Prevalence of Escherichia coli O157:H7 in Cattle and During Processing 1

FOOD SAFETY II- PATHOGEN INTERVENTION IN BEEF PROCESSING PLANTS Prevalence of Escherichia coli O157:H7 in Cattle and During Processing 1 Genevieve A. Barkocy-Gallagher* and Mohammad Koohmaraie Introduction-- E. coli Nomenclature Escherichia coli is a phenotypically and genomically diverse and versatile bacterial species. Commensal E. coli are valuable to warm-blooded animals (including humans) as part of the intestinal microflora, but pathogenic E. coli can cause a variety of diseases in the same hosts. Human illnesses associated with pathogenic E. coli include infections of the urinary tract, blood stream, meninges, or intestinal tract. The disease or virulence attributes (e.g., invasiveness) of pathogenic E. coli have been used to group them, using acronyms ending in EC for E. coli. For example, strains that cause diarrhea have been referred to as diarrheagenic E. coli, or DEC strains (Whittam et al., 1993). General information on pathogenic E. coli is available in Ørskov (1984) and Nataro and Kaper (1998). Of primary relevance to the red meat industry are the Shiga-toxigenic E. coli (STEC), not to be confused with enterotoxigenic E. coli (ETEC), which produce different types of toxin. STEC carry genes for at least one of two Shiga toxins: Stx1 or Stx2 (formerly SltI and SltII for Shiga-like toxins). STEC also have been referred to as VTEC, or Vero-toxic E. coli, due to the preliminary observation that the toxin(s) kill Vero cells in vitro. Some STEC have been implicated in human intestinal disease and are referred to as enterohemorrhagic E. coli (EHEC). EHEC first were associated with bloody diarrhea, but are now known to cause a range of symptoms including mild to bloody diarrhea, kidney failure associated with hemolytic uremic syndrome (HUS), and Genevieve A. Barkocy-Gallagher USDA, ARS Roman L. Hruska U.S. Meat Animal Research Center P.O. Box 166 Spur 18-D Clay Center, NE 68933-0166 gallagher@email.marc.usda.gov. 1 Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. Proceedings of the 55 th Reciprocal Meat Conference (2002) death. It is not clear whether all STEC, or even all STEC of a given serotype (see below), can cause human disease. A myriad of virulence factors probably are involved in pathogenesis, not all of which may be known or present in each STEC strain. Hence EHEC most likely comprise a subset of STEC. In the absence of information to differentiate diseasecausing strains, STEC of the same serotype as those previously associated with human disease sometimes are referred to as EHEC, particularly when they carry genes for additional known virulence factors. For reviews on STEC and EHEC pathogenesis see Bettelheim (2000), Donnenberg and Whittam (2001), Griffin and Tauxe (1991), and Nataro and Kaper (1998). Serotyping is the traditional method used to differentiate E. coli. It is based on the reaction of variable cell surface molecules with a battery of standardized antisera. The standard antigens recognized by serotyping are the K, O, H, and F antigens. K antigens are derived from capsular polysaccharides (Danish kapsul ). Capsules form a thick, gel-like layer around the cell and may be important in some extraintestinal infections. They generally cause autoagglutination, are produced in the laboratory only under certain environmental conditions (e.g., high salt), and may be removed by boiling. O antigens are derived from the oligosaccharide region of lipopolysaccharide (LPS), an outer membrane component comprised of three regions: the lipid A anchor, the core polysaccharide, and the variable oligosaccharide chain. LPS is a defining characteristic of Gramnegative bacteria. H antigens are derived from the structural proteins of flagella, the whip-like appendages that propel motile E. coli. Motility (and therefore H antigen expression) regularly must be induced in the laboratory by passage in semisolid agar or broth without added sugars (Smibert and Krieg, 1994; Strockbine et al., 1998). F antigens are proteins associated with pili (a.k.a. fimbriae). Pili are hair-like structures extending outward from the cell surface and often effect cellular adherence. Because some F antigens can cause auto-agglutination they were originally misidentified as K antigens, e.g., the capsular antigen K88 is actually fimbrial antigen F4. Ørskov (1984) and Ørskov et al. (1977) provide further details on serotyping. E. coli serotypes refer to the specific antigens identified; most often just the O and H types are determined. The vari- 55 th Annual Reciprocal Meat Conference 15

ants of each antigen have been assigned sequential numbers as they were recognized. Thus, for example, E. coli O157:H7 refers to strains expressing O antigen #157 and H antigen #7. Currently, 173 O antigens are recognized by standard typing (Ørskov et al., 1991), although additional O serotypes have been preliminarily identified (see http://ecoli.cas.psu.edu/). At least 56 H antigens have been identified (Ørskov, 1984). Certain O and H antigens may contribute to virulence in some E. coli associated diseases (Girón et al., 2002; Lerouge and Vanderleyden, 2002). However, the specific EHEC-associated O and H antigens such as O157 and H7 have not yet been shown to play significant roles in pathogenesis. E. coli O157:H7-- Background The most renowned EHEC in the U.S. are the serotype O157:H7 strains. These organisms have been connected with multiple illness outbreaks causing severe disease and death in children and the elderly, and they are the first and only microorganisms to have been declared an adulterant by the Food Safety and Inspection Service (FSIS). E. coli O157:H7 strains originally were identified as intestinal pathogens associated with ground beef during two outbreaks in 1982 (Riley et al., 1983). At that time, a retrospective examination of records from the Centers for Disease Control and Prevention (CDC) revealed only one other incident of E. coli O157:H7 infection associated with bloody diarrhea. In the following years, several more E. coli O157:H7 outbreaks were identified (Griffin and Tauxe, 1991), but the organisms achieved popular notoriety in 1993, following a multistate outbreak primarily affecting children in which four people died (CDC, 1993). This outbreak also was associated with undercooked ground beef. In 1994, E. coli O157:H7 was declared to be an adulterant in raw ground beef under the Federal Meat Inspection Act (FMIA). This action by the FSIS means that ground beef lots found to be contaminated with E. coli O157:H7 now must be disposed of or further processed. (Thorough cooking to 160 F will kill all of the E. coli O157:H7 present.) In 1999, the FSIS extended the policy to include nonintact beef, such as mechanically tenderized or reconstructed products (CFR, 1999). Both ground beef and nonintact beef products are considered higher risks than intact beef products due to the nature of E. coli O157:H7 contamination (CFR, 1999). The organisms contaminate carcass surfaces (not within the muscle; see below), and therefore they are found only on the exterior surfaces of intact beef. These areas are adequately cooked even when the meat is served rare. However, E. coli O157:H7 can be introduced to the inner regions of products such as hamburgers, etc. by grinding or other processing procedures, where the bacteria can survive the inadequate internal heating of rare cooking and thus have the potential to cause disease. E. coli O157:H7-- Culture Methods The presumed infectious dose of E. coli O157:H7 is very low, one of the reasons highly sensitive recovery methods are desirable for a variety of samples (see Nataro and Kaper, 1998). Enrichments generally are the first step for recovery of E. coli O157:H7 from cattle and beef samples. These broth cultures are used to increase the concentration of bacteria prior to plating, thereby improving assay sensitivity and permitting detection of few cells in large samples (e.g., 25 ml of carcass sponge sampling buffer or 25 g of ground beef). Generally, selective broths are used to inhibit growth of competing bacteria (Chapman, 2000), although nonselective broths may improve recovery of E. coli O157:H7 when used in combination with other selective techniques (Barkocy-Gallagher et al., 2002; Chapman, 2000; Fukushima and Gomyoda, 1999). For example, immunomagnetic separation (IMS) is frequently used to improve recovery of E. coli O157:H7. IMS is performed after enrichment and prior to plating. It serves two functions: to decrease recovery of background (non-e. coli O157:H7) bacteria and to concentrate the target E. coli O157:H7 cells. The basic principle of IMS is the specific binding of target bacteria to magnetic beads coated with antibodies that recognize the O157 antigen. Magnets then are used to retain the bound cells while unbound cells are washed away. Plating still must be done on selective screening media in order to differentiate E. coli O157:H7 from nonspecifically bound bacteria. No available technique will eliminate all background bacteria from samples or enrichments. Therefore, unusual characteristics of E. coli O157:H7 must be employed to help distinguish between these and other bacteria upon plating. E. coli (and other bacteria) commonly are differentiated on selective screening media with various sugars and ph indicator dyes incorporated. If an organism ferments a sugar to produce acid, ph changes in the media will result in differently colored colonies. Fortunately, most EHEC O157:H7 strains identified to date have the rare property of being unable to ferment sorbitol within 24 h (sor-); this property was exploited in the initial use of sorbitol Mac- Conkey agar (SMAC) for recovery of E. coli O157:H7 (March and Ratnam, 1986). However, since the absence of sorbitol fermentation is not a positive selection, finding the right colonies on a selective plate still may be like searching for a needle in a haystack. Other sor- bacteria, such as Hafnia species, complicate identification of the correct colonies. Additional distinctive properties of E. coli O157:H7 have since been discovered and exploited in a variety of selective screening media. Useful E. coli O157:H7 traits include the inability to ferment rhamnose, the inability to produce ß- glucuronidase, resistance to tellurite (a heavy metal), and resistance to antibiotics such as cefixime. Other antibiotics also may be incorporated into plating media to select against similar bacterial species; these antibiotics include cefsulodin, vancomycin, and novobiocin. However, no 16 American Meat Science Association

medium will permit growth of only E. coli O157:H7. Colonies suspected of being E. coli O157:H7 generally are screened using one of the commercially available O157 antigen detection kits, then picked for further confirmation. For comprehensive reviews of E. coli O157:H7 culture methods, see Clifton-Hadley (2000), Nataro and Kaper (1998), Reissbrodt (1998), and Strockbine et al. (1998). E. coli O157:H7-- Prevalence and Carcass Contamination There is no direct information demonstrating how E. coli O157:H7 arrives in beef products, although the obvious ultimate route is via carcass surface contamination during processing. Both hides and feces are potential sources of carcass contamination. Although little is known about the prevalence of E. coli O157:H7 on cattle hides, two recent reports suggest that they frequently are contaminated with E. coli O157:H7 (Elder et al., 2000; Keen and Elder, 2002). The hides could become contaminated by a variety of sources, including soil and feces from themselves, other cattle, and wild animals. Further research is needed to address the importance of hide contamination as a source of E. coli O157:H7 in beef. E. coli O157:H7 is shed intermittently in cattle feces (Besser et al., 1997; Conedera et al., 2001; Sargeant et al., 2000; Zhao et al., 1995). Initially, the organism seemed to be present in few of the cattle fecal samples tested in the U.S. and other countries, generally less than 1 to 2% (Centers for Epidemiology and Animal Health, 1997; Chapman et al., 1993; Chapman et al., 1994: Galland et al., 2001; Garber et al., 1999; Hancock et al., 1994; Hancock et al., 1997; Kobayashi et al., 2001; McDonough et al., 2000; Meyer-Broseta et al., 2001; Richards et al., 1998; Schurman et al., 2000; Zschöck et al., 2000). However, the sensitivity of E. coli O157:H7 assays improved substantially after these data were collected; the incorporation of enrichment and IMS, analysis of larger samples, and decreased shipping times in the absence of freezing or refrigeration all have been shown to have notable effects (Chapman et al., 1994; Elder et al., 2000; Heuvelink et al., 1998; Laegreid, 2000; Laegreid et al., 1999; McDonough et al., 2000; Sanderson et al., 1995; Zhao et al., 1995). New data obtained using more sensitive methods suggest that the fecal prevalence of E. coli O157:H7 in cattle at slaughter or on the farm ranges from 5 to 17% (Bonardi et al., 2001; Chapman et al., 2001; Conedera et al., 2001; Heuvelink et al., 1998; Laegreid et al., 1999; Meyer-Broseta et al., 2001; Paiba et al., 2002; Van Donkersgoed et al., 1999). E. coli O157:H7 are more frequently found in cattle fecal samples during the warmer months (Bonardi et al., 1999; Bonardi et al., 2001; Chapman et al., 1997; Chapman et al., 2001; Conedera et al., 2001; Garber et al., 1999; Hancock et al., 1997; Paiba et al., 2002; Sargeant et al., 2000; Van Donkersgoed et al., 1999). These organisms have been recovered from 17 to 37% of the fecal samples collected from cattle at slaughter in the summertime in several countries, including the U.S. (Bonardi et al., 1999; Chapman et al., 1997; Elder et al., 2000). In some cases, 70 to 100% of the animals tested within a lot had E. coli O157:H7 in their feces (Bonardi et al., 2001; Elder et al., 2000). E. coli O157:H7 prevalence on beef carcasses has been measured in a limited number of studies using various methods. In the U.S., the prevalence of E. coli O157:H7 on beef carcasses after dressing was determined to be 0.2% (4 of 2,081 carcasses; FSIS, 1994). Subsequently, by sampling in the summer months and using more sensitive detection methods, we found the prevalence rate to be 1.8% (6 of 330 carcasses sampled in the cooler; Elder et al., 2000). Two studies done in the U.K. have reported similar results: 0.47% and 1.4% of carcasses were found to be contaminated with E. coli O157:H7 (Chapman et al., 2001; Richards et al., 1998). Studies done in France and Hong Kong reported no carcass contamination with E. coli O157:H7 (851 and 986 carcasses sampled, respectively), but this may reflect the methodologies used (Leung et al., 2001; Rogerie et al., 2001). A study done in northern Italy found that 12 out of 100 carcasses sampled were contaminated with E. coli O157:H7 (Bonardi et al., 2001). The reason for such an apparently high prevalence is not clear, but 11 of the 12 positive samples were recovered during June. The carcasses and feces from individual cattle have been examined for E. coli O157:H7 contamination during studies done in Italy and the U.K. (Bonardi et al., 2001; Chapman et al., 1993). In both cases, one-third of the carcasses that were derived from animals carrying E. coli O157:H7 in their feces were contaminated (6 of 17 and 7 of 23). These data suggest that each carcass may have been contaminated directly by the same animal s feces. However, hides now have been implicated as a major contributor of E. coli O157:H7 and hides were not tested during these studies. Furthermore, both studies suggested that crosscontamination of adjacent carcasses occurred, but a cautious interpretation is warranted considering the low number of samples evaluated. The prevalence of E. coli O157:H7 on carcasses at different processing steps has been examined (Elder et al., 2000). This study was done in the U.S. in the summertime and 43% of the carcasses tested immediately after hide removal carried E. coli O157:H7. However, the prevalence rate dropped to 1.8% when the same carcasses were sampled in the cooler after processing. In-between these two samplings the carcasses were subjected to several antimicrobial steps, including trimming and some combination of hot water washes, organic acid washes, and/or steam pasteurization. The prevalence of E. coli O157:H7 on the same carcasses was 18% at an intermediate point during processing, after one wash, evisceration, and trimming. The data emphasize the value of multiple, consecutive antimicrobial interventions (such as two or more washes and/or steam pasteurization). 55 th Annual Reciprocal Meat Conference 17

E. coli O157:H7-- Genotyping The E. coli genome is highly plastic, or changeable (Brunder and Karch, 2000; Dougan et al., 2001; Lawrence, 1999; Ochman et al., 2000). This plasticity is due to the acquisition and loss of foreign DNA via horizontal transfer. Horizontal DNA transfer is propagated by phage, plasmids, transposable elements, etc., that become incorporated into the genome (Brunder and Karch, 2000; Lawrence, 1999; Ochman et al., 2000). Based on the two genome sequences published to date, E. coli O157:H7 strains undergo extensive horizontal DNA transfer (Hayashi et al., 2001; Perna et al., 2001). Different strains gain and lose DNA independently, so the genomes of E. coli O157:H7 strains are highly variable. This variability is exploited in genotyping or genomic fingerprinting strategies to identify specific E. coli O157:H7 strains (Strockbine et al., 1998). The gold standard for E. coli O157:H7 genotyping in the U.S. currently is restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis, or PFGE, for short. In PFGE analyses, the cells are gently broken open to release intact genomic DNA. The DNA is then restricted (digested) with an enzyme that cuts it into 20 to 30 large fragments. For E. coli O157:H7, the most commonly used enzyme is XbaI, which cuts the DNA into fragments of 10 to 500 kilobases (kb). Subsequently, the DNA fragments are size separated in an agarose gel. Due to the large size of the fragments, the electrical field used to mobilize the DNA is applied in pulses from six different angles over an extended period of time. The DNA is then stained for visualization and the resulting patterns ( fingerprints ) are compared for differences in the presence or absence of bands. Since E. coli genomes can change so rapidly, patterns with one to three band differences generally are interpreted to mean the strains are of the same type (Tenover et al., 1995). Among other purposes, PFGE is useful for studies that follow transfer of E. coli O157:H7 from one environment to another. However, PFGE data must be coupled with traditional epidemiology, because strains with similar or identical patterns can be recovered from geographically separate sources, and both animals and carcasses can carry E. coli O157:H7 of more than one PFGE type (Barkocy-Gallagher et al., 2001; Keen and Elder, 2002; Laegreid et al., 1999; Lee et al., 1996). One of the primary uses of PFGE is in epidemiological studies of the CDC via PulseNet (http://www.cdc.gov/pulsenet/). It has also been employed in other types of tracking studies (Barkocy-Gallagher et al., 2001; Besser et al., 1997; Bonardi et al., 2001; Lahti et al., 2002). Two of these studies used PFGE to demonstrate that E. coli O157:H7 on carcasses arrived in the plant via animals within the same lot, and possibly via the same animal. Summary E. coli O157:H7 are a significant foodborne health hazard for humans and are frequently associated with consumption of undercooked ground beef. Detection methods for recovering the organisms from various beef processing samples have improved substantially in recent years, contributing to the apparent rise in prevalence of the organisms. E. coli O157:H7 are more prevalent in cattle in the summer months, which can also affect apparent prevalence rates. In one study, the organisms were found on almost half of the beef carcasses analyzed immediately after hide removal, but prevalence on final carcasses (in the cooler) was relatively low (less than 2%). The drop in prevalence rates on carcasses sampled early in processing to carcasses sampled after processing probably can be attributed to the application of multiple antimicrobial interventions throughout processing. Specific types of E. coli O157:H7 can be identified by PFGE (genomic fingerprinting), which is useful in epidemiological and tracking studies. References Barkocy-Gallagher, G.A.; Arthur, T.M.; Siragusa, G.R.; Keen, J.E.; Elder, R.O.; Laegreid, W.W.; Koohmaraie, M. 2001. Genotypic analysis of Escherichia coli O157:H7 and O157 nonmotile isolates recovered from beef cattle and carcasses at processing plants in the Midwestern states of the United States. Applied and Environmental Microbiology 67:3810-3818. Barkocy-Gallagher, G.A.; Berry, E.D.; Rivera-Betancourt, M.; Arthur, T.M.; Koohmaraie, M. 2002. Development of methods for the recovery of Escherichia coli O157:H7 and Salmonella from beef carcass sponge samples and bovine fecal and hide samples. Journal of Food Protection (in press). Besser, T.E.; Hancock, D.D.; Pritchett, L.C.; McRae, E.M.; Rice, D.H.; Tarr, P.I. 1997. Duration of detection of fecal excretion of Escherichia coli O157:H7 in cattle. Journal of Infectious Diseases 175:726-729. Bettelheim, K.A. 2000. Role of non-o157 VTEC. Journal of Applied Microbiology 88:38S-50S. Bonardi, S.; Maggi, E.; Bottarelli, A.; Pacciarini, M.L.; Ansuini, A.; Vellini, G.; Morabito, S.; Caprioli, A. 1999. Isolation of verocytotoxinproducing Escherichia coli O157:H7 from cattle at slaughter in Italy. Veterinary Microbiology 67:203-211. Bonardi, S.; Maggi, E.; Pizzin, G.; Morabito, S.; Caprioli, A. 2001. Faecal carriage of verocytotoxin-producing Escherichia coli O157 and carcass contamination in cattle at slaughter in northern Italy. International Journal of Food Microbiology 66:47-53. Brunder, W.; Karch, H. 2000. Genome plasticity in Enterobacteriaceae. International Journal of Medical Microbiology 290:153-165. Centers for Disease Control and Prevention. 1993. Update: Multistate outbreak of Escherichia coli O157:H7 infections from hamburgers western United States, 1992-1993. Morbidity and Mortality Weekly Report 42:258-263. Centers for Epidemiology and Animal Health. 1997. An update: Escherichia coli O157:H7 in humans and cattle. U.S. Department of Agriculture, APHIS, VS. Chapman, P.A. 2000. Methods available for the detection of Escherichia coli O157 in clinical, food and environmental samples. World Journal of Microbiology & Biotechnology 16:733-740. Chapman, P.A.; Cerdan Malo, A.T.; Ellin, M.; Ashton, R.; Harkin, M.A. 2001. Escherichia coli O157 in cattle and sheep at slaughter, on beef and lamb carcasses and in raw beef and lamb products in South Yorkshire, UK. International Journal of Food Microbiology 64:139-150. 18 American Meat Science Association

Chapman, P.A.; Siddons, C.A.; Cerdan Malo, A.T.; Harkin, M.A. 1997. A 1-year study of Escherichia coli O157 in cattle, sheep, pigs and poultry. Epidemiology and Infection 119:245-250. Chapman, P.A.; Siddons, C.A.; Wright, D.J.; Norman, P.; Fox, J.; Crick, E. 1993. Cattle as a possible source of verocytotoxin-producing Escherichia coli O157 infections in man. Epidemiology and Infection 111:439-447. Chapman, P.A.; Wright, D.J.; Siddons, C.A. 1994. A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from bovine faeces. Journal of Medical Microbiology 40:424-427. Clifton-Hadley, F.A. 2000. Detection and diagnosis of Escherichia coli O157 and other verocytotoxigenic E. coli in animal feces. Reviews in Medical Microbiology 11:47-58. Code of Federal Regulations. Beef products contaminated with Escherichia coli O157:H7. United States Department of Agriculture, Food Safety and Inspection Service. Jan. 19, 1999. Federal Register 64(11):2803-2805. Conedera, G.; Chapman, P.A.; Marangon, S.; Tisato, E.; Dalvit, P.; Zuin, A. 2001. A field survey of Escherichia coli O157 ecology on a cattle farm in Italy. International Journal of Food Microbiology 66:85-93. Donnenberg, M.S.; Whittam, T.S. 2001. Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli. Journal of Clinical Investigation 107: 539-548. Dougan, G.; Haque, A.; Pickard, D.; Frankel. G.; O'Goara, P.; Wain, J. 2001. The Escherichia coli gene pool. Current Opinion in Microbiology 4:90-94. Elder, R.O.; Keen, J.E.; Siragusa, G.R.; Barkocy-Gallagher, G.A.; Koohmaraie, M.; Laegreid, W.W. 2000. Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing. Proceedings of the National Academy of Sciences of the United States of America 97:2999-3003. Food Safety and Inspection Service. 1994. Nationwide Beef Microbiological Baseline Data Collection Program: Steers and Heifers, October 1992-September 1993 (U.S. Dept. of Agriculture, http://www.aphis.usda.gov/vs/ceah/cahm). Fukushima, H.; Gomyoda, M. 1999. An effective, rapid and simple method for isolation of Shiga toxin-producing Escherichia coli O26, 0111 and 0157 from faeces and food samples. Zentralblatt fuer Bakteriologie 289:415-428. Galland, J.C.; Hyatt, D.R.; Crupper, S.S.; Acheson, D.W. 2001. Prevalence, antibiotic susceptibility, and diversity of Escherichia coli O157:H7 isolates from a longitudinal study of beef cattle feedlots. Applied and Environmental Microbiology 67:1619-1627. Garber, L.; Wells, S.; Schroeder-Tucker, L.; Ferris, K. 1999. Factors associated with fecal shedding of verotoxin-producing Escherichia coli O157 on dairy farms. Journal of Food Protection 62:307-312. Girón, J.A.; Torres, A.G.; Freer, E.; Kaper, J.B. 2002. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Molecular Microbiology 44:361-379. Griffin, P.M.; Tauxe, R.V. 1991. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiologic Reviews 13:60-98. Hancock, D.D.; Besser, T.E.; Kinsell, M.L.; Tarr, P.I.; Rice, D.H.; Paros, M.G. 1994. The prevalence of Escherichia coli O157:H7 in dairy and beef cattle in Washington State. Epidemiology and Infection 113:199-207. Hancock, D.D.; Besser, T.E.; Rice, D.H.; Herriott, D.E.; Tarr, P.I. 1997. A longitudinal study of Escherichia coli O157 in fourteen cattle herds. Epidemiology and Infection 118:193-195. Hayashi, T.; Makino, K.; Ohnishi, M.; Kurokawa, K.; Ishii, K.; Yokoyama, K.; Han, C.G.; Ohtsubo, E.; Nakayama, K.; Murata, T.; Tanaka, M.; Tobe, T.; Iida, T.; Takami, H.; Honda, T.; Sasakawa, C.; Ogasawara, N.; Yasunaga, T.; Kuhara, S.; Shiba, T.; Hattori, M.; Shinagawa, H. 2001. Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Research 8(1):11-22 (Erratum in: DNA Research 8(2):96, 2001.) Heuvelink, A.E.; Van Den Biggelaar, F.L.A.M.; De Boer, E.; Herbes, R.G.; Melchers, W.J.G.; Huis In 'T Veld, J.H.J.; Monnens, L.A.H. 1998. Isolation and characterization of verocytotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep. Journal of Clinical Microbiology 36:878-882. Keen, J.E.; Elder, R.O. 2002. Isolation of shiga toxigenic Escherichia coli O157 from hide surfaces and the oral cavity of finished beef feedlot cattle. Journal of the American Veterinary Medical Association 220:756-763. Kobayashi, H.; Shimada, J.; Nakazawa, M.; Morozumi, T.; Pohjanvirta, T.; Pelkonen, S.; Yamamoto, K. 2001. Prevalence and characteristics of Shiga toxin-producing Escherichia coli from healthy cattle in Japan. Applied and Environmental Microbiology 67:484-489. Laegreid, W.W. 2000. Diagnosis of infection by Escherichia coli O157:H7 in beef cattle. National Conference on Animal Production Food Safety Proceedings, pp. 101-103. Laegreid, W.W.; Elder, R.O.; Keen, J.E. 1999. Prevalence of Escherichia coli O157:H7 in range beef calves at weaning. Epidemiology and Infection 123:291-298. Lahti, E.; Eklund, M.; Ruutu, P.; Siitonen, A.; Rantala, L.; Nuorti, P.; Honkanen-Buzalski, T. 2002. Use of phenotyping and genotyping to verify transmission of Escherichia coli O157:H7 from dairy farms. European Journal of Clinical Microbiology and Infectious Diseases 21:189-195. Lawrence, J.G. 1999. Gene transfer, speciation, and the evolution of bacterial genomes. Current Opinion in Microbiology 2:519-523. Lee, M.-S.; Kaspar, C.W.; Brosch, R.; Shere, J.; Luchansky, J.B. 1996. Genomic analysis using pulsed-field gel electrophoresis of Escherichia coli O157:H7 isolated from dairy calves during the United States national dairy heifer evaluation project (1991-1992). Veterinary Microbiology 48:223-230. Lerouge, I.; Vanderleyden, J. 2002. O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions. FEMS Microbiology Reviews 26:17-47. Leung, P.H.M.; Yam, W.C.; Ng, W.W.S.; Peiris, J.S.M. 2001. The prevalence and characterization of verotoxin-producing Escherichia coli isolated from cattle and pigs in an abattoir in Hong Kong. Epidemiology and Infection 126:173-179. March, S.B.; Ratnam, S. 1986. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. Journal of Clinical Microbiology 23:869-872. McDonough, P.L.; Rossiter, C.A.; Rebhun, R.B.; Stehman, S.M.; Lein, D.H.; Shin, S.J. 2000. Prevalence of Escherichia coli O157:H7 from cull dairy cows in New York State and comparison of culture methods used during preharvest food safety investigations. Journal of Clinical Microbiology 38:318-322. Meyer-Broseta, S.; Bastian, S.N.; Arné, P.D.; Cerf, O.; Sanaa, M. 2001. Review of epidemiological surveys on the prevalence of contamination of healthy cattle with Escherichia coli serogroup O157:H7. International Journal of Hygiene and Environmental Health 203:347-361. Nataro, J.P.; Kaper, J.B. 1998. Diarrheagenic Escherichia coli. Clinical Microbiological Reviews. 11:142-201. Ochman, H.; Lawrence, J.G.; Groisman, E.A. 2000. Lateral gene transfer and the nature of bacterial innovation. Nature 405:299-304. Ørskov, F. 1984. Genus I. Escherichia, pp. 420-423. In N.R. Krieg; J.G. Holt (eds.), Bergey's Manual of Systematic Biology, vol. 1. Williams & Wilkins, Baltimore, Maryland. 55 th Annual Reciprocal Meat Conference 19

Ørskov, I.; Ørskov, F.; Jann, B.; Jann, K. 1977. Serology, chemistry, and genetics of O and K antigens of Escherichia coli. Bacteriological Reviews 42:667-710. Ørskov, I.; Wachsmuth, I.K.; Taylor, D.N.; Echeverria, P.; Rowe, B.; Sakazaki, R.; Ørskov, F. 1991. Two new Escherichia coli O groups: O172 from "Shiga-like" toxin II-producing strains (EHEC) and O173 from enteroinvasive E. coli (EIEC). Acta Pathologica Microbiologica et Immunologica Scandinavica 99:30-32. Paiba, G.A.; Gibbens, J.C.; Pascoe, S.J.S.; Wilesmith, J.W.; Kidd, S.A.; Byrne, C.; Ryan, J.B.M.; Smith, R.R.; McLaren, I.M.; Futter, R.J.; Kay, A.C.S.; Jones, Y.E.; Chappell, S.A.; Willshaw, G.A.; Cheasty, T. 2002. Faecal carriage of verocytotoxin-producing Escherichia coli O157 in cattle and sheep at slaughter in Great Britain. Veterinary Record 150:593-598. Perna, Nicole T.; Plunkett, G. III; Burland, V.; Mau, B.; Glasner, J.D.; Rose, D.J.; Mayhew, G.F.; Evans, P.S.; Gregor, J.; Kirkpatrick, H.A.; Pósfal, G.; Hackett, J.; Klink, S.; Boutin, A.; Shao, Y.; Miller, L.; Grotbeck, E.J.; Davis, N.W.; Lim, A.; Dimalanta, E.T.; Potamousis, K.D.; Apodaca, J.; Anantharaman, T.S.; Lin, J.; Yen, G.; Schwartz, D.C.; Welch, R.A.; Blattner, F.R. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:529-533. Reissbrodt, R. 1998. Enterohemorrhagic Escherichia coli: isolation and identification. Biotest Bulletin 6:65-74. Richards, M.S.; Corkish, J.D.; Sayers, A.R.; McLaren, I.M.; Evans, S.J.; Wray, C. 1998. Studies of the presence of verocytotoxic Escherichia coli O157 in bovine faeces submitted for diagnostic purposes in England and Wales and on beef carcasses in abattoirs in the United Kingdom. Epidemiology and Infection 120:187-192. Riley, L.W.; Remis, R.S.; Helgerson, S.D.; McGee, H.B.; Wells, J.G.; Davis, B.R.; Hebert, R.J.; Olcott, E.S.; Johnson, L.M.; Hargrett, N.T.; Blake, P.A.; Cohen, M.L. 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype. New England Journal of Medicine 308:681-685. Rogerie, F.; Marecat, A.; Gambade, S.; Dupond, F.; Beaubois, P.; Lange, M. 2001. Characterization of Shiga toxin producing E. coli and O157 serotype E. coli isolated in France from healthy domestic cattle. International Journal of Food Microbiology 63:217-223. Sanderson, M.W.; Gay, J.M.; Hancock, D.D.; Gay, C.C.; Fox, L.K.; Besser, T.E. 1995. Sensitivity of bacteriologic culture for detection of Escherichia coli O157:H7 in bovine feces. Journal of Clinical Microbiology 33:2616-2619. Sargeant, J.M.; Gillespie, J.R.; Oberst, R.D.; Phebus, R.K.; Hyatt, D.R.; Bohra, L.K.; Galland, J.C. 2000. Results of a longitudinal study of the prevalence of Escherichia coli O157:H7 on cow-calf farms. American Journal of Veterinary Research 61:1375-1379. Schurman, R.D.; Hariharan, H.; Heaney, S.B.; Rahn, K. 2000. Prevalence and characteristics of Shiga toxin-producing Escherichia coli in beef cattle slaughtered on Prince Edward Island. Journal of Food Protection 63:1583-1586. Smibert, R.M.; Krieg, N.R. 1994. Phenotypic characterization, ch. 25. In P. Gerhardt; R.G.E. Murray; W.A. Wood; N.R. Krieg (eds.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, D.C. Strockbine, N.A.; Wells, J.G.; Bopp, C.A.; Barrett, T.J. Overview of detection and subtyping methods, ch. 33. In J. B. Kaper; A. D. O'Brien (eds.), Escherichia coli O157:H7 and other Shiga toxin-producing E. coli strains. American Society for Microbiology, Washington, D.C. 1998. Tenovar, F.C.; Arbeit, R.D.; Goering, R.V.; Mickelsen, P.A.; Murray, B.E.; Persing, D.H.; Swaminathan, B. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. Journal of Clinical Microbiology 33:2233-2239. Van Donkersgoed, J.; Graham, T.; Gannon, V. 1999. The prevalence of verotoxins, Escherichia coli O157:H7, and Salmonella in the feces and rumen of cattle at processing. Canadian Veterinary Journal 40:332-338. Whittam, T.S.; Wolfe, M.L.; Wachsmuth, I.K.; Ørskov, F.; Ørskov, I.; Wilson, R.A. 1993. Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea. Infection and Immunity 61:1619-1629. Zhao, T.; Doyle, M.P.; Shere, J.; Garber, L. 1995. Prevalence of enterohemorrhagic Escherichia coli O157:H7 in a survey of dairy herds. Applied and Environmental Microbiology 61:1290-1293. Zschöck, M.; Hamann, H.P.; Kloppert, B.; Wolter, W. 2000. Shiga-toxinproducing Escherichia coli in faeces of healthy dairy cows, sheep and goats: prevalence and virulence properties. Letters in Applied Microbiology 31:203-208. 20 American Meat Science Association