PSVII-16 Galactosylated chitosan-oligosaccharides have anti-adhesive effect against enterotoxigenic Escherichia coli in piglets

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets. In vitro, high molecular weight β-galactosylated chitosan-oligosaccharides (Gal-COS) had strong anti-adhesive activity against ETEC-expressing K88 fimbriae (ETEC K88) binding to porcine erythrocytes. This study assessed the effects of Gal-COS differing in structure on anti-adhesive properties against ETEC in a small intestinal segment perfusion (SISP) model in 8 piglets (BW 10 kg; 5-wk old). With 10 jejunal segments in each pig, 5 segments were infected with ETEC K88, and the other 5 segments were infused with saline (non-ETEC). Every 2 paired segments (ETEC or non-ETEC) from the same pig were treated for 8 h with 64 ml of 10 g L-1 of one of the following test products: 1) α-Gal-COS; 2) β-Gal-COS; 3) exopolysaccharides produced by Lactobacillus reuteri; and 4) raffinose in a double 4 × 4 Latin square with a saline control. Infection by ETEC K88 was verified by quantitative PCR. Net fluid loss was calculated as difference of fluid loss between ETEC segment and its paired non-ETEC segment. Data were analyzed using the mixed model with segment and test product as fixed effects, and pig as random effect. Number of eubacterial rRNA genes was 10-fold greater (P < 0.001) in ETEC segments than non-ETEC segments, indicating that ETEC K88 accounted for > 90% of bacterial gene counts. Test product did not affect (P > 0.10) the number of ETEC bacteria in the outflow fluid. Furthermore, net fluid loss caused by ETEC tended (P = 0.08) to be decreased by β-Gal-COS compared to all other treatments. In conclusion, the in vivo SISP model confirmed that Gal-COS had anti-diarrheal effects, indicating that β-Gal-COS is a potential feed additive to reduce the ETEC-induced diarrhea in piglets.

Alleviation of enterotoxigenic Escherichia coli challenge by recombinant Lactobacillus plantarum expressing a FaeG- and DC-targeting peptide fusion protein

FaeG is the major subunit of K88 fimbriae. These cell surface attachments are considered to be the major virulence factor of enterotoxigenic Escherichia coli (ETEC), which causes diarrhoea in piglets. The use of dendritic cell-targeting peptide (DCpep) has been demonstrated to be an effective approach to enhance the immunity of vaccines. Lactobacillus plantarum is an attractive candidate for oral vaccination owing to its beneficial effects and safety. In this study, L. plantarum was employed to deliver a FaeG-DCpep fusion antigen, and the immune response in mice was evaluated. The synthesis of FaeG-DCpep dramatically increased the adhesion of recombinant L. plantarum (RLP) to IPEC-J2 cell surfaces, resulting in direct competition between L. plantarum and ETEC during adhesion assays. Significantly higher levels of body weight gain, sera immunoglobulin G and intestinal immunoglobulin A were observed in BALB/c mice immunised with RLP. In addition, the number of CD19+ B cells and CD11c+DC cells and the expression levels of several cytokines in the spleen and lymph nodes increased significantly compared to non-immunised mice. The oral administration of RLP also alleviated the symptoms of ETEC challenge, as shown by haematoxylin-eosin staining, indicating that RLP may be an efficient vaccine candidate.

Exopolysaccharides Synthesized by Lactobacillus reuteri Protect against Enterotoxigenic Escherichia coli in Piglets

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is a major cause of diarrhea in piglets; ETEC cells colonize the intestinal mucosa with adhesins and deliver toxins that cause fluid loss. This study determined the antiadhesive properties of bacterial exopolysaccharides (reuteran and levan) and related glycans (dextran and inulin) in a small intestinal segment perfusion (SISP) model. The SISP model used 10 jejunal segments from 5-week-old piglets. Five segments were infected with ETEC expressing K88 fimbriae (ETEC K88), while five segments were treated with saline. Every two segments (ETEC and non-ETEC infected) were infused with 65 ml of 10 g liter−1of glycans or saline (control) for 8 h. High-resolution melting-curve (HRM) quantitative PCR (qPCR) indicated thatE. coliis the dominant bacterium in infected segments, while other bacteria were predominant in noninfected segments. Infection by ETEC K88 was also verified by qPCR; gene copy numbers of K88 fimbriae and the heat-labile toxin (LT) in mucosal scrapings and outflow fluid of infected segments were significantly higher than those in noninfected segments. Genes coding for K88 fimbriae and LT were also detected in noninfected segments. LT amplicons from infected and noninfected segments were 99% identical over 481 bp, demonstrating the presence of autochthonous ETEC K88. All glycans reduced fluid loss caused by ETEC K88 infection. Reuteran tended (P= 0.06) to decrease ETEC K88 levels in mucosal scraping sample, as judged by qPCR. Fluorescentin situhybridization analysis demonstrated that reuteran significantly (P= 0.012) decreased levels of adherent ETEC K88. Overall, reuteran may prevent piglet diarrhea by reducing adhesion of ETEC K88.

Aptamer selection for the detection ofEscherichia coliK88

In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.

Involvement of Quorum Sensing and Heat-Stable Enterotoxin a in Cell Damage Caused by a Porcine EnterotoxigenicEscherichia coliStrain

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) strains with K88 fimbriae are often associated with the outbreaks of diarrhea in newborn and weaned piglets worldwide. In the present study, we observed that 108CFU/ml of K88+ETEC strain JG280 caused more death of pig intestinal IPEC-J2 cells than did 109CFU/ml, suggesting that ETEC-induced cell death was cell density dependent and that quorum sensing (QS) may play a role in pathogenesis. Subsequent investigations demonstrated a positive correlation between autoinducer 2 (AI-2) activity of JG280 and death of IPEC-J2 cells during the infection for up to 3 h. However, there was a negative correlation between AI-2 activity and expression of the JG280 enterotoxin genesestAandestBwhen IPEC-J2 cells were exposed to the pathogen at 108CFU/ml. We therefore cloned theluxSgene (responsible for AI-2 production) from JG280 and overexpressed it inE. coliDH5α, because deletion of theluxSgene was retarded by the lack of suitable antibiotic selection markers and the resistance of this pathogen to a wide range of antibiotics. The addition of culture fluid fromE. coliDH5α with the overexpressedluxSreduced cell death of IPEC-J2 cells by 108CFU/ml JG280. The addition also reduced theestAexpression by JG280. Nonpathogenic K88+strain JFF4, which lacks the enterotoxin genes, caused no death of IPEC-J2 cells, although it produced AI-2 activity comparable to that produced by JG280. These results suggest the involvement of AI-2-mediated quorum sensing in K88+ETEC pathogenesis, possibly through a negative regulation of STa production.

Proximity-Dependent Inhibition inEscherichia coliIsolates from Cattle

ABSTRACTWe describe a novel proximity-dependent inhibition phenotype ofEscherichia colithat is expressed when strains are cocultured in defined minimal media. When cocultures of “inhibitor” and “target” strains approached a transition between logarithmic and stationary growth, target strain populations rapidly declined >4 log CFU per ml over a 2-h period. Inhibited strains were not affected by exposure to conditioned media from inhibitor and target strain cocultures or when the inhibitor and target strains were incubated in shared media but physically separated by a 0.4-μm-pore-size membrane. There was no evidence of lytic phage or extracellular bacteriocin involvement, unless the latter was only present at effective concentrations within immediate proximity of the inhibited cells. The inhibitory activity observed in this study was effective against a diversity ofE. colistrains, including enterohemorrhagicE. coliserotype O157:H7, enterotoxigenicE. coliexpressing F5 (K99) and F4 (K88) fimbriae, multidrug-resistantE. coli, and commensalE. coli.The decline in counts of target strains in coculture averaged 4.8 log CFU/ml (95% confidence interval, 4.0 to 5.5) compared to their monoculture counts. Coculture of two inhibitor strains showed mutual immunity to inhibition. These results suggest that proximity-dependent inhibition can be used by bacteria to gain a numerical advantage when populations are entering stationary phase, thus setting the stage for a competitive advantage when growth conditions improve.

Gastrointestinal ecology response of piglets’ diets containing non-starch polysaccharide hydrolysis products and egg yolk antibodies following an oral challenge with Escherichia coli (k88)

The gastrointestinal ecology (GE) of piglets fed diets containing non-starch polysaccharide hydrolysis products (HP) and egg yolk antibodies against K88 fimbriae (EYA) following oral challenge with enterotoxigenic Escherichia coli K88 (ETEC) was investigated. The HP were products of incubating feedstuffs with a blend of carbohydrase enzymes. Forty, 21-d-old pigs (two pigs/pen) were assigned to four diets to give five pens per diet. The diets were: a control fed without or with 5 g kg-1 of HP and EYA either singly or in combination forming a 2 × 2 factorial arrangement. Following a 9-d adaptation period, all pigs were orally challenged with ETEC and killed at 24 and 48 h post-challenge (one pig/pen on each occasion). Feeding HP increased pre-challenge average daily gain (252 vs. 207 g d-1; P = 0.01). An interaction (P < 0.10) between EYA and HP was observed such that when fed in combination they resulted in higher ileal digesta lactic acid and cecal DM contents and lower ileal digesta ammonia. The main effects (P < 0.05) were such that pigs fed EYA-diets had shorter intestinal crypt whilst pigs fed HP-diets showed low gastric pH and high ileal mucosal adherent lactobacilli counts. In conclusion, HP and EYA influenced indices of fermentative characteristics and intestinal morphology in the gastrointestinal ecology of piglets orally challenged with enterotoxigenic E. coli (k88).Key words: Egg yolk antibodies, ETEC, gastrointestinal ecology, non-starch polysaccharides hydrolysis products, piglet

Acute phase response of piglets fed diets containing non-starch polysaccharide hydrolysis products and egg yolk antibodies following an oral challenge with Escherichia coli (k88)

Acute phase responses (APR) in pigs fed non-starch polysaccharides hydrolysis products (HP) and egg yolk antibodies against K88 fimbriae (EYA) following oral challenge with enterotoxigenic Escherichia coli K88 (ETEC) were evaluated. The HP were products of incubating feedstuffs with a blend of carbohydrase enzymes. Forty, 21-d-old pigs (two pigs/pen) were assigned to four diets to give five pens per diet. The diets were: a control fed without or with 5 g kg-1 of HP and EYA either singly or in combination forming a 2 × 2 factorial arrangement. Following a 9-d adaptation period, pigs were bled and given an oral dose of ETEC. Pigs were then bled and feed intake recorded within 48 h post-challenge. Pigs fed HP ate more (P < 0.05) than pigs not fed HP during the ETEC challenge. Interaction between additives and time was observed for packed cell volume (PCV, P = 0.0002), in which case pigs fed the control diet showed lower PCV at 6 h post-challenge than pigs fed additives. At 48 h post-challenge, main effects (P < 0.10) were such that pigs fed HP-diets had lower serum haptoglobin and pigs fed EYA-diets had higher interleukin-6 compared with pigs fed non-HP and non-EYA diets, respectively. In conclusion, HP and EYA reduced the severity of ETEC-enteritis in piglets with some evidence of synergistic effects. Key words: Acute phase responses, egg yolk antibodies, non-starch polysaccharides, hydrolysis products, piglet diarrhea