Ecology of Escherichia Coli O157:H7 in Beef Cow-Calf Operations from Ranch to Feedlot

NON-TECHNICAL SUMMARY: Escherichia coli O157 is an emerging cause of food borne illnesses in humans. Food products contaminated with cattle feces are a major source of E. coli O157 in outbreaks.

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APPROACH: We plan to collect samples from about 500 to 600 organically raised cattle from a Midwestern packing plant. Antimicrobial susceptibility determinations will be performed by microbroth dilution. There is some indication that the gall bladder may be a site for colonization, as it is for other enteric pathogens like Salmonella and Campylobacter. We will collect intact gall bladders from slaughtered cattle along with fecal samples from the rectum and swab the rectoanal mucosa of the same animals so that we could compare genotypes of isolates between gall bladder and rectum. The lymphoid tissue in the rectoanal junction of the gastrointestinal tract has been suggested as the principal colonization site for E. coli O157 in cattle. It is theorized that E. coli O157 in the feces is derived from the rectoanal junction as feces passed through this region. During the collection of samples, it is impossible to sample by both techniques without the risk of cross contamination from the feces to RAMS or vice versa. This cross contamination could potentially preclude the ability to clearly distinguish that strains from each source are in fact different. Therefore, we propose to test this hypothesis by obtaining intact rectum from slaughtered animals so that rectoanal mucosal region could be sampled without fecal contamination. A small proportion of cattle shed E. coli O157 in high concentration, generally > 104/g of feces, and is referred to as super shedders. Some naturally colonized cattle shed E. coli O157:H7 for a short duration, while other cattle are persistent shedders. Recently, important epidemiological questions have been raised due to the identification of the antiterminator Q gene that originates from the bacteriophage 933W (Q933) in 90% of the human disease isolates of E. coli O157. This gene has been identified upstream of the stx2 gene and has been associated with an increased production of Shiga toxin in isolates that possess the Q933 gene. Over the last 5 years, we have collected and evaluated over 1,500 E. coli O157 isolates in cattle from feces, feeds, and water by standard cultural and immunological methods, as well as by molecular epidemiological methods. We propose to evaluate them for the presence of Q933 gene using specific PCR primers for the gene and distinguishing them with PCR primers specific for the Q gene of bacteriophage 21 (Q21) that does not share sequence homology with Q933. House flies have been implicated in dissemination of several human and animal pathogens, including E. coli O157:H7 in several reports. We propose to assess the potential of house flies to transmit E. coli O157:H7 to cattle by exposing calves in screened pens to flies inoculated with nalidixic acid resistant E. coli O157:H7 and monitoring the shedding in the feces. Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen in the cattle population has the ability to induce immunosuppression during primary infection in immunocompetent cattle Further, BVDV can produce a state of persistent infection (PI) and permanently decrease immunocompetance in calves that are infected in-utero.

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PROGRESS: 2005/07 TO 2008/06<BR>
OUTPUTS: Escherichia coli O157 is a major cause of food borne illnesses in humans. Food products contaminated with cattle feces are a major source of E. coli O157 in outbreaks. Because hindgut is a major colonization site for E. coli O157 in cattle, we conducted a study to test an hypothesis that changes in hindgut could have an impact on E. coli O157. Cattle identified as being positive for fecal shedding of E. coli O157 were selected and randomly allocated to four dietary treatments: grain type (sorghum or wheat) and method of grain processing (steam-flaking or dry-rolling). Fecal samples were obtained to isolate and identify E. coli O157. Processing method (dry-rolled vs. steam-flaked) and the grain type x day interaction were significant (P < 0.05), but not the method of processing x grain type interaction. Results indicated that feeding dry-rolled grains compared to steam-flaked grains reduced fecal shedding of E. coli O157. Possibly, dry-rolling allowed more substrate to reach the hindgut where it was fermented, thus making the hindgut inhospitable to the survival of E. coli O157. In another study, culture methods for identifying cattle with high concentrations of fecal E. coli O157 were evaluated. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid resistant E. coli O157, and direct plating on selective medium containing nalidixic acid was considered the gold-standard method (GS). In experiment 1, methods evaluated were direct pre-enrichment streak on a selective medium, immunomagnetic separation (IMS) coupled with most probable number (MPN), and direct post-enrichment streaks coupled with MPN. The pre-enrichment streak detected samples having > 3.0 log CFU per g with a 74.4 percent sensitivity and 68.8 percent specificity. Direct post enrichment streak and IMS-MPN concentrations were significantly correlated with GS concentrations, but correlations (r = 0.53 and r = 0.39, respectively) were not strong. In experiment 2, direct pre- and post-enrichment streaks performed in triplicate and spiral plating on CT-sorbitol MacConkey agar (SMAC) were the methods evaluated. For pre-enrichment streaks, sensitivity was 79.7 percent and specificity was 96.7 percent for detecting > 3.0 log CFU per g when the criterion was positive cultures on at least two plates. For spiral plating, the sensitivity and specificity were 83.9 percent and 56.3 percent, respectively. Triplicate pre-enrichment streaks of 1:10 diluted feces on CT-SMAC may be a useful method for identifying cattle shedding high concentrations of E. coli O157. Little is known about the bison's status with respect to E. coli O157:H7. We conducted a study to determine the prevalence of E. coli O157:H7 in bison. Rectal contents samples were collected from a total of 342 bison at slaughter. An overall E. coli O157:H7 prevalence of 47.4 percent was observed. Fecal prevalence across sampling days ranged from 17 percent to 83 percent, with an average of 42.1 percent. The prevalence of E. coli O157:H7 in bison is variable and bison could serve an important reservoir for human infection. <BR> PARTICIPANTS: Nagaraja, T. G., Dave Renter, Richard Oberst, Mike Sanderson, and Ludek Zurek <BR> TARGET AUDIENCES: Scientists, Veterinarians, Food Safety Inspection Agency, Cattlemen, Packers, Students
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IMPACT: 2005/07 TO 2008/06<BR>
Escherichia coli O157 is a major food borne pathogen. Food products contaminated with cattle feces are a major source of E. coli O157 in outbreaks. Reducing the level and duration of fecal shedding in cattle will reduce the potential contamination of carcasses. The long term goal of our research is to identify means to control E. coli O157 at the farm level. Dietary intervention to influence hindgut fermentation offers a simple and practical mitigation strategy to reduce prevalence of E. coli O157 in feedlot cattle.

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PROGRESS: 2006/07/01 TO 2007/07/01<BR>
Escherichia coli O157 is a major cause of food borne illnesses in humans. Food products contaminated with cattle feces are a major source of E. coli O157 in outbreaks. Gallbladders and rectal contents were collected from cattle (n=933) at slaughter to determine whether the gallbladder harbors Escherichia coli O157. Both gallbladder mucosal swabs and homogenized mucosal tissues were used for isolation. Only 5 gallbladders (0.54 percent) were positive for E. coli O157. Fecal prevalence averaged 7.1 percent, however, none of the cattle that had E. coli O157 in the gall bladder was positive for feces. Therefore, gallbladder does not appear to be a common site of colonization for E. coli O157 in beef cattle. Previously, we have shown that house flies collected in a feedlot carried numerous viable and virulent E. coli O157. We conducted a study to assess whether house flies can transmit E. coli O157 to cattle. Eight calves were individually exposed to house flies orally inoculated with a mixture of four strains of nalidixic acid-resistant (NalR) E. coli O157 for 48 hours. Fresh cattle feces (rectal contents) and drinking water were sampled and analyzed for NalR E. coli O157 up to 19 days after the exposure. At the end of the experiment, all calves were euthanized and contents of rumen, cecum, colon, and rectum as well as swab samples of gall bladder and rectum wall were tested for NalR E. coli O157. On day 1 after the exposure, all fecal samples and 62.5 percent of drinking water samples of calves tested positive for NalR E. coli O157. Feces of all calves remained positive for NalR E. coli O157 up to 13 days after the exposure and majority (62.5 percent) of fecal samples were positive until the end of experiment. Contamination of drinking water was more variable and all samples were negative on day 19. Necropsy showed the highest prevalence of NalR E. coli O157 on the rectum wall, followed by the rectum and colon content. This data show that house flies can transmit E. coli O157 to cattle and their environment and likely play a role in the ecology of this human pathogen. Niche marketing of beef is an option that more producers have begun to consider because consumers are willing to pay premium price for beef that is produced by certain standards. The two main niches that exist in today's market for beef are organic and natural beef. There is no published report on the prevalence of E. coli 0157 in organic or natural-fed cattle. We conducted a study to determine the prevalence of E. coli 0157 in organic or natural-fed cattle that were delivered to a commercial abattoir in the Midwest. Samples from organic cattle (n=552) and natural-fed cattle (n=506) were collected on six different dates in the summer of 2005 and on three different days in the summer of 2006. The mean prevalence of E. coli 0157, based on fecal or RAMS sampling method in organic cattle was 9.3 and 8.7 percent, respectively. The mean prevalence of E. coli 0157, based on fecal or RAMS sampling method for naturally-fed cattle was 6.8 and 9.3 percent, respectively.
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IMPACT: 2006/07/01 TO 2007/07/01<BR>
Escherichia coli O157 is a major cause of food borne illnesses in humans. Food products contaminated with cattle feces are a major source of E. coli O157 in outbreaks. Reducing the level and duration of fecal shedding in cattle will reduce the potential contamination of carcasses. The long term goal of our research is to identify means to control E. coli O157 at the farm level.

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PROGRESS: 2005/07/01 TO 2008/06/30<BR>
OUTPUTS: Escherichia coli O157 is a major cause of food borne illnesses in humans. Food products contaminated with cattle feces are a major source of E. coli O157 in outbreaks. Because hindgut is a major colonization site for E. coli O157 in cattle, we conducted a study to test an hypothesis that changes in hindgut could have an impact on E. coli O157. Cattle identified as being positive for fecal shedding of E. coli O157 were selected and randomly allocated to four dietary treatments: grain type (sorghum or wheat) and method of grain processing (steam-flaking or dry-rolling). Fecal samples were obtained to isolate and identify E. coli O157. Processing method (dry-rolled vs. steam-flaked) and the grain type x day interaction were significant (P < 0.05), but not the method of processing x grain type interaction. Results indicated that feeding dry-rolled grains compared to steam-flaked grains reduced fecal shedding of E. coli O157. Possibly, dry-rolling allowed more substrate to reach the hindgut where it was fermented, thus making the hindgut inhospitable to the survival of E. coli O157. In another study, culture methods for identifying cattle with high concentrations of fecal E. coli O157 were evaluated. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid resistant E. coli O157, and direct plating on selective medium containing nalidixic acid was considered the gold-standard method (GS). In experiment 1, methods evaluated were direct pre-enrichment streak on a selective medium, immunomagnetic separation (IMS) coupled with most probable number (MPN), and direct post-enrichment streaks coupled with MPN. The pre-enrichment streak detected samples having > 3.0 log CFU per g with a 74.4 percent sensitivity and 68.8 percent specificity. Direct post enrichment streak and IMS-MPN concentrations were significantly correlated with GS concentrations, but correlations (r = 0.53 and r = 0.39, respectively) were not strong. In experiment 2, direct pre- and post-enrichment streaks performed in triplicate and spiral plating on CT-sorbitol MacConkey agar (SMAC) were the methods evaluated. For pre-enrichment streaks, sensitivity was 79.7 percent and specificity was 96.7 percent for detecting > 3.0 log CFU per g when the criterion was positive cultures on at least two plates. For spiral plating, the sensitivity and specificity were 83.9 percent and 56.3 percent, respectively. Triplicate pre-enrichment streaks of 1:10 diluted feces on CT-SMAC may be a useful method for identifying cattle shedding high concentrations of E. coli O157. Little is known about the bison's status with respect to E. coli O157:H7. We conducted a study to determine the prevalence of E. coli O157:H7 in bison. Rectal contents samples were collected from a total of 342 bison at slaughter. An overall E. coli O157:H7 prevalence of 47.4 percent was observed. Fecal prevalence across sampling days ranged from 17 percent to 83 percent, with an average of 42.1 percent. The prevalence of E. coli O157:H7 in bison is variable and bison could serve an important reservoir for human infection. <BR> PARTICIPANTS: Nagaraja, T. G., Dave Renter, Richard Oberst, Mike Sanderson, and Ludek Zurek <BR> TARGET AUDIENCES: Scientists, Veterinarians, Food Safety Inspection Agency, Cattlemen, Packers, Students <BR> <BR>
IMPACT: 2005/07/01 TO 2008/06/30<BR>
Escherichia coli O157 is a major food borne pathogen. Food products contaminated with cattle feces are a major source of E. coli O157 in outbreaks. Reducing the level and duration of fecal shedding in cattle will reduce the potential contamination of carcasses. The long term goal of our research is to identify means to control E. coli O157 at the farm level. Dietary intervention to influence hindgut fermentation offers a simple and practical mitigation strategy to reduce prevalence of E. coli O157 in feedlot cattle.
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PROGRESS: 2005/07/01 TO 2006/06/30<BR>
Escherichia coli O157 has many virulence factors and the most important virulence factor is a lambdoid prophage encoded Shiga toxin (Stx). There are two types, Stx1 and Stx2, the latter is more often associated with human infection. There is evidence that the amount of Stx production is related to severity of the infection. Antiterminator Q sequences, upstream of the stx2 gene, influence the activation of promoter sequences of the phage and influence the production of Shiga toxin. We investigated prevalence of two different sequences of the antiterminator Q, Q933 and Q21, in E. coli O157 strains. The Q gene plays important part in regulating toxin production, thus a possible explanation of why certain O157 genotypes are most often associated with human infections. We examined isolates from feces, water and feed samples from Kansas feedlots, as well as clinical isolates from human infections obtained from the Kansas Department of Public Health for eaeA (attaching and effacing gene), stx1 and stx2 genes using real-time PCR and multiplex PCR, respectively. Specifically, 336 E. coli O157 isolates were evaluated (278 bovine isolates recovered from Kansas cattle feedlots, and 58 isolates recovered from human cases in Kansas). E. coli O157 human isolates from clinical cases exhibited a higher prevalence of the Q933 allele than did E. coli O157 isolates from cattle feedlots (87.9 percent to 20.5 percent, respectively). In evaluating other types of samples from cattle feedlots isolates that contained only the Q933 allele were more frequently in feed or water samples, and to a lesser extent in cattle feces. Another study was conducted to determine whether an immunosuppressed animal will shed E. coli O157 at a higher and for a longer duration than a normal animal. Bovine viral diarrhea (BVDV) is an economically important viral disease in cattle and the virus causes immunosuppression in the animal, which often leads to secondary infection with other pathogens to result in severe illness. Nine calves, six to eight week old, persistently infected with non-cytopathic BVDV and eight normal calves obtained from three different cow-calf operations for use in the study. Persistent BVDV status of all calves was confirmed by repeated testing throughout the study. Both groups were inoculated orally with 10 billion CFUs of E. coli O157 (a mix of five nalidixic-acid resistant strains was used). All calves were examined daily and fecal samples were collected three times a week for 55 days for detection and enumeration of the nalidixic-acid resistant E. coli O157. All calves were then necropsied and samples from throughout the gastrointestinal tract were taken for the detection of the nalidixic-acid resistant E. coli O157. Analysis indicated no statistical difference in the concentration of E. coli O157 shed between the persistently infected BVDV calves and the control calves throughout the length of the study. This data suggests that immunosuppression caused by persistent infection with non-cytopathic BVDV infection does not play a role in the level or duration of shedding of E. coli O157 in calves.
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IMPACT: 2005/07/01 TO 2006/06/30<BR>
The higher prevalence of E. coli O157 isolates that contained the Q933 allele were from human clinical cases as opposed to cattle feces. We consider these findings to be significant in defining the ecology the E. coli O157 that are capable of causing infections in humans. Immunosuppression caused by persistent infection with a common viral disease in cattle does not play a role in the level or duration of shedding of E. coli O157.