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Rectoanal Mucosal Swab Culture Is More Sensitive Than Fecal Culture and Distinguishes Escherichia coli O157:H7-Colonized Cattle and Those Transiently Shedding the Same Organism

Abstract

Enrichment and direct (nonenrichment) rectoanal mucosal swab (RAMS) culture techniques were developed and compared to traditional fecal culture for the detection of Escherichia coli O157:H7 in experimentally infected and naturally infected cattle. Holstein steers (n = 16) orally dosed with E. coli O157:H7 were sampled after bacterial colonization starting 15 days postinoculation. Enrichment RAMS cultures (70.31% positive) were more sensitive than enrichment fecal cultures with 10 g of feces (46.88% positive) at detecting E. coli O157:H7 (P < 0.01). Holstein bull calves (n = 15) were experimentally exposed to E. coli O157:H7 by penning them with E. coli O157:H7-positive calves. Prior to bacterial colonization (1 to 14 days postexposure), enriched fecal cultures were more sensitive at detecting E. coli O157:H7 than enriched RAMS cultures (P < 0.01). However, after colonization (40 or more days postexposure), the opposite was true and RAMS culture was more sensitive than fecal culture (P < 0.05). Among naturally infected heifers, enriched RAMS or fecal cultures were equally sensitive (P = 0.5), but direct RAMS cultures were more sensitive than either direct or enriched fecal cultures at detecting E. coli O157:H7 (P < 0.01), with 25 of 144, 4 of 144, and 10 of 108 samples, respectively, being culture positive. For both experimentally and naturally infected cattle, RAMS culture predicted the duration of infection. Cattle transiently shedding E. coli O157:H7 for <1 week were positive by fecal culture only and not by RAMS culture, whereas colonized animals (which were culture positive for an average of 26 days) were positive early on by RAMS culture. RAMS culture more directly measured the relationship between cattle and E. coli O157:H7 infection than fecal culture.

Since Escherichia coli O157:H7 was first identified as a human pathogen (27, 33), investigations have demonstrated that human disease outbreaks are often linked to a bovine food source or bovine waste-contaminated water (2, 7, 30, 39). Although most of the known outbreaks of E. coli O157:H7-associated disease in humans are food borne or waterborne, several recent studies indicate that a significant number of human infections are acquired from direct contact with cattle, the environment, or unknown sources (1, 12, 30, 34). An accepted premise is that the reduction of the number of cattle infected with E. coli O157:H7 or the elimination of E. coli O157:H7 from cattle will effect a reduction in the rate of disease in humans. To this end, a great deal of research has focused on describing the ecology and epidemiology of E. coli O157:H7 in cattle, with the hope of identifying interventions to reduce its prevalence in cattle (11, 16, 17, 19, 20, 23, 24, 28, 29). Within this body of research, numerous methods for detecting E. coli O157:H7 in bovine fecal samples have been developed and used (8, 9, 22, 36, 37, 40). The reported sensitivities of detection by these various methods vary greatly, and problems occur when data from studies that have used methods with disparate sensitivities are compared. Nonetheless, fecal cultures show that E. coli O157:H7 occurs on the majority of cattle farms and that prolonged carriage by individual animals appears to be rare (4, 13, 16, 17, 28).

E. coli O157:H7 localization within the gastrointestinal tract and potential mechanisms of colonization in cattle have been the topics of several studies (4, 6, 13, 25, 32, 35). Numerous studies (4, 6, 13, 20, 28) have reported that animals are culture positive for both long durations (≥30 days) and short durations (≤10 days). Presumably, E. coli O157:H7 attaches and replicates at a site or sites in the gastrointestinal tracts of colonized animals and results in bacteria in the feces for a long duration. In contrast, ingestion of E. coli O157:H7 from an environmental source without establishment of a colonization site within the animal results in transient shedding of the bacteria in feces for a few days. Whether cattle on farms are typically colonized with E. coli O157:H7 or are only transiently shedding the bacteria is not known; however, several studies have documented the persistence of infection in individual cattle, and others have provided epidemiological evidence that at least some cattle are colonized (4, 6, 13, 16, 28).

A recent study provides compelling evidence that a primary site of E. coli O157:H7 colonization in cattle is the rectoanal junction of the gastrointestinal tract (32). This finding is supported by necropsy analyses of the gastrointestinal digesta and tissue of sheep colonized with E. coli O157:H7 (13). That study found the bacteria only in rectal tissue and feces (13). These findings are in contrast to those from previous studies that suggest that other sites in the gastrointestinal system are colonized, including the rumen and colon (6, 25, 35, 38). If the rectoanal junction is a site of colonization, the use of swab samples from this mucosal surface should be superior to the use of traditional fecal samples for the detection of E. coli O157:H7 and may have the potential to differentiate cattle that have been colonized over the long term from cattle that are transiently culture positive.

The present study used previously published methods of detecting E. coli O157:H7 but applied these to a novel type of sample, obtained by swabbing the rectoanal mucosal surface, rather than fecal material. Rectoanal mucosal swab (RAMS) samples and fecal samples were obtained from (i) experimentally infected steers, (ii) experimentally exposed calves, and (iii) naturally infected dairy heifers; and cultures with the two types of samples were compared for their abilities to detect E. coli O157:H7. Both selective enrichment and direct culture techniques were used, and the results were correlated to the durations that individual animals were culture positive for E. coli O157:H7.

MATERIALS AND METHODS

Animals.

Three groups of cattle were sampled in this study: artificially infected 8- to 10-month-old Holstein steers (n = 16), intentionally exposed 4- to 6-month-old Holstein bull calves (n = 15), and 4- to 14-month-old heifers (n = 40) located at the University of Idaho (UI) and the Washington State University (WSU) dairy farms. The artificially infected or exposed cattle were housed in large-animal isolation facilities and were demonstrated to be culture negative for E. coli O157:H7 at least three consecutive times over a 2-week period before being used in an experiment. The heifers at both dairies were not artificially inoculated or exposed to E. coli O157:H7. The UI Animal Care and Use Committee approved all experimental protocols with the animals.

E. coli O157:H7 experimental inoculation and experimental exposure.

The Holstein steers were orally inoculated one time with a four-strain mixture of E. coli O157:H7 (1010 CFU/strain) containing WSU isolates 180, 400, and 588 (14) and strain ATCC 43895 (American Type Culture Collection [ATCC], Manassas, Va.). This mixture is representative of human and cattle E. coli O157:H7 isolates. Two Holstein calves (referred to as Trojan calves) were orally inoculated with the same quantity and strains of E. coli O157:H7 as the steers, and the pen mates were exposed to E. coli O157:H7 by association with these dosed calves. In addition, a 200-liter tub of water was inoculated with 10 g of E. coli O157:H7-positive feces collected from the Trojan calves and was introduced into the pen 3 weeks after the Trojan calves were introduced. The water in this tub was in addition to the automatic water fountains that provided drinking water to the animals.

Sample collection.

A fecal sample and a RAMS sample were collected from each animal. Fecal samples were collected aseptically by rectal palpation or aseptically during defecation and placed into a Whirl-Pak bag (Nasco, Ft. Atkinson, Wis.). RAMS samples were obtained by inserting a sterile foam-tipped applicator (catalog no. 10812-022; VWR International, Buffalo Grove, Ill.) approximately 2 to 5 cm into the anus, and by using a rapid in-and-out motion, the entire mucosal surface of the rectoanal junction was swabbed. Each RAMS was placed into a culture tube containing 3 ml of Trypticase soy broth (TSB; Difco Laboratories, Detroit, Mich.) or TSB with cefixime (50 ng/ml; Wyeth-Ayerst, Pearl River, N.Y.), potassium tellurite (2.5 μg/ml; Sigma Chemical Co., St. Louis, Mo.), and vancomycin (40 μg/ml; Sigma Chemical Co.) (TSB-CTV). Both RAMS and fecal samples were kept on ice until they were processed in the laboratory within 2 h of collection.

Fecal and RAMS samples were obtained from the steers at 15, 22, 24, and 50 days postinoculation and then weekly for 78 days. The calves were sampled every 3 to 4 days between days 1 and 54 postexposure. Heifers from two university dairy farms were sampled, with 20 samples obtained from animals on each farm during each of two sampling visits during the late spring and early summer. An additional set of RAMS and fecal samples were collected weekly from 18 heifers at the UI dairy farm from late fall (the end of October) to early winter (mid-January).

Culture methods.

The samples were cultured by direct plating, which yielded quantitative culture data (number of CFU per gram), or were selectively enriched by incubation overnight prior to plating, which yielded qualitative (positive or negative) culture data.

(i) Direct (nonenriched) RAMS culture.

RAMS samples in TSB or TSB-CTV were vortexed for 1 min and spread plated (1, 10, and 100 μl) onto sorbitol MacConkey agar (SMac; Difco Laboratories, Detroit, Mich.) containing cefixime, potassium tellurite, vancomycin, and 4-methylumbelliferyl-beta-d-glucuronic acid dihydrayte (100 μg/ml; Biosynth Ag, Staad, Switzerland) (SMac-CTVM), and the plates were incubated at 37°C for 18 h. The numbers of sorbitol- and beta-glucuronidase-negative colonies on plates with a total of 30 to 300 colonies were counted, and a subset of the colonies was assayed for the O157 antigen by a latex agglutination test (Pro Lab Diagnostics, Richmond Hill, Ontario, Canada).

(ii) Enrichment RAMS culture.

RAMS samples were placed on a rotary shaker (150 rpm) and incubated at 37°C for 6 or 18 h. Samples were serially diluted, plated onto SMac-CTVM, and incubated at 37°C for 18 h. The E. coli O157:H7 colonies were then identified as described above for the direct RAMS culture.

(iii) Direct (nonenriched) fecal culture.

Ten grams of feces was suspended in 50 ml of TSB-CTV and spread plated (1, 10, and 100 μl) onto SMac-CTVM, the plates were incubated at 37°C for 18 h, and the E. coli O157:H7 colonies were identified as described above for the nonenriched RAMS culture.

(iv) Enrichment feces.

Ten grams of feces was cultured as described previously (23). Briefly, 10-g fecal samples were added to separate flasks containing 50 ml of TSB-CTV, the flasks were placed on a rotary shaker (150 rpm), and the samples were incubated at 37°C for 18 h. Sample enrichments were plated onto SMac-CTVM, and E. coli O157:H7 colonies were identified as described above for the enrichment RAMS culture.

Statistical analysis.

Fisher's exact test was used to determine significant differences between the culture methods used to detect E. coli O157:H7 in each group of cattle.

RESULTS

All animals remained healthy during the study period. At most samplings, the RAMS was collected first. A fecal sample of 10 g or more was then collected by aseptic rectal palpation or freely if the animal defecated. Very little visible fecal material was collected on the RAMS, and the amount of material on each RAMS ranged from 0.1 to 0.4 g, with an average of 0.24 g. Direct and enrichment RAMS cultures contained few non-O157 colonies, used low volumes of media, required minimal technician hands-on time, and could be completed within 1 day of sample collection. Enrichment fecal cultures required 48 h or more from the time of sample collection to the time that results were available. The enrichment fecal culture protocol used was as sensitive as selective enrichment with immunomagnetic bead capture of E. coli O157:H7, detecting 1 CFU/10 g (23).

Comparison of RAMS culture and fecal culture for detection of E. coli O157:H7 in experimentally colonized steers.

Steers were given a single oral dose of E. coli O157:H7, and during the first week postinoculation the highest E. coli O157:H7 titers in feces ranged from 2.1 × 104 to 9.5 × 106 CFU/g. During the second week postinoculation all steers remained fecal culture positive for E. coli O157:H7, but many samples contained less than 10 CFU/g and enrichment culture was required to detect the bacteria. Comparisons of RAMS and fecal cultures were started 2 weeks postinoculation to allow the bolus of inoculated bacteria to pass through the gastrointestinal tract and bacterial colonization to occur. Enrichment culture of RAMS samples (39 of 45; 70.31% positive) was more sensitive than enrichment culture of 10-g fecal samples (46.88% positive) (P < 0.01) at detecting E. coli O157:H7 2 or more weeks postinoculation (Table 1). The majority of these RAMS samples (39 of 45) were also positive by direct culture. The average duration that steers remained culture positive was 40 days (range, 15 days [one animal] to 71 days [three animals]).

TABLE 1.

Comparison of RAMS culture and fecal culture for E. coli O157 in three groups of cattle

Cattle sampled, age (mo) No. (%) of samples culture positive by:
Total Enrichment RAMS culture Enrichment fecal culture
Experimentally inoculateda steers, 8-10 (n = 16) 64 45 (70.31)d 30 (46.88)d
Experimentally exposedb calves, 4-6 (n = 15) 255 66 (25.88) 68 (26.67)
Naturally infectedc heifers, 3-14 80 25 (31.25) 26 (32.50)

Longitudinal comparison of RAMS and fecal culture with calves experimentally exposed to E. coli O157:H7.

To more closely simulate acquisition of a natural E. coli O157:H7 infection and avoid the large oral bolus of 1010 CFU of E. coli O157:H7 often used to generate experimental infections, Holstein bull calves were exposed to experimentally infected culture-positive calves by penning the animals together. The culture-positive calves, referred to as Trojan calves, carried the infection and spread it to the test animals. The exposed test calves were analyzed for E. coli O157:H7 by enrichment RAMS and fecal culture every 3 to 4 days for 54 days postexposure. The experimental exposure to the Trojan calves caused infections in almost all the test calves within the first 2 weeks postexposure, and both RAMS and fecal samples from 16 of 17 (94%) bull calves (including the Trojan calves) were culture positive at least once. When all culture results were analyzed together, the sensitivities of the enrichment cultures of RAMS and fecal samples appeared to be similar (Table 1). However, when the culture results were compared longitudinally over the course of the infections, the sensitivities differed. Early in the period of exposure (1 to 14 days) enrichment fecal cultures were more sensitive at detecting E. coli O157:H7 than enrichment RAMS culture (P < 0.01). Late in the period of exposure (40 days or longer), the opposite was true and enrichment RAMS cultures were more sensitive (P < 0.05) (Fig. 1). Presumably, early in the exposure period, the calves were ingesting E. coli O157:H7 from their environment and the bacteria were in the digesta and feces but may or may not have attached to the rectoanal junction mucosa (at this time, fecal cultures were the most sensitive); and late in the exposure period, the bacteria, if present, were colonizing the rectoanal junction (at this time, RAMS cultures were the most sensitive).

FIG. 1.

FIG. 1.

Detection by RAMS culture and fecal culture of E. coli O157:H7 from calves experimentally exposed to E. coli O157:H7 by penning them with culture-positive Trojan calves. Significant differences in sensitivities of detection exist early (P < 0.01) and late (P < 0.05) in the course of the infections.

Comparison of RAMS culture and fecal culture for detection of E. coli O157:H7 in naturally infected dairy heifers.

To show the sensitivity of RAMS culture for the detection of naturally infected cattle, heifers from two university dairies were sampled in the summer, a season that is associated with a higher prevalence of culture positivity among cattle. Unlike the results obtained with experimentally infected steers, enrichment RAMS culture and enrichment fecal cultures were equally sensitive (P = 0.50). Twenty-five of 80 RAMS samples and 26 of 80 fecal samples from heifers were culture positive (Table 1). To investigate natural infection in a season not usually associated with a high prevalence of culture positivity, 18 heifers from one university dairy were sampled weekly during the late fall and early winter. For these more frequently sampled animals, direct RAMS culture was compared to direct fecal and enrichment fecal cultures. Twenty-five of 144 RAMS samples tested by direct culture, 4 of 144 fecal samples tested by direct culture, and 10 of 108 fecal samples tested by enrichment culture were positive for E. coli O157:H7. Direct culture of RAMS samples was more sensitive at detecting E. coli O157:H7 than either enrichment or nonenrichment culture of fecal samples (P < 0.01) (Fig. 2). All positive samples with the exception of one sample tested by enrichment fecal culture were from the same five heifers. Samples obtained from these animals on a weekly basis were consistently positive for 34 to 41 days, and samples from two of the animals were positive at day 81. These data for naturally infected heifers sampled during the late fall are very striking. RAMS samples from 5 of 18 animals were repeatedly positive for 5 to 12 weeks, often in the absence of positive culture results for fecal samples, even when an enrichment culture technique was used. The number of E. coli O157:H7 CFU per gram in RAMS samples always exceeded that in fecal samples from these 18 heifers, ranging from 5.58 × 103 to 5.00 × 104 and 1.0 × 102 to 6.0 × 102, respectively. Although the number of E. coli O157:H7 organisms in RAMS and fecal samples consistently decreased at each subsequent sampling date, the number of E. coli O157:H7 organisms collected on the RAMS was significantly greater than that found in feces on all sampling dates (data not shown) (P < 0.01). These combined findings indicated that the rectoanal junction mucosae of these five cattle were colonized with E. coli O157:H7.

FIG. 2.

FIG. 2.

Prevalence of E. coli O157:H7 in 18 naturally infected dairy heifers determined by direct RAMS culture, direct fecal culture, or fecal culture with selective enrichment. Samples from the animals were cultured weekly for a 41-day period in the late fall, and the prevalence over that period of time was calculated. Direct RAMS culture was more sensitive than either enriched or nonenriched fecal culture (P < 0.01).

Association of RAMS and fecal culture status with the duration of E. coli O157:H7 infections in cattle.

The RAMS and fecal culture status of individual animals naturally infected or experimentally infected with E. coli O157:H7 were compared with the durations of infection. Animals that were positive for E. coli O157:H7 by RAMS culture remained positive for significantly longer periods (average, 26 days) and presumably represented colonized animals (Table 2). In contrast, the infections were rapidly cleared from animals that were positive for E. coli O157:H7 by fecal culture only and not by RAMS culture (all but one of the animals were positive on only one sampling day; Table 2), and presumably, these represented transiently shedding animals.

TABLE 2.

RAMS and fecal culture status and duration of E. coli O157:H7 infections in cattle

Type of infection and animal no. Status of first E. coli O157:H7-positive culture
Time (days) to first E. coli O157:H7-negative culture
RAMS Feces
Experimentala
    704 − + 3
    707 − + 3
    711 − + >28c
    712 − + 4
    717 − + 4
    718 − + 4
    725 − + 4
    703 + − 42
    709 + − 14
    710 + − 14
    712 + − 4
    718 + − >39c
    725 + − >18c
    728 + − 7
    705 + + 14
    707 + + >25c
    714 + + 18
    717 + + 39
    720 + + >43c
    721 + + 31
Naturalb
    2167 − + 2
    2160 + − 34
    2165 + − 34
    2175 + − 27
    2171 + + 49
    2172 + + 21

There were exceptions to the common pattern of RAMS culture-positive status and a long-term duration of E. coli O157:H7 infection. Animal 711 was consistently culture positive for 28 days and was then intermittently culture positive for 49 days through the last day of sampling. The animal was fecal culture positive on eight sampling occasions but was RAMS culture positive on only three sampling days. The highest E. coli O157:H7 titer shed by animal 711 was 1.0 × 103 CFU of E. coli O157:H7/g of feces. Also, animals 712 and 728 were RAMS culture positive but shed E. coli O157:H7 only briefly on only one or two sampling days, and then they cleared the infection (Table 2).

Variations to RAMS culture method.

The surprising finding that the direct RAMS culture was often more sensitive than either direct or enrichment fecal culture led us to more carefully analyze the RAMS culture protocol. Because there is so little fecal contamination on the RAMS samples, a subset of RAMS samples was collected in TSB without cefixime, potassium tellurite, and vancomycin and was subsequently enriched without cefixime, potassium tellurite, and vancomycin. The results obtained for four animals sampled on five different occasions over a 2-month period were compared. Direct and enriched RAMS cultures in TSB were as sensitive or more sensitive than RAMS cultures in TSB-CTV. Approximately 80% of the positive RAMS samples collected in TSB were also positive when the samples were cultured in TSB-CTV (data not shown). The colonies on enrichment RAMS plate cultures were ≥90% E. coli O157:H7, but enrichments in TSB had about 10-fold higher titers of E. coli O157:H7 than enrichments in TSB-CTV (data not shown). In addition, we compared enrichment incubation times of 6 and 18 h and found that 6 h was a sufficient incubation time and identified the same culture-positive samples identified by the 18-h incubation. Thus, collection and enrichment of RAMS samples in TSB without tellurite and antibiotics appeared to be more sensitive than collection and enrichment in TSB-CTV, and a 6-h incubation time could be substituted for the 18-h enrichment incubation time in the RAMS culture technique.

DISCUSSION

An important finding in this work was that culture of swabs of the rectoanal junction mucosa was, in almost all cases, as sensitive as and usually more sensitive than culture of feces at detecting E. coli O157:H7 in cattle. This is likely because the RAMS sampled the site of E. coli O157:H7 colonization, supporting the findings of Naylor et al. (32), and the RAMS samples were minimally contaminated with fecal material, which contains high titers of other bacteria. Also, the ability of RAMS culture to detect E. coli O157:H7 among naturally infected heifers whose fecal samples were not culture positive suggested that some animals harbor this pathogen much longer than previous estimates made on the basis of fecal culture data. These animals that are colonized for the long term are not shedding detectable levels of E. coli O157:H7 in their feces and may represent an on-farm reservoir not previously identified. Fecal cultures were more sensitive than RAMS cultures at detecting E. coli O157:H7 only initially following artificial exposure to E. coli O157:H7. This may indicate that upon exposure E. coli O157:H7 is present in digesta and feces and is only subsequently attached and colonizes the rectoanal junction mucosa, or it may indicate that bacterial division at that mucosal site took several days to create detectable numbers of the pathogens.

Recognition of which cattle have the greatest potential to negatively affect the safety of our food or contaminate the environment and successful intervention remain unrealized goals. One animal shedding 106 CFU of E. coli O157:H7/g of feces releases the same number of pathogens into the environment as 100,000 cattle shedding 10 CFU/g of feces. Therefore, techniques that only improve the sensitivity of E. coli O157:H7 detection may not be helpful in addressing the epidemiology of E. coli O157:H7. The technique with RAMS samples had several benefits, in addition to improved sensitivity. The technique required small volumes of media, minimal hands-on technician time, no expensive equipment, and a small number of reagents; and it produced easily interpreted plate cultures with few colonies other than E. coli O157:H7 within 1 day from sample collection. In addition, among both experimentally and naturally infected cattle, RAMS culture predicted the duration that the cattle remained culture positive for E. coli O157:H7. This is the first report of a technique with this ability. With few exceptions, animals that were RAMS culture positive remained positive for many weeks and were considered colonized. In contrast, cattle that were fecal culture positive only and RAM culture negative were most often culture positive on only one sampling day and were considered to be transiently shedding the pathogen. Among the cattle in this study we found only a few animals that did not follow this pattern. The finding of one experimentally infected animal that shed fecal E. coli O157:H7 over the long term without consistently being RAMS culture positive may indicate that a minority of animals may be colonized at some other gastrointestinal tract site or are particularly sensitive to ingesting E. coli O157:H7 from environmental sources and disseminating the bacteria in feces, or some combination of these attributes.

If E. coli O157:H7 primarily colonizes the mucosa of the rectoanal junction, RAMS samples should differentiate colonized from transiently positive cattle. Present fecal culture methods detect low levels of E. coli O157:H7, and thus, many animals deemed culture positive shed E. coli O157:H7 at low titers (<10 CFU/g of feces). Studies have demonstrated that most cattle are only transiently positive for E. coli O157:H7, and persistence in an animal appears to be rare (4, 6, 13, 16, 28). The existence of persistently colonized cattle on farms, even if rare, could be an important part of the maintenance of this organism on a farm and a source of contamination of otherwise culture-negative cattle at slaughter. The conventional idea that most of the E. coli O157:H7 contamination of beef carcasses at slaughter originates directly from the gastrointestinal contents of cattle being slaughtered is challenged by recent studies that show that the primary source of E. coli O157:H7 isolates that contaminate carcasses are the hides of cattle being slaughtered (3, 21). The existence of colonized cattle in pens of cattle awaiting slaughter could be important sources of contamination of the pen environment and, consequently, the hides of pen mates. It is possible that the majority of hide contamination results from a minority of cattle that are shedding E. coli O157:H7. Identification of colonized animals could allow elimination of carrier animals and promote pen environments with greatly reduced loads of this organism; consequently, this would reduce the levels of hide contamination.

Studies of fecal E. coli O157:H7 in cattle have led to a variety of conclusions regarding bacterial colonization of cattle. These range from uncertainty as to whether E. coli O157:H7 colonizes cattle to the identification of potential colonization sites, including the rectoanal junction and the rumen (10, 25, 32). Localization of E. coli O157:H7 on the rumen walls of mature cattle has been reported, and the occurrence of the organisms at this location appears to be associated with concurrent fecal shedding (25). E. coli O157:H7 persists in the rumens of 6- to 8-week-old calves that are not fully ruminant, but this persistence is not comparable to the situation for fully ruminant cattle going to slaughter (5, 10, 18, 35, 38). Other studies suggest that E. coli O157:H7 colonizes various sites in the intestinal tract, including the cecum and colon; however, those studies did not provide conclusive evidence that colonization occurs at these sites (6, 10, 38). A previous study (13) that evaluated the gastrointestinal location of E. coli O157:H7 in cattle and sheep rarely found E. coli O157:H7 in the rumen beyond 7 days postinoculation and often found the bacteria in fecal samples from long-term carriers. In addition, upon necropsy E. coli O157:H7 was found only in tissue from the distal rectum of sheep that were long-term carriers (colonized) (13). Recent evidence suggests that E. coli O157:H7 colonizes the rectoanal junction of cattle (32). The results reported here support the finding that the rectoanal mucosal junction is a site of E. coli O157:H7 colonization in cattle.

The association between season and RAMS culture sensitivity may be explained by changes in the environmental reservoirs of E. coli O157:H7. In samples from dairy heifers, enrichment RAMS and fecal cultures were equally sensitive at detecting E. coli O157:H7 during the summer months; however, RAMS cultures were much more sensitive than fecal cultures at detecting E. coli O157:H7 in the same group of cattle during the winter months. This observation may be due to increased E. coli O157:H7 titers in environmental reservoirs during the summer months that resulted in the ingestion of increased amounts of the organism and increased levels of transient shedding. The prevalence of E. coli O157:H7 in the feces of cattle and the environment has been demonstrated to be higher during the warm months of summer to fall (16, 26, 31). These E. coli O157:H7 reservoirs would likely be reduced or absent during the winter months. Environmental reservoirs likely exist in dairy heifer pens, although this has not been determined (15, 26, 29), and consequently, detection of E. coli O157:H7 only by enrichment fecal cultures (a method that detects as little as 1 CFU/g of feces [23]) and not by RAMS culture may reflect the passage of ingested E. coli O157:H7 rather than colonization.

In conclusion, the RAMS culture technique provides a new, easy method for the detection of E. coli O157:H7 in cattle and appears to delineate colonized from transiently shedding cattle. Large-scale epidemiological studies by the RAMS method are under way. These studies will potentially generate data on the prevalence of E. coli O157:H7 in cattle that differ from those from existing studies that used selectively enriched fecal cultures to detect the pathogen.

Acknowledgments

This work was supported, in part, by the Idaho Agriculture Experiment Station, U.S. Department of Agriculture NRICGP grant 99-35201-8539, and Public Health Service grants NO1-HD-0-3309 and P20RR15587 from the National Institutes of Health.

We thank D. D. Hancock for providing cefixime and L. Austin for assistance with handling of the cattle.

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