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Detection and Characterization of Dichelobacter Nodosus from Sheep with Different Clinical Manifestations of Ovine Footrot

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Detection and Characterization of Dichelobacter Nodosus from Sheep with Different Clinical Manifestations of Ovine Footrot UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO DE BIOLOGIA VEGETAL Detection and characterization of Dichelobacter nodosus from sheep with different clinical manifestations of Ovine Footrot Catarina Almeida de Albuquerque Mestrado em Biologia Molecular e Genética Dissertação orientada por: Doutora Ana Botelho Prof.ª Doutora Mónica V. Cunha 2019 This Dissertation was fully performed at Instituto Nacional de Investigação Agrária e Veterinária, I. P. (INIAV) under the direct supervision of Dr. Ana Botelho. Professor Mónica V. Cunha was the internal supervisor designated in the scope of the Master in Molecular Biology and Genetics of the Faculty of Sciences of the University of Lisbon. Agradecimentos Este último ano esteve repleto de novas experiências e todas contribuíram para que aprendesse a adaptar-me, afetando-me como pessoa de formas que eu tenho certeza terão um papel essencial no resto da minha vida, tanto profissional como pessoalmente. Este ano ensinou-me a verdadeira importância do trabalho de equipa na investigação. As pessoas que me acompanharam durante este processo (e as lições que com elas aprendi) foram essenciais durante esta etapa da minha vida e gostaria, por isso, agora de lhes agradecer. Gostaria, primeiro que tudo, de agradecer à minha orientadora de tese, Doutora Ana Botelho, acima de tudo por me ter oferecido esta oportunidade de desenvolver a tese a seu cargo e por me receber no seu departamento no INIAV, bem como por todo o apoio e aconselhamento que me concedeu. Agradeço também à Professora Doutora Mónica Cunha, a minha orientadora interna, pela disponibilidade e orientação. Também gostaria de agradecer à Doutora Sandra Cavaco por me dar confiança no laboratório, pela ajuda e pelos conselhos. Agradeço também à Doutora Célia Leão pelo apoio e aconselhamento científico. À Ana Reis e à Sofia Simões, obrigada por me fazerem sentir em casa no laboratório e por toda a ajuda. Agradeço à Professora Doutora Sandra Branco, professora da Universidade de Évora, e à Doutora Helena Monteiro da ACOS, pela oportunidade de integrar o projeto GEN-RES (ALT20-03- 0145-FEDER-000037) e contribuir para a sua finalização. Agradeço também ao Pedro Caetano o acesso aos seus resultados e por toda a ajuda e disponibilidade. À Ângela Dente e ao Filipe Rocha, obrigada pela amizade durante estes últimos dois anos. Agradeço também à Vanessa Guerra e à Laura Moura por terem tornado esta experiência muito mais rica e divertida e por serem o meu apoio (tanto científico como emocional!) todos os dias. Agradeço ainda à Rita Antunes, à Joana Ferreira e à Inês Guimarães, por serem amigas para a vida e por me mostrarem vezes sem conta que não importa o quão longe estamos e o quão diferentes as nossas vidas se possam tornar, a amizade mantém-se. Por último, obrigada aos meus pais por me terem dado a oportunidade de realizar os meus objectivos profissionais, por estarem sempre lá e por me apoiarem em tudo. Agradeço ainda ao meu irmão João por me ajudar a ver o que é realmente importante. I Resumo e Palavras-chave A Peeira é uma doença que afecta ovinos e outros ruminantes, sendo atualmente uma das principais causas de claudicação em ovelhas. Tem como principal agente patogénico a bactéria gram- negativa anaeróbica Dichelobacter nodosus. Esta doença tem grandes impactos económicos e relevância em termos de bem-estar animal, tornando-se por isso essencial o seu controlo, tratamento e erradicação. Existem essencialmente duas manifestações clínicas desta doença. A manifestação ligeira consiste na inflamação da epiderme interdigital, sendo por essa razão denominada Dermatite Interdigital (ID – Interdigital Dermatitis). Por outro lado, na manifestação mais grave, esta inflamação pode progredir e causar a separação do casco e do tecido subjacente, sendo por essa razão chamada de Peeira Grave (SFR – Severe footrot). A severidade desta doença está principalmente dependente de três fatores: a virulência da estirpe infectante de D. nodosus, as condições ambientais e a resistência natural do hospedeiro. Alguns dos fatores ambientais que contribuem para a progressão e transmissão da doença são a temperatura, a humidade e o tipo de solo. Foi também demonstrado que a coinfecção com bactérias de outros géneros desempenha um papel deveras importante no desenvolvimento e progressão da doença. Algumas das bactérias oportunistas que foram identificadas na pele interdigital de ovinos com Peeira são Fusobacterium necrophorum e Treponema spp.. O papel exercido por estas bactérias oportunistas na progressão da doença ainda não é totalmente compreendido. Verificou-se, no entanto, que F. necrophorum, também uma bactéria gram-negativa anaeróbica, contribui para o desenvolvimento e progressão da peeira e que exerce uma relação sinergística com D. nodosus, tornando esta bactéria oportunista um foco central no estudo da doença. Em relação à virulência de D. nodosus, os dois factores principais reconhecidos são as fímbrias do tipo IV e as proteases extracelulares. A análise das fímbrias permite a discriminação de dez serogrupos (A-I e M) com base na sua diversidade estrutural que é causada por variações na região carboxi-terminal da subunidade fimbrial, também chamada proteína FimA e codificada pelo gene fimA. A protease acídica 2 (AprB2/V2), em particular, é um importante fator de virulência responsável pela atividade de elastase presente nas estirpes virulentas que, por sua vez, é essencial para o desenvolvimento das lesões características da Peeira. Os genes aprV2 e aprB2 diferem apenas numa substituição de dois pares de base (TA/CG) na posição 661/662, o que resulta na alteração de um aminoácido (Tyr92Arg) na proteína madura, sendo que esta alteração determina a atividade de elastase. O principal objetivo do presente estudo foi determinar a prevalência de D. nodosus e F. necrophorum em ovelhas da região do Alentejo com diferentes manifestações de Peeira e isolar e caracterizar D. nodosus quanto à sua virulência e serogrupo. Foram utilizadas amostras de tecido da epiderme interdigital provenientes de ovelhas saudáveis (grau 0) e com Peeira (graus 1-5) de 17 explorações de 13 concelhos da região do Alentejo. Amostras de DNA das biópsias recolhidas (n=261) foram utilizadas para a deteção e identificação de D. nodosus e F. necrophorum e para determinar a virulência e os serogrupos de D. nodosus. Estes testes foram realizados através de PCR em tempo real. Os fatores de virulência foram ainda caraterizados com recurso a sequenciação de Sanger. A determinação dos serogrupos foi realizada por PCR em formato multiplex. O isolamento de D. nodosus a partir de amostras de biópsia foi realizado por cultura bacteriológica em meios seletivos. Os resultados deste estudo mostraram que, na região do Alentejo, existe uma prevalência de D. nodosus (51%, n= 132) e F. necrophorum (46,4%, n=121) elevada em casos clínicos de Peeira. Verificou-se também uma associação entre a prevalência destes dois agentes e a severidade da lesão, ou seja, com a progressão da doença para fases mais avançadas. D. nodosus não foi detetado em quatro das 17 explorações em estudo enquanto F. necrophorum apenas estava ausente numa dessas explorações. Observou-se ainda uma tendência para explorações com um maior número de animais infetados por D. II nodosus também apresentarem uma elevada prevalência de F. necrophorum. A coinfecção por ambas as espécies é a situação epidemiológica mais comum, estando associada a uma maior severidade da doença, quando comparada com as situações em que se registou infeção apenas com D. nodosus. Em relação à prevalência geográfica de D. nodosus, este foi detetado em 11 dos 13 concelhos, não se tendo detetado no Alandroal e Moura. As prevalências mais elevadas foram registadas em Almodôvar (83%), Portel (72,4%) e Évora (68%). A análise dos fatores de virulência nas amostras de DNA de biópsia onde D. nodosus foi detetado (n=132) revelou que a maioria dos isolados (n=127) era virulenta, com exceção de cinco cujo resultado foi indeterminado. Com base em critérios espaciais e qualitativos, das 132 amostras de DNA de biópsia positivas para a presença de D. nodosus foram selecionadas 53 para a determinação dos serogrupos, tendo-se confirmado o serogrupo em apenas 19 dos casos, distribuídos nas seguintes proporções: serogrupo B (90%), serogrupo C (5%) e serogrupo F (5%). Quanto à cultura bacteriológica, das 132 amostras processadas onde foi detetado D. nodosus foram obtidos 17 isolados identificados como D. nodosus por PCR em tempo real, tendo estes sido caraterizados quanto à virulência e serogrupo a partir de DNA extraído de culturas puras. Com a exceção de um isolado, os resultados dos testes de virulência foram concordantes com os resultados obtidos nas amostras de DNA de biópsia, confirmando os isolados como virulentos. O serogrupo de 15 dos 17 isolados foi identificado, tendo dois permanecido indeterminados. Os 15 isolados pertenciam a seis diferentes serogrupos, B, C, F, G, D e H, sendo que a maioria dos isolados pertencia ao serogrupo B (40%). O segundo serogrupo mais comum foi o serogrupo C (20%), seguindo-se, na mesma proporção, os serogrupos H e G (13%). Combinando os resultados provenientes da análise por PCR multiplex de DNA de biópsia total e de DNA de culturas puras, determinou-se os serogrupos de 29 (22%) das 132 amostras de ovelhas positivas para D. nodosus. Estes 29 serogrupos foram identificados em animais de explorações de oito dos 11 concelhos positivos para D. nodosus (73%), tendo o serogrupo B sido detetado em sete concelhos com a exceção de Odemira. Em contraste, os serogrupos D e H só foram encontrados num concelho cada (Portel e Ponte de Sôr, respectivamente). Em relação à localização geográfica, é importante mencionar a elevada prevalência em concelhos geograficamente próximos, incluindo o concelho de Évora (68%), Portel (72,4%) e Alvito (65,4%). Dois outros municípios com elevada prevalência são Almodôvar (83%) e Odemira (67%), os concelhos mais a sul incluídos neste estudo e também geograficamente próximos.
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