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Summary Previous 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.

Enterotoxigenic Escherichia coli (ETEC) remains a massive burden in developing countries with increasing morbidity and mortality rates; it is also an important pathogen in the farming industry and is a leading cause of bacterial diarrhea. Our previous study showed that nanometer-sized inclusion bodies (IBs) of the fimbrial adhesin subunit protein (FaeG), mutation heat-stable enterotoxin a (mSTa), heat-labile enterotoxin b (LTb), and STb (nontargeting) fusion protein as an oral vaccine induced both systemic and mucosal immune responses. In this study, to enhance the protective efficacy to ETEC, we used Yersinia enterocolitica adhesive and M-cell-targeting peptides to analyze high-efficiency antigen-specific immune presentation in the gut. Here, we showed that immunization with the IBs of ETEC-FaeG-mSTa-LTb-STb-induced a specific systemic and mucosal immune response in the gut, whereas the combination of both targeting peptides resulted in the highest titer, protective immune response against ETEC. A lymphocyte proliferation assay has shown that the IBs induced immunologic memory. The specific antibody of the targeting groups could effectively neutralize toxins, thereby protecting the cells of the small intestine and reducing the level of cAMP and cGMP, and the groups with double targeting showed the best effect. The most important finding was that the targeting peptides stimulate the T helper (Th) cells through Th17 and Th1 and that Th1 cells dominated the cellular immune response. We found that the targeting peptide could also activate CD11c+ on lymphoid dendritic cells, which processed and presented antigens to T cells through Th1-mediated IFN-γ and IL-12, thereby enhancing the antibody titers. The double-targeting peptide had a better effect on stimulating the immune cells to enhance the antibody titers.-Jiang, X., Xia, S., He, X., Ma, H., Feng, Y., Liu, Z., Wang, W., Tian, M., Chen, H., Peng, F., Wang, L., Zhao, P., Ge, J., Liu, D. Targeting peptide-enhanced antibody and CD11c+ dendritic cells to inclusion bodies expressing protective antigen against ETEC in mice.

This study investigated the occurrence of Escherichia coli pathotypes in sanitary wastewater and drinking water in a Bangladeshi urban slum and the potential associations between these sources. We examined 621 E. coli isolates from sanitary wastewater and stored drinking water by multiplex PCR and dual-index sequencing, classifying them into eight pathotypes based on 14 virulence genes and additionally evaluating the possession of the human-specific E. coli genetic biomarker H8. The proportions of pathogenic E. coli were significantly different (P

Vaccine delivery vehicles are just as important in vaccine efficiency. Through the progress in nanotechnology, various nanoparticles have been evaluated as carriers for these substances. Among them, alginate nanoparticles are a good choice because of their biodegradability, biocompatibility, ease of production, etc. In this study, feasibility of alginate nanoparticles (NPs) such as recombinant LTB from Enterotoxigenic Escherichia coli (ETEC) carrier was investigated. To do this, the eltb gene was cloned and expressed in E. coli BL21 (DE3) host cells, and a Ni-NTA column purified the protein. NPs were achieved through ion gelation method in the presence of LTB protein and CaCl2 as the cross-Linker and NPs were characterized physicochemically. Balb/C mice groups were immunized with LTB-entrapped NPs or LTB with adjuvant and immunogenicity was assessed by evaluating IgG titer. Finally, the neutralization of antibodies was evaluated by GM1 binding and loop assays. LTB protein was expressed and efficiently entrapped into the alginate NPs. The size of NPs was less than 50 nm, and entrapment efficiency was 80%. Western blotting showed maintenance of the molecular weight and antigenicity of the released protein from NPs. Administration of LTB-entrapped NPs stimulated antibody responses in immunized mice. Immunization induced protection against LT toxin of ETEC in ileal loops and inhibits enterotoxin binding to GM1-gangliosides. Alginate NPs are also appropriate vehicle for antigen delivery purpose. Moreover because of their astonishing properties, they have the potential to serve as an adjuvant.

Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti-CF and anti-enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti-CF and antitoxin immunogenicity was then assessed. To achieve high-level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti-CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion.

Enterotoxigenic Escherichia coli produces a long type 4 pilus called Longus. The regulatory elements and the environmental signals controlling the expression of Longus-encoding genes are unknown. We identified two genes lngR and lngS in the Longus operon, whose predicted products share homology with transcriptional regulators. Isogenic lngR and lngS mutants were considerably affected in transcription of lngA pilin gene. The expression of lngA, lngR and lngS genes was optimally expressed at 37°C at pH 7.5. The presence of glucose and sodium chloride had a positive effect on Longus expression. The presence of divalent ions, particularly calcium, appears to be an important stimulus for Longus production. In addition, we studied H-NS, CpxR and CRP global regulators, on Longus expression. The response regulator CpxR appears to function as a positive regulator of lng genes as the cpxR mutant showed reduced levels of lngRSA expression. In contrast, H-NS and CRP function as negative regulators since expression of lngA was up-regulated in isogenic hns and crp mutants. H-NS and CRP were required for salt- and glucose-mediated regulation of Longus. Our data suggest the existence of a complex regulatory network controlling Longus expression, involving both local and global regulators in response to different environmental signals.

The assembly of the class 5 CFA/I fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone-usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor-strand complementation and cleft-mediated anchorage. Mutagenesis indicated that donor-strand complementation controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus additionally assist in subunit refolding. Surprisingly, over-stabilization of the chaperone-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off-pathway self-polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly. This article is protected by copyright. All rights reserved.

Propagating epizootics due to Pilchard herpesvirus (PHV) occurred in the Australian population of pilchard, Sardinops sagax neopilchardus (Steindachner) (Clupeidae), in 1995 and 1998-99, with up to 60% losses. No mortality events have been evident in the ensuing 7 years, one reason for which could be that PHV is now endemic. During 2004, a survey was conducted to establish if PHV was present in pilchards in Australia. The pilchard is a highly active, pelagic schooling fish which is found in subpopulations, creating difficulties for the conduct of surveys. It occurs in Australian coastal waters and embayments below about 25°S latitude, feeds on plankton and is predated by birds, mammals and larger fish. It reaches sexual maturity at 2 years of age, spawns at sea, enters embayments when about 5 months old and returns to sea when about 1 year old. It may live for 6-9 years, reaching a maximum length of 200 mm. It forms schools and may travel up to 30 km per day. Pilchards aggregate in mobile shoals of fish containing large highly mobile schools, which interact randomly and exchange individuals. Four subpopulations were defined for the purposes of this survey based on differences in biological characteristics: south-eastern Queensland/northern New South Wales (NSW), Victoria/South Australia (SA), south coast Western Australia (SWA) and west coast Western Australia (WWA). Specimens were obtained from the catch of commercial fishermen using random sampling where possible. Polymerase chain reaction (PCR) for the detection of PHV was performed after appraising the suitability of all available tests according to their impact on sample size requirements, total survey costs and logistical constraints. In the analysis, estimates of true prevalence (TP) of infection and 95% confidence limits were adjusted from the apparent prevalence estimates provided by PCR results. Percentage TP of PHV and corresponding 95% confidence intervals for the four subpopulations: NSW, SA, SWA and WWA were thus estimated as 0 (0-1.5), 31 (22-43), 42 (31-55) and 29 (20-41), respectively. PHV is now endemic in Australian populations of pilchard. Implications of the findings for fisheries management are discussed.

Objective To evaluate the association between vitamin D status and diarrheal episodes by enterotoxigenic (ETEC) enteropathogenic (EPEC) and enteroaggregative (EAEC) E. coli in underweight and normal-weight children aged 6-24 months in urban Bangladesh. Methods Cohorts of 446 normal-weight and 466 underweight children were tested separately for ETEC EPEC and EAEC from diarrheal stool samples collected during 5 months of follow-up while considering vitamin D status at enrolment as the exposure. Cox proportional hazards models with unordered failure events of the same type were used to determine diarrheal risk factors after adjusting for sociodemographic and concurrent micronutrient status. results Vitamin D status was not independently associated with the risk of incidence of ETEC EPEC and EAEC diarrhea in underweight children but moderate-to-severe retinol de ciency was associated with reduced risk for EPEC diarrhea upon adjustment. Among normal-weight children insuf cient vitamin D status and moderate-to-severe retinol de ciency were independently associated with 44% and 38% reduced risk of incidence of EAEC diarrhea respectively. These children were at higher risk of ETEC diarrhea with vitamin D de ciency status when adjusted for micronutrient status only. Conclusion This study demonstrates for the rst time that normal-weight children with insuf cient vitamin D status have a reduced risk of EAEC diarrhea than children with suf cient status. Moderate-to-severe de ciency of serum retinol is associated with reduced risk of EPEC and EAEC diarrhea in underweight and normal-weight children.

The present study was performed to investigate the effects of dietary supplementation of bacteriophages (phages) against enterotoxigenic Escherichia coli (ETEC) K88 as a therapy against the ETEC infection in post-weaning pigs. Two groups of post-weaning pigs aged 35 days, eight animals per group, were challenged with 3.0 × 1010 colony forming units of ETEC K88, a third group given the vehicle. The unchallenged group and one challenged group were fed a basal nursery diet for 14 days while the remaining challenged group was fed the basal diet supplemented with 1.0 × 107 plaque forming units of the phage per kg. Average daily gain (ADG), goblet cell density and villous height:crypt depth (VH:CD) ratio in the intestine were less in the challenged group than in the unchallenged group within the animals fed the basal diet (p

Synthetic porcine beta-defensin-2 (pBD-2) was tested as an alternative to antimicrobial growth-promoters in pig production. Thirty 21-day weaned piglets were challenged with enterotoxigenic Escherichia coli, and orally dosed with either sterile water (CON), pBD-2 (BD) or neomycin sulphate (NS) twice daily for 21 days. pBD-2 and NS led to higher growth performance, jejunum villus height and increased expression of insulin-like growth factor-I compared with the CON group (P < 0.05). Hemolytic E. coli scores from rectal swabs, and copy numbers of E. coli, Bacteroides fragilis and Streptococcus in the cecal digesta of the BD- or NS-treated piglets were lower than those in the CON group (P < 0.05). Messenger RNA levels of toll-like receptor 4, tumor necrosis factor-α, interleukin (IL)-1β, and IL-8 in the jejunum mucosa of the BD and NS groups were lower than those in the CON group (P < 0.05). Copy numbers of Lactobacilli and Bifidobacteria in the cecal digesta of the BD group were higher than those of the CON and NS groups (P < 0.05). Therefore, pBD-2 has antimicrobial activity in piglets, and it can improve growth performance, reduce inflammatory cytokine expression and affect intestinal morphological indices in the same way as probiotics. © 2015 Japanese Society of Animal Science.

Enterotoxigenic Escherichia coli (ETEC) is a water and food-borne pathogen that infects the small intestine of the human gut and causes diarrhoea. Enterotoxigenic E. coli adheres to the epithelium by means of colonization factors and secretes two enterotoxins, the heat labile toxin and/or the heat stable toxin that both deregulate ion channels and cause secretory diarrhoea. Enterotoxigenic E. coli as all E. coli, is a versatile organism able to survive and grow in different environments. During transmission and infection, ETEC is exposed to various environmental cues that have an impact on survivability and virulence. The ability to cope with exposure to different stressful habitats is probably shaping the pool of virulent ETEC strains that cause both endemic and epidemic infections. This review will focus on the ecology of ETEC in its different habitats and interactions with other organisms as well as abiotic factors.

The most commonly used genome annotation processes are to a great extent based on computational methods. However, those can only predict genes that have been described earlier or that have sequence signatures indicative of a gene function. Here, we report a synonymous proteogenomic approach for experimentally improving microbial genome annotation based on label-free quantitative MS/MS. The approach is exemplified by analysis of cell extracts from in vitro cultured enterotoxigenic Escherichia coli (ETEC) strain TW10598, as part of an effort to create a new reference ETEC genome sequence. The proteomic analysis yielded identification of 2060 proteins, out of which 312 proteins were originally described as hypothetical. For 84% of the identified proteins we have provided description of their relative quantitative levels, among others, for 20 abundantly expressed ETEC virulence factors. Proteogenomic mapping supported the existence of four protein-coding genes that had not been annotated, and led to correction of translation start positions of another nine. The addition of the proteomic analysis into TW10598 genome re-annotation project improved quality of the annotation, and provided experimental evidence for a significant portion of ETEC expressed proteome. Data are available via ProteomeXchange with identifier PXD002473 (http://proteomecentral.proteomexchange.org/dataset/PXD002473).

The FaeG subunit is the major constituent of F4(+) fimbriae, associated with glycoprotein and/or glycolipid receptor recognition and majorly contributes to the pathogen attachment to the host cells. To investigate the key factor involved in the fimbrial binding of F4(+) Escherichia coli, both the recombinant E. coli SE5000 strains carrying the fae operon gene clusters that express the different types of fimbriae in vitro, named as rF4ab, rF4ac, and rF4ad, respectively, corresponding to the fimbrial types F4ab, F4ac, and F4ad, and the three isogenic in-frame faeG gene deletion mutants were constructed. The adhesion assays and adhesion inhibition assays showed that ΔfaeG mutants had a significant reduction in the binding to porcine brush border as well as the intestinal epithelial cell lines, while the complemented strain ΔfaeG/pfaeG restored the adhesion function. The recombinant bacterial strains rF4ab, rF4ac, and rF4ad have the same binding property as wild-type F4(+) E. coli strains do and improvement in terms of binding to porcine brush border and the intestinal epithelial cells, and the adherence was blocked by the monoclonal antibody anti-F4 fimbriae. These data demonstrate that the fimbrial binding of F4(+) E. coli is directly mediated by the major FaeG subunit.

The objectives of this study were to assess the effectiveness of an ultraviolet (UV‐C, 254 nm) irradiation system and the spray‐drying method as two independent safety steps on inactivation of Escherichia coli K88 and K99 spiked in porcine plasma at 6·46 ± 0·04 log10 ml−1 and 6·78 ± 0·67 log10 ml−1 respectively for UV‐C method, and at 7·31 ± 0·39 log10 ml−1 and 7·66 ± 0·11 log10 ml−1, respectively for the spray‐drying method. The UV‐C method was performed at different UV light doses (from 750 to 9000 J l−1) using a pilot plant UV‐C device working under turbulent flow. Spray‐drying treatment was done at inlet temperature 220 ± 1°C and two different outlet temperatures, 80 ± 1°C or 70 ± 1°C. Results indicated that UV‐C treatment induced a 4 log10 viability reduction for both E. coli at 3000 J l−1. Full inactivation of both E. coli strains was achieved in all spray‐dried samples dehydrated at both outlet temperatures. The special UV‐C system design for turbid liquid porcine plasma is a novel treatment that can provide an additional redundant biosafety feature that can be incorporated into the manufacturing process for spray‐dried animal plasma. Significance and Impact of the Study The safety of raw materials from animal origin such as spray‐dried porcine plasma (SDPP) may be a concern for the swine industry. Ultraviolet treatment at 254 nm (UV‐C) of liquid plasma has been proposed as an additional biosafety feature in the manufacturing process of SDPP. We found that UV‐C exposure in the liquid plasma at 3000 J l−1 reduces about 4 log10 ml−1 for E. coli K88 and K99. Full inactivation of both E. coli strains was achieved in all spray‐dried samples. The incorporation of UV‐C treatment to liquid plasma improves the robustness of the SDPP manufacturing process., Significance and Impact of the Study: The safety of raw materials from animal origin such as spray‐dried porcine plasma (SDPP) may be a concern for the swine industry. Ultraviolet treatment at 254 nm (UV‐C) of liquid plasma has been proposed as an additional biosafety feature in the manufacturing process of SDPP. We found that UV‐C exposure in the liquid plasma at 3000 J l−1 reduces about 4 log10 ml−1 for E. coli K88 and K99. Full inactivation of both E. coli strains was achieved in all spray‐dried samples. The incorporation of UV‐C treatment to liquid plasma improves the robustness of the SDPP manufacturing process.

Scope Immunobiotics are known to modulate intestinal immune responses by regulating Toll-like receptor (TLR) signaling pathways, which are responsible for the induction of cytokines and chemokines in response to microbial-associated molecular patterns. However, little is known about the immunomodulatory activity of compounds or molecules from immunobiotics. Methods and results We evaluated whether Lactobacillus delbrueckii subsp. delbrueckii TUA4408L (Ld) or its extracellular polysaccharide (EPS): acidic EPS (APS) and neutral EPS (NPS), modulated the response of porcine intestinal epitheliocyte (PIE) cells against Enterotoxigenic Escherichia coli (ETEC) 987P. The roles of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effects were also studied. ETEC-induced inflammatory cytokines were downregulated when PIE cells were prestimulated with both Ld or EPSs. Ld, APS, and NPS inhibited ETEC mediated mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation by upregulating TLR negative regulators. The capability of Ld to suppress inflammatory cytokines was diminished when PIE cells were blocked with anti-TLR2 antibody, while APS failed to suppress inflammatory cytokines when cells were treated with anti-TLR4 antibody. Induction of Ca2+ fluxes in TLR knockdown cells confirmed that TLR2 plays a principal role in the immunomodulatory action of Ld, while the activity of APS is mediated by TLR4. In addition, NPS activity depends on both TLR4 and TLR2. Conclusion Ld and its EPS have the potential to be used for the development of anti-inflammatory functional foods to prevent intestinal diseases in both humans and animals.

This study was aimed to evaluate the effect of dietary supplementation of lipid-encapsulated (coated) zinc oxide ZnO on post-weaning diarrhea (colibacillosis) in weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC). Thirty-two 35-day-old weaned piglets were orally challenged with 3 × 10(10) colony forming units of ETEC K88 while eight piglets received no challenge (control). Each eight challenged piglets received a diet containing 100 ppm ZnO (low ZnO), 2500 ppm ZnO (high ZnO) or 100 ppm of lipid (10%)-coated ZnO (coated ZnO) for 7 days; control pigs received the low ZnO diet. Daily gain, goblet cell density in the villi of the duodenum, jejunum and ileum, and villus height in the jejunum and ileum, which decreased due to the challenge, were equally greater in the coated ZnO and high ZnO groups versus low ZnO group. Fecal consistency score, serum interleukin-8 concentration, subjective score of fecal E. coli shedding, and digesta pH in the stomach, jejunum and ileum, which increased due to the challenge, were equally low in the coated ZnO and high ZnO groups versus low ZnO. Results suggest that a low level of coated ZnO might well substitute for a pharmacological level of native ZnO in dietary supplementation to alleviate colibacillosis of weaned piglets.

Enterotoxigenic Escherichia coli (ETEC) strains are the most common cause of bacterial diarrhea in children in developing countries and travelers to these areas. Enterotoxins and colonization factors (CFs) are two key virulence factors in ETEC pathogenesis, and the heterogeneity of the CFs is the bottleneck in reaching an effective vaccine. In this study, a candidate subunit vaccine, which is composed of CfaB, CssA and CssB, structural subunits of colonization factor antigen I and CS6 CFs, labile toxin subunit B, and the binding subunit of heat-labile and heat-stable toxoid, was designed to provide broad-spectrum protection against ETEC. The different features of chimeric gene, its mRNA stability, and chimeric protein properties were analyzed by using bioinformatic tools. The optimized chimeric gene was chemically synthesized and expressed successfully in a prokaryotic host. The purified protein was used for assessment of bioinformatic data by experimental methods.

Enterotoxigenic Escherichia coli (ETEC) colonize the human gut, causing severe cholera-like diarrhoea. ETEC utilize a diverse array of pili and fimbriae for host colonization, including the Type IVb pilus CFA/III. The CFA/III pilus machinery is encoded on the cof operon, which is similar in gene sequence and synteny to the tcp operon that encodes another Type IVb pilus, the Vibrio cholerae toxin co-regulated pilus (TCP). Both pilus operons possess a syntenic gene encoding a protein of unknown function. In V. cholerae, this protein, TcpF, is a critical colonization factor secreted by the TCP apparatus. Here we show that the corresponding ETEC protein, CofJ, is a soluble protein secreted via the CFA/III apparatus. We present a 2.6 A resolution crystal structure of CofJ, revealing a large β-sandwich protein that bears no sequence or structural homology to TcpF. CofJ has a cluster of exposed hydrophobic side-chains at one end and structural homology to the pore-forming proteins perfringolysin O and α-haemolysin. CofJ binds to lipid vesicles and epithelial cells, suggesting a role in membrane attachment during ETEC colonization.

Porcine edema disease (ED) is caused by Shiga toxin 2e-producing Escherichia coli (STEC). Post-weaned piglets often suffer from ED as a result of intestinal infection with STEC, which causes impaired growth performance and high mortality. Antimicrobial therapy is a curative treatment for piglets infected with STEC, but the emergence of antimicrobial-resistant STEC has become a serious problem for Japanese pig farmers. Therefore, an alternative strategy other than antimicrobial therapy is needed for the prevention or treatment of ED. In this study, we evaluated the effect of oral administration of Bacillus subtilis DB9011 (DB9011) to prevent the experimental infection of STEC in weaning piglets. Eight 21-day-old piglets were divided into two groups: STEC challenge with the basal diet, and STEC challenge with DB9011 supplemented diet. The challenge was carried out when the animals were 25, 26 and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 36 days of age. DB9011 improved the symptoms of ED and decreased the number of STEC in the ileal digesta and feces. Accordingly, oral administration of DB9011 in weaned piglets prevents ED through the suppression of the growth of STEC in the ileum.

Coli surface antigen 6 (CS6) is a widely expressed enterotoxigenic Escherichia coli (ETEC) colonization factor that mediates bacterial attachment to the small intestinal epithelium. CS6 is a polymer of two protein subunits CssA and CssB, which are secreted and assembled on the cell surface via the CssC/CssD chaperone usher (CU) pathway. Here, we present an atomic resolution model for the structure of CS6 based on the results of X-ray crystallographic, spectroscopic and biochemical studies, and suggest a mechanism for CS6-mediated adhesion. We show that the CssA and CssB subunits are assembled alternately in linear fibres by the principle of donor strand complementation. This type of fibre assembly is novel for CU assembled adhesins. We also show that both subunits in the fibre bind to receptors on epithelial cells, and that CssB, but not CssA, specifically recognizes the extracellular matrix protein fibronectin. Taken together, structural and functional results suggest that CS6 is an adhesive organelle of a novel type, a hetero-polyadhesin that is capable of polyvalent attachment to different receptors.

Recombinant antibodies have been proposed as invaluable tools for various therapeutic and diagnostic purposes. Here, we describe the development of a novel latex agglutination test (LAT) using single-chain Fv recombinant antibody fragment for the detection of K99(+) enterotoxigenic Escherichia coli strains. For the production of a single-chain Fv antibody fragment (scFv) against the major colonization factor (FanC) of K99 antigen, the scFv gene was integrated into a bacterial expression vector under the control of T7 promoter. After high-level expression of soluble scFv (approximately 50 mg/l) in flask cultivation of E. coli DE3 and purification, scFv was immobilized on different latex particles, and then, these sensitized beads were used in LAT. Results obtained with our latex reagents revealed that the recombinant antibody-coated particles were able to give a good agglutination signal with purified antigen, intact cells displaying this protein and clinical specimens. The strength of agglutination of scFv-coated beads for antigen was comparable to that of polyclonal anti-K99-coated particles. However, the assay proved to be simple and rapid, similar to conventional LATs, and owing to more convenient and economical production of recombinant antibodies, they can be considered as a useful reagent for replacing monoclonal antibodies in LATs.

Enterotoxigenic Escherichia coli (ETEC) is one of the most frequently isolated enteropathogens in production animals, especially pigs and calves. Economically, the swine industry is by far the most affected by infections with ETEC because of mortality, morbidity and decreased growth rate of newborn and early-weaned piglets. After ingestion by the animal, these bacteria attach themselves to specific receptors on the small intestinal epithelium by means of proteinaceous surface appendages, the fimbriae. The F4 fimbriae, which attach to the F4 receptor, are the most studied. The aim of our study was to investigate gene expression in the small intestine of piglets of MUC13 and MUC20 in relation to animals with a different treatment towards or a different reaction on ETEC-F4ac by means of quantitative reverse transcription chain reaction (qRT/PCR). MUC13 and MUC20 are positional candidate genes for this F4ac receptor and are located in the region on SSC13q41 that segregates with the susceptibility to ETEC-F4ac. The condition of the small intestine is crucial when examining expression differences between different samples. Therefore, the expression of two genes, fatty-acid binding protein 2, intestinal (FABP2) and pancreatitis-associated protein (PAP), now known as regenerating islet-derived 3 alpha (REG3A) in the small intestine was simultaneously checked. FABP2, a standard for epithelial content, reflects the state of damage, whereas REG3A is a measure for inflammation in the small intestine. The four different substudies presented here suggest that expression of MUC13 and MUC20 is not related to the susceptibility of piglets to ETEC-F4ac.

Enterotoxigenic Escherichia coli (ETEC) expressing K88 (F4) adhesins are associated with post-weaning diarrhoea in piglets. Different grain fractions from pea (Pisum sativum) and faba bean (Vicia faba) were tested in vitro for their capacity to counteract aetiological factors, which contribute to the development of diarrhoea. In detail, adhesion of E. coli O149:K91:K88ac (ETEC K88ac) to grain legume products, intended to impair the colonization of the host, was studied as well as interference with receptor binding of the pathogen’s heat-labile enterotoxin LT, intended to reduce toxin-inflicted gut cell damage. When comparing different pea and faba bean products tested for their binding capacity of ETEC K88ac, especially pea hulls, but also whole pea meal, starch-enriched and protein-enriched pea meal, and digestion-resistant pea hull and meal fractions showed a higher binding of ETEC K88ac than faba bean products. In contrast to the ETEC K88ac adhesion results, bean hulls proved more effective than pea hulls in preventing GM1 receptor binding of LT. Previous small intestinal segment perfusion experiments we performed with ETEC K88ac-challenged piglets indicated that both pea and bean hulls have the potential for successful application in diarrhoea prophylaxis and treatment, which is in agreement with and refined by our detection of their different modes of functioning.

Objective Determine the prevalence of the major enteric pathogens in dairy and dairy beef calves with diarrhoea in Australia. Design Cross-sectional study. Methods Faecal samples from 84 Australian dairy and dairy beef properties (597 samples) were screened for rotavirus and coronavirus using real-time reverse transcription polymerase chain reaction, for Salmonella spp. using selective enrichment faecal culture, and for enterotoxigenic Escherichia coli (K99) and Cryptosporidium parvum using a commercial enzyme-linked immunosorbent assay. A logistic regression with random effects model was used to compare prevalence of pathogens in dairy and dairy beef operations. Results Enteric pathogens were isolated from 97.6% of outbreaks and 95.0% of samples. Rotavirus was the most common pathogen identified (477/597, 79.9%) followed by C. parvum (349/597, 58.5%), Salmonella spp. (142/597, 23.8%), coronavirus (129/597, 21.6%) and E. coli K99 (104/597, 17.4%). Multiple pathogens were identified on 96.4% of farms and from 71.0% of samples. Samples from dairy beef properties were more likely to have multiple pathogens than dairy properties (P < 0.05), whereas rotavirus and Salmonella spp. were more likely to be identified in samples collected from dairy beef than dairy properties (P < 0.05). Conclusion Most outbreaks of calf diarrhoea in dairy and dairy beef operations involve multiple pathogens. Rotavirus and C. parvum were the most frequently identified pathogens across production systems. Salmonella spp. and rotavirus were more frequently identified in dairy beef operations.

Summary Enterotoxigenic Escherichia coli expressing F4 fimbriae is the major cause of diarrhoea in neonatal and post-weaning piglets. Previous studies have revealed that the loci controlling the F4ab/F4ac receptors are located on SSC13q41, between markers SW207 and S0283. In this study, we refined their positions in a two generation population containing 366 piglets of three breeds (Large White, Landrace, and Songliao Black). Nine microsatellite markers within this region were selected from the MARC (U.S. Meat Animal Research Center) porcine linkage map, and the pedigree disequilibrium test was employed for fine-mapping. The F4abR gene was located in the interval between S0283 and SW1833, a 4.8-cM region, and the F4acR gene was located in the interval between S0283 and SW1876, a 1.6-cM region. Our results also suggest that the F4ab/F4ac receptors might be controlled by two different but closely linked loci. The results of microsatellite-based haplotype analysis in the corresponding region show that some specific haplotypes were overwhelmingly present in the adhesive or non-adhesive animals, indicating that there are mutations within the identified regions that are strongly associated with the F4ab/ac phenotypes.

Enterotoxigenic Escherichia coli (ETEC) causes child and travelers' diarrhea and is presumed to be water- and food-borne. Sporadic outbreaks were traced to consumption of contaminated fresh produce, particularly salad leaves as lettuce and parsley. Importantly, the mechanism by which ETEC binds salad leaves is not known. In this study we investigated the ability of clinical ETEC isolates to adhere to Eruca vesicaria (commonly known as rocket). Towards this end we inoculated pieces of cut E. vesicaria leaves with clinical ETEC isolates grown in Luria broth at 20°C, conditions that are not permissive for expression of the plasmid-encoded colonization factors and hence mimic the actual transmission pathways of ETEC through intake of contaminated food. We found that ETEC strains bind E. vesicaria at various efficiencies. Examination of representative strains by scanning electron microscopy revealed that they adhere to the E. vesicaria surface in a diffuse pattern by extended filaments resembling flagella. Using the prototype ETEC strain H10407 we found that it also binds to lettuce, basil and spinach leaves. Binding of H10407 was dependent on flagella as a fliC mutant attached to leaves at a much lower efficiency. Interestingly, under the employed environmental conditions EtpA, which forms a flagellar tip structure, and colonization factor I are dispensable for leaf attachment. The results show that ETEC can bind specifically to salad leaves, which might represent an important, yet less recognized, source of infection.

Enterotoxigenic Escherichia coli (ETEC) is a main cause of diarrhoea in humans and piglets. In vitro, black tea extract (BTE) has anti-pathogenic properties. Anti-diarrhoeal properties of BTE were assessed in a pig model of gastrointestinal infection. At weaning (day 0), piglets (n = 96) were randomly assigned to a diet containing 0% (control), 0.4% or 0.8% (wt/wt) BTE during 27 days. Piglets were orally infected with 6.4 × 10(6) cfu of ETEC on day 6. Faecal consistency, feed intake and body weight were measured. In a sub-study (n = 30 piglets), the effect of BTE palatability on feed intake was assessed. Additionally, the effect of BTE on ETEC growth in the presence or absence of iron was studied in vitro. The 0.8% BTE diet reduced diarrhoea prevalence by 20% but also decreased feed intake by 16% and feed efficiency by 12% over the total period. The 0.4% BTE diet decreased feed efficiency and weight gain from day 13 onwards. The palatability study demonstrated that piglets preferred the control to the BTE diets. In vitro, BTE delayed ETEC exponential growth, which was reversed by iron addition. Although BTE had anti-diarrhoeal properties, this effect was accompanied by impaired performance. The absence of a correlation between diarrhoea prevalence and feed intake suggests that reduced diarrhoea directly results from BTE rather than from reduced feed intake caused by BTE astringency.

Summary Most of the porcine enterotoxigenic and drug resistant E. coli isolated in Hungary carry a conjugative R plasmid. In R and Ent plasmid transfer experiments 4 out of 11 donors (36%) transferred both R and Ent plasmid simultaneously when the J-53 strain was used as recipient. Treatment of E. coli diseases caused by these strains is quite difficult. The epidemiological importance of these observations is discussed. Zusammenfassung Vorkommen und Ubertragung von R- und Ent-Plasmiden bei enterotoxinbildenden E. coli-Stammen vom Schwein Die meisten der in Ungarn vom Schwein isolierten E. coli-Stamme weisen ein konjugiertes R-Plasmid auf. Durchgefuhrte R- und Ent-Plasmid Transfer-Experimente zeigten, das bei 4 von 11 Stammen (36%) gleichzeitig R- und Ent-Plasmid ubertragen wurden, wenn der Stamm J-53 als Empfanger diente. Die Behandlung von Erkrankungen, die durch solche Stamme hervorgerufen werden, ist ziemlich schwierig. Diskutiert wird die epidemiologische Bedeutung dieser Beobachtungen. Resume Presence et transmission de plasmides R et Ent chez des souches d'E. coli productrices d'enterotoxines d'origine porcine La plupart des souches d'E. coli isolees chez le porc en Hongrie presentent un plasmide R. conjugue. Des essais de transfert experimental de plasmides R et Ent ont montre que 4 des 11 souches (36%) etaient porteuses simultanement des plasmides R et Ent lorsque la souche J-53 servait de recepteur. Le traitement des affections dues a de telles souches est assez difficile. La signification epidemiologique de ces observations est discutee. Resumen Prevalencia y transmision de plasmides R y Ent en las estirpes E. coli enterotoxinogenas del cerdo La mayor parte de las estirpes E. coli aisladas en Hungria de los cerdos ostentan un plasmide R conjugado. Las experiencias de traspaso efectuadas con plasmides R y Ent muestran que en 4 de 11 estirpes (36%) se transmitieron al mismo tiempo plasmides R y Ent cuando la cepa J-53 servia como receptora. El tratamiento de las enfermedades ocasionadas por tales cepas es bastante dificil. Se discute la importancia epidemiologica de estas observaciones.

Summary Eighteen Caesarian-derived, colostrum-deprived calves were orally infected with either bovine rotavirus, enterotoxigenic Escherichia coli (ETEC) or with a combination of both enteropathogens within the first 24 h of life. Animals which received ETEC developed severe diarrhea when 1010organisms were given, but remained healthy after inoculation of 107organisms. Animals infected with four different field strains of bovine rotavirus developed mild, transient diarrhea after an average incubation time of 29 h. They usually recovered within a few hours after onset of the symptoms. A synergistic effect was observed after simultaneous application of rotavirus and 107ETEC, resulting in severe diarrhea after an average incubation time of 22 h. These calves had significantly more rotavirus in the intestinal samples than to samples from monoinfected calves. Those calves, which were infected with 107ETEC followed by rotavirus 15 to 36 hours later, had significantly less rotavirus in the intestinal samples and shed significantly less rotavirus in their feces than to monoinfected calves. The number of ETEC organisms demonstrated in the small intestine was more dependent on the time of sampling than on the mode of infection. Zusammenfassung Synergismus nach experimenteller Infektion neugeborener Kalber mit bovinem Rotavirus und enterotoxischen Escherichia coli (ETEC) Achtzehn Kalber wurden innerhalb der ersten 24 Lebensstunden oral entweder mit bovinem Rotavirus, enterotoxinbildenden E. coli (ETEC) oder mit einer Kombination aus beiden Erregern infiziert. Tiere, die 1010ETEC erhielten, entwickelten eine schwere Durchfallerkrankung, wahrend 107ETEC keine Durchfallsymptome mehr auslosten. Tiere, die mit vier verschiedenen Rotavirus-Feldstammen infiziert wurden, zeigten nach einer durchschnittlichen Inkubationszeit von 29 Stunden eine milde, kurzzeitige Diarrhoe. Sie erholten sich wenige Stunden nach Sistieren des Durchfalls. Nach gleichzeitiger Infektion von Rotavirus mit 107ETEC wurde eine synergistische Interaktion beider Erreger beobachtet. Diese Kalber entwickelten eine schwere Durchfallerkrankung nach einer durchschnittlichen Inkubationszeit von 22 Stunden. In den Darmabschnitten dieser Tiere wurden signifikant hohere Rotavirusinfektionsitatstiter gefunden als bei Tieren, die mit Rotavirus allein infiziert wurden. Dagegen war die Virusausscheidung im Kot und in den Darmabschnitten signifikant verringert bei Tieren, denen zuerst 107ETEC und 15 bis 36 Stunden spater Rotavirus verabreicht wurde. Die Zahl der im Dunndarm festgestellten ETEC-Keime war in allen Gruppen starker vom Zeitpunkt der Probengewinnung nach Infektion als von der Art der Infektion abhangig. Resume Synergisme apres une infection experimentale de veaux nouveaux-nes avec un Rotavirus bovin et Escherichia coli enterotoxique (ETEC) Des infections mixtes experimentales ont ete faites sur des veaux nouveaux-nes, prives de colostrum avec un Rotavirus bovin et E. coli enterotoxique (ETEC). Dix-huit veaux ont ete infectes oralement dans les premieres 24 heures de vie avec un Rotavirus bovin ou E. coli enterotoxique (ETEC) ou avec une combinaison des deux agents. Les animaux ayant recu 1010ETEC developperent une grave diarrhee alors que 107ETEC ne provoquerent aucun symptome de diarrhee. Les animaux qui furent infectes avec quatre souches differentes de Rotavirus du terrain presenterent une legere et breve diarrhee apres un temps d'incubation de 29 heures. Ils ont ete gueris quelques heures apres la cessation de la diarrhee. Une interaction synergique des deux agents a ete observee apres une infection simultanee de Rotavirus avec 107ETEC. Ces veaux developerent une grave diarrhee apres une periode d'incubation moyenne de 22 heures. On a trouve des titres infectieux de Rotavirus significativement plus eleves dans les portions d'intestin de ces animaux que chez les veaux infectes seulement avec du Rotavirus. L'excretion virale dans les matieres fecales et dans les portions intestinales fut par contre significativement diminuee chez les animaux qui avaient recu d'abord 107ETEC et Rotavirus 15 a 36 heures plus tard. Le nombre des germes ETEC etablis dans l'intestin grele avec tous les groupes a dependu plus du moment des prelevements apres l'infection que du genre de l'infection. Resumen Sinergismo tras infeccion experimental de terneros recien nacidos con rotavirus bovino y Escherichia coli enterotoxica (ETEC) Dieciocho terneros que no recibieron calostro fueron infectados dentro de las 24 horas primeras de vida por via oral bien con un rotavirus bovino, E. coli enterotoxinogena (ETEC) o bien con una combinacion de ambos agentes etiologicos. Los animales que recibieron 1010ETEC desarrollaron una enteritis grave, mientras que 107ETEC no desencadenaron sintomas diarreicos. Los animales que fueron infectados con cuatro estirpes campales diferentes de rotavirus mostraron tras un tiempo medio de incubacion de 29 horas una diarrea benigna de duracion breve. Los mismos se recuperaron pocas horas despues del cese de la descomposicion. Tras la infeccion simultanea de rotavirus con 107ETEC se observo una interaccion sinergica de ambos agentes etiologicos. Estos terneros desarrollaron una enteritis grave tras un periodo medio de incubacion de 22 horas. En los tramos entericos de estos animales se hallaron titulos de infecciosidad significantemente mas elevados que en los animales que habian sido infectados solo con rotavirus. Sin embargo, la eliminacion de virus con las heces y en los tramos intestinales se hallaba reducida de forma significativa en los animales que recibieron primero 107ETEC y 15 hasta 36 horas despues rotavirus. La cuantia de los germenes ETEC comprobada en el intestino delgado dependia en todos los grupos mas del momento en que se recogia la muestra tras la infeccion que del tipo de infeccion.

Zusammenfassung Von 503 E. coli-Stammen aus 105 Kotproben enteritiskranker Junghunde bildeten 21 Stamme (7,6%) — aus 8 Kotproben — hitzestabiles Enterotoxin (ETEC). Hitzelabiles Enterotoxin lies sich nicht nachweisen. Alle enterotoxinbildenden Stamme wiesen im Elektronenmikroskop Fimbrien auf. Mit Ausnahme von 4 Stammen agglutinierten alle Toxinbildner die Erythrocyten mehrerer Saugetierarten, wobei die Reaktion nur in einem Fall mannosestabil war. Die Fimbrien der mannosesensibel reagierenden Stamme wurden den Typ-I-Fimbrien zugeordnet. Die Frage der Identitat der isolierten ETEC wurde mit Hilfe der Chemotherapeutikasensibilitat und der Plasmidmuster uberpruft. Fast alle Stamme erwiesen sich als resistent gegen ein oder mehrere Chemotherapeutika; alle besasen Plasmide. Die Stamme aus 2 von 4 Kotproben, aus denen mehrere ETEC isoliert worden waren, zeigten gleiche Plasmidmuster und identisches Resistenzverhalten. Die ubrigen ETEC zeigten nur Teilidentitat. Summary Characteristics of Enterotoxigenic E. coli Strains Isolated from Young Dogs Suffering from Enteritis Twenty-one out of 503 E. coli strains isolated from 105 faecal samples of puppies suffering from enteritis produced heat-stable enterotoxin (7.6%). These enterotoxic strains (ETEC) were isolated from 8 faecal samples. Heat-labile enterotoxin was not detected. All ETEC were equipped with fimbriae as shown in the electron microscope. All but four strains were able to agglutinate erythrocytes of one or more mammalian species but only in one case in a mannose-resistant manner. The mannose-sensitive fimbriae were designed as type-I-fimbriae. It was shown that all ETEC harboured plasmids and that nearly all were resistant against one or more chemotherapeutics. The identity of the ETEC present in the same sample was checked by the aid of sensitivity to chemotherapeutics and their plasmidfingerprints. The strains isolated from two faecal samples out of 4, which harboured more than one ETEC, had respective identical plasmids and showed the same resistance pattern. The ETEC from the remaining samples were only partial identical. Resume Presence et proprietes de souches d'E. coli enterotoxiques chez des chiots atteints d'enterite 21 souches (7,6%) de 8 prelevements de matieres fecales sur 503 souches d'E. coli de 105 echantillons de feces de chiots atteints d'enterite ont forme une enterotoxine thermostable (ETEC). Une enterotoxine thermolabile n'a pas ete mise en evidence. Toutes les souches enterotoxiques ont montre des fimbries au microscope electronique. A l'exception de 4 souches, toutes les souches formant une toxine ont agglutine les erythrocytes de plusieurs especes de mammiferes, la reaction n'ayant ete qu'une fois mannose-stable. Les fimbries des souches mannose-sensibles ont ete classees sous type-I-fimbries. La question de l'identite des ETEC isolees a ete examinee a l'aide de la sensibilite aux chimiotherapiques et a l'echantillonnage plasmidique. Presque toutes les souches ont montre une resistance a l'egard d'un ou plusieurs chimiotherapiques; toutes possedaient des plasmides. Les souches de 2 echantillons de matieres fecales sur 4, a partir desquels plusieurs ETEC ont ete isoles, presenterent un echantillonnage plasmidique identique et un meme comportement de resistance. Les autres ETEC n'avaient qu'une identite partielle. Resumen La incidencia y propiedades de las estirpes enterotoxinogenas de Escherichia coli en el perro joven enfermo de enteritis Entre 503 estirpes de E. coli de 105 muestras de estiercol de perros jovenes enfermos de enteritis produjeron 21 estirpes (7,6%) — de 8 muestras de estiercol — enterotoxina termoestable (ETEC). No se pudo poner en evidencia ninguna enterotoxina termolabil. Todas las especies enterotoxinogenas ostentaban fimbrias en el microscopio electronico. Con excepcion de 4 estirpes, aglutinaban todas las estirpes toxinogenas los eritrocitos de varias especies de mamiferos, siendo la reaccion manosaestable en un solo caso. Las fimbrias de las estirpes que reaccionaban manosasensibles se encuadraron en el tipo I de fimbrias. La cuestion de la identidad de las ETEC aisladas se verificaron con ayuda de la sensibilidad frente a los quimioterapicos y el modelo de plasmidios. Casi todas las estirpes se mostraron como resistentes contra uno o varios quimioterapicos; todas poseian plasmidios. Las estirpes de 2 entre 4 muestras de estiercol, de las cuales se habian aislado varias ETEC, mostraron el mismo modelo de plasmidios e identico porte de resistencia. Las ETEC restantes exhibieron nada mas una identidad parcial.

In the intestine, enterotoxigenic Escherichia coli works against peristaltic forces, adhering to the epithelium via the colonization factor antigen I (CFA/I) fimbrial adhesin CfaE. The CfaE adhesin is similar in localization and tertiary (but not primary) structure to FimH, the type 1 fimbrial adhesin of uropathogenic E. coli, which shows shear-dependent binding to epithelial receptors by an allosteric catch-bond mechanism. Thus, we speculated that CfaE is also capable of shear-enhanced binding. Indeed, bovine erythrocytes coursing over immobilized CFA/I fimbriae in flow chambers exhibited low accumulation levels and fast rolling at low shear, but an 80-fold increase in accumulation and threefold decrease in rolling velocity at elevated shear. This effect was reversible and abolished by pre-incubation of fimbriae with anti-CfaE antibody. Erythrocytes bound to whole CfaE in the same shear-enhanced manner, but to CfaE adhesin domain in a shear-inhibitable fashion. Residue replacements designed to disrupt CfaE interdomain interaction decreased the shear dependency of adhesion and increased binding under static conditions to human intestinal epithelial cells. These findings indicate that close interaction between adhesive and anchoring pilin domains of CfaE keeps the former in a low-affinity state that toggles into a high-affinity state upon separation of two domains, all consistent with an allosteric catch-bond mechanism of CfaE binding.

Enterotoxigenic Escherichia coli (ETEC) F4ab and F4ac are major determinants of piglet diarrhoea. The locus for the ETEC F4ab/ac receptor has been mapped to SSC13q41. MUC13 is a transmembrane mucin expressed predominantly in the epithelial surface of the gastrointestinal tract and the MUC13 gene was assigned to SSC13q41, supporting it as a positional candidate gene for the ETEC F4ab/ac receptor. We herein determined the complete 2679-bp cDNA of pig MUC13, and proved that it was most highly expressed in the jejunum and moderately expressed in the trachea, stomach and liver. Furthermore, 13 MUC13 polymorphisms were identified in 19 founder animals of a White Duroc x Erhualian resource population, and a total of 727 F(2) animals with in vitro ETEC F4ab/ac adhesion phenotypes in this population were genotyped for three identified MUC13 polymorphisms including c.576C>T, c.908A>G and c.935A>C. The transmission disequilibrium test showed that the MUC13 alleles and haplotypes were significantly associated with susceptibility/resistance to ETEC F4ab/ac, especially between haplotype [C;G;A] and susceptibility to ETEC F4ac (P = 8.0e-18). Animals inheriting this haplotype were predominantly susceptible to ETEC F4ac (n = 291/303). Moreover, nearly all animals homozygous for haplotype [T;G;C] (n = 39/41) and a majority of those with the [C;A;A]/[T;G;C] haplotype pair (n = 79/88) were resistant to ETEC F4ab. Our results indicated that MUC13 is in strong linkage disequilibrium with the ETEC F4ab/ac receptor locus and provided potential markers for selection of ETEC F4ab/ac-resistant animals in the pig breeding scheme.

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 (F4ab, F4ac and F4ad) fimbriae is a significant cause of mortality and morbidity in newborn and weaned pigs. The locus controlling susceptibility towards ETEC F4ab/ac has been mapped to SSC13q41, in which TFRC (transferrin receptor) was localized and considered as a positional candidate gene for ETEC F4ab/ac receptor. In this study, we determined susceptibility/resistance to ETEC F4ab/ac in a total of 755 F2 animals from a White Duroc x Erhualian intercross using a microscopic enterocyte adhesion assay. We identified two TFRC polymorphisms (SNPs 591 A>G and 632 A>G) in a single exon after comparative sequencing analysis of 2371-bp amplicons containing the complete coding region of TFRC using RNA of eight full-sib F2 animals with susceptible and resistant phenotypes. The intron sequences flanking the two exon polymorphisms were obtained, revealing an intron polymorphism (SNP 291 C>T). We genotyped the 19 founder animals of the White Duroc x Erhualian intercross for the identified polymorphisms, showing that only the 291 C>T polymorphism is a highly informative marker. We further genotyped all 59 F1 and 755 F2 animals for the 291 C>T polymorphism, and the association of this polymorphism with susceptibility/resistance to ETEC F4ab/ac in these F2 animals was evaluated by the transmission disequilibrium test. The result showed that the 291 C>T polymorphism is not a causal mutation, however, has a significant linkage disequilibrium with the ETEC F4ab/ac, especially F4ac receptor locus.

Using a porcine radiation hybrid panel, we assigned the mucin 4 (MUC4) gene to SSC13q41, which harbours the enterotoxigenic Escherichia coli (ETEC) F4ab/ac receptor locus. In addition, we identified two SNPs in intron 17 of MUC4 (DQ124298:g.243A>G and DQ124298:g.334A>G) in the parental population of a White Duroc x Erhualian cross. Association analysis showed that the MUC4 g.243A>G mutation was strongly associated with ETEC F4ab/ac, and especially with F4ac adhesion phenotypes in the White Duroc x Erhualian resource population, indicating that this polymorphism was in a significant linkage disequlibrium with the ETEC F4ab/ac receptor locus. Because of different linkage disequlibrium values between the ETEC F4ab and F4ac adhesion phenotypes and the MUC4 g.243A>G mutation, we argue that the inheritance of F4ab and F4ac receptors might be under the control of two closely linked loci.

The families Enterobacteriaceae and Vibrionaceae encompass a heterogeneous group of gram-negative organisms. The organisms chosen for discussion in this chapter are recognized as major public health concerns because of their significant contribution to morbidity and mortality in both children and adults in less developed and industrialized countries. Cytotoxicity is usually examined under the microscope or can be quantified by colorimetric methods. Positive samples should be retested in the presence of Shiga toxins (Stx)-specific antibodies to confirm the specificity of the cytotoxic effect. The optimal concentration of antigen should be determined by standard enzyme-linked immunosorbent assay (ELISA) checkerboard titration, coating plates with different amounts of antigen and testing them with known positive and negative control sera during the standardization of the assay. Diagnosis of acute enterotoxigenic Escherichia coli (ETEC) infections requires the detection of labile toxin (LT), ST, or toxin genes in isolated strains. Typical enteropathogenic E. coli (EPEC) strains carry both a chromosomally encoded (the locus of enterocyte effacement (LEE), which mediates a complex signal transduction phenotype on the target epithelial cell, and the EPEC adherence factor plasmid (EAF), which encodes the principal EPEC adhesin.

The most common enteric colibacillosis in neonatal and newborns is caused by enterotoxigenic Escherichia coli (ETEC). Colonization of ETEC in the small intestine is associated with adhesions using fimbriae, which is known as a specific adhesion factor and provides highly specific means for anchoring and prerequisite for an infectious agent. In the present study we have engineered Lactobacillus acidophilus to produce recombinant K99 fimbriae, which is used for the colonization to the intestine of pigs. The expression of K99 fimbrial protein was confirmed using SDS‐PAGE, immunoblot and agglutination analyses. To evaluate a function of the K99 fimbrial protein, inhibition and competition tests were performed on pre‐screened intestinal brush border from pigs. The tests showed that recombinant L. acidophilus , not control L. acidophilus , had a significant inhibitory effect to and competition against K99+ E. coli in a dose dependent manner. In conclusion, we demonstrated that recombinant K99 fimbriae producing L. acidophilus was able to prevent E. coli binding to intestinal brush border.

Objective: To identify virulence genes in enterotoxigenic E coli (ETEC) isolates associated with diarrhoea in neonatal, 1 to 3 week-old and weaned pigs in south east Queensland. Design: Multiplex PCR and serotyping were applied to E coli isolates obtained over a 5-year period (1998-2002) from cases diagnosed at Toowoomba Veterinary Laboratory. Procedure: A total of 126 isolates from 25 different Queensland piggeries were tested for haemolytic activity on 5% sheep blood agar and by multiplex PCR for the presence of five commonly recognised fimbrial (F4, F5, F6, F41 and F18) and three enterotoxin genes (STa, STb, LT). A subset of 62 representative isolates were serotyped by slide agglutination. For comparative purposes, multiplex PCR was also performed on the DNA of 31 ETEC isolates from 9 serotypes originating from piggeries in southern New South Wales. Results: A total of 113 (89.7%) of the isolates from Queensland possessed ETEC virulence genes, including 14 of 15 isolates from neonatal pigs (93.3%), 18 of 23 isolates from 1 to 3 week old pigs (78.3%) and 81 of 88 isolates from weaned pigs (92.1%). F4:STa:STb:LT (serotype O149) was the most prevalent pathotype in neonatal and 1-3 week old pigs and F4:STa:STb:LT (serotype O149) and F18:STa:STb:LT (serotype O141) were most prevalent in weaned pigs. In comparison, isolates obtained from neonatal pigs from New South Wales belonged to a more diverse range of pathotypes and serotypes. Conclusion: Multiplex PCR was a rapid and specific method for detecting the presence of ETEC virulence genes in porcine E coli isolates. For isolates obtained from cases of suspected colibacillosis in Queensland, growth of a heavy pure culture of haemolytic E coli was a sensitive prognostic indicator of the presence of ETEC virulence genes in the isolate. ETEC pathotypes and serotypes remained stable in Queensland piggeries over the five-year study period and appear to have changed little over the last three decades.

Summary Susceptibility to enterotoxigenic Escherichia coli with fimbriae F4ac is dominantly inherited in the pig. A three-generation pedigree was created to refine the position of F4acR on chromosome 13 comprising 202 pigs: eight parents, 18 F1 and 176 F2 pigs. The 17-point analysis indicates that F4acR lies between Sw207 and S0283. Recombinant offspring specify that the most probable order is Sw207–S0075–F4acR–Sw225–S0283. We observed six phenotypes for the three fimbrial variants F4ab, F4ac and F4ad. The two missing phenotypes F4abR−/F4acR+/F4adR+ and F4abR−/F4acR+/F4adR− indicate that pigs susceptible to F4ac are always susceptible to F4ab. Furthermore, a weak and a strong adhesion of F4ab and F4ad bacteria was observed. The weak receptor F4abR (F4abRw) was present only in pigs devoid of the receptor F4acR (F4abR+/F4acR−). In contrast, in pigs with the phenotype F4abR+/F4acR+, F4ab bacteria adhered to the majority of enterocytes. F4abRw constitutes a frequently observed phenotype whose inheritance is still unclear. Strong adhesion of F4ab and F4ac bacteria is most likely influenced by the same receptor that we name F4bcR. The number of F4ad bacteria that adhered to enterocytes was very variable in the adhesion test. Moreover, expression of F4adR was independent of age. Our segregation analyses indicated a dominant inheritance of F4adR, although the number of susceptible pigs was smaller than expected. We examined four genes as candidates for the F4acR locus: the transferrin receptor gene (TFRC) and three genes members of the glucosyl/galactosyltransferase family (B3GnT5, B3GALT3 and B4GALT4). Comparison of sequences from resistant and homozygous susceptible F4ac pigs did not reveal any causative single nucleotide polymorphism in the four genes. Two silent mutations at the positions 295 (C/T) and 313 (T/C) in B3GALT3 were found. Using the somatic cell hybrid panel, B3GnT5 and B3GALT3 were assigned to the chromosomal region SSC13q23-q41. No mutations were found in the cDNA sequences of these genes associated with the F4acR genotypes.

The objectives of this work were to study the FA composition of milk gangliosides, as well as to gain further insight into the characterization of human milk gangliosides. The potential capacity of human milk gangliosides to adhere to human enterotoxigenic Escherichia coli (ETEC-strains) was also studied. Human milk gangliosides were isolated and identified by high-performance TLC or immunoassay. The latter also was used to assay bacterial adhesion. The FA composition of gangliosides was studied by GC. The presence of O-acetyl GD3 (Neu5,9Ac2alpha2-8 NeuAcalpha2-3Galbeta1-4GlcCer) and trace amounts of GM1 [Galgamma1]3-3GalNAcgamma1,-3(Neualpha2-3)Galbeta1-4GlcCerl in human milk was confirmed. Medium-chain FA were almost absent in colostrum, whereas in the subsequent stages they rose to 20%. The levels of long-chain FA decreased after colostrum. With respect to the degree of saturation, gangliosides from colostrum were richer in monounsaturated FA than gangliosides synthesized during the rest of the lactation period, opposite to the pattern for PUFA. A human-ETEC colonization factor antigen II-expressing strain showed binding capacity to human milk GM3 (NeuAcalpha2-3Gal[1-4GlcCer). New data on human milk gangliosides have been gathered. A thorough knowledge of their composition is needed since they may have important biological implications in regard to newborns' defense against infection.

The F17-G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N-acetyl-beta-D-glucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 A resolution crystal structure of the lectin domain of F17-G, both free and in complex with N-acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid-shaped protein in a conserved site around which all natural variations of F17-G are clustered. A model is proposed for the interaction between F17-fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17-G binding to N-acetylglucosamine (0.85 mM-1). Unexpectedly, the F17-G structure reveals that the lectin domains of the F17-G, PapGII and FimH fimbrial adhesins all share the immunoglobulin-like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C-terminal beta-strand G of the immunoglobulin-like fold and provides new insights into pilus assembly, function and adhesion.

Summary The gene cluster of the CS18 (PCFO20) fimbriae of human enterotoxigenic Escherichia coli (ETEC) was found to include seven genes (fotA to fotG) that are similar to each of the seven structural and export proteins of the 987P fimbriae. However, no analogous gene to the fasH regulatory gene, which is located at the 3′ end of the 987P gene cluster and encodes an AraC-like activator of transcription, could be detected. Surprisingly, two novel genes (fotS and fotT) encoding proteins similar to the site-specific recombinases of the type 1 fimbriae (FimB and FimE) were identified at the 5′ end of the fot gene cluster. These genes were shown to be required for the catalysis of a 312 bp-inversion just upstream of fotA. The inversion determines CS18 fimbrial phase variation. FotS participates in inverting the 312 bp-segment in both the ON and OFF orientation, whereas FotT has a bias for the OFF oriented recombination. Similar regulators of fimbriation by phase variation were described in uropathogenic and commensal Enterobacteriaceae. In contrast, only AraC-like transcriptional activators were previously described as regulators of the intestinal colonization factors of human ETEC isolates. Thus, the CS18 and 987P gene clusters encode similar components for fimbrial biogenesis but different types of regulators for fimbriation. The combination of blocks of genes encoding similar structural products but different regulatory proteins underlines how modular DNA rearrangements can evolve by serving pathogen diversification. Acquisition of a phase variation module to regulate fimbrial genes is proposed to be beneficial for the adaptation and transmission of pathogens.

The aim of this study was to refine the localization of the receptor locus for fimbriae F4ac. Small intestinal enterocyte preparations from 187 pigs were phenotyped by an in vitro adhesion test using two strains of Escherichia coli representing the variants F4ab and F4ac. The three-generation pedigree comprised eight founders, 18 F1 and 174 F2 animals, for a total of 200 pigs available for the linkage analysis. Results of the adhesion tests on 171 F2 pigs slaughtered at 8 weeks of age show that 23.5% of the pigs were adhesive for F4ab and non-adhesive for F4ac (phenotype F4abR+/F4acR-; R means receptor). Pigs of this phenotype were characterized by a weak adhesion receptor for F4ab. No pigs were found expressing only F4acR and lacking F4abR. Receptors for F4ab and F4ac (F4abR+/F4acR+) were expressed by 54.5% of the pigs. Animals of this phenotype strongly bound both F4ab and F4ac E. coli. In the segregation study, the serum transferrin (TF) gene and 10 microsatellites on chromosome 13 were linked with F4acR (recombination fractions (theta) between 0.00 and 0.11 and lod score values (Z) between 11.4 and 40.4). The 11-point analysis indicates the F4acR locus was located in the interval S0068-Sw1030 close to S0075 and Sw225, with recombination fractions (theta) of 0.05 between F4acR and S0068, 0.04 with Sw1030, and 0.00 with S0075 and Sw225. The lack of pigs displaying the F4abR-/F4acR+ phenotype and the presence of two phenotypes for F4abR (a strong receptor present in phenotype F4abR+/F4acR+ and a weak receptor in phenotype F4abR+/F4acR-) led us to conclude that the receptor for F4ac binds F4ab bacteria as well, and that it is controlled by one gene localized between S0068 and Sw1030 on chromosome 13.

Piglet diarrhea causes large economic losses to the swine industry. Epidemiological investigations show that piglet diarrhea is often caused by mixed infections, but the mechanisms by which multiple microorganisms cause disease are unclear. Because transmissible gastroenteritis virus (TGEV) and enterotoxigenic Escherichia coli K88 (ETEC K88) are important contributors to piglet diarrhea, coinfection experiments were conducted using porcine intestinal columnar epithelial cells (IPEC-J2) as a model system. LC–MS/MS coupled to iTRAQ was used to profile expressed proteins in IPEC-J2 cells infected by TGEV alone, ETEC K88 alone, and by both agents together. We identified 77, 89, and 136 differentially expressed proteins in TGEV infected, ETEC K88 infected, and coinfected cells, respectively. Based on these data, we suspected that integrin α5 might enable TGEV to promote ETEC K88 adhesion. In TGEV pre-infected IPEC-J2 cells, ETEC K88 adhesion was enhanced over uninfected cells. ETEC K88 was also found to inhibit the proliferation of TGEV. Additionally, cytokine levels (IL-1β, IL-6, IL-8, and TNF-α) in coinfected cells were lower than cells infected by TGEV alone, and higher than cells infected by ETEC K88 alone. Our study is the first to analyze piglet diarrhea caused by TGEV-ETEC K88 coinfection using high-throughput quantitative proteomics. The results advance our understanding of coinfection and its role in causing piglet diarrhea. This article is protected by copyright. All rights reserved

New technologies that do not rely on antibiotics are urgently needed to treat bacterial infections caused by multidrug-resistant bacteria. Herein, the feasibility of using alternating magnetic field (AMF) to selectively kill enterotoxigenic Escherichia coli strain K99 (EC K99) in the presence of multianchored glycoconjugate-functionalized magnetic nanoparticles is explored. Poly(ethylene oxide)-poly(acrylic acid)-dopamine functionalized magnetic nanoparticles (PEO-MNPs) are synthesized and functionalized with bacteria-specific glycoconjugate Neu5Ac(α2-3)-Gal-(β1-4)Glcβ-sp (GM3-MNPs) for specific adherence to EC K99. When such mixtures are exposed to an alternate magnetic field (31 kA m−1, 207 KHz), an ≈3-log reduction in colony forming units of EC K99 is achieved in 120 min. Moreover, in a mixed-bacterial culture environment, targeted killing of EC K99 is achieved with minimal damage to nontargeted bacterium. Electron microscopy images along with live/dead staining assays demonstrate visible membrane damage of EC K99 cells in the presence of GM3-MNPs and AMF. Additionally, intracellular adenosine triphosphate (ATP) levels of EC K99 are significantly diminished in the presence of GM3-MNPs and AMF. These results suggest that specific glycoconjugate-functionalized magnetic nanoparticles when mediated by AMF can be potentially used as a novel nonantibiotic treatment platform to inactivate/kill targeted bacterial pathogens, with minimal impact on normal microflora and the affected body region/tissue.

Thirty-six Lactobacillus strains were isolated from the small intestine of piglets and 14 of them were chosen as they had a good ability (>5 bacteria per cell) to attach to the epithelial cells of the small intestine. They were selected to investigate the antagonistic effects against enterotoxigenic Escherichia coli (ETEC) including K88ab, K88ac, K88+MB (a local strain of haemolytic (H) ETEC K88+ bacterium from Manitoba, Canada), K99 and 987P. Their effects on the adhesion of E coli K88+MB and K88ac to the intestinal mucus of piglets were also studied. The results indicated that six out of the 14 Lactobacillus isolates were able to moderately or strongly inhibit the growth of eight strains of ETEC through the production of organic acids, but not by bacteriocin or hydrogen peroxide. Well diffusion assays of the major organic acids of the Lactobacillus culture supernatants showed that both acetic acid and lactic acid inhibited the growth of E coli. Lactobacillus isolates did not affect the attachment of E coli K88+MB and K88ac to the small intestinal mucus of piglet. © 2000 Society of Chemical Industry

The assembly of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli requires the processing of CFA/III major pilin (CofA) by a peptidase, likely another type IV pilus formation system. Western blot analysis of CofA reveals that CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to 20.5-kDa mature pilin by a prepilin peptidase. This processing is essential for exportation of the CofA from the cytoplasm to the periplasm. In this experiment, the structural gene, cofP, encoding CFA/III prepilin peptidase which cleavages at the Gly-30-Met-31 junction of CofA was identified, and the nucleotide sequence of the gene was determined. CofP consists of 819 bp encoding a 273-amino acid protein with a relative molecular mass of 30,533 Da. CofP is predicted to be localized in the inner membrane based on its hydropathy index. The amino acid sequence of CofP shows a high degree of homology with other prepilin peptidases which play a role in the assembly of type IV pili in several gram-negative bacteria.

An enzyme-linked immunosorbent sandwich assay (ELISA) has been developed to detect and quantify both the K88+ fimbrial antigen and the concentration of bacterial cells in fecal and other samples using anti-fimbrial chicken egg-yolk and anti-fimbrial rabbit serum antibodies. The assay has a sensitivity of 40 ng ml−1 for K88+ fimbrial antigen and 107 CFU ml−1 for intact cells and cell homogenate. The inter- and intra-assay coefficients of variation (CV) for intact cells, homogenates, and fimbrial antigens were similar, suggesting that reproducible results can be obtained with any of the three preparations. In all cases the CV increased with decreasing concentration of the antigen, especially when very low concentrations of antigen were used. Fimbrial antigens had lower CV (values ranged from 2 to 37%, depending on its concentration) than standards prepared from either whole cells or homogenized cells (values ranged from 1 to 67%). The correlation between the actual number of cells as counted and the number as estimated from the ELISA using the amount of fimbrial antigen was r = 0.98, P