[Suppression of reproductive cyclicity by active immunization against GnRH in the adult ewe]. / Unterdrückung der Fortpflanzungsaktivität durch aktive Immunisierung gegen GnRH beim adulten weiblichen Schaf.

The objective of the present study was to evaluate the effect of an anti-GnRH vaccine on cycling activity in the adult ewe during the breeding season. For the experiments 22 cycling White Alpine sheep, aged between 2 and 4 years, were randomly divided into a treatment and control group of 11 animals, each. Sheep of the treatment group were immunized twice at an interval of 4 weeks with 2 ml (400microg GnRH-protein conjugate) of Improvac (Pfizer Animal Health, Australia) subcutanously in the neck. Sheep of the control group received the same amount of saline solution. Blood progesterone concentrations were measured weekly from 3 weeks before to 32 weeks after first immunization and anti-GnRH titers were determined monthly. All vaccinated ewes ceased cycling within 2 - 8 weeks after first immunization. Plasma progesterone was suppressed for a minimum of 12 weeks (2 ewes) with individual variation of 14 (1 ewe), 25 (1 ewe) and more than 25 weeks (7 ewes). Four animals resumed cyclicity while 7 animals remained suppressed until the end of the study. Antibody titers peaked one month after the booster injection and thereafter continuously dropped until the end of the study (8 months after first immunization) to values between 10.9 and 40.8 % binding. From our results it can be concluded that two vaccinations with Improvac 4 weeks apart suppress cycling activity in adult ewes for at least 12 weeks. The inhibitory effect on ovarian activity, however, varies individually and may last more than 31 weeks.

[Genomic methods for identification of traits and inherited disorders in farm animals]. / Genomik zur Identifikation von Merkmalen und Erbfehlern beim Nutztier.

In this review we demonstrate the interaction of the blueprint of an individual (the genome, genomic DNA), its phenotype and the environment. The phenotype consists of quantitative (e.g. growth, milk yield) or functional characteristics e.g. fitness, longevity, fertility and disease resistance. The latter characteristics influence the welfare of an animal substantially. As only the genetically determined part of a particular characteristic is transferred from one generation to the next, it is important to know what the genetic variants (alleles) of the parents at one or more gene loci are. New methods in molecular biology have made it possible to localize and characterize important genes which help to breed more efficient and healthy animals. The exact characterization of the phenotype is vital in identifying genes with major effects and therefore the cooperation with experts from veterinary medicine, biochemistry, and biology is indispensable. As well as an overview of available genetic tests in farm animals, we show various examples how to identify the molecular basis of a particular phenotype and how to use the results in practical breeding programs. Genetic diagnosis enables the breeder to identify undesired alleles early and hinders therefore its uncontrolled distribution in the population. In the long term this leads to a smaller number of affected animals and depending on the disease it may help to prevent animals from suffering.

Picogram cloning and direct in situ sequencing of DNA from gel pieces.

We describe a simple and rapid procedure for cloning and sequencing of DNA fragments separated by gel electrophoresis, using novel hydrophilic gels, Clearose BG, Spreadex, and Poly(NAT), that do not melt at 95 degrees C. For cloning, a band of interest is excised precisely and incubated in an extraction buffer containing 5-10 mM MgCl2 at 70 degrees C for 15-45 min. The eluted DNA is added directly to the plasmid solution. Using a topoisomerase-based ligation system, we were able to transform bacteria with a few picograms of DNA and isolate recombinant clones. For in situ sequencing, the DNA in the gel serves as the template. No treatment before cycle sequencing is necessary for fragments up to 500 bp.

Isolation of a porcine UDP-GalNAc transferase cDNA mapping to the region of the blood group EAA locus on pig chromosome 1.

UNLABELLED: In our studies of the genes constituting the porcine A0 blood group system, we have characterized a cDNA, encoding an alpha(1,3)N-acetylgalactosaminyltransferase, that putatively represents the blood group A transferase gene. The cDNA has a 1095-bp open reading frame and shares 76.9% nucleotide and 66.7% amino acid identity with the human ABO gene. Using a somatic cell hybrid panel, the cDNA was assigned to the q arm of pig chromosome 1, in the region of the erythrocyte antigen A locus (EAA), which represents the porcine blood group A transferase gene. The RNA corresponding to our cDNA was expressed in the small intestinal mucosae of pigs possessing EAA activity, whereas expression was absent in animals lacking this blood group antigen. The UDP-N-acetylgalactosamine (UDP-GalNAc) transferase activity of the gene product, expressed in Chinese hamster ovary (CHO) cells, was specific for the acceptor fucosyl-alpha(1,2)galactopyranoside; the enzyme did not use phenyl-beta-D-galactopyranoside (phenyl-beta-D-Gal) as an acceptor. Because the alpha(1,3)GalNAc transferase gene product requires an alpha(1,2)fucosylated acceptor for UDP-GalNAc transferase activity, the alpha(1,2)fucosyltransferase gene product is necessary for the functioning of the alpha(1,3)GalNAc transferase gene product. This mechanism underlies the epistatic effect of the porcine S locus on expression of the blood group A antigen. ABBREVIATIONS: CDS: coding sequence; CHO: Chinese Hamster Ovary; EAA: erythrocyte antigen A; FCS: foetal calf serum; Fucalpha(1,2)Gal: fucosyl-alpha(1,2)galactopyranoside; Gal: galactopyranoside; GGTA1: Galalpha(1,3)Gal transferase; PCR: polymerase chain reaction; phenyl-beta-D-Gal: phenyl-beta-D-galactopyranoside; R: Galbeta1-4Glcbeta1-1Cer; UDP-GalNAc: uridine diphosphate N-acetylgalactosamine

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

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.

The L-gulono-gamma-lactone oxidase gene (GULO) which is a candidate for vitamin C deficiency in pigs maps to chromosome 14.

Vitamin C deficient pigs, when fed a diet lacking L-ascorbic acid (AscA), manifest deformity of the legs, multiple fractures, osteoporosis, growth retardation and haemorrhagic tendencies. This trait was shown by others to be controlled by a single autosomal recessive allele designated as od (osteogenic disorder). The inability of AscA biosynthesis in primates and guinea pigs that exhibit similar symptoms, when they are not supplemented with AscA in the food, was traced to the lack of L-gulono-gamma-lactone oxidase, which catalyzes the terminal step in the biosynthesis of AscA. The non-functional GULOP was mapped to human chromosome 8p21 that corresponds to an evolutionarily conserved segment on either porcine chromosome 4 (SSC4) or 14 (SSC14). We investigated linkage between OD and SSC4- and 14-specific microsatellite loci in order to map the OD locus. Twenty-seven informative meioses in families from one sire and three dams revealed linkage of od with microsatellites SW857 and S0089, located in the subcentromeric region of SSC14. We isolated part of the GULO gene of the pig by screening a porcine genomic library using a pig GULO cDNA as a probe, and mapped it to SSC14q14 by fluorescence in situ hybridization (FISH). Thus, the porcine GULO gene is both a good physiological and positional candidate gene for vitamin C deficiency in pigs.

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

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.

[A molecular test for the detection of E. coli F18 receptors: a breakthrough in the struggle against edema disease and post-weaning diarrhea in swine]. / Ein molekularer Test für den Nachweis des E.-coli-F18-Rezeptors: ein Durchbruch im Kampf gegen Oedemkrankheit und Absetzdurchfall beim Schwein.

Oedema disease and post-weaning diarrhoea in swine are associated with the colonization of the intestine with toxigenic Escherichia (E.) coli bacteria of various serotypes. Colonization depends on specific binding between adhesive fimbriae and receptors on the enterocytes. The demonstration of these receptors allows the identification of susceptible and resistant pigs. Direct sequencing of the alpha (1,2) fucosyltransferase gene (FUT1) in swine being either susceptible or resistant to adhesion by F18 fimbriated E.coli revealed a mutation at basepair 307 (M307). Analysis of the mutation in Swiss Landrace and Large White families showed close linkage with the locus controlling resistance and susceptibility to E.coli F18 adhesion (ECF18R). The FUT1 (M307) mutation is a good marker for selection of E.coli of F18 adhesion resistant animals. The mutation is found with variable frequencies in Duroc, Hampshire and Pietrain pigs as well.

Partial characterization of porcine obesity gene (OBS) and its localization to chromosome 18 by somatic cell hybrids.

Degenerate primers based on human and mouse obesity gene (OBS) sequencing data were used in the reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA from pig white adipose tissue. Both strands of the resultant pig- specific 325 bp DNA fragment were sequenced. Comparison of the obtained sequence with known sequences revealed an 86% identity with the human and 84% identity with the mouse OBS cDNA. The OBS gene was physically mapped to pig chromosome 18 by PCR analysis of somatic cell hybrids, using pig-specific primers. This result is consistent with the recent assignment of the human OBS gene to chromosome 7 and the observation made by comparative mapping that by using a human chromosome 7 specific library two segments of conserved synteny were detected on porcine chromosomes 9 and 18. We conclude the border of conserved synteny to be in the 7q31-7q32 region of the human chromosome.