CARACTERIZAÇÃO DA INFECÇÃO POR EIMERIA SPP. EM CAPRINOS NO ALENTEJO TESE DE DOUTORAMENTO CHARACTERIZATION OF EIMERIA SPP INFECTIONS IN GOATS IN ALENTEJO Liliana Machado Ribeiro da Silva Tese apresentada à Universidade de Évora para obtenção do Grau de Doutor em Ciências Veterinárias ORIENTADOR (A/ES): Professor Helder Carola Espiguinha Cortes Professor Carlos Hermosilla Professora Anja Taubert ÉVORA, NOVEMBRO 2014 INSTITUTO DE INVESTIGAÇÃO E FORMAÇÃO AVANÇADA Aos meus Avós Ivete e Ircílio Machado por todo o apoio e amor que sempre me deram. “O sucesso é ter quem fique feliz com o meu sucesso, tão simples, e é mesmo assim, vale-me quem me abrace quando estou feliz, e no final das contas é mais por isso que estou feliz... todas as vitórias são colectivas, sobretudo as individuais.“ Pedro Chagas Freitas TABLE OF CONTENTS FIGURES ...................................................................................................................................... ii ABBREVIATIONS ......................................................................................................................... iii ABSTRACT .................................................................................................................................. iv RESUMO ...................................................................................................................................... v 1. INTRODUCTION ....................................................................................................................... 1 1.1. Social and economic importance of goat industry........................................................................ 1 1.2. Apicomplexan parasites ............................................................................................................... 3 1.3. Eimeria species ............................................................................................................................. 6 1.3.1. Epidemiology of Eimeria infections (coccidiosis) .................................................................... 8 1.3.2. Control of Eimeria spp. coccidiosis ........................................................................................ 11 1.4. Caprine coccidiosis ..................................................................................................................... 16 1.4.1. Eimeria arloingi: peculiarities of its life cycle and pathogenesis ............................................ 18 1.5. Host cell-parasite interactions .................................................................................................... 21 1.5.1. Host immune reactions against Eimeria .................................................................................. 22 1.6. Neutrophil extracellular traps ..................................................................................................... 27 2. EIMERIA INFECTION IN GOATS IN SOUTHERN PORTUGAL ....................................................... 31 3. SUITABLE IN VITRO EIMERIA ARLOINGI MACROMERONT FORMATION IN HOST ENDOTHELIAL CELLS AND MODULATION OF ADHESION MOLECULE, CYTOKINE AND CHEMOKINE GENE TRANSCRIPTION ......................................................................................................................... 39 4. THE APICOMPLEXAN PARASITE EIMERIA ARLOINGI INDUCES CAPRINE NEUTROPHIL EXTRACELLULAR TRAPS ............................................................................................................ 52 5. NEUTROPHIL EXTRACELLULAR TRAPS AS INNATE IMMUNE REACTION AGAINST THE EMERGING APICOMPLEXAN PARASITE BESNOITIA BESNOITI ......................................................................... 64 6. THE INTRIGUING HOST INNATE RESPONSE: NOVEL ANTI-PARASITIC DEFENCE BY NEUTROPHIL EXTRACELLULAR TRAPS ............................................................................................................ 74 7. DISCUSSION AND OUTLOOK .................................................................................................. 85 LIST OF OWN PUBLICATIONS AND CONFERENCE CONTRIBUTIONS ............................................ 106 REFERENCES ........................................................................................................................... 108 ACKNOWLEDGEMENTS ............................................................................................................ 118 SUPPLEMENTARY DATA ................................................................................................................ I i FIGURES FIG. 1. World population of goats distributed by continents (2012) .......................................... 2 FIG. 2. Life cycle of Eimeria arloingi ....................................................................................... 20 FIG. 3. Mechanisms of parasite-triggered neutrophil extracellular traps (NETs) release ........ 29 FIG. 4. First in vivo evidence of NETs in histological intestinal tissue slides of an Eimeria arloingi-infected goat kid .................................................................................................... I ii ABBREVIATIONS AMPs – antimicrobial peptides BPI – bacterial permeability increasing protein BUVEC – bovine umbilical vein endothelial cells CCL2 – CC-chemokine ligand 2 CCL5 – CC-chemokine ligand 5 CFSE – carboxyfluorescein succinimidyl ester COX-2 – prostaglandin-endoperoxide synthase 2 CXCL1 – CXC-chemokine ligand 1 CXCL8 – CXC-chemokine interleukine-8 CXCL10 – CXC-chemokine 10 DNA – deoxyribonucleic acid DNase – deoxyribonuclease DPI – diphenylene iodondium ECs – endothelial cells ETs – extracellular traps GM-CSF – granulocyte-macrophage colony-stimulating factor H3 – histone H3 ICAM-1 – intracellular adhesion molecule 1 IFN-γ – interferon gamma IgA – immunoglobuline A IgG2 – immunoglobuline G2 IgM – immunoglobuline M IMC – inner membrane complex MARC-145 – Rhesus monkey fetal kidney cells MDBK – Madin-Darby bovine kidney cells MHC – Major histocompatibility complex MPO – myeloperoxidase NADPH – nicotinamide adenine dinucleotide phosphate-oxidase NE – neutrophil elastase NET – neutrophil extracellular trap OPG – oocysts per gram of faeces PGRPs – peptido-glycan recognition proteins PMN – polymorphonuclear neutrophils PV – parasitophorous vacuole PVM – PV membrane RANTES – regulation upon activation normal T cell expressed and secreted RONs – rhoptry neck proteins ROS – reactive oxygen species SCID – severe combined immunodeficiency SEM – scanning electron microscopy VCAM-1 – vascular cellular adhesion molecule 1 WFB1 – wall forming bodies 1 WFB2 – wall forming bodies 2 iii ABSTRACT Coccidiosis is a major concern in goat industry leading to high economic losses. In Alentejo, the pathogenic Eimeria arloingi was one of most prevalent species identified. A new E. arloingi European strain was isolated and a suitable in vitro culture system for the first merogony in primary endothelial cells was established. Moreover, parasite-host endothelial cells interactions were investigated during macromeront formation. Polymorphonuclear neutrophils (PMN) release neutrophil extracellular traps (NETs). We have demonstrated for the first time NET formation performed by caprine PMN exposed to different stages of E. arloingi (sporozoites and oocysts), suggesting NETosis as an important effector mechanism in the early innate immune response to this infection in goats. The new E. arloingi (strain A) will be useful for better comprehension of early host innate immune reactions against this parasite in vitro/in vivo as well as to further our investigations in the complex Eimeria-host endothelial cell interactions. Key words: Coccidiosis; Eimeria arloingi; Endothelial cell modulation; Innate immune reactions; Neutrophil extracellular traps iv RESUMO A coccidiose constitui um enorme problema na indústria caprina, causando graves perdas económicas. No Alentejo, a espécie patogénica Eimeria arloingi foi uma das mais frequentemente identificadas. Uma nova estirpe europeia de E. arloingi foi isolada e um sistema de cultura in vitro foi estabelecido para o desenvolvimento da primeira merogonia em células endoteliais primárias e as interações parasita-células endoteliais do hospedeiro foram investigadas durante a formação dos macromerontes. Os neutrófilos (PMN) extrudem redes extracelulares (Nets). Pela primeira vez demonstramos a formação de NETs por PMN caprinos expostos a diferentes estádios de E. arloingi (esporozoitos e oocistos), sugerindo que a NETosis é um importante mecanismo da resposta imune inata nesta infecção em cabras. A nova E. arloingi (estirpe A) será útil para uma melhor compreensão das reações imunes inatas contra este parasita in vitro/in vivo, bem como para progredir nas nossas investigações sobre as complexas interações entre Eimeria-células endoteliais do hospedeiro. Palavras-chave: Coccidiose; Eimeria arloingi; Modulação das células endoteliais; Reações imunes inatas; Redes extracelulares de neutrófilos v 1. INTRODUCTION 1.1. Social and economic importance of goat industry According to FAOSTAT (2014), about one billion goats were reared for meat and milk production worldwide in 2012 (Fig. 1). In addition, to assure nutritional demands of small farmers in deprived regions and nourishment of growing ethnic populations, faith-based consumers and health-conscious people, goat production also guarantees working opportunities for a considerable number of people worldwide, apart from contributing to the cleaning of forest areas and fire prevention (Silva et al. 2014b). Portugal represents the eighth