A four-season longitudinal study of enterohaemorrhagic Escherichia coli in beef cow-calf herds in Mississippi and Nebraska.

Our objective was to describe the probability of detecting seven serogroups of enterohaemorrhagic Escherichia coli (EHEC-7) of public health importance in faecal samples from beef cow-calf herds and to test for factors associated with their detection. Fresh faecal samples (n = 85) from two Mississippi and two Nebraska herds were collected in each of four seasons. Samples were tested for each EHEC-7 serogroup by a molecular screening assay. Separate management groups within herds were sampled, and group-level factors were recorded. To measure the effects of factors on faecal shedding of EHEC-7, separate multivariable logistic regression models were used, accounting for the random effect of clustering by group within farm. Statistical significance was set α = 0.05. Fifty-nine samples (4.3%) were positive for EHEC O26, and Nebraska samples were more likely to be positive than Mississippi samples (OR = 12.4, 95% CI: 1.1, 139.2). Forty-four samples (3.2%) were positive for EHEC O45. Odds for detection were greater in the summer than all other seasons combined (OR = 4.2, 95% CI: 1.3, 14.0), and odds decreased if a precipitation event occurred (OR = 0.07, 95% CI: 0.006, 0.8). EHEC O103 was detected in 66 samples (4.9%) with increased probability to be detected at increased temperature. EHEC O111 was detected in 71 samples (5.2%), and 43 samples (3.2%) were positive for EHEC O145. Both EHEC O111 and O145 were associated separately with season, with greater probability for detection in the summer. Eighteen (1.3%) and 68 (5.0%) samples were positive for EHEC O121 and EHEC O157, respectively. We failed to detect significant explanatory factors associated with probability to detect EHEC O121 or O157. Factors that vary by time and place, such as precipitation, ambient temperature, region and season, are uniquely associated with the probability to detect EHEC-7 in fresh faeces collected from cow-calf herds.

Cross-sectional study to estimate the prevalence of enterohaemorrhagic Escherichia coli on hides of market beef cows at harvest.

Cattle hides are an important source of enterohaemorrhagic Escherichia coli (EHEC) carcass contamination at slaughter. Seven EHEC serogroups are adulterants in raw, non-intact beef: EHEC O26, O45, O103, O111, O121, O145 and O157. The objective of this study was to estimate the probability for hide contamination with EHEC among US market beef cows at slaughter and to test the effects of season and geographic region on prevalence of hide contamination. Hides (n = 800) of market cows were swabbed at slaughter immediately after exsanguination, prior to hide removal. Cows were sampled from two geographically distinct beef packing plants during four seasons of 2015. Cattle source was categorized by northern or southern region. Samples were tested for EHEC by a molecular screening assay. The effects of region, season and their interaction on the probability of hide contamination by each EHEC serogroup were tested in separate multilevel multivariable logistic regression models, accounting for the random effect of clustering by plant. Statistical significance was set α = .05. Of 800 total samples, at least one EHEC was detected on 630 (79%) hides. Enterohaemorrhagic E. coli O26 was detected on 129 (16%) of all hides sampled, EHEC O45 on 437 (55%), EHEC O103 on 289 (36%), EHEC O111 on 189 (24%), EHEC O121 on 140 (18%), EHEC O145 on 171 (21%) and EHEC O157 on 89 (11%). Detection of EHEC O26 and EHEC O121 was associated with season. Season and region were associated with detecting EHEC O45 and EHEC O157. Season-by-region interactions were associated with the outcome of detecting EHEC O103, EHEC O111 and EHEC O145. Season, region of origin and the interaction of these factors affect hide contamination of market beef cattle at slaughter by EHEC, and each serogroup responds to these factors uniquely.

A randomized controlled trial to evaluate the effects of dietary fibre from distillers grains on enterohemorrhagic Escherichia coli detection from the rectoanal mucosa and hides of feedlot steers.

Feeding high levels (≥40% dry matter) of distillers grains may increase the risk for cattle to carry enterohemorrhagic Escherichia coli (EHEC) O157. The mechanism for the increased risk is not known nor whether non-O157 EHEC are similarly affected. Our objective was to test whether the fibre content or other components of modified distillers grains plus solubles (MDGS) affects the probability for cattle to carry EHEC serogroups of public health importance. A 2 × 2 plus 1 factorial treatment arrangement within a randomized block design was utilized. Within each of four blocks, 25 feedlot pens (n = 8 steers/pen) were assigned randomly to (i) corn-based control diet; (ii) 20% dry matter (DM) MDGS; (iii) 40% DM MDGS; (iv) corn bran added to corn-based diet to match fibre of 20% MDGS or (v) 40% MDGS. Rectoanal mucosa swabs (RAMS) were collected on day (d)0, d35, d70 and d105; hide swabs were collected on the last feeding day. Samples were tested for EHEC by a molecular screening assay. The effects of fibre source and fibre level on EHEC carriage were tested using multilevel logistic regression (generalized linear mixed models; α = 0.05). EHEC O45 RAMS detection was associated with fibre level, source and sampling day. EHEC O103 RAMS detection increased by feeding 40% MDGS but not the corresponding corn bran diet. Hide contamination by EHEC O45 or O103 was less likely in cattle fed MDGS compared to corn bran diets. EHEC O111 RAMS detection decreased by feeding 40% MDGS but not by feeding the corresponding corn bran diet. Detection of EHEC O157 or O145 was not associated with dietary factors. Feeding 40% MDGS increased the probability for carriage of some EHEC serogroups but decreased probability of others, which indicated that EHEC serogroups have different risk factors associated with feeding MDGS and little association with dietary fibre.

Isotype-specific antibody responses against Escherichia coli O157:H7 locus of enterocyte effacement proteins in adult beef cattle following experimental infection.

Escherichia coli O157:H7 is an important food-borne pathogen and cause of hemorrhagic colitis and hemolytic uremic syndrome in humans. Cattle are an important reservoir of E. coli O157:H7, in which the organism colonizes the intestinal tract and is shed in the feces. Vaccination of cattle has significant potential as a pre-harvest intervention strategy for E. coli O157:H7; however, basic information about the bovine immune responses to important bacterial colonization factors resulting from infection has not been reported. The serum and fecal IgG and IgA antibody responses of adult cattle to E. coli O157:H7 intimin, translocated intimin receptor (Tir), E. coli-secreted proteins (Esp)A, EspB and O157 lipopolysaccharide (LPS) in response to infection were determined. All animals were seropositive for all five antigens prior to inoculation, with antibody titers to EspB and O157 LPS significantly higher (P<0.05) than those to Tir, intimin and EspA. After inoculation, the cattle became colonized and developed significant increases in their serum antibody titers to intimin, Tir, EspB, EspA and O157 LPS (P<0.05); however, by 42 days post-inoculation the titers to all except EspB were on the decline. In contrast, pre- and post-inoculation fecal IgG and IgA antibodies to these same antigens were not detected (<1:5). These results indicate that cattle respond serologically to E. coli O157:H7 type III secreted proteins, intimin and O157 LPS during the course of infection and the response is correlated with the extent of fecal shedding.

Efficacy of dose regimen and observation of herd immunity from a vaccine against Escherichia coli O157:H7 for feedlot cattle.

A clinical trial was conducted to test the effect of a vaccine product containing type III secreted proteins of Escherichia coli O157:H7 on the probability that feedlot steers shed E. coli O157:H7 in feces. Six hundred eight same-source steers were utilized. Of these, 480 steers were assigned randomly to 60 pens (eight head per pen) and to one of four vaccination treatments (120 cattle per treatment, two head per treatment per pen). The four treatments were (i) no vaccination; (ii) one dose, vaccinated once at reimplant (day 42); (iii) two doses, vaccinated on arrival (day 0) and again at reimplant (day 42); and (iv) three doses, vaccinated on arrival (day 0), on day 21, and again at reimplant (day 42). The remaining 128 steers were assigned randomly to 12 pens within the same feedlot to serve as unvaccinated external controls. The probability of detecting E. coli O157:H7 among cattle receiving different doses of vaccine was compared with that of unvaccinated external control cattle, accounting for clustering by repeated measures, block, and pen and fixed effects of vaccine, corn product, and test period. Vaccine efficacy of receiving one, two, and three doses of vaccine was 68, 66, and 73%, respectively, compared with cattle in pens not receiving vaccine. Cattle receiving three doses of vaccine were significantly less likely to shed E. coli O157:H7 than unvaccinated cattle within the same pen. Unvaccinated cattle housed with vaccinated cattle were 59% less likely to shed E. coli O157:H7 than cattle in pens not receiving vaccine, likely because they benefited from herd immunity. This study supports the hypothesis that vaccination with this vaccine product effectively reduces the probability for cattle to shed E. coli O157:H7. There was no indication that the vaccine affected performance or carcass quality. In addition, we found that vaccinating a majority of cattle within a pen offered a significant protective effect (herd immunity) to unvaccinated cattle within the same pen.

Effect of a vaccine product containing type III secreted proteins on the probability of Escherichia coli O157:H7 fecal shedding and mucosal colonization in feedlot cattle.

Preharvest intervention strategies to reduce Escherichia coli O157:H7 in cattle have been sought as a means to reduce human foodborne illness. A blinded clinical trial was conducted to test the effect of a vaccine product on the probability that feedlot steers, under conditions of natural exposure, shed E. coli O157:H7 in feces, are colonized by this organism in the terminal rectum, or develop a humoral response to the respective antigens. Steers (n = 288) were assigned randomly to 36 pens (eight head per pen), and pens were randomized to vaccination treatment in a balanced fashion within six dietary treatments of an unrelated nutrition study. Treatments included vaccination or placebo (three doses at 3-week intervals). Fecal samples for culture (n = 1,410) were collected from the rectum of each steer on pretreatment day 0 and posttreatment days 14, 28, 42, and 56. Terminal rectum mucosal (TRM) cells were aseptically collected for culture at harvest (day 57 posttreatment) by scraping the mucosa 3.0 to 5.5 cm proximal to the rectoanal junction. E. coli O157:H7 was isolated and identified with selective enrichment, immunomagnetic separation, and PCR confirmation. Vaccinated cattle were 98.3% less likely to be colonized by E. coli O157:H7 in TRM cells (odds ratio = 0.014, P < 0.0001). Diet was also associated with the probability of cattle being colonized (P = 0.04). Vaccinated cattle demonstrated significant humoral responses to Tir and O157 lipopolysaccharide. These results provide evidence that this vaccine product reduces E. coli O157:H7 colonization of the terminal rectum of feedlot beef cattle under conditions of natural exposure, a first step in its evaluation as an effective intervention for food and environmental safety.

Effect of Lactobacillus acidophilus strain NP51 on Escherichia coil O157:H7 fecal shedding and finishing performance in beef feedlot cattle.

A 2-year study was conducted during the summer months (May to September) to test the effectiveness of feeding Lactobacillus acidophilus strain NP51 on the proportion of cattle shedding Escherichia coli O157:H7 in the feces and evaluate the effect of the treatment on finishing performance. Steers (n = 448) were assigned randomly to pens, and pens of cattle were assigned randomly to NP51 supplementation or no supplementation (control). NP51 products were mixed with water and applied as the feed was mixed daily in treatment-designated trucks at the rate of 10(9) CFU per steer. Fecal samples were collected (n = 3,360) from the rectum from each animal every 3 weeks, and E. coli O157:H7 was isolated by standard procedures, using selective enrichment, immunomagnetic separation, and PCR confirmation. The outcome variable was the recovery of E. coli O157:H7 from feces, and was modeled using logistic regression accounting for year, repeated measures of pens of cattle, and block. No significant differences were detected for gain, intakes, or feed efficiency of control or NP51-fed steers. The probability for cattle to shed E. coli O157:H7 varied significantly between 2002 and 2003 (P = 0.004). In 2002 and 2003, the probability for NP51-treated steers to shed E. coli O157:H7 over the test periods was 13 and 21%, respectively, compared with 21 and 28% among controls. Over the 2 years, NP51-treated steers were 35% less likely to shed E. coli O157: H7 than were steers in untreated pens (odds ratio = 0.58, P = 0.008). This study is consistent with previous reports that feeding NP51 is effective in reducing E. coli O157:H7 fecal shedding in feedlot cattle.

Anti-capsular antibodies activate killing of Escherichia coli O8:K87 by the alternate complement pathway in porcine serum.

Enterotoxigenic Escherichia coli (ETEC) strains that produce K88 (F4)+ fimbria are important causes of diarrhea and post-diarrheal septicemia in swine. ETEC O8:K87, a serotype represented by a number of these strains, is typically serum resistant. Strain-specific antibodies are known to activate alternative C pathway-mediated killing of other serum-resistant E. coli [Hill, A.W., Shears, A.L., Hibbitt, K.G., 1978. The requirement of specific antibody for the killing of E. coli by the alternate complement pathway in bovine serum. Immunology 34, 131-136], but their antigenic targets have not been determined. We tested the hypothesis that anti-K87 antibodies activate alternative pathway-mediated killing of ETEC O8:K87. Pigs were immunized with ETEC O8:K87 strain 2534-86 cells or purified K87 polysaccharide. Post-, but not pre-immunization sera killed 2534-86 cells, and absorption with 2534-86 cells or by K87 affinity chromatography eliminated bactericidal activity. Complementation of absorbed serum with anti-K87 antibodies restored bactericidal activity, confirming the ability of these antibodies to activate C-mediated serum killing. Serum from age-matched, non-vaccinated control pigs also killed 2534-86. This activity was eliminated by absorption with 2534-86 cells, but not K87 affinity chromatography, indicating that specific non-capsular antibodies are also able to activate C-mediated killing. In all cases, Mg-EGTA-treated serum was as effective as non-treated serum in killing, suggesting that bactericidal activity was mediated predominantly if not exclusively via the alternative C pathway.

Induction of neutralizing antibodies to transmissible gastroenteritis virus by anti-idiotypic antibodies.

The potential of anti-idiotypic antibodies (anti-ids) as immunogens against transmissible gastroenteritis virus (TGEV) was tested in a heterologous system. A month-old pig was immunized with a neutralizing murine monoclonal antibody (MAb, 5A5) of the IgG2a isotype, specific for the E2 glycoprotein of TGEV. The anti-ids were isolated from the serum of the immunized pig by affinity chromatography, initially on a 5A5-Sepharose column, followed by repeated adsorption on a mouse IgG2a column. The swine anti-ids thus obtained bound to the MAb 5A5 (the idiotype), but not to MAbs of the same isotype IgG2a but of different idiotypes. The anti-ids also inhibited the binding of 5A5 to TGEV in a concentration-dependent manner. Mice immunized with the anti-ids produced antibodies to TGEV. These antibodies, neutralized TGEV in vitro and inhibited the binding of 5A5 to TGEV.

Escherichia coli O157:H7 induces attaching-effacing lesions in large intestinal mucosal explants from adult cattle.

Attaching-effacing (A/E) lesions following natural and experimental infection with Escherichia coli O157:H7 have been seen in neonatal and 3-4-month-old weanling but not older cattle. To test the hypothesis that the adult bovine large intestinal epithelium is resistant to the development of A/E lesions, colonic and rectal mucosal tissue explants from 18-month-old steers were inoculated with E. coli O157:H7 and examined. Epithelial cells of inoculated explants developed A/E lesions at the bacterial attachment sites, providing evidence that the large intestinal mucosal epithelium may be a site of infection that contributes to carriage of E. coli O157:H7 in adult cattle.