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Investigation Into Bacillus Anthracis Spore in Soil and Analysis of Environmental Parameters Related to Repeated Anthrax Outbreak in Sirajganj, Bangladesh

Md. Murshidul Ahsan
Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Faculty of Veterinary Science, Mymensingh-2202, Bangladesh and School of Biotechnology, Yeungnam University, Gyeongsan 712-749, South Korea.

Mohammad Ferdousur Rahman Khan
Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Faculty of Veterinary Science, Mymensingh-2202, Bangladesh.

Md. Bahanur Rahman
Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Faculty of Veterinary Science, Mymensingh-2202, Bangladesh.

Jayedul Hassan
Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Faculty of Veterinary Science, Mymensingh-2202, Bangladesh.

Shah Md. Ziqrul Haq Chowdhury
Bangladesh Agricultural Research Council (BARC), Dhaka, Bangladesh.

Md. Shafiullah Parvej
Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Faculty of Veterinary Science, Mymensingh-2202, Bangladesh.

Mueena Jahan
Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Faculty of Veterinary Science, Mymensingh-2202, Bangladesh.

K. H. M. Nazmul Hussain Nazir
School of Basic Studies, Yeungnam University, Gyeongsan 712-749, South Korea.

Abstract/ Executive Summary:

The study was conducted for the isolation and detection of Bacillus anthracis spores from soil collected from Sirajganj district (a north-western district of Bangladesh), and to assess the parameters that may relate to the repeated anthrax outbreak. A total of 48 soil samples were collected from the study area during January to November 2012. Endospores were extracted from soil and the Bacillus anthracis was identified using conventional bacteriological, biochemical and sensitivity test against Penicillin-G. The viable B. anthracis spores could be detected from 14 (29.17%) soil samples. Moisture content, pH, calcium and organic carbon contents of the soils were measured and the values of the endospore positive samples ranged from 6.31-28.37%, 5.17-7.22, 484.35-1372.35 ppm and 0.15-2.35%, respectively. All the endospore positive soil samples were of loamy type, while none of the clay type soil was found to be positive for B. anthracis, suggesting the influence of soil type on the occurrence of anthrax endospore in studied area. The mean pH of anthrax positive soil was weakly acidic (6.38±0.15), indicating that a suitable pH range for anthrax spore was present in the soil of Sirajganj. During the disease outbreak period (May and June) the average temperature of this area was 32⁰C and the average rainfall was 158 mm and 90 mm, respectively. Although the temperature variation had no significant influence on the occurrence of anthrax spore, rainfall was found to be significant.

Keywords: Anthrax, Bacillus anthracis spore, Bangladesh, Ecology

Location: Sirajganj District, Bangladesh

Start Date: 00-01-2012

End Date: 00-11-2012

Program Area: Crop-Soil-Water Management

Commodity: Land management

Objectives:

To investigate the association of anthrax spore in soils in Sirajganj district, and to evaluate the environmental parameters that might have positive influence on the survival of the bacterial spores in the soil.

Methodology:

Site selection and sample collection: The study was conducted in 3 Upazillas (sub-district) of Sirajganj (a north-western district of Bangladesh) namely Shahzadpur, Belkuchi and Ullapara over a period of January to November 2012. A total of 48 soil samples were collected randomly from anthrax reported areas. The place of sample collection in the study area was selected based on suspected carcass disposal or burial sites, comparatively low-lying area, livestock habitats and livestock pasturing sites. Approximately 400-gm of surface soil from a maximum depth of one-foot was collected in double layered plastic bags and transported to laboratory as early as possible. Isolation and identification of Bacillus anthracis: Isolation and identification of the bacteria were carried out at the Bacteriology and Molecular Microbiology Laboratory of the Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University (BAU). Physical and chemical parameters of the collected soils were examined at the Department of Soil Science, BAU. The isolation and identification of B. anthracis from the soil samples was performed according to the procedures described in the Manual for Laboratory Diagnosis of Anthrax and OIE. In brief, one gram of soil sample was blended in 10 ml of sterile distilled water and placed in a water bath at 62.5±0.50C for 30- 60 min. The heat will destroy all non-spore-forming bacteria. 10-fold dilution to 10-² or 10-³ was then prepared. From each dilution, 250-300 μl was plated on to Polymyxin B - Lysozyme - EDTA - Thallous acetate agar (PLET agar, Sigma-Aldrich, Switzerland) and incubated at 37⁰C for 40-48 hours. The PLET agar is a selective medium for B. anthracis that inhibits all Gram-negative and most Gram-positive bacteria including B. cereus. For confirmatory identification, the colonies were grown on Blood agar, Nutrient agar and Gelatin stab agar to observe the characteristic morphology. Microscopic examination was done after staining the bacteria by Gram’s Method and MacFadyean reaction. In addition, the bacteria were subjected to biochemical tests and antibiotic susceptibility test against Penicillin-G (10 IU/disc; Oxoid, UK). Physical and chemical analysis of soil: Soil type (e.g, sandy, loamy or clay) was determined by gross examination. Soil moisture content was determined by Gravimetric method. Soil pH was determined by glass electrode pH meter. Soil calcium content was determined. Organic carbon was determined by wet-oxidation method. Collection of the data regarding weather parameters (temperature and rainfall): Weather related data of the study was collected from the website of Accuweather (http://www.accuweather.com). Data analysis: Statistical analysis was performed using Statistical Package for Social Science (SPSS) commercial software packages (version 17). Frequency tables and cross tables were produced to present study findings. One-way ANOVA was used to see association among soil parameters followed by Dunkan’s Multiple Range Test (DMRT). Cramer’s V test was used to measure possible association between different subareas for presence of B. anthracis spore in the soil. A p value of < 0.05 was considered significant in all analysis.

Source of Information: Thai J Vet Med. 2013. 43(3): 449-454.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The repeated anthrax outbreak in livestock (mostly cattle) and subsequent infection to human has been considered as a nationwide alarming issue in Bangladesh. The study revealed, for the first time in Bangladesh, certain ecological factors that might be responsible for survival of anthrax spore in soil, e.g. soil type, Ca content, organic carbon content and soil pH.

Knowledge Management: Journal