[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 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.

[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.

[Dynamics of immunoglobulins M and G formation in experimental tularemia]. / Dinamika obrazovaniia immunoglobulinov M i G pri éksperimental'noi tuliaremii.

There were revealed certain differences in the structure and the level of immunoglobulins, the time of their persistence in the blood of the infected and vaccinated experimental animals in tularemia. IgG were characteristic chiefly of the infectious process and served as an index of the immunological response intensity. Vaccination conditioned the production of IgM. IgG were revealed in low titres and at earlier periods of study. The results coincided in using cystein hidrochloride and 2-mercaptoethanol (reducing agents).

[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.

[Higher fatty acid composition of the intraspecific taxa of Francisella tularensis]. / Sostav vysshikh zhirnykh kislot u vnutrividovykh taksonov Francisella tularensis.

The higher fatty acid composition of 27 F. tularensis strains differing in their biological properties and belonging to different intraspecific taxons has been determined with the use of gas and liquid chromatography. All the strains under study have been found to possess similar fatty acid spectra, characterized by the presence of long straight-chain fatty acids (saturated and unsaturated) with the number of carbon atoms varying from 10 to 26. Quantitative differences content of individual fatty acids (C10:0, C14:0, C24:0) have been revealed in various F. tularensis subspecies, which can serve as an additional criterion for their differentiation. The peculiar features of the higher acid composition of F. Tularensis give grounds for considering this species to be taxonomically independent and characterize its intraspecific taxons by the quantitative content of individual fatty acids.

[Results of the study of bird droppings collected in the Lithuanian SSR for the purpose of searching out tularemia epizootics]. / Rezul'taty issledovaniia pogadok ptits, sobrannykh na territorii Litovskoi SSR s tsel'iu poiska épizootii tuliaremii.

There are practically no records of cases of tularemia among humans in the Lithuanian SSR. Nevertheless, the mass sero-allergic survey of the population for tularemia, carried out 10-12 years ago, showed that 2.3% of the adult population in the Republic had had contacts with the causative agent of this infection. The work was aimed at the determination of the present activity of the foci of tularemia. During 6 years in 22 rural districts 2582 samples of avian excrements, containing bones and wool of small animals, were collected and studied by means of the antibody neutralization test (ANT). In 132 (5.1 +/- 0.4%) excrement samples collected on the territory of 12 districts Francisella tularensis antigen was detected. The average ANT titer was 45.2, the maximum titer (10 excrement samples) reached 1: 160. The study revealed the existence of the natural foci of tularemia in Lithuania at present, but their activity proved to be low. The most unfavorable situation was found to exist in western districts of the Republic.

[Experience with a bacteriologic and serologic study of a tularemia epizootic in rodents in a natural focus of the meadow and field type]. / Opyt izucheniia bakteriologicheskimi i seroligicheskimi metodami épizootii tuliaremii na gryzunakh v prirodnom ochage lugopolevogo tipa.

The autumn-winter (1977-1978) tularemia epizootic in small murine rodents was revealed and studied at the natural focus of the meadow and field type in the south of the Moscow Region. The efficacy of the serologic method (the antibody neutralization test) of studying the organs of the caught rodents and the bodies of dead rodents was found to be greater than that of the traditional bacteriologic methods (26.6% and 9.6%, respectively). The serologic study of 908 specimens of avian excrements collected during the period from autumn to spring (1977-1978) revealed that tularemia antigen could be constantly detected, starting from October. The serologic method was effective when used both for the early and retrospective detection of the infective agent and allowed to characterize the epizootic process in greater detail.

[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.

[Isolation of the causative agent of tularemia from an inoculum from skin lesions in the ulcerobubonic form of the disease]. / O vydelenii vozbuditelia tuliaremii posevom soderzhimogo kozhnogo affekta pri iazvenno-bubonnoi forme zabolevaniia.

A case of tularemia in a human patient infected through the sting of a gadfly (Tabanus) is described. The causative agent of the disease was isolated from the patient with the ulcerobubonic form of the disease by the method of the direct inoculation of the contents of the patient's cutaneous effect. The properties of the isolated culture were established; the strain thus obtained was classified as a representative of the geographical race Francisella tularensis holarctica 01s. The causative agent circulating in the human patients was found to be fully virulent.

[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.

[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.

[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.

[Exploration of natural foci of tularemia and plague in Armenia using the serological examination of bird droppings and excrements of predatory mammals]. / Rekognostsirovochnoe obsledovanie prirodnykh ochagov tuliaremii i chumy v Armenii putem serologicheskogo issledovaniia pogadok ptits i ekskrementov khishchnykh mlekopitaiushchikh.

Forty strains of tularemia and 619 of plague microbes were isolated in 1974 in bacteriological examination of natural plague and tularemia foci from a great number of small mammals and their ectoparasites. At the same time in serological examination (in the antibody neutralization test) of bird pellets, 52 mummified cadavers, and 34 excretion samples of mammalian beasts of prey collected in Armenia (its central and North-Western part) in 1973 the antigen of tularemia microbe was revealed in 73, 8, and 3, and of plagye--in 42, 5, and 1 cases, respectively. In one of the sites examined the number of positive findings failed to exceed 10--17%, this indicating a low intensity of the epizootic in the majority of the places. Judging by the mean titres of the serological test, which varied from 1:12 to 1:1428 in examination for tularemia and from 1:12 to 1428 in examination for plague, it was possible to detect both epizootics coursing during the examination, and those which occurred several monts ago. Tularemia and plague foci were not infrequently present at the same territories, and these diseases could involve the same individual animals of Microtus arvalis (Pall.). The data obtained pointed to the greater effectiveness of examination of bird's pellets and excretions of mammalian beasts of prey for the reconnaisance investigation of the natural foci of plague and tularemia in comparison with the bacteriological methods applied usually.