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 alpha (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.
The Pasteurella-like organism of porcine necrotizing pleuropneumonia described by Bertschinger and Seifert in 1978 was shown to be phenotypically similar to the V factor-requiring strains hitherto classified as Haemophilus pleuropneumoniae (Matthews and Pattison 1961) Shope 1964. Deoxyribonucleic acid-deoxyribonucleic acid hybridization experiments in which the optical method was used yielded 91% binding of the genomes of the type and reference strains of these two taxa, thus indicating that they belong to the same species. The levels of deoxyribonucleic acid binding between these strains and the type strain of Haemophilus influenzae indicated no measurable relatedness. Therefore, these organisms do not belong to the genus Haemophilus sensu stricto. On the other hand, they are closely related to Actinobacillus lignieresii, as determined by both phenotypic characteristics and deoxyribonucleic acid base sequence relatedness (72 to 75% binding). To take into account the different host ranges of these organisms, we propose to classify them as a species distinct from, but closely related to, A. lignieresii, under the new combination Actinobacillus pleuropneumoniae. The emended species A. pleuropneumoniae is composed of V factorrequiring and V factor-independent biovars. The type strain (strain Shope 4047 [= CCM 5869 = ATCC 270881) requires V factor; the V factor-independent biovar is represented by strain Bertschinger 2008/76 (= Frederiksen P 597 = HIM 677-3/4). granules or actinomycotic granules. Such granules were never observed in porcine infections with the new organism. Furthermore, the organism isolated by Bertschinger and Seifert (designated the BS organism) resembled Haemophilus pleuropneumoniae in many physiological properties; an exception was the V factor requirement (10). In this paper we describe the genetic relationships among the organisms mentioned above, as revealed by deoxyribonucleic acid (DNA)-DNA hybridization, and propose a classification of these organisms.
MATERIALS AND METHODSon the basis Of Phenotypic charac-studied are listed in Table 1. They were kept lyophi-
The alpha(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 alpha(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.
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