Molecular Typing of <em>Brucella</em> Species Strains from Georgia and Turkey

Special Article - Brucella

J Bacteriol Mycol. 2017; 4(3): 1054.

Molecular Typing of Brucella Species Strains from Georgia and Turkey

Sidamonidze K¹, Su W3, Zhgenti E¹, Buyuk F², Sahin M2, Trapaidze N4, Imnadze P1, Nikolich MP3,4, Obiso R5 and Kotorashvili A1*

¹National Center for Disease Control and Public Health, Tbilisi, Georgia

²Department of Microbiology, University of Kafkas, Turkey

²Walter Reed Army Institute of Research, USA

4US Army Medical Research Unit-Georgia, Georgia

5Avila Scientific, Virginia, USA

*Corresponding author: Kotorashvili A, National Center for Disease Control and Public Health, Tbilisi, Georgia

Received: July 28, 2017; Accepted: August 30, 2017; Published: September 06, 2017


The genus Brucella consists of several species that infect different mammalian hosts including humans. Brucella infection can lead to abortion in many domestic animals, which causes significant economic losses. Human brucellosis is one of the most globally widespread and debilitating zoonotic diseases, and it remains endemic in both Georgia and Turkey. Precise genetic characterization of Brucella isolates by molecular typing methods can provide useful analysis for examining strain diversity; detection of clonal groups; determining sources of infection; and discrimination of naturally occurring outbreaks from a bioterrorist event. Recent studies have demonstrated that Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) is a high resolution genetic subtyping tool that can provide valuable information for epidemiological investigations on brucellosis. In this study, we assessed the genetic variability of 30 isolates of Brucella isolated in Turkey and 65 Brucella isolates from Georgia. All samples were identified as B. melitensis or B. abortus based on microbiological and Bruce-ladder PCR results. Discriminatory genotypes were determined using the MLVA approach using 15 mini-satellite markers. Relationships between Turkish and Georgian isolates were revealed and showed that strains are grouped by origin of country and species; however, there were two strains of B. melitensis from Turkish strains mixed within the Georgian group. Our results suggest that Brucella spp. is homogenous, however, two strains have been introduced from Turkey into Georgia and that cross-border migration is possible.

Keywords: Mammalian hosts; Bacterial zoonosis; Human brucella


Brucellosis is globally the most common bacterial zoonosis, resulting in significant economic losses via the reduction of livestock productivity, and is a debilitating human disease. Although the picture of global brucellosis morbidity is not completely clear, brucellosis is found on almost every continent, with at least 500,000 people are infected annually [1]. Brucella melitensis is the most frequent cause of human brucellosis accounting for an estimated 96% of cases worldwide [2]. Human infection occurs from exposure to infected animal abortion products or contaminated dairy products, and it is accepted that control of the human disease can only occur through control and eradication of the disease in animals [3].

Brucellosis is currently under-diagnosed and under-reported in Turkey [4] and worldwide [5] due to the nonspecific clinical manifestations of the disease and the limitations of current diagnostic tests. Brucellosis infection in humans can be challenging to diagnose as its presentation is nondescript and mimics many other conditions [5]. In the early presentation (days to weeks after exposure), non-specific symptoms such as fever, fatigue/malaise, myalgia, and arthralgia predominate. Some patients proceed to develop focal complications, with osteoarticular complications being somewhat common and very debilitating [4]. Other patients may have chronic, mild symptoms for many months, making brucellosis difficult to recognize as the cause of their condition. These patients have traditionally been classified as having chronic brucellosis, but the utility of this classification is not established. Additionally, a certain percentage of patients relapse after treatment, usually with non-specific symptoms, even after an extensive course of initial therapy, however, the contribution of lack of treatment compliance and re-infection of these patients to relapse is not well known.

Human brucellosis is caused by five classical Brucella species (B. abortus, B. melitensis, B. suis, B. ovis, B. canis), of which B. melitensis is considered the most virulent for humans [2]. B. melitensis is highly infectious for humans with an ID50 of 10 cells per person, and it is virulent in other mammal hosts such as bovine cattle, though sheep and goats are its preferred host [3]. B. melitensis infections are especially problematic as the most commonly used attenuated B. abortus cattle vaccine (Strain 19) fails to protect from disease caused by B. melitensis [5]. Human cases of brucellosis are acquired through contact with infected animals and through consumption of contaminated dairy products. Adherence to traditional farming practices and the consumption of unpasteurized dairy products contribute to the high incidence of brucellosis in Turkey [4].

The B. abortus RB51 vaccine has also been used successfully in selected countries to aid in the control of bovine brucellosis (caused by B. abortus). A complicating factor of vaccination is that classical serologic diagnostic protocols cannot distinguish animals vaccinated with Strain 19 from infected animals and diagnosis during latent infection can be particularly challenging. Only the use of the rough B. abortus vaccine RB51 in bovine cattle addresses this issue because it lacks the dominant antigen, LPS O-side chain, but this vaccine is yet to be accepted worldwide. Meanwhile the utility of vaccination of livestock with Strain 19 is significantly decreased by the inability to distinguish between vaccinated animals and those animals with active infections without sophisticated laboratory assays [5]. These issues underscore the need for improved detection and diagnostic capabilities in the primary livestock hosts of Brucella.

Genetic characterization of a bacterium is often used for molecular epidemiology purposes and can be useful to observe population structures. Owning to the high genetic identity among members of the Brucella genus [6-12], the physiological typing methodologies of the past showed little success [13]. However, in recent years, the application of Multiple-Locus Variable Number Tandem Repeats Analysis (MLVA) with different sets of repeat loci has gained momentum in Brucella typing. The MLVA typing assay consists of two complementary panels, panel 1 (8 markers) and panel 2 (7 markers); fifteen markers clearly presents a discriminative power in typing these microorganisms [13].

Previous studies to examine the genetic diversity and regional distribution of Brucella species in Georgia found high genetic diversity between isolates, and geographic distribution was dependent on the genotype. A study of 35 isolates of human and animal Brucella obtained in Georgia in 2009 and 2010 using MLVA-8 analysis illustrated the high diversity between isolates [6]. It was found that only 2-3 strains were located in one genotype cluster, with some genotypes showing restricted geographical distribution, and others were disseminated among different regions of Georgia. One genotype cluster contained animal and human isolates from the same region, revealing a potential source of human infection [6].

A study similar study was conducted in Turkey with 162 human Brucella isolates collected from 2001-2008, which were analyzed using MVLA-16 [7]. A total of 105 genotypes were recorded, with 73 being represented by a unique isolate, and 32 including 2-8 isolates. As was found in Georgia, several the isolates had restricted geographic distribution. In addition, spanning tree analysis of published B. melitensis isolates, using MLVA-11 data, indicted that Turkish isolates were most closely related to the neighboring countries’ isolates included in the East Mediterranean group [7].

The aim of this study was to genetically characterize Brucella spp. from strains isolated from both Georgia and Turkey by applying high-resolution genetic characterization techniques such as Bruce- Ladder PCR and Multiple Locus Variable-Number Tandem Repeat Analysis (MLVA) to determine the genetic relationships between pathogens and potential migration pattern across the borders. This study was designed to evaluate the molecular epidemiology of Brucella spp. from human and livestock of the Georgia and Turkey by applying these findings to future planning for disease control in the both countries.

Materials and Methods

Samples and study design

All Brucella samples used in the study were provided from the bacterial repository of Kafkas University (Kars, Turkey) and the repository of the National Center for Disease Control and Public Health of Georgia (NCDC&PH, Tbilisi, Georgia). It was the first time applying high resolution molecular approaches (Bruce-Ladder PCR and MLVA typing) for the selected samples. In total, 30 Turkish Brucella samples (Table 1) and 65 Georgian Brucella samples (Table 2) were used.DNA samples from Kafkas University were transported to NCDC using iced containers and kept at -20°C. General Directorate of Food and Control, Republic of Turkey Ministry of Food Agriculture and Livestock, Permission number 55016929-604.01-13587. Public of Health Agency of Turkey, Republic of Turkey Ministry of health, Permission number: 80962070.