• The application of transcriptomic data in the authentication of beef derived from contrasting production systems

      Sweeney, Torres; Lejeune, Alex; Moloney, Aidan P; 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.
    • Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome.

      Morine, Melissa J; McMonagle, Jolene; Toomey, Sinead; Reynolds, Clare M; Moloney, Aidan P; Gormley, Isobel C; Gaora, Peadar Ó; Roche, Helen M.; Department of Agriculture, Food and the Marine; Irish Research Council for Science, Engineering and Technology; Science Foundation Ireland; 5254 (Biomed Central, 2010-10-07)
      Background Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Results Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p < 0.05), followed by muscle (601 genes) and adipose (16 genes). Results from modified GSEA showed that the high-CLA beef diet affected diverse biological processes across the three tissues, and that the majority of pathway changes reached significance only with the bi-directional test. Combining the liver tissue microarray results with plasma marker data revealed 110 CLA-sensitive genes showing strong canonical correlation with one or more plasma markers of metabolic health, and 9 significantly overrepresented pathways among this set; each of these pathways was also significantly changed by the high-CLA diet. Closer inspection of two of these pathways - selenoamino acid metabolism and steroid biosynthesis - illustrated clear diet-sensitive changes in constituent genes, as well as strong correlations between gene expression and plasma markers of metabolic syndrome independent of the dietary effect. Conclusion Bi-directional gene set enrichment analysis more accurately reflects dynamic regulatory behaviour in biochemical pathways, and as such highlighted biologically relevant changes that were not detected using a traditional approach. In such cases where transcriptomic response to treatment is exceptionally large, canonical correlation analysis in conjunction with Fisher's exact test highlights the subset of pathways showing strongest correlation with the clinical markers of interest. In this case, we have identified selenoamino acid metabolism and steroid biosynthesis as key pathways mediating the observed relationship between metabolic health and high-CLA beef. These results indicate that this type of analysis has the potential to generate novel transcriptome-based biomarkers of disease.
    • Characterisation and expression profile of the bovine cathelicidin gene repertoire in mammary tissue

      Whelehan, Cormac J; Barry-Reidy, Anne; Meade, Kieran G; Eckersall, P David; Chapwanya, Aspinas; Narciandi, Fernando; Lloyd, Andrew T; O'Farrelly, Cliona; Department of Agriculture, Food and the Marine (Biomed Central, 2014-02-13)
      Abstract Background Cathelicidins comprise a major group of host-defence peptides. Conserved across a wide range of species, they have several functions related to host defence. Only one cathelicidin has been found in humans but several cathelicidin genes occur in the bovine genome. We propose that these molecules may have a protective role against mastitis. The aim of this study was to characterise the cathelicidin gene-cluster in the bovine genome and to identify sites of expression in the bovine mammary gland. Results Bioinformatic analysis of the bovine genome (BosTau7) revealed seven protein-coding cathelicidin genes, CATHL1-7, including two identical copies of CATHL4, as well as three additional putative cathelicidin genes, all clustered on the long arm of chromosome 22. Six of the seven protein-coding genes were expressed in leukocytes extracted from milk of high somatic cell count (SCC) cows. CATHL5 was expressed across several sites in the mammary gland, but did not increase in response to Staphylococcus aureus infection. Conclusions Here, we characterise the bovine cathelicidin gene cluster and reconcile inconsistencies in the datasets of previous studies. Constitutive cathelicidin expression in the mammary gland suggests a possible role for these host defence peptides its protection. Background Cathelicidins comprise a major group of host-defence peptides. Conserved across a wide range of species, they have several functions related to host defence. Only one cathelicidin has been found in humans but several cathelicidin genes occur in the bovine genome. We propose that these molecules may have a protective role against mastitis. The aim of this study was to characterise the cathelicidin gene-cluster in the bovine genome and to identify sites of expression in the bovine mammary gland. Results Bioinformatic analysis of the bovine genome (BosTau7) revealed seven protein-coding cathelicidin genes, CATHL1-7, including two identical copies of CATHL4, as well as three additional putative cathelicidin genes, all clustered on the long arm of chromosome 22. Six of the seven protein-coding genes were expressed in leukocytes extracted from milk of high somatic cell count (SCC) cows. CATHL5 was expressed across several sites in the mammary gland, but did not increase in response to Staphylococcus aureus infection. Conclusions Here, we characterise the bovine cathelicidin gene cluster and reconcile inconsistencies in the datasets of previous studies. Constitutive cathelicidin expression in the mammary gland suggests a possible role for these host defence peptides its protection.
    • Comparative genomic identification and validation of β-defensin genes in the Ovis aries genome

      Hall, T. J; McQuillan, C.; Finlay, E. K; O’Farrelly, C.; Fair, S.; Meade, Kieran G.; Department of Agriculture, Food and the Marine; 11/S/104 (Biomed Central, 2017-04-04)
      Background β-defensins are small, cationic, antimicrobial peptides found in species across the plant and animal kingdoms. In addition to microbiocidal activity, roles in immunity as well as reproduction have more recently been documented. β-defensin genes in Ovis aries (domestic sheep) have been poorly annotated, having been identified only by automatic gene prediction algorithms. The objective of this study was to use a comparative genomics approach to identify and characterise the β-defensin gene repertoire in sheep using the bovine genome as the primary reference. Results All 57 currently predicted bovine β-defensin genes were used to find orthologous sequences in the most recent version of the sheep genome (OAR v4.0). Forty three genes were found to have close genomic matches (>70% similarity) between sheep and cattle. The orthologous genes were located in four clusters across the genome, with 4 genes on chromosome 2, 19 genes on chromosome 13, 5 genes on chromosome 20 and 15 genes on chromosome 26. Conserved gene order for the β-defensin genes was apparent in the two smaller clusters, although gene order was reversed on chromosome 2, suggesting an inversion between sheep and cattle. Complete conservation of gene order was also observed for chromosome 13 β-defensin orthologs. More structural differences were apparent between chromosome 26 genes and the orthologous region in the bovine reference genome, which is known to be copy-number variable. In this cluster, the Defensin-beta 1 (DEFB1) gene matched to eleven Bovine Neutrophil beta-Defensin (BNBD) genes on chromosome 27 with almost uniform similarity, as well as to tracheal, enteric and lingual anti-microbial peptides (TAP, EAP and LAP), suggesting that annotation of the bovine reference sequence is still incomplete. qPCR was used to profile the expression of 34 β-defensin genes, representing each of the four clusters, in the ram reproductive tract. Distinct site-specific and differential expression profiles were detected across the reproductive tract of mature rams with preferential β-defensin gene expression in the epididymis, recapitulating observations for orthologous genes in other species. Conclusions This is the first comprehensive analysis of β-defensin genes encoded by the ovine reference sequence, and the first report of an expanded repertoire of β-defensin genes in this species. The preferential expression of these genes in the epididymis suggests a role in fertility, possibly providing immunoprotection for sperm within the female reproductive tract.
    • Delaying pigs from the normal production flow is associated with health problems and poorer performance

      Calderón Díaz, Julia A.; Diana, Alessia; Boyle, Laura; Leonard, Finola C; McElroy, Máire; McGettrick, Shane; Moriarty, John; García Manzanilla, Edgar; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 14/S/832 (Biomed Central, 2017-07-05)
      Background Delaying pigs from advancing through the production stages could have a negative impact on their health and performance. The objective of this study was to investigate the possible implications of delaying pigs from the normal production flow on pig health and performance in a farrow-to-finish commercial farm with a self-declared All-In/All-Out (AIAO) management. Results Three flows of pigs were defined, flow 1 (i.e. pigs that followed the normal production flow; 8 weeks in the nursery stage, 4 weeks in the growing stage and 8 weeks in the finisher stage), flow 2 (i.e. pigs delayed 1 week from advancing to the next production stage) and flow 3 (i.e. pigs delayed >1 week from advancing to the next production stage). Flow 3 included higher proportions of pigs from first parity sows and of lighter birth weights. When the 3 flows were matched by parity and birth weight, pigs in flow 2 were 3.8 times more likely to be lame prior to slaughter compared with pigs in flow 1. Similarly, pigs in flow 3 were more likely to be lame prior to slaughter, 4.5 times more likely to present pleurisy, 3.3 times more like to present pericarditis and 4.3 times more likely to have their heart condemned at slaughter compared with pigs in flow 1. Additionally, carcasses from pigs in flow 3 were 10 kg lighter compared with carcasses from pigs in flow 1. Conclusion Delayed pigs were more affected by disease and were lighter at slaughter. Besides animal welfare issues, these findings could represent considerable economic loses for pig producers. In practice, delaying pigs from the normal production flow translates into higher feeding costs, increase number of days to slaughter and increased labour requirements reducing production efficiency for the pig operation. In farrow-to-finish farms an ‘all-forward’ policy (i.e. no pig is left behind from stage to stage and a split marketing approach is applied when sending pigs to slaughter) might be more easily adhered to.
    • Detection of selection signatures in dairy and beef cattle using high-density genomic information

      Zhao, Fuping; McParland, Sinead; Kearney, Francis; Du, Lixin; Berry, Donagh P.; Department of Agriculture, Food and the Marine; Agricultural Science and Technology Innovation Program; Natural Science Foundation of China; 11/S/112; ASTIP-IAS-TS-6 (Biomed Central, 2015-06-19)
      Background Artificial selection for economically important traits in cattle is expected to have left distinctive selection signatures on the genome. Access to high-density genotypes facilitates the accurate identification of genomic regions that have undergone positive selection. These findings help to better elucidate the mechanisms of selection and to identify candidate genes of interest to breeding programs. Results Information on 705 243 autosomal single nucleotide polymorphisms (SNPs) in 3122 dairy and beef male animals from seven cattle breeds (Angus, Belgian Blue, Charolais, Hereford, Holstein-Friesian, Limousin and Simmental) were used to detect selection signatures by applying two complementary methods, integrated haplotype score (iHS) and global fixation index (FST). To control for false positive results, we used false discovery rate (FDR) adjustment to calculate adjusted iHS within each breed and the genome-wide significance level was about 0.003. Using the iHS method, 83, 92, 91, 101, 85, 101 and 86 significant genomic regions were detected for Angus, Belgian Blue, Charolais, Hereford, Holstein-Friesian, Limousin and Simmental cattle, respectively. None of these regions was common to all seven breeds. Using the FST approach, 704 individual SNPs were detected across breeds. Annotation of the regions of the genome that showed selection signatures revealed several interesting candidate genes i.e. DGAT1, ABCG2, MSTN, CAPN3, FABP3, CHCHD7, PLAG1, JAZF1, PRKG2, ACTC1, TBC1D1, GHR, BMP2, TSG1, LYN, KIT and MC1R that play a role in milk production, reproduction, body size, muscle formation or coat color. Fifty-seven common candidate genes were found by both the iHS and global FST methods across the seven breeds. Moreover, many novel genomic regions and genes were detected within the regions that showed selection signatures; for some candidate genes, signatures of positive selection exist in the human genome. Multilevel bioinformatic analyses of the detected candidate genes suggested that the PPAR pathway may have been subjected to positive selection. Conclusions This study provides a high-resolution bovine genomic map of positive selection signatures that are either specific to one breed or common to a subset of the seven breeds analyzed. Our results will contribute to the detection of functional candidate genes that have undergone positive selection in future studies.
    • DNA sequence polymorphisms in a panel of eight candidate bovine imprinted genes and their association with performance traits in Irish Holstein-Friesian cattle

      Magee, David A; Sikora, Klaudia M; Berkowicz, Erik W; Berry, Donagh P.; Howard, Dawn J; Mullen, Michael Paul; Evans, Ross D; Spillane, Charles; MacHugh, David E; Department of Agriculture, Food and the Marine; Science Foundation Ireland; RSF-06-406; RSF-06-0353; RSF-06-0409; SFI/01/F.1/B028; SFI/08/IN.1/B1931; 07/SRC/B1156 (Biomed Central, 2010-10-13)
      Background: Studies in mice and humans have shown that imprinted genes, whereby expression from one of the two parentally inherited alleles is attenuated or completely silenced, have a major effect on mammalian growth, metabolism and physiology. More recently, investigations in livestock species indicate that genes subject to this type of epigenetic regulation contribute to, or are associated with, several performance traits, most notably muscle mass and fat deposition. In the present study, a candidate gene approach was adopted to assess 17 validated single nucleotide polymorphisms (SNPs) and their association with a range of performance traits in 848 progeny-tested Irish Holstein-Friesian artificial insemination sires. These SNPs are located proximal to, or within, the bovine orthologs of eight genes (CALCR, GRB10, PEG3, PHLDA2, RASGRF1, TSPAN32, ZIM2 and ZNF215) that have been shown to be imprinted in cattle or in at least one other mammalian species (i.e. human/mouse/pig/sheep). Results: Heterozygosities for all SNPs analysed ranged from 0.09 to 0.46 and significant deviations from Hardy-Weinberg proportions (P ≤ 0.01) were observed at four loci. Phenotypic associations (P ≤ 0.05) were observed between nine SNPs proximal to, or within, six of the eight analysed genes and a number of performance traits evaluated, including milk protein percentage, somatic cell count, culled cow and progeny carcass weight, angularity, body conditioning score, progeny carcass conformation, body depth, rump angle, rump width, animal stature, calving difficulty, gestation length and calf perinatal mortality. Notably, SNPs within the imprinted paternally expressed gene 3 (PEG3) gene cluster were associated (P ≤ 0.05) with calving, calf performance and fertility traits, while a single SNP in the zinc finger protein 215 gene (ZNF215) was associated with milk protein percentage (P ≤ 0.05), progeny carcass weight (P ≤ 0.05), culled cow carcass weight (P ≤ 0.01), angularity (P ≤ 0.01), body depth (P ≤ 0.01), rump width (P ≤ 0.01) and animal stature (P ≤ 0.01). Conclusions: Of the eight candidate bovine imprinted genes assessed, DNA sequence polymorphisms in six of these genes (CALCR, GRB10, PEG3, RASGRF1, ZIM2 and ZNF215) displayed associations with several of the phenotypes included for analyses. The genotype-phenotype associations detected here are further supported by the biological function of these six genes, each of which plays important roles in mammalian growth, development and physiology. The associations between SNPs within the imprinted PEG3 gene cluster and traits related to calving, calf performance and gestation length suggest that this domain on chromosome 18 may play a role regulating pre-natal growth and development and fertility. SNPs within the bovine ZNF215 gene were associated with bovine growth and body conformation traits and studies in humans have revealed that the human ZNF215 ortholog belongs to the imprinted gene cluster associated with Beckwith-Wiedemann syndrome--a genetic disorder characterised by growth abnormalities. Similarly, the data presented here suggest that the ZNF215 gene may have an important role in regulating bovine growth. Collectively, our results support previous work showing that (candidate) imprinted genes/loci contribute to heritable variation in bovine performance traits and suggest that DNA sequence polymorphisms within these genes/loci represents an important reservoir of genomic markers for future genetic improvement of dairy and beef cattle populations.
    • Evidence for genetic variance in resistance to tuberculosis in Great Britain and Irish Holstein-Friesian populations

      Bermingham, Mairead L; Brotherstone, Susan; Berry, Donagh P.; More, Simon J; Good, Margaret; Cromie, Andrew R; White, Ian MS; Higgins, Isabella; Coffey, Mike; Downs, Sara H; Glass, Elizabeth J; Bishop, Stephen C; Mitchell, Andy P; Clifton-Hadley, Richard S; Woolliams, John A; Welsh Assembly Government; Department of Agriculture, Food and the Marine; Eradication of Animal Disease Board; Biotechnology and Biological Sciences Research Council; The Roslin Institute Strategic Programme.; BB/E018335/2 (Biomed Central, 2011-06-03)
      Background: Here, we jointly summarise scientific evidence for genetic variation in resistance to infection with Mycobacterium bovis, the primary agent of bovine tuberculosis (TB), provided by two recent and separate studies of Holstein-Friesian dairy cow populations in Great Britain (GB) and Ireland. Methods: The studies quantified genetic variation within archived data from field and abattoir surveillance control programmes within each country. These data included results from the single intradermal comparative tuberculin test (SICTT), abattoir inspection for TB lesions and laboratory confirmation of disease status. Threshold animal models were used to estimate variance components for responsiveness to the SICTT and abattoir confirmed M. bovis infection. The link functions between the observed 0/1 scale and the liability scale were the complementary log-log in the GB, and logit link function in the Irish population. Results and discussion: The estimated heritability of susceptibility to TB, as judged by responsiveness to the SICTT, was 0.16 (0.012) and 0.14 (0.025) in the GB and Irish populations, respectively. For abattoir or laboratory confirmation of infection, estimates were 0.18 (0.044) and 0.18 (0.041) from the GB and the Irish populations, respectively. Conclusions: Estimates were all significantly different from zero and indicate that exploitable variation exists among GB and Irish Holstein Friesian dairy cows for resistance to TB. Epidemiological analysis suggests that factors such as variation in exposure or imperfect sensitivity and specificity would have resulted in underestimation of the true values.
    • Evolution of gut microbiota composition from birth to 24 weeks in the INFANTMET Cohort

      Hill, Cian J; Lynch, Denise B; Murphy, Kiera; Ulaszewska, Marynka; Jeffery, Ian B; O’Shea, Carol A; Watkins, Claire; Dempsey, Eugene; Mattivi, Fulvio; Tuohy, Kieran; Ross, R Paul; Ryan, C. A; O’ Toole, Paul W; Stanton, Catherine; Department of Agriculture, Food and the Marine; Health Research Board; APC Microbiome Institute; BIO-IT Platform; HRA_POR/2012/123 (Biomed Central, 17/01/2017)
      Background The gut is the most extensively studied niche of the human microbiome. The aim of this study was to characterise the initial gut microbiota development of a cohort of breastfed infants (n = 192) from 1 to 24 weeks of age. Methods V4-V5 region 16S rRNA amplicon Illumina sequencing and, in parallel, bacteriological culture. The metabolomic profile of infant urine at 4 weeks of age was also examined by LC-MS. Results Full-term (FT), spontaneous vaginally delivered (SVD) infants’ microbiota remained stable at both phylum and genus levels during the 24-week period examined. FT Caesarean section (CS) infants displayed an increased faecal abundance of Firmicutes (p < 0.01) and lower abundance of Actinobacteria (p < 0.001) after the first week of life compared to FT-SVD infants. FT-CS infants gradually progressed to harbouring a microbiota closely resembling FT-SVD (which remained stable) by week 8 of life, which was maintained at week 24. The gut microbiota of preterm (PT) infants displayed a significantly greater abundance of Proteobacteria compared to FT infants (p < 0.001) at week 1. Metabolomic analysis of urine at week 4 indicated PT-CS infants have a functionally different metabolite profile than FT (both CS and SVD) infants. Co-inertia analysis showed co-variation between the urine metabolome and the faecal microbiota of the infants. Tryptophan and tyrosine metabolic pathways, as well as fatty acid and bile acid metabolism, were found to be affected by delivery mode and gestational age. Conclusions These findings confirm that mode of delivery and gestational age both have significant effects on early neonatal microbiota composition. There is also a significant difference between the metabolite profile of FT and PT infants. Prolonged breastfeeding was shown to have a significant effect on the microbiota composition of FT-CS infants at 24 weeks of age, but interestingly not on that of FT-SVD infants. Twins had more similar microbiota to one another than between two random infants, reflecting the influence of similarities in both host genetics and the environment on the microbiota.
    • Genetic basis of benzimidazole resistance in Teladorsagia circumcincta in Ireland

      Keegan, Jason D; Good, Barbara; de Waal, Theo; Fanning, June; Keane, Orla M; Department of Agriculture, Food and the Marine (Biomed Central, 2017-02-13)
      Resistance to benzimidazole (BZ) anthelmintics is common in ovine nematodes of economic importance. Single nucleotide polymorphisms (SNP) at three positions in the isotype 1 β– tubulin gene have been associated with BZ resistance and molecular tests for the detection of BZ resistance have been developed. In order to determine if such tests are practicable in Ireland the polymorphisms associated with BZ resistance must be identified. To this end, BZ-resistant nematodes were recovered from four farms in Ireland. Resistant Teladorsagia circumcincta, Cooperia curticei and Trichostrongylus colubriformis were recovered, with resistant T. circumcincta the most common and the only species studied further. Sequencing of the isotype 1 β–tubulin gene from resistant T. circumcincta identified a T - A transition, resulting in an F200Y substitution known to be responsible for BZ-resistance, on three of the farms. However, on the fourth farm the frequency of the resistant A allele was only 0.33 indicating another BZ resistance mechanism may be present on this farm. An additional polymorphism resulting in a substitution of glutamate for leucine (E198L) was also found on this farm at low frequency (0.17). No polymorphisms at position 167 were identified on any farm. Therefore, molecular tests to detect BZ resistance in T. circumcincta in Ireland could prove useful; however, they may result in some instances of resistance remaining undetected.
    • Genome-wide association study for calving performance using high-density genotypes in dairy and beef cattle

      Purfield, Deirdre C; Bradley, Daniel G; Evans, Ross D; Kearney, Francis J; Berry, Donagh P.; Science Foundation Ireland; Department of Agriculture, Food and the Marine; 09/IN.1/B2642; RSF 11/S/112 (Biomed Central, 2015-06-12)
      Background Calving difficulty and perinatal mortality are prevalent in modern-day cattle production systems. It is well-established that there is a genetic component to both traits, yet little is known about their underlying genomic architecture, particularly in beef breeds. Therefore, we performed a genome-wide association study using high-density genotypes to elucidate the genomic architecture of these traits and to identify regions of the bovine genome associated with them. Results Genomic regions associated with calving difficulty (direct and maternal) and perinatal mortality were detected using two statistical approaches: (1) single-SNP (single nucleotide polymorphism) regression and (2) a Bayesian approach. Data included high-density genotypes on 770 Holstein-Friesian, 927 Charolais and 963 Limousin bulls. Several novel or previously identified genomic regions were detected but associations differed by breed. For example, two genomic associations, one each on chromosomes 18 and 2 explained 2.49 % and 3.13 % of the genetic variance in direct calving difficulty in the Holstein-Friesian and Charolais populations, respectively. Imputed Holstein-Friesian sequence data was used to refine the genomic regions responsible for significant associations. Several candidate genes on chromosome 18 were identified and four highly significant missense variants were detected within three of these genes (SIGLEC12, CTU1, and ZNF615). Nevertheless, only CTU1 contained a missense variant with a putative impact on direct calving difficulty based on SIFT (0.06) and Polyphen (0.95) scores. Using imputed sequence data, we refined a genomic region on chromosome 4 associated with maternal calving difficulty in the Holstein-Friesian population and found the strongest association with an intronic variant in the PCLO gene. A meta-analysis was performed across the three breeds for each calving performance trait to identify common variants associated with these traits in the three breeds. Our results suggest that a portion of the genetic variation in calving performance is common to all three breeds. Conclusion The genomic architecture of calving performance is complex and mainly influenced by many polymorphisms of small effect. We identified several associations of moderate effect size but the majority were breed-specific, indicating that breed-specific alleles exist for calving performance or that the linkage phase between genotyped allele and causal mutation varies between breeds.
    • A genome-wide association study for genetic susceptibility to Mycobacterium bovis infection in dairy cattle identifies a susceptibility QTL on chromosome 23

      Richardson, Ian W.; Berry, Donagh P.; Wiencko, Heather L; Higgins, Isabella M; More, Simon J; McClure, Jennifer; Lynn, David J; Bradley, Daniel G; Science Foundation Ireland; Department of Agriculture, Food and the Marine; 09/IN.1/B2642; RSF 11/S/112 (Biomed Central, 2016-03-09)
      Background Bovine tuberculosis (bTB) infection in cattle is a significant economic concern in many countries, with annual costs to the UK and Irish governments of approximately €190 million and €63 million, respectively, for bTB control. The existence of host additive and non-additive genetic components to bTB susceptibility has been established. Methods Two approaches i.e. single-SNP (single nucleotide polymorphism) regression and a Bayesian method were applied to genome-wide association studies (GWAS) using high-density SNP genotypes (n = 597,144 SNPs) from 841 dairy artificial insemination (AI) sires. Deregressed estimated breeding values for bTB susceptibility were used as the quantitative dependent variable. Network analysis was performed using the quantitative trait loci (QTL) that were identified as significant in the single-SNP regression and Bayesian analyses separately. In addition, an identity-by-descent analysis was performed on a subset of the most prolific sires in the dataset that showed contrasting prevalences of bTB infection in daughters. Results A significant QTL region was identified on BTA23 (P value >1 × 10−5, Bayes factor >10) across all analyses. Sires with the minor allele (minor allele frequency = 0.136) for this QTL on BTA23 had estimated breeding values that conferred a greater susceptibility to bTB infection than those that were homozygous for the major allele. Imputation of the regions that flank this QTL on BTA23 to full sequence indicated that the most significant associations were located within introns of the FKBP5 gene. Conclusions A genomic region on BTA23 that is strongly associated with host susceptibility to bTB infection was identified. This region contained FKBP5, a gene involved in the TNFα/NFκ-B signalling pathway, which is a major biological pathway associated with immune response. Although there is no study that validates this region in the literature, our approach represents one of the most powerful studies for the analysis of bTB susceptibility to date.
    • Genome-wide transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis reveals suppression of host immune genes

      Killick, Kate E; Browne, John A; Park, Stephen D.; Magee, David A; Martin, Irene; Meade, Kieran G; Gordon, Stephen V; Gormley, Eamonn; O'Farrelly, Cliona; Hokamp, Karsten; MacHugh, David E; Science Foundation Ireland; Department of Agriculture, Food and the Marine; European Union; Irish Research Council for Science, Engineering and Technology; SFI/01/F.1/B028; SFI/08/IN.1/B2038; RSF 06 405; KBBE-211602-MACROSYS (Biomed Central, 2011-12-19)
      Background: Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), a pathological infection with significant economic impact. Recent studies have highlighted the role of functional genomics to better understand the molecular mechanisms governing the host immune response to M. bovis infection. Furthermore, these studies may enable the identification of novel transcriptional markers of BTB that can augment current diagnostic tests and surveillance programmes. In the present study, we have analysed the transcriptome of peripheral blood leukocytes (PBL) from eight M. bovis-infected and eight control non-infected age-matched and sex-matched Holstein-Friesian cattle using the Affymetrix® GeneChip® Bovine Genome Array with 24,072 gene probe sets representing more than 23,000 gene transcripts. Results: Control and infected animals had similar mean white blood cell counts. However, the mean number of lymphocytes was significantly increased in the infected group relative to the control group (P = 0.001), while the mean number of monocytes was significantly decreased in the BTB group (P = 0.002). Hierarchical clustering analysis using gene expression data from all 5,388 detectable mRNA transcripts unambiguously partitioned the animals according to their disease status. In total, 2,960 gene transcripts were differentially expressed (DE) between the infected and control animal groups (adjusted P-value threshold ≤ 0.05); with the number of gene transcripts showing decreased relative expression (1,563) exceeding those displaying increased relative expression (1,397). Systems analysis using the Ingenuity® Systems Pathway Analysis (IPA) Knowledge Base revealed an over-representation of DE genes involved in the immune response functional category. More specifically, 64.5% of genes in the affects immune response subcategory displayed decreased relative expression levels in the infected animals compared to the control group. Conclusions: This study demonstrates that genome-wide transcriptional profiling of PBL can distinguish active M. bovis-infected animals from control non-infected animals. Furthermore, the results obtained support previous investigations demonstrating that mycobacterial infection is associated with host transcriptional suppression. These data support the use of transcriptomic technologies to enable the identification of robust, reliable transcriptional markers of active M. bovis infection.
    • Genome-wide transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis reveals suppression of host immune genes

      Killick, Kate E; Browne, John A; Park, Stephen D; Magee, David A; Martin, Irene; Meade, Kieran G; Gordon, Stephen V; Gormley, Eamonn; O'Farrelly, Cliona; Hokamp, Karsten; MacHugh, David E; Department of Agriculture, Food and the Marine; European Union; Science Foundation Ireland; Irish Research Council for Science, Engineering and Technology; RSF 06 405; KBBE-211602-MACROSYS; SFI/01/F.1/B028; SFI/08/IN.1/B2038 (2011-12-19)
      Background Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), a pathological infection with significant economic impact. Recent studies have highlighted the role of functional genomics to better understand the molecular mechanisms governing the host immune response to M. bovis infection. Furthermore, these studies may enable the identification of novel transcriptional markers of BTB that can augment current diagnostic tests and surveillance programmes. In the present study, we have analysed the transcriptome of peripheral blood leukocytes (PBL) from eight M. bovis-infected and eight control non-infected age-matched and sex-matched Holstein-Friesian cattle using the Affymetrix® GeneChip® Bovine Genome Array with 24,072 gene probe sets representing more than 23,000 gene transcripts. Results Control and infected animals had similar mean white blood cell counts. However, the mean number of lymphocytes was significantly increased in the infected group relative to the control group (P = 0.001), while the mean number of monocytes was significantly decreased in the BTB group (P = 0.002). Hierarchical clustering analysis using gene expression data from all 5,388 detectable mRNA transcripts unambiguously partitioned the animals according to their disease status. In total, 2,960 gene transcripts were differentially expressed (DE) between the infected and control animal groups (adjusted P-value threshold ≤ 0.05); with the number of gene transcripts showing decreased relative expression (1,563) exceeding those displaying increased relative expression (1,397). Systems analysis using the Ingenuity® Systems Pathway Analysis (IPA) Knowledge Base revealed an over-representation of DE genes involved in the immune response functional category. More specifically, 64.5% of genes in the affects immune response subcategory displayed decreased relative expression levels in the infected animals compared to the control group. Conclusions This study demonstrates that genome-wide transcriptional profiling of PBL can distinguish active M. bovis-infected animals from control non-infected animals. Furthermore, the results obtained support previous investigations demonstrating that mycobacterial infection is associated with host transcriptional suppression. These data support the use of transcriptomic technologies to enable the identification of robust, reliable transcriptional markers of active M. bovis infection.
    • High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

      O'Sullivan, Daniel J.; Fallico, Vincenzo; O’Sullivan, Orla; McSweeney, Paul L H; Sheehan, Jeremiah J; Cotter, Paul D.; Giblin, Linda; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine; 2012205 (Biomed Central, 17/11/2015)
      Background: The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. Results: Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. Conclusions: In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.
    • Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue

      Johnston, Dayle; Earley, Bernadette; Cormican, Paul; Murray, Gerard; Kenny, David A; Waters, Sinead M.; McGee, Mark; Kelly, Alan K; McCabe, Matthew S; Department of Agriculture, Food and the Marine; European Union; 11/S/131; 311,825 (Biomed Central, 2017-05-02)
      Background Bovine respiratory disease (BRD) is caused by growth of single or multiple species of pathogenic bacteria in lung tissue following stress and/or viral infection. Next generation sequencing of 16S ribosomal RNA gene PCR amplicons (NGS 16S amplicon analysis) is a powerful culture-independent open reference method that has recently been used to increase understanding of BRD-associated bacteria in the upper respiratory tract of BRD cattle. However, it has not yet been used to examine the microbiome of the bovine lower respiratory tract. The objective of this study was to use NGS 16S amplicon analysis to identify bacteria in post-mortem lung and lymph node tissue samples harvested from fatal BRD cases and clinically healthy animals. Cranial lobe and corresponding mediastinal lymph node post-mortem tissue samples were collected from calves diagnosed as BRD cases by veterinary laboratory pathologists and from clinically healthy calves. NGS 16S amplicon libraries, targeting the V3-V4 region of the bacterial 16S rRNA gene were prepared and sequenced on an Illumina MiSeq. Quantitative insights into microbial ecology (QIIME) was used to determine operational taxonomic units (OTUs) which corresponded to the 16S rRNA gene sequences. Results Leptotrichiaceae, Mycoplasma, Pasteurellaceae, and Fusobacterium were the most abundant OTUs identified in the lungs and lymph nodes of the calves which died from BRD. Leptotrichiaceae, Fusobacterium, Mycoplasma, Trueperella and Bacteroides had greater relative abundances in post-mortem lung samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Leptotrichiaceae, Mycoplasma and Pasteurellaceae showed higher relative abundances in post-mortem lymph node samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Two Leptotrichiaceae sequence contigs were subsequently assembled from bacterial DNA-enriched shotgun sequences. Conclusions The microbiomes of the cranial lung lobe and mediastinal lymph node from calves which died from BRD and from clinically healthy H-F calves have been characterised. Contigs corresponding to the abundant Leptotrichiaceae OTU were sequenced and found not to be identical to any known bacterial genus. This suggests that we have identified a novel bacterial species associated with BRD.
    • Inter- and intra-reproducibility of genotypes from sheep technical replicates on Illumina and Affymetrix platforms

      Berry, Donagh P.; O’Brien, Aine; Wall, Eamonn; McDermott, Kevin; Randles, Shane; Flynn, Paul; Park, Stephen; Grose, Jenny; Weld, Rebecca; McHugh, Noirin; Department of Agriculture, Food and the Marine; International Sheep Genomics Consortium (Biomed Central, 2016-11-10)
      Background Accurate genomic analyses are predicated upon access to accurate genotype input data. The objective of this study was to quantify the reproducibility of genotype data that are generated from the same genotype platform and from different genotyping platforms. Methods Genotypes based on 51,121 single nucleotide polymorphisms (SNPs) for 84 animals that were each genotyped on Illumina and Affymetrix platforms and for another 25 animals that were each genotyped twice on the same Illumina platform were compared. Genotypes based on 11,323 SNPs for an additional 21 animals that were genotyped on two different Illumina platforms by two different service providers were also compared. Reproducibility of the results was measured as the correlation between allele counts and as genotype and allele concordance rates. Results A mean within-animal correlation of 0.9996 was found between allele counts in the 25 duplicate samples that were genotyped on the same Illumina platform and varied from 0.9963 to 1.0000 per animal. The mean (minimum, maximum) genotype and allele concordance rates per animal between the 25 duplicate samples were equal to 0.9996 (0.9968, 1.0000) and 0.9993 (0.9937, 1.0000), respectively. The concordance rate between the two different Illumina platforms was also near 1. A mean within-animal correlation of 0.9738 was found between genotypes that were generated on the Illumina and Affymetrix platforms and varied from 0.9505 to 0.9812 per animal. The mean (minimum, maximum) within-animal genotype and allele concordance rates between the Illumina and Affymetrix platforms were equal to 0.9711 (0.9418, 0.9798) and 0.9845 (0.9695, 0.9889), respectively. The genotype concordance rate across all genotypes increased from 0.9711 to 0.9949 when the SNPs used were restricted to those with three high-resolution genotype clusters which represented 75.2% of the called genotypes. Conclusions and implications Our results suggest that, regardless of the genotype platform or service provider, high genotype concordance rates are achieved especially if they are restricted to high-quality extracted DNA and SNPs that result in high-quality genotypes.
    • A nationwide survey of anthelmintic treatment failure on sheep farms in Ireland

      Keegan, Jason D; Keane, Orla M; Good, Barbara; De Waal, Theo; Denny, Marian; Hanrahan, James P; Fitzgerald, William; Sheehan, Maresa; Department of Agriculture, Food and the Marine (Biomed Central, 2017-02-09)
      Background Between 2013 and 2015 the Department of Agriculture, Food and the Marine (DAFM) administered a sheep technology adoption programme (STAP), with the aim of increasing profitability on Irish sheep farms by encouraging the adoption of best management practices. One of the options available to STAP participants was to test the efficacy of the anthelmintic treatment (benzimadazole, levamisole or macrocyclic lactone) used in their flocks by means of a drench test, which is a modification of the faecal egg count reduction test; individual faecal samples were collected from the same group of lambs before and after anthelmintic treatment, the number of eggs present pre and post treatment was subsequently determined from a pooled sample. Results In total, 4211 drench tests were undertaken by farmers during the 3 years of the programme. Information on the anthelmintic product used was available for 3771 of these tests; anthelmintics from the classes benzimidazole (BZ), levamisole (LV) and macrocyclic lactone (ML) (avermectins (AVM) plus moxidectin (MOX)) were used in 42.0%, 23.4% and 32.5% of tests, respectively. The remaining 2.1% of tests involved an inappropriate product. The efficacy of treatment against ‘other trichostrongyles’ (excluding Nematodirus spp and Strongyloides papillosus.) could be established for 1446 tests, and 51% of these tests were considered effective (i.e. a reduction of faecal egg count (FEC) ≥ 95%). There was a significant difference among the drug groups in efficacy; 31.5%, 51.9%, 62.5% and 84% of treatments were considered effective for BZ, LV, AVM, MOX, respectively. The efficacy of treatment against Nematodirus spp. could be established for 338 tests and the overall efficacy was 96%. Conclusions Due to the significant difference among the anthelmintic classes for efficacy against ‘other trichostrongyles’ along with the high level of efficacy against Nematodirus spp., a genus for which anthelmintic resistance is rarely reported, it is concluded that anthelmintic resistance was responsible for the majority of the anthelmintic treatment failures observed.
    • Pig producer perspectives on the use of meat inspection as an animal health and welfare diagnostic tool in the Republic of Ireland and Northern Ireland

      Devitt, Catherine; Boyle, Laura; Teixeira, D. L; O’Connell, N. E; Hawe, M.; Hanlon, Alison; Department of Agriculture, Food and the Marine; RSF 11/S/107 (Biomed Central, 2016-02-09)
      Background Currently, there is growing interest in developing ante and post mortem meat inspection (MI) to incorporate measures of pig health and welfare for use as a diagnostic tool on pig farms. However, the success of the development of the MI process requires stakeholder engagement with the process. Knowledge gaps and issues of trust can undermine the effective exchange and utilisation of information across the supply chain. A social science research methodology was employed to establish stakeholder perspectives towards the development of MI to include measures of pig health and welfare. In this paper the findings of semi-structured telephone interviews with 18 pig producers from the Republic of Ireland and Northern Ireland are presented. Results Producers recognised the benefit of the utilisation of MI data as a health and welfare diagnostic tool. This acknowledgment, however, was undermined for some by dissatisfaction with the current system of MI information feedback, by trust and fairness concerns, and by concerns regarding the extent to which data would be used in the producers’ interests. Tolerance of certain animal welfare issues may also have a negative impact on how producers viewed the potential of MI data. The private veterinary practitioner was viewed as playing a vital role in assisting them with the interpretation of MI data for herd health planning. Conclusions The development of positive relationships based on trust, commitment and satisfaction across the supply chain may help build a positive environment for the effective utilisation of MI data in improving pig health and welfare. The utilisation of MI as a diagnostic tool would benefit from the development of a communication strategy aimed at building positive relationships between stakeholders in the pig industry.
    • Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples.

      Mullen, Michael Paul; Creevey, Christopher J.; Berry, Donagh P.; McCabe, Matthew; Magee, David A; Howard, Dawn J; Killeen, Aideen P.; Park, Stephen D.; McGettigan, Paul A.; Lucy, Matt C.; MacHugh, David E; Waters, Sinead M.; Science Foundation Ireland; Science Foundation Ireland (SFI) Stokes lecturer scheme; Department of Agriculture, Food and the Marine; 07/SRC/B1156; 07/SK/B1236A; RSF-06-0353; RSF-06-0409 (Biomed Central, 2012-01-11)
      Background: The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. Results: In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). Conclusions: The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait.