Animal Science Journal (2004) 75, 261–269 261 Correspondence: Yutaka Nakai, Graduate School of Agricul- tural Science, Tohoku University, Naruko-machi, Miyagi- ken, 989-6711, Japan. (Email: nakai@bios.tohoku.ac.jp) Received 26 November 2003; accepted for publication 25 December 2003. ORIGINAL ARTICLE Genetic analysis of shiga-toxigenic Escherichia coli isolates from cattle in a limited region Kenichi OTAWA, 1 Masaaki SATO, 1 Takako SASAKI, 1 Hiraku SASAKI, 1 Jun NONAKA, 1 Kikuji ITO, 2 Toshio KUROKI 3 and Yutaka NAKAI 1 1 Graduate School of Agricultural Science, Tohoku University, Naruko-machi, Miyagi-ken, 2 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo and 3 Kanagawa Prefectural Public Health Laboratory, Chigasaki-shi, Japan ABSTRACT The ecology of shiga-toxigenic Escherichia coli (STEC) is important in the animal production environment. We investigated fecal shedding of STEC in one town in Miyagi, Japan by multiplex polymerase chain reaction (PCR) targeting shiga toxin gene 1 (stx 1 ), gene 2 (stx 2 ) and malB promoter gene, and analyzed the PCR products of stx 1 or stx 2 (54 samples) by direct sequencing. Three of 46 (6.5%) beef cattle in the University Farm of Tohoku University (Kawatabi Farm) and 11 of 70 (15.7%) calves in neighboring dairy farms carried STEC. Rate of detecting genes of stx 1 , stx 2 and stx 1+2 was 3.4% (4/116), 8.6% (10/116) and 0.9% (1/116), respectively. Serotyping indicated that STEC contaminated farms at different times or through different routes. Isolates harbored no mutation among stx 1 , but six (Kawatabi Farm) and 38 (neighboring farms) base substitutions among stx 2 , respectively. The diversity of substitutions of stx 2 was observed among farms or even in a farm. Phylogenic analysis revealed that STEC detected in the area were classified into three clusters by the variety of stx 2 . Sequence analysis of stx 2 will be one of the tools for clarifying the source of outbreaks and the route of contamination of STEC. KEYWORDS: cattle, epidemiology, gene analysis, shiga-toxigenic Escherichia coli, stx. INTRODUCTION Shiga-toxigenic Escherichia coli (STEC) is associated with human diseases ranging from uncompli- cated diarrhea to hemorrhagic colitis (HC) and life- threatening hemolytic uremic syndrome (HUS) (Paton & Paton 1998). In Japan, STEC infection has received attention since 1990, when two kindergartners among 319 patients died from HUS in an outbreak of HC (Akashi et al. 1994). In 1996, the severe outbreak of STEC O157 : H7 occurred in junior high school chil- dren in Sakai city, Osaka, where more than 1000 patients were hospitalized for gastrointestinal symp- toms, and approximately 100 cases were complicated with HUS, resulting in three deaths (Yokoyama et al. 2000). Since then, outbreaks caused by STEC have been an important social issue in Japan. Outbreaks have frequently occurred in primary or nursery schools or nursery homes for aged people, and are mainly caused by contaminated foods (Kawamura et al. 1999). However, dairy and beef farm environ- ments are reported to be sources of sporadic cases of E. coli O157 : H7 and other STEC infections (Jackson et al. 1998; Locking et al. 2001). Although E. coli O157 : H7 is considered the most virulent type for humans (Beutin et al. 1997), it has been recognized that STEC strains causing human dis- ease belong to a broad range of O : H serotypes. Sero- types O5, O26, O111 and O113 are also recognized as a serious threat to public health (Griffin & Tauxe 1991; Fagan et al. 1999). Because O157 : H7 infections are often associated with the consumption of