ABSTRACT BOOK - IVIS 2019 · with COX-2 inhibitor in bovine leukemia virus infection Yamato Sajiki...

ivis2019.org

International Veterinary Immunology SymposiumAUG 13 – 16, 2019 | RENAISSANCE SEATTLE, SEATTLE, USA

13-16 AUGUST SEATTLE, USA2019INTERNATIONAL VETERINARY IMMUNOLOGY SYMPOSIUM

AmericanAssociationof VeterinaryImmunologists

ABSTRACT BOOK

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ORAL ABSTRACTS NUMBERS

O07Effect of adjuvant on cellular and humoral immune responses to an experimental subunit vaccine antigen in sheep Sean R Wattegedera (Penicuik, United Kingdom)

O08New adjuvant technologies to optimize mycoplasma hyopneumoniae vaccine Marie-Eve Koziol (Fairfield, United States)

REGULATION & MODULATION

O09Differential induction of innate training and tolerance in porcine monocytes by β-glucans or BCG Kristen A Byrne (Ames, United States)

O10Contribution of prostaglandin E2 to disease progression and enhancement of antiviral effects by anti-PD-L1 antibody combined with COX-2 inhibitor in bovine leukemia virus infection Yamato Sajiki (Sapporo, Japan)

O11Prophylactic digoxin treatment inhibits IL-17 production in vivo and reduces BRSV-associated disease in the neonatal calf Jodi L. McGill (Ames, United States)

O12Establishment of anti-bovine PD-1 chimeric antibody and a pilot clinical study in bovine leukemia virus infection Tomohiro Okagawa (Sapporo, Japan)

INNATE IMMUNITY

O01Establishment of a bovine C-type lectin map reveals both conserved and species-specific recognition patterns that impact on mycobacterial recognition Dirk Werling (Hatfield, United Kingdom)

O02Immunostimulatory effects of toll-like receptor (TLR) ligands on bovine peripheral blood dendritic cells (DCs) and monocytes Güliz Tuba Barut (Bern, Switzerland)

O03The role of dendritic cell in the pathogenesis of Bovine Herpesvirus-1 (BHV-1) in-vitro Christopher Chase (Brookings, United States)

O04Global profiling of innate immune responses by porcine blood conventional and plasmacytoid dendritic cell populations following stimulation with TLR ligands Gaël Auray (Mittelhäusern, Switzerland)

ENHANCING VACCINES

O05Mechanisms of action of adjuvants in pigs Heather L. Wilson (Saskatoon, Canada)

O06Parameters of protective immunity in swine induced by PCV2 vaccines with different antigen payload Massimo Amadori (Brescia, Italy)

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AVIAN IMMUNOLOGY

(CEVA SPONSORED)

O19Needle-free oral vaccination of surface flagellin coated Salmonella nanovaccine for chickens Sankar Renu (Wooster, United States)

O20Proof of concept studies for novel necrotic enteritis vaccine in broilers Lisa Bielke (Wooster, United States)

O21The importance of monitoring live vaccines application for obtaining immunological protection Irit Davidson (Bet Dagan, Israel)

γδ, NK, NKT CELLS

O22Functional profiling and dynamics of naïve porcine invariant Natural Killer T cells Alexander Schäfer (Greifswald - Insel Riems, Germany)

O23Swine γδ T cell subpopulations defined by WC1 and TCR gene expression Sabine Hammer (Vienna, Austria)

O24Characterization of goat gd T cells and responses of WC1+ gd T cells to pathogens Alehegne W Yirsaw (Amherst, United States)

O13Modulation of porcine macrophages susceptibility to viral infection by epithelial cell-derived alarmins Obdulio García-Nicolás (Mittelhäusern, Switzerland)

VIC MHC WORKSHOP

O14Ruminant MHC class II genotyping using cell free DNA recovered from archived plasma and serum samples Keith T. Ballingall (Edinburgh, United Kingdom)

O15Nomenclature for factors of the Swine Leukocyte Antigen (SLA) system – Update 2019 Sam Ho (Ann Arbor, United States)

O16New highly-contiguous cattle genome assemblies reveal the extent of genetic variation underlying differential and adaptive immune responses John Hammond (Woking, United Kingdom)

O17Characterization of expressed major histocompatibility complex class I and class II alleles and haplotypes in domestic cat (Felis catus) by NGS-based amplicon sequencing Masaharu Okano (Fujisawa, Japan)

O18Impaired SLA class I antigen presentation: an explanation for cellular immune dysregulation in ASFV infected pigs Ulrike Blohm (Greifswald, Germany)

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O25Porcine γδ T cells display unique phenotypes by intestinal compartment and time post-weaning Jayne E. Wiarda (Ames, United States)

O26Effect of early life exposure to adult microbiota on the function of intestinal NK cells in broiler chickens Nathalie Meijerink (Utrecht, Netherlands)

O27Classification of WC1 gene family in Sus scrofa and evaluation of individual SRCR domain affinity for Mycobacterium bovis and Leptospira spp. Janice C. Telfer (Amherst, United States)

MUCOSAL IMMUNITY

O28Potential role of Peyer´s Patch B cells in immune regulation in intestine of sheep Philip J. Griebel (Saskatoon, Canada)

O29Porcine small intestinal organoids as a model to study epithelial innate immune responses to enteropathogens Bert Devriendt (Merelbeke, Belgium)

TICK & PARASITE IMMUNITY

O30In silico characterization of multi-epitope anti-Rhipicephalus microplus tick vaccine constructions composed by epitopes from tick salivary proteins selected by phage display and immunoinformatics approaches Andressa Fisch (Ribeirao Preto, Brazil)

O31Protective adaptive immune transcriptomic evaluation of PBMC isolated from sheep after infection and challenge with E. ruminantium Welgevonden strain Tshifhiwa Nefefe (Onderstepoort, South Africa)

O32Investigation of the Theileria parva sporozoite surface glycan repertoire and binding C-type lectin receptors Jeannine Kolakowski (Hatfield, United Kingdom)

O33Molecular and metabolic changes in the proximal colon of pigs infected with Trichuris suis Harry D. Dawson (Beltsville, United States)

O34CD4 variant phenotype related to increased susceptibility to Babesia bovis Cintia H Okino (São Carlos, Brazil)

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VIC TOOLKIT WORKSHOP

O41The Immunological Toolbox website: a community resource to enable information exchange and access to reagents and techniques John Hammond (Woking, United Kingdom)

O42Development of a bovine/ovine cytokine 15-plex assay for immunoprofiling of the cellular response in ruminants (10 min.) Gilles Foucras (Toulouse, France)

O43CD40-targeted immunization as a method for rapid antiserum production and epitope mapping (10 min.) Christine N Vuong (Fayetteville, United States)

O44A fast screening method to obtain high producer CHO cells for the production of porcinized monoclonal antibodies Bert Devriendt, Merelbeke, Belgium

PIG ANTI-VIRAL IMMUNITY

O35Mechanisms of AFSV pathogenesis and immune evasion inferred from gene expression changes in infected macrophages James J. Zhu (Orient, United States)

O36Understanding the induction of adaptive immunity following PRRSV infection Volker Gerdts (Saskatoon, Canada)

O37The development of nanoparticles-entrapped porcine reproductive and respiratory syndrome virus (PRRSV) vaccine Puwich Chaikhumwang (Bangkok, Thailand)

O38Searching for markers of immunocompetence in blood: application to vaccination against Influenza A virus in pigs Claire Rogel-Gaillard (Jouy-en-Josas, France)

O39Nipah vaccine to eliminate porcine reservoirs and safeguard human health Miriam Pedrera (Pirbright, United Kingdom)

O40Co-infection of porcine deltacoronavirus and porcine epidemic diarrhea virus prolongs virus shedding and IFN-β up-regulation Kepalee Saeng-Chuto (Bangkok, Thailand)

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COMPANION ANIMALS

(ELANCO SPONSORED)

O45Combination chemo- and immunotherapy provides long term clinical improvement and sustained parasite clearance in L. infantum-infected dogs Maria Socorro Cruz (Teresina, Brazil)

O46Development of a sensitive PD-L1 immunohistochemistry of canine cancers and clinical efficacy of an anti-PD-L1 antibody in canine oral malignant melanoma with pulmonary metastasis Naoya Maekawa (Sapporo, Japan)

O47Food allergen-sublingual immunotherapy in dogs significantly reduces IL-17A and increases IL-10 and allergen-specific CD3+CD4-CD8- T cells Eric Cox (Merelbeke, Belgium)

O48Characterisation of the companion animal immune response to Mycobacterium bovis infection using cytokine profiling Conor O‘Halloran (Edinburgh, United Kingdom)

O49Evaluation of the efficacy of a canine influenza virus (H3N2) vaccine in SPF dogs Jeong Byoung Chae (Daejeon, Korea, Republic of)

O50Pre-conditioning of equine bone marrow-derived mesenchymal stromal cells: effect on the inhibition of lymphocyte proliferation Claudio Henriquez (Valdivia, Chile)

NOVEL SYSTEMS

O51Rousettus aegyptiacus cell lines enable analysis of Type I and III interferon responses to highly pathogenic viruses in reservoir species Virginia Friedrichs (Greifswald - Insel Riems, Germany)

O52The pig – a novel translational animal model for eosinophilic esophagitis Tobias Käser (Raleigh, United States)

O53Recombinant Lawsonia intracellularis outer membrane proteins confirmed as being neutralizing antibody targets selected for subunit vaccine development Heather L. Wilson (Saskatoon, Canada)

O54Good cells battling bad cells: cell-mediated immunity and transmissible cancer in Tasmanian devils Camila Espejo (Hobart, Australia)

O55Oral stimulation of rainbow trout with Aeromonas salmonicida and characterization of the adaptive immune response assembled: first steps to an oral vaccination model Bernd Köllner (Greifswald, Germany)

O56The role of IL-1R8 and Toll-like receptors in the pathogenesis of canine DLBCL Federica Riva (Milano, Italy)

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O63Immunogenomics of innate immune cells of the porcine lung during PRRSV infection Elisa Crisci (Jouy-en-Josas, France)

O64Whole blood transcriptome analysis reveals altered pathways related to inflammation and fatty acid metabolism in stocker calves that develop bovine respiratory disease Matthew Scott (Mississippi State, United States)

O65Transcriptomic analysis of devil facial tumour disease in non-immunised and immunised Tasmanian devils Amanda L Patchett (Hobart, Australia)

RUMINANT IMMUNITY

O66Intra-mammary vaccine delivery protects sheep against mastitis caused by mannheimia haemolytica infection Keith T. Ballingall (Edinburgh, United Kingdom)

O67Immunological responses and protection in dairy cows vaccinated with staphylococcal surface proteins Oudessa Kerro Dego (Knoxville, United States)

O68Phenotypic analysis of T cell subsets expressing Interferon-gamma, interleukin -17A and tumor necrosis factor-alpha across ruminant species Mahmoud M. Elnaggar (Pullman, WA, United States)

PREDICTING IMMUNITY

O57Resistance to Haemonchus contortus in Corriedale sheep is associated to high parasite-specific IgA titer and a systemic Th2 immune response Teresa Freire (Montevideo, Uruguay)

O58Characterisation of CXCL10 production and diagnostic potential for bovine TB in cattle Michael Coad (Weybridge, United Kingdom)

O59Predicting vaccine-induced heterologous protection via advanced analysis of PRRSV-specific memory immune cells Tobias Käser (Raleigh, United States)

IMMUNE GENE EXPRESSION

O60Epigenetic regulation in bovine monocytes upon LPS challenge Gilles Foucras (Toulouse, France)

O61Combined chromatin state and transcriptional analyses of pig alveolar macrophages reveal regulatory elements of the inflammatory response Juber Herrera Uribe (Ames, United States)

O62Immune fitness as a measure of animal health, welfare and productivity in the feedlot: transcriptomic analysis Auriol C Purdie (Camden, Australia)

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O69IFN-γ response to bacteria or vaccination is not correlated with protection against mycobacterial infection Gregers Jungersen (Kgs Lyngby, Denmark, Denmark)

O70Local Th17 immunity upon mammary immunization is protective against E. coli mastitis Gilles Foucras (Toulouse, France)

O71Type III interferon protects cattle against bovine viral diarrhea virus infection Alejandra V. Capozzo (CABA, Argentina)

POSTERS

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POSTERS

P013Gastro-intestinal helminths of free-ranging vervet monkeys (Chlorocebus pygerythrus) in Huye town, Rwanda – call for one health approachRichard S. Gashururu (Nyagatare, Rwanda)

P015Parallel measurement of IFNγ and IP-10 in QuantiFERON®-TB Gold plasma improves the detection of Mycobacterium bovis infection in African buffaloes (Syncerus caffer)Netanya Bernitz (Cape Town, South Africa)

P017Validating changes in expression of novel regulatory genes identified through GWAS for mastitis controlGina Pighetti (Knoxville, United States)

P019Comparing concentrations of natural antibodies and total immunoglobulin G in colostrum of beef and dairy cowsTess Altvater-Hughes (Guelph, Canada)

P021Preliminary heritability estimates of immune response in Canadian beef cattleShannon Beard (Guelph, Canada)

P023Isolation and preliminary characterization of bovine Th17 lymphocytesPierre Germon (Nouzilly, France)

P025Detection of zoonotic bacterial causes of bovine mastitic milk using nano gold-immunochromatographic lateral flow stripsRafik Sayed (Cairo, Egypt)

12:15-13:00

P001Selection for gastrointestinal parasite resistance reduces MHC allelic richness but favours functional diversity in Cashmere goat linesKeith T. Ballingall (Edinburgh, United Kingdom)

P003Immunomodulatory effects of race training at the level of the airway-derived macrophageAnna E. Karagianni (Edinburgh, United Kingdom)

P005Intensity of the local immune response reflects route of entry after intranasal H1N1pdm09 infection in pigsTheresa Schwaiger (Greifswald, Germany)

P007Chicken GM-CSF-differentiated dendritic cells can be separated into immature MHC-IIlow and spontaneously matured MHC-IIhigh subsetsRobin H G A Van Den Biggelaar (Utrecht, Netherlands)

P009Evaluation of conventional DEC205+dendritic cells response to porcine reproductive and respitarory syndrome virus Jesús Hernández (Hermosillo, Mexico)

P011Develop of lateral flow immunochromatographic test and PCR for detection of SalmonellaEnteritidis in poultry farm Rafik Dayed (Cairo, Egypt)

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POSTERS

13:00-13:45

P002Establishment and characterization of primary and hTERT immortalized sheep ileal epithelial cellsRadhey Shyam Kaushik (Brookings, United States)

P004Vitamin A deficiency impairs the mucosal immune response to respiratory syncytial virus vaccination and infection in neonatal calvesJodi L. McGill (Ames, United States)

P006Establishment of gnotobiotic pig model for studying the colonization of nasal microbiome and its role in respiratory immune system developmentRadhey Shyam Kaushik (Brookings, United States)

P008Diversity of the swine leukocyte antigen class II (SLA-II) in Thai commercial pig populationNavapon Techakriengkrai (Bangkok, Thailand)

P010Myeloid dendritic cells in in equine peripheral bloodFalko Steinbach (Guildford, United Kingdom)

P012Elimination of erroneous results related to bovine mononuclear cells immunophenotyping by antibody binding to Fc receptorsCintia H Okino (São Carlos, Brazil)

P014Development of a fluorescent scFv antibody to improve Rabies diagnosis Alejandra V. Capozzo (CABA, Argentina)

P027Development of bovine antibody reagents for the sorting and characterization of bovine plasmablastsKelcey D. Dinkel (Pullman, United States)

P029Development and characterization of immune reagents for swine health, vaccine and disease studiesJoan Lunney (Beltsville, United States)

P031Identification of a subset of bovine circulating CX3CR1 positive monocytes in blood and bone marrow through the novel application of fluorescently tagged recombinant CX3CL1Anna Raper (Edinburgh, United Kingdom)

P033Development and testing of common bottlenose dolphin (Tursiops truncatus)-specific cytokine ELISA reagents Randy E Sacco (Ames, United States)

P035The UK Immunological Toolbox: promoting veterinary immunology research using recombinant technologies to secure and engineer antibodies John Hammond (Woking, United Kingdom)

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POSTERS

P016Potential re-emergence of pathogenic tsetse–transmitted trypanosomes in the neighborhoods of Akagera National Park, RwandaRichard S. Gashururu (Nyagatare, Rwanda)

P018Akagera National Park buffalo surveillance for foot-and-mouth disease and other animal diseasesJean Claude Udahemuka (Nyagatare, Rwanda)

P020In Vivo and In Vitro Testing of African Clawed Frog Susceptibility to Aquatic RhabdovirusesEmma Bueren (Seattle, United States)

P022Predicted microRNA biomarkers of Mycobacterium avium subspecies paratuberculosis using an in-silico bioinformatics approachKathryn E Wright (Camden, Australia)

P024Measuring CMI responses using the PrimeFlow RNA assay; a potential new method of evaluating BVDV vaccination responseShollie Falkenberg (Ames, United States)

P026Effect of heat stress on Peripheral Blood Mononuclear Cell (PBMC) function of dairy cows ranked as high, average or low immune respondersShannon Cartwright (Guelph, Canada)

P028Effect of heat stress on body temperature and Peripheral Blood Mononuclear Cell (PBMC) function of beef cows ranked as high, average or low immune respondersNasrin Husseini (Guelph, Canada)

P032Analysis of immunological targets across multiple species demonstrates need for species-specific research reagentsJoanna LaBresh (Saint Paul, United States)

P034Advances for the Collaborative Immune Reagent Network for Aquacultured Species (CIRNAS)John Hansen (Seattle, United States)

P036Generation and characterization of monoclonal antibodies against porcine IgEArmin Saalmüller (Vienna, Austria)

P038Comparison of SepMateTM-50 tubes with lymphoprepTM and standard 50 mL tubes with Ficoll®-Paque Plus for PBMC isolation from Macaca fascicularis whole bloodDulce R. Rodriguez (Worcester, United States)

P040Porcine cytokines, chemokines and growth factors: 2019 UpdateHarry D. Dawson (Beltsville, United States)

P042The UK Immunological Toolbox: building novel reagents to further the understanding of livestock and avian immunologyJayne Hope , United Kingdom)

P044Unique features of water buffalo (Bubalus bubalis) γδ T cells and monocytesMahmoud M. Elnaggar (Pullman, WA, United States)

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POSTERS

P051e-PIG-enetic avenues: porcine mRNA and miRNA pathway analysis of highly pathogenic PRRSV InfectionLaura Miller (Ames, United States)

P052-BThe induction of immunosuppression via prostaglandin E2 and the enhancement of anti-bacterial effects by anti-PD-L1 antibody combined with COX-2 inhibitor in Mycoplasma bovis infectionShinya Goto (Sapporo, Japan)

P053The interferon gamma response to Mycobacterium avium in vitro can be correlated with a higher risk of clinical ketosis in dairy cowsMassimo Amadori (Brescia, Italy)

P055Differential local immune responses against Haemonchus contortus in a resistant Brazilian sheep breed (Morada Nova)Cintia H Okino (São Carlos, Brazil)

P057The identification of a porcine pneumovirus from an immunological perspectiveFrançois Meurens (Nantes, France)

P059Analysis ofpecific antibody and autoantibody repertoires between asymptomatic and symptomatic dogs in the Leishmania infantum infectionRamzi Boubaker Landolsi (Kalaat Landalous, Tunisia)

12:15-13:00

P037Cytokine assay: serum concentrations in apparently healthy calves in nigeriaJones S Akinbobola (Abuja, Nigeria)

P039The effect of lipid supplementation on macrophage activation plasticity in Mycobacterium avium subspecies paratuberculosis infectionKathryn E Wright (Camden, Australia)

P041Metabolic reprogramming of bovine classical and nonclassical monocytesStephanie C. Talker (Bern, Switzerland)

P043Cows selected for resistance to mastitis show contrasted immune responses compared to mastitis susceptible cowsPierre Germon (Nouzilly, France)

P045Atypical granuloma formation by Mycobacterium bovis in young calves Jacobo Carrisoza-Urbina (Mexico, Mexico)

P047Adjuvant modulated cytokine responses to infective larval stage of equine Strongyle parasites in vitroStina Hellman (Uppsala, Sweden)

P049Porcine interferon-omega subtype: differential expression and superior antiviral activityJordan Jennings (Nashville, United States)

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POSTERS

P075Identifying potential candidate genes regulating host response to gastrointestinal nematodes in Ontario grazing sheep using transcriptomicsSamantha Dixon (Guelph, Canada)

P077Modulation of Porcine Lung immune cells during PRRSV-2 infectionLakmini Premadasa (Raleigh, United States)

P079Toll-like receptor mediated responses to Leptospira spp. in dogsAndreja Novak (Utrecht, Netherlands)

13:00-13:45

P048Exploring species tropism of zoonotic Flaviviruses using Monocyte-derived Dendritic cells as an in vitro modelMarta Lewandowska (Mittelhäusern, Switzerland)

P050Bacterial outer membrane vesicles of aeromonas salmonicida induce a pro-inflammatory immune response in vitro and in vivoSven Ostermann (Greifswald, Germany)

P052-AThe study of immunogenicity in 25 RHDV strains (Lagovirus europeus GI.1 and GI.1a)Paulina Niedzwiedzka-Rystwej (Szczecin, Poland)

P061The effect of host genetic on the expression profile of inflammatory mediators in chickens Neda Barjesteh (Saint-Hyacinthe, Canada)

P063Gene expression in canine peripheral blood mononuclear cells infected with Mycobacterium avium subsp. hominissuisSuji Kim (Seoul, Korea, Republic of)

P065Relationship between survivability and immune response of swine classified by the HIR™ technology when exposed to common pig pathogens using a natural disease challenge modelJulie D Schmied (Guelph, Canada)

P067Characterizing biomarkers of inflammation in variable stress responding sheepDanielle Naylor (Guelph, Canada)

P069BVDV experimental infection in water buffaloes: dynamics of lymphocyte subsets variation evaluated by flow cytometryMassimo Amadori (Brescia, Italy)

P071Evaluation of bacterial killing in bovine milk samplesFederica Riva (Milano, Italy)

P073Evaluation of immune responses in post-weaning gnotobiotic pigs infected with human norovirus genogroup II genotype 4 by transcriptome analysisByung-Joo Park (Seoul, Korea, Republic of)

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POSTERS

P054Characterizing the global transcriptomic profile of bovine monocyte-derived macrophages classified based on nitric oxide response to escherichia coliMehdi Emam (Guelph, Canada)

P056Innate immune responses against Streptococcus suis infections in the porcine lungJean-Pascal Hoffmann (Greifswald, Germany)

P058Identification of natural disease resistance in Zebu and Holstein-Friesian (HF) cattle through a microbicidal assayOmar Alcaraz (Mexico City, Mexico)

P060Immunological tools to investigate myeloid cell differentiation in pigsJayne Hope United Kingdom

P062Systemic immune responses associated to Haemonchus contortus resistance in a resistant Brazilian sheep breed (Morada Nova)Cintia H Okino (São Carlos, Brazil)

P064Role of IgA antibodies in pig oral fluids for the control of PRRS virus infectionMassimo Amadori (Brescia, Italy)

P066Potential of the dual IFNγ/IL-2 fluorescence-immunospot assay to distinguish different stages in bovine tuberculosisSabine Steinbach (New Haw, United Kingdom)

P068Functional alterations in bovine monocytes during Theileria parva infectionReginaldo G. Bastos (Pullman, United States)

P070Programmed cell death of leukocytes of peripheral blood as an important element of rabbit haemorrhagic disease virus (Lagovirus europeus GI.1a) pathogenesisPaulina Niedzwiedzka-Rystwej (Szczecin, Poland)

P072Differential effects of rift valley fever virus MP-12 infection in two porcine macrophage-like cell linesLeela Noronha (Manhattan, United States)

P074Seroprevalence in broilers against two important foodborne pathogens Salmonella and Campylobacter jejuniHung-Yueh Yeh (Athens, United States)

P076Using NanoString transcriptomics to evaluate the fetal and placental response to congenital PRRSV infection Joan Lunney (Beltsville, United States)

P078Susceptibility of bovine macrophages to bovine viral diarrhea virus infection is dependent on macrophage phenotypeAlejandra V. Capozzo (CABA, Argentina)

P080Cross-species infection of rabbit with swine hepatitis E virus genotype 3 without induction of hepatitisSang-Hoon Han (Seoul, Korea, Republic of)

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POSTERS

P089Immunochemical study of protein profiles of Taleghan, Fars, and lorestan strains of mycoplasma agalactiaeHoma Orangi (Shiraz, Iran, Islamic Republic of)

P091Early transcriptional regulation of Th17 differentiation in nasopharyngeal-associated lymphoid tissue (NALT) of mice intranasally immunized with chitosan nanoparticles loaded with Brucella abortus malate dehydrogenaseSoojin Shim (Seoul, Korea, Republic of)

P093Immunogenicity of the local anthrax vaccine strain in animal modelsFarzana Islam Rume (Barishal, Bangladesh)

P095Formulation of A. salmonicida adjuvanted vaccines for rainbow trout: impact of the adjuvant oil originMarie-Eve Koziol (Fairfield, United States)

P097The humoral immune response and vaccine protection against paratuberculosis in sheepHannah Beth Pooley (Camden, Australia)

P099Vaccination with a recombinant PCV2-ORF2 vaccine does not interfere with the efficacy of a modified live vaccine against PRRSVArmin Saalmüller (Vienna, Austria)

P082A preliminary report on anthrax-like disease in cattle in Nigeria from seemingly odd pathogensMoses Odugbo (Vom- Jos, Nigeria)

P084Transfer of cellular immunity through colostrum studied in goat twinsLisa Robbers (Utrecht, Netherlands)

P086Elucidating the Immunoregulatory Functions of Exosomes Released from Tracheal Cells on Macrophages in ChickensWilliam Donnelly (Sainte-Hyacinthe, Canada)

12:15-13:00

P081Deletion of relA improves the efficacy of Mycobacterium bovis bacillus Calmette-Guerin as a vaccine against human and bovine tuberculosisAsmaa H Mahmoud (Pullman, United States)

P083Construction of a chimeric bovine herpesvirus 4 as a delivery system for ovine herpesvirus 2 glycoprotein BSmriti Shringi (Pullman, United States)P087Biocontamination in poultry vaccinesShaimaa Elsady (Cairo, Egypt)

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POSTERS

P103Interaction of the gamma delta TCR with the WC1 hybrid co-receptor / pattern recognition receptorCynthia L. Baldwin (Amherst, United States)

P105Advances towards a vaccine for ovine pneumoniaNatalie A Parlane (Palmerston North, New Zealand)

P107Intranasal immunization of swine with a porcine reproductive and respiratory syndrome virus inactivated whole-virus vaccine provides protective immunity against a homologous virus challengeFederico Zuckermann (Urbana, United States)

P109Bovine NK-lysin-derived peptide NK2A demonstrates a limited immunomodulatory activity on cattle leukocytesRohana P. Dassanayake (Ames, United States)

P111Functional analysis of bovine CTLA-4 in bovine leukemia virus infectionKei Watari (Sapporo, Japan)

P113Development of an equinized antibody to neutralize the insulin-like growth factor-1 receptor (IGF-1R): a potential immunotherapy for endocrinopathic/hyperinsulinemic equine laminitisNiveditha Vathsangam (Parkville, Australia)

P115Modulation of mycobacterium-macrophage interaction by a host factor, bovine conglutinin: implications for bovine tuberculosisAnthony G. Tsolaki (London, United Kingdom)

P117Probiotics delivered in milk replacer affect leukocytes collected from the lung lavage fluid of neonatal calvesCarol G. Chitko-McKown (Clay Center, United States)

P119Immunomodulation by catecholamines and catecholamine-treated Salmonella enterica cultures in pigs (sus scrofa)Lena Reiske (Stuttgart, Germany)

P121The influence of Interferon Lambda (IFN-λ) on STAT’ phosphorylation and ability canine mammary cancer cells to migrationJoanna Mucha (Warsaw, Poland)

P123The use of a liposome with MANα1-2MAN-PEG-PE molecule enhances the immunogenicity induced by a nanovaccine against BoHV-1 in the murine model and in cattleCecilia A Langellotti (Hurlingham, Buenos Aires, Argentina)

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POSTERS

P100Quantifying the persistence of vaccine T cell epitopes in circulating swine influenza strains from 2013-2017Swan Tan (Providence, United States)

P102A plant-produced immunoenhanced pig vaccine against PRRSVGitte Erbs (Kgs. Lyngby, Denmark)

P104Effective and protective immunization with a novel recombinant MERS-CoV expressed in CHO cells for developing subunit vaccineSoYoung Kim (Yesan, Korea, Republic of)

P106Targeting antigens to dendritic cells: Characterization of a full chimeric mouse x pig recombinant antibody anti-porcine DEC205 receptorJesús Hernández (Hermosillo, Mexico)

P108Characterization of protective humoral and cellular immune responses against RHDV2 induced by a new vaccine based on recombinant baculovirusClaudia Müller (Greifswald, Germany)

P110Head Start Immunity: characterising the early protection of C strain vaccine against subsequent classical swine fever virus infectionFalko Steinbach (Guildford, United Kingdom)

13:00-13:45

P088Toll-like receptor 1/2, 7/8 and 9 agonist combi as adjuvant in neonate pigs for inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccineSandra Vreman (Lelystad, Netherlands)

P090Comparative study evaluating the safety of type 2 porcine reproductive and respiratory syndrome (PRRS) modified-live virus (MLV) vaccines in pigsAdthakorn Madapong (Bangkok, Thailand)

P092Prevalence of avian influenza outbreak in Egypt 2018Hanan Ibrahim (Cairo, Egypt)

P094Preparation and evaluation of mucosal needles killed vaccine against infectious Coryza in chickenRafik Sayed (Cairo, Egypt)

P098A comparative study evaluating immune induction and protective efficacy of nanoparticles-entrapped porcine reproductive and respiratory syndrome virus (PRRSV) vaccine against co-challenge with PRRS1 and PPRS2Angkana Tantituvanont (Bangkok, Thailand)

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POSTERS

P122Indoleamine 2,3-dioxygenase and the tryptophan-kynurenine-nicotinamide pathway in human TuberculosisClement Gascua Adu-Gyamfi (Newlands, Pretoria, South Africa)

P124PD-1 / PD-L1 monoclonal antibody development for canine cancer therapyJin Wook Choi (Davis, United States)

P125Immunogenicity of two forms of cost-effective purified non-living anthrax vaccine candidate compared to Sterne live spore vaccine with concurrent penicillin G treatment in bovine Solomon Jauro (Pretoria, South Africa)

P126Animal vaccines for sustainable food security: creating new vaccines against porcine circovirusPeera Jaru-Ampornpan (Pathum Thani, Thailand)

P112Development of bovine rotavirus and bovine coronavirus combined vaccine of bovine diarrhea diseaseGijung Ryoo (Yesan, Korea, Republic of)

P114Effects of bovine tumor necrosis factor alpha decoy receptors on cell death and inflammatory cytokine kinetics: potential for bovine inflammation therapySotaro Fujisawa (Sapporo, Japan)

P116Immunomodulatory effects of sialostatin L and sialostatin L2 from Ixodes persulcatusSchulze, Taiga tickSatoru Konnai (Sapporo, Japan)

P118Porcine DCs derived from cryopreserved bone marrow cells for the in vitro evaluation of immunomodulatory compoundsMirelle Geervliet (Wageningen, Netherlands)

P120The activity of mitochondrial succinate dehydrogenase in the liver of recipient animals ande the influence the of mesenchymal stem cellAnatoliy Mazurkevych (Kyiv, Ukraine)

ORALABSTRACTS

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ORAL ABSTRACTS

INNATE IMMUNITY

O01Establishment of a bovine C-type lectin map reveals both conserved and species-specific recognition patterns that impact on mycobacterial recognition Werling D.1, Holder A.1, Jegouzo S.A.F.2, Hammond J.A.3, Taylor M.E.2, Drickamer K.2

1Royal Veterinary College, Hatfield, United Kingdom, 2Imperial College, Department of Life Science, London, United Kingdom, 3The Pirbright Institute, Pirbright, United Kingdom

C-type lectin receptors (CLRs) have been well defined in humans and mice, but are as yet not defined in ruminants. CLRs can play a role in homeostasis and recognise sugar-moieties expressed uniquely by a diverse range of pathogens. However, certain pathogens exploit CLRs for their entry into host cells to avoid immune recognition. To understand these mechanisms, a detailed analysis of the CLRs present within different mammalian genomes is necessary. Using different bovine genomes, we were able to show that the cow possesses orthologues to most human/murine CLRs, but altered by duplications/deletions. We present a summary our findings, which have now been included in the “Animal Lectin” database (http://www.imperial.ac.uk/research/animallectins/). A panel of cow CLRs have been cloned and their sugar-binding domains have been immobilized using biotin tags to generate a novel lectin array. Screening of the array with fluorescently labelled bacteria and viruses provides a rapid means for determining which receptors engage with different pathogens. Given the economic damage done by mycobacterial infection to the UK farming industry, we concentrated in

the first instance on CLRs that are involved in mycobacterial infection. Bovine DC-SIGN exists in the bovine genome only as a single copy gene, in contrast to murine and human, but its ligand-binding properties seem to be conserved. Bovine mincle, shares 84% sequence identity with human mincle, but there are subtle changes in a secondary binding site and adjacent hydrophobic surfaces that provide docking sites for acyl side chains of trehalose dimycolate (cord factor) and related compounds. Interestingly, we were also able to confirm a natural ligand for Mincle on T cells. Antibodies generated to the bovine Mincle carbohydrate-recognition domain and other CLRS as well as the development of small molecules potentially disrupting the interaction of mincle and the mycobacterium may provide a basis for designing new intervention strategies.

O02Immunostimulatory effects of toll-like receptor (TLR) ligands on bovine peripheral blood dendritic cells (DCs) and monocytes

Barut G.T.1,2, Keller I.3,4, Bruggmann R.3, Summerfield A.1,2, Talker S.C.1,2

1Institute of Virology and Immunology, Bern, Switzerland, 2Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland, 3Interfaculty Bioinformatics Unit and Swiss Institute of Bioinformatics, University of Bern, Bern, Switzerland, 4Department for BioMedical Research, University of Bern, Bern, Switzerland

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TLR-mediated activation of DCs triggers their maturation and migration to secondary lymphoid tissues, where DCs can initiate antigen-specific T-cell responses. Knowledge on TLR ligand responsiveness and ensuing function is essential for development of vaccine adjuvants and - due to species-specific differences - has great value for comparative DC biology.

We therefore aimed to characterize the effect of TLR ligands in detail, including transcriptomic analysis of stimulated bovine DC subsets. In a set of pilot experiments using bulk cultures of PBMC, bovine classical DC type 1 and 2 (cDC1, cDC2) and plasmacytoid DC (pDC) upregulated the chemokine receptor CCR7, MHC class II, and the co-stimulatory molecules CD40, CD80 and CD86 within 4 hours of stimulation with TLR ligands, indicating successful activation and maturation of all subsets in vitro. In order to analyze ligand responsiveness of individual subsets, we analyzed phosphorylation of p38 MAPK at 15 minutes post TLR ligand stimulation. With this early detection of activation we could avoid bystander activation in bulk cultures of PBMC and find subset-specific responses that correlated well with transcription of TLRs in bovine DC subsets.

Finally, to get detailed information on transcriptional changes upon activation, we have stimulated sorted bovine pDC (Flt3+CD4+CD13-), cDC1 (Flt3+CD4-CD13+), cDC2 (Flt3+CD4-CD13-), and monocytes (Flt3-CD172ahigh) with selected TLR ligands Gardiquimod, Poly(I:C), CpG D32 and Pam2CSK4 and isolated RNA at 3 hours post stimulation. Taken together, this transcriptomic analysis will not only confirm ligand responsiveness of bovine DC subsets and monocytes, but will provide valuable insights into subset- and species-specific differences

in transcriptional responses to pathogen-associated molecular patterns and DC/monocyte function.

O03The role of dendritic cell in the pathogenesis of Bovine Herpesvirus-1 (BHV-1) in-vitro

Abdelsalam K., Sobraske J., Chase C.

South Dakota State University, Veterinary and Biomedical Sciences, Brookings, United States

Background: BHV-1 is a major viral disease of cattle that has a severe economic impact on cattle industry world-wide. It is a part of bovine respiratory disease complex that cost $900 million loses annually in US. In the current research, we studied the role of monocyte-derived dendritic cells (MDDC) in the pathogenesis of BHV-1. Understanding this immune cell -virus interaction may help to improve the current control strategies followed in the US farms.

Method: MDDC we derived from blood monocytes after 5 days of incubation in the presence of cytokines and growth promoters. The MDDC were infected with either LA or Copper strains of BHV-1 in-vitro and the surface marker expression, viral growth kinetics as well as cytokine m RNA expression were measured over a 48 h period after infection.

Results: BHV-1 up-regulated interferon (IFN) alpha, beta and gamma whereas the rest of the examined cytokines, including IL-1b, IL-8, TNF-a, IL-1a, IL-10, and IL-12 were down-

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regulated. BHV-1 reduced the expression of MHC-I, MHC-II and CD86 on the surface of MDDC but had no effect on MDDC viability. BHV-1 succeeded to grow and multiply in MDDC and the peak of infectious virus progeny was achieved at 24 hours post infection prior to the peak of IFN response.

Conclusion: MDDC support the growth of BHV-1, however this growth is time dependent on the production of IFN that then blocks further replication. BHV-1 disrupted the immune response through down-regulation of MDDC surface markers and inflammatory cytokines that are important to recruit innate immune cells and initiate effector defense mechanism. MDDC immune dysfunction was maintained by the virus without affecting MDDC cell viability.

O04Global profiling of innate immune responses by porcine blood conventional and plasmacytoid dendritic cell populations following stimulation with TLR ligands

Auray G.1,2, Keller I.3, Python S.4, Gerber M.4, Bruggmann R.5, Ruggli N.1,2, Summerfield A.1,2

1Institute for Virology and Immunology, Mittelhäusern, Switzerland, 2Department of Infectious Diseases and Pathobiology, University of Bern, Bern, Switzerland, 3Department of Clinical Research, University of Berne, Bern, Switzerland, 4Institute for Virology and Immunology, University of Bern, Mittelhäusern, Switzerland, 5Interfaculty Bioinformatics Unit and Swiss Institute of Bioinformatics, University of Berne,, Bern, Switzerland

We previously identified the mononuclear phagocyte populations in the porcine blood (namely conventional dendritic cells cDC1 and cDC2, plasmacytoid pDC and monocyte) and characterized their transcription profiles using mRNA sequencing. We showed these subsets displayed different profiles of TLR expression and when PBMCs were stimulated with TLR ligands, pDCs were the main subset to produce cytokines and to be involved in the activation of cDC1 and cDC2. To assess further the immune response of these subsets to TLR ligands, we sorted DC from three different animals and stimulated them with a broad range of TLR ligands for 3h before harvesting the cells for mRNAseq. We studied the differential response of subsets to the same TLR ligand (cDC1, cDC2, pDC and monocytes stimulated with PAM3Cys, TLR1/2 ligand), but also the transcription profile of one subset after stimulation with different ligands (pDCs following PAM3Cys, Poly I:C, Gardiquimod, Resiquimod and CpG ODN stimulation or cDC2 following PAM3Cys, Poly I:C and Gardiquimod stimulation). This allowed us to precisely map the subset-specific immune response to TLR ligands in terms of cytokines, chemokines, chemokine receptor or co-stimulatory molecules expression, which represents important information to design improved vaccines and immunotherapeutics as well as to better understand host-pathogen interactions.

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ENHANCING VACCINES

O05Mechanisms of action of adjuvants in pigs

Magiri R.1, Wilson H.2, Mutwiri G.1

1University of Saskatchewan, School of Public Health & VIDO-InterVac, Saskatoon, Canada, 2University of Saskatchewan, VIDO Inter-Vac & School of Public Health, Saskatoon, Canada

Adjuvants are critical components of vaccines but the mechanisms of action (MOA) of most adjuvants are poorly understood. In fact, the MOA of adjuvants have not been systematically investigated in large animals. Knowledge gained from such mechanistic studies would provide a biologic explanation regarding their efficacy and safety. In recent investigations we demonstrated that pigs injected with a novel experimental adjuvant PCEP had increased expression of ‘adjuvant core response genes’, upregulation of pro-inflammatory cytokines (IL-1beta, IL-6 and IL-13) and developed inflammatory responses at the site of injection. These inflammatory responses were characterized by cell recruitment, granuloma formation and necrosis. Not surprisingly, the inflammatory response occurred in a dose-dependent manner. Pigs injected with 500 µg of PCEP+SIV (swine influenza virus) vaccine had significantly stronger inflammatory responses compared to those injected with 100, 20 and 4 µg of PCEP+SIV. However, when SIV-specific antibody responses were evaluated, pigs injected with 500 µg PCEP+SIV developed titers similar to those in the 100 and 20 µg groups. In contrast, pigs injected with 4 µg of PCEP+SIV developed little to no inflammatory responses and no significant

antibody responses were induced in this group. We conclude that induction of inflammatory responses is necessary for the development of antigen-specific immune responses, and the quality of the inflammation is important. Additionally, we propose that the sterile inflammation induced by the adjuvant PCEP is likely mediated through damage-associated molecular patterns (DAMPs) derived from the host following necrotic cell death, which are known to activate inflammatory pathways.

O06Parameters of protective immunity in swine induced by PCV2 vaccines with different antigen payload

Guarneri F.1, Sarli G.2, Boniotti M.B.1, Tresoldi E.T.1, Tudor C.2, Lelli D.1, D’Annunzio G.2, Barbieri I.1, De Tolla L.J.3, Amadori M.1

1IZSLER, Brescia, Italy, 2Bologna University, DIMEVET, Bologna, Italy, 3University of Maryland, School of Medicine, Baltimore, United States

Porcine circovirus associated disease (PCVAD) complex is caused by co-infections by PCV2 and other common pathogens and/or environmental stressors. Different vaccines have been developed to reduce PCV2 infections and PCVAD. Such vaccines are poorly standardized in terms of antigen payload and recognized correlates of protection. Therefore, we selected twenty, 40-day old piglets, and allocated them to 4 groups (5 animals each) with uniform levels of maternally-derived antibody to PCV2. Animals were vaccinated with 450/150/50/0 nanograms

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of an inactivated PCV2b strain, formulated in the same adjuvant of the commercial Circovac vaccine. Twenty-seven days later, all pigs were challenged intranasally with the homologous PCV2 strain. The main findings can be summarized as follows: 1) No clinical signs were observed in the pigs under study. 2) Viremia was observed in all the control pigs, as well as in 2 pigs of the 150 and 50 ng groups, respectively. No pigs of the 450-ng group developed viremia. 3) There was no correlation between protection and ELISA Ab titers in the single animals, even though the 450-ng group developed on average a stronger Ab response. 4) All the pigs with a PCV2-specific IFN-gamma response at 3 weeks after vaccination were fully protected against viremia. The IFN-gamma response at this time point was peculiar to CD4+, single positive T cells, whereas both CD8alpha and CD8 beta+ T cells were also positive after challenge infection. 5) In tissues (mainly tonsils and ileum) the presence of sparse reactive hystiocytes and multinucleated giant cells was the only PCV2-associated feature and, by immunohistochemistry, only 3 out of 20 subjects (still viremic at PID 35) had a low viral load (grade 1 in 10 samples and grade 2 in 2 samples). Our data point at the IFN-gamma release assay as a useful tool for monitoring the efficacy of PCV2 vaccines.

O07Effect of adjuvant on cellular and humoral immune responses to an experimental subunit vaccine antigen in sheep

Wattegedera S.R.1, Stronach K.2, Rampacci E.3, Villasol N.A.4, Tassi R.5, Thomson J.1, Rocchi M.6, Palarea-Albaladejo J.7, Aitchison K.6,

Livingstone M.6, Longbottom D.6, Entrican G.1

1Moredun Research Institute, Vaccines, Penicuik, United Kingdom, 2University of Edinburgh, School of Biological Sciences, Edinburgh, United Kingdom, 3Universita degli Studi di Perugia, Dipartmento di Medicina Veterinaria, Perugia, Italy, 4Universidad de Leon, Department of Animal Health, Leon, Spain, 5Moredun Research Institute, Disease Control, Penicuik, United Kingdom, 6Moredun Research Institute, Diagnostics, Penicuik, United Kingdom, 7Biomathematics and Statistics Scotland, Edinburgh, United Kingdom

Background: The desired goal of vaccines to protect against intracellular bacterial infections is usually the induction of cellular T-helper (Th)-1 type immunity, characterised by production of interferon (IFN)-gamma. This has been particularly challenging for subunit vaccine development in livestock. We have characterised cellular and humoral immune responses in sheep to an experimental chlamydial subunit vaccine antigen delivered in three adjuvants.

Method: Groups of 35 sheep were immunised with a single inoculation of the experimental vaccine antigen formulated in two water-in-oil adjuvants (Montanide ISA70 VG, Montanide ISA61 VG) or saponin-derived QuilA. Peripheral blood mononuclear cells (PBMC) were isolated pre- and post-immunisation and re-stimulated in vitro with both the vaccine antigen and whole killed chlamydial elementary bodies (EBs). Recall responses were measured by the presence of cytokines in the PBMC culture supernates, with IFN-γ as an indicator of Th1-type responses and interleukin (IL)-4 as an indicator of Th2-type responses. Humoral immune responses to chlamydial antigen were also measured.

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Results: All three adjuvants induced antigen-specific cellular immune responses that could be detected in recall assays to both the experimental vaccine antigen and whole chlamydial EBs. In each case, IFN-γ was the dominant cytokine in the post-immunisation antigen-specific recall responses, with almost no antigen-specific IL-4 being detected. Mitogen-induced IFN-γ and IL-4 was consistent pre- and post-immunisation. Of the three adjuvants, Montanide ISA61 VG induced slightly higher levels of IFN-γ. Measurement of specific antibody responses revealed a different profile for the humoral responses in all three adjuvants. Antibody responses were stimulated in all three adjuvants following vaccination, but interestingly, the saponin-derived QuilA produced the highest antibody titres.

Conclusion: Montanide and saponin-based adjuvants differentially elicit antigen-specific Th1-type cytokine and antibody responses to the same candidate subunit vaccine antigen in sheep. These immune responses correlate with vaccine-induced protection, demonstrating the crucial role of adjuvant in livestock vaccination.

O08New adjuvant technologies to optimize mycoplasma hyopneumoniae vaccine

Koziol M.-E.1, Versillé N.2, Xu B.2, Ben Arous J.2, Bertrand F.2

1Seppic Inc, Fairfield, United States, 2Seppic, Paris, France

Mycoplasma hyopneumoniae (M. hyo.) is the primary causative agent of enzootic pneumonia in pig herds. Inactivated vaccines are routinely used in the pig industry. They are considered to be safer compared to live vaccine but they are often less immunogenic. Consequently, adjuvants are required to improve vaccine. In this study, we compared the efficacy of different adjuvant technologies based on Mycoplasma vaccines for swine.

An aqueous micro-emulsion and an experimental water in oil emulsion (W/O) developed by Seppic, were formulated with inactivated swine M.hyo NJ strain. Both vaccines were tested in a pig trial and compared with commercial inactivated vaccine. Eight piglets per group were vaccinated once at D0 with the W/O adjuvant or twice at D0 and D14 for the other groups. Antibody titers were evaluated by ELISA detection at D0, 14, 28 and 35. Then, a heterologous challenge with an highly virulent strain of M hyo. was performed 3 weeks after the first injection. All surviving pigs were autopsied 28 days after challenge and lung lesions were scored.

All tested vaccines induced a good safety profile, no abnormality was observed at the injection site. Antibody titers showed that vaccines based on Seppic adjuvants induced a strong humoral immune response. After challenge, the lowest average lung lesion scores and the lowest frequency of lesions were observed with these adjuvants, especially with W/O based one-shot vaccine.

These results show that W/O adjuvants are efficient adjuvants for the formulation of one-shot inactivated Mycoplasma swine vaccines.

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REGULATION & MODULATION

O09Differential induction of innate training and tolerance in porcine monocytes by β-glucans or BCG

Byrne K.A.1, Palmer M.V.2, Tuggle C.K.3, Loving C.L.1

1USDA-ARS-National Animal Disease Center, Food Safety and Enteric Pathogen Research Unit, Ames, United States, 2USDA-ARS-National Animal Disease Center, Infectious Bacterial Diseases Research Unit, Ames, United States, 3Iowa State University, Animal Science, Ames, United States

Innate memory is characterized by altered (enhance or reduced) nonspecific responsiveness of monocytes after priming with various ligands. β-glucan from Candida albicans and the vaccine strain of Mycobacterium bovis (Bacillus Calmette-Guerin; BCG) induce innate memory in monocytes both in vitro and in vivo. As a method to enhance animal health, we interrogated the ability of β-glucans and BCG to alter the innate memory phenotype in pigs. In a series of in vitro studies, primary porcine monocytes were stimulated for 24h with various β-glucans or the Danish strain of BCG and restimulated after 5d resting withlipopolysaccharide (LPS; TLR 4 agonist) or Pam3CSK4 (synthetic triacylated lipopeptide; TLR 2 agonist) to determine induction of trained or tolerant phenotype (increase or decrease in cytokine production relative to unstimulated controls). Highly purified β-glucan from C. albicans or BCG enhanced IL-1β and TNFα production compared to media

stimulated monocytes when restimulated with either LPS or Pam3CSK4 (trained phenotype); however, monocytes primed with β-glucan from S. cerevisiae exhibited a tolerant phenotype (decreased cytokine production). Ex vivo analyses of innate memory were conducted whereby young pigs were fed dietary β-glucan or injected with BCG and 3-6wks later monocytes were isolated and stimulated in vitro with various TLR ligands. β-glucan from S. cerevisiae induced both a trained and tolerized phenotype ex vivo depending on the source/preparation of β-glucan. These data indicate that β-glucan or BCG can alter the monocyte innate memory phenotype; however, while BCG exclusively induced a trained phenotype, β-glucan can either train or tolerize depending on the source.

O10Contribution of prostaglandin E2 to disease progression and enhancement of antiviral effects by anti-PD-L1 antibody combined with COX-2 inhibitor in bovine leukemia virus infection

Sajiki Y.1, Konnai S.1,2, Okagawa T.2, Maekawa N.2, Goto S.1, Murata S.1,2, Ohashi K.1,2

1Hokkaido University, Department of Disease Control, Faculty of Veterinary Medicine, Sapporo, Japan, 2Hokkaido University, Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Sapporo, Japan

Bovine leukemia virus (BLV) infection is widespread in many countries. Previously, we demonstrated that prostaglandin E2 (PGE2) suppresses immune responses by the

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reduction of Th1 cytokine production and the upregulation of an immunoinhibitory molecule, programmed death-ligand 1 (PD-L1), in cattle, and contributes to the progression of Johne’s disease, a chronic bacterial infection. Additionally, a selective cyclooxygenase (COX)-2 inhibitor, which inhibits PGE2 production, activates Th1 responses in peripheral blood mononuclear cells (PBMCs) from cattle. However, little information is available on the association of PGE2 with chronic viral infection. Therefore, we analyzed the kinetics of PGE2 in BLV infection and antiviral effects of the COX-2 inhibitor with or without anti-PD-L1 antibody in vitro and in vivo.

First, we performed kinetic analysis of PGE2. Plasma PGE2 concentration was increased in BLV-infected cattle and positively correlated with BLV proviral load and PD-L1 expression in B cells. Next, we performed functional analysis of the COX-2 inhibitor. The COX-2 inhibitor promoted T-cell proliferation and the production of IFN-γ and TNF-α from PBMCs of BLV-infected cattle in vitro. Additionally, we conducted a clinical study of treatment of the COX-2 inhibitor using BLV-infected cattle, and this treatment significantly reduced BLV proviral load in vivo. Finally, we evaluated effects of the combined treatment of the COX-2 inhibitor with anti-PD-L1 antibody in vitro and in vivo. The combined treatment enhanced BLV-specific Th1 responses in vitro and dramatically reduced BLV proviral load in vivo.

Our results suggest that PGE2 contributes to the progression of BLV infection, and the COX-2 inhibitor activates Th1 responses and shows antiviral effects in BLV-infected cattle. Moreover, the combined treatment has the potent antiviral effects against BLV infection. Therefore, the present findings would contribute to the development of a novel strategy to control BLV infection.

O11Prophylactic digoxin treatment inhibits IL-17 production in vivo and reduces BRSV-associated disease in the neonatal calf

McGill J.L.1, Guerra-Maupome M.1, Schneider S.2

1Iowa State University, Department of Veterinary Microbiology and Preventive Medicine, Ames, United States, 2Kansas State University, Department of Diagnostic Medicine and Pathobiology, Manhattan, United States

Bovine respiratory disease (BRD) is a leading cause of morbidity and mortality in feedlot and dairy cattle. The pathogenesis of BRDC is not clearly understood. However, much of the lung pathology associated with the disease has been attributed to damage caused by the host immune response. Interleukin-17A (IL-17) is a pro-inflammatory cytokine that plays a critical role in the immune response in the respiratory tract and mucosa. While protective in some situations, IL-17 can also cause immunopathology, primarily through its role as a potent inducer of neutrophil recruitment and activation.Digoxin was recently identified to selectively inhibit IL-17 production by antagonizing its transcription factor, retinoid-related orphan receptor γ t (RORγt). Digoxin inhibits RORγt binding to IL-17 and Th17 associated genes, and suppresses IL-17 production in rodent models and human cells. Here, we demonstrate that in vitro and in vivo digoxin treatment also inhibits IL-17 production by bovine leukocytes. To determine the role of IL-17 in primary RSV infection, calves were treated with digoxin and infected with BRSV. Digoxin treated calves demonstrated reduced signs of clinical illness after BRSV infection, and reduced lung pathology compared to untreated control calves. Digoxin treatment

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did not adversely affect virus shedding or lung viral burden, but had a significant impact on pulmonary inflammatory cytokine expression on day 10 post infection. Together, our results suggest that exacerbated expression of IL-17 has a negative impact on acute viral infection, and that development of specific therapies targeting Th17 immunity may be a promising strategy to improve BRD.

O12Establishment of anti-bovine PD-1 chimeric antibody and a pilot clinical study in bovine leukemia virus infection

Okagawa T.1, Konnai S.1, Maekawa N.1, Nishimori A.1, Goto S.1, Sajiki Y.1, Watari K.1, Kohara J.2, Yamada S.3, Kato Y.3, Nakajima C.4, Suzuki Y.4, Murata S.1, Ohashi K.1

1Hokkaido University, Faculty of Veterinary Medicine, Sapporo, Japan, 2Hokkaido Research Organization, Animal Research Center, Shintoku, Japan, 3Tohoku University, Graduate School of Medicine, Sendai, Japan, 4Hokkaido University, Research Center for Zoonosis Control, Sapporo, Japan

Progressive exhaustion of T-cell functions is considered to facilitate the immunopathogenesis of several chronic infections, where pathogens evade immune elimination and establish persistent infection. We have reported that an immunoinhibitory signaling via programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) downregulates T-cell functions in cattle with bovine leukemia virus (BLV) infection, Johne’s disease, bovine anaplasmosis, and mycoplasmosis. We

therefore hypothesized that blockade of the PD-1/PD-L1 interaction restores the effector function of T cells and prevents disease progression in these diseases. To test this hypothesis, this study aimed to establish the blocking antibody targeting bovine PD-1 molecule, to characterize its biological function, and to confirm its clinical efficacy in a pilot clinical trial of BLV-infected cattle.

Anti-bovine PD-1 bovinized chimeric antibody (chAb) was stably expressed and produced with the use of the CHO-DG44 cell expression system and successfully purified from supernatants using Protein G/A resins. As expected, the heavy and light chains of th chAb were detected at approximately 50 and 25 kDa in SDS-PAGE, respectively.

Flow cytometric and Biacore analyses determined that the binding ability and affinity of the chAb were similar to that of the original anti-bovine PD-1 mAb. The chAb was also capable of blocking PD-1/PD-L1 binding at the same level as the original mAb.

The immunomodulatory and therapeutic effects of the chAb were evaluated by in vivo administration of the antibody to BLV-infected animals. The chAb inoculation resulted in activation of the proliferation of BLV-specific CD4+ T cells and decrease in the proviral load of BLV in the peripheral blood.

This study demonstrates that the established anti-bovine PD-1 chAb retains equivalent biochemical functions to that of the original mAb and anti-PD-1 immunotherapy is a significant strategy for the regulation of anti-viral T-cell response in cattle and prevention of the disease progression of BLV infection.

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O13Modulation of porcine macrophages susceptibility to viral infection by epithelial cell-derived alarmins

García-Nicolás O.1, Summerfield A.2

1Institute of Virology and Immunology, Immunology, Mittelhäusern, Switzerland, 2Institute for Virology and Immunology, University of Bern, Bern, Switzerland

Epithelial cells from either skin or mucosal surfaces as well as stromal cells in lymphoid tissue release “alarmins” in response to tissue and cell damage which act as “damage associated molecular pattern” and activate innate immune responses. Viruses themselves also cause cytopathogenic effects and thereby are expected to induce the release of alarmins. Here, we studied the effect of epithelial cell-derived IL-33, IL-25, and thymic stromal lyphoprotein (TSLP), which are known to activate immune cells like T cells, dendritic cells, and macrophages, normally driving a Th2 immune response which would potentially be counterproductive for a protective antiviral immune response. We therefore investigated the impact of such alarmins on porcine macrophages in terms of its susceptibility to the infection by different viruses with a high tropism for these cells including Japanese encephalitis virus (JEV) and porcine reproductive and respiratory syndrome virus (PRRSV). To this end, porcine monocyte-derived macrophages were stimulated with recombinant IL-25, IL-33 and TSLP, or any possible combination thereof, followed by infection with JEV or PRRSV. These experiments showed that IL-33, IL-25, and TSLP enhance viral infection in macrophages, suggesting that those alarmins could play a

negative role during virus infections. Current studies are underway to determine the putative mode of action of this virus promoting effect.

VIC MHC WORKSHOP

O14Ruminant MHC class II genotyping using cell free DNA recovered from archived plasma and serum samples

Ballingall K.T.1, Hanks E.1,2, McNeilly T.1, Smith D.3, Britton C.2

1Moredun Research Institute, Disease Control, Edinburgh, United Kingdom, 2University of Glasgow, Institute of Biodiversity, Animal Health and Comparative Medicine, Glasgow, United Kingdom, 3Moredun Research Institute, Edinburgh, United Kingdom

Analysis of genetic diversity in ruminant livestock populations has relied on the extraction of genomic DNA from blood or other fresh or archived tissue. Isolated DNA is stable and may be stored long term for retrospective analysis. However, for many large-scale in vivo disease control studies, such as testing novel vaccines or antimicrobials, genomic DNA or tissue samples are not often collected due to financial constraints. As such, retrospective analyses of the role of host genetic diversity in the target population was not thought possible. However, serum or plasma samples are often routinely collected during such trials for analysis of antibody responses, cytokine levels or other inflammatory mediators. With increasing interest in circulating cell free DNA

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(cfDNA) we wished to test whether plasma or serum collected during large scale vaccine trials could provide DNA of sufficient quality for population based genetic analysis. We demonstrated that cfDNA may be extracted from both archived serum and plasma following long term storage. Isolated cfDNA was used to analyse allelic diversity at the highly polymorphic MHC class II DRB1 locus in sheep using a sequence based genotyping method. This allowed a retrospective analysis of allelic diversity in a large cohort of vaccinated sheep. This approach was also successfully applied to sheep serum samples archived at -20 oC for up to 35 years and was equally applicable to archived serum samples from cattle and goats. This is the first such study to examine host MHC diversity using DNA extracted from archived serum and plasma samples, an approach that may be applied to retrospective analyses of genetic diversity and responses to vaccination, infection or inflammation across different species and populations.

O15Nomenclature for factors of the Swine Leukocyte Antigen (SLA) system - Update 2019

Ho S.1, Lunney J.K.2, Ando A.3, Rogel-Gaillard C.4, Lee J.-H.5, Schook L.B.6, Hammer S.E.7

1Gift of Life Michigan, Ann Arbor, United States, 2USDA, ARS, Beltsville, United States, 3Tokai University School of Medicine, Isehara, Japan, 4GABI, INRA, Jouy-en-Josas, France, 5Chungnam National University, Daejeon, Korea, Republic of, 6University of Illinois, Urbana, United States, 7University of Veterinary Medicine Vienna, Vienna, Austria

Background: The SLA system is among the most well characterized MHC systems in non-human animal species.

Method: The SLA Nomenclature Committee was formed in 2002 as a joint ISAG/IUIS-VIC committee, with the primary objectives: 1) to validate newly identified SLA sequences according to the guidelines established for maintaining high quality standards of the accepted sequences; 2) to assign appropriate nomenclatures for new alleles as they are validated; and 3) to serve as a curator of the IPD-MHC SLA sequence database (https://www.ebi.ac.uk/ipd/mhc/group/SLA), the repository for all recognized SLA genes, their allelic sequences and haplotypes.

Results: In 2016, the Committee began re-designating each allele an official number, adopting the HLA Nomenclature System with colons as field separators (e.g. SLA-1*01rh28 ª SLA-1*01:03). Phylogeny will remain the primary approach for assigning SLA-1, -2, -3, DRA, DRB1, DQA and DQB1 alleles into allele groups with similar sequence motifs, while alleles of the other loci including SLA-4, -5, -6, -7, -8, -9, -11, -12, DMA, DMB, DOA, DOB1, DOB2, DQB2, DRB2, DRB3, DRB4, DRB5, DYB, MIC1, MIC2, TAP1 and TAP2, are designated sequentially as they are discovered. There are currently 266 class I, 227 class II, 2 SLA-related and 2 non-SLA alleles officially designated. There are also 73 class I (SLA-1-3-2) and 52 class II (DRB1-DQB1) haplotypes designated at allele level resolution. Recent evidence has suggested certain loci in the SLA system, previously recognized as pseudogenes (e.g. SLA-9, SLA-11, DQB2, DOB2), may be expressed at the transcript level. The committee will consider reclassifying some of these loci as putative functional genes as additional evidence accumulates.

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Conclusions: Continuous efforts on characterizing SLA alleles and haplotypes and studying of their diversity in various pig populations will further deepen our understanding of the architecture and polymorphism of the SLA system and their role in disease, vaccine and transplant responses.

O16New highly-contiguous cattle genome assemblies reveal the extent of genetic variation underlying differential and adaptive immune responses

Schwartz J.C.1, Bickhart D.M.2, Smith T.P.L.3, Hammond J.1

1The Pirbright Institute, Woking, United Kingdom, 2Cell Wall Biology and Utilization Research, USDA-ARS, Madison, United States, 3Meat Animal Research Center USDA-ARS, Clay Centre, United States

Recent advances in whole genome sequencing and assembly have enabled the structure and gene content of repetitive immune-related gene complexes to be automatically resolved for the first time. This has allowed the first detailed examination of variation within and between immune-related gene complexes from four cattle genome assemblies, Hereford, angus, brahman and highland. This variation was then compared with the closely related domesticated yak, which shared a common ancestor with cattle approximately two million years ago.

We have studies the major histocompatibility complex (MHC), leukocyte receptor complex (LRC), natural killer complex (NKC), T cell

receptor (TCR) loci and B cell receptor (BCR) loci. These are all fundamentally important and rapidly evolving genomic regions that create and drive variable innate and adaptive immune responses. In total these complexes span over 6 mb and contain several hundred genes and gene segments.

Between the cattle genomes in this study, the gross organisation of all these genomic regions is largely conserved but gene content and levels of polymorphism vary significantly. The MHC class I region has the most dynamic size range as gene expansion or contraction appears to happen between relatively large blocks. Haplotypic variation is most variable in the LRC gene families including the leukocyte immunoglobulin-like receptors and the killer-cell immunoglobulin receptors. The NKC is more structurally conserved but contains highly expanded gene families in which polymorphism is concentrated. For the rearranging receptors (TCR and BCR) our preliminary data indicates a much higher level of polymorphism and V gene diversity than expected.

This data provides a unique and high-resolution window on the recent and dynamic evolution of the cattle immune system. Understanding and accounting for this variation when studying immune responses at the individual and population level will be an important future advance.

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O17Characterization of expressed major histocompatibility complex class I and class II alleles and haplotypes in domestic cat (Felis catus) by NGS-based amplicon sequencing

Okano M.1, Miyamae J.1, Nishiya K.1, Katakura F.1, Moritomo T.1, Shiina T.2

1Nihon University School of Bio Resource Science, Department of Veterinary Medicine, Fujisawa, Japan, 2Tokai University School of Medicine, Department of Molecular Life Science, Isehara, Japan

The major histocompatibility complex (MHC) genes are known to highly polymorphic genes and encode cell surface molecules linked antigen-presenting. The need of typing cat MHC (Feline Leukocyte Antigen, FLA) is increasing for association analysis with infectious diseases including zoonosis transmissible to humans and models for human viral immunity. However, information of FLA polymorphism is limited in comparison to other animals. In this study to better understand degree and types of FLA polymorphisms and haplotypes in domestic cats (Felis catus), we newly developed NGS-based genotyping methods for the FLA-class I and FLA-class II (FLA-DRB) loci and estimated haplotypes using four families of 20 domestic cats.

Complementary DNA samples were synthesized using total RNA that were extracted from peripheral blood cells. Primer pairs for the FLA-class I and FLA-DRB loci were designed in conserved exons of known FLA-class I and FLA-DRB allele sequences, respectively, and used separately for the amplification of FLA-class I and FLA-DRB loci. Sequence reads were generated from the

PCR products by amplicon sequencing method using the Ion PGM semiconductor sequencer. The FLA allele sequences were determined with a combination of de novo assembly among the reads and mapping the reads to known and newly determined FLA allele sequences.

In total 30 FLA-class I and 19 FLA-DRB allele sequences were identified by the amplicon sequencing analysis. Of them 17 FLA-class I and 9 FLA-DRB alleles were newly identified in this study. From the FLA allele sequence information 7 FLA-class I and 9 FLA-DRB haplotypes were estimated without discrepancy of familial relationships, and the haplotypes were composed of expressed 4 to 7 FLA-class I and 2 to 6 FLA-DRB loci.

Newly developed NGS-based FLA typing method is more outstanding and useful to detect novel alleles and resolve complicated copy number variation among FLA genes than previous Sanger sequencing method.

O18Impaired SLA class I antigen presentation: an explanation for cellular immune dysregulation in ASFV infected pigs

Blohm U.1, Hühr J.1, Hartmann L.1, Knittler M.1, Blome S.2

1Friedrich-Loeffler-Institute, Institute of Immunology, Greifswald, Germany, 2Friedrich-Loeffler-Institute, Institute of Diagnostic Virology, Greifswald, Germany

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Background: African swine fever virus (ASFV), a large double-stranded DNA virus, causes a devastating disease in pigs with mortality rates up to 100 percent. Since the disease first appeared in the Caucasus region in 2007, it has spread rapidly and is now present in several EU countries and Asia. While the African reservoir host, the common warthog, does not show clinical signs, a hemorrhagic fever like illness is seen in domestic pigs and European wild boar. The severity of clinical signs depends on strain virulence and different host factors. An effective and safe vaccine is not yet available. Therefore, research on immune dysfunction and pathogenesis is mandatory.

Methods: We established a flow cytometry-based multicolor analysis to identify T cell subtypes and their functional impairment in ASFV infected pigs. In in vitro analyses, we investigated the SLA I -mediated antigen presentation of macrophages infected with different ASFV strains (virulent and attenuated strains).

Results: Pigs infected with the highly virulent ASFV “Armenia08” show lymphopenia. Neither in the blood nor in the lymphoid organs a proliferation of CD8+ effector cells was observed. Furthermore, a T-bet or Eomes dependent activation of the remaining CD8 T cells did not occur.

In vitro experiments with ASFV “Armenia08” infected macrophages show a reduction of TAP1 and a restricted delivery of antigenic peptides into the ER. Furthermore, we detected a reduced maturation of peptide-loaded SLA I molecules, which leads to a measurable reduction of antigen presentation on the surface of the infected macrophages. These effects are specific for virulent ASFV and not (or to less extend) for an attenuated ASFV.

Conclusion: The immune dysregulation in acutely infected pigs can be explained by viral immune evasion, which leads to reduced SLA I loading and in consequence to disturbed antigen presentation.

AVIAN IMMUNOLOGY (CEVA

SPONSORED)

O19Needle-free oral vaccination of surface flagellin coated Salmonella nanovaccine for chickens

Renu S.1, Han Y.1, Lakshmanappa S.Y.1, Dhakal S.1, Feliciano-Ruiz N.1, Ghimire S.1, Selvaraj R.2, Renukaradhya G.1

1The Ohio State University, Food Animal Health Research Program, Wooster, United States, 2University of Georgia, Department of Poultry Science, Athens, United States

Background: Salmonella contamination in poultry meat and eggs causes severe economic impact and human health problems. Oral delivery of inactivated/subunit Salmonella vaccine targeting the intestinal lymphoid tissues is ideal, as it induces robust mucosal immunity and overcomes injection stress. Soluble antigens delivered oral gets degraded in stomach, thus identifying a suitable vaccine carrier is essential. A mucoadhesive chitosan nanoparticles (CS-NPs) based subunit Salmonella vaccine (nanovaccine) containing outer membrane proteins and flagellar (F) protein loaded and F-protein surface coated was formulated for oral delivery in poultry.

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Method: Salmonella nanovaccine was formulated using the ionic gelation method, and characterized by analytical techniques, biocompatibility and pH stability by hemolysis and turbidity assays. We found F-protein surface coated CS-NPs (CS-NPs-F) targeted to chicken intestinal Peyer’s patch sites by in vivo and ex vivo analyses by microscopy. Vaccinated layer chickens with nanovaccine by mixing in drinking water or feed challenged with live Salmonella were examined for induction of immune response and efficacy.

Results: Salmonella nanovaccine particles were spherical with average particle size 398 nm, and monodispersed with a high surface positive charge. Spectroscopic analyses showed CS-NPs compatible with chicken RBCs and stable in acidic pH condition. Fluorescent microscopy and F-protein specific immunostaining analysis confirmed the oral delivered CS-NPs-F specifically targeted to intestinal Peyer’s patches and up taken by chicken immune cells. Salmonella nanovaccine treated chicken immune cells upregulated the mRNA expression of TLR, Th1 and Th2 immune markers. In Salmonella nanovaccine immunized layer chickens, administered through drinking water and feed induced secretory IgA antibody response. Notably, drinking water and feed mixed nanovaccine induced significant and non-significant reduction in challenge bacterial load in the ceca of chickens, respectively.

Conclusion: Needle-free oral immunization of nanovaccine enhanced the local immune response associated with reduced bacterial load, thus likely be a potential alternate to injectable Salmonella vaccine for poultry.

O20Proof of concept studies for novel necrotic enteritis vaccine in broilers

Duff A.1, Searer K.1, Vuong C.2, Briggs W.1, Wilson K.1, Chasser K.1, Hargis B.2, Berghman L.3, Bielke L.1

1Ohio State University, Wooster, United States, 2University of Arkansas, Poultry Science, Fayetteville, United States, 3Texas A&M University, Depts of Veterinary Pathology and Poultry Science, College Station, United States

Clostridium perfringens (CP) possesses numerous toxins and virulence factors that affect onset of necrotic enteritis (NE). Importantly, glycoside hydrolases (GH) break down mucin in the host GIT as an energy source. Vaccination against GH may be an important component for controlling NE, and subunit vaccine technology targeting CP and Eimeria virulence factors may be a promising control strategy. Preliminary experiments evaluated antigenic peptides from CP mucolytic GH components. Based on in vitro CP growth inhibition assays and an in vivo NE vaccination test in broilers, 5 peptides were combined (MC) for proof of concept testing. In vivo experiments tested MC with previously developed alpha-toxin antigens (aTx) and an anticoccidial subunit vaccine targeting Eimeria proteins, TRAP and MPP (PTM). Treatments included a nonvaccinated control (NVCC), MC, PTM, PTM+MC, and PTM+MC+aTx groups. Vaccinated birds received vaccination on day of hatch and 14d prior to Eimeria maxima (EM) and CP induced NE. In Exp1, MC birds had the greatest numerical increase in body weight gain (BWG, 301.83±20.56g) amongst vaccinated groups. Lesion scores (LS) of PTM+MC+aTx group had a greater distribution of low scores. In Exp2, PTM+MC+aTx had a numerical

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increase in %ChangeBWG relative to NVCC (7.42±6.37%), and all vaccinated groups had a numerical increase in %ChangeBWG in Exp3. All vaccinated groups in Exp2 had a higher percentage of LS at 0-1, and PTM+MC+aTx was lower than NVCC with a mean score of 0.39 (p< 0.05). Exp3 failed to elicit differences in LS and contained a broader range of scores than captured in Exp2, but PTM+MC+aTx had a lower distribution of lesion scores. Throughout these proof of concept studies, PTM+MC+aTx vaccine provided the most encouraging results suggestive of alleviating performance losses associated with NE in broilers. These experiments provide evidence that merit future testing, development and evaluation of this NE vaccine and its components.

O21The importance of monitoring live vaccines application for obtaining immunological protection

Davidson I.

Kimron Veterinary Institute, Avian Diseases, Bet Dagan, Israel

Background: Protection against viral diseases is achieved by vaccination. Important factors for vaccination efficacy are the vaccine virus, its configuration, host immunological compatibility, but also the vaccine appropriate application. Vaccines can be administered by individual bird injections, manually or automatically, or mass-vaccination via drinking water, spray or egg inoculation. The live vaccine application issues attracted less scientific attention than vaccine development. The studied 4 avian live vaccine

viruses, two herpesviruses, Marek´s disease and Infectious laryngotracheitis, a circovirus, chicken anemia and a flavivirus, turkey meningoencephalitis virus in commercial flocks to reflect actual settings.

Method: The vaccine viruses were demonstrated in feathers, as a convenient organ, easy and not invasive collectible sample. Vaccine application monitoring was demonstrated innovatively by nested real-time amplification, as vaccine viruses are less abundant in vivo as compared to wild-type isolates.

Results: The Marek´s disease virus vaccine flock was detected from 4-7 dpv and for 3 months, until the survey stopped. Infectious laryngotracheitis vaccination, given in drinking water was detected in feathers between 2-20 dpv. The chicken anemia vaccine virus presence in feathers was demonstrated between 14-35 dpv. The Turkey meningoencephalitis vaccine virus was the first turkey virus amplified from feather-pulps, with kinetics that resemble those of the other 3 viruses, 3-21 dpv.

Conclusions: The study presented an innovative approach for evaluating the quality of vaccine application by reflecting the viremia following vaccination. The present approach was put in practice in several commercial flocks, where several problematic circumstances were identified.

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ΓΔ, NK, NKT CELLS

O22Functional profiling and dynamics of naïve porcine invariant Natural Killer T cells

Schäfer A.1, Schwaiger T.2, Dorhoi A.1, Mettenleiter T.C.3, Schröder C.2, Blohm U.1

1Friedrich-Loeffler-Institut, Institute of Immunology, Greifswald - Insel Riems, Germany, 2Friedrich-Loeffler-Institut, Department of Experimental Animal Facilities and Biorisk Management, Greifswald - Insel Riems, Germany, 3Friedrich-Loeffler-Institut, Institute of Molecular Virology and Cell Biology, Greifswald - Insel Riems, Germany

Background: Pigs are one of the economically most important domesticated species. To improve vaccines and therapies, it is pivotal to understand their immune responses to infection. Moreover, significant similarities between human and swine physiology suggest that pigs are a superior animal model for immunological studies. At present, studies in pigs are limited by a lack of research tools. Therefore, we aimed at increasing the knowledge of a population of unconventional lymphocytes, invariant Natural Killer T (iNKT) cells, and evaluating the pig as a biomedical model.

Methods: We established a flow cytometry-based multicolor analysis platform for the phenotypic characterization and functional analysis of cellular responses of porcine iNKT cells.

Results: Porcine iNKT cells in steady-state were CD3+/CD4-/CD8+ or CD3+/CD4-/CD8-

and exhibited an effector- and memory-like phenotype (CD25+/ICOS+/CD5hi/CD45RA-/CCR7±/CD27+). Using antibodies against the transcription factors Tbet and PLZF, we were able to differentiate functional iNKT cell subsets. Similar to iNKT cells in humans, porcine iNKT cells proliferated rapidly upon in vitro stimulation with the CD1d-specific ligand αgalactosylceramide, as shown by an increase in frequency among CD3+ lymphocytes and Ki67 expression. Activated porcine iNKT cells also displayed an upregulated expression of CD25, CD5, ICOS, and MHC II and induction of interferonγ and perforin. In naïve animals, iNKT cell frequency was regulated in an age-dependent manner.

Conclusion: Our detailed characterization of porcine iNKT cells paves the way to a more thorough understanding of porcine immune responses, enabling new innovative interventions against infectious diseases. Additionally, we provide further evidence that supports the suitability of pigs as a new biomedical model for iNKT cell research.

O23Swine γδ T cell subpopulations defined by WC1 and TCR gene expression

Gillespie A.1, Le Page L.1, Yirsaw A.1, Mair K.H.2,3, Gerner W.2,3, Saalmüller A.2, Baldwin C.L.1, Telfer J.C.1, Hammer S.E.2

1Department of Veterinary & Animal Sciences, University of Massachusetts Amherst, Amherst, United States, 2Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria,

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3CD Laboratory for Optimized Prediction of Vaccination Success in Pigs, Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria

Background: γδ T cells actively participate in protective immunity against infectious disease-causing organisms. In γδ-high species, such as ruminants and other artiodactyls, many γδ T cells bear the lineage-specific marker WC1. WC1 is a group B scavenger receptor and is encoded by a multigenic array. In swine, a subset of porcine γδ T cells can be identified by monoclonal antibodies recognizing a surface molecule of 180 kDa with the tentative designation Swine Workshop Cluster 5 (SWC5). We hypothesize that SWC5 may be one of the porcine orthologues of the WC1 molecules found in ruminants. In support of this, porcine CD2+ γδ T cells do not express SWC5, similar to the lack of WC1 by bovine CD2+ γδ T cells. In addition, CD2− γδ T cells in ruminants and swine express a shorter TRGC chain than the other TRGCs found in those species. There are 10 swine WC1 genes divided into three different types.

Method: Porcine CD2+ and CD2− γδ T cells were sorted by flow cytometry and evaluated for expression of TRG genes and WC1. Gene-specific primers were designed targeting TCR and WC1 genes as well as primer sets that were common to multiple genes. RT-PCR and subsequent cloning and sequencing was used to define transcription within the subpopulations.

Results: Phylogenetic analyses of TRG genes for cattle and swine showed striking homology between species for the genes expressed by bovine WC1+ and porcine CD2− γδ T cells. As we had hypothesized, CD2+SWC5− γδ T cells are negative for WC1 being consistent

with CD2+ cells in cattle; whereas within the CD2− subpopulations, WC1 molecules are differentially expressed.

Conclusions: Bovine and porcine γδ T cells still share much of the genetic layout of their common evolutionary ancestor, highlighting the benefits of comparative research efforts on this prominent T-cell subset in γδ-high species.

O24Characterization of goat gd T cells and responses of WC1+ gd T cells to pathogens

Yirsaw A.W.1, Gillespie A.1, Smith T.P.2, Telfer J.C.1, Baldwin C.L.1

1University of Massachusetts Amherst, Veterinary and Animal Science, Amherst, United States, 2USDA ARS, Clay Center, United States

Background: Vaccines are inadequate or not available for some goat diseases. Conventional B and T cells are the targets of past vaccines. Our focus is on next generation vaccines that target nonconventional lymphocytes including γδ T cells since their stimulation has been shown to contribute to protective immunity in mammals by bridging innate and adaptive responses. Our aims are to define WC1 gene family, determine the percentages of ϒδ T cell subpopulations in blood and their functions.

Method: Genome annotation was conducted to define the goat WC1 gene number and coding sequences. cDNA cloning and Sanger and PacBio sequencing was used to produce evidence for the WC1 genes and

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intracytoplasmic domain splice variants. MAbs were used to define γδ T cells population and subpopulations.

Results: 15 complete and 15 partials WC1 genes were annotated and full-length cDNA evidence presented for 12 of them. Goats are estimated to have 27 WC1 genes based on their unique a1 domains. They have 7 WC1 gene structures, 3 of them as cattle but 4 are unique. Goats WC1 endodomain structures have splice variants which is unique relative to cattle. The percentages of ϒδ, CD4 and CD8 T cells were similar in PBMC and there was no change between age groups, unlike in sheep and cattle. The WC1.1+ and WC1.2+

subpopulations account only for 50% and 20% of total gd T cells, respectively. Goat ϒδ T cells responded to leptospira and mycobacteria.

Conclusions: ϒδ T cells are similar in representation in the blood of goats as in cattle however they have more than twice in the number of WC1 genes. The presence of intracytoplasmic tails splice variants might indicate additional signaling pathways. The proliferation of WC1+ ϒδ T cells indicate a protective role for pathogens or vaccine response.

Keywords: ϒδ T cells, WC1, splice variants

O25Porcine γδ T cells display unique phenotypes by intestinal compartment and time post-weaning

Wiarda J.E.1,2, Byrne K.A.1, De Mille C.M.3, Gabler N.K.3, Loving C.L.1

1National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Food Safety and Enteric Pathogens Research Unit, Ames, United States, 2College of Veterinary Medicine, Iowa State University, Immunobiology Graduate Program, Department of Veterinary Microbiology and Preventative Medicine, Ames, United States, 3Iowa State University, Department of Animal Science, Ames, United States

Immune cells line the intestinal tract to block entry of environmental organisms. Piglet weaning stress can disrupt the balance between intestinal immune tolerance and activation, but dietary antimicrobials can modulate intestinal homeostasis and limit negative consequences of weaning. Alterations in porcine intestinal γδ T cell populations with respect to weaning and dietary antimicrobials have not been well characterized. Moreover, the study of single intestinal compartments is often generalized to the entire intestine, and investigation of γδ T cell populations within compartments of both the small and large intestine of pigs is lacking. To better characterize intestinal γδ T cells and changes associated with weaning, cells from jejunum, ileum, cecum, and colon of piglets fed control diets were isolated at 7, 21, and 35 days post-weaning (dpw) and characterized by flow cytometry. Ileal cells from piglets receiving dietary sub-therapeutic antibiotic or therapeutic zinc oxide were also evaluated. All tissues averaged 38% CD3+γδ+

cells at 7dpw, but by 21dpw, CD3+γδ+ cells rose to 53% and fell to 26% in large and small intestinal tissues, respectively. More than 60% of all intestinal γδ T cells were CD2+CD8α+, suggesting a predominately regulatory phenotype. However, the jejunum had a higher proportion of CD2+CD8α- γδ T cells than other tissues, suggesting a greater presence of pro-inflammatory γδ T cells. CD27, a co-stimulatory

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molecule down-regulated upon T cell activation, was detected on >60% of γδ T cells across intestinal compartments at 7dpw, but the proportion of CD27+ γδ T cells decreased over time, indicating increased activation status post-weaning. Ileal γδ T cells tended to remain CD27+overtime, suggesting reduced activation in the ileal compartment. Dietary antimicrobials had little to no effect on ileal γδ T cell phenotypes. Collectively, γδ T cells of different intestinal compartments display unique phenotypes that cannot be generalized to the entire intestine.

O26Effect of early life exposure to adult microbiota on the function of intestinal NK cells in broiler chickens

Meijerink N.1, Van Haarlem D.A.1, Kers J.G.2, Velkers F.C.2, Lamot D.M.3, De Oliveira J.E.3, Stegeman A.J.2, Rutten V.P.M.G.1, Jansen C.A.1

1Utrecht University, Infectious Diseases and Immunology, Utrecht, Netherlands, 2Utrecht University, Farm Animal Health, Utrecht, Netherlands, 3Cargill Animal Nutrition Innovation Center, Velddriel, Netherlands

Studies in mammals have shown that gut microbiota can affect natural killer (NK)-cells locally and systemically and thereby shape innate immunity. To assess whether this also applies to chickens, we investigated the association between gut microbiota establishment and NK-cell development in young broiler chickens. We hypothesize that administration of adult microbiota, as compared to naturally present microbiota,

will have differential effect on presence and differentiation of intestinal NK-cells.

Broiler chickens (Ross 308) were inoculated at hatch with Aviguard® (intestinal bacteria of adult SPF chickens; MSD Animal Health) or PBS as control. Chickens were sacrificed at day 3, 7, 14, 21 and 35; ileum, spleen and blood were collected, and lymphocytes were isolated. The presence of cytokine-producing (IL-2R+/28-4+) and cytotoxic (20E5+) NK-cells, γδ-T-cells and cytotoxic T(Tc)-cells was analysed by flowcytometry and NK activation assessed (increased surface expression of CD107). Furthermore, ileal and cecal content were collected for microbiota analysis.

At day 3, percentages of IL-2R+ NK-cells in the intestine of Aviguard inoculated birds tended to be higher compared to control birds (4.46±0.93% vs 2.50±0.84%,p=0.09). Also, NK-cell activation was slightly higher in Aviguard inoculated birds (10.52±0.70% vs 8.07±0.47%,p=0.09). Interestingly, this inoculation also resulted in increased CD8αα Tc-cell percentages in intestine at day 14 (14.40±0.88% vs 10.99±1.21%,p=0.03) and day 21 (16.36±1.24% vs 11.29±0.97%,p=0.01). These differences were only observed in the intestine and not in spleen and blood.

Inoculation with adult microbiota resulted in an increase in cytokine-producing and activated NK-cells in the intestine during the first week of life. Whether NK phenotype and activation is associated with microbiota composition will be determined based on microbiota analysis (results pending). Since cytokine-producing NK-cells have immune regulatory properties and can mature into cytotoxic NK-cells, early life exposure to adult microbiota may be a strategy to strengthen the first line of defence in young (broiler) chickens.

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O27Classification of WC1 gene family in Sus scrofa and evaluation of individual SRCR domain affinity for Mycobacterium bovis and Leptospira spp.

Le Page L., Gillespie A., Yirsaw A., Baldwin C.L., Telfer J.C.

University of Massachusetts Amherst, Veterinary & Animal Science, Amherst, United States

Background: WC1, a member of the group B Scavenger Receptor Cysteine Rich (SRCR) superfamily, is found in the genomes of most mammals and birds, and is expressed exclusively on γδ T cells in ruminants. Bovine WC1 molecules contain up to eleven extracellular SRCR domains, organized in the SRCR domain pattern of a1-[b2-c3-d4-e5-d6]-[b7-c8-d9-e10-d’11], where the alphabet designations indicate homology between genes and across species. We characterized 13 distinct genes in cattle. WC1-3, but not WC1-4, expressing γδ T cells respond to Leptospira. This is correlated with direct WC1-3, but not WC1-4, binding to Leptospira via its SRCR domains. Because WC1+ γδ T cells share a restriction in their γδ TCR and WC1 has TCR co-receptor activity, we hypothesize that WC1 co-ligation with the TCR plays the determining role in the activation of WC1+ γδ T cells by pathogens. Swine belong to the same order as cattle, Artiodactyl, and have WC1+ γδ T cells. WC1 is also closely related to the PRRSV receptor CD163A.

Method: We used 5’/ 3’ RACE PCR and RT-PCR to obtain full-length clones and expressed them. Maker was used to annotate the Sus scrofa 11.1 genomic assembly. Bacterial pull-down and far western assays were used to

assess bacterial binding.

Results: We obtained cDNA evidence for ten WC1 genes with the SRCR domain patterns of a1-[b-c-d-e-d’] or d1-[b-c-d-e-d’]. We confirmed the presence of seven of these genes in the genome and identified four unique exon-intron gene structures. Multiple SRCR domains from different WC1 genes bind to vaccine strain Leptospira spp, and freshly grown Pasteur and Danish strains of Mycobacterium bovis.

Conclusion: Classification of WC1 genes, and their role in the interaction of 𝛾δT cells with pathogens relevant to swine, will allow these cells to be recruited in next generation vaccines to pathogens that have significant negative economic impact.

MUCOSAL IMMUNITY

O28Potential role of Peyer´s Patch B cells in immune regulation in intestine of sheep

Jimbo S.1, Griebel P.J.2, Mutwiri G.2

1Government of Alberta, Alberta Environment and Parks, Vegreville, Canada, 2University of Saskatchewan, School of Public Health & VIDO Inter-Vac, Saskatoon, Canada

Regulation of Toll-like receptor (TLR) responses in the intestine is required to prevent unnecessary responses to commensal microorganisms and maintain tissue homeostasis. Peyer’s patches (PP) are the primary sites for the induction of immune

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responses in the intestine, but it is not known how immune responses are regulated in PP. We investigated the potential role of PP B cells in this regulation. We have identified two populations of B cells in PP of sheep: Effector (Beff) and regulatory B (Breg) cells . Beff cells secreted the cytokines IFN-gamma and IL-12, cytokines that play an important role in the generation of immune responses. In contrast, Breg cells secreted IL-10, a known immunoregulatory cytokine, which suppressed IFN-gamma and IL-12 responses in immune cells stimulated with the Toll-like receptor (TLR)-9 agonist, CpG DNA. We further showed that Breg cells existed in two forms: constitutive(c) and inducible(i). Constitutive Breg cells spontaneously secreted IL-10 and do not require stimulation with CpG DNA. In contrast, (i)Breg cells required stimulation with CpG DNA to secrete IL-10. Furthermore, Breg

cells were found in other lymphoid tissues; namely mesenteric lymph nodes and spleen, although their frequency varies. We conclude that Breg cells regulate immune responses to TLR stimulation in PP, and may play an important role in the maintenance of intestinal homeostasis.

O29Porcine small intestinal organoids as a model to study epithelial innate immune responses to enteropathogens

Vermeire B., Jansens R.J.J., Cox E., Devriendt B.

Ghent University, Laboratory of Veterinary Immunology, Merelbeke, Belgium

The small intestinal epithelium plays a pivotal role in gut homeostasis, acting both as a physical barrier and a regulating nexus for intestinal immunity to enteropathogens and the microbiota. Although intestinal epithelial cell lines have been valuable in unraveling epithelial responses during steady state and infection, they only offer a simplistic view on the intestinal epithelium as they lack a complex three-dimensional structure and represent a single intestinal epithelial cell type. Intestinal organoids represent a better model to study host-pathogen interactions at the epithelial surface. These stem cell-derived intestinal organoids comprise all six intestinal epithelial cell types and closely mimic the microarchitecture of the intestine. Here, we show the development of porcine intestinal organoids from stem cells isolated from different regions of the small intestine. Using live cell microscopy and a swelling assay, we further show that these porcine small intestinal organoids respond in a similar manner as in vivo gut tissues to infection with enterotoxigenic E. coli. This small intestinal enteropathogen causes postweaning diarrhea in piglets due to secretion of enterotoxin upon colonization of the gut. Upon stimulation with these enterotoxins, small intestinal organoids not only display a dysregulated electrolyte and water balance as shown by their acute swelling, but also secrete inflammation markers such as the chemokine interleukin 8. Hence, these enteroids closely mimic in vivo intestinal epithelial responses to enteropathogens and are a promising model to study host-pathogen interactions in the pig gut. The insights gained from this organoid model might accelerate the design of veterinary therapeutics to combat enteric diseases.

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TICK & PARASITE IMMUNITY

O30In silico characterization of multi-epitope anti-Rhipicephalus microplus tick vaccine constructions composed by epitopes from tick salivary proteins selected by phage display and immunoinformatics approaches

Fisch A., De Almeida L.G.N., Daher I.P., De Moraes A., Pena M.S., Brunato M.L., Santos I.K.F.M., Ferreira B.R.

University of Sao Paulo, Ribeirao Preto, Brazil

Background: Ticks are vectors of pathogens for cattle and promote losses in bovine production. Due to the importance of saliva during tick feeding, the development of multi-epitope vaccines that blocks salivary proteins activities could be a useful strategy to control R. microplus tick infestations.

Methods: Mimotopes from salivary tick proteins identified by phage display and in silico B-cell epitope mapping (Bebipred2.0 and Epitopia servers) were selected for vaccine constructions. They were fused together sequentially or with one of the following linkers between them: GGGGS, GSGSGS, GPGPG, KK or PAPAP. For purification, sequences received a c-terminal 6xhis-tag positioned after an EAAAK spacer. Predicted physicochemical parameters, protein solubility and allergenicity were accessed through Expasy ProtParam, Protein-Sol and AlgPred tools, respectively. MHC-II epitope prediction was performed with NetMHCIIpan3.2 server using a reference set of 15 HLA-DR and 6 HLA-DQ human alleles.

Results: 44 mimotopes were selected for vaccine design (6 distinct sequences). Molecular weight of the constructions ranges from 60 to 7.6 kDa. All of them presented estimated half-life higher than 10h in E. coli and 20h in yeast. Most constructions were predicted to be slightly acid (pI=6.65), while the KK-construction was predicted to be basic (pI=10,27). Inclusion of linkers promoted higher solubility of the multi-epitope constructions (average index=0.364) compared with the one without linkers (index=0.138), and KK-construction presented the higher solubility index (0.455). Only construction with GGGGS, GPGPG and KK linkers were predicted to be stable. None of the sequences were predicted to be allergenic. The construction without linkers depicted the higher occurrence of MHCII strong binders (95) predicted to bind a total of 16/21 alleles.

Conclusion: The constructions based in the fusion of the mimotopes without linkers or with KK-linkers can be recombinantly expressed and are potentially immunogenic, thus could be useful as anti-R. microplus vaccines. Financial support: FAPESP (2015/09683-9,2018/235798), CAPES.

O31Protective adaptive immune transcriptomic evaluation of PBMC isolated from sheep after infection and challenge with E. ruminantium Welgevonden strain

Nefefe T.1,2, Liebenberg J.1, Van Kleef M.1,2, Steyn H.1, Pretorius A.1,2

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1Agricultural Research Council Onderstepoort Veterinary Institute, Onderstepoort, South Africa, 2Department of Veterinary Tropical Diseases, University of Pretoria, Onderstepoort, South Africa

Heartwater is one of the most economically important tick-borne fatal disease of livestock. The disease is caused by the bacterium E. ruminantium transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma (IFNγ) controls E. ruminantium growth and that cellular immune responses could be protective, an effective recombinant vaccine for this disease is lacking. An overall analysis of which immune pathways are up- or downregulated in sheep peripheral blood mononuclear cells (PBMC) is expected to lead to a better understanding of protective-adaptive immune response of sheep to E. ruminantium infection. Consequently, a systems biology oriented approach following the infection with E. ruminantium was investigated from PBMC to aid recombinant vaccine development. This study reports on sheep experimentally infected with E. ruminantium infected ticks and transcriptome profiling of their PBMCs compared to their own naïve PBMC collected before infection using Illumina RNA-sequencing (RNASeq). Blood collected daily from sheep infected/uninfected with E. ruminantium was processed for flow cytometry, cytokine profiling by qRT-PCR and RNA-Seq. The behavioural pattern of lymphocytes cells (B and T-cells) and the expressed cytokines during primary and secondary infection (challenge) of sheep with E. ruminantium were determined. In addition, the circulation of CD4+ and CD8+ T cells was also linked to the production of cytokines. Cytokine qPCR indicated that IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-12, TGF-β and TNF-α were expressed during the febrile response of all the animals and IFN-γ and TNF-α after secondary infection. The dominancy of T and B cell pathways during

secondary infection in protected animals was determined using transcriptome sequencing. As per our knowledge, this is the first study that describes host transcriptomics of E. ruminantium infection coupled with activated adaptive immune related transcripts. The data provides useful information to further development of heartwater recombinant vaccine.

O32Investigation of the Theileria parva sporozoite surface glycan repertoire and binding C-type lectin receptors

Kolakowski J.1, Shiels B.2, Lepenies B.3, Ngugi D.1, Connelley T.K.4, Werling D.1

1Royal Veterinary College, Hatfield, United Kingdom, 2University of Glasgow, Institute of Biodiversity, Animal Health and Comparative Medicine, Glasgow, United Kingdom, 3University of Veterinary Medicine, Immunology Unit and Research Center for Emerging Infections and Zoonoses, Hannover, Germany, 4University of Edinburgh, The Roslin Institute, Edinburgh, United Kingdom

East Coast Fever is one of the most important diseases in sub-Saharan Africa and kills annually more than one million cattle. The causative agent, Theileria parva, is a tick-borne apicomplexan parasite with obligate differentiation steps in bovine blood cells. Successful treatment of the disease with the antiprotozoal drug buparvaquone is impaired by increasing resistances and the current prevention strategy, the so-called “Infection and Treatment Method”, is not cross-protective

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between different T. parva strains. High genetic complexity of the parasite, immunodominance as well as MHC class I restriction of the cytotoxic CD8+ T cell response significantly limit traditional vaccine development. One potential solution are glycoconjugate vaccines based on sugar moieties of T. parva sporozoites. A recent analysis of the parasite transcriptome has revealed a wide variety of glycosylated proteins and previous investigations of the cattle genome identified genes for more than ten different lectin receptors. One of these receptors seems to be essential for the development of experimental cerebral malaria, a closely related apicomplexan parasite. In contrast, almost nothing is known about potential carbohydrate-based targets of T. parva sporozoites and the C-type lectin receptors of bovine cells that are crucial for the initial infection process. Therefore, we will illustrate the glycan repertoire of T. parva sporozoites that we were able to identify and highlight the binding affinities of these targets to different C-type lectin receptors of the mouse. Methods used to gain these results include the transfection of T. parva sporozoites with liquid transfection reagents and electroporation systems to introduce a plasmid containing Green Fluorescent Protein. Such labelled sporozoites are then screened with C-type lectin receptor (CLR)-Fc fusion proteins in an ELISA-based glycan array and a subsequent flow cytometry-based binding study to verify the results. We will present the C-type lectin receptors that recognise surface glycan targets of T. parva sporozoites.

O33Molecular and metabolic changes in the proximal colon of pigs infected with Trichuris suis

Dawson H.D.1, Chen C.1, Li R.2, Bell N.3, Shea-Donohue T.4, Kringel H.5, Beshah E.1, Hill D.E.2, Urban Jr. J.F.1

1Agricultural Reseach Service, United States Department of Agriculture, Beltsville Human Nutrition Research Center, Diet, Genomics, and Immunology Laboratory, Beltsville, United States, 2Agricultural Reseach Service, United States Department of Agriculture, Beltsville Agricultural Research Center, Animal Parasitology Disease Laboratory, Beltsville, United States, 3Metabolon, Inc., Durham, United States, 4University of Maryland, School of Medicine, Baltimore, United States, 5University of Copenhagen, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, Copenhagen, Denmark

Background: The pig whipworm Trichuris suis is important to swine production because it negatively effects pig performance and to some humans with inflammatory bowel disease as a therapeutic agent that modulates inflammation. Protective immunity to whipworm conforms to a Th2-dependent paradigm characterized by the response to T. muris in mice; albeit similar features have also been observed in pigs infected with T. suis by measuring localized patterns of gene expression.

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Methods: We evaluated the transcriptome, by real-time PCR and RNASeq, of the proximal colon of T. suis infected pigs at day 21 and day 52 after inoculation. A metabolomic analysis of the luminal contents and tissue of the proximal colon was also used to characterize host and microbial metabolites that are altered by infection

Results: Infected pigs segregated into those with low versus high worm burden beginning around six weeks. The proximal colon of pigs with persistent worm infection showed a generally inflamed tissue beginning around day 21. About 3,000 differentially regulated genes are modulated in the proximal colon of pigs with a persistent adult worm infection at day 52, that is nearly 80% lower in pigs that have expelled worms as the tissue approaches a physiological normal. Prominent pathways represented at day 21 include genes involved in the Th2-response, de novo cholesterol synthesis, fructose and glucose metabolism, basic amino acid metabolism, and bile acid transport. Upstream regulatory factor analysis implicated the bile acid/farnesoid X receptor (NR1H4) in some of these processes. Metabolomic analysis indicated changes in fatty acids, antioxidant capacity and oxidative environment, biomolecules related to methylation, protein glycosylation, extracellular matrix structure, sugars and Krebs cycle intermediates, microbe-derived metabolites and altered metabolite transport.

Conclusions: This model can predict dietary nutrients that favorably alter the microbiome and improve host intestinal health in both pigs and humans exposed to Trichuris.

O34CD4 variant phenotype related to increased susceptibility to Babesia bovis

Okino C.H.1, Bassetto C.C.2, Giglioti R.3, Silva P.C.1, Toneli M.F.1, Simas P.V.2, Marcondes C.R.1, Oliveira H.N.2, Oliveira M.C.1

1Embrapa Southeastern Livestock, Lab of Animal Health, São Carlos, Brazil, 2Universidade Estadual Paulista “Júlio de Mesquita Filho”, Departamento de Zootecnia, Jaboticabal, Brazil, 3Instituto de Zootecnia, Centro de Pesquisa de Genética e Reprodução Animal, Nova Odessa, Brazil

Background: Due to the intra-erythrocyte nature of Babesia bovis infection, the adaptive immune response is dependent on the presentation of parasite antigens by antigen-presenting cells to CD4+ T lymphocytes. Even, destruction of infected erythrocytes by activated macrophages and neutralization of extracellular merozoites and infected erythrocytes by antibodies were also dependent on CD4+ T cells response. Three different allelic forms of bovine CD4 was previously described in cattle, by phenotyping cell populations using CC26 clone of mAb anti-CD4 bovine, although the functional relevance of these different CD4 phenotypes remains not elucidated.

Method: We have found approximately 12% calves of Canchim breed (5/8 Charolais + 3/8 Zebu) presenting no staining of mononuclear cells by phenotyping with anti-CD4 bovine clone CC8, and those animals were designated as CD4-, while the animals that present staining were designated as CD4+. Our study aimed to compare CD8β+, CD21+ and CD335+ populations (flow cytometry) and Babesia bovis loads (qPCR) in calves CD4- and CD4+ naturally infected with this hemoparasite. Thirty-

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one Canchim calves (6 of CD4- phenotype and 25 of CD4+ phenotype) were monitored every 15 days from the birth to 6 months of age. In this study, five samplings from each animal were selected for evaluation, the first (D0) was the sampling before calves become B. bovis infected, D15 was the first sampling with B. bovis detection, followed by D30, D45, and D60.

Results: Significant higher levels of CD335+ cells (D15) and B. bovisDNA (D60) were observed in calves CD4- compared to CD4+. Higher means were observed for CD8β+ cells in CD4+ group at D15 and D30, though no significant difference was observed.

Conclusion: CD4 variant phenotype was associated with increased susceptibility to B. bovis infection in Canchim calves, while these higher B. bovis levels seem to stimulate enhanced innate immunity by NK cells.

PIG ANTI-VIRAL IMMUNITY

O35Mechanisms of AFSV pathogenesis and immune evasion inferred from gene expression changes in infected macrophages

Zhu J.J.1, Ramanathan P.2, Bishop E.A.1, O’Donnell V.1, Gladue D.1, Borca M.1

1USDA-ARS-Plum Island Animal Disease Center, Foreign Animal Disease Research Unit, Orient, United States, 2University of Texas Medical Branch, Department of Pathology,

Galveston, United States

African swine fever (ASF) is a swine viral disease caused by African swine fever virus (ASFV). In domestic pigs, the clinical manifestations mostly depend on the virulence of virus strains. Acute infection by the highly virulent viruses causes hemorrhagic fever and death. It is widely considered that the pathogenesis is mainly due to cytokines produced by infected monocytes and macrophages. To understand the molecular mechanisms of ASF pathogenesis and immune evasion, we used transcriptome analysis to identify gene expression changes after ASFV infection in ex vivo macrophages for inferring mechanisms of pathogenesis and immune evasion caused by ASFV. Our results suggest that up-regulated expression of proinflammatory TNF cytokines including FASLG, LTA, LTB, TNF, TNFSF4, TNFSF10, TNFSF13B and TNFSF18 is major primary causative cytokine factors in ASF pathogenesis. Other up-regulated proinflammatory cytokines (IL17F and interferons) and down-regulated anti-inflammatory cytokine (IL10) could also significantly contribute to the pathogenesis. The differential expression also indicates that ASFV infection could (1) suppress MHC Class II antigen procession and presentation based on down-regulated expression of cathepsins, SLA-DMA and SLA-DMB and up-regulated SLA-DOA, SLA-DOB and CD74; (2) evade CD8+ cytotoxicity and neutrophil extracellular traps by decreasing expression of neutrophil/CD8+ T effector cell-recruiting chemokines; (3) reduce M1 activation by suppressing expression of C3a, C5a, IFN, IL17, Toll-like and TNF receptors and IL-1 signaling; and (4) inhibit autophagy and apoptosis via down-regulated ATG, BNIP3 and up-regulated NUPR1 expression. These results provide insightful information for further investigation to understand this devastating swine disease.

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O36Understanding the induction of adaptive immunity following PRRSV infection

Gerdts V., Darbellay J., Van Kessel J., Strom S.

University of Saskatchewan, Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac), Saskatoon, Canada

The porcine reproductive and respiratory syndrome virus (PRRSV) is the most economically devastating pathogen in the swine industry. The immune response to PRRSV is characterized by a rapid production of non-neutralizing antibodies and a delayed induction of T cell immunity. Depending on the strain, neutralizing antibodies appear > 4 weeks post-infection. The goal of our research is to understand the dysregulation of immunity following PRRSV infection, and in particular the role of dendritic cells and macrophages to promote T cell proliferation. Susceptibility to PRRSV infection was restricted to APCs that express CD163. Interestingly, CD163+ bone marrow-derived DCs, as well as monocyte-derived dendritic cells (MoDCs), were less susceptible to infection than monocyte-derived macrophages (MoMΦs). Furthermore, immunoprecipitation of MHCII from PRRSV infected APCs showed an altered association of gamma actin 1 with MHCII in MoDCs, but not in alveolar macrophages, suggesting that PRRSV is hijacking the actin cytoskeleton for its own replication, potentially interfering with the transport of peptide loaded MHCII to the plasma membrane. Lastly, we launched an animal trial to compare the immunostimulatory capacity of MoDCs and MoMΦs to induce T cell proliferation in vivo. T cell immunity to PRRSV was detected 14 days post-infection and was directed towards Th1 type immunity.

MoDCs were more potent inducers of central memory Th cell and CD4α+ effector T cell proliferation, whereas MoMΦs were more proficient at stimulating cytotoxic lymphocyte and γδ T cell proliferation. Overall, our data suggests that a delayed induction of T cell immunity causes an absence of CD4+ follicular T helper cells. As a result B cells would not undergo somatic hypermutation, potentially explaining the delayed induction of neutralizing antibodies to PRRSV.

O37The development of nanoparticles-entrapped porcine reproductive and respiratory syndrome virus (PRRSV) vaccine

Chaikhumwang P.1, Madapong A.2, Saeng-Chuto K.2, Nilubol D.2, Tantituvanont A.1

1Chulalongkorn University, Faculty of Pharmaceutical Sciences, Pharmaceutics and Industrial Pharmacy, Bangkok, Thailand, 2Chulalongkorn University, Faculty of Veterinary Science, Veterinary Microbiology, Bangkok, Thailand

The study was conducted to evaluate the induction of immunity and the efficacy of nanoparticles-entrapped porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (NPs-PRRS) against PRRS2 challenge. Forty-eight, 3 week-old, PRRSV-free, pigs, were allocated into 8 groups of 6 pigs each including Neg, Chal, NP1con, NP2con, NPsIN1, NPsIN2, KVIN, and MLV. Pigs in NPsIN1, NPsIN2 and KVIN were intranasally vaccinated with NP-PRRS system

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1 and 2, and killed PRRSV vaccine (KV), respectively. Pigs in MLV were intramuscularly vaccinated with a modified live vaccine (MLV). Neg, Chal, NP1con and NP2con were left as a negative, challenge and nanoparticles-entrapped system 1 and 2 controls. All pigs were challenged intranasally with PRRS2 at 28 days post-vaccination (DPV) and necropsied at 7 days post-challenge (DPC). Blood and nasal swabs were collected at 0, 14, 28 DPV and 7 DPC. Peripheral blood mononuclear cells (PBMC) were isolated and assayed for cell-mediated immunity as measured by the production of IFN-gand IL-10 using intracellular flowcytometry and ELISA, respectively. Nasal swabs were assayed for immunoglobulin (IgA) level. Severity of PRRSV-induced pneumonic lung lesion was evaluated and lungs were assayed for PRRSV RNA using RT-qPCR. Following vaccination, NPsIN2-vaccinated pigs had significantly higher level of IFN-g producing cells and IgA in nasal swabs at 28 DPV compared to that of other vaccinated groups. Following challenge, NPsIN2-vaccinated pigs exhibited significantly lower (P < 0.05) PRRSV-induced pneumonic lung lesion and PRRSV RNA in lungs, compared to that of other vaccinated groups. NPsIN2-vaccinated pigs had significantly lower IL-10 compared to that of other vaccinated groups. In conclusion, the results of the study demonstrated that the efficacy of NPsIN2 in term of eliciting immune responses against PRRSV and protecting PRRSV infections over NPsIN1, KV and commercially available MLV.

O38Searching for markers of immunocompetence in blood: application to vaccination against Influenza A virus in pigs

Blanc F.1, Estellé J.1, Lemonnier G.1, Leplat J.-J.1, Bouguyon E.2, Billon Y.3, Bouchez O.4, Pinard-Van Der Laan M.-H.1, Rogel-Gaillard C.1

1GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France, 2VIM, INRA, Université Paris-Saclay, Jouy-en-Josas, France, 3GenESI, INRA, Surgères, France, 4GeT-PlaGe, INRA, Castanet-Tolosan, France

Understanding why some animals respond better to vaccination than others is important to define biomarkers of immunocompetence and improve the sustainability of animal production. Our study focused on the response of pigs to Influenza A virus (IAV) vaccination to search for blood biomarkers from a prior-vaccination transcriptome that could differentiate animals with contrasted vaccine responses. Large White pigs without maternal IAV-specific IgG transmission (26 families, 98 animals) were vaccinated at weaning (around 28 days of age) with a booster 21 days post-vaccine (dpv). The humoral response was evaluated by measuring seric IAV-specific IgGs by ELISA and antibodies directed against HA antigens by hemagglutination inhibition assays (HAI) at three time-points corresponding to early response (21 dpv), maximum response intensity (28 or 35 dpv) or long-term persistence (118 dpv). At each time-point, high and low responders were selected and differential analyses of genes expressed in blood collected before vaccination (0 dpv) were conducted from RNA-Seq data. Ingenuity Pathway Analysis was performed to identify the biological functions enriched from the sets of differentially-expressed genes

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(DEGs). At the early response time-point, 91 DEGs were found between high and low responders for specific IgG levels (FDR < 0.01) with decreased functions related to blood cell migration, maturation and viability in low responders. At the persistence time-point, 269 DEGs mainly related to cell cycle were detected for the HAI titres (FDR< 0.05). Interestingly, a common network of genes related to humoral response involving immunoglobulins and IL-12 complex was obtained at both time-points. In conclusion, we show that gene expression levels in blood before vaccination correlate with vaccine response. Genes related to blood cell migration, maturation and viability seem to have an impact on the early response to IAV vaccination and other genes more related to cell cycle seem linked to the persistence of the response.

O39Nipah vaccine to eliminate porcine reservoirs and safeguard human health

Pedrera M.1, Bailey D.1, Barman N.N.2, Chang L.-Y.3, Chappell K.4, Gilbert S.5, Lambe T.5, Marsh G.6, McLean R.1, Mourino M.7, Raue R.8, Tchilian E.1, Thakur N.1, Watterson D.4, Young P.4, Graham S.1

1The Pirbright Institute, Pirbright, United Kingdom, 2Assam Agricultural University, Guwahati, India, 3University of Malaya, Kuala Lumpur, Malaysia, 4University of Queensland, Brisbane, Australia, 5Jenner Institute, University of Oxford, Oxford, United Kingdom, 6CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, Australia, 7Zoetis, Vall de Bianya, Spain, 8Zoetis, Zaventem, Belgium

Nipah virus (NiV) causes respiratory illness and severe, often fatal, encephalitis in humans. Fruit bats, considered the natural reservoir, were responsible for the most recent human outbreak in 2018 in India. NiV is highly contagious in pigs and causes respiratory and occasional neurological signs. Pig-to-human transmission was responsible for the first and most severe NiV outbreaks in Malaysia and Singapore in 1998-99 and caused significant economic costs to local pig industries. Despite the importance of NiV as an emerging pathogen, no vaccines are currently approved for human or livestock use.

We aim to develop a vaccine for use in pigs that would reduce the risk that NiV poses to public health, and the pig industries and livestock keepers in South and Southeast Asia. We have compared the immunogenicity of three NiV vaccine candidates in mice and pigs: i) a soluble NiV G (sG) protein subunit, ii) a molecular clamp stabilised NiV F (mcsF) protein subunit, and iii) an adenoviral (ChAdOx1) vectored NiV G protein. sG protein from the related Hendra virus (HeV), which is used in the licensed Equivac® HeV vaccine, was included as a ‘benchmark’ vaccine.

All vaccines stimulated NiV specific T cell and antibody responses in both mice and pigs. In pigs, the ChAdOx1 NiV G induced the strongest specific T cell response and was the only vaccine to stimulate a CD8+ T cell response; NiV sG induced the greatest NiV-neutralising antibody titres and NiV mcsF induced the strongest antibody response that neutralised the fusion of cells expressing NiV G and F proteins. Results from an ongoing efficacy trial in pigs to determine the levels of protection conferred by the vaccines against NiV challenge will additionally be presented.

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O40Co-infection of porcine deltacoronavirus and porcine epidemic diarrhea virus prolongs virus shedding and IFN-α up-regulation

Saeng-Chuto K., Madapong A., Watcharavongtip P., Jermsutjarit P., Temeeyasen G., Nilubol D.

Chulalongkorn University, Faculty of Veterinary Science, Bangkok, Thailand

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) causes clinical diseases characterized by diarrhea. Both viruses have been detected in diarrheic pigs. Understanding the co-habitation of these two viruses and outcomes following co-infection becomes interesting questions. The study, therefore, was conducted to investigate the severity of clinical diseases together with the antigen detection in epithelial cells in pigs experimentally co-infected with PEDV and/or PDCoV. Twenty-one genes associated with immunomodulation and cytokine expression were also evaluated. Twenty-four, 3-day-old pigs were divided into 4 groups of 6 pigs each including Neg, PDCoV, PEDV and co-infection groups. Pigs were orally inoculated with PDCoV and PEDV, either single or combination. Neg was left as no-challenge. Clinical signs including diarrhea and fecal shedding were monitored daily. Three pigs in each group were necropsied at 3- and 5-days post inoculation (dpi). The results demonstrated that clinical diseases induced by PEDV, either single or co-infected with PDCoV, were significantly more severe than a single PDCoV infection. There was no difference in clinical diseases a single PEDV infection and co-infected with PDCoV. PEDV shedding in the co-infection group was significantly longer than the single infection

group. However, there was no difference in PDCoV shedding between the single PDCoV infection and co-infected with PEDV groups. Immunohistochemistry (IHC) staining indicates that PEDV was more detected in villi of enterocytes while PDCoV was found in both villi and crypt enterocytes. PEDV infected cells were more density in the co-infection group than the single infection, especially in ileum. The up-regulation of IFN-α was observed until 5 dpi in the co-infection group, but not in either single infection group. In conclusion, co-infection of PEDV and PDCoV enhances the severity of clinical diseases. The co-infection prolongs PEDV shedding in fecal samples and up-regulation of IFN-α expression compared to either single infection.

VIC TOOLKIT WORKSHOP

O41The Immunological Toolbox website: a community resource to enable information exchange and access to reagents and techniques

Mwangi W., Maccari G., Hammond J.

The Pirbright Institute, Woking, United Kingdom

Since the 1980’s, enormous efforts by Institutes and individuals have produced the monoclonal antibodies and recombinant proteins that have driven the advances in veterinary immunological research. However, primarily because of more limited and less coordinated funding opportunities, reagent availability for veterinary species has always

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been limited compared to humans and mice. To address this gap, several Immunological Toolbox initiatives (from 2003 onwards; largely funded in the UK and USA) aimed to prioritise new reagent development against research gaps. Today, all these efforts have produced thousands of reagents and associated methods, but a global overview of availability and applicability is lacking.

After feedback from several national and international workshops, we have developed a searchable and highly scalable veterinary immunology reagent database, accessed through a website; immunologicaltoolbox.co.uk. This provides detailed knowledge on existing reagents, as well as reagents that are being developed. As such, we aim to highlight gaps in capability and avoid the duplication of effort, which itself can provide strong evidence to support investment in specific research and development. Information presented will include cross-reactivity between species and associated applications alongside a comments and rating feature to facilitate continual community scrutiny and improvement. Frameworks to facilitate collaboration, exchange or purchase from the owner or company will also be available.

The website also links directly to the practical components of the Pirbright/Roslin Toolbox. This includes the generation of new reagents based on community priorities, and the capability to recombinantly express and engineer monoclonal antibodies. Consequently, this first version of the website has largely been driven by the information available from Pirbright and Roslin antibody repositories. However, the quality and quantity of future information will rely on community engagement. Discussions at an international level will be extremely valuable in helping to remove barriers and promote veterinary immunological research.

O42Development of a bovine/ovine cytokine 15-plex assay for immunoprofiling of the cellular response in ruminants

Lesueur J.1, Barbey S.2, Cebron N.1, Walachowski S.1, Lefebvre R.3, Germon P.4, Boichard D.3, Corbiere F.1, Foucras G.1

1IHAP, Université de Toulouse, ENVT, INRA, Toulouse, France, 2UE Le Pin, INRA, Le Pin-aux-Haras, France, 3GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France, 4ISP, INRA, Université de Tours, Nouzilly, France

Evaluation of the immune status must be assessed in highly controlled conditions to be amenable for reproducibility. Furthermore, whereas the cellular response plays a major role, immunity has been measured for a long time through antibody production. For the purpose of assessing immune cell responsiveness in ruminants, we developed and validated a whole blood assay coupled to a high-throughput multiplex cytokine assay using the Luminex technology. A custom Milliplex assay was developed with MERCK-Millipore company for the measurement of 15 bovine/ovine cytokines of both the innate and adaptive immunity, and chemokines. Whole blood samples were collected from 110 dairy cows in an INRA experimental unit, and immediately stimulated during 24 hours with heat-killed bacteria (Escherichia coli, Staphylococcus aureus, and Streptococcus uberis), Toll-like receptor ligands (FSL-1 for TLR2, LPS for TLR4, and Gardiquimod for TLR7-8), or soluble anti-CD3/CD28 monoclonal antibodies. Cytokine secretion was determined and compared with a sample prepared in the same condition but without any stimulant. Using ANOVA and data mining methods (PCA, clustering…), we showed that the

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protocol is able to detect differences between bacteria, with S. uberis being the most potent to induce a response, compared to S. aureus and E. coli that were distinct but more closely related to each other. Similarly, TLR ligands could be distinguished, and Gardiquimod was significantly different from the MyD88-associated TLR2/4-activating ligands. LPS and E. coli provided very similar response profiles confirming previous data indicating that a large part of the E. coli response is mediated through LPS signaling. A high variability of response was detected amongst cattle suggesting environmental and genetic variations of the cytokine response. Altogether, results indicate that cytokine profiling in cattle is achievable and can be used in further work to delineate more precisely the responsiveness of cattle in various situations, including variability of the genetic background.

O43CD40-targeted immunization as a method for rapid antiserum production and epitope mapping

Vuong C.N.1, Chou W.-K.2, Hargis B.M.1, Bielke L.R.3, Berghman L.R.2

1University of Arkansas, Poultry Science, Fayetteville, United States, 2Texas A&M University, Poultry Science, College Station, United States, 3Ohio State University, Animal Science, Columbus, United States

The CD40 receptor is expressed on all professional antigen presenting cells and is integral to the activation and subsequent production/affinity-maturation of antibodies.

Previously published data from our laboratories demonstrated that targeting an antigen to chicken CD40 receptor induced more rapid and robust antibody production specific against the antigen. Using this method, a panel of oligopeptides of varying lengths ranging from 9 to 23 amino acids were designed to span an entire target toxin were synthesized for the purpose of epitope mapping. The target, Clostridium perfringens alpha toxin, possesses two enzymatic functions: hemolytic activity and phospholipase C activity. By loading this panel of peptides onto the CD40-targeting platform and immunizing chickens by subcutaneous injection (equivalent to 0.33ug of peptide antigen dosage per chicken), a panel of high titer peptide-specific antiserum samples was produced in a week. Peptide-specific antibody responses measured a minimum mean of 4.6-fold higher than pre-immune baseline, and as high as 13-fold higher. These serum samples were used to screen for the ability to neutralize the alpha toxin’s enzymatic functions. Results demonstrated multiple candidates for hemolytic neutralization, but not against phospholipase C. With this successful proof-of-concept, the CD40-targeted immunization method was able to produce functional antiserum for in vitro laboratory usage a week after a single immunization. This targeted method is more advantageous than the traditional style of antiserum production, which requires multiple immunizations over a span of several weeks, necessitating long-term animal care/housing and consumption of more antigen. Furthermore, because this platform uses chickens as a host, there is an increased chance of response against conserved mammalian domains. Additionally, this method of rapid antiserum production is a useful technique for cheaper, faster epitope mapping and has since been successfully used for candidate selection in development of a necrotic enteritis vaccine for poultry.

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O44A fast screening method to obtain high producer CHO cells for the production of porcinized monoclonal antibodies

Van Der Weken H., Cox E., Devriendt B.

Ghent University, Laboratory of Veterinary Immunology, Merelbeke, Belgium

Despite the recent expansion of the veterinary monoclonal antibody toolbox, veterinary reagents including swine-specific reagents are still limited. In addition to the actual development of these veterinary monoclonal antibodies, a major constraint is the large-scale production of the antibodies, which requires the generation and selection of a high producer clone. This process often takes longer than six months using standard limited dilution techniques and is very labor intensive. To speed up this process, we designed a tri-cistronic vector coding for green fluorescent protein (GFP), the light and the heavy chain of a monoclonal antibody, specific for porcine CD13. The original mouse IgG1 Fc domain was replaced by the porcine IgA Fc domain. These sequences were separated by a GT2A sequence, which allows the expression of these proteins under the control of a single promotor in equimolar ratios. Via single cell FACS sorting and live cell microscopy, we were able to show a strong correlation between GFP expression levels and the production of porcinized monoclonal antibodies by CHO cells. This workflow enabled the selection of high producer CHO clones within a time-frame of just four weeks after transfection. The highest producing CHO clone displayed a monoclonal antibody productivity of 2.32 pg/cell/day and >50 mg antibody/L culture supernatant were easily obtained using standard laboratory equipment and shake-flask batch culture. The

generated clones maintained these antibody production levels for more than 6 months and the produced porcinized monoclonal antibodies retained their specificity and showed minimal degradation. In conclusion, the designed workflow allows the production of large amounts of monoclonal antibodies, tailored to specific needs, and might further facilitate the expansion of the veterinary antibody toolbox.

COMPANION ANIMALS (ELANCO

SPONSORED)

O45Combination chemo- and immunotherapy provides long term clinical improvement and sustained parasite clearance in L. infantum-infected dogs

Nascimento L.1, Moura L.1, Miranda D.1, Pinho F.2, Reed S.3, Duthie M.3, Werneck G.4, Khouri R.2, Barral A.5, Barral-Netto M.5, Cruz M.S.1

1Federal University of Piaui, Teresina, Brazil, 2Federal University of Bahia, Salvador, Brazil, 3Infectious Disease Research Institute, Seatle, United States, 4Rio de Janeiro Federal University, Rio de Janeiro, Brazil, 5Fundação Oswaldo Cruz-Fiocruz BA, Salvador, Brazil

Backgroud:There is little evidence that current control strategies for canine visceral leishmaniasis (CVL), the veterinary disease caused by L. infantum infection, are having a positive impact and new measures are needed. Dogs are an important reservoir for L. infantum and support transmission to humans

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to cause VL, but the use drugs intended primarily for human use for the treatment of CVL is prohibited because of concerns over the propagation of resistant parasites. We therefore examined the therapeutic capacity of allopurinol, and its potential when combined in chemo- immunotherapeutic approaches involving defined subunit vaccines, for the treatment of dogs naturally infected with L. infantum.

Methods: As vaccines we used the Leish-F2 antigen formulated with stable emulasion (SE) and either second generation adjuvant (SLA; an agonist of Toll-like receptor (TLR) 4) or SLA with imiquimd (IMQ; and agonist of TLR7). Our study consisted of 28 dogs distributed into different groups: group 1 was not treated while all other animals received allopurinol therapy orally at a dose of 20 mg / kg, once each day for 90 days. Group 2 was treated with allopurinol alone; group 3 additionally received Leish-F2 + SLA-SE; and group 4 additionally received Leish-F2 + SLA/IMQ-SE.

Results: While the animals that did not receive treatment had a progressive decline in their clinical condition, treatment with allopurinol alone alleviated the clinical symptoms of CVL and provided long-term improvement in clinical condition. Concomitant vaccination with Leish-F2 and SLA-SE was, however, required to provide long-term clearance of L. infantum from the lymphoid tissues and systemic organs.

Conclusions: These results have important implications for both the management of CVL in individual animals and for limiting potential transmission of L. infantum from infected dogs to the general population.

O46Development of a sensitive PD-L1 immunohistochemistry of canine cancers and clinical efficacy of an anti-PD-L1 antibody in canine oral malignant melanoma with pulmonary metastasis

Maekawa N., Konnai S., Okagawa T., Murata S., Ohashi K.

Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan

Immunotherapy targeting immune checkpoint molecules, such as programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), represents a promising treatment for numerous cancer types in humans. It has been shown that various cancer types in dogs have aberrant expression of PD-L1, and a pilot clinical study has shown the therapeutic efficacy of a canine chimeric anti-PD-L1 monoclonal antibody (mAb) in a subset of dogs with cancers. In clinical studies performed on humans, tumor PD-L1 expression is assessed by immunohistochemistry (IHC) and it is considered a predictive biomarker of response to PD-1/PD-L1-inhibiting mAbs in certain circumstances. However, in dogs, the expression status of PD-L1 in some cancer types is still controversial and additional studies are needed to clarify the clinical efficacy of anti-PD-L1 mAb. In the present study, we established a novel mAb for accurate canine PD-L1 IHC. Interestingly, almost all canine oral malignant melanoma (OMM) samples were PD-L1-positive in the PD-L1 IHC. Subsequently, a therapeutic anti-PD-L1 mAb was administered to 13 dogs bearing OMM with pulmonary metastasis. Our goal was to evaluate its safety and clinical efficacy in this patient population. Treatment-related adverse events of any grade were observed in 4 of 13 patients (30.8%). One dog (7.7%) developed

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grade 3 pneumonitis, causing treatment discontinuation. Regression of lung metastasis was observed in three out of 13 dogs (23.1%). Additionally, significantly longer survival after the confirmation of pulmonary metastasis was seen in the treatment group vs. the control group (p = 0.003). Taken together, our results show that anti-PD-L1 antibody treatment is well-tolerated and effective against pulmonary metastatic OMM. The newly established PD-L1 IHC could be used to select eligible patients for anti-PD-L1 therapy.

O47Food allergen-sublingual immunotherapy in dogs significantly reduces IL-17A and increases IL-10 and allergen-specific CD3+CD4-CD8- T cells

Cox E.1, Devriendt B.1, Maina E.2

1Ghent University, Merelbeke, Belgium, 2DermaVetSuisse, Lausanne, Switzerland

Food-allergen-specific sublingual immunotherapy is a safe and effective therapy for humans with food allergy. Our lab demonstrated that this route of administration is safe and effective in decreasing clinical signs and pruritus. The mechanism of food allergy in dogs has not been elucidated yet. The correlation between results in provocative challenges and antigen specific IgE was low, but correlation with IL-17A production by allergen-restimulated PBMC´s of adverse food reactive dogs was high. Thirteen dogs with proven food allergy and no other concurrent allergies were randomized in a Treatment group (T; n = 7) and a Placebo group (P;n = 6) to

receive either FA-SLIT based on the results of the food trial or glycerinated saline, respectively. The treatment was continued daily for 6 months with fortnightly dosage escalations. Clinical signs were provoked by feeding the culprit diet before and at the end of the study. Peripheral blood samples taken during bood provocative food exposure phases and restimulated with the allergens showed that the treatment significantly increased the percentage of allergen-specific proliferating CD4-CD8- T cells, while the percentage of CD4-CD8+ and CD4+CD8+ T cells decreased. In addition, FA-SLIT also increased IL-10 and IFN-γ levels, while IL-17A levels were decreased. However, both interleukins and T cells did not correlate with clinical scores. Together our data show that FA-SLIT was safe and reduced clinical symptoms in food allergic dogs. This therapy thus triggers tolerance and may modulate the allergic T cell response toward Th1 and Treg phenotypes. Therefore, FA-SLIT may be a tool to desensitize dogs with food allergy.

O48Characterisation of the companion animal immune response to Mycobacterium bovis infection using cytokine profiling

O’Halloran C., Gunn-Moore D., Hope J.

University of Edinburgh, Edinburgh, United Kingdom

Background: Tuberculosis due to Mycobacterium (M.) bovis; termed “zoonotic TB” by the World Health Organisation, is a globally distributed disease of humans, companion animals, wildlife and livestock.

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Companion animal infections pose a health risk to their owners and can potentially lead to environmental contamination which may impede the control programmes implemented by countries such as the UK. Despite this, remarkably little is known regarding the immunopathogenesis of these infections.

Aim: To further our understanding of companion animal tuberculosis, we measured circulating cytokine concentrations in the serum of M. bovis-infected cats and in the supernatant of M. bovis-antigen stimulated peripheral blood mononuclear cells (PBMC) from infected dogs.

Methods: Cytokine concentrations for both species were measured using the appropriate Milliplex MAP multiplex assay according to the manufacturer’s instructions.

Feline blood samples were opportunistically obtained from 116 cats with a histological diagnosis of mycobacterial infection that were speciated by specialist mycobacterial culture or PCR. These were then compared to serum concentrations of 16 healthy control cats.

Antigen-stimulated canine PBMC supernatants from 90 Foxhounds (including a range of both test-positive and test-negative individuals) were evaluated in the context of clinical data from our previously published investigation into an outbreak of canine M. bovis-tuberculosis in England.

Results: Both species showed significant elevations in pro-inflammatory cytokines in the samples obtained from M. bovis infected animals when compared to those of uninfected controls e.g. GM-CSF, IL-2, IL-8, RANTES and TNF-α. As predicted, these are well described in the activity of cells from the monocyte-macrophage lineage. Similarly we found a reduction in cytokines which drive the Th-2

response in other species e.g. IL-13 and IL-4. This study shows a markedly conserved pattern of immune response to M. bovis infection within companion animals that could form the basis of future diagnostic test development.

O49Evaluation of the efficacy of a canine influenza virus (H3N2) vaccine in SPF dogs

Chae J.B., Lee B.H., Lee I.H., Lee D.U., Lee H.E., Min K.C., Yoon I.J.

Choong Ang Vaccine Laboratories Co. Ltd, Daejeon, Korea, Republic of

Background: Avian-origin H3N2 canine influenza viruses (CIV), which can cause acute respiratory diseases and be transmitted among dogs, have been detected in Korea. Since these viruses spread among local dog populations, the demand for a CIV vaccine has increased. This study aimed to evaluate the efficacy of an inactivated CIV vaccine in SPF beagle dogs.

Methods: Trial vaccines contained inactivated H3N2 CIV obtained from Korea, formulated in the adjuvant (CAvant eq oac(,R)R-PA, CAVAC, Daejeon, Korea). Six SPF beagles were intramuscularly administered the trial vaccine, and three SPF beagles constituted the non-vaccinated control group. A booster vaccine was administered 2 weeks later. Two weeks after the second vaccination, all dogs were challenged with 108.0 EID50/ml of H3N2 CIV (A/Canine/Korea/AS-11/2013), and clinical signs, body temperature, and viral shedding were examined daily. Viral detection and

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histopathological findings in the lung and trachea were examined after euthanizing the dogs at 6 days post-challenge (DPC). Serum hemagglutination-inhibition (HI) antibody titers were determined before vaccination, 2 weeks after initial vaccination, 2 weeks after second vaccination, and 6 DPC.

Result: After booster vaccination, the vaccinated group had a higher HI titer ≥ 28. All groups displayed seroconversion after the challenge. Clinical signs, body temperature, viral shedding, and viral detection in sampled tissue and histopathological findings displayed significant differences between the vaccinated and control groups (p< 0.05).

Conclusion: These results indicate that the inactivated CIV vaccine used herein provided sufficient immunogenicity and protection against virulent CIV challenges in SPF beagle dogs.

O50Pre-conditioning of equine bone marrow-derived mesenchymal stromal cells: effect on the inhibition of lymphocyte proliferation

Caffi V.1, Espinosa G.1, Plaza A.2, Gajardo G.3, Henriquez C.1

1Universidad Austral de Chile, Farmacología y Morfofisiología, Facultad de Cs. Veterinarias, Valdivia, Chile, 2Universidad Austral de Chile, Medicina, Facultad de Medicina, Valdivia, Chile, 3Universidad Austral de Chile, Ciencia Animal, Facultad de Cs. Veterinarias, Valdivia, Chile

Background: Mesenchymal stem/stromal cells (MSCs) are a heterogeneous cell population with a strong modulatory effect on the immune response. MSCs have shown to be able to inhibit lymphocyte proliferation, however, the mechanisms involved in this capacity vary between species. Here, we investigate the mechanisms involved in equine bone marrow-derived MSCs ability to inhibit lymphocyte proliferation, and what happens with the expression of immunomodulatory molecules when MSCs are exposed to pro-inflammatory cytokines

Methods: We determine the capacity of MSCs to inhibit lymphocyte proliferation after stimulation with Concanavalin A. Co-culture between PBMC and MSCs was performed at different ratios. Thereafter, we use pharmacological inhibition of cyclooxygenase (COX) using indomethacin, indoleamine 2,3-dioxygenase (IDO) using 1-MT, and inducible nitric oxide synthetase (iNOS), using aminoguanidine (AG) in order to determine the role of each in the inhibition of PBMC proliferation. Additionally, we stimulate MCSs with TNF-α, IFN-γ, or its combination to evaluate the expression of immunomodulatory molecules.

Results: MSCs were able to inhibit PBMC proliferation until 16:1 ratio (PBMC:MSCs). Indomethacin and 1-MT were able to revert the MSCs this capacity to inhibit the PBMC proliferation. On the other hand, no significant reversion of PBMC proliferation was produced by inhibition of iNOS. Additionally, the stimulation of MSCs with TNF-α, IFN-γ, or its combination show a strong increase in some modulatory molecules (IL-6, IDO, iNOS, and COX-2), while some pathways previously described in other species remains not modified (HGF and TGF-β). This pre-conditioning shows not modified the capacity of MSCs to inhibit PBMC proliferation.

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Conclusions: MSCs are capable to inhibit PBMC proliferation in a PGE2 and IDO dependent manner, while iNOS seems not to be critical. The exposure of MSCs to a pro-inflammatory environment increase their expression of immunomodulatory molecules, however, this not modify their capacity to inhibit PBMC proliferation.

NOVEL SYSTEMS

O51Rousettus aegyptiacus cell lines enable analysis of Type I and III interferon responses to highly pathogenic viruses in reservoir species

Friedrichs V., Dorhoi A., Köllner B.

Friedrich-Loeffler-Institut, Institute of Immunology, Greifswald - Insel Riems, Germany

Bats harbor many highly pathogenic viruses, including different Lyssaviruses, often in absence of severe clinical disease. The European Bat Lyssavirus (EBLV) was isolated from European bat species of the genera Myotis and Eptesicus, which display low mortality and seroconversion upon infection.

Whether global anti-viral resistance or EBLV-specific immunity prevents fatal disease in bats remains unclear. This is largely due to impracticability of in vivo studies in Myotis and Eptesicus bats as these are included in conservation programs for European species. To circumvent these limitations, we investigated

anti-Lyssavirus responses using the pteropid bat Rousettus aegyptiacus, which is currently not included in conservation programs, as model organism.

Cell lines originating from different tissues of R. aegyptiacus, i.e. nasal epithelium (RaNep), olfactory epithelium (RaOlf), nervus olfactorius (RaNol), Bulbus olfactorius (RaBulb) and the brain (RaCer), were generated and characterized. The patterns of susceptibility to EBLV infection as well as the mRNA profiles of type I (IFNα, IFNβ, IFNε, IFNκ, IFNω) and type III (IFNλ1-4) Interferons and Interferon-stimulated genes (IFIT1, IFIT3, Mx1) as well as major PRRs (MDA-5, RIG-I) were defined. Further, we employed these cellular systems to define impact of unique physiological features of bats on anti-viral immunity.

Bats are the only mammals capable of active flight and body temperatures in R. aegyptiacus vary from 35°C while resting to up to 41°C during flight. Considering that flight induces a fever situation regarding body temperature in bats and may subsequently restrict viral replication, R. aegyptiacus cell lines were incubated at various temperatures and ER stress responses, apoptosis and interferon expression were monitored under temperature variation. Thus, our novel cell lines offer unique opportunities to advance research focusing on immunology of infection in reservoir species.

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O52The pig - a novel translational animal model for eosinophilic esophagitis

Plundrich N.J.1, Smith A.R.2, Borst L.3, Edwards L.3, Pridgen T.4, Cortes L.M.3, Odle J.2, Blikslager A.4, Lila M.A.1, Käser T.3, Dellon E.S.5, Laster S.M.6

1NC State University, Food, Bioprocessing and Nutrition Services, Raleigh, United States, 2NC State University, Animal Sciences, Raleigh, United States, 3NC State University - College of Veterinary Medicine, PHP, Raleigh, United States, 4NC State University - College of Veterinary Medicine, DOCS, Raleigh, United States, 5University of North Carolina School of Medicine, Department of Medicine, Chapel Hill, United States, 6NC State University, Biological Sciences, Raleigh, United States

Food allergy affects ~8% of the world’s population and is caused by excessive immune responses against food allergens. These responses result in pathological consequences such as eosinophilic esophagitis (EoE) which has yearly associated costs in the US of $1B. EoE is a T-helper 2 cell driven disease leading to accumulation of eosinophils in the esophagus. The resulting inflammation and fibrosis cause failure to thrive, dysphagia and food impaction. There are no FDA-approved treatments for EoE partly explained by the limitations of the standard mouse model for translational research. Therefore, our goal was to develop the pig as a biologically relevant translational model for EoE.

Food allergy was induced in our sensitization + challenge group (sens+challenge) by intraperitoneal injection of hen egg white protein (HEWP) with cholera toxin as adjuvant followed by oral HEWP challenge.

Control pigs included non-treated and non-challenged pigs. Clinical signs of food allergy were monitored for 24h after challenge, then gastrointestinal (GI) tissues were collected and examined histologically for eosinophil content. Additionally, systemic food allergen specific T cells were examined by in vitro restimulation with the HEWP ovalbumin and subsequent flow cytometry analysis.

Sens+challenge pigs (5/6) showed clinical signs of food allergy (diarrhea, emesis, skin rash); and 4/6 pigs developed esophageal eosinophilia. Control pigs were healthy without signs of eosinophilia. Sens+challenge pigs also had a strong food allergen-specific T cell response. The majority of these ovalbumin-responding T cells expressed CD4 and GATA-3; and they produced IL-5. Therefore, the main T-cell subset in these pigs are IL-5 producing T-helper 2 cells, as described for EoE patients in mice and humans.

This study is the first step towards a new translational animal model of EoE. This model has the potential to highly improve the development of new diagnosis and treatment strategies for EoE.

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O53Recombinant Lawsonia intracellularis outer membrane proteins confirmed as being neutralizing antibody targets selected for subunit vaccine development

Wilson H.L., Obradovic M.

University of Saskatchewan, Vaccine and Infectious Disease Organization (VIDO)-International Vaccine Centre (InterVac), Saskatoon, Canada

Background: Lawsonia intracellularis (LI) are obligate intracellular microorganisms that cause proliferative enteropathy (PE). LI proteins involved in attachment to enterocytes are possibly immunogenic as they are recognized by the host immune system. We previously performed 2D electrophoresis and Western blot analysis coupled with Mass Spectrometry and identified four antigenic proteins important for attachment. They were cloned and their recombinant proteins were purified from E. coli. To evaluate their potential use as vaccine antigens, we developed a flow cytometry assay to detect in vitro inhibitory effects of rabbit antibodies on penetration of CFSE-stained LI into pig intestinal epithelial cell line, IPEC-1 cells.

Methods: Four rabbits were vaccinated with each of the recombinant proteins. Western blot analysis was performed to confirm target specificity. Next, CFSE-stained LI were pre-incubated with the hyperimmune sera (200 µg, 2000 µg and 4000 µg per ml concentration) and IPEC-1 cells were infected. Fluorescence was recorded by flow cytometry in FL1 channel and the inhibitory effect of serum antibodies on bacterial penetration into IPEC-1 cells was calculated.

Results: Western blot analysis confirmed that the rabbit hyperimmune sera bound to their respective targets and that the recombinant proteins were immunogenic. IPEC-1 cells infected with CFSE-stained LI showed increased fluorescence in FL-1 channel compared to non-infected cells. CFSE-stained LI preincubated with each of the four rabbit hyperimmune sera that was then used to infect IPEC-1 cells showed lowered fluorescence percentages in FL-1 channel indicating that less bacteria infected IPEC-1 cells. The inhibitory effect of all tested sera on bacterial penetration increased with increased serum concentration reaching over 80% inhibition in the highest concentration.

Conclusions: The four recombinant proteins were targeted by neutralizing antibodies in vitro indicating they may be good vaccine candidates.

Acknowledgments: University of Saskatchewan School of Public Health graduate teaching scholarship, NSERC ITraP-Create scholarship.

O54Good cells battling bad cells: cell-mediated immunity and transmissible cancer in Tasmanian devils

Espejo C.1, Lyons B.1, Woods G.2

1University of Tasmania, School of Medicine, Hobart, Australia, 2University of Tasmania, Menzies institute, Hobart, Australia

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The Tasmanian devil (Sarcophilus harrisii) is highly endangered in the wild because of a lethal transmissible cancer, Devil Facial Tumour Disease (DFTD). DFTD induces mortality in almost 100% of cases and has decimated devil numbers by up to 90%. The disease is caused by a clonal cancer allograft cell line which is transmitted by biting. DFTD is characterised by tumour growth on facial, oral and neck regions of the host. Devils with DFTD are driven to death within 12 months after clinical evidence of infection. This cancer has the capacity to avoid an immune response from the host by downregulating cell surface MHC class I molecules. A small percentage of devils in the wild develop antibodies against DFTD cells, indicating a humoral response against the disease. However, to ensure effective immunity against DFTD, a cell-mediated immune response is also required. Due to the lack of devil-specific immune reagents, it is currently not possible to fully analyse the cell-mediated immune responses and T-lymphocyte profiles of devils. This study reports the production of the first flow cytometry compatible anti-devil CD8 monoclonal antibody (mAb) and its use in the characterisation of devil CD8 T lymphocyte profiles and describes the patterns of CD4+ and CD8+ T cell subsets in healthy devils and devils with DFTD. Furthermore, assessment of cell-mediated immunity measured by the production of IFN-gamma by T cells in response to DFTD cells allows comparison between healthy and diseased wild devils. Since T cells have been shown to play a pivotal role in tumour and allograft rejection, this novel antibody will help determine the role T cells play in the disease and enhance our knowledge of how DFTD affects the devil immune system.

O55Oral stimulation of rainbow trout with Aeromonas salmonicida and characterization of the adaptive immune response assembled: first steps to an oral vaccination model

Montero R.1, Munkler L.2, Ostermann S.1, Köllner B.1

1Friedrich-Loeffler-Institut, Institute of Immunology, Greifswald, Germany, 2Hochschule Esslingen, Department of Applied Natural Sciences, Esslingen, Germany

Infectious diseases are one of the main risks in modern aquaculture, which can cause serious economic losses. Diverse type of vaccines have been developed, however they are administered by injection (intraperitoneal or intramuscular), causing stress and undesired side effects in the fish. Considering this, the present work is focused on developing a model for oral vaccination based on the pathogenic bacteria Aeromonas salmonicida, and the respective evaluation of the immune response assembled in the aquaculture species rainbow trout (Oncorhynchus mykiss).

In comparative vaccination trials, fish were stimulated with inactivated bacteria intra-peritoneally (ip) or orally using vaccine pellets. The distribution of B and T-cell populations were analyzed in gut, peritoneum, spleen and head kidney using monoclonal antibodies by flow cytometry. Additionally, cell populations were sorted for gene characterization of membrane and secreted markers, expressed cytokines and transcription factors.

The immune response was characterized by an early response of B and T-cells (24-48h post stimulation) in both target organs used,

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peritoneum and gut; a response was also observed in the spleen, the main systemic responder organ. Additionally, the percentage of cells detected was similar in the intraperitoneal and oral route, supporting the idea of oral vaccination, as the ip route is considered as an effective, but not ideal, immunization route for achieving fish protection. Further analysis like gene expression assays and ELISA will be done, in order to complete our first findings.

O56The role of IL-1R8 and Toll-like receptors in the pathogenesis of canine DLBCL

Riva F.1, Filipe J.1, Marconato L.2, Scanziani E.1, Iussich S.2, Giannuzzi D.2, Marini I.1, Aresu L.2

1Università degli Studi di Milano, DIMEVET, Milano, Italy, 2Università di Torino, Torino, Italy

Diffuse large B-cell lymphoma (DLBCL) is the most common canine aggressive B-cell lymphoma worldwide, and new recent molecular approaches have shown that DLBCL constitutes a heterogeneous tumor that cannot be unraveled by morphology and immunophenotype (1). DLBCL behaves aggressively, typically progressing over a short period of time. It is initially highly responsive to standard chemotherapy; however drug resistance occurs in most cases, resulting in disease recurrence. To date, DLBCL is the most investigated lymphoma histotype in veterinary medicine, and the role of the dog as animal model for human DLBCL was recently validated.

Recently, it has become clear that aberrant deregulated NF-kB activation is a major initiation mechanism both in human and canine B-cell malignancies, particularly in DLBCL (2). Given the relevance of IL-1R8 as a negative regulator of NF-kB (3), we investigated the role of this gene and others involved in the same pathway in canine DLBCL.

qPCR, NGS and immunohistochemistry were used to compare canine naïve nodal DLBCLs and normal nodes. Both gene expression and immunohistochemistry showed a significant down-regulation of IL-1R8 in affected dogs compared to controls, conversely TLR7 and TLR9 were upregulated, confirming a critical role of the proinflammatory pathways activated by Toll-like receptors in DLBCL pathogenesis. Further, IPA analysis identified different activated pathways in DLBCL dogs with high IL-1R8 levels compared to DLBCL dogs with low IL-1R8 levels.

The data presented here confirm the role of the dog as excellent model of human DLBCL. The deregulation of genes associated with pro-inflammatory signaling pathways opens a new scenario both in terms of pathogenesis and therapy of this tumor.

1) Aresu et al, 2018 Haematologica

2) Jost et al, 2017 Blood

3) Riva et al, 2012 Frontiers in Immunol

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PREDICTING IMMUNITY

O57Resistance to Haemonchus contortus in Corriedale sheep is associated to high parasite-specific IgA titer and a systemic Th2 immune response

Escribano C.1, Saravia A.1, Castells D.2, Giapessoni G.3, Riet-Correa F.4, Freire T.5

1Plataforma de Salud Animal, Instituto Nacional de Investigaciones Agropecuarias, Montevideo, Uruguay, 2Secretariado Uruguayo de Lana, Florida, Uruguay, 3Investigación en Genética y Mejoramiento Animal, Instituto Nacional de Investigaciones Agropecuarias, Canelones, Uruguay, 4Plataforma de Salud Animal, Instituto Nacional de Investigaciones Agropecuarias, Colonia, Uruguay, 5Grupo de Inmunomodulación y Desarrollo de Vacunas, Departamento de Inmunobiología, Facultad de Medicina, Universidad de La República, Montevideo, Uruguay

Gastrointestinal nematode infections are one of the main causes of economic losses to ovine farmers worldwide. In Uruguay, Haemonchus contortus is considered the most pathogenic and most economically infectious agent in this field. Aiming to better understand the mechanisms involved in host resistance against gastrointestinal nematode infections we selected resistant and susceptible Corriedale animal lines and evaluated the immune-mediated mechanisms by these animals against H. contortus infection. Resistance to H. contortus infection has been associated to Th2 response in sheep and presence of parasite specific antibodies in saliva. Here, to expand the knowledge about the adaptive immune

response generated in our resistant and susceptible lambs upon H. contortus infection, we evaluated IgA and IgG parasite-specific antibodies in saliva and plasma, together with the analyses of the systemic Th2 response. H. contortus infected resistant Corriedale lambs presented high levels of parasite-specific IgA in saliva and plasma since the beginning of the experiment, and they remained constant throughout the infection period. In contrast, susceptible Corriedale infected lambs presented parasite-specific IgA antibodies only after 21 days post infection, suggesting that a delayed antibody response is associated with susceptibility to infection. Furthermore, PBMC from resistant Corriedale lambs were capable of producing higher IL-4 and IL-13 levels than susceptible lambs, indicating the expansion of a Th2-type response associated with H. contortus resistance.

In conclusion, these results suggest that there is a parasite-specific local and systemic immune response (both humoral and cellular) in resistant animals, and that although susceptible lambs can produce high levels of IgA antibodies both in saliva and plasma, their antibody response is delayed that together with an impaired specific-Th2 response, does not contribute to initial parasite elimination.

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O58Characterisation of CXCL10 production and diagnostic potential for bovine TB in cattle

Coad M.1, Doyle M.2, Steinbach S.1, Gormley E.2, Vordermeier M.1, Jones G.1

1Animal and Plant Health Agency (APHA), Weybridge, United Kingdom, 2University College Dublin, School of Veterinary Medicine, Dublin, Ireland

Background: Bovine tuberculosis (BTB) is a disease of economic and zoonotic importance caused mainly by the intracellular pathogen Mycobacterium bovis (M. bovis). An interferon-gamma (IFN-γ) release assay (IGRA) blood test has been incorporated in the BTB control programs of numerous countries as an ancillary test but infected animals are still being missed. Thus, we have characterised the production of CXCL10 in M. bovis infection and investigated its potential as an additional readout in blood based diagnostic tests for bovine TB.

Methods: CXCL10 production from whole blood assays using known infected and uninfected animals stimulated with tuberculin reagents and defined mycobacterial antigens was quantified by ELISA and ROC curve analysis performed to generate appropriate cut off values for positive responses. The kinetics of CXCL10 response was investigated in cattle following experimental infection with M. bovis, whilst responding cell populations in naturally infected animals were characterised using flow cytometry. Lastly, the practical use of CXCL10 as an additional biomarker for the diagnosis of BTB was assessed in the setting of the current testing approach alongside IGRA.

Results: Studies in cattle experimentally infected with M. bovis revealed positive

CXCL10 responses could be detected in some animals as early as two weeks post infection. Flow cytometry data demonstrated CXCL10 production in both lymphocyte and monocyte cell populations. When using a tuberculin based whole blood assay, CXCL10 alone could not substitute for IFN-γ as the analyte measured in the test without reducing the sensitivity of detecting bovine TB animals. However, when used as an additional test readout, CXCL10 identified bovine TB animals that failed to induce IFN-γ responses.

Conclusion: The results demonstrate that in particular settings, measurement of CXCL10 has the potential to complement the current use of IFN-γ in blood assays to maximise the detection of BTB.

O59Predicting vaccine-induced heterologous protection via advanced analysis of PRRSV-specific memory immune cells

Kick A.R., Amaral A.F., Cortes L.M., Crisci E., Almond G.W., Käser T.

NC State University - College of Veterinary Medicine, PHP, Raleigh, United States

Immunological memory cells protect the host against homologous and heterologous strains of a pathogen as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Vaccination relies on the induction of these memory cells; but current testing of the vaccine-induced immune response in swine rarely evaluates these memory cells. To overcome this limitation, we developed an in

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vitro cell culture system combined with multi-color flow cytometry (FCM) to directly detect and distinguish various pathogen-specific memory immune cells. We also studied their cytokine production and homing patterns on a single cell level to predict their protective value.

In vivo, piglets were inoculated with either MOCK, a modified-live vaccine (MLV), a low-pathogenic (LP) or a high-pathogenic (HP) PRRSV-2 strain. For nine weeks, blood was collected to study - viremia, systemic anti-PRRSV IgG levels, neutralizing antibodies, and to isolate PBMC. At necropsy, draining lymph node cells were isolated to study the regional immune responses. Immune cells were in vitro re-stimulated with homologous and heterologous PRRSV strains to describe cross-reactive memory cells via FCM.

Both, PRRSV infection and vaccination resulted in similar viremia; while clinical disease was limited to LP and HP infected animals. The humoral response including neutralizing antibodies was delayed in vaccinated pigs (28dpi) compared to infected animals (10dpi); and it showed limited cross-protection against the LP and HP strains. The strongest homologous T-cell memory response was induced by HP followed by LP and then MLV inoculation. LP and HP induced memory cells had ~80% cross-reactivity to each other but only ~20% to the MLV; and ~20% of the MLV memory cells reacted to LP and HP strains. Immune cells reactive to PRRSV produced mainly TNF-α and/or IL-2 with IFN-γ ranking in third place.

This novel system provides a powerful tool to evaluate vaccine immunogenicity for PRRSV and predict cross-protection against various circulating PRRSV strains.

IMMUNE GENE EXPRESSION

O60Epigenetic regulation in bovine monocytes upon LPS challenge

Foucras G.1, Cebron N.1, Walachowski S.1, Chaulot-Talmon A.2, Pontelevoy C.2, Robcis R.1, de Boyer des Roches A.3, Ledoux D.3, Richard C.2, Gelin V.2, Kiefer H.2, Jammes H.2

1IHAP, Université de Toulouse, ENVT, INRA, Toulouse, France, 2BDR, INRA, ENVA, Université Paris-Saclay, Jouy-en-Josas, France, 3UMR H, Université Clermont Auvergne, VetAgro Sup, INRA, Saint- Genès-Champanelle, France

Responsiveness of the innate immune system in the long term is modified by previous exposure to microbes. This has been recently redefined as trained immunity. Monocytes can undergo functional changes upon contact with pathogens or their products, and then adapt their response to subsequent challenges. However, the molecular bases of long-term reprogramming are still poorly understood in cattle. Epigenetic marks were assessed after a LPS challenge in 16 cows, half of them being bovine clones of a single genotype. All cows had the same age and were raised together as a single group since birth in an experimental farm. Cows were challenged through an intravenous bolus injection of LPS (0.5 µg/kg BW ultrapure LPS, InVivogen). Cytokine production was measured sequentially (0, 3, 6, 12, and 24h) in plasma using a newly developed Custom bovine cytokine 15-plex

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assay. Blood samples were collected twice just before and 24 hours after LPS injection. Monocytes were isolated by Ficoll gradient and selected using CD14 magnetic beads. Genome-wide DNA methylation was analyzed by reduced representation bisulfite sequencing (RRBS; Perrier et al., 2018) using a dedicated pipeline, in order to assess epigenetic marks according to the genetic background and the response to the LPS challenge. Previous comparison between libraries from monocytes, PBMCs and fibroblasts identified the monocyte methylome. Using this new set of 16 libraries, we describe original differentially methylated cytosine (DMCs) highlighting methylation variations between the groups (cloned and control cows), and before/after the LPS challenge. Altogether, identified DMCs target genomic regions important to monocyte identity and functions potentially independent of genetic variation. Monocyte DNA undergoes epigenetic modifications after LPS challenge, indicating that previous exposure, at least to Gram-negative bacteria, may modify the later capacity of the cells to respond to an infection.

O61Combined chromatin state and transcriptional analyses of pig alveolar macrophages reveal regulatory elements of the inflammatory response

Herrera Uribe J.1, Haibo L.1, Olson Z.2, Byrne K.2, Loving C.2, Tuggle C.1

1Iowa State University, Animal Science, Ames, United States, 2USDA-ARS-National Animal Disease Center, Ames, United States

Transcription is regulated by transcription factor binding at gene regulatory elements, and histone modifications control the chromatin accessibility of these elements. Alveolar macrophages (AM) play critical roles in pulmonary host defense against invading pathogens. This study aims at integrating epigenetic and transcriptomic profiles of pig AM stimulated with bacterial and viral mimics, lipopolysaccharide (LPS) and double stranded RNA (Poly (I:C)), respectively, to contribute to identification of the functional components of the pig immune system. By combining RNA sequencing (RNA-seq) with chromatin immunoprecipitation and sequencing (ChIP-seq) for four histone modifications (H3K4me3, H3K4me1, H3K27me3 and H3K27ac), we investigated chromatin changes and the potential regulatory effect on the transcriptional program in macrophages stimulated with LPS, Poly (I:C), or vehicle for 2 or 6 hours. Differential expression gene (DEG) analysis and pairwise comparison between treatments and timepoints were performed. These analyses have shown the induction of differential expression of hundreds to thousands of genes at 2 and 6 hours after either stimulus. These gene lists are enriched for several biological functions related to inflammatory response. Pairwise comparison between treatments showed a similar pattern of gene expression; meanwhile, the time post-stimuli had a big impact on gene expression. Preliminary analysis of ChIP-seq peak distribution showed the permissive histone mark H3K4me3 was found primarily around promoter regions (34.3% detected from 0-3 kb around start site), and more so than the repressive histone mark H3K27me3 (19.9%). Further, through data integration of RNA-seq and ChIP-seq, we found a high correlation of peak signal intensity of H3K4me3 with levels of gene expression (high, middle-high, middle-low and low expression). Altogether, these

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combinatorial approaches present a powerful resource to develop a deeper understanding of the functional components of the pig immune system and provide information to develop a chromatin state map in macrophage immune response states.

O62Immune fitness as a measure of animal health, welfare and productivity in the feedlot: transcriptomic analysis

Purdie A.C., Plain K., de Silva K.

University of Sydney, Faculty of Science, Sydney School of Veterinary Science, Camden, Australia

Background: Animals have long been bred for production traits that enable high-yield but this does not take into account the overall immunological ‘fitness’ of production animals. Basic questions remain unanswered: what is the physiological basis that enables some animals to thrive when exposed to production stresses and are there any effective measures of the immune component to identify these animals? Conversely, what immune measures are correlated to or indicative of an animal at risk of poor performance? Further, are there commonly related genes associated with immune fitness?

Methods: 50 mixed breed beef cattle (Charolias, Droughtmaster, Red Angus, Charbray, Murray Grey, Angus, Hereford) were randomly selected at feedlot intake. Blood and saliva samples were collected from the cattle at arrival (high stress), post- acclimatization

and pre-slaughter (60 days post intake or ≥400kg). Body condition and other measures (weight, height, disposition, coat colour) were collected at each sampling and at slaughter. Samples were processed for cell counts, RNA (transcriptomics analysis) and serum (protein analysis). RNA samples were processed for transcriptomic analysis by 24k Bovine Affymetrix ST1 GeneChip.

Results and conclusion: Gene expression analysis correlated to outcome measures and previously published datasets identified genes associated with immune fitness including 227 genes correlated to animals with susceptibility to infection. These results suggest that there are immune measures that may be utilized to identify animals at risk of poor production performance and this may be utilized in the future to improve animal management to optimize animal health and welfare.

O63Immunogenomics of innate immune cells of the porcine lung during PRRSV infection

Crisci E.1,2,3, Mohammad A.4, Jourdren L.4, Moroldo M.1, Bourge M.5, Urien C.6, Bouguyon E.6, Bordet E.6, Schwartz I.6, Bertho N.6, Giuffra E.1

1UMR Génétique Animale et Biologie Intégrative, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France, 2North Carolina State University, College of Veterinary Medicine, Department of Population Health and Pathobiology, Raleigh, United States, 3North Carolina State University, Comparative Medicine Institute, Raleigh, United States,

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4Institut de Biologie de l’École normale supérieure (IBENS), École normale supérieure, CNRS, INSERM PSL Université Paris, Paris, France, 5Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France, 6Virologie et Immunologie Moléculaire, INRA, Université Paris-Saclay, Jouy-en-Josas, France

Porcine reproductive and respiratory syndrome virus (PRRSV) has an extensive impact on pig production and, due to its recombination properties, presents a vast genetic diversity worldwide. PRRSV is divided in two species, type 1 (European origin, PRRSV-1) and type 2 (North American origin, PRRSV-2) and within PRRSV-1 specie, PRRSV-1.3 strains such as Lena are more pathogenic and trigger a higher Th1 response than PRRSV-1.1 such as Lelystad or Flanders 13 (FL13).

To date, the molecular interactions of PRRSV with primary lung mononuclear phagocytes (MNP) subtypes such as conventional dendritic cells type 1 (cDC1), cDC2, monocyte-derived DCs (moDC) and parenchymal macrophages (AM-like/PIMs) have not been thoroughly investigated.

Here, we describe the transcriptome profiles of in vitro FL13- and Lena-infected parenchymal MNP and of in vivo FL13-infected parenchymal MNP subpopulations obtained using RNAseq.

In vitro, we found respectively 4,500 and 23 differentially expressed genes (DEGs) in Lena-infected MNP and FL13-infected MNP compared to mock-infected cells confirming the potent modulation induced by Lena. Considering the low number of DEGs obtained with conventional statistics in FL13 condition, we decide to use a machine learning approach to unravel other relevant genes in FL13-infected cells. Roughly, 500 additional

genes were predicted and enriched in 19 IPA canonical pathways; in particular, two of them, related to the oxidative phosphorylation and mitochondrial dysfunction, were not shared with the 202 pathways found in Lena-infected cells. Transcriptomic data from in vivo sorted cells (alveolar macrophages, AM-like/PIMs, cDC1, cDC2, moDC) delineated cell specific clusters and confirmed the low number of DEGs during FL13 infection.

These data indicate that, whereas Lena strongly triggers the innate lung immune system, FL13 keeps antiviral and inflammatory AM/DC functions silent. Some transcriptomic clues might relate FL13’ stealth to virus-induced mitochondrial dysfunctions. Validations of key DEGs are currently in progress.

O64Whole blood transcriptome analysis reveals altered pathways related to inflammation and fatty acid metabolism in stocker calves that develop bovine respiratory disease

Scott M.1, Woolums A.1, Swiderski C.2, Perkins A.3, Nanduri B.4, Smith D.1, Karisch B.5, Epperson W.B.1, Blanton Jr. J.5

1Mississippi State University College of Veterinary Medicine, Pathobiology and Population Medicine, Mississippi State, United States, 2Mississippi State University College of Veterinary Medicine, Department of Clinical Sciences, Mississippi State, United States, 3Mississippi State University, Department of Computer Science and Engineering, Mississippi State, United States, 4Mississippi State University College of Veterinary Medicine, Department of Basic Sciences, Mississippi

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State, United States, 5Mississippi State University, Department of Animal and Dairy Sciences, Mississippi State, United States

Bovine respiratory disease (BRD) is a multifactorial disease and the leading cause of morbidity and mortality in post-weaned beef cattle in North America. The complex interactions between infectious agents, host immune response, and environmental risk factors are poorly understood. The hypothesis of this study is that altered transcriptional profiles found in the blood of stocker calves will reveal regulatory pathways responsible for protection from and resistance to respiratory disease. Blood was collected from the jugular vein into Tempus tubes (ThermoFisher) from bull and steer calves (n=80, mean weight=206 kg) at stocker facility arrival. Animals were monitored for clinical signs of BRD based on an adapted version of the DART system. Cattle diagnosed with BRD within the first 14 days following arrival (n=6), and cattle without signs of BRD over the 84-day period of evaluation (n=5) were selected for blood RNA sequencing (Illumina HiSeq 3000). Sequencing reads (80M paired end/sample) were quality filtered and aligned to the bovine reference genome assembly ARS-UCD1.2. False discovery rate (FDR) adjusted p-values of 0.10 were applied to identify differentially expressed genes, utilizing the analysis tool edgeR. Fifty-two differentially expressed genes were identified between the healthy and diseased groups; thirty-six were downregulated and sixteen were upregulated in diseased calves when compared to healthy calves. The online resources WebGestalt 2019 (WEB-based Gene SeT AnaLysis Toolkit) and String v11.0 were utilized to identify biological functions, pathways, networks, and interactions represented by the differentially expressed genes. We identified pathways related to immune response, inflammatory mediation, metabolic processes,

and stress regulation. Using whole blood transcriptomic analysis, this study provides an insight to host immunity in response to BRD.

O65Transcriptomic analysis of devil facial tumour disease in non-immunised and immunised Tasmanian devils

Patchett A.L.1, Pye R.J.1, Woods G.M.1, Flies A.S.1, Lyons A.B.2

1Menzies Institute for Medical Research, University of Tasmania, Hobart, Australia, 2School of Medicine, University of Tasmania, Hobart, Australia

Background: Devil facial tumour disease (DFTD) is a transmissible Schwann cell cancer threatening the survival of the Tasmanian devil (Sarcophilus harrisii), a carnivorous marsupial native to the Australian state of Tasmania. DFTD is now endemic across most of Tasmania and has triggered a drastic population decline of around 80%. Advances in our understanding of DFTD have led to the development of a candidate prophylactic DFTD vaccine that induces tumour-specific immune responses in over 95% of immunised devils. However, these primed immune responses have not yet resulted in complete protection against DFTD transmission.

Method: To understand how DFT cells escape primed immune responses in immunised devils, we performed RNA-seq of DFTD tumour biopsies from non-immunised and immunised Tasmanian devils. Differential gene expression analysis was performed to identify differences

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between tumours arising among these devils. Candidate mechanisms of immune escape in immunised devils were determined and further investigated in vitro using recombinant technologies and cell culture assays.

Results: DFTD tumours from immunised devils exhibited increased expression of genes related to immune function, suggesting greater immune infiltration into these tumours. In comparison, many highly expressed DFTD genes including mature Schwann cell markers, were down-regulated in tumours taken from immunised devils. TGFβ was identified as a candidate driver of the phenotypic changes in these DFTD tumours, and a recombinant devil TGFβ protein has been produced to further assess these effects in vitro.

Conclusion: DFTD immunisations induce immune recognition of DFTD cells, but changes in the phenotype of DFTD tumours suggests that the cells can adapt to evade this primed response. A better understanding of the mechanisms promoting DFTD evasion of primed immunity will allow for optimisation of vaccines to induce protective responses against DFTD in Tasmanian devils.

RUMINANT IMMUNITY

O66Intra-mammary vaccine delivery protects sheep against mastitis caused by mannheimia haemolytica infection

Ballingall K.T., Todd H., Tassi R.

Moredun Research Institute, Disease Control, Edinburgh, United Kingdom

Bacterial intra-mammary infections are often the cause of mastitis in lactating ruminant livestock. While the cattle dairy industry is the principal target sector for interventions designed to reduce the impact of mastitis, the condition also impacts other ruminant livestock species including sheep. Mastitis adversely affects both dairy and meat/fibre sheep industries with losses un the UK alone in 2016 estimated to be over £120 million due to reductions in milk quality and quantity, treatment costs, early culling and slower lamb growth. Effective treatments including vaccines are therefore also required for mastitis control in sheep. However, the development of vaccines against mastitis has proved challenging due to the failure to target the response to the mammary gland. This study therefore aimed to test whether local administration directly into the mammary gland through the teat canal or subcutaneous immunisation targeting the local lymph nodes, of a vaccine originally developed against respiratory disease caused by Mannheimia haemolytica also confers protection against an intra-mammary infection with the same pathogen. Intra-mammary immunisation protected all ewes against experimental challenge seven days post immunisation while no protection was observed in control animals or those immunised over the draining lymph nodes. However, the protective effect at seven days was not detected fourteen days post immunisation. The response in both unvaccinated controls and immunised animals showed increases in the pro-inflammatory cytokines IL-1β and the anti-inflammatory IL-10 and IL-17A following challenge. This response was also induced following vaccination suggesting that the induction of a Th17 type response to the intra-

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mammary vaccine may be linked to the early protection but is not maintained for two weeks.

O67Immunological responses and protection in dairy cows vaccinated with staphylococcal surface proteins

Merrill C.1, Ensermu D.1, Abdi R.2, Gillespie B.1, Vaughn J.1, Headrick S.1, Hash K.1, Walker T.1, Kerro Dego O.2

1The University of Tennessee, Animal Science, Knoxville, United States, 2Th University of Tennessee, Animal Science, Knoxville, United States

Bovine mastitis is a significant cause of economic losses in the dairy industry. Staphylococcus aureus is one of the most common contagious mastitis pathogens, whereas Staphylococcus chromogenes increasingly became a significant cause of subclinical mastitis in dairy cows. Current mastitis control measures are not effective on all mastitis pathogens. There is no effective vaccine to control Staphylococcal mastitis in dairy cows. The objective of this study was to evaluate the immune responses and protection in dairy cows vaccinated with S. aureus surface proteins (SASP) and S. chromogenes surface proteins (SCSP). We divided eighteen Holstein dairy cows randomly into three groups of 6 animals each. We vaccinated group 1 and 2 animals with SASP and SCSP with Emulsigen-D adjuvant, respectively. We injected group 3 animals with PBS (pH 7.2) mixed with Emulsigen-D and served as a control. We vaccinated animals three times at 28 and 14 days before drying off, and at dry off. Two weeks after the third vaccination, we challenged each animal by dipping all teats

in S. aureus culture suspension once daily for 14 consecutive days. We evaluated milk or mammary secretion and serum antibody titers during vaccination and challenge periods. We evaluated milk samples for the number of bacteria shedding and somatic cell counts (SCC). Out of six cows vaccinated with SASP, one cow was removed from the study due to injury, two were infected clinically, another two were infected subclinically, and the remaining cow was not infected. No SCSP vaccinated cows developed clinical or subclinical mastitis. Out of six control cows, two developed clinical mastitis whereas four were infected subclinically. The SCSP vaccine cross-protected against S. aureus mastitis and reduced number of S. aureus shedding in milk. We concluded that the SCSP is an effective vaccine to control Staphylococcal mastitis in dairy cows.

O68Phenotypic analysis of T cell subsets expressing Interferon-gamma, interleukin -17A and tumor necrosis factor-alpha across ruminant species

Elnaggar M.M.1,2, Abdellrazeq G.S.1,2, Fry L.3, Dassanayake R.P.4, Mahmoud A.H.1,5, Hulubei V.1, Sacco R.E.4, Davis W.C.1

1College of Veterinary Medicine, Washington State University, Pullman, WA, United States, 2Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt, 3Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, WA, United States, 4Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, United

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States Department of Agriculture, Agricultural Research Service, Ames, IA, United States, 5General Organization for Veterinary Services, Ministry of Agriculture, Alexandria, Egypt

Interferon-gamma (IFN-γ), interleukin-17A (IL-17A) and tumor necrosis factor-alpha (TNF-α) are cytokines centrally involved in protective immune responses to pathogens. As part of our ongoing program to expand reagents available for animal research, we developed a set of monoclonal antibodies that recognize IFN-γ, IL-17A and TNF-α, and validated their use in intracellular flow cytometry staining in cattle, sheep and goats. As reported here, comparative flow cytometric analyses of lymphocyte subsets stimulated with phorbol 12-myristate 13-acetate and ionomycin revealed differences in expression of IFN-γ, IL-17A and TNF-α by CD4, CD8, and γδ T cells across ruminant species. Analysis of IFN-γ expression revealed the majority of IFN-γ expressing cells in cattle were CD4+ and CD8+ T cells, with little expression by γδ T cells which were mainly WC1-/CD2+. In sheep and goats, the majority of cells expressing IFN-γ were CD8+ and CD4+ T cells. Analysis of IL-17A expression in cattle showed the majority of cells expressing IL-17A were CD4+ and WC1+ γδ T cells. Results in sheep and goats revealed IL-17A is expressed mainly by CD4+ and CD8+ T cells, with little expression by WC1+ γδ T cells. Analysis of TNF-α expression in cattle, sheep and goats revealed the majority of cells expressing TNF-α were CD4+ and CD8+ T cells, and little expression by γδ T cells. The data presented in this study on the biology of IFN-γ, IL-17A and TNF-α expression among ruminant species expands our knowledge on the differential expression of cytokines by different T cell subsets, and should help clarify differences in their roles in response to pathogens.

O69IFN-γ response to bacteria or vaccination is not correlated with protection against mycobacterial infection

Jungersen G.1, Thakur A.2,3, Mikkelsen H.M.2

1Technical University of Denmark, Health Technology, Kgs Lyngby, Denmark, Denmark, 2Technical University of Denmark, National Veterinary Institute, Kgs Lyngby, Denmark, Denmark, 3Copenhagen University, Faculty of Health and Medical Sciences, Copenhagen, Denmark

Background: It is commonly perceived that protective immunity to mycobacteria is mediated by Th1 responses, and that vaccines against mycobacterial infections must induce Ag-specific IFN-γ, and possibly polyfunctional CD4+ T cells, to induce protection. Here we compile results from a number of vaccination studies in calves with Mycobacterium paratuberculosis (MAP) challenge, and correlate the early and late CMI responses to infection and vaccination with the final mycobacterial load in tissues.

Methods: Calves were vaccinated with commercial inactivated whole-cell MAP vaccine, recombinant multi-stage protein vaccines or non-vaccinated in pre- and post-exposure challenge studies and terminated 9 (n=31), 28 (n=48) or 52 (n=28) weeks post MAP challenge. Over the course of the experiments, whole blood samples were cultured with Johnin PPD and vaccine antigens and Ag-specific IFN-γ production measured in culture supernatants. Ag-specific polyfunctional (IFN-γ, TNFα, IL2) CD4+ T cells were monitored by flow cytometry. MAP in tissues were quantified by culture and qPCR at termination.

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Results: Vaccine induced protection and individual calf IFN-γ responses to vaccination were variable, allowing us to investigate the correlation between observed protection at slaughter and immune response to vaccination and challenge. Load of MAP in tissues at termination was not associated with level of Vaccine-specific IFN-γ production at early or late time points after vaccination. Map-specific (PPDj) IFN-γ production appeared as a surrogate of disease with a direct positive correlation with level of MAP in tissues at slaughter. Polyfunctional T cells were induced by vaccination, but were not correlated with protection and could not be sustained during the long-term infection although Vaccine- and MAP-specific IFN-γ levels increased throughout the study period. Conclusion: Ag-specific IFN-γ response to vaccination is not a reliable predictor of protection and MAP-specific IFN-γ response to PPDj is a predictor of level of infection rather than level of immunocompetent response to challenge.

O70Local Th17 immunity upon mammary immunization is protective against E. coli mastitis

Cebron N.1, Maman S.2, Rainard P.3, Foucras G.1

1IHAP, Université de Toulouse, ENVT, INRA, Toulouse, France, 2SIGENAE, INRA, Bio-informatics platform, Toulouse, France, 3ISP, INRA, Université de Tours, Nouzilly, France

Current vaccines to Escherichia coli mastitis have shown some albeit limited efficacy. Their

mode of action is poorly documented and the immune response protecting the mammary gland against E. coli is not completely understood. To improve our knowledge of mammary gland immune defenses, we compared the response of three groups of six cows that received either an intramuscular or an intramammary protocol of immunization against E. coli P4 before a homologous challenge. The control group received adjuvant only. Local immunization modified favorably the course of infection, by improving bacterial clearance while limiting inflammation. Systemic clinical score was also reduced and mammary secretion was preserved (Herry, 2017). High-throughput profiling using a newly developed cytokine 15-plex assay indicated a diminished TNFα production while increased IFNγ was detected in the immunized groups. Antibody response did not correlate with protection, but cellular immunity better related to protection of the mammary gland. Indeed, a transcriptome analysis performed using RNA sequencing on blood samples collected during immunization and infection phases shows that the lymphocyte response was activated in all groups 12 hours after inoculation and correlated with lower clinical scores as shown by weighted correlation network analysis. At the same time, neutrophils were produced and recruited with a moderate neutropenia in immunized groups. Type I interferon response correlated with intensity and persistence of inflammation during the late phase of mastitis. Furthermore, to assess the local T cell response in mammary tissues of locally immunized and control cows, CD4pos T cells were isolated by fluorescence cell sorting upon mammary tissue digestion of challenged and control glands, and their transcriptome determined by RNA sequencing. Results show that IL-17 expression was increased during E. coli infection and Th17 response was significantly enriched in immunized glands. These findings indicate

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that protective mechanisms linked to local immunization rely on IL-17-mediated immunity.

O71Type III interferon protects cattle against bovine viral diarrhea virus infection

Quintana M.E.1,2, Pereyra R.1,3, Cardoso N.P.1,2, Barone L.J.1,2, Barrionuevo F.M.2, Turco C.2, Mansilla F.C.2, Capozzo A.V.1,2

1Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), CABA, Argentina, 2Instituto de Virología, Centro de Investigaciones en Ciencias Veterinarias y Agronómicas (CICVyA), Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina, 3Área de Investigación en Sanidad Animal IIACS-CIAP- Estación experimental Agropecuaria EEA Cerrillos, Instituto Nacional de Tecnología Agropecuaria (INTA), Salta, Argentina

Background: Bovine viral diarrhea virus (BVDV) is an important pathogen of cattle that can cause significant economic losses in livestock industries, mainly due to reproduction failures and immunosuppression that predispose to opportunistic infections. It is known that infected animals elicit type-III interferon; however, there is no information on the antiviral activity of this cytokine in cattle.

Method: Recombinant bovine interferon lambda 3 (rIFNλ) was expressed in HEK-293 cells. Safety was assessed in 90-days-old calves (n=12) inoculated with two doses of 1.5, 3 and 6 U/kg, verifying the absence of hepatic and hematological toxicity. In a

second experiment, 21 days-old BVDV-free calves (n=6) were infected with a BVDV-2 non-cytopathic field strain which produces a well-characterized respiratory disease. Calves (n=4) were inoculated with 6 U/kg of rINFλ two days before and after infection, while another group (n=2) was mock-treated. Presence of BVDV-NS3 protein and cytokines in serum samples and nasal swabs were measured using commercial ELISA kits from 2 to 21 days post-infection (dpi).

Results: Untreated calves developed respiratory disease, clinical signs appeared at 2 dpi and reached a maximum score at 4 dpi. Shedding was detected from 4 to 7 dpi and viremia persisted from 2 to 7 dpi. Systemic TNF-α was induced at 2 dpi. Conversely, rIFNλ-treated calves did not develop clinical disease, none of them shedded the virus and viremia was completely abolished in 3 of the calves, while the other animal had significantly lower NS3-serum levels (p< 0.05) than untreated-calves at all time-points. TNF-α was not detected and systemic IL-10 levels were increased, suggesting a role of rIFNλ in controlling inflammation.

Conclusion: These results demonstrate that rIFNλ is safe in cattle and capable of protecting these animals against the clinical disease caused by BVDV. This is the first report of the efficacy of type-III INF therapy in cattle.

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P001Selection for gastrointestinal parasite resistance reduces MHC allelic richness but favours functional diversity in Cashmere goat lines

Ballingall K.T., McBean D., Todd H.

Moredun Research Institute, Disease Control, Edinburgh, United Kingdom

Many livestock breeds have undergone extensive selection for improved production. Selection is likely to result in a reduction in the genetic diversity of the selected population when compared with the original population. The Scottish Cashmere goat breed was derived from crossing Scottish feral goats with Cashmere goats originating from Iceland, Siberia, Tasmania and New Zealand. The progeny of these initial crosses acted as a nucleus flock for a large commercial breeding programme. Through this programme, goat lines were developed for improved production and helminth parasite resistance. An unselected line was also maintained providing an excellent opportunity to investigate the impact of selection. Two hundred goats from selected and unselected lines were genotyped by a novel sequence based method targeting the polymorphic MHC class II DRB1 locus. Twenty eight Cahi-DRB1 alleles were identified reflecting the composite origin of these animals. These alleles were used to populate a new goat section on the immunopolymorphism database IPD-MHC. Overall allelic richness was reduced by 25% in the selected line compared with the unselected line. Four Cahi-DRB1 alleles were significantly enriched through selection while six alleles were significantly reduced in frequency in the selected line but highly represented in the unselected line. Phylogenetic analysis grouped

all 28 alleles into seven clusters. Alleles favoured by selection were broadly distributed across the clusters indicating a high degree of nucleotide and amino acid diversity while those favoured in the unselected line were more limited in their distribution, indicating lower levels of diversity. These data suggest that while selection has reduced allelic richness, the enriched alleles represent a broader range of diversity than those at high frequency in the unselected line.

P002Establishment and characterization of primary and hTERT immortalized sheep ileal epithelial cells

Bhattarai S.1, Uprety T.1, Young A.2, Kaushik R.S.1

1South Dakota State University, Biology and Microbiology, Brookings, United States, 2South Dakota State University, Veterinary and Biomedical Sciences, Brookings, United States

Background: Intestinal epithelial cells are constantly exposed to both commensal and pathogenic microbes. These cells play important role in intestinal immunity and must maintain a balance between immunity and tolerance for homeostasis. Main goal of this study is to establish and characterize sheep ileal epithelial cell cultures for understanding intestinal innate immune responses of sheep.

Methods: Ileum tissue from 3-day-old lamb was obtained and after enzymatic digestion of tissue primary ovine ileal epithelial cell culture was established. Established primary

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cells were transfected with human telomerase reverse transcriptase (hTERT) gene and presence of the gene in the immortalized cells was confirmed by PCR. Immunocytochemistry was performed using cytokeratin, vimentin, alpha-smooth muscle actin or desmin as cell specific markers. Further characterization of ileal cultures was done by measuring trans-epithelial electrical resistance (TEER) of both primary and hTERT immortalized cell cultures. Indirect immunofluorescence assay (IFA) was used for the expression of tight junction proteins in both primary and hTERT immortalized polarized cells. Lectin binding profile of both cell types using 24 different lectins was also studied using flow cytometry.

Results: We successfully established both primary and hTERT immortalized ovine ileal epithelial cell cultures. Primary and immortalized cells strongly expressed cytokeratin protein confirming their epithelial phenotype. Both primary and immortalized cultures on transmembrane filters showed TEER in range of 720-1200 Ω indicating their polarization. IFA revealed the expression of tight junction proteins occludin, zonula occludin-1, and Claudin-3 in both primary and immortalized polarized cells. Lectins Solanum tuberosum, Datura stramonium, and Phaseolus vulgaris leucoagglutinin intensely stained (>80%) both cell types; however, Griffornia simplicifolia-II lectin did not stain either cell types.

Conclusion: Primary and immortalized sheep ileal epithelial cell lines were established in this study and these cell cultures may serve as good in-vitro model for studying innate immune responses and enteric disease pathogenesis.

P003Immunomodulatory effects of race training at the level of the airway-derived macrophage

Karagianni A.E., Cillán-Garcia E., Wishart T., Eaton S., Pirie S.R.

The Roslin Institute and the Royal (Dick) School of Veterinary Studies, Edinburgh, United Kingdom

The development of airway inflammation is very common in racehorses during training, with prevalence rates as high as 70-80%. Although rarely associated with overt clinical signs, this syndrome (mild to moderate equine asthma) can significantly impair athletic performance and impact welfare. However, its precise cause remains undetermined. In humans, an association between high intensity exercise and symptoms of respiratory infection amongst athletes is well recognised and has fuelled interest in the impact of training on immune function. We recently performed a gene expression scan using microarray technology on alveolar macrophages isolated from Standardbred racehorses prior to and following commencement of competition race training. Our results revealed a level of training-associated basal gene expression modulation consistent an immunological derangement with the potential to increase susceptibility to opportunistic infection. These data provided a clear justification for further defining the link between training and airway immunity in the horse.

To extend these studies, we aim to perform RNAseq and proteomic analysis of airway cells derived from routinely collected tracheal wash samples rather than bronchoalveolar-derived samples, which are less readily available.

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Preliminary studies have investigated the most appropriate means of (a) isolating airway macrophages from this sample set and (b) maximising RNA yield and quality. Moreover, current analysis has confirmed cross-reactivity of an anti-human CD163 antibody and successful application of magnetic bead separation to dithiothreitol-treated equine tracheal secretion samples.

This study will provide a necessary methodological platform for the planned RNAseq and proteomic analyses, which should subsequently provide a greater insight into the impact of training on equine pulmonary immunity. Furthermore, in light of the fact that the horse is a natural athlete and there are published similarities between equine and human alveolar macrophages, we consider our findings to have potentially valuable translational applications to human exercise immunology.

P004Vitamin A deficiency impairs the mucosal immune response to respiratory syncytial virus vaccination and infection in neonatal calves

McGill J.L.1,2, Kelly S.M.3, Guerra-Maupome M.1, Winkley E.4, Henningson J.4, Sacco R.E.5, Narasimhan B.2,3

1Iowa State University, Department of Veterinary Microbiology and Preventive Medicine, Ames, United States, 2Nanvaccine Institute, Iowa State University, Ames, United States, 3Iowa State University, Department of Chemical and Biological Engineering, Ames, United

States, 4Kansas State University, Department of Diagnostic Medicine and Pathobiology, Manhattan, United States, 5Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, USDA, Ames, United States

Bovine respiratory disease (BRD) is a leading cause of morbidity and mortality in feedlot and dairy cattle. Bovine respiratory syncytial virus (BRSV) causes acute viral infection in young calves and contributes to BRD. Micronutrients are essential components of livestock diets and are critical for supporting optimum animal health. Vitamin A (VA), or retinol, is a fat-soluble vitamin that plays a vital role in immunity at mucosal sites, impacting IgA production, mucosal innate immune function and tissue-resident T cell responses in both the gut and respiratory tracts. Dietary requirements for VA in cattle have been defined; however, there appears to be a clear distinction between the requirement for optimal growth, and levels required to support a strong immune system. Calves are naturally born VA deficient (VAD), and VA needs increase in stressed and morbid cattle. The recent worldwide shortage in synthetic VA has also increased the risk of deficiency in US herds. VA deficiency has been linked to increased susceptibility to disease, but its impact on BRD has not been examined. Here, we used the neonatal calf model to determine the impact of VAD on the immune response to an intranasal BRSV vaccine, and subsequent challenge with BRSV. Our results show that VAD calves are unable to respond to mucosal vaccination and are at increased risk for severe RSV disease. VAD animals also present with significant abnormalities in the inflammatory milieu in the infected lung, with alterations in Th1 and Th17 immune responses, and impaired mucin production. Our studies further show that acute respiratory infection has a significant negative impact on

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circulating and stored VA levels, causing even vitamin- and mineral-replete calves to become VA deficient. Our results suggest that VA status has a significant impact on the mucosal immune system and resistance to respiratory viral infection in young calves.

P005Intensity of the local immune response reflects route of entry after intranasal H1N1pdm09 infection in pigs

Schwaiger T.1, Sehl J.1,2, Karte C.3, Schäfer A.4, Hühr J.4, Mettenleiter T.C.2, Schröder C.1, Ulrich R.1, Köllner B.4, Blohm U.4

1Friedrich-Loeffler-Institute, Department of Experimental Animal Facilities and Biorisk Management, Greifswald, Germany, 2Friedrich-Loeffler-Institute, Institute of Molecular Virology and Cell Biology, Greifswald, Germany, 3Friedrich-Loeffler-Institute, Institute of Diagnostic Virology, Greifswald, Germany, 4Friedrich-Loeffler-Institute, Institute of Immunology, Greifswald, Germany

Background: Pigs represent a natural host for influenza A viruses (IAV) and play a significant role as a mixing vessel. The resulting reassorted viruses can render current antibody-based vaccines ineffective. Using the pig as a biomedical model, we investigated the systemic as well as the local cellular immune response upon two subsequent intranasal infections with H1N1pdm09.

Methods: Twenty-six German landrace pig were infected intranasally with H1N1pdm09 twice (day 0 and day 21). In irregular intervals,

blood was subjected to multicolor flow cytometric analysis. During necropsies at days 4, 7, 21, 25 and 31, bronchoalveolar lavage (BAL), mucosa from nasal cavity, lung and the tracheobronchial lymph node were collected. Isolated leukocytes from these tissues were analyzed to determine local immune responses along the respiratory tract.

Results: Pigs did not show any clinical signs during the study period, but peripheral blood lymphocyte counts decreased initially after the first infection but recovering until day 14. The simultaneous increase in the frequencies of proliferating cells correlated with an increase in infiltrating leukocytes in the lung. Enhanced perforin expression in αβ and γδ T cells in the respiratory tract indicated a cytotoxic T cell response in the nose, lung and BAL. Simultaneously, inhibitory CD8αα expressing αβ T cells were generated as early as day 4 after first infection.

Conclusion: This dual approach revealed that intranasal infection with H1N1pdm09 increased proliferative activity in lymphocytes systemically, but expression of effector as well as inhibitory molecules was mainly restricted to T cells along the route of virus entry. This in turn efficiently cleared the virus while preventing harmful immune responses. These characteristics and thus, the course of infection are similar to that of human seasonal IAV infections.

P006Establishment of gnotobiotic pig model for studying the colonization of nasal microbiome and its role in respiratory immune system development

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Uprety T.1, Bhattarai S.1, Antony L.2, Sreenivasan C.1, Ghimire S.2, Thomas M.2, Lawson S.2, Francis D.H.2, Li F.1, Scaria J.2, Kaushik R.S.1

1South Dakota State University, Biology and Microbiology, Brookings, United States, 2South Dakota State University, Veterinary and Biomedical Sciences, Brookings, United States

The goal of this study is to establish a gnotobiotic pig model for investigating the role of nasal microbiome in respiratory immune system development. We collected nasal microbiome from 2-3 week old piglets and a homogenous inoculum was prepared. In first experiment, colostrum-deprived gnotobiotic piglets on day 3 were intranasally inoculated with nasal microbiome. Surprisingly, all microbiome inoculated piglets died within 3-4 days due to infection with pathogens. In second experiment, to provide passive immunity, we injected sow serum intraperitoneally to colostrum-deprived gnotobiotic piglets on days 1-3 before inoculation of nasal microbiome. Nasal microbiome successfully colonized pig nasal cavity and gut; however, few piglets died or were euthanized by day 6 post inoculation as they showed signs of severe sickness. At 7 days post microbiome inoculation, we sacrificed remaining experimental piglets because of their poor health and collected nasal swabs, blood and tissue samples. The16s rRNA sequencing showed changes in bacterial diversity between inoculum and post colonization nasal samples. By 7th day Proteobacteria and Firmicutes were more abundant than Bacteroidetes. RT-PCR analysis of RNA from mediastinal lymph nodes, palatine tonsils, nasal mucosa and lungs showed no significant differences in expression of pattern recognition receptors and cytokines between control and nasal microbiome inoculated groups. ELISA showed no differences in serum IgG between control and nasal microbiome inoculated piglets. Based

on these observations, in third experiment, we inoculated gnotobiotic piglets with 10X lower dose of nasal microbiome and gave sow serum for longer duration (6 days) and bovine colostrum preparation for 4 days. Interestingly, all nasal microbiome inoculated piglets survived up to 3 weeks and were healthy. These piglets also showed the successful bacterial colonization in nasal cavity and gut. Thus, finally we successfully established a gnotobiotic pig model for studying the role of nasal microbiome in respiratory immune system development.

P007Chicken GM-CSF-differentiated dendritic cells can be separated into immature MHC-IIlow and spontaneously matured MHC-IIhigh subsets

Van Den Biggelaar R.H.G.A.1, Arkesteijn G.J.A.1, Rutten V.P.M.G.1,2, Van Eden W.1, Jansen C.A.1

1Utrecht University, Department of Infectious Diseases and Immunology, Utrecht, Netherlands, 2Pretoria University, Department of Veterinary Tropical Diseases, Pretoria, South Africa

Dendritic cells (DCs) play a central role in the immune system of both mammalian and non-mammalian species, including chickens. DC possess the ability to sense pathogens, using a wide variety of pattern-recognition receptors, and to present antigens resulting in T cell activation. Furthermore, they initiate events that lead to protective immunity after vaccination. Investigating chicken DCs is complicated by

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their limited cell numbers in vivo. To overcome this problem, in vitro culture methods have been developed to generate chicken DCs from bone marrow in the presence of 5% chicken serum and recombinant chicken GM-CSF and IL-4. These bone marrow-derived DCs (BM-DCs) have since then been employed to study chicken DC biology, DC-pathogen interactions, and the immunostimulatory effects of vaccines on DCs. Despite their use in many in vitro studies, a detailed characterization of chicken BM-DCs is still lacking. Here, we performed an elaborate phenotypical analysis of differentiated BM-DCs derived from 18-day-old chicken embryos using flow cytometry, fluorescent microscopy, and RT-qPCR. After 8 days of culture, chicken BM-DCs consisted of MHC-IIlow and MHC-IIhigh subsets that differed phenotypically, morphologically, and functionally from each other. MHC-IIhigh cells showed higher expression of maturation markers CD80, CCR7, and CD83 compared to MHC-IIlow cells, which suggests that the BM-DC culture contains spontaneously matured DCs. In parallel, we performed an ex vivo phenotypic analysis of splenic myeloid cells and showed the presence of similar MHC-IIlow CD80low and MHC-IIhigh CD80high subsets. Furthermore, we unexpectedly observed that the addition of chicken IL-4 to the BM-DC culture decreased the proportion of MHC-IIhigh cells. This demonstration of BM-DC subsets at different phases of maturation will have consequences for the interpretation of past studies and the design of future experiments with chicken BM-DCs.

This work has received support from the EU/EFPIA/Innovative Medicines Initiative 2 Joint Undertaking (VAC2VAC grant n° 115924).

P008Diversity of the swine leukocyte antigen class II (SLA-II) in Thai commercial pig population

Techakriengkrai N.1,2,3, Nedumpan T.1,3, Cui J.1, Suradhat S.1,3

1Chulalongkorn University, Faculty of Veterinary Science, Microbiology, Bangkok, Thailand, 2Chulalongkorn University, Diagnosis and Monitoring of Animal Pathogens Research Unit (DMAPRU), Bangkok, Thailand, 3Chulalongkorn University, Center of Excellence in Emerging Infectious Diseases in Animals (CU-EIDAs), Bangkok, Thailand

Among swine genetic markers, the highly polymorphic swine leukocyte antigen class II (SLA-II) genes are one of the key determinants, associate with not only the immune responses to infections and vaccines but also reproductive performance and meat quality. The objective of this study was to identify and characterize the SLA-II gene alleles in the commercial pig population in Thailand. In this study, a total number of 90 pigs (77 gilts and 13 boars) were randomly selected from different breeding herds of 4 major pig-producing companies. This represents approximately 70% of the commercial Thai swine production. Genomic DNA was extracted from buffy coat (gilts) and semen (boars) and used as template in a low-resolution SLA-II genotyping by sequence-specific PCR covering 17 DRB1, 11 DQB1 and 6 DQA allele groups. Overall, the frequency of SLA-II was highly variable among the pig herds in which DRB1*04XX, DQB1*04XX and DQA*03XX were the most common allele groups at a combined frequency of 29.44%, 25.44% and 31.60%, respectively. The most prevalent SLA-II haplotype in gilts was Lr-0.23 (DRB1*10XX-DQB1*06XX-DQA*01XX)

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at a frequency of 16.88%, followed by Lr-0.12 (DRB1*06XX-DQB1*07XX-DQA*01XX) and Lr-0.15b (DRB1*04XX-DQB1*0202-DQA*02XX) at 14.94% and 14.29%, respectively. In boars, Lr-0.2 (DRB1*02XX-DQB1*02XX-DQA*02XX) (42.31%) was the most frequent haplotype, followed by Lr-0.10 (DRB1*04XX-DQB1*08XX-DQA*03XX) and Lr-0.15b at 15.38% and 11.54%, respectively. While most gilts were heterozygous (70/77), almost all boars were homozygous (12/13), which might reflect their inbred nature. To our knowledge, this study is the first report on SLA-II diversity in the commercial pig population in Southeast Asia. Knowing the commonly occurring SLA-II gene allele(s) in the population will facilitate genetic improvement by selective breeding, establishment of genetically controlled animal model for further studies and identification of candidate T-cell vaccine, focusing on conserved antigenic epitopes shared by diverse strains of virus.

P009Evaluation of conventional DEC205+dendritic cells response to porcine reproductive and respitarory syndrome virus

Puebla-Clark L.1, Parra-Sánchez H.1, Reséndiz-Sandoval M.1, Valenzuela O.2, Hernández J.1

1Centro de Investigación en Alimentación y Desarrollo, A.C., Laboratorio de Inmunología, Hermosillo, Mexico, 2Universidad de Sonora, Hermosillo, Mexico

Background: Dendritic cells (cDCs) play a key role triggering naïve T cells, however, many

pathogens can infected or modulate their response and compromise T cell activation. Recently, an important progress was reported in the field of porcine conventional DCs (cDCs). Different groups described cDCs from skin, lung, blood and lymph tissues. Until few years ago, most of the studies evaluated monocyte or bone-marrow derived DCs. Now, is possible to evaluate the particpaiton of cDCs in the immunopathology of different diseasses. The aim of this work was to use the receptor DEC205 to characterize cDCs and evaluate their response against PRRSV.

Methods: PRRSV infected pigs were analyzed at 5 and 7 days post infection (dpi). Non-infected pigs were used as control. Tonsil DEC205+cDCs (MHCIIhighCADM1highDEC205+), CD163+cells (CD163+MHCII+) were sorted by flow cytometry and FLT3 and cytokine expression was analyzed by qRT-PCR. Blood and spleen from non-infected pigs were used to sort DEC205+cDCs and CD163+cells, and cultured with PRRSV at MOI of 0.1 during 24 h at 37°C. Later, cytokine expression was analyzed by qRT-PCR and ELISA.

Results: We demonstrated that DEC205 in combination witn MHCII and CAMD1 is suitable to identify bona fidecDCs. This result was confirmed with the mRNA expression of FLT3. Tonsil cDCs were not infected with PRRSV at 5 and 7 days post infection (dpi), in contrast to CD163+cells and PAMS. cDCs and tonsil CD163+number was not affected by PRRSV, in contrast to PAMS. At 5dpi, DEC205+cDCs increased the expression of IL-12. Spleen DEC205+cDCs in response to PRRSV produced more IL-12 than CD163+cells. PRRSV does not affect TNF-α production on DEC205+cDCs.

Conclusion: cDCs can be identifiy with the expresison of DEC205. Tonsil DEC205+cDCs

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are not infected with PRRSV, but can response through IL-12 production.

Disclosure: This project was supported by CONACYT, project No. 222973 and INFRA-2015-01-254826

P010Myeloid dendritic cells in in equine peripheral blood

Dakik D.1, Edwards J.2, Steinbach F.1,2

1Univ of Surrey, School of Veterinary Medicine, Guildford, United Kingdom, 2Animal and Plant Health Agency, Virology Department, Addlestone, United Kingdom

Dendritic cells (DCs) are the sentinels of the immune system and can be broadly divided into three populations, plasmocytoid DCs (pDCs), conventional DCs (cDCs), and monocyte-derived or inflammatory DCs (MoDC).. Recently, pDC have been identified in the blood of horses in CD14 CD172alow MHCIIlow CADM1low

Flt3+ cells. However, further work is required to assess the full phenotype of DCs in equine blood, firstly to demonstrate they represent the orthologues of the populations of DC described in other mammals and more importantly to permit equine DC to be potentially harnessed and targeted for improved therapeutics and vaccines

Conventional DCs (cDCs) can be further differentiated into at least two subsets; cDC1 and cDC2 based on their developmental pathways, phenotypical and functional differences. cDC1 have been shown to

express the highest level of CADM1 in other livestock species while cDC2 do not express a unique marker, but can be defined by the dim expression of CADM1 FLT3, IRF4 and CSF1R co-expression for example. Inflammatory DCs (infDC) are similar to MoDC that can be generated in vitro under the influence of GM-CSF & IL-4. Accordingly, the aim of this study was to identify the range of myeloid populations in equine blood using a panel of antibodies and multicolour flow cytometry such as applied in pigs and other species, complemented by equine specific mAbs such as eqWC1. Indeed, data obtained so far indicate the existence of two cDC populations, as well as some potential intermediates and inflammatory DC. Further characterisation using qPCR to determine the expression of genes for which there are no Abs is under way.

P011Develop of lateral flow immunochromatographic test and PCR for detection of SalmonellaEnteritidis in poultry farm

Elsady S.1, Dayed R.1, Latif A.1, El Jakee J.2

1Central Laboratory for Evaluation of Veterinary Biologics, Cairo, Egypt, 2Veterinary Medicine Cairo University, Cairo, Egypt

Salmonellae are responsible for considerable losses in the poultry industry through the death of birds and loss in production especially Salmonella Enteritidis (SE). Two diagnostic tools were developed and compared to detect SE in poultry farms. Lateral flow immunochromatographic test (LFIT) was

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considered a field and laboratory test while PCR was absolutely laboratory test. The minimal bacterial count that gave positive LFIT and PCR was 102CFU/0.1 ml and 10 CFU/ 0.1 ml respectively.For LFIT we could increase the sensitivity results to 1CFU/0.1ml by pre incubatingthe sample in trypticase soya broth at 37 C for 6 hour. About 100 samples were tested by the two developed methods and the results were compared with bacteriological methods. The sensitivity, specificity and accuracy of LFIT as compared to bacteriologicalexamination were calculated and were found to be 91%, 80% and 90% respectively and for PCR was 98.8%, 82.3% and 96% respectively.

P012Elimination of erroneous results related to bovine mononuclear cells immunophenotyping by antibody binding to Fc receptors

Okino C.H.1, Giglioti R.2, Bassetto C.C.3, Silva P.C.1, Oliveira H.N.3, Oliveira M.C.1

1Embrapa Southeastern Livestock, Lab of Animal Health, São Carlos, Brazil, 2Instituto de Zootecnia, Centro de Pesquisa de Genética e Reprodução Animal, Nova Odessa, Brazil, 3Universidade Estadual Paulista “Júlio de Mesquita Filho”, Departamento de Zootecnia, Jaboticabal, Brazil

Background: Fc receptors (Fc Rs) cell are surface structures that bind to the Fc region of antibodies. This binding of Fc part from IgG antibodies to Fc gamma receptors (FcγR) expressed on the surface of leukocytes, mainly

macrophages, and monocytes, is crucial for regulatory and effector functions of immune responses “in vivo”, but for immunoassays, as immunophenotyping by flow cytometry, it is a well-known cause of erroneous results. Blocking of IgG binding receptors on leukocytes has been established and highly recommended as a preventive procedure for immunological assays. Several studies testing different blocking reagents have been done in murine or human cells, but there are no specific studies for bovine cells.

Method: Our study aimed to investigate the efficiency of blocking reagents regarding the inhibition of nonspecific binding of mouse monoclonal antibodies (mAbs) to bovine peripheral blood cells.

Results: We observed nonspecific interactions of IgG2a and IgG2b isotypes to bovine leukocytes, but not IgG1. We demonstrated that these nonspecific bindings could be eliminated by blocking with purified mouse IgG, while scarce or no blocking effect was observed when murine or bovine serum or Mouse Seroblock Fc R were applied.

P013Gastro-intestinal helminths of free-ranging vervet monkeys (Chlorocebus pygerythrus) in Huye town, Rwanda - call for one health approach

Gashururu R.S.

University of Rwanda, School of Veterinary Medicine, Nyagatare, Rwanda

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Introduction: The effects of parasitism in non-human primates including vervet monkeys are likely to extend beyond their host populations. Patterns of parasitism in wildlife populations are influenced by host ranging patterns, density, intraspecific and interspecific contact rates and diet . There is an increasing population of vervet monkeys in Huye town. Owing to their close contact with people, there is a potential risk of transmitting pathogens to each other. Common intestinal parasites of human have been reported in primates suggesting a high potential for interspecies transmission.

Aim: The study was conducted in Huye city from February to May 2017 to determine the prevalence of gastrointestinal helminthes affecting vervet monkeys and to identify those of which that may be of public health importance.

Methods: 86 fecal samples were collected from four groups, selected using their living location. The samples were analyzed by using direct smear, floatation and Sedimentation techniques.

Results: Out of 76 faecal samples examined, 82.6% were found to harbour one or more gastrointestinal helmiths. The nematodes include Trichuris Trichiura, Strongyloides Stercoralis and Ascaris spp. The cestodes recovered were Bertiella mucronata and Taenia spp while the trematodes were Fasciola spp and Heterophyes sp (Haplorchis punilio). The most frequently detected helminth was Taenia spp (30.6 %) and the least found was Heterophyes spp (5.9 %). Most of the helminths found can affect other animals (wild and domestic) as well as humans.

Conclusion: This was a baseline information on the occurrence of helminthic infection in vervet monkeys in the area. There is need to do

conduct and deepen future studies in the area over a long period with more groups to identify definitely these parasites and determine the possible transmission patterns in order to confirm whether or not these are multi-host pathogens that can be shared.

P014Development of a fluorescent scFv antibody to improve Rabies diagnosis

Mansilla F.1, Turco C.1, Ferrero S.2, Alvarez I.1, Schammas J.M.1, Helguera G.2,3, Capozzo A.1,3

1Instituto de Virología, Centro de Investigaciones en Ciencias Veterinarias y Agronómicas (CICVyA), Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina, 2Laboratorio de Biotecnología Farmacéutica. Instituto de Biología y Medicina Experimental (IBYME), CABA, Argentina, 3Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), CABA, Argentina

Background: Rabies is the zoonotic infectious disease with the highest case-fatality rate. Its diagnostics relies on laboratory testing and makes use of specific antibodies. Several rabies virus (RABV) neutralizing mouse-derived monoclonal antibodies (mAbs) have been identified. However, mAb production in mammalian cell-culture has a considerable cost, and the use of mice to obtain mAbs from ascitic fluid is increasingly restricted. In this scenario, pursuing novel alternative biotechnological strategies is paramount.

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Method: We have developed a specific recombinant mini-antibody against RABV formed by the variable region of the heavy and light chains of a previously described neutralizing mAb (CR57) reactive against epitope-1 of the viral glycoprotein G (RV-GP). Both light and heavy chains variable regions of CR57 were covalently linked by a flexible peptide-linker and fused to the maltose binding protein (MBP) to improve its solubility. MBP-scFv57 expression was optimized using an E.coli expression system. Affinity-purified antibody was then covalently conjugated to fluorescein isothiocyanate (FITC) and its ability to recognize transfected and/or infected cells was determined by direct immunofluorescence test (DIF) and FACS.

Results: MBP-scFv57 was recovered, yielding 7.5g/l of initial culture. The integrity and stability was verified by SDS-PAGE/western blot, confirming that MBP fusion improves its stability. The scFv57 recognized RV-GP in the membrane of transiently-transfected HEK-293 cells. After conjugation, scFv57-FITC was able to detect 99% of the transfected cells by FACS. The presence of RV-GP was also determined by IFD on transfected HEK293 cells and on infected VERO and BHK cells with similar performance compared to standard reagents.

Conclussion: The scFv57 is efficiently produced as a soluble and stable recombinant fusion-protein in bacteria and can be affinity-purified in one step, preserving its binding activity. The fusion-protein has been FITC-conjugated, resulting in an inexpensive reagent capable of detecting the presence of virus in tissue samples or infected cells by DIF.

P015Parallel measurement of IFN-γ and IP-10 in QuantiFERON®-TB Gold plasma improves the detection of Mycobacterium bovis infection in African buffaloes (Syncerus caffer)

Bernitz N.1, Kerr T.J.1, Goosen W.J.1, Clarke C.1, Higgitt R.1, Roos E.O.1,2, Cooper D.3, Warren R.M.1, Van Helden P.D.1, Parsons S.D.C.1, Miller M.A.1

1Stellenbosch University, Biomedical Sciences, Cape Town, South Africa, 2The Pirbright Institute, Woking, United Kingdom, 3Ezemvelo KwaZulu-Natal Wildlife, Matubatuba, South Africa

The QuantiFERON®-TB Gold (QFT) stimulation platform for cytokine release is a novel approach for diagnosis of bovine tuberculosis in wildlife species. Plasma interferon gamma (IFN-γ) is routinely measured to detect immune sensitization to Mycobacterium bovis. However, the cytokine interferon gamma-inducible protein 10 (IP-10) has been proposed as an alternative, more sensitive, diagnostic biomarker. In this study, we investigated the use of the QFT system with measurement of IFN-γ and IP-10 in parallel to identify M. bovis-infected African buffaloes. The test results of either biomarker in a cohort of M. bovis unexposed buffaloes (n = 70) led to calculation of 100% test specificity. Furthermore, in cohorts of M. bovis culture-positive (n = 51) and M. bovis-suspect (n = 22) buffaloes, the IP-10 test results were positive in a greater proportion of animals than the proportion based on the IFN-γ test results. Most notably, when the biomarkers were measured in combination, the tests identified all M. bovis culture-positive buffaloes, a result neither the single comparative intradermal tuberculin test (SCITT) nor Bovigam® IFN-γ

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release assay (IGRA) achieved, individually or in parallel. Moreover, measurement of IFN-γ and IP-10 in parallel in the QFT system resulted in a positive predictive value (PPV) and negative predictive value (NPV) of 100%. These findings demonstrate the diagnostic potential of this blood-based assay to identify M. bovis-infected African buffaloes and a strategy to maximise the detection of infected animals while maintaining diagnostic specificity and simplifying test procedures.

P016Potential re-emergence of pathogenic tsetse-transmitted trypanosomes in the neighborhoods of Akagera National Park, Rwanda

Gashururu R.S.

University of Rwanda, School of Veterinary Medicine, Nyagatare, Rwanda

Background: The tsetse flies (Glossina) are the only biological vectors of the trypanosomes affecting humans and livestock. The Akagera National Park is the lone remaining home to tsetse flies in Rwanda. The wild game-livestock interface plays an important role in the epidemiology, being the reservoirs of the disease. The aim was to determine the current distribution of tsetse flies and the trypanosomes infection rates in field-captured tsetse flies.

Methods: Tsetse flies were collected from 3 districts surrounding the park between June and October 2018. Tsetse flies were trapped from different locations and were counted, sex-

determined. Live flies were dissected to find the trypanosomes in their predilection sites in the tsetse fly according to the distinct differential morphology.

Results: 2131 tsetse flies were collected around the park, among which 1169 were Glossina pallidipes and 962 were Glossina morsitans. Tsetse flies are more abundant in the district of Nyagatare (78%). 315 flies were dissected (203 Glossina pallidipes and 112 Glossina morsitans). The study revealed an overall infection rate of 9%. Trypanosoma congolense is more prevalent, followed by Trypanosoma brucei, Trypanosoma vivax and the mixed infections of T. congolense and T. vivax.

Conclusion: Both G. pallidipes and G. morsitans are potential efficient vectors of trypanosomes infection to livestock and humans but G. pallidipes appears to be the most important due to its high density. The study confirmed the presence of trypanosome infected tsetse flies and livestock infective trypanosomes. There is need for more accurate DNA based diagnosis to identify the blood meal sources and definitely differentiate the species and the sub-species or detect the new species to characterize even the human infective trypanosomes as we found the T. brucei-like species. There is also dire need to take human blood samples from the local communities to elucidate the tryapanosomes infection status.

P018Akagera National Park buffalo surveillance for foot-and-mouth disease and other animal diseases

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Udahemuka J.C.1,2, Beeton-Kempen N.3, Ndazigaruye G.1, Ingabire A.4, Lebea P.3, Obiero G.2, Aboge G.2

1University of Rwanda, Department of Veterinary Medicine, Nyagatare, Rwanda, 2University of Nairobi, Centre for Biotechnology and Bioinformatics, Nairobi, Kenya, 3TokaBio, Pretoria, South Africa, 4Rwanda Agriculture Board, Animal Resources Department, Kigali, Rwanda

Akagera National Park (ANP) harbours more than 2500 African Buffaloes and many other animal species susceptible to carry pathogens that can cross-infect domestic animals. The main objective of this study is to screen the health status of wild animals and compare the pathogens of the livestock with those found in wildlife.

In this study, seven African buffaloes from ANP were sampled. Animals were tranquillized with a 2 cc Pneudart in the hindquarters with 8 mg etorphine and 48 mg azaperone. Oropharyngeal samples were obtained using probang. The Buffaloes were woken up using up to 20 mg diprenorphine and 100 mg naltrexone given IV. Samples were transported in transport media (equal amounts of glycerine and 0.04 M phosphate buffer pH 7.2-7.6) in cooler boxes. To diagnose the presence of any pathogen, full sequencing was performed.

There was no Foot-and-Mouth Disease Virus (FMDV) detected in the seven African buffaloes. However, other pathogens were detected. Several parasites and commensals have been found in the oropharyngeal part of the sampled animals. The most predominant was P. ruminicola and M. bovoculi giving an idea also on the face fly presence. More works are

recommended to sample more buffaloes and from different locations to speak out on the role played by the wildlife in the cross-infection of infectious animal diseases.

It was observed that the oropharyngeal medium of the ANP sampled African buffaloes contains many of the known commensals of the African buffaloes and other parasites most probably originating from different water sources. The results didn’t show the presence of FMDV and this might give an explanation to the endemicity of this virus in this region from other factors such as transboundary movements, the circulation of the virus within domestic animals (small and large ruminants).

P019Comparing concentrations of natural antibodies and total immunoglobulin G in colostrum of beef and dairy cows

Altvater-Hughes T., Hodgins D., Wagter-Lesperance L., Mallard B., Read L., Gallo N.

University of Guelph, Pathobiology, Guelph, Canada

In cattle transfer of passive immunity from cow to calf occurs following ingestion and absorption of colostrum containing protective immunoglobulins, including IgM, and IgG natural antibodies (NAb). The minimum recommended concentration of IgG in colostrum is 50 g/L. Guidelines for NAb remain undetermined. In the dairy industry failure of passive transfer (FPT, calf serum IgG < 10g/L) occurs 6 -19% of the time. Less is known about FPT in beef. NAb are produced by the dam

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without contact with infectious agents; NAb in colostrum contribute to immediate protection of the calf. The objective of this study was to investigate differences in concentrations of total IgG, IgM NAb, and IgG NAb in colostrum between beef and dairy cows. Colostrum was collected within 12 hours of calving from Holstein dairy cows (n=50) and crossbred beef cows (n=62) at the university dairy and beef innovation centres. Radial immunodiffusion (RID) was used to quantify colostral IgG concentrations, and IgM NAb and IgG NAb are quantified by ELISA. Concentrations of IgG from RID were evaluated using SAS general linear model (GLM) and LS Means are reported. Preliminary results show that on average beef cows had significantly greater concentrations of IgG in colostrum (148.4 ± 6.7 SEM g/L) than dairy cows (94.5 ±5.8 SEM g/L, p < 0.01). It is noteworthy that 100% of the colostral samples from beef cows had over 50 g/L of IgG, but FPT still occurred in 4.5% of calves, indicating environmental effects. For dairy, 87% of colostral samples had over 50 g/L; historically, FPT occurred in 12% of calves in this herd. Preliminary data show substantial individual variation in NAb among cattle. These results indicate that FPT remains a problem in beef and dairy but for different reasons - colostrum quality in dairy versus cow/calf behavior and FcRn of beef calves.

P020In Vivo and In Vitro Testing of African Clawed Frog Susceptibility to Aquatic Rhabdoviruses

Bueren E.1, Emmenegger E.1, Bui-Marinos M.2, Robert J.3, Katzenback B.2

1USGS Western Fisheries Research Center, Seattle, United States, 2University of Waterloo, Department of Biology, Waterloo, Canada, 3University of Rochester, Department of Microbiology & Immunology, Rochester, United States

The African clawed frog (Xenopus laevis) is an exotic invasive species that are known to decimate native amphibian and fish populations when introduced. They are extremely adept at colonizing both wild and anthropogenic habitats, voracious predators, and potential carriers of three lethal amphibian pathogens that are notifiable to the World Organization for Animal Health (OIE): chytrid fungi (Bd and Bsal) and ranavirus (Iridoviridae). In Washington state, a breeding population of African clawed frogs was first discovered in 2015 and subsequently another population was found in a different western Washington watershed. Also, in 2015 sick ornamental Chinese firebelly newts (Cynops orientalis) just imported into the U.S. tested negative for Bsal, but positive for an exotic OIE-listed aquatic fish rhabdovirus, spring viremia of carp virus, which appeared to be responsible for their morbidity. This was the first detection of a rhabdovirus in an amphibian species.

To better assess the susceptibility of amphibians to aquatic rhabdoviruses, we conducted in vitro testing of Xenopus-derived cell lines and in vivo challenges of X. laevis with infectious hematopoietic necrosis virus (IHNV),

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viral hemorrhagic septicemia virus (VHSV), and SVCV. These three viruses are OIE-listed rhabdoviruses and thought to exclusively infect fish species. In vitro replication of these viruses was assessed by cytopathic effect and plaque assay. In vivo exposure of IHNV and VHSV was performed in wild-caught and lab-bred X. laevis juveniles and tadpoles via intraperitoneal injection and immersion respectively. Viable IHNV and VHSV were detected and clinical signs of disease observed at 7 days post-injection in juveniles and no mortality occurred out to 13 days post-exposure. These results suggest that X. laevis may serve as a reservoir for IHNV or VHSV. This could be a resource management concern if X. laevis begins colonizing habitats utilized by salmonid populations which are highly vulnerable to these rhabdoviral diseases.

P021Preliminary heritability estimates of immune response in Canadian beef cattle

Beard S.1,2, Hodgins D.C.1, Husseini N.1, Schmied J.1, Mallard B.A.1,2

1University of Guelph, Department of Pathobiology, Guelph, Canada, 2University of Guelph, Centre for the Genetic Improvement of Livestock, Department of Animal Biosciences, Guelph, Canada

Control of animal health in the beef industry has long relied on management, medication, and vaccination. Immune response (IR) traits have not yet been incorporated into breeding programs for beef cattle. Inclusion of such traits has been suggested to improve overall

animal health and enhance disease resistance. The High Immune Response (HIR™) technology identifies animals with superior heritable immunity by evaluating antibody-mediated (AMIR) and cell-mediated (CMIR) immune response, whereby an estimated breeding value for total IR is calculated. Before incorporating IR traits into beef breeding schemes, heritabilities for AMIR and CMIR must be estimated. To achieve this objective, IR phenotypes were measured using the patented HIR™ test on 290 crossbred beef cattle. On day 0, cattle were immunized intramuscularly with a preparation of type 1 (CMIR) and type 2 (AMIR) antigens with adjuvant. On day 14, baseline double skinfold thickness of the left and right tail fold was measured. Cattle received an intradermal injection of 0.1 mL of the CMIR test antigen in the right tail fold, and 0.1 mL PBS in the left tail fold. Cutaneous double skinfold thickness after 24 hours was used to assess delayed-type hypersensitivity (type 1 response). Blood was collected on Day 0 and 14 of the immunization protocol to evaluate serum antibody to the type 2 antigen by ELISA. Pearson’s correlation was estimated between AMIR and CMIR residuals using R, and variance components and heritabilities were calculated using ASReml. The Pearson’s correlation between AMIR and CMIR was not significant (p = 0.71). Preliminary heritabilities for AMIR and CMIR residuals were estimated at 0.47 (SE = 0.12) and 0.18 (SE = 0.13), respectively. The results of this study provide preliminary estimates of heritability of IR in beef cattle and indicate a potential for its inclusion into beef breeding schemes.

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P022Predicted microRNA biomarkers of Mycobacterium avium subspecies paratuberculosis using an in-silico bioinformatics approach

Wright K.E., Plain K.M., Purdie A.C., de Silva K.

University of Sydney, Sydney School of Veterinary Science, Camden, Australia

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s Disease (JD) or paratuberculosis, a chronic granulomatous enteritis affecting ruminants. Typified by long and silent subclinical disease phases, there is a defined need for the early detection and diagnosis of MAP infection. Biomarkers are defined as objectively measurable indicators of normal or disease processes and may provide further insight into the interplay between host immune systems and pathogenic bacteria. microRNA (miRNA), a small non-coding subset of RNA, have been proposed as potential biomarker targets as they are highly stable and exert control over protein translation and gene expression. Previous studies have shown modulation of miRNA responses to mycobacterial infection, making them ideal candidates for biomarkers for the diagnosis and differentiation between disease states of paratuberculosis. This study aimed to generate a list of candidate biomarkers for the diagnosis of paratuberculosis in cattle and sheep using bioinformatic predictions and Ingenuity Pathway Analysis software (IPA).

A full literature search was carried out for the terms “Mycobacteria + paratuberculosis + miRNA + microRNA + mycobacterium” using the Scopus database and Abcam’s Firefly Discovery Engine. A list of potential

miRNAs was compiled and uploaded to IPA. A core analysis was run, and new miRNAs added based on suggested function and commonalities between networks. Key networks were generated based on function and relevance to MAP pathogenesis.

Eight key networks were obtained from the original core analysis and a list of candidate biomarkers for identifying MAP infection was generated.

This list of candidate biomarkers for MAP infection provides potential miRNA biomarkers that may aid in the diagnosis of paratuberculosis in sheep and cattle. These candidates will not only provide the basis for future investigations, but may also further elucidate the molecular mechanisms of MAP pathogenesis through their associated modulation of target molecules and immunological pathways.

P023Isolation and preliminary characterization of bovine Th17 lymphocytes

Cunha P.1, Gitton C.1, Germon P.1, Foucras G.2, Rainard P.1

1ISP, INRA, UMR 1282, Université de Tours, Nouzilly, France, 2IHAP, Université de Toulouse, ENVT, INRA, Toulouse, France

Interleukin 17A-producing T helper cells (Th17) are effector memory CD4+ T cells that are crucial to adaptive immunity to extracellular bacteria. The activities of these cells in the bovine species are not yet defined for want

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of straightforward cultivation and isolation procedures. We have developed a method to cultivate, expand, sort and maintain in culture bovine Th17 cells from circulating CD4+ T cells of adult cows. Using polyclonal stimulation (antibodies to CD3 and CD28), we expanded positive CD4+ IL-17A+ T (Th17) cells in a cell culture medium without serum supplemented with TGF-β1 and IL-6. We used IL-2 to expand the cells, which were characterized by intracellular labeling for IL-17A and IFN-γ. Then, we isolated populations of CD4+ T cells producing IL-17A, IFN-γ or both by labeling surface IL-17A with either a complex of biotinylated anti-CD45 antibodies-streptavidin-biotinylated anti-IL-17A antibodies or by direct surface labeling with antibodies to IL-17A, followed by flow cytometry cell sorting. The percentages of surface-labeled IL-17A-secreting cells were quite similar to the percentages of intracellular-labeled IL-17A-producing cells of the same cultures, and the two labeling procedures of live cells yielded similar results. The sorted IL-17A+ cells were restimulated and expanded. After expansion, 80% of the isolated Th17 cells were positive for IL-17A intracellular labeling. The sorted IL-17A+ cells can be frozen, stored and expanded again. The sorted Th17 cells secreted much more IL-17A and IL-17F than did CD4+ IL-17- cells. Notably, most of Th17 cells secreted IFN-γ, although in lower amounts than did CD4+ IL-17- cells. Sorted cells were characterized by transcriptomic profiling. Genes coding for Th17 signature cytokines (IL-17A, IL-17F, IL-26) and transcription factors (RORγt, RORa) were overexpressed in Th17 cells. The techniques developed will make it possible to investigate the phenotypic and functional profiles of bovine Th17 cells along with their stability or plasticity.

P024Measuring CMI responses using the PrimeFlow RNA assay; a potential new method of evaluating BVDV vaccination response

Falkenberg S.1, Dassanayake R.1, Walz P.2, Neill J.1, Ridpath J.1, Roth J.3

1USDA-ARS-National Animal Disease Center, Ruminant Diseases and Immunology Unit, Ames, United States, 2Auburn University, College of Veterinary Medicine, Department of Pathobiology, Auburn, United States, 3Iowa State University, College of Veterinary Medicine, Department of Veterinary Microbiology and Preventive Medicine, Ames, United States

Current methods for evaluating bovine viral diarrhea virus (BVDV) vaccination response typically rely on measurement of humoral responses as determined by neutralizing antibody titers against BVDV. While virus neutralization titers (VNT) are correlated with increased protection, research has shown that T-cell mediated immunity (CMI) is an important component of a protective response against BVDV. For example, improved protection to BVDV by modified-live viral (MLV) vaccines as compared to killed vaccines for BVDV is thought to be due to better CMI induced by MLV. The goal of this work was to evaluate the immune response in vaccinated calves using methods that quantitated both humoral and CMI responses. Classic VNT was used to measure the humoral response while a new method based on the PrimeFlow RNA assay was used for measuring CMI. PBMC from both vaccinated (MLV) and non-vaccinated calves were isolated and stimulated with BVDV. The frequency of IFN-γ, IL-2, and BVDV positive

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CD4+, CD8+, and γδ T cells, were assessed to evaluate CMI responses using the PrimeFlow RNA assay. Concurrently collected serum samples were assessed for VNT. Similar results were observed for both IL-2 and IFN-γ, only IFN-γ results will be reported. Vaccinated calves had 2 times fewer BVDV positive T cells and 2 times more IFN-γ producing T cells than the non-vaccinates. Vaccinated calves had CMI values, expressed as the ratio IFN-γ and BVDV positive cells, 5 times greater than non-vaccinates. Subsequently the CMI value and VNT [expressed as Log(2)] were added together to give the protective index score, and the vaccinated calves were 5 times greater than the non-vaccinates. Future studies will compare the correlation between the protective index score and response to viral challenge. Benefits of using this method is the ability to quantify both CMI and humoral responses and understand the contribution of each to protection.

P025Detection of zoonotic bacterial causes of bovine mastitic milk using nano gold-immunochromatographic lateral flow strips

Sayed R.

Central Laboratory for Evaluation of Veterinary Biologics, Cairo, Egypt

Bovine Mastitis continues to be the most economically important disease of dairy cattle, and is caused mainly by certain highly human significance pathogens which were determined for the three species Staphylococcus auras, Streptococcus pyogens (group A), and E. coli. They have worldwide economic and public health significanc.

nano gold-immunochromatographic lateral flow strips were developed.

Rabbit polyclonal antibody against different specific antigens of different bacteria in mastitic milk were be generated to immunization of rabbit. Rabbit specific to different antigens were be selected to conjugated with nano gold as the detector antibody was be laid on a conjugated pad. Different PAbs specific to different antigens were be used as the capture antibody at the test line and goat anti-rabbit IgG antibody (GAR) used as the capture antibody at the control line of nitrocellulose strip. The ready-to-use strips were consists of 3 strips for different bacteria. The sensitivity, specificity and accuracy of parepared strips as compared to bacteriologically examination were calculate. the minimal bacterial count can be detected was ‹103 CFU/ml for each strips. If the test sample was pre-enriched in brain heart infusion broth for 6 h before application to the strip, the sensitivity would increase to 1 CFU/ml. The sensitivity, specificity and accuracy were found to be 93.7%, 97.6% and 96%, respectively, for Staphylococcus auras 82.6%, 94.2 % and 90.6% respectively, for Streptococcus pyogens . It was 93.7 %, 98.3 % and 97.3%, respectively, for E. coli. The developed strips are a simple field rapid test of high sensitivity, specificity, and accuracy that could be used to detect of selected bacteria in mastitic milk in order to rapid application of the correct method of treatment in the fastest time with subsequent reduction of the complication and economic losses additionally reduction of this pathogens incidence for transmit to human.

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P026Effect of heat stress on Peripheral Blood Mononuclear Cell (PBMC) function of dairy cows ranked as high, average or low immune responders

Cartwright S.1, Livernois A.1,2, McKenchnie M.1, Schmied J.1, Mallard B.1,2

1University of Guelph, Pathobiology, Guelph, Canada, 2University of Guelph, Centre of Genetic Improvement of Livestock, Guelph, Canada

Greenhouse gas emissions, among other factors, are contributing to a warmer climate. This may make it difficult for livestock to adapt to heat stress. The ability of Holstein cows, identified as high, average and low immune responders, to regulate body temperature during exposure to hot summer temperatures was observed to be variable, such that 50% of the cows measured had rectal temperatures above normal. It is known that cows identified as high immune responders display reduced incidence of disease and better response to vaccination than average or low responders. The objective of this study was to determine the effect of heat stress on PBMCs from dairy cattle, previously ranked for immune response, using estimated breeding values. Blood was taken from 15 (5 high, 5 average and 5 low) lactating Holstein cows and PBMCs were isolated and cultured. PBMCs were stimulated with ConA to measure cell proliferation or LPS to assess nitric oxide (NO) production. Control cells and cells from which HSP70 was measured were not stimulated. The cells were subjected to three different treatments; thermoneutral: incubation at 37, one heat stress treatment: incubation at 42 for 4 hours and two heat stress treatments: previously heat treated cells were subjected to

a second heat treatment of 42 for 4 hours. All cells were incubated for 72 hours at 37°C except when indicated. Cell proliferation was determined by MTT assay. A stimulation index was calculated as a ratio of OD measured by the MTT assay for stimulated and unstimulated PBMCs. Analysis of results using a mixed model indicated that high immune responders had significantly greater (P = 0.0443) cell proliferation across all treatments compared to average and low. Preliminary data on NO production and HSP70 expression indicate variability in response of PBMCs from individual cows to the heat stress treatments.

P027Development of bovine antibody reagents for the sorting and characterization of bovine plasmablasts

Dinkel K.D.1, Madsen-Bouterse S.A.1, Bastos R.G.1, Herndon D.R.2, Knowles D.P.1, Fry L.M.2

1Washington State University, Department of Veterinary Microbiology and Pathology, Pullman, United States, 2United States Department of Agriculture, Agricultural Research Service, Animal Disease Research Unit, Pullman, United States

Objective: Plasmablasts are rapidly proliferating, short-lived, antibody-secreting cells that dominate the early stages of the humoral immune response. In humans, plasmablasts can be isolated from other leukocyte populations by the unique expression levels of multiple surface proteins. Of these proteins, the leukocyte differentiation molecules (LDM) CD19, CD38, and CD138 are of primary

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interest due to roles in lymphocyte activation, proliferation, and signaling. In an effort to identify bovine plasmablasts, antibodies to these LDMs were developed.

Methods: Gene gun immunization was performed on a minimum of three mice with DNA encoding bovine CD19, CD38, or CD138, all codon-optimized for expression in mice. Mouse sera and hybridoma supernatants were screened by flow cytometry, ELISA, and/or immunoblot using transfected HEK 293, bovine spleen leukocytes, and/or bovine peripheral blood mononuclear cells (PBMC). Plasmablast enrichment was performed using fluorescent-activated cell sorting, and enriched populations were examined by microscopy.

Results: Screening of selected hybridoma supernatants using spleen leukocytes revealed distinct populations by flow cytometry. Differences in CD38+ and CD138+ populations were observed when compared between PBMC and spleen leukocytes. Antibody specificity was demonstrated by detection of LDM in transfected HEK 293 by flow cytometry, ELISA, and/or immunoblot. Microscopic analysis of multi-labeled, sorted spleen leukocyte populations suggested the enrichment of blasting cell populations.

Conclusions: The use of these newly developed antibody reagents, together and in conjunction with additional antibodies, will contribute to the immune reagents necessary for the isolation of bovine plasmablasts and the subsequent study of early immune responses to diseases of cattle.

P028Effect of heat stress on body temperature and Peripheral Blood Mononuclear Cell (PBMC) function of beef cows ranked as high, average or low immune responders

Husseini N., Hodgins D.C., Tabatabaei S.S., Altvater-Hughes T., McKechnie M., Livernois A.M., Schmied J., Mallard B.A.

University of Guelph, Pathobiology, Guelph, Canada

Livestock need to efficiently dissipate heat load to maintain health and production. This is becoming more challenging in the face of climate change and may be especially difficult for feedlot cattle that consume higher energy feeds. Ambient temperature-humidity indices (THI) are used as a guide to indicate heat stress, with THIs above 74 reported to cause decreased ability to regulate core body temperature, lack of appetite and in turn, weight loss. In this study, rectal temperatures in 32 beef cows classified with High, Average, or Low antibody and cell-mediated immune response (IR) phenotypes were measured twice/day (am and pm) during normal and above normal THI. Results indicated substantial individual variation in thermo-regulation ability, with 50% showing increases in body temperature above 39.2°C.Nonetheless, a small percentage of cows showed no change in body temperature as a result of heat stress. Preliminary analysis did not indicate association with IR phenotypes. In vitroheat stress experiments indicated variability in PBMC’s functions among individuals. PBMCs were stimulated with concanavalin A to measure cell proliferation or LPS to assess nitric oxide (NO) production. Control cells and cells from which HSP70 expression was measured were not stimulated. The cells were subjected to three different

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treatments; thermoneutral: incubation at 37°C, one heat stress treatment: incubation at 42°Cfor 4 hours and two heat stress treatments: previously heat-treated cells were subjected to a second heat treatment of 42°Cfor 4 hours. All cells were incubated for 72 hours at 37°C except when indicated. Preliminary results indicate that HSP70 from unstimulated PBMCs increased after one or two heat stress treatments. Conversely, thermoneutral (control) PBMCs challenged with LPS produced more NO in comparison to PBMCs after one or two heat treatments. No differences in cell proliferation were noted.

P029Development and characterization of immune reagents for swine health, vaccine and disease studies

Lunney J.1, Bailey M.2, Manirarora J.1, Gourapura R.3, Kenney S.3, LaBresh J.4, Sang Y.5, Francis O.2, Wooldridge L.2

1USDA ARS NEA BARC, Animal Parasitic Diseases Laboratory, Beltsville, United States, 2University of Bristol, Bristol Veterinary School, Bristol, United Kingdom, 3The Ohio State University, Food Animal Health Research Program, Wooster, United States, 4Kingfisher Biotech, Inc., St. Paul, United States, 5Tennessee State University, Department of Agricultural and Environmental Sciences, Nashville, United States

Background: The US-UK Collaborative Swine Immune Toolkit Initiative has as its goal to generate priority reagents for swine immunity studies and pipeline them for marketing.

Priorities are based on international input.

Method: US efforts are aimed at expression of soluble proteins and swine CD molecules, and production of panels of monoclonal antibodies (mAbs). The team has set up collaborations with commercial partners for protein expression and mAb production, and has updated protocols to evaluate reagent specificity. UK researchers have focused on mucosal targets, including production of mAbs to chemokine receptors and IgE.

Results: Immunizations for mAbs reactive with BAFF and IL-28 are in production. Panels of mAbs reactive with IFNb, CXCL10, CX3CL1 (fractalkine), IL-6, IL-13, IL-17A, and IFNg, have been produced. Each of these panels of mAbs has been tested for reactivity on yeast expressed swine proteins and orthologues from other species. Epitope reactivity was assigned based on that data and inhibition ELISAs. Selected mAbs were then screened for intracellular staining of stimulated cells and immunohistochemistry on immune tissues. Sets of mAbs are being screened for best pairs to develop new multiplex assays. Plans for producing mAbs to porcine CD1d are in progress. In the UK, target peptides were used to probe phage display libraries with commercial partners. Several mAbs reactive with CCR3, CCR9 and CCR10 peptides have been identified with further validation planned using transfected cells. New screens for anti-IgE mAbs are underway.

Conclusion: Our goal is to provide the veterinary community with new commercial reagents and techniques for their research efforts. Tools and reagents generated by this project will undoubtedly advance swine immune, disease and biomedical research efforts. Supported by USDA ARS, NIFA AFRI grant #2015-67015-23216 and BBSRC grant BB/M028232/1.

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P031Identification of a subset of bovine circulating CX3CR1 positive monocytes in blood and bone marrow through the novel application of fluorescently tagged recombinant CX3CL1

Raper A.1, Gregory B.2, Hume D.3, Hope J.1

1Roslin Institute, Infection and Immunity, Edinburgh, United Kingdom, 2University of Edinburgh, Edinburgh, United Kingdom, 3M Mater Research Institute-UQ, Brisbane, Australia

Background: CX3CR1 is the receptor for CX3CL1 (fractalkine). Previous murine and human studies have shown that CX3CR1 is expressed in lymphoid and neuronal tissues and mediates adhesive and migratory functions. Studies of CX3CR1 in veterinary species have been impeded due to the lack of reagents.

Method: A fusion protein was generated from the secreted bovine CX3CL1 and human IgG1 Fc sequences. The recombinant protein was fluorescently tagged and its binding profile in bovine blood was characterised using flow cytometry. Sequence alignments were carried out with murine, porcine, ovine, equine and human CX3CR1 and cross-reactivity determined using blood and bone marrow.

Results: The CX3CL1-Fc protein bound a subset of CD14, CD16, CSF1R positive circulating monocytes in bovine blood. Cross-reactivity to murine and caprine CX3CR1 was observed by binding of CX3CL1 to a subset of circulating monocytes in murine blood and a subset of progenitor cells in murine and caprine bone marrow. However, no binding was observed in porcine or equine blood. The

sequence alignment of CX3CR1 identified that the presence of aspartic acid residues in the CX3CL1 binding site of the porcine and equine receptor may prevent binding.

Conclusions: This study shows the generation of the recombinant bovine CX3CL1-Fc protein as a novel molecular marker, enabling detection of CX3CR1 expression. Characterisation identified a subset of CD14, CD16, CSF1R positive circulating monocytes in bovine blood, and subpopulations of bone marrow progenitors were shown to bind CX3CL1 suggesting subset specific expression of CX3CR1.

P032Analysis of immunological targets across multiple species demonstrates need for species-specific research reagents

LaBresh J., Sullivan Y.B.

Kingfisher Biotech, Saint Paul, United States

Historically, there have been limited immunological reagents available for many of the veterinary species. Due to this limited availability, animal model and veterinary health studies were either not performed or were forced to utilize non-species-specific reagents to evaluate immune responses. Over the past ten years, Kingfisher Biotech has developed recombinant proteins and antibodies across over twenty species to various immunological targets. Due to this recent increase in species-specific reagents for animal model and veterinary health, we have been able to evaluate the protein homology and antibody

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cross-reactivity across multiple species. By utilizing amino acid sequence analysis tools, we determined the percentage of homology of protein sequences across multiple species to various immunological targets. We then evaluated the recombinant proteins to these multiple targets in protein gel electrophoresis to visualize the similarities and differences of the recombinant proteins. Utilizing these recombinant proteins, we developed antigen-purified, species-specific polyclonal antibodies. Finally, we evaluated these antigen-purified, species-specific polyclonal antibodies’ cross-reactivity across multiple species in ELISA. Our data highlight the need for species-specific tools for immunological research.

P033Development and testing of common bottlenose dolphin (Tursiops truncatus)-specific cytokine ELISA reagents

Sacco R.E.1, Harsla T.R.2, LaBresh J.3, Sullivan Y.B.3, Hulubei V.4, Elnaggar M.M.4, Le-Bert C.R.5, Jensen E.D.6, Davis W.C.4

1National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, United States, 2Iowa State University, Ames, United States, 3Kingfisher Biotech, Inc., St Paul, United States, 4Washington State University, Pullman, United States, 5National Marine Mammal Foundation, San Diego, United States, 6U.S. Navy Marine Mammal Program, San Diego, United States

Earlier prediction of onset and/or progression of illness in cetaceans might allow for treatment prior to development of other clinical signs. An

important early contributor to many disease conditions is the inappropriate activation of inflammatory processes, which are regulated by cytokines and other immunomediators. Thus, monitoring changes in the expression of immunomediators could serve as a screen for alterations in marine mammal health status. While a number of cytokine assays are readily available for humans and mice, specific assays for many veterinary species, including cetaceans such as bottlenose dolphins (Tursiops truncatus), are more limited. We have previously described the development of sandwich ELISAs based on the use of polyclonal antibodies specific to bottlenose dolphin cytokines. Utilizing these assays, we have monitored the immune status of bottlenose dolphins from a managed population. In our published research, IFN-γ and TNF-α concentrations in bottlenose dolphin sera or in culture supernatants from PBMCs stimulated with differing concentrations of mitogens concanavalin A (ConA) or phytohemagglutinin (PHA) were determined. It was noted that these mitogens varied in their ability to induce maximal levels of IFN-γ and TNF-α. More recently, the reagent tool kit of available dolphin-specific ELISA assays has been expanded to include IL-1RA, IL-2, IL-4, CXCL8 (IL-8), and IL-17A. These assays are currently being tested on bottlenose dolphin sera, mitogen-stimulated cell culture supernatants, and BAL fluid samples. Furthermore, we have developed monoclonal antibodies for bottlenose dolphin IFN-γ, TNF-α, and CXCL8 with a goal of optimizing their use in the sandwich ELISAs. These cytokine assays provide an important first step in the development of a broader immunological reagent tool kit for common bottlenose dolphins, which could be utilized to monitor changes in health status, as an adjunct to currently available diagnostic tests.

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P034Advances for the Collaborative Immune Reagent Network for Aquacultured Species (CIRNAS)

Hansen J.1, Sunyer O.J.2, Salinas I.3

1USGS Western Fisheries Research Center, Fish Health, Seattle, United States, 2University of Pennsylvania, Department of Pathobiology, School of Veterinary Medicine, Philadelphia,, United States, 3University of New Mexico, Department of Biology, Albuquerque, United States

Aquacultured fish are a critical food resource for human consumption. Importantly, disease is a major hurdle limiting production. To advance basic and applied research for fish health we are developing specific immunological tools for the fish health community. These reagents and associated assays are needed for evaluating shifts in immunity during infection and vaccination. CIRNAS is a collaborative network that is focused on serving the aquaculture community by advancing the availability of immunological resources and knowledge base for fish health. We are focused on four fish species—Atlantic salmon, rainbow trout, channel catfish and Nile tilapia. Specific focus areas include the development of reagents for mucosal immunity and vaccinology. Importantly, one of the major goals of CIRNAS is to establish an international, collaborative network for improving health, safety and production of aquacultured fish by partnering with other fish health researchers from throughout the world. By doing so, the project will advance the development of collaborative research projects for diseases impacting production and fish health on a global scale. To accelerate the process of developing tools, we have been using a high throughput immunization and

screening approach. Results from this and standard approaches will be presented.

P035The UK Immunological Toolbox: promoting veterinary immunology research using recombinant technologies to secure and engineer antibodies

Mwangi W.1, Nettleship J.2,3, Lokhman E.1, Crossley S.1, Maccari G.1, Waddell L.A.4, Raper A.4, Hope J.C.4, Owens R.J.2,3, Hammond J.1

1The Pirbright Institute, Woking, United Kingdom, 2University of Oxford, Research Complex at Harwell, Didcot, United Kingdom, 3University of Oxford, The Division of Structural Biology, Oxford, United Kingdom, 4The Roslin Institute, Edinburgh, United Kingdom

The UK Immunological Toolbox project is an integrated UKRI-BBSRC strategically funded initiative aiming to remove barriers to veterinary immunology research with complementary activities at The Pirbright and Roslin Institutes. While the Roslin is focussed on generating new reagents and assays to address gaps in our capabilities, Pirbright has developed the capability to express and engineer recombinant antibodies.

Sequencing and recombinantly expressing mouse monoclonal antibodies can have many benefits. The most immediate being the permanent securing of important reagents (while potentially reducing storage costs) and removing cell line drift of antibody sequences in growing hybridomas producing consistent performance. Recombinant expression systems

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also allow flexibility in labelling, production and purification methods. This can drive down cost at different scales and create very simple mechanisms to share reagents and ultimately promote research.

The availability of antibody sequences also present opportunities to engineer antibodies that better suit research needs. As well as making Fab fragments without the Fc component to eliminate cellular responses, the Fc class can be switched between different mouse Ig subclasses, or indeed species. We now have cassettes that allow the expression of antibodies with all mouse, cattle, pig or chicken Ig subclasses. These cassettes also allow the cloning of antibodies isolated directly from these host species.

The UK Immunological Toolbox welcomes project proposals and enquires for the creation of new reagents and assays. A steering committee comprising of veterinary immunology experts review project proposals, which are prioritised based on the nature of the tool(s) requested, community requirements, their utility, and accessibility. For access and more information please visit immunologicaltoolbox.co.uk or email [email protected].

P036Generation and characterization of monoclonal antibodies against porcine IgE

Haiden E., Patzl M., Hammer S.E., Mair K.M., Saalmüller A.

University of Veterinary Medicine Vienna, Vienna, Austria

Introduction: There is huge interest in IgE-mediated immune response of pigs against parasites and in using pigs as large animal models for allergic diseases. But the lack of specific antibodies against porcine IgE (pIgE) is limiting these studies and generation of IgE specific monoclonal antibodies (mAb) has been a long lasting effort in veterinary immunology.

Methods: BALB/c mice were immunized with HEK 293T cell derived 6xHIS-tagged recombinant porcine IgE heavy chain (CH2-CH4; HIS-IgE) of about 50 kDa. Spleen cells of immunized mice were fused with SP2/0 cells. Clones producing mAb were screened on HEK 293T cells expressing intracellular Flag-tagged pIgE heavy chain (CH2-CH4; Flag-IgE) by flow cytometry (FCM). Specific mAb were further characterized by ELISA with 15-mer peptides of the respective pIgE sequence overlapping in five amino acids to identify linear epitopes. The same peptides were also used in blocking assays on Flag-IgE expressing HEK 293T cells by FCM to identify potential conformational epitopes. Cross-reactivity with other porcine immunoglobulins was tested by ELISA and FCM blocking assays.

Results: Three specific mAb producing clones were identified, two with IgG1 isotype and one IgM. The peptide recognition patterns of the two IgG1 clones exhibited similarities in ELISA whereas the mAb of IgM class showed a quite different reactivity pattern. In Immuno-blots all mAb bound to HIS-IgE and none to the HIS-protein. Cross-reactivity with pIgG or pIgM was not detected. This was confirmed in silico by matching the 15-mer peptides with the sequences of pIgG or pIgM where not any identical sequence longer than four amino acids was found.

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Conclusion: These newly generated mAb provide a new approach to study IgE responses in pigs and to use swine as large animal model for allergies.

P037Cytokine assay: serum concentrations in apparently healthy calves in nigeria

Akinbobola J.S.1, Jeremiah O.T.2, Akinbobola R.I.A.3, Oguntade E.S.4

1University of Abuja, Veterinary Teaching Hospital, Abuja, Nigeria, 2University of Ibadan, Veterinary Medicine, Ibadan, Nigeria, 3University of Abuja, Veterinary Parasitology and Entomology, Abuja, Nigeria, 4University of Abuja, Statistics, Abuja, Nigeria

Diagnostic assays involving cytokines is on the increase in clinical and research practice. The aim of the study was to create normal references ranges, which is lacking in apparently healthy calves. Using body condition score, appropriate hematology and parasitology techniques, apparently healthy calves were distinguished from the unhealthy ones. Waters - 616/626 High Performance Liquid Chromatography was used to analyze the profile of TH1 and TH2 cytokines in the serum of 330 apparently healthy White Fulani calves (≤6 months old) from selected farms in Ibadan (Oyo State), Nigeria. Among the cytokines assayed, interferon gamma had the highest serum concentration (167.9 ± 6.9pg/L) while Interleukin-4 was the least (3.4 ±0.1pg/L). This study provides information on the baseline serum concentrations of some cytokines, which is of value in the comprehension of disease

mechanism and progress in calves. It also brings to bare, the implication of the existence of an inverse relationship between Interferon gamma and Interleukin 4.

P038Comparison of SepMateTM-50 tubes with lymphoprepTM and standard 50 mL tubes with Ficoll®-Paque Plus for PBMC isolation from Macaca fascicularis whole blood

Rodriguez D.R.1, Grant C.2, Bell C.1

1Biomere, IN Vitro, Worcester, United States, 2Biomere, Large Animal, Worcester, United States

Peripheral blood mononuclear cells (PBMCs) are widely used in biomedical research to support pharmacokinetic/pharmacodynamic endpoints in drug discovery and development. However, PBMC isolation from non-human primates (NHP) can be problematic. In this study, we compared two different methods of PBMC isolation: Stemcell Technologies’ SepMateTM-50 tubes with LymphoprepTM and standard 50 mL tubes with Ficoll®-Paque Plus, both using whole blood collected from female Macaca fascicularis. Using these two methods in a series of experiments, cell viability

and cell recovery were measured using a Nexcellom K2 Cellometer and cell purity was measured using an IDEXX ProCyte Dx analyzer. A lipid profile was performed on all samples using IDEXX Catalyst Dx analyzer to investigate a possible relationship between the amount of lipid in samples and higher PBMC contamination. Results showed that

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cell viability and cell recovery was remarkably better with SepMateTM tubes than with standard 50 mL tubes and Ficoll®-Paque Plus. Red blood cell contamination of the final PBMC preparation was significantly reduced; however, the removal of platelets and reticulocyte were similar between both methods. Moreover, blood samples that had above normal total protein showed higher PBMC contamination with red blood cells in both methods. Blood samples that had above normal total protein also showed lower PBMC contamination with reticulocytes using the standard Ficoll® isolation method and higher contamination with reticulocyte using the SepMateTM method. This needs further investigation. The results from these experiments indicate that the SepMateTM

method offered several advantages over the standard Ficoll® isolation method.

P039The effect of lipid supplementation on macrophage activation plasticity in Mycobacterium avium subspecies paratuberculosis infection

De Silva K., Mizzi R., Wright K., Purdie A., Plain K.

University of Sydney, Camden, Australia

Background: Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular parasite of macrophages, causes a granulomatous enteritis and chronic wasting in ruminants. The granuloma is an aggregation of mononuclear phagocytes accompanied by lymphocytes as disease progresses. Manipulation of host macrophage lipid metabolism is involved in the pathogenesis

of other mycobacterial diseases such as tuberculosis. We have previously shown aberrant expression of host lipid metabolism genes (Thirunavukkarasu et al 2014) and accumulation of host intracellular cholesterol in MAP infected animals or cells (Johansen et al 2018 and 2019). Monocyte/macrophage phenotype, characterized by distinct functional properties, is different in the peripheral blood monocyte population of MAP nonexposed and exposed animals (Thirunavukkarasu et al 2015). Macrophage phenotype is also divergent in the different types of intestinal granuloma (Fernandez et al 2017). The aim of this study was to investigate the effect of lipid on plasticity of macrophage activation during MAP infection.

Method: Macrophages were differentiatied into M1 and M2 phenotypes, exposed to lipid supplementation and infected with MAP in vitro. Phenotype was confirmed by gene expression and nitric oxide secretion. Infection was assessed microscopically, lipid metabolism by expression of ABCA1 and LDLR genes and microRNA by quantitative PCR.

Results: Lipid supplementation increases NO secretion and expression of iNOS by macrophages in nonpolarized macrophages. In these cells, lipid supplementation and MAP infection does not alter macrophage activation state. In M1 macrophages lipid supplementation and MAP infection supports persistence of the M1 state while in M2 macrophages these factors shift the cells away from an M2 state. There were no significant differences in the lipid metabolism genes assessed in this study.

Conclusion: MAP infection drives an inflammatory activation state in both M1 and M2 macrophages. Lipid supplementation and MAP infection shifts M2 macrophages towards an inflammatory phenotype.

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P040Porcine cytokines, chemokines and growth factors: 2019 Update

Dawson H.D.1, Lunney J.K.2

1Agricultural Reseach Service, United States Department of Agriculture, Beltsville Human Nutrition Research Center, Diet, Genomics, and Immunology Laboratory, Beltsville, United States, 2Agricultural Reseach Service, United States Department of Agriculture, Beltsville Agricultural Research Center, Animal Parasitology Disease Laboratory, Beltsville, United States

Background: Pigs are a major food source worldwide as well as major biomedical models for human physiology and therapeutics. A thorough understanding of porcine immunity is essential for a safe and secure food supply, to prevent and treat infectious diseases, and develop effective vaccines and therapeutics. The use of pigs as biomedical models is dependent on the growing molecular and immune toolbox. Research progress has been limited because the immune toolkit is not robust.

Methods: We have summarized current knowledge of swine cytokines, chemokines and growth factors, identifying 229 pig immune proteins, characterizing knowledge of their gene structures and families. A broad-based literature and vendor search was conducted to identify defined sets of monoclonal (mAbs) and polyclonal Abs (pAbs) reacting with porcine cytokines, chemokines, growth factors along with availability of cloned recombinant proteins and assays for their quantitation. We identified areas in the current swine genome build that need to be clarified.

Results: This process identified numerous reagents that are reportedly reactive with 115 pig cytokines, chemokines, growth factors: 95 have at least one commercial Ab reagent, 64 a cloned recombinant peptide, and 42 with quantitative assays.

Conclusions: We affirmed the great need to develop and characterize additional reagents for pig immunity research. There are panels of reagents for numerous high priority targets that are essential reagents for characterizing porcine immunity, disease and vaccine responses, and factors regulating development of innate immune response, polarized macrophages and lymphoid cells, and T regulatory cells. Yet there are many areas requiring investment of efforts to more fully explore the pig immune system. Use of improved technologies and genomics will provide such tools and increase our knowledge of the pig immune system. The development of more reagents to understand the complex of cytokines, chemokines, and growth factors will clearly advance these initiatives.

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P041Metabolic reprogramming of bovine classical and nonclassical monocytes

Talker S.C.1,2, Barut G.T.1,2, Rufener R.3, Summerfield A.1,2

1Institute of Virology and Immunology, Bern, Switzerland, 2Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland, 3Institute of Parasitology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland

Monocytes in peripheral blood of cattle can be phenotypically split into CD14+CD16- classical monocytes and CD14-CD16+ nonclassical monocytes. Their transcriptomic profiles differ considerably, with classical monocytes displaying a pro-inflammatory profile and nonclassical monocytes expressing genes associated with tissue regeneration. Metabolism has been shown to govern function of immune cells. Accordingly, murine macrophage subsets M1 and M2 have been shown to rely on aerobic glycolysis and oxidative phosphorylation, respectively.

Aim of the current study is to characterize the metabolic status of sorted bovine monocyte subsets and the influence of stimulation and activation on metabolic reprogramming. By measuring extracellular acidification and oxygen consumption, Agilent Seahorse XF technology allows for differentiation between the two main metabolic pathways: glycolysis and oxidative phosphorylation. According to macrophage subsets in mice, it is expected that activated classical monocytes switch to aerobic glycolysis, whereas activated nonclassical

monocytes rely on oxidative phosphorylation to exert their regulatory function. Differing metabolic programs in these bovine monocyte subsets would support fundamentally different functions as indicated by transcriptomic analysis.

P042The UK Immunological Toolbox: building novel reagents to further the understanding of livestock and avian immunology

Waddell L.A.1, Raper A.1, Wu Z.1, Mwangi W.2, Lokhman E.2, Crossley S.2, Lefevre L.1, Hume D.A.1, Hammond J.2, Hope J.C.1

1The Roslin Institute, Edinburgh, United Kingdom, 2The Pirbright Institute, Pirbright, United Kingdom

The UK Immunological Toolbox project is an integrated UKRI-BBSRC strategically funded initiative aiming to remove barriers to veterinary immunology research with complementary activities at the Pirbright and The Roslin Institutes. While Pirbright has developed the capability to express and engineer recombinant antibodies, at Roslin the focus is on the development of new monoclonal antibodies, recombinant proteins, immunological tools and assays to address gaps in capability.

At The Roslin Institute we have already produced a number of novel monoclonal antibodies and recombinant proteins which we aim to commercialise and make available to the wider community. Examples of projects successfully completed include development of an ELISA for chicken IL-10 (Wu et al., 2016)

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and production of a novel monoclonal antibody recognising ADGRE1 (F4/80) on porcine macrophages (Waddell et al., 2018).

The Immunological Toolbox welcomes project proposals and enquires for the creation of new reagents and assays. The facility boasts expertise which allows projects to be individually tailored according to requirements, with advice on procedures and protocols also available. A steering committee comprising of veterinary immunology experts review project proposals, which are prioritised project proposals on the basis of the nature of the tool(s) requested, community requirements, their utility, and accessibility. For access and more information please visit www.immunologicaltoolbox.co.uk or email [email protected]

P043Cows selected for resistance to mastitis show contrasted immune responses compared to mastitis susceptible cows

Germon P.1, Barbey S.2, Lefebvre R.3, Boichard D.3, Foucras G.4, Rainard P.1

1ISP, INRA, UMR 1282, Université de Tours, Nouzilly, France, 2DEP, INRA, UE 0326 Domaine Expérimental du Pin-Au-Haras, Le-Pin-Au-Haras, France, 3GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France, 4IHAP, Université de Toulouse, ENVT, INRA, Toulouse, France

Mastitis remains an important disease in dairy farming nowadays. Genetic selection of animals for increased resistance to mastitis is

a relevant strategy to reduce the burden that mastitis entails on dairy cows. A divergent selection scheme based on somatic cell counts and clinical mastitis records was set-up on Prim’Holstein cows at the INRA experimental unit of Le Pin-au-Haras. In order to better define the mechanism underlying genetic resistance to mastitis, inflammatory challenges as well as immunization with a model antigen were performed on cows from these two different lines.

LPS was infused in one healthy udder quarter of each cow approx. 1 month post-partum. Milk was collected 4, 8, 12 and 24h post-infusion. Somatic cells recruited in milk were counted and the cytokines/chemokines CXCL8, IL-6 and IL-1β were measured by ELISA. For adaptive response studies, cows were immunized 15 days before dry-off with ovalbumin and the response was evaluated 15 days after dry-off. Response to immunization was evaluated by measuring IFNγ and IL-17A and upon antigen stimulation in whole blood assays.

Although LPS triggered mastitis in all infused quarters, mastitis resistant cows (n= 16) showed decreased inflammatory response compared to more susceptible cows (n= 25) at t=8h post-infusion. No differences were observed between the two groups in terms of response to immunization.

Altogether, these results suggest that, in response to intra-mammary LPS challenge, cows selected for resistance to mastitis are better able to control the inflammatory response.

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P044Unique features of water buffalo (Bubalus bubalis) γδ T cells and monocytes

Elnaggar M.M.1,2, Grandoni F.3, Abdellrazeq G.S.1,2, Fry L.4, Hulubei V.1, Davis W.C.1

1College of Veterinary Medicine, Washington State University, Pullman, WA, United States, 2Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt, 3Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia agraria - CREA-ZA, Research Centre for Animal Production and Aquaculture, Monterotondo (RM), Italy, 4Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, WA, United States

Although buffaloes (Bubalus bubalis) are a major component of the livestock industry in many countries, progress has been slow in characterizing the immune response to infectious agents affecting their productivity and wellbeing. This is mainly attributable to the limited availability of monoclonal antibody (mAb) reagents needed to study the immune response to pathogens in buffaloes. As part of our mission to address this limitation and enhance buffalo immunology research, a survey study was conducted with mAbs developed against bovine leukocyte differentiation molecules to identify mAbs cross reactive with orthologues in buffalo. The study yielded a large set of mAbs that show the similarities and differences in the immune systems of cattle and buffalo. Flow cytometric analysis of buffalo peripheral blood mononuclear cells labelled with these mAbs revealed unique features in expression of some molecules on buffalo γδ T cells and monocytes in comparison to expression in cattle. Analysis showed high expression of CD8 on WC1+

γδ T cells when compared to expression in cattle. Analysis also revealed differences in the expression of CD16 on buffalo monocytes compared to expression on bovine monocytes. The similarities and differences in the expression of buffalo CD molecules suggest there may be associated differences in the function of these cell subsets in the immune response against pathogens in buffaloes and could explain their noted greater resistance to diseases. The identification of these cross reactive mAbs opens the way for extensive studies to be conducted in buffaloes.

P045Atypical granuloma formation by Mycobacterium bovis in young calves

Carrisoza-Urbina J.1, Morales Salinas E.1, Bedolla Alva M.A.1, Hernández Pando R.2, Gutiérrez-Pabello J.Á.3

1Universidad Nacional Autónoma de México, Mexico, Mexico, 2Instituto Nacional de Ciencias Médicas y Nutrición Salvador Subirán, Mexico, Mexico, 3Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Mexico City, Mexico., Mexico, Mexico

Bovine tuberculosis is a chronic inflammatory disease that causes granuloma formation. Several studies have been focused on characterizing granulomas in experimentally infected cattle, which has been useful to understand the pathogenesis of the infection and to allow assessment of the protection of prototype vaccines. Nevertheless, few studies

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have reported macroscopic and microscopic characteristics of these lesions in cattle naturally infected by M. bovis. Therefore, in the present study granulomas from 32 Holstein Friesian cattle of a dairy area in central Mexico were studied. In this opportunity sampling 46.8% (15/32) cattle were less than 4 months of age (calves) and 53.2% (17/32) were more than one year old (adults). Macroscopically, 100% (32/32) of the animals included, developed lesions suggestive of tuberculosis in mediastinal lymph nodes and 50% (16/32) of lesions were identified in lungs. A total of 1,143 granulomatous lesions were microscopically identified and analyzed, from which 34.6% (396/1143) belonged to adults and were classified according to the nomenclature of Wangoo et al., 2005. Surprisingly, calves showed an unusual pattern that interfered with classification. These granulomas features include large areas of necrosis extending in most of the affected organ, central calcification, absence of connective tissue capsule, few giant cells and a higher percentage of lesions with AFBs. The presence of M. bovis was confirmed in 84.3% (27/32) of the cases analysed by PCR. These observations suggest that cattle under 4 months of age are more susceptible to naturally M. bovis infection. This knowledge can be useful in understanding natural host resistance to mycobacterial infections.

P047Adjuvant modulated cytokine responses to infective larval stage of equine Strongyle parasites in vitro

Hellman S., Tydén E., Hjertner B., Morein B., Fossum C.

Swedish University of Agricultural Sciences, BVF, Uppsala, Sweden

A Th1 polarizing effect of a new adjuvant, G3, was recently demonstrated in cultures of equine PBMC (Hellman et al., 2018). The adjuvant response was potentiated by co-exposing PBMC to G3 and synthetic cytokine inducers in vitro. In particular, G3 together with the TLR2 agonist Pam3CSK4 markedly enhanced the production of IFN-γ while decreasing the production of IL-10. This methodology to in vitro assess adjuvant/antigen stimulation was applied to study cytokine responses to antigen preparations of the gastro-intestinal helminths Cyathostominae spp. and Strongylus vulgaris (S. vulgaris), with the long-term objective to develop a vaccine against S. vulgaris.

Procedures for decontamination, culturing and attenuation by UV-irradiation were developed using infective stage larvae (L3) of Cyathostominae and thereafter applied to S. vulgaris. These L3 preparations showed no bacterial growth and contained less than 0.70 IU/mL of endotoxin. Between 12-30 UV-irradiated L3 were added to cultures of equine PBMC and incubated for 18 hours followed by qPCR analysis of cytokine mRNA. In parallel, PBMCs were stimulated with the G3 adjuvant, chitin or endotoxin (1 IU/mL).

Both Cyathostominae and S. vulgaris L3 induced gene expression of IL-4, IL-5, IL-10 and IL-13. In PBMC stimulated with chitin the gene encoding IL-5 was up-regulated whereas endotoxin up-regulated the genes for IL-1β, IL-6 and IL-10. In accordance with previous data, G3 induced the genes encoding IFN-γ, IL-1β, IL-6, IL-12p40, IL-17A and IL-23p19. Interestingly, when G3 was added simultaneously as L3 to the cell cultures, the gene expression of IFN-γ was enhanced whereas the expression of IL-10 was reduced. This suggests that the G3

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adjuvant exert a similar modulating effect on cytokine responses to biological antigens as previously seen using synthetic inducers.

Reference: Hellman, S., Hjertner, B., Morein, B., Fossum, C. 2018. The adjuvant G3 promotes a Th1 polarizing innate immune response in equine PBMC. Vet Res. 49(1):108.

P048Exploring species tropism of zoonotic Flaviviruses using Monocyte-derived Dendritic cells as an in vitro model

Lewandowska M.1,2, García-Nicolás O.1, Python S.1, Summerfield A.1,3

1Institute of Virology and Immunology, Immunology, Mittelhäusern, Switzerland, 2Graduate School for Cellular and Biomedical Sciences, Bern, Switzerland, 3University of Bern, Vetsuisse Faculty, Department of Infectious diseases and Pathobiology, Bern, Switzerland

The Flavivirus genus contains a large group of mosquito-transmitted zoonotic pathogens such as Zika (ZIKV), dengue (DENV), yellow fever (YFV), Japanese encephalitis (JEV) and West Nile viruses (WNV) and many other less studied viruses such as Wesselsbron (WESSV), Ilheus (ILHV), Spondweni (SPOV), Usutu (USUV), Kokobera (KOKV), or Bagaza viruses (BAGV). Animal species living in close proximity to humans may play an important role for virus/host coevolution, transmission routes and possible reservoirs or maintenance hosts. By assessing the ability of a large collection of flaviviruses to infect target cells from various

animal species, this study aimed to collect information on potential host susceptibility. To this end, we utilized monocyte derived dendritic cells (MoDCs) from several species such as human, pig, cattle and sheep. For each species, monocytes were sorted and cultivated for 4 days with GM-CSF and IL-4. Cells were then challenged with the different viruses and the infectivity (flow cytometry), viral replication (immunoperoxidase monolayer assay) and cytokines responses (IL-1β, IFN-β and TNF measured by ELISA) were determined at 24h and 48h.

Our results show innate immune responses correlated with early infectivity and cytokine production, demonstrating species-dependent differences in infectivity of MoDCs. This study could help in exploring potential domestic animal hosts involvement and to predict which flaviviruses could emerge in particular species.

P049Porcine interferon-omega subtype: differential expression and superior antiviral activity

Jennings J., Sang Y.

Tennessee State University, Nashville, United States

Background: Interferons (IFNs) are critical cytokines in regulation of animal immune response. However, substantial IFN research has been centered on the classical IFN-α and IFN-β subtypes of type I IFNs, leaving much to be understood on functional diversity of other unconventional subtypes. Previous studies

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have placed emphasis on the porcine model to study such as IFN-δ and ω subtypes; and this study is to characterize the differential expression and antiviral superiority of porcine IFN-ω subtype.

Method: Porcine macrophages (MΦs) and dendritic cells (mDCs) were infected with porcine Arterivirus (PRRSV) with different virulence, or treated with different stimuli for IFN response. The expression of IFN-ω genes was determined by a TaqMan- or SYBR Green-based RT-PCR assays. Antiviral activity of porcine IFN-ω peptides were determined in several virus-cell systems. Ongoing tests are to determine different dependence of IFN-ω ligands on IFN receptors (IFNARs) in regulation of IFN-stimulated genes (ISGs).

Results: Two types of quantitative RT-PCR assays were used to analyze differential expression of porcine IFN-ω genes. Data showed that IFN-ω3, IFN-ω4 and IFN-ω5 were generally more responsive than other IFN-ω genes in the treatments above. Correspondingly, we showed that some IFN-ω members, for example IFN-ω5, has evolved superior antiviral potency in most of our antiviral tests.

Conclusion: Porcine IFN complex, which contains the most functional gene numbers compared with any other vertebrate species, represents a signature surge in IFN evolution. In addition to the classical IFN-α and IFN-β subtypes that are common in most amniotic species, the unconventional porcine IFN subtypes, such as IFN-ω subtype, evolve diverse molecular and functional properties in terms of expression profiling and antiviral activity.

Disclosure: Supported by grants from USDA NIFA 2018-67016-28313.

P051e-PIG-enetic avenues: porcine mRNA and miRNA pathway analysis of highly pathogenic PRRSV Infection

Fleming D.1,2, Miller L.2

1ORAU/ORISE, Oak Ridge, United States, 2National Animal Disease Center-ARS-USDA, Virus and Prion Research Unit, Ames, United States

Background: Studies of the host-pathogen interaction between pigs and highly pathogenic Porcine respiratory and reproductive syndrome virus (HP-PRRSV) have tried to uncover the process that allows the virus to evade the immune response to its benefit. The virus infects pigs through nasal passages, replicates within lung macrophages disrupting their normal function. Research has examined post-transcriptional expression, unfortunately, looking at differences in expression alone does not give information on the pathways and networks that are affected and that contribute to homeostatic imbalances due to infection. Therefore this study examined the effects mRNA and miRNA expression has on host biological networks.

Methods: Analysis was carried using whole blood from both mock infected and HP-PRRSV infected pigs. Transcriptomic techniques were then applied to examine the differential expression of miRNAs using Hisat2 for alignment to the Sus scrofa 10.2 genome and changes in expression analyzed using DeSeq2. Statistical significance for pathway analysis was set at FDR ⫹ 0.5. The resulting list of miRNAs were examined for their effects on porcine biological networks using the mirPATH software to generate KEGG pathways affected by changes in miRNA differential expression.

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Results: The results of the statistically significant (FDR ⫹ 0.15) miRNAs were divided by dpi and used to examine biological networks targeted to be possibly inhibited or activated by the identified miRNAs. Post-transcriptional control of host gene expression appeared to take place over several host biological pathway groupings which effect homeostasis that included immune, metabolic, and structural pathways. This included pathways such as the proteoglycan in cancer pathways that showed multiple genes targeted for inhibition that would cause host dysregulation and impair the ability to properly maintain homeostasis during infection.

Conclusion: The analyses suggests inhibition and activation of networks involved in viral entry, proliferation, and pro-inflammatory signaling may underlie the ability of HP-PRRSV to hinder homeostasis.

P052-AThe study of immunogenicity in 25 RHDV strains (Lagovirus europeus GI.1 and GI.1a)

Tokarz-Deptuła B.1, Niedzwiedzka-Rystwej P.1, Hukowska-Szematowicz B.1, Deptuła W.2

1University of Szczecin, Department of Immunology, Szczecin, Poland, 2University of Szczecin, Department of Microbiology, Szczecin, Poland

Background: Immune system has an enormous impact on pathogenicity of RHD virus. One of the most important element of innate immunity is phagocytosis, which is underestimated in course of this infection.

Objectives: To assess the immunogenicity of 25 strains (GI.1) based on 11 indicators of phagocytosis of PMN cells in rabbits.

Methods: The following innate immunity tests were performed: ingesting capacity of PMN cells (% of ingesting cells, ingestion index) and cidal capacity (NBT test - spectrophotometric, spontaneous, stimulated, metabolic activity coefficient of neutrophilic granulocytes spontaneous and stimulated, stimulation index, myeloperoxidase activity) and also concentration and activity of lysozyme in serum of rabbits.

Results: 25 examined strains G.I.1, including 5 G.I.1a, may be divided into 3 immunotypes. It can be concluded that immunogenicity of examined RHDV strains (G.I.1 and G1.1a) is correlated with their positive haemagglutination capacity, as HA+ strains are 100% in immunotype I, 75% immunotype III and 56% immunotype II.

P052-BThe induction of immunosuppression via prostaglandin E2 and the enhancement of anti-bacterial effects by anti-PD-L1 antibody combined with COX-2 inhibitor in Mycoplasma bovis infection

Goto S.1, Konnai S.1,2, Okagawa T.1,2, Maekawa N.1,2, Sajiki Y.1, Murata S.1,2, Ohashi K.1,2


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Mycoplasma bovis causes chronic pneumonia, mastitis, arthritis and otitis in cattle. We previously demonstrated that the programmed cell death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway was involved in immunosuppression during M. bovis infection. Recently, several reports showed prostaglandin E2 (PGE2) suppressed immune responses and upregulated PD-L1 expression in bovine chronic diseases, such as Johne’s disease and bovine leukemia virus infection. In this study, we investigated the role of PGE2 in immunosuppression during M. bovis infection.

In the initial experiments, we stimulated monocytes from healthy cattle with live-M. bovis in vitro. Live-M. bovis induced PGE2 production and the upregulation of PD-L1 expression in monocytes from healthy cattle. We next investigated plasma PGE2 production in M. bovis-infected cattle. Plasma PGE2 production in infected cattle was significantly increased compared to that in uninfected cattle. Interestingly, plasma PGE2 production was positively correlated with the proportion of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in M. bovis-infected cattle was negatively correlated with M. bovis-specific IFN-γ production from PBMCs. These results suggested that PGE2 was a inducer of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. On the other hand, in clinical test for M. bovis-infected cattle with pneumonia, combined treatment with cyclooxygenase 2 inhibitor and anti-bovine PD-L1 antibody significantly upregulated M. bovis-specific IFN-γ production and reduced bacterial loads in bronchoalveolar lavage. Thus, our study opens up a new perspective in the therapeutic strategy for M. bovis infection by targeting both PGE2 and the PD-1/PD-L1 pathways.

P053The interferon gamma response to Mycobacterium avium in vitro can be correlated with a higher risk of clinical ketosis in dairy cows

Minuti A.1, Amadori M.2, Mezzetti M.1, Lovotti G.1, Piccioli-Cappelli F.1, Trevisi E.1

1Università Cattolica del Sacro Cuore, Department of Animal Science, Food and Nutrition, Piacenza, Italy, 2IZSLER, Brescia, Italy

Background: Ketosis is a metabolic disease associated with reduction of immune competence. The aim of this study was to evaluate the association between clinical ketosis after calving and adaptive immune responses.

Material and methods: Thirteen pluriparous Friesian dairy cows were monitored from 21 days before till 28 days after calving. Blood was collected from the jugular vein at different days from calving (DFC) from -21 till 28 DFC. Plasma samples were analyzed for energy parameters (glucose, NEFA and BHBA). Moreover, at -21 and 28 DFC an IFN-gamma (IFNG) release assay for Mycobacterium avium was carried out on heparinized whole blood. Results were evaluated in terms of Delta OD (difference between avian PPD-stimulated and control wells). Cows were retrospectively grouped according to their plasma BHBA concentrations after calving in Control (CTR, BHBA< 1.4 mmol/L; 7 cows) and Ketosis (KET, BHBA > 1.4 mmol/L; 6 cows). Data were analysed as a repeated measures study using the MIXED procedure of SAS considering the group (CTR or KET) as fixed effect.

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Results: The IFNG response showed differences, both before (-21 DFC) and after calving (28 DFC), delta OD being higher in KET compared to CTR cows (P< 0.05). In practice, a higher IFNG response to avian PPD was associated with a higher risk of ketosis after calving.

Conclusions: Considering that IFNG is related to metabolic pathways of energy use, a vigorous IFNG response to environmental microbial stressors like M. avium may represent a risk after calving, when homeostatic control circuits are less effective.

P054Characterizing the global transcriptomic profile of bovine monocyte-derived macrophages classified based on nitric oxide response to escherichia coli

Emam M.1,2, Canovas A.2, Islas-Trejo A.D.3, Fonseca P.2, Medrano J.F.3, Mallard B.1,2

1Ontario Veterinary College, University of Guelph, Pathobiology, Guelph, Canada, 2University of Guelph, Animal Biosciences, Guelph, Canada, 3University of California, Animal Science, Davis, United States

Macrophages are among the first responders to infections with a notable role in eliminating pathogens via phagocytosis and producing microbicidal components, such as nitric oxide (NO-). They are key regulators of inflammation, producing a wide range of cytokines and chemokines. Recently, we have shown that the variation in the magnitude of NO- response of bovine Monocyte-Derived Macrophages

(MDMs) to Escherichia coli (E. coli) in vitro is highly associated with the genetic structure of these cells (genomic heritability = 0.46). Although producing NO- is an important function of MDMs, the inflammatory profiles of MDMs associated with extreme high (H) and low (L) NO- responders remained to be investigated. In the current study, MDMs from H and L responders (n = 3 per phenotype) were challenged in a highly controlled in vitro environment with E. coli, followed by mRNA sequencing at 3 and 18 hours (24 libraries in total) by Illumina Hiseq-4000. The sequence reads were trimmed and filtered for quality. Then, they were aligned to the bovine reference genome (UMD 3.1, release 94). Differentially expressed (DE) genes were identified by comparing the challenged versus untreated controls and then by subtracting L from H responders. DE genes were further investigated in functional annotation and pathway inference analysis. The results showed that 413 and 853 genes were DE (absolute fold change difference between phenotypes > 2 and FDR < 0.05) at 3 and 18 hrs, respectively. The pathway enrichment analysis revealed that “cytokine-cytokine receptor interaction” was significantly enriched at both time points. Specifically, the expression of some key cytokines (TNF-α, IL12B, and IL23A), chemokines (CXCL10, and CXCL8), and co-stimulatory molecules (CD40 and CD80) were positively associated with macrophage classification based on NO- production. These results suggest that MDM classification based on NO- production extends beyond just microbicidal functions to overall cell inflammatory profiles.

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P055Differential local immune responses against Haemonchus contortus in a resistant Brazilian sheep breed (Morada Nova)

Okino C.H.1, Toscano J.H.B.2, Giraldelo L.A.3, Von Haehling M.B.2, Dos Santos I.B.2, Esteves S.N.4, Ferraz-Júnior R.S.5, Chagas A.C.S.4

1Embrapa Southeastern Livestock, Lab of Animal Health, São Carlos, Brazil, 2Universidade Estadual Paulista “Júlio de Mesquita Filho”, Departamento de Medicina Veterinária Preventiva, Jaboticabal, Brazil, 3Centro Universitário Central Paulista, São Carlos, Brazil, 4Embrapa Southeastern Livestock, São Carlos, Brazil, 5Embrapa Southeastern Livestock, Lab of Veterinary Parasitology, São Carlos, Brazil

Background: Effective mucosal immune response is essential for the development of resistance to Haemonchus contortus in sheep. This immune response is predominantly characterized by activation and development of Th2 responses, eosinophilia, mastocytosis and increased IgE and IgG1 levels. However, the immune mechanisms associated to increased host resistance against this parasite remains poorly elucidated.

Method: This study was carried out, aiming to better elucidate the immune mechanisms involved on the host resistance against H. contortus. Briefly, two groups of ten lambs of Morada Nova Brazilian breed, previously characterized as resistant or susceptible to infection by this parasite, were submitted to challenge with 4000 H. contortus L3, and euthanized at 7 days post-infection. Blood samples were collected for blood count, including differential analysis for granulocytes. Fragments from abomasal pyloric and fundic

regions were collected and subjected to relative quantification of gene expression of immune-related mediators (RT-qPCR), and were also evaluated for eosinophils and mast cell counts.

Results: Significant higher levels were observed in resistant group for eosinophils counts in fundic abomasal region, occurring the same for erythrocytes, hemoglobin and total leucocytes in the whole blood. Regarding the gene expression results, for the fundic region, increased levels of IL1β and TNFα were found in the susceptible group, while up-regulation of MS4A2 (high affinity IgE receptor, Fε CRI) and IL33 genes were observed in resistant group. Even, TLR2 and CFI transcripts were up-regulated in pyloric region of resistant group.

P056Innate immune responses against Streptococcus suis infections in the porcine lung

Hoffmann J.-P.1, Schäfer A.2, Schwaiger T.1, Blohm U.2, Schröder C.1, Köllner B.2

1Friedrich-Löffler-Institut, Department of Experimental Animal Facilities and Biorisk Management, Greifswald, Germany, 2Friedrich-Löffler-Institut, Institute of Immunology, Greifswald, Germany

Streptococcus suis (S. suis) is a pathobiontic bacterium endemic in domestic pigs. Virulent strains not only can initiate inflammatory processes in the porcine lung and brain, they also display zoonotic potential. Due to the high homology between human and porcine physiology, pigs are chosen as a biomedical

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model to investigate immune responses against these bacteria in the lung.

Established cell lines from nasal epithelia to the mucosal surfaces of lungs are used to study Type I and III interferon responses to infection with different S. suis serotypes in vitro. Furthermore, the influence of different virulent S. suis strains on functional activities of lung derived myeloid cells is investigated. Finally, the composition of different functional myeloid cell populations (monocytes, alveolar macrophages, dendritic cells) in lungs of common, untreated pigs regularly obtained from a slaughterhouse are characterized by flow cytometry and their cytokine pattern analyzed by PCR with regard to the observed pathological stage.

The data will provide a first overview of the interactions of S. suis, epithelial cells and functional myeloid cell populations in porcine innate immunity.

P057The identification of a porcine pneumovirus from an immunological perspective

Hervet C.1, Richard C.-A.2, Gutsche I.3, Normand V.4, Leblanc-Maridor M.1, Renois F.1, Bertho N.1, Simon G.5, Eléouët J.-F.2, Meurens F.1

1BIOEPAR, INRA, Oniris, Nantes, France, 2Virologie et Immunologie Moléculaire, INRA, Université Paris-Saclay, Jouy-en-Josas, France, 3University of Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France, 4Porc.Spective Swine Vet Practice, Chêne Vert Conseil Veterinary Group,

Noyal-Pontivy, France, 5Anses, Ploufragan-Plouzané-Niort laboratory, Swine Virology Immunology Unit, Ploufragan, France

The presence of pneumoviruses in pigs is extremely poorly documented. To inquire about these viruses in French pig farms, we used the published sequence of the nucleoprotein (N) of the recently identified Swine Orthopneumovirus (SOV) to express and purify SOV N as a recombinant protein in Escherichia coli. Then, SOV N was purified as nanorings and used to set up an enzyme-linked immunosorbent assay (ELISA). Using that ELISA we analysed the presence of anti-SOV N Immunoglobulins G (IgG) in various swine sera to inquire about the circulation of the virus in France. Since the virus has never been isolated, real positive controls were unavailable and negative controls challenging to obtain. Sera collected from specific pathogen free new born piglets before colostrum uptake were used as negative controls. These sera were negative for anti-SOV N IgG while most of the other sera collected in older animals from different pig farms in Brittany (France) were positive at various levels. To further confirm the validity of our ELISA assay cross-reactivity with bovine sera, human respiratory syncytial virus (hRSV) N, and whole inactivated bovine RSV (bRSV) particles were tested. Bovine sera were negative for IgG anti-SOV N and porcine sera did not react with inactivated bRSV. Interestingly, in two pig farms presenting respiratory clinical signs and negative or under control for common respiratory pathogens such as influenza virus and Mycoplasma spp, pigs were detected positive for anti-SOV N IgG. Several field and laboratory studies will be required to further characterize the circulation of the virus amongst pigs, to isolate the circulating virus, and to determine its potential pathogenicity and interactions with the porcine immune system. SOV may prove to be an overlooked actor of

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the porcine respiratory complex, important on its own or in association with other viral and bacterial micro-organisms.

P058Identification of natural disease resistance in Zebu and Holstein-Friesian (HF) cattle through a microbicidal assay

Alcaraz O.1, Villarreal-Ramos B.2, Vordermeier M.2, Benítez A.3, Werling D.4, Esquivel H.5, Carrisoza J.1, Cortéz O.6, Vigueras G.6, Salguero J.7, Gutiérrez J.Á.6

1UNAM, Microbiology and Immunology, Mexico City, Mexico, 2APHA, Bovine tuberculosis, Addlestone, United Kingdom, 3UNAM, Microbiology and Immunology, Ciudad de Mexico, Mexico, 4London College, London, United Kingdom, 5CIATEJ, Biotechnology, Guadalajara, Mexico, 6UNAM, Microbiology and Immunology, Mexico city, Mexico, 7University of Surrey, London, United Kingdom

Background: Natural resistance to disease has been defined as the innate capacity of an animal to resist disease when exposed to pathogens, without previous exposure or immunization. Previous studies indicated that Zebu (Bos indicus) cattle are more resistant to external, internal parasites and showed lower bovine tuberculosis prevalence than HF cattle. This would suggest that Zebu cattle are more resistant to M. bovis infection. However, there is no evidence about microbicidal activity

in macrophages from Zebu cattle against mycobacterial infection. Therefore, the aim of this work was to identify and compare the natural resistance between Zebu and HF through a microbicidal assay.

Methods: To identify bovine resistance we performed microbicidal assay on macrophages from HF (n=31) and Zebu (n=32). Percentage of BCG survival was determined with animals exhibiting ≤ 65 % being classified as resistant (R) whilst animals being > 65 % were classified as susceptible (S); the data was used to determine the phagocytosis index (PI) and ROC curves.

Results: Microbicidal assay allows us to segregate 9 and 11 R HF and Zebu cattle respectively with BCG survival ≤ 65 % (p≤ 0.05). The remaining cattle showed S phenotype. Although HF cattle showed higher PI than Zebu cattle (p≤0.01), ROC analysis shows area under the curve of 1.00, p-value < 0.0001. When reevaluating the R cut off it shows % ≤ of 67 and 68, with a sensitibity of 100 %, with specificity of 95.45, 95.24 % for HF and Zebu respectively. Further experiments will have to be carried out in order to confirm the cut-off in a blind experiment.

Conclusion: Through microbicidal assay we identified a resistance trait in Zebu and HF macrophages. Zebu cattle shows a higher number of R animals than HF suggesting that Zebu cattle have a great potential to resist intracellular parasites as mycobacteria.

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P059Analysis ofpecific antibody and autoantibody repertoires between asymptomatic and symptomatic dogs in the Leishmania infantum infection

Sassi A.1, Boubaker Landolsi R.2, Chaabouni A.1, Mhadhbi M.3, Gharbi M.3

1University Tunis El Manar Pasteur Institute Tunis, Tunis, Tunisia, 2University of Manouba, Higher Institute of Biotechnology, Kalaat Landalous, Tunisia, 3University National School of Veterinary Medecine, SIDI THABET, Tunisia

Leishmania (L.) infantum-infected dogs may present with a large range of clinical signs, from apparently healthy with no or few (asymptomatic dogs, AD) to several clinical signs indicators of active infection (symptomaticdogs, SD).

On the basis of clinical evaluation and laboratory testing (IFAT, parasitological examination of Giemsa-stained lymph node smears, L. infantum antigens-ELISA of total (Tot) IgG), 131 dogs were categorized as SD, asymptomatic seronegative (AND) or seropositive dogs (APD) from surrounding areas, and as negative control dogs

(CTD). ELISA based on leishmanial native antigens or recombinant LACK and LeIF proteins showed that SD produce higher levels of specific Tot IgG, IgG1 and IgG2 antibodies than APD, and that for both clinical stages, the antibody titers of IgG2 isotype were constantly higher than those of the IgG1. The seroprevalences of Tot IgG,

IgG2 did not differ between APD and SD groups (97 and 97% in SD; 100 and 96% in APD, respectively) whereas that of IgG1 was

slightly lower in SD (88% of APD versus 82% of SD). The autoantibody repertoires were analyzed by ELISA using HEp-2 extracts, ds-DNA, human albumin and transferrin as self-antigens and by Western blot using HEp-2 proteins. ELISA results’ indicated that APD develop higher levels of IgG1 autoantibodies, and higher seroprevalence (50% and 26% in APD and SD, respectively), contrasting with lower levels and seroprevalences of Tot IgG and IgG2 (43 and 68% for APD; 100 and 74% for SD). Interestingly, SD showed a stronger IgG1 and particularly IgG2 reactivity with transferrin, an iron-binding protein, than APD and AND. Western blotting experiments produced heterogeneous IgG1 and IgG2 inter- and intra-groups reactivity profiles towards HEp-2 proteins, to identify a specific antigenic profile.

P060Immunological tools to investigate myeloid cell differentiation in pigs

Waddell L.A., Lefevre L., Sauter K., Hume D.A., Hope J.

The Roslin Institute, Edinburgh, United Kingdom

Background: Pigs are an important livestock species worldwide for a range of economic, agricultural and food supply reasons. A number of diseases which affect pigs have no suitable small animal models, and we hypothesise that this is, in part, due to differences in species’ macrophage populations. Increasing our knowledge of porcine macrophage biology provides the opportunity to further investigate

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the susceptibility of pigs to disease and their response to pathogens. This is an important area of study as the understanding of livestock disease processes underpins the development of disease control strategies.

A limitation of progress in this field is the availability of specific reagents. A key component of this project therefore aims to produce and characterise novel immunological reagents which recognise previously unidentifiable cell specific molecules.

Method: ADGRE1 (EMR1, F4/80) is a long-established macrophage marker in rodents, but its function in pigs remains unstudied. CSF1 is a fundamental growth factor for the differentiation of monocytes to macrophages. Monoclonal antibodies against each of these molecules were raised and used to study the process of monocyte to macrophage differentiation, in addition to the expression pattern on other macrophage populations and classical dendritic cells. This project provides novel tools for studying porcine myeloid cell biology and differentiation. The existence of these reagents will provide opportunities to answer previously unanswered questions within the field of porcine macrophage biology.

Results: ADGRE1 and CSF1 were optimised and characterised on a number of cell types by flow cytometry. Both were shown to be suitable markers for monocyte-macrophage differentiation and mature alveolar macrophages. ADGRE1 also bound specifically to lung and liver macrophages in tissue sections.

Conclusion: The availability of these novel reagent will provide an opportunity to further progress the understanding within the field of myeloid cell differentiation and the porcine immune response.

P061The effect of host genetic on the expression profile of inflammatory mediators in chickens

Emam M.1, Choiniere W.2, Mehrabani-Yeganeh H.3, Nikbakht G.3, Charkhkar S.4, Barjesteh N.5

1University of Guelph, Guelph, Canada, 2Université de Sherbrooke, Sherbrooke, Canada, 3University of Tehran, Tehran, Iran, Islamic Republic of, 4Azad University, Tehran, Iran, Islamic Republic of, 5Université de Montréal, Pathology and Microbiology, Saint-Hyacinthe, Canada

Poultry industry incurs an enormous cost for challenging with outbreaks of infectious diseases despite the vaccination and biosecurity protocols. In addition, new regulations to reduce the use of antibiotics is also encouraging the industry to seek alternative approaches. Improving host defense through direct selection for resistance to pathogens or immunocompetence is an alternative approach. Comparing the immune response profile of chickens with distinct genetic architecture can shed light on the genetic regulation of immunocompetence and molecular mechanisms that lead to variation. Previously, we demonstrated the effect of the genetic background on the magnitude of antibody and cellular immune response in chickens. To understand the mechanisms that are associated with variation of immune responses in chickens, the expression of interleukin (IL)-4, IL-6, IL-18, IFN-γ and iNOS in spleen after exposure to Brucella abortus was investigated in the current study. B. abortus is not a natural pathogen of chickens, which would eliminate the effect of previous exposure. The cytokine expression was measured by TaqMan real-time PCR in five populations

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(Ross, Cobb and three Iranian indigenous strains) of chicken (n = 7 per experimental unit (2 levels in exposure time* 3 levels in sample collection time* 2 levels in treatment), in total 84 chickens per population). The expression of IL6, IL18, iNOS, and IFN-γ was higher in Ross. The kinetics of expression of iNOS and IFN-γ was similar with a peak at 6 hours post challenge while the expression of IL6 and IL18 peaked at 2 hours with gradual decrease up to 10 hours post-challenge, in Ross. The overall profile of the expression was associated with each population genetic structure. However, our data did not demonstrate a distinct difference between indigenous versus commercial populations.

P062Systemic immune responses associated to Haemonchus contortus resistance in a resistant Brazilian sheep breed (Morada Nova)

Toscano J.H.B.1, Okino C.H.2, Von Haehling M.B.1, Dos Santos I.B.1, Giraldelo L.A.3, Figueiredo A.1, Esteves S.N.4, Ferraz-Júnior R.S.5, Bassetto C.C.6, Amarante A.F.T.7, Chagas A.C.S.4

1Universidade Estadual Paulista “Júlio de Mesquita Filho”, Departamento de Medicina Veterinária Preventiva, Jaboticabal, Brazil, 2Embrapa Southeastern Livestock, Lab of Animal Health, São Carlos, Brazil, 3Centro Universitário Central Paulista, São Carlos, Brazil, 4Embrapa Southeastern Livestock, São Carlos, Brazil, 5Embrapa Southeastern Livestock, Lab of Veterinary Parasitology, São Carlos, Brazil, 6Universidade Estadual Paulista

“Júlio de Mesquita Filho”, Departamento de Zootecnia, Jaboticabal, Brazil, 7Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, Brazil

Background: Haemonchus contortus infection is the greatest cause of economic losses in sheep production worldwide. In face of widespread anthelmintic resistance, there is increasing number of studies regarding host resistance to parasite infections. The immune response against this parasite is predominantly Th2-polarized, with blood eosinophilia, and increased IgG and IgA serum levels. However, the immune mechanisms associated to host resistance are incompletely understood, even; studies regarding systemic mediators directly associated are scarce.

Method: This study aimed to evaluate systemic immune response profile related H. contortus resistance in Morada Nova sheep. 287 lambs were characterized through FEC and PCV, during two challenges with 4000 H. contortus L3. 20% of the flock were classified as resistant (lowest FEC and highest PCV) and 20% as susceptible (highest FEC and lowest PCV). At weaning (natural infection, -14 dpi), and days 0 and 14 of each challenged, plasma samples were collected to evaluate IgG and IgA levels against H. contortus L3 (ELISA). The ten most resistant and ten most susceptible lambs were submitted to a third parasitic challenge, and blood samples were collected (0 and 7 dpi) for TNFα, IL1β, IL4 and IL13 mRNA quantification.

Results: Higher antibody levels were found in resistant animals, for both IgG and IgA, during different evaluated intervals. On the third challenge, susceptible animals had greater levels of TNFα at 0dpi and higher IL4 levels on 7 dpi. No difference between groups occurred for the other genes.

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Conclusion: IgG and, more intensely, IgA levels were higher in resistant animals, even on -14 dpi, in which FEC and PCV did not differ between groups. Therefore, systemic humoral profile seems to be a good resistance marker in this sheep breed. Susceptible lambs had exacerbated pro-inflammatory response (TNFα), which is commonly associated to parasitic susceptibility. Interestingly, IL4 levels were greater on susceptible animals.

P063Gene expression in canine peripheral blood mononuclear cells infected with Mycobacterium avium subsp. hominissuis

Kim S., Park W.B., Park H.-E., Park H.-T., Yoo H.S.

College of Veterinary Medicine, Seoul National University, Department of Infectious Diseases, Seoul, Korea, Republic of

Background: An opportunistic intracellular pathogen Mycobacterium avium subsp. hominissuis, a member of the nontuberculous mycobacteria (NTM) cluster, causes respiratory disease in immunosuppressed hosts. In particular, infected companion dogs are a potential role to transmit the agent to children or immunosuppressed peoples. However, an underlying mechanism on the pathogeneses of M. avium hominissuis in dogs is not well known. Therefore, the purpose of this study is to investigate a host-M. avium hominissuis interactome in canine PBMCs during the infection.

Method: Canine peripheral blood mononuclear cells were infected with MOI of 1:1 for 2h into 6-well plates. Total RNAs were extracted from the cells at 2h, 6h, 12h, and 24h after infection and gene expressions were quantified using real-time PCR.

Results: M. avium hominissuis induce different cytokine gene expression in canine PBMCs. Our results showed the higher secretion of Th1-associated cytokines, together with the lower secretion of anti-microbial Th2-Th-17-associated cytokines by M. avium hominissuis in canine PBMCs. Also, expressions of the genes related to macrophage activation showed different expression patterns in canine PBMCs compared with humans.

Conclusion: This result could reveal a specific interaction between M. avium hominissuis and the immune system in canine. As increasing interaction between human and companion animals, this study would contribute to prevent or treat M. avium subsp. hominissuis infection in dog and human. This work was carried out with the support of “Cooperative Research Program of Center for Companion Animal Research (Project NO. PJ013985012018)” RDA, the BK21 PLUS and RIVS, SNU, Korea.

P064Role of IgA antibodies in pig oral fluids for the control of PRRS virus infection

Ruggeri J., Ferlazzo G., Boniotti M.B., Capucci L., Amadori M.

IZSLER, Brescia, Italy

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Background: Porcine Reproductive and Respiratory Syndrome (PRRS) is a complex model of host/virus relationship. Disease control is often pursued by “acclimatization” of gilts, i.e. the exposure of PRRS-naïve gilts to PRRSV-infected pigs before the breeding period. In this respect, we had repeatedly observed an association between IgA responses in oral fluids (OF) and block of PRRSV spread through OF.

Material and methods: We investigated the neutralization of PRRSV by OF samples with different titers of PRRSV-specific IgA and IgG antibody, using Real-time RT PCR.

Results: PRRSV yield reduction in macrophages was dependent on the Ig isotypes in OF samples, the IgG-rich samples being sometimes associated with Antibody-dependent Enhancement (ADE) of PRRSV replication. These effects varied as a function of the susceptibility to PRRSV replication of macrophage batches, and also of the PRRSV strains under study. We could discriminate between ADE-positive and ADE-negative PRRSV strains. Next, we separated IgG and IgA in OF samples of PRRSV-infected pigs by means of protein A and size exclusion chromatography. The above results were confirmed by using separated Ig isotypes. In general, the combination of dimeric and monomeric IgA was associated with the strongest reduction of PRRSV replication. Also, the treatment of pig macrophages with separated OF antibodies before PRRSV infection was associated with PRRSV yield reduction, along with a down-regulation of both CD163 and CD169 surface expression.

Conclusions: OF IgA could control PRRSV replication by extra or intracellular interaction with PRRSV, as well as by induction of signals leading to a reduced susceptibility of macrophages to PRRSV infection.

P065Relationship between survivability and immune response of swine classified by the HIR™ technology when exposed to common pig pathogens using a natural disease challenge model

Schmied J.D.1, Putz A.2, Dekkers J.2, Dyck M.3, Fortin F.4, Harding J.5, Plastow G.3, Canada P.6, Mallard B.A.1,7

1Ontario Veterinary College, University of Guelph, Pathobiology, Guelph, Canada, 2Iowa State University, Ames, United States, 3University of Alberta, Edmonton, Canada, 4Centre de Development du Porc du Quebec, Quebec City, Canada, 5University of Saskatchewan, Saskatoon, Canada, 6PigGen Canada, Guelph, Canada, 7Centre for the Genetic Improvement of Livestock, Guelph, Canada

Societal concern regarding food safety and animal health are increasing, encompassing issues including the presence of antibiotic residues in meat, antimicrobial resistant organisms and the risk of zoonotic disease. Therefore, effective economic alternatives, with the potential to improve animal health while maintaining productivity are essential to the livestock industry’s continued success. Previously, when Yorkshire pigs were selectively bred for immune response (IR) it was found that high (H) IR pigs had improved responses to vaccination and pathogen challenge compared to control line and low responders. Although previous research in pigs demonstrated favourable responses to breeding pigs for HIR, the method has not been tested in commercial herds. Here, differences in mortality rate and likelihood of survival, of IR phenotyped Yorkshire x Landrace F1 barrows (n=1915), provided by seven different Canadian

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swine companies, comprising PigGen Canada, were examined. It was hypothesized that when naturally exposed to common swine pathogens, a higher proportion of pigs classified as H immune responders would survive disease challenge and be more likely to reach slaughter than pigs classified as mixed-H, average and low immune responders. Results indicated that H-IR pigs and mixed-H phenotypes died less frequently than those with average or low IR (p=0.0015, Chi-square test for trend). Moreover, pigs classified as having a high antibody-mediated IR died less frequently than those with an average or low antibody-mediated IR (p=0.0402, Chi-square test for trend). Analysis of survival curves revealed a positive association between likelihood of death and phenotype, (p=0.0012, Log-rank test for trend) such that, pigs classified as low and average immune responders were more likely to die before reaching slaughter, than pigs classified as having a mixed-H or H-IR. Therefore, IR phenotyping of pigs clearly identifies animals with the potential to withstand pathogen-challenge by making strong and appropriate IRs.

P066Potential of the dual IFN-γ/IL-2 fluorescence-immunospot assay to distinguish different stages in bovine tuberculosis

Steinbach S., Vordermeier H.M., Jones G.J.

Animal and Plant Health Agency (APHA), Bacteriology: TB Immunology and Vaccines, New Haw, United Kingdom

Human studies have identified the potential of measuring Mycobacterium tuberculosis specific IFNγ and/or IL2 secreting T cell subsets to distinguish different clinical stages of human tuberculosis (TB). To assess these functional T cell subsets in different states of bovine TB we have established a bovine dual IFNγ/IL2 fluorescence-immunospot (FluoroSpot) assay and analysed the frequencies of Mycobacterium bovis (M. bovis) specific IL2 and/or IFNγ producing cells in PBMC from 30 cattle naturally infected with M. bovis. Depending on their post mortem results the animals were grouped in 22 cattle with visible lesions (VL) and 8 cattle without visible lesions (NVL). In response to bovine tuberculin purified protein derivative (PPDB) the frequencies of cytokine producing cells and proportions of IL2 single producers were significantly higher in VL compared to NVL while PWMinduced cytokine responses were similar between the two groups. Dual IL2+IFNγ+ T cells could be identified as the largest PPDB responsive T cell subset in both cattle groups. In conclusion, our FluoroSpot is a valid method to enumerate individual antigen-specific IFNγ+ and IL2+ T cell subsets ex vivo. The greater levels of single IL-2 producing T cells associated with the presence of pathology could be a potential biomarker for active TB in cattle.

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P067Characterizing biomarkers of inflammation in variable stress responding sheep

Naylor D.1, Sharma A.1, Li Z.1, Canovas A.1, Baes C.1, Mallard B.A.2, Karrow N.A.1

1University of Guelph, Animal Biosciences, Guelph, Canada, 2University of Guelph, Pathobiology, Guelph, Canada

Climate change is predicted to exacerbate microbial and environmental stressors on livestock. These stressors have negative impacts on animal health and production that may be mitigated by breeding more resilient livestock. Since the cortisol response to stress is variable and moderately heritable, there is an opportunity to investigate this phenotype for improved stress resilience. Therefore, we sought to characterize biomarkers of inflammation in variable cortisol responding sheep. Intravenous challenge with Escherichia coli lipopolysaccharide (LPS) (400 ng/kg) is a well-documented model of bacterial endotoxemia and implicated in heat stress due to gut leakage. In this study, an LPS stress challenge was performed in 18 ewe-lambs classified as high (H, >300 nmol/L, n=5), middle (M, between 127-208 nmol/L, n=5), and low (L, < 100 nmol/L, n=5) stress responders based on their peak LPS-induced serum cortisol response. Serum pro- and anti-inflammatory cytokines, regulatory microRNAs (miR-132, miR-223, and miR- 1246), and rectal temperature were measured at 0, 2, 4, and 6 hr post LPS challenge to characterize the acute-phase response in these stress phenotyped sheep. Rectal temperatures coincided with cortisol levels and were distinct among the groups at all time points except at 0 hr. Pro- (IL-6, IFN-y, IP-10, TNF-a, CCL3) and anti- (IL-10 and IL-1RA) inflammatory cytokines had a stronger response in H, compared to L, stress responders. The

expression of miR-132, known to inhibit LPS-induced inflammation, was 186-fold upregulated in H stress responders, and 75-fold upregulated in the M and L stress responders 2 hr post LPS challenge. Our results demonstrate that differential cytokine and miRNA profiles are observed in sheep that have been classified according to their stress response. Future monitoring of these serum biomarkers during heat stress may provide insight to mechanisms of resilience to climate-associated stressors.

P068Functional alterations in bovine monocytes during Theileria parva infection

Bastos R.G.1, Knowles D.P.1, Fry L.M.1,2

1Washington State University, Vet Micro Path, Pullman, United States, 2USDA, ARS, Animal Disease Research Unit, Pullman, United States

Background: Theileria parva is the causative agent of East Coast Fever (ECF), a tick-borne disease that is the leading cause of mortality of cattle in sub-Sahara Africa. Evidence shows that immune protection against T. parva involves development of CD8+ cytotoxic T-cell response to parasite-infected cells. However, there is currently a paucity of knowledge regarding the role that innate immune cells play during infection. In this study, we addressed the hypothesis that T. parva infection induces functional changes in bovine monocytes, resulting in altered cell activation.

Method: We evaluated functional alterations in classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical

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(CD14+CD16+) monocytes of cattle during T. parva lethal infection (inoculation with parasite stabilate) and non-lethal infection (inoculation with parasite stabilate plus long-acting oxytetracycline).

Results: Results demonstrated significant up regulation (p < 0.05) of mRNA for IL-1β and TNFα, and increased (p < 0.05) nitric oxide (NO) production during T. parva lethal infection compared to non-lethal infection at 12 days post-infection (DPI). Interestingly, no significant difference in parasite load, assessed by quantitative PCR, was observed in the peripheral blood of lethally infected and non-lethally infected animals at 12 DPI. In vitro stimulation of monocytes with T. parva schizont-infected cells resulted in significant (p < 0.05) up-regulation of both soluble IL-1β and IL-10 mRNA in monocytes from lethally infected cattle compared to cells from non-lethally infected animals.

Conclusion: Our data demonstrates that T. parva infection causes functional alterations in bovine monocytes leading to dysregulation in the expression of pro- and anti-inflammatory cytokines and in the production of NO. These alterations were more pronounced during lethal infection than in non-lethal infection and may be involved in the pathogenesis of ECF. In addition, by comparing lethal and non-lethal T. parva infection, our results revealed functional changes in monocytes that can serve as a biomarkers for ECF progression.

P069BVDV experimental infection in water buffaloes: dynamics of lymphocyte subsets variation evaluated by flow cytometry

Grandoni F.1, Martucciello A.2, Amadori M.3, Grassi C.4, De Matteis G.1, Petrini S.5, Feliziani F.5, Davis W.C.6, De Carlo E.7

1CREA - Consiglio per la Ricerca in Agricoltura e l’Analisi Dell’Economia Agraria (Council for Agricultural Research and Economics) - Centro di Ricerca Zootecnia e Acquacoltura -Monterotondo (RM) - Italy, Monterotondo, Italy, 2Istituto Zooprofilattico Sperimentale del Mezzogiorno-Centro di Referenza Nazionale Sull’igiene e le Tecnologie Dell’Allevamento e delle Produzioni Bufaline -Salerno- Italy, Salerno, Italy, 3Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna -Brescia- Italy, Brescia, Italy, 4Istituto Zooprofilattico Sperimentale del Mezzogiorno-Centro di Referenza Nazionale Sull’igiene e le Tecnologie dell’Allevamento e delle Produzioni Bufaline -Salerno- Italy, Salerno, Italy, 5Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche -Perugia- Italy, Perugia, Italy, 6College of Veterinary Medicine, Washington State University, USA, Pullman, United States, 7Istituto Zooprofilattico Sperimentale del Mezzogiorno-Centro di Referenza Nazionale sull’igiene e le Tecnologie dell’Allevamento e delle Produzioni Bufaline -Salerno- Italy, Salerno, Italy

Several reports show that Bovine Viral Diarrhea Virus (BVDV) infects water buffaloes (Bubalus bubalis). In cattle, BVDV is lymphotropic and, to our knowledge, the effect of acute infection on the buffalo immune system has not been reported. In this study, we evaluated the effect of BVDV infection on the frequency of lymphocyte subsets in pregnant buffaloes using

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Flow Cytometric (FC) analyses. Two healthy pregnant buffaloes seronegative for BVDV were experimentally inoculated intranasally with BVDV on day 81 of the gestation. Blood samples were collected on days 0 and 2, 3, 4, and 14 days after infection (a.i.). FC was used to determine infection effect on frequency of CD4+, CD8+, WC1+ γδ T cells, NK and B cells.

The blood leucocyte counts showed a decrease in lymphocyte numbers at 2, 3 and 4 days a.i. but it was not significant. The most interesting results of FC analyses were at T0 vs T3, they showed: a significant lowering (P< 0.01) of T-lymphocytes, due a significant decrease of CD4+ T lymphocytes (37.2% vs 10.1%, P< 0.01) whileT-CD8+WC1- lymphocytes showed a not significant decrease.

There was an associated transient increase in the frequency of WC1+ γδ T cells (8.3% vs 33.6%, T0 to T3 respectively). No differences were observed in the frequency of cell subsets by day 14 a.i.

Identification of mAbs cross reactive with buffalo lymphocyte subsets has provided the first opportunity to examine the effect of BVDV on lymphocyte subsets in blood during the first 14 days of infection. Consistent with observations in cattle, there was an apparent change in the frequency of CD4 T cells. Follow up studies with a larger number of animals are needed to validate these initial observations and determine whether changes in the frequency of CD4 T cells is associated with alteration in their effector activity.

P070Programmed cell death of leukocytes of peripheral blood as an important element of rabbit haemorrhagic disease virus (Lagovirus europeus GI.1a) pathogenesis

Niedzwiedzka-Rystwej P.1, Tokarz-Deptuła B.1, Deptuła W.2

1University of Szczecin, Department of Immunology, Szczecin, Poland, 2University of Szczecin, Department of Microbiology, Szczecin, Poland

Background: Apoptosis of hepatocytes has a documented role in pathogenesis of RHDV, moreover this process of granulocytes and lymphocytes B and T and their subpopulations in peripheral blood in rabbits infected with RHDV (Lagovirus europeus GI.1) has also been shown. In this paper we undertook the problem of apoptosis of leukocytes in peripheral blood of rabbits infected with RHDVa (GI.1a).

Objectives: The assesment of the percentage of apoptotic granulocytes and lymphocytes in rabbits infected with European strains of RHDVa (Lagovirus europeus GI.1a).

Methods: ApoFluor®Green Caspase (MP Biomedicals, USA) kit was used on FACScan Canto II (Becton Dickinson) cytometer with the Cell Quest (USA) programme. Total amount of caspases 1,3,4,5,6,7,8 and 9 was measured in granulocytes and lymphocytes in peripheral blood in rabbits infected with RHDVa (GI.1a). The study was performed in 0, 4, 8, 12, 24, 36 h p.i. The results was shown as percentage of apoptotic granulocytes and lymphocytes.

Results: The results showed that apoptosis of examined cells in peripheral blood of rabbits

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infected with RHDVa (GI.1a) is more intensive in granulocytes comparing to lymphocytes. Moreover, the apoptosis in granulocytes begins 8 h p.i. and lasts till 36 h p.i, to death of rabbits. In lymphocytes this process begins a bit earlier in 4 and 8 h p.i. and also last until 36 h p.i. Summing up it may be stated infection of rabbits with RHDV activates apoptosis in granulocytes and lymphocytes and it is, as it seems, a new important element of pathogenic mechanism of RHDV (GI.1a).

P071Evaluation of bacterial killing in bovine milk samples

Riva F.1, Ferlazzo G.2, Filipe J.1, Herrera V.1, Piccinini R.1, Amadori M.2

1Università degli Studi di Milano, DIMEVET, Milano, Italy, 2Istituto Zooprofilattico Sperimentale della Lombardia e Emilia Romagnao- Italy , Brescia, Italy

Dairy cow mastitis causes heavy losses due to reduced milk yield, discarded milk and early culling. Autochtonous dairy cattle often show low mastitis prevalence. In order to investigate mastitis resistance in autochtonous breeds, milk samples were collected to evaluate differences of mastitis prevalence between two cattle breeds, and relevant innate immune parameters in milk.

Holstein Friesian (HF) and Modenese (MO) native breed cows were reared under the same conditions inside the same farm. Quarter samples were collected from MO and HF cows at 4 different time points and frozen at -80°C.

Killing of S. aureus and E. coli strains isolated from field mastitis samples was evaluated on milk samples by flow cytometry. Moreover, NAGase was evaluated as previously described (Kitchen et al. 1978) on a microplate fluorometer.

Fewer infected quarters were observed in MO compared with HF breed. Our flow cytometry procedure revealed samples with very low to high killing activity. Within the ten milk samples with the highest killing for either S. aureus or E. coli, eight belonged to the MO native breed. MO cows showed a higher level of killing for S. aureus at T2 (colostral phase, P< 0.05). No correlation was found between % killing and NAGse levels. Our data could partially explain the higher resistance to S. aureus of the autochtonous breed, as demonstrated by bacteriological analysis on MO cows , that turned negative without treatment within a month after pathogen isolation.

P072Differential effects of rift valley fever virus MP-12 infection in two porcine macrophage-like cell lines

Smolensky D.1, Wilson W.1, Cox V.1, Schirtzinger E.1, Chitko-McKown C.2, Fawver Z.1, Noronha L.1

1United States Department of Agriculture, Agricultural Research Service, Center for Grain and Animal Health Research, Arthropod-Borne Animal Diseases Research Unit, Manhattan, United States, 2United States Department of Agriculture, Agricultural Research Service, U.S. Meat Animal Research Center, Genetics,

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Breeding, and Animal Health Research Unit, Clay Center, United States

Rift Valley fever virus (RVFV) is a zoonotic pathogen that primarily affects domestic ruminants, but can also cause severe disease and death in humans. Symptomatic disease from RVFV infection has not been observed in domestic pigs (Sus scrofa); however, two serosurveys have detected circulating antibodies to RVFV among wild and domestic members of the pig family (Suidae). Experimentally, RVFV has been shown to replicate in some porcine cells in vitro, and induce viremia in a fraction of pigs inoculated in vivo. It is unclear how once infected, pigs are apparently able to avert clinical disease. To perform initial mechanistic investigations of porcine innate immune cell responses to infection with RVFV, we used two porcine monocyte-derived cell lines, CΔ2+ and CΔ2-, which have previously been demonstrated to exhibit phenotypic characteristics consistent with macrophages, with CΔ2− cells thought to be less differentiated than CΔ2+ cells. Following 12, 24, and 48-hour infections with MP-12, an attenuated form of RVFV, at 0.01, 0.1, and 1.0 MOI, CΔ2+ cells demonstrated lower viability than CΔ2- cells at intermediate time/dose combinations as measured by MTS assay. Apoptosis was measured in both cell lines at 24 hours post-infection by flow cytometric analysis using Annexin V and propidium iodide labeling. Apoptosis was observed in CΔ2+, but not CΔ2- cells, at 0.1 and 1.0 MOI. Transcription of inflammatory mediators was measured by RT-qPCR and the observed patterns differed between the two cell lines. IL-12 and IFNβ expression was upregulated in CΔ2+ cells, but not CΔ2- cells. Expression of IL-1α increased immediately following a 1 hour virus adsorption in CΔ2+ cells, but only after 12 hours post-adsorption in CΔ2- cells. This work serves as a proof-of-concept starting point to warrant

additional studies on the dynamics of RVFV infection in suids, including whether monocyte-lineage differentiation states may affect outcomes in infected cells.

P073Evaluation of immune responses in post-weaning gnotobiotic pigs infected with human norovirus genogroup II genotype 4 by transcriptome analysis

Park B.-J., Ahn H.-S., Go H.-J., Lyoo E.-L., Kim D.-H., Lee J.-B., Park S.-Y., Song C.-S., Lee S.-W., Choi I.-S.

Konkuk University, Department of Infectious Diseases, Seoul, Korea, Republic of

Human norovirus (HuNoV) is a major pathogen causing serious gastroenteritis in humans. The pathogenesis of HuNoV is poorly understood because there are no well-established cell culture systems and animal models. However, gnotobiotic pigs have been used as an affordable animal model for studying the pathogenesis of HuNoV. The transcriptome has been widely used to analyze complex immune responses in hosts infected with pathogenic viruses.

In this study, we determined the expression profiles of mRNA in post-weaning gnotobiotic pigs infected with HuNoV. A total of 10 post-weaning gnotobiotic pigs were used: 5 pigs were orally inoculated with 1 x 106 genomic equivalent copies of HuNoV GII.4 and the remaining 5 pigs were used as negative controls. All pigs were euthanized 3 days after inoculation and their ileum samples were

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collected. Total mRNA was extracted from the samples and expression levels of 3,911 genes were determined by transcriptome analysis.

Expression levels of 623 genes among 3,911 ones were significantly up-regulated or down-regulated in the HuNoV-infected pigs when compared with negative control pigs (P < 0.05). Among the 623 genes, 62 genes were mainly associated with innate and adaptive immune responses and 28 genes were involved in inflammations. Notably, expression levels of interferon-a and -d were significantly elevated, however, Jak/STAT pathway was down-regulated leading to the suppression of 2’-5’ oligoadenylate synthetase. Overexpression of NK cell receptors (KIR, Nkp46) and their ligands (ITGAL, ITGB2) indicated the increase of NK cell-mediated immune responses. Significant decrease of swine leucocyte antigen (SLA) class I and increase of CTLA-4 expression indicated suppression of cytotoxic CD8+ T cell responses. Expression of TNF-a was significantly increased. These data collectively indicate that HuNoV infection induces stimulation of early innate immunity and inflammation but suppresses adaptive cellular immunity. We expect these results contribute for understanding of pathogenesis of HuNoV.

P074Seroprevalence in broilers against two important foodborne pathogens Salmonella and Campylobacter jejuni

Yeh H.-Y.

USDA ARS, PMSPRU, Athens, United States

Background: Poultry production is one of important components in agricultural output worldwide. It is critical that higher quality and microbiologically safer poultry products be produced for human consumption. Outbreaks of human salmonellosis and campylobacteriosis have often been linked to consumption and handling of unsanitary poultry products. Both Salmonella and Campylobacter jejuni are regarded as commensals in the broiler gastrointestinal tracts. It was demonstrated that maternal antibodies against whole cell lysates of these bacteria were detected. Additionally, the antibodies in sera against these two bacteria could be detected in naturally infected poultry flocks. However, antigenicity of individual proteins using broiler sera has not been extensively studied. The aim of this study was to explore the antigenicity of flagellar and chemotactic proteins to identify potential novel antigens for broilers.

Methods: The genes of flagella and chemotactic proteins of Campylobacter jejuni and Salmonella were amplified by PCR. The amplicons were ligated into a pETite vector and transformed into E. coli cells. The proteins were over-expressed in Escherichia coli cells harboring the genes. The recombinant proteins were purified by a nickel-chelating affinity chromatography, and confirmed by SDS-PAGE analysis and the His tag detection. The recombinant proteins were tested for their antigenicity with immunoblot using chicken sera from several geographical locations.

Results: The recombinant proteins were purified by six-His tag affinity chromatography and had a respective, relative mobility of relevant sizes and positions in SDS-PAGE. The immunoblot results show that each broiler serum reacted to various numbers of recombinant flagellar proteins and chemotactic proteins.

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Conclusion: These results suggest that anti-Salmonella and anti-Campylobacter antibodies be prevalent in the poultry population. These screening results of antibodies in broiler sera may provide a rationale for further evaluation of these recombinant proteins as potential vaccines for broilers to improve human food safety.

P075Identifying potential candidate genes regulating host response to gastrointestinal nematodes in Ontario grazing sheep using transcriptomics

Dixon S.1, Karrow N.1, Menzies P.2, Suarez-Vega A.1, Kennedy D.3, Peregrine A.4, Borkowski E.4, Mallard B.4, Canovas A.1

1University of Guelph, Animal Biosciences, Guelph, Canada, 2Ontario Veterinary College, University of Guelph, Population Medicine, Guelph, Canada, 3Ontario Ministry of Agriculture and Rural Affairs, Guelph, Canada, 4Ontario Veterinary College, University of Guelph, Pathobiology, Guelph, Canada

Background: Gastrointestinal nematode (GIN) infections are considered the most important disease of grazing sheep. Genetically selecting for animals more adept at managing GIN infection will help to control these. The liver is an important participant in the host defense against GINs, and transcriptomics is a powerful method that provides information on gene expression. Therefore, the objective of this study was to identify differentially expressed genes (DEG) in the liver of antibody and stress responding sheep naturally exposed to GIN infection.

Methods: Liver tissue was collected from sheep exposed to Haemonchus contortus, Teladorsagia circumcincta andTrichostrongylus GINs. These sheep were identified as HIGH and MEDIUM stress responding sheep (n=29), based on cortisol levels following E. Colilipopolysaccharide exposure,HIGH and LOW antibody responding sheep (n=29), based on immunoglobulin G levels after vaccination with hen egg white lysozyme, and control sheep (n=16). Based on abomasum worm count post-slaughter, RNA-Sequencing was performed on 29 liver tissues using Illumina HiSeq 2000 analyzer; HIGH stress responders (n=5) with low worm count, MEDIUM stress responders (n=6) with high worm count, HIGH antibody responders (n=4) with high worm count, LOW antibody responders (n=4) with low worm count, and control animals (n=7) with no worm count.

Results: Differential expression analysis revealed 146 DEG between HIGH stress and control sheep and 159 DEG between MEDIUM stress and control sheep. Functional pathway enrichment analysis of these genes showed an increase in immune system processes and a decrease in metabolic processes. 36 DEG between HIGH antibody and control sheep were found and 43 DEG between LOW antibody and control sheep. Pathway analysis showed a decrease in both immune system and metabolic processes. Further pathway and network analysis will identify a list of potential candidate genes associated with resistance to GINs in the stress and antibody responding sheep.

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P076Using NanoString transcriptomics to evaluate the fetal and placental response to congenital PRRSV infection

Lunney J.1, Pasternak A.2, Malgarin C.2, Walker K.1, Ko H.3, Venner L.4, Hong L.5, Harding J.2

1USDA ARS NEA BARC, Animal Parasitic Diseases Laboratory, Beltsville, United States, 2Univ. Saskatchewan, Western College of Veterinary Medicine, Saskatoon, Canada, 3National Institute of Animal Science, Animal Genomics & Bioinformatics Division, Wanju-Gun, Korea, Republic of, 4The Ohio State University, CVM, Columbus, United States, 5South China Agricultural Univ., College of Animal Science, Guangzhou, China

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) infection of 3rd trimester pregnant gilts results in congenitally PRRSV infected fetuses with fetal mortality, disruption of placental function and variable viral burden within each litter.

Methods: We evaluated transcriptomic responses in fetal and placental (PLC) tissues, following maternal PRRSV challenge at day 86 ±0.4 of gestation, using NanoString based transcriptomics. Gilts (3 infected and 1 control) were euthanized at 2, 5, 8, 12 and 14 days post infection (dpi) and tissues collected from the gilts and every fetus. Tissues were snap frozen and PRRSV load quantitated. RNA was extracted and integrity affirmed using an Agilent 2200 Bioanalyzer. Differential expression (DE) of genes was evaluated using a 230 gene NanoString array (designed on biomarkers previously predicted to alter PRRS resistance and susceptibility). Based on log viral load PLC and fetal thymus (THY) samples were assigned to 3 experimental groups: ND (none detected),

LOW and HI viral load. The resulting data were normalized, and a univariate analysis conducted using a Generalized Least Squares (GLS) model with false discovery rate (FDR) correction.

Results: In the PLC a total of 52 genes were found to be significantly upregulated between ND and HI. In the THY a total of 197 and 84 genes were found to be differentially expressed between ND-LOW and ND-HI, respectively. 35 genes were found to be commonly DE for PLC and THY, with response to type 1 interferons (IFNs) including upregulation of STAT1-3 and IFN response genes (IFIT1-3, IFIHI, IRF1&5 and GBP).

Conclusions: Gene selection for the NanoString array was effective in distinguishing PLC and THY immune responses to PRRSV infection. An expanded set of samples from 12 dpi are currently being evaluated to distinguish the effect of viral infection and cross placental transmission on fetal survival and local immune responses.

P077Modulation of porcine lung immune cells during PRRSV-2 infection

Premadasa L., Crisci E.

North Carolina State University, College of Veterinary Medicine, Department of Population Health and Pathobiology, Raleigh, United States

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an RNA virus

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that participates in a variety of polymicrobial syndromes. PRRSV is divided into two species, type 1 (European) and type 2 (North American). Here we study the immunological modulations in primary porcine immune cells induced by two PRRSV-2 strains isolated from North Carolina (USA): strain 1-7-4 generally associated with high pathogenicity (HP) and strain 1-3-4 classified as low pathogenic (LP). PRRSV targets innate immune cells of the monocytic lineage, specifically, porcine alveolar (PAM) and parenchymal macrophages. We used an ex vivo model using primary PAMs isolated from bronchoalveolar lavage and lung parenchyma cells (PAR) obtained with enzymatic digestion. The cells were infected with PRRSV strains NC-1-7-4, NC-1-3-4 and the modified live vaccine (MLV) prototype strain while uninfected cells were used as controls. Cell lysates and supernatants were collected at 6h and 24h post-infection and cytokines levels were measured at transcriptional and translational levels using RT-qPCR and ELISA, respectively. Compared to strain NC-1-7-4, strain NC-1-3-4 showed significantly higher transcripts levels of IL-6, IL-8, IFN-, and TNF- in PAR cells, and significantly higher IL-8 and TNF- expression in PAM cells at 24h post-infection. On the other side, ELISA results showed that both strains induced a significantly higher production of TNF-α and IL-8 in PAM and PAR cells at both time points compared to control cells. IL-1β showed non-significant increase in infected PAR/PAM cells exposed to both strains at 24h, but the level in PAM were lower than PAR. PRRSV ORF5 mRNA expression was similar in PAM and PAR cells infected with all strains. Thus, preliminary data indicate that NC-1-3-4 and 1-7-4 PRRSV strains differently modulate primary lung innate immune cells.

P078Susceptibility of bovine macrophages to bovine viral diarrhea virus infection is dependent on macrophage phenotype

Barone L.J.1,2, Cardoso N.P.1,2, Quintana M.E.1,2, Forlenza M.B.2, Capozzo A.V.1,2

1Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), CABA, Argentina, 2Instituto de Virología, Centro de Investigaciones en Ciencias Veterinarias y Agronómicas (CICVyA), Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina

Background: Bovine viral diarrhea virus (BVDV) (family Flaviviridae, genus Pestivirus) is the etiological agent of a high-prevalent infectious disease in worldwide cattle herds. Macrophage (Mφ) plays a key role in innate immunity and in BVDV immune pathogenesis. Mφ are usually described as classically activated, with pro-inflammatory features (M1) or alternatively activated (M2) with anti-inflammatory and tissue repairs functions. Here we assessed BVDV tropism in peripherical blood mononuclear cells (PBMC) and the role of polarized Mφ in BVDV immunopathogenesis.

Method: PBMC were isolated from adult bovines and infected with BVDV (MOI= 2 and 0.2). Infected cells were identified by flow cytometry analysis, staining live cells (LIVE/DEAD™ ThermoFisher) with anti-BVDV FITC antibody (VMRD). Lineages were labelled using CD172a-RPE-Alexa 647, CD21-RPE, CD4-RPE and CD8-RPE (Abd Serotec). Mφ were differentiated from positively-selected CD14+-PBMC (MACS Miltenyi Biotec) cultured by adherence for 72 h and polarized to M1, M2 and Mα using LPS (20 ng/ml), IL-4 (20 ng/ml) or IFN-α (10 U/ml), respectively. Infection

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was performed at a MOI= 2. Newly produced virus was titrated in MDBK-cells and replication was tested by NS3-detection (Priocheck-BVDV ELISA).

Results: BVDV revealed a marked tropism for the monocytic lineage, followed by B-cells. Infected cells were not killed by the virus. M2-Mφ sustained higher viral replication levels than M1 (p< 0.05) and produced more infective viral particles (p< 0.05). BVDV did not replicate in IFN-α-treated Mφ.

Conclusion: BVDV infects the monocytic lineage, being M2-Mφ more susceptible than M1. Treatment with IFN-α impedes infection. These findings may have implications in BVDV immune pathogenesis, because systemic M2 polarization is predominant in pregnancy and young age, when animals are more susceptible to BVDV infection.

P079Toll-like receptor mediated responses to leptospira spp. in dogs

Novak A.1,2, Sloots A.2, Rutten V.1,3, van Eden W.1, Broere F.1

1Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands, 2Institute for Translational Vaccinology (Intravacc), Bilthoven, Netherlands, 3Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa

Toll-like receptors (TLR) play a key role in the innate defense against pathogens. Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. and affects many

mammals including dogs. The recognition of LPS from Gram-negative bacteria is mediated by TLR4, cofactor MD2 and CD14. However, in human macrophages leptospiral LPS is recognised by TLR2, the lipoprotein receptor, and not by TLR4. It is not known which TLRs are activated by Leptospira in dogs. In the present study, we set up a canine monocyte-derived dendritic cell (moDC) culture and HEK-Blue reporter system to study TLR2- and TLR4-mediated recognition of inactivated Leptospira in dogs.

Canine CD14+ monocytes were cultured in RPMI supplemented with FCS, canine GM-CSF and IL-4 for 6 days to differentiate into moDC. Cells were stimulated for 24 hours with E. coli LPS or Pam3CSK4 (positive controls) or whole-inactivated L. canicola or L. icterohaemorrhagiae. The cytokine and surface marker expression was assessed on mRNA and protein level by RT-PCR and flow cytometry. Additionally, TLR2, TLR4 and MD2 were cloned from canine cDNA and transiently transfected in HEK-Blue-Null1 cell line. The activation of TLR2 and TLR4 was determined by the presence of secreted alkaline phosphatase (SEAP) in the supernatant of stimulated HEK-Blue-Null1 cells.

Both Leptospira serovars upregulated mRNA expression of IL-6, TNF-α, IL-12p40, CD80 and CD83 as well as protein expression of MHC-II and CD80 on canine moDC. Pam3CSK4 and Leptospira serovars activated canine TLR2 in transiently transfected HEK-Blue-Null1 cells. Interestingly, L. canicola showed higher activation potency of moDC and TLR2 in vitro compared to L. icterohaemorrhagiae. The expression and activation of canine TLR4 in transiently transfected HEK-Blu-Null1 cells could not be confirmed yet.

Data suggest TLR2 is involved in the innate

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recognition of Leptospira spp. in dogs. However, whether TLR2 is activated by leptospiral LPS or lipoproteins remains to be determined.

P080Cross-species infection of rabbit with swine hepatitis E virus genotype 3 without induction of hepatitis

Han S.-H., Ahn H.-S., Park B.-J., Go H.-J., Lyoo E.-L., Kim D.-H., Lee J.-B., Park S.-Y., Song C.-S., Lee S.-W., Choi I.-S.

Konkuk University, Seoul, Korea, Republic of

Hepatitis E virus (HEV) belonging to the genus Orthohepevirus in the family Hepeviridae is non-enveloped, positive-sense single stranded RNA virus. Rabbit HEV has been detected in wild, farmed, and pet rabbits in several countries including Korea. Analysis of full genomic sequences indicates that rabbit HEV is classified into HEV genotype 3 (HEV-3). In this study, the possibility of cross-species infection of HEV was determined in rabbits. A total of twenty 8-week old SPF New Zealand white rabbits were divided into four experimental groups. Four rabbits were used as negative controls and four were infected with rabbit HEV as positive controls. Six rabbits each group were inoculated with 1 x 106 genomic equivalent copies of swine HEV-3 and HEV-4 through intravenous route. Serum and fecal samples were collected every week until 8 weeks post infection. Viral RNA was extracted from those samples with Patho Gene-spin DNA/RNA kit (Intron, Korea) according to manufacturer’s instructions. Nested RT-PCR was performed to detect partial genomic

sequence of HEV ORF2 in the serum and fecal samples. Enzyme-linked immunosorbent assay was used to detect serum anti-HEV antibodies and pro-inflammatory cytokines (IL-1, IL-6, and TNF-a) in the serum samples. As expected, infection was identified in rabbits infected with rabbit HEV. HEV RNA and anti-HEV antibodies were identified in rabbits inoculated with swine HEV-3 but not in rabbits inoculated with swine HEV-4. However, the levels of liver enzymes (ALT and AST) and pro-inflammatory cytokines (IL-1, IL-6, TNF-α) were not significantly increased in the rabbits inoculated with swine HEV-3. These results suggest that swine HEV-3 could cause cross-species transmission from pigs to rabbits but not induce acute hepatitis in the infected rabbits.

P081Deletion of relA improves the efficacy of Mycobacterium bovis bacillus Calmette-Guerin as a vaccine against human and bovine tuberculosis

Mahmoud A.H.1,2, Abdellrazeq G.S.1,3, Park K.-T.4, Fry L.M.5, Elnaggar M.M.3,6, Hulubei V.1, Davis W.C.1

1Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, United States, 2Veterinary Quarantine of Alexandria, General Organization for Veterinary Services, Ministry of Agriculture, Alexandria, Egypt, 3Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt, 4Department of Biotechnology, Inje University, Injero 197, Kimhae-si, Gyeongsangnam-do, Gyeongsangnam-do, Korea, Republic of,

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5USDA, ARS, Animal Disease Research Unit, Pullman, United States, 6Department of Veterinary Microbiology and Pathology, Washington State University, pullman, United States

Background: Mycobacterium bovis (Mbv) bacillus Calmette-Guerin (BCG), a mutant derived from Mbv, is currently the only vaccine available for eliciting protective immunity against tuberculosis, caused by M. tuberculosis (Mtb) and Mbv in humans. Results from its extensive use show efficacy is limited. Attempts to use BCG in livestock have not been successful and may explain the variable results obtained in humans. Similar to infection with Mtb or Mbv, infection with BCG leads to development of a persistent infection under immune control. The immune response elicited in cattle is not protective and is not fully protective in humans.

Objective: The objective of the study described herein was to determine if deletion of the gene relA, a global regulator, would alter the immune response to BCG. Studies with M. avium subsp. paratuberculosis (Map) revealed deletion of relA abrogated the capacity of Map to establish a persistent infection. An immune response developed that cleared infection with the mutant. Analysis of the immune response ex vivo showed infection with the mutant led to development of CD8 cytotoxic T cells with the ability to kill intracellular bacteria.

Methods: A BCG relA deletion mutant (ΔBCG/relA) was developed and used with a novel ex vivo tissue culture assay to compare the immune response to BCG and ΔBCG/relA. A novel bacterium viability assay was used to demonstrate killing of intracellular bacteria by CD8 T cells.

Results: Ex vivo stimulation of PBMC with ΔBCG/relA elicited development of CD8 CTL with ability to kill intracellular bacteria.

Conclusion: Deletion of relA disrupts the mechanisms used by mycobacteria to dysregulate the immune response to establish a persistent infection. Now, studies need to be conducted in cattle to show the ΔBCG/relA mutant elicits the same CTL response in vivo similar to the response observed with the Map relA mutant.

P082A preliminary report on anthrax-like disease in cattle in Nigeria from seemingly odd pathogens

Idachaba S.E., Rimfa A.G., Abiayi E.A., Agada G.O., Dashe Y.M., Odugbo M.

Research Institute, Vom- Jos, Nigeria

Anthrax is a neglected zoonotic disease of the poor and vulnerable population that is rarely reported. An outbreak of anthrax-like disease was reported in April 2016 in three herds of cattle in North Central Nigeria. Clinically, the signs and symptoms observed in the diseased cattle were anthrax-like resulting in four deaths. Microbiologically, the phenotypic characteristics of the pathogens incriminated a co-infection of Bacillus laterosporus (now Brevibacillus laterosporus) and Bacillus pantothenticus ( now Virgibacillus pantothenticus) isolated from spleen, blood, and nasal swabs of diseased cattle. Bacillus anthracis, the established causative agent of anthrax was not isolated either microscopically, culturally or with use of USA Navy field ELISA test kits for antigen

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detection; sensitivity of both Bacillus species to ciprofloxacin and penicillin were noted. There were reports of skin ulcers on humans that came in contact with infected blood of carcasses athough.no fatality, perhaps due to intervention using procaine penicillin and local herbs. Anthrax aetiology is now been represented by quasi-species of Bacillus, other than the traditional B. anthracis. Similar reports of anthrax symptoms due to Bacillus cereus was recently reported in neighboring Cameroon and Cote d’Ivoire. Genomic characterization and the virulence of these pathogens in laboratory animals are currently underway. Although vaccination of farm animals with Bacillus anthracis spore vaccine strain 34F2 has long been the hub of control programmes against anthrax, the recent discoveries of other Bacillus species as aetiology of anthrax could undermine the current vaccines in use.

P083Construction of a chimeric bovine herpesvirus 4 as a delivery system for ovine herpesvirus 2 glycoprotein B

Shringi S.1, Donofrio G.2, Li H.1,3, Cunha C.W.1,3,4

1Washington State University, Department of Veterinary Microbiology and Pathology, Pullman, United States, 2Università di Parma, Facoltà di Medicina Veterinaria, Parma, Italy, 3USDA, ARS, Animal Disease Research Unit, Pullman, United States, 4Washington State University, Paul G. Allen School for Global Animal Health, Pullman, United States

Ovine herpesvirus 2 (OvHV-2) causes a frequently fatal disease called malignant catarrhal fever in several ungulates, such

as bison, cattle, pigs and deer. The inability to propagate OvHV-2 in vitro systems has hindered the development of a vaccine. Since the virus cannot be modified or attenuated in vitro, alternative approaches for delivering OvHV-2 antigens for immunization are of utmost importance. OvHV-2 glycoproteins, gB, gH and gL, can stimulate neutralizing antibody responses capable of protecting animals from infection/disease. Therefore, these glycoproteins are considered as important vaccine candidates for OvHV-2. Bovine herpesvirus 4 (BoHV-4) has been evaluated as a vaccine vector, with promising results in delivering heterologous antigen that confer immunity. The objective of this study was to construct and evaluate a chimeric BoHV-4 for the expression of OvHV-2 gB. We used a recombination-mediated genetic engineering system with galK selection to insert a codon optimized sequence of the OvHV-2 gB gene at the TK location of a BoHV-4-A previously cloned as a bacterial artificial chromosome (pBAC-BoHV-4-A). This resulted in the chimeric pBAC-BoHV-4-A-ΔTK/OvHV-2-gB, which was confirmed by sequencing. We evaluated the ability of chimeric pBAC-BoHV-4-A-ΔTK/OvHV-2-gB to reconstitute infectious virus and express OvHV-2 gB in mammalian cells. Transfection of mammalian cells with pBAC-BoHV-4-A-ΔTK/OvHV-2-gB DNA resulted in formation of plaques indicating the reconstitution of infectious viruses. The expression OvHV-2 gB by mammalian cells transfected with pBAC-BoHV-4-A-ΔTK/OvHV-2-gB DNA was confirmed by OvHV-2 gB specific antibodies in immuno-fluorescence assay and western blot. The construction of chimeric pBAC-BoHV-4-A-ΔTK/OvHV-2-gB will allow us to test its potential as an OvHV-2 gB delivering viral vector for vaccine development and as an diagnostic tool to assess neutralizing antibody responses against OvHV-2 gB.

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P084Transfer of cellular immunity through colostrum studied in goat twins

Robbers L.1, van de Mheen R.1, Jorritsma R.1, Nielen M.1, Koets A.2

1Utrecht University, Farm Animal Health, Utrecht, Netherlands, 2Wageningen Bioveterinary Reserach, Lelystad, Netherlands

New-born ruminants are for first immune protection dependent on colostrum, since the epitheliochorial placenta prevents transfer of immunoglobulins and immune cells from mother to neonate. Colostrum contains many immunoglobulins and recently, studies have shown colostrum contains cellular components of the immune system as well. In our study, our aim was to investigate whether lymphocytes in maternal colostrum provide functional immune responses in neonates. The study was carried out in twenty twin pairs of goat kids, from which their mothers were vaccinated against Mycobacterium avium paratuberculosis strain 316 F. Straight after birth, twin pairs were separated and randomly assigned to one of two groups. The first group (n = 20) received mothers’ own colostrum, followed by maternal milk for four weeks. Group 2 (n = 20) were fed cow colostrum followed by artificial milk, both of which are commonly used in dairy goat farming practices. We expected that by providing group 1 with maternal colostrum, immune cells generated in the mother during M. avium vaccination would be transferred and would be functional in the goat kids. A delayed type hypersensitivity (DTH) skin test was performed to test maternal lymphocyte activation in response to purified protein derivative of M. avium. As it appears, kids receiving maternal colostrum showed significant higher DTH response after 24h in comparison to their

corresponding twins who received cow colostrum. These first clinical results imply the colostral transfer of maternal immune compounds to kids and that these compounds respond specifically to M. avium. Further results are to be expected, since blood, colostrum and faecal samples are currently being analysed in the lab.

P086Elucidating the Immunoregulatory Functions of Exosomes Released from Tracheal Cells on Macrophages in Chickens

Barjesteh N.1, Douanne N.1, Emam M.2, Fernandez Prada C.1, Segura M.1, Lavoie J.-P.3

1Université de Montréal, Faculty of Veterinary Medicine, Pathology and Microbiology, Sainte-Hyacinthe, Canada, 2Ontario Veterinary College, University of Guelph, Pathobiology, Guelph, Canada, 3Université de Montréal, Faculty of Veterinary Medicine, Clinical Sciences, Sainte-Hyacinthe, Canada

Innate immune responses at mucosal surfaces provide the first line of defence against viruses, aiming to block the entry of these pathogens and their replication. The effective elimination of the virus depends on the coordinated communication between different cells of the innate system and the epithelial cells in the mucosal surfaces. The release of exosomes from host cells is a newly discovered mechanism possibly contributing to the immune response to viruses. The exosomal content can be affected by different stimuli. It is critical to first characterize the composition of exosome cargos released from different cell-types to

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identify immunoregulatory functions of these extracellular vesicles.

In this study, we hypothesized that chicken tracheal epithelial cells secrete exosomes whose content is modified in response to different TLR ligands and inactivated avian influenza virus (AIV).

To investigate this, tracheal organ culture (TOC) was prepared using 20-days-old-chicken embryos. Exosomes released from chicken tracheal rings treated with TLR-ligands and inactivated AIV and untreated tracheal rings were isolated. We are in the process of characterizing the differences in exosomal contents following the treatment with TLR ligands and UV-inactivated H9N2 low-pathogenic AIV. We will then identify which exosomal miRNA is induced by TLR ligands or influenza virus stimulation. In addition, we will determine the effect of exosomes released from TOCs on the chicken macrophages.

The results of this study will provide insight into the complex interplay of the immune system and the induction of antiviral responses in chickens.

P087Biocontamination in poultry vaccines

Elsady S.1, Latif A.1, El Jakee J.2

1Central Laboratory for Evaluation of Veterinary Biologics, Cairo, Egypt, 2Veterinary Medicine Cairo University, Cairo, Egypt

Freedom of the poultry vaccines from biocontaminants (bacterial and viral contaminants) should be secured throughout the process of production and before market release to avoid infection of immunized poultry. The principle cause of contamination of poultry vaccines is using embryonated chicken eggs in production of these vaccines. In this study a total of 577 different live viral vaccine batches were screened for detection of some bacterial and viral contaminants. The detection of biocontaminats by the classical methods is expensive, time consuming and need specific equipments, so in the present study we are going to use tests that are simple, sensitive, specific and rapid give results in short possible time. In this study a total of 577 vaccine samples were to detect bacterial and viral contaminants, one vaccine sample was contaminated with Salmonella Enteritidis, seven vaccine samples were contaminated with Newcastle disaease virus (NDV) and two vaccine samples were contaminated with avian leukosis virus (ALV)

P088Toll-like receptor 1/2, 7/8 and 9 agonist combi as adjuvant in neonate pigs for inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine

Vreman S.1, Rebel J.M.2, Barnier-Quer C.3, Moore A.4,5, Stockhofe-Zurwieden N.1

1Wageningen University, Wageningen Bioveterinary Research, Lelystad, Netherlands, 2Wageningen Bioveterinary Reserach, Wageningen Livestock Research, Wageningen,

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Netherlands, 3University of Lausanne, Lausanne, Switzerland, 4University College Cork, School of pharmacy, Cork, Ireland, 5Xeolas Pharmaceuticals, Dublin, Ireland

Background: Neonates show differences compared to adults regarding their immune development, which complicates vaccine responses at young age. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important porcine pathogen inducing health and welfare problems and economic losses to swine industry. Vaccination at an early age is pivotal to prevent disease. In this study different adjuvant systems and skin vaccination with dissolving microneedle (DMN) patches, were investigated to enhance inactivated (i)PRRSV vaccine responses in neonates.

Method: Three-day old pigs received a prime vaccination and a booster vaccination four weeks later. The adjuvant system was composed of a mixture of TLR1/2 (Pam3Cys), TLR7/8 (R848) and TLR9 (CpG ODN) agonists (TLRaCombi) with squalene based adjuvant (SWE) and combined with iPRRSV strain 07V063. This vaccine was delivered intramuscular and into the skin with DMN patches. An O/W adjuvant (Montanide™) applied intramuscular was used as reference adjuvant and one group received a placebo vaccination. The TLRaCombi-only and the SWE-only group were included as additional control groups. All animals received a homologous challenge with PRRSV three weeks after the booster vaccination. Specific antibodies, IFN-γ production and viremia were measured at several time-points after vaccination and/or challenge, and lung pathology at necropsy.

Results and conclusion: Whereas some animals in the O/W reference group developed antibodies, none of the other vaccines induced

a specific antibody response after booster. Interestingly, the SWE-only group was able to induce a specific IFN-Ƴ response after booster vaccination, which was lower in the O/W group, and absent in the other groups. Overall, none of the vaccines were effective when first applied at three-days days of age and there was no difference between intramuscular and skin vaccination. This indicates that iPRRSV 07V063 in combination with the investigated adjuvant system is not protective in neonate piglets in a prime and booster vaccination regimen.

P089Immunochemical study of protein profiles of Taleghan, Fars, and lorestan strains of mycoplasma agalactiae

Orangi H.1,2

1Shiraz University, Shiraz, Iran, Islamic Republic of, 2Veterinary School of Shiraz University, Shiraz, Iran, Islamic Republic of

Agalactia is a contagious disease in sheep and goats. To prevent the disease, a vaccine containing three strains of Mycoplasma agalactiae namely Taleghan (T), Fars (F), and Lorestan (L) is currently used in Iran. In this study, the protein profiles of T, F, and L strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 8.5% gel and Western blotting were studied. Antigens were prepared from these strains, and the amounts of protein were measured using Lowry method9. Protein profiles of the three strains studied by SDS-PAGE revealed only minor differences with

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as many as 8, 14, and 10 bands detected in T, F, and L strains, respectively. Majority of the proteins were shared by all of the three strains. Western blotting with rabbit antisera against T, F, and L strains detected nine, nine, and eight immunodominant antigenic bands. All of the above strains revealed more or less similar protein profiles with major common immunodominant antigens at 117, 110, 98, 80, 50, and 5KDa positions. Keywords Mycoplasma agalactiae -Protein profile-SDS-PAGE-Western blotting

P090Comparative study evaluating the safety of type 2 porcine reproductive and respiratory syndrome (PRRS) modified-live virus (MLV) vaccines in pigs

Madapong A., Saeng-Chuto K., Meebumroong S., Nilubol D.

Chulalongkorn University, Faculty of Veterinary Science, Bangkok, Thailand

Porcine reproductive and respiratory syndrome (PRRS) modified-live virus (MLV) vaccines have been used to control PRRS worldwide. The safety of MLV are of interest due to the potential for vaccinated pigs to continue to shed virus for prolonged periods. Therefore, the study was conducted to evaluate the safety of MLV in terms of virus persistence and shedding pattern using sentinel pigs. PRRSV-free, weaned sentinel pigs (n=56) were allocated into 2 groups, G1 (n=21) and G2 (n=21). G1 was vaccinated with PrimePac® PRRS (MSD Animal Health, The Netherlands). G2 was vaccinated with Ingelvac® PRRS MLV (Boehringer Ingelheim, Germany). Following vaccination,

age-matched sentinels were introduced weekly into vaccinated groups (n=1/group), from 0 to 42 days post vaccination (DPV). Each batch of sentinel was commingled with vaccinated pigs for 3 weeks and monitored for seroconversion using ELISA. Three vaccinated pigs of each group were necropsied on a weekly basis from 7 to 42 DPV. Tonsils and nasal swabs were collected and assayed for virus using virus isolation and PCR. The results demonstrated that sentinel pigs introduced to G2 at 14 to 42 DPV seroconverted, but sentinel pig introduced to G1 seroconverted at 28 DPV only. For tonsils, PRRSV was detected in only 1 G1 pig each at 28 and 35 DPV. In contrast, PRRSV was detected in 2/3, 2/3, and 1/3 of pigs in G2 at 28 to 42 DPV. For nasal swabs, PRRSV was detected in 2/3, 3/3, 2/3, and 2/3 of pigs in G2 at 21 to 42 DPV, respectively. In comparison, PRRSV was detected in 1/3, 2/3, and 1/3 of pigs in G1 at 21 to 35 DPV, respectively. In conclusion, pigs vaccinated with PrimePac® PRRS shed vaccine virus shorter than did Ingelvac® PRRS MLV. The persistence of infection in tonsils and nasal swabs from vaccinated pigs was also lesser.

P091Early transcriptional regulation of Th17 differentiation in nasopharyngeal-associated lymphoid tissue (NALT) of mice intranasally immunized with chitosan nanoparticles loaded with Brucella abortus malate dehydrogenase

Shim S., Im Y.B., Park H.-T., Park H.-E., Kim S., Yoo H.S.

Seoul National University, Seoul, Korea, Republic of

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Background: Brucella abortus malate dehydrogenase (Mdh) is promising vaccine candidate. To develop subunit vaccine, Mdh was loaded to mucoadhesive chitosan nanoparticles (CNs) and it was intranasally immunized to mice. The NALT plays an important role in inducing a first immune response against pathogens. We evaluated immunogenicity of Mdh and elucidated adjuvant ability of CNs, by studying the comprehensive innate immune responses in NALT after intranasal immunization of mice.

Method: CNs were generated with chitosan dissolved in D.W (0.5 w/v %) and mixed with 150ul of 8% Tween 80. 6-week-old BALB/c mice were intranasally immunized with of 30 µg of CNs loaded with the Mdh. NALT was isolated from immunized mice and transcriptomic analysis was conducted using RNA from the NALT. The production of IgA was detected using serum, nasal washes, genital secretions, and fecal excretions.

Results: As an adjuvant activity, CNs triggered activation of PRRs and chemotaxis of innate immune cells. The pathway analysis revealed that intranasal immunization of Mdh induced maturation of APCs such as dendritic cells and T lymphocytes eliciting Th17 cell differentiation. Nasal vaccination of CNs-Mdh in mice also resulted in higher levels of mucosal secretory IgA in digestive and genital mucosa.

Conclusion: These results suggest that CNs-Mdh could be promising delivery system and nasal vaccine candidate that can trigger activation of APCs, Th17 cell response and production of secretory IgA through NALT. This work was supported by Korea Health Industry Development Institute (No. HI16C2130) and the Brain Korea 21 PLUS program for Creative Veterinary Science Research and the Research Institute of Veterinary Science, SNU, ROK.

P092Prevalence of avian influenza outbreak in Egypt 2018

Ibrahim H.

Central Laboratory for Evaluation of Veterinary Biologics, Bacterial Strility, Cairo, Egypt

Avian influenza virus is highly contagious disease affect poultry and resulted human cases with deaths.

Our aim is isolating circulating viruses in Egyptian environment by using current research data and comparison using available software.

73 isolates of AIV were collected from 13 Egyptian governorates during the period from 2016 to 2018 from different poultry flocks, the mortality rate from examined flocks ranged from 25 - 35% regardless to vaccination regime or vaccine type (homologus or heterologous).

Our data recorded gradual increase in rates of death caused by H5N1 AIV in ducks and geese.

Samples subjected for RT-PCR and sequencing. The results revealed 43/73(58.9%) positive when using M gene primer while 26/ 73 (35.6%) positive when using H gene primer whereas cleaveage site sequence revealed 17 / 73(23.2%) positive.

Phylogenetic analysis revealed two major genetic groups of H5N1 AIVs were being co-circulating. Continuous evolution of H5N1 viruses and emergence of new antigenic variants may be partially by immune pressure caused by massive use of vaccines, which such variants can circumnavigate vaccine-

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induced immunity and are called escape mutants.

Phylogenetic tree HA gene reported two major antigenic and genetic distinct groups of H5N1, the variant group 2.2.1.1 and the classic group 2.2.1/C.

AI strain (A/chicken/Egypt/M7217B/2017(H5N1) represent the most common clusters of AIV was reassorted at National research center and then adapted on MDCK cell line for preparation of Tissue culture (TC) vaccine in parallel with egg adapted one. The AIV had a titer of 10 9.5 TCID50/ml, after adaptation on MDCK cell culture while the egg adapted vaccine had titer 1012.

P093Immunogenicity of the local anthrax vaccine strain in animal models

Rume F.I.1,2, Karim M.R.3, Biswas P.K.4, Yasmin M.2, Ahsan C.R.2

1Patuakhali Science and Technology University, Microbiology and Public Health, Barishal, Bangladesh, 2University of Dhaka, Microbiology, Dhaka, Bangladesh, 3Bangabandhu Sheikh Mujibur Rahman Agricultural University, Medicine, Gazipur, Bangladesh, 4Chittagong Veterinary and Animal Sciences University, Microbiology and Veterinary Public Health, Chattogram, Bangladesh

Background: Anthrax is considered an endemic disease in Bangladesh since 2009. There are many factors, including the immunogenicity of the present anthrax

vaccine strain of Bangladesh, are thought to be associated with the recent outbreaks of anthrax. Therefore, the present study was undertaken to evaluate the immunogenicity of the present anthrax vaccine in animal models.

Methods: A total of four cattle were immunized with local anthrax vaccine and six mice were immunized intraperitoneally with the sonicated extract of that vaccine (85 µg protein/mouse) following standard protocol. Sera were collected four weeks after the last dose of immunization and Western blot technique was applied to check the immunogenicity of the vaccine and the sonicated extract. Sera collected from similar number of cattle or mice, without any immunization, were used as control sera.

Results: A 36 kDa prominent antigenic band was obtained from immunized cattle sera, whereas, at least three antigenic bands, 30, 36 and 83 kDa, were obtained from the sonicate extract immunized mice sera. However, control sera from both cattle or mice showed no such bands.

Conclusion: The present anthrax vaccine showed immunogenicity in the cattle model. However, the sonicated extract immunized mice sera showed some more bands, including an 83 kDa, the molecular weight similar to the protective antigen (PA). Recently our group reported the presence of four different genotypes with remarkable genetic variations of B. anthracis in the soil of numerous outbreaks. The absence of the 83 kDa band in the sera of the vaccine immunized cattle or the presence of new genetic variants of B. anthracis in the environment, could be related to the recent outbreaks of anthrax. However, this genetic mismatching between the vaccine strain and the strains circulating in the environment is contributing to vaccine failure needs to be studied further.

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P094Preparation and evaluation of mucosal needles killed vaccine against infectious Coryza in chicken

Sayed R.

Central Laboratory for Evaluation of Veterinary Biologics, Cairo, Egypt

Infectious Coryza (IC) is an acute respiratory disease of chickens which characterized by nasal discharge, facial swelling, lacrimation, conjunctivitis and reduced egg production. Avibacterium paragallinarum (Ap) is known to be the causative agent of IC which infect the sinus mucosa this area is lake vascularity that is lead to difficult treatment and protection. In Egypt is found of three different serovars, of Ap termed A, B and C. Protection of chicken flocks against IC involves inactivated killed vaccine ,thus killed vaccination programs may not totally protect chicks that are to lake of vascularity in the infraorbital sinus that lead to the low amount of antibodies against AP reach this region. So in this work we use the poly (D,L lactide-co-glycolic acid) PLGA nano emulsion for preparation of mucosal IC vaccine in . The immunizing purified hemagglutinin antigen of three serotypes was prepared and emulsified with PLGA nano emulsion. About 180 SPF chicks (5weeks ago) were divided into four groups. The first group was dropped with mucosal IC vaccine at nasal and second groups were injected with the traditional gel adjuvant killed vaccine while the third groups were injected with traditional oil adjuvant killed vaccine. The forth group was considered a control group. Serum sample were collected weekly and intranasal challenge test was carried out with

virulent AP at 3 weeks post vaccination. ELISA antibody titers for mucosal and killed vaccine reached to 750 and 2398 respectively at 7 weeks post vaccination. The protection rate for mucosal and killed vaccine was 95% and 80% respectively.

P095Formulation of A. salmonicida adjuvanted vaccines for rainbow trout: impact of the adjuvant oil origin

Koziol M.-E.1, A. Veenstra K.2, Xu B.3, Versillé N.3, Ben Arous J.3, J. Secombes C.2

1Seppic Inc, Fairfield, United States, 2University of Aberdeen, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK, a Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences,, Aberdeen, United Kingdom, 3Seppic, Paris, France

Injectable inactivated fish vaccines require adjuvants to enhance the immune response. Water-in-oil emulsion adjuvants are widely used in the aquaculture industry due to their cost effectiveness, stability, and long-term effect. However, oil-adjuvanted vaccines can be reactogenic and induce side effects in fish. In this study, we analyzed the impact of adjuvant oil origins in the safety and immunogenicity of Aeromonas salmonicida vaccines.

Two different adjuvants were tested, one based on a non-mineral oil (Montanide™ ISA 763A VG) and one based on a mineral oil (Montanide™ ISA 761 VG). Following intraperitoneal vaccination of rainbow trout, blood samples were taken at 42 and 53 days

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post vaccination (dpv) to assess antibody response, and adipose tissue samples were collected at 3, 14 and 28 dpv for RT-qPCR analysis of immune genes implied in the pro-inflammatory and adaptive responses. Side effects in the peritoneum were scored at 7, 14, 28, 42 and 53 dpv.

Both vaccines induced a high antibody response against A. salmonicida with a significant increase in titre between 42 dpv and 53dpv. Vaccination-induced adhesion scores for the vaccine groups fell within industry-accepted limits as per Speilberg Standardized Extended Post-Vaccine Scoring. However scores were lower for the fish vaccinated with the non-mineral oil adjuvant. Compared to the control group (antigen alone), a clear upregulation of immune genes occurred in response to both vaccine groups, which persisted over time. This upregulation was higher for fish vaccinated with the mineral oil adjuvant. Furthermore, a strong correlation between gene expression, modulated by the oil origin, and vaccine safety was observed.

These results showed that oil origin of fish adjuvants has an important impact on the immunogenicity and safety profile of fish vaccines, and that Montanide™ ISA 763A VG and Montanide™ ISA 761 VG are efficient adjuvants for the formulation of inactivated A. salmonicida vaccines.

P097The humoral immune response and vaccine protection against paratuberculosis in sheep

Pooley H.B., Begg D., Plain K., Whittington R., Purdie A., De Silva K.

Th University of Sydney, Farm Animal Health, Sydney School of Veterinary Science, Camden, Australia

Background: B cell responses are often considered unimportant in diseases of intracellular pathogens. Recently, there has been evidence to suggest a significant role for the humoral immune response in mycobacterial diseases. Paratuberculosis caused by Mycobacterium avium subspeciesparatuberculosis (MAP) is a chronic wasting disease of ruminants and significant financial burden to producers globally. In this study we examinedthe B cell response in ileal tissue (the site of predilection) as well as the longitudinal peripheral antibody in sheep that were successfully protected against paratuberculosis by vaccination, and those that were not.

Methods: Fifty merino wethers in 4 treatment groups:vaccinated unexposed (n=5) and exposed (n=20), non-vaccinated unexposed (n=5) and exposed (n=20) were studied. Sheep exposed to MAP were retrospectively classified as infected or uninfected based on tissue culture at necropsy, 52 weeks post MAP exposure. Blood samples were taken every 3-4 months for MAP specific serum IgG1 and IgG ELISA. Ileal tissue taken at necropsy was used for analysis of gene expression related to B cell function by qPCR.

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Results: B cell activating factor and CD40L were both downregulated in vaccinated uninfected sheep compared to vaccinated infected animals. MAP-specific IgG1 was significantly higher in vaccinated uninfected sheep compared to the infected animals. Differences in serum IgG1 were evident as early as 4 weeks following vaccination, prior to MAP exposure.

Conclusions: B cell functionality plays a role in vaccine protection in paratuberculosis. MAP-specific IgG antibodies have potential to be used to screen for sheep that are not protected by vaccination against paratuberculosis.

P098A comparative study evaluating immune induction and protective efficacy of nanoparticles-entrapped porcine reproductive and respiratory syndrome virus (PRRSV) vaccine against co-challenge with PRRS1 and PPRS2

Tantituvanont A.1, Chaikhumwang P.1, Madapong A.2, Saeng-Chuto K.2, Nilubol D.2

1Chulalongkorn University, Faculty of Pharmaceutical Sciences, Pharmaceutics and Industrial Pharmacy, Bangkok, Thailand, 2Chulalongkorn University, Faculty of Veterinary Science, Veterinary Microbiology, Bangkok, Thailand

The study was conducted to compare the induction of immunity and the protective efficacy of nanoparticles-entrapped porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (NP-PRRS) against PRRS2

challenge. Thirty, 3 week-old, PRRSV-free, pigs, were allocated into 5 groups of 6 pigs each including Chal, NP, KV, Subunit and MLV. Pigs in the NP group were intranasally vaccinated with NP-PRRS system three times. Pigs in the KV, subunit and MLV were intramuscularly vaccinated with KV, subunit and modified live vaccine (MLV), respectively. The Chal group was left as challenge control. All pigs were challenged intranasally with PRRS2 (HP-PRRSV) at 28 days post-vaccination (DPV) and necropsied at 7 days post-challenge (DPC). Blood samples were collected at 0, 14, 28 DPV and 7 DPC. Peripheral blood mononuclear cells (PBMC) were isolated and assayed for cell-mediated immunity as measured by the production of IFN-g and IL-10 using intracellular flow cytometry and ELISA, respectively. The severity of PRRSV-induced pneumonic lung lesion was evaluated and lungs were assayed for PRRSV RNA using RT-qPCR. Following vaccination, NP-vaccinated pigs had significantly higher level of IFN-g producing cells at 28 DPV compared to that of other vaccinated groups. No IL-10 was detected in the NP-vaccinated group. Following challenge, NP-vaccinated pigs exhibited significantly lower (P < 0.05) PRRSV-induced pneumonic lung lesion and PRRSV RNA in lungs, compared to that of other vaccinated groups. NP-vaccinated pigs had significantly lower IL-10 compared to that of other vaccinated groups. In conclusion, the results of the study suggested NP vaccine administered intranasally, can provide protection against challenge with HP-PRRSV, either alone or in conjunction with PRRSV type 1 as demonstrated by reduced lung lesion and viremia. NP vaccine might represent an alternative to improve vaccine efficacy.

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P099Vaccination with a recombinant PCV2-ORF2 vaccine does not interfere with the efficacy of a modified live vaccine against PRRSV

Saalmüller A.1, Koinig H.C.1,2, Stadler M.1, Glatthaar-Saalmüller B.3, Gerner W.1, Ladinig A.1

1University of Veterinary Medicine Vienna, Vienna, Austria, 2Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria, 3Labor Dr. Glatthaar, Ochsenhausen, Germany

Background: Vaccines against Porcine Circovirus 2 (PCV2) belong to the most efficient vaccines for pigs. Besides the reduction of clinical signs of PCV2-related diseases, this vaccination is able to lower viral and bacterial co-infections, including a reduction of PRRSV loads during PRRSV infection. However, PRRSV vaccines are widely applied and the majority of them are modified live viruses (MLV). With the knowledge of a reduced viremia after PRRSV infection by PCV2 vaccination one might postulate that a simultaneous PCV2 and PRRSV vaccination has a negative influence on the efficacy of a PRRSV-MLV.

Methods: 24 four week-old PRRSV-negative piglets were divided into 5 groups: a control group, a group vaccinated against PCV2 (CircoFLEX®, Boehringer Ingelheim), a group vaccinated with a MLV-PRRSV vaccine (PRRSFLEX®, BI), a group immunized with PRRSFLEX® + Carbopol, and one group immunized with PRRSFLEX® together with CircoFLEX®. Blood was taken after vaccination (days 0, 7, 10, 14, 21, 28, 35) and the following parameters analyzed: antibodies against PRRSV (IDEXX X3), PRRSV neutralizing antibodies, PRRSV-specific lymphocytes in IFN-γ ELISPOT assays and PRRSV-specific T

cells identified by intracellular cytokine staining (IFN-γ, IL-2 and TNF-α).

Results: Independent of a co-vaccination with CircoFLEX® all PRRSFLEX® vaccinated swine developed antibodies against PRRSV starting 10 days after vaccination. 14 days after vaccination most of the pigs showed neutralizing antibodies and developed antigen-specific IFN-γ lymphocytes. PRRSV-specific T cells showed both CD4 and CD8β phenotypes and especially in CD4+ T cells multifunctional PRRSV-specific cells producing IFN-γ, IL-2 and TNF-α could be detected.

Conclusions: PRRSFLEX® is able to induce PRRSV-specific antibodies within 10 days after vaccination, within 14 days neutralizing antibodies could be detected as well as PRRSV-specific IFN-γ producing T cells and multifunctional TH1 cells. Co-vaccination with CircoFLEX® had no influence on the efficacy of the PRRSV-MLV and the induction of a PRRSV-specific immune response.

P100Quantifying the persistence of vaccine T cell epitopes in circulating swine influenza strains from 2013-2017

Tan S.1, Gutiérrez A.H.2, Gauger P.C.3, Opriessnig T.3, Moise L.1,2, De Groot A.S.1,2

1University of Rhode Island, Institute for Immunology and Informatics, Providence, United States, 2EpiVax, Inc., Providence, United States, 3Iowa State University, College of Veterinary Medicine, Veterinary Diagnostic and Production Animal Medicine, Ames, United States

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Background: When swine influenza vaccine and circulating strains are poorly matched, vaccine-induced antibodies may not protect, but highly conserved T-cell epitopes may still have a disease-mitigating effect. Conservation of T cell epitopes between vaccine and novel swine influenza A virus strains is variable and may explain variability in vaccine efficacy. Methods for estimating the degree of epitope conservation between vaccines and outbreak strains are needed. Here, we examine class I and II T cell epitope conservation of a T cell-directed DNA vaccine among swine influenza isolates over five years by immunoinformatics methods.

Method: Twenty-eight class I and 20 class II epitopes in the prototype vaccine are published (Gutiérrez et al. 2016). We obtained 1272 circulating strains from 2013 to 2017 consisting of H1N1, H1N2 and H3N2 subtypes from the Influenza Research Database. Two algorithms were used to evaluate conservation of vaccine epitopes among these isolates: T cell epitope content comparison (EpiCC), and JanusMatrix (JMX). Pairwise comparisons between prototype and circulating strains were conducted with EpiCC to analyze overall vaccine epitope cross-conservation; an epitope-by-epitope assessment was conducted using JMX.

Results: Epitopes in the prototype vaccine are fairly conserved over five years despite extensive variability across proteins and subtypes. Higher EpiCC scores are thought to be associated with greater protection by vaccines against challenge strains (Gutiérrez et al. 2017). The prototype vaccine had the highest EpiCC cross-conservation with H1N1 viruses. Internal proteins are well conserved across all subtypes, suggesting internal proteins might contribute to vaccine efficacy. This vaccine showed encouraging efficacy

against H1N1 challenge (Hewitt et al. 2019, submitted).

Conclusions: These complementary immunoinformatics methods suggest that the prototype vaccine could stimulate CD4 and CD8 T cells that recognize swine influenza epitopes conserved over many years and contribute to protection. These approaches can be applied to analyze other viral vaccines.

P101Linear B-cell epitope mapping of tick antigens by PEPSCAN immunoassay and in silico construction seeking an anti-tick multi-epitope vaccine for cattle

Moraes A.1, Fisch A.2, Almeida L.G.N.1, Daher I.P.1, Peña M.S.2, Pupin M.A.F.2, Brunato M.L.2, Santos I.K.F.M.1, Ferreira B.R.1,2

1University of Sao Paulo, Department of Biochemistry and Immunology, Ribeirão Preto, Brazil, 2University of Sao Paulo, Department of Maternal-Infant Nursing and Public Health, Ribeirão Preto, Brazil

Background: Ticks are vectors of lethal diseases to animals, causing negative impact on livestock activity worldwide. Tick salivary proteins are protective against Rhipicephalus microplus ticks and induce antibody responses. Linear B-cell epitopes from these targets could be useful for an anti-tick multi-epitope vaccine.

Method: Sera from cattle immunized with nine tick salivary antigens were used for linear B-cell epitope mapping through PEPSCAN. The epitopes identified in vitro were used to

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construct seven chimeric proteins containing flexible (GGGGS, 3xGGGGS and GGGGGG), rigid (EAAK and 3xEAAK), double lysine (KK) linkers or no linker at all. Nterminal 6xHis-tag was added to sequences. Constructions were evaluated in silico regarding to predicted physicochemical parameters (ExPASy ProtParam), solubility in Escherichia coli (PROSO, EXPROPRIATOR), allergenicity (AlgPred), and MHCII binding to 26 human HLA and 3 mice H2 alleles (NetMHCIIpan).

Results: Thirty-five linear B cell epitopes were identified in vitro by PEPSCAN, while in silico analysis indicated that only constructs containing 3xEAAK and KK linkers were feasibly stable; all constructs were possibly insoluble when expressed in E. coli, with estimated half-life >10h in this expression system. Only the construct without linkers was predicted to be allergenic. Regarding to the presence of peptides that bind MHCII molecules, the constructs with flexible linkers had predicted strong binders to only 9 alleles, while the constructions containing EAAK, 3xEAAK, KK linkers presented strong binders to 18, 10 and 18 alleles, respectively; the construction without linkers, however possibly allergenic, presented strong binders to 24 alleles.

Conclusion: The in silico construct inclosing KK-linkers was considered the best among the tested constructs. This construct has potential to induce humoral responses, has a structure compatible with heterologous expression and is non-allergenic, thus can be tested as an anti-tick multi-epitope based vaccine to control R. microplus infestations in animals.

Financial Support: FAPESP (2015/09683-9), CNPq, CAPES.

P102A plant-produced immunoenhanced pig vaccine against PRRSV

Erbs G.1, Thomsen J.2, Munch D.2, Kvisgaard L.K.3, Larsen L.E.3, Pedersen F.S.2, Jungersen G.1

1DTU, Department of Health Technology, Kgs. Lyngby, Denmark, 2Aarhus University, Department of Molecular Biology & Genetics, Aarhus, Denmark, 3DTU, National Veterinary Institute, Kgs. Lyngby, Denmark

Porcine Reproductive Respiratory Syndrome Virus (PRRSV) is an enveloped, positive-stranded RNA virus that causes one of the most infectious diseases in pigs worldwide. It has high impact on animal welfare and agricultural economics, and the extensive use of antibiotics to control PRRSV-associated respiratory problems may endanger public health. Thus, control of PRRSV is top priority in all pig producing countries. Contrary to most other virus infections, PRRSV persists in the blood for weeks after antibodies are formed due to dominant decoy antigens masking the subdominant epitopes that provide immunity at later stages. PRRSV is extremely difficult to control, as existing vaccines are not effective in preventing infection or excretion of virus. State-of-the-art vaccine research has identified non-infectious subunit vaccines such as virus-like-particles (VLPs) to be the most efficient entities developed to date, triggering both humoral and cell-mediated immune responses. We aim to develop an efficient vaccine by altering the immunosuppressive (ISD) motifs in the major target for neutralizing antibodies of PRRSV (GP5) and to produce the VLPs presenting the modified GP5 in plants to gain advantages in terms of safety, speed, cost, and efficacy.

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To investigate immunogenicity of modified PRRSV GP5, mice were DNA immunized with different PRRSV GP5 encoding plasmids with mutations in the ISD domain and with recombinant produced VLPs based on a PRRSV full length GP5, the GP5 ectodomain and transmembrane region with or without a fused Rhabdovirus glycoprotein. In addition, more than 40 genes, several of them involved in the JAK-STAT signaling pathway, were analyzed by high-throughput gene expression in Porcine Alveolar Macrophage (PAM) cells infected with wild type PRRSV (LoN94), a type 1 PRRSV (strain 18794) and a GP5 single point mutated PRRSV 18794 clone, where a threonine at position 76 was substituted with an alanine (T76A).

P103Interaction of the gamma delta TCR with the WC1 hybrid co-receptor / pattern recognition receptor

Gillespie A.1, Gervasi M.G.1, Yirsaw A.1, Connelley T.2, Hope J.2, Telfer J.1, Baldwin C.L.1

1UMass Amherst, Amherst, United States, 2Roslin Institute, Edinburgh, United Kingdom

Background: γδ T lymphocytes are found in high numbers within the blood of young ruminants such as cattle where they have been shown to be first responders to a number of different pathogens including Leptospira and Mycobacterium. Bovine γδ T cells express particular members of a unique γδ T-cell specific multigenic array known as WC1 which are hybrid pattern recognition receptors and signaling co-receptors. Using

the Leptospira model, we show that the γδ T cells that are dividing in both bovine and caprine models express WC1 molecules. In cattle WC1 has been shown to bind several pathogens, corresponding with proliferation of γδ T cells, and production of cytokines while shRNA silencing of WC1 results in inhibition of response. These data indicate that both WC1 and TCR are crucial for activation of the cells.

Method: Amnis imaging flow cytometry and STORM high resolution microscopy was used to visualize interactions between WC1, TCR and leptospira.

Results: While ab T cells have a well-characterized immune synapse formed upon T cell stimulation by an antigen presenting cell (APC) with central supramolecular activation clusters (cSMACs), immune synapse formation of γδ T cells is not well characterized. We showed using Amis imaging flow cytometry that FRET occurs between the TCR and WC1 molecules after stimulation with Leptospira. Using STORM high resolution microscopy we showed that activated γδ T cells do not exhibit a cSMAC but rather that while WC1 and TCR are found in separate protein islands before activation they are found together in protein islands after stimulation with Leptospira only in responsive cells. We also showed binding of leptospira to WC1 expressing cells.

Conclusions: This information regarding pathogen interaction with WC1 molecules and TCR can be used in the development of next generation vaccines that promote a cellular response.

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P104Effective and protective immunization with a novel recombinant MERS-CoV expressed in CHO cells for developing subunit vaccine

Kim S., Park Y., Lee E., Kim S., Jang J., Ryoo G., Jang H.

WOOGENEBIO, Yesan, Korea, Republic of

Middle East respiratory syndrome coronavirus (MERS-CoV) is emerging a viral respiratory disease that was first identified in Saudi Arabia, 2012. Its spread of their reservoir host (camels) widely, that issue is ongoing for public health security. Mortality is approximately 35% of reported patients with MERS, there is no available vaccine and specific treatment for human use. MERS-CoV Spike (S) protein is composed of 2 subunits; the receptor binding domain (RBD) containing S1 subunit and the fusion subunit S2. Several vaccines candidates are based on the full-length or truncated S protein and some reports have shown that RBD of MERS-CoV spike (S) is a good candidate antigen for a MERS-CoV subunit vaccine. We focused on the spike protein (S) of MERS-CoV to improve immunogenicity and two vaccine candidates have been investigated. We exploited a recombinant MERS-CoV spike protein containing a 770-amino acid fragment (residues 19-770) fused with human IgG Fc fragment (WG-770-Fc) and expressed in the culture supernatant of transfected CHO cells transiently. To induce stability additionally, we constructed mutant type and confirmed that it augmented the immunogenicity of WG-S770-Fc with inducing neutralizing antibodies. Because the spike protein (S) of MERS-CoV targets the cellular receptor dipeptidyl peptidase 4 (DPP4) on the host-cell surface, we examined the immune response of subunit vaccine against MA MERS-CoV

lethal challenge in human DPP4 knock-in mice, WG-S770-Fc vaccinated group exhibited 100% survival compared to 100% mortality observed in PBS control group. In sum, these results indicate that vaccine expressing mutant type WG-S770-Fc could represent a good target of the developing MERS-CoV vaccine.

P105Advances towards a vaccine for ovine pneumonia

Parlane N.A., Gupta S.K., Bridgeman B.J., Wedlock D.N.

AgResearch, Animal Health, Palmerston North, New Zealand

Pneumonia in lambs causes a reduction in growth-rate and often death. Over 20% of animals within flocks can be affected, with disease prevalent in animals that are stressed. The disease is complex, involving multiple pathogens, including bacteria (Mannheimia haemolytica), mycoplasmas (Mycoplasma ovipneumoniae) and viruses (Parainfluenza virus type 3). While vaccines have been used in the past in New Zealand, these have not been effective and there is a need for better antigens and adjuvants. Multiple serotypes (S) of M. haemolytica are involved in the disease and S2 and S1 are the most frequent isolates associated with lung lesions in sheep. Our earlier studies showed the S1 strain of M. haemolytica vaccine provided a degree of cross-protection against a S2 strain. We hypothesized that this protection can be enhanced by adding mycoplasmas to the vaccine formulation. There is motivation from

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farmers, meat processors and animal welfare groups to use vaccine adjuvants that cause minimal vaccination site reactions. We have conducted adjuvant trials in lambs to identify potent and safe adjuvants for formulating a combination of M. haemolytica and M. ovipneumoniae antigens. Significant antibody responses were measured to these organisms following vaccination. Higher antibody titres were observed in animals vaccinated with QuilA or Montanide ISA61 adjuvants compared to Alum. The use of Montanide resulted in vaccination site lesions in a proportion of animals. Other adjuvants are currently under investigation.

A comprehensive measurement of immune responses to pneumonia in sheep is restricted due to limited availability of immunological reagents. We are investigating new approaches to measure a wide range of immune responses in vaccinated sheep using Nanostring technology. A multiplex Nanostring technology-based assay has been established to measure expression of 20 key genes involved in various immune related pathways including T-cell, B-cell immunity and monocyte/macrophage activation.

P106Targeting antigens to dendritic cells: Characterization of a full chimeric mouse x pig recombinant antibody anti-porcine DEC205 receptor

Bustamante-Córdova L.1, Parra-Sánchez H.1,2, Reséndiz-Sandoval M.1, Burgara-Estrella A.2, Arvizu-Flores A.2, Hernández J.3

1Centro de Investigación en Alimentación y Desarrollo, A.C., Hermosillo, Mexico, 2Universidad de Sonora, Hermosillo, Mexico, 3Centro de Investigación en Alimentación y Desarrollo, A.C., Laboratorio de Inmunología, Hermosillo, Mexico

Background: Antigen targeting toward dendritic cells (DCs) is considered a promising vaccination strategy in veterinary medicine. Antigen targeting using recombinant antibodies (rAb) has been performed in mice, chicken, and pigs. The aim was to produce a chimeric mouse x pig rAb anti-porcine DEC205 and to prove its immunogenicity when fused with peptides of the porcine respiratory and reproductive syndrome virus (PRRSV).

Methods: Western blot and flow cytometry analyses were used to confirm the recognition ability of the chimeric rAb toward porcine DEC205 receptor and DEC205+DCs. An immunization assay in pigs was performed to evaluate the immunogenicity of the antigenized chimeric antibody. Prime and boost were applied three weeks apart intradermally in the neck area. Challenge was performed intranasally one week after the boost. Euthanasia was done three weeks post-challenge. We evaluated the presence of CD4+IFN-γ+cells, cell proliferation, anti-PRRSV antibodies, viremia, viral load in lungs and tonsil, and microscopic lung lesions.

Results: The resulting mouse x pig rAb anti-porcine DEC205 maintained a similar binding capacity to DEC205 receptor from the original monoclonal antibody and recognized blood DEC205+DCs. The immunogenicity assay showed that the use of this antigen fused with the mouse x pig rAb anti-porcine DEC205 induced a robust production of anti-PRRSV antibody (p< 0.05), lower viral load in tonsil and the presence of CD4+IFN-γ+cells in response

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to some peptides of the M and Nsp2 peptides, compared to the non-vaccinated control group. No differences were observed in viremia, viral load in lungs, microscopic lung lesions and cell proliferation response between control and vaccine groups.

Conclusion: This mouse x pig rAb anti-porcine DEC205 can be used for antigen targeting and its immunogenic properties can be improved to offer a more complete level of protection against PRRS or other porcine diseases.

Disclosure: This work was funded by projects 248922 and INFRA-2015-01.

P107Intranasal immunization of swine with a porcine reproductive and respiratory syndrome virus inactivated whole-virus vaccine provides protective immunity against a homologous virus challenge

Chen W., Husmann R., Villamar M., Zuckermann F.

University of Illinois, Pathobiology, Urbana, United States

Intranasal immunization against respiratory viral pathogens is known to provide a superior level of protective immunity as compared to vaccines delivered by other routes. Hence, we investigated the immunogenicity and efficacy a porcine reproductive and respiratory syndrome (PRRS) virus inactivated whole-virus (IWV) vaccine administered intranasally as a spray. The PRRS IWV vaccine was adjuvanted with a whole cell lysate (WCL) of a saprophytic

Mycobacteria, and it was sprayed twice intranasally at a 2-week interval. Sixteen days after the booster vaccination, the animals were challenged intranasally with 2x104 TCID50 of the same virulent virus that was used to prepare the vaccine, namely the PRRS virus strain LTX1, which belongs to lineage 1 of the North American virus genotype. For comparison, groups of animals were similarly vaccinated using the PRRS IWV vaccine without the Mycobacterial adjuvant or with the Mycobacterial WCL adjuvant alone. At the time of euthanasia (13 days after virus challenge), the presence of GP5-specific IgA in the lung lavage was evident in animals immunized with adjuvated PRRS IWV vaccine. Most notably, the substantial extent of gross-lung pathology observed in mock-vaccinated pigs at 13 days after the virulent virus challenge (90% of lung involvement), was reduced by half in animals immunized with the IWV vaccine administered with or without the mycobacterial WCL adjuvant. Further, immunization with the adjuvanted IWV vaccine reduced the level of viremia by >10-fold as compared to the level of viremia observed in the unvaccinated and challenged controls, or the animals immunized with the IWV vaccine without the adjuvant. Although much work remains to be done, these results provide encouraging evidence that an efficacious intranasal IWV vaccine-based against PRRS virus is feasible.

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P108Characterization of protective humoral and cellular immune responses against RHDV2 induced by a new vaccine based on recombinant baculovirus

Müller C.1, Ullrich R.2, Schinköthe J.2, Müller M.3, Köllner B.2

1Friedrich-Loeffler-Institut, Institute of Immunology, Greifswald, Germany, 2Friedrich-Loeffler-Institut, Greifswald, Germany, 3IDT Biologika, Greifswald, Germany

Rabbit hemorrhagic disease (RHD) is a lethal disease in rabbits caused by RHD virus (RHDV). Protection is only possible through vaccination. A new virus variant (RHDV-2) which emerged in 2010 in France differed from the classical RHDV1 variant in certain aspects and vaccines against RHDV1 induced limited cross protection only. In a previous study, we designed a recombinant baculovirus based RHDV2-VP1 vaccine, which provided a protective immunity in rabbits against RHDV2. In the present study this newly created vaccine is characterized with regard to onset and duration of protection, and possible cross protection against classical RHDV1. Furthermore, humoral and cellular immune mechanisms in vaccinated and infected rabbits were analyzed. In all experiments, the recombinant vaccine was compared to a conventional liver-based RHDV2 vaccine.

The RHDV2-VP1 vaccine induced a protective immune response already seven days after single vaccination and fully protected for at least 14 months. A booster vaccination 21 days after the first had a negative influence on long-term protection. The cross protection provided by the RHDV2-VP1 vaccine against classical RHDV1 was limited since only 50 %

of vaccinated rabbits survived the infection. Conclusively, the new, baculovirus-based RHDV2-VP1 vaccine has the potential to protect rabbits against the infection with RHDV2, blocks completely the disease progression and prevents the spread of RHDV-2 at the population level.

P109Bovine NK-lysin-derived peptide NK2A demonstrates a limited immunomodulatory activity on cattle leukocytes

Dassanayake R.P., Porter T.J., Falkenberg S.M., Sacco R.E., Lippolis J.D., Reinhardt T.A.

Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, Agricultural Research Service, United Sates Department of Agriculture, Ames, United States

Bovine NK-lysins (bNK-lysins), a type of antimicrobial proteins, are primarily produced by cytotoxic T-lymphocytes and NK-cells. We and others have previously demonstrated the potent antimicrobial activities of synthetic bNK-lysin-derived peptides (NK1, NK2A, NK2B, and NK2C) against a variety of microbial pathogens. While immunomodulatory properties of several antimicrobial peptides including chicken NK-lysin have recently been reported, it is unknown whether bNK-lysins also display such immunomodulatory properties. Given the structural and functional similarities of bNK-lysins with chicken NK-lysin, we hypothesized that bNK-lysins also display immunomodulatory activities. To test this hypothesis, one peptide corresponding to the functional region helices 2 and 3 of bNK-

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lysin NK2A (bNK2A) was synthesized. The antimicrobial activity of bNK2A peptide was evaluated on Mannheimia haemolytica, which is a major contributor to the bovine respiratory disease complex in cattle. The immune responses of cattle PBMC to bNK2A peptide were assessed for increase in intracellular calcium concentration ([Ca2+]i) and induction of chemokine/cytokine transcript expression levels (mRNA) by flow cytometry and quantitative RT-PCR, respectively. As expected, bNK2A showed a potent antimicrobial activity against M. haemolytica. Immediate elevation of [Ca2+]

i, which is a key early feature in immune cell activation, was observed with Fluo-4-AM loaded PBMC when incubated with bNK2A. Contrary to our expectations, bNK2A had a modest effect on the expression levels of chemokines (CCL4, CCL5, CCL20 and IL-8), and cytokines, (IL-1β, IL-6, and TNF-α) in monocytes. Cells incubated with PBS (negative control) did not show any changes in [Ca2+]i or chemokine/cytokine expression levels. These findings suggest that bNK-lysins appear to have limited immunomodulatory properties as compared to strong antimicrobial activities.

P110Head Start Immunity: characterising the early protection of C strain vaccine against subsequent classical swine fever virus infection

McCarthy R.1,2, Everett H.3, Graham S.1,4, Steinbach F.5,6, Crooke H.3

1Animal and Plant Health Agency (APHA), Virology Department, Addlestone, United Kingdom, 2Brunel University London, Uxbridge,

United Kingdom, 3Animal and Plant Health Agency, Virology Department, Addlestone, United Kingdom, 4Univ of Surrey, Guildford, United Kingdom, 5Animal and Plant Health Agency (APHA), Addlestone, United Kingdom, 6Univ of Surrey, School of Veterinary Medicine, Guildford, United Kingdom

Classical Swine Fever Virus (CSFV) is a single stranded RNA virus belonging to the Flaviviridae family. It is an ongoing threat to the livestock industry due to its high transmission and mortality rates or high mortality associated with infection. The primary sites of replication are the tonsils and oropharangyeal lymph nodes. Interferon signalling is a key component of how the innate immune system responds to challenge with CSFV, where high levels of interferon-α (IFN-α) are a characteristic feature of acute disease that are not protective, but lead to the development of disease-associated immunopathology.

Live attenuated vaccines such as the CSFV C strain vaccine are capable of protecting against infection within 5 days of vaccination, where IFNg+ CD8+ cells precede the detection of a humoral, virus neutralising response; but the molecular mechanisms through which this early protection is mediated have yet to be established. In this study, we compared the response of pigs vaccinated with the C strain to non-vaccinated pigs both challenged with a pathogenic strain of CSFV. Analysis of transcriptomic data from the tonsils of these animals during the early stages after vaccination and challenge reveals a set of regulated genes that appear throughout the analysis. Many of these are linked to the ISG15 antiviral pathway that is upregulated suggesting it plays a key role in the rapid and early response protection conferred by C strain vaccination.

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P111Functional analysis of bovine CTLA-4 in bovine leukemia virus infection

Watari K.1, Konnai S.1,2, Okagawa T.2, Maekawa N.2, Murata S.1,2, Ohashi K.1,2

1Hokkaido University, Disease Control, Sapporo, Japan, 2Hokkaido University, Advanced Pharmaceutics, Sapporo, Japan

Background: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is known as an immune inhibitory receptor expressed on activated effector T cells and regulatory T cells. Interaction of CTLA-4 with its ligands CD80 or CD86 delivers an inhibitory signal that regulate excessive immune response. Recent studies have reported that CTLA-4 is related to evasion from host immune response in chronic infections and malignant neoplasms. On the other hand, CTLA-4 blockade restores the immune response against these diseases. In a previous report, we indicated that the upregulation of CTLA-4 was closely associated with disease progression in cattle infected with bovine leukemia virus (BLV). In this study, we established an anti-bovine CTLA-4 monoclonal antibody (mAb) to confirm effect of CTLA-4 blockade on the activation of bovine immune response.

Method: Recombinant bovine CTLA-4-Ig protein was expressed by mammalian cells and immunized to mouse to establish anti-bovine CTLA-4 mAb. The binding of CTLA-4-Ig and anti-CTLA-4 mAb was analyzed by flow cytometry. Bovine peripheral blood mononuclear cells (PBMCs) were cultivated with anti-CTLA-4 mAb in the presence of CTLA-4-Ig and Staphylococcal enterotoxin B (SEB), or BLV antigens. IFN-γ production from PBMCs was measured by ELISA to evaluate

T-cell response.

Results: Bovine CTLA-4-Ig binds to bovine CD80- and CD86-expressing cells and inhibited IFN-γ production from bovine PBMCs activated by SEB. An established anti-bovine CTLA-4 mAb specifically recognized bovine CTLA-4, not CD28, and blocked the binding of CTLA-4-Ig to both CD80 and CD86 in a dose-dependent manner. Additionally, suppression of IFN-γ production from CTLA-4-Ig-treated PBMCs was restored by anti-CTLA-4 mAb. Finally, anti-CTLA-4 mAb enhanced IFN-γ production by CTLA-4-expressing PBMCs activated by SEB and PBMCs isolated from BLV-infected cattle in response to BLV antigens.

Conclusions: Collectively, these results suggest that anti-bovine CTLA-4 mAb can reactivate T-cell function and could be applied for a new therapy against chronic diseases of cattle.

P112Development of bovine rotavirus and bovine coronavirus combined vaccine of bovine diarrhea disease

Ryoo G., Kim S., Kim S., Jang J., Park Y., Lee E., Jang H.

Woogenebio CO., Yesan, Korea, Republic of

Bovine rotavirus and bovine coronavirus are major cause of bovine viral diarrhea disease.The primary aim of experiment is development of combined vaccine of them. Virus seed Bovine rotavirus G6P[5](KVCC No. VR1800005), Bovine corona virus Group

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1b (spike gene) is received from QIA. Figure out factor of increase virus tier by titrating according to virus infection condition with TCID50,FA. After the increase of virus titer is confirmed, inject inactivated virus into mouse. And figure out antibody efficatious by virus neutralization test with the mouse serum.

Bovine coronavirus titer was increased up to 10 6.0 TCID50 /ml. Bovine rotavirus titer was increased up to 107.5 TCID50 /ml and result of neutralization test is up to 29.

For further study clinical trial with cattle is scheduled.

P113Development of an equinized antibody to neutralize the insulin-like growth factor-1 receptor (IGF-1R): a potential immunotherapy for endocrinopathic/hyperinsulinemic equine laminitis

Vathsangam N.1, Busfield S.2, Strietzel C.J.3, Sillence M.4, Bailey S.1

1The University of Melbourne, Faculty of Veterinary and Agricultural Sciences, Parkville, Australia, 2Nexvet Ltd., Bundoora, Australia, 3Zoetis Inc., Kalamazoo, United States, 4Queensland University of Technology, Earth, Environmental and Biological Sciences School, Brisbane, Australia

Equine laminitis is a devastating disease of the foot that results in lameness and, in severe cases, require euthanasia. Insulin toxicity appears to have a key role in the pathogenesis of the disease, and further research has

indicated that it may cause lamellar damage through excessive stimulation of insulin-like growth factor-1 receptors (IGF-1R). The objective of this study was to modify a clinically-tested human anti-IGF-1R antibody, to develop a neutralizing antibody with high-affinity for equine IGF-1R, while avoiding immuno-intolerance when administered in vivo. Development of the equine antibody was challenging due to the limited availability immunoglobulin sequences (EquCab 2.0 database), in addition to the predominance of circulating light chain isotypes (λ), unlike the case in humans. However, by utilizing the database and established effector function data, a speciation process, “PETization”, was adopted to generate chimeric equine-human and fully-equine monoclonal antibodies. The antibodies were further engineered to abolish immunogenic effector functions, while retaining receptor binding functionality. The PETized equine antibodies were purified at a yield of 0.3 g/L, comparable to the chimeric and human antibodies. A comparison of binding characteristics by Biacore and direct immunosorbent assay, to immobilized soluble IGF-1R, demonstrated similar affinities (3.3 x 10 -10 and an EC50 = 0.127 nM respectively) between equine and chimeric antibodies. Additionally, flow cytometric analysis confirmed that the equine antibodies bound to native receptors presented in equine fibroblasts. Preliminary immunoblotting data of fibroblasts treated with the equine antibody under hyperinsulinemic conditions suggested a marked reduction in downstream signalling protein Ribosomal protein s6 (RPS6) phosphorylation. These data enabled the best candidate to be selected for scale-up production, for testing in a hyperinsulinemic equine model in vivo. A successful immunotherapy for equine laminitis would reduce the economic and welfare costs of this destructive disease, while advancing our understanding of antibody therapeutics in the horse.

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P114Effects of bovine tumor necrosis factor alpha decoy receptors on cell death and inflammatory cytokine kinetics: potential for bovine inflammation therapy

Fujisawa S.1, Konnai S.1,2, Okagawa T.2, Maekawa N.2, Murata S.1,2, Ohashi K.1,2


Background: In dairy farming and the livestock industry worldwide, numerous refractory diseases including bacterial infections threaten animal health and productivity. Despite efforts to prevent and treat these diseases in cattle, including the use of antimicrobials, it is not well controlled in the field. Several inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), play important roles in disease progression; thus, blocking these cytokines may be a novel strategy for treatment. In humans, a soluble TNF receptor 2 (sTNFR2) IgG1 fusion protein is commonly used as a decoy receptor to treat rheumatoid arthritis. However, biological drugs targeting inflammatory cytokines have not been used in cattle.

Method: Bovine sTNFR1 and sTNFR2 IgG1 Fc-fusion proteins (TNFR1-Ig and TNFR2-Ig) were prepared using mammalian cells, and their anti-inflammatory functions were analyzed in vitro, using recombinant bovine TNF-α　based assays.

Results: Both TNFR1-Ig and TNFR2-Ig were shown to bind with TNF-α, and TNFR2-Ig showed higher affinity toward TNF-α than TNFR1-Ig. We next stimulated murine

fibroblast-derived cells (L929 cells) with TNF-α to induce apoptosis and analyzed cell viability in the presence of TNFR-Ig proteins. Both TNFR1-Ig and TNFR2-Ig significantly improved cell viability. In addition, apoptosis induced by TNF-α was suppressed, even at low TNFR2-Ig concentrations, suggesting TNFR2-Ig has higher activity to suppress TNF-α functions than TNFR1-Ig. Finally, to examine TNFR2-Ig’s anti-inflammatory properties, we cultured peripheral blood mononuclear cells from cattle with TNF-α in the presence of TNFR2-Ig. TNFR2-Ig significantly reduced the gene expression and protein production of the inflammatory cytokines IL-1β and TNF-α. Our results suggest that TNFR2-Ig inhibits inflammatory cytokine kinetics by blocking TNF-α to transmembrane TNFR, thereby attenuating excessive inflammation induced by TNF-α.

Conclusions: Collectively, the findings of this study demonstrated the potential of TNFR2-Ig as a novel therapeutic for bovine mastitis. Further investigation is required for future clinical application.

P115Modulation of mycobacterium-macrophage interaction by a host factor, bovine conglutinin: implications for bovine tuberculosis

Tsolaki A.G., Mehmood A., Kishore U.

Brunel University London, Biosciences, Department of Life Sciences, College of Health and Life Sciences, London, United Kingdom

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Collectins are collagen-containing C-type lectins, which are potent pattern recognition innate immune molecules. Bovine conglutinin was the first animal collectin to be discovered, and is structurally very similar to Surfactant Protein D (SP-D). SP-D is known to interact with Mycobacterium tuberculosis, and the closely-related Mycobacterium bovis pathogen, the causative agent of bovine tuberculosis. Because of this similarity and presence in serum, conglutinin may have a protective influence in bovine tuberculosis. We show that a recombinant form of conglutinin (rfBC), composed of homotrimeric neck and C-type lectin domains, binds specifically and in a dose-dependent manner to the model organism Mycobacterium bovis BCG. rfBC showed a direct bacteriostatic effect on the growth of M. bovis BCG in culture. In addition, rfBC inhibited the uptake of M. bovis BCG by THP-1 macrophages and suppressed the subsequent pro-inflammatory response. Conglutinin is also a well-known binder of the complement activation product, iC3b. rfBC was able to inhibit the uptake of complement-coated M. bovis BCG by THP-1 macrophages, whilst down-regulating the pro-inflammatory response. It is likely that rfBC inhibits the phagocytosis of mycobacteria by two distinct mechanisms: firstly, rfBC interferes with mannose receptor-mediated uptake by masking lipoarabinomannan (LAM) on the mycobacterial surface. Secondly, since conglutinin binds iC3b, it can interfere with complement receptor-mediated uptake via CR3 and CR4, by masking interactions with iC3b deposited on the mycobacterial surface. rfBC was also able to modulate the pro-inflammatory response in THP-1 cells, which is important for mobilizing the adaptive immune response, facilitating containment of mycobacterial infection. Thus, conglutinin has complement-dependent as well as complement-independent anti-mycobacterial activities, interfering with

both known mechanisms of mycobacterial uptake by macrophages. As mycobacteria are specialized intracellular pathogens, conglutinin may therefore inhibit M. bovis from establishing an intracellular niche within macrophages, negatively affecting the long-term survival of the pathogen in the bovine host.

P116Immunomodulatory effects of sialostatin L and sialostatin L2 from Ixodes persulcatusSchulze, Taiga tick

Konnai S., Sajiki Y., Okagawa T., Maekawa N., Murata S., Ohashi K.

Hokkaido University, Faculty of Veterinary Medicine, Sapporo, Japan

The inhibitors of cysteine protease, sialostatin L (sL1) and sialostatin L2 (sL2), are tick salivary glands proteins, derived from Ixodes scapularis. In Japan, I. persulcatusis a major vector for Lyme disease agent, such as Borrelia burgdorferi, and B. miyamotoiwhich causes relapsing fever, but only a few information is available for the molecules including sL1 and sL2 of this tick. Thus, in this study, homologues of sL1 (Ip-sL1) and sL2 (Ip-sL2) were identified in I. persulcatus, and the biological functions were studied. The cDNA clones, Ip-sL1 and Ip-sL2, showed a high homology with sL1 and sL2 sequences from the salivary glands of I. scapularis. Quantitative PCR analysis indicated that Ip-sL1 and Ip-sL2 were expressed in the salivary glands, and throughout life cycle stages of the ticks. In addition, Ip-sL1 and Ip-sL2 were expressed even before feeding of ticks, and continuously

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expressed during blood feeding of the ticks. To investigate the functions of Ip-sL1 and Ip-sL2, recombinant Ip-sL1 and Ip-sL2 were prepared, and immunological analyses were performed using these recombinant proteins. Both Ip-sL1 and Ip-sL2 inhibited the production of IP-10, TNFαand IL-6 from bone-marrow-derived dendritic cells (BMDCs) stimulated with LPS. Additionally, Ip-sL1 significantly impaired the maturation of BMDCs. Taken together, Ip-sL1 and Ip-sL2 have the immunosuppressive functions and seem to be involved in the transmission of pathogens by suppressing host immune responses such as the production of cytokines and the maturation of dendritic cells. It will be necessary to further investigate immunosuppressive functions of Ip-sL1 and Ip-sL2 in detail to develop effective vaccines against I. persulcatus.

P117Probiotics delivered in milk replacer affect leukocytes collected from the lung lavage fluid of neonatal calves

Eicher S.D.1, Chitko-McKown C.G.2, Kritchevsky J.E.3, Bryan K.A.4

1USDA-ARS, Livestock Behavior Research Unit, West Lafayette, United States, 2USDA-ARS, USMARC, Clay Center, United States, 3Purdue University College of Veterinary Medicine, Department of Veterinary Clinical Sciences, West Lafayette, United States, 4Chr. Hansen, Inc., Milwaukee, United States

We designed a series of experiments to determine if the feeding of probiotics effects respiratory immunity of neonatal dairy calves

as well as on their respiratory microbiome. Twenty dairy calves were assigned to one of two treatments: milk replacer with or without probiotics (0.5g/d Bovamine, Chr. Hansen, Inc.) beginning 2 days after birth (n = 10/treatment). Calves were weaned to 1 bottle at day 42 and were completely weaned by day 52 at which time lung lavages were performed on a subset of the calves (n = 5/treatment). The objective of this study was to determine if the oral feeding of probiotics to neonatal dairy calves would affect the phenotype and function of leukocytes collected from lung lavage fluids. Leukocytes were aliquoted for flow cytometric analysis and were stained for bovine α-CD3, α-CD4, α-CD8, α-CD11b, α-CD14, and α-CD205; unstained controls were included for each animal. To measure phagocytosis and oxidative burst, leukocytes were exposed to opsonized E. coli fluorescent bioparticles. Percent positive (PP) cells and mean fluorescent intensity (MFI) were determined. Data were analyzed by un-paired T-test using GraphPad Prism. Due to our small sample size and variation among the calves, none of the differences between Control and Probiotic-fed calves were significant at P = 0.05. However, the following trends were found: Percent positive cells for CD3, CD11b, and CD205 were higher, and PP cells were lower for CD4, CD8, CD14, phagocytosis, and oxidative burst in calves fed probiotics. In contrast, MFI tended to be lower for all measures with the exception of CD14 in Probiotic-fed calves. We hypothesize that probiotic bacteria may affect overall immunity in calves by their effect on immune cells in the nasal mucosa and tonsils of the respiratory system in addition to overall changes in the microbiota. Additional experiments are underway to identify these changes.

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P118Porcine DCs derived from cryopreserved bone marrow cells for the in vitro evaluation of immunomodulatory compounds

Geervliet M.1, Jansen C.A.2, Rutten V.P.M.G.2, Tijhaar E.1, Savelkoul H.F.J.1

1Wageningen University, Cell Biology and Immunology, Wageningen, Netherlands, 2Faculty of Veterinary Medicine, Utrecht University, Department of Infectious Diseases and Immunology, Utrecht, Netherlands

Background: Morbidity and mortality is a major issue for the pig sector worldwide, with the weaning period being the most critical period in a pigs life. One of the novel and promising strategies to enhance developmental processes that impact adaptability and resilience around weaning is immunomodulation by feed (e.g. pre- and probiotics) in early life. To determine the potential of immunomodulatory components, an in vitro pre-screening tool was set up using porcine dendritic cells (DCs) derived from both cryopreserved and fresh bone marrow cells.

Materials and methods: Fresh and cryopreserved bone marrow cells from four pigs were differentiated into bone marrow derived DCs by culturing these cells in the presence of porcine granulocyte macrophage colony stimulating factor (GM-CSF) for 3 days. On day 4, the differentiated cells were stimulated with different concentrations of either yeast-derived β-glucans or the gram negative probiotic E.coli Nissle 1917 for 24 hours. Upregulation of DC maturation factors (CD80/86 and MHC class II) were analysed by flowcytometry and induction of cytokine production by ProcartaPlex immunoassay.

Results: Yeast-derived β-glucans and E.coli Nissle 1917 are both able to mature porcine DC derived from fresh and cryopreserved bone marrow cells, in a dose-dependent, but differential, manner. They induce differential upregulation of cell surface markers (CD80/86 and MHC class II) and production of cytokines (e.g. TNF, IL-6, IL-10).

Conclusion: Yeast derived β-glucans and E.coli Nissle 1917 both demonstrate to possess exerting, but differential, immunomodulatory properties. To determine the potential of bone marrow derived DCs as a pre-screening tool for immunomodulatory (feed) components, the results from this in vitro study are currently being compared to data collected from a large in vivo study which aims to investigate the immune modulatory in vivo effects of β-glucan and E.coli Nissle 1917 added as a feed additive in the postnatal period.

P119Immunomodulation by catecholamines and catecholamine-treated Salmonella enterica cultures in pigs (sus scrofa)

Reiske L., Schmucker S., Toulouse C., Steuber J., Stefanski V.

University of Hohenheim, Stuttgart, Germany

In addition to being an important farm animal, pigs play an increasing role as model organism for human physiology. However, knowledge about the porcine immune system is still fragmentary, particularly its modulation by stress hormones. Catecholamine effects on the porcine immune system have not been

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investigated at all. In acute stress situations, catecholamines can reach high concentrations in various tissues including the gastrointestinal tract. Here they can affect immune cells but also pathogenic microorganisms like salmonellae, a common threat in pig farms. It has been shown previously that some bacteria can sense catecholamines and subsequently enhance their pathogenicity but it remains to be investigated whether they can also modulate the host immune system in a stress situation. The aim of this study was thus to investigate the effect of catecholamines and products from catecholamine-treated Salmonella enterica cultures on porcine lymphocyte function. Blood from catheterized barrows was used to isolate mononuclear leukocytes and investigate mitogen-induced lymphocyte proliferation and number of TNF-alpha producing cells among different lymphocyte subsets. If treated with adrenaline or noradrenaline, proliferation was enhanced whereas supernatants from catecholamine-treated Salmonella cultures had the opposite effect. TNF-alpha producers were reduced by both adrenaline and supernatants from adrenaline- or noradrenaline-treated salmonellae while noradrenaline had no effect. If supernatant of hormone-free Salmonella cultures was added, lymphocyte function was enhanced. The results show for the first time that salmonellae can cause an immunosuppression if catecholamine levels are enhanced, probably leading to a higher risk of infection. This is particularly interesting as we could show that both investigated catecholamines lead to a stimulation of lymphocyte proliferation in pigs, preparing them for a quick immune reaction in a fight-or-flight situation with enhanced risk of injury and infection. Future studies will investigate Salmonella-derived molecules probably responsible for the observed effects.

P120The activity of mitochondrial succinate dehydrogenase in the liver of recipient animals ande the influence the of mesenchymal stem cell

Mazurkevych A.1, Kladnytska L.2

1National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine, 21National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine

It has been shown that transplantation of allogenic mesenchymal stem cells modulates the immune response of the recipient through the production of paracrine factors and through direct interaction with cells. There is an assumption that one of the most important mechanisms for restoring the functions of damaged cells is the transfer of mitochondria from MSCs.

The studies were conducted on 2-3-months-old males of C57BL/6 mice. MSC were isolated and cultured using standard protocols The following groups of animals were formed: 1 - intact animals; 2 - animals, to whom 0.5 ml of 0.89% NaCl solution (placebo) were injected into the caudal vein; 3 - animals, to whom were injected 104 of allogenic AD MSCs, 4 - animals, to whom were injected 104 of allogenic BM MSCs. On 12th day after transplantation of MSCs activity of succinate dehydrogenase in animals were determined.

It was found that at the 12 day of the study, the activity of the succinate dehydrogenase in the third experimental group was 57.7 ± 1.6 mmol/l K3[Fe(CN)6]/ mg*min (p < 0.001), which is significantly higher than in the animals of the first and second group - 45.9 ± 0.7 and 43.3 ± 1.2 mmol/l K3[Fe(CN)6]/mg*min, respectively.

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The activity of the enzyme in animal of the fourth experimental groups was also reliable higher compared to the first two groups and was 53.3 ± 1.4 mmol/l K3[Fe (CN)6]/mg*min (p < 0.01). It should be noted that mitochondrial succinate dehydrogenase activity in the liver of recipient animals after transplantation of MSCs from adipose tissue is significantly higher than after transplantation of MSCs, obtained from bone marrow (p < 0.05).

Thus, reliable raising of mitochondrial succinate dehydrogenase activity in the liver of recipient animals after transplantation of allogenic mesenchymal stem cells from adipose tissue and bone marrow has been determined.

P121The influence of Interferon Lambda (IFN-λ) on STAT’ phosphorylation and ability canine mammary cancer cells to migration

Mucha J.1, Pingwara R.2

1Warsaw University of Life Sciences, Warsaw, Poland, 2Warsaw University of Life Sciences, Faculty of Veterinary Medicine, Department of Physiological Sciences, Warsaw, Poland

Changes in immune system during cancer development are confirmed fact. Researches focused on restoration of immune response. Immunotherapy becomes a buzzword of human oncology and many types of immunotherapies have been already successfully applied. Interferon Lambda is a interferon from III generation of interferon, and is showing antiviral activity. Recent study showed antitumor/ protumor activity. Literature data are

not clear. It’s seems that it can show opposite featured dependent on dose and tumor type. It is still poorly examined in veterinary medicine.

For our study we used human recombinant interferon-λ1 and -2, as its 80% of identity with canine interferon-λ to examine and comparison their influence on canine mammary cancer cell lines. Moreover, we have examined canine mammary tumors (n=20) for presence of IFN-λ and IFNλ1- receptor.

The expression of INL-λ and IFNLR1 were examined by real time PCR.

Cell respiration and proliferation were analyzed through MTT assay and CV, retrospectively.

Western blot method was used to study JAK/ STAT signaling pathway activation.

Also “scratch test” were used to investigate of cancer cell migration.

We have shown higher expression IFNLR1 both in canine mammary tumors and in 3 canine mammary tumor cell lines (P114,CMT U-27,CMT U-309)both there was low expression of the ligand. We have shown no effect of INF λ 1 and 2 on proliferation and respiration of these cells. However, IFN-λ was activated JAK/STAT signaling pathway, interestingly show double effect on ability of tumor cells to migration in dose-depending maner.

IFN-λ plays an underestimated and important role in canine mammary tumor development but the molecular mechanism of its action require for further investigation.

The research was founded by grant NCN no UMO-2016/23/D/NZ5/03276. Participation was funded by KNOW (Leading National Research

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Centre) Scientific Consortium “Healthy Animal - Sade Food”, decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015

P122Indoleamine 2,3-dioxygenase and the tryptophan-kynurenine-nicotinamide pathway in human Tuberculosis

Adu-Gyamfi C.G.1, Snyman T.2, Makhathini L.3, Chaisson R.E.4, Hoffmann C.4, Scriba T.J.5, Martinson N.6, George J.2, Suchard M.S.3

1National Institute for Communicable Diseases, Centre fro Vaccines and Immunology, Newlands, Pretoria, South Africa, 2University of the Witwatersrand, Johannesburg, Chemical Pathology, Johannesburg, South Africa, 3National Institute for Communicable Diseases, Centre for Vaccines and Immunology, Johannesburg, South Africa, 4John Hopkins Medical School, Department of Medicine, Baltimore, United States, 5University of Cape Town, South Africa Tuberculosis Vaccine Initiative, Department of Immunology, Cape Town, South Africa, 6University of the Witwatersrand, Johannesburg, Perinatal HIV Research Unit, Johannesburg, South Africa

Mycobacterium tuberculosis (M. TB) relies on its ability to counteract host immunity to cause Tuberculosis (TB). Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the conversion of tryptophan to kynurenine metabolites and nicotinamide. IDO activity mediates immune tolerance at the placenta and in alternatively activated macrophages.

We evaluated IDO activity in plasma from patients with active TB disease, compared with latently infected controls, both with and without HIV. We measured plasma tryptophan and kynurenine using enzyme-linked immunosorbent assay (ELISA). IDO activity was calculated as the product-to-substrate ratio.

In the HIV infected group, patients with active TB disease had significantly higher IDO activity than controls (median, 0.101[0.091-0.140] versus 0.061[0.034-0.077], P < 0.0001). At a cut-off 0.080 IDO activity gave a diagnostic sensitivity 89% [CI 64-98%], specificity 80% [CI 56-94%], PPV 81% and NPV of 84%. In a receiver operating characteristic (ROC) analysis, plasma IDO activity had t area under curve 0.89 (P = 0.0001). In the HIV uninfected group, patients with active TB disease had significantly elevated IDO activity than latent TB infection (median, 0.064[0.040-0.088] versus 0.022[0.016-0.027], P < 0.0001).

Our results suggest that plasma IDO activity is a suitable plasma biomarker of active TB disease, in HIV-infected and uninfected patients. IDO activity can be measured using low-cost ELISA methods. Further, results point to an integral role of the tryptophan-kynurenine-nicotinamide pathway in TB pathogenesis.

P123The use of a liposome with MANα1-2MAN-PEG-PE molecule enhances the immunogenicity induced by a nanovaccine against BoHV-1 in the murine model and in cattle

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Kornuta C.1,2, Bidart J.1,2, Soria I.1, Gammella M.1, Quattrocchi V.1, Moore D.P.2,3, Hecker Y.2,3, Cheuquepán F.3, Pappalardo J.S.4, Salmaso S.5, Torchilin V.6, Zamorano P.I.1,2,7, Langellotti C.A.1,2

1Instituto de Virología-CICVyA, INTA, Hurlingham, Buenos Aires, Argentina, 2CONICET, Research Council, Ciudad de Buenos Aires, Argentina, 3EEA Balcarce, INTA, Balcarce, Argentina, 4EEA Bariloche, INTA, Bariloche, Argentina, 5Universita degli Studi di Padova, Padova, Italy, 6Northeastern University, Boston, United States, 7Universidad del Salvador, Pilar, Argentina

Bovine herpesvirus-1 (BoHV1) is an etiological agent of the bovine respiratory disease complex. In this work, BALB / c mice and cattles were used; they were immunized with a DNA vaccine that carries the secreted version of the BoHV1 glycoprotein D using as a pCIneo vector (pCIgD). A liposome containing the Manα1-2Man-PEG-PE molecule was formulated together with LPS and pCIgD (pCIgD-Man-L) was also evaluated.

Groups of BALB/c mice (n = 5) were inoculated on days 0 and 20 with: pCIgD; pCIgD-MAN-L; pCIneo-MAN-L. At 20, 40 and 120dpv, the group pCIgD-MAN-L presented significant differences in the level of total antibodies against BoHV1 in contrast to the group that did not include liposome in the vaccine. When isotypes were evaluated, IgG1 levels were significantly increased (p < 0.001) in the animals that received the pCIgD-Man-L in contrast to the other groups. There was also an increase in isotypes IgG2a and IgG2b (p< 0.01)

Groups of cattle (n=5) were inoculated intradermally on days 0, 30 and 60 with pCIgD; pCIgD-MAN-L; pCIneo. At 90 dpv levels of antibodies in pCIgD and pCIgD-Man-L groups

were significantly higher than those in the pCIneo group.

At 90dpv the animals were challenged with 1x106.81 TCID50/ml of BoHV1. Nasal swabs were obtained over 12days for virus excretion evaluation. The titers in pCIgD-Man-L were significantly lower (p< 0.01) than titers in the others groups at 5dpc. When calculating the area under the curve of excretion during the 12dpc, it was lower in pCIgD-Man-L group (4,77x106TICD50/ml in pCIneo group, 1,12x106TICD50/ml in pCIgD group and 2,62x105TICD50/ml in pCIgD-Man-L group).

pCIgD-Man-L was capable of improving humoral immune response and diminishing viral excretion. It is the first time that a BoHV1 DNA vaccine is combined with Manα1-2Man-PEG-PE liposome. These results could be useful to design a vaccine for the control of bovine rhinotracheitis.

P124PD-1 / PD-L1 monoclonal antibody development for canine cancer therapy

Choi J.W.1, Withers S.2, De La Trinidad V.1, Fife B.3, Kent M.2, Moore P.4, Sciammas R.1, Rebhun R.2, McSorley S.1

1UC Davis, Center for Comparative Medicine, School of Veterinary Medicine, Davis, United States, 2UC Davis, Center for Companion Animal Health, School of Veterinary Medicine, Davis, United States, 3University of Minnesota, Department of Medicine, Medical School, Minneapolis, United States, 4UC Davis, Department of Pathology, Microbiology and

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Immunology, School of Veterinary Medicine, Davis, United States

Background: PD-1 is an inhibitory receptor that binds to PD-L1 on the surface of tumor and antigen presenting cells in the tumor microenvironment, causing suppression of T-cell activity and tumor progression. Human clinical trials with anti PD-1 monoclonal antibodies, nivolumab and pembrolizumab, showed an objective

response rate (ORR) of 30% to 40% with melanoma and 87% with relapsed or refractory Hodgkin’s lymphoma. Here, we have explored the development of canine PD-1/PD-L1 reagents for potential veterinary applications.

Method: Recombinant canine PD-1Ig and PD-L1Ig were prepared from S2 cells and antigen-specific B cells were enriched using PD-1 and PD-L1 tetramers. Enriched B cells were fused and resulting hybridomas were screened for specific reactivity to PD-1Ig and PD-L1Ig in ELISA. Positive hybridomas were further screened on PD-1 and PD-L1 expressed on CHO and DH82 cells as well as canine PBMC. Then, Ifn-γ from PBMC culture was measured by ELISA to evaluate the effectiveness of each antibody in reverting T-cell suppression. The highest Ifn-γ producing antibody was sequenced and caninized for therapeutic application.

Result: One PD-1 and four PD-L1 monoclonal antibodies were selected after multiple steps of ELISAs and flow cytometry. At least one of them was capable of staining a few frozen tissues. All four anti-PDL1s were able to produce high titer Ifn-γ in a ConA activated canine PBMC culture. One of anti-PDL1s was sequenced and caninized to generate a chimeric IgGD for therapeutic application.

Conclusion: All 5 monoclonal antibodies can be used for flow cytometry and ELISA. At least one of anti-PD-L1s can be used for frozen tissue staining. And all four anti-PDL1s have therapeutic potential to revive suppressed T-cells in the tumor environment.

P125Immunogenicity of two forms of cost-effective purified non-living anthrax vaccine candidate compared to Sterne live spore vaccine with concurrent penicillin G treatment in bovine

Jauro S.1,2, Ndumnego O.C.3, Ellis C.4, Buys A.4, Beyer W.5, van Heerden H.1

1University of Pretoria, Department of Veterinary Tropical Diseases, Pretoria, South Africa, 2University of Maiduguri, Department of Veterinary Microbiology, Maiduguri, Nigeria, 3Africa Health Research Institute, Durban, South Africa, 4Design Biologixcc, Pretoria, South Africa, 5University of Hohenheim, Department of Livestock Infectiology and Environmental Hygiene, Stuttgart, Germany

Sterne live spore vaccine (SLSV) is effective and the OIE recommended anthrax vaccine in veterinary practice in most countries. However, it´s incompatible with antibiotic treatment during anthrax outbreaks. Therefore, this study focused on evaluating the immunogenicity of non-living vaccine candidates consisting of recombinant protective antigen (rPA) purification and formalin-inactivated spores (FIS). The non-living vaccine was formulated using purified rPA (PrPA) or crude rPA (CrPA) with FIS and emulsigen-D/alhydrogen adjuvant.

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Bovine were vaccinated along with Penicillin-G treatment. The vaccinated animals’ groups consisted of PrPA+FIS, CrPA+FIS with adjuvant and SLSV with penicillin-G treatment as well as SLSV without penicillin-G and were vaccinated twice (week 0 and week 3). ELISA (IgM, IgG1, IgG2, and IgG), Toxin neutralization assay (TNA) and Opsonophagocytic assay were employed for the humoral immunogenicity analysis using blood samples collected before each vaccination and at week 5. The ELISA IgG titre for PrPA+FIS and CrPA+FIS with penicillin-G had shown a significant increase (P< 0.0413 and P< 0.0341), whereas the SLSV with penicillin-G group titres were low with poor immune response compared to SLSV without penicillin-G treatment group (P< 0.0001). Anti-FIS IgG titre followed same pattern of titre increase as seen with PrPA+FIS and CrPA+FIS with penicillin-G (P< 0.0173 and 0.0010) whereas SLSV with penicillin-G vaccinate group showed significant antibody development without significant difference (P>0.3648). The toxin neutralization was high in PrPA+FIS and CrPA+FIS with penicillin-G. The immunoglobulin isotypes(IgM, IgG1, IgG2) response demonstrated good trend with different time points, IgM and IgG1 showing sudden elevation 2 week following vaccination revealing the possibility of PrPA+FIS and CrPA+FIS stimulating Innate immune response in vaccinated heifers. The antibodies generated against PrPA+FIS and CrPA+FIS raised macrophages spore phagocytosis significantly. Therefore, the PrPA and more cost-effective CrPA non-living vaccines with penicillin-G treatment were able to stimulate high immune titre and will be tested for protection using passive protection mouse model.

P126Animal vaccines for sustainable food security: creating new vaccines against porcine circovirus

Jaru-Ampornpan P.1, Peswani A.2, Mikaliunaite L.3, Liwnaree B.1, Muensaen K.1, Narkpuk J.1, Jongkaewwattana A.1, Robinson C.2

1National Center for Genetic Engineering and Biotechnology, Pathum Thani, Thailand, 2University of Kent, Canterbury, United Kingdom, 3University College London, London, United Kingdom

Pork consumption in South East Asia has doubled over the past 20 years. However, many pig farms in the region are vulnerable to porcine circovirus type 2 (PCV2). This virus devastates pig farms by reducing farrowing rates to below 60% and can cause up to 30% fatality in piglets, creating strains on pork supply and prices. Currently, Thai farmers rely on imported PCV2 commercial vaccines which come with high cost, valued at 14M GBP in 2017. The imported vaccine risks reduced effectiveness due to mismatches with the local strains. Additionally, recently-discovered PCV type 3 (PCV3) has been detected in Thai farms as well as elsewhere worldwide. Despite unclear causal links, PCV3 has been associated with many diseases and currently has no commercial vaccines available. Our strategy is to work collaboratively between the UK and Thailand and create a new PCV vaccine that is produced regionally and therefore is more widely affordable, is better matched to local PCV2 strains, and protects against PCV3.

AUTHOR INDEX

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Barral-Netto M. ............................................O45 Barrionuevo F.M. .........................................O71 Barut G.T. ......................................... O02, P041 Bassetto C.C. ......................... O34, P012, P062 Bastos R.G. .....................................P027, p068 Beard S. ....................................................P021 Bedolla Alva M.A. ......................................P045 Beeton-Kempen N. ................................... P018 Begg D. .....................................................P097 Bell C. .......................................................P038 Bell N. .........................................................O33 Ben Arous J...................................... O08, P095 Benítez A. .................................................P058 Berghman L. ......................................O20, O43 Bernitz N. .................................................. P015 Bertho N. ......................................... O63, P057 Bertrand F. ..................................................O08 Beshah E. ...................................................O33 Beyer W..................................................... P125 Bhattarai S. .....................................P002, P006 Bickhart D.M. ..............................................O16 Bidart J...................................................... P123 Bielke L. .............................................O20, O43 Billon Y. .......................................................O38 Bishop E.A. .................................................O35 Biswas P.K. ...............................................P093 Blanc F. .......................................................O38 Blanton Jr. J. ...............................................O64 Blikslager A. ................................................O52 Blohm U. ....................... O18, O22, P005, P056 Blome S. .....................................................O18 Boichard D. ...................................... O42, P043 Boniotti M.B. ..................................... O06, P064 Borca M. .....................................................O35 Bordet E. .....................................................O63 Borkowski E. .............................................P075 Borst L. .......................................................O52 Boubaker Landolsi R. ...............................P059 Bouchez O. .................................................O38 Bouguyon E. ......................................O38, O63 Bourge M. ...................................................O63 Bridgeman B.J. ......................................... P105 Briggs W. .....................................................O20 Britton C. .....................................................O14 Broere F. ...................................................P079

A A. Veenstra K. ...........................................P095 Abdellrazeq G.S. ....................O68, P044, P081 Abdelsalam K. .............................................O03 Abdi R. ........................................................O67 Abiayi E.A. ................................................P082 Aboge G. ................................................... P018 Adu-Gyamfi C.G. ....................................... P122 Agada G.O. ...............................................P082 Ahn H.-S. ........................................P073, P080 Ahsan C.R. ...............................................P093 Aitchison K. .................................................O07 Akinbobola J.S. .........................................P037 Akinbobola R.I.A. ......................................P037 Alcaraz O. .................................................P058 Almeida L.G.N. ......................................... P101 Almond G.W. ...............................................O59 Altvater-Hughes T. ...........................P019, P028 Alvarez I. ................................................... P014 Amadori M. .......O06, P053, P064, P069, P071 Amaral A.F...................................................O59 Amarante A.F.T. .........................................P062 Ando A. .......................................................O15 Antony L. ...................................................P006 Aresu L. ......................................................O56 Arkesteijn G.J.A. .......................................P007 Arvizu-Flores A. ........................................ P106 Auray G. ......................................................O04

B Baes C. .....................................................P067 Bailey D. ......................................................O39 Bailey M. ...................................................P029 Bailey S. .....................................................P113 Baldwin C.L. .....................O23, O24, O27, P103 Ballingall K.T. ...........................O14, O66, P001 Barbey S. ......................................... O42, P043 Barbieri I. ....................................................O06 Barjesteh N. ....................................P061, P086 Barman N.N. ...............................................O39 Barnier-Quer C. .........................................P088 Barone L.J........................................ O71, P078 Barral A. ......................................................O45

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Connelley T.K. .................................. O32, P103 Cooper D. .................................................. P015 Corbiere F. ..................................................O42 Cortes L.M. ........................................O52, O59 Cortéz O. ..................................................P058 Cox E. .......................................O29, O44, O47 Cox V. ........................................................P072 Crisci E. .................................. O59, O63, P077 Crooke H. ...................................................P110 Crossley S. ......................................P035, P042 Cruz M.S. ....................................................O45 Cui J. .........................................................P008 Cunha C.W. ...............................................P083 Cunha P. ...................................................P023

D Daher I.P. ..........................................O30, P101 Dakik D...................................................... P010 D‘Annunzio G. .............................................O06 Darbellay J. .................................................O36 Dashe Y.M. ................................................P082 Dassanayake R.P. ............................ O68, P024 , P109 Davidson I. ..................................................O21 Davis W.C. ........O68, P033, P044, P069, P081 Dawson H.D. .................................... O33, P040 Dayed R. ....................................................P011 De Almeida L.G.N. ......................................O30 De Boyer des Roches A. .............................O60 De Carlo E. ...............................................P069 De Groot A.S. ............................................ P100 De La Trinidad V. ....................................... P124 De Matteis G. ............................................P069 De Mille C.M. ..............................................O25 De Moraes A. ..............................................O30 De Oliveira J.E. ...........................................O26 De Silva K. .................. O62, P022, P039, P097 De Tolla L.J. ................................................O06 Dekkers J. .................................................P065 Dellon E.S. ..................................................O52 Deptuła W. ......................................P052, P070 Devriendt B. ..............................O29, O44, O47 Dhakal S. ....................................................O19 Dinkel K.D. ................................................P027

Bruggmann R.....................................O02, O04 Brunato M.L. .....................................O30, P101 Bryan K.A...................................................P117 Bueren E. ..................................................P020 Bui-Marinos M...........................................P020 Burgara-Estrella A. .................................... P106 Busfield S. ..................................................P113 Bustamante-Córdova L. ............................ P106 Buys A. ...................................................... P125 Byrne K.A. ................................O09, O25, O61

C Caffi V. .........................................................O50 Canada P. .................................................P065 Canovas A. ............................P054, P067, P075 Capozzo A. ............................ P014, O71, P078 Capucci L. .................................................P064 Cardoso N.P. .................................... O71, P078 Carrisoza J. .....................................P045, P058 Cartwright S. .............................................P026 Castells D. ...................................................O57 Cebron N. .................................O42, O60, O70 Chaabouni A. ............................................P059 Chae J.B. ....................................................O49 Chagas A.C.S. ................................P055, P062 Chaikhumwang P. ............................ O37, P098 Chaisson R.E. ........................................... P122 Chang L.-Y. .................................................O39 Chappell K. .................................................O39 Charkhkar S. .............................................P061 Chase C. .....................................................O03 Chasser K. ..................................................O20 Chaulot-Talmon A. ......................................O60 Chen C. .......................................................O33 Chen W. .................................................... P107 Cheuquepán F. ......................................... P123 Chitko-McKown C. .......................... P072, P117 Choi I.-S. .........................................P073, P080 Choi J.W. ................................................... P124 Choiniere W. .............................................P061 Chou W.-K. ..................................................O43 Cillán-Garcia E. .........................................P003 Clarke C. ................................................... P015 Coad M. ......................................................O58

169 IVIS2019.ORG

AUTHOR INDEX

Ferreira B.R. .....................................O30, P101 Ferrero S. .................................................. P014 Fife B. ........................................................ P124 Figueiredo A. .............................................P062 Filipe J. ............................................. O56, P071 Fisch A. .............................................O30, P101 Fleming D. .................................................P051 Flies A.S. .....................................................O65 Fonseca P. ................................................P054 Forlenza M.B. ...........................................P078 Fortin F. .....................................................P065 Fossum C. .................................................P047 Foucras G. ........... O42, O60, O70, P023, P043 Francis D.H. ..............................................P006 Francis O...................................................P029 Freire T. .......................................................O57 Friedrichs V. ................................................O51 Fry L.M. ..............O68, P027, P044, P068, P081 Fujisawa S. ................................................ P114

G Gabler N.K. .................................................O25 Gajardo G. ..................................................O50 Gallo N. ..................................................... P019 Gammella M. ............................................ P123 García-Nicolás O. ............................ O13, P048 Gashururu R.S. ............................... P013, P016 Gauger P.C................................................ P100 Geervliet M. .............................................. P118 Gelin V. ........................................................O60 George J. .................................................. P122 Gerber M. ....................................................O04 Gerdts V. .....................................................O36 Germon P. ..............................O42, P023, P043 Gerner W.......................................... O23, P099 Gervasi M.G. ............................................. P103 Gharbi M. ..................................................P059 Ghimire S. ........................................ O19, P006 Giannuzzi D. ...............................................O56 Giapessoni G. .............................................O57 Giglioti R. ..........................................O34, P012 Gilbert S. .....................................................O39 Gillespie A. .......................O23, O24, O27, P103 Gillespie B. ..................................................O67

Dixon S. ....................................................P075 Donofrio G. ...............................................P083 Dorhoi A. ............................................O22, O51 Dos Santos I.B. ...............................P055, P062 Douanne N. ...............................................P086 Doyle M. ......................................................O58 Drickamer K. ...............................................O01 Duff A. .........................................................O20 Duthie M. ....................................................O45 Dyck M. .....................................................P065

E Eaton S. ....................................................P003 Edwards J. ................................................ P010 Edwards L. ..................................................O52 Eicher S.D. .................................................P117 El Jakee J. ...................................... P011, P087 Eléouët J.-F. ..............................................P057 Ellis C. ....................................................... P125 Elnaggar M.M. ............ O68, P033, P044, P081 Elsady S. ......................................... P011, P087 Emam M. ............................. P054, P061, P086 Emmenegger E. ........................................P020 Ensermu D. .................................................O67 Entrican G. ..................................................O07 Epperson W.B. ............................................O64 Erbs G. ...................................................... P102 Escribano C. ...............................................O57 Espejo C. ....................................................O54 Espinosa G. ................................................O50 Esquivel H. ................................................P058 Estellé J. .....................................................O38 Esteves S.N. ...................................P055, P062 Everett H. ...................................................P110

F Falkenberg S.M. ..............................P024, P109 Fawver Z. ..................................................P072 Feliciano-Ruiz N..........................................O19 Feliziani F. .................................................P069 Ferlazzo G. .....................................P064, P071 Fernandez Prada C. ..................................P086 Ferraz-Júnior R.S. ...........................P055, P062

170IVIS2019.ORG

AUTHOR INDEX

Hernández J. ..................................P009, P106 Hernández Pando R. ................................P045 Herndon D.R. ............................................P027 Herrera V. ..................................................P071 Herrera Uribe J. ..........................................O61 Hervet C. ...................................................P057 Higgitt R. ................................................... P015 Hill D.E. .......................................................O33 Hjertner B. ................................................P047 Ho S. ...........................................................O15 Hodgins D.C. .........................P019, P021, P028 Hoffmann C. .............................................. P122 Hoffmann J.-P. ...........................................P056 Holder A. .....................................................O01 Hong L. ..................................................... P076 Hope J.C. O48, P031, P035, P042, P060, P103 Hühr J. ............................................. O18, P005 Hukowska-Szematowicz B. .......................P052 Hulubei V. .................... O68, P033, P044, P081 Hume D.A. ................................................P031 , P042, P060 Husmann R. .............................................. P107 Husseini N. .....................................P021, P028

I Ibrahim H. .................................................P092 Idachaba S.E. ...........................................P082 Im Y.B. .......................................................P091 Ingabire A. ................................................. P018 Islas-Trejo A.D. ..........................................P054 Iussich S. ....................................................O56

J J. Secombes C. .........................................P095 Jammes H. ..................................................O60 Jang H. ........................................... P104, P112 Jang J. ............................................ P104, P112 Jansen C.A. ............................ O26, P007, P118 Jansens R.J.J. ............................................O29 Jaru-Ampornpan P. ................................... P126 Jauro S. ..................................................... P125 Jegouzo S.A.F. ............................................O01 Jennings J. ................................................P049

Giraldelo L.A. ..................................P055, P062 Gitton C. ....................................................P023 Giuffra E. .....................................................O63 Gladue D. ....................................................O35 Glatthaar-Saalmüller B. .............................P099 Go H.-J. ..........................................P073, P080 Goosen W.J. ............................................. P015 Gormley E. ..................................................O58 Goto S. .....................................O10, O12, P052 Gourapura R. ............................................P029 Graham S. ........................................O39, P110 Grandoni F. .....................................P044, P069 Grant C. ....................................................P038 Grassi C. ...................................................P069 Gregory B. ................................................P031 Griebel P.J. ..................................................O28 Guarneri F. ..................................................O06 Guerra-Maupome M. ........................O11, P004 Gunn-Moore D. ...........................................O48 Gupta S.K. ................................................ P105 Gutiérrez A.H. ........................................... P100 Gutiérrez-Pabello J.Á. .....................P045, P058 Gutsche I. .................................................P057

H Haibo L. ......................................................O61 Haiden E. ..................................................P036 Hammer S.E. ...........................O15, O23, P036 Hammond J. .........O01, O16, O41, P035, P042 Han S.-H. ..................................................P080 Han Y. ..........................................................O19 Hanks E. .....................................................O14 Hansen J. ..................................................P034 Harding J. ........................................P065, P076 Hargis B.M. ........................................O20, O43 Harsla T.R. ................................................P033 Hartmann L. ................................................O18 Hash K. .......................................................O67 Headrick S. .................................................O67 Hecker Y. ................................................... P123 Helguera G. .............................................. P014 Hellman S. ................................................P047 Henningson J. ...........................................P004 Henriquez C. ...............................................O50

171 IVIS2019.ORG

AUTHOR INDEX

Konnai S. ....O10, O12, O46, P052, P111, P114, P116 Kornuta C. ................................................. P123 Koziol M.-E....................................... O08, P095 Kringel H. ....................................................O33 Kritchevsky J.E. .........................................P117 Kvisgaard L.K............................................ P102

L LaBresh J. ............................ P029, P032, P033 Ladinig A. ..................................................P099 Lakshmanappa S.Y. ....................................O19 Lambe T. .....................................................O39 Lamot D.M. .................................................O26 Langellotti C.A. .......................................... P123 Larsen L.E. ............................................... P102 Laster S.M...................................................O52 Latif A. ............................................. P011, P087 Lavoie J.-P. ................................................P086 Lawson S. .................................................P006 Le Page L. .........................................O23, O27 Lebea P. .................................................... P018 Le-Bert C.R. ..............................................P033 Leblanc-Maridor M. ...................................P057 Ledoux D. ....................................................O60 Lee B.H. ......................................................O49 Lee D.U. ......................................................O49 Lee E. ............................................. P104, P112 Lee H.E. ......................................................O49 Lee I.H. .......................................................O49 Lee J.-B...........................................P073, P080 Lee J.-H. .....................................................O15 Lee S.-W. ........................................P073, P080 Lefebvre R. ...................................... O42, P043 Lefevre L. ........................................P042, P060 Lelli D. .........................................................O06 Lemonnier G. ..............................................O38 Lepenies B. .................................................O32 Leplat J.-J. ..................................................O38 Lesueur J. ...................................................O42 Lewandowska M. ......................................P048 Li F. ...........................................................P006 Li H. ..........................................................P083 Li R. ............................................................O33

Jensen E.D. ..............................................P033 Jeremiah O.T. ............................................P037 Jermsutjarit P. .............................................O40 Jimbo S. ......................................................O28 Jones G.J. ........................................ O58, P066 Jongkaewwattana A. ................................. P126 Jorritsma R. ..............................................P084 Jourdren L. ..................................................O63 Jungersen G. ................................... O69, P102

K Karagianni A.E. .........................................P003 Karim M.R. ................................................P093 Karisch B. ...................................................O64 Karrow N.A. ..................................... P067, P075 Karte C. .....................................................P005 Käser T. ..............................................O52, O59 Katakura F. ..................................................O17 Kato Y. .........................................................O12 Katzenback B. ...........................................P020 Kaushik R.S. .................................. P002, P006 Keller I. ...............................................O02, O04 Kelly S.M. ..................................................P004 Kennedy D. ...............................................P075 Kenney S. .................................................P029 Kent M. ..................................................... P124 Kerr T.J. ..................................................... P015 Kerro Dego O. .............................................O67 Kers J.G. .....................................................O26 Khouri R. .....................................................O45 Kick A.R. .....................................................O59 Kiefer H. ......................................................O60 Kim D.-H. ........................................P073, P080 Kim S. .....P063, P091, P104, P104, P112, P112 Kishore U. ..................................................P115 Kladnytska L. ............................................ P120 Knittler M. ....................................................O18 Knowles D.P. ....................................P027, p068 Ko H. ......................................................... P076 Koets A. ....................................................P084 Kohara J. .....................................................O12 Koinig H.C. ................................................P099 Kolakowski J. ..............................................O32 Köllner B. ............O51, O55, P005, P056, P108

172IVIS2019.ORG

AUTHOR INDEX

McKechnie M. ...........................................P028 McKenchnie M. .........................................P026 McLean R. ..................................................O39 McNeilly T. ...................................................O14 McSorley S. .............................................. P124 Medrano J.F. .............................................P054 Meebumroong S. ......................................P090 Mehmood A. ...............................................P115 Mehrabani-Yeganeh H. .............................P061 Meijerink N. .................................................O26 Menzies P. .................................................P075 Merrill C. .....................................................O67 Mettenleiter T.C. ............................... O22, P005 Meurens F. ................................................P057 Mezzetti M. ................................................P053 Mhadhbi M. ...............................................P059 Mikaliunaite L. ........................................... P126 Mikkelsen H.M. ...........................................O69 Miller L. .....................................................P051 Miller M.A. ................................................. P015 Min K.C. ......................................................O49 Minuti A. ....................................................P053 Miranda D. ..................................................O45 Miyamae J. ..................................................O17 Mizzi R. .....................................................P039 Mohammad A. .............................................O63 Moise L. .................................................... P100 Montero R. ..................................................O55 Moore A. ...................................................P088 Moore D.P.................................................. P123 Moore P. .................................................... P124 Moraes A. .................................................. P101 Morales Salinas E. ....................................P045 Morein B....................................................P047 Moritomo T. .................................................O17 Moroldo M. ..................................................O63 Moura L. ......................................................O45 Mourino M. ..................................................O39 Mucha J. ................................................... P121 Muensaen K. ............................................. P126 Müller C. ................................................... P108 Müller M. ................................................... P108 Munch D. ................................................... P102 Munkler L. ...................................................O55

Li Z. ...........................................................P067 Liebenberg J. ..............................................O31 Lila M.A. ......................................................O52 Lippolis J.D................................................ P109 Livernois A.M. .................................P026, P028 Livingstone M. .............................................O07 Liwnaree B. ............................................... P126 Lokhman E. .....................................P035, P042 Longbottom D. .............................................O07 Loving C.L. ................................O09, O25, O61 Lovotti G. ...................................................P053 Lunney J.K. ..................O15, P029, P040, P076 Lyons A.B. ...................................................O65 Lyons B. ......................................................O54 Lyoo E.-L.........................................P073, P080

M Maccari G. ....................................... O41, P035 Madapong A...................O37, O40, P090, P098 Madsen-Bouterse S.A. ..............................P027 Maekawa N. ......... O10, O12, O46, P052, P111, P114, P116 Magiri R. .....................................................O05 Mahmoud A.H. ................................. O68, P081 Maina E. ......................................................O47 Mair K.H. .....................................................O23 Mair K.M. ..................................................P036 Makhathini L. ............................................ P122 Malgarin C. ............................................... P076 Mallard B.A. ....P019, P021, P026, P028, P054, P065, P067, P075 Maman S. ...................................................O70 Manirarora J. .............................................P029 Mansilla F. .........................................O71, P014 Marconato L. ...............................................O56 Marcondes C.R. ..........................................O34 Marini I. .......................................................O56 Marsh G. .....................................................O39 Martinson N. ............................................. P122 Martucciello A. ..........................................P069 Mazurkevych A. ......................................... P120 McBean D. ................................................ P001 McCarthy R. ...............................................P110 McGill J.L. ........................................ O11, P004

173 IVIS2019.ORG

AUTHOR INDEX

Oliveira H.N. .....................................O34, P012 Oliveira M.C. .....................................O34, P012 Olson Z. ......................................................O61 Opriessnig T. ............................................. P100 Orangi H. ..................................................P089 Ostermann S. ..............................................O55 Owens R.J. ...............................................P035

P Palarea-Albaladejo J. ..................................O07 Palmer M.V. .................................................O09 Pappalardo J.S. ......................................... P123 Park B.-J. ........................................P073, P080 Park H.-E. .......................................P063, P091 Park H.-T. ........................................P063, P091 Park K.-T. ..................................................P081 Park S.-Y. ........................................P073, P080 Park W.B. ..................................................P063 Park Y. ............................................. P104, P112 Parlane N.A. ............................................. P105 Parra-Sánchez H. ...........................P009, P106 Parsons S.D.C. .......................................... P015 Pasternak A. ............................................. P076 Patchett A.L. ................................................O65 Patzl M. .....................................................P036 Pedersen F.S. ............................................ P102 Pedrera M. ..................................................O39 Peña M.S. .........................................O30, P101 Peregrine A. ..............................................P075 Pereyra R. ...................................................O71 Perkins A. ....................................................O64 Peswani A. ................................................ P126 Petrini S. ...................................................P069 Piccinini R. ................................................P071 Piccioli-Cappelli F. .....................................P053 Pinard-Van Der Laan M.-H. ........................O38 Pingwara R. .............................................. P121 Pinho F. .......................................................O45 Pirie S.R. ...................................................P003 Plain K.M.. .................. O62, P022, P039, P097 Plastow G. ................................................P065 Plaza A. .......................................................O50 Plundrich N.J. .............................................O52 Pontelevoy C. ..............................................O60

Murata S. ....O10, O12, O46, P052, P111, P114, P116 Mutwiri G. ..........................................O05, O28 Mwangi W. .............................O41, P035, P042

N Nakajima C. ................................................O12 Nanduri B. ...................................................O64 Narasimhan B. ..........................................P004 Narkpuk J. ................................................. P126 Nascimento L. .............................................O45 Naylor D. ...................................................P067 Ndazigaruye G. ......................................... P018 Ndumnego O.C. ........................................ P125 Nedumpan T. .............................................P008 Nefefe T. ......................................................O31 Neill J. .......................................................P024 Nettleship J. ..............................................P035 Ngugi D. ......................................................O32 Niedzwiedzka-Rystwej P. ................P052, P070 Nielen M. ...................................................P084 Nikbakht G. ...............................................P061 Nilubol D. .......................O37, O40, P090, P098 Nishimori A. ................................................O12 Nishiya K. ....................................................O17 Normand V. ...............................................P057 Noronha L. ................................................P072 Novak A. ...................................................P079

O Obiero G. .................................................. P018 Obradovic M. ..............................................O53 Odle J. ........................................................O52 O‘Donnell V. ................................................O35 Odugbo M. ................................................P082 Oguntade E.S. ..........................................P037 O‘Halloran C. ..............................................O48 Ohashi K. ....O10, O12, O46, P052, P111, P114, P116 Okagawa T. .O10, O12, O46, P052, P111, P114, P116 Okano M. ....................................................O17 Okino C.H. ...................O34, P012, P055, P062

174IVIS2019.ORG

AUTHOR INDEX

Rodriguez D.R. .........................................P038 Rogel-Gaillard C. ...............................O15, O38 Roos E.O. ................................................. P015 Roth J........................................................P024 Rufener R..................................................P041 Ruggeri J. .................................................P064 Ruggli N. .....................................................O04 Rume F.I. ...................................................P093 Rutten V.P.M.G. .............O26, P007, P079, P118 Ryoo G. ........................................... P104, P112

S Saalmüller A. ..........................O23, P036, P099 Sacco R.E. ...................O68, P004, P033, P109 Saeng-Chuto K. .............O37, O40, P090, P098 Sajiki Y. ...........................O10, O12, P052, P116 Salguero J. ................................................P058 Salinas I. ...................................................P034 Salmaso S. ............................................... P123 Sang Y. ............................................P029, P049 Santos I.K.F.M. ..................................O30, P101 Saravia A. ...................................................O57 Sarli G. ........................................................O06 Sassi A. .....................................................P059 Sauter K. ...................................................P060 Savelkoul H.F.J. .........................................P118 Sayed R. .........................................P025, P094 Scanziani E. ................................................O56 Scaria J. ....................................................P006 Schäfer A. ..............................O22, P005, P056 Schammas J.M. ........................................ P014 Schinköthe J. ............................................ P108 Schirtzinger E. ..........................................P072 Schmied J. ........................... P021, P026, P028 Schmied J.D. .............................................P065 Schmucker S. .............................................P119 Schneider S. ...............................................O11 Schook L.B. ................................................O15 Schröder C. ............................O22, P005, P056 Schwaiger T. ..........................O22, P005, P056 Schwartz I. ..................................................O63 Schwartz J.C. ..............................................O16 Sciammas R. ............................................ P124 Scott M. .......................................................O64

Pooley H.B. ...............................................P097 Porter T.J. .................................................. P109 Premadasa L. ............................................P077 Pretorius A. .................................................O31 Pridgen T. ....................................................O52 Puebla-Clark L. .........................................P009 Pupin M.A.F. .............................................. P101 Purdie A.C. .................. O62, P022, P039, P097 Putz A. ......................................................P065 Pye R.J. ......................................................O65 Python S. ......................................... O04, P048

Q Quattrocchi V. ............................................ P123 Quintana M.E. .................................. O71, P078

R Rainard P................................O70, P023, P043 Ramanathan P. ...........................................O35 Rampacci E. ...............................................O07 Raper A. ............................... P031, P035, P042 Raue R. .......................................................O39 Read L. ..................................................... P019 Rebel J.M. .................................................P088 Rebhun R. ................................................. P124 Reed S. .......................................................O45 Reinhardt T.A. ........................................... P109 Reiske L. ................................................... P119 Renois F. ...................................................P057 Renu S. .......................................................O19 Renukaradhya G. ........................................O19 Reséndiz-Sandoval M. ....................P009, P106 Richard C. ...................................................O60 Richard C.-A. ............................................P057 Ridpath J. ..................................................P024 Riet-Correa F. ..............................................O57 Rimfa A.G. ................................................P082 Riva F. .............................................. O56, P071 Robbers L. ................................................P084 Robcis R. ....................................................O60 Robert J. ...................................................P020 Robinson C. .............................................. P126 Rocchi M. ....................................................O07

175 IVIS2019.ORG

AUTHOR INDEX

T Tabatabaei S.S. .........................................P028 Talker S.C. ....................................... O02, P041 Tan S. ........................................................ P100 Tantituvanont A. ................................O37, P098 Tassi R. ...............................................O07, O66 Taylor M.E. ..................................................O01 Tchilian E. ...................................................O39 Techakriengkrai N. ....................................P008 Telfer J.C. .........................O23, O24, O27, P103 Temeeyasen G. ...........................................O40 Thakur A. ....................................................O69 Thakur N. ....................................................O39 Thomas M. ................................................P006 Thomsen J. ............................................... P102 Thomson J. .................................................O07 Tijhaar E. ...................................................P118 Todd H. ............................................ O66, P001 Tokarz-Deptuła B. ...........................P052, P070 Toneli M.F. ...................................................O34 Torchilin V. ................................................. P123 Toscano J.H.B. ................................P055, P062 Toulouse C. ................................................P119 Tresoldi E.T. ................................................O06 Trevisi E. ...................................................P053 Tsolaki A.G. .............................................. P115 Tudor C. ......................................................O06 Tuggle C.K. ........................................O09, O61 Turco C. .............................................O71, P014 Tydén E. ....................................................P047

U Udahemuka J.C. ....................................... P018 Ullrich R. ................................................... P108 Ulrich R. ..........................................P005, P108 Uprety T. ..........................................P002, P006 Urban Jr. J.F. ...............................................O33 Urien C. .......................................................O63

V Valenzuela O. ............................................P009 Van de Mheen R. ......................................P084 Van Den Biggelaar R.H.G.A. ....................P007

Scriba T.J. ................................................. P122 Searer K. .....................................................O20 Segura M. .................................................P086 Sehl J. .......................................................P005 Selvaraj R. ..................................................O19 Sharma A. .................................................P067 Shea-Donohue T. ........................................O33 Shiels B. ......................................................O32 Shiina T. ......................................................O17 Shim S. .....................................................P091 Shringi S. ..................................................P083 Sillence M. .................................................P113 Silva P.C. ...........................................O34, P012 Simas P.V. ...................................................O34 Simon G. ...................................................P057 Sloots A. ....................................................P079 Smith A.R. ...................................................O52 Smith D. .............................................O14, O64 Smith T.P.L. .................................................O16 Smith T.P. ....................................................O24 Smolensky D. ............................................P072 Snyman T. ................................................. P122 Sobraske J. .................................................O03 Song C.-S. ......................................P073, P080 Soria I. ...................................................... P123 Sreenivasan C. .........................................P006 Stadler M. .................................................P099 Stefanski V. ................................................P119 Stegeman A.J..............................................O26 Steinbach F. .....................................P010, P110 Steinbach S. .................................... O58, P066 Steuber J....................................................P119 Steyn H. ......................................................O31 Stockhofe-Zurwieden N. ...........................P088 Strietzel C.J. ...............................................P113 Strom S. ......................................................O36 Stronach K. .................................................O07 Suarez-Vega A. .........................................P075 Suchard M.S. ............................................ P122 Sullivan Y.B. ....................................P032, P033 Summerfield A. .... O02, O04, O13, P041, P048 Sunyer O.J. ...............................................P034 Suradhat S. ...............................................P008 Suzuki Y. .....................................................O12 Swiderski C. ................................................O64

176IVIS2019.ORG

AUTHOR INDEX

Withers S. ................................................. P124 Woods G. ....................................................O54 Woods G.M. ................................................O65 Wooldridge L. ............................................P029 Woolums A. .................................................O64 Wright K.E. ......................................P022, P039 Wu Z. ........................................................P042

X Xu B. ................................................ O08, P095

Y Yamada S....................................................O12 Yasmin M. .................................................P093 Yeh H.-Y. ................................................... P074 Yirsaw A.W.. .....................O23, O24, O27, P103 Yoo H.S. ..........................................P063, P091 Yoon I.J. ......................................................O49 Young A. ....................................................P002 Young P. ......................................................O39

Z Zamorano P.I. ............................................ P123 Zhu J.J. .......................................................O35 Zuckermann F. .......................................... P107

Van Der Weken H. ......................................O44 Van Eden W. ................................... P007, P079 Van Haarlem D.A. .......................................O26 Van Heerden H. ........................................ P125 Van Helden P.D. ........................................ P015 Van Kessel J. ..............................................O36 Van Kleef M. ...............................................O31 Vathsangam N. ......................................... P113 Vaughn J. ....................................................O67 Velkers F.C. .................................................O26 Venner L. .................................................. P076 Vermeire B. .................................................O29 Versillé N. ......................................... O08, P095 Vigueras G. ...............................................P058 Villamar M. ................................................ P107 Villarreal-Ramos B. ...................................P058 Villasol N.A. ................................................O07 Von Haehling M.B. ..........................P055, P062 Vordermeier H.M. ...................O58, P058, P066 Vreman S. .................................................P088 Vuong C.N..........................................O20, O43

W Waddell L.A.......................... P035, P042, P060 Wagter-Lesperance L. ............................... P019 Walachowski S. ..................................O42, O60 Walker K. .................................................. P076 Walker T. .....................................................O67 Walz P. ......................................................P024 Warren R.M. .............................................. P015 Watari K. ........................................... O12, P111 Watcharavongtip P. .....................................O40 Wattegedera S.R. ........................................O07 Watterson D. ...............................................O39 Wedlock D.N. ............................................ P105 Werling D. ................................O01, O32, P058 Werneck G. .................................................O45 Whittington R. ...........................................P097 Wiarda J.E. .................................................O25 Wilson H.L. ........................................O05, O53 Wilson K. .....................................................O20 Wilson W. ..................................................P072 Winkley E. .................................................P004 Wishart T. ..................................................P003

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ABSTRACT BOOK - IVIS 2019 · with COX-2 inhibitor in bovine leukemia virus infection Yamato Sajiki...

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