Multicellular living organisms and unmodified parts thereof and – Nonhuman animal
Reexamination Certificate
1999-11-19
2003-07-22
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
C536S023100, C536S023500, C424S009100
Reexamination Certificate
active
06596923
ABSTRACT:
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. “Oedema 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. coli
” (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
(“colonization test”), 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 “Searches are underway for appropriate genetic markers . . . ” (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 “marker” 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 Lewis
b
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
on-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 r
Bosworth Brad T.
Vögeli Peter
Barnes & Thornburg
Biotechnology Research & Development Corp.
Crouch Deborah
Martin Alice O.
Woitach Joseph
LandOfFree
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