H.U. Bertschinger

Two α(1,2) fucosyltransferase genes on porcine Chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci

Abstract. The Escherichia coli F18 receptor locus (ECF18R) has been genetically mapped to the halothane linkage group on porcine Chromosome (Chr) 6. In an attempt to obtain candidate genes for this locus, we isolated 5 cosmids containing the α(1,2)fucosyltransferase genes FUT1, FUT2, and the pseudogene FUT2P from a porcine genomic library. Mapping by fluorescence in situ hybridization placed all these clones in band q11 of porcine Chr 6 (SSC6q11). Sequence analysis of the cosmids resulted in the characterization of an open reading frame (ORF), 1098 bp in length, that is 82.3% identical to the human FUT1 sequence; a second ORF, 1023 bp in length, 85% identical to the human FUT2 sequence; and a third FUT-like sequence thought to be a pseudogene. The FUT1 and FUT2 loci therefore seem to be the porcine equivalents of the human blood group H and Secretor loci. Direct sequencing of the two ORFs in swine being either susceptible or resistant to adhesion and colonization by F18 fimbriated Escherichia coli (ECF18) revealed two polymorphisms at bp 307 (M307) and bp 857 (M857) of the FUT1 ORF. Analysis of these mutations in 34 Swiss Landrace families with 221 progeny showed close linkage with the locus controlling resistance and susceptibility to E. coli F18 adhesion and colonization in the small intestine (ECF18R), and with the locus of the blood group inhibitor S. A high linkage disequilibrium of M307–ECF18R in Large White pigs makes the M307 mutation a good marker for marker-assisted selection of E. coli F18 adhesion-resistant animals in this breed. Whether the FUT1 or possibly the FUT2 gene products are involved in the synthesis of carbohydrate structures responsible for bacterial adhesion remains to be determined.

A DNA polymorphism influencing α(1,2)fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18 adhesion

Abstract. The α(1,2)fucosyltransferases (FUT1 and FUT2) contribute to the formation of blood group antigen structures, which are present on cell membranes and in secretions. In the present study we demonstrate that both FUT1 and FUT2 are expressed in the pig small intestine. FUT1 polymorphisms influence adhesion of F18 fimbriated Escherichia coli (ECF18) to intestinal mucosa, and FUT2 is associated with expression of erythrocyte antigen 0. The FUT1 polymorphisms result in amino acid substitutions at positions 103 (Ala→Thr) and 286 (Arg→Glu). Tightly controlled expression of the FUT2 gene results in either an abundance or an absence of mRNA in small intestinal mucosa. ECF18-resistant animals were shown to be homozygous for threonine at amino acid 103 of the FUT1 enzyme. Susceptibility to ECF18 adhesion appeared to be solely dependent on the activity of FUT1 in intestinal epithelia. In intestinal mucosae of ECF18-resistant pigs which expressed FUT1 but not FUT2 RNA, the levels of α(1,2)fucosyltransferase activity were significantly lower (28- to 45-fold, P<0.001) than in susceptible pigs. Moreover, lysates of CHO cells transfected with FUT1 constructs encoding threonine at amino acid position 103 also showed significantly reduced enzyme activity compared with constructs encoding alanine at this position. Our genetic and enzymatic studies support the hypothesis that the FUT1 enzyme, and particularly the amino acid at position 103, is likely important in the synthesis of a structure that enables adhesion of ECF18 bacteria to small intestinal mucosa.

Inheritance of resistance to oedema disease in the pig: Experiments with an Escherichia coli strain expressing fimbriae 107

Abstract Inheritance of resistance to intestinal colonization with E. coli causing oedema disease is hypothetized to be under the control of one locus consisting of two alleles with susceptibility (S) dominating resistance (s). This mode of inheritance was investigated by matting pigs, resistant and susceptible to the disease, and examining the offspring. Weaned piglets were repeatedly inoculated orally with 5 × 105 CFU per pig per day of a streptomycin resistant strain of E. coli serotype O139:K12(B):H1:F(107) and susceptibility determined by daily semiquantitative cultural examination of rectal swabs. Using results obtained from offspring. 5 boars were retrospectively assigned the genotype ss, 1 was assigned Ss, and 2 were assigned SS. Nine sows were designated ss, 8 classified Ss and 4 SS. Ninety two pigs resulted from matings regarded as ss×ss; 89 (97%) of these were resistant to colonization and oedema disease. Of the 168 pigs from Ss×ss matings, 83 (49%) were resistant, while only 13 (9%) of 146 pigs from matings with at least one SS parent were classified resistant. The results are compatible with inheritance being controlled by one locus and with susceptibility dominating resistance to oedema disease.

Prevalence of F107 fimbriae on Escherichia coli isolated from pigs with oedema disease or postweaning diarrhoea

The study comprises fifty 4 to 12 weeks old pigs that died from oedema disease or severe diarrhoea. Smears were prepared from the mucosa of duodenum, jejunum and ileum, and by immunofluorescence F107 fimbrial antigens were detected. E. coli strains were isolated from the intestines and were characterised by slide agglutination (serogroup and F107 fimbriae production), by their cytotoxicity for Vero cells, and by gene amplification (genes coding for the major F107 subunit FedA, the toxin causing oedema disease SLT-IIv, and enterotoxins LTI, STIa and STII). F107 fimbriae were demonstrated in association with E. coli of serogroups O139:K12 and O141:K85a,b but not of serogroup O149:K91:F4a,c. Expression in culture of F107 fimbriae by some isolates gave additional evidence for production of these fimbriae by ETEC strains. The genetic determinant of SLT-Ilv was found in association with F107, and could not be detected in serogroup O149:K91:F4a,c. Gene fedA was demonstrated in two isolates which were devoid of SLT-IIv. Most isolates from cases of oedema disease belonged to serogroup O139:K12 and did not contain enterotoxin genes. Isolates from pigs that suffered from diarrhoea were serotyped O141:K85a,b or O149:K91:F4a,c, and carried at least two enterotoxin genes in their genomes. In a small proportion of the cases F107 antigens were demonstrated in intestinal smears although gene fedA was not detected in the corresponding isolates. The results confirm the importance of F107 fimbriae as virulence factor in oedema disease E. coli strains, but also demonstrate that F107 fimbriae can be found in association with postweaning diarrhoea isolates. In these latter strains enterotoxins were always demonstrated, irrespective of the presence of toxin SLT-IIv.

Active oral immunization of suckling piglets to prevent colonization after weaning by enterotoxigenic Escherichia coli with fimbriae F18

Immunoprophylaxis of porcine oedema disease and post-weaning diarrhoea caused by strains of Escherichia coli expressing fimbriae F18 is an unsolved problem. The study was designed to examine whether vaccination with a live F18ac vaccine of unweaned pigs born to sows with F18ac antibody in the colostrum requires preformed fimbriae in the vaccine, and whether protection against the heterologous fimbrial variant F18ab is induced as well. Genetically susceptible pigs were vaccinated orally on three consecutive days, beginning 10 days before weaning with 10(11) CFU of an F18ac culture. Challenge with a dose of 10(7) CFU of E. coli F18 on three consecutive days was initiated 9 or 11 days after weaning. Eighteen pigs given the fimbriated F18ac vaccine and challenged with a strain of the homologous fimbrial variant were protected against colonization; mean faecal viable counts of the challenge strain were >3 log10 lower than those from the 17 non-vaccinated control pigs. The vaccinated pigs developed a significant rise of F18ac IgA serum antibodies. The 23 pigs which had received the non-fimbriated vaccine showed no significant protection and exhibited much lower serum F18ac IgA ELISA reactivities. Eighteen pigs vaccinated with the fimbriated F18ac and challenged with an F18ab strain had faecal viable counts nearly as high as those from 16 non-vaccinated control pigs. It is concluded that only oral vaccines having preformed fimbriae induce protection limited to the homologous fimbrial variant.

Fimbrial Colonisation Factors F18ab and F18ac of Escherichia coli Isolated from Pigs with Postweaning Diarrhea and Edema Disease

During the last 5 years at least four new types of colonisation factors have been described in association with porcine postweaning diarrhea and edema disease strains of E. coli. Recently, evidence was presented that these fimbrial factors are closely related to each other, and therefore the common denomination F18 was proposed. Until now, two variants F18ab and F18ac were identified that can be distinguished by serology. Alternatively, to circumvent elaborate growth conditions for the optimal expression of F18 fimbriae in vitro, PCR and subsequent restriction enzyme digestion of the amplification product can be used to differentiate F18ab from F18ac positive isolates. Reports that studied the prevalence of F18 positive E. coli show that this factor is present in about 30% to more than 50% of the PWD or ED strains negative for F4, F5, F6 or F41. Susceptibility of pigs to colonisation depends on the availability of intestinal receptors, and is under the control of a chromosomal locus. In young pigs susceptibility increases with age. Intestinal infection with F18 positive E. coli induces protection against repeated colonisation with E. coli bearing the homologous or the heterologous fimbrial variant of F18. Finally, preliminary passive protection studies suggest that F18 antibodies inhibit the colonisation of the pigs intestine by F18ab and F18ac positive strains.

The role of adhesive F107 fimbriae and of SLT-IIv toxin in the pathogenesis of edema disease in pigs

Colonization of the small intestine and the excretion of a toxin are important steps in the pathogenesis of edema disease in pigs. Although much is known about the chemical and biological characteristics of SLT-IIv toxin, its mode of action and its genetic determinant, F107 fimbriae were only recently described as colonization factors. Here we summarize our current knowledge about the virulence factors F107 fimbriae and SLT-IIv toxin.