[THE EPIZOOTIC AND EPIDEMIC ACTIVITY OF NATURAL TULAREMIA FOCI OF DIFFERENT LANDSCAPE EPIDEMIOLOGICAL TYPES IN 2009-2014].

OBJECTIVE: to assess the present state of the natural tularemia foci of different landscape epidemiological types, by using individual focal areas as an example. MATERIALS AND METHODS: Epizootological monitoring and epidemiological analysis were conducted in the areas of natural tularemia foci of tundra (Wrangel Island), meadow-field (Central Federal District of the Russian Federation), flood-swamp (Arkhangelsk Region, Khanty-Mansi Autonomous District), and steppe (Mongolii) types. Small mammals (organs, blood), tularemia patients' sera, and environniental objects were examined. Molecular genetic and immune serological diagnostic assays were used. The incidence of tularemia in the past decade was analyzed using the maps for the epidemiological examinations of tularemia cases and medical reports. RESULTS: The natural foci of tularemia were established to continue to actively operate. There were 2913 cases of tularemia in the Russian Federation in 2001 to 2014. The flood-swamp natural foci, in which there were summer transmissive tularemia outbreaks, the largest of high occurred in Khanti-Mansiysk in 2013 when a total of 1005 people fell ill, are a special epidemic hazard. Analysis of the tularemia outbreaks suggests that there is a need for continuous epizootological monitoring of the areas of natural tularemia foci for the timely prediction and prevention of epidemic complications. It is noted that there is an unfounded reduction in the scope of preventive measures, and immunoprevention in particular, and a weaker control of the antitularemia immune status in the population residing in the area of active natural foci of tularemia.

FIRST MOLECULAR IDENTIFICATION OF THE TULAREMIA AGENT IN THE TICKS IXODES TRIANGULICEPS BIR. IN RUSSIA.

The ticks Ixodes trianguliceps (140 nymph pool and 211 adults) collected from small forest mammals in the forests of the Middle Urals (Chusovskoy district of the Perm Region) were tested using real-time PCR for the presence of Francisella tularensis DNA. Using the target gene 16S rRNA, the locus size 1165-1170 bp Francisella DNA was detected in 12 adults and 4 pools of nymphs. DNA-positive samples from 17 individuals from 128 adults and in 16 of 89 nymph pools were additionally detected by amplification of a shorter locus of the same gene (221-222 bp). All 49 16S rRNA gene-positive samples of real-time Taqman PCR assays directed against the tul4 (lpnA) gene locus and ISFtu2 element were identified as F. tularensis. These data suggest the possible involvement of the ticks I. trianguliceps in the circulation of the causative agent of tularemia in the natural foci of the forest type.

[REAL TIME POLYMERASE CHAIN REACTION IN TULAREMIA LABORATORY DIAGNOSTICS].

AIM: Enhancement of tularemia laboratory diagnostics by F. tularensis DNA determination in blood sera of patients using real time polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: 39 blood sera of patients obtained during transmissive epidemic outbreak of tularemia in Khanty-Mansiysk in 2013 were studied in agglutination reaction, passive hemagglutination, RT-PCR. Specific primers and fluorescent probes were used: ISFTu2F/R+ISFTu2P, Tu14GF/R+tul4-PR2. RESULTS: Advantages of using RT-PCR for early diagnostics of tularemia, when specific antibodies are not detected using traditional immunologic methods, were established. Use of a combination of primers and ISFTu2F/R+ISFTu2P probe allowed to detect F. tularensis DNA in 100% of sera, whereas Tul4G F/R+tul4-PR2 combination--92% of sera. The data were obtained when DNA was isolated from sera using "Proba Rapid" express method. Clinical-epidemiologic diagnosis oftularemia was confirmed by both immune-serologic and RT-PCR methods when sera were studied 3-4 weeks after the onset of the disease. CONCLUSION: RT-PCR with ISFTu2F/R primers and fluorescent probe ISFTu2P, having high sensitivity and specificity, allows to determine F. tularensis DNA in blood sera of patients at both the early stage and 3-4 weeks after the onset of the disease.

[The molecular and genetic characterization of Francisella tularensis strains of different taxonomic status and virulence].

Typing of Francisella strains collection by means of PCR on the basis of tul4 and RDI genes was carried out. The identification of the species and subspecies of the 112 strains of Francisella tularensis was reashed. The PCR on DNA-targets loci of type IV pili genes: pilA, pilE2, pilE3, pilE4, pilE5, pilF, pilT, pilD and pilQ for differentiation of F. tularensis strains on virulence was carried out. It was demonstrated the possibility of differentiation of F. tularensis strains in PCR (primers A-B) on the basis of the revelation of the gene pilA in virulent strains of third subspecies F. tularensis and F. tularensis subsp. novicida. This gene pilA was not detected in the vaccine strain 15/10 and its variants, as well as in the most of avirulent F. tularensis subsp. holarctica strains. However, the fragment gene pilA was found in the attenuated strains F. tularensis subsp. tularensis and mediasiatica. It was not revealed any differences on other targets of pili genes between of F. tularensis strains, with the exception of the strain F. tularensis subsp. novicida Utah 112, which had not a fragment of the gene pilE2. The use of PCR to target the locus of the pilA gene allows to discriminate virulent F. tularensis subsp. holarctica strains from the vaccine 15/10, its variants and avirulent strains.

[Monitoring the natural foci of tularemia on Wrangel Island].

Long-term annual monitoring of the natural foci of tularemia was first made on Wrangel Island. The objects of the investigation were pellets of birds-myophages, blood samples from rodents, and excrements from carnivorous mammals. A total of 2626 biological samples were examined in the period 2002 to 2011. A serological test was ascertained to be the most effective method for the detection of tularemia epizooties; polymerase chain reaction should be used as an additional technique to examine blood samples, as well as rodent tubular bone debris taken from the pellets. Tularemia epizooties were registered in the populations of two species of lemmings every year, except in 2003. An intensive diffuse tularemia epizooty was first detected in this area, which emerged in 2019, peaked by spring 2011, and covered most of the island. The antigen of tularemia pathogen was identified in 43.46% of the samples under examination,which is a high quantitative indicator of the intensity of an epizootic process. The fact that positive samples are annually found in the same areas of the island suggests that the causative agent is steadily and long preserved in the parasitic system. The availability of stable and active natural tularemia foci on Wrangel Island calls for preventive measures, particularly vaccination of risk groups coming to the island to conduct researches.

[Detection of natural tularemia foci in Mongolia].

AIM: Study of the current spread of natural tularemia foci in Mongolia and its epizootic activity evaluation for consequent substantiation of the recommendations for prophylaxis of this disease. MATERIALS AND METHODS: Study of 1119 pellet specimens from predatory birds obtained in 6 aimag in Mongolia in 2008--2010 was performed. Tularemia antigen was detected by using antibody neutralization reaction (ANR) and passive hemagglutination reaction (PHR) with tularemia diagnosticums. Tularemia DNA was detected by PCR by using strain specific primers. Presence of plague antigen in PHR with plague immunoglobulin diagnosticum was also studied in all the samples. RESULTS: Epizootologic monitoring allowed the detection of natural tularemia foci in 5 of the 6 studied aimags in Mongolia. PHR was the most effective study method that allowed to detect tularemia antigen in the environmental objects in high quantities (up to 9.2% of positive samples) and high titers (up to 1:1600). PCR was less effective. Plague antigen was detected in 9 samples in 2010 for the first time, and in 3 cases together with tularemia antigen, which indicates a presence of combined natural foci of tularemia and plague in this territory. CONCLUSION: In the studied regions of Mongolia natural tularemia foci were detected, their epizootic activity was determined and recommendations for future study tactics of natural tularemia foci were given.

[Natural foci of tularemia on the Wrangel island].

The subjects of the study were snowy owl castings (611 samples), polar fox litters (148 samples), and water samples of outdoor tundra water reservoirs. Tularemia antigen was sought in the castings and litters by the antibody neutralization test. The water was examined by bioassays. Tularemia antigen was annually detected in the study samples. Epizootically active autonomous natural foci of tundra-type tularemia were ascertained to continue to exist on the Wrangel island. The major vectors of the causative agent of tularemia were two types of lemmings (Siberian lemming and Vinogradov's one). The availability of epizootically active natural foci determines the need for vaccination against tularemia of persons who are long engaged in researches who are epidemiologically a risk group.

[The ultrastructural characteristics of Francisella tularensis interaction with Tetrahymena pyriformis]. / Ul'trastrukturnye osobennosti vzaimodeistviia Francisella tularensis s Tetrahymena pyriformis.

The electron-microscopic study of the interaction of F. tularensis virulent and attenuated strains with infusoria of the species T. pyriformis was dynamically studied. In this study the structural changes of F. tularensis and T. pyriformis cells, as well as their capacity for survival, were revealed. The data on their ultrastructure correlated with the dynamics of the number of both F. tularensis and T. pyriformis: during the whole term of observation the tendency to a slow decrease in the number of F. tularensis was registered with the concentration of T. pyriformis remaining stable. The interaction of F. tularensis with T. pyriformis may be regarded as a variant of commensal, but not antagonistic interactions.

[Characterization of the recombinant strain Francisella tularensis R1A]. / Kharakteristika rekombinatnogo shtamma Francisella tularensis R1A.

For the first time F.tularensis recombinant strain R1A, obtained by the transfer of genes responsible for virulence in F.tularensis strain B 399 A-Cole into recipient cells of the R-form, was studied. Strain R1A was characterized by morphological, tinctorial and biochemical properties, similar to those of F.tularensis virulent strains, and became resistant to the bactericidal action of animal sera, as well as heat resistant (tr42). The results of animal experiments revealed that strain R1A proved to be highly virulent for noninbred white mice, faintly virulent for guinea pigs and avirulent for rabbits. In contrast to the R-form, recombinant strain R1A exhibited high antigenic activity, as well as partially protected guinea pigs from 100 DCL of F.tularensis highly virulent strain B A-Cole. The totality of its properties places recombinant strain R1A in an intermediate position between F.tularensis virulent and vaccine strains.

[A comparative analysis of Francisella protein antigens]. / Sravnitel'nyi analiz belkovykh antigenov Francisella.

Comparative analysis of protein antigens of cells lysates and outer membranes of different Francisella species (F. tularensis, F. novicida(-like), and F. philomigaria) by immunoblotting showed similarity and detected the individual features of each species as regards the protein antigen spectrum. Protein antigen with molecular weight of 22.5 kD localized on the outer membrane and coded for by plasmid pFNL10 was discovered for F. novicida-like stain F6168. The significance of new data for characterization of Francisella genus is discussed.

The new vaccine strains (or variants) of Francisella tularensis.

A colony (N83) of the vaccine strain of Francisella tularensis (15/10) and a strain (N268) isolated from a water sample in nature were revealed for susceptibility to cultivation at 42 degrees C. Both strains had low virulence for white mice and were avirulent for guinea pigs but possessed high immunogenicity in these animals. The spontaneous mutant of vaccine strain 15/10 showed resistance to doxicycline and rifampicine (15/10 Dox (r)40 Rif (r)40). The obtained mutant had biological characteristics similar to the parent vaccine strain. It provided immunity in experimental animals when vaccination and antimicrobial agents were used in combination.

[The characteristics of a natural attenuated isolate of Francisella tularensis]. / Kharakteristika prirodnogo attenuirovannogo izoliata Francisella tularensis.

The natural isolate of F. tularensis subsp. holarctica 268 was detected and studied. The isolate possessed the properties of the vaccine strain: residual virulence for white mice, avirulence for guinea pigs and high immunogenicity for experimental animals. A significant decrease in its virulence for animals, highly sensitive to tularemia, was noted. In contrast to most virulent strains circulating in natural foci, F. tularensis strain 268 was characterized by the absence of growth at a cultivation temperature of 42 degrees C and had stable biological properties after 10-fold passage through white mice.

[The characteristics of new species of pathogenic microorganisms in the genus Francisella]. / Kharakteristika novykh vidov patogennykh mikroorganizmov roda Francisella.

The comparative study of newly discovered pathogenic bacteria of the genus Francisella was carried out with the use of a complex of microbiological and serological methods. While having great similarity to the causative agent of tularemia, F. novicida, F. novicida-like bacteria and F. philomiragia had lesser growth requirements, some specific morphological and structural features, were capable of fermenting sucrose and exhibited low pathogenicity to experimental animals. The strains under study proved to be virulent with regard to golden hamsters, who were for this reason proposed as an adequate model for the isolation of these bacteria from environmental objects and pathological material obtained from patients. The use of immunoblotting made it possible to find out that all Francisella species had protein antigens, similar to their electrophoretic mobility and serological activity.

[Biological characteristics of spectinomycin-resistant strains of the tularemia pathogen]. / Biologicheskaia kharakteristika rezistentnykh k spektinomitsinu shtammov tuliaremiinogo mikroba.

Sensitivity of 2 subspecies of the tularemia causative agent to spectinomycin, an aminoglycoside antibiotic, was studied in vitro. The MIC of the antibiotic with respect to strains 503/847 and Schu was 40 micrograms/ml and with respect to strain A-Cole 20 micrograms/ml. The frequency of spontaneous spectinomycin resistant mutants was low. The mutants grown on a medium containing spectinomycin in a concentration of 100 micrograms/ml were highly resistant to the antibiotic (at least 10000 micrograms/ml). By the main biological properties and virulence the spectinomycin resistant mutants did not differ from the initial strains.