Identification of polymorphisms in the ovine Shadow of prion protein (SPRN) gene and assessment of their effect on promoter activity and susceptibility for classical scrapie.

Shadow of prion protein (SPRN) is an interesting candidate gene thought to be involved in prion pathogenesis. In humans, an association has already been discovered between mutations in SPRN and the incidence of variant and sporadic Creutzfeldt-Jakob disease. However, in sheep, the effect of mutations in SPRN is largely unknown. Therefore, we analysed the presence of mutations in the entire ovine SPRN gene, their association with scrapie susceptibility and their effect on SPRN promoter activity. In total, 26 mutations were found: seven in the promoter region, four in intron 1, seven in the coding sequence and eight in the 3' untranslated region. The mutations detected in the coding sequence and the promoter region were subsequently analysed in more detail. In the coding sequence, a polymorphism causing a deletion of two alanines was found to be associated with susceptibility for classical scrapie in sheep. Furthermore, a functional analysis of deletion constructs of the ovine SPRN promoter revealed that the region 464 to 230 bp upstream of exon 1 (containing a putative AP-2 and putative Sp1 binding sites) is of functional importance for SPRN transcription. Six mutations in the SPRN promoter were also found to alter the promoter activity in vitro. However, no association between any of these promoter mutations and susceptibility for classical scrapie was found.

Correlation between porcine PPARGC1A mRNA expression and its downstream target genes in backfat and longissimus dorsi muscle.

Knowledge of in vivo relationship between the coactivator PPARGC1A and its target genes is very limited, especially in the pig. In this study, a real-time PCR experiment was performed on longissimus dorsi muscle (MLD) and backfat with 10 presumed PPARGC1A downstream target genes, involved in energy and fat metabolism, to identify possible relationships with PPARGC1A mRNA expression in vivo in the pig (n = 20). Except for UCP3 and LPL, a very significant difference in expression was found between MLD and backfat for all genes (P < 0.01). Hierarchical cluster analysis and the significant pairing of mRNA expression data between sampling locations suggested a genetic regulation of the expression of several target genes. A positive correlation with PPARGC1A was found for CPT1B, GLUT4, PDK4, and TFAM (P < 0.0001). A negative correlation was found for UCP2, FABP4, LEP (P < 0.0001), and TNF (P = 0.0071). No significant correlation was detected for UCP3 and LPL. This study provides evidence for a clear difference in mRNA expression of crucial genes in fat and energy metabolism between 2 important tissues. Our data suggest a clear impact of PPARGC1A on energy and lipid metabolism in vivo in the pig, through several of these downstream target genes.

Monitoring the efficacy of ivermectin and albendazole against gastro intestinal nematodes of cattle in Northern Europe.

Faecal egg count reduction tests (FECRT) using ivermectin (IVM) and benzimidazole (BZ) were conducted to investigate the prevalence of anthelmintic resistance in gastro-intestinal nematodes on cattle farms in Germany, Belgium and Sweden in 2006 and 2007. Based on sufficient numbers of eggs prior to the study, between 3 and 10 farms per country were selected. 10-15 animals were randomly selected per farm and subcutaneously treated with 0.2 mg IVM/kg bodyweight (Ivomec, Merial). Faecal samples were collected individually from every animal on day 0 (treatment), day 7 (Belgium & Sweden) or 14 (Germany), and day 21 (Germany, Belgium and Sweden). Faecal egg counts (FEC) were performed at each sampling occasion to estimate the eggs per gram of faeces (EPG) and the reduction of eggs after treatment. The FECRT using IVM in 2006 revealed mean reduction of egg counts between 69-100% on day 7/14 (95% confidence interval (CI) 19-102) and 35-96% (95% CI 0-102) on day 21. Farms with a suggested problem of anthelmintic resistance have been re-visited in 2007 and except for one case all results obtained in 2006 were confirmed in 2007. Larvae obtained from faecal cultures were identified using microscopic identification keys or genus-specific real time PCR. Cooperia oncophora was the predominant species detected after treatment, but Ostertagia ostertagi was found in samples on 3 farms in Germany and 3 farms in Sweden post-treatment. In 2007 additionally a FECRT using benzimidazoles was conducted in Germany and Sweden. In Germany oral Valbazen (albendazole, 10%, Pfizer) was used at a concentration of 7.5 mg albendazole/kg bodyweight; in Sweden Valbazen Vet (albendazole, 10%, Orion Pharma) at a dose of 8 mg/kg was used. For benzimidazoles an efficacy of 100% was obtained on all tested farms in both countries. This is the first report of a multinational anthelmintic efficacy investigation in cattle in Europe. The results suggest that testing of anthelmintic efficacy should be performed more intensively due to possible insufficient efficacy of current drugs.

An abortion of monozygotic twins in a warmblood mare.

Naturally occurring monozygotic twins are extremely rare in the horse. This paper describes an abortion in a mare after 260 days of pregnancy with monozygotic twins, one a fresh foal and the other a mummified foal.

Gender-enriched transcription of activation associated secreted proteins in Ostertagia ostertagi.

Activation associated secreted proteins (ASP) are members of a nematode-specific protein family belonging to the SCP/Tpx-1/Ag5/PR-1/Sc7 family. Three different types of molecules have been identified in this family: two-domain ASPs and single-domain ASPs showing homology to either the C-terminal or N-terminal domain of the two-domain ASP. The function of these proteins is still unclear, but a role in transition to parasitism and a role as allergen are often suggested. Here we report that the abomasal cattle parasite Ostertagia ostertagi produces at least 15 ASPs, including two-domain and C- and N-type single-domain ASPs. Ten of these are highly transcribed in the L4 stage, whereas others are highly enriched in adult male worms. The latter was especially the case for the N-type single-domain ASPs Oo-ASP1 and Oo-ASP2 and also for Oo-ASP3, which is homologous with the Haemonchus contortus and Ancylostoma caninum C-type single-domain ASPs. Immunohistochemistry showed that Oo-ASP3 was localised in the oesophagus. Oo-ASP1 and Oo-ASP2 on the other hand were localised in the reproductive tract of both male and female worms, suggesting a role in reproduction or in the development of the reproductive tract.

Experimental selection for ivermectin resistance in Ostertagia ostertagi in cattle.

Recent reports of suspected ivermectin (IVM) resistance in Ostertagia ostertagi have highlighted the need for research into the mechanisms of IVM resistance. However, there are no reports of resistant field isolates of O. ostertagi, which have been characterized for molecular research. Therefore, an anthelmintic susceptible O. ostertagi population was selected for IVM resistance by repeatedly exposing the population to subtherapeutic and therapeutic levels of IVM over 10 generations. In each selection round, a group of calves was infected with the progeny of the previous IVM-selected O. ostertagi population. In the last selection round a therapeutic IVM dose (0.2 mg/kg BW) only reduced the faecal egg counts by 57% and 65% on days 7 and 14 after treatment, respectively. In contrast, the therapeutic IVM dose was 100% effective at eliminating the parental IVM-susceptible isolate.

Porcine PPARGC1A (peroxisome proliferative activated receptor gamma coactivator 1A): coding sequence, genomic organization, polymorphisms and mapping.

We report here the characterisation of porcine PPARGC1A. Primers based on human PPARGC1A were used to isolate two porcine BAC clones. Porcine coding sequences of PPARGC1A were sequenced together with the splice site regions and the 5' and 3' regions. Using direct sequencing nine SNPs were found. Allele frequencies were determined in unrelated animals of five different pig breeds. In the MARC Meishan-White Composite resource population, the polymorphism in exon 9 was significantly associated with leaf fat weight. PPARGC1A has been mapped by FISH to SSC8p21. A (CA)n microsatellite (SGU0001) has been localised near marker SWR1101 on chromosome 8 by RH mapping and at the same position as marker KS195 (32.5 cM) by linkage mapping. The AseI (nt857, Asn/Asn489) polymorphism in exon 8 was used to perform linkage analysis in the Hohenheim pedigrees and located the gene in the same genomic region. Transcription of the gene was detected in adipose, muscle, kidney, liver, brain, heart and adrenal gland tissues, which is in agreement with the function of PPARGC1A in adaptive thermogenesis.

Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence.

Aim of our study was to clarify if the occurrence of apoptosis in oocytes and cumulus cells is correlated to bovine oocyte developmental competence. The cumulus-oocyte complexes (COCs) were selected according to cumulus status: G1 with more than five layers of compact cumulus cells, G2 with one to five layers of compact cumulus cells and G3 with expanded cumulus cells. The degree of apoptosis in cumulus cells and oocytes measured by caspase staining and TUNEL assay before and after maturation, and 24 h post-insemination was compared to the cleavage, blastocyst formation and hatching rates of each group. Highest cleavage, blastocyst and hatching rates were found in cumulus-oocyte complexes with more than five layers of compact cumulus cells, but no apoptosis was detected in immature or in vitro matured oocytes, regardless of the cumulus status. Many cumulus cells contained active caspases before maturation, but caspase activity declined dramatically after maturation. TUNEL positive cells were rarely observed in each cumulus-oocyte complex upon oocyte recovery, but a huge increase of them was seen after in vitro maturation. Significantly more TUNEL and caspase positive cells were found in G2 cumulus-oocyte complexes. Our results suggest that: (i) oocyte apoptosis does not account for the inferior oocyte quality of G2 and G3; (ii) apoptosis occurs in cumulus cells regardless of the number and compactness of cumulus cells; and (iii) the degree of apoptosis in the compact cumulus-oocyte complexes (G1 and G2) is negatively correlated to the developmental competence of oocyte.

A refined comparative map between porcine chromosome 13 and human chromosome 3.

We report here the localisation of BAIAP1 (13q24), HTR1F (13q45), PTPRG (13q23) and UBE1C (13q24) by fluorescence in situ hybridisation (FISH), and BAIAP1 (Swr2114; 21 cR; LOD = 11.03), GATA2 (Sw2448; 37 cR; LOD = 8.26), IL5RA (Swr2114; 64 cR; LOD = 3.85), LMCD1 (Sw2450; 61 cR; LOD = 4.73), MME (CP; 50 cR; LOD = 7.75), RYK (Swc22; 12 cR; LOD = 18.62) and SGU003 (Sw1876; 6 cR; LOD = 16.99) by radiation hybrid (RH) mapping to porcine chromosome 13 (SSC13). The mapping of these 10 different loci (all mapped to human chromosome 3; HSA3) not only confirms the extended conservation of synteny between HSA3 and SSC13, but also defines more precisely the regions with conserved linkage. The syntenic region of the centromeric part of SSC13 was determined by isolating porcine bacterial artificial chromosome (BAC) clones (842D4 and 1031H1) using primers amplifying porcine microsatellite markers S0219 and S0076 (mapped to this region). Sequence comparison of the BAC end sequences with the human genome sequence showed that the centromeric part of SSC13 is homologous with HSA3p24.

Protein disulphide isomerase of Ostertagia ostertagi: an excretory-secretory product of L4 and adult worms?

A pepstatin A-agarose column was used in an attempt to purify a previously described antibody-degrading aspartyl proteinase from excretory-secretory material from the L4 and the adult stages of the bovine abomasal nematode Ostertagia ostertagi. However, no aspartyl proteinase activity was detected in the eluted fractions (L4Pepst and AdPepst). Screening of cDNA libraries with polyclonal antibodies raised against L4Pepst and AdPepst showed that a protein disulphide isomerase (Ost-PDI2) was present in both antigen fractions. This multifunctional enzyme was detected in extracts of L3, L4 and adult parasites and, interestingly, also in excretory-secretory material of L4 and adult O. ostertagi. By immunohistochemistry, the Ost-PDI2 enzyme was localised in some parts of the hypodermis of L4 and adult worms and in the intestinal cells of all three parasitic life stages. Two-dimensional Western blot analysis indicated that Ost-PDI2 is recognised by calves during a natural O. ostertagi infection, which suggests that Ost-PDI2 could be used for immunological control of ostertagiosis.

Influence of oxygen tension on apoptosis and hatching in bovine embryos cultured in vitro.

Various oxygen tensions are employed for in vitro embryo production. Since it is known that oxygen tension can influence the efficiency of embryo production and embryo quality, the aim of our study was to define an optimal oxygen concentration for bovine embryo production in vitro in synthetic oviduct fluid (SOF). Embryo quality criteria were hatching ability and the degree of apoptosis as assessed by TUNEL staining and Bax gene expression. In Experiment 1, the effects of 2, 5 and 20% O(2) tensions on embryo development were compared. The highest rate of eight-cell embryos (47%) at 72 hpi was obtained under 20% O(2). However, it seemed that 2 and 5% O(2) were also suitable as assessed by embryo survival rates at 144 hpi (29 and 30% at morula stage), 168 hpi (21 and 19% at blastocyst stage) and 216 hpi (14 and 17% at hatched blastocyst stage). In Experiment 2, comparisons were made between effects of 5, 20% and alternating O(2) (20% O(2) to 72 hpi and then changed to 5% O(2) up to 216 hpi) on embryo development. Alternating the O(2) tension significantly reduced the number of hatching blastocysts to 7%. Staining with TUNEL revealed that apoptosis occurred in all tested hatched blastocysts, but a significantly lower apoptotic cell ratio was found in embryos cultured under 5% O(2) (P<0.05). Total cell number of embryos cultured under 5% and alternating oxygen was significantly higher than that of other groups (P<0.05). Bax gene expression was detected by means of RT-PCR in only 2 of 66 hatched blastocysts. It can be concluded that 5% oxygen is optimal for bovine embryo culture in cell free media. Moreover, it is very likely that the apoptosis detected by TUNEL staining in this study is Bax-independent.

Bioavailability of two ibuprofen oral paste formulations in fed or nonfed ponies.

The bioavailability and pharmacokinetics of ibuprofen, a nonsteroidal antiinflammatory drug, was studied in healthy Shetland ponies. Ibuprofen was administered IV, as a suspension, and as a solid solution oral paste to ponies from which food was withheld. The suspension paste was also administered to ponies that received hay and water ad libitum. Both formulations had an absolute bioavailability of about 80%. Bioavailability was not influenced by feeding.

Distribution of the Shadoo protein in the ovine brain assessed by immunohistochemistry.

Shadow of prion protein is a gene potentially involved in the pathogenesis of prion diseases. However, the Shadoo protein encoded by this gene has not yet been studied in sheep, an important species in prion matters. Therefore, we developed a polyclonal antibody against ovine Shadoo and assessed the presence and distribution of this protein in the ovine brain by immunohistochemistry. The strongest staining level was found in the cerebellum (especially in the Purkinje cells) and in the pons, but cerebrum, hippocampus, pituitary gland, medulla oblongata, thalamus and hypothalamus were also immunopositive. Remarkably, a typical granular pattern was seen in most of the tested brain tissues, which might indicate that Shadoo is primarily expressed at synapses. The results of this study and the availability of an ovine anti-Shadoo antibody can contribute to future research on the function of Shadoo and on its potential involvement in prion diseases.

Altered avr-14B gene transcription patterns in ivermectin-resistant isolates of the cattle parasites, Cooperia oncophora and Ostertagia ostertagi.

Ivermectin (IVM) resistance is an emerging problem for the control of gastrointestinal nematodes of cattle such as Cooperia oncophora and Ostertagia ostertagi. Although there is still a poor understanding of the molecular basis of macrocyclic lactone (ML)-resistance, it is clear that IVM exerts its activity by binding to glutamate-gated chloride (GluCl) channels within the parasite's neuromuscular system. One of the GluCl genes (avr-14) encodes, via alternative splicing, two subunits, AVR-14A and AVR-14B; the latter is suggested to be the main target for IVM. The genomic DNA (gDNA) sequence of avr-14 in C. oncophora contains 21 exons separated by 20 introns and spans approximately 10 kb of gDNA. Intron 13 contains a sequence with high homology to a mammalian mariner transposase. The L256F polymorphism in the avr-14 gene, which was shown to be associated with IVM resistance in a UK isolate of C. oncophora, was not found in the IVM-resistant C. oncophora and O. ostertagi isolates investigated in this study. However, genetic analyses on C. oncophora indicated a loss in allelic diversity of the avr-14 gene in the resistant isolates compared with the susceptible isolate. This suggests that the avr-14 gene, or another genetically linked locus, is under selection in these Belgian C. oncophora isolates. Comparison of the full-length avr-14B coding sequence in the susceptible and resistant C. oncophora isolates did not show any polymorphisms specifically linked to IVM resistance, although a decrease in the number of avr-14B isoforms was observed in the resistant isolates compared with the susceptible one. Measuring the transcription levels of avr-14B in adult male and female C. oncophora and O. ostertagi worms showed significantly lower levels in resistant worms compared with susceptible ones. Whether the down-regulation of this IVM target actually contributes to the resistance mechanism in these worms remains unclear.

Estimation of phenotypic and genetic parameters for weight gain and weight at fixed ages in the double-muscled Belgian Blue Beef breed using field records.

In the double-muscled Belgian Blue beef (DM-BBB) breed, selection focuses on muscular conformation and not on weight gain and higher weight. There are very few studies on growth in the DM-BBB using field records. Therefore, farms have no available useful figures on weight at fixed ages and weight gain for the DM-BBB. This study describes and evaluates live weights of DM-BBB animals. All the data were gathered on farms in Belgium. It was found that a male DM-BBB weighs an average of 51 kg at birth, 98 kg at 3 months, 242 kg at 7 months, 430 kg at 13 months and 627 kg at 20 months. Between the age of 7 and 20 months, weight gain is more than 1200 g a day. Females weigh 47 kg at birth, 96 kg at 3 months, 189 kg at 7 months and 332 kg at 13 months. For males, estimates of heritability for weights at 7, 13 and 20 months were between 0.21 and 0.36. The heritability for weight gain between 13 and 20 months was 0.13. This demonstrates that it is possible to select for higher weights and for increased growth between 13 and 20 months. Animals having high weights at a young age (7 and 13 months) tend to have also high weight at slaughtering age (20 months; r(g) between 0.81 and 0.98), but no additional growth between 13 and 20 months (r(g) between -0.09 and 0.00). High weight at 20 months is partially due to growth between 13 and 20 months (r(g) = 0.49).

Effectiveness of bovine microsatellites in resolving paternity cases in American bison, Bison bison L.

A set of 33 cattle microsatellite primer pairs was tested with the DNA of American bison from a captive population in Belgium and evaluated for usefulness in parentage testing. Two primer sets did not amplify and three were monomorphic. Among the polymorphic markers, the number of alleles ranged from two to nine. Heterozygosity, polymorphism information content (PIC) and probability of exclusion (PE) values were low by comparison with those obtained with the same markers in cattle. Two methods of estimating PE were used, one which assumed equal allele frequencies between parental sexes and another which took into account differences in allele frequencies between parental sexes. An internationally accepted set of nine microsatellites gives cumulative PE values of 0.98 and 0.97, respectively, for the two methods. The potential of this marker set to identify bison x cattle hybrids is discussed. Because bison and cattle have a common ancestor, these microsatellites are a useful way to establish genetic distances and can lead to the construction of phylogenetic trees.

A contribution to the D system in horses.

The inheritance of a new D system red cell antigen, factor 22, is described. It has also been possible to discriminate more efficiently between D system phenogroups enabling genotypes to be identified from phenotypes in the majority of cases. This improves the accuracy of animal identification and gene frequency estimates.

Characterization and mapping of bovine dopamine receptors 1 and 5.

A cDNA encoding the bovine dopamine receptor 1 (DRD1) was isolated from a bovine cDNA library, cloned and completely sequenced. The coding region showed 93 and 91% sequence identity on DNA level and 96 and 94% on protein level with its respective porcine and human orthologs. The bovine DRD1 and dopamine receptor 5 (DRD5) were mapped, respectively, to BTA10 and 6 by radiation hybrid mapping. One SNP was found in DRD1 and four in DRD5. Using polymerase chain reaction-restriction fragment length polymorphism, 11 different European cattle breeds were screened for the presence of the DRD1 and DRD5 substitutions. Allele frequencies for DRD1 and DRD5 alleles were very similar across all the breeds examined. Allele frequency discrepancies were found between Belgian Blue beef breed and the other breeds.