[Role of laboratory methods in epidemic control of brucellosis outbreaks in Moscow zoo nursery].

AIM: Evaluation of the role of contemporary methods in epidemic control of brucellosis outbreaks among employees of auxiliary facilities in Moscow zoo nursery. MATERIALS AND METHODS: During 2003-2013, biannually, sera from more than 200 employees of the nursery, that work during periods of epizootics of small and large cattle in nursery auxiliary facilities, were studied. A complex of laboratory methods for brucellosis was used: variations of traditional serologic agglutination method in tubes--Wright's reaction (WR), on glass--Huddleston's reaction, Coombs' anti-globulin reaction; enzyme immunoassay with immune-dominant S-LPS; realtime polymerase chain reaction with primers based on genus target of BCSP31 protein gene. RESULTS: After eradication of sheep brucellosis in 2003, the percentage of nursery facility employees, that react positively to brucellosis, has decreased from 42.7% to 15.9-17.2% in 2005-2006. RT-PCR detected human infection during epizootics 5.8 times more effectively compared with EIA. The repetition of the brucellosis epizootics in 2007 and 2009 among large cattle and a 2-year yak had initiated a rise in infection rate among employees, that had not previously (2003-2006) reacted to brucellosis. Acute clinical forms of brucellosis were not detected in 2012-2013 and antibody titers in EIA in previously infected employees have decreased in the absence ofbrucellosis epizootics in the nursery. 30 employees, infected with brucellosis causative agent, were detected for the entire period of examination, among those 10 individuals had developed clinical forms of brucellosis (6--acute, 4--chronic). CONCLUSION: Epidemic control for 11 years, based on a complex of laboratory methods, allowed to register infection and outbreaks of brucellosis in the nursery employees during epizootics of 2003, 2007 and 2009. RT-PCR--effective method of detection of infection in humans during brucellosis periods in the nursery. EIA--sensitive method during chronic forms of brucellosis in the periods between and after epizootics compared with RT-PCR. A variation of a traditional serologic method of diagnostics has shown a lower sensitivity, informativity with a larger duration of analysis compared with RT-PCR and EIA.

[Molecular genetics typing of Brucella circulating in several provinces of Mongolia].

AIM: Comparative molecular-genetic typing of Brucella strains isolated in Mongolia from different animal species as well as from humans. MATERIALS AND METHODS: Twenty-one strains of Brucella isolated from different hosts in 7 provinces of Mongolia were typed. Conventional phenotypic methods, genotyping by PCR with primers for genus- and species-specific differentiating targets of Brucella genes as well as multiple locus variable number tandem repeats analysis (MLVA) with 12 pairs of primers bounding locus variable tandem repeats of different length (from 134 bp to 8 bp). RESULTS: Phenotypic identification and genotyping by PCR using primers for differentiating DNA markers allowed to attribute 14 isolates to B. melitensis biovar 2, and 7 - to B. abortus biovar 3. By using the MLVA method, connection of MLVA genotypes of 9 Brucella isolates with their reservoir hosts (sheep, cows) was shown providing their circulation in Khentii, Bulqan, and Khubsgul provinces bordering with Russia. Nine isolates from different hosts (camel, yaks, goats, sheep) isolated in Ovorkhangai, Dundgovi, and Dornogovi provinces, which have not border with Russia, had closely related MLVA genotypes indicating an opportunity of migration of pathogenic Brucella species to not-typical hosts. CONCLUSION: Molecular-genetic typing of Brucella isolated in Mongolia was done for the first time; levels of their genetic relation and diversity were demonstrated. Circulation of Brucella isolated with specific MLVA genotypes was connected to territories of specific Mongolian provinces. The study proved migration of Brucella to not-typical hosts. Comparative study of isolates circulating in frontier with Mongolia areas of Russia (Irkutsk region, Tyva and Buryat Republics) are necessary to perform.

[Highly sensitive enzyme-linked assay based on monoclonal antibodies for detection of Brucella antigens].

Mice monoclonal antibodies against lypopolysaccharides (LPS) of Brucella abortus has been obtained and characterized. The antibodies detected LPS of B. abortus, B. melitensis and B. suis with high sensivity and specificity and did not react with LPS of Yersinia enterocolitica O:3, Y. enterocolitica O:9, Salmonella typhimurium, and Francisella tularensis. It has been shown that interaction of monoclonal antibodies and LPS of Brucella species can be critically dependent from buffer system. Obtained monoclonal antibodies allowed to develop highly sensitive assay which was able to detect antigens of Brucella species in concentrations 0.05 - 0.1 ng/ml. The assay can be used for detection and identification of Brucella species.

[Enzyme immunoassay and polymerase chain reaction for evaluation of brucella persistence]. / Ispol'zovanie immunofermentnogo analiza i polimeraznoi tsepnoi reaktsii dlia otsenki persistentsii vozbuditelia brutselleza.

The complex approach, including the use of traditional bacteriological and serological methods, as well as the polymerase chain (PCR) reaction and the enzyme immunoassay (EIA), was used for evaluation of Brucella (the causative agents of brucellosis) persistence in the dynamics of the infectious process in patients with the acute and chronic forms of brucellosis as well as in experimentally infected laboratory animals. Sick humans and experimental animals were found to have positive PCR and EIA reactions at different periods of the disease. The use of these methods makes it possible to evaluate indirectly the persistence of Brucella.

[Use of molecular-biological methods for identifying brucella in a comparative analysis of strains, isolated from sick dogs]. / Ispol'zovanie molekuliarno-biologicheskikh metodov identifikatsii brutsell v sravnitel'nom analize shtammov, vydelennykh ot bol'nykh sobak.

Ten strains isolated from sick dogs in 1998 in St. Petersburg were studied by traditional and molecular biological methods of Brucella identification. PCR study confirmed that the isolated cultures were Brucellae, and comparative study of the traditional phenotypical characteristics and protein and antigenic composition allowed referring all the isolated strains to B. canis. Traditional identification showed similarity of 7 strains with the reference B. canis strain RM6/66, and 3 strains were similar to B. canis Mex 51 strain. These results confirmed the division of B. canis into two biovars. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate demonstrated the identity of protein profiles of 10 strains isolated from dogs to the reference B. canis RM6/66 strain. Immunoblotting analysis with S- and R-specific rabbit antisera also demonstrated the identity of antigens binding IgG antibodies in the strains isolated from dogs to the reference B. canis RM6/66 strain.

[A rapid immunofluorescent method of determining the antibiotic sensitivity of brucella]. / Immunofliuorestsentnyi ékspress-metod opredeleniia antibiotikochuvstvitel'nosti brutsell.

Optimal conditions for rapid assay of Brucella antibiotic sensitivity with the immunofluorescent method were developed. With this method high sensitivity of the main Brucella species to tetracycline, doxycycline and rifampicin was confirmed. It was found actually possible to use the immunofluorescent method for rapid assay of Brucella antibiotic sensitivity in practice.

[Trial of different nutrient media in studying the antibiotic sensitivity of brucellae]. / Ispytanie razlichnykh pitatel'nykh sred pri izuchenii antibiotikochuvstvitel'nosti brutsell.

The possible use of the Unimicon-s nutrient medium for determination of antibiotic sensitivity of Brucella by the methods of serial dilutions in dishes and agar diffusion with paper disks was studied. Previously this method was not used in manipulations with Brucella. The results indicated that the dry Unimicon-s nutrient medium was applicable for determination of Brucella antibiotic sensitivity. It was shown that the agar diffusion method with the use of paper disks may be recommended for determination of antibiotic sensitivity of Brucella. The optimal time for recording the results of the laboratory assays is 48 hours.

[Immunity status of guinea pigs infected with Brucella L forms]. / Izuchenie sostoianiia immuniteta u morskikh svinok, zarazhennykh L-formami Brucella.

B. abortus L-forms injected subcutaneously into guinea pigs adapt in the lymph nodes of the animals in the absence of reversion to normal cells. Complete and incomplete antibodies belonging to macro- and microglobulins (IgM and IgG) were synthetized. The allergic transformation of the organism is faintly pronounced. After this form of infection guinea pigs become resistant to B. melitensis infection for 6 months (the term of observation).

[Cell structure and the pathogenicity of Brucella at different stages of L transformation]. / Struktura kletki i patogennost' brutsell na raznykh étapakh L-transformatsii.

On the basis of changes in the biological properties and morphology of Br. abortus culture under the action of penicillin 3 stages of L-transformation in Brucella were determined. The prevalence of first bacilliform and then typical L-cells and rapid reversion hampering the determination of virulence were characteristic of the initial stage (passages 1-4). Typical L-cells with the wrinkled surface, deep depressions and holes as well as a decrease in virulence and slight pathomorphological changes in the organs of the infected animals were characteristics of the intermediate stage (passages 5-10). Typical L-cells and amorphous masses, a further decrease in virulence, pathomorphological changes of toxic character (only after the injection of L-culture in large doses) were characteristic of the late stage (from passage 11 and further on). At all stages of L-transformation Brucella cultures showed a high reproductive capacity, binary division, the formation of elementary bodies by budding both inside and on the surface of L-cells.

On the pathogenicity of nonstable Brucella L-forms and their revertants.

The pathogenicity of B. abortus 870 L-forms obtained by long-term passaging of virulent culture on media with penicillin and of revertants obtained in vitro and in vivo was studied. L-form cultures stimulated only a mild response of the reticulo-endothelial system of the animal organism, at the same time displaying a certain level of toxicity. In vitro revertants approximated to L-forms, while in vivo revertants stood closer to the initial virulent culture, as regards pathogenicity. This seems to be evidence of a potential danger of brucella L-forms for the human and animal organisms.

[Biological properties and ultrastructure of brucellae during L-transformation and reversion]. / Biologicheskie svoistva i ul'trastruktura brutsell v protsesse L-transformatsii i reversii

There was shown a difference in the biological properties and the ultrastructure of two strains of brucellae, spheroplasts obtained from them under the action of penicillin, L-form and revertants obtained from the L-form. Spheroplasts formation was characterized by a change of brucellae into R-form and some virulence reduction. The cells had an outer and a cytoplasmic membranes, and usually lost their capacity to binary division. L-forms were obtained during the 9th and the 35th passage on a medium with penicillin; their formation was accompanied by the change in serological properties of the culture and significant reduction of the virulence; the cells were characterized by a marked polymorphism and the capacity to budding; they had 2 membranes on the cell surface and an intensively developed system of intracytoplasmic membranes. The revertants formed on the medium without penicillin during the 16th-30th passage or spontaneously on the medium with penicillin. They differed from the initial strains of brucella culture by a marked increase in penicillin-resistance, by the changes in serological properties, and also by polymorphism of cells, capable, however, of binary division.

[Study of the L-transforming action on Brucella cells of tetracycline, streptomycin, rifampicin and their combinations with penicillin in in vitro experiments]. / Izuchenie L-transformiruiushchego deistviia na kletki brutsell tetratsiklina, streptomitsina, rifampitsina i ikh kombinatsii s penitsillinom v opytakh in vitro

The in vitro study of the possible formation of L-forms of Brucella under the effect of tetracycline, streptomycin, rifampicin or their combinations with penicillin showed that passages of various Brucella species on media containing increasing concentrations of the antibiotics resulted in insignificant morphological changes in the cells. Passages of the same cultures on media containing combinations of the above antibiotics with penicillin resulted in more significant changes in the cell morphology, up to formation of spheroplasts. No L-transformation was observed under the experimental conditions.