Rectal Administration of Escherichia coli O157:H7: Novel Model for Colonization of Ruminants

Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening complications. Because healthy cattle are reservoirs for the bacterium, ruminant infection models have applications in analyzing the relationship between cattle and this human pathogen and in testing interventions to reduce or prevent bovine colonization with this bacterium. Current approaches often do not reliably mimic natural, long-term bovine colonization with E. coli O157:H7 in older calves and adult animals (ages that enter our food chain). Based on the recent identification of the bovine rectoanal junction mucosa as a site of E. coli O157:H7 colonization, we developed a novel rectal swab administration colonization model. We compared this method with oral dosing and direct contact transmission (Trojan) methods. E. coli O157:H7 carriage status was determined by fecal or rectoanal mucosa swab culture. High (~10¹⁰ CFU) and low (~10⁷ CFU) oral doses of E. coli O157:H7 in sheep and cattle resulted in variable infection with the bacterium. Some animals became colonized with the bacteria and remained culture positive for several weeks, and some animals did not become colonized and rapidly cleared the bacteria in a few days. Pen mates of E. coli O157:H7 culture-positive Trojan cattle had a low infection rate and variable colonization status. However, rectal swab administration of E. coli O157:H7 to cattle resulted in consistent long-term colonization in all animals. The surprising ease with which long-term infections resulted from a single application of bacteria to the rectoanal mucosa also strongly supported this location as a site of E. coli O157:H7 colonization in cattle.     

Application of bacteriophages to control intestinal Escherichia coli O157:H7 levels in ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios > or = 10(2) terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10(10) PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be > or = 10(2). In addition, phages were maintained at 10(6) PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.     

Escherichia coli O157:H7 colonization at the rectoanal junction of long-duration culture-positive cattle

Long-duration consistently Escherichia coli O157:H7 culture-positive cattle were euthanized and necropsied. Tissue and digesta from along the gastrointestinal tract (GIT) were cultured for the bacteria and examined histologically for lymphoid character. E. coli O157:H7 was detected only at the rectoanal junction mucosa and not at any other GIT location.     

Variable colonization of chickens perorally inoculated with Escherichia coli O157:H7 and subsequent contamination of eggs.

Challenging 1-day-old White Leghorn chicks perorally with 2.6 x 10(1) to 2.6 x 10(5) Escherichia coli O157:H7 bacteria per chick resulted in cecal colonization at all levels. Two of six chicks inoculated with only 2.6 x 10(1) E. coli O157:H7 bacteria carried 10(3) to 10(4) E. coli O157:H7 bacteria per g of cecal tissue when sacrificed 3 months postinoculation. E. coli O157:H7 colonization persisted at least 10 to 11 months when chicks were administered 10(8) E. coli O157:H7 bacteria. Eggs from five hens that were fecal shedders of E. coli O157:H7 until the termination of the study (10 to 11 months) were assayed for E. coli O157:H7. The organism was isolated from the shells of 14 of 101 (13.9%) eggs but not from the yolks and whites. Considering that chicks can be readily colonized by small populations of E. coli O157:H7 and continue to be long-term shedders, it is possible that chickens and hen eggs can serve as vehicles of this human pathogen.     

Rectal carriage of enterohemorrhagic Escherichia coli O157 in slaughtered cattle

Escherichia coli O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for E. coli O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for E. coli O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of E. coli O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with E. coli O157 levels of <1 x 10(3) CFU g(-1) or <1 x 10(3) CFU ml(-1) and animals with E. coli O157 levels of > or =1 x 10(3) CFU g(-1) or > or =1 x 10(3) CFU ml(-1) in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for E. coli O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (> or =1 x 10(3) CFU g of feces(-1)) of enterohemorrhagic E. coli O157 results from colonization of this site.     

Effect of diet on the shedding of Escherichia coli O157:H7 in a sheep model.

The purpose of this study was to develop a sheep model to investigate reproduction, transmission, and shedding of Escherichia coli O157:H7 in ruminants. In addition, we investigated the effect of diet change on these parameters. Six groups of twin lambs given oral inoculations of 10(5) or 10(9) CFU of E. coli O157:H7 and their nondosed mothers were monitored for colonization by culture of fecal samples. A modified selective-enrichment protocol that detected E. coli O157:H7 at levels as low as 0.06 CFU per g of ovine feces was developed. Horizontal transmission of infection occurred between the lambs and most of the nondosed mothers. When animals were kept in confinement and given alfalfa pellet feed, lambs receiving the higher dose shed the bacteria sooner and longer than all other animals. However, when the animals were released onto a sagebrush-bunchgrass range, every animal, regardless of its previous status (dosed at one of the inoculum levels tested or nondosed) shed E. coli O157:H7 uniformly. Shedding persisted for 15 days, after which all animals tested negative. E. coli O157:H7 reproduction and transmission and the combined effect of diet change and feed withholding were also investigated in a pilot study with experimentally inoculated rams. Withholding feed induced animals to shed the bacteria either by triggering growth of E. coli O157:H7 present in the intestines or by increasing susceptibility to infection. Introduction of a dietary change with brief starvation caused uniform shedding and clearance of E. coli O157:H7, and all animals then tested negative for the bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Escherichia coli O157:H7 colonization in small domestic ruminants

Enterohaemorrhagic Escherichia coli O157:H7 was first implicated in human disease in the early 1980s, with ruminants cited as the primary reservoirs. Preliminary studies indicated cattle to be the sole source of E. coli O157:H7 outbreaks in humans; however, further epidemiological studies soon demonstrated that E. coli O157:H7 was widespread in other food sources and that a number of transmission routes existed. More recently, small domestic ruminants (sheep and goats) have emerged as important sources of E. coli O157:H7 human infection, particularly with the widespread popularity of petting farms and the increased use of sheep and goat food products, including unpasteurized cheeses. Although the colonization and persistence characteristics of E. coli O157:H7 in the bovine host have been studied intensively, this is not the case for small ruminants. Despite many similarities to the bovine host, the pathobiology of E. coli O157:H7 in small domestic ruminants does appear to differ significantly from that described in cattle. This review aims to critically review the current knowledge regarding colonization and persistence of E. coli O157:H7 in small domestic ruminants, including comparisons with the bovine host where appropriate.     

Comparison of rectoanal mucosal swab cultures and fecal cultures for determining prevalence of Escherichia coli O157:H7 in feedlot cattle

We compared fecal samples with samples collected with rectoanal mucosa swabs (RAMS) to determine the prevalence of Escherichia coli O157 in feedlot cattle (n = 747). Escherichia coli O157 was detected in 9.5% of samples collected with RAMS and 4.7% of samples tested by fecal culture. Pulsed-field gel electrophoresis analysis of isolates suggested that the strains colonizing the rectoanal junction were the same as those from the feces. Mucosal swab sampling was more sensitive than fecal sampling for determining the prevalence of E. coli O157 in feedlot cattle.     

Rumen contents as a reservoir of enterohemorrhagic Escherichia coli

Abstract We investigatedthe role of the rumen fermentation as a barries to the foodborne pathogen, Escherichia coli O157:H7. Strains of E. coli , including several isolates of O157:H7, grew poorly in media which simulated the ruminal environment of a well-fed animal. Strains of E. coli O157:H7 did not display a superior tolerance to ruminal conditions which may facilitate their colonization of the bovine digestive tract. Unrestricted growth of E. coli was observed in rumen fluid collected from fasted cattle. Growth was inhibited by rumen fluid collected from well-fed animals. Well-fed animals appear less likely to become reservoirs for pathogenic E. coli . These results have implications for cattle slaughter practices and epidemiological studies of E. coli O157:H7.     

Experimental and field studies of Escherichia coli O157:H7 in white-tailed deer

Studies were conducted to evaluate fecal shedding of Escherichia coli O157:H7 in a small group of inoculated deer, determine the prevalence of the bacterium in free-ranging white-tailed deer, and elucidate relationships between E. coli O157:H7 in wild deer and domestic cattle at the same site. Six young, white-tailed deer were orally administered 10(8) CFU of E. coli O157:H7. Inoculated deer were shedding E. coli O157:H7 by 1 day postinoculation (DPI) and continued to shed decreasing numbers of the bacteria throughout the 26-day trial. Horizontal transmission to an uninoculated deer was demonstrated. Although E. coli O157:H7 bacteria were recovered from the gastrointestinal tracts of deer necropsied from 4 to 26 DPI, attaching and effacing lesions were not apparent in any deer. Results are similar to those of inoculation studies in calves and sheep. In field studies, E. coli O157 was not detected in 310 fresh deer fecal samples collected from the ground. It was detected in feces, but not in meat, from 3 of 469 free-ranging deer in 1997. In 1998, E. coli O157 was not detected in 140 deer at the single positive site found in 1997; however, it was recovered from 13 of 305 dairy and beef cattle at the same location. Isolates of E. coli O157:H7 from deer and cattle at this site differed with respect to pulsed-field gel electrophoresis patterns and genes encoding Shiga toxins. The low overall prevalence of E. coli O157:H7 and the identification of only one site with positive deer suggest that wild deer are not a major reservoir of E. coli O157:H7 in the southeastern United States. However, there may be individual locations where deer sporadically harbor the bacterium, and venison should be handled with the same precautions recommended for beef, pork, and poultry.     

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.     

Application of Bacteriophages To Control Intestinal Escherichia coli O157:H7 Levels in Ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios ≥10² terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10¹⁰ PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be ≥10². In addition, phages were maintained at 10⁶ PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.     

Survival of Escherichia coli O157:H7 in feces from corn- and barley-fed steers

The survival of Escherichia coli O157:H7 in feces from steers fed corn (CO) or barley (BA) was evaluated at -10, +4 and +22 degrees C. Fecal pats were inoculated with a four-strain mixture of nalidixic-acid resistant E. coli O157:H7 at two levels: 10(3) CFU g(-1) (low, L) and 105 CFU g(-1) (high, H). At -10 degrees C, duration of survival of E. coli O157:H7 was reduced (p < 0.05) in CO-L (35 days) compared to BA-L (49 days), likely due to the effects of fecal volatile fatty acids in combination with a fecal pH of <6.5. At 4 degrees C, E. coli O157:H7 was detected in BA-H, CO-H, CO-L and BA-L for 77, 77, 56 and 63 days, respectively, with no difference (p > 0.05) observed in the duration of survival or rate of decline of E. coli O157:H7 among treatments. Survival of E. coli O157:H7 was twice as likely (p < 0.05) at 22 degrees C than at 4 degrees C and -10 degrees C. While pH and dry matter content increased, and volatile fatty acid concentrations decreased over 84 days at all three temperatures, these changes were most pronounced at 22 degrees C. Survival of E. coli O157:H7 for extended periods of time in feces from both corn- and barley-fed animals was demonstrated, thus fecal material may serve as a vector for the transmission of the organism. The greater survival of E. coli O157:H7 at 22 degrees C suggests that temperature may play a role in the seasonality of transmission and prevalence of this bacterium in feedlot cattle. The reported greater prevalence of E. coli O157:H7 in cattle fed barley as compared to those fed corn does not appear to be related to elevated risk of transmission arising from differential survival of the bacterium in feces.     

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

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.     

Expression profiles of bovine genes in the rectoanal junction mucosa during colonization with Escherichia coli O157:H7

A bovine-specific cDNA microarray was used to characterize gene expression in the bovine rectoanal junction mucosa in response to Escherichia coli O157:H7 colonization, and results were confirmed using quantitative real-time PCR. The results showed involvement of cell processes including immune response, cell structure/dynamics, signal transduction, intercellular communication, and metabolism.     

Escherichia coli O157:H7 Colonization at the Rectoanal Junction of Long-Duration Culture-Positive Cattle

Long-duration consistently Escherichia coli O157:H7 culture-positive cattle were euthanized and necropsied. Tissue and digesta from along the gastrointestinal tract (GIT) were cultured for the bacteria and examined histologically for lymphoid character. E. coli O157:H7 was detected only at the rectoanal junction mucosa and not at any other GIT location.     

Effect of cattle diet on Escherichia coli O157:H7 acid resistance.

The duration of shedding of Escherichia coli O157 isolates by hay-fed and grain-fed steers experimentally inoculated with E. coli O157:H7 was compared, as well as the acid resistance of the bacteria. The hay-fed animals shed E. coli O157 longer than the grain-fed animals, and irrespective of diet, these bacteria were equally acid resistant. Feeding cattle hay may increase human infections with E. coli O157:H7.     

Mucosal antibody responses of colonized cattle to Escherichia coli O157-secreted proteins, flagellin, outer membrane proteins and lipopolysaccharide

The aim of this work was to characterize adaptive mucosal immune responses to Escherichia coli O157:H7 at the principal site of colonization in the bovine species. Following experimental infection, extracts from terminal rectum mucosal samples were tested for IgA antibodies by immunoblotting against different bacterial antigens including: whole-cell E. coli O157:H7 with and without proteinase treatment, outer membrane and cytoplasmic preparations, secreted protein supernatants and purified E. coli O157 lipopolysaccharide and H7 flagellin. Lipopolysaccharide and H7 flagellin preparations were also used to coat enzyme-linked immunosorbent assay plates to determine mucosal IgG1 and IgA antibody titers. In this work, evidence is presented of strong local IgA immune responses induced following infection at the bovine terminal rectal mucosa directed against multiple antigens including type III secretion-dependent proteins, O157 lipopolysaccharide, H7 flagellin and OmpC.     

Association of Escherichia coli O157:H7 with houseflies on a cattle farm

The ecology of Escherichia coli O157:H7 is not well understood. The aims of this study were to determine the prevalence of and characterize E. coli O157:H7 associated with houseflies (HF). Musca domestica L. HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively. E. coli O157:H7 counts ranged from 3.0 x 10(1) to 1.5 x 10(5) CFU among the positive HF. PCR analysis of the E. coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment.     

Gastrointestinal tract location of Escherichia coli O157:H7 in ruminants

Experimentally inoculated sheep and cattle were used as models of natural ruminant infection to investigate the pattern of Escherichia coli O157:H7 shedding and gastrointestinal tract (GIT) location. Eighteen forage-fed cattle were orally inoculated with E. coli O157:H7, and fecal samples were cultured for the bacteria. Three distinct patterns of shedding were observed: 1 month, 1 week, and 2 months or more. Similar patterns were confirmed among 29 forage-fed sheep and four cannulated steers. To identify the GIT location of E. coli O157:H7, sheep were sacrificed at weekly intervals postinoculation and tissue and digesta cultures were taken from the rumen, abomasum, duodenum, lower ileum, cecum, ascending colon, descending colon, and rectum. E. coli O157:H7 was most prevalent in the lower GIT digesta, specifically the cecum, colon, and feces. The bacteria were only inconsistently cultured from tissue samples and only during the first week postinoculation. These results were supported in studies of four Angus steers with cannulae inserted into both the rumen and duodenum. After the steers were inoculated, ruminal, duodenal, and fecal samples were cultured periodically over the course of the infection. The predominant location of E. coli O157:H7 persistence was the lower GIT. E. coli O157:H7 was rarely cultured from the rumen or duodenum after the first week postinoculation, and this did not predict if animals went on to shed the bacteria for 1 week or 1 month. These findings suggest the colon as the site for E. coli O157:H7 persistence and proliferation in mature ruminant animals.