Liu et al. Infectious Diseases of Poverty (2017) 6:14 DOI 10.1186/s40249-016-0218-6 RESEARCHARTICLE Open Access Genetic source tracking of an anthrax outbreak in Shaanxi province, China Dong-Li Liu1†, Jian-Chun Wei2,3,4†, Qiu-Lan Chen5†, Xue-Jun Guo6†, En-Min Zhang2,3,4,LiHe7, Xu-Dong Liang2,3,4, Guo-Zhu Ma1, Ti-Cao Zhou5, Wen-Wu Yin5, Wei Liu7, Kai Liu5, Yi Shi1, Jian-Jun Ji7, Hui-Juan Zhang2,3,4, Lin Ma1, Fa-Xin Zhang7, Zhi-Kai Zhang2,3,4, Hang Zhou5, Hong-Jie Yu5,BiaoKan2,3,4,Jian-GuoXu2,3,4,FengLiu1* and Wei Li2,3,4* Abstract Background: Anthrax is an acute zoonotic infectious disease caused by the bacterium known as Bacillus anthracis. From 26 July to 8 August 2015, an outbreak with 20 suspected cutaneous anthrax cases was reported in Ganquan County, Shaanxi province in China. The genetic source tracking analysis of the anthrax outbreak was performed by molecular epidemiological methods in this study. Methods: Three molecular typing methods, namely canonical single nucleotide polymorphisms (canSNP), multiple-locus variable-number tandem repeat analysis (MLVA), and single nucleotide repeat (SNR) analysis, were used to investigate the possible source of transmission and identify the genetic relationship among the strains isolated from human cases and diseased animals during the outbreak. Results: Five strains isolated from diseased mules were clustered together with patients’ isolates using canSNP typing and MLVA. The causative B. anthracis lineages in this outbreak belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-31 genotype (the 31 genotype in MLVA15 scheme). Because nine isolates from another four provinces in China were clustered together with outbreak-related strains by the canSNP (A.Br.001/002 subgroup) and MLVA15 method (MLVA15-31 genotype), still another SNR analysis (CL10, CL12, CL33, and CL35) was used to source track the outbreak, and the results suggesting that these patients in the anthrax outbreak were probably infected by the same pathogen clone. Conclusions: It was deduced that the anthrax outbreak occurred in Shaanxi province, China in 2015 was a local occurrence. Keywords: Anthrax, Outbreak, Bacillus anthracis, Molecular typing, canSNP, MLVA, SNR, Shaanxi province, China Multilingual abstracts can form spores resistant to extreme environmental con- Please see Additional file 1 for translations of the ab- ditions and can persist for long periods of time in soil or stract into the five official working languages of the hay. Susceptible herbivores, such as cows, mules, sheep, United Nations. horses, and donkeys, can become infected through contact with B. anthracis spores. Humans can become infected in- cidentally through contact with diseased animals or by in- Background haling spores from contaminated animal products [1]. Anthrax is an acute infectious zoonotic disease caused There are three primary forms of the disease in humans, by the spore-forming, aerobic, gram-positive, non-motile namely inhalation, gastrointestinal, and cutaneous an- bacterium Bacillus anthracis (B. anthracis). B. anthracis thrax. The most common form, cutaneous anthrax, ac- * Correspondence: [email protected]; [email protected] counts for 95% of cases worldwide [1]. It is mainly caused †Equal contributors by the handling of infected animal carcasses or products 1 Shaanxi Provincial Center for Disease Control and Prevention, Shaanxi of diseased animals. A new infection form, injection an- province, China 2National Institute for Communicable Disease Control and Prevention, China thrax, had recently been discovered [2]. CDC, Changping, Beijing, China Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Infectious Diseases of Poverty (2017) 6:14 Page 2 of 8 Efficient genotyping methods are required not only to determine the genetic relationship among the human assist microbial forensics to track outbreaks back to their cases and investigate the possible routes of transmission, origins, but also for facilitating epidemiological studies the canSNP and the MLVA15 typing methods were in order to reduce the risk of an outbreak or control it employed, and their profiles were compared with the B. effectively if it does occur [3]. B. anthracis is a relatively anthracis strains in the Chinese B. anthracis Genetic In- homogeneous bacteria species with a demonstrated lack formation Database (CBaGID, Chinese Center for Dis- of polymorphism, which may be due to the long periods ease Control and Prevention, CDC). In addition, a more its endospores spend being dormant during its lifecycle precise method, the SNR analysis, was used to source [4]. Genetic markers such as single-nucleotide polymor- track the outbreak. phisms (SNPs) have been used to illustrate the phylogen- etic and evolutionary relationship of B. anthracis strains, Methods which allows us to better understand how they fit into Epidemiological investigation regional and global phylogeographic patterns [4, 5]. An In early August 2015, a suspected anthrax outbreak was approach termed canonical single nucleotide polymor- reported to China’s National Health and Family Planning phisms (canSNP), which includes 13 selected SNPs Commission. A national field team including clinical, located at the key phylogenetic junctions in the B. epidemiologic, and laboratory personnel carried out an anthracis SNP tree, could replace the genome-wide SNP investigation and attempted to control the disease to- analysis [4]. Other molecular typing methods, such as gether with provincial and local CDC staff. A total of 20 the multiple-locus variable-number tandem repeat ana- clinical samples, including serum and blister fluid, were lysis (MLVA) [6–8], have also been used to illustrate the collected from each suspected individual during the in- phylogenetic relationship of B. anthracis strains and for vestigation. A suspected cutaneous anthrax case in source tracking in the event of a bioterrorist attack or humans was defined as an acute onset of skin vesicles or anthrax outbreak [3]. The MLVA15 scheme described by skin ulceration with a raised margin and central black Van Ert et al. in 2007 [4] includes eight markers that eschar and having corresponding exposure history (con- were initially described by Keim et al. [9] and seven tacting infected animal product, contaminated fomite, markers newly included in the study of Van Ert et al. [4]. etc.). Confirmed cases were determined through sup- The global genetic population structure of B. anthracis portive laboratory tests, including isolation of bacilli, a has previously been defined using canSNP typing and four-fold increase in anti-protective antigen (PA) anti- MLVA. It was found that the A.Br.001/002 subgroup body titer. We also categorized suspected cutaneous an- was a major part of the B. anthracis population in China thrax cases as confirmed cases if they were evidenced by [4, 10]. Single-nucleotide repeats (SNRs), also called at least two of the following evidence: Morphology mononucleotide nucleotide repeats, are a special type of under the microscope with traditional Gram’s and M’Fa- variable-number tandem repeats (VNTRs) that display dyean’s (polychrome methylene blue) stained smears in very high mutation rates [11, 12]. A previous study fresh tissue or blood samples, positive serology tests showed that seven strains of B. anthracis recovered from (ELISA or colloidal gold test), and positive real-time Northeast China from 2010–2014 were distinguished as polymerase chain reaction (RT-PCR). This met the diag- A.Br.001/002 (n = 5) or A.Br.Ames (n = 2) sublineages, nostic criteria recommended by the World Health and the two A.Br.Ames strains were further identified as Organization (WHO) [16]. same SNR pattern [13]. Because SNR markers can pro- All procedures performed in studies were in accord- vide additional genetic resolution among B. anthracis ance with the ethical standards of the institutional re- strains of the same MLVA genotype [14, 15], the SNR search committee in China CDC. The clinical samplings analysis has therefore been used as a means of source were obtained with the consent of related patients. The tracking anthrax outbreaks [14]. blood and meat from diseased and dead animals were Anthrax is an endemic disease and cases have been re- sampled by local Animal Husbandry and Veterinary In- ported every year in China, especially in the northern spection organization after permission was obtained and western provinces of mainland China. A strength- from animal owners. ened national surveillance system covering seven prov- inces (expanded to 11 provinces in 2014) in mainland Laboratory diagnosis China has been in place since 2005. On 8 August 2015, Two types of clinical specimens (blood and blister fluid) an anthrax outbreak with 20 cutaneous anthrax cases were collected from patients in hospital. Blister fluid was was reported in Shaanxi province, which resulted in streaked on nutrient LB agar plates or blood agar plates more than 300 people contacted with the contaminated and cultured at 28 °C for 24 h. Then, specimens were animal products were under medical observation, and, at stained on slides with Gram and M’Fadyean staining. Fi- least 15 mules and two donkeys died. In order to nally, vesicular fluid samples of suspected cases were Liu et al. Infectious Diseases of Poverty (2017) 6:14 Page 3 of 8 used to extract DNA templates according to the et al. [11] were used as the sequencing primers to iden- DNeasy Blood & Tissue Kit (QIAGEN Germany) manual.