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Very few antigens have been described that induce an intestinal immunity when given orally. Our laboratory demonstrated that oral administration of isolated F4 (K88) fimbriae of Escherichia coli to F4-receptor positive (F4R(+)) pigs induces protective mucosal immunity against challenge infection. However, presence of F4-receptors (F4R) on villous enterocytes is a prerequisite for inducing the immune response, as no F4-specific antibody-secreting cells (ASC) can be induced in F4R(-) pigs. In this study, the in vivo binding of isolated F4 fimbriae (F4) to the gut epithelium was examined in F4R(+) and F4R(-) pigs. It was further investigated whether binding of F4 to the F4R results in endocytosis in and translocation across the gut epithelium using microscopy. F4 did not adhere to the intestinal epithelium of F4R(-) pigs, whereas it strongly adhered to the villous epithelium and the follicle-associated epithelium (FAE) of the jejunum and ileum of F4R(+) pigs. Following binding to F4R, F4 was endocytosed by villous enterocytes, follicle-associated enterocytes and M cells. Transcytosis of F4 across the epithelium resulted in the appearance of F4 in the lamina propria and dome region of the jejunal and ileal PP. This is the first study showing transcytosis of fimbriae across the gut epithelium. This receptor-dependent transcytosis can explain the success of F4 fimbriae as oral immunogen for inducing protective immunity in F4R(+) pigs strengthening the importance of receptor-dependent endocytosis and translocation in oral vaccine strategies. Further identification of the receptor responsible for this transport is in progress.

Currently, no vaccines are available for edema disease and post-weaning diarrhoea (PWD) in pigs. In the present study, a subunit vaccine containing the F18 fimbrial adhesin FedF was studied. Hereto, recombinant FedF was produced as a fusion protein with maltose-binding protein. Even though the produced MBPFedF was shown to attach in vitro to enterocytes, almost no FedF-specific immune response could be detected after oral administration to piglets. The delivery of FedF to the intestinal mucosa was improved by conjugating the MBPFedF to F4 fimbriae. Indeed, this conjugation induced a systemic and local FedF-specific immune response and led to a reduction in excretion after infection with F18+ E. coli. Although complete protection was not observed, the conjugation between FedF and F4 fimbriae can be considered as a first step towards the development of a combined vaccine against F4+ and F18+ E. coli infections.

F18(+)Escherichia coli infections causing post-weaning diarrhoea and/or oedema disease are a major cause of economic losses in pig industry. To date, no preventive strategy can protect pigs from F18(+)E. coli infections. One of the most attractive approaches to eliminate F18(+)E. coli infections is the selection for pigs that are resistant to F18(+)E. coli infections. However, this strategy was not believed to be favourable because of reports of genetic association with the stress-susceptibility gene in the Swiss Landrace. To investigate this potential association more thoroughly, 131 randomly selected Belgian hybrid pigs were genotyped for both the F18(+)E. coli resistance alleles (FUT1(A)) and the stress-susceptibility alleles (RYR1(T)) and their association was investigated by determining the linkage disequilibrium. This linkage disequilibrium (LD=-0.0149) is close to zero and does not differ significantly from 0 (likelihood ratio test chi(1)(2)=1.123, P=0.29), demonstrating no association between the FUT1(A) and RYR1(T) alleles. Furthermore, only a small fraction (4.6%) of the Belgian pigs was found to be resistant to F18(+)E. coli infections. Our results suggest that selection for F18(+)E. coli resistant pigs might be an attractive approach to prevent pigs from F18(+)E. coli infections, unlike to what has previously been postulated.

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 alpha(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.

F18+ Escherichia coli can cause post-weaning diarrhoea and oedema disease in pigs. These diseases are responsible for substantial economic losses, but a vaccine is not available. A good knowledge of the characteristic of the fimbriae is useful for the development of a vaccine composed of the fimbrial virulence factor. F18 fimbriae are composed of the major subunit FedA and the minor subunits FedE and the adhesin FedF. In the present study monoclonal antibodies (mAbs) against FedA and FedF were produced. In addition to their diagnostic value, these mAbs revealed a weaker interaction between FedA and FedF compared to the subunit-subunit interactions in other fimbriae, like type 1 and P pili. Further experiments are needed to investigate if this weak interaction could be one of the reasons for the slow colonisation of the small intestinal mucosa by F18+ E. coli.

One of the problems hindering the development of DNA vaccines is the relatively low immunogenicity often seen in humans and large animals compared to that in mice. In the present study, we tried to enhance the immunogenicity of a pcDNA1/faeG19 DNA vaccine in pigs by optimizing the FaeG expression plasmid and by coadministration of the plasmid vectors encoding the A and B subunits of the Escherichia coli thermolabile enterotoxin (LT). The insertion of a Kozak sequence and optimization of vector (cellular localization and expression) and both vector and codon usage were all shown to enhance in vitro FaeG expression compared to that of pcDNA1/faeG19. Subsequently, pcDNA1/faeG19 and the vector-optimized and the vector-codon-optimized construct were tested for their immunogenicity in pigs. In line with the in vitro results, antibody responses were better induced with increasing expression. The LT vectors additionally enhanced the antibody response, although not significantly, and were necessary to induce an F4-specific cellular response. These vectors were also added because LT has been described to direct the systemic response towards a mucosal immunoglobulin A (IgA) response in mice. Here, however, the intradermal FaeG DNA prime-oral F4 boost immunization resulted in a mainly systemic IgG response, with only a marginal but significant reduction in F4+ E. coli fecal excretion when the piglets were primed with pWRGFaeGopt and pWRGFaeGopt with the LT vectors.

F18+Escherichia coli cause postweaning diarrhoea and oedema disease in newly weaned piglets. Protection against these diseases can be established by preventing the fimbrial adhesion of these bacteria to the enterocytes of the porcine intestine. To test a vaccine against F18+E. coli consisting of the adhesin of F18 fimbriae, FedF, the conservation of the FedF subunit had to be examined. Therefore, the fedF sequence of 37 F18+E. coli isolates from different countries was determined and compared to the fedF gene of the F18ab reference strain F107/86. The amino acid sequence of the mature FedF from the individual F18+E. coli isolates was 96-100% identical to that from E. coli F107/86, but the overall homology was 90.4%. Hyper variable regions were not found in the FedF sequence. The FedF sequence was conserved over the different countries and between the two antigenic variants, F18ab and F18ac, suggesting that F18ab and F18ac strains have the same receptor. Furthermore, the conserved C-terminal region in the FedF adhesin suggests that the F18 fimbriae, in analogy with type 1 and P pili, are assembled by a donor strand mechanism. In conclusion, the reported conservation of FedF supports the usefulness of the fimbrial adhesin as a subunit vaccine against F18+E. coli infection.

Receptor-mediated uptake of orally administered antigen can lead to an antigen-specific immune response, whereas oral administration of most other non-replicating soluble antigens results in the induction of oral tolerance. In the present study, it is shown that fimbriae purified from an F4(K88)(+) enterotoxigenic Escherichia coli strain can function as a mucosal carrier molecule for the model antigen human serum albumin (HSA). Glutaraldehyde-coupled F4/HSA conjugates were able to bind F4 receptor positive (F4R(+)) enterocytes, but not to F4R(-) enterocytes. Moreover, oral immunization of F4R(+) pigs with F4/HSA conjugates induced a HSA-specific immune response, whereas oral immunization with HSA/HSA conjugates did not. This mucosal carrier function of F4 fimbriae was improved following oral co-administration of the F4/HSA conjugates with the mucosal adjuvant cholera toxin (CT) to F4R(+) pigs, since both humoral and cellular HSA-specific responses were significantly increased. In comparison with F4R(+) pigs, the HSA-specific response was reduced following oral F4/HSA+CT immunization of F4R(-) pigs. This indicates that F4 fimbriae as mucosal carrier and CT as adjuvant synergistically improve the induction of a HSA-specific immune response following oral immunization of pigs. These results could open new perspectives in the development of vaccines against enteropathogens.

Piglets are susceptible to F4 (K88)+ enterotoxigenic Escherichia coli (ETEC)-induced neonatal and post-weaning diarrhoea. The F4 fimbriae are composed of some minor subunits and the major subunit FaeG that also constitutes the adhesin. Parenteral vaccination of sows with an F4-containing vaccine protects the suckling piglets against neonatal F4+ ETEC-induced diarrhoea, but no commercial (mucosal) vaccine exists against F4+ ETEC-induced weaning diarrhoea. To develop a vaccine, a bioactive F4-receptor (F4R) binding FaeG molecule is required that binds to the F4R following oral immunization and induces a FaeG-specific immune response. The present study reports the altered binding of the FaeG-specific monoclonal antibody IMM01 with bioactive versus non-bioactive F4 fimbrial adhesin FaeG. The correlation of altered IMM01 binding with altered FaeG bioactivity permits the use of an IMM01-based ELISA as a fast, specific and sensitive in vitro selection for potent F4 or (recombinant) FaeG antigen formulations, useful in an F4+ ETEC vaccine.

Oral immunization of newly weaned piglets with recombinant F4 (K88) fimbrial adhesin FaeG induces a F4-specific immune response, significantly reducing F4+ Escherichia coli excretion following challenge. In order to use FaeG subunits in an oral vaccine against F4+ enterotoxigenic E. coli, it is necessary to determine the conservation of the adhesin subunit. Hereto, the faeG sequence was determined of 21 F4ac+ E. coli field isolates from piglets with diarrhoea and subsequently compared with these of the reference strain GIS26 and previously reported FaeG sequences from F4ab, F4ac and F4ad antigenic variant strains. The FaeG amino acid sequence was 96-100% homologous within each F4 serotype, but only 92 and 88% when the F4ab and F4ad antigenic variants were compared with the F4ac antigenic variant. Furthermore, the conserved regions of the adhesin suggest a donor strand mechanism in F4 fimbriae assembly as reported for type 1 and P pili. In conclusion, the results of the reported experiments support the usefulness FaeG in an oral subunit vaccine against F4+ E. coli infections or as a mucosal carrier since the adhesin is conserved among F4+ E. coli field isolates.

Enterotoxigenic and verotoxigenic F18+ Escherichia coli colonising the pig small intestine, adhere to receptors on intestinal villous enterocytes by F18 fimbriae. The aim of the present study was to define the F18R nature. The knowledge on the nature of this receptor could be important for the development of receptor-based treatments against F18+ E. coli-induced disease. The adhesion of F18+ E. coli to pig intestinal villous enterocytes was analysed in an in vitro assay. The adhesion of F18+ E. coli but not of F4ac+ E. coli was strongly inhibited by monoclonal antibodies (mAb) with blood group H-2 specificity. Conversely, blood group H-1 specific mAb could not inhibit the adhesion of F18+ E. coli nor F4ac+ E. coli. Moreover, the blood group H-2 trisaccharide strongly inhibited the adhesion of F18+ E. coli, but only partially the adhesion of F4ac+ E. coli. These data demonstrate that the F18 receptor contains the blood group antigen H-2 (alpha-fuc-(1-2)-beta-Gal-(1-4)-GlcNAc) as major carbohydrate.

Early vaccination is necessary to protect pigs against postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). However, at present no commercial vaccine allows successful vaccination. This is partly due to the presence of maternally derived antibodies. Since DNA vaccines are suggested to be superior to protein vaccines in young animals with maternal antibodies, we determined whether the fimbrial adhesin (FaeG) of F4ac(+) ETEC could be used as a plasmid DNA vaccine to prime piglets in a heterologous prime-boost approach. Hereto, pcDNA1/faeG19 was constructed and expression of rFaeG in Cos-7 cells was demonstrated. Thereafter, pigs were immunised (days 0, 21 and 42) intramuscularly by injection or intradermally by gene gun and humoral and cellular immune responses were analysed. Even though responses were low, results demonstrated that intramuscular injection was superior to gene gun delivery for priming the humoral immune response since higher antibody titres were raised, whereas gene gun delivery better induced a cellular response, evaluated by a lymphocyte proliferation assay. Effective priming of the humoral immune response was evidenced by high IgG titres 1 week after a protein boost with purified F4. The low responses to the pcDNA1/faeG19 DNA vaccination suggest that delivery of the DNA and/or the expression of the faeG gene should be improved.

Newly weaned piglets can be orally immunised against F4+ enterotoxigenic Escherichia coli (ETEC) infection with F4 fimbriae. However, to efficiently develop a vaccine against ETEC induced postweaning diarrhoea, knowledge of the stability of the F4 fimbriae to different pH and gastric digestion is needed. The gastrointestinal pH in suckling and recently weaned piglets was measured and the stability of F4 fimbriae to different pH and to pepsin was assessed in vitro. In the stomach the lowest pH was found in the fundus gland region. Gastric pH values below 2.5 were not found in suckling piglets or at the day of weaning, in contrast to piglets 1 and 2 weeks postweaning. Along the first half of the small intestine and in the caecum, a negative correlation was found between pH and age. The F4 fimbriae were stable to pH 1.5 and 2 for 2 h, whereas longer incubation periods resulted in conversion of the multimeric forms into monomers. The F4 fimbriae were partially degraded by incubation for 15-30 min in simulated gastric fluid at pH 1.5 and 2, and completely digested from 3 h onwards. At pH 3, the fimbriae maintained their antigenicity for at least 4h. The results demonstrate that gastric digestion will only have a limited impact on oral immunisation since liquid passes through the stomach relatively quickly (50% within 2 h). However, we previously demonstrated that the transit times are prolonged shortly after weaning. Shortly after weaning it could be necessary to protect the F4 fimbriae against gastric digestion to obtain efficient oral immunisation of the piglets.

An overwhelming number of infectious diseases in both humans and animals are initiated by bacterial adhesion to carbohydrate structures on a mucosal surface. Most bacterial pathogens mediate this adhesion by fimbriae or pili which contain an adhesive lectin subunit. The importance of fimbriae as virulence factors led to research elucidating the regulation of fimbrial expression and their molecular assembly process. This review provides an overview of the current knowledge of induction, expression and assembly of F4 (K88) fimbriae and discusses its unique as well as its identical characteristics compared to other intensively studied fimbriae or pili expressed by Escherichia coli., (Copyright 2004 S. Karger AG, Basel)

In this study it was analysed whether intramuscular (IM) immunisation of piglets with F4 during the suckling period could protect against oral challenge with F4(+)-Escherichia coli and whether addition of 1alpha,25(OH)(2)D(3) or CpG-ODN could improve this protection.F4-seronegative F4-receptor positive pigs were divided into four groups of five pigs each. The pigs were intramuscularly injected with F4 fimbriae only or supplemented with 1alpha,25(OH)(2)D(3) (D(3)-group) or CpG-ODN (CpG-group). The control group received PBS in IFA. Seven days after the second immunisation, all pigs were intragastrically inoculated with 1 x 10(10) CFU of F4(+)-E. coli. All F4-injected groups, showed a reduced faecal excretion of F4(+)-E. coli. However, this reduction was only statistically significant in the D(3)-group 2 days post challenge. Pigs in the latter group showed a secondary antibody response upon challenge, indicating that F4-primed memory B-cells were present in the gut-associated lymphoid tissues at that moment.CpG-ODN, on the other hand, did not enhance the F4-specific antibody response. However, CpG-ODN significantly increased the F4-specific as well as mitogen-induced proliferation of peripheral blood monomorphonuclear cells indicating a direct or indirect overall effect on T-lymphocytes. In conclusion, supplementation with 1alpha,25(OH)(2)D(3) or CpG-ODN improved protection against an F4(+)-E. coli infection. This protection was most obvious for 1alpha,25(OH)(2)D(3) and indicates its potential use in veterinary vaccines against enteropathogens.

The intestinal mucosal immune system can discriminate actively between harmful pathogenic agents and harmless food antigens resulting in different immune responses namely IgA production and oral tolerance, respectively. Recently, a pig model has been developed for studying intestinal mucosal immune responses in which F4 fimbrial antigens of enterotoxigenic Escherichia coli (F4 ETEC) are used as oral antigens. A unique feature of this model is that soluble F4 antigens can be administered to pigs which have a receptor for this fimbriae (F4R(+)) on their small intestinal villous enterocytes and pigs which do not have this receptor (F4R(-)). Oral administration of F4 to the F4R(+) pigs results in an intestinal mucosal immune response that completely protects the pigs against a challenge infection. In F4R(-) pigs such an intestinal mucosal immune response does not occur. However, a priming of the systemic immune system can be seen similar to the priming in pigs fed with the same dose of a food antigen, suggesting that F4 in F4R(-) pigs behaves as a food antigen. The fact that different mucosal immune responses can be induced with soluble F4, makes it an interesting model to study mucosal immune mechanisms in the pig.

To develop a vaccine against Escherichia coli-induced post-weaning diarrhea and edema disease, insights in the induction of the protective immune response following infection with these pathogenic E. coli is needed. Therefore, the fimbriae-specific antibody response of newly weaned pigs following infection with the Shiga-like toxin type II variant (SLT-IIv) producing F18(+) verotoxigenic E. coli (VTEC) (strain 107/86) was compared with the response following an infection with LT producing F4(+) enterotoxigenic E. coli (ETEC) (strain GIS 26). F4(+) ETEC were able to colonize the gut very soon after infection, as peak excretion of F4(+) E. coli bacteria was seen 2 days post-infection (dpi), but had already disappeared 7dpi. On the other hand, F18(+) VTEC infection resulted in a slower colonization of the gut as the peak excretion of F18(+) E. coli was observed between 3 and 5dpi, but this colonization remained longer as F18(+) E. coli were detected till 9dpi in feces. Furthermore, this fast colonization pattern of F4(+) ETEC is accompanied with the presence of F4-specific antibodies in mucosal tissues and serum from 4dpi onward, with maximal amounts of F4-specific IgA in the jejunal lamina propria and serum 7dpi. In contrast, F18-specific IgA was only readily detected in the jejunal lamina propria 15dpi and showed a maximum serum titer 21dpi. Besides this faster induction and higher antibody response, the switch from IgM to IgA and IgG was also earlier following the F4(+) ETEC infection.

Abstract: Post-weaning diarrhoea and oedema disease in weaned piglets are caused by infection with F4+ or F18+ Escherichia coli strains. There is no commercial vaccine available, but it is shown that oral immunization of weaned piglets with purified F4 fimbriae induces a protective mucosal immune response. In the present study, piglets were orally and nasally immunized with purified F18 fimbriae in the presence of the mucosal adjuvant LT(R192G) or CTA1-DD, respectively. This immunization could not lead to protection against F18+ E. coli infection. The induced F18-specific immune response was directed towards the major subunit FedA and weakly towards the adhesive subunit FedF. The results of these experiments demonstrate that it is difficult to induce protective immunity against F18+ E. coli using the whole fimbriae due to the low response against the adhesin. [Copyright &y& Elsevier]

Abstract: The present study analysed quantitatively the mucin 4 polymorphism for determing the F4ac/ab receptor status of a total of 63 pigs by comparing it with the in vitro villous adhesion assay. The probability of a susceptible genotype for the mucin 4 increases significantly with increasing F4ab or F4ac ETEC adhesion per 250μm villi (P =0.029 for F4ab, P =0.030 for F4ac), with the odds ratio for each unit increase of F4ab or F4ac equal to, respectively, 1.036 (95% CI [1.004–1.069]) and 1.018 (95% CI [1.002–1.034]). In the phenotypic in vitro villous adhesion test, a cut-off value of 5 bacteria was chosen as a criteria for the distinction between an F4R positive and F4R negative pig. The sensitivity and specificity for the in vitro villous adhesion test, with the genotyping test for mucin 4 as golden standard, is 100% and 24%, respectively, for F4ab as well as F4ac. Absence of adhesion of F4ac and F4ab ETEC to the villous brush borders was not associated with genotypic resistance suggesting that there is at least one other receptor for F4ab/ac Escherichia coli. As a consequence, not only mucin 4 gene polymorphism but also expression of these other receptor(s) has to be included in a screening assay for F4ac/ab receptor negative pigs. [Copyright &y& Elsevier]

Abstract: 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 α(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. [Copyright &y& Elsevier]

Abstract: Transgenic plants are attractive bioreactors to large-scale production of recombinant proteins because of their relatively low cost. This study reports for the first time the use of transgenic plants to reduce enterotoxigenic Escherichia coli (ETEC) excretion in its natural host species. The DNA sequence encoding the major subunit and adhesin FaeG of F4+ ETEC was transformed into edible alfalfa plants. Targeting of FaeG production to chloroplasts led to FaeG levels of up to 1% of the total soluble protein fraction of the transgenic alfalfa. Recombinant plant-produced FaeG (pFaeG) remained stable for 2 years when the plant material was dried and stored at room temperature. Intragastric immunization of piglets with pFaeG induced a weak F4-specific humoral response. Co-administration of pFaeG and the mucosal adjuvant cholera toxin (CT) enhanced the immune response against FaeG, reflected a better induction of an F4-specific immune response. In addition, the intragastric co-administration of CT with pFaeG significantly reduced F4+ E. coli excretion following F4+ ETEC challenge as compared with pigs that had received nontransgenic plant material. In conclusion, transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against F4+ ETEC infections. [Copyright &y& Elsevier]

Abstract: F18+ Escherichia coli cause postweaning diarrhoea and oedema disease in newly weaned piglets. Protection against these diseases can be established by preventing the fimbrial adhesion of these bacteria to the enterocytes of the porcine intestine. To test a vaccine against F18+ E. coli consisting of the adhesin of F18 fimbriae, FedF, the conservation of the FedF subunit had to be examined. Therefore, the fedF sequence of 37 F18+ E. coli isolates from different countries was determined and compared to the fedF gene of the F18ab reference strain F107/86. The amino acid sequence of the mature FedF from the individual F18+ E. coli isolates was 96–100% identical to that from E. coli F107/86, but the overall homology was 90.4%. Hyper variable regions were not found in the FedF sequence. The FedF sequence was conserved over the different countries and between the two antigenic variants, F18ab and F18ac, suggesting that F18ab and F18ac strains have the same receptor. Furthermore, the conserved C-terminal region in the FedF adhesin suggests that the F18 fimbriae, in analogy with type 1 and P pili, are assembled by a donor strand mechanism. In conclusion, the reported conservation of FedF supports the usefulness of the fimbrial adhesin as a subunit vaccine against F18+ E. coli infection. [Copyright &y& Elsevier]

Abstract: Oral immunization of both humans and animals with non-replicating soluble antigens often results in the induction of oral tolerance. However, receptor-dependent uptake of orally administered soluble antigens can lead to the induction of an antigen-specific immune response. Indeed, oral immunization of pigs with recombinant FaeG (rFaeG), the adhesin of the F4(K88) fimbriae of enterotoxigenic Escherichia coli (ETEC), induces an F4-specific humoral and cellular immune response. This response is accompanied with a reduction in the excretion of F4+ E. coli following challenge. To improve the immune response against F4, rFaeG was orally co-administered with the mucosal adjuvant cholera toxin (CT). Oral immunization of pigs with rFaeG and CT significantly improved the induction of an F4-specific humoral and cellular immune response and also significantly reduced the faecal F4+ E. coli excretion following F4+ ETEC challenge as compared to rFaeG-immunized pigs. Therefore, the present study demonstrates that CT can act in pigs as a mucosal adjuvant for antigens that bind to the intestinal epithelium by a CT-receptor-independent mechanism. [Copyright &y& Elsevier]

The importance of adhesins in the pathogenicity of several bacteria resulted in studies on their usefulness in vaccines. In this study, the gene of the F4(K88)-fimbrial adhesin FaeG of the pathogenic enterotoxigenic Escherichia coli (ETEC) strain GIS26 was cloned in the pET30Ek-LIC vector and expressed with an N-terminal His- and S-tag in the cytoplasm of BL21(DE3). Recombinant FaeG (rFaeG) subunits were isolated from insoluble cytoplasmic aggregates and refolded into a native-like F4 receptor (F4R)-binding conformation. Indeed, the presence of conformational epitopes was shown by ELISA and the ability to bind the F4R was observed by inhibiting the adhesion of F4+ ETEC to F4R+ villi with increasing concentrations of native-like refolded rFaeG subunits. The rFaeG subunits appear as monomers, whereas the purified F4 fimbriae are multimers. Oral immunization of newly weaned piglets with native-like rFaeG induced a mucosal and systemic F4-specific immune response, significantly reducing F4+ E. coli excretion from 2 till 5 days following challenge infection. However, improvement of stability and immunogenicity of rFaeG is necessary since a higher F4-specific response was obtained following immunization with purified F4 fimbriae. Furthermore, the N-terminal fusion of a His- and S-tag was not detrimental for binding the F4R, supporting the use of FaeG as mucosal carrier. In conclusion, oral immunization with a recombinant fimbrial adhesin subunit of Escherichia coli induces a mucosal and systemic fimbriae-specific immune response. [Copyright &y& Elsevier]

Enterotoxigenic and verotoxigenic F18+ Escherichia coli colonising the pig small intestine, adhere to receptors on intestinal villous enterocytes by F18 fimbriae. The aim of the present study was to define the F18R nature. The knowledge on the nature of this receptor could be important for the development of receptor-based treatments against F18+ E. coli-induced disease. The adhesion of F18+ E. coli to pig intestinal villous enterocytes was analysed in an in vitro assay. The adhesion of F18+ E. coli but not of F4ac+ E. coli was strongly inhibited by monoclonal antibodies (mAb) with blood group H-2 specificity. Conversely, blood group H-1 specific mAb could not inhibit the adhesion of F18+ E. coli nor F4ac+ E. coli. Moreover, the blood group H-2 trisaccharide strongly inhibited the adhesion of F18+ E. coli, but only partially the adhesion of F4ac+ E. coli. These data demonstrate that the F18 receptor contains the blood group antigen H-2 (α-fuc-(1-2)-β-Gal-(1-4)-GlcNAc) as major carbohydrate. [Copyright &y& Elsevier]

F18+ enterotoxigenic and verotoxigenic Escherichia coli (E. coli) are widespread and induce post-weaning diarrhoea and oedema disease, respectively. No commercial vaccine exists. Therefore, the F18 herd seroprevalence of 178 randomly selected pig-breeding farms in northern Belgium was evaluated by determining F18-specific IgG antibody titres in pooled sera of sows per breeding farm. This survey reported the first data concerning F18 herd seroprevalence and showed that F18+ E. coli seroprevalence was common in northern Belgium. Significantly more open pig-breeding farms were F18 seropositive (96.4%) compared to closed (88.8%). Furthermore, no clear association was determined between the F18 herd seroprevalence and the number of sows per breeding farm (rsp=0.19), or with the number of fattening pigs per breeding farm (rsp=0.17). [Copyright &y& Elsevier]

In this study it was analysed whether intramuscular (IM) immunisation of piglets with F4 during the suckling period could protect against oral challenge with F4+-Escherichia coli and whether addition of 1α,25(OH)2D3 or CpG-ODN could improve this protection.F4-seronegative F4-receptor positive pigs were divided into four groups of five pigs each. The pigs were intramuscularly injected with F4 fimbriae only or supplemented with 1α,25(OH)2D3 (D3-group) or CpG-ODN (CpG-group). The control group received PBS in IFA. Seven days after the second immunisation, all pigs were intragastrically inoculated with 1×1010 CFU of F4+-E. coli. All F4-injected groups, showed a reduced faecal excretion of F4+-E. coli. However, this reduction was only statistically significant in the D3-group 2 days post challenge. Pigs in the latter group showed a secondary antibody response upon challenge, indicating that F4-primed memory B-cells were present in the gut-associated lymphoid tissues at that moment.CpG-ODN, on the other hand, did not enhance the F4-specific antibody response. However, CpG-ODN significantly increased the F4-specific as well as mitogen-induced proliferation of pheripheral blood monomorphonuclear cells indicating a direct or indirect overall effect on T-lymphocytes. In conclusion, supplementation with 1α,25(OH)2D3 or CpG-ODN improved protection against an F4+-E. coli infection. This protection was most obvious for 1α,25(OH)2D3 and indicates its potential use in veterinary vaccines against enteropathogens. [Copyright &y& Elsevier]