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Identification And Molecular Characterization Of Shiga Toxin Producing

By: Khawar Ali Shahzad | Prof.Dr.Khushi Muhammad.
Contributor(s): Dr.Tahir Yaqub | Mr.Tanveer Hussain | FVS.
Material type: materialTypeLabelBookPublisher: 2011Subject(s): Department of MicrobiologyDDC classification: 1221,T Dissertation note: Escherichia coli is normal inhabitant of all the animals and human beings. Its non-sorbitol fermenting biotype (SNF) was detectable in buffalo (90 percent), cattle (80 percent) or rarely in sheep (20 percent) and goat (30 percent). However, SNF E. coli were un-detectable in droppings of rural chickens and feces of donkeys. The SNF E. coli was detected in 100, 92, 71 and 80 percent of the market milk samples and 100, 83, 83 and 53 percent beef samples from Multan, Sandha, Wagha and Sheikhupura Roads, of Lahore city, respectively. However, SNF E. coli was not detected from freshly aseptically collected milk and beef samples but was detectable in over all 96 percent of market raw milk and 82 percent market beef samples. White colored colonies of each positive sample on Sorbitol MacConkey's Agar (SMA) contained gram negative, rods with round ends non spore former. Each of the isolates was sorbitol non fermenter, lactose fermenter, indole positive, Methyle Red positive, Voges Prauskaur negative and citrate negative. However, each of such isolates showed green metallic sheen on Eosin Methylene Blue (EMB) agar. Each of the isolate was further characterized using polymerase chain reaction (PCR). Bacterial DNA was extracted easily by boiling method when the isolates were grown on Sorbitol MacConkey's agar or EMB agar at 370C for 12-24 hours. The DNA was recovered when the culture was boiled for 5-10 minutes. The DNA of each sample remained stable on storage at 40C for 48 hours or at -200C for 7 days. The isolated DNA (100 samples) when amplified using universal, Stx1, Stx2 and O157 specific primers showed that 82 percent samples were positive for universal primers, 50 percent for O157, 60 % for Stx1 and 51 percent for Stx2. The filtrate of each isolate when diluted as 1:10 dilution and sterilized by filtration induced cyto-pathogenic effect (CPE) on Vero cell line. It is concluded that SNF E. coli O157 normally exists in intestinal tract of buffalo, cattle, sheep and goat. Counts of SNF E. coli O157 were higher in milk samples as compared to beef samples. More than 80 percent samples of milk or beef were contaminated with SNF E. coli O157. Feces of the animals are presumably main source of SNF E. coli contamination of raw milk and beef. PCR is a quick, reliable, and sensitive technique for confirmation of SNF E. coli O157 in the samples.
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Escherichia coli is normal inhabitant of all the animals and human beings. Its non-sorbitol fermenting biotype (SNF) was detectable in buffalo (90 percent), cattle (80 percent) or rarely in sheep (20 percent) and goat (30 percent). However, SNF E. coli were un-detectable in droppings of rural chickens and feces of donkeys. The SNF E. coli was detected in 100, 92, 71 and 80 percent of the market milk samples and 100, 83, 83 and 53 percent beef samples from Multan, Sandha, Wagha and Sheikhupura Roads, of Lahore city, respectively. However, SNF E. coli was not detected from freshly aseptically collected milk and beef samples but was detectable in over all 96 percent of market raw milk and 82 percent market beef samples. White colored colonies of each positive sample on Sorbitol MacConkey's Agar (SMA) contained gram negative, rods with round ends non spore former. Each of the isolates was sorbitol non fermenter, lactose fermenter, indole positive, Methyle Red positive, Voges Prauskaur negative and citrate negative. However, each of such isolates showed green metallic sheen on Eosin Methylene Blue (EMB) agar.

Each of the isolate was further characterized using polymerase chain reaction (PCR). Bacterial DNA was extracted easily by boiling method when the isolates were grown on Sorbitol MacConkey's agar or EMB agar at 370C for 12-24 hours. The DNA was recovered when the culture was boiled for 5-10 minutes. The DNA of each sample remained stable on storage at 40C for 48 hours or at -200C for 7 days. The isolated DNA (100 samples) when amplified using universal, Stx1, Stx2 and O157 specific primers showed that 82 percent samples were positive for universal primers, 50 percent for O157, 60 % for Stx1 and 51 percent for Stx2. The filtrate of each isolate when diluted as 1:10 dilution and sterilized by filtration induced cyto-pathogenic effect (CPE) on Vero cell line.
It is concluded that SNF E. coli O157 normally exists in intestinal tract of buffalo, cattle, sheep and goat. Counts of SNF E. coli O157 were higher in milk samples as compared to beef samples. More than 80 percent samples of milk or beef were contaminated with SNF E. coli O157. Feces of the animals are presumably main source of SNF E. coli contamination of raw milk and beef. PCR is a quick, reliable, and sensitive technique for confirmation of SNF E. coli O157 in the samples.

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