The development of molecular tools for detection and typing of unicellular intestinal parasites has revealed genetic diversities in species that were previously considered as distinct entities. Of great importance is the genetic distinction found between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar, two morphologically indistinguishable species. Blastocystis sp. is a ubiquitous intestinal parasite with unsettled pathogenicity. Molecular studies of Blastocystis sp. have identified 17 genetic subtypes, named ST1-17. Genetically, these subtypes could be considered as different species, but it is largely unknown what phenotypic or pathogenic differences exist between them. This thesis explores molecular methods for detection and genetic subtyping of unicellular intestinal parasites, with special focus on Blastocystis. We found that PCR-based methods were highly sensitive for detection of unicellular intestinal parasites, but could be partially or completely inhibited by substances present in faeces. A sample transport medium containing guanidinium thiocyanate was shown to limit the occurrence of PCR inhibition. The prevalence of Blastocystis in Swedish university students was over 40%, which is markedly higher than what was previously estimated. Blastocystis ST3 and ST4 were the two most commonly found Blastocystis subtypes in Sweden, which is similar to results from other European countries. Blastocystis sp. and Giardia intestinalis were both commonly detected in Zanzibar, Tanzania, each with a prevalence exceeding 50%. Blastocystis ST1, ST2, and ST3 were common, but ST4 was absent. While G. intestinalis was most common in the ages 2-5 years, the prevalence of Blastocystis increased with increasing age, at least up to young adulthood. We found no statistical association between diarrhoea and Blastocystis sp., specific Blastocystis subtype or G. intestinalis. Metagenomic sequencing of faecal samples from Swedes revealed that Blastocystis was associated with high intestinal bacterial genus richness, possibly signifying gastrointestinal health. Blastocystis was also positively associated with the bacterial genera Sporolactobacillus and Candidatus Carsonella, and negatively associated with the genus Bacteroides. Blastocystis ST4 was shown to have limited intra-subtype genetic diversity and limited geographic spread. ST4 was also found to be the major driver behind the positive association between Blastocystis and bacterial genus richness and the negative association with Bacteroides.

Em 2005 o primeiro caso de Febre Maculosa Brasileira (FMB) foi reconhecido no estado do Rio Grande do Sul (RS), Brasil. Desde então até abril de 2016 doze casos foram confirmados, sendo que quatro destes são autóctones do município de Cerro Largo, área considerada endêmica para a enfermidade riquetsial. Neste mesmo período o RS também notificou 58 casos suspeitos ao Ministério da Saúde. No RS são encontrados dois biomas, Mata Atlântica e Pampa, este último não ocorrendo em outras unidades federativa do país. Até o momento, inexistem estudos relatando a infecção por riquétsias na ixodofauna gaúcha. Desta forma, o presente estudo teve como objetivo pesquisar a infecção de riquétsias do Grupo da Febre Maculosa (GFM) em cães, pequenos mamíferos e carrapatos; através da Reação de Imunofluorescência Indireta (RIFI), qPCR e tentativa de isolamento de riquétsias em cultivo celular dos ixodídeos coletados em área endêmica (Cerro Largo) e áreas não endêmicas nos biomas Pampa e Mata Atlântica no RS. Na sorologia (RIFI), 33,5 % (55/164), 2,9% (1/33), 40% (16/40) dos pequenos mamíferos e 8,3% (3/36), 13,9% (5/36) e 20,4 (28/137) dos cães coletados na Mata Atlântica, Pampa e área endêmica para FMB, respectivamente, foram sororreagentes para pelo menos um dos seis antígenos de riquétsias testados. Oito espécies de carrapatos foram coletadas no local de Mata Atlântica, sendo: Amblyomma aureolatum, Amblyomma brasiliense, Amblyomma incisum, Amblyomma ovale, Haemaphysalis juxtakochi, Ixodes loricatus, Rhipicephalus microplus e Amblyomma yucumense com destaque para esta última que foi descrita como uma espécie nova. Já para o fragmento de Pampa quatro espécies foram encontradas (A. aureolatum, A. ovale, Amblyomma tigrinum e Rhipicephalus sanguineus) e na área endêmica para FMB no RS, coletamos cinco espécies de carrapatos, Amblyomma dubitatum, Amblyomma longirostre, A. ovale, I. loricatus e R. microplus. Detectamos molecularmente pela primeira vez no RS, Candidatus Rickettsia amblyommii em A. longirostre e A. brasiliense, Candidatus Rickettsia andeanae em A. aureolatum e A. tigrinum, Rickettsia bellii em I. loricatus, Rickettsia rhipicephali em A. yucumense e H. juxtakochi, Candidatus Rickettsia asemboensis em pulgas no Brasil e de extrema importância para o conhecimento da epidemiologia da FMB no RS, detectamos Rickettsia sp. cepa Mata Atlântica em A. ovale oriundos de Cerro Largo (área endêmica para FMB no RS). Como primeiros isolamentos de riquétsia para o estado do RS, isolamos R. bellii de I. loricatus coletados na Mata Atlântica e área endêmica para FMB no RS. O presente estudo é pioneiro em relação a investigação comparativa sobre FMB nos biomas Pampa e Mata Atlântica e também o primeiro estudo molecular e isolamento de riquétsias em cultivo celular de amostras coletadas no RS, Brasil / In 2005 the first Brazilian Spotted Fever (BSF) case was confirmed in the Rio Grande do Sul (RS) state, Brazil. Ever since until April 2016, twelve cases have been confirmed, and four of them are native from Cerro Largo city, an area considered endemic to the riquetsial illness. During the same period, RS also notified 58 suspected cases to the Ministry of Health. There are two distinct biomes in RS, Atlantic Forest and Pampa, the later restricted to RS within the Brazilian land. Until now, no studies reported rickettsial infection in the ixodofauna of RS. Thus, this study aimed to investigate the rickettsial infection of Spotted Fever Group (SFG) in dogs, small mammals and ticks; by immunofluorescence assay (IFA), qPCR and attempted rickettsial isolation in cell culture from ticks collected in an endemic area (Cerro Largo) and non-endemic areas in Pampa and Atlantic Forest biomes of RS. Through serology (IFA), 33.5% (55/164), 2.9% (1/33) and 40% (16/40) small mammals, and 8.3% (3/36), 13.9 % (5/36) and 20.4 (28/137) dogs from the Atlantic Forest, Pampa, and BSF-endemic area, respectively, were seroreactive (titer ≥64) to at least one of the six rickettsia antigens tested. Eight species of ticks were collected in the Atlantic Forest: Amblyomma aureolatum, Amblyomma brasiliense, Amblyomma incisum, Amblyomma ovale, Haemaphysalis juxtakochi, Ixodes loricatus, Rhipicephalus microplus and Amblyomma yucumense, the later described as a new species. In the Pampa fragment, four tick species were found (A. aureolatum, A. ovale, Amblyomma tigrinum and Rhipicephalus sanguineus), and five tick species (Amblyomma dubitatum, Amblyomma longirostre, A. ovale, I. loricatus, R. microplus) in the BSF-endemic area of RS. We performed the first molecular detection in the RS state of "Candidatus Rickettsia amblyommii" in A. longirostre and A. brasiliense; "Candidatus Rickettsia andeanae" in A. aureolatum and A. tigrinum; Rickettsia bellii in I. loricatus; Rickettsia rhipicephali in A. yucumense and H. juxtakochi; and "Candidatus Rickettsia asemboensis" in fleas in Brazil. Noteworthy, we detected the pathogen Rickettsia sp. strain Atlantic rainforest in A. ovale ticks from Cerro Largo (endemic area for BSF in RS). We also performed the first isolation in cell culture of rickettsia from RS state, which comprised R. bellii from I. loricatus collected in the Atlantic Forest and the BSF-endemic area. This study is the first comparative research about rickettsiae among Pampa and Atlantic Forest biomes, and also the first molecular detection and isolation in cell culture of rickettsiae from Rio Grande do Sul, Brazil

A criptosporidiose já foi descrita em várias espécies animais, incluindo várias espécies de aves. É considerada uma das principais infecções por protozoários em aves das ordens Anseriformes, Charadriformes, Columbiformes, Galliformes, Passeriformes, Psittaciformes e Struthioniformes. Três espécies de Cryptosporidium infectam aves: Cryptosporidium baileyi, Cryptosporidium galli e Cryptosporidium meleagridis, além de vários genótipos distintos geneticamente, como os genótipos I, II, III, IV e V de aves. Cryptosporidium galli e Cryptosporidium genótipo III de aves estão relacionados com infecções crônicas no proventrículo, de forma semelhante à infecção por Cryptosporidium serpentis, em serpentes. Vários métodos de diagnóstico são utilizados para detecção do parasito, mas somente aqueles com base em biologia molecular são capazes de determinar a espécie de Cryptosporidium. O projeto teve com objetivo o desenvolvimento da PCR duplex em tempo real, tendo como alvo o gene da subunidade 18S do rRNA, por meio de ensaio TaqMan, para detecção de DNA de C.galli e Cryptosporidium genótipo III de aves, e avaliar os atributos diagnósticos da PCR duplex em tempo real quando comparada à nested PCR, utilizando 1027 amostras fecais de aves das ordens Passeriformes e Psittaciformes. A PCR duplex em tempo real apresentou positividade em 580/1027 (56,47%) para C. galli, enquanto que a nested PCR resultou em positividade para Cryptosporidium spp. em 104/1027 (10,13%) amostras. Para Cryptosporidium genótipo III de aves, houve positividade em 21/1027 (2,04%). A PCR duplex em tempo real resultou em alta especificidade analítica quando foram utilizadas amostras de DNA de outras espécies e genótipos de Cryptosporidium. A única exceção foi a amplificação de DNA de C. serpentis, com a utilização de primers e sonda para detecção de Cryptosporidium genótipo III de aves, porém, com uma baixa eficiência de amplificação. Foram identificados novos hospedeiros aviários para ambas as espécies gástricas, assim como foi possível a identificação de C. baileyi e, pela primeira vez no Brasil, de Cryptosporidium genótipo V de aves. Conclui-se que a PCR duplex em tempo real desenvolvida neste estudo é um recurso que apresenta rapidez e confiabilidade para diagnóstico de criptosporidiose gástrica em aves / Cryptosporidiosis has been described in several animal species, including many species of birds, and is considered a major protozoan infection of the orders Anseriformes, Charadriformes, Columbiformes, Galliformes, Passeriformes, Psittaciformes and Struthioniformes. Three species of Cryptosporidium are valid in birds: Cryptosporidium baileyi, Cryptosporidium meleagridis and Cryptosporidium galli; beside these species, there are several genotypes genetically distinct, as avian genotypes I, II, III, IV and V. Cryptosporidium galli and Cryptosporidium avian genotype III are related to chronic infections in proventriculus, similar to Cryptosporidium serpentis infection in snakes. Several methods are used for cryptosporidiosis diagnosis, but only those based on molecular biology are able to determine the Cryptosporidium species and genotypes. The aim of this project was the development of a duplex real-time PCR targeting 18S subunit of rRNA gene, by TaqMan assay, to detect DNA of C. galli and Cryptosporidium avian genotype III, and to evaluate the diagnostic attributes of the duplex real-time PCR compared to nested PCR, using 1027 fecal samples from birds of the orders Passeriformes and Psittaciformes. The duplex real time PCR showed positivity in 580/1027 (56.47%) for C. galli, whereas nested PCR was positive for Cryptosporidium spp. in 104/1027 (10.13%) samples. For Cryptosporidium avian genotype III, there was positivity in 21/1027 (2.04%) samples. The duplex real time PCR resulted in high analytical specificity when tested using DNA samples from other Cryptosporidium species and genotypes. The only exception was the amplification of DNA from C. serpentis with primers and probe for the detection of Cryptosporidium avian genotype III, although with lower efficiency. New avian hosts were identified for both gastric species, as well as it was possible to identify C. baileyi and, for the first time in Brazil, Cryptosporidium avian genotype V. It was concluded that the duplex real time PCR developed in this study is a fast and reliable tool for diagnosis of gastric cryptosporidiosis in birds.

Made available in DSpace on 2017-07-25T19:29:51Z (GMT). No. of bitstreams: 1 EdilaineGrabicoski.pdf: 3057508 bytes, checksum: 91f718973e4409c8611259602683583d (MD5) Previous issue date: 2012-02-09 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / The white mold disease, caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary, responsible for large losses in soybean (Glycine max (L.) Merrill). The pathogen has more than 400 hosts and presents the formation of a resistance structure (sclerotia), difficulting the disease control, thus increasing the importance of the pathogen in Brazilian agriculture. The seeds are the main spreading disease means, a quickly and correct presence detection of the pathogen is important to control the white mold, especially for non-infested areas by the pathogen. In this study, 57 S. sclerotiorum isolates were morphological and pathologically characterized to assess variations within the specie. Also was developed a method to detect the pathogen directly from seed-soak liquor of infected soybean seeds, through PCR. The proposed method was compared to the traditional recommended methods. The pathogen isolates showed some morphological variations among themselves, as the number and weight of the formed sclerotia. Pathologically, were observed two and seven pathogenic levels second, respectively, the soybean inoculation on the stem and on the detached trifoliate leaves. By the detection method, it was possible to detect one seed artificially contaminated in the sample with 400 total seeds. In naturally contaminated seeds, was possible to detected the pathogen presence in a sample that, according to the method of incubation on paper roll and on Neon-S medium, respectively, presented 1 and 3 seeds contaminated in four hundred seeds. The proposed methodology is promising for the detection of S. sclerotiorum, however, requires optimization because many false negative results were obtained, mainly due to substances released by the seeds that inhibited the PCR reaction. The use of filter in the samples has presented as a possible tool to overcome the PCR inhibitors. For the optimization of the method, increase the size of seed samples, use of filters, others techniques such as nested PCR, Real-Time PCR and DNA probes can help overcome these difficulties. / O Mofo branco, doença causada pelo fungo Sclerotinia sclerotiorum (Lib.) de Bary, vem causando grandes prejuízos na cultura da soja (Glycine max (L.) Merrill) e, por apresentar mais de 400 hospedeiros e a formação de uma estrutura de resistência (escleródios), o seu controle torna-se difícil, aumentando a importância do patógeno para a agricultura brasileira. As sementes são o principal meio de disseminação da doença, detectar rápido e corretamente a presença do patógeno nas mesmas é importante no controle do Mofo branco, principalmente, para não infestar áreas isentas do patógeno. No presente trabalho, 57 isolados de S. sclerotiorum foram caracterizados morfológica e patologicamente para avaliar variações dentro da espécie. Também se desenvolveu uma metodologia de detecção do patógeno em sementes de soja embebidas, através de PCR diretamente do líquido de embebição das mesmas. Comparou-se a metodologia proposta com as tradicionais recomendadas para o patógeno. Os isolados do patógeno apresentaram algumas variações morfológicas entre si, como quantidade e peso dos escleródios formados. Patologicamente, foram verificados dois e sete níveis de patogenicidade segundo, respectivamente, a inoculação dos isolados na haste e em trifólios destacados de plantas de soja. Diretamente a partir do líquido de embebição das sementes, foi possível detectar uma semente artificialmente contaminada em 400 sementes totais. Em sementes naturalmente contaminadas, detectou-se a presença do patógeno em amostra que, segundo o método de incubação em rolo de papel e em Neon-S apresentava, respectivamente, 1 e 3 sementes contaminadas em quatrocentas totais. A metodologia proposta é promissora para a detecção de S. sclerotiorum, no entanto, necessita de otimização, pois muitos resultados falsos-negativos foram obtidos, principalmente devido a substâncias liberadas pelas próprias sementes que inibiram a reação de PCR. A utilização de um filtro nas amostras apresentou-se como uma possível ferramenta para driblar os inibidores de PCR. Para a otimização do método, técnicas como Nested-PCR, Real-Time PCR, utilização de sondas de DNA, aumento no tamanho de amostras de sementes e a utilização dos filtros podem ajudar a superar essas dificuldades.

Submitted by Valquíria Barbieri (kikibarbi@hotmail.com) on 2018-04-18T20:33:59Z No. of bitstreams: 1 DISS_2015_Belgath Fernandes Cardoso.pdf: 3865065 bytes, checksum: 3b7d79b4224429f58cb6ca5c3d96d7e9 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-04-27T17:29:40Z (GMT) No. of bitstreams: 1 DISS_2015_Belgath Fernandes Cardoso.pdf: 3865065 bytes, checksum: 3b7d79b4224429f58cb6ca5c3d96d7e9 (MD5) / Made available in DSpace on 2018-04-27T17:29:40Z (GMT). No. of bitstreams: 1 DISS_2015_Belgath Fernandes Cardoso.pdf: 3865065 bytes, checksum: 3b7d79b4224429f58cb6ca5c3d96d7e9 (MD5) Previous issue date: 2015-03-26 / CAPES / O gênero Orthobunyavirus, família Bunyaviridae, alberga arbovírus de importância médica. Estes estão envolvidos em epidemias de doença febril em áreas tropicais. No Brasil, o vírus Oropouche (OROV) é considerado o arbovírus mais frequente após o vírus da dengue (DENV). O objetivo deste estudo foi investigar a circulação de orthobunyavírus em Mato Grosso (MT). 529 amostras de soro obtidas entre outubro de 2011 e julho de 2012 de pacientes com doença febril aguda suspeita de dengue com até cinco dias do início dos sintomas em MT e 387 pools de mosquitos Cx quinquefasciatus capturados entre janeiro e abril de 2013 foram submetidos à nested RT-PCR para o segmento S de orthobunyavírus pertencentes ao sorogrupo Simbu. Amostras positivas foram testadas em pelo menos duas reações independentes e submetidas a sequenciamento nucleotídico para análise filogenética. Inoculou-se as amostras positivas em células vero. Dentre os 529 pacientes, 5 (0,94%) foram positivos para orthobunyavirus do sorogrupo Simbu por nested RT-PCR e isolamento viral. O vírus foi isolado de 3/8 pools. O segmento S do OROV foi identificado em cinco pacientes, quatro do sexo feminino, com 14-62 anos. Dois pacientes encontravam-se co-infectados com DENV-4. Estes pacientes são oriundos das cidades de Cuiabá (n=3), Várzea Grande (n=1) e Nova Mutum (n=1), todos residentes em área urbana, que apresentavam febre, cefaleia, dor retroorbital, mialgia, artralgia, prostação e náuseas. 8/387 pools de Cx. quinquefasciatus foram positivos para o segmento S do OROV por nested-RT-PCR, com taxa mínima de infecção (MIR) de 2,3 por 1000 mosquitos. As sequências obtidas do segmento S apresentaram 98% a 100% de homologia com o mesmo segmento das cepas do OROV verificadas no GenBank. A análise filogenética indica que as amostras de humanos e mosquitos pertencem ao subgenótipo Ia, similares a cepas do Pará obtidas de humanos, preguiças, Aedes (Ochlerotatus) serratus e Cx. quinquefasciatus. O genótipo I é o mais conservado e frequente no Brasil dentre os genótipos do OROV. Cx. quinquefasciatus, culicídeo de maior abundância em Cuiabá, é considerado vetor secundário do OROV em área urbana. Culicoides paraensis, principal vetor do OROV em áreas urbanas na região amazônica, não foi capturado neste estudo. Sorologia para o OROV foi identificada em residentes de cidades do Pará afetadas pela rodovia Cuiabá-Santarém e em primatas do Pantanal Sul-matogrossense, corroborando com a identificação do OROV em cidades do MT geograficamente interligadas por esta mesma rodovia. Infecções esporádicas por um orthobunyavirus do sorogrupo Simbu, possivelmente o OROV, foram identificadas em pacientes de MT, além de oito pools de Cx. quinquefasciatus em Cuiabá, indicando que este mosquito possui capacidade vetorial e pode estar envolvido no ciclo urbano de transmissão do vírus no estado. / The genus Orthobunyavirus, family Bunyaviridae, contains medically importante arboviruses. These are involved in epidemics of febrile illness in tropical areas. In Brasil, Oropouche virus (OROV) is considered the most frequent arbovirus after dengue virus (DENV). The aim of this study was to investigate the circulation of orthobunyaviruses in Mato Grosso (MT). 529 serum samples collected between October, 2011 and July, 2012 of patients with acute febrile illness suspected of dengue for up to five days of symptom onset from MT and 387 pools of Cx. quinquefasciatus mosquitoes captured between January and April, 2013, were subjected to nested RT-PCR for the segment S of orthobunyaviruses belonging to Simbu serogroup. Positive samples were tested in at least two independent reactions and subjected to nucleotide sequencing for phylogenetic analysis. Positive samples were inoculated in vero cells. Among the 529 patients, five (0.94%) were positive for serogroup Simbu by nested RT-PCR and viral isolation. The virus was isolated from 3/8 pools. The OROV segment S was identified in five patients, four females, with 14-62 years-old. Two patients were co-infected with DENV-4. These patients are from Cuiabá (n=3), Várzea Grande (n=1) and Nova Mutum (n=1), all residents in urban areas, presenting fever, headache, retroorbital pain, myalgia, arthralgia, prostration and nausea. 8/387 pools of Cx. quinquefasciatus were positive for OROV segment S by nested-RT-PCR with a minimum infection rate (MIR) of 2.3 per 1,000 mosquitoes. The segment S nucleotide sequences presented 98% to 100% of homology with the same segment of OROV strains available in GenBank. Phylogenetic analysis indicate the human and mosquito samples belong to genotype Ia, similar to strains obtained in Pará from humans, sloths, Aedes (Ochlerotatus) serratus and Cx. quinquefasciatus. The genotype I is the most conserved among OROV genotypes. Cx. quinquefasciatus, the most abundant culicidae in Cuiabá, is considered a secondary vector for OROV in urban areas. Culicoides paraensis, the main vector for OROV in urban areas in the Amazon region, was not captured in the study. Serology for OROV was identified in humans in cities of Pará affected by the Cuiabá-Santarém highway and in primates in South Pantanal, corroborating for the findings in cities of MT geographically linked by the same highway. Sporadic infections by an orthobunyavirus from Simbu serogroup, possibly OROV, were identified in patients from MT, also eight Cx. quinquefasciatus pools from Cuiabá, indicating that has vectorial capacity and may be involved in the urban cycle of virus transmission in the state.

Made available in DSpace on 2016-12-08T16:24:22Z (GMT). No. of bitstreams: 1 PGCA15MA178.pdf: 1072214 bytes, checksum: 2feb2bb4cea985aa369f44b04592defd (MD5) Previous issue date: 2015-08-14 / Chicken anemia virus (CAV) is an important virus that belongs to Circoviridae family, Gyrovirus genre, known to lead to significant losses in poultry, the viruses can cause immunosuppression and disease especially in young birds. CAV was the only virus of this family known to infect chickens by 2011, after the discovery of a new virus, the Avian Gyrovirus type 2 (AGV2). According to the literature, AGV2 is closely related to CAV, as well as being widely distributed in chickens in Brazil and other countries, but its pathogenic potential is unknown and studies on molecular epidemiology are still scarce. The present study aimed to detect CAV (Nested-PCR), and AGV2 (PCR) on DNA extracted from samples of thymus tissue of broiler chickens housed on two states of southern Brazil. In addition, perform phylogenetic analysis of AGV2 positive samples. Results showed that of 180 birds tested, 105 (58.33%) were positive for CAV and 39 (21.67%) positive for AGV2. On the positive material for AGV2 were selected eleven amplicons for sequencing and phylogenetic analysis, where it became clear that there were no differences among the samples. The sequences obtained were compared with samples from other countries, many of these detected in humans (HGyV). It was not possible in the present work, observe correlation between geographical distribution and genetic variability among most samples AGV2 now studied / O vírus da anemia das galinhas (CAV) é um importante vírus pertencente a família Circoviridae, gênero Girovirus, conhecido por levar a perdas significativas na avicultura, este vírus pode causar imunossupressão e doença principalmente em aves jovens. O CAV era o único vírus desta família conhecido por infectar galinhas até 2011, após a descoberta de um novo vírus, o Girovírus Aviário tipo 2 (AGV2). Segundo consta na literatura, o AGV2 está estreitamente relacionado ao CAV, além de estar amplamente distribuído em frangos no Brasil e em outros países, mas seu potencial patogênico ainda é desconhecido e trabalhos sobre sua epidemiologia molecular ainda são bastante escassos. Assim, o presente trabalho teve por objetivo realizar a detecção de CAV (Nested-PCR), e AGV2 (PCR) em amostras de DNA extraídas de tecido do timo, de aves alojadas em plantéis de frango de corte de dois estados da região Sul do Brasil. Além disso, realizar a análise filogenética de amostras positivas para AGV2. Os resultados apontaram que das 180 aves testadas 105 (58,33%)foram positivas para CAV e 39 (21,67%) positivas para AGV2. Do material positivo para AGV2 foram selecionados onze amplicons para sequenciamento e análise filogenética, onde foi possível evidenciar que não houve diferenças entre o material analisado. As sequências obtidas foram comparadas com amostras provenientes de outros países, muitas destas detectadas em humanos (HGyV), não tendo sido possível, no presente trabalho, observar correlação entre distribuição geográfica e variabilidade genética entre a maioria das amostras de AGV2 ora estudadas

A criptosporidiose já foi descrita em várias espécies animais, incluindo várias espécies de aves. É considerada uma das principais infecções por protozoários em aves das ordens Anseriformes, Charadriformes, Columbiformes, Galliformes, Passeriformes, Psittaciformes e Struthioniformes. Três espécies de Cryptosporidium infectam aves: Cryptosporidium baileyi, Cryptosporidium galli e Cryptosporidium meleagridis, além de vários genótipos distintos geneticamente, como os genótipos I, II, III, IV e V de aves. Cryptosporidium galli e Cryptosporidium genótipo III de aves estão relacionados com infecções crônicas no proventrículo, de forma semelhante à infecção por Cryptosporidium serpentis, em serpentes. Vários métodos de diagnóstico são utilizados para detecção do parasito, mas somente aqueles com base em biologia molecular são capazes de determinar a espécie de Cryptosporidium. O projeto teve com objetivo o desenvolvimento da PCR duplex em tempo real, tendo como alvo o gene da subunidade 18S do rRNA, por meio de ensaio TaqMan, para detecção de DNA de C.galli e Cryptosporidium genótipo III de aves, e avaliar os atributos diagnósticos da PCR duplex em tempo real quando comparada à nested PCR, utilizando 1027 amostras fecais de aves das ordens Passeriformes e Psittaciformes. A PCR duplex em tempo real apresentou positividade em 580/1027 (56,47%) para C. galli, enquanto que a nested PCR resultou em positividade para Cryptosporidium spp. em 104/1027 (10,13%) amostras. Para Cryptosporidium genótipo III de aves, houve positividade em 21/1027 (2,04%). A PCR duplex em tempo real resultou em alta especificidade analítica quando foram utilizadas amostras de DNA de outras espécies e genótipos de Cryptosporidium. A única exceção foi a amplificação de DNA de C. serpentis, com a utilização de primers e sonda para detecção de Cryptosporidium genótipo III de aves, porém, com uma baixa eficiência de amplificação. Foram identificados novos hospedeiros aviários para ambas as espécies gástricas, assim como foi possível a identificação de C. baileyi e, pela primeira vez no Brasil, de Cryptosporidium genótipo V de aves. Conclui-se que a PCR duplex em tempo real desenvolvida neste estudo é um recurso que apresenta rapidez e confiabilidade para diagnóstico de criptosporidiose gástrica em aves / Cryptosporidiosis has been described in several animal species, including many species of birds, and is considered a major protozoan infection of the orders Anseriformes, Charadriformes, Columbiformes, Galliformes, Passeriformes, Psittaciformes and Struthioniformes. Three species of Cryptosporidium are valid in birds: Cryptosporidium baileyi, Cryptosporidium meleagridis and Cryptosporidium galli; beside these species, there are several genotypes genetically distinct, as avian genotypes I, II, III, IV and V. Cryptosporidium galli and Cryptosporidium avian genotype III are related to chronic infections in proventriculus, similar to Cryptosporidium serpentis infection in snakes. Several methods are used for cryptosporidiosis diagnosis, but only those based on molecular biology are able to determine the Cryptosporidium species and genotypes. The aim of this project was the development of a duplex real-time PCR targeting 18S subunit of rRNA gene, by TaqMan assay, to detect DNA of C. galli and Cryptosporidium avian genotype III, and to evaluate the diagnostic attributes of the duplex real-time PCR compared to nested PCR, using 1027 fecal samples from birds of the orders Passeriformes and Psittaciformes. The duplex real time PCR showed positivity in 580/1027 (56.47%) for C. galli, whereas nested PCR was positive for Cryptosporidium spp. in 104/1027 (10.13%) samples. For Cryptosporidium avian genotype III, there was positivity in 21/1027 (2.04%) samples. The duplex real time PCR resulted in high analytical specificity when tested using DNA samples from other Cryptosporidium species and genotypes. The only exception was the amplification of DNA from C. serpentis with primers and probe for the detection of Cryptosporidium avian genotype III, although with lower efficiency. New avian hosts were identified for both gastric species, as well as it was possible to identify C. baileyi and, for the first time in Brazil, Cryptosporidium avian genotype V. It was concluded that the duplex real time PCR developed in this study is a fast and reliable tool for diagnosis of gastric cryptosporidiosis in birds.

Em 2005 o primeiro caso de Febre Maculosa Brasileira (FMB) foi reconhecido no estado do Rio Grande do Sul (RS), Brasil. Desde então até abril de 2016 doze casos foram confirmados, sendo que quatro destes são autóctones do município de Cerro Largo, área considerada endêmica para a enfermidade riquetsial. Neste mesmo período o RS também notificou 58 casos suspeitos ao Ministério da Saúde. No RS são encontrados dois biomas, Mata Atlântica e Pampa, este último não ocorrendo em outras unidades federativa do país. Até o momento, inexistem estudos relatando a infecção por riquétsias na ixodofauna gaúcha. Desta forma, o presente estudo teve como objetivo pesquisar a infecção de riquétsias do Grupo da Febre Maculosa (GFM) em cães, pequenos mamíferos e carrapatos; através da Reação de Imunofluorescência Indireta (RIFI), qPCR e tentativa de isolamento de riquétsias em cultivo celular dos ixodídeos coletados em área endêmica (Cerro Largo) e áreas não endêmicas nos biomas Pampa e Mata Atlântica no RS. Na sorologia (RIFI), 33,5 % (55/164), 2,9% (1/33), 40% (16/40) dos pequenos mamíferos e 8,3% (3/36), 13,9% (5/36) e 20,4 (28/137) dos cães coletados na Mata Atlântica, Pampa e área endêmica para FMB, respectivamente, foram sororreagentes para pelo menos um dos seis antígenos de riquétsias testados. Oito espécies de carrapatos foram coletadas no local de Mata Atlântica, sendo: Amblyomma aureolatum, Amblyomma brasiliense, Amblyomma incisum, Amblyomma ovale, Haemaphysalis juxtakochi, Ixodes loricatus, Rhipicephalus microplus e Amblyomma yucumense com destaque para esta última que foi descrita como uma espécie nova. Já para o fragmento de Pampa quatro espécies foram encontradas (A. aureolatum, A. ovale, Amblyomma tigrinum e Rhipicephalus sanguineus) e na área endêmica para FMB no RS, coletamos cinco espécies de carrapatos, Amblyomma dubitatum, Amblyomma longirostre, A. ovale, I. loricatus e R. microplus. Detectamos molecularmente pela primeira vez no RS, Candidatus Rickettsia amblyommii em A. longirostre e A. brasiliense, Candidatus Rickettsia andeanae em A. aureolatum e A. tigrinum, Rickettsia bellii em I. loricatus, Rickettsia rhipicephali em A. yucumense e H. juxtakochi, Candidatus Rickettsia asemboensis em pulgas no Brasil e de extrema importância para o conhecimento da epidemiologia da FMB no RS, detectamos Rickettsia sp. cepa Mata Atlântica em A. ovale oriundos de Cerro Largo (área endêmica para FMB no RS). Como primeiros isolamentos de riquétsia para o estado do RS, isolamos R. bellii de I. loricatus coletados na Mata Atlântica e área endêmica para FMB no RS. O presente estudo é pioneiro em relação a investigação comparativa sobre FMB nos biomas Pampa e Mata Atlântica e também o primeiro estudo molecular e isolamento de riquétsias em cultivo celular de amostras coletadas no RS, Brasil / In 2005 the first Brazilian Spotted Fever (BSF) case was confirmed in the Rio Grande do Sul (RS) state, Brazil. Ever since until April 2016, twelve cases have been confirmed, and four of them are native from Cerro Largo city, an area considered endemic to the riquetsial illness. During the same period, RS also notified 58 suspected cases to the Ministry of Health. There are two distinct biomes in RS, Atlantic Forest and Pampa, the later restricted to RS within the Brazilian land. Until now, no studies reported rickettsial infection in the ixodofauna of RS. Thus, this study aimed to investigate the rickettsial infection of Spotted Fever Group (SFG) in dogs, small mammals and ticks; by immunofluorescence assay (IFA), qPCR and attempted rickettsial isolation in cell culture from ticks collected in an endemic area (Cerro Largo) and non-endemic areas in Pampa and Atlantic Forest biomes of RS. Through serology (IFA), 33.5% (55/164), 2.9% (1/33) and 40% (16/40) small mammals, and 8.3% (3/36), 13.9 % (5/36) and 20.4 (28/137) dogs from the Atlantic Forest, Pampa, and BSF-endemic area, respectively, were seroreactive (titer ≥64) to at least one of the six rickettsia antigens tested. Eight species of ticks were collected in the Atlantic Forest: Amblyomma aureolatum, Amblyomma brasiliense, Amblyomma incisum, Amblyomma ovale, Haemaphysalis juxtakochi, Ixodes loricatus, Rhipicephalus microplus and Amblyomma yucumense, the later described as a new species. In the Pampa fragment, four tick species were found (A. aureolatum, A. ovale, Amblyomma tigrinum and Rhipicephalus sanguineus), and five tick species (Amblyomma dubitatum, Amblyomma longirostre, A. ovale, I. loricatus, R. microplus) in the BSF-endemic area of RS. We performed the first molecular detection in the RS state of "Candidatus Rickettsia amblyommii" in A. longirostre and A. brasiliense; "Candidatus Rickettsia andeanae" in A. aureolatum and A. tigrinum; Rickettsia bellii in I. loricatus; Rickettsia rhipicephali in A. yucumense and H. juxtakochi; and "Candidatus Rickettsia asemboensis" in fleas in Brazil. Noteworthy, we detected the pathogen Rickettsia sp. strain Atlantic rainforest in A. ovale ticks from Cerro Largo (endemic area for BSF in RS). We also performed the first isolation in cell culture of rickettsia from RS state, which comprised R. bellii from I. loricatus collected in the Atlantic Forest and the BSF-endemic area. This study is the first comparative research about rickettsiae among Pampa and Atlantic Forest biomes, and also the first molecular detection and isolation in cell culture of rickettsiae from Rio Grande do Sul, Brazil

Mefenoxam has been a premier compound for Phytophthora disease control in the nursery industry for 30 years. The primary objectives of this research were to examine whether Phytophthora species have developed resistance to this compound and to investigate fungicide resistance management strategies. Phytophthora nicotianae, a destructive pathogen of numerous herbaceous and some woody ornamental plants, was used as a model system. P. cinnamomi, a major pathogen of a wide range of tree species and shrub plants, was also included for comparison. Twenty-six isolates of P. nicotianae were highly resistant to mefenoxam with a mean EC50 value of 326.5 µg/ml while the remaining 70 were sensitive with an EC50 of <0.01 µg/ml (Label rate: 0.08µg/ml). All resistant isolates were recovered from herbaceous annuals and irrigation water in 3 Virginia nurseries. Resistant isolates were compared with sensitive ones using seedlings of Lupinus "Russell Hybrids" in the absence of mefenoxam for relative competitive ability. Resistant isolates out-competed sensitive ones within 3 to 6 sporulation cycles. Resistant isolates exhibited greater infection rate and higher sporulation ability than sensitive ones. No mefenoxam resistant isolates were identified in P. cinnamomi. All 65 isolates of P. cinnamomi were sensitive to mefenoxam with an EC50 of < 0.04 ï ­g/ml. Attempts to generate mutants with high resistance to mefenoxam through UV mutagenesis and mycelial adaptation were not successful. However, there were significant reductions in sensitivity to mefenoxam; those slightly resistant mutants carried fitness penalties, which may explain why P. cinnamomi remains sensitive to mefenoxam. The effect of propamocarb hydrochloride on different growth stages of Phytophthora nicotianae was evaluated in search for an alternative fungicide. Propamocarb greatly inhibited sporangium production, zoospore motility, germination and infection. However, it has little inhibition of mycelial growth and infections. Propamocarb can be used as an alternative fungicide to mefenoxam where mefenoxam resistance has become problematic. However, it must be used preventively; i.e. before infections occur. The genetic inheritance of mefenoxam resistance in P. nicotianae was studied using F1 progenies of a cross between resistant and sensitive isolates. The F1 progenies segregated for mefenoxam resistance in ratio of 1R:1S, indicating the mefenoxam resistance is controlled by a single dominant gene. One RAPD marker putatively linked to resistant locus in repulsion phase was obtained by bulked segregant analysis and was converted to the SCAR marker. This marker is capable of differentiating mefenoxam resistant populations from sensitive populations included in this study. / Ph. D.

Arrière-plan: les cellules tumorales circulantes (CTC) sont détectables dans de nombreux cancers et peuvent être utiles cliniquement pour le pronostic de la maladie, pour mesurer la récidive et pour prédire la sensibilité aux medicaments chimiothérapeutiques. Au cours des dernières années, l’études des CTC dans de nombreux cancers tels que le cancer du sein, du poumon, du côlon et de la prostate a grandement évolué. Alternativement, il y peu d'études à ce sujet concernant le cancer du col de l’utérus (CCU). Objectifs: Notre objectif est d’optimiser le processus d'enrichissement des CTC dans le CCU et la détection moléculaire des biomarqueurs E6 et E7. Matériel et Méthodes: Dans l’optique de mimer la présence de CTC dans le sang, nous avons dilué des cellules cancéreuses CaSki VPH16-positif provenant d’un CCU dans du sang humain prélevé sur des volontaires sains. Les CaSki ont été collectées suite à une centrifugation par densité avec le Ficoll, la lyse des globules rouges (RBC) et la lyse des RBC combinée avec un enrichissement positif et négatif à l’aide de marqueurs de surface cellulaire. Les CTC ont été détectées par la mesure d’expression des oncogènes E6 et E7 du virus du papillome humain (VPH), de la cytokératine 19 (CK19) et de la cycline p16INK4 en utilisant la technique quantitative en temps réel de Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). Pour valider notre méthode de détection des CTC in vivo, nous avons recruté dix patientes atteintes d’un CCU VPH16 positif et six contrôles sains. Résultats: Dans le modèle de dilutions de cellules CaSki, la lyse des RBC seule ou combinée avec l'enrichissement négatif ou positif suggèrent des limites de détection de 1 CTC par mL de sang pour tous les biomarqueurs moléculaires utilisés. La sensibilité de détection est accrue lors de l'utilisation de l’enrichissement positif et négatif en réduisant le bruit de fond causé par les monocytes sanguins. Contrairement aux oncogènes E6 et E7, les marqueurs CK19 et p16INK4A ont été détectés chez des individus sains, les niveaux d'expression de base appropriés doivent donc être déterminés avec précision par rapport aux patientes CCU. Le gradient de densité par Ficoll a une limite de détection de seulement environ 1000 cellules par mL de sang. Enfin, les CTC ont été détectées dans 2/10 patientes en utilisant le marqueur CK19. Cependant, ces patientes étaient négatives pour les oncogènes E6/E7. Le marqueur p16INK4A était exprimé au même niveau dans tous les échantillons (CCU et normaux). Conclusion: Notre étude suggère que les oncogènes E6 et E7 du VPH16 sont les marqueurs biologiques les plus sensibles et spécifiques en qRT-PCR pour détecter les CTC dans le modèle de dilution de cellules de CCU dans le sang. Chez les patientes atteintes d’un CCU de stade précoce, seulement CK19 a révélé la présence potentielle de CTC, ce qui suggère que ces cellules sont rares à ce stade de la maladie. Mots clés: cancer du col de l’utérus, cellules tumorales circulantes, RT-qPCR, E6 et E7, CK19, p16INK4A, enrichissement immunomagnétique, détection moléculaire. / Background: Circulating tumor cells (CTCs) have been detected in many cancers and are used in multiple clinical applications including disease prognosis, tumor recurrence prediction and prediction of tumor sensitivity to chemotherapeutic drugs. Studies in most major solid cancer(s) (breast, lung, colon and prostate) are progressing rapidly, but there has been very little progress concerning uterine cervical cancer (UCC).Objective: our aim is to optimize enrichment processes and the molecular biomarker-based detection of human circulating tumor cells (CTCs) in uterine cervical cancer (UCC). Material & Methods: To mimic CTCs in patients, we designed an experimental spiking model where the CaSki HPV16-positive UCC cell line was serially diluted and spiked into human blood collected from healthy volunteers. CaSki CTCs were enriched using either Ficoll density centrifugation, red blood cell (RBC) lysis or RBC lysis combined with cell surface markers negative or positive enrichment. CTCs were detected using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure the gene expression of human papillomavirus (HPV) viral oncogenes (E6 and E7), cytokeratin 19 (CK19), or the cyclin dependent kinase inhibitor p16INK4A. Finally, ten HPV16- positive UCC patients and six healthy controls were recruited to validate CTCs detection in vivo. Result: In the spiking model, RBC lysis alone or combined with negative or positive enrichment suggests detection limits close to 1 CTC per mL of blood for all molecular biomarkers used. The sensitivity of detection increased when using positive and negative enrichment probably by reducing the peripheral blood mononuclear cell-derived RNA background. Unlike HPV oncogenes, CK19 and p16INK4A were detected in normal individuals, thus appropriate basal expression levels need to be accurately determined compared to cancer patients. Alternatively, Ficoll density gradient had a detection limit of only about 1000 cells per mL of blood. Finally CTCs were detected in 2/10 patients using CK19. None of the patients had E6/E7 transcripts and p16INK4A was expressed at similar level across all samples (cancer and healthy). Conclusion: qRT-PCR of HPV16 E6 and E7 is the most sensitive and specific biomarker used to detect CTCs in the spiking model. In early disease UCC patients, only CK19 revealed the presence of CTCs suggesting that these cells are rare at that stage of the disease. Keywords: uterine cervical cancer, circulating tumor cells, qRT-PCR, E6 and E7 oncoprotein, CK19, p16INK4A, immune-magnetic enrichment, molecular detection.