Enterotoxigenic Escherichia Coli Research Articles

Influence of hydrometeorological risk factors on child diarrhea and enteropathogens in rural Bangladesh.

A number of studies have detected relationships between weather and diarrhea. Few have investigated associations with specific enteric pathogens. Understanding pathogen-specific relationships with weather is crucial to inform public health in low-resource settings that are especially vulnerable to climate change. Our objectives were to identify weather and environmental risk factors associated with diarrhea and enteropathogen prevalence in young children in rural Bangladesh, a population with high diarrheal disease burden and vulnerability to weather shifts under climate change. We matched temperature, precipitation, surface water, and humidity data to observational longitudinal data from a cluster-randomized trial that measured diarrhea and enteropathogen prevalence in children 6 months-5.5 years from 2012-2016. We fit generalized additive mixed models with cubic regression splines and restricted maximum likelihood estimation for smoothing parameters. Comparing weeks with 30°C versus 15°C average temperature, prevalence was 3.5% higher for diarrhea, 7.3% higher for Shiga toxin-producing Escherichia coli (STEC), 17.3% higher for enterotoxigenic E. coli (ETEC), and 8.0% higher for Cryptosporidium. Above-median weekly precipitation (median: 13mm; range: 0-396mm) was associated with 29% higher diarrhea (adjusted prevalence ratio 1.29, 95% CI 1.07, 1.55); higher Cryptosporidium, ETEC, STEC, Shigella, Campylobacter, Aeromonas, and adenovirus 40/41; and lower Giardia, sapovirus, and norovirus prevalence. Other associations were weak or null. Higher temperatures and precipitation were associated with higher prevalence of diarrhea and multiple enteropathogens; higher precipitation was associated with lower prevalence of some enteric viruses. Our findings emphasize the heterogeneity of the relationships between hydrometeorological variables and specific enteropathogens, which can be masked when looking at composite measures like all-cause diarrhea. Our results suggest that preventive interventions targeted to reduce enteropathogens just before and during the rainy season may more effectively reduce child diarrhea and enteric pathogen carriage in rural Bangladesh and in settings with similar meteorological characteristics, infrastructure, and enteropathogen transmission.

178 Effects of L-glutamate and L-aspartate supplementation on systemic and intestinal immune responses of weaned piglets challenged with F18 enterotoxigenic Escherichia coli challenge

Abstract L-glutamate (Glu) and L-aspartate (Asp) are pivotal components in the cellular metabolic pathways and immune regulation of pigs. This study aimed to investigate the effects of Glu and Asp on systemic and intestinal immune responses of weaned piglets challenged with an enterotoxigenic Escherichia coli (ETEC) F18. A total of 49 weaned pigs [initial body weight (BW) = 8.18 ± 1.54 kg] were randomly assigned to one of seven treatments, with seven piglets per treatment. The treatments included negative control (NC) and positive control (PC) that were fed with control diet without or with F18 enterotoxigenic Escherichia coli (ETEC) challenge, respectively. The other five dietary treatments were supplemented with either 1% or 2% Glu or Asp, or 50 mg/kg of Carbadox, and were challenged with ETEC. All pigs except for NC were challenged with F18 ETEC orally for three consecutive days at the dose of 1010 CFU•dose-1•d-1. The study period lasted for 3 wk, with a 7-d adaptation period and 14 d post-inoculation (PI). Blood samples were collected on d 0 before ETEC inoculation, and on d 2, 5, and 14 PI to analyze systemic inflammatory markers by the enzyme-linked immunosorbent assays. On d 14 PI, intestinal mucosa from jejunum and ileum were collected to analyze the mRNA expression of inflammatory cytokines by quantitative polymerase chain reaction. All data were analyzed with ANOVA using the PROC MIXED of SAS with pig as the experimental unit. Among all treatments, pigs fed with carbadox had the least (P < 0.05) serum TNF-α, haptoglobin, and C-reactive protein throughout the experiment. Pigs supplemented with 1% Glu had similar concentrations of TNF-α, haptoglobin, and C-reactive protein, while pigs fed with 2% Asp had similar serum haptoglobin compared with pigs fed with carbadox. Compared with PC, supplementing Glu and Asp reduced (P < 0.05) the mRNA expression of IL12 in jejunal mucosa. Pigs fed with carbadox tended to have greater (P < 0.10) expression of IFNG in ileal mucosa than pigs in PC, while no difference was observed in IFNG expression among 1% Glu, 1% or 2% Asp, and carbadox groups. In addition, pigs fed with 1% Glu or 2% Asp had greater (P < 0.05) expression of IL12 in ileal mucosa than pigs in PC. However, dietary treatments did not affect the gene expression of TNFA, IL1B and TGFB in jejunal and ileal mucosa of pigs. In conclusion, results of the present study indicate that dietary supplementation of 1% Glu or 2% Asp affected both systemic and intestinal immune responses of ETEC challenged weaned pigs with similar trend as the supplementation of carbadox.

PSIV-A-3 Effect of fiber and stimbiotic on growth performance and the gut microbiome in pigs challenged with F18 enterotoxigenic Escherichia coli (ETEC)

Abstract Dietary fiber has been shown to promote a healthy gut microbiota, while stimbiotics enhance fermentable substances and boost the expansion of the commensal microbiome, improving host health by alleviating deleterious effects of proinflammatory responses. The intended purpose of this study was to gain an understanding of the effectiveness of dietary fiber sources and stimbiotic on the sustainability of F18 enterotoxigenic Escherichia coli (ETEC) challenge. A total of 216 piglets were blocked (n = 8) by weaning body weight (BW) and randomly assigned to 1 of 4 dietary treatments, including corn-SBM based control diet (CC), high fiber diet (HF: 5.0, 3.8. 2.5% sugar beet pulp and 4.0, 3.0 and 2.0% soy hulls for phases 1, 2, and 3, respectively), CC plus 0.01% stimbiotic (CS: Signis, ABVISTA Marlborough, UK), and HF plus 0.01% stimbiotic (FS). Pigs were fed a four-phase feeding regimen: phase 1 (d 0-9), phase 2 (d 9-16), phase 3 (d 16-22), phase 4 (d 22-41). Dietary fiber and stimbiotic were provided from weaning (d 0) to d 22, and then a common diet was fed from d 22 to the end of the trial. On d 5 postweaning, all pigs were orally inoculated with 10 mL of F18 ETEC (108 CFU/mL). One median BW pig from each pen was selected at weaning for fecal sampling. Before the challenge, the same pigs were sampled on d 5 and again on d 8 and d12. Samples were used to quantify the E. Coli using 3M Petrifilm (3M Food Safety, St. Paul, MN) and virulence genes of the isolate were qualified using multiplex PCR. Colony-forming-count was log-transformed. All data normality was verified using Univariate procedure and analyzed using Mixed procedure of SAS (Cary, NC). Pigs fed both CS and HF increased average daily gain (ADG) in phase 2 and phase 1-3 compared with pigs fed CC diets, while adding stimbiotic had no additional benefit to HF (Fiber×Stimbotic P = 0.03 and 0.06, respectively). This response led to heavier BW in pigs fed CS at the end of phase 3 compared with pigs fed CC with HF and FS as intermediate (Fiber×Stimbotic P = 0.06, respectively). Overall, average daily feed intake (ADFI) was greater in pigs fed CS than pigs fed FS while no difference was found between CC and CF (Fiber×Stimbotic P = 0.02). Dietary fiber did not impact gain to feed ratio (G:F); however, G:F was improved in stimbiotic fed pigs in phase 2 (Stimbiotic P = 0.01). Total coliform count was reduced from d 2 to d 7 (day; P < 0.01), but neither fiber nor stimbiotic altered the coliform count. Results of this study demonstrated that both high fiber and stimbiotic help restore ADG when pigs were challenged with ETEC. Additionally, stimbiotic also benefited feed efficiency and resulted in the heaviest BW.

206 Evaluation of the effect of oral administration of bivalent VHH constructs in weaned piglets challenged with enterotoxigenic Escherichia coli F4+

Abstract Following the European restriction on the use of antibiotics and zinc oxide (ZnO) in livestock, the search for novel and sustainable strategies to reduce the incidence of enteric infections in piglets caused by Escherichia coli (colibacillosis) has increased. Among them, the ongoing development of bivalent VHH constructs designed to bind toxins and/or virulence factors of pathogens, appears to hold potential. The aim of this study was to evaluate the effect of in-feed administration of bivalent VHH constructs designed to bind to heat-labile enterotoxin (LT) and F4 fimbria of enterotoxigenic E. coli (ETEC) in orally challenged piglets. A total of 96, 21-d old, weaned piglets were assigned to three experimental diets: control diet (CTR); diet supplemented with 3,100 ppm ZnO (ZnO); or diet supplemented with 1.71 kg/MT bivalent VHH constructs (VHH). Animals on the VHH treatment also received an oral booster dose (15 mgּ pig-1ּ d-1) of the same product just before the challenge and one d later. Pigs were orally challenged with ETEC F4+ (4.62×109 colony forming unit/animal), 1 wk after weaning (d 28). Data on growth, feed intake and clinical health signs of the animals were recorded throughout the 15 d of the study. On d 4 and 8 post inoculation (PI), fecal samples, digestive contents, and ileal scrapings were taken for microbiological plate counts of lactobacilli and ETEC in selective media. Ileal tissue samples were also taken for histomorphology. After the challenge, animals in the supplemented groups (ZnO and VHH) showed statistically greater growth rates than the CTR (28, 112 and 106 g/d (0-4 PI period) for CTR, ZnO and VHH, respectively, P = 0.018; Table 1). The experimental ZnO and VHH groups showed numerical reductions in the incidence of diarrhea at d 2 PI (60, 29 and 40 % for CTR, ZnO and VHH, P = 0.107), in agreement with reduced fecal ETEC counts also registered at d 8 PI in the VHH group compared with CTR (5.77 vs. 7.21 cfu/g, P = 0.046), and also greater lactobacilli/ETEC ratio (d 8 PI: 1.52 vs. 1.20, P = 0.071). Histomorphology of ileal samples showed significantly greater numbers of intraepithelial lymphocytes cells (IEL) with VHH supplementation compared with ZnO, with CTR showing intermediate values (8.06, 5.80 and 9.46 cell nº/villi for CTR, ZnO and VHH respectively, P = 0.045). The results from this study suggest that bivalent VHH constructs can reduce the negative effects of an oral ETEC F4+ challenge through reducing their growth in the gut, improving intestinal mucosa architecture and function, and increasing piglet growth rate. With results similar to those observed in pigs supplemented with ZnO, bivalent VHH constructs appear as a promising alternative technology for the management of post-weaning colibacillosis.

307 Awardee Talk: Effects of dietary valerate glycerides on weanling piglet diarrhea and immunity

Abstract The use of various feed additives for their non-nutrient, immunomodulatory qualities is under investigation for application in swine production, especially during the post-weaning period. Butyrate (C4) is a known modulator of intestinal physiology, produced endogenously by commensal microbes and found in other organic sources. However, the understanding of the role of valerate (C5) as a feed additive for pigs has not been well-evaluated. Two experiments were conducted utilizing glycerides of butyrate or valerate as feed additives for weanling piglets under enterotoxigenic Escherichia coli (ETEC) infection conditions. In both experiments, daily diarrhea scores were recorded to monitor the frequency of diarrhea. The first pilot experiment utilized 20 weanling piglets (21 to 24 d of age) housed individually and assigned to either control diet, or control diet supplemented with 0.10% butyrate glycerides or 0.10% valerate glycerides. This experiment also aimed to establish a coinfection disease model, administering two strains of ETEC at a dose of 0.5 × 109 CFU/1.5 mL for each strain after a 7-d adaptation period (F4 and F18; two highly prevalent strains in swine production). Overall, piglets had significantly (P < 0.05) decreased frequency of diarrhea in either treatment group compared with control, suggesting similar modulatory effects by dietary valerate glycerides and butyrate glycerides. In addition, pigs fed valerate glycerides tended (P = 0.061) to have less neutrophils and had significantly (P < 0.05) less serum TNF-α on d 4 post-inoculation (PI). Moving forward, a full-scale experiment was performed using 60 weanling piglets (21 to 24 d of age) to further investigate the effect of dietary valerate glycerides on piglet health under ETEC F18 infection conditions. Treatments included nursery basal diet (control), 0.075% or 0.10% monovalerin, or 0.1% trivalerin added to control. Pigs fed 0.1% trivalerin exhibited significantly (P < 0.05) reduced frequency of diarrhea and fecal shedding of ETEC F18. Serum samples were collected throughout the experimental period and six replicate pigs per treatment group were sacrificed on d 7 PI to collect intestinal samples for morphological analysis and intestinal mucosa RT-qPCR analysis. Pigs fed trivalerin had reduced (P < 0.05) serum TNF-α on d 0 before inoculation and d 6 PI compared with pigs in control. The mRNA expression of TNFA in ileal mucosa from pigs fed 0.1% monovalerin tended (P < 0.10) to be lower compared with control. In addition, ZO1 mRNA expression tended (P < 0.10) to be increased in jejunal mucosa from pigs fed 0.1% trivalerin. Overall, the second experiment indicated that dietary supplementation of valerate glycerides reduced diarrhea and fecal shedding of ETEC F18 in piglets infected with ETEC. In conclusion, both experiments provide evidence in support of supplementing 0.075 or 0.10% valerate glycerides could enhance disease resistance of weanling piglets experiencing ETEC-associated diarrhea.

177 Modulation of fecal microbiome by phytogenics, a postbiotic, and mix of organic acids with essential oils in weaned pigs subjected to F18 Escherichia coli challenge

Abstract This study evaluated the effects of bioactive feed ingredients on fecal microbiome of weaned pigs subjected to F18 enterotoxigenic Escherichia coli (ETEC) challenge. Two blocks of weaned pigs [n = 72; Yorkshire/Landrace/Duroc; initial body weight (BW) = 6.62 ± 1.37 kg] were balanced by gender and litter and randomly allocated to one of the six treatments with 12 pens/block and 6 pigs/pen. Treatments included: 1) basal diet (NC), 2) NC + 0.1% phytogenic (Fresta Protect, Delacon, Austria; PFP), 3) NC + 0.2% postbiotic (Dia-V Nursery, Diamond V, IA; PB), 4) NC + 0.2% new phytogenic prototype (Delacon, Austria; NPP), 5) NC + 0.2% organic acid and essential oil blend (OA+EO), and 6) NC + 0.1% phytogenic (Fresta Protect) + 0.2% postbiotic (Dia-V Nursery; PFP+PB). The trial lasted 15 d, with a 7-d pre-challenge and 8-d post-challenge periods. Pigs were orally inoculated with 5 x 1010 CFU/mL of F18 ETEC (O147:H2, LT:STb:STa:Stx2:F18) on d 8 and 9 of study after 5-h fasting. Fecal swabs were collected before challenge (d 8) and 3- and 8-d post-challenge (d 10 and 15 of study) and subjected to shotgun metagenomics. Diarrhea score and E. coli shedding (CFU/mL) were monitored daily and analyzed using a multi-level mixed effect ordinal logistic regression with block and pen as random effects. Microbiome data were center-log-ratio transformed and analyzed for diversity, abundance, and interaction networks. At 3-d post-challenge, the OA+EO, PFP+PB and NPP supplemented pigs had reduced (P < 0.001) E. coli shedding compared with NC. Diarrhea score tended to be less (P = 0.054) in PFP+PB fed pigs 5-d post-challenge and was less (P = 0.02) in PFP+PB, OA+EO, and NPP 6-d post-challenge than NC. Alpha-diversity declined (P < 0.01) 3-d post-challenge with most reduction in PFP and OA+EO but recovered 8-d post-challenge except for PFP and OA+EO (P < 0.01). At the baseline and 8-d post-challenge, the largest number of differentially abundant taxa (P < 0.05) were observed in PFP+PB compared with NC. The PFP+PB reduced the abundances of Escherichia.sp000208585 and Campylobacter.P585 which were negatively correlated (P < 0.01) with average daily gain (ADG) and BW, and positively correlated (P < 0.01) with diarrhea score and E. coli shedding. The PFP+PB increased beneficial species including Prevotella.sp900543585, Mitsuokella jalaludinii, and Faecalibacterium prausnitzii with the first two positively correlated (P < 0.01) with ADG and BW, and negatively correlated (P < 0.01) with diarrhea score and E. coli shedding. By d-8 post-challenge, PFP+PB microbial network had the highest degree of negative connections suggestive of a more competitive microbial ecosystem against opportunistic microorganisms compared with NC. In conclusion, co-supplementation of Fresta Protect phytogenic and postbiotic promoted a microbiome with greater beneficial and reduced detrimental bacteria resulting in low diarrhea score post-challenge.

Protecting the piglet gut microbiota against ETEC-mediated post-weaning diarrhoea using specific binding proteins

Post-weaning diarrhoea (PWD) in piglets presents a widespread problem in industrial pig production and is often caused by enterotoxigenic E. coli (ETEC) strains. Current solutions, such as antibiotics and medicinal zinc oxide, are unsustainable and are increasingly being prohibited, resulting in a dire need for novel solutions. Thus, in this study, we propose and evaluate a protein-based feed additive, comprising two bivalent heavy chain variable domain (VHH) constructs (VHH-(GGGGS)3-VHH, BL1.2 and BL2.2) as an alternative solution to manage PWD. We demonstrate in vitro that these constructs bind to ETEC toxins and fimbriae, whilst they do no affect bacterial growth rate. Furthermore, in a pig study, we show that oral administration of these constructs after ETEC challenge reduced ETEC proliferation when compared to challenged control piglets (1-2 log10 units difference in gene copies and bacterial count/g faeces across day 2–7) and resulted in week 1 enrichment of three bacterial families (Prevotellaceae (estimate: 1.12 ± 0.25, q = 0.0054), Lactobacillaceae (estimate: 2.86 ± 0.52, q = 0.0012), and Ruminococcaceae (estimate: 0.66 ± 0.18, q = 0.049)) within the gut microbiota that appeared later in challenged control piglets, thus pointing to an earlier transition towards a more mature gut microbiota. These data suggest that such VHH constructs may find utility in industrial pig production as a feed additive for tackling ETEC and reducing the risk of PWD in piglet populations.

A Novel Multiepitope Fusion Antigen as a Vaccine Candidate for the Prevention of Enterotoxigenic E. coli-Induced Calf Diarrhea

Calf diarrhea caused by enterotoxigenic E. coli (ETEC) poses an enormous economic challenge in the cattle industry. Fimbriae and enterotoxin are crucial virulence factors and vaccine targets of ETEC. Since these proteins have complicated components with large molecular masses, the development of vaccines by directly expressing these potential targets is cumbersome Therefore, this study aimed to develop a multiepitope fusion antigen designated as MEFA by integrating major epitopes of FanC and Fim41a subunits and a toxoid epitope of STa into the F17G framework. The 3D modeling predicted that the MEFA protein displayed the epitopes from these four antigens on its surface, demonstrating the desired structural characteristics. Then, the MEFA protein was subsequently expressed and purified for mouse immunization. Following that, our homemade ELISA showed that the mouse antiserum had a consistent increase in polyclonal antibody levels with the highest titer of 1:217 to MEFA. Furthermore, the western blot assay demonstrated that this anti-MEFA serum could react with all four antigens. Further, this antiserum exhibited inhibition on ETEC adhesion to HCT-8 cells with inhibitory rates of 92.8%, 84.3%, and 87.9% against F17+, F5+, and F41+ ETEC strains, respectively. Additionally, the stimulatory effect of STa toxin on HCT-8 cells was decreased by approximately 75.3% by anti-MEFA serum. This study demonstrates that the MEFA protein would be an antigen candidate for novel subunit vaccines for preventing ETEC-induced diarrhea in cattle.

Construction of the ETECFinder database for the characterization of enterotoxigenic Escherichia coli (ETEC) and revision of the VirulenceFinder web tool at the CGE website.

Detecting colonization factors in enterotoxigenic Escherichia coli (ETEC) is challenging due to their large number, heterogeneity, and lack of standardized tests. Therefore, it is important to include these ETEC-related genes in a more comprehensive VirulenceFinder database in order to obtain a more complete coverage of the virulence gene repertoire of pathogenic types of E. coli. ETEC vaccines are of great importance due to the severity of the infections, primarily in children. A tool such as this could assist in the surveillance of ETEC in order to determine the prevalence of relevant types in different parts of the world, allowing vaccine developers to target the most prevalent types and, thus, a more effective vaccine.

Carrier state of enterotoxigenic Escherichia coli virulence markers in pigs: Effects on gut microbiota modulation and immune markers transcription

Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in pigs at early age, leading to high mortality rates and significant economic losses in the swine industry. ETEC effect on gut microbiota and immune system is mostly studied in diarrheic model under controlled laboratory conditions, however its impact on asymptomatic carriers remains unknown. Thus, we investigated whether ETEC can modulate gut microbiota or regulate the transcription of immune markers in asymptomatic pigs in farm environment. Stool samples from newborn piglets, nursery and growing pigs, and sows were screened for ETEC markers, then submitted to 16S-rDNA sequencing to explore gut microbiota composition in carriers (ETEC+) and non-carriers (ETEC-) animals. We observed a reduced α-diversity in ETEC+ animals (p < 0.05), while bacterial compositions were mostly driven by ageing (p > 0.05). Prevotella marked ETEC-carrier group, while Rikenellaceae RC9 gut group was a marker for a healthy gut microbiota, suggesting that they might be biomarker candidates for surveillance and supplementation purposes. Furthermore, we observed transcription regulation of il6 and tff2 genes in ETEC+ in newborn and nursery stages, respectively. Our findings indicate that ETEC presence modulate gut microbiota and the immune response in asymptomatic pigs; nevertheless, further studies using a probabilistic design must be performed to assess the effect of ETEC presence on gut imbalance in pigs despite the age bias.

Field evaluation of a simple and rapid diagnostic test, RLDT to detect Shigella and enterotoxigenic E. coli in Indian children

The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected additional 12% Shigella and 13% ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.

Validation of a Human Challenge Model Using an LT-Expressing Enterotoxigenic E. coli Strain (LSN03-016011) and Characterization of Potential Amelioration of Disease by an Investigational Oral Vaccine Candidate (VLA1701).

Controlled human infection models are important tools for the evaluation of vaccines against diseases where an appropriate correlate of protection has not been identified. Enterotoxigenic Escherichia coli (ETEC) strain LSN03-016011/A (LSN03) is an LT enterotoxin and CS17-expressing ETEC strain useful for evaluating vaccine candidates targeting LT-expressing strains. We sought to confirm the ability of the LSN03 strain to induce moderate-to-severe diarrhea in a healthy American adult population, as well as the impact of immunization with an investigational cholera/ETEC vaccine (VLA-1701) on disease outcomes. A randomized, double-blinded pilot study was conducted in which participants received two doses of VLA1701 or placebo orally, one week apart; eight days after the second vaccination, 30 participants (15 vaccinees and 15 placebo recipients) were challenged with approximately 5 × 109 colony-forming units of LSN03. The vaccine was well tolerated, with no significant adverse events. The vaccine also induced serum IgA and IgG responses to LT. After challenge, 11 of the placebo recipients (73.3%; 95%CI: 48.0-89.1) and 7 of the VLA1701 recipients (46.7%; 95%CI: 24.8-68.8) had moderate-to-severe diarrhea (p = 0.26), while 14 placebo recipients (93%) and 8 vaccine recipients (53.3%) experienced diarrhea of any severity, resulting in a protective efficacy of 42.9% (p = 0.035). In addition, the vaccine also appeared to provide protection against more severe diarrhea (p = 0.054). Vaccinees also tended to shed lower levels of the LSN03 challenge strain compared to placebo recipients (p = 0.056). In addition, the disease severity score was lower for the vaccinees than for the placebo recipients (p = 0.046). In summary, the LSN03 ETEC challenge strain induced moderate-to-severe diarrhea in 73.3% of placebo recipients. VLA1701 vaccination ameliorated disease severity, as observed by several parameters, including the percentage of participants experiencing diarrhea, as well as stool frequency and ETEC severity scores. These data highlight the potential value of LSN03 as a suitable ETEC challenge strain to evaluate LT-based vaccine targets (NCT03576183).

Development and application of one-step multiplex Real-Time PCR for detection of three main pathogens associated with bovine neonatal diarrhea.

Neonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive. In this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+. The established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves' diarrhea in China. The established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​.

The Incidence and Risk Factors for Enterotoxigenic E. coli Diarrheal Disease in Children under Three Years Old in Lusaka, Zambia.

This study aimed to estimate the incidence and risk factors for Enterotoxigenic Escherichia coli (ETEC) diarrhea. This was a prospective cohort study of children recruited in a household census. Children were enrolled if they were 36 months or below. A total of 6828 children were followed up passively for 12 months to detect episodes of ETEC diarrhea. Diarrheal stool samples were tested for ETEC using colony polymerase chain reaction (cPCR). Among the 6828 eligible children enrolled, a total of 1110 presented with at least one episode of diarrhea. The overall incidence of ETEC diarrhea was estimated as 2.47 (95% confidence interval (CI): 2.10-2.92) episodes per 100 child years. Children who were HIV-positive (adjusted Hazard ratio (aHR) = 2.14, 95% CI: 1.14 to 3.99; p = 0.017) and those whose source of drinking water was public tap/borehole/well (aHR = 2.45, 95% CI: 1.48 to 4.06; p < 0.002) were at increased risk of ETEC diarrhea. This study found that children whose mothers have at least senior secondary school education (aHR = 0.49, 95% CI: 0.29 to 0.83; p = 0.008) were at decreased risk of ETEC diarrhea. Our study emphasizes the need for integrated public health strategies focusing on water supply improvement, healthcare for persons living with HIV, and maternal education.

Preparation of Bispecific IgY-scFvs Inhibition Adherences of Enterotoxigenic Escherichia coli (K88 and F18) to Porcine IPEC-J2 Cell.

Enterotoxigenic Escherichia coli (ETEC) strains are significant contributors to postweaning diarrhea in piglets. Of the ETEC causing diarrhea, K88 and F18 accounted for 92.7%. Despite the prevalence of ETEC K88 and F18, there is currently no effective vaccine available due to the diversity of these strains. This study presents an innovative approach by isolating chicken-derived single-chain variable fragment antibodies (scFvs) specific to K88 and F18 fimbrial antigens from chickens immunized against these ETEC virulence factors. These scFvs effectively inhibited adhesion of K88 and F18 to porcine intestinal epithelial cells (IPEC-J2), with the inhibitory effect demonstrating a dose-dependent increase. Furthermore, a bispecific scFv was designed and expressed in Pichia pastoris. This engineered construct displayed remarkable potency; at a concentration of 25.08 μg, it significantly reduced the adhesion rate of ETEC strains to IPEC-J2 cells by 72.10% and 69.11% when challenged with either K88 or F18 alone. Even in the presence of both antigens, the adhesion rate was notably decreased by 57.92%. By targeting and impeding the initial adhesion step of ETEC pathogenesis, this antibody-based intervention holds promise as a potential alternative to antibiotics, thereby mitigating the risks associated with antibiotic resistance and residual drug contamination in livestock production. Overall, this study lays the groundwork for the development of innovative treatments against ETEC infections in piglets.

Epidemiology of enterotoxigenic Escherichia coli and impact on the growth of children in the first two years of life in Lima, Peru.

Enterotoxigenic E. coli (ETEC) is a leading cause of diarrheal morbidity and mortality in children, although the data on disease burden, epidemiology, and impact on health at the community level are limited. In a longitudinal birth cohort study of 345 children followed until 24 months of age in Lima, Peru, we measured ETEC burden in diarrheal and non-diarrheal samples using quantitative PCR (LT, STh, and STp toxin genes), studied epidemiology and measured anthropometry in children. About 70% of children suffered from one or more ETEC diarrhea episodes. Overall, the ETEC incidence rate (IR) was 73 per 100 child-years. ETEC infections began early after birth causing 10% (8.9-11.1) ETEC-attributable diarrheal burden at the population level (PAF) in neonates and most of the infections (58%) were attributed to ST-ETEC [PAF 7.9% (1.9-13.5)] and LT + ST-ETEC (29%) of which all the episodes were associated with diarrhea. ETEC infections increased with age, peaking at 17% PAF (4.6-27.7%; p = 0.026) at 21 to 24 months. ST-ETEC was the most prevalent type (IR 32.1) with frequent serial infections in a child. The common colonization factors in ETEC diarrhea cases were CFA/I, CS12, CS21, CS3, and CS6, while in asymptomatic ETEC cases were CS12, CS6 and CS21. Only few (5.7%) children had repeated infections with the same combination of ETEC toxin(s) and CFs, suggested genotype-specific immunity from each infection. For an average ETEC diarrhea episode of 5 days, reductions of 0.060 weight-for-length z-score (0.007 to 0.114; p = 0.027) and 0.061 weight-for-age z-score (0.015 to 0.108; p = 0.009) were noted in the following 30 days. This study showed that ETEC is a significant pathogen in Peruvian children who experience serial infections with multiple age-specific pathotypes, resulting in transitory growth impairment.

Screening Traditional Foods for the Prevention of Enterotoxigenic Escherichia coli K88ac (F4ac) Attachment to IPEC-J2 Cells.

Enterotoxigenic Escherichia coli (ETEC) is the major diarrhoea-causing pathogen world-wide. Fimbria-receptor recognition is the primary step when attachment of ETEC to the intestine occurs. This study aims to evaluate the potential of some traditional foods, particularly those rich in β-glucans, as analogues for fimbriae or receptors in reducing ETEC colonisation. The adhesion test (AT) demonstrated that aqueous extracts of highland barley (EHB), black rice (EBR) and little millet (ELT) at concentrations of 2% and 1% could attach to more ETEC K88ac (p < 0.001), as well as aqueous extracts of shiitake (EST) (p < 0.01). The competition test (CT) revealed that EHB and EST significantly prevented ETEC K88ac from adhering to intestinal epithelial cells (IPEC-J2) at 2% (p < 0.01) and 1% (p < 0.05). In the Exclusion Test (ET) and the displacement test (DT), the food samples were unable to impair ETEC colonisation in terms of blocking receptors or removing attached pathogens. These results demonstrate how some traditional foods such as highland barley and shiitake contain bioactive compounds that interfere with the attachment of ETEC to the intestinal epithelium, and their potential in the prevention and treatment of ETEC diarrhoea.

Characteristics of Pathogenic Escherichia coli Associated with Diarrhea in Children under Five Years in Northwestern Ethiopia.

Diarrheagenic Escherichia coli (DEC) are the leading cause of infectious diarrhea and pose a significant global, regional, and national burden of disease. This study aimed to investigate the prevalence of six DEC pathotypes in children with diarrhea and determine their antibiotic resistance patterns. Samples from 107 diarrheagenic children were collected and processed for Escherichia coli (E. coli). Single-plex PCR was used to detect target virulence genes as well as characterize and categorize DEC pathotypes. Antibiotic resistance patterns were determined by the Kirby-Bauer disk diffusion method. E. coli was detected in 79 diarrheal stool samples, accounting for 73.8% of the samples collected. Additionally, 49.4% (39 out of 79) of the isolates harbored various typical virulence factors. Results revealed six pathotypes of virulence: enterotoxigenic E. coli (ETEC) (53.8%), enteropathogenic E. coli (EPEC) (12.8%), enteroaggregative E. coli (EAEC) (10.3%), Heteropathotypes (7.8%), Shiga toxin-producing E. coli (STEC), and enterohemorrhagic E. coli (EHEC) (7.7% each). The isolates exhibited high antibiotic resistance against trimethoprim/sulfamethoxazole (82.1%), amoxicillin (79.5%), ampicillin (74.4%), gentamicin (69.2%), and streptomycin (64.1%). An overall occurrence of 84.6% of multiple-drug resistance was observed in the isolates, with resistance ranging from three to four antibiotic classes. Our findings revealed a high level of pathogenic E. coli that were highly resistant to multiple categories of antibiotics among children in the Awi zone. These findings highlight the potential role of pathogenic E. coli in childhood diarrhea in tropical low-resource settings and underscore the need for continued research on the characteristics of pathogenic and antibiotic-resistant strains.

Ovomucin Hydrolysates Reduce Bacterial Adhesion and Inflammation in Enterotoxigenic Escherichia coli (ETEC) K88-Challenged Intestinal Epithelial Cells.

Enterotoxigenic Escherichia coli (ETEC) K88 is the most common cause of diarrhea in neonatal and postweaning pigs. After adhering to small intestinal epithelial cells via glycoprotein receptor recognition, the pathogen can produce enterotoxins, impair intestinal integrity, trigger watery diarrhea, and induce inflammation via nuclear factor κB (NF-κB) and mitogen-activated protein kinase phosphatase (MAPK) pathways. Inhibiting ETEC K88 adhesion to cell surfaces by interfering with the receptor-fimbriae recognition provides a promising strategy to prevent the initiation and progression of infection. Ovomucin is a highly glycosylated protein in chicken egg white with diverse bioactivities. Ovomucin hydrolysates prepared by the enzymes Protex 26L (OP) and pepsin/pancreatin (OPP) were previously revealed to prevent adhesion of ETEC K88 to IPEC-J2 cells. Herein, we investigated the protective effects of ovomucin hydrolysates on ETEC K88-induced barrier integrity damage and inflammation in IPEC-J2 and Caco-2 cells. Both hydrolysates inhibited ETEC K88 adhesion to cells and protected epithelial cell integrity by restoring transepithelial electronic resistance (TEER) values. Removing sialic acids in the hydrolysates reduced their antiadhesive capacities. Ovomucin hydrolysates suppressed ETEC-induced activation of NF-κB and MAPK signaling pathways in both cell lines. The ability of ETEC K88 in activating calcium/calmodulin-dependent protein kinase 2 (CaMK II), elevating intracellular Ca2+ concentration, and inducing oxidative stress was attenuated by both hydrolysates. In conclusion, this study demonstrated the potential of ovomucin hydrolysates to prevent ETEC K88 adhesion and alleviate inflammation and oxidative stress in intestinal epithelial cells.

Application of Recombinant Lactic Acid Bacteria (LAB) Live Vector Oral Vaccine in the Prevention of F4+ Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in piglets. The current primary approach for ETEC prevention and control relies on antibiotics, as few effective vaccines are available. Consequently, an urgent clinical demand exists for developing an effective vaccine to combat this disease. Here, we utilized food-grade Lactococcus lactis NZ3900 and expression plasmid pNZ8149 as live vectors, together with the secreted expression peptide Usp45 and the cell wall non-covalent linking motif LysM, to effectively present the mutant LTA subunit, the LTB subunit of heat-labile enterotoxin, and the FaeG of F4 pilus on the surface of recombinant lactic acid bacteria (LAB). Combining three recombinant LAB as a live vector oral vaccine, we assessed its efficacy in preventing F4+ ETEC infection. The results demonstrate that oral immunization conferred effective protection against F4+ ETEC infection in mice and piglets lacking maternal antibodies during weaning. Sow immunization during late pregnancy generated significantly elevated antibodies in colostrum, which protected piglets against F4+ ETEC infection during lactation. Moreover, booster immunization on piglets during lactation significantly enhanced their resistance to F4+ ETEC infection during the weaning stage. This study highlights the efficacy of an oral LAB vaccine in preventing F4+ ETEC infection in piglets by combining the sow immunization and booster immunization of piglets, providing a promising vaccination strategy for future prevention and control of ETEC-induced diarrhea in piglets.