H7 Shedding Research Articles

Epidemiological studies on Escherichia coli O157:H7 in Egyptian sheep.

In the present work, the epidemiological role of apparently healthy sheep in transmission of Escherichia coli O157:H7 in different seasons was investigated. Fecal samples (convenience sampling) of apparently healthy farmed sheep (three farms, n = 70) and from 15 wandering flocks fed on city wastes (n = 80) in the Giza governorate were examined. The samples were collected in spring under mild weather conditions and during hot summer to be compared. Out of the 150 animals, 13 (8.7%) were E. coli O157 shedders. The 13 ovine sorbitol-negative E. coli O157 were characterized by different PCR sets. The eae gene was detected in 11 isolate (85%), stx1 in 3 isolates (23%), stx2 in 8 isolates (62%), and finally the hlyA in 11 isolate (85%). Among the 13 isolates, 2 strains (15%) were positive for eae, stx1, stx2, and hlyA as gene combination, one isolate (8%) for eae, stx1, and hlyA, 5 isolates (38%) for eae, stx2, and hlyA, 1 isolate (8%) for eae and stx2, 2 isolates (15%) contained eae and hlyA, 1 isolate (8%) contained hlyA only, and finally, 1 isolate (8%) did not contain any of these genes. None of the isolates showed the gene combination eae stx1, stx1 hlyA, or stx2 hlyA. The results indicated significant association of unfavorable weather and management conditions on O157:H7 shedding while the age or sex did not play any role in this process.

Impact of Infection Dose and Previous Serum Antibodies against the Locus of Enterocyte Effacement Proteins on Escherichia coli O157:H7 Shedding in Calves following Experimental Infection.

Escherichia coli O157:H7 is the main causative agent of haemolytic uremic syndrome. Cattle are the main reservoir of these bacteria, and have been shown to develop immune response to colonization. Our aim was to investigate the faecal shedding pattern of E. coli O157:H7 in calves challenged intragastrically with either 108 or 1010 CFU, as well as the ability of specific preexisting antibodies to reduce shedding of the pathogen. Shedding was analysed by direct counting as well as enrichment of rectoanal mucosal swabs. Statistical analysis was performed using a linear model for repeated measures with and without the inclusion of preexisting antibodies against the carboxy-terminal fraction of intimin-γ (γ-intimin C280) as a covariable. Results suggest that there is a statistical difference in the area under the shedding curves between both doses for 14 as well as 28 days after challenge (p = 0.0069 and 0.0209, resp.). This difference is increased when the prechallenge antibodies are taken into account (p = 0.0056 and 0.0185). We concluded that the bacterial dose influences shedding on calves experimentally challenged and that preexisting antibodies against E. coli O157:H7 γ-intimin C280 could partially reduce faecal excretion.

Perspectives on super-shedding of Escherichia coli O157:H7 by cattle.

Escherichia coli O157:H7 is a foodborne pathogen that causes illness in humans worldwide. Cattle are the primary reservoir of this bacterium, with the concentration and frequency of E. coli O157:H7 shedding varying greatly among individuals. The term "super-shedder" has been applied to cattle that shed concentrations of E. coli O157:H7 ≥ 10⁴ colony-forming units/g feces. Super-shedders have been reported to have a substantial impact on the prevalence and transmission of E. coli O157:H7 in the environment. The specific factors responsible for super-shedding are unknown, but are presumably mediated by characteristics of the bacterium, animal host, and environment. Super-shedding is sporadic and inconsistent, suggesting that biofilms of E. coli O157:H7 colonizing the intestinal epithelium in cattle are intermittently released into feces. Phenotypic and genotypic differences have been noted in E. coli O157:H7 recovered from super-shedders as compared to low-shedding cattle, including differences in phage type (PT21/28), carbon utilization, degree of clonal relatedness, tir polymorphisms, and differences in the presence of stx2a and stx2c, as well as antiterminator Q gene alleles. There is also some evidence to support that the native fecal microbiome is distinct between super-shedders and low-shedders and that low-shedders have higher levels of lytic phage within feces. Consequently, conditions within the host may determine whether E. coli O157:H7 can proliferate sufficiently for the host to obtain super-shedding status. Targeting super-shedders for mitigation of E. coli O157:H7 has been proposed as a means of reducing the incidence and spread of this pathogen to the environment. If super-shedders could be easily identified, strategies such as bacteriophage therapy, probiotics, vaccination, or dietary inclusion of plant secondary compounds could be specifically targeted at this subpopulation. Evidence that super-shedder isolates share a commonality with isolates linked to human illness makes it imperative that the etiology of this phenomenon be characterized.

Variable Efficacy of a Vaccine and Direct-Fed Microbial for ControllingEscherichia coliO157:H7 in Feces and on Hides of Feedlot Cattle

To evaluate the efficacy of a type-III secreted proteins vaccine and a Lactobacillus-acidophilus-based direct-fed microbial (DFM) for controlling Escherichia coli O157:H7, cattle (n=864) were allocated to the following groups: DFM, finishing diets containing 10(9) colony-forming units (CFU)/animal/day L. acidophilus and Propionibacterium freudenreichii; VAC, finishing diets and 2 mL intramuscular injection of vaccine at allocation and 28 days later; or CON, finishing diets only. Cattle within replicates were stratified by initial levels of E. coli O157:H7 and randomized to experimental groups, with 30 pens allocated on June 15, 2011 (AS1), 18 pens allocated on June 28, 2011 (AS2), and 18 cattle per pen. Rectal fecal samples and perineal swabs were collected at 28-day intervals until shipment to slaughter (103-145 days on trial). Numbers of cattle with enumerable E. coli O157:H7 (≥1.6 CFU/g feces) were reduced in AS1 and AS2 by VAC (p=0.008), although interventions had no impact on numbers of E. coli O157:H7 shed. For AS1, VAC reduced prevalence of E. coli O157:H7 in feces (p=0.03) and perineal swabs (p=0.04) in the feeding period but not at shipment to slaughter. For AS2, prevalence of E. coli O157:H7 was not reduced in either feces or perineal swabs by VAC at any time. For AS1, DFM reduced prevalence of E. coli O157:H7 in perineal swabs (p=0.01) during the feeding period. For AS2, DFM increased E. coli O157:H7 detection in feces (p=0.03) and perineal swabs (p=0.01) at shipment to slaughter. Seventy-five percent of AS1 E. coli O157:H7 isolates had only stx1, while 87% of AS2 isolates had stx1 and stx2 genes. Of the two interventions, VAC shows the most potential for pre-harvest control of E. coli O157:H7, but due to variable efficacy of both DFM and VAC, additional product development is necessary to ensure more consistent pre-harvest control of E. coli O157:H7.

Underlying Mechanism of Antimicrobial Activity of Chitosan Microparticles and Implications for the Treatment of Infectious Diseases

The emergence of antibiotic resistant microorganisms is a great public health concern and has triggered an urgent need to develop alternative antibiotics. Chitosan microparticles (CM), derived from chitosan, have been shown to reduce E. coli O157:H7 shedding in a cattle model, indicating potential use as an alternative antimicrobial agent. However, the underlying mechanism of CM on reducing the shedding of this pathogen remains unclear. To understand the mode of action, we studied molecular mechanisms of antimicrobial activity of CM using in vitro and in vivo methods. We report that CM are an effective bactericidal agent with capability to disrupt cell membranes. Binding assays and genetic studies with an ompA mutant strain demonstrated that outer membrane protein OmpA of E. coli O157:H7 is critical for CM binding, and this binding activity is coupled with a bactericidal effect of CM. This activity was also demonstrated in an animal model using cows with uterine diseases. CM treatment effectively reduced shedding of intrauterine pathogenic E. coli (IUPEC) in the uterus compared to antibiotic treatment. Since Shiga-toxins encoded in the genome of bacteriophage is often overexpressed during antibiotic treatment, antibiotic therapy is generally not recommended because of high risk of hemolytic uremic syndrome. However, CM treatment did not induce bacteriophage or Shiga-toxins in E. coli O157:H7; suggesting that CM can be a potential candidate to treat infections caused by this pathogen. This work establishes an underlying mechanism whereby CM exert antimicrobial activity in vitro and in vivo, providing significant insight for the treatment of diseases caused by a broad spectrum of pathogens including antibiotic resistant microorganisms.

C-di-GMP signaling regulates E. coli O157:H7 adhesion to colonic epithelium

Escherichia coli O157:H7 is an important foodborne pathogen that causes serious illness in humans at low infectious doses. The main source of infections is beef or greens contaminated with E. coli O157:H7 shed by cattle. Here we investigated the role of c-di-GMP-dependent signal transduction in cattle gut colonization of E. coli O157:H7. To manipulate intracellular c-di-GMP levels, we introduced into E. coli O157:H7 a c-di-GMP specific phosphodiesterase (PDE). Liquid chromatography tandem mass spectrometry analysis confirmed that in E. coli O157:H7, over-expression of PDE decreased c-di-GMP level. Consistent with the altered c-di-GMP level, PDE overexpression resulted in decreased biofilm formation in E. coli O157:H7. Furthermore, this diminished c-di-GMP levels reduced adhesion of E. coli O157:H7 to both cultured HT-29 cells and cattle colon explants. Consistently, mRNA levels of genes involved in adhesion were down-regulated including genes encoding E. coli common pili, long polar fimbriae 1, hemorrhagic coli pilus, as well as intimin and tir. We further observed decreased curli fimbriae synthesis in the strain with decreased c-di-GMP levels, which was supported by the reduction in the transcription of curli large subunit gene csgA and the curli expression regulator gene csgD. Genes for enterocyte effacement encoded regulator (Ler) and type III secretion system effectors, EspA and EspB, were also down-regulated. Collectively, data indicated that c-di-GMP signaling positively regulates E. coli O157:H7 intestinal epithelial cell and tissue colonization and expression of associated adhesion factors.

Efficacy of a vaccine and a direct-fed microbial against fecal shedding of Escherichia coli O157:H7 in a randomized pen-level field trial of commercial feedlot cattle

Our primary objective was to determine the efficacy of a siderophore receptor and porin proteins-based vaccine (VAC) and a Lactobacillus acidophilus-based direct-fed microbial (DFM) against fecal shedding of Escherichia coli O157:H7 in commercial feedlot cattle fed a corn grain-based diet with 25% distiller's grains. Cattle projected to be on a finishing diet during the summer were randomly allocated into 40 study pens within ten blocks based on allocation dates. Blocks were complete; each of the four pens within a block was randomly assigned one treatment: control, VAC, DFM, or VAC+DFM. The DFM was fed (106CFU/animal/day of Lactobacillus) throughout the study periods (84–88 days) and cattle were vaccinated at enrollment and again three weeks later. Fresh fecal samples (30/pen) from pen floors were collected weekly for four consecutive weeks (study days 52–77). Two concurrent culture procedures were used to enable estimates of E. coli O157:H7 shedding prevalence and prevalence of high shedders. From 4800 total samples, 1522 (31.7%) were positive for E. coli O157:H7 and 169 (3.5%) were considered high shedders. Pen-level linear mixed models were used for data analyses. There were no significant interactions among treatments and time of sampling. However, vaccinated pens had lower (P<0.01) overall prevalence of E. coli O157:H7 (model-adjusted mean ±SEM=17.4±3.95%) and lower (P<0.01) prevalence of high shedders (0.95±0.26%) than unvaccinated pens (37.0±6.32% and 4.19±0.81%, respectively). There was no evidence of a DFM effect on either measure of E. coli O157:H7 shedding. Results indicate that a two-dose regimen of the vaccine significantly reduces fecal prevalence of E. coli O157:H7 (vaccine efficacy of 53.0%) and prevalence of E. coli O157:H7 high shedders (vaccine efficacy of 77.3%) in commercial feedlot cattle reared in the summer on a finishing diet with 25% distiller's grains.

Effects of wheat or corn distillers dried grains with solubles on feedlot performance, fecal shedding, and persistence of Escherichia coli O157:H7

Distillers dried grains with solubles (DDGS) are a coproduct of the ethanol industry and are often used as a replacement for grain in livestock production. Feeding corn DDGS to cattle has been linked to increased fecal shedding of Escherichia coli O157:H7, although in Canada, DDGS are often produced from wheat. This study assessed the effects of including 22.5% wheat or corn DDGS (DM basis) into barley-based diets on performance, carcass characteristics, animal health, and fecal E. coli O157:H7 shedding of commercial feedlot cattle. Cattle (n = 6,817) were randomly allocated to 10 pens per treatment group: WDDGS (diets including 22.5% wheat DDGS), CDDGS (diets including 22.5% corn DDGS), or CTRL (barley substituted for DDGS). Freshly voided fecal pats (n = 588) were collected and pooled monthly for fecal pH measurement and screened for naturally occurring E. coli O157:H7 by immunomagnetic separation (IMS) and direct plating (DP). Hide swabs (n = 367) were collected from randomly selected cattle from each pen before slaughter. Pen-floor fecal samples (n = 18) were collected from treatment groups at entry to the feedlot (<14 d on the finishing diet) and after adapting to the finishing diet for ≥ 14 d, inoculated (10(9) cfu of a 5 strain naldixic acid-resistant E. coli O157:H7 mixture), incubated (20°C) and evaluated weekly (IMS and DP) to assess fecal E. coli O157:H7 persistence. The WDDGS group had 3.0% poorer ADG (P = 0.007), 5.3% poorer G:F (P < 0.001), and a decreased proportion of Canada Quality Grade AAA carcasses (P = 0.022) compared with CTRL cattle. The CDDGS group had a similar ADG (P = 0.06), a decreased proportion of Canada Yield Grade (YG) 1 (P < 0.001), and greater proportions of Canada YG 2 (P = 0.003) and YG 3 (P < 0.001) carcasses compared with the CTRL group. There were no differences among groups in any of the animal health parameters assessed. Inclusion of DDGS in cattle finishing diets had no effect on fecal shedding (P = 0.650) or persistence (P = 0.953) of E. coli O157:H7. However, feces from cattle on starter diets <14 d had longer persistence of E. coli O157:H7 (week) than cattle on finishing diets ≥ 14 d (P < 0.003). Inclusion of DDGS in feedlot diets depends on commodity pricing relative to that of barley and for WDDGS must also include the risk of feedlot performance and carcass grading disadvantages. Feeding cattle barley based-diets with 22.5% corn or wheat DDGS did not affect fecal shedding of E. coli O157:H7.

Vaccination of pregnant cows with EspA, EspB, γ-intimin, and Shiga toxin 2 proteins from Escherichia coli O157:H7 induces high levels of specific colostral antibodies that are transferred to newborn calves

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of intestinal disease and hemolytic uremic syndrome, a serious systemic complication that particularly affects children. Cattle are primary reservoirs for EHEC O157:H7 and the main source of infection for humans. Vaccination of cattle with different combinations of bacterial virulence factors has shown efficacy in decreasing EHEC O157:H7 shedding. It is, therefore, important to demonstrate whether vaccination of pregnant cows with EHEC O157:H7 induces high titers of transferable antibodies to avoid early colonization of calves by the bacteria. In this study we evaluated the ability of EspA, EspB, the C-terminal fragment of 280 amino acids of γ-intimin (γ-intimin C280) and inactivated Shiga toxin (Stx) 2 proteins to induce specific antibodies in colostrum and their passive transference to colostrum-fed calves. Friesian pregnant cows immunized by the intramuscular route mounted significantly high serum and colostrum IgG responses against EspB and γ-intimin C280 that were efficiently transferred to their calves. Antibodies to EspB and γ-intimin C280 were detected in milk samples of vaccinated cows at d 40 postparturition. Significant Stx2-neutralizing titers were also observed in colostrum from Stx2-vaccinated cows and sera from colostrum-fed calves. The results presented showed that bovine colostrum with increased levels of antibodies against EHEC O157:H7 may be obtained by systemic immunization of pregnant cows, and that these specific antibodies are efficiently transferred to newborn calves by feeding colostrum. Hyperimmune colostrum and milk may be an alternative to protect calves from early colonization by EHEC O157:H7 and a possible key source of antibodies to block colonization and toxic activity of this bacterium

Influence of vitamin D on fecal shedding of Escherichia coli O157:H7 in naturally colonized cattle.

Three experiments were conducted to evaluate the influence of vitamin D on fecal shedding of Escherichia coli O157:H7 in cattle. In the first experiment, two groups of cattle (beef and dairy) were assigned to a control treatment or to receive 0.5 × 10(6) IU vitamin D per day via oral bolus for 10 days. Fecal samples were collected before and throughout the dosing period for culture of E. coli O157:H7. No differences were observed for fecal shedding of E. coli O157:H7 among treatments for either beef or dairy animals. Serum concentrations of vitamin D were markedly higher (P < 0.0001) in treated beef cattle but only tended to be higher (P = 0.09) in the dairy cattle. In the second experiment, three successive vitamin D dosages (2,400, 4,800, and 9,600 IU/day; 14 days each) were administered to 14 dairy steers (7 steers served as controls), fecal samples were collected daily, and serum samples were collected weekly throughout the 42-day experimental period. No significant differences in fecal prevalence or serum vitamin D concentrations were observed for any of the vitamin D dosages. A third experiment sampled feedlot cattle (winter and summer) to determine whether serum vitamin D concentrations were correlated with fecal shedding of E. coli O157:H7. A fecal sample and a blood sample were obtained in each season from 60 randomly selected animals (total of 120 fecal samples and 120 corresponding blood samples). As expected, season was highly correlated (r = 0.66) with serum vitamin D concentration with higher concentrations (P < 0.01) observed in the summer. E. coli O157:H7 prevalence (percentage of positive samples) was not highly correlated (r = 0.16) with season, although the correlation tended to be significant (P = 0.08). The proportion of cattle shedding E. coli O157:H7 was 16.7 and 6.7% for the summer and winter collections, respectively. Results of this research do not support a correlation between vitamin D intake and E. coli O157:H7 shedding in cattle.

Effect of transportation on E. coli O157:H7 prevalence and coliform concentrations in feces of feedlot cattle (2012)

Foodborne illness from Escherichia coli O157:H7 is a major concern for the food industry. Contamination of food products can occur at slaughter by contact with hide or feces. Limiting E. coli O157:H7 shedding is important to prevent outbreaks. Previous studies have demonstrated a relationship between stress and levels of pathogens shed in feces. During transport to the slaughterhouse, animals are subjected to large amounts of stress. This stress could increase shedding of E. coli O157:H7 prior to slaughter, and in so doing increase the risk of contamination of beef products by contact with hides or feces. Our objective in this study was to evaluate the effects of transportation on fecal shedding of E. coli 4 and 24 hours after transport compared with non-transported animals.

Effect of a nutritional reconditioning program for thin dairy cattle on body weight, carcass quality, and fecal pathogen shedding

To assess changes in body weight, carcass quality, and fecal pathogen shedding in cull dairy cows fed a high-energy ration for 28 or 56 days prior to slaughter. Randomized clinical trial. 31 adult Holstein dairy cows. Cows were randomly assigned to a control (immediate slaughter) group or a 28-day or 56-day feeding group. Cows in the feeding groups received a high-energy feed and were weighed every 7 days. Carcasses were evaluated by USDA employees. Fecal and blood samples were collected at the start and end of the feeding periods. Body condition score and adjusted preliminary yield grade were significantly increased in both feeding groups, compared with values for the control group; body weight, hot carcass weight, dressing percentage, and ribeye area were significantly increased after 56 days, but not after 28 days, compared with values for the control group. Average daily gain and marbling score were significantly lower after feeding for 28 days versus after 56 days. Prevalence of Escherichia coli O157:H7 shedding in feces decreased from 14% to 5.6%, but this difference was not significant. Cows seropositive for antibodies against bovine leukemia virus that had signs of lymphoma and lame cows had a low average daily gain. Net loss was $71.32/cow and $112.80/cow for the 28-day and 56-day feeding groups, respectively. Feeding market dairy cows improved body condition and carcass quality. Cows seropositive for antibodies against bovine leukemia virus that have signs of lymphoma and lame cows might be poor candidates for reconditioning.

Escherichia coli O157:H7 populations in ruminants can be reduced by orange peel product feeding.

Foodborne pathogenic bacteria such as Escherichia coli O157:H7 are threats to the safety of beef. Citrus peel and dried orange pulp are by-products from citrus juice production that have natural antimicrobial effects and are often incorporated into least-cost ration formulations for beef and dairy cattle. This study was designed to determine if orange peel and pulp affected E. coli O157:H7 populations in vivo. Sheep (n = 24) were fed a cracked corn grain-based diet that was supplemented with a 50-50 mixture of dried orange pellet and fresh orange peel to achieve a final concentration (dry matter basis, wt/wt) of 0, 5, or 10% pelleted orange peel (OP) for 10 days. Sheep were artificially inoculated with 10(10) CFU of E. coli O157:H7 by oral dosing. Fecal shedding of E. coli O157:H7 was measured daily for 5 days after inoculation, after which all animals were humanely euthanized. At 96 h postinoculation, E. coli O157:H7 shedding was reduced (P < 0.05) in sheep fed 10% OP. Populations of inoculated E. coli O157:H7 were reduced by OP treatment throughout the gastrointestinal tract; however, this reduction reached significant levels in the rumen (P < 0.05) of sheep fed 10% OP diets. Cecal and rectal populations of E. coli O157:H7 were reduced (P < 0.05) by inclusion of both 5 and 10% OP diets. Our results demonstrate that orange peel products can be used as a preharvest intervention strategy as part of an integrated pathogen reduction scheme.

European Starlings (Sturnus vulgaris) challenged with Escherichia coli O157 can carry and transmit the human pathogen to cattle

European starlings (Sturnus vulgaris) are an invasive species in the United States and are considered a nuisance pest to agriculture. The goal of this study was to determine the potential for these birds to be reservoirs and/or vectors for the human pathogen Escherichia coli O157:H7. Under biosecurity confinement, starlings were challenged with various doses of E. coli O157:H7 to determine a minimum infectious dose, the magnitude and duration of pathogen shedding, and the potential of pathogen transmission among starlings and between starlings and cattle. Birds transiently excreted E. coli O157:H7 following low-dose inoculation; however, exposure to greater than 10(5.5) colony-forming units (CFUs) resulted in shedding for more than 3 days in 50% of the birds. Colonized birds typically excreted greater than 10(3) CFU g(-1) of faeces, and the pathogen was detected for as long as 14 days postinoculation. Cohabitating E. coli O157:H7-positive starlings with culture-negative birds or 12-week-old calves resulted in intra- and interspecies pathogen transmission within 24 h. Likewise, E. coli O157:H7 was recovered from previously culture-negative starlings following 24-h cohabitation with calves shedding E. coli O157:H7. European starlings may be a suitable reservoir and vector of E. coli O157:H7. Given the duration and magnitude of E. coli O157:H7 shedding by European starlings, European starlings should be considered a public health hazard. Measures aimed at controlling environmental contamination with starling excrement, on the farm and in public venues, may decrease food-producing animal and human exposure to this pathogen.

Escherichia coli O157:H7 shedding in vaccinated beef calves born to cows vaccinated prepartum with Escherichia coli O157:H7 SRP vaccine.

Extensive research, intervention equipment, money, and media coverage have been directed at controlling Escherichia coli O157:H7 in beef cattle. However, much of the focus has been on controlling this pathogen postcolonization. This study was conducted to examine the performance, health, and shedding characteristics of beef calves that were vaccinated with an E. coli O157:H7 SRP bacterial extract. These calves had been born to cows vaccinated prepartum with the same vaccine. Cows and calves were assigned randomly to one of four treatments: (i) neither cows nor calves vaccinated with E. coli O157:H7 SRP (CON), (ii) cows vaccinated with E. coli O157:H7 SRP prepartum but calves not vaccinated (COWVAC), (iii) calves vaccinated with E. coli O157:H7 SRP but born to cows not vaccinated (CALFVAC), (iv) cows vaccinated with E. coli O157:H7 SRP prepartum and calves also vaccinated (BOTH). Calves born to vaccinated cows had significantly higher titers of anti-E. coli O157:H7 SRP antibodies (SRPAb) in circulation at branding time (P < 0.001). Upon entry to the feedlot, overall fecal E. coli O157:H7 prevalence was 23 % among calves, with 25 % in the CON treatment group, 19 % in the CALFVAC group, 32 % in the COWVAC group, and 15 % in the BOTH group (P > 0.05). Fecal shedding of E. coli O157 on arrival to the feedlot was not correlated with fecal shedding at slaughter (Spearman's rho = -0.02; P = 0.91). No significant effects of cow or calf E. coli O157:H7 SRP vaccination treatment were found on feedlot calf health or performance (P > 0.05), prevalence of lung lesions or liver abscess (P > 0.05), or morbidity, retreatment, or mortality numbers (P > 0.05). The findings of this study indicate that the timing of vaccination of calves against E. coli O157:H7 may be an important consideration for maximizing the field efficacy of this vaccine.

Evaluation of hha and hha sepB mutant strains of Escherichia coli O157:H7 as bacterins for reducing E. coli O157:H7 shedding in cattle

Escherichia coli O157:H7 colonizes cattle intestines by using the locus of enterocyte effacement (LEE)-encoded proteins. The induction of systemic immune response against LEE-encoded proteins, therefore, will prove effective in reducing E. coli O157:H7 colonization in cattle. The previous studies have demonstrated that a hha (encodes for a hemolysin expression modulating protein) deletion enhances expression of LEE-encoded proteins and a sepB (encodes an ATPase required for the secretion of LEE-encoded proteins) deletion results in intracellular accumulation of LEE proteins. In this study, we demonstrate the efficacy of the hha and hha sepB deletion mutants as bacterins for reducing fecal shedding of E. coli O157:H7 in experimentally inoculated weaned calves. The weaned calves were injected intramuscularly with the bacterins containing 109 heat-killed cells of the hha+ wild-type or hha or hha sepB isogenic mutants, and boosted with the same doses 2- and 4-weeks later. The evaluation of the immune response two weeks after the last booster immunization revealed that the calves vaccinated with the hha mutant bacterin had higher antibody titers against LEE proteins compared to the titers for these antibodies in the calves vaccinated with the hha sepB mutant or hha+ wild-type bacterins. Following oral inoculations with 1010 CFU of the wild-type E. coli O157:H7, the greater numbers of calves in the group vaccinated with the hha or hha sepB mutant bacterins stopped shedding the inoculum strain within a few days after the inoculations compared to the group of calves vaccinated with the hha+ wild-type bacterin or PBS sham vaccine. Thus, the use of bacterins prepared from the hha and hha sepB mutants for reducing colonization of E. coli O157:H7 in cattle could represent a potentially important pre-harvest strategy to enhance post-harvest safety of bovine food products, water and produce.

Subcutaneous and intranasal immunization with Stx2B–Tir–Stx1B–Zot reduces colonization and shedding of Escherichia coli O157:H7 in mice

The type III secretion system of Escherichia coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E. coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXφ of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B–Tir–Stx1B–Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST·Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs. intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against Stx2B–Tir–Stx1B–Zot, Stx2B–Tir–Stx1B and control agent (GST/PBS) were also monitored. Subcutaneous immunization of mice with Stx2B–Tir–Stx1B–Zot induced significant Stx2B–Tir–Stx1B–Zot-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant Stx2B–Tir–Stx1B–Zot-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Mice that were immunized intranasally with Stx2B–Tir–Stx1B–Zot showed dramatically decreased E. coli O157:H7 shedding compared to those of Stx2B–Tir–Stx1B and control agent following experimental infection. Mice immunized subcutaneously with Stx2B–Tir–Stx1B–Zot or Stx2B–Tir–Stx1B both showed reduced shedding in feces, moreover, Stx2B–Tir–Stx1B–Zot did better. These results demonstrate the perspective for the use of Stx2B–Tir–Stx1B–Zot to prevent colonization and shedding of E. coli O157:H7.

Reduction of Escherichia coli O157:H7 Shedding in Cattle by Addition of Chitosan Microparticles to Feed

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.

Vaccination against EHEC: insights from model in sheep

Enterohaemorrhagic Escherichia coli O157:H7 are zoonotic pathogens associated with haemorrhagic colitis (HC) and the haemolytic uremic syndrome (HUS). Ruminants are the main reservoir of this organism and most outbreaks of E. coli O157:H7 infections are food borne. Food contamination by ruminant manure has been reported as the primary source of human infection, therefore inhibition of E. coli O157:H7 colonization and shedding in ruminants could control the risk of human exposure to this pathogen. In the present study a vaccine based on the translocon proteins EspA and EspB and the outer membrane adhesion factor intiminγ significantly reduced faecal shedding of E. coli O157:H7 by orally infected sheep.

Immunization of sheep with a combination of intiminγ, EspA and EspB decreases Escherichia coli O157:H7 shedding

Enterohaemorrhagic Escherichia coli O157:H7 are zoonotic pathogens associated with haemorrhagic colitis (HC) and the haemolytic uremic syndrome (HUS). Ruminants are the main reservoir of this organism and most outbreaks of E. coli O157:H7 infections are food borne. Food contamination by ruminant manure has been reported as the primary source of human infection, therefore inhibition of E. coli O157:H7 colonization and shedding in ruminants could control the risk of human exposure to this pathogen. In the present study a vaccine based on the translocon proteins EspA and EspB and the outer membrane adhesion factor intiminγ significantly reduced faecal shedding of E. coli O157:H7 by orally infected sheep. Protection correlates with serum antibody responses to the defined antigens and validates the targeting of these colonization factors. Whereas vaccination has been described in cattle, this is the first study describing a significant decrease in faecal shedding following systemic immunization of sheep.