Brucellosis is a zoonotic disease of major public health, animal welfare, and economic significance worldwide. In humans, infection with Brucella can lead to a chronic debilitating infection; in domesticated animals, the main symptom is a reproductive failure. Among the 12 species currently proposed in this genus, Brucella melitensis, Brucella abortus, and Brucella suis are highly dangerous for humans and cause diseasewith severe complications and chronic processes (Meyer, 1990; Godfroid et al., 2011). Rare cases of infection of people with other Brucella species are also recorded (Ficht, 2010). Despite its low mortality rates, brucellosis is a very important public health problem in Kazakhstan. Brucellosis causes great economic damage to livestock. The disease is accompanied by mass abortions and barrenness of animals suffering from brucellosis, joint damage, inflammation of mucous bags (in horses at the withers and the nape of the neck), the sex glands, the appearance of abscesses (more often in pigs), a decrease in the viability of offspring, a decrease in animal productivity. At the same time, elimination of brucellosis requires great effort and cost (Demirev, 2013; Whatmore et al., 2006; Whatmore, 2009). The development of discriminatory molecular tools for the identification and typing of Brucella has been problematic, reflecting the lack of genetic polymorphism in Brucella.

To address the issue of epizootology and understanding the pathogenesis of the disease, an accurate and timely diagnosis is necessary. The success of the fight against brucellosis depends on the rate of detection of all infected animals, on the effectiveness of diagnostic studies. The traditional system of identification and systematization of isolated field strains of Brucella is imperfect. None of the existing molecular tools provide adequate resolution to confidently permit epidemiological traceback in the case of accidental import or deliberate release. The phenotypic methods used allow differentiation of the causative agent of brucellosis to the species-specific level, but they are characterized by laboriousness, duration, and non-specificity of the analysis (Whatmore, 2009; Alton et al., 1988; Le Flèche et al., 2006). These methods do not meet modern typing requirements. The solution to this problem is seen in the use of modern methods of molecular genetic typing of Brucella isolates.

Using molecular methods of analysis, genomic polymorphism is simply detected – quantitative changes in mini- and microsatellite DNA sequences (decrease or increase in the number of repeats), creating a series of unique alleles for each variable locus. These types of polymorphic loci are convenient markers. The phenomenon of polymorphism underlies the common method of identifying mutant DNA fragments – analysis of microsatellite markers (Ayala and Kiger, 1980; Gershkovich, 1968; Al Dahouk et al., 2007, Kattar et al., 2008). In this regard, the use of information about the causative agent of brucellosis obtained in the analysis of multilocus provides high-quality, effective elimination and prevention of the infectious process.

Since there were essentially no molecular tools that could provide useful discrimination beyond the level of biovar, the first aim of this study was to characterize highly discriminatory loci that would facilitate the development of tools to permit epidemiological traceback of Brucella to the source of infection The initial stages of the work focused on: 1. Isolation of DNA from microorganism cultures; 2. Determination of DNA nucleotide sequence; 3. Sequencing steps; 4. Cleared the amplification from residual reagents with CleanSweep reagent, mix composition (Table 1).

Then a sequencing reaction was performed. Each PCR product was pipetted into the 2 wells of a strip of 1 μL (the primers were excavated separately in the wells, to the first was added straight, to the second – the reverse) + the reaction mixture of the following composition (Table 2).

Put on the amplification according to the following program (Table 3). The rate of increase or decrease in temperature – 1 °C/sec.

Purification (precipitation) before sequencing (performed in a plate with cooling) was performed.

45 μL Sam Solution and 10 μL Xterminator Solution had been added to the amplification obtained after the sequential reaction, shaking the reagent strongly, as it quickly precipitated. The resulting mixture was vortexed at 1800 rpm for 20 minutes using Digital Vortex-Genie 2. After that, the plate was centrifuged at 1000g for 2 minutes. 15 μL of the supernatant was collected and the plate was loaded into the sequencer. As a result of sequencing the cultures of microorganisms, the following results were obtained (Table 4, Figure 1).

The success of brucellosis control depends on the speed of detection of all infected animals and the effectiveness of diagnostic tests (Alton et al., 1988; Meyer, 1990; Yagupsky, 1999). Up-to-date disease detection helps to take steps to isolate the infected animal early as a source of infection (Bricker and Ewalt, 2005; Ridler et al., 2005). The following serological tests for animal brucellosis are officially recognized in Kazakhstan: RBS, AR, CBD, and ELISA. The rate of serological reactions in brucellosis is unstable (Ficht, 2010; Scholz and Vergnaud, 2013). While each method is complex with other methods, it does not isolate all sick animals. Traditional phenotypic differentiation methods make it difficult to standardize the situation, which is the reason for erroneous differentiation (Pappas et al., 2006; Godfroid et al., 2011, Borriello et al., 2013). It is known that the phenotypic characteristics of bacteria can vary depending on the growing conditions and the allelic state responsible for gene expression. The most conservative structure that characterizes the type of microorganism in its gene (Ayala and Kiger, 1980; Gershkovich, 1968; Al Dahouk et al., 2007, Kattar et al., 2008). In this regard, the most promising is the assessment based on the nature of the pathogen genome.

The solution of the problem made it possible to determine the genotype of the studied strains using cluster analysis and to start an investigation of the outbreak in the regions endemic for brucellosis (Frachetti et al., 2017). Timely provision of information on the causative agent of brucellosis, obtained using multilocus analysis, for the use of scientific research results in veterinary practice, which ensures high-quality monitoring of the epidemic process, effective eradication, and prevention (Le Flèche et al., 2006; Whatmore, 2009; Demirev, 2013). The expediency of the introduction of 16S rRNA is an accurate and timely diagnosis of brucellosis in animals since the timely detection of the disease will help to take measures for the early isolation of the animal as a source of infection and prevent its further development and spread (Garofolo et al., 2013; Shevtsov et al., 2015).

The region of the 16S rRNA gene sequence among the primers used helps to determine 100% affinity, but the specificity is not enough to identify the species. The obtained data are consistent with bacteriological data (colony shape – S, bluish-transparent in daylight; microscopy: round negative rods with rounded ends, located immediately; all characteristics correspond to Brucella abortus). It can be said that the Brucella family is present in the studied samples of microorganisms (no. 4, 5, 7, 8). It turned out that the study of the genetic diversity of brucellosis using Brucella spp. The genetic structure of the Kazakh population has not been studied enough.

Multiple-locus analysis genotyping constitutes a convenient medium-resolution classification assay for large-scale investigations. Follow-up studies based on the whole genome sequencing of selected strains will be necessary to more precisely decipher the dynamics of strain circulation both within Kazakhstan and with Kazakhstan’s neighboring countries. Such studies will also permit to design of new low-cost genotyping assays tailored for Central Asia.