Porcine Post-weaning Diarrhoea Research Articles

Survey on resistance occurrence for F4+ and F18+ enterotoxigenic Escherichia coli (ETEC) among pigs reared in Central Italy regions.

Porcine Post Weaning Diarrhoea (PWD) is one of the most important swine disease worldwide, caused by Enterotoxigenic Escherichia coli (ETEC) strains able to provoke management, welfare and sanitary issues. ETEC is determined by proteinaceous surface appendages. Numerous studies conducted by now in pigs have demonstrated, at the enterocytes level, that, the genes mucin 4 (MUC4) and fucosyltransferase (FUT1), coding for ETEC F4 and F18 receptors respectively, can be carriers of single nucleotide polymorphisms (SNPs) associated with natural resistance/susceptibility to PWD. The latter aspect was investigated in this study, evaluating the SNPs of the MUC4 and FUT1 genes in slaughtered pigs reared for the most in Central Italy. Genomic DNA was extracted from 362 swine diaphragmatic samples and then was subjected to the detection of known polymorphisms on MUC4 and FUT1candidate target genes by PCR-RFLP. Some of the identified SNPs were confirmed by sequencing analysis. Animals carrying the SNPs associated with resistance were 11% and 86% for the FUT1 and MUC4 genes respectively. Therefore, it can be assumed that the investigated animals may be an important resource and reservoir of favorable genetic traits for the breeding of pigs resistant to enterotoxigenic E.coli F4 variant.

Easy and reliable assessment of the prevalence of porcine post-weaning diarrhoea.

Post-weaning diarrhoea is a condition of increasing importance due to recent restrictions and bans on the preventive use of antimicrobials and medicinal zinc oxide. For various purposes, it is valuable to monitor the occurrence of post-weaning diarrhoea. The aim of this paper was to propose a protocol for easy and reliable assessment of the prevalence of post-weaning diarrhoea within a section of pigs as an alternative to clinical examination of a random sample of pigs. Two datasets were collected in two different observational field investigations, including more than 4000 individual clinical examinations of newly weaned pigs. First we identified a clinical marker for post-weaning diarrhoea. Second, we drew samples by simulation from our two dataset using different simplified sampling strategies and compared these to conventional random sampling strategies. The prediction error for estimates of the diarrhoea prevalence within a section was compared for the different sampling strategies. The study showed that pigs with diarrhoea had an increased risk of displaying diarrheic soiling of the hind part as well as dull, long, and bristly hair coat, and possibly also hollow flanks and a suboptimal body condition score. Diarrheic soiling of the hind part was the best clinical predictor of diarrhoea; our best estimate of the diagnostic sensitivity was 76.1% (95% credible interval: 72.0, 79.8), and the specificity was 97.1% (95% credible interval: 96.5 0.97.7). Diarrheic soiling of the hind is only a valid clinical predictor of diarrhoea for the first 14 days after insertion into the nursery unit. The precision and accuracy of prevalence estimates were similar for haphazard sampling compared to random sampling of the pigs. Likewise, sampling from a restricted number of pens produced prediction errors similar to sampling across all pens. Yet, the study had limitations regarding sample sizes, and furthermore it is difficult to provide certainty for absence of effects. We recommend sampling pigs haphazardly within at least three randomly selected pens for post-weaning diarrhoea prevalence surveys in order to easily obtain a reliable prevalence estimate. Based on our findings, we conclude the paper by proposing a simple four-step protocol for surveys of the within-section prevalence of post-weaning diarrhoea.

An observational field study of porcine post-weaning diarrhea: clinical and microbiological findings, and fecal pH-measurements as a potential diagnostic tool

BackgroundRecently, in-feed medicinal zinc has been phased out in pig production in the European Union. This makes updated knowledge about porcine post-weaning diarrhea (PWD) crucial. The objectives of the present study were to investigate (i) the clinical presentation of PWD in pigs housed in Danish herds that did not use medicinal zinc, specifically the prevalence of diarrhea and whether PWD was associated to clinical signs of dehydration or altered body temperature; (ii) which microorganism are associated to PWD; and iii) whether measurements of the fecal pH have a potential to be used diagnostically to differentiate between infectious etiologies in cases of PWD.ResultsThe prevalence of diarrhea varied considerably between the outbreaks in the nine studied herds (median = 0.58, range = 0.10; 0.94). In a cross-sectional design (n = 923), diarrhea was associated with reduced rectal temperature and alkaline feces. Diarrhea was also associated with observably reduced skin elasticity, possibly indicating dehydration. In both diarrheic case pigs (n = 87) and control pigs (n = 86), the presence of Brachyspira pilosicoli, Clostridium perfringens, Cryptosporidium spp., Cystoisopora suis, enterotoxigenic Escherichia coli, Lawsonia intracellularis, porcine circovirus types 2 and 3, rotavirus A, B, C, and H, Samonella enterica spp. enterica, and Trichuris suis was described. PWD was associated with high levels of enterotoxigenic E. coli shedding (odds ratio versus no E. coli detection = 4.79 [CI 1.14; 12.62]). Diarrhea was associated with high levels of rotavirus A shedding (odds ratio versus no/low rotavirus A = 3.80 [CI 1.33; 7.97]). The association between microbiological findings in diarrheic pigs and fecal pH was negligible.ConclusionsEnterotoxigenic E. coli was confirmed to be a cause of PWD; however, cases of PWD where enterotoxigenic E. coli was not detected in high levels occurred commonly, and this adds to the increasing evidence suggesting that PWD is not necessarily a result of enteric colibacillosis. Rotaviral enteritis might be a differential diagnosis of PWD. pH-measurements cannot be used to differentiate between differential diagnoses for PWD.

Evidence-Based Recommendations for Herd Health Management of Porcine Post-Weaning Diarrhea.

Simple SummaryIn this paper, you will find recommendations on how to prevent post-weaning diarrhea in pigs kept in indoor pig herds. The recommendations are based on the scientific knowledge that is currently available. The authors first validated that Danish veterinarians working with pigs demanded such recommendations. Then, we collected papers written by other researchers who had summarized the scientific knowledge on different topics related to post-weaning diarrhea. From the papers, we extracted and synthesized 79 specific recommendations that may help veterinarians and pig producers make good decisions for their pig herd. The paper exemplifies a novel approach to summarizing and transferring science into practice that may be of interest to people that are not involved with pigs and post-weaning diarrhea.Aided by their advising veterinarians, pig producers need to make difficult decisions regarding herd health management strategies. For instance, the preventive use of antimicrobials and medicinal zinc oxide must be substituted with more sustainable preventive approaches to porcine post-weaning diarrhea. Veterinarians and pig producers may find assistance in knowledge based on evidence in this regard; however, the overwhelming scientific literature is not always readily available. The overall aim of this paper is to suggest herd health management decision-support tools that can aid veterinary-assisted decision making in the control of porcine post-weaning diarrhea at a tactical level. The first objective was to validate the need for a herd health management concept, including two decision-support tools. The second objective was to develop evidence-based recommendations that can aid veterinary-assisted decision-making for the herd health management of post-weaning diarrhea. The first objective was investigated by a questionnaire-based study among veterinary pig practitioners in Denmark. For the second objective, we conducted a scientific summary based on scientific review papers identified through a systematic search in three databases. From the papers, we synthesized and extracted 79 specific recommendations. In this paper, we report comprehensive evidence-based recommendations for the herd health management of post-weaning diarrhea.

Post-weaning diarrhea in pigs weaned without medicinal zinc: risk factors, pathogen dynamics, and association to growth rate

BackgroundPorcine post-weaning diarrhea (PWD) has reemerged as an important topic in pig production, as common control strategies based on prophylactic use of antimicrobials and zinc oxide have been deemed unsustainable. The objectives of this study were to estimate the cumulative incidence of porcine post-weaning diarrhea with different etiologies in production systems weaning without zinc oxide and prophylactic antimicrobials, to assess risk factors for post-weaning diarrhea, and to estimate the impact of post-weaning diarrhea on growth rate. A cohort study was conducted at two commercial indoor producers weaning without medicinal zinc oxide and prophylactic antimicrobials.ResultsPiglets were included at birth (n = 300) and 272 survived until weaning. After insertion to the nursery units, the piglets were clinically examined every day for 14 days, and rectal swabs were collected and analyzed for enterotoxigenic Escherichia coli (ETEC) and rotavirus A. The cumulative incidences of PWD the first 14 days after insertion to the nursery units were 41.8% (CI 33.6, 50.4) and 51.1% (CI 42.3, 60.0) at the two producers, respectively. We found a low incidence of cases associated to ETEC, and detected a substantial proportion of cases associated to rotavirus. We observed a biphasic pattern in the assumed etiology with rotavirus occurring first, and then a shift towards cases associated to ETEC/non-ETEC hemolytic E. coli. Being offspring of older sows was a protective factor for the development of PWD (Hazard ratio = 0.88 [CI 0.78, 0.99] per unit increase in parity of the dam). Low birth weight reduced the post-weaning growth rate (− 5.2 g/day [CI − 7.5, − 2.9] per 100 g decrease in birthweight) and increased the hazard of developing PWD (Hazard ratio for birthweight below 1100 g: 2.30 [CI 1.41–3.74]). The combined effect of having diarrhea for 2 days or more and receiving antimicrobial treatment was associated with an increased average daily weight gain.ConclusionsThis study suggests novel insights regarding pathogen dynamics and risk factors for PWD in productions not using prophylactic antimicrobials and medicinal zinc. The findings may have important implications for both antimicrobial usage and prevention strategies.

Fecal Microbiota Transplantation Shows Marked Shifts in the Multi-Omic Profiles of Porcine Post-weaning Diarrhea.

Weaning is the most critical phase in pig production and is generally associated with significant impacts on intestinal morphology, structure, physiology, and immune responses, which can lead to subsequent production inefficiencies such as decreases in growth and intake and increases in morbidity and mortality. In the present study, we attempted to explore the effects of fecal microbiota transplantation (FMT) on the fecal microbiota, fecal metabolites, and transcriptome in the jejunum, colon, liver, spleen, and oral mucosa in piglets with post-weaning diarrhea and to evaluate the therapeutic potential of FMT in piglets with post-weaning diarrhea. We found that FMT partially relieved the symptoms of diarrhea in piglets, and microbiota analysis results indicated that unclassified_f_Prevotellaceae was identified as an FMT-associated bacterial family at 66 day and that the Shannon index in the healthy group at 34, 38, and 66 days were higher than that at 21 day. Functional enrichment analysis of the oral mucosa, liver, jejunum, and colon showed that most of the differentially expressed genes (DEGs) were enriched in the terms metabolic process, immune response, and inflammatory response. Moreover, the enriched fecal metabolites focused mostly on apoptosis, beta-alanine metabolism, glutathione metabolism, and sphingolipid metabolism. We tried to detect specific “metabolite-bacterium” pairs, such as “g_Catenisphaera-stigmastentriol,” “p_Bacteroidetes-(6beta,22E)-6-hydroxystigmasta-4,22-dien-3-one,” and “g_Prevotellaceae_NK3B31_group-stenocereol.” Overall, the present study provides a theoretical basis for the alleviation of weaning stress and contributes to the realization of effective and sustainable application of FMT in the pig production industry in the future.

A multivalent vaccine candidate targeting enterotoxigenic Escherichia coli fimbriae for broadly protecting against porcine post-weaning diarrhea

Fimbriae-mediated initial adherence is the initial and critical step required for enterotoxigenic Escherichia coli (ETEC) infection. Therefore, vaccine candidates have been developed that target these fimbriae and induce specific anti-fimbriae antibodies to block initial ETEC attachment. While this vaccine effectively protects against ETEC-associated post-weaning diarrhea (PWD), developing a broadly effective vaccine against initial ETEC attachment remains a challenging problem, owing to the immunological heterogeneity among these antigens. Here, we applied multi-epitope fusion antigen (MEFA) technology to construct a FaeG–FedF–FanC–FasA–Fim41a MEFA using the adhesive subunits of predominant fimbriae K88 and F18 as the backbone, which also integrated epitopes from adhesive subunits of the rare fimbriae K99, 987P, and F41; we then generated a MEFA computational model and tested the immunogenicity of this MEFA protein in immunized mice. We next evaluated the potential of the fimbriae-targeted MEFA as a vaccine candidate to effectively prevent PWD using in vitro assessment of its anti-fimbriae, antibody-directed inhibition of bacterial adherence. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface and mice subcutaneously immunized with the MEFA protein developed IgG antibodies to all five fimbriae. Moreover, anti-fimbriae antibodies induced by the MEFA protein significantly inhibited the adhesion of K88+, F18+, K99+, 987P+, and F41+ ETEC strains to piglet small intestinal IPEC-1 and IPEC-J2 cell lines. Taken together, these results indicate that FaeG–FedF–FanC–FasA–Fim41a MEFA protein induced specific anti-fimbriae neutralizing antibodies against the five targeted fimbriae. Critically, these results show the potential of fimbriae-targeted MEFA and indicate their promise as a broad, effective vaccine against PWD.

Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.

F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.

Expression fusion immunogen by live attenuated Escherichia coli against enterotoxins infection in mice.

SummaryPrevious epidemiological studies have shown that enterotoxins from enterotoxigenic Escherichia coli (ETEC) appear to be the most important causes of neonatal piglet and porcine post‐weaning diarrhoea (PWD). Thus, it is necessary to develop an effective vaccine against ETEC infection. In the present study, the Kil cassette was inserted into the pseudogene yaiT by homologous recombination to create an attenuated E. coli double selection platform O142(yaiT‐Kil). After that, PRPL‐Kil was replaced with a fusion gene (LTA1‐STa13‐STb‐LTA2‐LTB‐STa13‐STb) to establish oral vaccines O142(yaiT::LTA1‐STa13‐STb‐LTA2‐LTB‐STa13‐STb) (ER‐T). Subsequently, BALB/c mice were orally immunized with ER‐T. Results showed that serum IgG and faecal sIgA responded against all ETEC enterotoxins and induced F41 antibody in BALB/c mice by orogastrically inoculation with recombinant E. coli ER‐T. Moreover, the determination of cellular immune response demonstrated that the stimulation index (SI) was significantly higher in immunized mice than in control mice, and a clear trend in the helper T‐cell (Th) response was Th2‐cell (IL‐4) exceed Th1‐cell (IFN‐γ).Our results indicated that recombinant E. coli ER‐T provides effective protection against ETEC infection.

Pathogenic potential and virulence genotypes of intestinal and faecal isolates of porcine post-weaning enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are frequent causes of diarrhoea in infants and in young mammals by inducing attaching effacing (AE) lesions of the intestinal epithelium. EPEC bacteria have also been implicated in cases of porcine post-weaning diarrhoea but their pathogenicity for conventional weaned pigs remains less elucidated.This present study investigates differences in pathogenic potential and virulence genotypes of intestinal and faecal isolates of EPEC from newly-weaned pigs. For this we inoculated ligated ileal loops of four weeks old weaned pigs to assess EPEC adherence to enterocytes by histology and immunohistology. Virulence gene patterns were identified by using a PCR-microarray. Intestinal EPEC isolates of sero−/intimin types O45:H11:eae-β, O49:NM:eae-β, O84:H7:eae-γ, and O123:H11:eae-β formed adherent microcolonies of EPEC with AE lesions on ileal villi more frequently than faecal isolates of O28:H28:eae-NT, O108:H9:eae-β, O145:H28:eae-γ and O157:H2:eae-β (p≤0.05). The PCR-array analysis of both groups detected all together 25 virulence genes of LEE (Locus of Enterocyte Effacement), and of non-LEE pathogenicity islands, of plasmids and phages characteristic to EPEC. Intestinal isolates carried significantly more virulence genes than faecal isolates (p≤0.05). Intestinal isolates possessed efa1, lpfA, and tsh genes most likely contributing to enterocyte adhesion while faecal isolates did not carry these genes (p≤0.05). Overall, the ileal loop model in weaned pigs combined with virulence genotyping PCR-array indicated a greater pathogenic potential of intestinal isolates over faecal isolates of porcine post-weaning EPEC. Differing virulence genotypes of the intestinal and faecal isolates as demonstrated here suggests dynamic evolutionary events within the population of porcine EPEC.

Expression Level of FUT1 Gene in Different Pig Populations and its Relationship with ETEC F18 Resistance

F18- fimbriated Escherichia coli are associated with porcine post-weaning diarrhea. The presence of E. coli F18 receptor on small intestinal villi is the essential requirement for the adhesion and colonization.

Oral immunization of a live attenuated Escherichia coli strain expressing a holotoxin-structured adhesin–toxoid fusion (1FaeG-FedF-LTA2:5LTB) protected young pigs against enterotoxigenic E. coli (ETEC) infection

ETEC strains expressing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of porcine post-weaning diarrhea (PWD). PWD continues causing significant economic losses to swine producers worldwide. Vaccines effectively protecting against PWD are needed. Our recent study revealed that a tripartite adhesin–toxin monomer (FaeG-FedF-LTA2–B) elicited protective antibodies. In this study, we constructed a new adhesin–toxoid fusion, expressed it as a 1A:5B holotoxin-structured antigen (1FaeG-FedF-LT192A2:5LTB) in an avirulent Escherichia coli strain, and evaluated its vaccine potential in pig challenge studies. Piglets orally inoculated with this live strain showed no adverse effects but developed systemic and mucosal antibodies that neutralized cholera toxin and inhibited adherence of K88 and F18 fimbriae in vitro. Moreover, the immunized piglets, when were challenged with ETEC strain 3030-2 (K88ac/LT/STb), had significant fewer bacteria colonized at small intestines and did not develop diarrhea; whereas the control piglets developed severe diarrhea and died. These results indicated the 1FaeG-FedF-LT192A2:5LTB fusion antigen induced protective antiadhesin and antitoxin immunity in pigs, and suggested a live attenuated vaccine can be potentially developed against porcine ETEC diarrhea. Additionally, presenting antigens in a holotoxin structure to target host local mucosal immunity can be used in vaccine development against other enteric diseases.

Production of a Subunit Vaccine Candidate against Porcine Post-Weaning Diarrhea in High-Biomass Transplastomic Tobacco

Post-weaning diarrhea (PWD) in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC) is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD. In this study we used a transplastomic approach in tobacco to produce a recombinant variant of the FaeG protein, rFaeGntd/dsc, engineered for expression as a stable monomer by N-terminal deletion and donor strand-complementation (ntd/dsc). The generated transplastomic tobacco plants accumulated up to 2.0 g rFaeGntd/dsc per 1 kg fresh leaf tissue (more than 1% of dry leaf tissue) and showed normal phenotype indistinguishable from wild type untransformed plants. We determined that chloroplast-produced rFaeGntd/dsc protein retained the key properties of an oral vaccine, i.e. binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. Additionally, the plant biomass matrix was shown to delay degradation of the chloroplast-produced rFaeGntd/dsc in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeGntd/dsc protein could be used for production and, possibly, delivery of an oral vaccine against porcine F4+ ETEC infections. Our findings therefore present a feasible approach for developing an oral vaccination strategy against porcine PWD.

Study on the age-dependent tissue expression of FUT1 gene in porcine and its relationship to E. coli F18 receptor

Escherichia coli (E. coli) that produces adhesin F18 is the main pathogen responsible for porcine post-weaning diarrhea and edema disease. The receptor for E. coli F18 has not been described in pigs, however the alpha (1,2)-fucosyltransferase (FUT1) gene on chromosome 6 has been proposed as a candidate. The objective of this study, therefore, was to investigate the relationship between FUT1 gene expression and E. coli F18 receptor in Sutai pigs of different ages (8-, 18-, 30- and 35-day-old). FUT1 gene expression was detected in 11 pig tissues with the highest level in lung, and expressed consistently at the four time points. In most tissues, FUT1 gene expression levels decreased from days 8 to 18, then continually increased on days 30 and 35, with expression around weaning time higher than that on day 8. Gene ontology and pathway analysis showed that FUT1 was involved in 32 biological processes, mainly those integral to the membrane, or involved in glycosylation, as well as regulation of binding, interestingly participating in three pathways related to glycosphingolipid biosynthesis. From this analysis and the high linkage disequilibrium between the FUT1 gene and the E. coli F18 receptor locus, we can speculate that higher expression of the FUT1 gene in small intestine is beneficial to the formation of receptors to the E. coli F18 strain and is related to the sensitivity to the pathogen.

Investigation of the relationship between SLA-1 and SLA-3 gene expression and susceptibility to Escherichia coli F18 in post-weaning pigs

Porcine post-weaning diarrhea and edema disease are principally caused by Escherichia coli strains that produce F18 adhesin. FUT1 genotyping and receptor binding studies divided piglets into E. coli F18-resistant and -sensitive groups, and the roles of SLA-1 and SLA-3 were investigated. SLA-1 and SLA-3 expression was detected in 11 pig tissues, with higher levels of SLA-1 in lung, immune tissues and gastrointestinal tract, and higher levels of SLA-3 also in lung and lymphoid tissues. Both genes were expressed higher in F18-resistant piglets, and their expression was positively correlated in different tissues; a negative correlation was observed in some tissues of F18-sensitive group, particularly in lung and lymphatic samples. Gene ontology and pathway analyses showed that SLA-1 and SLA-3 were involved in 37 biological processes, including nine pathways related to immune functions. These observations help to elucidate the relationship between SLA class I genes and E. coli F18-related porcine gastrointestinal tract diseases.

Antimicrobial resistance and virulence gene profiles in multi-drug resistant enterotoxigenic Escherichia coli isolated from pigs with post-weaning diarrhoea

This study aimed to characterize antimicrobial resistance and virulence genes in multi-drug resistant enterotoxigenic Escherichia coli (ETEC) isolates ( n = 117) collected from porcine post-weaning diarrhoea cases in Australia (1999–2005). Isolates were serotyped, antibiogram-phenotyped for 12 antimicrobial agents and genotyped by PCR for 30 plasmid-mediated antimicrobial resistance genes (ARGs), 22 intestinal and 38 extraintestinal E. coli virulence genes (VGs). Nine serogroups were identified, the most prevalent being O149 (46.2%), O141 (11.2%) and Ont (31.6%). None of the isolates showed resistance to ceftiofur or enrofloxacin and 9.4% were resistant to florfenicol. No corresponding extended-spectrum/AmpC β-lactamase, fluoroquinolone or floR ARGs were detected. An antimicrobial resistance index (ARI) was calculated from the combined data with a weighting for each antimicrobial agent dependent upon its significance to human health. Serogroup O141 isolates had a significantly higher ARI due to an elevated prevalence of aminoglycoside ARGs and possession of more virulence genes (VGs), including ExPEC or EHEC adhesins ( bmaE, sfa/focDE, fimH, ihA) in toxin-producing strains that lacked the normally associated F4 and F18 fimbriae. Few associations between ARGs and VGs were apparent, apart from tetC, sfa/focDE and ompT which, for a sub-set of O141 isolates, suggest possible plasmid acquisition from ExPEC. The multi-drug resistant ETEC ARG/VG profiles indicate a high probability of considerable strain and plasmid diversity, reflecting various selection pressures at the individual farm level rather than emergence and lateral spread of MDR resistant/virulent clones.

The Escherichia coli O149 rfb gene cluster and its use for the detection of porcine E. coli O149 by real-time PCR

PCR or hybridization assays are widely used for the identification and detection of various Escherichia coli serogroups and serotypes. This study explored this approach for the detection of E. coli O149 in pigs, a dominant serogroup among those associated with porcine post-weaning diarrhea (PWD) worldwide. For this purpose, we sequenced the O antigen (rfb) gene cluster of a representative enterotoxigenic E. coli (ETEC) O149:H10 strain and identified a 2kbp region specific for the O149 rfb gene cluster. This region lacked similarity with sequences in GenBank, except for the rfb gene cluster of the human pathogen Shigella boydii O1 with which it shares 99% sequence identity. Southern blot hybridization with 91 E. coli strains belonging to 62 serogroups confirmed the specificity of the identified region. Restriction analysis of the rfb gene cluster of 38 geographically and chronologically diverse E. coli O149 strains with the endonucleases HindIII, PstI and XhoI demonstrated that this cluster is highly conserved across the serogroup O149. Analysis of the O antigen of a representative O149:H10 ETEC strain by nuclear magnetic resonance confirmed the identity of the O antigen structure in recent and older O149 ETEC strains. Finally, we developed a real-time PCR assay for the detection of E. coli O149 in pig fecal samples by targeting the part of the rfb sequence specific for this serogroup. This method was suitable for a sensitive qualitative detection of O149 E. coli in pig fecal samples after enrichment, but further investigations are needed to find a suitable method for a direct, reproducible and sensitive quantification of O149 E. coli in pig feces.

Effect of plasmid pTENT2 on severity of porcine post-weaning diarrhoea induced by an O149 enterotoxigenic Escherichia coli

A particularly virulent O149:H10 enterotoxigenic Escherichia coli clone harbours a newly characterized plasmid pTENT2 carrying the tetracycline-resistance tetA and the virulence genes estA, paa, and sepA that were not present in less virulent clones. The objectives of this study were to assess whether the additional genes on pTENT2 played a role in the increased severity of post-weaning diarrhoea and if they provided any potential advantage for the emergence of the highly virulent clone. Groups of pigs were dosed orally with isogenic pTENT2-positive and pTENT2-negative ETEC strains, and the clinical and pathological changes were compared between the groups. Two additional groups were given the pTENT2-positive strains and maintained on feed with or without chlortetracycline to assess the effect of subtherapeutic levels of tetracycline on the short-term persistence of the ETEC O149:H10 clone. The severity of diarrhoea within the first few hours post-inoculation was significantly increased ( p = 0.0408) in animals receiving pTENT2-positive strains as compared to animals receiving pTENT2-negative strains. There were no consistent or significant histopathological differences between any of the groups and no significant difference in the persistence of ETEC between groups.

Isolation and characterization of nine bacteriophages that lyse O149 enterotoxigenic Escherichia coli

The goal of this study was to isolate and characterize phages that might be used in prevention and treatment of porcine post-weaning diarrhea due to O149 enterotoxigenic E. coli (ETEC). Serotype O149:H10:F4 was especially targeted because this is the dominant ETEC serotype. Mixtures of 10 strains of O149:H10:F4 ETEC and of 10 O149:H43:F4 ETEC were used as hosts for isolation of phages in sewage from 38 Ontario pig farms. Six phages (GJ1–GJ6) that lysed O149:H10:F4 ETEC and three (GJ7–GJ9) that lysed O149:H43:F4 ETEC were isolated. All phages produced large, clear plaques. All nine phages had necks and contractile tails and therefore belonged to the Myoviridae. Their estimated genome sizes were 48.3–50.7 kb and their restriction enzyme fragments suggested that they were closely related. Phages GJ1–GJ6 lysed 99–100% of 85 O149:H10:F4 ETEC, 0–12% of 42 O149:H43:F4 ETEC, 3–35% of 37 non-O149 porcine ETEC, and 6–68% of the 72 strains of the ECOR collection. Phages GJ7–GJ9 lysed 86–98% of the O149:H43:F4 ETEC, 2–53% of the O149:H10:F4 ETEC, and 24–41% of the non-O149 porcine ETEC. Titres of the nine phages were unaffected by exposure for 16 h to pH 5–9. Among phages GJ1–GJ6, resistance of O149:H10:F4 ETEC to one phage was generally not accompanied by resistance to other phages. It is concluded that the nine phages are suitable candidates for prophylaxis and therapy of porcine post-weaning diarrhea due to O149 ETEC.

Glycosylated F4 (K88) Fimbrial Adhesin FaeG Expressed in Barley Endosperm Induces ETEC-neutralizing Antibodies in Mice

The F4-positive enterotoxigenic Escherichia coli (ETEC) strains are a frequent cause of porcine post-weaning diarrhea. Orally administered F4 fimbriae or FaeG, the major subunit and adhesin of F4, induce a protective mucosal immune response in F4 receptor-positive piglets. Feed plants carrying immunogenic subunit proteins can offer great advantages for oral vaccination of domestic animals. Here, we describe high-level endosperm-specific production (1% of total soluble proteins) of FaeG in the crop plant barley. The endoplasmic reticulum-targeted recombinant endospermic FaeG (erFaeG) was shown to be heterogeneously glycosylated. The erFaeG showed resistance at digestive conditions simulating piglet gastric fluid. Glycosylation did not abolish the immunogenic character of the FaeG protein, since erFaeG was able to induce F4 fimbria-specific antibodies in mice. Biological activity of these anti-F4 antibodies was demonstrated in vitro by blocking the attachment of the F4+ ETEC to the F4 receptors present on porcine intestinal enterocytes.