Monoclonal antibodies reveal a weak interaction between the F18 fimbrial adhesin FedF and the major subunit FedA.

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.

The age-dependent expression of the F18+ E. coli receptor on porcine gut epithelial cells is positively correlated with the presence of histo-blood group antigens.

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.

Mucosal immunization of piglets with purified F18 fimbriae does not protect against F18+ Escherichia coli infection.

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.

The F18 fimbrial adhesin FedF is highly conserved among F18+Escherichia coli isolates.

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.

Maltose-binding protein is a potential carrier for oral immunizations.

Maltose binding protein (MBP) is often fused to a relevant protein to improve its yield and facilitate its purification, but MBP can also enhance the immunogenicity of the fused proteins. Recent data suggest that MBP may potentiate antigen-presenting functions in immunized animals by providing intrinsic maturation stimuli to dendritic cells through TLR4. The aim of this study was to examine if an MBP-specific immune response can be elicited by oral administration of MBP. Therefore, in a first experiment the MBP specific immune response was analyzed after oral immunization with MBP or MBP+CT to piglets and both the systemic and mucosal immune responses were examined Although no high systemic response was observed in the MBP-group, a local mucosal IgM MBP-specific response in the jejunal Peyer's patches was observed. In the second experiment MBPFedF was orally administered to piglets. A significant systemic response against MBP and a weak response against FedF were found after oral administration of MBPFedF+CT. Also the presence of MBP-specific IgA ASC in the lamina propria indicates that a local intestinal immune response against MBP was induced. Our data suggests that MBP can cross the epithelial barrier reaching the gut-associated lymphoid tissue after oral administration to pigs, which implicates that MBP could act as a carrier and delivery system for fused proteins to target the vaccine antigens to intestinal immune cells.

The excretion of F18+ E. coli is reduced after oral immunisation of pigs with a FedF and F4 fimbriae conjugate.

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.

MET15 as a visual selection marker for Candida albicans.

To develop better molecular genetic tools for the diploid yeast Candida albicans, the suitability of the MET15 gene as a visual selection marker was studied. Both MET15 alleles of C. albicans CAI-4 were isolated by functional complementation of a Saccharomyces cerevisiae strain lacking the MET15 gene. Growth of this complemented strain on Pb(2+)-containing medium was associated with a colour shift of brown into white colonies. The MET15 alleles of C. albicans were located on chromosome 4 by pulsed-field gel electrophoresis and Southern blotting. A met15-deficient strain of C. albicans CAI-4 was generated using the ura blaster technique. This strain showed a brown colony colour on Pb(2+)-containing medium, which corresponded with the colony colour of a S. cerevisiae strain lacking the MET15 gene. Unexpectedly, the met15-deficient strain of C. albicans still grew on methionine-depleted medium. However, this growth was severely delayed. In addition, complementation of this strain with an integrative or replicative plasmid containing either of the MET15 alleles resulted in the formation of white transformants on Pb(2+)-containing medium. These transformants grew very well on methionine-depleted medium. Colony sectoring was obtained with the replicative plasmid and not with the integrative one. This study demonstrates that the MET15 gene of C. albicans is suitable as a visual marker and therefore can be used to identify transformants and study plasmid stability. GenBank Accession Nos for MET15 nucleotide sequences are AF188273, AF188274 and AF188275.