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Universidade Nova de Lisboa Instituto de Higiene e Medicina Tropical Differential expression and functional characterization of cattle tick genes in response to pathogen infection (Babesia bigemina) Sandra Isabel da Conceição Antunes DISSERTAÇÃO PARA A OBTENÇÃO DO GRAU DE DOUTOR EM CIÊNCIAS BIOMÉDICAS, ESPECIALIDADE DE PARASITOLOGIA MÉDICA Outubro, 2013 Universidade Nova de Lisboa Instituto de Higiene e Medicina Tropical Thesis: “Differential expression and functional characterization of cattle tick genes in response to pathogen infection (Babesia bigemina)” Author: Sandra Isabel da Conceição Antunes Supervisor: Investigadora Doutora Ana Isabel Amaro Gonçalves Domingos, (IHMT, UNL) Co-supervisor: Professor Doutor Virgílio Estólio do Rosário (IHMT,UNL) Professor Doutor José de la Fuente (IREC, UCLM) Dissertation presented to obtain a PhD degree in Biomedical Sciences, speciality of Medical Parasitology. Financial support was provided by Fundação para a Ciência e Tecnologia (FCT) with a PhD fellowship Grant SFRH / BD / 48251 / 2008. Universidade Nova de Lisboa Instituto de Higiene e Medicina Tropical Tese: Expressão diferenciada e caracterização funcional de genes de carraças em resposta a infecção por agentes patogénicos (Babesia bigemina) Autor: Sandra Isabel da Conceição Antunes Orientador: Investigadora Doutora Ana Isabel Amaro Gonçalves Domingos (IHMT, UNL) Co-orientador: Professor Doutor Virgílio Estólio do Rosário (IHMT,UNL) Professor Doutor José de la Fuente (IREC, UCLM) Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Doutor em Ciências Biomédicas, especialidade em Parasitologia Médica. Apoio financeiro pela Fundação para a Ciência e Tecnologia (FCT) com a bolsa de doutoramento SFRH / BD / 48251 / 2008. The results obtained during the development of this PhD project incited the following original research articles: Antunes S, Galindo RC, Almazán C, Rudenko N, Golovchenko M, Grubhoffer L, Shkap V, do Rosário V, de la Fuente J, Domingos A. 2012. “Functional genomics studies of Rhipicephalus (Boophilus) annulatus ticks in response to infection with the cattle protozoan parasite, Babesia bigemina.” Int J Parasitol: 42(2):187-95 Antunes S, Merino O, Mosqueda J, Moreno-Cid JA, Bell-Sakyi L, Fragkoudis R, Weisheit S, Pérez de la Lastra JM, Alberdi P, Domingos A, de la Fuente J. 2014. “Characterization of tick proteins involved in tick-pathogen interactions as potential antigens for the control of tick infestation and pathogen infection.” Parasit Vectors: 7: 42 Merino O, Antunes S, Mosqueda J, Moreno-Cid JA, Pérez de la Lastra JM, Rosario- Cruz R, Rodríguez S, Domingos A, de la Fuente, J. 2013. “Vaccination with proteins involved in tick-pathogen interactions reduces vector infestations and pathogen infection.” Vaccine: 31(49):5889-5896 Dedico esta tese á minha Mãe… Uma guerreira desconhecida pelo Mundo mas reconhecida pelos que a rodeiam! Vou sempre acreditar na tua força! Acknowlegments This thesis would not have been realized without the support and collaboration from a great many people over the years and I would like to take this opportunity to thank them all. First and foremost, I would like to express my deep gratitude to Ana Domingos and Jose de la Fuente my research supervisors, for their patient guidance, enthusiastic encouragement, useful critiques that pushed me further and also for their friendship. Also I want to express my appreciation to Prof. Virgílio do Rosário for his help; whenever necessary he was always present. When I began this long journey I already suspected that this would be a bumpy road… But what a ride it was!!! If not for anything else the experience of working with so many different people alone was so worthy. This project allowed me to meet and work with amazing persons in different places. A special thanks to Doctor Varda Shkap that kindly provided ticks, fundamental to all the work developed in this project. Also I want to express my gratitude to all the technical support in the histological cuts given by Doctor Jorge Correia and Maria do Rosário from FMV. So now: first in Ciudad Real, Ruth Galindo: thank so so much for the help and patience in the lab; I will be forever grateful for all you teach me and for your sincere friendship! Tell your Mum that I remember her often and I always smile when thinking of the time I spent with you. Mario Canales: thank you for sharing your family time with me! Jose Manuel: I´m grateful for all the advices and guidance you gave me during my stays at IREC. Juan Moreno-Cid: thanks for the friendship. To all the staff in IREC thanks, I really enjoyed working in IREC, it as a good energy!!! In České Budějovice: I´m so grateful to Doctor Libor Grubhoffer that found time in his busy schedule to take me to inspiring places, like Český Krumlov. Natasha and Marina you both are so special to me. I will forever treasure your friendship and genuine interest in my life; moreover you helped me to reach my first big goal in the PhD project so indeed you have a special place in my heart. Again to all the people in the Academy of Sciences that in some way helped me I leave here my sincere gratitude. My Mexican adventure before and after João: in Ciudad Victória a special acknowledgement to Chelo for hers professional guidance, valuable support and friendship and to Octávio my research buddy! If not for both of you I would be still looking to cows and ticks not knowing what to do, like a donkey looking to a palace!!! i Lorena, Uriel, Sarahy and Orlando you were not forgot! Thanks for the smiles and rides along the city! In my second time around in Mexico, now in Querétaro, I had the pleasure of meeting Doctor Juan Mosqueda and Gabriela and to work again with Octávio. Doctor Juan, I’m grateful for all the advices and support you gave me in this last part of the project. Octávio again thank you so much for sharing the work and your good vibes! Finally, aos meus colegas e amigos portugueses! Esta viagem começou no INETI no grupo da UTPAM onde conheci a Ana Domingos, a Bé, o Tiago, Ana Armada, Dr. Carlos, o Fernando, a Ângela, a Maria, a Raquel e a Cristina. Este grupo maravilhoso aceitou-me de braços abertos e apesar de já não existir deixa muitas saudades. No IHMT tenho de agradecer a todos, os quais me ajudaram directa ou indirectamente a terminar este projecto de doutoramento: Renato, Cátia, Tiago, Pedro, Isabel, Ana Afonso, Manuela, Prof. Paulo Almeida, Prof. Silvana Belo, obrigado pelo interesse no meu trabalho e pela amizade. À Fábia, Joana e Lara o meu sincero obrigado por partilharem comigo algum do trabalho! Quero deixar um agradecimento especial a três pessoas que de facto foram muito importantes para mim neste percurso: Cristina, muito obrigado por seres minha amiga apesar de eu ser relaxada e esquecida por sorrires mesmo when you´re feeling down. Raquel és uma pessoa extraordinária com muita força e determinação e adorei estar grávida contigo. Ana, o apoio que me dá em todas as alturas e em todos os campos tem sido fundamental, obrigado por partilhar comigo os bons e maus momentos. Não posso deixar de agradecer à minha família por todo o apoio: Aos meus pais, Alberto e Susete, pelos sacrifícios que fizeram e ainda fazem para me darem esta oportunidade. Ao meu irmão Hugo, pelo apoio sempre discreto. À minha irmã Zélia e ao meu cunhado Zé por acreditarem e me incentivarem a lutar pelo que quero. Claro aos meus sobrinhos adorados Sara e Pedro! Maria Inês, não me esqueci de ti, nem dos teus pais e avós! À Carina (que já é da família!) obrigado porque sim… Não podia sonhar com uma família melhor! Comparo muitas vezes a minha família a um circo onde há espaço para todos, alegria todos dias e força para enfrentar os desafios da vida! Ao Pedro por amar os meus defeitos tal como amo os dele, por tudo e por nada, por existirmos juntos… Ao João o melhor acidente de todos os tempos, o filho mais perfeito! Que felicidade construir e partilhar a minha vida com ambos! ii Resumo Expressão diferenciada e caracterização funcional de genes de carraças em resposta a infecção por agentes patogénicos (Babesia bigemina) Sandra Isabel da Conceição Antunes Palavras-chave: Carraça, Vacina, Babesia, RNA de interferência, TROSPA O conceito “One health” reconhece a necessidade do trabalho conjunto de veterinários, profissionais de saúde e cientistas, dada a interface dinâmica entre pessoas, animais e ambiente. Este conceito é muito importante em zoonoses, tais como doenças associadas a carraças (DAC´s) que dependem de animais como reservatório. Os protozoários do género Babesia são agentes patogénicos transmitidos por carraças que causam a doença denominada babesiose num variado número de animais incluindo o Homem. Particularmente a B. bovis e B. bigemina são transmitidas por carraças, relacionadas com gado, Rhipicephalus (Boophilus) annulatus e R. microplus sendo estas consideradas os ectoparasitas de maior importância, com largo impacto económico na produção animal. O uso tradicional de acaricidas no controlo de carraças apresenta diversas desvantagens incluindo a seleção de carraças acaricido-resistentes e contaminação ambiental com resíduos químicos. As vacinas destacam- se como uma alternativa ao uso de acaricidas. O objetivo destas vacinas é a proteção contra DAC´s através do controlo das infestações pelos vectores e redução de transmissão de doença. As proteínas envolvidas nas interações carraça-agente patogénico podem ser bons candidatos para essas novas vacinas mas a sua identificação e validação continuam a ser obstáculos. Os objetivos do presente estudo foram, primeiro, a identificação de genes de R. annulatus diferenciadamente expressos em resposta à infeção por B. bigemina, segundo, a validação da influência destes genes no processo de infeção e finalmente a caracterização dos antigénios identificados, a fim de selecionar os melhores candidatos, para o desenvolvimento de uma potencial nova vacina.
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  • A Portuguese Isolate of Borrelia Lusitaniae Induces Disease in C3H/Hen Mice

    A Portuguese Isolate of Borrelia Lusitaniae Induces Disease in C3H/Hen Mice

    J. Med. Microbiol. Ð Vol. 50 72001), 1055±1060 # 2001 The Pathological Society of Great Britain and Ireland ISSN 0022-2615 BACTERIAL PATHOGENICITY A Portuguese isolate of Borrelia lusitaniae induces disease in C3H/HeN mice NORDIN S. ZEIDNER, MARIA S. NU NCIOÃ,BRADLEYS.SCHNEIDER,LISEGERN{, JOSEPH PIESMAN, OTILIA BRANDAÄ O{ and ARMINDO R. FILIPEà Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Fort Collins, CO 80522, USA, ÃInstituto Nacional de Sau de, Dr. Ricardo Jorge, Centro de Estudos de Vectores e Doencas Infecciosas, A guas de Moura, Portugal, {Institut de Zoologie, University of Neuchaà tel, Neuchaà tel, Switzerland and {Department of Pathology, SaÄo Bernardo Hospital, Setu bal, Portugal A low-passage, Portuguese isolate of Borrelia lusitaniae, strain PotiB2, was inoculated into C3H/HeN mice and disease was monitored by histopathology at 8weeks after spirochaete challenge. Ear, heart, bladder, femoro-tibial joint, brain and spinal cord were examined. B. lusitaniae strain PotiB2 &6 of 10 mice) and B. burgdorferi sensu stricto strain N40 &9 of 10 mice) induced similar lesions in the bladder of infected mice characterised as a multifocal, lymphoid, interstitial cystitis. Moreover, both B. lusitaniae PotiB2 and B. burdorferi N40 induced lesions in the heart of infected mice. The lesions induced by B. lusitaniae PotiB2 &2 of 10 mice) were characterised as a severe, necrotising endarteritis of the aorta, with a minimal, mixed in¯ammatory in®ltrate &neutrophils, macrophages and lymphoid cells) extending into the adjacent myocardium. In contrast, B. burgdorferi N40 induced a periarteritis of the pulmonary artery &7 of 10 mice), with no involvement of the endothelium and more extensive in¯ammation and subsequent necrosis of the adjacent myocardium.
  • Prevalence of Borrelia Anserina in Argas Ticks

    Prevalence of Borrelia Anserina in Argas Ticks

    Pakistan J. Zool., vol. 47(4), pp. 1125-1131, 2015. Prevalence of Borrelia anserina in Argas Ticks Bilal Aslam,1* Iftikhar Hussain,2 Muhammad Asif Zahoor,1 Muhammad Shahid Mahmood2 and Muhammad Hidayat Rasool1 1Department of Microbiology, Government College University, Faisalabad 2Institute of Microbiology, University of Agriculture, Faisalabad Abstract.- Borrelia anserina is a pathogen of high importance in poultry industry which causes fowl spirocheatosis. This study was designed to determine the prevalence of Borrelia anserina in poultry soft ticks, Argas persicus collected from birds and poultry farms. A total of 1500 ticks were collected from poultry farms located in Faisalabad and Kamalia, Pakistan. B. anserina was isolated using BSK-H medium and confirmed by dark field microscopy and indirect immunoflourescence. In addition, B. anserina was characterized using polymerase chain reaction by employing specific primer set of fla B gene. Of 750 tick samples collected from poultry birds, 144 (19.2%) were positive for B. anserina; whereas 131 (17.4%) were positive collected from poultry farms. The data indicated that the Argas ticks had the significant prevalence of B. anserina. Furthermore, the data may warrant future studies towards the vector control and/or immunoprophylaxsis against B. anserina which might indirectly be helpful for the eradication of this threatening disease. Key words: Borrelia anserina, Argas persicus, poultry industry. INTRODUCTION 2007). Morphologically, B. anserina bacterium is 0.2-0.5 µm in diameter and 10-50 µm in length. It has a very complex morphological structure, so it Poultry products are a good source of gains poor staining and only observed by dark field protein; as eggs contain superior quality protein or phase contrast microscope (Barbour, 1984).