Compositions and non-invasive methods are provided for the identification of swine genetically resistant to E. Coli bacteria supplied with fimbriae F18. DNA polymorphisms in the swine alpha (1,2) fucosyltransferase (FUT1) gene were identified that differentiate resistant from susceptible swine and provide a diagnostic test useful for swine breeders.
A major problem in breeding swine is to keep them disease-free. Intestinal disorders postweaning are a particular problem. A limited number of serotypes of toxigenic Escherichia (E.) Coli strains are the causative agents of oedema disease and postweaning diarrhea in swine which induce serious economic losses, especially among piglets aged 4 to 12 weeks, in swine breeding farms all over the world. The typical symptoms of oedema disease are neurological signs such as ataxia, convulsions and paralysis. At post mortem examination, oedema is typically present at characteristic sites such as eyelids and forehead, stomach wall and mesocolon. The diseases are cause be Shiga-like toxin-II variant and enterotoxins LT, Sta, Stb respectively, produced by E. coli that colonize the surface of the small intestine without effecting major morphological changes of the enterocytes (cells in the intestine). Certain types of bacterial E. coli strains, F18, F4 and K88 are major lethal villains in this regard. xe2x80x9cOedema disease of pigs is an enterotoxaemia characterized by generalized vascular damage. The latter is cause by a toxin, Shiga-like toxin II variant, produced by certain strains of E. colixe2x80x9d (Bertschinger et al., 1993). The E. coli are distinguished by their pili types, a group of adhesive fimbriae that are related are designated e.g., K88 or F18 (Vogeli et al., 1997).
Not all swine succumb to E. coli infections. Colonization depends on adherence of the bacteria to the enterocytes which is mediated by the bacterial fimbriae designated e.g., K88 or F18. Susceptibility to adhesion, i.e. expression of receptors in swine for binding the fimbriae, has been shown to be genetically controlled by the host and is inherited as a dominant trait with, in the case of F18, B being the susceptibility allele and b the resistance allele. (Vogeli et al., 1996; Meijerink et al., 1996). The genetic locus for this E. coli F18-receptor (ECF18R) has been mapped to porcine chromosome 6 (SSC6), based on its close genetic linkage to the S locus and other loci of the halothane (HAL) linkage group on chromosome 6. The receptor for K88 E. coli is on chromosome 13.
The mechanism for resistance appears to be that intestinal borders in resistant animals are not colonized by E. coli, i.e., the bacteria do not adhere to intestinal walls of resistant swine. Glycoprotein receptors in the brush border membrane of the intestine were shown to be responsible for the differences between adhesive and non-adhesive phenotypes related to some E. coli, therefore, the genotype of the host swine determines resistance. The fimbriated bacteria also have been studied. (WO 9413811).
Current methods of identifying swine that are resistant to F18 E. coli associated diseases are either to 1) collect intestinal samples from swine at slaughter and perform the microscopic adhesion test, 2) challenge the animals with virulent E. coli (xe2x80x9ccolonization testxe2x80x9d), or 3) perform blood typing of the A-O(S) blood group system. The first two methods are not practical for identifying resistant animals for use as breeding stock. Although the blood typing method does identify resistant animals, the test is unable to determine whether susceptible animals are homozygous or heterozygous for susceptibility. Knowledge of the genotype of animals with regard to these alleles (conditions of a gene) is essential to develop a successful breeding program. The purpose of the breeding program is to produce swine that are resistant to F18 E. coli associated diseases that decimate stock post-weaning.
In one publication the authors stated, in reference to oedema disease in swine, that xe2x80x9cSearches are underway for appropriate genetic markers . . . xe2x80x9d (Bertschinger et al., 1993, page 87) and, citing Walters and Sellwood, 1982:
Breeding resistant swine is an attractive method for prevention of diseases for which an effective prophylaxis is not available. The feasibility of this approach will depend on the prevalence of the gene(s) encoding resistance in the pig population improved methods for the detection of resistant pigs, and absence of negative genetic traits co-selected with this resistance.
A genetic xe2x80x9cmarkerxe2x80x9d locus is a coding or non-coding locus that is close to a genetic locus of interest, but is not necessarily the locus itself. Detectable phenotypes include continuous or discontinuous traits, e.g. restriction length fragment polymorphisms, production traits, bacterial adhesion traits, colorimetric or enzymatic reactions, and antibiotic resistance. The S locus controls expression of the A and O blood group antigens. Swine homozygous recessive at the S locus do not express either A or O blood group antigens. A similar condition exists in humans and is due to mutations in the alpha (1,2) fucosyltransferase gene which encodes the human blood group H (Kelly et al., 1994; see also WO 9628967). The porcine alpha (1,2) fucosyltransferase gene of swine has recently been sequenced (Cohney et al., 1996). This gene is very likely the gene present at the S locus in swine.
The blood group H and Se loci have been mapped genetically and physically to human chromosome 19q13.3. This region is evolutionarily conserved, containing genes homologous to the HAL linkage group of genes in pigs. The blood group H encoding gene is the so called FUT1 whereas the Se gene is equivalent to the FUT2 gene. FUT1 determines H antigen expression in the erythroid cell lineage, whereas FUT2 regulates expression of the H antigen in the secretory epithelia and saliva. Conservation of the FUT1 gene has been shown in lower mammals such as rat and rabbit, and mRNA expression has been shown in rabbit brain tissue and rat colon. In all these species two types of alpha (1,2) fucosyltransferase genes have been reported which are structurally very similar to the human FUT1 and FUT2 genes, but in particular the FUT1 homologous genes show a species specific expression pattern. In humans the FUT1 gene is responsible for synthesis of H antigens in the precursors of erythrocytes. However, in pigs erythrocytes passively adsorb H-like antigens from the serum, as is the case for the human Lewis antigens. In pigs all H-like antigens are related to exocrine secretory tissues, and expression of the FUT2 (Secretor) gene is seen in secretory tissue of other animal species. Therefore, expression of the porcine A-O blood group determinants which cross-react with anti-human blood group H and A antibodies might be influenced by the FUT2 gene.
Further information about blood groups and E. coli swine diseases include that carbohydrate structures of blood group antigens mediate the adhesion of some pathogenic microorganisms to host tissues, e.g. Helicobacter pylori adhere to Lewisb blood group antigens, and E. coli causing urinary tract infections adhere to blood group P substance. Genes encoding glycosyltransferases that are responsible for the formation of the blood group specific carbohydrate structures, therefore, represent candidate genes for the control of bacterial colonization by the host. The localization of these genes is in the same chromosomal region as the locus responsible for adhesion/non-adhesion of F18 positive E. coli in the swine small intestine. Swine do not express blood group antigens A and O until after weaning, this is the same time that they become susceptible to disease caused by F18 E. coli. 
New methods of diagnosis and treatment are needed for E. coli related intestinal diseases in swine. Detection of a genetic mutation was proposed as a diagnostic test for some swine disorders (malignant hypothermia) (Fujii et al., 1991; U.S. Pat. No. 5,358,649), but polymorphic markers were not reported for diagnosis. Vaccines to develop resistance to E. coli colonization were described (U.S. Pat. No. 5.552,144; WO 8604604), but are unlikely to be a preferred method to prevent the E. coli disease because of difficulties in administering live vaccine orally to newborn swine, and because of regulatory restrictions. Antibiotics are available for treatment, but there is no successful prophylaxis.
The compositions and non-invasive methods of the present invention provide detection and elimination of swine that are susceptible to E. coli associated diseases. A non-invasive method for identifying a swine that is resistant to intestinal colonization by E. coli F18 includes the following steps: determining whether a genetic polymorphism associated with resistance to colonization is in a biological sample from the swine; and inferring that the swine is resistant if the swine is homozygous for the polymorphism.1 
1 A polymorphism is a change in a nucleotide sequence that exists in a population due to mutation. 
More particularly, the method is determining in a biological sample from the swine whether the nitrogen base at position 307 in the alpha (1,2) fucosyltransferase gene of the swine is only adenine or only guanine; and identifying the swine as resistant if the only nitrogen base at position 307 is adenine.
To determine whether a polymorphism is present in a biological sample, restriction fragment length polymorphisms are analyzed on a gel that separates them by molecular weight. Restriction endonucleases are enzymes that reproducibly cut nucleic acid molecules at specific sites, resulting in nucleic acid fragments of different molecular weights, depending on the location of the cuts.
The invention also relates to a method for breeding swine to be resistant to E. coli associated diseases by selecting for breeding swine that have a genetic polymorphism in the alpha (1,2) fucosyltransferase 1 gene that identifies them as swine that are resistant to E. coli related intestinal diseases; and breeding the selected swine.
An aspect of the invention is a DNA molecule which is polymorphic for the alpha (1,2) fucosyltransferase 1 gene in swine, in particular a sequence in accordance with FIG. 1. Other aspects of the invention are molecules with nucleotide sequences complementary to that of FIG. 1.
An aspect of the invention is an isolated DNA molecule with a substitution of adenine for guanine in position 307. This molecule may also bond a substitution of adenine for guanine in position 857. Other isolated DNA molecules of the present invention include those with a mutation at nucleotide position 229 of the sequence of FIG. 1, wherein the codon CTT is changed to TTT, encoding for the amino acid phenylalanine instead of leucine. A mutation at nucleotide position 714 is from GATxe2x86x92GAC, but there is no accompanying amino acid substitution in the encoded product.
Polypeptides encoded by the DNA molecules of the present invention and having alpha (1,2) fucosyltransferase activity are also aspects of the invention.
A molecular assay for detecting E. coli F18 receptors in swine is to (a) isolate DNA from porcine nucleated cells; (b) amplify the DNA in a polymerase chain reaction (PCR) using oligonucleotides as primers which are complementary to a DNA sequence of the porcine alpha (1,2) fucosyltransferase gene 1; (c) perform a restriction enzyme digest with at least one restriction enzyme e.g., CfoI; (d) separate the resulting fragments by gel electrophoresis; and (e) determine the respective numbers and lengths of fragments on the gel; and (f) determine from the numbers and length of fragments of F18, which receptors are present in the porcine cells. Use of the larger amplified fragments disclosed herein for restriction length polymorphism analysis (RFLP), rather than smaller fragments, is less expensive because the DNA bands can be run on agarose gels of relatively low concentration. Also, to produce some of the fragments, only one restriction enzyme is needed for a constant restriction site adjacent to the variable diagnostic site.
A kit for detecting polymorphisms associated with E. coli F18 receptors uses oligonucleotides in separate containers which are complementary to a DNA sequence of the porcine alpha (1,2) fucosyltransferase gene 1 that distinguishes resistant from sensitive swine. The test can be performed on swine of any age.
The polymorphisms are also useful to develop drugs to treat swine that have E. coli-associated disease. A mutated form of porcine alpha 1,2 fucosyltransferase could interfere with the normal enzyme, preventing it from producing the intestinal receptor for F18.
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