Oral feed-based passive immunization can be a promising strategy to prolong maternal lactogenic immunity against postweaning infections. Enterotoxigenic Escherichia coli (ETEC)-caused postweaning diarrhea in piglets is one such infection that may be prevented by oral passive immunization and might avert recurrent economic losses to the pig farming industry. As a proof of principle, we designed anti-ETEC antibodies by fusing variable domains of llama heavy chain-only antibodies (VHHs) against ETEC to the Fc part of a porcine immunoglobulin (IgG or IgA) and expressed them in Arabidopsis thaliana seeds. In this way, four VHH-IgG and four VHH-IgA antibodies were produced to levels of about 3% and 0.2% of seed weight, respectively. Cotransformation of VHH-IgA with the porcine joining chain and secretory component led to the production of light-chain devoid, assembled multivalent dimeric, and secretory IgA-like antibodies. In vitro analysis of all of the antibody-producing seed extracts showed inhibition of bacterial binding to porcine gut villous enterocytes. However, in the piglet feed-challenge experiment, only the piglets receiving feed containing the VHH-IgA-based antibodies (dose 20 mg/d per pig) were protected. Piglets receiving the VHH-IgA-based antibodies in the feed showed a progressive decline in shedding of bacteria, significantly lower immune responses corroborating reduced exposure to the ETEC pathogen, and a significantly higher weight gain compared with the piglets receiving VHH-IgG producing (dose 80 mg/d per pig) or wild-type seeds. These results stress the importance of the antibody format in oral passive immunization and encourage future expression of these antibodies in crop seeds. molecular farming | mucosal immunity | nanobody | enteric infections | antibiotic replacement

F18-fimbriated Escherichia coli are associated with porcine postweaning diarrhea and edema disease. Adhesion of F18-fimbriated bacteria to the small intestine of susceptible pigs is mediated by the minor fimbrial subunit FedF. However, the target cell receptor for FedF has remained unidentified. Here we report that F18-fimbriated E. coli selectively interact with glycosphingolipids having blood group ABH determinants on type 1 core, and blood group A type 4 heptaglycosylceramide. The minimal binding epitope was identified as the blood group H type 1 determinant (Fuc␣2Gal␤3GlcNAc), while an optimal binding epitope was created by addition of the terminal ␣3-linked galactose or N-acetylgalactosamine of the blood group B type 1 determinant (Gal␣3(Fuc␣2)Gal␤3GlcNAc) and the blood group A type 1 determinant (GalNAc␣3(Fuc␣2)-Gal␤3GlcNAc). To assess the role of glycosphingolipid recognition by F18-fimbriated E. coli in target tissue adherence, F18-binding glycosphingolipids were isolated from the small intestinal epithelium of blood group O and A pigs and characterized by mass spectrometry and proton NMR. The only glycosphingolipid with F18-binding activity of the blood group O pig was an H type 1 pentaglycosylceramide (Fuc␣2Gal␤3GlcNAc-␤3Gal␤4Glc␤1Cer). In contrast, the blood group A pig had a number of F18-binding glycosphingolipids, characterized as A type 1 hexaglycosylceramide (GalNAc␣3(Fuc␣2)Gal␤3Glc-NAc␤3Gal␤4Glc␤1Cer), A type 4 heptaglycosylceramide (GalNAc␣3(Fuc␣2)Gal␤3GalNAc␤3Gal␣4Gal␤4Glc␤1Cer), A type 1 octaglycosylceramide (GalNAc␣3(Fuc␣2)Gal␤3GlcNAc␤3-Gal␤3GlcNAc␤3Gal␤4Glc␤1Cer), and repetitive A type 1 nonaglycosylceramide (GalNAc␣3(Fuc␣2)Gal␤3GalNAc␣3-(Fuc␣2)Gal␤3GlcNAc␤3Gal␤4Glc␤1Cer). No blood group antigen-carrying glycosphingolipids were recognized by a mutant E. coli strain with deletion of the FedF adhesin, demonstrating that FedF is the structural element mediating binding of F18-fimbriated bacteria to blood group ABH determinants. Enterotoxigenic (ETEC)4 and verotoxigenic Escherichia coli are important causes of disease in man and animal (1, 2). In newly weaned pigs, F18-fimbriated E. coli producing enteroand/or Shiga-like toxins induce diarrhea and/or edema disease, which accounts for substantial economical losses in the pig industry (3). Two virulence factors are of major importance, namely the F18 fimbriae, the adhesive polymeric protein surface appendages of F18-fimbriated E. coli, and the Shiga-like toxin (SLT-IIv), or enterotoxins (STa or STb), that are produced by the bacterium. F18 fimbriae are expressed by the fed (fimbriae associated with edema disease) gene cluster, with fedA encoding the major subunit, fedB the outer membrane usher, fedC the periplasmic chaperone, whereas fedE and fedF encode minor subunits (4). FedF is the adhesive subunit and is presumably located at the tip of the fimbrial structure (5, 6). Typically, tip adhesins consist of two domains: an N-terminal carbohydrate-specific lectin domain and a C-terminal pilin domain (7), which needs to be donor-strand-complemented ...

F18+ Escherichia coli have the ability to colonize the gut and cause oedema disease or post-weaning diarrhoea by adhering to specific F18 receptors (F18R) on the porcine epithelium. Although it is well established that a DNA polymorphism on base pair 307 of the FUT1 gene, encoding an a(1,2)fucosyltransferase, accounts for the F18R phenotype, the F18R nature is not elucidated yet. The aim of the present study was to investigate the correlation between the presence of H-2 histo-blood group antigens (HBGAs) or its derivative A-2 HBGAs on the porcine gut epithelium and F18 + E. coli adherence. A significant positive correlation was found between expression of both the H-2 (r = 0.586, P < 0.01) and A-2 (r = 0.775, P < 0.01) HBGAs and F18 + E. coli adherence after examination of 74 pigs aged from 0 to 23 weeks. The majority of the genetically resistant pigs (FUT1M307 A/A ) showed no HBGA expression (91.7%) and no F18 + E. coli adherence (83.3%). In addition, it was found that F18R expression levels rise with increasing age during the first 3 weeks after birth and that F18R expression is maintained in older pigs (3-23 weeks old). Taken together, these data suggest that, apart from H-2 HBGAs, A-2 HBGAs might be involved in F18 + E. coli adherence.

SummaryF18-positive enterotoxigenic and Shiga toxinproducing Escherichia coli are responsible for postweaning diarrhoea and oedema disease in pigs and lead to severe production losses in the farming industry. F18 fimbriae attach to the small intestine of young piglets by latching onto glycosphingolipids with A/H blood group determinants on type 1 core. We demonstrate the N-terminal domain of the F18 fimbrial subunit FedF to be responsible for ABH-mediated attachment and present its X-ray structure in ligandfree form and bound to A and B type 1 hexaoses. The FedF lectin domain comprises a 10-stranded immunoglobulin-like b-sandwich. Three linear motives, Q 47-N50, H88-S90 and R117-T119, form a shallow glycan binding pocket near the tip of the domain that is selective for type 1 core glycans in extended conformation. In addition to the glycan binding pocket, a polybasic loop on the membrane proximal surface of FedF lectin domain is shown to be required for binding to piglet enterocytes. Although dispensable for ABH glycan recognition, the polybasic surface adds binding affinity in the context of the host cell membrane, a mechanism that is proposed to direct ABH-glycan binding to cell-bound glycosphingolipids and could allow bacteria to avoid clearance by secreted glycoproteins.

F4 enterotoxigenic Escherichia coli (F4 ETEC) are an important cause of diarrhea in neonatal and newly-weaned pigs. Based on the predicted differential O-glycosylation patterns of the 2 MUC13 variants (MUC13A and MUC13B) in F4ac ETEC susceptible and F4ac ETEC resistant pigs, the MUC13 gene was recently proposed as the causal gene for F4ac ETEC susceptibility. Because the absence of MUC13 on Western blot from brush border membrane vesicles of F4ab/acR+ pigs and the absence of F4ac attachment to immunoprecipitated MUC13 could not support this hypothesis, a new GWAS study was performed using 52 non-adhesive and 68 strong adhesive pigs for F4ab/ac ETEC originating from 5 Belgian farms. A refined candidate region (chr13: 144,810,100–144,993,222) for F4ab/ac ETEC susceptibility was identified with MUC13 adjacent to the distal part of the region. This candidate region lacks annotated genes and contains a sequence gap based on the sequence of the porcine GenomeBuild 10.2. We hypothesize that a porcine orphan gene or trans-acting element present in the identified candidate region has an effect on the glycosylation of F4 binding proteins and therefore determines the F4ab/ac ETEC susceptibility in pigs.

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Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO3-3Galß1Cer), sulf-lactosylceramide (SO3-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).

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