Anthrax is a zoonosis caused by the pathogenic bacterium Bacillus anthracis (1). When nutrients are exhausted, B. anthracis can form a resistant spore that can survive for decades in soil. The natural hosts, herbivores, can be infected by contact with spore-contaminated soil. Humans can usually become infected when they touch animals infected by B. anthracis or products of infected animals (2). When endospores of B. anthracis enter the body by ingestion or inhalation, or through skin abrasions, the fatal anthrax often occurs (1, 3).

Humans commonly suffer anthrax by handling infected animals, carcasses of animals, or animal products (4). In addition, anthrax is a potential agent of bioterrorism and biowarfare, as spores of B. anthracis can be stable for decades and can be readily used as biological weapons (5).

B. anthracis is most closely related to Bacillus thuringiensis and Bacillus cereus, which belongs to the B. cereus group (2). The plasmids pXO1 and pXO2 are the major virulence factors of B anthracis. The plasmid pXO1 codes for lethal factor, edema factor, and protective antigen, which cause hemorrhage, edema, and necrosis, respectively. The plasmid pXO2 encodes cap B, cap C, and cap A gene, which is involved in the polyglutamyl capsule synthesis. Both plasmids pXO1 and pXO2 are necessary for the full virulence of B. anthracis, and loss of either leads to an attenuated strain (2). Anthrax infects humans through the skin, respiratory tract, or the gastrointestinal tract, which results in the cutaneous, inhalational, or gastrointestinal form of the disease, respectively (1). Cutaneous anthrax is the most common form of anthrax, which is caused by bacterial spore contamination of the skin with abrasion or damage (6).

It has been reported that an anthrax-like disease has occurred in China more than 5,000 years ago, and the symptoms and epidemiology of anthrax had been depicted (7). The results of an anthrax surveillance and control project that had been carried out in 10 provinces of China between 1990 and 1994 have been reported in 1995 (7). A more recent report showed that the distribution geographically declined overall from west to east, but the cumulative number of counties affected by anthrax ascended from 2005 to 2014. About 86% of anthrax cases took place in farmers or herdsmen, which indicated the shift of disease from industrial workers to farmers (8). Guizhou Province is one of the historical centers of anthrax in the southwest of China. A total of 17,975 cases of anthrax were reported in Guizhou Province from 1957 to 1999 (9), and a total of 32 outbreaks of anthrax occurred in Guizhou Province from 2001 to 2003 (10).

In September 2016, a suspected human cutaneous anthrax epidemic occurred in Zhijin County, which was reported to Guizhou Provincial Center for Disease Control and Prevention (CDC), and the suspected anthrax epidemic was then epidemiologically investigated. To provide an etiologic basis for the diagnosis of patients and control of the epidemic as well as to enhance our understanding of the genetic characteristics of B. anthracis in Guizhou Province, the skin blister and blood samples collected from patients and the soil samples collected from butchering locations were used for B. anthracis isolation. The isolates were identified using conventional methods and PCR and were subsequently genotyped using multiple-locus variable-number tandem repeats (VNTRs) analysis (MLVA-15) and canonical single-nucleotide polymorphism (canSNP). The genetic relationship between the epidemic strains and isolates collected from other regions was described in this study.

Anthrax is a worldwide zoonosis, and sporadic anthrax cases have been reported occasionally in all countries of the world (16, 17). Some farming regions in China are in naturally infectious areas prone to anthrax outbreaks; livestock are commonly infected by B. anthracis during farming, so many recurrent animal anthrax cases are reported (17).

Guizhou Province is a historical center of anthrax in China, and sporadic human anthrax epidemics have happened frequently in Guizhou Province in recent years (9, 10, 18, 19). In this study, a case of suspected anthrax case was reported to the Guizhou Provincial CDC in September 2016. Two suspected B. anthracis strains were isolated from the blood and blister samples collected from the patient in this epidemic. Conventional identification, which included Gram staining, a phage lysis test, and a penicillin inhibition test for anthrax diagnosis, identified the two isolates as B. anthracis (Table 4).

B. anthracis belongs to the B. cereus group, which includes B. cereus, B. thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus, and B. anthracis (20). B. anthracis spores can keep stable for decades. The ecology and evolution may be greatly influenced by this longevity. The evolutionary change rate of B. anthacis may be reduced by the dormant stage which may result in the extremely homogeneous feature of B. anthracis populations (5, 21).

It has been revealed that the chromosome of B. anthracis resembles those of non-B. anthracis members in group of the B. cereus, whereas the two plasmids were identical to plasmids pXO1 and pXO2, which represent the anthrax virulence (22). The anthrax toxins encoded by plasmid pXO1 and capsules encoded by the plasmid pXO2 are the major virulence factors (23). To confirm whether the two isolates were virulent strains, we used PCR to detect the PA and CAP genes, the target genes for the pXO1 and pXO2 plasmids, respectively. The PCR detection results revealed that the two isolates of suspected B. anthracis from the epidemic (Table 4) were positive for the PA and CAP genes, which indicates that the two isolates are virulent B. anthracis strains.

Previous studies have verified the lack of molecular polymorphisms of B. anthracis, which result in the difficulty to subtype it (5, 21, 24, 25). Commonly used genotype methods, including pulsed-field gel electrophoresis (PFGE) and multiple-locus sequence typing (MLST), are not able to distinguish closely related species. By using PFGE and MLST genotyping, many strains within a species show identical patterns (5). Two molecular approaches, MLVA-15 and whole-genome SNP discovery and analysis, have greatly enhanced the identification of genetic markers that help to establish the phylogenetic relationships among B. anthracis isolates (5, 15). For the present study, in order to further demonstrate that the infection was caused by B. anthracis and to reveal the genetic relationship of the two isolates with strains from other regions, we used MLVA-15 and canSNP to genotype the two isolates. MLVA-15 showed that the two B. anthracis isolates, based on the unique MLVA profile that belonged to one MT (GZGT8), were distinct from the 55 profiles of B. anthracis collected from other regions in Guizhou Province and representative strains from other countries (Figure 1). SNP analysis based on 13 markers showed that the two isolates belonged to the A.BR.001/002 subgroup. The cluster tree and MST indicated that the two isolates were closely related to strains from other regions of Guizhou Province, which suggest that this epidemic was a local epidemic but not imported from other province or other countries.

The field epidemic investigation conducted by the Guizhou Provincial CDC and the Zhijin County CDC showed that a cow, owned by a villager from Wuan Village, died suddenly, and a total of four people from the village participated in butchering the dead animal. Among the four people, one developed a fever, with red pimples and blisters on the arm. The local county doctor diagnosed it as a suspected case of anthrax, and the patient was hospitalized. Aside from the four people who participated in the butchering, there were 13 people who ate the cooked stomach collected from the cow. No other people displaying anthrax-related symptoms were observed, except for the one who participated in butchering the cow. Based on the clinical symptoms, including red papules, pimples, blisters with tissue swelling and infiltrate, skin ulcers with necrosis and black scabs, in combination with the epidemiological history of the patient, the patient was diagnosed with anthrax.

In this epidemic investigation, soil samples from the environment where the dead cattle were butchered were also collected for B. anthracis isolation. However, no strains suspected to be B. anthracis were isolated, which may be due to the cleaning of the contaminated soil environment with water after butchering, incorrect collection of samples, or distribution of the bacteria in the soil environment. To avoid spread of the epidemic due to possible environmental contamination, several rounds of disinfection of the environment were performed by the local CDC. The soil samples for B. anthracis isolation were collected after disinfection, and no B. anthracis was detected from the soil samples. During the investigation, investigators attempted to collect meat samples from the dead cow for B. anthracis isolation and identification, but the meat had been sold to other people who subsequently resold the meat in the market. Therefore, no meat samples were successfully collected for B. anthracis detection.

Although no B. anthracis was successfully isolated from the soil environment and the dead cow, B. anthracis was isolated from the blood and skin blister fluid samples. Both conventional and molecular techniques identified the two bacterial strains as virulent B. anthracis, which provided the etiological basis for the control and prevention of the epidemic. Additionally, as anthrax is zoonotic and the patient had a history of contact with the sick cow, it can be determined that the patient's case was one of cutaneous anthrax caused by butchering a sick cow. Because the spores are resistant to many disinfectants, disinfection by drying and radiation, the spores can remain surviving for decades in soil (26). The local husbandry and health department of the government should strengthen its surveillance of animal anthrax and environmental contamination caused by B. anthracis and take measures to disinfect the environment of the natural foci, which could reduce the risk of broader epidemic of anthrax. From a bioterrorism and biowarfare perspective, B. anthracis could be used as the agent of bioterrorism and biowarfare. The government should attach extremely great importance on the storage, transportation, and handling of B. anthracis. All of the activities have to meet the requirement of the biosafety regulation, which could reduce the risk of bioterrorism and biowarfare.

The results from the molecular investigation suggested that the epidemic was caused by B. anthracis infection, which provided an etiologic basis for the diagnosis of patients and the investigation of this epidemic. Additionally, the two B. anthracis isolates recovered from the epidemic were genetically distinct from isolates from other regions of China and other countries. Our results will not only aid in understanding disease origins and transmission patterns but also facilitate the development of various measures to control anthrax.

The SNP data generated has been uploaded to the European Variation Archive (EVA), Project: PRJEB36719, Analyses: ERZ1300463. Other raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Ethical approval was not required in accordance with institutional guidelines and national legislation. All data have been anonymized, so written informed consent was not required.

SL conceived and designed the experiments, wrote and edited the manuscript. QM and YL performed the experiments. SL, QM, and HC analyzed the data. SL, GY, and GT performed the epidemiological investigation. SL, GT, and DW contributed reagents, materials, and analysis tools.