RESUMEN
Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3' untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT-) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R-) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R- pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R- pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.
Asunto(s)
Antígenos CD13/metabolismo , Escherichia coli Enterotoxigénica/clasificación , Escherichia coli Enterotoxigénica/fisiología , Infecciones por Escherichia coli/veterinaria , Regulación Enzimológica de la Expresión Génica/fisiología , Porcinos/genética , Animales , Adhesión Bacteriana , Antígenos CD13/genética , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Predisposición Genética a la Enfermedad , Genotipo , Mutación , Porcinos/metabolismoRESUMEN
F4(+) enterotoxigenic Escherichia coli (F4(+) ETEC) are an important cause of diarrhoea and mortality in piglets. F4(+) ETEC use their F4 fimbriae to adhere to specific receptors (F4Rs) on small intestinal brush borders, resulting in colonization of the small intestine. To prevent pigs from post-weaning diarrhoea, pigs should be vaccinated during the suckling period. Previously, we demonstrated that F4acR(+), but not F4acR(-) piglets could be orally immunized with purified F4 fimbriae resulting in a protective immunity against F4(+) ETEC infections, indicating that this immune response was F4R dependent. Recently, aminopeptidase N has been identified as a glycoprotein receptor important for this oral immune response. However, in some oral immunization experiments, a few F4acR(+) piglets did not show an antibody response upon oral immunization, suggesting additional receptors. Therefore, the binding profile of F4 to brush border membrane (glyco)proteins was determined for pigs differing in F4-specific antibody response upon oral immunization, in in vitro adhesion of F4(+)E. coli to small intestinal villi, and in Muc4 genotype. Six groups of pigs could be identified. Only two groups positive in all three assays showed two high molecular weight (MW) glycoprotein bands (>250kDa) suggesting that these high MW bands are linked to the MUC4 susceptible genotype. The fact that these bands were absent in the MUC4 resistant group which showed a positive immune response against F4 and was positive in the adhesion test confirm that at least one or perhaps more other F4Rs exist. Interestingly, two pigs that were positive in the villous adhesion assay did not show an immune response against F4 fimbriae. This suggests that a third receptor category might exist which allows the bacteria to adhere but does not allow effective immunization with soluble F4 fimbriae. Future research will be necessary to confirm or reveal the identity of these receptors.
Asunto(s)
Antígenos Bacterianos/metabolismo , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Fimbrias/metabolismo , Mucina 4/metabolismo , Enfermedades de los Porcinos/metabolismo , Adhesinas de Escherichia coli/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Escherichia coli Enterotoxigénica/genética , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Proteínas Fimbrias/genética , Proteínas Fimbrias/inmunología , Genotipo , Inmunización/métodos , Inmunización/veterinaria , Mucina 4/genética , Mucina 4/inmunología , Polimorfismo de Nucleótido Simple , Porcinos , Enfermedades de los Porcinos/genética , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/microbiologíaRESUMEN
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.
Asunto(s)
Vacunas Bacterianas/inmunología , Diarrea/veterinaria , Infecciones por Escherichia coli/veterinaria , Escherichia coli/genética , Proteínas Fimbrias/inmunología , Adhesinas Bacterianas/inmunología , Administración Oral , Animales , Vacunas Bacterianas/administración & dosificación , Diarrea/prevención & control , Edematosis Porcina/inmunología , Escherichia coli/inmunología , Escherichia coli/metabolismo , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/inmunología , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/inmunología , Fimbrias Bacterianas/metabolismo , Inmunización , Porcinos , Vacunas Conjugadas/inmunología , DesteteRESUMEN
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.
Asunto(s)
Edematosis Porcina/microbiología , Infecciones por Escherichia coli/veterinaria , Escherichia coli/fisiología , Antígenos de Histocompatibilidad/inmunología , Receptores Inmunológicos/metabolismo , Adhesinas de Escherichia coli/inmunología , Adhesinas de Escherichia coli/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Adhesión Bacteriana/inmunología , Adhesión Bacteriana/fisiología , Edematosis Porcina/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Fucosiltransferasas , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Receptores Inmunológicos/inmunología , Porcinos , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
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.