Prevalence of Bacillus anthracis Spores in Soil of District Badin

Bacillus anthracis is the etiological agent of anthrax, a serious and worldwide disseminated zoonotic disease affecting a wide array of wild and domestic animals, consistently also humans. The disease may cause high mortality, especially in herbivores. B. anthracis is a spore-forming, Gram positive, non-motile, rod-shaped, and obligate pathogen of genus Bacillus. Spores formation only happens when the bacterium is exposed to extreme environmental conditions that are totally opposed for the continued multiplication of the vegetative form. B. anthracis spores are highly resilient to chemical disinfectants and environmental conditions including high temperature (Dey et al., 2012; OIE, 2008), thus could be persist in soil for decades, and considered very difficult to eliminate from the environment. Therefore, epidemiological surveys of different geographical locations for determination of disease prevalence and circumstances that favour B. anthracis persistence in soil are the most important areas of anthrax research (Dragon et al., 2001).

It has been suggested that the ecology and epidemiology of anthrax is largely influenced by environmental factors that probably effect the spore concentrations at various sites and regions (Ganz et al., 2014). Though, the ecobiology of B. anthracis entails more research to completely illuminate the mechanism responsible for transmission and spread of organism in various geographical locations. It will progress effective means for eradication of the disease from contaminated areas, and cut the risk of future epidemics (Dey et al., 2012).

The qualitative and quantitative estimation of microbes in a certain portion of soil are determined by a multifaceted interaction of variable quantities of moisture, temperature, sunlight, nutrients, redox potential and soil pH. Microorganisms may be native or enter the soil indirectly through manure application, animal deposits, or contaminated sewage water (Mongoh et al., 2008). Factors like, predation, infection and competition by other microorganisms (viruses, bacteria, fungi, protozoa and archaea) further preferred the major species, mainly in diverse rhizosphere (nutrient rich soil zone adjacent a plant root). It has hypothesized that strategies that bacteria use to exist in soil competition may, in turn, offer the competence to infect individuals (Cheun et al., 2003).

Keeping in understanding the above scenario, the current study was planned to explore the prevalence of B. anthracis spores contamination in soil of different areas of district Badin. It further identified the association of anthrax risk with various soil types.

Among infectious diseases, anthrax is a disease itemized in the World Organization for Animal Health (OIE) Terrestrial Animal Health Code and mandatory to report to the OIE (OIE Terrestrial Animal Health Code). According to this manual, while dealing with anthrax bacteria appropriate safety precautions must be strictly followed and laboratory work must be done with a minimum health hazard to the environment (biocontainment) and staff (biosafety) (OIE, 2008).

A total of 100 samples were collected for this study from five different talukas (n= 20 from each talukas) such as Golarchi, Tando Ghulam Ali, Tando Bago, Talhar and Matli of district Badin, Sindh, Pakistan. The place of sample collection in the study area were selected based on suspected carcass disposal or burial sites, comparatively low-lying area, livestock habitats and livestock pasturing sites. Approximately 400gm of superficial soil from a maximum depth of one-foot were collected in double layered plastic bags and were transported in cooling box to the Vaccine Production Unit (VPU) Tandojam in Anthrax Spore section for further evaluation process.

The physical analysis of soil samples for soil type (clay, silt and loam) were physically analyzed through methods as suggested by FAO, (1974). Whereas, for chemical analysis, the soil samples were investigated through the methods suggested by Eckert and Sims (1995) for pH, whereas, electrical conductivity (EC dS/m) was calculated through the method as suggested by Richards (1954). Calcium carbonate (CaCO3%) were observed by method developed by Wolf and Beegle (1995), while, organic Matter (OM%) were determined in the soil samples through oxidation-reduction titration with ferrous sulphate as suggested by Walkley, (1947).

Bacterial spore extraction of soil samples was carried out under careful precautions at Vaccine Production Unit (VPU) Tandojam in Anthrax Spore Section according to OIE manual (2008). Extraction of spores was done using 10 g of soil sample that was suspended overnight in 100 ml of sterile distilled water. Supernatants were sieved through 0.45μm pore cellulose nitrate, and deposits were suspended in 5 ml sterilized phosphate-buffered solution (PBS, pH: 6.6 ± 0.2), aliquoted and heated at temperature regimen of 70°C for 20 min in water bath. Sample suspension was centrifuged at 4000 rpm for 20 minutes then it was pelleted and re-suspended in 2ml PBS. Then the aliquot was plated onto heart infusion (BHI) agar, nutrient agar, MacConKey’s agar, Muller Hinton agar, egg yolk agar and blood agar. Cultured plates were incubated at 37 degree Celsius, in an aerobic incubator. Colonies evolving at the end of 24–48h incubation period were studied for cultural characteristics and morphological features defined to B. anthracis. Confirmation of B. anthracis was done on the basis of biochemical properties and antibiotic penicillin-G (10 IU/disc; Difco, Labarotries Inc) susceptibility test as described previously (Rajput et al., 2017a).

All the resulted data were entered a computer database and analyzed using analysis of variance (ANOVA) with statistical analytical system software (SAS, 2009). Correlation analysis was also performed to determine the relationship among chemical properties of soil and prevalence of B. anthracis spores.

The chemical properties of soil samples collected from district Badin were evaluated and the results reported in Table 1. The lowest EC was calculated in the soil samples of Taluka Matli (3.72 dS/m) and highest was calculated in the soil samples of Taluka Talhar (4.55 dS/m). The overall mean EC (dS/m) of soil samples of district Badin was found 4.21. The lower pH value was observed in the soil samples of Taluka Talhar (8.34) and higher value was found for the soil samples of Taluka Matli and Golarchi (8.52). The overall pH of soil of district Badin was found 8.46. The higher OM% was calculated in the soil samples of Taluka Tando Bago (0.57) and lower OM% was observed in Taluka Talhar (0.48). The overall OM% of district Badin was 0.536. The higher CaCO3% was found in the soil sample of Taluka Matli (7.8%) and lower was found in Taluka Talhar (6.65%). The overall CaCO3% was recorded in soil sample of district Badin was 7.04. The highest number of B. anthracis positive soil samples were recorded for Taluka Talhar, Golarchi and Tando Bago (05), followed by Taluka Badin (03), while lowest was found in Taluka Matli (02).

Table 1: Chemical Properties of Soil Samples Collected from different Talukas of district Badin

As shown in Table 2, among 100 soil samples of district Badin, 46 were silty loam type, 10 were clay loam type and 44 were silty clay loam type in physical nature. The higher prevalence of B. anthracis spores was observed in clay loam type soil samples (7/10; 70%) followed by silty loam type soil (8/44; 18.18%) and silty clay loam type soil samples (5/46; 10.86%) respectively. The higher B. anthracis spores were found in the soil samples of Taluka Talhar, Golarchi and Tando Bago (25%) followed by Taluka Badin (15%) and Taluka Matli (10%), respectively (Table 2).

The correlation among chemical properties of soil samples and prevalence of B. anthracis were evaluated and presented in Table 3. The results indicated that positive and lower correlation was observed among OM% and prevalence of B. anthracis (0.0276) and CaCO3% and prevalence of B. anthracis (0.1775). While negative correlation was observed among soil pH and prevalence of B. anthracis and EC and prevalence of B. anthracis.

Although great advances in understanding the biology and ecology of B. anthracis, anthrax sustained to cause significant losses to livestock industry throughout the globe. Spread of B. anthracis is environmentally facilitated and such ecological communications in the environment probably effect its spread to consequent animal hosts that swallow spores during grazing (Ganz et al., 2014). However, although outbreaks are more or less infrequently occurring today, especially in developing countries, anthrax is still of relevance and in order to control outbreaks it is of major importance to gain further understanding of the ecology of B. anthracis (Dragon et al., 2001). Epidemiological studies have declared that in the higher and middle latitudes of Europe, B. anthracis infection in animals is now either absent or present only in very intermittent level, however it is found comparatively common in countries adjacent the Mediterranean Sea.

In Pakistan, B. anthracis infection is still one of the important infectious diseases of man and livestock animals because of its extensive dispersal. In some parts of the country, the disease is sporadic during the rainy season (Shabbir et al., 2015). Unfortunately, no efficient studies of this disease in Pakistan have carried out yet, although controlling the disease through immunization has been adopted for many years. The strategic plans for effective anthrax control and its prevention must need precise epidemiology of the disease. In current decade, some epidemics of anthrax have been reported by the veterinarians in some regions of district Badin, Sindh (unpublished data). Therefore, we decided to carry out this epidemiological study to record the presence of the B. anthracis spores in soil of district Badin.

In current investigation a total of 100 soil samples from different talukas of district Badin were examined and among those 20 were found positive for B. anthracis spores. Another study reported 15% prevalence of anthrax spores

Table 2: Prevalence of B. anthracis spores in different types of soil samples at district Badin

Table 3: Correlation among chemical properties of soil samples and prevalence of B. anthracis spores in district Badin

EC: electrical conductivity (dS/m; deciSiemens per meter); OM: organic matter (%); CaCO3: calcium carbonate (%); prevalence (%)

in Isfahan, Iran using similar culture technique (Jula et al., 2004), while a Canadian study reported 1.9% prevalence of anthrax spores in soil samples of Northern Canada (Zaidan et al., 2015). A study investigating the soil-borne zoonotic pathogens in Pakistan through real time PCR reported the 37.90% prevalence of B. anthracis in Lahore (Shabbir et al., 2015), whereas, our earlier work demonstrated the 40% prevalence of anthrax spores in soil (Rajput et al., 2017a) and 28% in hair/wool samples of small ruminants (Rajput et al., 2017b) in endemic areas of Tharparker. These variations in prevalence rate of anthrax spores could be justified because of geographical differences (Mshelia et al., 2016), ecological factors including soil pH, presence of certain cations in soil, particularly calcium, and oxygen contents of soil (Hammerstrom et al., 2011).

Our study has further indicated significant differences in the percentage prevalence of B. anthracis spores in different talukas of district Badin. The highest number of B. anthracis positive soil samples were recorded for Taluka Talhar, Golarchi and Tando Bago (05), followed by Taluka Badin (03), while lowest was found in Taluka Matli (02). Studies have suggested a positive correlation between the animal market chain and/or highly populated animal areas with the geographical spread of diseases.

Environmental factors, such as humidity and high ambient temperature could provide a milieu for spread and survival of B. anthracis spores (Kracalik et al., 2013). District Badin of Sindh province have temperature as high as 40°C and relative humidity almost 75% during most times of the year (Leghari et al., 2000), which favoured the high incidence of anthrax spores in the present study area. The current study area (Badin district) is a drought affected area of Sindh province, having least human development index (HDI), where scarcity of both water and food are quite common (Leghari et al., 2000). The factors including nutritional deficiencies suppress the development of animal’s immunity, thus play a vital role in the onset of infection (Rakib et al., 2016; Mondal and Yamage 2014).

The moisture content of soil mainly depends on its physical properties and type, which may influence the spore viability and persistency in soil. The soil of district Badin was found loamy type and highest prevalence (70%) was recorded in clay loam soil. These results are in accordance with a previous study from Bangladesh that reported that all positive soil samples were of loamy type (Ahsan et al., 2013).

The soil pH directly related with the ratio of many soil nutrients (Pabian and Brittingham, 2012). Alkaline soil containing high Ca, nitrogen, and organic matter that provide favorable conditions to the spore growth and/or survival in soil (Jula et al., 2007; Hugh-Jones and Blackburn, 2009). It is also well recognized that a slight alkaline pH (7.2-8.7) and soil that rich in calcium and organic matter promotes the survival of resilient B. anthracis spores (Moazeni-Jula et al., 2004; Dey et al., 2012). These afore mentioned facts were in agreement to our current findings as we found the positive correlation between organic matter and prevalence of B. anthracis and CaCO3% and prevalence of B. anthracis, however we observed a negative correlation between soil pH and prevalence of B. anthracis. Our result regarding negative correlation of pH and spores occurrence needs further investigation, as it is not inconsistent with the studies of Dey et al. (2012).

From the current report, it is concluded that B. anthracis spores were prevailing (20%) in soil samples of all talukas of district Badin. A comparatively higher prevalence was recorded in Taluka Talhar, Golarchi and Tando Bago (25%), that followed by Badin (15%), and Matli (10%). When positive soil samples were analyzed for physical properties, it was noted that the highest prevalence of anthrax spores were observed in clay loam type soil (70%), that followed by silty clay loam (18.18%) and silty loam type soil (10.86%). Furthermore, a positive correlation was recorded between organic matter and prevalence of B. anthracis spores and calcium contents of soil and prevalence of B. anthracis spores.

All staff of the Badin Veterinary Hospital and Anthrax section, VPU is highly appreciated for support in sample collection and analysis respectively.

Authors declare no conflict of interest.

ASM and AAK conceived the experimental design. ASM done experiments. KRB and IAS helped in experiments and paper writing. AAK and SHA helped in statistical analysis and proof reading.