• 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.
    • Anthelmintic-resistant nematodes in Irish commercial sheep flocks- the state of play

      Good, Barbara; Hanrahan, James P; de Waal, Daniel Theodorus; Patten, Thomas; Kinsella, Andrew; Lynch, Ciaran Oliver (Biomed Central, 2012-12-22)
      Anthelmintic resistance has been reported in most sheep producing countries. Prior to the mid 1990s, reports of anthelmintic resistance in Ireland were sparse and focused on benzimidazole, one of the three classes of anthelmintic available during this period. This evidence for efficacy issues on Irish farms combined with awareness that anthelmintic resistance was increasingly being reported in other countries prompted the need for more comprehensive investigations on Irish farms. Faecal egg count reduction and micro-agar larval development tests were employed to investigate resistance to benzimidazole, levamisole and macrocyclic lactone. There is compelling evidence for resistance to both benzimidazole (>88% of flocks) and levamisole (>39% of flocks). Resistance of nematode populations to macrocyclic lactone was suspected on a small number of farms (11%) but needs to be confirmed. The recent introduction of two new classes of anthelmintics, after over a 25 year interval, together with the evidence that anthelmintic resistance is reported within a relatively short time following the introduction of a new anthelmintic compound means that the challenge to the industry is immediate. Actions are urgently required to manage anthelmintic resistance so as to prolong the lifespan of anthelmintics.
    • 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.
    • Direct Evidence on the Contribution of a Missense Mutation in GDF9 to Variation in Ovulation Rate of Finnsheep

      Mullen, Michael P.; Hanrahan, James P; Teagasc Walsh Fellowship Programme (PLOS, 2014-04-21)
      The Finnish Landrace (Finnsheep) is a well known high-prolificacy sheep breed and has been used in many countries as a source of genetic material to increase fecundity of local breeds. Analyses to date have indicated that mutations with a large effect on ovulation rate are not responsible for the exceptional prolificacy of Finnsheep. The objectives of this study were to ascertain if: 1) any of 12 known mutations with large effects on ovulation rate in sheep, or 2) any other DNA sequence variants within the candidate genes GDF9 and BMP15 are implicated in the high prolificacy of the Finnish Landrace breed; using material from lines developed by divergent selection on ovulation rate. Genotyping results showed that none of 12 known mutations (FecBB, FecXB, FecXG, FecXGR, FecXH, FecXI, FecXL, FecXO, FecXR, FecGE, FecGH, or FecGT) were present in a sample of 108 Finnsheep and, thus, do not contribute to the exceptional prolificacy of the breed. However, DNA sequence analysis of GDF9 identified a previously known mutation, V371M, whose frequency differed significantly (P<0.001) between High and Low ovulation rate lines. While analysis of ovulation rate data for Finnsheep failed to establish a significant association between this trait and V371M, analysis of data on Belclare sheep revealed a significant association between V371M and ovulation rate (P<0.01). Ewes that were heterozygous for V371M exhibited increased ovulation rate (+0.17, s.e. 0.080; P<0.05) compared to wild type and the effect was non-additive (ovulation rate of heterozygotes was significantly lower (P<0.01) than the mean of the homozygotes). This finding brings to 13 the number of mutations that have large effects on ovulation rate in sheep and to 5, including FecBB, FecGE, FecXO and FecXGR, the number of mutations within the TGFβ superfamily with a positive effect on prolificacy in the homozygous state.
    • 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.
    • Effects of extended grazing during mid, late or throughout pregnancy, and winter shearing of housed ewes, on ewe and lamb performance

      Keady, Tim; Hanrahan, James P; Flanagan, S. (Teagasc, Oak Park, Carlow, Ireland, 2007)
      A flock of March-lambing ewes was used to evaluate the effects of (i) extended (deferred, winter) grazing of pasture during mid, late or throughout pregnancy, and (ii) winter shearing of ewes housed during mid and late pregnancy, on lamb birth weight and subsequent growth to weaning. Ewes (n = 265) were allocated at random to five treatments for the period from 7 December (~ day 47 of pregnancy) to lambing. The treatments were: housed shorn (HS), housed unshorn (HU), grazing throughout (EG), grazing to 20 January followed by housing (EGH), housed to 20 January followed by grazing (HEG). From 1 March to lambing the HEG and EG ewes were dispersed on the paddocks intended for grazing post lambing. All ewes were offered a concentrate supplement during the final 6 weeks of pregnancy. Housed ewes were offered grass silage while ewes on extended grazing were allocated 1.3 kg herbage dry matter per head per day from swards that had been closed for approximately 10 weeks. Ewes plus lambs (except triplet-rearing ewes which were grazed separately) from all treatments were grazed together post lambing, grouped according to lambing date. For treatments HS, HU, EGH, HEG and EG gestation lengths were 147.0, 145.6, 146.3, 146.6 and 146.9 (s.e. 0.34, P < 0.001) days, lamb birth weights were 4.9, 4.3, 4.4, 4.6 and 5.0 (s.e. 0.10, P < 0.001) kg, and lamb weaning weights were 34.6, 32.1, 33.3, 33.8 and 34.9 (s.e. 0.66, P < 0.001) kg, respectively. Extended grazing in mid and late pregnancy resulted in 35% and 65%, respectively, of the increase in lamb birth weight associated with extended grazing throughout. Treatment effects on lamb birth weight were associated with those on weaning weight (P < 0.01, R2 = 0.93). It is concluded that extended grazing or shearing of housed ewes increased lamb birth weight and subsequent weaning weight. The increased lamb birth weight from deferred grazing in mid pregnancy was probably due to improved protein utilisation from the grazed herbage. Meanwhile, the increased
    • An evaluation of two grassland-based systems of mid-season prime lamb production using prolific ewes of two genotypes

      Keady, Tim; Hanrahan, James P; Flanagan, S. (Teagasc, Oak Park, Carlow, Ireland, 2009)
      A 4-year study was undertaken to evaluate the effects of two contrasting management systems [year-round grazing (YRG) and normal seasonal grazing followed by indoor feeding during winter (GWF)] on performance of mid-season lambing ewes. On the GWF system, the annual stocking rate was 14.4 ewes/ha, grass silage was conserved for winter feeding indoors, and the ewes were lambed indoors and were then turned out to pasture. The YRG system was stocked at 10.5 ewes/ha, was grazed during the winter, had outdoor lambing and the animals had access to all the farmlet for summer grazing. The ewes were Belclare and Cheviot × Belclare which were balanced across systems. Mean lambing dates and fertiliser N application rates were 20 and 30 March, and 85 and 92 kg/ha, for the GWF and YRG systems, respectively. Concentrate supplementation during late pregnancy was similar on both systems. For the GWF and YRG systems, litter size, lamb mortality, number of lambs reared, birth weight (kg), weaning weight (kg) and lamb carcass output (kg/ha) were 2.17 and 2.24 (s.e. 0.038), 10.1 and 13.8% (P = 0.05), 1.77 and 1.78 (s.e. 0.042), 4.0 and 4.7 (s.e. 0.05, P < 0.001), 27.9 and 30.8 (s.e. 0.25, P < 0.001) and 469 and 348, respectively. Belclare ewes had a higher litter size (2.34 v 2.07; s.e. 0.038, P < 0.001) and number of lambs reared per ewe joined (1.86 v 1.69; s.e. 0.048, P < 0.01) than the Cheviot × Belclare ewes. There were no significant interactions between system and ewe breed type. It is concluded that the YRG system of prime lamb production was sustainable using prolific ewes but at a reduced stocking rate (−26%) and with greater lamb mortality relative to the GWF system. Ewe genotypes with a mean litter size of up to 2.34 lambs are suitable for both systems. Lamb carcass output of 501 kg/ha was achieved from a primarily grass-based system of mid-season prime lamb production using prolific ewes (Belclare).
    • Gastrointestinal nematode control practices on lowland sheep farms in Ireland with reference to selection for anthelmintic resistance

      Patten, Thomas; Good, Barbara; Hanrahan, James P; Mulcahy, Grace; de Waal, Theo (Biomed Central, 2011-03-31)
      Gastrointestinal parasitism is a widely recognised problem in sheep production, particularly for lambs. While anthelmintics have a pivotal role in controlling the effects of parasites, there is a paucity of data on how farmers use anthelmintics. A representative sample of Irish lowland farmers were surveyed regarding their parasite control practices and risk factors that may contribute to the development of anthelmintic resistance. Questionnaires were distributed to 166 lowland Irish sheep producers. The vast majority of respondents treated their sheep with anthelmintics. Lambs were the cohort treated most frequently, the majority of farmers followed a set programme as opposed to treating at sign of disease. A substantial proportion (61%) administered four or more treatments to lambs in a 'normal' year. Departures from best practice in anthelmintic administration that would encourage the development of anthelmintic resistance were observed. In conclusion, in the light of anthelmintic resistance, there is a need for a greater awareness of the principles that underpin the sustainable use of anthelmintics and practices that preserve anthelmintic efficacy should be given a very high priority in the design of helminth control programmes on each farm. To this end, given that veterinary practitioners and agricultural advisors were considered to be the farmer's most popular information resource, the capacity of these professions to communicate information relating to best practice in parasite control should be targeted.
    • Investigation of Prolific Sheep from UK and Ireland for Evidence on Origin of the Mutations in BMP15 (FecXG, FecXB) and GDF9 (FecGH) in Belclare and Cambridge Sheep

      Mullen, Michael P.; Hanrahan, James P; Dawn, J. Howard; Powell, Richard; Teagasc Walsh Fellowship Programme; Genesis Faraday SPARK award; Science Foundation Ireland; 07/SRC/B1156 (PLoS, 2013-01-02)
      This paper concerns the likely origin of three mutations with large effects on ovulation rate identified in the Belclare and Cambridge sheep breeds; two in the BMP15 gene (FecXG and FecXB) and the third (FecGH) in GDF9. All three mutations segregate in Belclare sheep while one, FecXB, has not been found in the Cambridge. Both Belclare and Cambridge breeds are relatively recently developed composites that have common ancestry through the use of genetic material from the Finnish Landrace and Lleyn breeds. The development of both composites also involved major contributions from exceptionally prolific ewes screened from flocks in Ireland (Belclare) and Britain (Cambridge) during the 1960s. The objective of the current study was to establish the likely origin of the mutations (FecXG, FecXB and FecGH) through analysis of DNA from Finnish Landrace and Lleyn sheep, and Galway and Texel breeds which contributed to the development of the Belclare breed. Ewes with exceptionally high prolificacy (hyper-prolific ewes) in current flocks on Irish farms were identified to simulate the screening of ewes from Irish flocks in the 1960s. DNA was obtained from: prolific ewes in extant flocks of Lleyn sheep (n = 44) on the Lleyn peninsula in Wales; hyper-prolific ewes (n = 41); prolific Galway (n = 41) ewes; Finnish Landrace (n = 124) and Texel (n = 19) ewes. The FecXG mutation was identified in Lleyn but not in Finnish Landrace, Galway or Texel sheep; FecXB was only found among the hyper-prolific ewes. The FecGH mutation was identified in the sample of Lleyn sheep. It was concluded from these findings that the Lleyn breed was the most likely source of the FecXG and FecGH mutations in Belclare and Cambridge sheep and that the FecXB mutation came from the High Fertility line that was developed using prolific ewes selected from commercial flocks in Ireland in the 1960′s and subsequently used in the genesis of the Belclare.
    • 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.
    • Response to Teladorsagia circumcincta infection in Scottish Blackface lambs with divergent phenotypes for nematode resistance

      McRae, Kathryn M.; Good, Barbara; Hanrahan, James P; Glynn, Assumpta; O'Connell, Mary J.; Keane, Orla M; Teagasc Walsh Fellowship Programme; Allan and Grace Kay Overseas Scholarship (Elsevier, 2014-10-24)
      The objective of this study was to identify Scottish Blackface lambs that were at the extremes of the spectrum of resistance to gastrointestinal nematodes and characterise their response to an experimental nematode challenge. Lambs (n = 90) were monitored for faecal egg count (FEC) (2 samples from each of 2 independent natural infections). The most resistant (n = 10) and susceptible (n = 10) individuals were selected and challenged with 30,000 Teladorsagia circumcincta larvae (L3) at 9 months of age. Response to infection was monitored by measuring FEC, plasma pepsinogen, serum antibodies against nematode larval antigens and haematology profile, until necropsy at 71 days post infection. Worm burden, worm fecundity and the level of anti-nematode antibodies in abomasal mucosa were determined at necropsy. FEC was consistently higher in susceptible animals (P < 0.05), validating the selection method. Worm fecundity was significantly reduced in resistant animals (P = 0.03). There was also a significant correlation (r = 0.88; P < 0.001) between the number of adult worms and FEC at slaughter. There was no effect of phenotype (resistance/susceptibility) on plasma pepsinogen or on haematology profile. Phenotype had a significant effect on the level of anti-nematode IgA antibodies in serum (P < 0.01), reflecting a higher peak in resistant animals at day 7 post infection. It is concluded that significant variation in the response to gastrointestinal nematode challenge exists within the Scottish Blackface population with resistant animals displaying significantly lower FEC, lower worm fecundity and higher concentration of anti-nematode IgA antibodies in serum.
    • 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.