Refined localization of the Escherichia coli F4ab/F4ac receptor locus on pig chromosome 13

Diarrhoea in newborn and weaned pigs caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae leads to considerable losses in pig production. In this study, we refined the mapping of the receptor locus for ETEC F4ab/F4ac adhesion ( F4bcR ) by joint analysis of Nordic and Swiss data. A total of 236 pigs from a Nordic experimental herd, 331 pigs from a Swiss experimental herd and 143 pigs from the Swiss performing station were used for linkage analysis. Genotyping data of six known microsatellite markers, two newly developed markers ( MUC4gt and HSA125gt ) and an intronic SNP in MUC4 ( MUC4-8227 ) were used to create the linkage map. The region for F4bcR was refined to the interval SW207 – S0075 on pig chromosome 13. The most probable position of F4bcR was in the SW207 – MUC4 region. The order of six markers was supported by physical mapping on the BAC fingerprint contig from the Wellcome Trust Sanger Institute. Thus, the region for F4bcR could be reduced from 26 to 14 Mb.     

Refined mapping of the Escherichia coli F4ab/F4ac receptor gene(s) on pig chromosome 13

Enterotoxigenic Escherichia coli expressing F4 fimbriae is the major cause of diarrhoea in neonatal and post-weaning piglets. Previous studies have revealed that the loci controlling the F4ab/F4ac receptors are located on SSC13q41, between markers SW207 and S0283. In this study, we refined their positions in a two generation population containing 366 piglets of three breeds (Large White, Landrace, and Songliao Black). Nine microsatellite markers within this region were selected from the MARC (U.S. Meat Animal Research Center) porcine linkage map, and the pedigree disequilibrium test was employed for fine-mapping. The F4abR gene was located in the interval between S0283 and SW1833, a 4.8-cM region, and the F4acR gene was located in the interval between S0283 and SW1876, a 1.6-cM region. Our results also suggest that the F4ab/F4ac receptors might be controlled by two different but closely linked loci. The results of microsatellite-based haplotype analysis in the corresponding region show that some specific haplotypes were overwhelmingly present in the adhesive or non-adhesive animals, indicating that there are mutations within the identified regions that are strongly associated with the F4ab/ac phenotypes.     

Refined candidate region specified by haplotype sharing for Escherichia coli F4ab/F4ac susceptibility alleles in pigs

Infection of the small intestine by enterotoxigenic Escherichia coli F4ab/ac is a major welfare problem and financial burden for the pig industry. Natural resistance to this infection is inherited as a Mendelian recessive trait, and a polymorphism in the MUC4 gene segregating for susceptibility/resistance is presently used in a selection programme by the Danish pig breeding industry. To elucidate the genetic background involved in E. coli F4ab/ac susceptibility in pigs, a detailed haplotype map of the porcine candidate region was established. This region covers approximately 3.7 Mb. The material used for the study is a three generation family, where the founders are two Wild boars and eight Large White sows. All pigs have been phenotyped for susceptibility to F4ab/ac using an adhesion assay. Their haplotypes are known from segregation analysis using flanking markers. By a targeted approach, the candidate region was subjected to screening for polymorphisms, mainly focusing on intronic sequences. A total of 18 genes were partially sequenced, and polymorphisms were identified in GP5, CENTB2, APOD, PCYT1A, OSTalpha, ZDHHC19, TFRC, ACK1, MUC4, MUC20, KIAA0226, LRCH3 and MUC13 . Overall, 227 polymorphisms were discovered in the founder generation. The analysis revealed a large haplotype block, spanning at least 1.5 Mb around MUC4 , to be associated with F4ab/ac susceptibility.     

Gene Silencing of Porcine MUC13 and ITGB5: Candidate Genes towards Escherichia coli F4ac Adhesion

Background

Integrin beta-5 (ITGB5) and mucin 13 (MUC13) genes are highly expressed on the apical surface of intestinal epithelia and are thought to be candidate genes for controlling the expression of the receptor for enterotoxigenic Escherichia coli (ETEC) F4ac. Human MUC13 protein has an expected role in protecting intestinal mucosal surfaces and porcine ITGB5 is a newly identified potential receptor for ETEC F4ac.

Methodology/Principal Findings

To test the hypothesis that ITGB5 and MUC13 both play key roles in protection of the intestinal mucosa against pathogenic bacterium, porcine intestinal epithelial cells (IPEC-J2) were transfected with ITGB5-targeting, MUC13-targeting or negative control small interfering RNA (siRNA), respectively. Firstly, we measured mRNA expression levels of mucin genes (MUC4, MUC20), pro-inflammatory genes (IL8, IL1A, IL6, CXCL2), anti-inflammatory mediator SLPI, and PLAU after RNAi treatments with and without ETEC infection. Secondly, we compared the adhesions of ETEC to the pre- and post-knockdown IPEC-J2 cells of ITGB5 and MUC13, respectively. We found that ITGB5 and MUC13 knockdown both had small but significant effects in attenuating the inflammation induced by ETEC infection, and both increased bacterial adhesion in response to F4ac ETEC exposure.

Conclusions/Significance

Our current study first reported that ITGB5 and MUC13 are important adhesion molecules of mucosal epithelial signaling in response to Escherichia coli in pigs. These data suggest that both ITGB5 and MUC13 play key roles in defending the attachment and adhesion of ETEC to porcine jejunal cells and in maintaining epithelial barrier and immunity function.     

Investigation of the porcine MUC13 gene: isolation, expression, polymorphisms and strong association with susceptibility to enterotoxigenic Escherichia coli F4ab/ac

Enterotoxigenic Escherichia coli (ETEC) F4ab and F4ac are major determinants of piglet diarrhoea. The locus for the ETEC F4ab/ac receptor has been mapped to SSC13q41. MUC13 is a transmembrane mucin expressed predominantly in the epithelial surface of the gastrointestinal tract and the MUC13 gene was assigned to SSC13q41, supporting it as a positional candidate gene for the ETEC F4ab/ac receptor. We herein determined the complete 2679-bp cDNA of pig MUC13, and proved that it was most highly expressed in the jejunum and moderately expressed in the trachea, stomach and liver. Furthermore, 13 MUC13 polymorphisms were identified in 19 founder animals of a White Duroc x Erhualian resource population, and a total of 727 F(2) animals with in vitro ETEC F4ab/ac adhesion phenotypes in this population were genotyped for three identified MUC13 polymorphisms including c.576C>T, c.908A>G and c.935A>C. The transmission disequilibrium test showed that the MUC13 alleles and haplotypes were significantly associated with susceptibility/resistance to ETEC F4ab/ac, especially between haplotype [C;G;A] and susceptibility to ETEC F4ac (P = 8.0e-18). Animals inheriting this haplotype were predominantly susceptible to ETEC F4ac (n = 291/303). Moreover, nearly all animals homozygous for haplotype [T;G;C] (n = 39/41) and a majority of those with the [C;A;A]/[T;G;C] haplotype pair (n = 79/88) were resistant to ETEC F4ab. Our results indicated that MUC13 is in strong linkage disequilibrium with the ETEC F4ab/ac receptor locus and provided potential markers for selection of ETEC F4ab/ac-resistant animals in the pig breeding scheme.     

Polymorphisms of three gene-derived STS on pig chromosome 13q41 are associated with susceptibility to enterotoxigenic Escherichia coli F4ab/ac in pigs

Neonatal diarrhea caused by enterotoxigenic Escherichia coli(ETEC)F4 is a common and serious disease,resulting in significant economical loss in the pig industry.The locus encoding ETEC F4 receptor has been mapped to pig chromosome(SSC)13q41,and one of the most significantly linked markers is S0075.In this study,we selected three genes including SLC12A8,MYLK and KPNA1 from a chromosomal region flanking S0075 on SSC13 to develop pig specific sequence tagged sites(STS). Seven single nucleotide polymorphisms were identified in the three pig STS using DNA of four full-sib susceptible and resistant animals in a White Duroc×Erhualian intercross.All grandparents,parents and 755 offspring in the intercross were genotyped for three polymorphisms,including SLC12A8 g.159A>G,MYLK g.1673A>G and KPNA1 g.306A>G.Family-based transmission disequilibrium test(TDT) revealed that all polymorphisms and the corresponding haplotypes are significantly associated with ETEC F4ab/ac(especially F4ac)brush border adhesion phenotypes,indicating that these polymor- phism are in linkage disequlibrium with causal mutation(s)of the gene encoding ETEC F4ab/ac receptor. Our results strengthen the evidence for the involvement of SSC13q41 in high acquiring risk of ETEC F4ab/ac infection,and provide novel polymorphic markers for fine mapping of the ETEC F4ab/ac receptor locus.     

A genome-wide association study identifies two novel promising candidate genes affecting Escherichia coli F4ab/F4ac susceptibility in swine

Enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbria is the major pathogenic bacteria causing diarrhoea in neonatal and post-weaning piglets. Previous studies have revealed that the susceptibility to ETEC F4ab/F4ac is an autosomal Mendelian dominant trait and the loci controlling the F4ab/F4ac receptor are located on SSC13q41, between markers SW207 and S0283. To pinpoint these loci and further validate previous findings, we performed a genome-wide association study (GWAS) using a two generation family-based population, consisting of 301 piglets with phenotypes of susceptibility to ETEC F4ab/F4ac by the vitro adhesion test. The DNA of all piglets and their parents was genotyped using the Illumina PorcineSNP60 BeadChip, and 50,972 and 50,483 SNPs were available for F4ab and F4ac susceptibility, respectively, in the association analysis after quality control. In summary, 28 and 18 significant SNPs (p<0.05) were detected associated with F4ab and F4ac susceptibility respectively at genome-wide significance level. From these significant findings, two novel candidate genes, HEG1 and ITGB5, were firstly identified as the most promising genes underlying F4ab/F4ac susceptibility in swine according to their functions and positions. Our findings herein provide a novel evidence for unravelling genetic mechanism of diarrhoea risk in piglets.     

Fimbrial Subunit Protein FaeG Expressed in Transgenic Tobacco Inhibits the Binding of F4ac Enterotoxigenic Escherichia coli to Porcine Enterocytes

Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed. Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants. In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein. Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment. For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast. The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein. The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions. Plant-produced FaeG proved to be stable up to 2 h under these conditions. The binding and inhibition properties were tested with isolated piglet villi. These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner. Thus, the first two prerequisites for the development of an oral vaccine have been met.     

Fine-mapping of the intestinal receptor locus for enterotoxigenic Escherichia coli F4ac on porcine chromosome 13

The aim of this study was to refine the localization of the receptor locus for fimbriae F4ac. Small intestinal enterocyte preparations from 187 pigs were phenotyped by an in vitro adhesion test using two strains of Escherichia coli representing the variants F4ab and F4ac. The three-generation pedigree comprised eight founders, 18 F1 and 174 F2 animals, for a total of 200 pigs available for the linkage analysis. Results of the adhesion tests on 171 F2 pigs slaughtered at 8 weeks of age show that 23.5% of the pigs were adhesive for F4ab and non-adhesive for F4ac (phenotype F4abR+/F4acR-; R means receptor). Pigs of this phenotype were characterized by a weak adhesion receptor for F4ab. No pigs were found expressing only F4acR and lacking F4abR. Receptors for F4ab and F4ac (F4abR+/F4acR+) were expressed by 54.5% of the pigs. Animals of this phenotype strongly bound both F4ab and F4ac E. coli. In the segregation study, the serum transferrin (TF) gene and 10 microsatellites on chromosome 13 were linked with F4acR (recombination fractions (theta) between 0.00 and 0.11 and lod score values (Z) between 11.4 and 40.4). The 11-point analysis indicates the F4acR locus was located in the interval S0068-Sw1030 close to S0075 and Sw225, with recombination fractions (theta) of 0.05 between F4acR and S0068, 0.04 with Sw1030, and 0.00 with S0075 and Sw225. The lack of pigs displaying the F4abR-/F4acR+ phenotype and the presence of two phenotypes for F4abR (a strong receptor present in phenotype F4abR+/F4acR+ and a weak receptor in phenotype F4abR+/F4acR-) led us to conclude that the receptor for F4ac binds F4ab bacteria as well, and that it is controlled by one gene localized between S0068 and Sw1030 on chromosome 13.     

Isolation of K88 Antigen Variants (ab,ac, ad) from Porcine Enterotoxigenic Escherichia coli Belonging to Different Serotypes

Fimbriae isolation by means of thermal shock was applied to fifteen K88-positive (three K88ab, nine K88ac and three K88ad) Escherichia coli reference strains belonging to serotypes O8:K87, O32, O45, O138:K81, O141:K85, O147:K89, O149:K91, and O157, as well as to ten K88-positive enterotoxigenic strains isolated from porcine diarrhea in Spain, all of them belonging to the O149 serogroup. Fimbriae were removed from the bacterial cells by thermal shock at 60 C and then precipitated using ammonium sulfate. The final amount of K88 antigen and the purification degree were not related to the serogroup of the bacteria or to the antigen variant but were related to the buffer used for isolation. Phosphate buffer containing urea was shown to be more effective than Tris-HCl for isolation of K88 antigen. The molecular weights by SDS-PAGE for K88ab, K88ac, and K88ad were 28.5, 29.2, and 31.0 kDa, respectively. All enterotoxigenic E. coli strains isolated in Spain showed the K88ac variant.     

The g.243A>G mutation in intron 17 of MUC4 is significantly associated with susceptibility/resistance to ETEC F4ab/ac infection in pigs

Using a porcine radiation hybrid panel, we assigned the mucin 4 (MUC4) gene to SSC13q41, which harbours the enterotoxigenic Escherichia coli (ETEC) F4ab/ac receptor locus. In addition, we identified two SNPs in intron 17 of MUC4 (DQ124298:g.243A>G and DQ124298:g.334A>G) in the parental population of a White Duroc x Erhualian cross. Association analysis showed that the MUC4 g.243A>G mutation was strongly associated with ETEC F4ab/ac, and especially with F4ac adhesion phenotypes in the White Duroc x Erhualian resource population, indicating that this polymorphism was in a significant linkage disequlibrium with the ETEC F4ab/ac receptor locus. Because of different linkage disequlibrium values between the ETEC F4ab and F4ac adhesion phenotypes and the MUC4 g.243A>G mutation, we argue that the inheritance of F4ab and F4ac receptors might be under the control of two closely linked loci.     

Effective genetic markers for identifying the Escherichia coli F4ac receptor status of pigs

The F4ac receptor locus (F4acR), which encodes susceptibility or resistance to Escherichia coli diarrhoea, is inherited as an autosomal recessive monogenetic trait. F4acR is localized on pig chromosome 13 (SSC13q41–q44) near the MUC13 gene. Two flanking markers (CHCF1 and ALGA0106330) with a high linkage disequilibrium (LD) with F4acR were found to be effective for the genetic identification of F4ac‐resistant pigs in the Swiss Large White breed (one recombinant out of 2034 genotyped pigs). Three recombinant boars, one each from the Duroc, Swiss Landrace and Piétrain breeds, were genotyped with seven different markers and phenotyped by means of a microscopic adhesion test. Only ALGA0072075, CHCF1 and CHCF3 indicated the correct phenotype. To test the effect of the resistance allele on production traits, 530 Large White pigs from the national test station were investigated. A significant difference existed among the F4acR locus genotypes in the intramuscular fat content of the longissimus dorsi muscle, whereas no other production traits were influenced by the resistance allele. The frequency of the CHCF1‐C and ALGA0106330‐A alleles associated with resistance in the Swiss Large White population was 60%, which is advantageous for implementing this trait in a breeding programme to select for E. coli F4ac‐resistant animals. The selection of resistant pigs should start on the male side due to the inability of resistant sows to produce sufficient amounts of protecting antibodies in the colostrum. Selection of genetically F4ac‐resistant pigs is a sustainable and suitable alternative to decreasing animal loss and antibiotic use due to diarrhoea.     

Evidence for linkage between K88ab, K88ac intestinal receptors to Escherichia coli and transferrin loci in pigs

Segregation at the loci coding for the K88ab and K88ac small intestinal receptors to E. coli adhesins (K88abR, K88acR) and at the transferrin (TF) locus was studied in 38 pig families including 273 piglets. The TF locus showed a segregation deviation towards the B variant while each of the K88 receptors behaved as a single autosomal dominant gene. Recombinants between K88abR and K88acR provide evidence that they are under the control of two different loci. Thirty-two triple backcross families were selected to test linkage and estimate recombination rates (θ). Our results demonstrate that the two K88 receptor loci are closely linked (θ= 0.02) with a maximum lod score value (Z_m) of 46.0. In addition, they are linked to the TF locus, θ= 0.14, Z_m= 19.6 for the K88abR locus and θ= 0.16, Z_m= 17.9 for the K88acR locus. The estimated recombination rates, smaller in males than in females, are consistent with the order TF-K88abR-K88acR. This linkage thus localizes the K88 loci, as the TF locus, on chromosome 13.     

Linkage and comparative mapping of the locus controlling susceptibility towards E. COLI F4ab/ac diarrhoea in pigs

In 1995, Edfors-Lilja and coworkers mapped the locus for the E. COLI K88ab (F4ab) and K88ac (F4ac) intestinal receptor to pig chromosome 13 (SSC13). Using the same family material we have refined the map position to a region between the microsatellite markers Sw207 and Sw225. Primers from these markers were used to screen a pig BAC library and the positive clones were used for fluorescent in situ hybridization (FISH) analysis. The results of the FISH analysis helped to propose a candidate gene region in the SSC13q41-->q44 interval. Shotgun sequencing of the FISH-mapped BAC clones revealed that the candidate region contains an evolutionary breakpoint between human and pig. In order to further characterise the rearrangements between SSC13 and human chromosome 3 (HSA3), detailed gene mapping of SSC13 was carried out. Based on this mapping data we have constructed a detailed comparative map between SSC13 and HSA3. Two candidate regions on human chromosome 3 have been identified that are likely to harbour the human homologue of the gene responsible for susceptibility towards E. COLI F4ab/ac diarrhoea in pigs.     

The porcine intestinal receptor for Escherichia coli K88ab,K88ac: regional localization on chromosome 13 and influence of IgG response to the K88 antigen

The loci encoding the porcine intestinal receptors for Escherichia coli K88ab and K88ac (K88abR and K88acR) were firmly assigned to chromosome 13 by linkage analysis using a three-generation pedigree. The linear order of these loci and seven other markers on chromosome 13 was determined by multipoint analyses. The K88abR and K88acR loci were tightly linked with the K88abR locus localized 7·4 cM (sex average) proximal to the transferrin locus. The results, together with previous reports from two other groups, provide an unequivocal assignment of the K88 receptor loci to chromosome 13, and reject a previous assignment to chromosome 4. Pigs possessing the receptor had a slightly higher specific IgG response to the K88 antigen after an intramuscular immunization with an E. coli vaccine.     

Inheritance of porcine receptors for enterotoxigenic Escherichia coli with fimbriae F4ad and their relation to other F4 receptors

Enteric Escherichia coli infections are a highly relevant cause of disease and death in young pigs. Breeding genetically resistant pigs is an economical and sustainable method of prevention. Resistant pigs are protected against colonization of the intestine through the absence of receptors for the bacterial fimbriae, which mediate adhesion to the intestinal surface. The present work aimed at elucidation of the mode of inheritance of the F4ad receptor which according to former investigations appeared quite confusing. Intestines of 489 pigs of an experimental herd were examined by a microscopic adhesion test modified in such a manner that four small intestinal sites instead of one were tested for adhesion of the fimbrial variant F4ad. Segregation analysis revealed that the mixed inheritance model explained our data best. The heritability of the F4ad phenotype was estimated to be 0.7±0.1. There are no relations to the strong receptors for variants F4ab and F4ac. Targeted matings allowed the discrimination between two F4ad receptors, that is, a fully adhesive receptor (F4adR^FA) expressed on all enterocytes and at all small intestinal sites, and a partially adhesive receptor (F4adR^PA) variably expressed at different sites and often leading to partial bacterial adhesion. In pigs with both F4ad receptors, the F4adR^PA receptor is masked by the F4adR^FA. The hypothesis that F4adR^FA must be encoded by at least two complementary or epistatic dominant genes is supported by the Hardy–Weinberg equilibrium statistics. The F4adR^PA receptor is inherited as a monogenetic dominant trait. A comparable partially adhesive receptor for variant F4ab (F4abR^PA) was also observed but the limited data did not allow a prediction of the mode of inheritance. Pigs were therefore classified into one of eight receptor phenotypes: A1 (F4abR^FA/F4acR⁺/F4adR^FA); A2 (F4abR^FA/F4acR⁺/F4adR^PA); B (F4abR^FA/F4acR⁺/F4adR⁻); C1 (F4abR^PA/F4acR⁻/F4adR^FA); C2 (F4abR^PA/F4acR⁻/F4adR^PA); D1 (F4abR⁻/F4acR⁻/F4adR^FA); D2 (F4abR⁻/F4acR⁻/F4adR^PA); E (F4abR⁻/F4acR⁻/F4adR⁻).     

Increased number of intestinal villous M cells in levamisole -pretreated weaned pigs experimentally infected with F4ac+ enterotoxigenic Escherichia coli strain

Immunoprophylaxis of porcine postweaning colibacillosis (PWC) caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae is an unsolved problem. Just as ETEC strains can exploit intestinal microfold (M) cells as the entry portal for infection, their high transcytotic ability make them an attractive target for mucosally delivered vaccines, adjuvants and therapeutics. We have developed a model of parenteral/oral immunization of 4-weeks-old pigs with either levamisole or vaccine candidate F4ac⁺ non-ETEC strain to study their effects on de novo differentiation of antigen-sampling M cells. Identification, localization and morphometric quantification of cytokeratin 18 positive M cells in the ileal mucosa of 6-weeks-old pigs revealed that they were: 1) exclusively located within villous epithelial layer, 2) significantly numerous (P< 0.01) in levamisole pretreated/challenged pigs, and 3) only slightly, but not significantly numerous in vaccinated/challenged pigs compared with non-pretreated/challenged control pigs. The fact that levamisole may affect the M cells frequency by increasing their numbers, makes it an interesting adjuvant to study development of an effective M cell-targeted vaccine against porcine PWC.Key words: M cells, levamisole, E. coli vaccine, weaned pigs.