Shahzad et al., J. Anim. Plant Sci. 23(6):2013 1618 ISOLATION AND MOLECULAR CHARACTERIZATION OF SHIGA TOXIN PRODUCING E. COLI O157 K. A. Shahzad, K. Muhammad, A. A. Sheikh * , T. Yaqub * , M. Rabbani * , T. Hussain ** , A. A. Anjum and M. Anees Department of Microbiology, Faculty of Veterinary Science, University of Veterinary and Animal Sciences, Lahore, Pakistan * University Diagnostic Laboratory, University of Veterinary and Animal Sciences, Lahore, Pakistan ** Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan Corresponding Author e-mail address: drkhushi.muhammad@uvas.edu.pk ABSTRACT Escherichia coli (E. coli) is normal inhabitant of all domestic animals and human beings. Its Sorbitol-non fermenting (SNF) biotype was detected in dung of buffalo (92 %), cattle (84 %), sheep (52 %) and goat (56 %). However, E. coli (SNF) was not detected in droppings of rural chickens, feces of donkeys and fresh aseptically collected milk and beef samples, whereas, the E. coli was detected in 96 percent market raw milk and 82 percent samples from beef shops. Eighty percent milk and 53 percent beef samples contained SNF isolates of E. coli. All isolates of SNF E. coli showing green metallic sheen on eosin methylene blue (EMB) agar were further characterized using polymerase chain reaction (PCR). The isolated DNA of 100 samples when amplified using universal, Stx1, Stx2 and O157 specific primers confirmed that 82 percent samples were positive for universal primers, 50 percent for O157, 60 percent for Stx1 and 51 percent for Stx2. It is concluded that E. coli O157 is normal inhabitant of intestinal tract of domestic ruminants and presumably animal feces are acting as main source of contamination of raw milk and beef. Key words: Sorbitol, Eosin Methylene Blue (EMB) agar, E. coli O157, PCR, Buffalo, Sorbitol Non Fermenters INTRODUCTION Escherichia coli is an important member of enterobacteriacae which contains gram negative bacilli. It is a large group of germs that are normal inhabitant of animal and human intestinal tract. On the basis of somatic (O), capsular (K) and flagellar (H) antigen, more than 700 serotypes of E. coli have already been identified. On the basis of sorbitol fermentation, its species are also bio- typed as sorbitol non- fermenting E. coli that are cause of human food poising. This biotype results a wide range of clinical manifestations, including mild illness, vomiting, sudden onset of diarrhea, hemolyticuremic syndrome and death (Su and Brandt, 1995). The incidence of food poisoning outbreaks with E. coli O157:H7 has increased in recent years. The disease is an increasing public health concern. The infective dose of E. coli O157:H7 in humans is very low (Jones, 1999). Cattle are believed to be the primary reservoir of E. coli O157:H7 (Garber et al., 1995). Contamination of swimming pools, water supplies, raw vegetables and more commonly dairy and beef products with the cattle feces are associated with an increasingly wide variety of food poisoning (Jones, 1999). Raw or improperly pasteurized milk and meat can be source of E. coli O157:H7 and cause of food poisoning. Fresh beef can be contaminated with Salmonellae, Campylobacter species, and enterohaemorrhagic E. coli especially O157:H7 serotype and also by other enteric pathogens (Keene et al., 1997). Currently, E. coli are classified into distinct groups on the bases of toxigenicity, viz. Entero-toxin Producing E. coli (ETEC), which causes diarrhea by producing heat-stable and/or heat-labile enterotoxins. Shiga toxin or verocytotoxin-producing E. coli (STEC or VTEC) is an important foodborne pathogen, which may results in sporadic cases to serious outbreaks in whole world. Morbidity and mortality rates due to several recent outbreaks of STEC have proved it as a significant threat to public health. Verotoxin or Shiga toxin is the most important virulent attribute for the STEC (Staats et al., 2003). Shiga toxins producing E. coli is an etiological agent for individual cases and outbreaks in the developed nations (Armstrong et al., 1996). The resistance of powerful virulence factors to acid is mainly associated with the pathogenicity of E. coli O157:H7. The pathogenic E. coli adhere efficiently to the intestinal epithelial cells thus causing lesions (Sherman et al., 1988). The present study was designed with the following objectives: (1) Isolation and cultural/biochemical characteristics of sorbitol non fermenting E. coli from fecal samples of healthy domestic animals (2) Monitoring of raw milk and beef samples from four outlets/roads of Lahore city for sorbitol non fermenting E. coli (3) Molecular characterization of E. coli O157 using universal, specific, Stx1 and Stx2 primers. The Journal of Animal & Plant Sciences, 23(6): 2013, Page: 1618-1621 ISSN: 1018-7081