYV) in the northwestern focus. The intensity of an epidemic process is not associated with the presence of the plasmid 82 mD (pVM). The presence of the latter statistically significantly determines the severity of the clinical course of pseudotuberculosis infection, which seems to be caused by the immunodepressive action of the plasmid pVM.

[The seasonal dynamics of blocking in the flea Citellophorus tesquorum altaicus from the Tuva natural plague focus]. / Sezonnaia dinamika blokoobrazovaniia u blokhi Citellophorus tesquorum altaicus iz Tuvinskogo prirodnogo ochaga chumy.

Seasonal changes in the frequency of block formation have been established in C. tesquorum altaicus from the Tuva natural plague [correction of plaque] focus. Distinctions have been observed in the blocking of fleas born in different calendar years. "Young" fleas born the current year block more frequently than old ones born the previous year.

[Monospecific agglutinating sera for the identification of Yersinia strains]. / Monospetsificheskie aggliutiniruiushchie syvorotki dlia identifikatsii shtammov iersinii.

An original scheme of rabbit immunization with Yersinia O-antigen is suggested. Technology for preparation of monospecific Yersinia sera to be used in drop agglutination test on the glass has been developed. Field and laboratory trials evidence that the prepared sera are fit for serologic identification of Y. enterocolitica.

[Paradoxopsyllus scorodumovi (Aphaniptera) flea, an effective vector of plague in a Gorno-Altai natural focus]. / Blokha Paradoxopsyllus scorodumovi (Aphaniptera)--éffektivnyi perenoschik chumy v Gorno-Altaiskom prirodnom ochage.

A study was conducted on the fleas of P. scorodumovi and five local strains of the plague microbe, one of which is typical of the strains of the Altai subspecies and four are non-typical of this nidus. The fleas of this species are capable to transmit not only the plague agent of the strains typical of this nidus but also non-typical ones which differ in some biological properties and are avirulent for most carriers but Pallas's pika. Biological peculiarities of fleas of P. scorodumovi in addition to their high efficiency as vectors of the plague microbe enable us to associate the more active autumn epizooty with fleas of this species.

[Epizootiological importance of Frontopsylla hetera (Siphonaptera) fleas in the Gorno-Altai natural plague focus]. / Epizootologicheskoe znachenie blokh Frontopsylla hetera v Gorno-Altaiskom prirodnom ochage chumy (Siphonaptera).

Experiments conducted during all seasons have established that F. hetera, one of the mass species of fleas in Mountain Altai, can be infected both by the strain of selective virulence typical to this nidus and by the non-typical non-virulent mountain-altai strain of plague agent. The non-virulent strain does not form in fleas the block of proventriculus and within 1.5-2 months they become free from the microbe. At the infection with the typical strain of the altai subspecies rare transmissions of the agent to Pallas' pika can take place as well as its long preservation in fleas.

[Flea Ceratophyllus fasciatus as the vector of the Altai-mountain strain of plague microbe]. / Blokha Ceratophyllus fasciatus kak perenosnik gornoaltaiskogo shtamma chumnogo mikroba.

The work was conducted with a typical strain of the plague agent, which is virulent to white mice and little virulent to guinea pigs (subcutaneous infection), and with C. fasciatus. The fleas of this species can be infected, form the block of proventriculus within 4 to 35 days, transmit the agent during bloodsucking to healthy animals and cause the death both white mice and guinea pigs.