Os vírus da influenza aviária têm provocado epidemias e pandemias através dos tempos, a pandemia mais devastadora que se tem notícia, a gripe espanhola em 1918, teve sua origem no vírus aviário do tipo A subtipo H1N1. Desde 2003 o vírus aviário do subtipo H5N1 infectou 442 pessoas e levou a morte 262. Além do aspecto de saúde os vírus da gripe aviária causam grande impacto econômico. O Brasil como maior exportador de frango do mundo tem muito a perder caso a gripe aviária chegue ao país. Devido às aves selvagens serem o reservatório natural influenza A, é que se faz necessário a execução do monitoramento. Apesar de existir programas de monitoramento contínuo de aves selvagens na Europa, EUA, Canadá, Japão entre outros, pouco foi feito no Brasil. Amostras coletadas de 671 aves foram testadas por meio das técnicas de GeneScan, PCR em tempo real e RT-PCR e Duplex Nested-PCR. / The avian influenza virus has caused epidemics and pandemics through the ages, the most devastating pandemic that we know, the Spanish flu in 1918, had its origin in the avian virus type A subtype H1N1. Since 2003 the avian virus subtype H5N1 has infected 442 people and led to death 262. Besides the health aspect of the avian influenza viruses cause major economic impact. Brazil as the largest exporter of chicken in the world has much to lose if bird flu reaches the country. Because wild birds are the natural reservoir of influenza A, is that it is necessary to implement the monitoring. Although programs exist for continuous monitoring of wild birds in Europe, USA, Canada, Japan and others, little has been done in Brazil. Samples collected from 671 birds were tested by GeneScan techniques, real-time PCR and RT-PCR and nested-PCR Duplex.

This master thesis is focused on the molecular characterization of the eight red yeasts species. For molecular characterisation, the most variable rDNA regions ITS1, 5,8S ITS2 and the region encoding the large ribosomal subunit (26S) were amplified. This long region of the yeasts DNA was sequenced and compared by NCBI database for identification. The red yeasts identification was confirmed by data from DGGE method. Another aim of this thesis was to select the best yeasts producer of carotenoids and triacylglycerols. Rhodosporidium toruloides was found as the best producer and, thus, this strain was subjected to random mutagenesis by UV irradiation. The results of the production of metabolites by R. toruloides were compared with mutant strains, which were also adapted to the glycerol and waste whey substrates. The mutant strain G33 was found as the best producer of total carotenoids with a yield of 7.14 mg.g-1 of biomass. The highest production of ergosterol was demonstrated by the mutant strain Y34, the ergosterol yield was 47.72 mg.g-1 of biomass. The wild type of R. toruloides was able to produce the highest amount of both carotene (2.42 mg.g-1 of biomass) and TAG (76.32 mg.g-1 of biomass) on glucose medium.

Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. They represent one of the widest group of natural antioxidants with significant biological effects and numerous of industrial applications. There is an increased interest in carotenoids as natural antioxidants for their ability to reduce chronic diseases, various pathological stages and aging. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. In presented thesis carotenogenic yeast belonging to the genus Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates and also random mutagenesis in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. As alternative nutrient sources derived from waste substrates from agricultural and food production (rapeseed substrate, rice, wheat, apple fiber, pasta and lignocellusic materials) were tested. To selected production media extracellular hydrolytic enzymes or commercial enzymes degrading polysaccharide were added. All tested red yeast strains were able to utilize these substrates as the only carbon source and simultaneous produce carotenoid enriched biomass. In this work, characterization of carotenogenic yeast using molecular techniques was studied. For this usage, interspecific variables of strongly conserved sequences of genomic DNA, especially rDNA D1/D2 large ribosomal subunit and ITS1 and 5,8-ITS2 rDNA regions were amplified. These sequences were subjected analysed by DGGE method to compare differences of carotenogenic yeasts. Isolation procedure of the intact DNA were optimized for caryotypic yeast characterization by pulsed field gel electrophoresis (PFGE). The karyotype of tested yeasts contain visible differences between yeast species and genera.

This diploma thesis deals with the molecular characterization of carotenogenic yeasts. The techniques used for the analysis of the conserved regions of the D1/D2 rDNA region of the 26S ribosomal large subunit region and the ITS1 and 5,8-ITS2 regions were nested PCR and DGGE. The results of DGGE show that all analyzed yeast strains have very similar sequences of these regions The yeast Rhodotorula mucilaginosa with the collection number CCY 20-7-28 showed differences from the other carotenogenic yeast strains. As a part of melucular characterisation using ribosomal gene sequences, eight yeast strains were examinated for substrate utilisation tests using different substrates. Characterisation of growth and metabolite production was tested in each strain too. The next aim of this thesis was to prepare a carotenoid yeast strain characterized by overproduction of metabolites, in particular carotenoids and lipids,. Yeasts were subjected to a random mutation caused by UV irradiation and the influence of this mutantagen onthe production of metabolites was evaluated. As a candidate yeast strain C. capitatum CCY 10-1-2 was selected. This selection was based on previous studies due to its good production of lipids using waste glycerol as asubstrate. This strain was subsequently adapted to waste whey, glycerol, and a glucose as a basic carbon source.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health concern in many developing countries. It mainly infects young adults and has a mortality of up to 25% in pregnant women. Although hepatitis E is only sporadic in industrialized countries including the United States, a relative high seroprevalence rate has been reported in healthy individuals. Evidence suggests that there exist animal reservoirs for HEV and HEV transmission is zoonotic. Animal strains of HEV, swine HEV and avian HEV have been identified from a pig and a chicken, respectively, in the United States. Studies showed that swine HEV and avian HEV are genetically and antigenically related to human HEV, and that pigs and chickens are useful animal models to study HEV replication, pathogenesis and cross-species infection. The objectives of this dissertation were to genetically characterize both avian HEV and swine HEV, to determine their serological and molecular epidemiology in the United States, to assess the ability of avian HEV cross-species infection in non-human primates, to determine the full-length genomic sequence and genome organization, and to construct an infectious cDNA clone of avian HEV. The prevalence of swine HEV infections in US swine herds and the heterogeneity of swine HEV isolates from different geographic regions of the United States were determined. We found that 35% pigs and 54% swine herds were positive for swine HEV RNA. Partial capsid gene region of twenty-seven US swine HEV isolates was sequenced and was showed to share 88%-100% nucleotide sequence identity to each other and 89-98% identity with the prototype US swine HEV, but only <79% identity with Taiwanese swine HEV isolates and most known human strains of HEV worldwide. All US swine HEV isolates belong to the same genotype 3 with the prototype US swine HEV and the two US strains of human HEV. Similarly, the prevalence of avian HEV infections in US chicken flocks and the heterogeneity of avian HEV isolates were also determined. Helicase gene region of eleven field isolates of avian HEV from chickens with hepatitis-splenomegaly (HS) syndrome was sequenced and was found to share 78-100% nucleotide sequence identities with each other, 79-88% identities with the prototype avian HEV, 76-80% identities with Australian chicken big liver and spleen disease virus (BLSV), and 56-61% identities with other known strains of mammalian HEV. A relative high prevalence of anti-avian HEV antibodies was found in apparently healthy chicken flocks in 5 states. Like swine HEV, the seropositivity of avian HEV in adult chickens was higher than that in young chickens. To genetically characterize the avian HEV genome, we determined the full-length genomic sequence of avian HEV, which is 6,654 bp in length excluding the poly (A) tail, and 600 bp shorter than that of mammalian HEVs. Avian HEV has similar genomic organization with human and swine HEVs, but shared only about 50% nucleotide sequence identity with mammalian HEVs in the complete genome. Significant genetic variations such as deletions and insertions, particularly in the ORF1 of avian HEV, were observed, but motifs in the putative functional domains of the ORF1 were relatively conserved between avian HEV and mammalian HEVs. Phylogenetic analyses based on the full-length genomic sequence revealed that avian HEV represents a branch distinct from human and swine HEVs. Since swine HEV infects non-human primates and possibly humans, the ability of avian HEV cross-species infection in non-human primates was also assessed. However, unlike swine HEV, avian HEV failed to infect two rhesus monkeys under experimental conditions. With the availability of the complete genome sequence of avian HEV, we constructed three full-length cDNA clones of avian HEV and tested their infectivity by in vitro transfection of the LMH chicken liver cells and by in vivo intrahepatic inoculation of specific-pathogen-free (SPF) chickens. The results showed that all 3 cDNA clones of avian HEV were infectious both in vitro and in vivo, as the capped RNA transcripts from each of the clones were replication-competent in transfected LMH cells and developed active infection in inoculated SPF chickens. In summary, avian HEV and swine HEV infections are enzootic in chicken flocks and in swine herds in the United States, respectively. Like human HEV, swine HEV and avian HEV isolates from different geographic regions are also genetically heterogenic. Complete genomic sequence analyses showed that avian HEV is related to, but distinct from, human and swine HEVs. Unlike swine HEV, avian HEV is probably not transmissible to non-human primates. Infectious cDNA clones of avian HEV have been successfully constructed. The availability of the infectious clones for a chicken strain of HEV now affords us an opportunity to study the mechanisms of HEV replication, pathogenesis and cross-species infection. / Ph. D.

Ammonia-oxidizing bacteria (AOB) are chemolithotrophs that oxidize ammonia/ammonium to nitrite in a two-step process to obtain energy for survival. AOB are difficult to isolate from the environment and iso lated strains may not represent the diversity in soil. A genetic database and molecular tools were developed based on the ammonia monooxygenase (AMO) encoding genes that can be used to assess the diversity of AOB that exist in soil and aquatic environments without the isolation of pure cultures. The amo genes have excellent potential as molecular markers; since AMO is only found in the AOB and is essential for their metabolism, AOB must carry at least one functional copy of the amo operon. The operon is composed of at least three genes, amoC, amoA. and amoB (encoding for the subunits AmoC, AmoA, and AmoB). The amoC gene was first discovered and its sequence was obtained from Nitrosospira sp. NpA V. The amooperon is found in several copies within AOB genomes in the β-subdivision but as a single copy in y-subdivision genomes. In Southern analysis, cross-hybridization was only observed between amo genes within a subdivision. They-subdivision amo sequences have higher identity values to the genes encoding the related particulate methane monooxygenase than to the β-subdivision amo sequences. Since amoA encodes the subunit containing the active site, it was sequenced entirely for all the strains studied (16 amoA sequences total). The amoC and amoB genes were also sequenced for several strains. The amo genes allow for better discrimination between closely related strains than the 16S rRNA genes. In all cases, the amo operon consists of amoC, followed by a variable length intergenic region, and then by amoAB. The variability in length of the intergenic region is strain specific, and is therefore potentially useful for profiling AOB communities. The amo-gene database was the basis for the design of conserved oligonucleotide primers for the polymerase chain reaction (PCR). These primers were used to amplify amo sequences from a mixed template of DNA extracted directly from soil. Results indicate that the amo genes are excellent molecular markers for the assessment of AOB communities in the environment.

Cercarial dermatitis (CD) is manifested as a strong itchy rash that usually occurs after bathing in the natural water bodies where it makes recreation uncomfortable. As a consequence, the natural swimming areas and be closed because of inconvenient water quality, what subsequently leads to the financial losses. The infection can also affect people working in the natural water bodies as lifeguards (at the natural swimming areas), people monitoring water organisms or water quality etc.). Nowadays, CD in Czech Republic occurs more frequently than in the past. For this reason, the topic of avian schistosomes and CD is very attractive not only for scientists, but newly also for employees of hygienic stations and health institutes. Based on the Act No. 258/2000 Coll. on protection of public health and related executive Decree No. 238/2011 Coll. approved this year their duty will be regular monitoring of official natural swimming areas for causative agents of CD. For the comprehensive overview about the occurrence of the avian schistosomes and CD, up to now, at the localities in the Czech Republic the specialized overview map with the marked catches of avian schistosomes and CD was created in the program ArcGIS Online. This map was made with use the records obtained from the scientific articles, final...

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Este trabalho teve como objetivos a identificação de sequências 18S rDNA amplificadas de Cryptosporidium spp. de diversas espécies de hospedeiros e avaliar variabilidade em sequências gênicas deste lócus, com vistas ao desenho de sondas moleculares com melhor eficiência diagnóstica para detecção e identificação deste parasito. Foram coletadas 392 amostras de animais domésticos (bovinos, eqüinos, suínos, ovinos, cães e felinos) de 98 propriedades rurais do município de Teodoro Sampaio, Estado de São Paulo, 474 de aves silvestres de cativeiro de diversas famílias, provenientes de criadouros comerciais do Estado de São Paulo e criadas como estimação, 141 de sagüis de cativeiro do Estado de São Paulo, e 24 de humanos imunodeprimidos provenientes de hospital do município de São Paulo. As amostras foram submetidas a prova coproparasitológica e molecular para detecção e identificação de Cryptosporidium. Alinhamentos múltiplos obtidos de seqüências 18S rDNA de Cryptosporidium spp. determinadas neste estudo e de sequências recuperadas do Genbank foram analisados visualmente para a definição das regiões polimórficas. Após a definição das regiões polimórficas, foram realizadas análises filogenéticas empregando-se separadamente cada uma delas. Pelo exame coproparasitológico foi encontrado positividade em amostras de nove (4,57%) bovinos, três (11,11%) cães, 41 (8,64%) aves silvestres, 13 (9,20%) sagüis e todas as de humanos. As outras espécies de animais domésticos não apresentaram positividade para o parasita no exame coproparasitológico. Nos bovinos foi encontrado o Cryptosporidium Andersoni, em cães o Cryptosporidium canis, em sagüis o Cryptosporidium parvum e em humanos, C. parvum, Cryptosporidium hominis, Cryptosporidium felis e C. canis. Dentre as amostras de aves nenhuma foi identificada como Cryptosporidium meleagridis. As amostras de curiós (Oryzoborus angolensis) foram classificadas como Cryptosporidium galli, com exceção de uma, identificada como Cryptosporidium baileyi. C. galli foi encontrado também em um Sabiá Laranjeira (Turdus rufiventris), um Picharro (Saltator similis), dois canários e um Pintassilgo (C. carduelis). C. baileyi foi encontrado em um pintassilgo (Carduelis carduelis) um Pichochó (Sporophila frontalis), um Galo da Campina (Paroaria dominicana) e dois Canários (Sicalis flaveola). Pelos resultados, duas regiões polimórficas em sequência 18S rDNA de Cryptosporidium spp. (denominadas regiões 1 e 3) permitiram discriminar as diferentes espécies neste gênero de parasita, podendo ser utilizadas isoladamente como marcadores moleculares para identificação molecular dentro deste gênero. Saguis (Chalitrix spp.) de cativeiro são espécies susceptíveis a infecção por Cryptosporidium parvum apresentando-se como um hospedeiro de importância epidemiológica para esta zoonose. Curiós (O. angolensis) de cativeiro são espécies susceptíveis a infecção por Cryptosporidium galli apresentando-se como hospedeiro de importância epidemiológica para esta espécie de parasito. A não detecção de Cryptosporidium parvum em animais domésticos na região de Teodoro Sampaio, Estado de São Paulo, mostra uma condição sanitária favorável, já que este agente é causador de importante zoonose. A presença de espécies de Cryptosporidium spp. adaptadas a animais domésticos (como o C. felis e o C. canis) em humanos na cidade de São Paulo mostra que estes animais podem desempenhar importante papel na cadeia epidemiológica da criptosporidiose humana. / The objectives of this study were to identify 18S rDNA sequences of Cryptosporidium spp. From various species of hosts and to avaluate the variability in gene sequences of this locus, aiming the design of molecular probes with better diagnostic efficiency for the detection and identification of this parasite. It was collected 392 samples of domestic animals (cattle, horses, pigs, sheeps, dogs and cats) of 98 rural properties of Teodoro Sampaio city, São Paulo State, 474 captive wild birds of various families, from pet and comercial breeding in São Paulo State, 141 captive marmosets, of São Paulo State, and 24 immunossupressed humans from a São Paulo city hospital. The samples were submitted to coproparasitological and molecular tests for the detection and identification of Cryptosporidium. Multiple alignment of Cryptosporidium 18S rDNA sequences, that was determinated in this study and other download from GenBank were visually inspected in order to define polymorphic regions. After the definition of polymorphic regions, phylogenetic trees were reconstructed using each polymorphic region. Cryptosporidium spp. Were found by using coproparasitological tests in nine (4,57%) samples of cattle, tree (11,11%) dogs, 41 (8,64%) wild birds, 13 (9,20%) marmosets and all human samples. The other animal species were negative by coproparasitological tests. In cattle it was found Cryptosporidium andersoni, in dogs Cryptosporidium canis, in marmosets Cryptosporidium parvum and in humans, C. parvum, Cryptosporidium hominis, Cryptosporidium felis e C. canis. Among the samples of birds Cryptosporidium meleagridis was not found. All the samples of lesser seed-finch (Oryzoborus angolensis) were classified as Cryptosporidium galli, except for that from one individual with was identified as Cryptosporidium baileyi. Cryptosporidium galli was also found in one rufous-bellied thrush (Turdus rufiventris), one green-winged saltator (Saltator similis), two saffron finch (Sicalis flaveola) and one eurasian goldfinch (Carduelis carduelis). C. baileyi was found in one eurasian goldfinch (C. carduelis), one buffy-fronted seedeater (Sporophila Frontalis), one red-cowled cardinal (Paroaria dominicana) and two saffron finch (S. flaveola). From the results two polymorphic regions within 18S rDNA sequences of Cryptosporidium spp. (named as regions 1 and 3) enabled the discrimination of the different species in this genera, and then could be used alone as molecular markers for identification within this genera. Captive marmosets (Chalitrix spp.) are susceptible species for Cryptosporidium infection, presenting itself as an important source of infection for this zoonosis. Captive lesser seed-finch (Oryzoborus angolensis) are susceptible species for Cryptosporidium galli infection presenting itself as an epidemologic important host for this parasite. The absence of Cryptosporidium parvum in domestic animals of Teodoro Sampaio, São Paulo State, is indicative of a favorable health condition, as C. parvum is an agent causative of an important zoonosis. The presence of Cryptosporidium spp. species adapted to domestic animals (as C. felis and C. canis) in humans at São Paulo State indicate that these animals could play an important role in the epidemiology of human cryptosporidiosis.

Giardia duodenalis é um protozoário entérico de distribuição mundial responsável por causar a giardíase em uma grande variedade de mamíferos, incluindo os humanos. É considerada uma espécie complexa, no qual os isolados podem ser classificados em sete agrupamentos genéticos distintos apesar de serem morfologicamente indistinguíveis. O presente trabalho teve como objetivo avaliar a variabilidade genotípica de isolados de G. duodenalis provenientes de humanos naturalmente infectados, residentes em cidades do litoral de São Paulo, na Baixada Santista. A caracterização molecular de 43 isolados pelo seqüenciamento parcial de genes codificadores da enzima glutamato-desidrogenase (gdh) e da proteína beta-giardina (bg) mostrou que o assemblage B da G. duodenalis é o mais freqüente na região litorânea, ocorrendo em 53,5% (n=23) das amostras, sendo o assemblage A identificado em 34,8% (n=15). A maioria das seqüências obtidas pelo gene gdh se mostrou polimórfica, caracterizada por picos duplos de nucleotídeos em algumas posições em cromatograma e quando a análise dos dois genes foi combinada cinco isolados apresentaram identidades diferentes. A esses fenômenos duas explicações são atribuídas, infecção mista ou heterozigose de seqüência alélica. As análises filogenéticas mostraram que o gene bg é mais conservado que o gene gdh, não sendo capaz de discriminar os sub-agrupamentos que constituem os Assemblages do parasita. Com base nos resultados apresentados podemos concluir que a participação de genótipos zoonóticos é relevante na epidemiologia das giardíases nos indivíduos residentes das cidades litorâneas do estado de São Paulo e que estudos de caracterização molecular da G. duodenalis são indispensáveis para melhor conhecimento da epidemiologia desta infecção. / Giardia duodenalis is an enteric protozoa of global distribution responsible for causing giardiasis in a wide range of mammals including humans. Is considered complex specie in which the isolates can be classified in different genetic groups despite to be morphologically indistinguishable. The purpose of this study was evaluating the genetic variability of G. duodenalis isolates from humans naturally infected residents in the coast cities of Sao Paulo, in Baixada Santista. The molecular characterization of 43 isolates using the gene encoding glutamate-desidrogenasi enzyme (gdh) and beta-giardin protein (bg) showed that the assemblage B is the most common in the coast region, occurring in 53.5% (n=23) samples, the assemblage A was identified in 34.8% (n=15). Most sequences obtained by the gdh gene showed polymorphism, characterized for double peaks of nucleotides in some chromatogram positions and when the analysis of two genes was combined five isolates were differently identified. For this phenomena can be attributed two explanations, mixed infections or heterozygosis allelic sequence. The phylogenetic analysis showed that bg gene is more conserved than gdh gene, not being able to discriminate the sub-groups of parasite assemblages. Based on the presented results we can conclude the participation of zoonotic genotypes is important in the epidemiology of giardiasis in residents of the coast cities of Sao Paulo state and molecular characterization studies of G. duodenalis are essential for better understanding the epidemiology of this infection.