Haemorrhagic Colitis Associated with Enterohaemorrhagic \u3ci\u3eEscherichia coli\u3c/i\u3e O165:H25 Infection in a Yearling Feedlot Heifer

Introduction: Enterohaemorrhagic Escherichia coli (EHEC) cause haemorrhagic colitis and haemolytic uraemic syndrome in humans. Although EHEC infection typically results in haemorrhagic colitis in all ages of human patients, in cattle it is usually limited to 1- to 5-week-old nursing calves. Case Presentation: A 1-year-old feedlot beef heifer was moribund with neurological signs and bloody diarrhoea. At necropsy, the colonic mucosa contained multiple grossly visible haemorrhagic erosions, each measuring \u3c1 mm in diameter. Histologically, foci corresponding to the gross erosions had E. coli O165 antigen-positive bacterial rods adherent to the apical surfaces of degenerate and necrotic colonic mucosal epithelial cells in association with attaching and effacing lesions, and also within cytoplasmic vacuoles in some of these cells. An E. coli O165:H25 strain was isolated from the colonic mucosal tissue, and by microarray analysis was found to contain virulence genes corresponding to type III secretion system (T3SS) structure and regulation (cesD, cesT, escD, escF, escN/escV, escR, escT, ler, sepL, sepQ), T3SS effectors (espA, espB, espC, espD, espD, espF, espH, espJ, nleB, nleC, nleD, nleH, tir), serine proteases (eatA, espC, espP), Shiga toxin (stx2), EHEC-haemolysin (ehxA), and adhesins [intimin-ε (eae-ε), type 1 fimbria (fimA, fimB, fimH), type IV pili (pilA, pilB, pilC, pilM, pilP, pilQ) and non-fimbrial adhesin (efa1/lifA)]. Conclusion: To the best of our knowledge, this is the first report of disease in cattle associated with EHEC O165:H25 infection, the oldest bovine EHEC disease case with isolation of the pathogen and the first bovine case to demonstrate grossly evident, haemorrhagic, colonic mucosal erosions associated with EHEC infection

Haemorrhagic Colitis Associated with Enterohaemorrhagic \u3ci\u3eEscherichia coli\u3c/i\u3e O165:H25 Infection in a Yearling Feedlot Heifer

Introduction: Enterohaemorrhagic Escherichia coli (EHEC) cause haemorrhagic colitis and haemolytic uraemic syndrome in humans. Although EHEC infection typically results in haemorrhagic colitis in all ages of human patients, in cattle it is usually limited to 1- to 5-week-old nursing calves. Case Presentation: A 1-year-old feedlot beef heifer was moribund with neurological signs and bloody diarrhoea. At necropsy, the colonic mucosa contained multiple grossly visible haemorrhagic erosions, each measuring \u3c1 mm in diameter. Histologically, foci corresponding to the gross erosions had E. coli O165 antigen-positive bacterial rods adherent to the apical surfaces of degenerate and necrotic colonic mucosal epithelial cells in association with attaching and effacing lesions, and also within cytoplasmic vacuoles in some of these cells. An E. coli O165:H25 strain was isolated from the colonic mucosal tissue, and by microarray analysis was found to contain virulence genes corresponding to type III secretion system (T3SS) structure and regulation (cesD, cesT, escD, escF, escN/escV, escR, escT, ler, sepL, sepQ), T3SS effectors (espA, espB, espC, espD, espD, espF, espH, espJ, nleB, nleC, nleD, nleH, tir), serine proteases (eatA, espC, espP), Shiga toxin (stx2), EHEC-haemolysin (ehxA), and adhesins [intimin-ε (eae-ε), type 1 fimbria (fimA, fimB, fimH), type IV pili (pilA, pilB, pilC, pilM, pilP, pilQ) and non-fimbrial adhesin (efa1/lifA)]. Conclusion: To the best of our knowledge, this is the first report of disease in cattle associated with EHEC O165:H25 infection, the oldest bovine EHEC disease case with isolation of the pathogen and the first bovine case to demonstrate grossly evident, haemorrhagic, colonic mucosal erosions associated with EHEC infection

Prevalence and Level of Enterohemorrhagic \u3ci\u3eEscherichia coli\u3c/i\u3e in Culled Dairy Cows at Harvest

The primary objective of this study was to determine the prevalence and level of enterohemorrhagic Escherichia coli (EHEC) O26, O45, O103, O111, O121, and O145 (collectively EHEC-6) plus EHEC O157 in fecal, hide, and preintervention carcass surface samples from culled dairy cows. Matched samples (n=300) were collected from 100 cows at harvest and tested by a culture-based method and two molecular methods: NeoSEEK STEC (NS) and Atlas STEC EG2 Combo. Both the culture and NS methods can be used to discriminate among the seven EHEC types (EHEC-7), from which the cumulative prevalence was inferred, whereas the Atlas method can discriminate only between EHEC O157 and non-O157 EHEC, without discrimination of the serogroup. The EHEC-7 prevalence in feces, hides, and carcass surfaces was 6.5, 15.6, and 1.0%, respectively, with the culture method and 25.9, 64.9, and 7.0%, respectively, with the NS method. With the Atlas method, the prevalence of non-O157 EHEC was 29.1, 38.3, and 28.0% and that of EHEC O157 was 29.1, 57.0, and 3.0% for feces, hides, and carcasses, respectively. Only two samples (a hide sample and a fecal sample) originating from different cows contained quantifiable EHEC. In both samples, the isolates were identified as EHEC O157, with 4.7 CFU/1,000 cm2 in the hide sample and 3.9 log CFU/g in the fecal sample. Moderate agreement was found between culture and NS results for detection of EHEC O26 (k=0.58,

Detection Methods and Intestinal Adherence of non-O157 Shiga Toxin-Producing \u3ci\u3eEscherichia coli\u3c/i\u3e

Shiga toxin-producing Escherichia coli (STEC) are enteric pathogens of humans. Cattle serve as a reservoir and harbor STEC in their intestines. Intimin-positive STEC are referred to as enterohemorrhagic E.coli (EHEC). Seven serogroups (O26, O45, O103, O111, O121, O145, and O157) account for the majority of illness due to STEC and are hereafter referred to as STEC/EHEC-7. To improve detection, enrichment broths were compared for supporting growth of STEC-7 and STEC O104:H4 (STEC-8). In pure culture, STEC enriched in trypticase soy broth (TSB) had significantly greater growth compared to TSB containing antimicrobials. In fecal samples, E. coli broth enrichment yielded growth of STEC-8 that was significantly greater than in TSB. Optimized culture conditions allow for greater detection of EHEC-7 in cattle. To determine the prevalence of EHEC-7 in feedlot cattle, culture-based methods and molecular screening assays were used. In 576 feedlot cattle, EHEC-7 prevalence in hide samples as detected by NeoSEEK (NS) was 80.7% compared to 1.2% by culture. The prevalence of EHEC-7 on carcasses was 6.0% detected by NS. Additionally, EHEC-7 prevalence was determined in 100 culled dairy cows. The EHEC-7 prevalence in feces, hides, and carcasses, respectively, was 6.5%, 15.6%, and 1.0% by culture, and 25.9%, 64.9%, and 7.0% by NS. These studies provide evidence that EHEC are ubiquitous on cattle hides and to a lesser extent feces and carcasses. Given the discordant results, continued improvement in EHEC-7 detection methods is needed. Comparison studies were performed using CHROMagar STEC, Possé differential agar (Possé), Possé with modified antimicrobials, STEC heart infusion washed blood agar with mitomycin C (SHIBAM), and SHIBAM with modified antimicrobials. CHROMagar STEC performed better than Possé or SHIBAM for detection of EHEC-7 in cattle feces, but modifications of the antimicrobials in the latter two media resulted in significant improvements. STEC attachment facilitates colonization of the intestine. All STEC-8 strains tested adhered to bovine colonic explants and Caco-2 cells. One strain invaded both bovine colonic epithelial cells and Caco-2 cells. STEC O104:H4 had significantly higher levels of adherence on Caco-2 cells compared to most STEC. Interventions which block adherence may be effective for pre-harvest control. Advisor: Rodney A. Moxle

Detection methods and intestinal adherence of non-O157 shiga toxin-producing Escherichia coli

DETECTION METHODS AND INTESTINAL ADHERENCE OF NON-O157 SHIGA TOXIN-PRODUCING ESCHERICHIA COLI Zachary R. Stromberg, Ph.D. University of Nebraska, 2015 Advisor: Rodney A. Moxley Shiga toxin-producing Escherichia coli (STEC) are enteric pathogens of humans. Cattle serve as a reservoir and harbor STEC in their intestines. Intimin-positive STEC are referred to as enterohemorrhagic E.coli (EHEC). Seven serogroups (O26, O45, O103, O111, O121, O145, and O157) account for the majority of illness due to STEC and are hereafter referred to as STEC/EHEC-7. To improve detection, enrichment broths were compared for supporting growth of STEC-7 and STEC O104:H4 (STEC-8). In pure culture, STEC enriched in trypticase soy broth (TSB) had significantly greater growth compared to TSB containing antimicrobials. In fecal samples, E. coli broth enrichment yielded growth of STEC-8 that was significantly greater than in TSB. Optimized culture conditions allow for greater detection of EHEC-7 in cattle. To determine the prevalence of EHEC-7 in feedlot cattle, culture-based methods and molecular screening assays were used. In 576 feedlot cattle, EHEC-7 prevalence in hide samples as detected by NeoSEEK (NS) was 80.7% compared to 1.2% by culture. The prevalence of EHEC-7 on carcasses was 6.0% detected by NS. Additionally, EHEC-7 prevalence was determined in 100 culled dairy cows. The EHEC-7 prevalence in feces, hides, and carcasses, respectively, was 6.5%, 15.6%, and 1.0% by culture, and 25.9%, 64.9%, and 7.0% by NS. These studies provide evidence that EHEC are ubiquitous on cattle hides and to a lesser extent feces and carcasses. Given the discordant results, continued improvement in EHEC-7 detection methods is needed. Comparison studies were performed using CHROMagar STEC, Possé differential agar (Possé), Possé with modified antimicrobials, STEC heart infusion washed blood agar with mitomycin C (SHIBAM), and SHIBAM with modified antimicrobials. CHROMagar STEC performed better than Possé or SHIBAM for detection of EHEC-7 in cattle feces, but modifications of the antimicrobials in the latter two media resulted in significant improvements. STEC attachment facilitates colonization of the intestine. All STEC-8 strains tested adhered to bovine colonic explants and Caco-2 cells. One strain invaded both bovine colonic epithelial cells and Caco-2 cells. STEC O104:H4 had significantly higher levels of adherence on Caco-2 cells compared to most STEC. Interventions which block adherence may be effective for pre-harvest control

A recombinant multi-antigen vaccine with broad protection potential against avian pathogenic Escherichia coli.

Chickens are a major source of protein worldwide, yet infectious diseases continue to threaten the poultry industry. Avian pathogenic Escherichia coli (APEC), a subgroup of extraintestinal pathogenic E. coli (ExPEC), causes colibacillosis in chickens resulting in economic loss because of treatment, condemnation of products, and death. In this study, we evaluated a recombinant antigens (rAg) vaccine combining common ExPEC surface proteins EtsC, OmpA, OmpT, and TraT for broad protective potential against APEC infections in chickens. The specific objectives were to evaluate antibody (serum) and cytokines (lymphoid organs) responses to vaccination; in vitro bactericidal ability of serum and splenocytes against multiple APEC serotypes; and in vivo protection against APEC challenge in chickens. Groups of four-day old chickens (N = 10) were vaccinated twice (two-week interval) subcutaneously with rAgs alone or in combination and CpG adjuvant or PBS (control). IgY antibody in the serum and mRNA expression of IL-1β, IL-6, IL-18, IFN-γ, IL-4, IFN-β, and IL-8 in bursa, spleen, and thymus were measured using ELISA and RT-qPCR, respectively. Serum and splenocytes were tested for their bactericidal ability in vitro against multiple APEC isolates. Vaccinated and non-vaccinated chickens were challenged with 108 CFU of APEC-O2 via air sac at 31 days post first vaccination. Vaccine protection was determined by the decrease of bacterial loads in blood and organs (lung, heart, spleen, and liver), as well as gross colibacillosis lesion scores in air sac, heart, and liver. Vaccination significantly (P < 0.05) elicited IgY against specific antigens, induced immune related mRNA expression in the spleen and bursa, reduced in vitro growth of multiple APEC serotypes, and decreased bacterial loads in the heart and spleen, and gross lesion scores of the air sac, heart and liver in chickens. The vaccine reported may be used to provide broad protection against APEC strains, increasing animal welfare and food production

Nucleic Acid-Based Sensing Techniques for Diagnostics and Surveillance of Influenza

Influenza virus poses a threat to global health by causing seasonal outbreaks as well as three pandemics in the 20th century. In humans, disease is primarily caused by influenza A and B viruses, while influenza C virus causes mild disease mostly in children. Influenza D is an emerging virus found in cattle and pigs. To mitigate the morbidity and mortality associated with influenza, rapid and accurate diagnostic tests need to be deployed. However, the high genetic diversity displayed by influenza viruses presents a challenge to the development of a robust diagnostic test. Nucleic acid-based tests are more accurate than rapid antigen tests for influenza and are therefore better candidates to be used in both diagnostic and surveillance applications. Here, we review various nucleic acid-based techniques that have been applied towards the detection of influenza viruses in order to evaluate their utility as both diagnostic and surveillance tools. We discuss both traditional as well as novel methods to detect influenza viruses by covering techniques that require nucleic acid amplification or direct detection of viral RNA as well as comparing advantages and limitations for each method. There has been substantial progress in the development of nucleic acid-based sensing techniques for the detection of influenza virus. However, there is still an urgent need for a rapid and reliable influenza diagnostic test that can be used at point-of-care in order to enhance responsiveness to both seasonal and pandemic influenza outbreaks

Presence of pathogenic \u3ci\u3eEscherichia coli\u3c/i\u3e is correlated with bacterial community diversity and composition on pre-harvest cattle hides

Background: Since 1982, specific serotypes of Shiga toxin-producing Escherichia coli (STEC) have been recognized as significant foodborne pathogens acquired from contaminated beef and, more recently, other food products. Cattle are the major reservoir hosts of these organisms, and while there have been advancements in food safety practices and industry standards, STEC still remains prevalent within beef cattle operations with cattle hides implicated as major sources of carcass contamination. To investigate whether the composition of hide-specific microbial communities are associated with STEC prevalence, 16S ribosomal RNA (rRNA) bacterial community profiles were obtained from hide and fecal samples collected from a large commercial feedlot over a 3-month period. These community data were examined amidst an extensive collection of prevalence data on a subgroup of STEC that cause illness in humans, referred to as enterohemorrhagic E. coli (EHEC). Fecal 16S rRNA gene OTUs (operational taxonomic units) were subtracted from the OTUs found within each hide 16S rRNA amplicon library to identify hide-specific bacterial populations. Results: Comparative analysis of alpha diversity revealed a significant correlation between low bacterial diversity and samples positive for the presence of E. coli O157:H7 and/or the non-O157 groups: O26, O111, O103, O121, O45, and O145. This trend occurred regardless of diversity metric or fecal OTU presence. The number of EHEC serogroups present in the samples had a compounding effect on the inverse relationship between pathogen presence and bacterial diversity. Beta diversity data showed differences in bacterial community composition between samples containing O157 and non-O157 populations, with certain OTUs demonstrating significant changes in relative abundance. Conclusions: The cumulative prevalence of the targeted EHEC serogroups was correlated with low bacterial community diversity on pre-harvest cattle hides. Understanding the relationship between indigenous hide bacterial communities and populations may provide strategies to limit EHEC in cattle and provide biomarkers for EHEC risk assessment

Characterization of Spleen Transcriptome and Immunity Against Avian Colibacillosis After Immunization With Recombinant Attenuated Salmonella Vaccine Strains

Avian pathogenic Escherichia coli (APEC) causes extraintestinal infections in poultry. Vaccines targeting APEC in chickens have been partially successful, but many lack heterologous protection. Recombinant attenuated Salmonella vaccine (RASV) strains can induce broad immunity against Salmonella and be modified to deliver E. coli antigens. Along with vaccine characteristics, understanding the host response is crucial for developing improved vaccines. The objectives of this study were to evaluate host responses to vaccination with an RASV producing E. coli common pilus (ECP) and assess protection against APEC infection in chickens. Four-day-old White Leghorn chickens were unvaccinated or orally vaccinated and boosted 2 weeks later with RASV χ8025(pYA3337), RASV χ8025(pYA4428) carrying ecp operon genes, or a combination of χ8025(pYA3337) and χ8025(pYA4428) (Combo). To assess host responses, serum IgY and intestinal IgA antibody titers were measured, and spleen samples (n = 4/group) were collected from unvaccinated and Combo vaccinated 4-week-old chickens for RNA-seq. Vaccine protection potential against Salmonella and APEC was evaluated in vitro using bacterial inhibition assays. Five-week-old chickens were challenged via air sac with either an APEC O2 or O78 strain. E. coli was enumerated from internal organs, and gross colibacillosis lesions were scored at necropsy. RASV immunized chickens elicited anti-E. coli antibodies. The spleen transcriptome revealed that 93% (89/96) of differentially expressed genes (DEG) were more highly expressed in Combo vaccinated compared to unvaccinated chickens, with signal as the most significantly impacted category. RNA-seq analysis also revealed altered cellular and metabolic processes, response to stimulus after vaccination, and immune system processes. Six DEG including genes linked to transcription regulation, actin cytoskeleton, and signaling were highly positively correlated with antibody levels. Samples from RASV immunized chickens showed protection potential against Salmonella strains using in vitro assays, but a variable response was found for APEC strains. After APEC challenges, significant differences were not detected for bacterial loads or gross lesions scores, but χ8025(pYA3337) immunized and χ8025(pYA4428) immunized chickens had significantly fewer number of APEC-O2-positive samples than unvaccinated chickens. This study shows that RASVs can prime the immune system for APEC infection, and is a first step toward developing improved therapeutics for APEC infections in chickens