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Characterization of Salmonella Isolated from Pigs of Sadar Upazila of Mymensingh District

S C Paul
Department of Microbiology and Hygiene, Banagladesh Agricultural University, Mymensingh, Bangladesh.

M S R Khan
Department of Microbiology and Hygiene, Banagladesh Agricultural University, Mymensingh.

M M Amin
Department of Microbiology and Hygiene, Banagladesh Agricultural University, Mymensingh

F Begum
Department of Microbiology and Hygiene, Banagladesh Agricultural University, Mymensingh.

J Hassan
Veterinary Training Institute, Khakdahor, Mymensingh.

M S Islam
Department of Microbiology and Hygiene, Banagladesh Agricultural University, Mymensingh.

Abstract/ Executive Summary:

Characterization of Salmonella isolated from pigs was conducted between January to November, 2008 in the district of Mymensingh. Out of 23 pigs examined, only 4 (17.39) pigs had Salmonella infection. Genome analysis using PCR revealed that ST gene of enterotoxin was absent in some strains of pigs Salmonella but it’s not clear whether the serovars belongs to Salmonella (S.) choleraesuis, S. typhimurium or S. derby. The isolates were found to be highly sensitive to ciprofloxacin, kanamycin, nalidixic acid, co-trimoxazole and cephalexin but highly resistant to chloramphenicol.

Keywords: Salmonella, Isolation, Characterization, PCR, ST gene, Pig, Antibiogram

Location: Sadar Upazila of Mymensingh district, Bangladesh

Start Date: 00-01-2008

End Date: 00-11-2008

Program Area: Animal Health and Management

Commodity: Pig

Objectives:

To isolate and characterize the Salmonellae spp. of pig from Mathorpotty, Sadar upazilla, Mymensingh and compare these with the isolates of chicken and human by the application of molecular tool, PCR.

Methodology:

A total number of 23 field samples comprising rectal swab and faeces samples from apparently healthy and diarrhoeic pigs were aseptically collected and carried to the laboratory in Bacto selenite broth (BSB) for the characterization of Salmonellae. The samples were then cultured on to Salmonella-Shigella (SS) agar, MacConkey (MC) agar, Brilliant Green agar (BGA) and Eosin Methylene Blue (EMB) agar. Individual colonies were picked up and stained with Gram stain for morphological study and subjected to biochemical tests (sugar fermentation tests, Voges Proskauer test, Indole test and Methyl red test). Salmonella polyvalent antiserum (Poly “O” and Poly “H”) was used for the serological identification of Salmonella spp. The isolated Salmonella spp. of pig and previously isolated Salmonella of chicken and human were preserved in 20% glycerin. The isolated Salmonella preserved in 20% glycerin were placed in ice box and transported to ICDDR,B for performing molecular characterization by PCR. DNA template was prepared by inoculation a single colony from LA plate to 3 ml of LB broth and incubated at 37⁰C with agitation (1200 rpm). Then 1.5 ml of sample was taken in an eppendorf tube and centrifuged at 13000 rpm for 10 min. After then supernatant was discarded and pellet was dissolved in the sterile normal saline and again sample was centrifuged at 13000 rpm for 10 minutes. The supernatant was discarded and the pellet was dissolved in 200μl of normal saline. The samples was boiled for 10 minutes and cooled in ice for 30 minutes. Then the sample was centrifuged at 13000 rpm for 10 minutes and supernatant was transferred to a fresh eppendorf tube and stored at -20⁰C for use template DNA in PCR. PCR amplification was performed in a final volume of 20μl containing 3μl of DNA template, Tap polymerase 0.2μl, dNTP 10mM, F primer (GCCTGA GCG AGA AGGT) 1.0μl, R primer (CAG TCC CACCCA CTT C) 1.0μl, MgCl2 50mM and Deionized Distilled water 8.8μl. The required number of PCR tubes were labeled and kept on ice. Then 17μl reaction mixture was dispensed into each of the PCR tubes and 3μl of DNA from each sample was added to that respective tube and mixed properly with the help of the micropipette. The tubes were placed in peltier thermocycler. The initial denaturation was at 95⁰C for 5 minutes, 95⁰C for 1 minute, annealing at 60⁰C for 1 minute and extension at 72⁰C for 10 minutes for total 7 cycles and held for 4⁰C for maximum 18 hours. After completion of PCR, PCR products were kept in 0⁰C for Agarose Gel Electrophoresis. The fingerprint in the gel was analyzed using a computer software package, Quantity one version 3.0 (Bio-Red, USA). After background subtraction and gel normalization, the fingerprint patterns were subjected to typing based on banding similarity and dissimilarity. Two methods for similarity measuring, one based on binary data of occurrence of the band (band- based) calculated using the Dice coefficient and another method based on overall densitometry profile (curve-based) of the banding pattern calculated using Pearson’s product moment correlation were compared. Eight different antibacterial discs, manufactured by Span Diagnostics Limited, India were selected for the antibacterial sensitivity study against isolated Salmonella that were marketed by Stimulus Speciality Diagnostics, 173-B, New Industrial Estate, Udhna-394210 (Suart) India. The antibacterial agents corresponding eight different discs are commonly used in the field condition for the treatment of salmonellosis.

Source of Information: Bangl. J. Vet. Med. (2009). 7(1) : 281 – 286

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The study also opined that the isolated Salmonellae were highly sensitive to ciprofloxacin, kanamycin, nalidixic acid, co-trimoxazole and cephalexin, moderately sensitive to kanamycin, nalidixic acid, co-trimoxazole, cephalexin, amoxicillin and erythromycin. These are less sensitive to erythromycin, amoxicillin and chloramphenicol while resistant to chloramphenicol. The antibacterial resistance observed here in the isolated Salmonella might be due to indiscriminate use of those antibacterial agents in field condition in study areas and/or rapid chromosomal mutation and presence of specific plasmid DNA. The antibiotic sensitivity tests were performed by disc diffusion method using eight different antibiotic discs. The isolates were found to be highly sensitive to ciprofloxacin, kanamycin, nalidixic acid, co-trimoxazole and cephalexin but highly resistant to chloramphenicol.

Knowledge Management: Journal