Veterinary World, EISSN: 2231-0916 Available at www.veterinaryworld.org/Vol.6/Oct-2013/15.pdf RESEARCH ARTICLE Open Access

Aim: Present study was undertaken to find out the frequency of few virulent genes and prevalence of related strains of Escherichia coli isolated from chicken meat obtained from chicken retail shops by Polymerase Chain Reaction (PCR). Materials and Methods: 66 samples of freshly slaughtered chicken meat were collected from 22 identified retail shops located at Mumbai city, randomly. Processed meat samples were cultured in EMB agar and presumptive colonies were confirmed by various biochemical tests. PCR method was accustomed for identification of the genes coding for heat-stable enterotoxin a (STa), heat labile enterotoxin (LT), shiga-like toxins 1 and 2 (SLT1 and SLT2). E. coli isolates were sent to National Salmonella and Escherichia Centre, CRI, Kasauli, HP, India for serotyping. Results: 11 (16.67%) E. coli strains were isolated from 66 chicken meat samples. 3 (27.27%) out of 11 harbored the gene for SLT2, and 2 (18.18%) for STa. None of the strain contains SLT1 and LT genes. Serotypes detected were rough, O2, O20, O22, O102 each for one isolate and 6 isolates were untypable (UT). Conclusion: The results concluded that chicken meat samples analysed harbored genes for shiga like toxins and enterotoxins and different serotypes of E. coli. These findings indicating that regular monitoring of chicken meat is essential for this pathogen to prevent potential public health problems.


Introduction
fever, watery diarrhea and nausea [5].Heat-stable (ST) and heat labile (LT), these two enterotoxins, produced Escherichia coli strains are usually recovered by this category plays a distinct role in the from intestinal tracts of human, poultry, animal and pathogenesis.Two classes of heat-stable toxins, STa commonly present in soil, water and foods due to faecal (STI) and STb (STII), differ structurally and contamination or contamination during food animal functionally.The genes encoding both these toxins are slaughter.They are commonly present in nature and present on plasmids [6].Shiga-like toxin-producing E. has major role in maintaining equilibrium of intestinal coli strains, responsible for HUS, HC, diarrhea and physiology of poultry [1].Some E. coli strain causes renal failure in children, are zoonotic water and food several human diseases like haemolytic uremic borne pathogens [7].Previous reports revealed that syndrome (HUS), haemorrhagic colitis (HC) etc. and most outbreaks of HUS and HC are caused by serotype various isolates established as major pathogen for E. coli O157:H7 [8].STEC produces two types of poultry colibacillosis [2,3].It is recognized as major shiga-toxins, SLT1 and SLT2.These toxins have pathogen for public health problems in developing similar biological functions, molecular structure and countries and represents leading etiological agent of encode different set of genes in which most of genes are diarrhea [4].
virulence associated so that STEC contaminated food Several classes of E. coli, specifically could be at risk for public health.There are several enteropathogenic E. coli (EPEC), enteroinvasive E.
techniques for detection of toxins in which PCR widely coli (EIEC), enterotoxigenic E. coli (ETEC), Shigataken for targeting SLT-specific genes in many studies l i k e t o x i n -p r o d u c i n g E .c o l i ( S T E C ) o r [9,10].Therefore, this study was designed taken to find enterohaemorrhagic E. coli (EHEC) or verotoxin out the frequency of ETEC and STEC virulent genes producing E. coli (VTEC), enteroaggregative E. coli isolated from chicken meat slaughtered in the retail (EAggEC) have been recognized [4].Enterotoxigenic chicken shops by PCR method.E. coli infection owing to ingestion of contaminated food or water produces abdominal cramps, low-grade and 66 samples of chicken meat were collected was dried by keeping tubes open and resuspended in 50 randomly (Random Sampling Method).From these µl of elution buffer.Finally, DNA integrity was samples, muscle pieces were separated into sterile bags assessed by running in 1.7% agarose (HiMedia, India) 0 by using sterile forceps and scissors and transported by gel.Extracted DNA was stored at -20 C till further use.maintaining cold.
Primers: The presence of virulence associated genes Identification of E. coli: The surfaces of chicken i.e. shiga-like toxin (SLT1and SLT2), heat stable toxin muscles were incised with scissor and 10 gm muscle (STa) and heat labile toxin (LT) of E. coli were was mixed with 90 ml of NSS in sterile bag.Thereafter, determined by PCR.Primers used for associated genes -1 -5 serially diluted the sample from 10 to 10 then are depicted in Table -1

. cultured on Eosin Methylene Blue (EMB) agar
Detection of virulent genes by PCR: The PCR reaction (HiMedia, India) and incubated for 18 to 24 h at 37 °C.
for amplification of SLT1, SLT2, STa and LT genes was Colonies with green metallic sheen were considered E.
were used for each reaction.The mixture containing DNA extraction: DNA of E. coli was isolated by Cetyl PCR tubes were quickly spun at 10000 rpm for few Trimethyl Ammonium Bromide (CTAB) method as seconds and placed in gradient thermal cycler described by Wilson [11] with slight modifications.
(Eppendorf, Germany).The cycling conditions for o Five ml LB broth (HiMedia, India) culture of E. coli amplification included 94 C for 3 minute (initial o o were incubated at 37 C overnight.Three ml culture was denaturation), 30 cycles of 94 C for 1 minute taken in microcentrifuge tube and pelleted by (denaturation), annealing for 1 minute (annealing centrifugation at 10,000 rpm for 5 minutes.Pellet was temperature shown in Table 1) and 72°C for 1 minute resuspended in 567 µl of TE buffer, added 0.5% SDS (polymerization) followed by 72°C for 10 minute (final (SRL, India) and 100 µg/ml proteinase K (SRL, India), extension).The positive amplification was visualized o mixed thoroughly and incubated for 1 hour at 37 C. by electrophoresis of the product in 1.7% agarose Added 100 µl of 5 M NaCl (SRL, India) to cell lysate (HiMedia, India) gel stained with ethidium bromide in and mixed thoroughly.Thereafter, 80µl of CTAB/NaCl a submarine horizontal electrophoresis apparatus (SRL, India) solution was added and incubated at 65°C (Bangalore Genei).for 10 minutes.Equal volume (0.7 to 0.8ml) of Serotyping of E. coli: E. coli isolates obtained from Phenol/Chloroform/Isoamyl-alcohol (25:24:1) was chicken meat were sent to Central Research Institute, added and centrifuged for 5 minutes at 13000 rpm.
Kasauli, HP, for serotypes differentiation.Thereafter, the upper aqueous, viscous supernatant was

Results
transferred to fresh tubes then 0.5 volume of 7.5 M ammonium acetate (SRL, India) and double amount of Out of 66 chicken meat samples analysed, 11 chilled absolute ethanol were added.The tubes were (16.66%)

Conclusion
isolates (Figure 1).In other words, frequency of shiga like toxin and heat stable toxin gene was 27.27% and The results concluded that chicken meat samples 18.18% respectively.None of the heat labile toxin gene analyzed were harbored virulent genes for shiga like associated E. coli isolates was identified in this study.
toxins and enterotoxins.It pointed out that chicken The total identified virulent strains were 5 among 11 meat contaminated with E. coli strains in such level investigated strains (45.45%).As a result, overall could produce potential hazard for consumers.Next to incidence of virulent strains of E. coli was 7.57% that, further exploration of other virulent strains like (ETEC, 3.03% and STEC, 4.54%) (5 virulent strains enteroinvasive E. coli (EIEC), enteropathogenic E. coli out of 11 E. coli strains isolated from 66 samples).In (EPEC), enteroaggregative E. coli (EAggEC) are addition, all E. coli strains were serotyped in which 5 needed to know the particular situation of E. coli isolates were confirmed as rough, O2, O20, O22 and contaminated chicken meat at local market.O102 while rest of the 6 isolates were untypable (UT). in chicken meat was reported.In traditional isolated.But in this study incidence of virulent strains 7.
Coombes, K.B., Wickham, E.M., Mascarenhas, M., of E. coli showed that there is need for necessary Gruenheid, S., Finlay, B.B. and Karmali, A.M. (2008) measures to protect consumer health by forwarding Molecular analysis as an aid to assess the public health risk of these necessary information to higher authorities and non-O157 shiga toxin-producing Escherichia coli strains.
coli strains isolated from hemolytic uremic syndrome

Table - 1
. Oligonucleotide primer sets used in PCR