[Assessment of rate of infection with agents of bacterial infections in ticks captured on one of the Moscow park terrains].

AIM: To study the rate of infection of ticks captured one of the Moscow park terrains with bacteria (agents of tick borreliosis and anaplasmosis). MATERIALS AND METHODS: Rates of infection of dried ticks with agents of main tick-borne bacterial infections (tick borreliosis and anaplasmosis) were determined by nested PCR. RESULTS: In May-June 2006, 76 ticks (40 adult females, 36 adult, males) belonged to Ixodes ricinus species were captured by the method "on flag". Number of ticks on the chosen terrain was 1.77 ticks per km2. 22.4% of ticks (12 females and 5 males) were positive for the agent of tick borreliosis--spirochete Borrelia burgdorferi sensu lato which is pathogenic for humans. The main detected pathogen was Euro-Asian genovariant of B. garinii--7 female and 5 male ticks (70.6% from total number of infected ticks) were infected with it. Five female ticks were infected with genovariant of B. afzelii. One female tick (1.2%) was infected with B. valaisiana. CONCLUSION: Anaplasma A. phagocytophilum causing human granulocytic anaplasmosis was not detected in captured adult ticks.

Effect of proteins from the spirochete Borrelia burgdorferi sensu lato on myelinated nerve excitability.

We studied the effect of spirochete Borrelia burgdorferi sensu lato cell membrane proteins on excitability of myelinated nerve fiber. It was found that cell surface proteins of spirochetes B. burgdorferi s. s. bind to Ranvier nodes of the axon and to Schwann cells. Binding of B. burgdorferi s. s. and B. garinii to the nerve fiber modulates the amplitude and conduction velocity of the action potential, while B. afzelii had no effect on these parameters. The decrease in the spike amplitude and conduction velocity during sorption of B. burgdorferi s. s. or cell wall proteins was accompanied by desorption of membrane-bound calcium.

Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus.

The tick Ixodes ricinus is responsible for the transmission of a number of bacterial, protozoan and viral diseases to humans and animals in Europe and Northern Africa. Female I. ricinus from England, Switzerland and Italy have been found to harbour an intracellular alpha-proteobacterium, designated IricES1, within the cells of the ovary. IricES1 is the only prokaryote known to exist within the mitochondria of any animal or multicellular organism. To further examine the distribution, prevalence and mode of transmission of IricES1, we performed polymerase chain reaction screening of I. ricinus adults from 12 countries across its geographic distribution, including tick colonies that have been maintained in the laboratory for varying periods of time. IricES1 was detected in 100% of field-collected female ticks from all countries examined (n = 128), while 44% of males were found to be infected (n = 108). Those males that are infected appear to harbour fewer bacteria than females. Sequencing of fragments of the 16S rRNA and gyrB genes revealed very low nucleotide diversity among various populations of IricES1. Transmission of IricES1 from engorged adult females to eggs was found to be 100% (n = 31). In tick colonies that had been maintained in the laboratory for several years, a relatively low prevalence was found in females (32%; n = 25). To our knowledge, IricES1 is the most widespread and highly prevalent of any tick-associated symbiont.

[Designing and clinical testing of immune-enzyme and immunofluorescence test systems for serodiagnosis of ixodes borreliosis]. / Razrabotka i klinicheskaia aprobatsiia immunofermentnykh i immunoliuminestsentnykh test-sistem dlia serodiagnostiki iksodovykh kleshchevykh borreliozov.

The methods of immune enzyme assay (MIEA) and of lanthanide immunofluorescence analysis (LIFA) were used to work out the test systems for the detection (in blood serum of patients) of specific IgM IgG antibodies to the B. burgdorferi spirochete--a causative agent of ixodic borrelioses. The test system was clinically tested versus the indirect immunofluorescence reaction (IIFR) and commercial immune enzyme test system (CIET). The results of antibodies' detection were shown to correlate with the analysis data for the same sera in IIFR and to be in line with a real presence or absence of the disease. Test systems based on LIFA were proven to be most sensitive and specific.

Epitope mapping of the outer surface protein A (OspA) of the spirochete Borrelia burgdorferi using a panel of monoclonal antibodies and lanthanide competition fluoroimmunoassay.

A panel of fourteen different monoclonal antibodies was used for detection and analysis of antigenic determinants located on the outer surface protein A (OspA) of the spirochete Borrelia burgdorferi, which is a causative agent of tick-borne borreliosis (Lyme disease). Two main and several minor partially overlapping antigenic determinants have been found on the surface of the OspA protein of Borrelia burgdorferi sensu stricto (strain 297) by lanthanide competition fluoroimmunoassay. One of the main antigenic determinants is located in the N- and the other in the C-half of the OspA molecule. The involvement of the OspA protein in intact Borrelia burgdorferi sensu stricto (four bacterial strains have been analyzed: 297, B31, FR90-594, and CA90-742) is associated with retention of the above-mentioned two major antigenic determinants, but unlike the case of the isolated OspA they are partially overlapping with each other and with other antigenic determinants. The protein of the spirochete Borrelia afzelii (two bacterial strains have been analyzed: Ip-21 and Pko) contains only one antigenic determinant, which is the same as the main determinant of the OspA protein of Borrelia burgdorferi sensu stricto located in the N-half of the OspA molecule.

Alteration in the antigenic structure of M1 protein of influenza A virus mutant resistant to a new antiviral compound mopyridone.

Using 14 monoclonal antibodies (MoAbs) in solid-phase ELISA it was found that influenza virus A/Hong Kong/1/68 (H3N2) mutants resistant to the antiviral compound mopyridone as compared to the mopyridone-sensitive mutant manifested significant changes in the antigenic structure (sites 1A, 2 and 3) of M1 protein. No differences in M1 were found between rimantadine-resistant and rimantadine-sensitive mutants of influenza virus A(H3N2).

Detection of tick-borne encephalitis virus in ixodid ticks collected in natural foci by time-resolved fluoroimmunoassay.

Time-resolved fluoroimmunoassay (TR-FIA) was used for the first time for evaluation of infestation of ixodid ticks with tick-borne encephalitis virus. Comparison of TR-FIA results with those obtained in enzyme immunoassay and by virus isolation confirmed the high efficacy of the method in question. Positive results of TR-FIA coincided with the data of virus isolation in 83.6% cases, the level of false-negative results did not exceed 1.2%, the overall time consumption amounted to about 1.2 hr.

Analysis of internal proteins of influenza A (H2N2) viruses isolated from birds in East Germany in 1983.

Proteins and RNAs of influenza A (H2N2) viruses isolated from birds in 1983 in East Germany were compared antigenically with those of H2N2 human strains. The electrophoretic mobility of the viral proteins and of the S1-treated double-stranded RNAs from two human and six avian strains, as well as the results of EIA-tests using monoclonal antibodies to their matrix protein and nucleoproteins indicate an antigenic relationship between the avian isolates and human strains of H2N2 subtype. One of the avian strains had a reduced amount of matrix protein.

[The use of lanthanide immunofluorescence analysis for demonstrating the antigen of viral hepatitis A]. / Primenenie lantanidnogo immunofliuorestsentnogo analiza dlia indikatsii antigena virusnogo gepatita A.

The study dealt with the development of an express method for detection of hepatitis A virus (HAV) antigen employing lantanides, especially europium. The new time-resolved fluoroimmunoassay (TRFIA), alongside with a significant shortening of the time for reaction, was also 8 times as sensitive as the analogous enzyme immunoassay. The TRFIA may be used effectively both for control of the antigen amplification in HAV-infected cell culture and for early diagnosis of acute forms of viral hepatitis A.

[The detection of antibodies to HIV protein p24 in human blood sera by the method of lanthanide immunofluorescent analysis]. / Vyiavlenie antitel k belku p24 VICh v syvorotkakh krovi cheloveka metodom lantanidnogo immunofliuorestsentnogo analiza.

A competitive time-resolved fluoroimmunoassay (TR-FIA) system for the detection of antibodies to protein p24 of HIV was developed on the basis of monoclonal antibodies. The advantages of this test system over analogous enzyme immunoassay system and commercial test system "Antigen" (USSR) were demonstrated. The newly developed test system of TR-FIA was used for examination of sera from HIV-infected persons.

[The effect of detergents on the physicochemical and immunochemical properties of the isolated M1 protein of the influenza virus]. / Vliianie detergentov na fiziko-khimicheskie i immunokhimicheskie svoistva izolirovannogo belka M1 virusa grippa.

The degree of solubility of influenza virus protein M1 preparations isolated from virions by acid chloroform-methanol extraction was studied under the effect of a wide spectrum of detergents of different origin. The same detergents were used for solution of a lipid comprising a part of artificially formed liposomes. Only some of the detergents used (sodium dodecyl sulfate, SDS, triton X-100, and disintegron-B) were shown to be optimal for solution of both influenza virus protein M1 and lipid. The degree of effect on the immunochemical properties of protein ML isolated from influenza virus virion of the above-mentioned detergents optimal for solution was also studied. For this purpose, a panel of 18 monoclonal antibodies with different determinant specificity to protein M1 was used. Two of the three detergents (SDS and disintegron-B) were shown not to change the antigenic profile of protein M1. The immunochemical properties of protein M1 of influenza virus isolated from virions by two methods: chloroform-methanol extraction and preparative polyacryl amide gel electrophoresis, were studied. These two methods of protein M1 isolation were shown not to alter its immunochemical properties.

[The demonstration of the Venezuelan equine encephalomyelitis virus by the method of indirect lanthanide immunofluorescent analysis]. / Indikatsiia virusa venesuél'skogo éntsefalomielita loshadei metodom nepriamogo lantanidnogo immunofliuorestsentnogo analiza.

A test system of indirect time-resolved fluoroimmunoassay (TR-FIA) was developed and tested on an alpha-virus, Venezuelan equine encephalomyelitis virus. The indirect TR-FIA test system was shown to be highly effective in the detection of antigens of this virus. Not differing in specificity from the direct TR-FIA, the newly developed test system was 4 times as sensitive.

[The strain-specific diagnosis of influenza by using lanthanide immunofluorescence analysis based on monoclonal antibodies to the hemagglutinin of the influenza A virus]. / Shtammospetsificheskaia diagnostika grippa s ispol'zovaniem lantanidnogo immunofliuorestsentnogo analiza na osnove monoklonal'nykh antitel k gemaggliutininu virusa grippa A.

Nine monoclonal antibodies (MCA) to hemagglutinin of influenza A/Taiwan/1/86 (H1N1) virus and 5 MCA to influenza A/Mississippi/1/85 (H3N2) virus were generated and characterized. The MCA were used for the development of diagnostic test systems on the basis of time-resolved fluoroimmunoassay. The same MCA were used as primary and detecting antibodies in the test system specific for HA of the H1 serosubtype, whereas in the test system specific for influenza A serosubtype H3 virus MCA of different epitope appurtenance were used as primary and secondary antibodies. The sensitivity of the test system for HA of serosubtype H1 was found to be 10 ng/ml and that for serosubtype H3 5 nh/ml. The developed test systems were tried on the clinical material collected during the epidemic periods of 1983-1989.

[The differentiation of viruses in the Venezuelan equine encephalomyelitis complex by using monoclonal antibodies and lanthanide immunofluorescence analysis]. / Differentsiatsiia virusov kompleksa venesuél'skogo éntsefalomielita loshadei s primeneniem monoklonal'nykh antitel i lantanidnogo immunofliuorestsentnogo analiza.

Potentialities of differentiation between Venezuelan equine encephalomyelitis (VEE) complex viruses by time-resolved fluoroimmunoassay and enzyme immunoassay were studied. For this, 4 test systems were used based on different combinations of native and labeled polyclonal antibodies to VEE virus, strain Trinidad, and monoclonal (MCA) antibody MAK 14-7 to protein EL of this virus. The maximal sensitivity and specificity was achieved in the test system formed from native MCA MAK 14-7 for sensitization of the solid phase and labeled polyclonal immunoglobulins for demonstration of the test results. This combination of antibodies allowed to differentiate the epidemic variant of VEF/Trinidad (IA) from epizootic variants of Mucambo (III), Pixuna (IV) and attenuated strain No. 230.

[The design and trial of conjugates for performing lanthanide immunofluorescence analysis]. / Konstruirovanie i aprobatsiia kon''iugatov pri postanovke lantanidnogo immunofliuorestsentnogo analiza.

The possibility of using a number of complexons for labeling of antibodies to Venezuelan equine encephalomyelitis virus and to adenovirus with europium ions was studied. The resultant conjugates, irrespective of the type of complexon, were shown to retain their immunochemical activity and could be used for lanthanide immunofluorescence analysis of virus-specific antigens.

[The use of sectional polystyrene plates in the set-up of lanthanide immunofluorescence analysis]. / Ispol'zovanie razbornykh polistirolovykh planshetov pri postanovke lantanidnogo immunofliuorstsentnogo analiza.

The authors have examined the possibility of using sectional polystyrene plates, made in this country, in time-resolved fluoroimmunoassay (tr-FIA) with Venezuelan equine encephalomyelitis and tick-borne encephalitis arboviruses, and with influenza A virus. The plates presensitized with specific antibodies were found fit for the detection of the antigens of the above viruses. These plates are not recommended for the detection of influenza A virus-specific proteins adsorbed directly onto the microplate surface.