[SUBSPECIES DIFFERENTIATION OF YERSINIA PESTIS STRAINS BY PCR WITH HYBRIDIZATION-FLUORESCENT DETECTION].

AIM: Develop a method of differentiation of Y.pestis strains of different subspecies based on PCR with hybridization-fluorescent detection in real-time. MATERIALS AND METHODS: DNA target search for differentiation of subspecies of plague causative agent was carried out by Mauve 2.3.1, Mega 5.0 and BLAST algorithm based on comparison of full-genome sequenc- es of Y.pestis strains. Primers and TAqMan probes were calculated for the DNA targets found, conditions of PCR with hybridization-fluorescent detection - optimized. RESULTS: DNA targets carrying marker mutations for the caucasus, altai, gissar, ulegei subspecies, strains from Talass alpine plague reservoir were detected. The effectiveness of the DNA targets found and the developed approach of subspecies differentiation is confirmed on 101 Ypestis strains of different subspecies, isolated from natural foci of Russia, near and far abroad. CONCLUSION: The developed approach based on PCR with real-time detection allows for a rapid and effective differentiation of Ypestis strains of various subspecies.

[IMPACT OF CASPIAN SEA LEVEL FLUCTUATIONS ON THE EPIZOOTIC ACTIVITY OF THE CASPIAN SANDY NATURAL PLAGUE FOCUS].

There is evidence that in 1923-2014 the sharp aggravations of the epizootic situation of plague in the area of its Caspian sandy natural focus after long interepizootic periods are in time with the ups of the Caspian Sea in the extrema of 11-year solar cycles. There were cases of multiple manifestations of plague in the same areas in the epizootic cycles of 1946-1954, 1979-1996, 2001, and 2013-2014. The paper considers the possible role of amebae of the genus Acanthamoeba and nematodes, the representatives of the orders Rhabditida and Tylenchida in the microfocal pattern of plague manifestations.

[Analysis of the Nucleotide Sequence of a Cryptic Plasmid from Yersinia pestis Strains in the Central Caucasian High-Mountain Plague Focus].

An analysis of a 5.4-kbp cryptic plasmid detected in the course of whole-genome sequencing of the Yersinia pestis medieval biovar isolated in the Russian Central Caucasian high-mountain plague focus was performed. The identification of the nucleotide sequence of this cryptic plasmid and its structural and functional analysis revealed that it contained eight open reading frames, among which the following genes were identified: the rep gene of a replication protein, the virB6 gene of a type-IV secretion system inner membrane protein, the virB5gene of the type-IV secretion system minor pilin, and a number of genes probably associated with secretion and transport. A general analysis of the pCKF plasmid DNA showed that the adenine content was 28.34%, the cytosine content was 20.5%, the guanine content was 17.87%, and that of thymine was 33.28%, while the total G+C content appeared to be 38.38%. The G+C content of the chromosome of the Y pestis strain C-627 is 47.6%, which indicates that the pCKF plasmid was obtained from a microorganism equally-phylogenetically distant from the Yersinia bacteria andany other bacteria from the Enterobacteriaceae family. A comparison of the amino acid sequences.of hypothetical proteins encoded by pCKF plasmid with analogous proteins encoded by other bacteria was carried out. The possible contribution of the pCKF plasmid to the maintenance of the most ancient known phylogenetic line of Y. pestis medieval biovar, 2.MEDO, was discussed.

[Analysis of diversity and identification of the genovariants of plague agent strains from Mongolian foci].

The genetic diversity of Yersinia pestis strains from the Mongolian natural plague foci has been investigated. A total of 32 strains isolated from western, eastern, and central aimaks, as well as from the territory of the Gobi region, have been studied. Twenty-four strains belong to the main Y. pestis subspecies, while eight belong to other subspecies. There is only one strain of biovar medievalis (genovariant 2.MED1) among the strains of the main subspecies, while the rest of the subspecies belong to the biovar antiqua. Biovar antiqua strains are split into three groups. Strains from the eastern part of the country were classified as genovariant 2.ANT3, and those from the western and central regions were classified as genovariant 3.ANT2, which was endemic for Mongolia. One strain from the Bayan-Ulegeiskii aimak had the rare genovariant 4.ANT. None of the strains of the biovar antiqua belonged to its ancient 0.ANT branch, which is inconsistent with the commonly accepted idea that ancient marmot's plague agent race originates from Mongolia. Six out of eight strains of the minor subspecies belonged to the ulegeica subspecies, which are endemic to Mongolia, one strain belonged to the microtus group, and the last belonged to a previously uncharacterized variant of the minor subspecies.

[A study on the taxonomy of soil amoebas from Caspian plague foci based on an analysis of ribosomal operon sequences].

The results of a study on the taxonomy and quantitative abundance of free-living amoebas in soil samples from the Russian plague foci of the northwestern Caspian steppe, the Caspian sand, and the Volga-Ural steppe are presented. Amoebas of the Willaertia and Hartmanella genera, as well as representatives of myxomycetes, were isolated from samples. From these, amoebas of the Acanthamoeba genus predominated and could be as abundantas 300000 cells per 1 g of soil. Sequencing of the 18S rRNA gene region showed that Acanthamoeba from the Volga-Ural steppe focus belonged to the A. castellanii species. Phylogenetic analysis confirmed that amoebas from two other Caspian foci belonged to the species of Acanthamoeba spp.

Phylogenetic analysis of entomoparasitic nematodes, potential control agents of flea populations in natural foci of plague.

Entomoparasitic nematodes are natural control agents for many insect pests, including fleas that transmit Yersinia pestis, a causative agent of plague, in the natural foci of this extremely dangerous zoonosis. We examined the flea samples from the Volga-Ural natural focus of plague for their infestation with nematodes. Among the six flea species feeding on different rodent hosts (Citellus pygmaeus, Microtus socialis, and Allactaga major), the rate of infestation varied from 0 to 21%. The propagation rate of parasitic nematodes in the haemocoel of infected fleas was very high; in some cases, we observed up to 1,000 juveniles per flea specimen. Our study of morphology, life cycle, and rDNA sequences of these parasites revealed that they belong to three distinct species differing in the host specificity. On SSU and LSU rRNA phylogenies, these species representing three genera (Rubzovinema, Psyllotylenchus, and Spilotylenchus), constitute a monophyletic group close to Allantonema and Parasitylenchus, the type genera of the families Allantonematidae and Parasitylenchidae (Nematoda: Tylenchida). We discuss the SSU-ITS1-5.8S-LSU rDNA phylogeny of the Tylenchida with a special emphasis on the suborder Hexatylina.

[Genetic basis of the variability of nitrate reduction trait in Yersinia pestis strains].

The genetic basis of the varying ability to reduce nitrate in strains belonging to different biovars and subspecies of plague-causing microbe has been investigated and the inability to reduce nitrate observed in different intraspecies groups of Yersinia pestis has been shown to stem from mutations in different genes involved in the expression of this trait. The absence of denitrifying activity in strains of altaica and hissarica subspecies was not due to a mutation at position 613 of the periplasmic reductase napA observed in the strains of the biovar medievalis of the main subspecies, but rather was due to a mutation in the sequence encoding the nitrate-binding domain of the ABC transporter protein SsuA; a thymine insertion (+T) was detected at position 302 from the start of the ssuA gene. Five strains of biovar antiqua isolated at different times in Mongolia, China, and Africa were shown to lack the ability to reduce nitrate. A PCR test targeting two chromosomal regions containing deletions of 19 and 24 bp in size has been developed for the identification of strains of the biovar medievalis. This test can be combined with the test for the marker mutation in the napA gene for a more reliable detection of Y. pestis strains belonging to this biovar.

[Genetic characterization of toxigenic Vibrio cholerae non-O1/non-O139 strains, isolated in the Middle Asia].

Here, we report the characterization of 22 clinical toxigenic V. cholerae non-O1/non-O139 strains isolated in the Middle Asia (Uzbekistan) in 1971-1990. PCR analysis has revealed that these strains contain the main virulence genes such as ctxA, zot, ace (CTXφ); rstC (RS1φ); tcpA, toxT, aldA (pathogenicity island VPI), but they lack both pandemic islands VSP-I and VSP-II specific to epidemic strains of O1 serogroup of El Tor biotype and O139 serogroup. Only two of the twenty two toxigenic strains have tcpA gene of El Tor type, one strain has tcpA gene of classical type, while nineteen other strains carry a new variant of this gene, designated as tcpA(uzb).. Nucleotide sequences analysis of virulence genes in toxigenic V. cholerae non-O1/non-O139 strains from Uzbekistan showed that they differ significantly from the sequences of these genes in epidemic O1 and O139 strain indicating that they belong to a separate line of evolution of virulent V. cholerae strains. For the first time it is shown that V. cholerae non-O1/non-O139 toxigenic strains of different serogroups may belong to the same clone.

[Improvement of typification of natural foci of plague based on ecological-genetic analysis of Yersinia pestis].

Contemporary features of distribution of various subspecies and biovars of plague causative agent by landscape-geographical zones and mountain belts on the territory of Russia and other CIS countries are examined. The most widely spread in plain and mountain natural foci were noted to be Yersinia pestis main subspecies medieval biovar strains. Strains of Y. pestis non-main subspecies are spread in mountain landscapes of Altai, Caucasus, Tian Shan. Change of dominating species of rodents considered as the main carriers of plague was noted not to result in change of genetic and biochemical characteristics of Y. pestis strains. Perspectives of study of "micro-focality" of plague are emphasized for deciphering the mechanism of the enzootic.

[Genetic analysis of biochemical differences of Yersinia pestis strains].

Literature data and results of our experimental studies on genetic base of biochemical differentiation of Yersinia pestis strains of various subspecies and biovars are summarized in the review. Data on variability of genes coding biochemical features (sugar and alcohol fermentation, nitrate reduction), the differential development of which are the base of existing phenotypic schemes of Y. pestis strains classification, are presented. Variability of these genes was shown to have possible use for the development of genetic classification of Y. pestis strains of various subspecies and biovars.

[Standard algorithm of molecular typing of Yersinia pestis strains].

AIM: Development of the standard algorithm of molecular typing of Yersinia pestis that ensures establishing of subspecies, biovar and focus membership of the studied isolate. Determination of the characteristic strain genotypes of plague infectious agent of main and nonmain subspecies from various natural foci of plague of the Russian Federation and the near abroad. MATERIALS AND METHODS: Genotyping of 192 natural Y. pestis strains of main and nonmain subspecies was performed by using PCR methods, multilocus sequencing and multilocus analysis of variable tandem repeat number. RESULTS: A standard algorithm of molecular typing of plague infectious agent including several stages of Yersinia pestis differentiation by membership: in main and nonmain subspecies, various biovars of the main subspecies, specific subspecies; natural foci and geographic territories was developed. The algorithm is based on 3 typing methods--PCR, multilocus sequence typing and multilocus analysis of variable tandem repeat number using standard DNA targets--life support genes (terC, ilvN, inv, glpD, napA, rhaS and araC) and 7 loci of variable tandem repeats (ms01, ms04, ms06, ms07, ms46, ms62, ms70). The effectiveness of the developed algorithm is shown on the large number of natural Y. pestis strains. Characteristic sequence types of Y. pestis strains of various subspecies and biovars as well as MLVA7 genotypes of strains from natural foci of plague of the Russian Federation and the near abroad were established. CONCLUSION: The application of the developed algorithm will increase the effectiveness of epidemiologic monitoring of plague infectious agent, and analysis of epidemics and outbreaks of plague with establishing the source of origin of the strain and routes of introduction of the infection.

[Determination of genetic bases of auxotrophy in Yersinia pestis ssp. caucasica strains].

Based on the results of computer analysis of nucleotide sequences in strains Yersinia pestis and Y. pseudotuberculosis recorded in the files of NCBI GenBank database, differences between genes argA, aroG, aroF, thiH, and thiG of strain Pestoides F (subspecies caucasica) were found, compared to other strains of plaque agent and pseudotuberculosis microbe. Using PCR with calculated primers and the method of sequence analysis, the structure of variable regions of these genes was studied in 96 natural Y. pestis and Y. pseudotuberculosis strains. It was shown that all examined strains of subspecies caucasica, unlike strains of plague-causing agent of other subspecies and pseudotubercolosis microbe, had identical mutations in genes argA (integration of the insertion sequence IS100), aroG (insertion of ten nucleotides), aroF (inserion of IS100), thiH (insertion of nucleotide T), and thiG (deletion of 13 nucleotides). These mutations are the reason for the absence in strains belonging to this subspecies of the ability to synthesize arginine, phenylalanine, tyrosine, and vitamin B1 (thiamine), and cause their auxotrophy for these growth factors.

[Genetic bases of methionine dependence in Yersinia pestis strains of major and non-major subspecies].

Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and minor subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metB, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and minor subspecies revealed that the reason for the methionine dependence of strains belonging to the major subspecies is a deletion of a single nucleotide (-G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis.

[Analysis of insect toxin complex gene variability of Yersinia pestis and Yersinia pseudotuberculosis strains].

The nucleotide sequences of the Tc's insect toxin complex genes have been analyzed in 18 natural strains of the main and non-main subspecies of Yersinia pestis isolated in different natural foci in the Russian Federation, as well as neighboring and more remote countries, as compared to the data on Y. pestis and Y. pseudotuberculosis strains stored in the NCBI GenBank database. The nucleotide sequences of these genes in plague agent strains have been found to be highly conserved, in contrast to those of the pseudotuberculosis agent. The sequences of two genes, tcaC and tccC2, have been found to be almost identical in Y. pestis strains, whereas other three genes (tcaA, tcaB, and tccC1) contain a few mutations, which, however, are not common for all strains of the plague agent. Exceptions are only strains of the Y. pestis biovar orientalis, whose tcaB gene is in a nonfunctional state due to a nucleotide deletion. The results suggest that the formation of the species Y. pestis as an agent of a natural focal infection with a transmissive mechanism has not resulted in degradation of the Tc's complex genes. Instead, these genes are likely to have been altered as the plague agent have been adapting to the new environment.

[Structural analysis of genes participating in melibiose fermentation and isocitrate lyase production in Yersinia pestis strains of main and non main subspecies].

Comparative analysis of nucleotide sequences of genes participating in melibiose fermentation and isocitrate lyase production was conducted in 90 natural Yersinia pestis strains of main and non main subspecies. It was ascertained that the lack of the ability to utilize disaccharide melibiose in strains of the main subspecies is caused by integration of the insertion sequence IS285 at 73 bp from the beginning of the structural gene melB that encodes the transport protein galactoside permease. In contrast, strains of non main subspecies (caucasica, altaica, and ulegeica) contain the intact gene melB and are capable of fermenting melibiose. Differences in the manifestation of the other differential trait, production of isocitrate lyase, are connected with the presence of mutation (insertion of two nucleotides +CC) in the regulatory gene iclR encoding repressor protein of the acetate operon, which is the reason for constitutive synthesis of this enzyme. Strains of non main subspecies do not contain mutations in gene iclR, and this correlates in these strains with their capacity for inducible synthesis of isocitrate lyase.

[The biofilm formation in Yersinia pestis strains isolated in Astrakhan region].

AIM: To study biofilm formation in strains of Yersinia pestis isolated in 2009 in Astrakhan region. MATERIALS AND METHODS: Study of biofilm formation was performed on abiotic surfaces as well as on cuticle of nematode Caenorhabditis elegans. Detection of genes was performed by PCR with specific primers. RESULTS: Study of phenotypic (fermentation of sugars and alcohols) as well as genetic (presence of plasmids, genes of chromosome region of pigmentation) characteristics of Y. pestis strains showed that they are typical for strains isolated in Astrakhan region. All isolated in 2009 strains formed well developed biofilm on abiotic surfaces and cuticle of C. elegans nematode. They contained genes of hms operon and regulatory genes hmsT and hmsP, which are necessary for formation of pigmented colonies on Congo red medium as well as biofilm on abiotic and biotic surfaces. CONCLUSION: Strains of Y. pestis isolated in 2009 in Astrakhan region formed well developed biofilm on different types of surfaces, which could facilitate their survival in complex parasitic biocenosis of plague natural focus.

[Sequence analysis of the yadA, inv, and ail genes and their expression in the main and nonmain Yersinia pestis subspecies and Yersinia pseudotuberculosis].

The nucleotide sequences of the inv, yadA, and ail adhesin-invasin genes were analyzed in 24 strains of the main and nonmain Yersinia pestis subspecies, which were isolated from natural plague foci in Russia and neighbor countries, and ten Y. pseudotuberculosis strains. All of the five plague agent subspecies (main, caucasica, altaica, ulegeica, and hissarica) had the inv and yadA genes altered by insertion of the IS element and a single nucleotide deletion, respectively, as was earlier observed for the Y. pestis strains KIM and CO92. Consequently, the strains lacked functional activity of the Inv and YadA proteins. The ail gene of the main and ulegeica subspecies had a missense mutation, which replaced Val138 with Phe in the Ail protein. The strains of the caucasica subspecies had an AGT insertion in the ail gene, resulting in Ser148 insertion in the polypeptide chain. The changes in the ail sequence probably exerted no effect on ail expression, since the strains of all subspecies were resistant to blood serum complement.

[Structural and functional analysis of araN gene in the Yersinia pestis strains of various origin].

Structural and functional analysis of the araN gene involved in regulation of expression of diagnostically significant symptom (arabinose fermentation) was performed in the Yersinia pestis microorganism. Lack of arabinose fermentation in the Altai substrain, Hissar substrain, and Talas strains was shown to be due to solitary nucleotide insert into the araN gene. The insert is in the position 763 bp. The strains of the main, Caucasian, and Ulege substrains do not contain this mutation of the araN gene. The absence of the mutation correlates with ability to ferment arabinose.

[Comparative genetic characteristic of vaccine strain of Yersinia pestis EV and its putative "virulent derivatives"].

AIM: To perform a comparison of genetic characteristics of vaccine strain EV and its putative "virulent derivatives", obtained after passages through highly susceptible animals, in order to identify the strains-"revertants" and establish their possible origin. MATERIALS AND METHODS: Yersinia pestis EV strains and its putative "virulent derivatives" were used in the study. Polymerase chain reaction and DNA-DNA hybridization were used for genetic analysis. RESULTS: Comparison of several genetic characteristics of vaccine strain EV and its putative "virulent derivatives" allowed to establish that virulent "revertants" are not derivatives of vaccine strain EV because they do not belong to East biovar, do not have ribotype characteristic for EV strain and contain pigmentation area, which is absent in vaccine strain. CONCLUSION: Obtained results evidence against possibility of reversion of vaccine EV strain to virulent forms in organisms of highly susceptible animals and confirm its safety for vaccination.

[Comparative analysis of distribution of pseudogenes in the genome of strains of basic and supplementary species of the plague infection agent].

Comparative analysis of distribution of pseudogenes (YPO1582, YPO1728, YPO1967, and YPO4008) of strains of basic and supplementary species of the plague infection agent and pseudotuberculosis infection agent was performed. The genome of basic subspecies of plague infection agent species strain contains 3 different variants: intact genes, genes with IS-element inserts, or individual fragments. The pseudogene profile can be used as genetic marker of the Y. Pestis strains of basic subspecies from natural foci of plague. Strains of supplementary subspecies of Y. Pestis and Y. pseudotuberculosis contain these genes as the wild-type gene alleles. In addition to other factors, this fact can be regarded as evidence of ancient origin of plague infection agent supplementary subspecies and their similarity to pseudotuberculosis infection agent.