• Analysis of DRB1 exon 2 genotyping by STR size analysis in Suffolk and Texel sheep breeds

      Sayers, Gearoid; Mitchel, S; Ryan, Marion T; Stear, Michael J.; Hanrahan, James P; Sweeney, Torres; Department of Agriculture, Food and the Marine; Wellcome Trust; RSF16; 061354 (Teagasc (Agriculture and Food Development Authority), Ireland, 2004)
      Alleles of the DRB1 exon 2 locus of the major histocompatibility complex have recently been associated with genetic resistance to gastrointestinal nematodes in sheep. While sequence-based typing is the standard method for allele discrimination, a rapid, high throughput method for DRB1 exon 2 genotyping is required if such information is to be incorporated into national breeding programmes. Previous studies have highlighted a simple tandem repeat (STR) located within intron 2 of the DRB1 gene, which could potentially be used to accurately assess the allele present within the adjacent exon 2. The aims of this study were firstly to compare two methods of STR analysis, Genescan™ and autoradiography, and secondly to investigate if STR analysis of DRB1 intron 2 could be used to accurately assess the profile of DRB1 exon 2. Six DRB1 exon 2 alleles were identified by sequence-based typing in Suffolk (n = 31) and eight in Texel (n = 60) sheep. The results indicated that Genescan™ was a more accurate method of STR analysis than autoradiography. The expected 1:1 correspondence between STR size, analysed by Genescan™ and DRB1 exon 2 allele, determined by sequence-based typing, was not observed. However, the correspondence was found to be degenerate, whereby some alleles were associated with two STR sizes. Thus, irrespective of the STR size identified, STR analysis by Genescan™ identified the correct allele in all cases within both populations of animals studied. However, the Genescan™ method of allele identification cannot be used for Suffolk × Texel crossbred progeny or in other breeds where the relationship between STR size and DRB1 exon 2 allele is not known.
    • The application of transcriptomic data in the authentication of beef derived from contrasting production systems

      Sweeney, Torres; Lejeune, Alexandre; Moloney, Aidan; Monahan, Frank J; Gettigan, Paul M; Downey, Gerard; Park, Stephen D. E.; Ryan, Marion T; Department of Agriculture, Food and the Marine (Biomed Central, 2016-09-21)
      Background Differences between cattle production systems can influence the nutritional and sensory characteristics of beef, in particular its fatty acid (FA) composition. As beef products derived from pasture-based systems can demand a higher premium from consumers, there is a need to understand the biological characteristics of pasture produced meat and subsequently to develop methods of authentication for these products. Here, we describe an approach to authentication that focuses on differences in the transcriptomic profile of muscle from animals finished in different systems of production of practical relevance to the Irish beef industry. The objectives of this study were to identify a panel of differentially expressed (DE) genes/networks in the muscle of cattle raised outdoors on pasture compared to animals raised indoors on a concentrate based diet and to subsequently identify an optimum panel which can classify the meat based on a production system. Results A comparison of the muscle transcriptome of outdoor/pasture-fed and Indoor/concentrate-fed cattle resulted in the identification of 26 DE genes. Functional analysis of these genes identified two significant networks (1: Energy Production, Lipid Metabolism, Small Molecule Biochemistry; and 2: Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry), both of which are involved in FA metabolism. The expression of selected up-regulated genes in the outdoor/pasture-fed animals correlated positively with the total n-3 FA content of the muscle. The pathway and network analysis of the DE genes indicate that peroxisome proliferator-activated receptor (PPAR) and FYN/AMPK could be implicit in the regulation of these alterations to the lipid profile. In terms of authentication, the expression profile of three DE genes (ALAD, EIF4EBP1 and NPNT) could almost completely separate the samples based on production system (95 % authentication for animals on pasture-based and 100 % for animals on concentrate- based diet) in this context. Conclusions The majority of DE genes between muscle of the outdoor/pasture-fed and concentrate-fed cattle were related to lipid metabolism and in particular β-oxidation. In this experiment the combined expression profiles of ALAD, EIF4EBP1 and NPNT were optimal in classifying the muscle transcriptome based on production system. Given the overall lack of comparable studies and variable concordance with those that do exist, the use of transcriptomic data in authenticating production systems requires more exploration across a range of contexts and breeds.
    • A catalogue of validated single nucleotide polymorphisms in bovine orthologs of mammalian imprinted genes and associations with beef production traits

      Magee, David A; Berkowicz, Erik W; Sikora, Klaudia M; Berry, Donagh; Park, Stephen D. E.; Kelly, Alan K; Sweeney, Torres; Kenny, David A.; Evans, R. D.; Wickham, Brian W.; et al. (Cambridge University Press, 2010-06)
      Genetic (or ‘genomic’) imprinting, a feature of approximately 100 mammalian genes, results in monoallelic expression from one of the two parentally inherited chromosomes. To date, most studies have been directed on imprinted genes in murine or human models; however, there is burgeoning interest in the effects of imprinted genes in domestic livestock species. In particular, attention has focused on imprinted genes that influence foetal growth and development and that are associated with several economically important production traits in cattle, sheep and pigs. We have re-sequenced regions in 20 candidate bovine imprinted genes in order to validate single nucleotide polymorphisms (SNPs) that may influence important production traits in cattle. Putative SNPs detected via re-sequencing were subsequently re-formatted for high-throughput SNP genotyping in 185 cattle samples comprising 138 performance-tested European Bos taurus (all Limousin bulls), 29 African B. taurus and 18 Indian B. indicus samples. Analysis of the resulting genotypic data identified 117 validated SNPs. Preliminary genotype–phenotype association analyses using 83 SNPs that were polymorphic in the Limousin samples with minor allele frequencies >0.05 revealed significant associations between two candidate bovine imprinted genes and a range of important beef production traits: average daily gain, average feed intake, live weight, feed conversion ratio, residual feed intake and residual gain. These genes were the Ras proteinspecific guanine nucleotide releasing factor gene ( RASGRF1) and the zinc finger, imprinted 2 gene ( ZIM2). Despite the relatively small sample size used in these analyses, the observed associations with production traits are supported by the purported biological function of the RASGRF1 and ZIM2 gene products. These results support the hypothesis that imprinted genes contribute significantly to important complex production traits in cattle. Furthermore, these SNPs may be usefully incorporated into future marker-assisted and genomic selection breeding schemes.
    • A differential interplay between the expression of Th1/Th2/Treg related cytokine genes in Teladorsagia circumcincta infected DRB1*1101 carrier lambs

      Hassan, Musa; Hanrahan, James P; Good, Barbara; Mulcahy, Grace; Sweeney, Torres; Teagasc Walsh Fellowship Programme (Biomed Central, 2011-03-08)
      Substantial debate exists on whether the immune response between sheep resistant and susceptible to gastrointestinal nematodes can be differentiated into a Th1 and Th2 phenotype. The present study addresses the hypothesis that variation in resistance to Teladorsagia circumcincta between DRB1*1101 (associated with reduced faecal egg count and worm burden) carriers and non-carriers is due to a differential interplay in the expression of Th1/Th2 and regulatory T (Treg) related cytokine genes. Lambs from each genotype were either slaughtered at day 0 (un-infected control) or infected with 3 × 104 Teladorsagia circumcincta L3 and slaughtered at 3, 7, 21, and 35 days later. Lambs carrying the DRB1*1101 allele had a significantly lower worm burden (P < 0.05) compared to the non-carriers. Abomasal mucosal cytokine gene expression was evaluated by quantitative real-time PCR and comparison made for time and genotype effects. The response generated varied through the course of infection and was affected by genotype. DRB1*1101 carriers had an up-regulated expression of the Th1-related cytokine genes (IL-1β, TNFα, and IFN-γ) at day 3, but this was replaced by an up-regulated expression of Th2-related cytokine genes (IL-10 and IL-13) and Treg-related cytokine genes (IL-2RA-CD25, TGFα, TGFβ, Arg2, MIF and FOXP3) by day 7. Conversely, in the non-carriers these changes in gene expression were delayed until days 7 and 21 post infection (pi), respectively. It is concluded that resistance to Teladorsagia circumcincta in animals carrying the DRB1*1101 allele is influenced by an earlier interplay between Th1, Th2 and T regulatory immune response genes.
    • The dynamic influence of the DRB1*1101 allele on the resistance of sheep to experimental Teladorsagia circumcincta infection

      Hassan, Musa; Good, Barbara; Hanrahan, James P; Campion, Deirdre P; Sayers, Gearoid; Mulcahy, Grace; Sweeney, Torres; Teagasc Walsh Fellowship Programme (Biomed Central, 2011-03-08)
      Suffolk sheep carrying the DRB1*1101 (previously referred to as-DRB1*0203 or G2) allele have been reported to show increased resistance to natural Teladorsagia circumcincta infection compared to non-carriers. The objective of this study was to compare the biochemical and physiological responses of DRB1*1101 carrier and non-carrier twin lambs to an experimental infection with 3 × 104 L3 Teladorsagia circumcincta. The variables studied included worm burden, faecal egg count, abomasal mast cells, IgA, IgE, IgG1 plus IgG2 and haematological parameters at 0, 3, 7, 21 and 35 days post infection (dpi), and duodenal smooth muscle contractility at 0 and 35 dpi. DRB1*1101 carrier lambs had significantly lower worm burden, higher mast cell and plasma platelet counts than the DRB1*1101 non-carriers (P < 0.05). Before infection, the non-carrier lambs exhibited significantly higher mucosal levels of all antibody isotypes measured compared to the carriers; these levels remained relatively stable over the course of infection in the non-carriers while there was a slow build up of these antibodies in the carriers up to day 21 post infection (pi). The DRB1*1101 non-carrier lambs had a significantly higher plasma lymphocyte count, and produced greater duodenal contractile force relative to the carrier lambs (P < 0.05). There was no significant difference between genotypes in the level of plasma eosinophils, monocytes, neutrophils or FEC. This evidence suggests that resistance conferred by DRB1*1101 is acquired rather than innate, depends on worm expulsion rather than fecundity and is dependent on mucosal mast cell proliferation, platelet activation, and IgA and IgE antibody responses.
    • Long-term stability of RNA in post-mortem bovine skeletal muscle, liver and subcutaneous adipose tissues

      Bahar, Bojlul; Monahan, Frank J; Moloney, Aidan P; Schmidt, Olaf; MacHugh, David E; Sweeney, Torres; National Development Plan 2000-2006; Teagasc Walsh Fellowship Programme (Biomed Central, 2007-11-29)
      Background: Recovering high quality intact RNA from post-mortem tissue is of major concern for gene expression studies in animals and humans. Since the availability of post-mortem tissue is often associated with substantial delay, it is important that we understand the temporal variation in the stability of total RNA and of individual gene transcripts so as to be able to appropriately interpret the data generated from such studies. Hence, the objective of this experiment was to qualitatively and quantitatively assess the integrity of total and messenger RNA extracted from bovine skeletal muscle, subcutaneous adipose tissue and liver stored at 4°C at a range of time points up to 22 days post-mortem. These conditions were designed to mimic the environment prevailing during the transport of beef from the abattoir to retail outlets. Results: The 28S and 18S rRNA molecules of total RNA were intact for up to 24 h post-mortem in liver and adipose tissues and up to 8 days post-mortem in skeletal muscle. The mRNA of housekeeping genes (GAPDH and ACTB) and two diet-related genes (RBP5 and SCD) were detectable up to 22 days post-mortem in skeletal muscle. While the mRNA stability of the two housekeeping genes was different in skeletal muscle and liver, they were similar to each other in adipose tissue. After 22 days post-mortem, the relative abundance of RBP5 gene was increased in skeletal muscle and in adipose tissue and decreased in liver. During this period, the relative abundance of SCD gene also increased in skeletal muscle whereas it decreased in both adipose tissue and liver. Conclusion: Stability of RNA in three tissues (skeletal muscle, subcutaneous adipose tissue and liver) subjected to long-term post-mortem storage at refrigeration temperature indicated that skeletal muscle can be a suitable tissue for recovering biologically useful RNA for gene expression studies even if the tissue is subjected to post-mortem storage for weeks, whereas adipose tissue and liver should be processed within 24 hours post-mortem.
    • Peripheral and gastrointestinal immune systems of healthy cattle raised outdoors at pasture or indoors on a concentrate-based ration

      Lejeune, Alexandre; Monahan, Frank J; Moloney, Aidan P; Earley, Bernadette; Black, Alistair D; Campion, Deirdre P; Englishby, Tanya; Reilly, Petrina; O'Doherty, John V.; Sweeney, Torres (Biomed Central, 2010-03-31)
      Background: Despite an increasing preference of consumers for beef produced from more extensive pasture-based production systems and potential human health benefits from the consumption of such beef, data regarding the health status of animals raised on pasture are limited. The objective of this study was to characterise specific aspects of the bovine peripheral and the gastrointestinal muscosal immune systems of cattle raised on an outdoor pasture system in comparison to animals raised on a conventional intensive indoor concentrate-based system. Results: A number of in vitro functional tests of immune cells suggested subtle differences between the animals on the outdoor versus indoor production systems. There was a decrease in the number of neutrophils and monocytes engaged in phagocytosis in outdoor cattle (P < 0.01 and P < 0.05, respectively) in comparison to those indoors. Following mitogen stimulation, a lower level of interferon-γ was produced in leukocytes from the outdoor animals (P < 0.05). There was evidence of a gastrointestinal nematode infection in the outdoor animals with elevated levels of serum pepsinogen (P < 0.001), a higher number of eosinophils (P < 0.05) and a higher level of interleukin-4 and stem cell factor mRNA expression (P < 0.05) in the outdoor animals in comparison to the indoor animals. Lower levels of copper and iodine were measured in the outdoor animals in comparison to indoor animals (P < 0.001). Conclusion: Despite distinctly contrasting production systems, only subtle differences were identified in the peripheral immune parameters measured between cattle raised at pasture in comparison to animals raised on a conventional intensive indoor concentrate-based production system.
    • Temporal patterns of inflammatory gene expression in local tissues after banding or burdizzo castration in cattle

      Pang, Wanyong; Earley, Bernadette; Sweeney, Torres; Gath, Vivian; Crowe, Mark A (Biomed Central, 2009-09-23)
      Background: Castration of male cattle has been shown to elicit inflammatory reactions and acute inflammation is initiated and sustained by the participation of cytokines. Methods: Sixty continental × beef bulls (Mean age 12 ± (s.e.) 0.2 months; Mean weight 341 ± (s.e.) 3.0 kg) were blocked by weight and randomly assigned to one of three treatments (n = 20 animals per treatment): 1) untreated control (Con); 2) banding castration at 0 min (Band); 3) Burdizzo castration at 0 min (Burd). Samples of the testis, epididymis and scrotal skin were collected surgically from 5 animals from each group at 12 h, 24 h, 7 d, and 14 d post-treatment, and analysed using real-time PCR. A repeated measurement analysis (Proc GLM) was performed using SAS. If there was no treatment and time interaction, main effects of treatment by time were tested by ANOVA. Results: Electrophoresis data showed that by 7 d post-castration RNA isolated from all the testicle samples of the Burd castrated animals, the epididymis and middle scrotum samples from Band castrates were degraded. Transitory effects were observed in the gene expression of IFN-γ, IL-6, IL-8 and TNF-α at 12 h and 24 h post treatment. Burd castrates had greater (P < 0.05) testicular IFN-γ mRNA levels compared with Band and Con animals, but lower (P < 0.05) testicular TNF-α mRNA levels compared with Con animals. Band castrates had greater (P < 0.05) testicular IL-6 mRNA levels than Burd castrates at 12 h post-castration. Burd castrates had greater (P < 0.05) testicular IL-8 mRNA levels than Band and Con animals at 24 h post-castration. In the epididymis, Burd castrates had greater (P < 0.05) IL-6 mRNA (both at 12 h and 24 h post treatment) and IL-8 mRNA (12 h post treatment) levels compared with Band and Con animals; Burd castrates had greater (P = 0.049) IL-10 mRNA levels than Band castrates at 12 h post-castration. Conclusion: Banding castration caused more inflammatory associated gene expression changes to the epididymis and scrotum than burdizzo. Burdizzo caused more severe acute inflammatory responses, in terms of pro-inflammatory cytokine gene expression, in the testis and epididymis than banding.
    • Variation in the Ovine Abomasal Lymph Node Transcriptome between Breeds Known to Differ in Resistance to the Gastrointestinal Nematode

      Ahmed, Albin M.; Good, Barbara; Hanrahan, James P; McGettigan, Paul; Browne, John A; Keane, Orla M; Bahar, Bojlul; Mehta, Jai; Markey, Bryan; Lohan, Amanda; et al. (PLoS, 2015-05-15)
      Texel lambs are known to be more resistant to gastrointestinal nematode (GIN) infection than Suffolk lambs, with a greater ability to limit infection. The objectives of this study were to: 1) profile the whole transcriptome of abomasal lymph node tissue of GIN-free Texel and Suffolk lambs; 2) identify differentially expressed genes and characterize the immune-related biological pathways and networks associated with these genes. Abomasal lymph nodes were collected from Texel (n = 6) and Suffolk (n = 4) lambs aged 19 weeks that had been GIN-free since 6 weeks of age. Whole transcriptome profiling was performed using RNA-seq on the Illumina platform. At the time of conducting this study, a well annotated Ovine genome was not available and hence the sequence reads were aligned with the Bovine (UMD3.1) genome. Identification of differentially expressed genes was followed by pathway and network analysis. The Suffolk breed accounted for significantly more of the differentially expressed genes, (276 more highly expressed in Suffolk v 162 in Texel; P < 0.001). The four most significant differentially expressed pathways were all related to immunity and were classified as: Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses, Activation of IRF by Cytosolic Pattern Recognition Receptors, Role of RIG-I-like Receptors in Antiviral Innate Immunity, and Interferon Signaling. Of significance is the fact that all of these four pathways were more highly expressed in the Suffolk. These data suggest that in a GIN-free environment, Suffolk lambs have a more active immune profile relative to the Texel: this immune profile may contribute to the poorer efficiency of response to a GIN challenge in the Suffolk breed compared to the Texel breed.