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Isolation and Identification of Bacteria from the Workers of Live Bird Markets at Mymensingh, Bangladesh

S Sarker
Department of Pathology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

S Talukder
Department of Animal Science & Nutrition, Chittagong Veterinary & Animal Sciences University, Chittagong-4202, Bangladesh

E H Chowdhury
Department of Pathology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

P M Das
Department of Pathology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Context: Identification of bacteria from the workers of live bird markets is important factor for zoonotic aspects and for implementing appropriate control strategies. Objectives: To determine the occurrence of bacteria especially Salmonella sp. and Escherichia coli from the workers of live bird markets. Materials and Methods: A total of 40 samples were collected from hand washes (n=20) and nasal swabs (n=20) of the associated workers in urban and suburban live bird markets. Bacteria were isolated in different media, and identification was performed based on the staining, cultural and some biochemical tests. For Salmonella sp., DNA was extracted using a DNA isolation kit and rfbs gene was amplified by using commercial PCR kit. Results: The bacteria such as Salmonella sp. and E. coli were detected in the samples by several microbial tests. The prevalence of Salmonella sp. was 40% and 30%, and E. coli was 70% and 40% in the hand washes and nasal swabs respectively of the workers of urban and periurban live bird markets. Conclusion: The results obtained in this study suggest that the appropriate precautions should be taken during and subsequent to the handling of live birds to minimize the risk of zoonotic diseases.

Keywords: Salmonella, Escherichia coli, Live bird markets, Isolation and identification

Location: BAU, Mymensingh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

To determine the occurrence of bacteria especially Salmonella sp. and Escherichia coli from the workers of live bird markets.

Methodology:

Collection and culture of samples: The total of 40 samples of which twenty (20) hand wash (10 from urban and 10 from suburban) and 20 nasal swabs (10 from urban and 10 from suburban) were collected from the people working in live bird markets. Test tubes containing samples were incubated for 24 h at 37 ºC. From the cultured samples, subcultures were also made on nutrient agar, BGA agar, EMB agar and blood agar and incubated at 37ºC for overnight. The identification of the organisms was performed by following the tests of Merchant and Packer (1967), Carter (1979), Freeman (1985) and Cheesbrough (2000). On the basis of colony, and staining characters and biochemical tests, the organisms were isolated and identified. The representative bacteria were stained using modified Gram’s staining (Lillie 1928). Biochemical tests were performed according to the methods described by Merchant and Packer (1967). Cultivation of Salmonella isolates and DNA extraction: The purity of Salmonella sp. was verified by cultural, staining and biochemical tests (Cheesbrough 2000). One colony from each sample was inoculated into 10 ml of LB broth (Oxoid Ltd. Bahingstoke, Hamshire, England) and incubated at 37ºC for 18 h. One ml from each culture was taken into a sterile eppendorf tube for extraction of genomic DNA. DNA was extracted from five field samples culturing in LB broth by using Wizard® Genomic DNA Purification Kit (Promegra Corporation. 2800 Woods Hollow Road. Madison, USA). The extracted DNA was quantified using a spectrophotometer’s (Spectronic® GeneticsTM New York, USA) and expressed in ng/ μl. Oligonucleotide Primer and Amplification of DNA by PCR: One pair of oligonucleotide primer (SG1: 5?-tca-cga-ctt-aca-tcc-tac -3? and SG2: 5?-ctg-cta-tat-cag-cac-aac-3?) was used to amplify the rfbS gene of Salmonella sp. that is genus specific. Primers were diluted with appropriate amount of TE Buffer (pH: 8.0) and stored at -20ºC until use. The expected product size of this primer is 720 bp. PCR amplification was performed in a final volume of 10μl containing 2μl (50 ng/ μl) of DNA template, Taq buffer-A 1μl, dNTPs 1μl, primer-F 0.5μl, primer-R 0.5μl, Taq DNA polymerase 0.2μl and 4.8μl nuclease free water. Three independent reactions with the primers were made for DNA template. Amplification was carried out in Gene amplification PCR system 9600 Thermocycler (eppendorf, Germany), using condition modified from Doran et al., (1996). The pre-mix was then mixed well through spinning. Initial denaturation was at 94°C for 1 min, 94°C for 60 sec, annealing at 50°C for 60 seconds and extension at 72°C for 21 seconds, with a final extension at 72°C for 7 minutes for total 33 cycles and held for 4°C. The amplified products were separated by electrophoresis on 1.5% agarose gel containing 5 μl ml-1 ethidium bromide with a 100 bp ladder (Promega, Madison, WI, USA) as molecular weight marker (Oliveira et al. 2003).

Source of Information: J. bio-sci. 17: 135-138, 2009 ISSN 1023-8654

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The results obtained in this study suggest that the appropriate precautions should be taken during and subsequent to the handling of live birds to minimize the risk of zoonotic diseases.

Knowledge Management: Journal