title: Enhancing the toolbox to study IL-17A in cattle and sheep.
abstract: The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs [CD4+ve, CD8+ve and Workshop Cluster 1 (WC-1)+ve gamma-delta (γδ)] we show that distinct T cell subsets in cattle (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.
authors.affiliation: Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, UK. Sean.Wattegedera@moredun.ac.uk.
authors.forename: Sean R
authors.identifier: 0000-0002-2218-5144
authors.initials: SR
authors.lastname: Wattegedera
authors.affiliation: Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, UK.|The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush, Midlothian, Scotland, EH25 9RG, UK.
authors.forename: Yolanda
authors.identifier: 0000-0002-1344-7405
authors.initials: Y
authors.lastname: Corripio-Miyar
authors.affiliation: Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, UK.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Pang
authors.affiliation: Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, UK.
authors.forename: David
authors.initials: D
authors.lastname: Frew
authors.affiliation: Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, UK.
authors.forename: Tom N
authors.identifier: 0000-0001-6469-0512
authors.initials: TN
authors.lastname: McNeilly
authors.affiliation: Biomathematics and Statistics Scotland, JCMB, The King's Buildings, Peter Guthrie Tait Road, Edinburgh, Scotland, EH9 3FD, UK.
authors.forename: Javier
authors.initials: J
authors.lastname: Palarea-Albaladejo
authors.affiliation: Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, UK.
authors.forename: Colin J
authors.identifier: 0000-0002-1465-9901
authors.initials: CJ
authors.lastname: McInnes
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush, Midlothian, Scotland, EH25 9RG, UK.
authors.forename: Jayne C
authors.identifier: 0000-0001-6573-6172
authors.initials: JC
authors.lastname: Hope
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush, Midlothian, Scotland, EH25 9RG, UK.
authors.forename: Elizabeth J
authors.identifier: 0000-0002-6183-0185
authors.initials: EJ
authors.lastname: Glass
authors.affiliation: Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, UK.|The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush, Midlothian, Scotland, EH25 9RG, UK.
authors.forename: Gary
authors.identifier: 0000-0002-8822-2331
authors.initials: G
authors.lastname: Entrican
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D016466:CHO Cells; D002417:Cattle / Q000276:immunology / Q000502:physiology*; D003001:Cloning, Molecular; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D020381:Interleukin-17 / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000662:veterinary; D012756:Sheep / Q000276:immunology / Q000502:physiology*; D013601:T-Lymphocytes / Q000737:chemistry
chemical_list: D000906:Antibodies; D020381:Interleukin-17