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Overconditioning is a risk factor for upregulated pre- and postpartum fat mobilization. Therefore, we hypothesized that overconditioning at the end of pregnancy leads to the accumulation of lipids in the liver and modifications of the hepatic gene expression pattern. The aim of this study was to evaluate the effect of normal- versus overconditioning on the hepatic transcriptomic profile of dairy cows at the end of pregnancy. Ten dry multiparous Holstein cows were killed 2 wk before expected calving. Body condition score (BCS) and backfat thickness (BFT) were evaluated, and blood samples for nonesterified fatty acids (NEFA) were taken before cows were killed. After cows were killed, liver biopsy samples were collected for further assessment of total lipids and RNA sequencing. Five cows were classified as normal-conditioned (median BCS = 3, range 2.75-3.5) and 5 as overconditioned (median BCS = 4, range 4-5). Regression models confirmed that normal-conditioned cows had lower BFT (1.29 +/- 0.29 cm; least squares means +/- standard error) and serum NEFA (0.16 +/- 0.04 mmol/L) in comparison to overconditioned cows (3.14 +/- 0.43 cm and 0.38 +/- 0.07 mmol/L for BFT and NEFA, respectively). Total liver lipid percentage tended to be lower in normal- versus overconditioned cows (4.63 +/- 0.40% and 6.06 +/- 0.44%, respectively). In comparison to the mean liver lipid percentage of the normal- and overconditioned cows, 1 overconditioned cow had a relatively low (5.21%) and 1 normal-conditioned cow had a relatively high (6.07%) liver lipid percentage. Differentially expressed genes analysis (edgeR quasi-likelihood method) showed that normal-conditioned cows presented 11 upregulated and 12 downregulated genes in comparison to overconditioned cows. Linear discriminant analysis effects size revealed 133 differentially expressed genes between normal- versus overconditioned cows. Notably, the liver of normal-conditioned cows had upregulated genes associated with liver functionality (ALB, SELENOP, IGF1, and IGF2). On the other hand, overconditioned cows had upregulated genes associated with the acute-phase response (C3, HPX, and, LBP). High basal lipolysis in overconditioned cows at the end of pregnancy increased liver lipid content, and this may alter the hepatic gene expression pattern to a pro-inflammatory state.

In neutrophils, toll-like receptor and complement component 5a (C5a) signaling are critical pathways regulating innate immunity. In cows, not much is known about the second C5a receptor, complement component 5a receptor 2 (C5AR2). It is an interesting player in sepsis treatment because it is considered to have an anti-inflammatory effect during normal inflammation. Periparturient cows are prone to severe infections, and the objectives of this study were to investigate the expression and functionality of C5AR2 during peripartum. We investigated the effect of 2 major inflammatory stimuli, C5a and lipopolysaccharide (LPS), on the expression of a selected number of genes (C5AR1, C5AR2, TLR4, ITGAM, COX2, and CXCL8) and functions linked to these receptors. Overall, TLR4, ITGAM, and C5AR2, all of which are involved in early inflammation, showed a lower expression in periparturient cows. However, an overall lower expression seems not to be the only explanation for the increased risk of sepsis in periparturient cows. Normally, in response to inflammation and as seen in the mid-lactation group, the expression of these genes increases after stimulation with LPS. However, in periparturient cows, stimulation with LPS led to a decrease in expression of these receptors, indicating a different response of neutrophils in response to LPS during this period. A decrease in ITGAM (coding for CD11b) expression complicates correct neutrophil localization and phagocytosis. Its downregulation upon stimulation might be detrimental for adequate eradication of the pathogen and might increase the risk of an imbalanced inflammation; C5AR2 seems to play a central role in this altered response. In addition, myeloperoxidase (MPO) activity in periparturient cows is lower in response to C5a stimulation. It has been suggested that MPO plays an important role in neutrophil shutdown and, thereby, timely resolution of inflammation. A decreased MPO activity might thus prolong the inflammatory reaction of the neutrophils. This finding was supported by the increased viability of the neutrophils obtained from periparturient cows. Even after stimulation, we found a lower caspase-3 activity in this group, indicating that they might be activated for a longer time compared with the neutrophils from mid-lactation cows. Accordingly, these alterations might contribute to a temporal mismatch in inflammatory responses, as often seen in severe periparturient infections.

The aim of this study was to determine the number of adipose tissue macrophages (ATM) and the mRNA expression of adipokines [adiponectin (ADIPOQ), leptin (LEP), interleukin 6 (IL6), tumor necrosis factor (TNF), and interleukin 10 (IL10)] in different adipose depots from cows with a variable body condition score (BCS) at the end of the dry period. We hypothesized that the number of ATM and the expression of these adipokines depend on adipocyte size and the anatomical location of the adipose depot. Subcutaneous, omental, mesenteric, perirenal, and intrapelvic adipose tissue samples were taken immediately after euthanasia of 10 Holstein Friesian dairy cows (upcoming parity 2 to 5, age 3.9 ± 1.4 yr; mean ± standard deviation) at the end of pregnancy (actual days of pregnancy at the moment of euthanasia: 269 ± 5 d). During the dry period, all animals received similar diets to meet but not exceed requirements. Five animals were considered to have a normal BCS (2.5-3.5) and 5 animals were considered to be over-conditioned (BCS = 3.75-5). Body weight of the animals at the moment of euthanasia was 717 ± 77 kg. Expression of the different genes was determined by reverse transcription quantitative real-time PCR. Adipocyte size was determined by measuring the area of 100 adipocytes on histological sections. Average adipocyte area was 10,475 ± 1,019, 8,500 ± 780, 10,383 ± 1,227, 11,466 ± 1,039, and 11,087 ± 1,632 µm2 for the subcutaneous, mesenteric, omental, intrapelvic, and perirenal adipose depot, respectively. Immunohistochemistry using anti-bovine CD172a antibodies was performed to determine the proportion of ATM (the number of CD172a-positive cells per 100 adipocytes, given as a percentage). Expression of LEP, IL6, and TNF was positively associated with adipocyte size, whereas no association could be detected between ADIPOQ and IL10 with the size of the adipocytes. The omental adipose depot was especially infiltrated with ATM (1.92 ± 0.55, 1.10 ± 0.33, and 8.28 ± 2.24% for the subcutaneous, mesenteric, and omental adipose depot, respectively). The proportion of ATM was positively associated with the size of the adipocytes in the omental and mesenteric adipose depot. Expression of ADIPOQ, LEP, IL6, TNF, and IL10 differed among depots, which suggests differences in inflammatory characteristics depending on the anatomical location of depots. In conclusion, the results of the present study confirm the adipose tissue as a potential source of inflammatory mediators and demonstrate ATM infiltration, especially in the omental adipose depot.

Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3′ untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT–) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R–) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R– pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R– pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.

The piperidine ring in the title compound is in the chair conformation, whereas the diketopiperazine ring has a boat form. The benzyl group is axially positioned and folded above the diketopiperazine ring. No dimerisation is observed.

Tubular joints are intensively used in off-shore structures for shallow waters. Depending on the sea conditions and the type of structure, the design can be fatigue driven. This is particularly the case for off-shore wind turbines, where turbulences are generating a fatigue loading. Any improvement of the fatigue performance of the tubular joint would be beneficial to reduce the weight and the cost of the structure. To assess efficiently the fatigue resistance of the tubular joint, a testing method has been developed based on the resonance principle. The complete circumference of the welded joint can be loaded, successively in the in-plane and out-of-plane modes at a frequency close to 20Hz. Finite element computations were used to investigate the feasibility of the concept. Then, an X-node was made and successfully tested to investigate the stress distribution along the weld. The experimental results were compared with finite element computations, giving a good agreement.

Summary The lamins are components of nuclear lamina and they have a profound influence on nuclear structure and functions. They are encoded by three genes, LMNA, LMNB1 and LMNB2. A genomic fragment of the porcine LMNA gene (822 bp; from exons 7 to 9) was amplified by polymerase chain reaction and comparatively sequenced. Four single nucleotide polymorphisms (SNPs) were identified in intronic sequences: G162A, G208A, T367G and C618T. The SNPs are within the restriction sites for enzymes Bsh1236I, HpaII, AluI and Bsh1236I respectively. Allele frequencies at SNPs G208A, T367G and C618T were determined by using eight pig breeds. Linkage analysis in the Hohenheim Meishan × Pietrain family placed the LMNA gene in the chromosome 4q linkage group, between MEF2D and GBA (MEF2D– 3.0 cM –LMNA– 0.2 cM –GBA). In radiation hybrid mapping LMNA was most significantly linked to SW270 on chromosome 4 (39 cR; LOD = 7.86). The LMNA gene is located in the quantitative trait loci region for some carcass traits on chromosome 4q.

A porcine bacterial artificial chromosome (BAC) clone, containing the melanocortin 2 receptor gene (MC2R) was isolated. The complete coding sequence of the MC2R gene, contained in 1 exon, was determined. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was performed on a 241-bp coding fragment. An AluI polymorphism, detecting a silent mutation, was found and typed on unrelated animals of five different pig breeds. The Meishan, Pietrain and Large White breeds differ significantly in allele frequencies from the Landrace and Czech Meat Pig breeds. The melanocortin 5 receptor gene (MC5R) was detected by PCR in the same BAC clone, as could be expected from the human and porcine mapping data. PCR-SSCP was performed on a 200-bp coding of MC5R, but no polymorphisms were detected. The BAC clone was mapped to Sscr6q27 by fluorescent in situ hybridization (FISH). A (CA)n microsatellite (SGU0002), isolated from the BAC, was localized on chromosome 6 by RH mapping near marker SW1473 and by linkage mapping on the MARC reference family at the same position as the marker SW2173 (97 cM). Allele frequencies, heterozygosity and polymorphism information contents (PIC) values were calculated for the five different pig breeds examined. The transcription of both genes in porcine liver, heart, kidney, fat, brain, pancreas, stomach, bladder, ovaries, lung, spleen, skin, adrenal gland and muscle tissues was examined by reverse transcriptase-polymerase chain reaction. Transcription was detected in skin and adrenal gland tissues for MC2R, while a positive signal was detected for MC5R in kidney, fat, pancreas, skin, adrenal gland and spleen tissues.

The porcine major histocompatibility complex, also called swine lymphocyte antigen (SLA) complex, is of particular interest not only because of its central role in the immune response, but also because of its influence on many traits such as reproduction, fatness and meat quality. The porcine FABGL (FabG (beta-ketoacyl-[acyl-carrierprotein] reductase, Escherichia coli) like) gene, coding for a 17beta-hydroxysteroid dehydrogenase (17beta-HSD), is a candidate gene for these traits. The complete gene was sequenced and compared with human and mouse FABGL sequences. The deduced amino acid sequence showed 85 and 83% sequence identity to human and mouse sequences, respectively. Polymorphicic BbvI and DdeI restriction sites were found in the porcine FABGL gene. The promoter was compared with the promoter regions of human and mouse FABGL sequence in order to identify putative regulatory elements. The transcription profile of the porcine gene was determined and showed a widespread tissue distribution.

*Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Liběchov, Czech Republic. Department of Genetics, Mendel University of Agriculture and Forestry, Zemědělska 1, 613 00 Brno, Czech Republic. Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium. Department of Animal Breeding and Biotechnology, Institute of Animal Husbandry and Breeding, University of Hohenheim, Garbenstrasse 17, D-70593 Stuttgart, Germany

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.

An Alw 26I PCR-RFLP in exon 1 of the porcine SKI oncogene and mapping the gene to the RYR1 (CRC ) linkage group on chromosome 6

Similar to other countries, heifer mastitis is highly prevalent in Belgium. Marker assisted genetic selection against certain disadvantageous genotypes could offer possibilities to enhance host resistance. The main objective of this study was to screen CXCR1 for polymorphisms and to analyze their association with udder health during first lactation. This potential candidate gene encodes the homonymous receptor binding interleukin 8, a crucial chemokine in the innate immunity of the mammary gland. Eighty-six heifers originating from 20 Belgian farms were genotyped for the entire coding region. Fifteen single nucleotide polymorphisms (SNPs) were detected of which 10 have not been described before. Four distinct haplotypes were detected. The association between SNP at position +735 relative to the start codon (c.735C>G) and subclinical mastitis which has already been reported by other research groups could not be confirmed in our study. Instead, SNP c.642G>A was found to be associated with udder health. Prevalence of intramammary infection (IMI) in early lactation was significantly lower in heifers expressing genotype AA compared to heifers expressing genotype AG or GG. Additionally, SNP c.982A>G tended to be associated with pathogen group specific IMI. Heifers with genotype GG on this locus were more susceptible specifically to major pathogens compared to those expressing genotype AG or AA. Numerical differences in somatic cell count in early lactation and throughout lactation by CXCR1 genotype were observed but were not statistically significant. Results in this study are promising and stimulate to further elucidate the role of CXCR1 in mastitis susceptibility.

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.

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.

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.

Bovine papillomavirus (BPV), the causative agent of papillomas in cattle, has been shown to play a major role in the pathogenesis of equine sarcoids in horses. BPV has also been detected occasionally in normal equine skin. In this study, presence and activity of BPV in normal skin and peripheral blood of 4 groups of horses were evaluated: sarcoid-affected horses, horses living in contact with sarcoid-affected horses, horses living in contact with papilloma-affected cattle and control horses. From each horse, 3 samples on 4 locations were collected: a swab of the intact skin surface and both a swab and a biopsy after decontamination. BPV DNA was found in the normal skin of 24 of 42 horses (57%). Mainly sarcoid-affected horses and horses living in contact with cattle were carriers (73%), but BPV DNA was also detected in 50% of the horses living in contact with sarcoid-affected horses and in 30% of the control population. BPV mRNA was detected in 58% of the samples positive for BPV DNA, although in a much lower quantity compared to sarcoids. In most of the BPV DNA positive samples mild acanthosis, slight basophilic cytoplasmic swelling of the epidermal layers and/or thickening of the basal membrane were noticed, but these observations were also present in several BPV DNA negative normal skin samples. BPV DNA could not be detected in peripheral blood. These findings suggest latent infection and a wide-spread occurrence of BPV in the horse population. (C) 2007 Elsevier B.V. All rights reserved.

Mapping of the porcine FBN2, YWHAQ, CNN3, DCN, POSTN, SPARC, RBM39 and GNAS genes, expressed in foetal skeletal muscles A. Stratil*, A. Knoll*, P. Horak*, K. Bilek, R. Bechyňova*, H. Bartenschlager, M. Van Poucke, L. J. Peelman, K. Svobodova and H. Geldermann *Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Liběchov, Czech Republic. Department of Animal Morphology, Physiology and Genetics, Mendel University of Agriculture and Forestry, Zemědělska 1, 613 00 Brno, Czech Republic. Department of Animal Breeding and Biotechnology, Institute of Animal Husbandry and Breeding, University of Hohenheim, Garbenstrasse 17, D-70593 Stuttgart, Germany. Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium

Equine sarcoids, the most common skin tumours in horses, are induced by bovine papillomavirus (BPV). Their clinical appearance varies from small stable patches to aggressively growing masses. Differences in BPV load and mRNA expression and Ki67 and p53 immunostaining among four clinical types (fibroblastic, occult, nodular and verrucous sarcoids) were evaluated to test the hypothesis that the clinical behaviour of equine sarcoids correlates with BPV activity. Viral load and expression of the BPV E2, E5, E6 and E7 genes were determined using quantitative real-time PCR. The proliferative fraction (PF) of the tumours was determined by Ki67 immunostaining and expression of p53 was analysed by immunohistochemistry. Nodular sarcoids showed a significantly higher viral load than the other types. A significant overall difference among the four types was observed for E2, E5, E6 and E7 mRNA expression. Nodular sarcoids showed the highest expression level for each BPV gene examined, followed by verrucous, fibroblastic and occult tumours. Viral DNA and mRNA outcomes correlated with each other, indicating a similar transcription pattern in each type of sarcoid. The PF was significantly higher in the superficial layers of verrucous and fibroblastic sarcoids compared with occult and nodular types. No significant difference was observed for the PF in the deep layers and for p53 expression. These results clearly demonstrate the omnipresence and active transcription of BPV in equine sarcoids. However, the hypothesis that the clinical behaviour of an equine sarcoid can be explained on the basis of differences in BPV activity could not be demonstrated.

Summary The porcine orthologues of human chromosome HSA9q22.31 genes osteoglycin (OGN) and asporin (ASPN) were mapped to porcine chromosome SSC3 using linkage analysis and a somatic cell hybrid panel. This mapping was refined to SSC3q11 using fluorescence in situ hybridization. These results confirm the existence of a small conserved synteny group between SSC3 and HSA9. Polymorphisms were revealed in both genes, including a pentanucleotide microsatellite (SCZ003) in OGN and two single nucleotide polymorphisms (AM181682.1:g.780G>T and AM181682.1:g.825T>C) in ASPN. The two genes were included in a set of markers for quantitative trait loci (QTL) mapping on SSC3 in the Hohenheim Meishan × Pietrain F2 family. Major QTL for growth and carcass traits were centred in the ASPN–SW902 region.

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.

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).

*Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic. Institute of Animal Husbandry and Breeding, Department of Animal Breeding and Biotechnology, University of Hohenheim, Garbenstrasse 17, D-70593 Stuttgart, Germany. Ghent University, Faculty of Veterinary Medicine, Department of Animal Nutrition, Genetics, Breeding and Ethology, Heidestraat 19, 9820 Merelbeke, Belgium. DIPROVAL Sezione di Allevamenti Zootecnici, Faculty of Agriculture, University of Bologna, Via F.lli Rosselli 107, 42100 Reggio Emilia, Italy

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.

A porcine bacterial artificial chromosome (BAC) clone, containing the melanocortin 2 receptor gene (MC2R) was isolated. The complete coding sequence of the MC2R gene, contained in 1 exon, was determined. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was performed on a 241-bp coding fragment. An AluI polymorphism, detecting a silent mutation, was found and typed on unrelated animals of five different pig breeds. The Meishan, Piétrain and Large White breeds differ significantly in allele frequencies from the Landrace and Czech Meat Pig breeds. The melanocortin 5 receptor gene (MC5R) was detected by PCR in the same BAC clone, as could be expected from the human and porcine mapping data. PCR-SSCP was performed on a 200-bp coding of MC5R, but no polymorphisms were detected. The BAC clone was mapped to Sscr6q27 by fluorescent in situ hybridization (FISH). A (CA)n microsatellite (SGU0002), isolated from the BAC, was localized on chromosome 6 by RH mapping near marker SW1473 and by linkage mapping on the MARC reference family at the same position as the marker SW2173 (97 cM). Allele frequencies, heterozygosity and polymorphism information contents (PIC) values were calculated for the five different pig breeds examined. The transcription of both genes in porcine liver, heart, kidney, fat, brain, pancreas, stomach, bladder, ovaries, lung, spleen, skin, adrenal gland and muscle tissues was examined by reverse transcriptase-polymerase chain reaction. Transcription was detected in skin and adrenal gland tissues for MC2R, while a positive signal was detected for MC5R in kidney, fat, pancreas, skin, adrenal gland and spleen tissues.

Protein aggregates, red cell or white cell fragments are known to interfere with platelet counts in automated blood analysers, both by aperture impedance and optical technologies. When a falsely high value is suspected, interference by pseudo-platelet particles can be confirmed by systematic examination of stained blood films. The method that best avoids these sources of interference is the reference, immunological platelet count. We describe a case of treated malaria with a false normal platelet count. The blood smear revealed small red cells, infected by trophozoites of Plasmodium falciparum, that interfered with the platelet count. The Cell Dyn 4000 shows different patterns of interference by infected red cells in its impedance and optical counts, and thrombocytopenia was suspected immediately. This was confirmed by a phase-contrast microscopic platelet count.

The porcine major histocompatibility complex, also called swine lymphocyte antigen (SLA) complex, is of particular interest not only because of its central role in the immune response, but also because of its influence on many traits such as reproduction, fatness and meat quality. The porcine FABGL (FabG (beta-ketoacyl-[acyl-carrierprotein] reductase, Escherichia coli) like) gene, coding for a 17beta-hydroxysteroid dehydrogenase (17beta-HSD), is a candidate gene for these traits. The complete gene was sequenced and compared with human and mouse FABGL sequences. The deduced amino acid sequence showed 85 and 83% sequence identity to human and mouse sequences, respectively. Polymorphicic BbvI and DdeI restriction sites were found in the porcine FABGL gene. The promoter was compared with the promoter regions of human and mouse FABGL sequence in order to identify putative regulatory elements. The transcription profile of the porcine gene was determined and showed a widespread tissue distribution.