UNIVERSIDADE ESTADUAL PAULISTA INSTITUTO DE BIOCIÊNCIAS CÂMPUS DE BOTUCATU ECOLOGIA MOLECULAR DE FUNGOS PATOGÊNICOS ONYGENALES EM ANIMAIS SILVESTRES DO INTERIOR DO ESTADO DE SÃO PAULO VIRGÍNIA BODELÃO RICHINI PEREIRA Tese apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, para obtenção do título de Doutor no Programa de Pós-Graduação em Biologia Geral e Aplicada. BOTUCATU - SP 2009 UNIVERSIDADE ESTADUAL PAULISTA INSTITUTO DE BIOCIÊNCIAS CÂMPUS DE BOTUCATU ECOLOGIA MOLECULAR DE FUNGOS PATOGÊNICOS ONYGENALES EM ANIMAIS SILVESTRES DO INTERIOR DO ESTADO DE SÃO PAULO Virgínia Bodelão Richini Pereira Orientador: Profº Drº Eduardo Bagagli Co-Orientadora: Profª Drª Sandra de Moraes Gimenes Bosco Tese apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, para obtenção do título de Doutor no Programa de Pós-Graduação em Biologia Geral e Aplicada. BOTUCATU - SP 2009 DD ee dd ii cc aa tt óó rr iiaa :: AA DD ee uu ss Por se mostrar presente em todos os momentos da minha vida, iluminando meu caminho, perdoando meus erros, e me concedendo sempre novas oportunidades de acertar e também por enviar pessoas maravilhosas em minha vida. AA oo mm ee uu oo rr ii ee nn tt aa dd oo rr :: PP rr oo ff oo DD rr oo EE dd uu aa rrdd oo BB aa gg aa gg ll ii Pelas palavras de incentivo e elogio que foram fundamentais para tornar esse trabalho muito prazeroso, pela valiosa orientação, amizade, paciência, e acima de tudo por contribuir para meu enriquecimento profissional e pessoal. AA mm iinn hh aa cc oo -- oo rr ii ee nn tt aa dd oo rr aa ee aa mm iigg aa :: PP rr oo ff ªª DD rr ªª SS aa nn dd rr aa dd ee MM oo rr aa ee ss GG iimm ee nn ee ss BB oo ss cc oo Obrigada por me ensinar a Biologia Molecular, pela paciente leitura tantas vezes realizada, pela atenção em todo o tempo, inclusive priorizando a vida profissional mesmo na véspera do nascimento da Beatriz. AA oo ss mm ee uu ss ff aa mm ii ll iiaa rr ee ss :: Que mesmo à distância torcem por mim, nunca faltando palavras de apoio. AA ss mm iinn hh aa ss mm ãã ee ss :: NN ii ll cc aa ee HH éé ll iiaa Exemplos de luta e dedicação, que com muito amor foram responsáveis por minha educação e valores. Por sempre torcerem para que minha vida fosse repleta de realizações. AA oo mm ee uu aa mm aa dd oo mm aa rr iidd oo :: SS éé rr gg iioo PP ee rr ee ii rr aa Pelo apoio constante em todos esses anos de nossas vidas, por todos os momentos compartilhados que guardarei sempre na memória, pelo amor, carinho e preocupação principalmente nos momentos mais decisivos desta etapa. Palavras não são suficientes para expressar minha gratidão Eu amo muito vocês! AA gg rr aa dd ee cc iimm ee nn tt oo ss Agradeço a todos que me auxiliaram para que este trabalho se concretizasse, especialmente: Ao corpo docente do Programa de Pós Graduação em Biologia Geral e Aplicada pelo estímulo na busca pelo aperfeiçoamento profissional; Ao Departamento de Microbiologia e Imunologia do Instituto de Biociências da UNESP – Botucatu por possibilitar a realização deste projeto; A Profa Dra Terezinha Serrão Peraçoli pela confiança em me orientar “burocraticamente” no início, sem me conhecer; Aos docentes do Departamento de Microbiologia e Imunologia, especialmente a Profa Dra Vera Lúcia Moraes Rall�pela oportunidade de aprendizado no estágio de docência; Aos funcionários do departamento, Pedro, Isaltino, Luiz, Ademival e, especialmente, ao “Lula” pelo auxílio com os tatus capturados; Às secretárias do departamento, Sônia e Nice, pela atenção; Às funcionárias da limpeza, por tornar agradável o ambiente de trabalho; Aos funcionários da Seção de Pós-Graduação, Sérgio, Herivaldo, Luciene e Maria Helena, pelos serviços e esclarecimentos sempre quando solicitados; Ao Serviço de Biblioteca e Documentação da UNESP – Botucatu, pela elaboração da ficha catalográfica e revisão das referências; Aos amigos de laboratório Ariane Nascimento, Assis Macoris, Gabriela Reis, Gisela Terçarioli, Hélio Jr., Keila Siqueira, Lígia Barrozo, Raquel Sanzovo, Raquel Theodoro, Sandra Bosco, Sandra Olbrich e Sílvia Pedrini pelos momentos de aprendizado e descontração no dia a dia; Aos funcionários do Departamento de Estradas de Rodagem de São Paulo (DER), pelo aviso sobre os animais silvestres atropelados; À Lígia Barrozo pelo auxílio no Sistema de Informação Geográfica (SIG), na elaboração dos mapas e amizade; Ao Profº Drº Reinaldo José da Silva e Juliana Griese pela parceria de trabalho no aproveitamento dos animais atropelados; Ao Instituto Lauro de Souza Lima (ILSL), especialmente, às pesquisadoras Patrícia Sammarco Rosa e Silvia Pedrini que apoiaram a pesquisa com os tatus dessa região; A Profa Dra Márcia Guimarães, pela oportunidade de estágio no laboratório de Patologia; Ao Departamento de Anatomia, especialmente ao Profº Drº Sérgio Pereira por confeccionar as lâminas do histopatológico; Ao Setor de Sequenciamento da USP, pelo sequenciamento das amostras de DNA; Ao Departamento de Recursos Naturais/Ciências do Solo, pela análise físico-química do solo das tocas de tatus; Aos proprietários dos Sítios em Cerqueira César, Srº Benedito Pinto Cardoso, Srº Eder Ferreira da Silva e Srº Rui Correa, que gentilmente permitiram os trabalhos de campo; Ao Profo Dro Eduardo Bagagli, Raquel Cordeiro e Nilca Richini, por compartilharem as aventuras de campo e pelo auxílio na coleta dos tatus; Aos amigos, Raquel Domeniconi, Carol Luchini, Justulin, Priscila Martins, Shirlei e a “Turma da Dança de Salão”, pela amizade, alto astral e conversas agradáveis. À Assessoria científica da FAPESP pelas sugestões durante a realização do projeto; Ao IBAMA e Comitê de Ética pela autorização na captura e coleta dos animais silvestres; Auxílio financeiro Processo nº 0015006 Processos nº 05/56771-9 e nº 06/03597-4 SUMÁRIO Resumo Abstract Introdução ............................................................................................................................... 11� Fungos patogênicos ............................................................................................................... 11 Paracoccidioides brasiliensis ............................................................................................... 13 P. brasiliensis e animais ........................................................................................................ 14 Animais silvestres atropelados .............................................................................................. 15 Biologia molecular ................................................................................................................ 16 Sistema de Informação Geográfica (SIG) ............................................................................. 17 Capítulo I: Molecular approaches for eco-epidemiological studies of Paracoccidioides brasiliensis ................................................................................................................................ 18 Capítulo II: Molecular detection of Paracoccidioides brasiliensis in road-killed wild animals .................................................................................................................................................. 37 Capítulo III: Detecção de Paracoccidioides brasiliensis em tatus (Dasypus novemcinctus) provenientes de uma reserva de cerrado do Instituto Lauro de Souza Lima (Bauru-SP) ......... 51 Capítulo IV: Role of the Xenarthra Superorder in the epidemiology of Paracoccidioidomycosis ........................................................................................................... 66 Capítulo V: Animais silvestres atropelados: um problema atual de preservação, mas com grande potencial para estudos eco-epidemiológicos de patógenos .......................................... 79 Conclusões ............................................................................................................................... 98 Referências .............................................................................................................................. 99 RR ee ss uu mm oo Resumo A Paracoccidioidomicose (PCM) é uma micose sistêmica e a de maior ocorrência na América Latina, causada pelo fungo Paracoccidioides brasiliensis. Apesar dos esforços contínuos de diversos grupos de pesquisa principalmente do Brasil, Colômbia, Venezuela e Argentina, a fase ambiental produtora de propágulos infectantes, seu nicho ecológico e outros aspectos fundamentais da biologia deste patógeno ainda representa um enigma. Sabe-se, no entanto, que há alguns indicadores biológicos onde se constata a infecção natural do P. brasiliensis em espécies de tatu Dasypus novemcinctus em áreas endêmicas. Assim, o isolamento sistemático deste patógeno nesta espécie animal tem despertado o interesse em avaliar outras espécies animais cuja distribuição geográfica seja coincidente com a da PCM. O conhecimento de reservatórios naturais de fungos patogênicos e o mapeamento de regiões habitadas pelos fungos são dados fundamentais para elucidar a eco-epidemiologia da PCM. O presente projeto focalizou a detecção ambiental do P. brasiliensis, bem como de outros fungos patogênicos geneticamente relacionados, em animais silvestres, pelo emprego de métodos moleculares em amostras de tecido de animais atropelados em beiras de estradas. Sabe-se que esse material é muitas vezes negligenciado, sendo utilizado em estudos de conservação biológica e de parasitologia. Esta abordagem é inédita e útil no estudo da eco-epidemiologia da PCM. Foram avaliados também tatus (D. novemcintus) capturados em regiões geograficamente definidas, visando o isolamento fúngico do P. brasiliensis, bem como de outros fungos patogênicos, por cultura de órgãos, histopatologia e técnicas moleculares. A integração dos dados micológicos, moleculares e a aplicação de técnicas de geoprocessamento permitiu a caracterização da área geográfica dos animais avaliados e contribuiu para um melhor conhecimento sobre a ocorrência do patógeno no hospedeiro animal e dos fatores bióticos e abióticos associados. Palavras-chave: Paracoccidioides brasiliensis, Paracoccidiodomicose, animais silvestres atropelados, biologia molecular, Dasypus novemcinctus AA bb ss tt rraa cc tt Abstract Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis of Latin America, caused by fungus Paracoccidioides brasiliensis. Despite the continuous efforts by several research groups mainly from Brazil, Colombia, Venezuela and Argentina, the saprobic form producing infective propagula, its exact niche in nature and other fundamental aspects of the biology of this pathogen still remains enigmatic. It is known however that some biological indicators where there is a natural infection of P. brasiliensis from the nine-banded armadillo Dasypus novemcinctus in an endemic area. Thus, the systematic isolation of this pathogen in this species has increased the necessity in evaluating several wild mammals whose geographical distribution is coincident of PCM. The knowledge about natural reservoirs of pathogenic fungi and mapping landscape of areas inhabited by fungi may be clarified the eco- epidemiology of PCM. This work focused on the environmental detection of P. brasiliensis, as well as other related pathogenic fungi in wild animals, the use of molecular tools in tissue samples from road-killed wild animals. While road-killed animals proved to be usefull both form parasitological and conservative studies, these materials have been negleted in the area of infections disease. This approach is new and has been useful to elucidate PCM eco- epidemiology. It was evaluated armadillos (D. novemcinctus) captured is some restricted areas, such as from Savanna and a County considered to be hyperendemic for PCM, by culture, histopathology and molecular techniques, in order to confirm if P. brasiliensis also occurs in such defined ecological conditions. The use the Geographical Information Systems (GIS), thus contributing to a better understanding about the occurrence of the pathogen in the host animal and the associated biotic and abiotic factors. Key words: Paracoccidioides brasiliensis, Paracoccidiodomycosis, road-killed wild animals, molecular biology, Dasypus novemcinctus II nn tt rr oo dd uu çç ãã oo 11 Introdução Fungos patogênicos A distribuição cosmopolita dos fungos sugere a adaptação em diversas condições ambientais (Figura 1, adaptada de MOSS, 1987). Figura 1: Resumo das principais atividades fúngicas. A diversidade fúngica é estimada em 1,5 milhões de espécies, porém cerca de 100 mil espécies são conhecidas e menos de 200 espécies são consideradas patogênicas para humanos e outros mamíferos. Infecções causadas por fungos constituem um importante problema de saúde pública e uma das principais causas de morte em todo o mundo. Os principais fungos de importância médica, causadores de micoses sistêmicas, apresentam várias características micológicas e ecológicas em comum como: dimorfismo (forma filamentosa em condições saprofíticas e leveduriforme na condição patogênica), distribuição geográfica restrita limitada às chamadas áreas endêmicas, ocorrência saprofítica em micro- hábitats com produção de propágulos infectantes, que penetram no hospedeiro principalmente pelo trato respiratório (HAWKSWORTH, 2001; CHAKRABARTI, 2005). Dentre eles destacam-se: Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, Emmonsia spp. e Paracoccidioides brasiliensis. Trata-se de fungos ascomicetos, pertencentes à Ordem Onygenales e Família Onygenaceae (PETERSON & SIEGLER, 1998; BIALEK et al., 2000; HERR et al., 2001; SAN-BLAS et al., 2002). Essa - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - fungos Doenças de Plantas e Animais Micotoxinas Alergia Deterioração Agricultura • fertilidade do solo • controle biológico de pragas • micorrizas Industrial •produção álcool, cerveja, vinho, fármacos, enzimas Alimentação •cogumelos comestíveis •queijo •pães Ambiental • reciclagem de nutrientes • biorremediação • interação com plantas e animais II nn tt rr oo dd uu çç ãã oo 12 Família apresenta duas características micológicas importantes: a) presença de artroconídias, as quais se apresentam intercaladas por células estéreis que facilitam sua liberação, e aleuroconídias (formas terminais ou laterais, de parede espessa) e b) crescimento em substratos e hábitats relacionados com solo enriquecido com queratina (pele, pelo, unha de animais e penas de aves) ou fezes de animais ou aves (ALEXOPOULOS et al., 1996). Além de compartilhar características micológicas e moleculares semelhantes, os membros desta família apresentam uma ecologia normalmente associada a uma fase saprofítica ambiental em solos e fase parasitária associada a hospedeiros vertebrados (TAYLOR et al., 2000; BIALEK et al., 2000; HERR et al., 2001; UNTEREINER et al., 2004). Um clado distinto da Família Onygenaceae foi reconhecido como uma nova Família denominada Ajellomycetaceae que inclui os gêneros B. dermatitidis H. capsulatum, Emmonsia spp. e o P. brasiliensis. Estudos filogenéticos avaliando as relações de Lacazia loboi com outros membros da Família Ajellomycetaceae mostraram que esta espécie está contida dentro desta Família e que é grupo-irmão de P. brasiliensis (HERR et al., 2001; UNTEREINER et al., 2004) (Figura 2). Figura 2: Relações filogenéticas da Família Ajellomycetaceae (extraído de Herr, et al. (2001) com adaptações de Untereiner et al. (2004)). II nn tt rr oo dd uu çç ãã oo 13 Relacionando filogenia com ecologia, curiosamente observa-se que as espécies P. brasiliensis, L. loboi e B. dermatitidis, além de serem próximas filogeneticamente, são também as menos compreendidas ecologicamente. Importante ressaltar que L. loboi é espécie ainda não cultivável em meios tradicionais, sendo considerado um parasita obrigatório (TABORDA et al., 1999). Torna-se importante o conhecimento desses fungos e em particular o P. brasiliensis por ser o agente etiológico da micose sistêmica mais importante na América Latina (TAYLOR et al., 2000; BIALEK et al., 2000; HERR et al., 2001; UNTEREINER et al., 2004). Paracoccidioides brasiliensis P. brasiliensis é o fungo causador da Paracoccidioidomicose (PCM), uma micose sistêmica endêmica na América Latina. Esse microrganismo apresenta-se na forma de levedura em condições de parasitismo ou quando cultivados a 35-37ºC, e micelial quando na forma saprofítica ambiental ou cultivo a 22-25ºC (LACAZ et al., 2002). As leveduras apresentam forma arredondada a oval, caracterizadas por apresentarem brotamentos múltiplos originados por evaginações da célula-mãe, apresentando um aspecto de “roda de leme”. A forma micelial é caracterizada pela formação de hifas alongadas, geralmente ramificadas, com produção de conídias (Figura 3). Figura 3: A) Fase leveduriforme do P. brasiliensis, coradas com lactophenol azul algodão, aumento 40X. B) Fase micelial com produção de conídias, coradas com lactophenol azul algodão, aumento 100X. A PCM usualmente infecta o hospedeiro humano pela inalação de propágulos em suspensão que vão se alojar predominantemente nos pulmões. Ao atingir o alvéolo pulmonar, o fungo transforma-se em células leveduriformes adaptadas à temperatura corporal (RESTREPO, 1986; MEDOFF et al., 1987). Uma vez no organismo o fungo pode ser completamente destruído ou então persistir e multiplicar-se, podendo disseminar por vias hematogênica ou II nn tt rr oo dd uu çç ãã oo 14 linfática acometendo outros órgão e sistemas como fígado, baço, glândula adrenal, ossos e sistema nervoso central (SAN-BLAS, 1993; CAMARGO & FRANCO, 2000; VALERA et al., 2008). A evolução e as consequências da infecção vão depender da interação de fatores relacionados ao fungo, como virulência e composição antigênica; do hospedeiro, como características genéticas e imunidade e ao meio ambiente (CALICH et al., 1985 e 1987). O fungo e as manifestações clínicas da infecção vêm merecendo atenção de vários grupos de pesquisadores (FRANCO et al., 1993; RESTREPO et al., 2001; SAN-BLAS et al., 2002; FELIPE et al., 2005; CAMARGO, 2008; PUCCIA et al., 2008) principalmente pelas características peculiares da doença: a) longo período de latência, b) ausência de surtos epidêmicos e c) pequeno número de casos agudos (BRUMMER et al., 1993; RESTREPO, et al., 2000; LACAZ et al., 2002). Essas circunstâncias dificultam a determinação da origem do foco de infecção, assim como o hábitat do fungo, além disso, a repercussão da infecção primária e o tempo real do período de incubação ainda continuam intrigando os pesquisadores (RESTREPO, 1985; WANKE & LONDERO, 1994; RESTREPO et al., 2000). P. brasiliensis e animais A PCM foi pouco avaliada em animais, os quais podem representar importantes indicadores biológicos da ocorrência do fungo em uma determinada área (RESTREPO, 1985). Em 1965, Grose & Tamsitt isolaram P. brasiliensis de trato intestinal de morcegos. Estudos epidemiológicos empregando-se a paracoccidioidina mostraram que tanto animais domésticos quanto silvestres apresentam-se positivos para o P. brasiliensis (CONTI-DÍAZ et al., 1972; COSTA et al., 1995). Testes sorológicos (ELISA) realizados em cães (ONO et al., 2001; FAGUNDES, 2002) e macacos (CORTE et al., 2007) mostraram que estes animais apresentam- se naturalmente infectados. Ricci et al. (2004) e Farias et al., 2005 relataram dois casos de PCM em cães, sendo obtida cultura fúngica apenas para o segundo caso. O isolado foi caracterizado molecular e morfologicamente (BOSCO et al., 2005). Porém, somente após a constatação de que os tatus (D. novemcinctus) apresentam-se infectados pelo P. brasiliensis em alta frequência nas áreas endêmicas de PCM, é que novas perspectivas de estudos eco-epidemiológicos se consolidaram, pois esta espécie vem sendo apontada como um seguro indicador epidemiológico para rastreamento dos locais de ocorrência do patógeno (BAGAGLI et al., 2003). Este fato foi inicialmente confirmado por Naiff et al. (1986, 1989) na região Amazônica e, posteriormente em outras regiões (BAGAGLI et al., 1998, 2003; CORREDOR et al., 1999; MACEDO et al., 1999; RESTREPO et al., 2000; SILVA- VERGARA et al., 2000). Além da espécie D. novemcinctus, o fungo também foi isolado do tatu II nn tt rr oo dd uu çç ãã oo 15 Cabassous centralis, em Caldas, Colômbia, indicando que outras espécies além do tatu-de- nove-bandas também podem estar infectadas com o patógeno (CORREDOR et al., 2005). Estes animais possuem hábito escavatório, cuja distribuição geográfica costuma coincidir com a observada na PCM (RESTREPO-MORENO, 1994). O contato com estes animais esta associado a um aumento dos fatores de riscos para a infecção em pessoas residentes nas áreas endêmicas da doença (CADAVID & RESTREPO, 1993). Por apresentar temperatura corporal e imunidade celular relativamente baixa (PURTILO et al., 1975; ULRICH et al., 1976), o tatu parece favorecer o desenvolvimento de infecções, podendo inclusive ter desempenhado algum papel na evolução do P. brasiliensis a condição zoofílica (adaptada ao tecido animal) (BAGAGLI et al., 2006), proporcionando uma tendência ao estabelecimento de lesões crônicas nos hospedeiros e a baixa produção de conídias (McEWEN et al., 1987), o que poderia explicar também o difícil isolamento ambiental deste fungo a partir da sua fase saprofítica (FRANCO et al., 2000). Assim, a pesquisa de fungos patogênicos avaliados em diversos exemplares de animais silvestres mortos por atropelamento, bem como de tatus (D. novemcinctus) capturados em áreas ainda não avaliadas, possibilita um impacto positivo no melhor entendimento da PCM. Animais silvestres atropelados As pesquisas com animais silvestres são cada vez mais restritivas, principalmente tratando-se de métodos que necessita de anestesia e/ou eutanásia do animal, assim a detecção molecular de patógenos fúngicos em animais silvestres mortos por atropelamento atende a necessidade de se buscar alternativas para o uso de animais em pesquisa, como indicado pelos comitês de ética em experimentação animal. O número e a diversidade de animais mortos em rodovias brasileiras aumenta a cada ano e o problema se agrava principalmente pelo aumento do fluxo de automóveis e também porque a rodovia corta áreas potencialmente ricas em fauna e flora, interferindo no deslocamento natural da espécie (FORMAN & ALEXANDER, 1998; RODRIGUES et al., 2002; PRADA, 2004). Desta maneira, os atropelamentos de mamíferos silvestres contribuem para o declínio e dificuldades na recuperação de populações, principalmente aquelas em risco de extinção (TROMBULAK & FRISSEL, 2000). Estudos de conservação biológica, principalmente de levantamentos quantitativos e qualitativos das ocorrências de atropelamento da fauna são realizados (VIEIRA, 1996; CANDIDO Jr. et al., 2002; RODRIGIES et al., 2002; PRADA, 2004; PINOWSKI, 2005), porém são poucos os trabalhos que aproveitam as carcaças dos animais para outros estudos, como por exemplo, II nn tt rr oo dd uu çç ãã oo 16 helmintológico, morfológico e genético (COYNER et al., 1996; FOSTER et al., 2003; NELDER & REEVES 2005; GRIESE, 2007). Mesmo com as dificuldades para o cultivo e análise histopatológica das amostras de tecido de animais atropelados, técnicas moleculares podem ser utilizadas na identificação e tipagem de patógenos nestes materiais. Biologia molecular Os recentes avanços nas técnicas de Biologia Molecular proporcionaram o surgimento de várias técnicas de identificação e tipagem de patógenos, através da PCR (reação em cadeia da polimerase), com o uso de primers específicos, que apresentam alta especificidade e sensibilidade para a amplificação de um determinado fragmento de DNA específico do patógeno (BOWMAN et al., 1993). Já a Nested-PCR é um avanço da técnica de PCR, pois trata-se de duas amplificações seguidas, utilizando-se primeiramente outer primers e depois inner primers. Diversos alvos dentro do genoma de fungos têm sido avaliados, em especial áreas da sequência dentro do DNA ribossomal (rDNA) (ANDERSON et al., 2001; UETAKE et al., 2002). Essa região do genoma é importante para detecção e identificação de fungos patogênicos, pois apresenta sequências de nucleotídeos relativamente conservadas entre os fungos e também regiões variáveis chamadas ITS (internal transcribed spacer), sendo utilizadas na diferenciação de espécies ou entre linhagens da mesma espécie (ESTEVE- ZARZOSO et al., 1999; HENRY et al., 2000; IWEN et al., 2002). Por apresentar cerca de centenas de cópias por genoma, esta região confere maior sensibilidade e especificidade, representando um importante alvo para identificação fúngica. Várias metodologias moleculares são empregadas para a identificação do P. brasiliensis tanto para materiais clínicos como amostras ambientais. Motoyama et al. (2000) desenvolveram um método de identificação do P. brasiliensis amplificando e sequenciando regiões de DNA ribossomal 5.8S e 28S e regiões intergênicas, o que possibilitou diferenciar o P. brasiliensis de outros fungos patogênicos. Imai et al. (2000) amplificaram e sequenciaram a região ITS (sequências que separam os genes 18S, 5.8S e 28S e são transcritas e processadas para dar origem ao DNA ribossômico maduro), incluindo o gene 5.8S, e deduziram um par de primers que amplifica um fragmento de 418 pares de bases, específico de P. brasiliensis, em 29 isolados testados, provenientes do Brasil, Costa Rica, Japão, Argentina. Gomes et al. (2000) avaliaram várias combinações de primers, todos derivados do gene da gp43, e observaram que o par de primers (PC2-PC6) apresentou alta sensibilidade, tanto em II nn tt rr oo dd uu çç ãã oo 17 amplificação direta, como em reação de nested PCR, após um primeiro ciclo com PC1-PC5 (outer primers). Bialek et al. (2000) também desenharam dois pares de primers do gene da gp43, para uso em Nested-PCR, cuja sensibilidade é alta, em amostras de tecidos infectados com conídias de P. brasiliensis, e também específico pois não amplificam DNA de H. capsulatum. Em 2005, San-Blas et al. desenharam primers derivados de sequências randômicas do DNA que foram específicos para P. brasiliensis na detecção em amostras clínicas. Em nosso laboratório foram desenhados primers que se anelam na região do rDNA, particularmente nas regiões espaçadoras ITS1 e ITS2 conservadas para os isolados de P. brasiliensis, e polimórficas para B. dermatitidis, segundo o sequenciamento feito por Hebeler- Barbosa et al. (2003). Estes primers (PbITSE e PbITSR) são utilizados como inner primers, em condição de Nested-PCR com os outer primers (ITS4 e ITS5) universais para fungos e mostraram ser promissores para estudos de ocorrência ambiental do patógeno (THEODORO et al., 2005). Sistema de Informação Geográfica (SIG) Outra potente ferramenta para estudos eco-epidemiológicos é a utilização do Sistema de Informação Geográfica (SIG), a qual já vem sendo amplamente utilizada na análise da distribuição geográfica e dinâmica de diversas doenças, das mais variadas etiologias (LINTHICUM et al., 1987; ROGERS & RANDOLPH, 1991; BECK et al., 1994), inclusive no estudo da Coccidioiodomicose (BAPTISTA-ROSA et al., 2007), Blastomicose (REED et al., 2008) e PCM, realizado em nosso laboratório (SIMÕES et al., 2004). A aplicação de Geoprocessamento no estudo da ecologia de P. brasiliensis pode auxiliar na identificação do seu hábitat, além de estabelecer indicadores ambientais facilmente mapeáveis, permitindo a identificação das áres de risco à infecção. Desta forma, a pesquisa de novos hospedeiros utilizando-se da combinação das técnicas de Biologia Molecular e de SIG são um diferencial nos estudos de eco-epidemiologia do P. brasiliensis, bem como de outros patógenos Onygenales. CC aa pp íí tt uu lloo II 18 Molecular approaches for eco-epidemiological studies of Paracoccidioides brasiliensis Submitted: Memórias do Instituto Oswaldo Cruz Virgínia Bodelão Richini-Pereira1, Sandra de Moraes Gimenes Bosco1, Raquel Cordeiro Theodoro1, Severino Assis da Graça Macoris1, Eduardo Bagagli1* 1 Dept. Microbiologia e Imunologia, Instituto de Biociências, UNESP-Botucatu. Summary Medical mycology has greatly benefited from the introduction of molecular techniques. The advances in our understanding about several aspects of pathogenic fungi have been notable in the latest years, providing both theoretical and practical support. Considering Paracoccidioides brasiliensis in particular, important eco-epidemiological aspects, such as environmental distribution, and new hosts were learnt through molecular approaches. These methodologies also contributed to a better understanding about the genetic variability of this pathogen; thus, P. brasiliensis is now assumed to represent a species complex. The present review focuses on some recent findings about the current taxonomic status of P. brasiliensis species complex, its phylogenetic and speciation processes, as well as on some practical applications for the molecular detection of this pathogen in environmental and clinical materials. Key words: Paracoccidioides brasiliensis, eco-epidemiology, PCR Sponsorships: Fapesp (05/56771-9 and 06/03597-4) Paracoccidioides brasiliensis Paracoccidioidomycosis (PCM) is the most important and prevalent systemic mycosis in Latin America, mainly in Brazil, Colombia and Venezuela (Wanke & Londero 1994). It is caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus that grows as a yeast-like structure in the host tissues or when cultured at 35-37ºC and as a mycelium under saprobic conditions or when cultured at room temperature, 18-23ºC (Lacaz 1994). Similarly to other systemic mycosis also caused by dimorphic fungi in which the lung is the main compromised organ, PCM-infection is believed to be acquired through the CC aa pp íí tt uu lloo II 19 inhalation of conidia present in the environment (Gonzales-Ochoa 1956, Bustamante et al. 1985). Although PCM is considered an endemic disease in many regions, there are few reports in literature about the isolation of this pathogen from the environment, which has made ecological studies on P. brasiliensis a hard work for mycologists (Shome & Batista 1963, Negroni 1966, Albornoz 1971, Restrepo 1985, Silva-Vergara et al. 1998, Franco et al. 2000). There is evidence of P. brasiliensis isolation from bat and penguin feces and dog food. However, these were casual remarks with no reproducibility (Grose & Tamsitt 1965, Gezuele 1989, Ferreira et al. 1990). Besides, the lack of outbreaks and the prolonged latency period of this disease, associated with human migration, lead the exact infection source to remain unknown (Restrepo 1985). An important clue for ecological studies on P. brasiliensis was the finding that the nine-banded armadillo (Dasypus novemcinctus) is naturally infected with this pathogen (Naiff et al. 1986, Bagagli et al. 1998, 2003, Corredor et al. 1999, Silva-Vergara 1999). The fungus was also isolated from another armadillo species, Cabassous centralis, reinforcing that armadillos are in constant contact with the pathogen in the environment (Corredor et al. 2005). The systematic recovery of P. brasiliensis from armadillo tissues has demonstrated the importance of this animal in PCM endemic areas, helping locate hot spots of the fungus occurrence in some environments, and suggested valuable insights about the pathogen evolutionary aspects (Bagagli et al. 2006, 2008). The environment represented by the armadillo burrow and its surroundings, associated with biotic and abiotic features, may contribute to the development of the fungus saprobic stage in nature, as already demonstrated by Terçarioli et al. (2007). For a long time, armadillos had been the unique animal species in which the fungus recovery from tissue cultures was possible, although many attempts have been made in other animal species from PCM endemic areas. Recently, other wild mammals such as guinea pig (Cavia aperea), porcupine (Sphiggurus spinosus), raccoon (Procyon cancrivorus) and grison (Gallictis vittata) have been recognized through molecular methods as new hosts for this infection (Richini-Pereira et al. 2008). Learning about the natural reservoirs of this pathogenic fungus may contribute to map endemic areas and to better understand its eco-epidemiological features. However, some questions still need to be elucidated, such as: Does the fungus need some special substrate or a specific period to grow? Which are the most appropriate CC aa pp íí tt uu lloo II 20 environmental conditions, climate and soil type? Which kind of host-parasite relationship does the fungus establish? Is there another infection route, besides the airborne route? Medical mycology has extensively benefited from the great development of molecular biology in the latest years. Thus, the aim of this review is to present some recent advances in the understanding about the eco-epidemiology of P. brasiliensis and related species through molecular approaches. Molecular Biology demonstrated that P. brasiliensis belongs to a peculiar Ascomycota Family Morphological and molecular findings have suggested that the main pathogenic fungi causing systemic mycosis had been classified into Ajellomycethaceae, a new family of vertebrate-associated Onygenales, which includes fungi of the genera Histoplasma, Blastomyces, Emmonsia and Paracoccidioides (Untereiner et al. 2004). The correct taxonomic position of these fungi has opened new possibilities for the study and understanding about their eco-epidemiological relationships with their respective hosts. This fungal group (Onygenales, Onygenaceae sensu lato) presents several common mycological and ecological features such as dimorphism, arthroconidia and restricted geographic distribution. In addition, the natural affinity of some of its members, such as Blastomyces dermatitidis and Histoplasma capsulatum, for animal product derivatives or remnants like feces and uric acid is well documented (Baumgardner & Paretsky 1999, Restrepo et al. 2000, Untereiner et al. 2004). Several evidence lines indicate that P. brasiliensis is phylogenetically closer to Lacazia loboi than to B. dermatididis, E. parva and H. capsulatum (Bialek et al. 2000, Herr et al. 2001). It must be emphasized that L. loboi could not yet be cultured on standard media and is therefore considered an obligate parasite (Taborda et al. 1999, Herr et al. 2001). This suggests a possible tendency for reduction or extinction of the fungus saprobic form in nature. Lacaziosis, a mycosis caused by L. loboi, is acquired through traumatic route and has a typical chronic form, occurring in a restricted region of South America; it has also been observed in some wild aquatic mammals, specially dolphins (Herr et al. 2001). It must be considered that this phylogenetic group (Onygenales) includes the species Emmonsia parva and E. crescens which cause adiaspiromycosis, a localized lung infection of cosmopolite distribution, both in wild animals and humans (Hubalék et al. 1998). Some aspects of their saprobic and parasitic stages have led Emmonsia species to be considered close relatives of dimorphic fungi (Emmons & Ashburn 1942, Sigler 1996, Peterson & Sigler CC aa pp íí tt uu lloo II 21 1998). Their occurrence has been related to the agroecosystem, and the highest abundance was observed in plant remnants and rodent burrows (Hubalék et al. 1998). The presence of this fungus has been reported in domestic and wild rodents (Zlatanov & Genov 1975, Hubalék et al. 1998), carnivores (Krivanec et al. 1980), and armadillos (Santos 1999). Studies have also indicated that the different Onygenaceae (sensu lato) species may originated in the Americas around 3-20 million years ago (Fisher et al. 2000), and some of the most important ones, such as Histoplasma and Paracoccidioides species, had certainly evolved in South America, prior to the Panamanian Isthmus connection (Kasuga et al. 1999, 2003). Molecular Phylogenetics indicates that P. brasiliensis is a species complex Like all living organisms, P. brasiliensis has been shown to present variable characters (both morphologic and molecular), and the correlation of these different features with its clinical manifestations (in animals and humans) is the new challenge for several studies on PCM. Isolates obtained from armadillos and humans presented a significant diversity in virulence (Calcagno et al. 1998, Sano et al. 1999, Hebeler-Barbosa et al. 2003a, 2003b). Hebeler-Barbosa et al. (2003a, 2003b) compared these isolates with those obtained from human patients, concerning virulence (in hamster model), through RAPD and ITS1-5.8S- ITS2 sequencing. They did not recognize any separation between armadillo and human isolates, concluding that the host, in this case, was not a differentiation factor among them. Therefore, human and animals can be infected with the same ecopathogenotypes (Bagagli et al. 1998, Franco et al. 2000, Restrepo et al. 2000), which makes this aspect useful for mapping P. brasiliensis genotypes that cause human infections in risk areas. Recent phylogenetic studies have revealed cryptic speciation for P. brasiliensis through Multi-Locus Sequence Type (MLST). Differently from the morphological and biological recognition of species, which is rarely successfully applicable to separate fungus species (Taylor et al. 2000), MLST is more reliable as it deals with several nuclear genes and detects the species limits through genealogical concordance (the clade must be present in the majority of the single-locus genealogies) with high bootstrap and posterior probability values supporting the clades. Matute et al. (2006) analyzed eight regions of five nuclear coding genes: chitin synthase (promoter-exon1 and exon 2 to 4), �-glucan synthase (exon2 and exon 3), �-tubulin (exons 2-4), adenyl ribosylation factor (exons 2-3) and PbGP43 (promoter-exon 1 and exon 2), and detected three distinct, previously unrecognized species: S1 (species 1 CC aa pp íí tt uu lloo II 22 from Brazil, Argentina, Paraguay, Peru and Venezuela), PS2 (phylogenetic species 2 from Brazil and Venezuela) and PS3 (phylogenetic species from Colombia). They also observed that S1 and PS2 were recombining sexual species, whereas PS3 was shown to be clonal. It is interesting to note that S1 and PS2 are sympatric and reproductively isolated, although they have sexual reproductive attributes (Matute et al. 2006). These data were corroborated by microsatellite analysis of the same isolates used in the previous gene genealogy (Matute et al. 2006), and the divergence time between PS2 and PS3 was estimated as 8.04-8.37mya through chitin synthase gene analysis (Matute et al. 2007). Those authors suggested that the speciation of PS3, geographically restricted to Colombia, could be attributed to dispersal, leading to genetic isolation of PS3 from S1 (allopatric speciation). The other speciation event that originated PS2 is still poorly understood. S1 and PS2 may be the result of sympatric speciation, the initial step of which is the existence of polymorphism, much more abundant in recombining species such as PS2 and S1. Therefore, two forms of one single species can be adapted to different conditions in their niche, leading to negatively selected interbreeding due to the low adaptive value of the hybrids (Coyne & Orr 2004). Recently, more P. brasiliensis isolates from the central region of Brazil were included in a new MLST analysis and showed a significant genetic divergence when compared with other S1, PS2 and PS3 isolates (Carrero et al. 2008, Teixeira 2008). This cluster of isolates was named Pb01-like, since the first studies that revealed such divergence were carried out with Pb01 isolate. Phylogenetic analyses with several nuclear encoding regions such as GP43, CH4, Actin, ODC, URA3, CHS2, FKS1, HSP70, Hydrophobin, Kex, Catalase A, Catalase P, Formamidase, and Glyoxalase showed a significant genetic distance between Pb01 and the remaining genetic groups (Carrero et al. 2008). Pb01-like isolates form a well supported clade for several nuclear coding genes, suggesting the existence of one more cryptic species of P. brasiliensis (Teixeira 2008, Carrero et al. 2008). The divergence time between Pb01-like and the remaining clades was estimated as 20mya, a long evolutionary time (Teixeira 2008). However, it is not clear yet which factors (geographic, biotic and abiotic) could have contributed to the genetic isolation of this group right in the central region of Brazil. This Pb01 isolate, which has been the subject of genome and transcriptome projects (Felipe et al. 2005), also substantially differs from the others as to the hsp70 gene that encodes a conservative heat shock protein (Teixeira et al. 2005, Theodoro et al. 2008). Four large insertions: one of 4 nucleotides, one of 16, a third one of 23, and another one of 21 (the latter corresponds to a CT microsatellite), were observed in the hsp70 gene of Pb01, relative to P. brasiliensis isolates from the other species (Teixeira et al. 2005, Theodoro et al. 2008). CC aa pp íí tt uu lloo II 23 An additional molecular marker, prp8 gene intein, was used to recognize P. brasiliensis isolates of the different genetic groups (Theodoro et al in press). Inteins are coding sequences that are transcribed and translated with flanking sequences (exeins). After translation, inteins are excised by an autocatalytic process; then, the host protein assumes its normal conformation and can develop its function (Cooper et al. 1995). These parasitic genes have been found in several vital proteins in all three life domains and are largely spread in fungi; among them, PRP8 intein is known to occur in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), C. gattii, Histoplasma capsulatum, and P. brasiliensis (Butler & Poulter 2005, Butler et al. 2006). There are two intein types, mini-inteins and full-length inteins, both of which have a Splicing domain. Full- length inteins additionally present an Endonuclease domain that can play a homing function (Homing Endonuclease Gene or HEG), which makes intein a mobile genetic element, resulting in both the occupation of empty alleles and the duplication of parasitic genetic element (Liu 2000). These large inteins are expected to have more sequence variation in the Endonuclease domain than in the Splicing domain due to a more relaxed selection, especially if the HEG is no longer active (Gorgaten & Hilario 2006), which constitutes thus a promising source of phylogenetic information. In this assay, PRP8 intein was sequenced for 22 P. brasiliensis isolates belonging to all four previously recognized species. The phylogenetic analysis clearly separated the isolates from the four species and revealed a significant difference between Pb01-like and the remaining species (Theodoro et al. in press). All evaluated P. brasiliensis isolates presented full-length intein in prp8 gene. The HEG domain of PRP8 intein from P. brasiliensis appears to be inactive due to a substitution in the second aspartic acid residue, which is indispensable to its functionality. Although the polymorphism degree in PRP8 intein from P. brasiliensis was not as high as that in the commonly used nuclear-coding, this sequence contains sufficient phylogenetic information to separate cryptic species of P. brasiliensis, constituting therefore a reliable additional molecular marker for this pathogen. Distinct cryptic species have been recognized through molecular techniques, which also facilitate the detection of other biological features that could be associated with the different genetic groups. Theodoro et al. (2008) detected some microscopic features of S1 and PS2 isolates that could be important candidates for a morphological differentiation between these two species. The isolates T10 and Bt84, representing PS2, seemed to have elongated yeast cells and presented slower M-L transition when compared with the other isolates. In addition, Terçarioli et al. (2007) studied fungal growth and conidia production on CC aa pp íí tt uu lloo II 24 Soil Extract Agar and observed that most isolates of S1 group produced large quantities of conidia; the same was not observed in PS2 group isolates. Both S1 and PS2 genetic groups are sympatric in the same endemic area; however, S1 is curiously much more frequent in both patients and armadillos, probably due to its higher conidium production (Bagagli et al. 2008). As a large number of isolates from different areas are molecularly characterized, the geographical distribution of such strains becomes clearer. Thus, S1 appears to be widely distributed, sympatrically occurring with PS2 in several endemic areas; PS3 group is centralized in Colombia; and Pb01-like, in the central region of Brazil. The geographic distribution of the different genetic groups of P. brasiliensis species complex is summarized in Figure 1, according to the currently available data. Figure 1 - Geographic distribution of the different genetic groups of P. brasiliensis species complex, according to the actual available data. Since prophylactic or treatment measures must preferentially include all genotypes and/or phenotypes of P. brasiliensis, which causes PCM, the discovery of cryptic species CC aa pp íí tt uu lloo II 25 increased the importance of comparative studies aim at detecting some phenotypic differences among species. These studies will provide important evidence for correct diagnosis, specially regarding antigen variation, clinical presentation and distinct responses to antifungal drugs. Pathogen detection – clinical and environmental sources PCM diagnosis can be traditionally made through culture, microscopic detection of yeast cells in clinical specimens, histopathology, and serological tests, specially including gp43, a reference P. brasiliensis antigen (Lacaz 1994). Each of these procedures has advantages and disadvantages. Although a definitive diagnosis can be made through fungal culture, it must be considered that such procedure involves high contamination risk, long incubation period and low sensitivity due to the fungus scarcity in clinical samples (Salina et al. 1998, Sano et al. 2001). In histological sections, the etiological agent can be lost or confused with other dimorphic fungi (Bialek et al. 2000). Several serology techniques using different P. brasiliensis antigen types have been employed for both diagnosis and monitoring therapy of patients; however, some problems such as anergy, cross reactivity, and the technique specificity and sensitivity must be considered (Albuquerque et al. 2005). Recent advances in molecular biology, mainly based on polymerase chain reaction (PCR), provided powerful tools for the detection, identification and typing of different pathogen groups. The main genomic regions employed for primer designs that have been applied for P. brasiliensis, both clinical and environmental samples, are listed in Table 1. T ab le 1 - P ri m er u se d fo r de te ct io n of P . b ra si li en si s pr im er s ge ne am pl ic on s iz e pr im er s se qu en ce s ev al ua te d sa m pl es re fe re nc es P ri m er 1 β -a ct in 62 bp 5’ -T C G T T A T C C T C A T C G A A -3 ’ fu ng al c ul tu re se ru m ( m ic e in oc ul at ed ) G ol da ni e t a l. (1 99 5) G ol da ni & S ug ar ( 19 98 ) P ri m er 2 5’ -A A G A G T C T T C C C T C G C -3 ’ P C 1 gp 43 10 30 bp 5� -T C A T C T C A C G T C G C A T C T C A C A T T -3 � cl in ic al ( sp ut um ) G om es e t a l. (2 00 0) P C 5 5� -A G C G C C A G A T G G T T T G C C C G C T A G G A A C G A A -3 ’ P C 2 60 0b p 5� -A T A G A G G G A G A G C C A T A T G T A C A A G G T -3 � P C 6 5� -G G C T C C T C A A A G T C T G C C A T G A G G A A G 3 ’ pa ra I gp 43 35 5 bp 5’ -A A C T A G A A T A T C T C A C T C C C A G T C C -3 ’ fu ng al c ul tu re ti ss ue ( m ic e in oc ul at ed a nd pa ra ff in e m be dd ed ) B ia le k et a l. (2 00 0) R ic ci e t a l. (2 00 7) pa ra I I 5’ -T G T A G A C G T T C T T G T A T G T C T T G G G -3 ’ pa ra I II 19 6b p 5’ -G A T C G C C A T C C A T A C T C T C G C A A T C -3 ’ pa ra I V 5’ -G G G C A G A G A A G C A T C C G A A A T T G C G -3 ’ L O p2 7 53 6b p 5’ -C A A C T C T T G G C T T T G G T T G A A G -3 ’ ti ss ue ( ar m ad il lo s, m ic e in oc ul at ed ) so il ar ti fi ci al ly c on ta m in at ed D ié z et a l. (1 99 9) C or re do r et a l. (1 99 9) U P 5’ -C T G T T G T T T C C G T C C T T G C G C -3 ’ M G 2( 1) F ge no m ic D N A 28 5 bp 5’ -G G G A T T C C C T A G G C A A A C A C T T G T G T G A - 3’ fu ng al c ul tu re cl in ic al ( sp ut um , C SF ) Sa n- B la s et a l. (2 00 5) M G 2( 1) R 5’ -G T G C A G T T A T C C A C A A G C C A T A T A T T C -3 ’ M G 2( 2) F 28 8 bp 5’ -G G A G A T G A T C T G A C G T T A G T A C G T G A T G - 3’ M G 2( 2) R 5’ -A T G C T A A T T T A T G T C A T T C C G C G T C T G -3 ’ O L 3 rD N A 20 3b p 5’ -C T C A G C G G G C A C T T -3 ’ fu ng al c ul tu re M ot oy am a et a l. (2 00 0) U N I- R 5’ -G G T C C G T G T T T C A A G A C G -3 ’ P bI T S1 s* rD N A 41 8 bp 5’ -C C G C C G G G G A C A C C G T T G -3 ’ fu ng al c ul tu re Im ai e t a l. (2 00 0) P bI T S3 a* 5’ -A A G G G T G T C G A T C G A G A G -3 ’ P b- IT S- E * rD N A 38 7 bp 5’ -G A G C T T T G A C G T C T G A G A C C -3 ’ fu ng al c ul tu re ti ss ue ( ar m ad ill os a nd r oa d- ki ll ed w il d an im al s) so il T he od or o et a l. (2 00 5) T er ça ri ol i e t a l. (2 00 7) R ic hi ni -P er ei ra e t a l. (2 00 8) P b- IT S- R * 5’ -A A G G G T G T C G A T C G A G A G A G -3 ’ *T he f ir st P C R w as c ar ri ed o ut u si ng p an fu ng al p ri m er s su ch a s IT S 1, I T S 4 an d IT S5 ( W hi te e t a l. 19 90 ). CC aa pp íí tt uu lloo II 27 Goldani et al. (1995) designed specific primers based on β-actin gene which amplified a 62bp-fragment not detected in other fungi such as H. capsulatum, B. dermatitidis, Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus, Sacharomyces cerevisiae, and Pneumocystis carinii. Later, Goldani & Sugar (1998) used the same set of primers and obtained positivity in sera from five experimentally infected mice. Those authors suggested further evaluation of different PCM clinical forms to assess the true value of this diagnostic approach. PCR assays targeting gp43 gene (PbGP43) have been widely used for the molecular detection of P. brasiliensis DNA in several samples. Cisalpino et al. (1996) cloned and characterized the entire coding region of gp43 gene, which is constituted of 2 exons interrupted by a 78bp-intron. After this study, several sets of primers were designed and employed for molecular diagnosis. Gomes et al. (2000) combined five primer pairs and suggested a PCR using PC2-PC6 primer pair for direct amplification from clinical material (sputum) or Nested- PCR following the PC1-PC5 primer set amplification. In an experimental model infected with the yeasts H. capsulatum and P. brasiliensis, Bialek et al. (2000) employed a Nested-PCR, also based on gp43 gene, with high sensitivity and specificity for the sets of primers para I/II and para III/IV. Using these same sets of primers, Ricci et al. (2007) demonstrated 30% positivity in pathogen detection in biopsies from PCM-patients. Those authors emphasized that this low positivity might be due to the procedures used for fixation, paraffin embedding and storage of the material, which favored DNA degradation, and the use of primers of gp43 regions that have been proved to be polymorphic. This fact may have interfered with the perfect annealing of the employed primers (San-Blas et al. 2002, Morais et al. 2000). Since gp43 showed to be one of the most polymorphic genes that clearly separates the four cryptic species of P. brasiliensis (Matute et al. 2006, Carrero et al. 2008, Teixeira 2008), PCR based on this genomic region is now indicated not only for the pathogen detection, but also for its genotyping in clinical samples. By using this strategy, Ricci et al. (2008) evaluated and distinguished between S1 and PS2 from paraffin-embedded tissue. Besides gp43 glycoprotein, a 27-kDa antigen protein was cloned, sequenced and characterized by McEwen et al. (1996). The use of LO and UP primer combination allowed the detection of P. brasiliensis DNA in tissue samples of armadillos, inoculated mice and artificially contaminated soil (Corredor et al. 1999, Díez et al. 1999). Internal transcribed spacer (ITS) regions, including the 5.8S ribosomal DNA of P. brasiliensis, are also employed for molecular detection and distinction from other fungal CC aa pp íí tt uu lloo II 28 species. It is known that ribosomal DNA (rDNA) genes are present in all microorganisms and have regions that accumulate mutations both at slow (28S, 5.8S and 18S) and high (ITS1 and ITS2) rates over time. The first regions provide molecular basis for establishing phylogenetic relationships among taxonomic levels above genus, and the second regions are useful for the separation between genera and species. Besides, this target provides higher sensitivity to PCR due to its several copies per genome (more than 100 copies) (White et al. 1990, Iwen et al. 2002). This region has been considered the main candidate for Barcoding system in fungal identification (Buckley 2008). PCR of rDNA (5.8S-ITS) can be amplified by panfungal primers and the sensitivity and specificity of this reaction may be significantly improved by Nested-PCR with species- specific inner primers. Using this method, Imai et al. (2000) designed a set of primers, PbITS1s and PbITS3a, for Nested-PCR and identified 29 P. brasiliensis strains by means of a specific 418bp-fragment, which was not detected in A. fumigatus, B. dermatididis, C. albicans, C. neoformans, H. capsulatum and P. marneffei. Motoyama et al. (2000) employed the OL3 and UNI-R primer combination for PCR and obtained a 203bp-fragment when P. brasiliensis (Pb01) DNA was used as the template. These primers were capable of discriminating between P. brasiliensis and H. capsulatum. Nested-PCR, also using species-specific inner primers (PbITSE and PbITSR) derived from ITS-5.8S rDNA, was developed for P. brasiliensis detection in soil (Theodoro et al. 2005, Terçarioli et al. 2007). This technique also proved to be useful for fungal detection in road-killed wild animals (Richini-Pereira et al. 2008). Since P. brasiliensis actually consists of a species complex with distinct genetic groups, it must be emphasized that all molecular protocols should be re-evaluated both in silico and experimentally to detect any genotype that cause PCM in endemic areas. References Albornoz MB 1971. Isolation of Paracoccidioides brasiliensis from rural soil in Venezuela. Sabouraudia 9: 248-253. Albuquerque CF, Silva SHM, Camargo ZP 2005. Improvement of the specificity of na enzyme-linked immunosorbent assay for diagnosis of Paracoccidioidomycosis. J Clin Microbiol 43(4): 1944-1946. Bagagli E, Sano A, Coelho KIR, Alquati S, Miyaji M, Camargo ZP, Gomes G, Franco M, Montenegro MR 1998. Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus CC aa pp íí tt uu lloo II 29 novemcinctus) captured in an endemic area of paracoccidioidomycosis. Am J Trop Med Hyg 58: 505-512. Bagagli E, Franco M, Bosco SMG, Hebeler-Barbosa F, Trinca LA, Montenegro MR 2003. High frequency of Paracoccidioides brasiliensis infection in armadillo (Dasypus novemcinctus): an ecological study. Med Mycol 41: 217-223. Bagagli E, Bosco SMG, Theodoro RC, Franco M 2006. Phylogenetic and evolutionary aspects of Paracoccidioides brasiliensis reveal a long coexistence with animal hosts that explain several biological features of the pathogen. Infect Genet Evol 6(5): 344-351. Bagagli E, Theodoro RC, Bosco SMG, McEwen J 2008. Paracoccidioides brasiliensis: phylogenetic and ecological aspects. Mycopathologia 165: 197-207. Baumgardner DJ, Paretsky DP 1999. The in vitro isolation of Blastomyces dermatitidis from a woodpile in north central Wisconsin, USA. Med Mycol 37(3): 163-168. Bialek R, Ibricevic A, Aepinus C, Najvar Lk, Fothergill AW, Knobloch J, Graybill JR 2000. Detection of Paracoccidioides brasiliensis in tissue samples by a Nested PCR assay. J Clin Microbiol 38: 2940-2942. Buckley M 2008. The fungal kingdom diverse and essential roles in earth’s ecosystem. American Academy of Microbiology, Washington, 44p. Bustamante B, McEwen JG, Tabares, AM, Arango M, Restrepo A 1985. Characteristics of the conidia produced by the mycelial form of Paracoccidioides brasiliensis. Sabouraudia 23: 407-414. Butler ML, Poulter RTM 2005. The PRP8 inteins in Cryptococcus are a source of phylogenetic and epidemiological information. Fungal Genet Biol 42: 452-463. Butler ML, Gray J, Goodwin TJD, Poulter RTM 2006. The distribution and evolutionary history of the PRP8 intein. BMC Evol Biol 6:1-26. Calgagno AM, Nino-Veja G, San-Blas F, San-Blas G 1998. Geographic discrimination of Paracoccidioides brasiliensis strains by randomly amplified polymorphic DNA analysis. J Clin Microbiol 36: 1733-1736. CC aa pp íí tt uu lloo II 30 Carrero LL, Niño-Vega G, Teixeira MM, Carvalho MJA, Soares CMA, Pereira M, Jesuíno RSA, McEween JG, Mendoza L, Taylor J W, Felipe MS, San-Blas G 2008. New Paracoccidioides brasiliensis isolate reveals unexpected genomic variability in this human pathogen. Fung Genet Biol 45(5): 605-612. Cisalpino PS, Puccia R, Yamauchi LM, Cano MI, Silveira JF, Travassos LR 1996. Cloning, characterization, and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis. J Biol Chem 271: 4553-4560. Cooper AA, Stevens TH 1995. Protein splicing: self-splicing of genetically mobile elements at the protein level. Trends Biochem Sci 20: 351-356. Corredor GG, Castaño JH, Peralta LA, Díez S, Arango M, McEwen J, Restrepo A 1999. Isolation of Paracoccidioides brasiliensis from the nine-banded armadillo Dasypus novemcinctus, in an endemic area for paracoccidioidomycosis in Colombia. Rev Iberoam Micol 16: 216-220. Corredor GG, Peralta LA, Castaño JH, Zuluaga JS, Henao B, Arango M, Tabares AM, Matute DR, McEwen JG, Restrepo A 2005. The naked-tailed armadillo Cabassous centralis (Miller 1899): a new host to Paracoccidiodes brasiliensis. Molecular identification of the isolate. Med Mycol 43(3): 275-280. Coyne JA, Orr HA 2004. Speciation. Sinauer Associates, Sunderland, MA. Díez S, Garcia EA, Pino PA, Botero S, Corredor GG, Peralta LA, Castaño JH, Restrepo A, McEwen JG 1999. PCR with Paracoccidioides brasiliensis specific primers: potential use in ecological studies. Rev Inst Med Trop Sao Paulo 41(6): 351-358. Emmons CW, Ashburn LL 1942. The isolation of Haplosporangium parvum n. sp. and Coccidioides immitis from wild rodents. Public Health Rep 57: 1715-1727. Felipe MS, Torres FA, Maranhão AQ, Silva-Pereira I, Poças-Fonseca MJ, Campos EG, Moraes LM, Arraes FB, Carvalho MJ, Andrade RV, Nicola AM, Teixeira MM, Jesuíno RS, Pereira M, Soares CM, Brígido MM 2005. Functional genome of the human pathogenic fungus Paracoccidioides brasiliensis. Fems Immunol Med Microbiol 45(3): 369-381. CC aa pp íí tt uu lloo II 31 Ferreira MS, Freitas LS, Lacaz CS, Del Negro GM, Aielo NT, Garcia NM, Assis CM, Salebian A, Heris-Vaccari EM 1990. Isolation and characterization of a Paracoccidioides brasiliensis strain in dogfood probably contaminated with soil in Uberlândia, Brazil. J Med Vet Mycol 28: 253-256. Fisher MC, Koenig GL, White TJ, Taylor JW 2000. A test for concordance between the multilocus genealogies of genes and microsatellites in the pathogenic fungus Coccidioides immitis. Mol Biol Evol 17:1164-1174. Franco M, Bagagli E, Scapolio S, Lacaz CS 2000. A critical analysis of isolation of Paracoccidioides brasiliensis from soil. Med Mycol 38: 185-191. 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Paracoccidioides brasiliensis recovered from the intestinal tract of three bats (Artibeus lituratus) in Colombia, S.A. Sabouraudia 4: 124-125. Hebeler-Barbosa F, Montenegro MR, Bagagli E 2003a. Virulence profiles of ten Paracoccidioides brasiliensis isolates obtained from armadillos (Dasypus novemcinctus). Med Mycol 41: 89-96. CC aa pp íí tt uu lloo II 32 Hebeler-Barbosa F, Morais FV, Montenegro MR, Kuramae EE, Montes B, McEwen JG, Puccia R, Bagagli E 2003b. Sequence comparison of the internal transcribed spacer regions and gp43 in Paracoccidioides brasiliensis for patients and armadillos Dasypus novemcinctus. J Clin Microbiol 41: 5735-5737. Herr RA, Tarcha EJ, Taborda PR, Taylor JW, Ajello L, Mendoza L 2001. Phylogenetic analysis of Lacazia loboi places this previously uncharacterized pathogen within the dimorphic Onygenales. J Clin Microbiol 39 (1): 309-314. Hubalék Z, Nesvadbova J, Halouzka J 1998. Emmonsiosis of rodents in an agroecosystem. Med Mycol 36(6): 387-390. Imai T, Sano A, Mikami Y 2000. A new PCR primer for the identification of Paracoccidioides brasiliensis based on rRNA sequences coding the internal transcribed spacers (ITS) and 5.8 regions. Med Mycol 38: 323-326. Iwen PC, Hinrichs SH, Rupp ME 2002. Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens. Med Mycol 40(1): 87- 109. Kasuga T, Taylor JW, White TJ 1999. Phylogenetics relationships of variets and geographical groups of the human pathogenic fungus Histoplasma capsulatum darling. J Clin Microbiol 37: 653-663. Kasuga T, White TJ, Koenig G, McEwen J, Restrepo A, Castañeda E, Lacaz CS, Heins- Vaccari, EM, Freitas RS, Zancopé-Oliveira RM, Qin Z, Negroni R, Carter DA, Mikami Y, Tamura M, Taylor ML, Miller GF, Poonwan N, Taylor JW 2003. Phylogeography of the fungal pathogen Histoplasma capsulatum. Mol Ecol 12: 3383-3401. Krivanec K, Otcenasek M, Slais J 1980. Adiaspiromycosis in large free living carnivores. Mycopathologia 71: 125-126. Lacaz CS 1994. Historical evolution or the knowledge on Paracoccidioidomycosis and its etiologic agent, Paracoccidioides brasiliensis In: Franco M, Lacaz CS, Restrepo A, Del Negro G eds. Paracoccidioidomycosis. Boca Raton, FL: CRC Press, 410p. CC aa pp íí tt uu lloo II 33 Liu XQ 2000. Protein-splicing intein: genetic mobility, origin, and evolution. Ann Rev Genet 34: 61-76. Matute DR, McEween JG, Montes BA, San-Blas G, Bagagli E, Rauscher JT, Restrepo A, Morais F, Nino-Veja G, Taylor JW 2006. Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies. Mol Biol Evol 23: 65-73. Matute DR, Torres IP, Salgado-Salazar C, Restrepo A, McEwen JG 2007. Background selection at the chitin synthase II (chs2) locus in Paracoccidioides brasiliensis species complex. Fungal Genet Biol 44(5): 357-367. McEwen JG, Ortiz BL, Garcia AM, Florez AM, Botero S, Restrepo A 1996. Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. Fungal Genet Biol 20: 125-131. Morais FV, Barros TF, Fukada MK, Cisalpino PS, Puccia R 2000. Polymorphism in the gene coding for the immunodominant antigen gp43 from the pathogenic fungus Paracoccidioides brasiliensis. J Clin Microbiol 38(11): 3960-3966. Motoyama AB, Venancio EJ, Brandão GO, Petrofeza-Silva S, Pereira IS, Soares CM, Felipe MS 2000. Molecular identification of Paracoccidioides brasiliensis by PCR amplification of ribosomal DNA. J Clin Microbiol 38(8): 3106-3109. Naiff RD, Ferreira LCP, Barret TV, Naiff MF, Arias JR 1986. Paracoccidioidomicose enzoótica em tatus (Dasypus novemcinctus) no Estado do Pará. Rev Inst Med Trop Sao Paulo 28: 19-27. Negroni P 1966. El Paracoccidioides brasiliensis vive saprofiticamente en el suelo Argentino. Prensa Med Argent 53: 2381-2382. Peterson SW, Sigler L 1998. Molecular genetic variation in Emmonsia crescens and Emmonsia parva, etiologic agents of adiaspiromycosis, and their phylogenetic relationship to Blastomyces dermatitidis (Ajellomyces dermatitidis) and other systemic fungal pathogens. J Clin Microbiol 36: 2918-2925. Restrepo A 1985. The ecology of Paracoccidioides brasiliensis: a puzzle still unsolved. J Med Vet Mycol 23: 323-334. CC aa pp íí tt uu lloo II 34 Restrepo A 2000. Morphological aspects of Paracoccidioides brasiliensis in lymph nodes: implications for the prolonged latency of paracoccidioidomycosis? Med Mycol 38: 317-322. Ricci G, Silva ID, Sano A, Borra RC, Franco M 2007. Detection of Paracoccidioides brasiliensis by PCR in biopsies from patients with paracoccidioidomycosis: correlation with the histopathological pattern. Pathologica 99(2): 41-45. Ricci G, Zelck U, Mota F, Lass-Flörl C, Franco MF, Bialek R 2008. Genotyping of Paracoccidioides brasiliensis directly from paraffin embedded tissue. Med Mycol 43(1): 31- 34. Richini-Pereira VB, Bosco SMG, Griese J, Theodoro RC, Macoris SAG, Silva RJ, Barrozo L, Tavares PMS, Zancopé-Oliveira RM 2008. Molecular detection of Paracoccidioides brasiliensis in road-killed wild animals. Med Mycol 46(1): 35-40. Salina MA, Shikanai-Yasuda MA, Mendes RP, Barraviera B, Mendes-Giannini MJ 1998. Detection of circulating Paracoccidioides brasiliensis antigen in urine of paracoccidioidomycosis patients before and during treatment. J Clin Microbiol 36: 1723- 1728. San-Blas G, Nino-Veja G, Iturriaga T 2002. Paracoccidioides brasiliensis and Paracoccidioidomycosis: molecular approaches to morphogenesis, diagnosis, epidemiology, taxonomy and genetics. Med Mycol 40: 225-242. Sano A, Tanaka R, Yokoyama K, Franco M, Bagagli E, Montenegro MR, Mikami Y, Miyaji M, Nishimura K 1999. Comparison between human and armadillo Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis. Mycopathologia 143(3): 165-169. Sano A, Yokoyama K, Tamura M, Mikami Y, Takahashi I, Fukushima K, Miyaji M, Nishimura K 2001. Detection of gp43 and ITS1-5.8S-ITS2 ribosomal RNA genes of Paracoccidioides brasiliensis in paraffin-embedded tissue. Nippon Ishinkin Gakkai Zasshi 42: 23-27. Santos VM 1999. Comportamento em cultura e diagnóstico morfológico da Emmonsia crescens em tatus. Rev Soc Bras Med Trop 32(3): 307. CC aa pp íí tt uu lloo II 35 Shome SK, Batista AC 1963. Occurrence of Paracoccidioides brasiliensis in the soil of Recife, Brazil. Rev Fac Med Fed Ceará 3: 90-94. Sigler L 1996. Ajellomyces crescens sp. nv., taxonomy of Emmonsia spp., and relatedness with Blastomyces dermatitidis (teleomorph Ajellomyces dermatitidis) J Med Vet Mycol 34: 303-314. Silva-Vergara ML, Martínez R, Chadu A, Madeira M, Freitas-Silva G, Leite Maffei CM 1998. Isolation of Paracoccidioides brasiliensis strain from the soil of a coffee plantation in Ibiá, State of Minas Gerais, Brazil. Med Mycol 36: 37-42. Silva-Vergara ML, Martínez R 1999. Role of the armadillo Dasypus novemcinctus in the epidemiology of Paracoccidioidomycosis. Mycopathologia 144: 131-133. Taborda PR, Taborda VA, McGinnis MR 1999. Lacazia loboi gen. nov., comb. nov., the etiologic agent of lobomycosis. J Clin Microbiol 37(6): 2031-2033. Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC 2000. Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 31(1): 21- 32. Teixeira MM, Carvalho MJA, Dantas AS, Felipe MSS 2005. The hsp70 gene presents sequence diffrences among isolates of Paracoccidioides brasiliensis. Rev Inst Med Trop S Paulo 47(suppl14): 48. Teixeira MM 2008. Tipagem molecular e evolução do gênero Paracoccidioides. Master Thesis, Universidade de Brasília, Faculdade de Medicina, 154pp. Terçarioli GR, Bagagli E, Reis GM, Theodoro RC, Bosco SMG, Macoris SAG, Richini- Pereira VB 2007. 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CC aa pp íí tt uu lloo II II 37 Molecular detection of Paracoccidioides brasiliensis in road-killed wild animals Medical Mycology: 46(1): 35-40, 2008 Short title: Paracoccidioides brasiliensis in road-killed animals Virgínia Bodelão Richini-Pereira*, Sandra de Moraes Gimenes Bosco*, Juliana Griese†, Raquel Cordeiro Theodoro*, Severino Assis da Graça Macoris1, Reinaldo José da Silva†, Lígia Barrozo‡, Patrícia Morais e Silva Tavares§, Rosely Maria Zancopé-Oliveira§, Eduardo Bagagli* * Departamento de Microbiologia e Imunologia, Instituto de Biociências de Botucatu, Universidade Estadual Paulista, Botucatu, SP, † Departamento de Parasitologia, Instituto de Biociências de Botucatu, Universidade Estadual Paulista, Botucatu, SP, ‡ Departamento de Geografia, Faculdade de Filosofia, Letras e Ciências Humanas, Universidade de São Paulo, São Paulo, SP and § Serviço de Micologia, Departamento de Micro-Imuno-Parasitologia, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil. Summary Paracoccidioides brasiliensis infection have been little studied in wild and/or domestic animals, which may represent an important indicator of the presence of the pathogen in nature. Road-killed wild animals have been used for surveillance of vectors of zoonotic pathogens and may offer new opportunities for eco-epidemiological studies of paracoccidioidomycosis (PCM). The presence of P. brasiliensis infection was evaluated by Nested-PCR in tissue samples collected from 19 road-killed animals; 3 Cavia aperea (guinea pig), 5 Cerdocyon thous (crab-eating-fox), 1 Dasypus novemcinctus (nine-banded armadillo), 1 Dasypus septemcinctus (seven-banded armadillo), 2 Didelphis albiventris (white-eared opossum), 1 Eira barbara (tayra), 2 Gallictis vittata (grison), 2 Procyon cancrivorus (raccoon) and 2 Sphiggurus spinosus (porcupine). Specific P. brasiliensis amplicons were detected in (a) several organs of the two armadillos and one guinea pig, (b) the lung and liver of the porcupine, and (c) the lungs of raccoons and grisons. P. brasiliensis infection in wild animals from endemic area might be more common than initially postulated. Molecular techniques can be used for detecting new hosts and mapping “hot spot” areas of PCM. CC aa pp íí tt uu lloo II II 38 Keywords: Paracoccidioides brasiliensis, Paracoccidiodomycosis, road-killed animals, molecular epidemiology Introduction Paracoccidioides brasiliensis is the etiological agent of Paracoccidioidomycosis (PCM), the most important and prevalent systemic mycosis in Latin America, mainly in Brazil, Colombia and Venezuela [1]. The fungus is thermo-dimorphic, growing as multi- budding yeast cells in the hosts or when cultured at 35-37ºC and as a mold under saprobic condition of 28-30ºC. It is in the latter phase that the fungus produces its infective propagula [2]. Since the recovery of P. brasiliensis from environmental sources is a rare event and since the disease has a prolonged latency period (with no outbreaks), its exact niche in nature remains a mystery. The observation that the nine-banded armadillo Dasypus novemcinctus, an primitive mammal that evolved in the same geographic area as P. brasiliensis, is naturally infected by the fungus opened up new research opportunities. The animal has been used as both a sentinel for locating risk areas of the disease and for supplying insights about the pathogen’s evolution [3]. Could the pathogen’s association with animal hosts be a primary strategy for the fungus to survive in nature or is the infection just a blind alley? These questions point to the necessity of looking for the fungus in several wild mammals. Wild animals, by living outdoors all the time and being constantly exposed to airborne pathogens, have been considered better environmental indicators of human risk than companion animals [4]. Since preservation of wildlife is a real concern, we are proposing herein to use highly sensitive molecular techniques with road-killed wild animals to detect the fungus. This approach, which has already been applied in parasitological studies, could also be useful to elucidate PCM eco-epidemiology. The use of sensitive and specific molecular tools would overcome the difficulties encountered in detecting the pathogen in host tissue through either culture or histopathological analysis. PCR reaction with panfungal primers from rDNA genomic region can be considered more sensitive because they usually target a multicopy gene [5]. The combined use of specific primers derived from this genomic region with Nested-PCR may increase the specificity of molecular detection of P.brasiliensis [6]. The present work aimed to describe possible new hosts of PCM by using molecular tools to detect P. brasiliensis in tissues of road-killed animals that had lived in endemic areas of the disease. CC aa pp íí tt uu lloo II II 39 Materials and Methods Study Area and Animals: The road-killed animals were collected in the Botucatu endemic PCM area by the Departamento de Estradas de Rodagem do Estado de São Paulo (DER) team that routinely patrol the roads. Only animals that appeared to have been recently killed (1-7 hours) and had not completely disfigured were placed into plastic bags, labelled as to dates, hour and geographic location and sent to the laboratory where they were necropsied. The animal organs were collected and processed for DNA extraction at necropsy or preserved at -80ºC. The taxonomical data of the animals, including their home ranges, sex and the tissue analysed are summarised on Table 1. The geographic positions of the road-killed animals, established through GPS (Global Positioning System), were plotted on a digital map using a geographic database by the IDRISI32®GIS and Surfer (Figure 1). This study was developed after receiving authorization from the Brazilian Protection Agency (IBAMA) and Animal Ethics Committees (CEEA) at the Institute of Biosciences/UNESP-Botucatu, SP, Brazil. Molecular analyses: The DNA extraction was performed by grinding the liquid-nitrogen frozen tissue sample with mortar and pestle as proposed by Corredor et al. [8]. The DNA pellet was suspended in 100μL of ultra-pure water and the quality was checked by 1% agarose gel electrophoresis using Low Mass DNA Ladder (Invitrogen) as molecular marker. The molecular detection was carried out by Nested-PCR reactions, using as outer primers the panfungal primers ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS5 (5’- GGAAGTAAAAGTCGTAACAACG-3’), annealing temperature of 60ºC [5] and inner primers PbITSE (5’-GAGCTTTGACGTCTGAGACC-3’) and PbITSR (5’- AAGGGTGTCGATCGAGAGAG-3’), annealing temperature of 62ºC [6], and matching from 162 to 548 nucleotides at GenBank (AY374339) access. The specificity of the Nested-PCR was evaluated in a blind test against a panel of 16 DNA samples from Emmonsia parva, Histoplasma capsulatum, P. brasiliensis, Renispora spp. and Sporothrix schenckii, provided by Setor de Imunodiagnóstico do Serviço de Micologia (IPEC/FIOCRUZ). The samples code numbers and identities were revealed only after amplifications. The Nested PCR amplicons were purified by the commercial kit GFX PCR DNA and Gel Band Purification (Amersham CC aa pp íí tt uu lloo II II 40 Biosciences) and the sequencing reactions were carried out in both strands in a MegaBaceTM 1000 (Amersham Biosciences). The sequences were compared to the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST). T ab le 1 :T ax on om ic al d at a of th e ro ad -k il le d an im al s, in cl ud in g th e sp ec ie s ho m e ra ng e, s ex , e va lu at ed ti ss ue s an d ne st ed -P C R r es ul ts . O rd er F am ily Sp ec ie s H om e ra ng e* ( ha ) A ni m al Se x T is su e/ N es te c- P C R ( + or - ) C ar ni vo ra C an id ae C er do cy on th ou s 0. 1 C t1 M al e lu ( -) , s ( -) , l ( -) , k ( -) , h ( -) , m ln ( -) C t2 M al e lu ( -) , s ( -) , l ( -) , k ( -) , h ( -) , m ln ( -) C t3 N a lu ( -) , s ( -) , l ( -) , k ( -) , h ( -) , m ln ( -) C t4 M al e lu ( -) , s ( -) , l ( -) , k ( -) , h ( -) C t5 M al e lu ( -) , s ( -) ,h ( -) , m ln ( -) M us te lid ae E ir a ba rb ar a 2. 44 E b1 M al e lu ( -) , s ( -) , l ( -) ,h ( -) , m ln ( -) G al li ct is v it ta ta 0. 4 G v1 M al e lu ( +) , s ( -) , l ( -) , k ( -) , h ( -) G v2 M al e lu ( -) , s ( -) , l ( -) , h ( -) P ro cy on id ae P ro cy on c an cr iv or us N A Pc 1 M al e lu ( +) , l ( -) , k ( -) , h ( -) , m ln ( -) Pc 2 Fe m al e lu ( -) , s ( -) , l ( -) , k ( -) , h ( -) , m ln ( -) D id el ph im or ph ia D id el ph id ae D id el ph is a lb iv en tr is 0. 57 D a1 Fe m al e lu ( -) , s ( -) , l ( -) , m ln ( -) D a2 M al e lu ( -) , s ( -) , l ( -) , m ln ( -) R od en ti a C av id ae C av ia a pe re a 0. 1 C a1 Fe m al e lu ( +) , s (+ ), l ( -) , k ( +) , h ( -) , m ln (+ ), ag ( +) C a2 M al e lu ( -) , s ( -) , h ( -) , m ln ( -) C a3 Fe m al e lu ( -) , s ( -) , l ( -) , k ( -) , h ( -) , m ln ( -) E re th iz on ti da e Sp hi gg ur us s pi no su s 15 -2 0 Ss 1 Fe m al e lu ( -) , s ( -) , l ( +) , k ( -) , h ( -) Ss 2 M al e lu ( +) , s ( -) , k ( -) X en ar th ra D as yp od id ae D as yp us n ov em ci nc tu s 3. 4- 15 D n1 Fe m al e lu ( +) , s (+ ), l ( +) , k (+ ), h (- ), m ln ( +) D as yp us s ep te m ci nc tu s N a D s1 M al e lu ( +) , s (+ ), l ( +) , k ( -) , h ( -) , m ln (+ ) lu , l un g; s , s pl ee n; l, liv er ; k , k id ne y; h , h ea rt ; m ln , m es en te ri c ly m ph n od e; a g, a dr en al g la nd . *A cc or di ng to E is en be rg & R ed fo rd 1 99 9 [7 ]. N A , n ot a va il ab le . CC aa pp íí tt uu lloo II II 42 Results Area of discovery of animals Fig. 1 illustrates the geographic location of all the road-killed animals evaluated. Fig. 2 illustrates the road-killed animals evaluated (*figura não consta no artigo publicado). Fig. 1- Geographic location of the road-killed animals employed for Paracoccidioides brasiliensis molecular detection. CC aa pp íí tt uu lloo II II 43 Fig. 2: Road-killed wild animals, A) C. thous - Ct1, B) C. thous - Ct2, C) C. thous - Ct3, D) C. thous - Ct4, E) C. thous - Ct5, F) E. barbara - Eb1, G) G. vittata - Gv1, H) G. vittata - Gv2, I) P. cancrivorus - Pc1, J) P. cancrivorus – Pc2, K) C. aperea - Ca1, L) C. aperea - Ca3, M) S. spinosus - Ss1, N) S. spinosus - Ss2, O) D. novemcinctus - Dn1 and P) D. septemcintus - Ds1. CC aa pp íí tt uu lloo II II 44 DNA Amplification The specificity of the PbITSE/PbITSR primers were successfully tested against a panel of DNA samples from several fungi such as S. schenckii, Renispora spp., H. capsulatum, E. parva as well as from P. brasiliensis. Overall, the panfungal PCR with ITS4/ITS5 primers amplified a 650bp DNA fragment in all fungi tested, but the PbITSE/PbITSR primers in the PCR showed an amplicon of 387bp only in DNA samples obtained from P. brasiliensis (Fig. 3). With respected to animal samples, predictive specific amplicons of P. brasiliensis were detected by Nested-PCR reactions in tissue fragments from:(i) several organs of the two armadillo species (Dn1 and Ds1) and a guinea pig (animal Ca1), (ii) porcupine liver (Ss1) and (iii) the lungs of raccoon (Pc1), grison (Gv1) and porcupine (Ss2) (Table 1). The molecular identities of the amplicons from the guinea pig (Ca1), raccoon (Pc1) and porcupine (Ss1) were confirmed by direct double-strand sequencing. The latter studies produced unambiguous fragments, varying from 332 to 340 bp, which showed 100% similarity with P. brasiliensis DNA sequences deposited at Gen Bank, with included 44 different accession numbers, obtained both from armadillo (AY374339) and human isolates (AF416745, ATCC 32069). Fig. 4 illustrates the Nested-PCR sensitivity with ITS4/ITS5 as outer primers and PbITSE/PbITSR as inner primers (*figura não consta no artigo publicado). Fig. 3 - Specificity testing of the Nested-PCR using DNA from several fungi: Lane 1-19: (1) 100bp DNA ladder (Invitrogen), (2-6) S. schenckii, (7-11) H. capsulatum, (12) Renispora spp. (13-17) P. brasiliensis, (18) Emmonsia spp. and (19) negative control. CC aa pp íí tt uu lloo II II 45 Fig. 4 - Nested-PCR sensitivity in decreasing concentrations of P. brasiliensis DNA. Discussion PCM in both in domestic and wild animals has been reported in the literature based on the use of intradermal reactions with paracoccidioidin. It was shown that some of these animals showed high rates of infection, especially those whose habitats are related to soil [9, 10]. Serological surveys have also been employed to determine PCM in dogs [11], equines [2], bovines [13], free-living monkeys [14] and armadillos [15], thus showing that a wide variety of mammals can be infected by P. brasiliensis. The systematic isolation of P. brasiliensis in armadillo tissues demonstrated the importance of this animal as a natural of the etiologic agent in endemic areas. It has been suggested that armadillos were the only animals that could acquire PCR [16,17]. However, the natural occurrence of PCM was confirmed in two dogs with generalized lymphadenithis by the recovery of P. brasiliensis in culture, as well as by histopathological, immunohistochemical and molecular detection of the gp43 gene [18,19]. In order to increase our knowledge on the ecology of P. brasiliensis and the epidemiology of PCM, we developed a new approach that combined molecular tools to identify P. brasiliensis in other hosts by demonstrating fungal DNA in animal tissue. In the present study, panfungal primers ITS4/ITS5 amplified an amplicon of around 650 bp in lungs of C. aperea (guinea pig), D. albiventris (white-eared opossum), P. cancrivorus (raccoon) and S. spinosus (porcupine), thus demonstrating previous contact and the existence of fungi, but not necessarily P. brasiliensis. This data corroborates that the CC aa pp íí tt uu lloo II II 46 airborne route is the major means of transmission of several pathogenic and non-pathogenic fungi in animals [20]. A serious limitation for any molecular protocol is the occurrence of nonspecific annealing when a common DNA sequence is present. Since it has been estimated that there are approximately 1.5 million fungal species [21], it is possible that DNA sequences of phylogenetically closely related fungi, mainly environmental ones, have not yet been deposited in the Gen Bank. P. brasiliensis has been recently classified as a member of Ajellomycethaceae, a new family of saprobic and pathogenic vertebrate-associated fungi, which includes Histoplasma, Blastomyces, Emmonsia and Paracoccidioides [22]. Up to now, the use of Nested-PCR with PbITSE/PbITSR primers has proven to be specific for the detection of only P. brasiliensis since any amplification occurs for the several other related genera. Concerning the members of the Carnivora order evaluated, which are biologically related to the domestic dog, P. brasiliensis DNA was detected only in the lung of P. cancrivorus (raccoon) and G. vittata (grison), while all organs of C. thous (crab-eating-fox) and E. barbara (tayra) remained negative. It is known that high temperatures are limiting factors for the growth of major fungal species and that domestic dogs have a body temperature around 37.5-38.5oC [23]. This might be a factor contributing to the limited number of cases involving P. brasiliensis in Carnivora members. It appears that the members of the Didelphidae Family, are also not frequently infected by fungi as already reported by Silva-Vergara et al.[24]. P. brasiliensis infections does not necessarily indicate systemic PCM. The histopathological studies of armadillo tissues showed that if the disease occured, it was a mild form [25]. The molecular detection of P. brasiliensis DNA in organs of C. aperea (guinea pig) demonstrated that the fungus indeed disseminated from the lung. We also detected the pathogen in the liver and lung of S. spinosus (porcupine). The importance of rodents as a reservoir of other fungal pathogens such as in Coccidioides immitis [26], Emmonsia spp. [27] and Penicillium marneffei [28,29] has previously been demonstrated. As expected, the two armadillos evaluated in this investigation provided positive amplification in several organs. The molecular detection of P. brasiliensis in tissues from organs such as the adrenal gland, liver, spleen, kidney and mesenteric lymph node also might exclude the possibility of the presence of non-pathogenic fungi because the latter do not have the capacity to disseminate to extra-pulmonary organs. Since in all evaluated animals the integrity of the organs was preserved we believe that the risk of cross-contamination in this investigation was CC aa pp íí tt uu lloo II II 47 low. While problems with PCR sensitivity and inhibition can not be excluded completely, we have observed a DNA detection limit of 1.0 ρg. In addition, the specific amplification of the target sequence of P.brasiliensis was not inhibited even when using only one ρg of fungal DNA mixed with a relatively large amount of animal DNA (around 100-200 μg). The detection of P. brasiliensis in different organs from different species show that the fungus could have different dissemination profiles. This in turn may indicate different interactions of P. brasiliensis with several host species studied and possibly different genotypes of this etiologic agent. It is known that P. brasiliensis presents at least three cryptic species [30]. This divergence must be studied in order to evaluate how some genetic differences may indicate distinct host-pathogen interactions as well as distinct ecological niches. Without the necessity of applying a laborious sampling effort, it was possible to evaluate nine different wild species, belonging to seven different taxonomic families. In fact, the numbers and diversity of road-killed animals are considerably higher and, in general, they are killed in their own habitat, because the roads invade their natural habitats [31]. In this manner, the geographic coordinates of the places where the infected animals might be are well-integrated in databases that use the Geographical Information Systems (GIS), thus contributing to a better understanding of pathogen distribution and the associated biotic and abiotic factors. In summary, our results show that road-killed animals can be important in the eco-epidemiologal study of P. brasiliensis. Acknowledgements This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-nº 05/56771-9 and 06/03597-4) and Fundação para o Desenvolvimento da Unesp (FUNDUNESP-nº 0015006). The authors thank the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA) for permission to collect road-killed animals. 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Cienc Cult 1996, 48(4): 270-271. CC aa pp íí tt uu lloo II II II 51 Detecção de Paracoccidioides brasiliensis em tatus (Dasypus novemcinctus) provenientes de uma reserva de Cerrado do Instituto Lauro de Souza Lima (Bauru-SP) Boletim Epidemiológico Pau