Escherichia coli O157:H7 in Environments of Culture-Positive Cattle

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2005, p Vol. 71, No /05/$ doi: /aem Copyright 2005, American Society for Microbiology. All Rights Reserved. Escherichia coli O157:H7 in Environments of Culture-Positive Cattle Margaret A. Davis, Karen A. Cloud-Hansen, John Carpenter, and Carolyn J. Hovde* Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, Idaho Received 15 February 2005/Accepted 20 July 2005 Outbreaks of Escherichia coli O157:H7 disease associated with animal exhibits have been reported with increasing frequency. Transmission can occur through contact with contaminated haircoats, bedding, farm structures, or water. We investigated the distribution and survival of E. coli O157:H7 in the immediate environments of individually housed, experimentally inoculated cattle by systematically culturing feed, bedding, water, haircoat, and feed bunk walls for E. coli O157:H7 for 3 months. Cedar chip bedding was the most frequently culture-positive environmental sample tested (27/96 or 28.15%). Among these, 12 (44.0%) of positive bedding samples were collected when the penned animal was fecal culture negative. Survival of E. coli O157:H7 in experimentally inoculated cedar chip bedding and in grass hay feed was determined at different temperatures. Survival was longest in feed at room temperature (60 days), but bacterial counts decreased over time. The possibility that urine plays a role in the environmental survival of E. coli O157:H7 was investigated. Cedar chip bedding moistened with sterile water or bovine urine was inoculated with E. coli O157:H7. Bedding moistened with urine supported growth of E. coli O157:H7, whereas inoculated bedding moistened with only water yielded decreasing numbers of bacteria over time. The findings that environmental samples were frequently positive for E. coli O157:H7 at times when animals were culture negative and that urine provided a substrate for E. coli O157:H7 growth have implications for understanding the on-farm ecology of this pathogen and for the safety of ruminant animal exhibits, particularly petting zoos and farms where children may enter animal pens. Disease in humans caused by Escherichia coli O157:H7 is primarily transmitted by the food-borne route (3). In recent years, however, outbreaks associated with animal exhibits have been reported with increasing frequency (2, 4, 6, 13, 19, 20, 22, 23, 27, 28). The environments of domestic ruminants may be an important reservoir for E. coli O157:H7 and pose a continued risk for human exposure. Visitors, particularly young children, at open farms, petting zoos, and agricultural fairs can become exposed by direct animal contact or by contact with contaminated bedding, farm structures, or soil. Little work has been done to investigate E. coli O157:H7 survival in the environment and studies primarily focused on bacterial survivability in farm animal manure, manure slurry, or water (7, 16, 30). It is known that E. coli O157:H7 survives in raw manure for many months ( 21 months in one study) but that the pathogen can be eliminated with composting conditions similar to those required to eliminate coliforms (12, 16). The low prevalence of culture-positive animals and the inability to culture E. coli O157:H7 routinely from the farm environment has hampered efforts to study the ecology of this human pathogen. These limitations led us to take advantage of the conditions in a research barn housing cattle experimentally dosed with E. coli O157:H7. In this setting, mature cattle were * Corresponding author. Mailing address: Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID Phone: (208) Fax: (208) E- mail: cbohach@uidaho.edu. Present address: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA Present address: Department of Plant Pathology, University of Wisconsin, Madison, WI Present address: Department of Anesthesiology, University of Arizona, Tucson, AZ individually housed, and records of their carriage of E. coli O157:H7 and the conditions of their immediate environment were available. Because the research barn is similar to many private cattle facilities and is open air, it was representative of the microcosm typical of domestic cattle. In this study, we (i) investigated the distribution and survival of E. coli O157:H7 in the environments of fecal culture-positive animals by systematically culturing feed, bedding, water, haircoat, and feed bunk walls for E. coli O157:H7; (ii) determined the ability of E. coli O157:H7 to survive in feed and bedding inoculated with laboratory-grown strains under various temperature and sunlight conditions; and (iii) investigated the role of urine in environmental survival and replication of E. coli O157:H7. MATERIALS AND METHODS Animals and animal housing. Eight two-year-old Holstein steers were penned individually without contact with other animals in covered stalls with a cement floor, plywood sides, wooden feed bunk, and a 15-gallon galvanized steel water bucket. The back of each stall was open to the environment and closed with a steel gate. Nose-to-nose contact between individual animals was prevented, and no water or urine could pass between pens. Pen-to-pen contamination was further prevented by using separate shovels, boots, and coveralls in each pen. Bedding consisted of cedar chips and was removed and replaced with clean bedding each week. Visible fecal material was removed daily. A grass hay growing ration was fed daily, and animals had continual access to drinking water, which was changed daily and originated from the municipal drinking water supply. Animals were prescreened by culture for E. coli O157:H7 before use in the study. Oral inoculations of steers with E. coli O157:H7. Each steer was orally dosed with to CFU of E. coli O157:H7, ATCC strain 43894, as previously described (8). This strain was chosen because of its relevance to human disease and much previous work with this strain in cattle. Seventeen weeks after this first oral dose of E. coli O157:H7, when all animals were culture negative for the bacteria, the steers were redosed once with to CFU of E. coli O157:H7 (ATCC 43894). Environmental sampling. Before their use in the facility, fresh feed, water, and bedding were tested for the presence of E. coli O157:H7 (culture methods are 6816

2 VOL. 71, 2005 E. COLI O157 IN ENVIRONMENTS OF CATTLE 6817 given below). To determine the distribution of E. coli O157:H7 in the steers environments at a time when fecal counts most closely reflected field conditions, environmental sampling was begun 7 weeks following their initial oral dose of bacteria. Fecal samples and each type of environmental sample were collected weekly. Samples of bedding (2 to 3 g each) that were free of visible feces were collected from 10 sites in each pen, for a total of 25 g of bedding per sample. Sterile cotton swabs were used to swab a 400-cm 2 area of the interior feed bunk surface. Haircoat samples (1 g of matted hair) were removed from the abdomen or perianal region with a scalpel. If no matted hair was present, 1 g of loose hair was removed by rubbing a gloved hand over the entire animal. Three milliliters of saliva was collected from each animal 4 days postdose. A piece of wide Tygon tubing was offered to chew, and dripping saliva was collected in a sterile tube. The saliva samples were cultured as described below in Culture of E. coli O157:H7. Water samples (100 ml each) were collected in a sterile container from each bucket before the water was changed and the bucket was cleaned. Water sediment samples (0.1 g each) were removed from the bottom of the emptied bucket with a sterile cotton swab. Experimentally contaminated feed and bedding. To determine the survival of E. coli O157:H7 in feed and bedding under a variety of conditions, E. coli O157:H7 at high or low concentrations was inoculated into samples of feed and bedding and held inside a barn, outside a barn in sunlight, or inside the laboratory (25 C) and sampled periodically for 60 days. High and low concentrations were chosen based on accounts of naturally occurring coliform and E. coli counts. Cattle feeds were frequently found to contain 10 4 to 10 5 CFU E. coli/g and as high as 10 7 CFU E. coli/g. Also, similar studies of sawdust bedding frequently found coliform counts in the range of 10 6 to 10 7 CFU/g. An overnight culture of E. coli O157:H7 (ATCC 43894) grown in Luria broth (LB) was pelleted, washed with sterile saline solution, and resuspended. To approximate a natural level of contamination, the initial concentrations of 10 3 CFU/g of bedding and 10 3 CFU/g of feed were used and referred to as low inoculation. For the bedding experiment, this cell suspension was diluted in saline (60 ml) and mixed with 2.0 kg of cedar chip bedding in a 5-gallon plastic container. For the feed experiment, washed cells were suspended in saline (80 ml) and mixed with 1.41 kg of chopped grass hay. Postinoculation samples of bedding and feed were collected for culture daily for the first 4 days, then every 3 or 4 days for 3 weeks, and then every 10 days until day 60. To determine the survival of E. coli O157:H7 in feed and bedding after inoculation with higher concentrations of bacteria, 1.45 kg of bedding was mixed with stationary cells suspended in a total volume of 100 ml in a 5-gallon plastic container to give an initial concentration near 10 6 CFU/g. Similarly, a 900-g sample of feed was mixed with stationary cells to give an initial concentration near 10 7 CFU/g. Inoculated feed and bedding were held inside a barn at ambient temperatures. All experimentally inoculated feed and bedding samples were collected aseptically by taking 1- to 1.5-g portions of feed or bedding from 10 different sites in the bucket being sampled and placing them into a sterile Whirlpak bag. From this mixture, a 10-g sample was cultured for E. coli O157:H7 as described below for fecal samples. Culture of E. coli O157:H7. (i) Fecal samples. Feces were tested by a culture method comparable in sensitivity to O157-specific immunomagnetic bead capture and culture (9). Samples were cultured by direct plating that yielded quantitative culture data (in CFU per gram) or selectively enriched by incubation overnight prior to plating that yielded qualitative (positive or negative) culture data. Briefly, feces (10 g) were suspended in Trypticase soy broth (50 ml; BBL/ Becton Dickinson) supplemented with cefixime (50 g/liter; Lederle Laboratories, Pearl River, NY), potassium tellurite (2.5 mg/liter; Sigma Chemical Co., St. Louis, MO), and vancomycin (40 mg/liter; Sigma) (TSB-CTV). Dilutions of each sample were prepared in sterile saline (0.15 NaCl) before (direct culture) and after (enrichment culture) overnight incubation with aeration at 37 C. Before incubation, dilutions were spread plated on sorbitol MacConkey agar containing 4-methylumbelliferyl- -D-glucuronide (100 mg/liter; Biosynth Ag Biochemica and Synthetica, Stokie, IL) (SMAC-MUG; direct culture). After incubation, dilutions were plated on sorbitol MacConkey agar supplemented with cefixime (50 g/ml), potassium tellurite (2.5 mg/liter), and MUG (100 mg/liter) (designated SMAC-CTM; enrichment culture). Colonies that did not ferment sorbitol or utilize MUG were confirmed to be E. coli O157 by a latex agglutination test (Pro-Lab Diagnostics, Toronto, Canada). (ii) Environmental samples. Bedding samples (each, 25 g) were suspended in TSB-CTV (500 ml). Dilutions of each sample were prepared in sterile saline (0.15 M NaCl) before and after overnight incubation with aeration at 37 C. Dilutions from bedding samples were cultured as described for fecal samples (above). Feed bunk samples (swab of 400 cm 2 ) were streaked directly onto SMAC-MUG plates and then placed in 50-ml culture tubes; TSB-CTV (10 ml) was added. After overnight incubation, dilutions were made and plated on SMAC-CTM. The hair samples (1 g) were each suspended in TSB-CTV (50 ml) and processed as described for fecal samples (above). The water samples (100 ml) were added to 2 TSB-CTV (100 ml) and processed as for fecal samples. Water bucket sediment swabs were streaked onto SMAC-MUG plates then placed into 50-ml culture tubes, and TSB-CTV (10 ml) was added. Isolation of E. coli O157:H7 from this point was identical to that for fecal samples. Urine and cedar chip bedding experiments. To determine the effect of urine on E. coli O157:H7 survival and growth, the bacteria were mixed with urine, dilute urine, or mixtures of cedar chip bedding with or without urine. Before each experiment, urine and bedding samples were tested for the presence of E. coli O157:H7 as described above. Bedding samples (10 g) were moistened with either sterile deionized water (100 ml), undiluted urine (100 ml), or 10% urine (100 ml). Moistened bedding was inoculated with 10 3 CFU of E. coli O157:H7 and incubated without aeration at room temperature (25 C) or at 37 C. Six strains of E. coli O157:H7 were tested: ATCC 43894; ATCC 43895; ATCC 43888; 905, a clinical isolate from a patient with hemolytic-uremic syndrome and a gift from M. K. Waldor, Tufts-New England Medical Center and Howard Hughes Medical Institute, Boston, MA; WSU180, a natural bovine isolate from a dairy heifer at Washington State University, Pullman, WA; and , a clinical isolate and a gift from P. Tarr, Washington University, St. Louis, MO. At 0-, 3-, 6-, 9-, 12-, and 24-h intervals postinoculation, samples were plated onto SMAC-CTVM, and the number of E. coli O157:H7 was determined as described above for direct culture of bedding samples. Experiments were done with bovine urine from cattle fed a grass hay ration or human urine from a consenting volunteer. Urease activity was tested by culture in urea medium (Diagnostic Systems, Sparks, MD). RESULTS All animals were healthy throughout the study. The oral inoculations of E. coli O157:H7 resulted in animals that passed the bacteria in fecal material for various lengths of time ranging from 1 week to 1 month. The number of E. coli O157: H7/g feces ranged from CFU/g to levels detectable only by enrichment culture (defined as 10 1 CFU/g). The pattern of carriage of E. coli O157:H7 by the experimentally inoculated steers was similar to that of cattle carrying the bacteria naturally, showing wide variation in duration of fecal positive status and in fecal counts of E. coli O157:H7 (1, 11, 14, 18, 24, 26). Environmental components were frequently culture positive when cattle feces were culture negative for E. coli O157:H7. The environment around cattle with high or low numbers of fecal E. coli O157:H7 were analyzed (Tables 1 and 2). Seven weeks after a single oral dose of the bacteria, animals were culture negative or were shedding low numbers of fecal bacteria (Table 1). Two animals, numbers 1 and 4, were consistently culture negative for E. coli O157:H7 for the next 8 weeks, and the environmental samples from their pens were also culture negative except for one water column (WC) sample (Table 1). The environmental samples had low numbers of E. coli O157:H7 and required enrichment culture to detect the bacteria. Bedding was the most common environmental sample to be culture positive. Interestingly, one bedding sample was positive by direct culture and contained 10 3 E. coli O157: H7/g, even though the steer in that pen had been fecal culture negative for the bacteria for 2 weeks (animal 6 at week 12). To determine if a different pattern of environmental contamination would be seen when cattle had higher numbers of fecal E. coli O157:H7, the animals were redosed (17 weeks after the first dose), and environmental samples were analyzed for four consecutive weeks while animals had high numbers of E. coli O157:H7 in their feces (Table 2). Regardless of the

3 6818 DAVIS ET AL. APPL. ENVIRON. MICROBIOL. TABLE 1. Fecal and environmental culture results for eight individually penned steers with low numbers of fecal E. coli O157:H7 long after oral inoculation Time Sample type Animal no Day 1 a Fecal b Wk 7 Fecal c d Other e B e Fb e Wk 8 Fecal Other B H e B Wk 9 Fecal Other WS e B B, H B Wk 10 Fecal Other B B B Wk 11 Fecal Other WS B B WC Wk 12 Fecal Other WS WS, B f WS Wk 13 Fecal Other B WC e,ws WC B H WS Wk 14 Fecal Other B B B a Steers were given an oral dose of 10 9 to CFU of E. coli O157:H7 (ATCC 43894) on day 0. b Number of CFU of E. coli O157:H7 per gram. c, culture negative for E. coli O157:H7. d, culture positive by selective enrichment only. e Environmental samples included bedding (B); feed bunk surface (Fb); hair coat (H); water column from drinking water bucket (WC); and sediment from drinking water bucket (WS). Use of these letters in the table represents culture-positive status for that sample. f This bedding sample had CFU of E. coli O157:H7 per gram of sample. number of fecal E. coli O157:H7 organisms, the type of environmental sample most frequently cultured positive was bedding (Tables 1 and 2). Overall, 27/96 (28.1%) bedding samples were culture positive. Among these, 12 (44.0% of positive bedding samples, or 12.5% of all bedding samples) were collected during the same week that fecal samples from the animal in that pen were culture negative. Samples from the water buckets were the next most frequently positive sample (Tables 1 and 2): overall, 13/96 (13.5%) water sediment samples were culture positive and 4/96 (4.2%) water column samples were culture positive. Two of the water column samples (50.0% of the positive samples and 2.1% of the total) and four of the water sediment samples (30.8% of the positive samples and 4.2% of total samples) were collected when animals in those pens were fecal culture negative. Three haircoat samples were Time culture positive, one of which was collected when the fecal sample from the same animal was culture negative. The single feed bunk sample that was culture positive was collected when fecal sample from the animal in that pen was also positive. Although not sampled more than once, no saliva sample was culture positive (data not shown). E. coli O157:H7 can survive for 35 days in cedar chip bedding and for 60 days in grass hay feed. To determine the survivability of E. coli O157:H7 in bedding and feed without contribution of an animal, bedding and feed samples held in the laboratory or in the barn but without animal contact were inoculated with the bacteria, and samples were cultured at various times. All of the experimentally inoculated samples showed decreasing numbers of E. coli O157:H7 over time (Tables 3 and 4). Cedar chip bedding samples inoculated with TABLE 2. Fecal and environmental E. coli O157:H7 culture results for eight individually penned steers early after oral inoculation Sample type Animal no Day 1 a Fecal b Wk 1 Fecal c d Other B e WS e WS Wk 2 Fecal Other B, WS B B B B Wk 3 Fecal Other B Wk 4 Fecal Other WC e B WS B WS B, WS a Steers were given an oral dose of 10 9 to CFU of E. coli O157:H7 (ATCC 43894) on day 0. b Number of CFU of E. coli O157:H7 per gram. c, culture negative for E. coli O157:H7. d, culture positive by selective enrichment only. e Environmental samples included: bedding (B); feed bunk surface (Fb); hair coat (H); water column from drinking water bucket (WC); sediment from drinking water bucket (WS). Use of these letters in the table represents culture-positive status for that sample.

4 VOL. 71, 2005 E. COLI O157 IN ENVIRONMENTS OF CATTLE 6819 Location and inoculation TABLE 3. Survival of E. coli O157:H7 in cedar chip bedding No. of days postinoculation Indoors (high) a b NS c NS NS NS d NS NS e NS NS Indoors (low) f NS NS NS Barn g b NS NS NS NS NS NS NS Outside h c NS NS NS a Bedding (1.45 kg) was inoculated with a total of CFU of E. coli O157:H7 (ATCC 43894) and held at room temperature inside the laboratory. b Expressed as CFU per gram as detected by nonenrichment. c NS, not sampled on that day. d, culture positive by selective enrichment only. e, culture negative. f Bedding (2.0 kg) was inoculated with a total of CFU of E. coli O157:H7 (ATCC 43894) and held at room temperature inside the laboratory. g Bedding inoculated with CFU of E. coli O157:H7 (ATCC 43894) was held at outside temperatures inside a barn. h Bedding inoculated with CFU of E. coli O157:H7 (ATCC 43894) was held outside exposed to sunlight and weather conditions. high or low concentrations of E. coli O157:H7 and held at room temperature (25 C) were culture positive for 35 or 18 days, respectively. Bedding samples inoculated with low concentrations of E. coli O157:H7 were culture positive for shorter durations of at least 11 or 14 days when held inside or outside the barn, respectively. Core temperatures of bedding samples sheltered inside the barn ranged from 16 C to 35 C and of bedding samples held outside ranged from 16 C to 36 C. After inoculation with high concentrations of E. coli O157:H7, chopped grass hay feed held inside the laboratory was culture positive for the duration of the study (42 days). After inoculation with low concentrations of E. coli O157:H7, chopped grass hay feed samples were culture positive for at least 14 days when held inside the barn, 31 days when held outside, and 60 days when held in the laboratory (Table 2). Core temperatures of feed samples sheltered inside the barn ranged from 17 C to 32 C; core temperatures of exposed feed samples held outside ranged from 17 C to 44 C. Cedar chip bedding supports growth of E. coli O157:H7 when moistened with bovine urine. Urine from cattle fed a typical forage-based growing diet of grass hay had a ph of To determine whether the presence of bovine urine in the steer bedding might support the growth of E. coli O157:H7 and therefore explain the number of positive bedding samples in the absence of detectable fecal E. coli O157:H7, bedding samples were experimentally inoculated after being moistened with sterile water or bovine urine. E. coli O157:H7 ATCC replicated in cedar chip bedding moistened with dilute bovine urine (10%), in contrast to the declining bacterial numbers in the bedding moistened with sterile water when incubated at 25 C or 37 C (Fig. 1). The averages of the log 10 bacterial counts between growth at 25 C with urine compared to sterile water were well over 2 standard deviations apart from each other at 9 through 24 h postinoculation. The differences we observed in the single experiment at 37 C between the two substrates were even greater at 12 h. In addition, E. coli O157:H7 ATCC and five different strains were tested for the ability to grow in bedding moistened with 10% urine or undiluted urine at 25 C or 37 C (Fig. 2). For every strain, the addition of urine to the bedding and incubation at the cooler temperature resulted in bacterial growth. Likewise, for every strain incubated in bedding moistened with undiluted urine and incubated at the warmer temperature, bacterial death resulted. Interestingly, strain variation in the ability to grow in bedding moistened with dilute urine and incubated at the 37 C was seen. All E. coli O157:H7 strains incubated in bedding moistened with sterile water and incubated at 37 C declined in number by 2 logs in 24 h (data not shown). Cedar chip bedding is acidic, and bedding moistened with water had a ph of 5.4, while bedding moistened with the bovine urine was neutralized to ph 6.8. However, this ph change did not account for the observed bacterial replication because similar experiments, done with mixtures of bedding and acidic human urine (ph near 6.0), also resulted in E. coli O157:H7 replication (data not shown). Also, cedar chip bedding moistened and neutralized with a phosphate buffer to ph 7.0 did not support the growth of E. coli O157:H7 (data not shown). The ability of the six E. coli O157:H7 strains to grow in urine or buffered solutions of urea (without bedding) were assessed (Table 5). Although E. coli O157:H7 was negative for urease activity, as determined by a standard urease assay (data not shown), all strains grew in undiluted or dilute urine at 25 C Location and inoculation TABLE 4. Survival of E. coli O157:H7 in grass hay feed No. of days postinoculation Indoors (high) a NS NS NS NS NS NS NS Indoors (low) b NS NS Barn c NS NS NS NS NS NS NS NS NS Outside d NS NS a Feed (900 g) was inoculated with a total of CFU of E. coli O157:H7 (ATCC 43894) and held at room temperature inside the laboratory. b Feed inoculated with CFU of E. coli O157:H7 (ATCC 43894) was held at room temperature inside the laboratory. c Feed inoculated with CFU of E. coli O157:H7 (ATCC 43894) was held at outside temperatures inside a barn. d Feed inoculated with CFU of E. coli O157:H7 (ATCC 43894) was held outside exposed to sunlight and weather condition.

5 6820 DAVIS ET AL. APPL. ENVIRON. MICROBIOL. FIG. 1. Growth of E. coli O157:H7 in bedding moistened with water or dilute bovine urine. Average of the log 10 CFU of E. coli O157:H7/g of inoculated bedding moistened with sterile water or 10% bovine urine. Samples were incubated at 25 C for 24 h or at 37 C for 12 h. Results from three experiments are shown and standard errors were 0.3 log 10 CFU/g. (Table 5). Temperature played a role in this ability, with less growth or no growth seen with incubations at 37 C, depending on the strain. Taken together, these data indicated that E. coli O157:H7 was able to utilize urine as a growth substrate and used it best in cooler temperatures. DISCUSSION This study was unique because it took advantage of experimentally inoculated animals, penned individually and cared for without cross-contamination, to systematically study the effect of individual animals on the environment. In addition, to complement the environmental samples in this setting, we compiled detailed histories of each animal for the number and persistence of fecal E. coli O157:H7 organisms. Three important findings of this study include the following. (i) Samples from the pen environments of inoculated animals were frequently positive for E. coli O157:H7 at times when E. coli O157:H7 was not detected in feces. (ii) Evidence was obtained that bovine urine provided a necessary substrate for growth of E. coli O157:H7. (iii) Enhanced bacterial growth in the presence of bovine urine was temperature and urine concentration dependent. These findings have major implications for the ecology of this human pathogen and for the safety of animal exhibits that feature ruminants, particularly petting zoos and open farms where children are allowed to enter pens for contact with ruminant animals. Among the types of environmental samples examined, E. coli O157:H7 was isolated most frequently from cedar chip bedding. The steers in this study were maintained separately, so that contamination of bedding in a pen was unlikely to come from any source other than the animal within that pen. Because of the open-air nature of the housing facility, contamination from avian sources could not be ruled out, but surveys of bird feces have detected only a very low prevalence of E. coli O157:H7 (10, 15, 29). Furthermore, many animal exhibits featuring ruminants are not bird proof but do pen animals individually, similar to this experimental situation. We did not find routine or gross contamination of the animal hides which may be attributed to the individual penning arrangement, a situation very different from feedlot group penning. Survival of pathogenic E. coli strains in several types of animal bedding, other than cedar chips, has been documented. A field study of environmental sources of E. coli O157:H7 on a heifer raiser farm in Italy found 6 of 16 straw bedding samples to be positive for E. coli O157:H7 (5). A longitudinal study of commercial dairy farms showed the use of sawdust, rather than sand, bedding was associated with a higher prevalence of fecal E. coli O157:H7 among dairy cows (17). The ability of E. coli to survive and replicate in different types of bedding was examined for a strain of E. coli isolated from a clinical bovine mastitis case (31). In pine sawdust bedding at 37 C, counts of this strain of E. coli underwent rapid log reductions during the first 12 h. In the present pen study, we were unable to study the long-term survival of E. coli O157:H7 in bedding or feed in the setting of the barn because all used bedding was removed and replaced on a weekly basis and the feed was completely consumed by the steers on a daily basis. Although we observed lengthy survival times of E. coli O157:H7 in cedar chip bedding (at least 34 days) and in grass hay feed ( 42 days) into which the bacteria were inoculated, the bacterial counts decreased over time. However, this is the first report that the addition of bovine urine to bedding promoted replication of E. coli O157: H7. This finding may account for our ability to culture E. coli O157:H7 from the environment of steers that were not shedding detectable numbers of E. coli O157:H7 in their feces. Understanding the mechanisms by which urine contributes to survival of E. coli O157:H7 in the farm environment may suggest containment strategies to reduce bacterial numbers or

6 VOL. 71, 2005 E. COLI O157 IN ENVIRONMENTS OF CATTLE 6821 Downloaded from FIG. 2. E. coli O157:H7 strain variation in growth in bedding moistened with urine. Sterilized cedar chip bedding was moistened with 10% (A and C) or 100% (B and D) solutions of fresh, filter-sterilized bovine urine. Moistened bedding was inoculated with 10 2 CFU E. coli O157:H7/g with each of the following strains: ATCC 43894, ATCC 43895, ATCC 43888, 905, WSU180, or After thorough mixing, samples were incubated without aeration at 25 C (A and B) or 37 C (C and D) for 24 h. The CFU of E. coli O157:H7 per gram of bedding was determined by plating serial dilutions on selective medium (SMAC-CTVM) and was confirmed by latex agglutination tests. Experiments were done in triplicate, and representative results are shown. Strain TABLE 5. Growth of E. coli O157 in bovine urine and urea solutions 100% 10% urine b urine 37 C a 25 C a 2% 0.2% urea c urea 100% urine 10% urine 2% urea 0.2% urea d e f WSU a Cultures were incubated without aeration at 37 C or 25 C for 24 h. b Fresh, filter-sterilized bovine urine. c Urea dilutions made with 50 mm phosphate-buffered saline. d, no growth or decline in CFU per milliliter. e, growth of 1 log CFU/ml. f, growth between 1 and 2 log CFU/ml. provide insight into the epidemiology of this organism. The main available carbon and nitrogen source in bovine urine from cattle fed grass hay would be urea. Urea can be hydrolyzed to ammonia and carbon dioxide by urease, a nickelcontaining metalloenzyme. E. coli O157:H7 contains the ure operon encoding urease, although urease activity, as our results showed, is usually undetectable on conventional media. Recently, Nakano et al. (21) reported that strains do not express urease activity, due to an amber stop codon (UAG) that prematurely terminates the UreD protein at 245 amino acids rather than at the full-length 274 amino acids. It is possible that E. coli O157:H7 expresses urease activity in urine-soaked bedding if readthrough of the amber codon is occurring. Likewise, its growth in 0.2% urea suggests expression of urease activity. Alternatively, E. coli O157:H7 may be using something other than urea in urine or urine-soaked bedding as a substrate. For example, many uropathogenic E. coli strains are able to metabolize D-serine in urine (25); its presence and capability in on November 19, 2018 by guest

7 6822 DAVIS ET AL. APPL. ENVIRON. MICROBIOL. E. coli O157:H7 could be tested in future work. Also, E. coli O157:H7 growth at some temperatures was enhanced only with dilute urine and not with nondilute urine which, although not tested in this study, may implicate rain as a factor that promotes E. coli O157:H7 replication in the environment. In conclusion, water, feed, and bedding from the environments of animals shedding E. coli O157:H7 can be a source of exposure to persons attending or participating in animal exhibits that involve ruminants, most commonly, state and county agricultural fairs. This is true even when animals are not shedding E. coli O157:H7 at detectable levels in their feces. The presence of urine-soaked bedding in a ruminant stall or dilution of that urine with rainwater may provide a growth medium for E. coli O157:H7. Because eliminating urine-soaked bedding from the environment is not feasible, future research into the role of bedding, as well as other components of the ruminant s environment in disease transmission, should include an examination of the affects of the presence of urine. ACKNOWLEDGMENTS This work was supported, in part, by the Idaho Agriculture Experiment Station; the National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service, grant numbers and ; Public Health Service grants NO1-HD , U54-AI-57141, P20-RR16454, and P20-RR15587 from the National Institutes of Health; and grants from the United Dairymen of Idaho and the Idaho Beef Council. We thank Hannah Knecht, Carl Hunt, Lonie Austin, and Paula Austin for technical expertise and assistance with handling the cattle. REFERENCES 1. Besser, T. E., D. D. Hancock, L. C. Pritchett, E. M. McRae, D. H. Rice, and P. I. 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