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Study the role of LPS in induction of anti-tularemia immunity in humans and animals.Activity of various antigenic preparations of tularemia microbe, including highly purified from protein and S- and R-LPS, was studied using leukocytolysis reaction with blood of vaccinated humans and guinea pigs and skin allergy test (guinea pigs).Only the whole cells of Francisella tularensis, killed in protein non-denaturating conditions and conserving full S-LPS structure (tularin⁺) were shown to be inductors of delayed-type hypersensitivity reaction. Alterations in LPS structure (tularin⁻) results in a significant decrease, and denaturation of bacterial proteins (during boiling) results in a complete loss of immune stimulating properties of the preparations. Purified LPS preparations and O-polysaccharide fraction of S-LPS are not able to activate cell-mediated immunity.The presence of LPS with the full structure affects the ability of antigenic preparations of F. tularensis to cause allergic reactions, and thus, form cell-mediated antitularemia immunity. LPS of F. tularensis can not be excluded as an adjuvant and provides the most effective presentation of epitopes of protein molecules for interaction with receptors of T-lymphocytes.

The two subspecies of Francisella tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. palaearctica (type B), differ from each other in biochemistry and virulence. Strains of F. tularensis subsp. tularensis are believed to be confined to North America, whereas strains of F. tularensis subsp. palaearctica occur in Europe, in Asia, and in North America. Moreover, the existence of two other subspecies, designated F. tularensis subsp. mediaasiatica and F. tularensis subsp. palaearcitica japonica, has been suggested for strains of F. tularensis isolated in the central Asian focus of the Soviet Union and in Japan, respectively. In the present study, strains biochemically classified as F. tularensis subsp. mediaasiatica or F. tularensis subsp. palaearctica japonica have been investigated by hybridization with probes specific to 16S rRNAs of the two main subspecies. Furthermore, the virulence and biochemical characteristics of the strains were compared with those of strains belonging to F. tularensis subsp. palaearctica and F. tularensis subsp. tularensis. It was found that 16S rRNAs of F. tularensis subsp. mediaasiatica and F. tularensis subsp. palaearctica japonica hybridize with the probe specific to a genotype proposed herein, genotype A (F. tularensis subsp. tularensis), which shows that strains genetically related to this subspecies are found outside North America. However, the central Asian strains differed from F. tularensis subsp. palaearctica and F. tularensis subsp. tularensis strains when investigated by fermentation of glucose. The results of the biochemical tests could not be unambiguously used for differentiation of strains into F. tularensis subsp. palaearctica or F. tularensis subsp. tularensis. These drawbacks suggest that classification of strains of Francisella on the basis of 16S rRNA analysis may be preferable to classification on the basis of biochemical analysis.

Retrospective VNTR-analysis of 159 Francisella tularensis subsp. holarctica strains isolated in December 1988 - February 1989 in former USSR and some European countries was carried out. Analysis of heterogenic genotypes of strains allow to subdivide them into 30 groups of variants by individual genotypes, while cluster analysis--to subdivide them in 7 clusters with different number of compositions. The predominance of genotype C1 strains isolated on the Rostov and Archangelsk regions and the Crimea was established. F. tularensis strains isolated in winter time 1988 - 1989 in different geographic regions were supposed to be resident cultures typical for their biotope in natural focus of disease.

It was demonstrated that the lipopolysaccharides (LPS) preparations, which were isolated from all representatives of Francisella Genus bacteria, i.e. F. tularensis, F. novicida, F. novicida-like and F. philomiragia by using the method of R.P. Darveau, R.E. Hancock (1983), were not toxic for white rats and white mice. A comparative study of toxicity of live F. tularensis bacteria (both wild and LPS-defective strains) made it possible to establish a direct correlation between the toxicity of microbes and LPS chemotype. It was found that only typical strains, which synthesize the wild-type S-LPS, caused the death of white rats and white mice in 24 hours after intraperitoneal contamination (10(9), 10(10) CFU/animal). Live bacteria of F tularensis R-mutants were not able to induce a lethal infection of rats and retained only residual virulence for mice. Other representatives of Francissela genus possessed less pronounced pathogenic properties. Thus, the toxic effect was registered, in case of white rats, only for F. novicida but not for F. novicida-like or F. philomiragia. At the same time, the two last mentioned species displayed a certain degree of virulence at high challenge doses (10(9), 10(10) CFU/animal) in respect to white mice. F. philomiragia, which generated lipoolygosaccharide (LOS) with an unusual structure, was found to be least pathogenic (25-75% of dead mice). The toxicity of bacteria, killed experimentally by different means (heating, UV-light, chloroform, acetone and formalin), was studied to define the role of bacterial proteins in the realisation of F. tularensis toxic potential in vivo. No lethal effect was exerted on experimental animals by killed microbes or purified LPS preparations. Finally, the study results show a priority role of the LPS molecule in the toxic effect of F. tularensis, which is possible in vivo only if structurally valuable molecules of live bacterial cells are available.

Serum antibodies were analyzed in rabbits immunized with live and formalin-killed Francisella (F. tularensis, F. novicida, F. novicida-like, and F. philomiragia). Passive hemagglutination test with erythrocytes sensitized by these bacteria' LPS showed much higher titers of species-specific antibodies in all sera to live microorganisms than sera to killed bacteria. The results of immunoblotting with purified LPS and bacterial lysates indicate that sera to live bacteria contained mainly immunoglobulins to species-specific antigenic epitopes of LPS O-polysaccharide chain and few antibodies to the protein component of the cell. By contrast, killed bacterial cells induced weak production of antibodies to S-LPS and a pronounced antibody response to protein antigens. Besides the quantitative differences, live and killed bacteria differed by the qualitative spectrum of immunodominant proteins. Serum to live F. tularensis 15/10 contained antibodies to at least 3 immunodominant antigens of the cell, while serum to killed bacteria contained antibodies to only two of these. Immunoglobulins to protein antigens, absent in homologous sera to live bacteria, were detected in the sera to killed F. novicida and F. novicida-like bacteria. Both sera to F. philomiragia had antibodies reacting with LPS epitopes and immunodominant complex containing protein. In contrast to other Francisella, F. philomiragia was found to synthesize an uncommon LPS representing two major lipooligosaccharides with different molecular weights and antigenic specificity. Therefore, immune response of the host to live and killed Francisella is different: live cells more effectively induce the production of antibodies to S-LPS epitopes, while killed ones to protein antigens.

To determine antitularemia antibodies in the sera of humans and animale, the possibility of using dot immunoassay with the use of F. tularensis lipopolysaccharide (LPS) as antigen-containing preparation was ascertained. Experiments demonstrated that this method made it possible to determine specific antitularemia antibodies in the sera of sick and immunized humans and animals. Investigetions carried out with the use of heterologous antisera to F. novicida, F. novicida-like and F. philomiragia, as well as Brucellf abortus, Vibrio cholerae and Yersinia enterocolitica, revealed that F. tularensis S-LPS was highly specific. The results obtained in this investigation are indicative of good prospects of using F. tularensis LPS in dot blotting for the laboratory diagnostics of tularemia in humans.

The study of the biological properties of lipopolysaccharides (LPS) of bacteria of the genus Francisella (F. tularensis, F. novicida, F. novicida-like, F. philomiragia) revealed that the preparation of Francisella LPS possessed immunomodulating and antitoxic properties in the absence of toxicity. At the same time the structure of LPS (S or R) was found to produce an essential effect of the immunobiological activity of this molecule. Thus, the S-forms of LPS proved to be more effective as immunomodulators and the R-forms of LPS, as antagonists of classical endotoxins.

To estimate the in vitro susceptibility of the plague microbe to chemotherapeutics, various experimental models with the maximum closeness to the host conditions were tested. The tests included the assay of the drug antibacterial activity against the plague microbe by the method of two-fold dilutions in biological fluids i.e. human normal (nonimmune) serum (HNS) and guinea pig heparinized blood. Hottinger broth was used as the control. It was shown that any system used for estimation of the drug MIC influenced the plague microbe susceptibility. Thus, the serum complement increased the antibacterial activity of cefotaxime, rifampicin, doxycycline, erythromycin and polymyxin B. In the blood of a susceptible host (guinea pigs) the activity of quinoxydine and dioxydine against the plague microbe markedly increased while the effect of benzylpenicillin, cefotaxime and furazolidone decreased. The data on the in vitro activity of the antibiotics in blood were comparable with those on their in vivo therapeutic efficacy.

Therapeutic efficacies of various drugs were studied comparatively in the treatment of experimental plague in albino mice at the stage of the infection generalization. It was shown that out of the tested drugs such as ciprofloxacin, amikacin, gentamicin, rifampicin and polymyxin B only ciprofloxacin provided a rather high therapeutic effect in the treatment of the plaque septic form. The in vitro and in vivo experiments demonstrated that ciprofloxacin had an antitoxic action on lipopolysaccharide (LPS) and the plague microbe toxin. In comparison to ciprofloxacin, polymyxin B had a higher neutralizing activity. It was found that the efficacy of the experimental plague treatment at the stage of the infection generalization increased with the use of combinations of the drugs with antitoxic and antibacterial activities (ciprofloxacin and polymyxin B).

Comparative study of the properties of initial capsular (cap+) and non-capsular (cap-) F. tularensis strains has revealed that the non-capsular variants have their specific biological features. The characteristic features of these variants are avirulence and inability to produce an immunogenic effect in sensitive laboratory animals. Cap- mutants, depending on the method of their obtaining, may exhibit different sensitivity to some antibacterial preparations. In contrast to the initial virulent strains, cap- variants are intensively phagocytosed by peritoneal macrophages of white mice and capable of multiplication and survival in these macrophages. F. tularensis non-capsular forms are characterized by higher superoxide-dismutase and neuraminidase activity than the initial strains, which may be the explanation of the phenomenon of their intracellular survival. At the same time the main biochemical signs of cap- strains (citrullinureidase, phosphatase and penicillinidase) remain unchanged, which determines the possibility of their classification with this subspecies.

Natural strains of F. tularensis were characterized by sensitivity to 2,3,5-triphenyl tetrazolium chloride (TTC). Development of TTC resistance in the cells of F. tularensis was accompanied by changes in the biological properties of the culture, i.e. the colony morphology, antigenic structure, virulence and immunogenicity for laboratory animals. Moreover, there was a direct correlation between the levels of TTC resistance and resistance to chloramphenicol, erythromycin, tetracycline, furazolidone and rifampicin. The antibiotic resistant mutants of F. tularensis were in turn more resistant to TTC than the initial strains. This could be useful in isolation of polymarked strains of F. tularensis for genetic studies and investigation of the nature of the phenomenon of virulence in F. tularensis.

In the causative agent of tularemia new markers correlating with different subspecies of this microbe have been detected. Thus, F. tularensis strains belonging to the American and Central Asian subspecies are characterized by phosphatase activity, which makes it possible to use the phosphatase test for their differentiation from the strains of the holarctic variety. F. tularensis subsp. mediasiatica are incapable of producing beta-lactamase which differentiates them from the representatives of the varieties holarctica and tularensis. These newly discovered signs are stable and do not depend on the virulence of the cultures under study and on the conditions of the cultivation of F. tularensis.

With a large collection of the strains of F. tularensis isolated it has been recently shown that cultures belonging to holarctica and mediaasiatica circulate in the endemic foci of the USSR. By their biological and genetic properties the natural strains of F. tularensis were homogeneous and represented type cultures of F. tularensis. Various ecological conditions in the natural environment did not change within the last 20 years the sensitivity of the tularemia microbe to the antibacterial drugs.

Fifteen stable variants of Yersinia pestis strains exhibiting different degrees of virulence for white mice and guinea pigs were obtained. Multiple passages of the organisms at 37 degrees C in a fluid nutrient medium containing antiplague agglutinating serum were found to be the most efficient method for obtaining noncapsular forms of the plague agent. Acquisition of the Fra(-) phenotype both by wild and laboratory strains was not associated with a loss of the high-molecular plasmid by cells but was probably a result of mutational alterations in the plasmid genes.

In vitro study on antibacterial activity of amikacin in comparison to that of streptomycin revealed a high sensitivity of tularemia microbes of three geographical races to it. Amikacin showed a high therapeutic activity in treatment of albino mice infected with tularemia. The prospects of amikacin use in prophylaxis and treatment of tularemia are defined by its antibiotic activity against streptomycin-resistant forms of the tularemia causative agent.