key: cord-265765-zpf1bla8
authors: Holland, Robert E; Wilson, Richard A; Holland, Margo S; Yuzbasiyan-Gurkan, Vilma; Mullaney, Thomas P; White, David G
title: Characterization of eae(+)Escherichia coli isolated from healthy and diarrheic calves
date: 1999-05-14
journal: Vet Microbiol
DOI: 10.1016/s0378-1135(99)00013-9
sha: 
doc_id: 265765
cord_uid: zpf1bla8

Strains of Escherichia coli from 101 healthy and 114 diarrheic calves were screened by PCR for the eae (intimin) gene and Shiga toxin genes (stx). Each eae(+) and eae/stx(+) strain was examined for antimicrobial susceptibility, enterohemolysin activity, and the somatic O antigen was determined. An immunoassay was used to detect Shiga toxin antigens for the eae/stx(+)E. coli. Significantly more (p = 0.005) of the healthy calves carried eae(+) and eae/stx(+)E. coli in their feces when compared to strains from diarrheic calves. Moreover, Shiga toxin antigens were detected significantly more (p = 0.001) often among the eae/stx(+) strains from healthy calves when compared to eae/stx(+) strains from diarrheic calves. However, significantly more (p = 0.001) of the eae(+) and eae/stx(+) strains from diarrheic calves were resistant to at least one of the antimicrobials tested, and the strains from diarrheic calves had a significantly (p = 0.05) higher rate of antimicrobial resistance to at least two different antimicrobial classes. No significant difference (p ≥ 0.05) was detected among the eae(+) and eae/stx(+) strains from healthy and diarrheic calves for enterohemolysin production. Serogroups O-negative, O5, O26, and O111 were predominate among both healthy and diarrheic calves.

Calf strains of Escherichia coli possessing the eae gene and having the capacity to induce the attaching and effacing lesion are designated as attaching and effacing E. coli (AEEC) (Moon et al., 1983) . Intimin, a 94 kDa outer membrane protein encoded by the eae gene, mediates adherence of AEEC to intestinal epithelial cells; an event critical for the formation of the attaching and effacing lesion (Jerse and Kaper, 1991; Donnenberg and Kaper, 1992) . Thus, the pathophysiologic mechanism by which AEEC cause the attaching and effacing lesion in the intestinal tract of calves may follow the three-stage model proposed for human strains of enteropathogenic E. coli (EPEC) (Donnenberg and Kaper, 1992; Donnenberg et al., 1993a, b) . The three-stage model for the attaching and effacing lesion consists of localized adherence, signal transduction, and intimate adherence. There is an initial nonintimate attachment of the bacteria to intestinal epithelial cells leading to effacement of the underlying microvilli, followed by intimate attachment, and then the accumulation of cytoskeletal proteins beneath the areas of bacterial attachment.

Based on expression of intimin, Shiga toxins, and the ability to elicit the attaching and effacing lesion, some bovine AEEC strains are equivalent to human enterohemorrhagic E. coli (EHEC) (Levine, 1987; Knutton, 1994) . Thus, two classes of bovine AEEC have been described (Mainil et al., 1993) . One class, in addition to carrying the eae gene, carry stx genes; and the second class do not carry stx genes. In calves, the eae gene and intimin have a defined role in causing the attaching and effacing lesion, whereas Shiga toxins do not appear to have a role in causing the attaching and effacing lesion (Tzipori et al., 1987; Hall et al., 1990) . Further, the importance of Shiga toxins in calf diarrhea and systemic disease is not well defined. In humans, Shiga toxins are an essential virulence attribute of EHEC associated with severe systemic disease such as hemolytic uremic syndrome (Karmali, 1989; Tesh and O'Brien, 1991) . In pigs and rabbits, Shiga toxins have a role in inducing vascular lesions and have a direct effect on the intestinal epithelium (Gyles, 1994; Nataro and Kaper, 1998) .

Numerous studies have documented the importance of AEEC in calf diarrhea (Chanter et al., 1984 (Chanter et al., , 1986 Sherwood et al., 1985; Hall et al., 1985 Hall et al., , 1990 Moxley and Francis, 1986; Mohammad et al., 1985 Mohammad et al., , 1986 Pospischil et al., 1987; Blanco et al., 1988 Blanco et al., , 1993 Schoonderwoerd et al., 1988; Wray et al., 1989; Janke et al., 1990; Synge and Hopkins, 1992; Dorn et al., 1993; Mainil et al., 1993; Knutton, 1994; Fisher et al., 1994; Wieler et al., 1996; Saridakis et al., 1997) . Few reports have examined the occurrence of AEEC infections concurrently in both healthy and diarrheic calves (Mohammad et al., 1985; Wieler et al., 1992; Blanco et al., 1993 Blanco et al., , 1994 . The objectives in this research were to determine the presence of certain virulence properties associated with eae E. coli isolated from healthy and diarrheic calves and to assess the role of AEEC in calf diarrheal disease.

Fecal specimens were obtained from 215 Holstein calves after digital stimulation of the rectal mucosa. At the time of fecal collection, 114 calves had diarrhea as determined by visual assessment of the specimen and the calf. On each farm, the diarrheic calves were matched to an approximate number of healthy calves. Thus, fecal specimens were collected from 101 calves that did not have diarrhea and appeared healthy. Depending on the number of diarrheic calves sampled, 10 to 18 calves (both diarrheic and healthy) were sampled on six farms, and 19 to 24 calves were sampled on six farms. The 12 dairy farms were in Michigan.

Feces were collected into sterile cups, packed on ice packs in chests, and were transported to the laboratory. Upon arrival to the laboratory, feces were immediately cultured. At the time of fecal collection, historical and clinical examination data were recorded on each calf.

For primary culture, 1.0 g of feces was homogenized in 9.0 ml of peptone-saline solution. An inoculating loop was used to transfer 10 ml of the fecal/peptone-saline suspension to MacConkey (MAC) agar plates, and the plates were streaked for isolation of lactose fermenting colonies (LFC). The MAC plates were incubated aerobically at 378C for 12 to 16 h. Ten LFC that exhibited E. coli growth characteristics were randomly harvested with sterile toothpicks from the last streaking area of each MAC plate. Total bacterial DNA was extracted and then prepared for PCR analysis. Each eae and eae/stx colony from each calf was verified as E. coli by the API 20E system (bioMerieux Vitek, Hazelwood, MO).

Ten individual LFC from each calf were suspended in individual 500 ml microcentrifuge tubes (Elkay Products, Shrewsbury, MA) containing 200 ml of lysis buffer (1.0% Triton X-100, 20 mM Tris±HCl, 2.0 mM EDTA, pH 8.5), mixed by vortexing for 30 s, heated at 1008C for 3 min, then centrifuged at 12,000 Â g for 3 min. Amplification of bacterial DNA was performed with 5 ml of bacterial DNA in 45 ml of PCR reaction mixture. The PCR reaction mixture contained 3 ml of double distilled water, 5 ml of 10 Â PCR buffer, 2 mM MgCl 2 , 10 mM dNTP mix, 0.8 mM of each primer, and 1.5 U of Taq DNA polymerase (Gibco BRL, Life Technologies, Inc., Grand Island, NY).

Prior to testing each LFC for the eae gene, three sets of published oligonucleotide sequences (Gannon et al., 1993; Donnenberg and Kaper, 1991; Karch et al., 1993) were evaluated for detection of eae sequences among well characterized strains of E. coli that have been determined to possess various eae allelic sequences or lacked the eae gene (Table 1 ). These strains were of multiple serotypes and were isolated from human and animal sources. Based on this study, the AE 13±AE 14 oligonucleotide sequences {(5 H GTG GCG AAT ACT GGC GAG ACT 3 H ) and (5 H CCC CAT TCT TTT TCA CCG TCG 3 H )} were chosen to amplify the eae gene (Gannon et al., 1993) . The size of the amplified DNA product was 890 bp. The amplification method was 948C for 1 min for denaturing, 578C for 1 min for annealing, and 728C for 1 min for extension for 30 cycles.

For amplification of stx genes, the MK1±MK2 oligonucleotide primers {(5 H TTT ACG ATA GAC TTC TCG AC 3 H ) and (5 H CAC ATA TAA ATT ATT TCG CTC 3 H )} were used (Karch and Meyer, 1989; Schmidt et al., 1993) . The MK1±MK2 primer pair was demonstrated to amplify a 230 bp fragment for both stx1 and stx2 loci (Karch and Meyer, 1989) . The thermocycling program was 948C for 1 min for denaturation, 438C for 1 min for annealing, and 728C for 1 min for extension for 30 cycles.

All eae and stx amplifications were performed in a programmable thermal controller (PTC-100, MJ Research, Inc., Watertown, MA). 16 ml of amplified products were analyzed by gel electrophoresis in 1.5% agarose. Products were visualized by ethidium bromide staining followed by UV transillumination. A 100 bp DNA ladder (GibcoBRL, Grand Island, NY) was used as the molecular weight marker. For each PCR run, DNA from EHEC O157:H7 and a calf strain of AEEC (serotype 05:NM) were used as positive controls, and DNA from a strain of E. coli and Salmonella dublin that lacked eae genes were negative controls. The control strains had been previously evaluated for eae and stx genes by colony blot hybridization and PCR.

Each eae and eae/stx E. coli was assessed for enterohemolysin activity on 5% defibrinated washed sheep blood agar plates as described (Beutin et al., 1988) . Colonies surrounded by clear zones of hemolysis were defined as exhibiting enterohemolysin activity.

Serotyping was performed by standard methods at The E. coli Reference Center, The Pennsylvania State University (Wilson and Francis, 1986 ). E. coli strains were grown overnight in tryptic soy broth, then heated to 1008C for 2 h. Agglutinations were performed by using preselected dilutions of each of 183 O group antisera in microtitre plates and the positive reactions were confirmed by microtitre titrations against monovalent antisera (Glantz, 1971) . If there was no agglutination in the preliminary screening assay, the bacterial suspension was reheated at 1218C for an additional 1 h and the microtitre agglutination assays were repeated.

Susceptibility to different classes of antimicrobial agents was performed using standard disk diffusion methods in Mueller-Hinton agar (Bauer et al., 1966) . Results were interpreted according to (National Committee for Clinical Laboratory Standards, 1997) guidelines. The disks containing the following amounts of antibiotics were used: amikacin 30 mg, ampicillin 10 mg, ceftiofur 30 mg, cephalothin 30 mg, chloramphenicol 30 mg, enrofloxacin 5 mg, gentamicin 10 mg, tetracycline 30 mg, trimethoprim/sulfamethoxazole 1.25/23.75 mg, and nalidixic acid 30 mg.

To demonstrate Shiga toxin antigens, the Premier EHEC assay (Meridian Diagnostics Inc., Cincinnati, OH) was performed on eae/stx E. coli strains known to produce Shiga toxins ( Table 1) . As additional controls, 10 eae only and 10 stx only strains were examined by the enzyme immunoassay. After 12 h growth at 378C on MAC plates, a single colony of each strain was suspended in 200 ml of sample diluent for testing. The immunoassay was performed according to the manufacturer's recommendation. The reaction mixture was read spectrophotometrically at 450 nm.

Postmortem and microbiologic examinations were performed on 45 of the 114 diarrheic calves. Postmortem examinations were performed by attending veterinary pathologists. Tissue segments from the duodenum, jejunum, ileum, cecum, spiral colon, transverse colon, descending colon, and rectum were immediately fixed in neutral buffered 10% formalin. After 24 h in the fixative, tissues were trimmed, routinely processed, and embedded in paraffin. Thin sections were cut, mounted on slides, and stained with H & E. Slides were examined by light microscopy.

Intestinal contents or feces from each necropsied calf were examined for Cryptosporidium parvum, enterotoxigenic E. coli, Salmonella sp., enteric viruses, and BVDV by routine procedures. Briefly, C. parvum oocysts were detected by light microscopic examination of concentrated oocysts (Garcia et al., 1983) . 1 g of intestinal contents was dispensed in 9.0 ml of peptone-saline and 10 ml was plated onto MacConkey and brilliant green agar plates. Selenite enrichment broth was inoculated with 1.0 ml of the fecal/peptone-saline suspension and was subcultured onto brilliant green agar for detection of Salmonella sp. Suspect colonies were confirmed as Salmonella after differential biochemical and serological testing. Strains of E. coli were screened for heatstable enterotoxin type A by PCR using published oligonucleotide sequences (Woodward et al., 1992) . The K99 fimbrial antigen was detected by agglutination in K99 monospecific antibody after enhancement on Minca Isovitalex agar. The presence of enteric viruses was detected by electron microscopy and group A rotavirus antigen was detected by ELISA (Pathfinder Rotavirus, Kallestad, Chaska, MN). Bovine viral diarrhea virus was identified in lysed mononuclear cells or sera by the immunoperoxidase monolayer assay (Houe et al., 1995) .

To determine significance of the E. coli properties between healthy and diarrheic calves, the chi-square test of independence was used to evaluate the association between illness and the characteristics of the E. coli strains (Snedecor and Cochran, 1989) .

Based on interviews with calf caretakers and review of individual calf records (when available), 4 of the 101 healthy calves and 32 of the 114 diarrheic calves had received antimicrobial therapy. Twenty of these 32 calves had received antimicrobial therapy for their diarrheal disease and 12 calves had been treated for medical problems unrelated to and prior to the onset of diarrhea. Eight of the 23 diarrheic calves that carried eae or eae/ stx E. coli had received antimicrobial therapy. Gentamicin sulfate, ceftiofur sodium, oxytetracycline (LA 200), and procaine penicillin were the antimicrobials most often used.

The mean age of the 101 healthy calves was 22.0 AE 15.3 days (range 1 to 90 days) and the mean age of the 40 healthy calves infected with eae E. coli was 25.5 AE 14.6 days (range 6 to 79 days). The mean age of the 114 diarrheic calves was 14.4 AE 11.6 days (range 1 to 83 days), whereas, the mean age of the 23 diarrheic calves infected with eae E. coli was 24.3 AE 19.0 days (range 3 to 83 days).

In preliminary experiments, correlation between previously determined eae and stx profiles for select strains (Table 1 ) and our PCR results was 100%. Based on these results, the selected primers and the PCR assays were considered definitive for detection of E. coli eae and stx genes.

In this study, the Premier EHEC assay was used to detect Shiga toxin antigens associated with eae/stx strains. The assay accurately detected Shiga toxin antigens for the eae/stx strains (Table 1) , no false positives of eae only strains were detected, and 9 of 10 stx only strains were detected. Under the conditions used in this study, the Premier EHEC assay was considered a sensitive assay to detect Shiga toxin antigens.

Overall, 63 of the 215 (29.3%) calves carried eae E. coli in their feces. Forty of the 101 (40.0%) healthy calves and 23 of the 114 diarrheic calves (20.2%) carried eae E. coli in their feces. Infection with eae E. coli was significantly higher (p 0.001) in the healthy calves when compared to the diarrheic calves.

The stx gene was identified in approximately equal percentages of eae E. coli in healthy and diarrheic calves (Table 2) . Among the 40 eae strains from healthy calves, 20 (50.0%) possessed stx genes, whereas, among the 23 eae strains from diarrheic calves, 11 (48.0%) possessed the stx gene. When examined in the immunoassay, Shiga toxin antigens were detected for all 20 eae/stx (100%) strains from healthy calves and in 8 of 11 (73.0%) strains from diarrheic calves.

The enterohemolytic phenotype was expressed by 50.0% of the eae and eae/stx strains from healthy calves and by 52.0% of the strains from diarrheic calves. Enterohemolysin activity was not restricted to one particular serogroup and was detected in both eae and eae/stx strains (Table 2) .

Distribution of eae and eae/stx E. coli among serogroups is shown in Table 3 . Forty six of the 63 (73.0%) eae and eae/stx E. coli strains belonged to 17 different typable serogroups and 17 (27.0%) eae E. coli and eae/stx strains were O-negative. Serogroups O5, O8, O26, O50, and O111 were detected in both healthy and diarrheic calves. However, strains belonging to serogroups O5, O26 and O111 accounted for 45.0% and 39.0% of the eae and eae/stx E. coli from healthy and diarrheic calves, respectively. The O-negative strains accounted for 22.5% and 35.0% of the strains from healthy and diarrheic calves, respectively.

Shiga toxin antigens were detected among E. coli belonging to serogroups 05, O26, O84/172, O98, O103, O111, O157 and O171 from healthy calves, and from serogroups 05, 055, 069, and 0111 from diarrheic calves. 

All strains were sensitive to amikacin, enrofloxacin, and nalidixic acid (Table 4 ). Significantly more of the eae and eae/stx strains from diarrheic calves, when compared to strains from healthy calves, were resistant to at least one of the antimicrobials tested. Moreover, significantly more of the strains from diarrheic calves had multiple antibiotic resistance patterns. Four eae/stx strains recovered from diarrheic calves were resistant to chloramphenicol. Antimicrobial resistance was not restricted to any one serogroup, nor was resistance associated with enterohemolysin production. Most resistance patterns included resistance to tetracycline, ampicillin, and gentamicin (Table 4) .

Among the 45 calves that had postmortem examinations, 15 (33.3%) had eae or eae/ stx E. coli in their feces. In five calves infected with eae or eae/stx E. coli, and in the tissue sections examined did not have histopathologic evidence of the attaching and effacing lesion, C. parvum, coronavirus, and rotavirus were identified in one calf each. C. parvum and coronavirus were identified in one additional calf. Histopathologic lesions consistent with the attaching and effacing phenotype (Moon et al., 1983; Hall et al., 1985 Hall et al., , 1990 Chanter et al., 1984 Chanter et al., , 1986 Moxley and Francis, 1986; Pospischil et al., 1987; Janke et al., 1990) were detected in 10 calves. The lesion was detected in the colon only of six calves and in the ileum and colon of four calves. Among these 10 calves, C. parvum was Table 3 Distribution of eae and eae/stx E. coli among serogroups and relationship to the attaching and effacing lesion detected in five calves, Salmonella typhimurium in one calf, and BVDV and C. parvum were detected in one calf. The pathogens screened for by our assays were not detected in feces from 3 of the 10 calves.

In this study, significantly more of the healthy calves (40.0%) carried eae and eae/ stx E. coli in their feces when compared to diarrheic calves (20.0%). However, significantly more eae and eae/stx E. coli that exhibited single and multiple antimicrobial resistances were isolated from diarrheic calves. Selection for resistance among the strains from diarrheic calves may have been due to a greater use of antimicrobials among the diarrheic calves. Based on limited observations made with 10 calves on one farm, a relation between antimicrobial use and selection for resistance was observed. Three of 10 calves developed diarrhea after being treated for respiratory disease. All three calves were subsequently positive for eae (one calf) and eae/stx E. coli (two calves). Two of the three strains were resistant to the antimicrobial used to treat the respiratory disease. Examination of E. coli from the seven healthy calves did not reveal any eae , eae/stx E. coli or commensal E. coli strains that were resistant to that antimicrobial. In this case, exposure to the particular antimicrobial appeared to select for resistance. However, environmental sources of the resistant strains could not be excluded (Linton, 1986) .

Our data show that the stx genes were equally distributed among eae E. coli from both healthy (50.0%) and diarrheic calves (48.0%). Furthermore, Shiga toxin antigens were detected in more strains from healthy calves (100.0%) than in strains from diarrheic calves (73.0%). Although the immunoassay used detects Shiga toxin antigens, the data are consistent with that of others who have demonstrated the prevalence of Shiga toxin producing E. coli in the feces of young calves (Mainil et al., 1993; Blanco et al., 1988 Blanco et al., , 1993 Butler and Clarke, 1994; Dorn et al., 1993; and Wieler et al., 1992) . Enterohemolysin, a distinctively different hemolysin from -hemolysin, is synthesized by calf strains of E. coli that produce Shiga toxins (Beutin et al., 1986 (Beutin et al., , 1988 (Beutin et al., , 1989 . Although the influence of enterohemolysin on calf intestinal disease has not been defined, it has been suggested that enterohemolysins may compliment the effects of Shiga toxins (Nataro and Kaper, 1998) . Furthermore, screening for enterohemolysin has been proposed as a diagnostic marker for Shiga toxin producing E. coli (Beutin et al., 1989; Wieler et al., 1995) , since its presence is strongly correlated with Shiga toxins. Among bovine Shiga toxin producing E. coli, enterohemolysin activity has been reported to range from 57.6% to 70.8% (Beutin et al., 1989; Wieler et al., 1992) . Our data show that 50.0% and 52.0% of the eae and eae/stx strains from healthy and diarrheic calves, respectively, had enterohemolysin activity.

Strains of AEEC belonging to the same serogroups were often isolated from healthy and diarrheic calves. Strains belonging to serogroups O5, O26, and O111 accounted for 43.0% of all the typable strains, and for 45.0% and 39.0% of the typable strains from healthy and diarrheic calves, respectively. Characterization of AEEC by O group only is less powerful than O:H typing since strains of the same O group but possessing different H antigens may have different virulence properties. Nevertheless, determination of specific virulence attributes and the association with specific O groups provide valuable epidemiologic information (Wieler et al., 1992) . Attaching and effacing E. coli belonging to serogroups O5, O26, and O111 are widely distributed among calves and have been demonstrated in natural outbreaks and experimentally (with some strains) to cause diarrhea and dysentery (Chanter et al., 1984 (Chanter et al., , 1986 Hall et al., 1985 Hall et al., , 1990 Schoonderwoerd et al., 1988; Wray et al., 1989; Moxley and Francis, 1986; Mainil et al., 1993; Janke et al., 1990; Knutton, 1994; Butler and Clarke, 1994; Dorn et al., 1993) . As calf AEEC have a similar pathophysiologic mechanism to human EPEC and EHEC, strains carrying both eae and stx genes, and belonging to serogroups O negative, O5, O8, O26, O55, O69, O84/172, O98, O103, O111, O118, O157 and 0171 would be of concern to human health.

The age at which calves are most susceptible to colonization by eae E. coli and the age at which they are most likely to develop attaching and effacing lesions are not known. Based on the distribution of the eae gene among calves of various ages, it appears that an age-associated resistance does not occur during the first week of life as is recognized with ETEC. Furthermore, the mean ages of the healthy (25.5 days) and diarrheic (24.3 days) calves infected with eae E. coli would support the notion that calves older than 21 days are more susceptible to infection with eae E. coli. However, this does not mean that the infected calves would develop attaching and effacing lesions. The age of calves infected with eae E. coli and those that had demonstrable attaching and effacing lesions ranged from 8 to 83 days. In natural infections, calves as young as 2 days and as old as 4 months have been described with attaching and effacing lesions (Janke et al., 1990) .

Antibiotic susceptibility testing by a standardized single disk method

Close association of verotoxin (shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli

Characterization of hemolytic strains of Escherichia coli belonging to classical enteropathogenic O-serogroups

Entero-hemolysin, a new type of hemolysin produced by some strains of enteropathogenic E. coli (EPEC)

Genes coding for Shiga-like toxins in bovine verotoxin-producing Escherichia coli (VTEC) strains belonging to different O : K : H serotypes

Production of toxins by Escherichia coli strains isolated from calves with diarrhoea in Galicia

Enterotoxigenic, verotoxigenic, and necrotoxigenic Escherichia coli isolated from cattle in Spain

Diarrhoea and dysentery in calves

Dysentery in gnotobiotic calves caused by atypical Escherichia coli

Dysentery in calves caused by an atypical strain of Escherichia coli

Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector

Enteropathogenic Escherichia coli

Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection

A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells

Characteristics of verocytoxin producing Escherichia coli associated with intestinal colonization and diarrhea in calves

Pathogenicity of a bovine attaching effacing Escherichia coli isolate lacking shiga-like toxins

Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens

Detection and characterization of the eae gene of shiga-like toxin-producing Escherichia coli using polymerase chain reaction

Serotypes of Escherichia coli associated with colibacillosis in neonatal animals

Escherichia coli verotoxins and other cytotoxins

Dysentery caused by Escherichia coli (S102-9) in calves: natural and experimental disease

Attaching and effacing lesions in vivo and adhesion to tissue culture cells of vero-cytoxin-producing Escherichia coli belonging to serogroup O5 and O103

Prevalence of cattle persistently infected with bovine viral diarrhea virus in 20 dairy herds in two counties in central Michigan and comparison of prevalence of antibody-positive cattle among herds with different infection and vaccination status

Attaching and effacing Escherichia coli infection as a cause of diarrhea in young calves

The eae gene of enteropathogenic Escherichia coli encodes a 94-kilodalton membrane protein, the expression of which is influenced by the EAF plasmid

Clonal structure and pathogenicity of shiga-like toxin-producing, sorbitol-fermenting Escherichia coli O157:H

Single primer pair for amplifying segments of distinct shiga-like toxin genes by polymerase chain reaction

Infection by verocytotoxin-producing Escherichia coli

Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic and enteroadherent

Flow of resistance genes in the environment and from animals to man

Association between the effacing (eae) gene and the shiga-like toxin-encoding genes in Escherichia coli isolates from cattle

Role of verocytogenic Escherichia coli in cattle and buffalo calf diarrhoea

Serotypes of verocytotoxigenic Escherichia coli isolated from cattle and buffalo calf diarrhoea

Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines

Natural and experimental infection with an attaching and effacing strain of Escherichia coli in calves

Performance standards for antimicrobial disk susceptibility tests. Approved Standard M2-A4

Attaching and effacing bacteria in the intestine of calves with diarrhea

Virulence properties of Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups isolated from calves with diarrhea

Shiga-like toxin II-related cytotoxins in Citrobacter freundii strains from humans and beef samples

Colitis in calves: Natural and experimental infection with a verotoxin-producing strain of Escherichia coli O111:NM

Shiga-like toxin production from Escherichia coli associated with calf diarrhea

Analysis of frequencies in one-way and two-way classifications

Verotoxigenic Escherichia coli O.157 in Scottish calves

The pathogenic mechanisms of Shiga and Shiga-like toxins

Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli O157:H7 in gnotobiotic piglets

Characterization of shiga-like toxin producing Escherichia coli (SLTEC) isolated from calves with and without diarrhea

Association of enterohemolysin and nonfermentation of rhamnose and sucrose with Shiga-like toxin-genes in Escherichia coli from calves

Shiga toxin-producing Escherichia coli strains from bovines: association of adhesion with carriage of eae and other genes

Fimbriae and enterotoxins associated with Escherichia coli serogroups isolated from pigs with colibacillosis

Detection of entero-and verocyto-toxin genes in Escherichia coli from diarrhoeal disease in animals using polymerase chain reaction

Occurrence of`attaching and effacing' lesions in the small intestine of calves experimentally infected with bovine isolates of verocytotoxic E. coli

Financial support from The Agricultural Experiment Station, National Food Safety and Toxicology Center, and The College of Veterinary Medicine, Michigan State University.