[Detection of Babesia canis (Piroplasmida) DNA in the blood samples and lysates of the ticks Dermacentor reticulatus (Ixodidae) collected in the Tula and Moscow Regions].

Chimeric primers, the sensitivity and specificity of which allow them to be used in both the clinical setting and the epizootological assessment of tick infection by a real-time polymerase chain reaction (RT-PCR) assay, have been designed against Babesia canis infection. The findings suggest that a large number of Babesia DNA copies are detectable in the blood in acute babesiosis. Some animals that had experienced babesiosis developed blood B. canis carriage--a small number oftrophozoites remained alive for a long time. When babesiosis was suspected, its diagnosis could be confirmed by RT-PCR in half of dogs with subclinical signs. The tick concentration of Babesia ranged from several hundred to a few thousand parasites. There were no significant differences in the number of Babesia parasites in the infected ticks in relation to their collection site. However, the occurrence of infected ticks was significantly higher in the places of constant contact with a canine population, which is indicative of the decisive role of dogs in the intensity of an epizootic process in the foci of B. canis infection.

[Immunomodulating effect of an Ixodes persulcatus (Ixodidae) tick salivary gland extract on BALB/c mice lymphocytes in an in vitro system].

The immunomodulating effect of the components of an Ixodes persulcatus (Ixodidae) tick salivary gland extract (SGE) on BALB/c mice lymphocytes was evaluated. SGE of partially engorged ticks at a concentration of 50 microg/ml causes the maximum suppression ofT- and B-lymphocyte subpopulations. SGE of hungry ticks at the same concentration induces the suppression of only CD69+ T cells and TLR-2+ B cells, but produces no suppressive effect on CD69+ B lymphocytes, TLR-2+ T lymphocytes, and TLR-4+ T and B lymphocytes. SGE shows different effects on the synthesis of IFN-gamma and IL-4 by T helper cells. SGE of hungry ticks stimulated the increase of IFN-gamma and IL-4 synthesis by 4.7 and 2.6 times, respectively, as compared to the control. The findings may be of value in studying the pathogenesis of transmissible infections and in designing the vaccines based on tick gland components.

[Search for protective antigens in Ixodes persulcatus (ixodidae) salivary gland extracts].

RT-PCR evaluation of the activity of eight Ixodes persulcatus salivary gland genes shows clear distinctions in their expression depending of the stage of tick feeding. Out of them, only Salp 10 and Salp 15 proteins may be regarded as candidates for protective antigens to develop anti-tick and anti-Borrelia vaccines. Firstly they play an important role in feeding a tick and modifying a host's immune response. Secondly, the increasing expression of the salp 10 and salp 10 genes begins at early tick feeding stages. Thirdly, the activity of these genes increases with the beginning of feeding by tens and hundreds times and keeps at this level until the third tick feeding stage is over.

[Changes in the expression of salivary gland genes in Ixodes persulcatus (Ixodidae) depending on the stage of tick feeding].

By using the guanidine-isothiocyanate test, the authors isolated a summary RNA preparation from Ixodes persulcatus salivary gland extracts. Activity products of the genes responsible for the expression of some salivary proteins were first identified using the RT-PCR. It has been shown that, firstly, I. persulcatus synthesizes at least 3 transcripts homologous to the respective salivary components of the related species I. scapularis, the translation product of which is likely to be immunodominant antigens; secondly, the number of each of these transcripts, as in I. scapularis, depends on the stage of tick feeding. The changes in the expression of each transcript are specific: monotonously increasing changes in Salp 17 and cyclic ones in Salp 16, and synthesis, only when the ticks are fully ingested, in Salp 25.

[Evaluation of in vitro antibacterial activity of fosmidomycin and its derivatives].

Unlike the mammals, some species of pathogenic microorganisms synthesize isoprenoids by the mevalonate-independent pathway known as the methyl-erythritol phosphate pathway (MEP). The macromolecules of the polyprenyl compounds play an essential role in the metabolism of the microbial cell. Therefore, the MEP enzymes can be targets for new antibiotics. Antibacterial activity of fosmidomycin, an inhibitor of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), the key enzyme of MEP in isoprenoid biosynthesis was estimated. By the results of the in vitro experiments the tested microorganisms were divided into susceptible and resistant to fosmidomycin. Vaccinal strains of B. anthracis and practically all the strains of P. aeruginosa were included into the first group. The minimum inhibitory concentrations of fosmidomycin for them determined by the method of serial dilutions were 1-8 mcg/ml. The second group for which the MICs were 16-64 mcg/ml included representatives of Listeria, Yersinia and Burkholderia. The tested species of enteric bacteria, Mycobacterium, Corynebacterium, Campylobacterium and the tularemia vaccinal strain were fosmidomycin resistant. The MICs for them varied from 128 to 512 mcg/ml. Since all the above mentioned bacteria have DXR, resistance to fosmidomycin was conjectured with the difficulty of its delivery to the target in the microbial cell. To increase penetrability of fosmidomycin, various functional groups modifing its hydrophoby were added to the antibiotic molecule. However, no expected increase of the susceptibility to the derivatives was achieved probably because their affinity to DXR lowered. Penetrability of fosmidomycin to the cell was facilitated by using its combinations with compounds influencing the integrity of the bacterial cell membrane. Combined use of fosmidomycin with polymyxin B, chlorhexidine and cetrimide 4-64 times lowered its MICs for the strains of Listeria, Burkholderia and Yersinia.

[Isolation and characteristics of a temperature-sensitive plasmid pRP19.6--an RP1 derivative containing the duplicated IS21 sequence]. / Poluchenie i kharakteristika termochuvstvitel'noi plazmidy pRP19.6--proizvodnoi RP1, soderzhashchei duplitsirovannuiu posledovatel'nost' IS21.

When analysing the antibiotic resistant, temperature-independent derivatives of Proteus mirabilis cells, carrying the plasmid RP1ts12, a derivative of the latter (pRP19.6) with an elevated frequency of integration into E. coli K12 chromosome, has been isolated. The structure and properties of pRP19.6 was studied. As revealed from the data of structural and genetic analyses pRP19.6 is identical to the factor R68.45 described earlier by Haas and Holloway. Similarly to R68.45, the plasmid under study contains two copies of IS21 sequence and mobilises nonconjugative plasmid pBR325 with high efficiency. Using the temperature sensitive replication of pRP19.6, frequency of it's integration into the chromosomes of E. coli rec+ and recA- stains is determined. It is demonstrated that the clones carrying the plasmid in integrated state are Hfr-strains. The possibilities to use the temperature sensitive R68.45 like plasmid for isolation of Hfr-strains in the broad range of gram-negative bacteria and for insertional inactivation of chromosomal genes are discussed.

[Deletions of plasmid pRP3.1ts12 derived from RP1 leading to the suppression of the thermosensitive ts12 mutation and mucoid phenotype induction in Escherichia coli K-12]. / Deletsii plazmidy pRP3.1ts12--proizvodnoi RP1, privodiashchie k supressii termochuvstvitel'noi mutatsii ts12 i induktsii mukoidnogo fenotipa u Escherichia coli K-12.

Properties of a temperature-sensitive in replication mutant pRP3.1ts12 derived from the broad host range RP1 plasmid have been studied. pRP3.1ts12 is a shortened variant of the temperature-sensitive RP1ts12 mutant carrying a deletion in a region from 2.3 to 7.6 MD. In contrast to RP1ts12, the plasmid pRP3.1ts12 is a leaky ts mutant and is characterized by an elevated frequency of reversions to the temperature-independent phenotype. Temperature-independent derivatives of pRP3.1ts12 were studied. Approx. 15% of these were found to induce mucoid growth of the host cells. As revealed from restriction endonuclease analysis, most of the latter derivatives contain deletions of small DNA segments in the region 0.56 to 2.3 MD of the RP1 map. The possible nature of the gene(s), whose deletions suppress the temperature-sensitive ts12 mutation and results in superproduction of Escherichia coli capsular poly-saccharide is discussed.

[Comparative analysis of use of two strains of various genotypes of Borrelia burgdorferi sensu lato as antigens for antibody identification in Ixodes tick borreliosis by indirect immunofluorescence]. / Sravnitel'nyi analiz rezul'tatov ispol'zovaniia dvukh shtammov razlichnykh genovidov Borrelia burgdorferi sensu lato v kachestve antigenov dlia opredeleniia antitel pri iksodovykh kleshchevykh borreliozakh metodom nepriamoi immunofluorestsentsii.

The results of the indirect reaction of immune-fluorescence (IRIF) was studied in testing 49 sera of 19 patients with Lyme-borreliosis with antigens of genotypes Borrelia afzeli (strain Jp-21) and Borrelia burgdorferi sensu stricto (strain No. 17); rabbit fluorescini-sothiocyanate-marked conjugates to human immunoglobulins M and G as well as polyvalent conjugate were used of. No reliable differences were found between all positive and all negative results. The biggest portion of positive results was registered in tests with anti-G-conjugate (up to 92% with strain No 17).

[Borrelia burgdorferi s.s. length and its variability]. / Dlina Borrelia burgdorferi s.s. i ee izmenchivost'.

The length of 469 Borreliae burgdorferi s.I. from the Ixodes ricinus and I. persulcatus images collected in the Moscow Region, that of 5433 B. burgdorferi s.s from the I. persulcatus nymphs and images cultured at a laboratory, and B. burdorferi s.s. grown on the BSK-II (1 and 10 passages) were measured. There was a wide range of variations in the length of specimens (3-74 microns) and in those of this group average sizes (10.7-24.8 microns). The lengths of Borelliae from natural and laboratory ticks after their molt were 17-18 microns. When the ticks were kept in the refrigerator as long as 1-2 years, the length of Borreliae decreased to 10.7-10.9 microns, upon multiple (10) passages on the BSK-II medium, their lengths increased to 24.8 microns (the differences being significant). When the length of Borreliae reduced due to their keeping in the refrigerator, their pathogenicity for albino mice diminished. This disappeared after multiple BSK-II medium passages. It is suggested that the length of Borreliae may serve as a marker of their pathogenicity.

Prevalence of borreliosis, anaplasmosis, ehrlichiosis and Dirofilaria immitis in dogs and vectors in Voronezh Reserve (Russia).

Most of the dogs studied for the prevalence of CVBD have previously received acaricidal and insecticidal treatments. In the present work, a very specific population of dogs (Group 1) that had never been treated against ticks and mosquitoes was studied. Moreover, the territory occupied by this population has also never been treated, because it is a protected area--Voronezh Natural Reserve. Canine patients from veterinary clinics (Group 2) that had been treated against VBD vectors were studied for comparison. Eighty-two dogs (Group 1) were enrolled in June, 2008. Blood samples were tested using the IDEXX SNAP(®) 4Dx(®) test. A specific heartworm antigen was detected in 12.2% samples. The seroprevalence for Anaplasma phagocytophilum was found to be 34.1%. The antibodies to Borrelia C6 peptide and to Ehrlichia canis were detected in 2.4% of the samples. Almost all dogs with infections had no clinical signs. Only 3 mixed-infected dogs showed non-specific clinical signs. During the tick season, 358 Ixodes ricinus were collected; the prevalence of Borrelia burgdorferi s.l. and Anaplasma phagocytophilum was 21.9% and 0.6%, respectively. Four hundred and forty dogs (Group 2) were studied for comparison. Antibodies to B. burgdorferi s.l. were detected only in one dog, seroprevalence for A. phagocytophilum represented 1.1%, no E. canis seropositive dogs were identified, and 8.2% dogs were found infected with Dirofilaria immitis. Fifty-six percent of dogs with dirofilariosis had clinical signs. All dogs with anaplasmosis showed specific clinical signs--fever, anemia, splenitis. Three dogs died within a few days.

Quantitative analysis of C-bands based on optical density profiles in human chromosomes.

A method of quantitative analysis of C segments in human chromosomes 1, 9, and 16 based on longitudinal densitometry has been developed. The way of fitting apparatus data to visual estimates is represented. Density curve parameters not dependent on stain intensity were used. The general C band length error is approx. 0.05 micrometer. Heteromorphic chromosome 16 pairs have been investigated with this method. A significant difference (about 0.18 micrometer) between C bands of the homologues has been detected in chromosome 16. It has been calculated that C bands can be distinquished if the mean difference between the length of the homologues is more than 15%.