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    Dendrogram based on the MLVA genotyping assay showing relationships of the B. anthracis isolates in this study. The dendrogram was constructed using unweighted pair-group method arithmetic average cluster analysis of diverse B. anthracis genotype scores from the China mainland, Hong Kong Special Administrative Region, and well-known reference strains, including Ames and Sterne by BioNumerics version 4.0 software (Applied Maths, Sint-Martens-Latem, Province of East Flanders, Belgium). Genotype numbers are listed as previously described.9,12 GER = Germany; INDO = Indonesia; SAF = South Africa.

  • 1.

    Hicks CW, Sweeney DA, Cui X, Li Y, Eichacker PQ, 2012. An overview of anthrax infection including the recently identified form of disease in injection drug users. Intensive Care Med 38: 10921104.

    • Search Google Scholar
    • Export Citation
  • 2.

    Dong S, 1999. Progress on the study of prevention and control of anthrax in China. Zhonghua Liu Xing Bing Xue Za Zhi 20: 135137.

  • 3.

    Zhang M, Yao W, Tian J, Yu W, Mao L, Sun B, Qin C, Sun Y, Chen D, Wang Y, 2008. Isolation and identification of Bacillus anthracis in a epidemic situation. Chin J Health Lab Technol 18: 905906.

    • Search Google Scholar
    • Export Citation
  • 4.

    Qin Y, Zhao Z, Liu D, Zhang Y, Zhang Y, Dong D, Zhang L, You Y, Liu R, Cui R, Guo J, 2012. Source tracing and epidemiological characteristics of a cutaneous anthrax outbreak. Chin J Zoonoses 28: 11481151.

    • Search Google Scholar
    • Export Citation
  • 5.

    Zhang T, Cui L, Li L, Zhang M, Qi F, Ying L, Bao C, 2012. Investigation of an outbreak of cutaneous anthrax in Banlu village, Lianyungang, China, 2012. Western Pac Surveill Response J 3: 1215.

    • Search Google Scholar
    • Export Citation
  • 6.

    Sohni Y, Kanjilal S, Kapur V, 2008. Performance evaluation of five commercial real-time PCR reagent systems using TaqMan assays for B. anthracis detection. Clin Biochem 41: 640644.

    • Search Google Scholar
    • Export Citation
  • 7.

    Matero P, Hemmilä H, Tomaso H, Piiparinen H, Rantakokko-Jalava K, Nuotio L, Nikkari S, 2011. Rapid field detection assays for Bacillus anthracis, Brucella spp., Francisella tularensis and Yersinia pestis. Clin Microbiol Infect 17: 3443.

    • Search Google Scholar
    • Export Citation
  • 8.

    Rantakokko-Jalava K, Viljanen M, 2003. Application of Bacillus anthracis PCR to simulated clinical samples. Clin Microbiol Infect 9: 10511056.

    • Search Google Scholar
    • Export Citation
  • 9.

    Keim P, Price LB, Klevytska AM, Smith KL, Schupp JM, Okinaka R, Jackson PJ, Hugh-Jones ME, 2000. Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J Bacteriol 182: 29282936.

    • Search Google Scholar
    • Export Citation
  • 10.

    Cheung DT, Kam KM, Hau KL, Au TK, Marston CK, Gee JE, Popovic T, Ert MN, Kenefic L, Keim P, Hoffmaster AR, 2005. Characterization of a Bacillus anthracis isolate causing a rare case of fatal anthrax in a 2-year-old boy from Hong Kong. J Clin Microbiol 43: 19921994.

    • Search Google Scholar
    • Export Citation
  • 11.

    Wang B, Smith KL, Keys C, Coker P, Keim P, Jones MH, 2002. Molecular epidemiology of Bacillus anthracis in China. Prog Microbiol Immunol 30: 1417.

  • 12.

    Hoffmaster AR, Fitzgerald CC, Ribot E, Mayer LW, Popovic T, 2002. Molecular subtyping of Bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, United States. Emerg Infect Dis 8: 11111116.

    • Search Google Scholar
    • Export Citation
  • 13.

    Welkos SL, Lowea JR, Eden-McCutchanb F, Vodkina FM, Lepplaa SH, Schmidta JJ, 1988. Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis. Gene 69: 287300.

    • Search Google Scholar
    • Export Citation
  • 14.

    Price LB, Hugh-Jones M, Jackson PJ, Keim P, 1999. Genetic diversity in the protective antigen gene of Bacillus anthracis. J Bacteriol 181: 23582362.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

Molecular Characterization of Bacillus anthracis Directly from Patients' Eschar and Beef in an Anthrax Outbreak in Jiangsu Province, China, 2012

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  • Key Laboratory of Enteric Pathogenic Microbiology, Ministry of Health, Jiangsu Provincial Centre for Disease Control and Prevention, Nanjing, Jiangsu Province, China

An outbreak of anthrax was reported in Lianyungang, Jiangsu Province, China in 2012. Laboratory confirmation of cases was made by detection of Bacillus anthracis genes rpoB, pagA, and cap using real-time polymerase chain reaction (PCR); source tracking was conducted by multiple locus variable-number tandem-repeat analysis (MLVA) and pagA sequencing using DNA extracted from case specimens and meat from a suspected slaughtered cow. The genotypes were MLVA type 57 and pagA genotype I. Combined with the field epidemiological data, the four cutaneous anthrax cases most likely were caused by butchering of the sick cow. Backward tracing of animal cases identified the region of origin, and some public health measures, such as reactive or preventative animal vaccination for cattle, intersectoral cooperation, ensuring proper pre-slaughter inspection, and educating butchers and villagers about this disease, could be used to prevent B. anthracis infection.

Bacillus anthracis, the etiological agent of anthrax, is a Gram-positive, spore-forming bacterium that can cause serious and often fatal disease among livestock and humans. Animals are infected by contact with contaminated grass or soil. Human infections may result from contact with infected animals or products made from or derived from those animals, including hair, wool, leather, hide, hoof, and bone. Four types of human anthrax are currently recognized: cutaneous, gastrointestinal, pulmonary (inhalation), and injectional anthrax.1 The most common form, cutaneous anthrax, generally results from infection through abrasions of the skin that often occur when handling animal products contaminated with B. anthracis spores. In China, the main form of infection is cutaneous anthrax, resulting from butchering and subsequent consumption of infected livestock.2

On July 25, 2012, 200 cattle were transported to the slaughter house by trucks in Lianyungang, Jiangsu Province. The cattle originated from a farm in Liaoning Province in the northern part of China, where cutaneous anthrax has been previously reported.3,4 On the same day, a sick cow from this transported cohort from Liaoning was found just after arriving in Ganyu County, unloaded from the truck, and slaughtered by several villagers in Lianyungang. On July 26, four individuals who participated in the slaughter presented to local medical facilities with a variety of symptoms. These symptoms included lesions, fever > 39°C, dizziness, headache, cough, chest pain, fatigue, and armpit lymphadenectasis or submaxillary lymph node intumescence after onset of the disease. An additional four cases presented with mild symptoms. On August 2, a hospital doctor reported a case of anthrax (case 1) with three lesions on both of his hands after slaughtering a sick cow in Banlu village. After confirming the information by telephone, an outbreak response team from Lianyungang Municipal Center for Disease Control and Prevention (CDC) went to the village to conduct an investigation as previously reported.5

Clinical specimens (including blood and eschar) from the four severe cases and uncooked meat from the slaughtered cow were collected, total DNA was extracted from each sample, and the DNA was assayed for the rpoB, pag, and cap genes of B. anthracis by real-time polymerase chain reaction (PCR) using previously described methods.68 Escher samples from all four cases as well as the meat sample were positive for all three B. anthracis genes; none of the blood samples from cases were real-time PCR-positive.

Although B. anthracis is a genetically conserved species, a previous study of 426 isolates showed that multiple locus variable-number tandem-repeat analysis (MLVA) could separate isolates into two main groups (A and B); these groups could be further separated into six unique clusters consisting of 89 distinct genotypes.9 Group A isolates formed four of these clusters (A1–A4) and were found to be responsible for most epidemics and outbreaks. In this study, MLVA was used to provide a genotype of B. anthracis directly from the human and meat samples. All of them, independent of origin, were identified as type 57 (vrrA = 313, vrrB1 = 229, vrrB2 = 162, vrrC1 = 583, vrrC2 = 532, CG3 = 158, pXO1 = 123, and pXO2 = 141); MLVA also confirmed that all of the B. anthracis associated with this outbreak harbored both plasmids pXO1 and pXO2.

Additional analysis of the MLVA type from the outbreak-associated B. anthracis with isolates from mainland China, Hong Kong Special Administrative Region,10,11 and the well-described reference strains Ames12 and Sterne9 confirmed that MLVA type 57 belongs to the dominant branch A3.b, which includes almost all B. anthracis isolates characterized from China11 and strains Ames and Sterne (Figure 1). MLVA type 57 has been associated with B. anthracis isolates recovered from Beijing, Guangxi, Hebei, Shandong Province, and Xinjiang Uygur Autonomous Region. However, B. anthracis isolates from Liaoning, Jiangxi, and Shandong Provinces have previously been typed as MLVA type 58,11 and MLVA type 59 isolates have been recovered from Gansu, Guangxi, Hebei, Henan, Shandong Province, and Inner Mongolia Autonomous Region. Additional accumulation of MLVA data, including data from other provinces, might reveal the potential relationship between strains isolated during the same time period in China or rapidly and easily distinguish non-outbreak isolates, helping to elucidate the geographical distribution of B. anthracis.

Figure 1.
Figure 1.

Dendrogram based on the MLVA genotyping assay showing relationships of the B. anthracis isolates in this study. The dendrogram was constructed using unweighted pair-group method arithmetic average cluster analysis of diverse B. anthracis genotype scores from the China mainland, Hong Kong Special Administrative Region, and well-known reference strains, including Ames and Sterne by BioNumerics version 4.0 software (Applied Maths, Sint-Martens-Latem, Province of East Flanders, Belgium). Genotype numbers are listed as previously described.9,12 GER = Germany; INDO = Indonesia; SAF = South Africa.

Citation: The American Society of Tropical Medicine and Hygiene 91, 3; 10.4269/ajtmh.13-0633

Protective antigen (PA) is encoded by the pagA gene and one of three toxins produced by isolates of B. anthracis; it is a critical component in the development of immunity against anthrax.13 Previous studies have documented six pagA genotypes.14 PagA (2,294 bp) was amplified from real-time PCR-positive samples and sequenced as described.14 All of them in this study were pagA genotype I, identical to B. anthracis strains Ames and Sterne.

Collectively, the genotyping results provided evidence that four human cases and the meat obtained from the slaughtered sick cow may be infected from a common source. Together with the epidemiological investigation, our evidence indicated the link between the four human cases and the slaughtered sick cow. In conclusion, our results provide evidence supporting the usefulness of genotyping for epidemiological investigation.

We recommend that some public health measures should be taken to prevent B. anthracis infection, including identification of the region of origin by backward tracing of animal cases, reactive or preventative animal vaccination for cattle, intersectoral cooperation between medical and veterinary authorities, ensuring proper pre-slaughter inspection to ensure that only healthy animals are used for meat sources, and providing education to butchers and villagers about this disease. This latter point addresses refraining from butchering and eating meat from sick livestock or carcasses and leaving the disposal of these animals to veterinary or other trained personnel.

ACKNOWLEDGMENTS

The authors thank Dr. John Klena from the US Centers for Disease Control and Prevention for modifying our manuscript.

  • 1.

    Hicks CW, Sweeney DA, Cui X, Li Y, Eichacker PQ, 2012. An overview of anthrax infection including the recently identified form of disease in injection drug users. Intensive Care Med 38: 10921104.

    • Search Google Scholar
    • Export Citation
  • 2.

    Dong S, 1999. Progress on the study of prevention and control of anthrax in China. Zhonghua Liu Xing Bing Xue Za Zhi 20: 135137.

  • 3.

    Zhang M, Yao W, Tian J, Yu W, Mao L, Sun B, Qin C, Sun Y, Chen D, Wang Y, 2008. Isolation and identification of Bacillus anthracis in a epidemic situation. Chin J Health Lab Technol 18: 905906.

    • Search Google Scholar
    • Export Citation
  • 4.

    Qin Y, Zhao Z, Liu D, Zhang Y, Zhang Y, Dong D, Zhang L, You Y, Liu R, Cui R, Guo J, 2012. Source tracing and epidemiological characteristics of a cutaneous anthrax outbreak. Chin J Zoonoses 28: 11481151.

    • Search Google Scholar
    • Export Citation
  • 5.

    Zhang T, Cui L, Li L, Zhang M, Qi F, Ying L, Bao C, 2012. Investigation of an outbreak of cutaneous anthrax in Banlu village, Lianyungang, China, 2012. Western Pac Surveill Response J 3: 1215.

    • Search Google Scholar
    • Export Citation
  • 6.

    Sohni Y, Kanjilal S, Kapur V, 2008. Performance evaluation of five commercial real-time PCR reagent systems using TaqMan assays for B. anthracis detection. Clin Biochem 41: 640644.

    • Search Google Scholar
    • Export Citation
  • 7.

    Matero P, Hemmilä H, Tomaso H, Piiparinen H, Rantakokko-Jalava K, Nuotio L, Nikkari S, 2011. Rapid field detection assays for Bacillus anthracis, Brucella spp., Francisella tularensis and Yersinia pestis. Clin Microbiol Infect 17: 3443.

    • Search Google Scholar
    • Export Citation
  • 8.

    Rantakokko-Jalava K, Viljanen M, 2003. Application of Bacillus anthracis PCR to simulated clinical samples. Clin Microbiol Infect 9: 10511056.

    • Search Google Scholar
    • Export Citation
  • 9.

    Keim P, Price LB, Klevytska AM, Smith KL, Schupp JM, Okinaka R, Jackson PJ, Hugh-Jones ME, 2000. Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J Bacteriol 182: 29282936.

    • Search Google Scholar
    • Export Citation
  • 10.

    Cheung DT, Kam KM, Hau KL, Au TK, Marston CK, Gee JE, Popovic T, Ert MN, Kenefic L, Keim P, Hoffmaster AR, 2005. Characterization of a Bacillus anthracis isolate causing a rare case of fatal anthrax in a 2-year-old boy from Hong Kong. J Clin Microbiol 43: 19921994.

    • Search Google Scholar
    • Export Citation
  • 11.

    Wang B, Smith KL, Keys C, Coker P, Keim P, Jones MH, 2002. Molecular epidemiology of Bacillus anthracis in China. Prog Microbiol Immunol 30: 1417.

  • 12.

    Hoffmaster AR, Fitzgerald CC, Ribot E, Mayer LW, Popovic T, 2002. Molecular subtyping of Bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, United States. Emerg Infect Dis 8: 11111116.

    • Search Google Scholar
    • Export Citation
  • 13.

    Welkos SL, Lowea JR, Eden-McCutchanb F, Vodkina FM, Lepplaa SH, Schmidta JJ, 1988. Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis. Gene 69: 287300.

    • Search Google Scholar
    • Export Citation
  • 14.

    Price LB, Hugh-Jones M, Jackson PJ, Keim P, 1999. Genetic diversity in the protective antigen gene of Bacillus anthracis. J Bacteriol 181: 23582362.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Yefei Zhu, Key Laboratory of Enteric Pathogenic Microbiology, Ministry of Health, Department of Acute Infectious Disease Control and Prevention, Jiangsu Provincial Center for Disease Control and Prevention, 172 Jiangsu Road, Nanjing 210009, China. E-mail: jszyf@jscdc.cn

Financial support: This study was supported by Jiangsu Province Health Development Project with Science and Education Grants ZX201109 and RC2011085 and Jiangsu Province Preventive Medicine Research Project No. Y2012093.

Authors' addresses: Zhongming Tan, Xian Qi, Ling Gu, Changjun Bao, Fenyang Tang, and Yefei Zhu, Key Laboratory of Enteric Pathogenic Microbiology, Ministry of Health, Jiangsu Provincial Centre for Disease Control and Prevention, Nanjing, Jiangsu Province, China, E-mails: jstzm@jscdc.cn, qixiansyc@hotmail.com, jcgl126@126.com, bao_2000cn@163.com, tfyepi@jscdc.cn and jszyf@jscdc.cn.

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