Farm animal manure or manure slurry may disseminate, transmit, or propagate Escherichia coli O157:H7. In this study, the survival and growth of E. coli O157:H7 in ovine or bovine feces under various experimental and environmental conditions were determined. A manure pile collected from experimentally inoculated sheep was incubated outside under fluctuating environmental conditions.E. coli O157:H7 survived in the manure for 21 months, and the concentrations of bacteria recovered ranged from <102 to 106 CFU/g at different times over the course of the experiment. The DNA fingerprints of E. coli O157:H7 isolated at month 1 and month 12 were identical or very similar. A second E. coli O157:H7-positive ovine manure pile, which was periodically aerated by mixing, remained culture positive for 4 months. An E. coliO157:H7-positive bovine manure pile was culture positive for 47 days. In the laboratory, E. coli O157:H7 was inoculated into feces, untreated slurry, or treated slurry and incubated at −20, 4, 23, 37, 45, and 70°C. E. coliO157:H7 survived best in manure incubated without aeration at temperatures below 23°C, but it usually survived for shorter periods of time than it survived in manure held in the environment. The bacterium survived at least 100 days in bovine manure frozen at −20°C or in ovine manure incubated at 4 or 10°C for 100 days, but under all other conditions the length of time that it survived ranged from 24 h to 40 days. In addition, we found that the Shiga toxin type 1 and 2 genes in E. coli O157:H7 had little or no influence on bacterial survival in manure or manure slurry. The long-term survival of E. coli O157:H7 in manure emphasizes the need for appropriate farm waste management to curtail environmental spread of this bacterium. This study also highlights the difficulties in extrapolating laboratory data to on-farm conditions.

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios >10 2 terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10 10 PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be >10 2 . In addition, phages were maintained at 10 6 PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.Since its association with disease in 1982 (33), Escherichia coli O157:H7 has become a worldwide threat to public health and is one of today's most troubling food-borne pathogens. Human illness with E. coli O157:H7 ranges from self-limited watery diarrhea and hemorrhagic colitis to life-threatening manifestations such as the hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Cattle and sheep, important domestic ruminants, are the primary reservoirs for this human pathogen and are the most common sources for foodborne, waterborne, and direct-animal-contact infections (9-11, 18, 48, 51). In contrast to humans, the carrier ruminants remain healthy, and E. coli serotype O157:H7 is a transient member of the ruminant gastrointestinal flora. Persistence of E. coli O157:H7 carriage in naturally and experimentally infected animals varies from days to months (6,8,12,13,19,35,38).Successful strategies to control or reduce the carriage and prevalence of E. coli O157:H7 in live ruminants would reduce the risk of human exposure to this pathogen. There is currently no reliable intervention or animal vaccine available to curb carriage of E. coli O157:H7. Probiotics aimed at creating an intestinal microenvironment inhibitory to E. coli O157:H7 have been tested, but without consistent success (14,22,28,29,50). A lytic bacteriophage that would specifically target the E. coli O157 serotype is another appealing approach because phage therapy has been successful in animal trials against a broad range of bacterial pathogen...

Experimentally inoculated sheep and cattle were used as models of natural ruminant infection to investigate the pattern of Escherichia coli O157:H7 shedding and gastrointestinal tract (GIT) location. Eighteen forage-fed cattle were orally inoculated with E. coli O157:H7, and fecal samples were cultured for the bacteria. Three distinct patterns of shedding were observed: 1 month, 1 week, and 2 months or more. Similar patterns were confirmed among 29 forage-fed sheep and four cannulated steers. To identify the GIT location of E. coli O157:H7, sheep were sacrificed at weekly intervals postinoculation and tissue and digesta cultures were taken from the rumen, abomasum, duodenum, lower ileum, cecum, ascending colon, descending colon, and rectum. E. coli O157:H7 was most prevalent in the lower GIT digesta, specifically the cecum, colon, and feces. The bacteria were only inconsistently cultured from tissue samples and only during the first week postinoculation. These results were supported in studies of four Angus steers with cannulae inserted into both the rumen and duodenum. After the steers were inoculated, ruminal, duodenal, and fecal samples were cultured periodically over the course of the infection. The predominant location of E. coli O157:H7 persistence was the lower GIT. E. coli O157:H7 was rarely cultured from the rumen or duodenum after the first week postinoculation, and this did not predict if animals went on to shed the bacteria for 1 week or 1 month. These findings suggest the colon as the site for E. coli O157:H7 persistence and proliferation in mature ruminant animals.For many years it has been known that healthy ruminants transiently harbor the human pathogen Escherichia coli O157:H7 in their gastrointestinal tract; however, the conditions that lead to its acquisition, persistence, and clearance from that site are not clearly understood (18,30). Elucidating the relationship between cattle and E. coli O157:H7 may impact the development of interventions to curb its presence in ruminants and thereby reduce the incidence of human infections with this pathogen.The hallmark of human disease with E. coli O157:H7 is a hemorrhagic colitis that is most often self-limiting (2, 18). However, among reported outbreaks, as many as 25% of infected individuals have been hospitalized, 6% have developed life-threatening sequelae, the hemolytic uremic syndrome, and 1% have died from their infection. For children infections are even more severe, with 5 to 10% progressing to hemolytic uremic syndrome and a 3 to 5% mortality rate (2, 6, 18). E. coli O157:H7 is transmitted by ingestion of contaminated bovine food products, contaminated drinking water or fruit juice, contact with contaminated recreation water or culture-positive animals, and in rare cases, human-to-human transmission via the fecal-oral route (2,3,18,24).Although healthy cattle (5, 9, 15, 21, 37), sheep (9, 26, 28), deer (16, 34), and goats (9, 32) have been shown to transiently harbor E. coli O157:H7 naturally, cattle are the main source of human infections ...

Hodve CJ. Characterization of Escherichia coli O157:H7 and other shigatoxin-producing E coli serotypes isolated from sheep. J Clin Microbiol 1997;35:892-899. 5. Sidjabat-Tambunan H, Bensink JC. Verotoxin-producing E coli from the faeces of sheep, calves and pigs. Aust Vet J 1997;75:292-293. 6. Bettelheim KA. The isolation of Escherichia coli from a sheep slaughtering line in an abattoir. Comp Imm Microbiol Infect Dis 1981;4:93-100. 7. Huis in't Veld JHJ, Mulder RWAW, Snijders JMA Impact of animal husbandry and slaughter technologies on microbial contamination of meat: monitoring and control. Meat Sci 1994;36:123-154. 8. Brooks HJL, Bettelheim KA, Todd B Holdaway MD. Non-O157 verocytotoxin producing Escherichia coli: Aetiological agents of diarrhoea in children in Dunedin, New Zealand. Comp Immunol Microbiol Infect Dis 1997;20:163-170. 9. Dorn CR, Scotland SM, Smith HR, Willshaw GA, Rowe B. Properties of verocytotoxin-producing Escherichia coli of human and animal origin belonging to serotypes other than O157.H7. Epidemiol Infect Dis 1989;103:83-95. 10. Willshaw GA, Scotland SM, Smith HR, Rowe B. Properties of verocytotoxinproducing Escherichia coli of human origin of O serogroups other than O157. J Infect Dis 1992;166:797-802. 11. Bockermhl J, Aleksic S, Karch H. Serological and biochemical properties of shiga-like toxin (verotoxin) -producing strains of Escherichia coli O157 from patients in Germany. Zbl Bakt 1992;276:189-195. 12. Bettelheim KA, Brown JE, Lolekha S, Escheverria P. Serotypes of Escherichia coli that hybridized with DNA probes for genes encoding shiga-like toxin I, shiga-like toxin II, and serogroup O157 enterohaemorrhagic E coli fimbriae isolated from adults with diarrhoea in Thailand. J Clin Microbiol 1990;25:293-295. 13. Scotland SM, Rowe B, Smith HR, Willshaw GA, Gross RJ. Verocytotoxinproducing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes. J Med Microbiol 1988;25:237-243. 14. Willshaw GA, Thirlwell J, Jones AP et al. Verocytotoxin-producing Escherichia coli O157 in beefburgers linked to an outbreak of diarroea, haemorrhagic colitis and haemolytic uraemic syndrome in Britain.

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coliO157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4°C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. colicultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4°C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.

Aim:  To develop a new adherence assay, using cattle recto‐anal junction squamous epithelial (RSE) cells, for evaluating bacterial adherence to cells of bovine origin. Methods and Results:  Proof of concept was demonstrated using the human gastrointestinal pathogen Escherichia coli O157:H7, for which cattle are reservoirs. Adherence assays were conducted using both RSE and HEp‐2 cells, in the presence and absence of D+Mannose. E. coli O157 specifically adhered in a type I fimbriae‐independent manner to RSE cells in significantly higher numbers and also bound significantly higher numbers of RSE cells than diverse laboratory strains of nonpathogenic E. coli. Conclusion:  The RSE cell adhesion assay output highly reproducible and interpretable results that compared very well with those obtained using the more extensively used HEp‐2 cell adherence assay. Significance and Impact of the study:  The RSE cell adhesion assay provides a convenient means of directly defining and evaluating pathogen factors operating at the bovine recto‐anal junction. The RSE cell adhesion assay further has the potential for extrapolation to diverse bacteria, including food‐borne pathogens that colonize cattle via adherence to this particular anatomical site.

The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture of 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx 1 , stx 2 , and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx 1 , stx 2 , and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens.

Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as “super-shedders” (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.