Heather Finlayson

A genome-wide linkage analysis for reproductive traits in F2 Large White × Meishan cross gilts

Female reproductive performance traits in pigs have low heritabilities thus limiting improvement through traditional selective breeding programmes. However, there is substantial genetic variation found between pig breeds with the Chinese Meishan being one of the most prolific pig breeds known. In this study, three cohorts of Large White × Meishan F2 cross-bred pigs were analysed to identify quantitative trait loci (QTL) with effects on reproductive traits, including ovulation rate, teat number, litter size, total born alive and prenatal survival. A total of 307 individuals were genotyped for 174 genetic markers across the genome. The genome-wide analysis of the trait-recorded F2 gilts in their first parity/litter revealed one QTL for teat number significant at the genome level and a total of 12 QTL, which are significant at the chromosome-wide level, for: litter size (three QTL), total born alive (two QTL), ovulation rate (four QTL), prenatal survival (one QTL) and teat number (two QTL). Further support for eight of these QTL is provided by results from other studies. Four of these 12 QTL were mapped for the first time in this study: on SSC15 for ovulation rate and on SSC18 for teat number, ovulation rate and litter size.

Application of Sus scrofa microsatellite markers to wild suiformes

Stewart Lowden1∗, H.A. Finlayson2, A.A. Macdonald1, A.C. Downing2, S.J. Goodman3, K. Leus4, L. Kaspe5, E. Wahyuni5 & A.L. Archibald2 1Department of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh EH9 1QH, UK; 2Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UK; 3The Zoological Society of London, Regent’s Park, London NW1 4RY, UK; 4Centre for Research and Conservation, Royal Zoological Society of Antwerp, Koningin Astridplein 26, 2018 Antwerpen, Belgium; 5Kebun Binatang Surabaya, Jl. Setail 1, Surabaya, Indonesia (*Author for correspondence: E-mail: s.lowden@ed.ac.uk; Fax: 0131 650 6576)

Functional analysis of the porcine USP18 and its role during porcine arterivirus replication.

Emerging evidence places deubiquitylation at the core of a multitude of regulatory processes, ranging from cell growth to innate immune response and health, such as cancer, degenerative and infectious diseases. Little is known about deubiquitylation in pig and arterivirus infection. This report provides information on the biochemical and functional role of the porcine USP18 during innate immune response to the porcine respiratory and reproductive syndrome virus (PRRSV). We have shown that UBP gene is the ortholog of the murine USP18 (Ubp43) gene and the human ubiquitin specific protease 18 (USP18) gene and encodes a biochemically functional de-ubiquitin enzyme which inhibits signalling pathways that lead to IFN-stimulating response element (ISRE) promotor regulation. Furthermore we have demonstrated that overexpression of the porcine USP18 leads to reduced replication and/or growth of PRRSV. Our data contrast with the conclusion of numerous reports demonstrating that USP18-deficient mice are highly resistant to viral and bacterial infections and to oncogenic transformation by BCR-ABL, and highlight USP18 as a potential target gene that promotes reduced replication of PRRSV.

Lawsonia intracellularis infection of intestinal crypt cells is associated with specific depletion of secreted MUC2 in goblet cells.

The expression patterns of secreted (MUC2 and MUC5AC) and membrane-tethered (MUC1, MUC4, MUC12 and MUC13) mucins were monitored in healthy pigs and pigs challenged orally with Lawsonia intracellularis. These results showed that the regulation of mucin gene expression is distinctive along the GI tract of the healthy pig, and may reflect an association between the function of the mucin subtypes and different physiological demands at various sites. We identified a specific depletion of secreted MUC2 from goblet cells in infected pigs that correlated with the increased level of intracellular bacteria in crypt cells. We concluded that L. intracellularis may influence MUC2 production, thereby altering the mucus barrier and enabling cellular invasion.

Lawsonia intracellularis exploits β-catenin/Wnt and Notch signalling pathways during infection of intestinal crypt to alter cell homeostasis and promote cell proliferation

Lawsonia intracellularis is an obligate intracellular bacterial pathogen that causes proliferative enteropathy (PE) in pigs. L. intracellularis infection causes extensive intestinal crypt cell proliferation and inhibits secretory and absorptive cell differentiation. However, the affected host upstream cellular pathways leading to PE are still unknown. β-catenin/Wnt signalling is essential in maintaining intestinal stem cell (ISC) proliferation and self-renewal capacity, while Notch signalling governs differentiation of secretory and absorptive lineage specification. Therefore, in this report we used immunofluorescence (IF) and quantitative reverse transcriptase PCR (RTqPCR) to examine β-catenin/Wnt and Notch-1 signalling levels in uninfected and L. intracellularis infected pig ileums at 3, 7, 14, 21 and 28 days post challenge (dpc). We found that while the significant increase in Ki67+ nuclei in crypts at the peak of L. intracellularis infection suggested enhanced cell proliferation, the expression of c-MYC and ASCL2, promoters of cell growth and ISC proliferation respectively, was down-regulated. Peak infection also coincided with enhanced cytosolic and membrane-associated β-catenin staining and induction of AXIN2 and SOX9 transcripts, both encoding negative regulators of β-catenin/Wnt signalling and suggesting a potential alteration to β-catenin/Wnt signalling levels, with differential regulation of the expression of its target genes. We found that induction of HES1 and OLFM4 and the down-regulation of ATOH1 transcript levels was consistent with the increased Notch-1 signalling in crypts at the peak of infection. Interestingly, the significant down-regulation of ATOH1 transcript levels coincided with the depletion of MUC2 expression at 14 dpc, consistent with the role of ATOH1 in promoting goblet cell maturation. The lack of significant change to LGR5 transcript levels at the peak of infection suggested that the crypt hyperplasia was not due to the expansion of ISC population. Overall, simultaneous induction of Notch-1 signalling and the attenuation of β-catenin/Wnt pathway appear to be associated with the inhibition of goblet cell maturation and enhanced crypt cell proliferation at the peak of L. intracellularis infection. Moreover, the apparent differential regulation of apoptosis between crypt and lumen cells together with the strong induction of Notch-1 signalling and the enhanced SOX9 expression along crypts 14 dpc suggest an expansion of actively dividing transit amplifying and/or absorptive progenitor cells and provide a potential basis for understanding the development and maintenance of PE.

An intronic polymorphism in the porcine IRF7 gene is associated with better health and immunity of the host during Sarcocystis infection, and affects interferon signalling

Interferon regulatory factor 7 (IRF7), as a key regulator of type I interferon response, plays an important role during innate response against viral infection. Although well conserved across species, the structure of IRF7 and its function during parasite infection are not well documented in farm animals, such as the pig. To bridge this gap, we have determined the porcine IRF7 gene structure and identified two intronic single nucleotide polymorphisms (SNPs), SNP g.748G>C and SNP g.761A>G, in commercial pig breeds. The distribution of SNP g.761A>G in multiple breeds suggested that it was in Hardy-Weinberg equilibrium and allowed us to map it at the top of SSC2. We found that during Sarcocystis miescheriana infection, the G allele was associated with high lymphocyte levels (P < 0.02), reduced drop in platelet levels (P < 0.002) and IgG1-Th2-dominated response (P < 0.05). This suggests that the G allele was associated with better health and immunity of the host during Sarcocystis infection. Furthermore, we have also provided suggestive evidence that the G allele of SNPc.761A>G enhances the transactivation activity of IRF7, possibly by improving IRF7 transcript splicing of intron-3. These findings would suggest that IRF7, as a transcriptional regulator, is involved in the defence mechanism against a larger spectrum of pathogens, and in more host species, than initially anticipated.

Balancing selection at a premature stop mutation in the myostatin gene underlies a recessive leg weakness syndrome in pigs

Balancing selection provides a plausible explanation for the maintenance of deleterious alleles at moderate frequency in livestock, including lethal recessives exhibiting heterozygous advantage in carriers. In the current study, a leg weakness syndrome causing mortality of piglets in a commercial line showed monogenic recessive inheritance, and a region on chromosome 15 associated with the syndrome was identified by homozygosity mapping. Whole genome resequencing of cases and controls identified a mutation coding for a premature stop codon within exon 3 of the porcine Myostatin (MSTN) gene, similar to those causing a double-muscling phenotype observed in several mammalian species. The MSTN mutation was in Hardy-Weinberg equilibrium in the population at birth, but significantly distorted amongst animals still in the herd at 110 kg, due to an absence of homozygous mutant genotypes. In heterozygous form, the MSTN mutation was associated with a major increase in muscle depth and decrease in fat depth, suggesting that the deleterious allele was maintained at moderate frequency due to heterozygous advantage. Knockout of the porcine MSTN by gene editing has previously been linked to problems of low piglet survival and lameness. This MSTN mutation is an example of putative balancing selection in livestock, providing a plausible explanation for the lack of disrupting MSTN mutations in pigs despite many generations of selection for lean growth.