M. Van Poucke

Integration of porcine chromosome 13 maps

In order to expand the comparative map between human chromosome 3 (HSA3) and porcine chromosome 13 (SSC13), seven genes from HSA3 were mapped on SSC13 by fluorescence in situ hybridisation (FISH), viz. ACAA1, ACPP, B4GALT4, LTF, MYLK, PDHB and RARB. With a view to integrating this expanded comparative map with the existing SSC13 linkage map, we used the INRA-University of Minnesota porcine Radiation Hybrid panel (IMpRH) to localize more precisely and to order 15 genes on the SSC13 map, viz. ACPP, ADCY5, APOD, BCHE, CD86, DRD3, GAP43, PCCB, RAF1, RHO, SI, TF, TFRC, TOP2B and ZNF148. In this way, we were able to create an integrated map, containing 38 type I and 81 type II markers, by correlating the linkage, radiation hybrid (RH) and cytogenetic maps of SSC13. This integrated map will give us the opportunity to take maximal advantage of the comparative mapping strategy for positional candidate cloning of genes responsible for economically important traits.

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.

Anaphylatoxin C5a-induced toll-like receptor 4 signaling in bovine neutrophils.

It is well known that signaling in neutrophils through both the complement component 5a (C5a) and C5a receptor (C5aR) and the toll-like receptor 4 (TLR4) pathways plays an essential role in innate defense. Neutrophil dysfunction, as seen during sepsis in severe mastitis during the periparturient period, is correlated with elevated concentrations of anaphylatoxin C5a. The aim of the current study was to elucidate the effect of C5a on TLR4 signaling in bovine neutrophils. Neutrophils were incubated with a high (but physiological) dose of purified C5a, and mRNA was extracted from neutrophils at different time points postincubation (PI). The incubation with C5a resulted in a biphasic C5aR expression profile, a phenomenon that might be explained by internalization (at 10 min PI) with subsequent reconstitution (starting at 40 min PI) of this receptor. The expression of TLR4, as well as its coreceptor, CD14, showed a similar biphasic change as observed with C5aR. In addition, changes in the mRNA expression levels of several genes belonging to the TLR4 pathway, such as TICAM-1, IKKα, and MAP3K7 were noted. The maximal expression of TLR4, CD14, and C5aR mRNA at 80 min PI was accompanied by a peak in IL8 mRNA, indicating that C5a is able to induce IL-8 production in neutrophils in vitro without the need of a costimulatory factor such as lipopolysaccharide. Moreover, a relatively constant expression of RELA was accompanied by increased expression of ATF3, an endogenous inhibitor of nuclear factor-κB mediated transcription, implying that C5a regulates TLR4 signaling and IL-8 synthesis independently. A significant time-dependent correlation was found between C5aR and TLR4, with the majority of the selected TLR4-dependent genes showing a significant correlation with C5aR at 80 min PI, when C5aR and TLR4 mRNA expression reached its maximum, suggesting crosstalk between both receptors. Taken together, this study showed that C5a is able to (1) alter the expression of genes belonging to the TLR4 pathway and (2) induce IL8 gene expression in bovine neutrophils. In addition, indications for cross-talk between complement activation and TLR4 signaling were found in the present study.

Differentiation of F4 receptor profiles in pigs based on their mucin 4 polymorphism, responsiveness to oral F4 immunization and in vitro binding of F4 to villi

F4(+) enterotoxigenic Escherichia coli (F4(+) ETEC) are an important cause of diarrhoea and mortality in piglets. F4(+) ETEC use their F4 fimbriae to adhere to specific receptors (F4Rs) on small intestinal brush borders, resulting in colonization of the small intestine. To prevent pigs from post-weaning diarrhoea, pigs should be vaccinated during the suckling period. Previously, we demonstrated that F4acR(+), but not F4acR(-) piglets could be orally immunized with purified F4 fimbriae resulting in a protective immunity against F4(+) ETEC infections, indicating that this immune response was F4R dependent. Recently, aminopeptidase N has been identified as a glycoprotein receptor important for this oral immune response. However, in some oral immunization experiments, a few F4acR(+) piglets did not show an antibody response upon oral immunization, suggesting additional receptors. Therefore, the binding profile of F4 to brush border membrane (glyco)proteins was determined for pigs differing in F4-specific antibody response upon oral immunization, in in vitro adhesion of F4(+)E. coli to small intestinal villi, and in Muc4 genotype. Six groups of pigs could be identified. Only two groups positive in all three assays showed two high molecular weight (MW) glycoprotein bands (>250kDa) suggesting that these high MW bands are linked to the MUC4 susceptible genotype. The fact that these bands were absent in the MUC4 resistant group which showed a positive immune response against F4 and was positive in the adhesion test confirm that at least one or perhaps more other F4Rs exist. Interestingly, two pigs that were positive in the villous adhesion assay did not show an immune response against F4 fimbriae. This suggests that a third receptor category might exist which allows the bacteria to adhere but does not allow effective immunization with soluble F4 fimbriae. Future research will be necessary to confirm or reveal the identity of these receptors.

Detection of polymorphisms in the prion protein gene in the Belgian sheep population: Some preliminary data

Summary The development of clinical signs of TSE/scrapie in sheep has been linked to polymorphisms in the prion protein (PRNP) gene. The most important polymorphisms appear to be at codons 136, 154, and 171. The objective of this study was to investigate the polymorphisms at these codons in the Belgian sheep population, including clinical healthy animals, healthy animals at the slaughterhouse and animals in TSE/scrapie positive farms (including a Nor98 farm).

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.

Estimating internal pelvic sizes using external body measurements in the double-muscled Belgian Blue beef breed

In the double-muscled (DM) Belgian Blue beef (BBB) breed, caesarean section (CS) is being applied systematically as a management tool to prevent dystocia. As a matter of fact, CS is the only possible way of calving in the breed. High birth weight and a relatively small pelvic area are the main causes of dystocia and, in the DM-BBB breed, the reasons for the systematically applied CS. Selection for lower birth weight and larger pelvic sizes might reduce dystocia and routine CS. Few data on inner pelvic sizes of pedigree animals are available. Using external measurements to estimate the inner pelvic sizes might be an option to resolve this problem. In this study, animals of the DM-BBB breed were measured and weighed on farms and in abattoirs. External and internal pelvic sizes increased with live weight and age of the animals. Gender had a significant influence on inner pelvic traits. Increased muscular conformation was associated with decreased inner pelvic dimensions. Models with weight, gender, age, withers height and outer pelvic width (TcTc) can be used to estimate inner pelvic sizes ( R 2 between 0·35 and 0·77). The estimated inner pelvic sizes can then be used to genetically evaluate pelvic traits in the DM-BBB breed. Improving weight, withers height and TcTc width in combination with lowering muscular conformation may help to decrease the high rate of caesarean section in the DM-BBB.

Cell-specific localization of oestrogen receptor beta (ESR2) mRNA within various bovine ovarian cell types using in situ hybridization.

The localization of oestrogen receptor beta (ESR2) mRNA, in this article denominated as (ERβ) mRNA, was examined using in situ hybridization in the ovaries of randomly selected cows, irrespective of the cycle stage of the animals. A 602‐bp fragment of ERβ mRNA was cloned, sequenced and digoxigenin (DIG)‐labelled. Semi‐quantitative evaluation showed that the scores for ERβ mRNA were moderate to high in the follicle cells of both primordial and primary follicles, but lower in granulosa cells of secondary follicles. In vital tertiary follicles, the total ERβ mRNA expression was low but varied between the different animals. In both obliterative and cystic atretic follicles, high to moderate ERβ mRNA scores were noticed in the granulosa cells. The stroma cells surrounding primordial and primary follicles and the theca cells of secondary follicles showed moderate ERβ mRNA levels, whereas the ERβ mRNA score in theca interna and theca externa cells of vital tertiary follicles was distinctly higher. In the theca cells of atretic follicles the score was even higher. Cells of corpora hemorrhagica and corpora lutea had moderate ERβ mRNA scores, while higher scores were seen in cells of corpora albicantia. Cells of the surface epithelium had a moderate score for ERβ mRNA, whereas cells of the tunica albuginea and deep stroma showed high ERβ mRNA scores. The present findings have clearly established a cell‐specific localization of ERβ mRNA in several cell types in the bovine ovary.

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.

Mapping of the ATP2B2 and PCCB genes on porcine chromosome 13.

We report the cloning and mapping of two genes on porcine Chromosome (Chr) 13: the ATP2B2 gene, coding for a plasma membrane Ca2+-ATPase, and the PCCB gene, coding for the propionylCoA carboxylase beta-subunit. Plasma membrane Ca2+-ATPases play an essential role in the regulation of the free cytosolic Ca 2§ concentration in eukaryotes, by pumping free cytosolic Ca 2+ out of the cell, while hydrolyzing ATP. The pumps are encoded by a highly conserved and widely dispersed multigene family, consisting of at least four different genes (ATP2B1, ATP2B2, ATP2B3, and ATP2B4; Wang et al. 1994). Propionyl-CoA carboxylase (PCC) is a biotin-dependent, mitochondrial enzyme that catalyzes the carboxylation of propionyl-CoA to o-methylmalonyl-CoA in an ATP-dependent process. The enzyme is involved in the catabolism of branched-chain amino acids, fatty acids of odd-numbered chain lengths, and other metabolites. PCC is composed of c~and [ 3 s u b u n i t s (Os 4 o r ff61~6), encoded by the PCCA and the PCCB gene, respectively (Lamhonwah et al. 1986). cDNA clones of human ATP2B2 (phPMCA2-1) and PCCB (pPCC41A2) were obtained from the American Type Culture Collection. A 1100-bp BamHI fragment from phPMCA2-1 and a 1350-bp BamHI fragment from pPCC41A2 were labeled with 32p using the Ready To Go DNA labeling Kit (-dCTP) (Pharmacia) and hybridized to 6 x 105 tofu of a porcine phage h library (Peelman et al. 1991). The isolated hATP2B2 clone contains a 17-kb insert, and the isblated kPCCB clone contains a 15-kb insert (inserts sized using pulsed field gel electrophoresis). Southern analysis of the KATP2B2 clone, with the 1100-bp BamHI fragment from phPMCA2-1 as probe, revealed a 3100-bp and a 3300-bp BamHI fragment, and a 2800-bp EcoRI fragment. The 3100-bp BamHI fragment was cloned in pUC 18 and partially sequenced with the TTSequencingTm Kit. A 352-bp sequence was compared with the databases using BLAST and FASTA. A 91-bp fragment shows 93% sequence identity with the human phPMCA2-1 clone (X63575; from position 2387), with only one amino acid difference, whereas the sequence identity of the other part is lower than 40%. Since the 91-bp fragment is flanked by the consensus AG . . . GT splice sites, it probably forms a complete exon. This 91-bp fragment shows also 91% sequence identity with the rat ATP2B2 gene. The clone contains ATP2B2 and not the related ATP2B1, since it shows only 78% sequence identity with the porcine ATP2B 1 sequence published by De Jaegere and colleagues (1990). Southern analysis of the hPCCB clone, with the 1350-bp BamHI fragment from pPCC41A2 as probe, revealed a 1500-bp and a 3200-bp BamHI fragment, and a 2000-bp EcoRI fragment. The 1500-bp BamHI fragment was cloned in pUC18, and 452 bp respectively 460 bp from both ends was sequenced. Comparison of