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This paper studies a full nucleotide sequence of cryptic plasmid pTP33, which was isolated from the typical plague strain of the Tuvinian natural focus, Yersinia pestis I-2638. Sequencing was carried out using the 454 GS Junior platform (Roche). In analysis using the software package GS De Novo Assembler v. 2.7 (Roche) and the algorithm Newbler v. 2.7, 1855 nucleotide reads, which contained 1101246 nucleotides, were assembled to a contig of 33 978 bp. The GC content of the obtained nucleotide sequence was 50.25%. During annotation, we found 56 open reading frames. Homologs of the predicted reading frames were sought in the BLAST databases. We detected 22 reading frames coding hypothetical proteins, 23 frames coding phagerelated proteins, and 11 frames coding proteins with known functions, including toxin–antitoxin system YefM-YoeB, nucleic acids and polysaccharides metabolism proteins (exopolysaccharide production protein ExoZ, exodeoxyribonuclease VIII), and replication proteins (ParA). Some predicted pTP33 proteins were found to be homologs (from 45 to 75%) with sequences of phage-related proteins of certain microorganisms—endosymbionts of insects (Sodalis glossinidius) and endosymbionts of entomopathogenic nematodes (Photorhabdus luminescens, P. asymbiotica, Xenorhabdus bovienii).

It was experimentally established that plague pathogen strains with different plasmid composition variously suppressed the viability of Frontopsylla luculenta luculenta fleas. Dead insects were most frequently observed among those infected with a virulent strain having the cryptic plasmid pTP33. The presence of the avirulent and apesticinogenic plasmid I-3480 in the fleas less deteriorated their state. Biofilm formation by different F.l.luculenta strains in the body was characterized by quantitative and qualitative differences. The strains that had the cryptic plasmid and were able to form the biofilm in the F.l.Iuculenta fleas surpassed the three-plasmid strain I-3230 and their formned aggregates achieved very large sizes and frequently persisted until the end of the experiment. Small solitary masses were generally observed in the insects infected with the three-plasmid strain. Thus, the pTP33 plasmid potentiated the pYT plasmid-encoded ability to colonize the F.l.Iuculenta fleas with the plague pathogen; in this case the products of the pYV and pYP plasmids (or one of them) are toxic to ectoparasites.

The comparative analysis was applied concerning antimicrobic action of different groups of antibacterial preparations on the plague agent strains isolated from Siberian natural focuses of disease. The analysis was applied to results obtained using such different methods as disco-diffusive technique, serial dilution and HiCOMB MIC test. It is established that freshly isolated cultures of Yersinia pestis have high sensitivity to antibacterial preparations of different groups. The results obtained using three technical approaches intercorrelate in significant degree. The HiCOMB MIC test was applied for the first time for analysis Y. pestis strains from natural focuses of plague of Siberia.

Data obtained during feeding of Citellophilus tesquorum aitaicus Ioff, 1936 infested females and males (Siphonaptera: Ceratophyllidae), the main vectors of plague in Tuva natural plague locus, on the natural host and laboratory animal was analyzed. It was found that sexual differences in fleas depended on the type of the host. Females fed more actively on the longtailed ground Citellus undulatus than on white mouse. Alimentary activity of males on these animals was similar. Higher mortality of fed females and males was noted during feeding on mice. Frequency of formation of the "block" and transmission of the pathogen in males was higher during bloodsucking on the ground squirrel; in females, during feeding on mice. Thus, differences in the transmission of the plague pathogen, revealed in laboratory on white mice, can be quite different in nature. So, extrapolation of experimental data on natural processes of interrelations between plague pathogen and ectoparasites must be performed taking into account revealed peculiarities.

The interaction of two Yersinia pestis strains varying in plasmid composition with the fleas Xenopsylla cheopis was studied. The reference virulent strain I-2638 having four plasmids (pCad, pPst, pFra, and pTP 33) and its selected avirulent strain I-3480 that had lost the plasmids pCad and pPst formed a proventricular block in the fleas with equal frequency. There were no differences in the block-forming capacity of these strains among the infected females; however, the stock strain was more active in blocking the proventriculus in females in spring than was the mutant one. in summer. The fleas infected with a defect strain failed to transmit the pathogen. It follows that the presence of a proventricular block is not an indicator of how effectively the fleas transmit the causative agent of plague. While being in the insect, both strains of plague microbe did not alter their biological properties. In experiments, the death rate for insects infected with different strains was similar, but higher in spring than that in summer. The males naturally died more frequently than the females.

The data of a long-term study of the relationships of the plague pathogen with male and female fleas, the vectors of this infection in Siberian natural foci, were analyzed. Gender differences were established in the rate of block formation and vector activity of fleas. In female and male fleas, these indices depend on both the species-specific features of ectoparasites and the pro-feeders used in the experiments, the season of their performance, and keeping conditions. The blocks of the proventriculus more frequently form in males of the majority of flea species, they more actively transmit a plague microbe to animals. The differences in both the rate of proventricular block formation and the pathogen transmission in different seasons were variously shown in males and females. They were clearly marked in one season and insignificant in another. Apparently, flea gender differences may affect the development of an epizootic situation since the sex ratio in these insects may vary in the natural foci of this infection depending on a season.

The results of experiments held in 1982-1983 in Tuva plague natural focus with flea Amphipsylla primaris primaris (Jordan et Rothschild, 1915) from natural populations, whish were inflected and fed on specific host--flat-headed vole (Alticola strelzovi), are analyzed. The initial infectivity of the insects in autumn was higher than in spring: 90 and 50 % respectively. Accumulation of the agent in aggregated form in the organism of A. p. primaris, estimated by the quantity of fleas with <> and partial blocks, was more active in imago of both sexes in autumn than in spring, while sucking flea were observed in spring more often than in autumn. Irrespective of season, the part of males with visible accumulations of Y. pestis was more, and their alimentary activity was higher than that of females. Fleas died much more quickly in spring. Part of the males with proventriculus block exceeded that of females in spring experiment. Females with alimentary canal obstruction prevailed in autumn. Thus, sex of the insect and season of the experiment conducting influenced on all studied indices. Besides that, Y pestis ability for the proventriculus block formation in fleas during different seasons can change by the opposite way depending on sex of the ectoparasites.

Some aspects of relationships of the flea Citellophilus tesquorum altaicus and bacterium Yersinia pestis of two strains isolated from different parts of the Tuva natural plague focus were studied. Peculiarities of elimination and blood meal activity of fleas infected with two strains of the plague agent were not revealed. Differences in mortality and alimentary activity are considerably determined by the sex of insects. The ability of examined strains to form a proventriculus block was not identical in the strains examined. This ability was expressed higher in the strain I-3428, which originated from the same part of the natural focus as the insectarium flea culture, than in the strain I-3327. During the spring and first half of summer, the proventriculus block appeared more frequently in females. The increasing of the fraction of blocked individuals was observed in both sexes from spring to summer. As for the ability to transmit the plague agent, similar seasonal increasing was noted in males, but in females, the ability to inoculate the plague microbe was always maintained at the same level.

Citellophilus tesquorum altaicus fleas from two natural (Karginsky and Barlyksky) populations have been studied. There are differences between the fleas of these populations in susceptibility to plague microbe. The adaptability and abundance of the causative organism in the body are higher in the fleas of the Barlyksky population. The sizes of the head are significantly higher in the females of the Karginsky population. There are no differences in this index between males. According to all 5 studies signs of chaetotaxia, the chaeta abundance is greater in both females and males of the Karginsky population. The fluctuating asymmetry is higher in the Barlyksky fleas that are more susceptible to the studied type of the causative agent of plague.

Frequency of block formation was studied in Xenopsylla cheopis infected with the triptophan-dependent and the typical strains of Yersinia pestis from the Mountain-Altai natural plague focus. It was shown that the rate of blocked males in comparison with that of females was statistically higher. In experiments with the triptophan-dependent strain the frequency of blocking in males was 40.9% and exceeded that in females at four times. The highest rate of blocked males was marked at 7-10th and that of females at 21st days. The tryptophan-dependent strain did not concede to the typical strain in the ability to form a proventricular block in fleas.