[Evaluation of modern epizootic activity of natural tularemia foci in Voronezh region using immune-serological and molecular-genetic study of main carriers of the disease].

AIM: Improvement of monitoring and prognosis of epidemic manifestations of natural foci of tularemia on the territory of Voronezh region using immune-serological and molecular-genetic study of main carriers of the disease. MATERIALS AND METHODS: 539 small mammals captured during summer period of 2011 in 4 districts of North-Eastern part of Voronezh region were studied. Animal organs were studied by serologic (search for Francisella tularensis antigens) and molecular-biologic (detection of F. tularensis DNA) methods. Tularemia antigen was detected using passive hemagglutination reaction (PHAR) with erythrocytic tularemia immunoglobulin diagnosticum. Real-time polymerase chain reaction (RT-PCR) was applied for detection of tularemia causative agent DNA. RESULTS: Complex study revealed epizootic activity of natural foci of tularemia in the examined territory. F. tularensis antigen and/or DNA were detected in 82 objects (15.2%). Use of RT-PCR allowed to additionally detect samples with relatively low content of F. tularensis DNA substrate, when antigen was not detected in samples. High sensitivity and specificity of the RT-PCR was ensured by inclusion of specific probes (tu14-PR2 and ISFTu2P). CONCLUSION: The results obtained give evidence on functioning and epizootic activity of natural foci of tularemia in Voronezh region that requires constant monitoring of the territory and prophylaxis measures, first of all vaccination of risk groups by live tularemia vaccine.

[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.

[Specific effect of attenuated strain of Francisella tularensis on the development of radiation induced lesions in experimental animals and effictiveness of antibacterial therapy for lesions induced by radiation combined with virulemt strain of the bacteria].

For study of the effects of whole-body gamma-radiation (1 and 4 Gy) on the response of the body to administration of vaccines and virulent strains of tularemia 206 outbred white mice were used. The results of the study shown that the administration of attenuated bacterial cells in 5 days after exposure to radiation (1 and 4 Gy) caused more severe post-radiation effects and the increase in the number of died animals. The severity of the disease was less if mice were vaccinated in 26 days after irradiation (4 Gy). The treatment of tularemia in irradiated mice twith Riphampicin (daily peroral administration, 5 mg/mouse, duration of treatment--7 days) administered in 4 hours after infection was effective and caused high survival of affected mice. The results show effectiveness of the riphampicin treatment of tularemia in the animals exposed to sublethal dose of radiation.

[Immunization with cellulose-immobilized antigens. The development of A. E. Gurvitch concept].

A highly purified TUL4-CBD chimeric protein was obtained by one stage purification method. TUL4-CBD protein consists of TUL4 Francisella tularensis mature peptide sequence, Gly-Ser spacer and cellulose binding domain (CBD) of Anaerocellum thermophilum. The TUL4-CBD protein was shown to induce production of specific antibodies to TUL4 protein in laboratory animals.

[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.

[A system for the automated chemotaxonomic determination of salmonellae and other pathogenic bacteria]. / Sistema avtomatizirovannogo khemotaksonomicheskogo obnaruzheniia sal'monell i drugikh patogennykh bakterii.

On the basis of the data of the gas-chromatographic analysis of the fatty acids of 208 bacterial strains, representatives of 16 different microbial genera, the algorithm of decision taking, necessary for the program provision of investigations, was worked out. In working out the algorithm the characteristics of 30 fatty acids were used, making it possible to classify bacteria with their genera and in some cases their species. The groups of fatty acids with the number of carbon atoms in their molecules ranging from 10 to 25, their melting temperatures and the dependence of relative characteristics of binding from the number of carbon atoms in the molecule of the acid, its chemical composition and the presence of double bonds were taken into consideration. To indicate salmonellae by their fatty acid profiles, a chromatographic system on the basis of a type Crystal 2000 M gas chromatograph is proposed. In addition, the complex method for the determination of bacteria, combining the determination of salmonellae by changes in the medium resistance (impedance) with the use of an electrochemical analyzer and the subsequent identification of the infective agent by its fatty acid profile in the common system of gas-chromatographic investigation, is proposed.

[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 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.

[Isolation and molecular-genetic characteristic of a cryptic plasmid from the Francisella novicida like F6168 strain]. / Vydelenie i molekuliarno-geneticheskaia kharakteristika kripticheskoi plazmidy iz shtamma Francisella novicida like F6168.

A 4 Kb plasmid DNA has been isolated from Francisella novicida like strain F6168. Restriction map of the plasmid was constructed for restriction endonucleases HindIII, XbaI, EcoRV, BgIII. The plasmid pFN10 has been shown to be stably inherited by F. tularensis. The use of pFN10 for the construction of plasmid vectors for microorganisms of the genus Francisella is discussed.

[Detection of tularemia pathogens in patients with the use of an immunofluorescence reaction]. / Obnaruzhenie vozbuditelia tuliaremii u bol'nykh s pomoshch'iu reaktsii immunofliuorestsentsii.

For the first time three cases of the detection of Francisella tularensis, made by means of the direct immunofluorescence test in the fluid obtained from punctured buboes or in purulent matter taken from patients with the ulcerous bubonic form of tularemia, are presented. The simplicity of the test and its capacity of yielding rapid results make it possible to recommend this test, together with other diagnostic methods, for the clinical diagnosis of tularemia.

[Use of the ELISA immunoenzyme method for the detection of the causative agent of tularemia]. / Ispol'zovanie immunofermentnogo metoda ELISA dlia vyiavleniia vozbuditelia tuliaremii.

The conditions of the enzyme-linked immunosorbent assay (ELISA) for the detection of Francisella tularensis were worked out. In the study of 27 strains differing in their biological characteristics, the sensitivity of the assay was determined, varying within the range of 1 X 10(4)--5 X 10(4) million cells/ml and exceeding the sensitivity of the currently used methods for the immunodiagnosis of tularemia by 1-2 orders. ELISA also proved to be a highly effective technique for the detection of the specific antigen in the organs of infected animals. The antigen was regularly detected in the organs of white mice, beginning from day 3 after their infection with the minimal doses of F. tularensis. The method may be recommended both for the identification of isolated cultures and for the early diagnosis of tularemia infection.

[Development of an immunoenzyme method for detecting Francisella tularensis]. / Razrabotka immunofermentnogo metoda dlia vyiavleniia Francisella tularensis.

The conditions permitting the determination of F. tularesis cells by means of the enzyme immunoassay (EIA) in 3-5 hours have been established. Ways for enhancing the reliability of results obtained in the assay of the least possible amount of the test material have been proposed. The sensitivity and specificity of the rapid EIA technique permitting the determination of F. tularensis cells at a concentration of 20 000 cells/ml in the presence of other bacterial cells in 100-fold excess have been shown.

[Use of an immunoenzyme method on a solid-phase carrier for determining the tularemia antigen]. / Primenenie immunofermentnogo metoda na tverdofaznom nositele dlia opredeleniia tuliaremiinogo antigena.

The method of the highly sensitive (up to 10 ng/ml) and specific determination of soluble tularemia antigen, based on the use of "sandwich" type ELISA techniques, has been developed. The dependence of the specificity and sensitivity of the method on the degree of purification of antibodies and their peroxidase conjugates used in the assay has been studied. The study has revealed that the best results can be obtained with the use of purified IgG and its conjugate free of unbound peroxidase. Both foreign peroxidase preparations and type A enzyme manufactured in the USSR can be equally used as enzymatic labels.

[Determination of tularemia antibodies by an immunoenzyme method on a solid-phase carrier (ELISA)]. / Opredelenie tuliaremiinykh antitel immunofermentnym metodom na tverdofaznom nositele (ELISA).

ELISA "sandwich" techniques have been developed and the optimum assay conditions for detecting specific antibodies in human serum samples have been determined. The possibility of using these techniques for the determination of the level of antibodies to tularemia antigens in the sera of persons immunized with live tularemia vaccine has been shown. Statistically significant differences in the level of antibodies to tularemia antigen in the sera of immunized and nonimmunized persons have been established. The comparative study of five serological methods - ELISA, the agglutination test, the passive hemagglutination test, the immunofluorescence test and the defined antigen substrate sera ( DASS ) techniques - has revealed the advantage of ELISA, whose sensitivity has proved to be considerably higher than that of all other methods used in our work.