Detection of vero toxin, cytolethal distending toxin and intimin gene by real time polymerase chain reaction and characterization of E. coli isolated from mutton

Bhong, Chandrakant (2006) Detection of vero toxin, cytolethal distending toxin and intimin gene by real time polymerase chain reaction and characterization of E. coli isolated from mutton. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.

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Abstract

This study was undertaken on E. coli isolates obtained from market mutton to find out prevalence of virulence genes, VT 1, VT 2, CDT 1 and eae and characterization by rep-PCR, enterohemolysin production, antibiogram and mannose sensitive and mannose resistance haemagglutination assay, to judge the hygienic level and diversity among the isolates at phenotypic and genotypic level. Total 93 samples belonging to 35 serotypes were tested during the experiment. Real Time PCR based detection was carried out for virulence genes using SYBR Green II fluorescence dye. Fluorescence was measured at extension step of each cycle. Amplified product was confirmed by melt curve analysis. These amplified product and 2 per cent agarose eletrophoresis. Prevalence of VT 1 gene in these isolates was high (38.70 per cent) on the other hand, that of VT 2 gene was nil (0 per cent) while that of eae and CDT 1 was very less (3.22 ) and 0 per cent respectively Repetitive element PCR (rep-PCR) uses outward facing primers to amplify multiple segments of DNA located between conserved repeated sequences interspersed along the bacterial chromosome. Polymorphism of rep-PCR amplification products can serve as strain specific molecular fingerprints. In this study, capability of repetitive BOX elements, enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) was tested to detect genetic diversity among Escherichia coli strains recovered from market mutton. DNA from 93 E. coli isolates were extracted and used to amplify BOX, ERIC and REP sequences. DNA from E. coli strains produced 1-11 bands by BOX-PCR, 2-13 bands by ERIC-PCR, where as 1-13 bands by REP-PCR. All these primers showed good discriminating power. BOX-PCR produced 23 different products where as ERIC-PCR and REPPCR produced 26 and 25 products, respectively. All bands were showing 100 per cent polymorphism. These results suggest intense heterogeneity or genetic diversity among E. coli strains studied. Also, rep-PCR has discriminating capacity that could improve the studies needed to understand genetic makeup of E. coli strains. 31.18 per cent isolates were found positive for enterohemolysin production when tested on washed sheep blood agar supplemented with CaCl2. All enterohemolysin producing isolates were also positive for VT 1 gene. Further, the isolates were tested against for 12 antimicrobial agents. Majority of the isolates were while Pefloxacin (93.55 per cent) and lowest sensitivity to Amikacin (67.74). Out of 93 isolates 32.25 per cent isolates showed Mannose Sensitive Haemagglutination Assay and 62.38 per cent showed Mannose Resistance Haemagglutination Assay.

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