[Molecular-genetic characterization of canine and rangiferine brucella isolates from different regions of Russia].

GOAL: Comparative molecular-genetic characterization of Brucella isolates from dogs and reindeers in Russia by molecular-genetic typing methods. MATERIALS AND METHODS: 19 canine and 2 rangiferine Brucella isolates were studied by molecular typing methods based on PCR for differential species and biovar specific molecular targets and MLVA (multiple locus variable number tandem repeats analysis) using primers to 12 known variable loci. RESULTS: Using PCR for differential molecular targets, canine Brucella isolates were characterized as B. canis and rangiferine isolates as B. suis biovar 4. MLVA revealed 5 identical and 7 variable MLVA loci. Using the dendrogram. all the isolates on the data of 12 loci were classified into the close related cluster. On the other hand, high discrimination power of MLVA with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.9842 was shown to reveal genetic diversity for the isolates of 17 MLVA genotypes. CONCLUSION: B. canis and B. suis isolates from different geographical regions in Russia were genetically close related, thereby confirming known genetic relationship between these species. Related MLVA genotypes of isolates were connected to certain regions of preliminary isolation in Russia. To improve the system ofbrucellosis surveillance in Russia MLVA typing of more canine and rangiferine Brucella isolates having epidemiological danger for humans is required to be studied.

[Use of multiple locus variable number tandem repeats analysis for the Brucella systematization].

UNLABELLED: The methods of molecular-genetic differentiation to strain level acquire increasing significance in the current system of struggle with brucellosis. MLVA (multiple locus variable number tandem repeats analysis) was selected for molecular-genetic differentiation to strain level and simultaneous establishment of the genetic relationship of investigated Brucella strains. The goal of this work was MLVA typing of three pathogenic Brucella species strains with the analysis of stability of chosen loci, discrimination power and concordance to conventional phenotypic methods of the Brucella differentiation for use in systematization of brucellosis causing agents. MATERIALS AND METHODS: Twenty six Brucella strains representing reference (n = 15), vaccine (n = 2) and field strains of three pathogenic Brucella species were tested: B. melitensis (n = 3), B. abortus (n = 2), B. suis (n = 2), and isolates (n = 2) with unidentified taxonomic position using MLVA with 9 pairs primers on known variable loci of Brucella genome. The analysis of the stability of chosen loci, discrimination power on Hunter-Gaston discrimination index (HGDI) and consistency to phenotypic methods of identification was performed. RESULTS: MLVA was confirmed for the results of phenotypic methods of identification, stability of the chosen loci in majority reference, and vaccine strains with a high index of variability HGDI 0.9969 for all loci. A dendrogram was plotted on the basis of MLVA data on distributed Brucella strains in related clusters according to its taxonomic species and biovar positions and construction of 25 genotypes. B. melitensis strains formed cluster related to the reference strain of B. melitensis 63/9 biovar 2. Australian isolates of Brucella 83-4 and Brucella 83-6 isolated from rodents formed a cluster distant from other strains of Brucella. CONCLUSION: MLVA is a promising method for differentiation of Brucella strains with known and unresolved taxonomic status for their systematization and creation of MLVA genotype catalogue that will promote qualitative improvement of brucellosis surveillance system in Russia.

[Genetic characterization of the Brucella melitensis isolates from Mongolia, Russia, and Azerbaijan].

UNLABELLED: The goal of this work was to provide comparative genetic characterization of the human and animal Brucella melitensis isolates from Mongolia, Russia and Azerbaijan using current molecular-genetic typing methods. MATERIALS AND METHODS: Twenty eight Mongolian (n = 18), Russian (n = 6), and Azerbaijan (n = 4) human and animal Brucella melitensis isolates were studied using 2 molecular typing methods based on PCR for differential species and biovar specific ORF (open reading frames) molecular targets and MLVA (multiple locus variable number tandem repeats analysis) using primers to 12 known loci. RESULTS: The PCR was used for differential molecular targets (all B. melitensis isolates) were characterized as the B. melitensis biovar 2. The MLVA revealed 7 identical and 5 variable MLVA loci. All the isolates were classified into 25 genotypes using the dendrogram on the data of 12 loci and the cluster related to reference strain B. melitensis 63/9 biovar 2. The B. melitensis isolates having related MLVA genotypes were connected to Mongolian, Russian and Azerbaijan regions. The circulation for two B. melitensis isolates to not typical hosts as camel and yak was demonstrated using molecular typing methods. CONCLUSION: The genetic characterization of twenty eight B. melitensis isolates from different geographical regions in Mongolia, Russia, and Azerbaijan recognized genetic relationships. On the other hand, the MLVA has high discrimination power with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.9841 revealing genetic diversity for the isolates forming of 25 MLVA genotypes. To improve the system of the brucellosis surveillance in Russia MLVA typing of B. melitensis isolates are necessary to investigate from the Siberian (Republics Tuva, Buryatia, and Irkutsk region) and South (Republics Dagestan, Kalmykia, and Stavropol region) Districts having frontier areas with Mongolia and Azerbaijan.

[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.

[Modeling of Brucella persistence in macrophage-like cells in vitro].

AIM: To develop model of chronic brucellosis infection in macrophage-like cells in vitro and to study properties of persistence of Brucella in them. MATERIALS AND METHODS: Infection of macrophage-like cells U937 and phagocytes B10.MLM with analysis of B. melitensis 16M intracellular growth and persistence. RESULTS: Dependence of intracellular growth and persistence of B. melitensis 16M strain in macrophages U937 from infection's multiplicity (IM) and activation of U937 cells, but notfrom preliminary intracellular adaptation of Brucella was demonstrated. Main parameters of infection (IM, centrifugation during phagocytosis, and time of phagocytosis) with B. melitensis 16M strain was modeled and their influence on persistence of the strain in B10.MLM phagocytes was studied. Centrifugation during phagocytosis resulted in development of long-lasting persistence of B. melitensis 16M in B10.MLM phagocytes. Differences in persistence of Brucella in B10.MLM phagocytes compared with U937 macrophages were demonstrated. CONCLUSION: Intracellular persistence of Brucella in B10.MLM phagocytes depends from high antibacterial activity of the latter. Phagocytes B10.MLM could be used in assays for testing chemical compounds' activities against intracellular invasion and persistence of Brucella on early stages of infection.

[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.

[Molecular basis of Brucella persistence].

This review focuses on Brucella persistence. Data on Brucella--macrophage interaction and the role of molecular-genetic systems including homologues of Mos operon Rhizobium, a type IV secretion system, a flagellum apparatus and a "quorum sensing"--virulence factors using signal or effector molecules are updated. Brucella enters macrophages through lipid raft microdomains, avoids its bactericidal attacks, phagolysosome fusion, expressing a set of virulence genes and inhibits TNF-alpha secretion and apoptosis for persistence in macroorganisms. Comparative whole-genome microarray analyses reveal genomic islands, limited genome diversity in Brucella species and also alterations and deletions of genes responsible for virulence.

[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.

[Molecular bases for brucella virulence]. / Molekuliarnye osnovy virulentnosti brutsell.

The authors review published reports on the molecular bases of Brucella virulence, including type IV secretion proteins, S-lipopolysaccharide biosynthesis enzymes (O-antigen), regulatory proteins of various systems, and cellular metabolism proteins. High efficiency of modified transposon mutagenesis technique (selective labeled transposon mutagenesis) in search for virulence genes is shown. Analysis of DNA sequences of Brucella genome promotes identification of new virulence factors.

[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.

[Sensitivity and adaptation of various Brucella species to acid shock]. / Chuvstvitel'nost' i adaptatsiia razlichnykh vidov Brutsell k kislotnomu shoku.

The sensitivity to acid shock (pH 2.5, 2.8, 3.0, and 3.3) and the capacity to adaptive acid tolerance response (ATR) is studied in the reference Brucella strains differing by the origin, biological, and virulent properties. The ability of Brucella to survive under conditions of acid pH depended on the medium composition, growth phase, and preliminary adaptation at pH 5.6. B. suis biovar 1 and B. melitensis biovar 3 were more resistant to low pH than other Brucella species. Adaptation by overnight growth at pH 5.6 (stationary phase) induced ATR to the acid shock (pH 3.3-3.0) in all Brucella species except B. abortus biovar 1. ATR induced in B. melitensis biovar 2, B. canis, and B. neotomae was more marked, with more than an 100-fold increase in survival of adapted cultures, while adapted B. suis biovar 1, B. melitensis biovars 1 and 2 showed a 2-24-fold increase in survival rates. The detected differences in acid sensitivity and ATR of various Brucella species may be useful for identification and characterization of Brucella strains.

[Increased production of the protein antigen of Brucella melitensis in Escherichia coli K-12 cells]. / Povyshennaia produktsiia belkovogo antigena Brucella melitensis v kletkakh Escherichia coli K-12.

The production of Brucella melitensis protein antigen with molecular weight of 38 kD in Escherichia coli K-12 cell lysates has been studied by immunoblotting with various antisera. E. coli strains differed by the vector plasmid and the size of B. melitensis 565 DNA fragment with 38 kD protein gene, cloned in this plasmid. The immunoblotting analysis detected increased production of 38 kD protein in the recombinant GSE830 strain in comparison with the B. melitensis strain 565, from which the gene was cloned, and other E. coli strains containing this protein gene. The production of 38 kD protein was determined by the size of the cloned B. melitensis 565 DNA fragment with this protein gene, but not by the conditions of culturing.

[Use of Brucellar protein antigen synthesized in Escherichia coli cells in the enzyme immunoassay]. / Ispol'zovanie v immunofermentnom analize belkovykh antigenov brutsell, sintezirovannykh v kletkakh Escherichia coli.

The possibility of using brucellar protein antigens with mol. weights of 18 and 38 kD, synthesized in E.coli cells, as sensitins on polystyrene plates in the enzyme immunoassay (EIA) for the detection of antibodies in the blood sera of patients with different forms of brucellosis. The use of antigens with mol. weights of 18 and 38 kD made it possible to detect antibodies in patients with the acute form of the disease in 68.97% and 75.86% of cases, and in patients with the chronic form in 33.3% and 22.9% of cases respectively. The main advantage of using antigen with a mol. weight of 38 kD in EIA was its specificity. Antibodies to heterologous microorganisms (Yersinia enterocolitica O:9) having common antigenic determinants with Brucella were not detected. Still the titers of antibodies isolated with the use of these antigens were lower than those obtained in reactions with B.abortus 99 S-lipopolysaccharide.

[Comparative analysis of antigens from different Brucella species using immunoblotting with antisera from immunized rabbits]. / Sravnitel'nyi analiz antigenov razlichnykh vidov Brucella metodom immunoblota s antisyvorotkami immunizirovannykh krolikov.

Brucella antigens recognized by IgG antibodies in cell lysates from various Brucella species differing by the origin, biological, and virulent properties (including the reference, vaccine, and newly isolated strains) were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in SDS-cell lysates were separated by 12% SDS-PAGE and protein gels were stained with Coomassie brilliant blue R-250 and Silver reagent. SDS-PAGE showed differences in the protein profiles of 15 strains of different species. Immunoblotting revealed that rabbit S-antisera contained IgG reacting with S-LPS and identical proteins of 90 to 16 kDa belonging to B, melitensis, B. suis, B. abortus, and B. neotomae strains. B. canis strains had 4 antigens reacting with these antisera, whereas B. ovis had none. No agglutinating antibody were detected by the standard tube agglutination test with smooth Brucella strains in rabbit R-antisera. By contrast, immunoblotting analysis with these sera demonstrated common 90-16 kDa antigens in the strains of B. melitensis, B. suis, B. abortus, B. neotomae, and B. canis. B. ovis possessed none of these antigens. These results confirm that all Brucella species except B. ovis possess common protein antigens reacting with IgG.

[Comparative study of the antigenic composition of Brucella canis strains by immunoblotting]. / Sravnitel'noe izuchenie antigennogo sostava shtammov Brucella canis metdom immunoblottinga.

Brucella cultures isolated from sick dogs have not been properly studied in Russia up to the present time. In 1994 a culture has been isolated from aborted fetus of a dog. Investigations by the traditional methods referred it to Brucella genus, species canis (strain K-01). Reference strain B. canis RM6/66 and B. canis K-01 were identical by the profiles of protein antigens in immunoblotting with a set of antibrucellosis sera. B. canis, B. suis, B. abortus, and B. melitensis. However, immunoblotting with sera to B. canis showed the similarity of B. canis cultures with the reference strain B. suis 1330, and use of sera to B. suis, B. abortus, and B. melitensis helped differentiate between the reference B. suis 1330 strain and B. canis strains. All the antisera used permitted the differentiation of Brucella strains from Yersinia enterocolitica 0:9, Escherichia coli 0:157, and Salmonella typhimurium cross reacting with Brucella in serological tests. Immunoblotting is a promising taxonomic criterion for identification of newly detected representatives of the Brucella genus.

[A genetic and molecular biological study of the pathogenicity factors in Brucella]. / Geneticheskoe i molekuliarno-biologicheskoe izuchenie faktorov patogennosti brutsell.

The use of gene engineering techniques made it possible to obtain strain GSE830, capable of a higher level of expression of the gene of 38-kD protein in immunoblotting with sheep and rabbit antibrucellar sera in comparison with the expression of this gene of other Escherichia coli strains, containing recombinant plasmids with this gene. Due to the presence of the gene of 38-kD protein, recombinant E.coli strains were capable of survival in macrophage-like cell line U937 3.6-6.3 times more effectively. The model of interaction of Brucella pathogenic and nonpathogenic species with HeLa cells was studied. The bank of insertion mutants of B.suis virulent strain 1330 was studied with the use of transposon TnblaM. Out of 380 insertion mutants, 7 clones expressing beta-lactamase and having decreased capacity for multiplication in HeLa cells 48 hours after inoculation were selected. Detailed analysis revealed that 3 of them had lower adhesive capacity, 1 of them had lower invasive capacity and 3 other mutants were less capable of intracellular multiplication in HeLa cells than the initial B.suis strain 1330. All these 7 mutants had different sites of TnblaM insertion into the chromosome of B.suis strain 1330.

[A highly sensitive non-isotopic system of DNA hybridization using amplification (PCR) for identifying and indicating presence of Brucella]. / Vysokochuvstvitel'naia neizotopnaia sistema gibridizatsii DNK s primeneniem amplifikatsii (PTsR) dlia identifikatsii i indikatsii brutsell.

A non-isotopic amplification system was used to identify and indicate Brucella. The terminal sequences of a protein gene fragment in Brucella outer membrane were identified and direct and reverse primers were chosen for a polymerase chain reaction. (PCR). PCR amplifies a specific DNA fragment, 700 kb in size, only in representatives of the Brucella genus. A probe was design, which is the central part of the amplified DNA fragment, 550 kb in size. Single Brucella cells were detectable with an unlabelled probe in the analyzed samples during hybridization reactions. The system can be recommended for a rapid and reliable analysis in medical and veterinary practice.

[Cloning of Brucella genes in Escherichia coli K12 cells and analysis of products of the cloned genes]. / Klonirovanie genov Brucella v kletkakh Escherichia coli K12 i analiz produktov klonirovannykh genov.

The libraries of Brucella melitensis 565 and Brucella abortus 99 in Escherichia coli cells have been constructed. Some clones of Escherichia coli producing the specific brucella antigens have been found in immunological tests with brucella antiserum. Two strains producing antigens have been characterized, one being from Brucella melitensis 565 and another from Brucella abortus 99 clone libraries . Both strains synthesize two antigens that were studied by immunoelectrophoresis, immunoblotting after treatment of antigen preparations with different physical and chemical agents substrate specific enzymes. Both strains are found to synthesize the specific brucella antigens of protein nature. One of them has the mol mass about 15 kD, another--31-32 kD. The 31-32 kD antigen can be, evidently, referred to as the main protein of an outer membrane of brucella.

[Functional properties of the pOV13 plasmid as a vector for DNA cloning in a broad spectrum of gram negative bacteria]. / Funktsional'nye svoistva plazmidy pOV13 kak vektora dlia klonirovaniia DNK v shirokom kruge gramotritsatel'nykh bakterii.

The birepliconed plasmid pOV13 possesses all the properties of a vector for DNA cloning in a broad host range of bacterial cells. pOV13 is transfered by transformation and stably inherited by Escherichia coli, Brucella, Pseudomonas cells determining the resistance to streptomycin, tetrocycline and kanamycin in these bacteria. The plasmid pOV13 is a multicopy plasmid optimal in replication capacity (23kb). The plasmid carries single sites for some restriction endonucleases that are used for DNA cloning, including some restriction sites in antibiotic resistance genes. The examples of DNA cloning with the selection of recombinant clones by the insertional inactivation of kanamycin or tetracycline resistance and expression of the cloned DNAs are presented.

[Structural rearrangements of the plasmid R68,45 in cells of Brucella suis]. / Strukturnye perestroiki plazmidy R68,45 v kletkakh Brucella suis.

The mobilizing activity of the plasmid R68,45 in Escherichia coli and Brucella abortus has been studied. The plasmid R68,45 has been found to lose its ability to mobilize the chromosome in Brucella suis cells. The experiments on the conjugational transfer of R68,45 were confirmed by restriction analysis of the plasmid DNA. R68,45 has been shown to lose Cma in brucella cells via deletion. The molecular mechanisms of deletion process in brucella and other bacteria are discussed.