ORIGINAL RESEARCH published: 18 January 2017 doi: 10.3389/fmicb.2017.00006 MLVA Genotyping Characteristics of Human Brucella melitensis Isolated from Ulanqab of Inner Mongolia, China Zhi-Guo Liu 1, 2 †, Dong-Dong Di 3 †, Miao Wang 2, Ri-Hong Liu 2, Hong-Yan Zhao 4, Dong-Ri Piao 4, Guo-Zhong Tian 4, Wei-Xing Fan 3, Hai Jiang 4*, Bu-Yun Cui 4* and Xian-Zhu Xia 1, 5* 1 College of Veterinary Medical Inner Mongolia Agriculture University, Hohhot, China, 2 Ulanqab Centre for Endemic Disease Prevention and Control, Health and Family Planning Commission of Ulanqab, Jining, China, 3 Laboratory of Zoonoses, China Animal Health and Epidemiology Center, MOA, Qingdao, China, 4 State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China, 5 Institute of Military Veterinary AMMS, Changchun, China Edited by: Adrian Whatmore, Brucellosis is a serious public health problem in Ulanqab, which is a region located in Animal and Plant Health Agency, UK the middle of the Inner Mongolia Autonomous Region adjacent to Shanxi and Hebei Reviewed by: provinces. The disease is prevalent in both the latter provinces and Ulanqab with the Giuliano Garofolo, Istituto Zooprofilattico Sperimentale highest prevalence of brucellosis occurring in Inner Mongolia. The MLVA-16 scheme dell’Abruzzo e del Molise G. Caporale, is a genotyping tool for assessing genetic diversity and relationships among isolates. Italy Jeffrey T. Foster, Moreover, this genotyping tool can also be applied to epidemiological trace-back University of New Hampshire, USA investigations. This study reports the occurrence of at least two B. melitensis biovars *Correspondence: (1 and 3) in Ulanqab, encompassing 22 and 94 isolates, respectively. B. melitensis biovar Hai Jiang 3 was the predominant biovar in the area examined. Panel 1 (MLVA-8) identified three [email protected] Bu-Yun Cui genotypes (42, 63, and 114), with genotype 42 (n = 101) representing 87% of the tested [email protected] strains. MLVA-11 identified eight genotypes (116, 111, 297, 291, and 342–345) from Xian-Zhu Xia [email protected] 116 of the analyzed isolates. All of these isolates were identified as belonging to the † = These authors have contributed East Mediterranean group. Genotype 116 (n 94) was the predominant genotype and equally to this work. represented 81% of the isolates. The isolates pertaining to this genotype were distributed throughout most of Ulanqab and neighboring regions. The MLVA-16 scheme showed the Specialty section: This article was submitted to presence of 69 genotypes, with 46 genotypes being represented by single isolates. This Infectious Diseases, analysis revealed that Ulanqab brucellosis cases had epidemiologically unrelated and a section of the journal sporadic characteristics. The remaining 23 genotypes were shared (between a total of Frontiers in Microbiology 70 isolates) with each genotype being represented by two to eight isolates. These data Received: 17 August 2016 Accepted: 03 January 2017 indicate that these cases were epidemiologically related. MLVA genotyping confirmed Published: 18 January 2017 the occurrence of a multipoint outbreak epidemic and intrafamilial brucellosis. Extensive Citation: genotype-sharing events were observed among isolates from different regions of Ulanqab Liu Z-G, Di D-D, Wang M, Liu R-H, and from other provinces of China. These findings suggest either a lack of control of Zhao H-Y, Piao D-R, Tian G-Z, Fan W-X, Jiang H, Cui B-Y and animal movement between different regions or the circulation of contaminated animal Xia X-Z (2017) MLVA Genotyping products in the market. Our study is the first comprehensive genotyping and genetic Characteristics of Human Brucella melitensis Isolated from Ulanqab of analysis of B. melitensis in Ulanqab. We believe that this study will help to improve the Inner Mongolia, China. effectiveness of brucellosis control programs. Front. Microbiol. 8:6. doi: 10.3389/fmicb.2017.00006 Keywords: Brucella melitensis, MLVA, molecular epidemiology, trace-back analysis, Ulanqab, Inner Mongolia Frontiers in Microbiology | www.frontiersin.org 1 January 2017 | Volume 8 | Article 6 Liu et al. MLVA Genotyping Characteristics of B. melitensis INTRODUCTION and Prevention and the Chinese Center for Disease Control and Prevention. Informed consent was obtained from all of Brucella is a genus of Gram-negative facultative intracellular the patients prior to diagnosis. Brucella spp. were isolated from pathogens that infect humans and a variety of animal species patients’ blood samples following confirmation of their consent. (Shevtsov et al., 2015). Brucellosis is transmitted to humans following direct contact with infected animals or indirectly Bacterial Strains through the consumption of unpasteurized animal products A total of 116 strains were examined. These strains were (Deshmukh et al., 2015). The disease causes severe morbidity obtained from 11 Ulanqab counties and three neighboring in humans and results in many medical challenges globally, regions [Suniteyouqi (Xilin Hot City of Inner Mongolia), Datong especially in poor regions (Corbel, 1997; Pappas et al., 2006; (Shanxi province), and Shangyi (Hebei province)] from 2011 to Ducrotoy et al., 2015). Furthermore, animal brucellosis causes 2015. A total of 115 strains were recovered from 114 patients, abortion and infertility in livestock, resulting in serious economic and one strain was recovered from sheep. Two strains (ws091 losses (Araj, 1999). and ws101) were obtained from the same patient. The Brucella Since 2000, the incidence of brucellosis has increased rapidly strains were isolated and biotypes were identified using standard in many regions in China (Deqiu et al., 2002; Jiang et al., procedures (Alton et al., 1975; Al Dahouk et al., 2003). B. 2011). The incidence of brucellosis in Inner Mongolia ranks melitensis 16M, B. abortus 544, and B. suis 1330 reference strains in the top three incidence rates in Chinese provinces. The were used as control strains. Species-level identification was incidence of brucellosis in Ulanqab was 177.1/100,000 cases undertaken using B. abortus, B. melitensis, B. ovis, B. suis PCR in 2011, 42.2/100,000 cases in 2012, 17.2/100,000 cases in (AMOS-PCR; Bricker and Halling, 1994). 2013, 24.1/100,000 cases in 2014, and 26.5/100,000 cases in 2015. Ulanqab is a relatively small region in Inner Mongolia, DNA Preparation 2 covering a geographical area of 545,000 km , and comprises DNA was extracted with a Nucleic Acid Automatic Extraction 11 regions that are extensively pastoral and semi-pastoral. The System (LLXBIO China Ltd., China) using a single loop of people that inhabit these regions are economically dependent on fresh bacterial cells that were grown for 48 h on Brucella agar. small ruminant (sheep) livestock (Jiang et al., 2013). Livestock DNA concentrations were measured by UV spectrophotometry exchange occurs frequently in this region, and includes import (NanoDrop 2000, Thermo, US). from other areas, internal exchange, livestock foster care, and shifting field grazing. Due to excessive poverty and limitations in Brucella MLVA-16 Genotyping Scheme relation to control measures, brucellosis is endemic among sheep MLVA was performed as described previously (Le Flèche et al., and humans in these regions. Technical difficulties have meant 2006; Al Dahouk et al., 2007). The 16 primer pairs were that previous studies only concentrated on Brucella infection divided into three groups: Panel 1 (MLVA-8: eight loci including surveillance in this region, and adequate attention was not paid bruce06, bruce08, bruce11, bruce12, bruce42, bruce43, bruce45, to the molecular typing of Brucella species. However, rapid and bruce55), panel 2A (three loci including bruce18, bruce19, and accurate identification and typing procedures are important and bruce21), and panel 2B (five loci including bruce04, bruce07, for epidemiologic surveillance, investigation of outbreaks, and bruce09, bruce16, and bruce30); MLVA-11 (panels 1 and 2A), control program follow-up (Whatmore et al., 2006; Al Dahouk and MLVA-16 (panels 1, 2A, and 2B). PCR amplifications were et al., 2007; García-Yoldi et al., 2007). Previous studies have performed in 20 µL reaction volumes. The PCR conditions confirmed that MLVA (Multiple-locus variable-number tandem were as follows: initial denaturation at 94◦C for 3 min, and repeat analysis) is a useful tool for identifying and genotyping then 30 cycles of 94◦C for 30 s, 60◦C for 30 s, and 72◦C Brucella isolates and the resultant data can be used for genetic for 50 s, with a final extension of 72◦C for 3 min. PCR diversity analysis and epidemiology trace-back investigations products for the 16 loci were denatured and resolved by capillary (Whatmore et al., 2006; Kattar et al., 2008; Ferreira et al., electrophoresis on an ABI Prism 3130 automated fluorescent 2012; Garofolo et al., 2013). In this study, the MLVA-16 capillary DNA sequencer (Applied Biosystems). Fragments were scheme was used to type samples and determine the genotyping sized following comparison with a ROX (carboxy-X-rhodamine)- characteristics and epidemiological links of associated strains. labeled molecular ladder (MapMaker 1000; Bioventures Inc., The results of this study contribute to our understanding of the Murfreesboro, TN, USA) and Gene Mapper software version main transmission routes associated with this