Among bovine fecal and recto-anal mucosal swab samples cultured in our laboratory for Escherichia coli O157:H7, we frequently isolated E. coli organisms that were phenotypically similar to the O157:H7 serotype as non-sorbitol fermenting and negative for -glucuronidase activity but serotyped O nontypeable:H25 (ONT: H25). This study determined the prevalence and virulence properties of the E. coli ONT:H25 isolates. Among dairy and feedlot cattle (n ؍ 170) sampled in Washington, Idaho, and Alberta, Canada, the percentage of animals culture positive for E. coli ONT:H25 ranged from 7.5% to 22.5%, compared to the prevalence of E. coli O157:H7 that ranged from 0% to 15%. A longitudinal 8-month study of dairy heifers (n ؍ 40) showed that 0 to 15% of the heifers were culture positive for E. coli O157:H7, while 15 to 22.5% of the animals were culture positive for E. coli ONT:H25. As determined by a multiplex PCR, the E. coli ONT:H25 isolates carried a combination of virulence genes characteristic of the enterohemorrhagic E. coli, including intimin, translocated intimin receptor, Stx2, and hemolysin (eae-, tir, stx 2vh-a , and hly). E. coli ONT:H25 isolates from diverse geographic locations and over time were fingerprinted by separating XbaI-restricted chromosomal DNA by pulsed-field gel electrophoresis (PFGE) separation. Two strains of E. coli ONT:H25 were highly similar by PFGE pattern. Experimental inoculation of cattle showed that E. coli ONT:H25, like E. coli O157:H7, colonized the bovine recto-anal junction mucosa for more than 4 weeks following a single rectal application of bacteria.Shiga toxin (Stx)-producing Escherichia coli (STEC) organisms that cause hemorrhagic colitis and the potentially fatal hemolytic uremic syndrome (HUS) are classed as enterohemorrhagic E. coli (EHEC) (16,28,45). Although the majority of HUS cases in humans have been associated with the EHEC serotype O157:H7 (27, 32), non-O157 serotypes also cause disease and are more common than E. coli O157:H7 in some geographical areas (18,22,36,46). There are over 200 different serotypes of STEC (32), and the number associated with human illness exceeds 100 (6). Domestic ruminants are important reservoirs of STEC and are the most common source for foodborne and animal-contact infections (13). Investigations throughout the world reveal that 10 to 80% of cattle are infected with STEC (5, 6, 49; http://www.who.int/emc-documents /zoonoses/whocsraph988c.html). Non-O157 STEC strains are more prevalent in animals and as contaminants in foods and water than E. coli O157 and, therefore, presumably humans are more frequently exposed to them (9, 10, 27). In the United States, it is estimated that 20% to 50% of STEC human infections are caused by non-O157:H7 serotypes (1, 2).The recto-anal junction (RAJ) mucosa has recently been identified as a site of E. coli O157:H7 colonization in the bovine gastrointestinal tract (33). This finding was supported by earlier observations in mature cattle that E. coli O157:H7 persists in the lower gastrointestinal tract an...