Thomas E. Besser

Lymphoid Follicle-Dense Mucosa at the Terminal Rectum Is the Principal Site of Colonization of Enterohemorrhagic Escherichia coli O157:H7 in the Bovine Host

ABSTRACT Escherichia coli O157:H7 causes bloody diarrhea and potentially fatal systemic sequelae in humans. Cattle are most frequently identified as the primary source of infection, and E. coli O157:H7 generally colonizes the gastrointestinal tracts of cattle without causing disease. In this study, persistence and tropism were assessed for four different E. coli O157:H7 strains. Experimentally infected calves shed the organism for at least 14 days prior to necropsy. For the majority of these animals, as well as for a naturally colonized animal obtained from a commercial beef farm, the highest numbers of E. coli O157:H7 were found in the feces, with negative or significantly lower levels detected in lumen contents taken from the gastrointestinal tract. Detailed examination demonstrated that in these individuals the majority of tissue-associated bacteria were adherent to mucosal epithelium within a defined region extending up to 5 cm proximally from the recto-anal junction. The tissue targeted by E. coli O157:H7 was characterized by a high density of lymphoid follicles. Microcolonies of the bacterium were readily detected on the epithelium of this region by immunofluorescence microscopy. As a consequence of this specific distribution, E. coli O157:H7 was present predominately on the surface of the fecal stool. In contrast, other E. coli serotypes were present at consistent levels throughout the large intestine and were equally distributed in the stool. This is a novel tropism that may enhance dissemination both between animals and from animals to humans. The accessibility of this site may facilitate simple intervention strategies.

Iha: a novel Escherichia coli O157:H7 adherence-conferring molecule encoded on a recently acquired chromosomal island of conserved structure.

ABSTRACT The mechanisms used by Shiga toxin (Stx)-producingEscherichia coli to adhere to epithelial cells are incompletely understood. Two cosmids from an E. coliO157:H7 DNA library contain an adherence-conferring chromosomal gene encoding a protein similar to iron-regulated gene A (IrgA) ofVibrio cholerae (M. B. Goldberg, S. A. Boyko, J. R. Butterton, J. A. Stoebner, S. M. Payne, and S. B. Calderwood, Mol. Microbiol. 6:2407–2418, 1992). We have termed the product of this gene the IrgA homologue adhesin (Iha), which is encoded by iha. Iha is 67 kDa in E. coliO157:H7 and 78 kDa in laboratory E. coli and is structurally unlike other known adhesins. DNA adjacent toiha contains tellurite resistance loci and is conserved in structure in distantly related pathogenic E. coli, but it is absent from nontoxigenic E. coli O55:H7, sorbitol-fermenting Stx-producing E. coli O157:H−, and laboratory E. coli. We have termed this region the tellurite resistance- and adherence-conferring island. We conclude that Iha is a novel bacterial adherence-conferring protein and is contained within an E. coli chromosomal island of conserved structure. Pathogenic E. coli O157:H7 has only recently acquired this island.

Multiple sources of Escherichia coli O157 in feedlots and dairy farms in the Northwestern USA

Samples from cattle, other domestic and wild animals, flies, feeds, and water-troughs were collected from 12 cattle farms and tested for Escherichia coli O157. E. coli O157 was isolated from bovine fecal samples on all 12 farms with a within herd prevalence ranging from 1.1% to 6.1%. E. coli O157 was also found in 1 of 90 (1.1%) equine fecal samples, 2 of 65 (3.1%) canine fecal samples, 1 of 200 pooled bird samples (0.5%), 2 of 60 pooled fly samples (3.3%), and 10 of 320 (3.1%) water-trough sample sets (biofilm and water). No E. coli O157 were isolated from 300 rodents, 33 cats, 34 assorted wildlife, or 335 cattle feed samples. Indistinguishable pulsed-field gel electrophoresis patterns of XbaI digested chromosomal DNA and Shiga toxin types were observed for bovine and water-trough isolates from two farms and for one equine and two bovine isolates from one farm.

Cattle Water Troughs as Reservoirs of Escherichia coli O157

ABSTRACT Environmental survival of Escherichia coli O157 may play an important role in the persistence and dissemination of this organism on farms. The survival of culturable and infectious E. coli O157 was studied using microcosms simulating cattle water troughs. Culturable E. coli O157 survived for at least 245 days in the microcosm sediments. Furthermore, E. coli O157 strains surviving more than 6 months in contaminated microcosms were infectious to a group of 10-week-old calves. Fecal excretion ofE. coli O157 by these calves persisted for 87 days after challenge. Water trough sediments contaminated with feces from cattle excreting E. coli O157 may serve as a long-term reservoir of this organism on farms and a source of infection for cattle.

PRP GENOTYPES OF CAPTIVE AND FREE-RANGING ROCKY MOUNTAIN ELK (CERVUS ELAPHUS NELSONI) WITH CHRONIC WASTING DISEASE

The PrP gene encodes the putative causative agent of the transmissible spongiform encephalopathies (TSEs), a heterogeneous group of fatal, neurodegenerative disorders including human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, ovine scrapie and chronic wasting disease (CWD) of North American deer and elk. Polymorphisms in the PrP gene are associated with variations in relative susceptibility, pathological lesion patterns, incubation times and clinical course of TSEs of humans, mice and sheep. Sequence analysis of the PrP gene from Rocky Mountain elk showed only one amino acid change (Met to Leu at cervid codon 132). Homozygosity for Met at the corresponding polymorphic site (Met to Val) in humans (human codon 129) predisposes exposed individuals to some forms of Creutzfeldt-Jakob disease. In this study, Rocky Mountain elk homozygous for PrP codon 1 32 Met were over-represented in both free-ranging and farm-raised CWD-affected elk when compared to unaffected control groups.

Rectoanal Junction Colonization of Feedlot Cattle by Escherichia coli O157:H7 and Its Association with Supershedders and Excretion Dynamics

ABSTRACT Feedlot cattle were observed for fecal excretion of and rectoanal junction (RAJ) colonization with Escherichia coli O157:H7 to identify potential “supershedders.” RAJ colonization and fecal excretion prevalences were correlated, and E. coli O157:H7 prevalences and counts were significantly greater for RAJ samples. Based on a comparison of RAJ and fecal ratios of E. coli O157:H7/E. coli counts, the RAJ appears to be preferentially colonized by the O157:H7 serotype. Five supershedders were identified based on persistent colonization with high concentrations of E. coli O157:H7. Cattle copenned with supershedders had significantly greater mean pen E. coli O157:H7 RAJ and fecal prevalences than noncopenned cattle. Cumulative fecal E. coli O157:H7 excretion was also significantly higher for pens housing a supershedder. E. coli O157:H7/E. coli count ratios were higher for supershedders than for other cattle, indicating greater proportional colonization. Pulsed-field gel electrophoresis analysis demonstrated that isolates from supershedders and copenned cattle were highly related. Cattle that remained negative for E. coli O157:H7 throughout sampling were five times more likely to have been in a pen that did not house a supershedder. The data from this study support an association between levels of fecal excretion of E. coli O157:H7 and RAJ colonization in pens of feedlot cattle and suggest that the presence of supershedders influences group-level excretion parameters. An improved understanding of individual and population transmission dynamics of E. coli O157:H7 can be used to develop preslaughter- and slaughter-level interventions that reduce contamination of the food chain.

Epidemiology of Escherichia coli O157 in feedlot cattle

Fecal samples from cattle in 100 feedlots in 13 states were bacteriologically cultured for Escherichia coli O157 that did not ferment sorbitol, lacked beta-glucuronidase, and possessed genes coding for Shiga-like toxin. In each feedlot 30 fresh fecal-pat samples were collected from each of four pens: with the cattle shortest on feed, with cattle longest on feed, and with cattle in two randomly selected pens. E. coli O157 was isolated from 210 (1.8%) of 11,881 fecal samples. One or more samples were positive for E. coli O157 in 63 of the 100 feedlots tested. E. coli O157 was found at roughly equal prevalence in all the geographical regions sampled. The prevalence of E. coli O157 in the pens with cattle shortest on feed was approximately threefold higher than for randomly selected and longest on feed pens. Of the E. coli O157 isolates found in this study, 89.52% expressed the H7 flagellar antigen. E. coli O157 was found to be widely distributed among feedlot cattle, but at a low prevalence, in the United States.

Evaluation of Pulsed-Field Gel Electrophoresis as a Tool for Determining the Degree of Genetic Relatedness between Strains of Escherichia coli O157:H7

ABSTRACT Pulsed-field gel electrophoresis (PFGE) has been used extensively to investigate the epidemiology of Escherichia coli O157:H7, although it has not been evaluated as a tool for establishing genetic relationships. This is a critical issue when molecular genetic data are used to make inferences about pathogen dissemination. To evaluate this further, genomic DNAs from 62 isolates of E. coli O157:H7 from different cattle herds were digested with XbaI and BlnI and subjected to PFGE. The correlation between the similarity coefficients for these two enzymes was only 0.53. Four additional restriction enzymes (NheI, PacI, SfiI, and SpeI) were used with DNAs from a subset of 14 isolates. The average correlations between similarity coefficients using sets of one, two, and three enzymes were 0.405, 0.568, and 0.648, respectively. Probing with lambda DNA demonstrated that some DNA fragments migrated equal distances in the gel but were composed of nonhomologous genetic material. Genome sequence data from EDL933 indicated that 40 PFGE fragments would be expected from complete XbaI digestion, yet only 19 distinguishable fragments were visible. Two reasons that similarity coefficients from single-enzyme PFGE are poor measures of relatedness (and hence are poorly correlated with other enzymes) are evident from this study: (i) matching bands do not always represent homologous genetic material and (ii) there are limitations to the power of PFGE to resolve bands of nearly identical size. The findings of the present study indicate that if genetic relationships must be inferred in the absence of epidemiologic data, six or more restriction enzymes would be needed to provide a reasonable estimate using PFGE.

Role of Calf-Adapted Escherichia coli in Maintenance of Antimicrobial Drug Resistance in Dairy Calves

ABSTRACT The prevalence of antimicrobial drug-resistant bacteria is typically highest in younger animals, and prevalence is not necessarily related to recent use of antimicrobial drugs. In dairy cattle, we hypothesize that antimicrobial drug-resistant, neonate-adapted bacteria are responsible for the observed high frequencies of resistant Escherichia coli in calves. To explore this issue, we examined the age distribution of antimicrobial drug-resistant E. coli from Holstein cattle at a local dairy and conducted an experiment to determine if low doses of oxytetracycline affected the prevalence of antimicrobial drug-resistant E. coli. Isolates resistant to tetracycline (>4 μg/ml) were more prevalent in <3-month-old calves (79%) compared with lactating cows (14%). In an experimental trial where calves received diets supplemented with or without oxytetracycline, the prevalence of tetracycline-resistant E. coli was slightly higher for the latter group (P = 0.039), indicating that drug use was not required to maintain a high prevalence of resistant E. coli. The most common resistance pattern among calf E. coli isolates included resistance to streptomycin (>12 μg/ml), sulfadiazine (>512 μg/ml), and tetracycline (>4 μg/ml) (SSuT), and this resistance pattern was most prevalent during the period when calves were on milk diets. To determine if prevalence was a function of differential fitness, we orally inoculated animals with nalidixic acid-resistant strains of SSuT E. coli and susceptible E. coli. Shedding of SSuT E. coli was significantly greater than that of susceptible strains in neonatal calves (P < 0.001), whereas there was no difference in older animals (P = 0.5). These data support the hypothesis that active selection for traits linked to the SSuT phenotype are responsible for maintaining drug-resistant E. coli in this population of dairy calves.

PRP GENOTYPES AND EXPERIMENTAL SCRAPIE IN ORALLY INOCULATED SUFFOLK SHEEP IN THE UNITED STATES

One-hundred and three Unites States Suffolk sheep were inoculated orally with a scrapie agent preparation and monitored for clinical disease and histopathological lesions characteristic of scrapie. A retrospective study of the polymorphisms at codon 171 of the prion protein (PrP) gene was performed on these sheep. All 63 sheep that developed scrapie during the observation period were homozygous for the glutamine 171 (171-QQ) PrP allele. Twelve 171-QQ sheep failed to develop disease. All 5 sheep homozygous for arginine (171-RR) and all 23 heterozygous (171-QR) sheep remained free of scrapie.