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University of Idaho, Department of Microbiology, Molecular Biology, and Biochemistry, Moscow, Idaho 83844-3052
Washington State University, Department of Veterinary Medicine, Pullman, Washington
Veterinary Services Inc., Lacombe, Alberta, Canada
ABSTRACT
Among bovine fecal and recto-anal mucosal swab samples cultured in our laboratory for Escherichia coli O157:H7, we frequently isolated E. coli organisms that were phenotypically similar to the O157:H7 serotype as non-sorbitol fermenting and negative for -glucuronidase activity but serotyped O nontypeable:H25 (ONT:H25). This study determined the prevalence and virulence properties of the E. coli ONT:H25 isolates. Among dairy and feedlot cattle (n = 170) sampled in Washington, Idaho, and Alberta, Canada, the percentage of animals culture positive for E. coli ONT:H25 ranged from 7.5% to 22.5%, compared to the prevalence of E. coli O157:H7 that ranged from 0% to 15%. A longitudinal 8-month study of dairy heifers (n = 40) showed that 0 to 15% of the heifers were culture positive for E. coli O157:H7, while 15 to 22.5% of the animals were culture positive for E. coli ONT:H25. As determined by a multiplex PCR, the E. coli ONT:H25 isolates carried a combination of virulence genes characteristic of the enterohemorrhagic E. coli, including intimin, translocated intimin receptor, Stx2, and hemolysin (eae-, tir, stx2vh-a, and hly). E. coli ONT:H25 isolates from diverse geographic locations and over time were fingerprinted by separating XbaI-restricted chromosomal DNA by pulsed-field gel electrophoresis (PFGE) separation. Two strains of E. coli ONT:H25 were highly similar by PFGE pattern. Experimental inoculation of cattle showed that E. coli ONT:H25, like E. coli O157:H7, colonized the bovine recto-anal junction mucosa for more than 4 weeks following a single rectal application of bacteria.
INTRODUCTION
Shiga toxin (Stx)-producing Escherichia coli (STEC) organisms that cause hemorrhagic colitis and the potentially fatal hemolytic uremic syndrome (HUS) are classed as enterohemorrhagic E. coli (EHEC) (16, 28, 45). Although the majority of HUS cases in humans have been associated with the EHEC serotype O157:H7 (27, 32), non-O157 serotypes also cause disease and are more common than E. coli O157:H7 in some geographical areas (18, 22, 36, 46). There are over 200 different serotypes of STEC (32), and the number associated with human illness exceeds 100 (6). Domestic ruminants are important reservoirs of STEC and are the most common source for food-borne and animal-contact infections (13). Investigations throughout the world reveal that 10 to 80% of cattle are infected with STEC (5, 6, 49; http://www.who.int/emc-documents/zoonoses/whocsraph988c.html). Non-O157 STEC strains are more prevalent in animals and as contaminants in foods and water than E. coli O157 and, therefore, presumably humans are more frequently exposed to them (9, 10, 27). In the United States, it is estimated that 20% to 50% of STEC human infections are caused by non-O157:H7 serotypes (1, 2).
The recto-anal junction (RAJ) mucosa has recently been identified as a site of E. coli O157:H7 colonization in the bovine gastrointestinal tract (33). This finding was supported by earlier observations in mature cattle that E. coli O157:H7 persists in the lower gastrointestinal tract and feces but is not detected in the upper gastrointestinal tract after a few days post-oral inoculation (12, 23) and by the detection of the bacterium only in the distal rectal tissue in experimentally inoculated sheep (23). Our laboratory recently demonstrated that recto-anal mucosal swab (RAMS) culture is a more sensitive detection method for E. coli O157:H7 in cattle than traditional fecal culture (40), and a method to reliably colonize animals is application of the bacteria to the recto-anal junction mucosa (44).
Routine identification of E. coli O157:H7 from cattle samples includes culture on sorbitol MacConkey (SMAC) agar containing cefixime, potassium tellurite, vancomycin, and 4-methylumbelliferyl-beta-D-glucuronic acid dihydrate (MUG),a molecule that fluoresces when cleaved and therefore is used to detect -glucuronidase activity. Differentiation of the O157:H7 serotype on this medium is easy because colonies not fermenting sorbitol (colorless) and not hydrolyzing MUG (no fluorescence at 363 nm) are presumptive E. coli O157:H7. The serotype is confirmed by a commercially available latex agglutination assay. While culturing bovine feces and RAMS samples for E. coli O157:H7, we frequently isolated an E. coli phenotypically similar to E. coli O157:H7 in that it was non-sorbitol fermenting and did not exhibit -glucuronidase activity and it contained the gene for Stx type 2 (stx2) but serotyped as O untypeable and H25 (ONT:H25). Because STEC ONT:H25 isolation from both human patients and cattle had been previously reported in Europe (7, 9, 50, 51), we further characterized the E. coli ONT:H25 isolates from representative dairy, feedlot, and research settings in our region. In this study we analyzed E. coli ONT:H25 for (i) prevalence among dairy and feedlot cattle, (ii) virulence factors and DNA fingerprint, and (iii) the ability to colonize the bovine recto-anal junction mucosa.
MATERIALS AND METHODS
Animals. All animals in this study were apparently healthy and had fully developed ruminant gastrointestinal tracts. Animals from three distinct settings provided representative populations of each group.
Dairy heifers. Forty Holstein heifers (2 months to 4 months old) in eastern Washington were sampled once per month, except for the month of September. Forty Holstein heifers (6 months to 14 months old) in southern Washington were sampled once.
Steers. Twenty-three Holstein steers at a University of Idaho research barn in northern Idaho were sampled twice, and 27 Holstein steers on a farm in central Washington were sampled once. The steers were 4 to 8 months old.
Feedlot cattle. A total of 40 cattle from four different feedlots in Alberta, Canada, were sampled; 30 animals were sampled twice, 1 month apart, and 10 animals were sampled once.
Research animals. Eight 7-month-old Holstein steers were used in the inoculation study. Animals were housed in quarantined facilities in which feed, water, bedding, manure, and other waste were not in contact with any nonexperimental animals. All personnel followed strict biosafety procedures, and all procedures were approved by the Institutional Animal Care and Use and Biosafety Committees. Steers were fed alfalfa hay twice and pellet grain feeds once daily, with free access to drinking water.
RAMS and fecal sample collection. The RAMS samples were collected from dairy heifers and Holstein steers as previously described (44). Briefly, a sterile foam-tipped applicator (catalog number 10812-022; VWR International, Buffalo Grove, IL) was inserted into the anus approximately 2 to 5 cm and the entire mucosal circumference of the recto-anal junction was swabbed using a rapid in-and-out motion. Each RAMS was placed into a culture tube containing 3 ml of Trypticase soy broth (TSB; Difco Laboratories, Detroit, Mich.). All RAMS samples in TSB tubes were kept on ice until they were processed in the laboratory within 6 h of collection. RAMS samples from the feedlot cattle in Alberta, Canada, could not be obtained. Instead, feces (0.2 to 0.5 g) from each animal were put into tubes containing 3 ml TSB and shipped on ice to the laboratory and processed within 24 h.
Bacterial culture. RAMS samples and fecal samples were processed by direct and enrichment (if direct culture was negative) culture methods as previously described (40). SMAC agar (Difco Laboratories) containing cefixime (50 ng/ml; Wyeth-Ayerst, Pearl River, N.Y.), potassium tellurite (2.5 mg/liter; Sigma Chemical Co., St. Louis, MO), vancomycin (40 mg/liter; Sigma), and MUG (100 μg/ml; Biosynth Ag, Staad, Switzerland) (SMAC-CTVM) was used for isolation of E. coli O157:H7 and ONT-H25 strains from RAMS and fecal samples. Sorbitol-nonfermenting and MUG-negative (SMN) colonies were assayed for the O157 antigen by a latex agglutination test (Pro-Lab Diagnostics, Toronto, Canada). Bacterial counts were estimated from plates inoculated with serial dilutions of samples by multiplying the colony counts by the appropriate dilution factor. Non-O157 SMN colonies were stabbed into Luria-Bertani agar until further analysis. All or five isolates (if 5 colonies were found on the plate) from each animal positive by direct culture and two isolates from each animal positive by enrichment culture were preserved at each sampling time.
Api test. The Api 20E identification system (BioMerieux, France) was used according to the manufacturer's instructions for bacterial genus species designation based on biochemical reactions.
Serotyping. Non-O157 SMN isolates were serotyped by the Gastroenteric Disease Center (Pennsylvania State University, State College) by standard technique. A total of 180 O-antigen-specific and 53 H-antigen-specific antisera were used for O and H typing, respectively. The O157 antigen was detected using a latex slide agglutination test (Pro Lab Diagnostics, Canada).
Chromosomal DNA fingerprinting. To analyze the genetic relationship between isolates of E. coli ONT:H25 and E. coli O157:H7, pulsed-field gel electrophoresis (PFGE) separation of digested chromosomal DNA was performed as previously described (15). DNA was restricted with XbaI, and fragments were resolved in 1% agarose gels with 0.5x Tris-borate-EDTA electrophoresis buffer using a contour-clamped homogeneous electric field DR-III electrophoresis chamber (Bio-Rad, Richmond, Calif.). Pulse times were ramped from 2.2 to 54.2 seconds over a run time of 20 h at 6 V/cm.
Detection of virulence genes. A multiplex PCR was designed to detect stx1, stx2, eaeA, tir, EHEC hlyA, and fliCh7 gene sequences and performed with a GeneAmp PCR System 2400 (Applied Biosystems/Perkin-Elmer, Norwalk, Conn.). Oligonucleotide primers were commercially manufactured (Invitrogen, Carlsbad, CA). Primers and the predicted amplicon lengths are listed in Table 1. A single colony was transferred into a 50-μl reaction mixture containing 10x PCR buffer (5 μl), deoxynucleoside triphosphates (0.2 mM), MgCl2 (2.0 mM), primer sets (0.2 mM of each), and Taq polymerase (0.5 U; Invitrogen). Initial denaturation was done at 95°C for 3 min, followed by 35 cycles composed of incubation at 94°C for 1 min, 58°C for 45 s, and 72°C for 90 s. Amplified DNA fragments were resolved by gel electrophoresis in 1.5% (wt/vol) agarose gels. DNA was stained with ethidium bromide, visualized with UV illumination, and imaged with an AlphaDigiDoc gel documentation and analysis system (Alpha Innotech Corporation, San Leandro, CA). E. coli O157:H7 (ATCC 43894) and E. coli K-12 (MG1655) were used as positive and negative controls, respectively.
Shiga toxin expression. To compare production of Stx by E. coli isolates, cells were grown in Luria-Bertani broth and an enzyme immunoassay (Premier EHEC; Meridian Diagnostics Inc., Cincinnati, Ohio) was performed according to the manufacturer's instructions. Results were analyzed spectrophotometrically at two wavelengths, 450 and 630 nm, in a PowerWave XS reader (Bio-Tek, Winooski, Vermont). E. coli K-12 (MG1655) and E. coli O157:H7 (ATCC 43894) were used as negative and positive controls, respectively.
Enterohemolysin expression. Hemolysin expressed by the E. coli isolates was detected on tryptose agar plates (Difco Laboratories, Detroit, Mich.) supplemented with 10 mM CaCl2 and 5% defibrinated sheep blood cells, as described previously (8). The inoculated plates were observed for hemolysis after 4 h of incubation (for detection of alpha-hemolysis) and after overnight incubation in ambient air at 37°C (for detection of hemolysis or nonhemolysis). E. coli K-12 (MG1655) and O157:H7 (ATCC 43894) were used as negative and positive controls, respectively.
Intimin subtyping. To subtype the eae genes, primers and conditions for amplification of 13 eae variants (, , 1, /, , , , 2/, , , μ, , and ) were used according to those described by Blanco et al. (9). Amplified DNAs were separated by electrophoresis through 2% agarose gels and visualized by UV fluorescence after staining with ethidium bromide. A DNA size standard 100-bp ladder (Invitrogen) was used in each gel.
stx2 subtyping. To identify the stx2 types of the E. coli O157:H7 and ONT:H25 isolates, restriction fragment length polymorphism-PCR (RFLP-PCR) was used, as previously reported (3, 4, 11, 39, 48). The primers, restriction endonucleases, and expected sizes of DNA products are listed in Table 1. PCRs were performed with a GeneAmp PCR System 2400 (Applied Biosystems/Perkin-Elmer). Initial denaturation at 95°C for 3 min was followed by 35 cycles of incubation at 94°C for 2 min, 55°C for 2 min, and 72°C for 1 min. All PCRs were performed on isolated single colonies. PCR products were digested with the corresponding restriction enzymes: HincII, HaeII, PvuII, and RsaI (Invitrogen). DNA fragments were electrophoresed through 2% agarose gels and visualized as described above. E. coli O157:H7 (ATCC 43894) and E. coli K-12 (MG1655) were used as positive and negative controls, respectively.
Experimental inoculation. Two groups containing four animals each were penned separately and inoculated with E. coli O157:H7 ATCC 43894 or E. coli ONT:H25 strain SH2 by using a rectal application method, as previously described (44). Briefly, feces were manually removed from the terminal rectum. A 10-cm by 3.5-cm-diameter cylindrical sponge (Rubbermaid, St. Francis, WI) with a wooden handle was saturated with 10 ml of the E. coli O157:H7 or E. coli ONT:H25 overnight culture (1.9 x 109 CFU/ml or 1.7 x 109 CFU/ml, respectively) and then inserted into the anus and gently and rapidly rubbed against the recto-anal junction mucosa. After swabbing, defecation was prevented for 10 min by holding the steer tail firmly against the anus. This presumably ensured that the inocula were retained in the rectal lumen for a sufficient period of time for bacteria to attach to the mucosa.
Data analysis. To test the difference between the prevalence of E. coli ONT:H25 and that of E. coli O157:H7 in dairy heifers, the McNemar's chi-square P values for each sampling month were calculated using GraphPad Quickcalcs (http://www.graphpad.com/quickcalcs/McNemar1.cfm). The error bars presented below in Fig. 2 were also calculated using GraphPad based on the exact 95% confidence intervals around a binomial proportion. Bacterial counts were log transformed, and averages were taken of the log-transformed data for purposes of comparison between the two groups in the inoculation study. Error bars in Fig. 4, below, were calculated based on the standard errors of the means.
RESULTS
This study was spurred by our observation that in both bovine fecal and RAMS samples cultured for E. coli O157:H7, we frequently found non-O157 SMN colonies on SMAC-CTVM agar. Although very similar in morphology to E. coli O157:H7 colonies, the non-O157 SMN colonies were visually distinguishable on the SMAC-CTVM plates because of a lighter center and an overall slightly lighter color. From a collection of 433 RAMS and 70 fecal samples from healthy cattle in four locations of Washington and Idaho and from four feedlots in Alberta, Canada, we preserved 258 representative non-O157 SMN isolates. Also, among the bovine E. coli O157:H7 isolates recovered from these same animals during this study, two were designated as E. coli O157:H7 W1 and W2 and used for comparisons with the ONT:H25 isolates.
The non-O157 SMN isolates serotyped as ONT-H25 and were STEC. All non-O157 SMN isolates had the same API profile (0044112), indicating they were E. coli. Serotyping by the Gastroenteric Disease Center at the University of Pennsylvania and PFGE profiles of chromosomal DNA indicated that all SMN E. coli isolates belonged to a single serotype: ONT:H25. All isolates were designated as having no reaction with any O antisera. However, a weak and unusual reaction was noticed with O15 antisera, unlike the standard O15 control strain. Following heat treatment, strains showed a weak agglutination after 6 to 8 h that became flaky after 24 h. This unusual reaction was deemed nontypeable. All isolates were positive for stx2 by PCR and expressed Stx2 by enzyme-linked immunosorbent assay, indicating they were STEC (Table 1 and data not shown).
The E. coli ONT:H25 isolates had three different PFGE DNA fingerprints. DNA from 87 of the 258 E. coli ONT:H25 isolates, which represented different animals, sampling times, and locations, were digested and analyzed by PFGE. Among the 87 E. coli ONT:H25 isolates there were three distinct PFGE profiles that differed by two or more bands and were designated as SH1, SH2, and SH3 (Fig. 1). SH1 was isolated from eastern, central, and southern Washington. SH2 was isolated from Idaho and Alberta, Canada. SH3 was isolated from both southern Washington and Alberta, Canada, locations separated by more than 800 miles. Each PFGE type (SH1, -2, and -3) was represented in the subset of isolates that were serotyped (Table 2). Two natural bovine E. coli O157:H7 isolates (W1 and W2) from this study and E. coli O157:H7 ATCC 43894 had PFGE profiles distinct from each other and from E. coli ONT:H25 (Fig. 1).
E. coli ONT:H25 was prevalent among cattle. Among 170 cattle from the northwestern United States and Alberta, Canada, the percentage of animals positive for STEC ONT:H25 ranged from 7.5% to 22.5% (Table 2). In a longitudinal study the percentage of heifers positive for E. coli O157:H7 ranged from 0 to 15% (n = 40), whereas the percentage of animals positive for E. coli ONT:H25 ranged from 15 to 22.5% during the 8-month sampling (Fig. 2). E. coli ONT:H25 prevalence was significantly higher than E. coli O157:H7 prevalence in the months of October (E. coli ONT:H25 prevalence was 22.5% while E. coli O157:H7 prevalence was 5%; McNemar's chi-square P value = 0.05) and March (E. coli ONT:H25 prevalence was 20%, while E. coli O157:H7 prevalence was 0; McNemar's chi-square P value = 0.01). Most of the culture-positive heifers carried E. coli ONT:H25 transiently during the 8-month period, but three animals were consistently culture positive for the bacteria. In only one dairy heifer at one sampling time were both E. coli O157:H7 and E. coli ONT:H25 simultaneously isolated. Among the 40 feedlot animals, three (7.5%) were positive for E. coli ONT:H25 (Table 2) and four (10%) were positive for E. coli O157:H7 (data not shown).
E. coli ONT:H25 carried eae-, stx2, tir, and EHEC hlyA genes. Multiplex PCR results from testing 258 individual SMN isolates (representing SH1, SH2, and SH3) and two E. coli O157:H7 strains showed that STEC in this study (E. coli O157:H7 W1 and W2 and E. coli ONT:H25 strains SH1, SH2, and SH3) carried eae, stx2, tir, and EHEC hlyA gene sequences (Table 1). All three E. coli ONT:H25 strains carried only stx2 and not stx1, whereas the E. coli O157:H7 W1 and W2 isolates possessed both stx1 and stx2.
Among the five STEC strains characterized, eae gene subtyping results showed that E. coli O157:H7 W1 and W2 isolates carried eae-1, which were detected by the primer pair 5'-AAAACCGCGGAGATGACTTC-3' and 5'-AGAACGCTGCTCACTAGATGTC-3'. The size of that PCR product matched the expected 804 bp (data not shown). Subtyping of eae variants from all three E. coli ONT:H25 isolates showed that they possessed eae-, which was detected by the primer pair 5'-AAAACCGCGGAGATGACTTC-3' and 5'-CTTGATACACTTGATGACTGT-3'. The sizes of the PCR-amplified products matched the expected 830 bp (data not shown).
E. coli ONT:H25 isolates expressed enterohemolysin and Stx2vh-a. All E. coli ONT:H25 strains displayed a typical EHEC-enterohemolytic phenotype with small, clear hemolytic zones along the sides of bacterial growth on blood agar plates after overnight incubation at 37°C and no visible hemolysis after 46 h of incubation at 37°C (8).
To test Stx production, the E. coli ONT:H25 strains SH1, SH2, and SH3 and the E. coli O157:H7 bovine strains W1 and W2 were compared by enzyme-linked immunosorbent assay to positive control strains: E. coli O157:H7 strain 905, a clinical isolate from a patient with HUS (a gift from M. K. Waldor, Tufts-New England Medical Center and Howard Hughes Medical Institute, Boston, MA) which possesses only stx2 (41), and E. coli O157:H7 ATCC 43894, which possesses both stx1 and stx2. Toxin production by all ONT:H25 isolates and the bovine O157:H7 isolates was similar to that of the positive control strains (Fig. 3). All three strains of E. coli ONT:H25 (SH1, SH2, and SH3) produced Stx at levels similar to the clinical E. coli O157:H7 isolates 905 and ATCC 43894. The toxin-negative control E. coli K-12 did not produce toxin. On the basis of RFLP-PCR analysis, all five STEC strains, three E. coli ONT:H25 strains and two E. coli O157:H7 strains found in this study, carried the Stx2vh-a subtype (Table 3).
STEC ONT:H25 colonized the RAJ mucosa in a manner similar to E. coli O157:H7. Because STEC ONT:H25 strains were found in RAMS and fecal samples and carried a set of virulence genes similar to the O157:H7 serotype, we speculated that STEC ONT:H25 might colonize the same niche as E. coli O157:H7 in the bovine gastrointestinal tract. To test whether E. coli ONT:H25 was able to colonize the RAJ mucosa, one of the primary sites of colonization for E. coli O157:H7 (33), two groups of four steers were inoculated with a rectal application of the ONT:H25 or the O157:H7 E. coli serotypes. All animals remained healthy and had normal weight gain throughout the study. Figure 4, shows the average of the log10 bacteria counts detected by RAMS culture in each group. Both E. coli O157:H7 and E. coli ONT:H25 continued to be detected throughout the 35-day experimental period, though the numbers of bacteria declined from 103 to 104 CFU/swab at day 4 to 101 CFU at day 35. The pattern of E. coli O157:H7 detected by RAMS was consistent with our previous study of steers following rectal application of E. coli O157:H7 (44), and E. coli ONT:H25 showed the same pattern of colonization. There was no significant difference in the average log10 bacteria counts/RAMS between the two groups (Fig. 4). These results indicated that E. coli ONT:H25 can colonize at the RAJ as well as E. coli O157:H7.
DISCUSSION
This is the first detailed report of a non-O157 bovine STEC, E. coli ONT:H25, which is characterized by genotypic and phenotypic traits usually associated with disease-causing EHEC. Like E. coli O157:H7, E. coli ONT:H25 does not ferment sorbitol and lacks -glucuronidase activity. These characteristics allowed easy screening for both of these E. coli serotypes and were the initiating factor in our observations of E. coli ONT:H25. In addition, culture from RAMS samples and the persistence of E. coli ONT:H25 in cattle for more than 35 days after a single rectal application of the bacteria identify it as an E. coli serotype (in addition to the O157:H7 serotype) that can attach to the bovine RAJ mucosa.
An unexpected and striking finding in this study was the high prevalence of E. coli ONT:H25 in cattle from several locations in the Pacific Northwest. Our findings suggest that E. coli ONT:H25 may be more prevalent among cattle than E. coli O157:H7. We compared the prevalence of ONT:H25 to O157:H7 in dairy heifers sampled longitudinally over 8 months, using the same sampling method, and consistently found E. coli ONT:H25 more frequently over time in both RAMS samples and fecal samples. The lower prevalence (7.5%) found among feedlot animals may have been due to the fecal rather than RAMS sample, the 24-h delay in culture due to shipping distance, and the smaller number of animals tested only twice. Studies of total STEC in cattle populations have reported variable prevalence, although sampling and detection methods have been different and individual serotypes are not always reported (14). The ease with which we were able to visually distinguish E. coli ONT:H25 from other E. coli serotypes made the study of a STEC serotype other than O157 possible without elaborate culture and detection techniques. Development of new techniques and more careful study of other STEC may find a serotype(s) even more prevalent in the bovine RAJ mucosa microbiota.
In this study we identified three E. coli ONT:H25 strains with different chromosomal DNA patterns following restriction and PFGE separation. Although the three strains differed by at least two bands, very slight differences were observed between strains SH1 and SH3. These high similarities between banding patterns within a serotype may be interpreted as indicating a close genetic relatedness. Further, these PFGE data suggest a broad geographic distribution of the SH2 and SH3 strains over >800 miles among both dairy and feedlot animals.
In addition to its relatively high prevalence, E. coli ONT:H25 shares many genotypic and phenotypic traits with E. coli O157:H7. It is a slow sorbitol fermenter and lacks -glucuronidase activity. Some investigators have reported an association between the sorbitol-negative phenotype and human disease (34). It has genes for traits that are considered important for the disease-causing ability of E. coli O157:H7, including Shiga toxin 2, the eae gene encoding intimin, the tir gene encoding the translocated intimin receptor, and the hly gene encoding hemolysin. Among the four well-characterized STEC virulence factors, Stx1, Stx2, intimin, and hemolysin, Stx2 is the most frequently associated with HUS (10, 24, 35) Eleven distinct variants of Stx2 have been identified (4, 26, 37-39, 42, 43), the most frequently reported being Stx2c (subdivided into Stx2vha or Stx2vhb), Stx2d, and Stx2e (39, 42). The E. coli ONT:H25 strains tested in this study carried the Stx2vha type. This toxin has also been called Shiga-like toxin (SLT) II-vha (31, 47), verotoxin (VT) 2vha (26), or stx2d1 (17) and is the mucus-activatable toxin found in strain B2F1 (31, 47). Readers are referred to Schultz et al. for a complete compilation of Stx nomenclature (41). Finally, although the small numbers of animals limited our ability to detect a difference, the results of the animal inoculation study indicate that E. coli ONT:H25 shares the ability to colonize the bovine RAJ mucosa with E. coli O157:H7 and that temporal patterns of shedding by animals rectally inoculated with E. coli ONT:H25 closely parallel those with E. coli O157:H7.
Although E. coli ONT:H25 carries all of the known genetic factors associated with disease-causing EHEC, and although it appears to be a common finding among cattle in our region, there is little evidence of an association with human disease. We have not ruled out the possibility that the presence of this strain has been missed from clinical stool specimens associated with bloody diarrhea or HUS. This is plausible, because clinical and hospital laboratories often do not screen for STEC other than E. coli O157:H7. However, a strong association with hemorrhagic colitis and HUS or involvement with outbreaks of human disease is unlikely to have gone unnoticed. One recent prospective study of post-diarrhea HUS in the United States found that STEC was isolated from 30 of 70 specimens and that of these 30, 25 were O157:H7. None of the non-O157 STEC was ONT:H25 (2). This raises the possibility that E. coli ONT:H25 may be a strain lacking in specific genes required to cause disease in humans. E. coli ONT:H25 may be an important tool in studies using close genetic comparisons to identify unique elements of the E. coli O157:H7 genome that are critical for causing human disease.
ACKNOWLEDGMENTS
We thank Lonie Austin and Dan Rice for assistance with sampling and handling the cattle.
This work was supported, in part, by the Idaho Agriculture Experiment Station, U.S. Department of Agriculture NRICGP grants 99-35201-8539 and 04-04562, Public Health Service grants NO1-HD-0-3309, U54-AI-57141, P20-RR16454, and P20-RR15587 from the National Institutes of Health, and by grants from the United Dairymen of Idaho and the Idaho Beef Council.
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