O presente trabalho esteve inserido em um projeto mais amplo de cooperação internacional intitulado Conservation and Sustainable Management of Below-Ground Biodiversity CSMBGBD/ BiosBrasil, implementado pela United Nations Environment Programmer (UNEP) na bacia do Alto Solimões, Amazônia Ocidental, estado do Amazonas. Esta região é território remanescente de povos indígenas e permanece conservada, sendo um importante hotspot de biodiversidade. Ainda assim, as áreas de estudo foram caracterizadas por paisagens antropizadas com diferentes sistemas de usos da terra. Amostras de solos foram coletadas em um período de elevado índice pluviométrico, nos anos de 2008 e 2009, em áreas caracterizadas por floresta primária tropical, cultivos semiperenes de mandioca manejados por prática agrícola de corte-equeima, pastagens implantadas nos anos de 1970 e áreas florestais em estádios avançados de regeneração (>10 anos de abandono). As amostras foram analisadas pelas técnicas de DGGE, ARISA, clonagem e sequenciamento a fim de obter uma caracterização das estruturas de comunidades de Archaea, Bacteria e microfungos e da composição e diversidade de um grupo funcional de Archaea envolvido no processo de oxidação de amônia nos ambientes do solo. Os resultados permitiram concluir que o uso da terra tem um grande efeito sobre as estruturas de comunidades de Archaea, Bacteria e microfungos presentes no solo e sugerem que longo período de abandono das áreas é necessário para cumprir com a resiliência dos ecossistemas amazônicos no contexto de recomposição da paisagem. Adicionalmente, os dados revelaram que a riqueza e a diversidade de comunidades de Archaea oxidadoras de amônia foram capazes de refletir sensivelmente as alterações percebidas nos ambientes do solo em decorrência do desmatamento de áreas de floresta primária e uso subsequente com cultivo agrícola tradicional e pastagem na região do Alto Solimões, Amazônia Ocidental. / The present study was part of a wider project of international cooperation entitled Conservation and Sustainable Management of Below-Ground Biodiversity CSMBGBD/ BiosBrasil, established by the United Nations Environment Programmer (UNEP) at the basin of Alto Solimões, Western Amazon, Amazonas State. This region is a remanescent territory of indigenous people and remains conserved, being considered an important hotspot of biodiversity. Even though, the studied areas were characterized by mosaic landscapes under different land use systems. Soil samples were collected in a period of high pluviometric index in the years 2008 and 2009 in areas characterized by tropical rainforest, semi permanent manioc cultivation under agricultural management of slash-and-burn, pasture established in the 1970s and forested areas at a higher stage of regeneration (>10 years abandoned). The samples were analyzed by DGGE, ARISA techniques, cloning and sequencing in order to obtain a characterization of the community structure of Archaea, Bacteria and microfungi and the composition and diversity of an archaeal functional group involved in the process of ammonia oxidation in the soil environments. The results allowed to conclude that land use has a great effect on the community structure of Archaea, Bacteria and microfungi present in the soil and suggest that long period of abandon of the areas is necessary to accomplish with the resilience of Amazonian ecosystems in the context of landscape recomposition. Additionally, the data revealed that richness and community diversity of ammonia oxidizing Archaea were able to sensitively reflect the changes observed in the soil environment due to deforestation of rainforest areas and subsequent use with traditional agriculture cultivation and pasture in the region of Alto Solimões, Western Amazon.

Tecidos vegetais, incluindo as sementes, são habitados por microrganismos denominados endofíticos, cuja interação com a planta pode conferir características vantajosas ao hospedeiro. Sabe-se que o crescimento de plantas é influenciado por fatores como a síntese de ácido-indolacético (AIA), solubilização de fosfato, fixação de nitrogênio e controle de fungos fitopatogênicos. Antes da comercialização, as condições de armazenamento de sementes de soja podem restringir o desenvolvimento de microrganismos devido à baixa temperatura e umidade. Esse fato leva ao interesse de exploração de microrganismos endofíticos resistentes a essas condições. O estudo e a caracterização dessas comunidades são de grande interesse agronômico e biotecnológico, sendo possível sua aplicação em sementes, introduzindo no campo plantas com superior potencial de produção. Com os objetivos de comparar a comunidade bacteriana endofítica de sementes de soja geneticamente modificadas e convencionais; e de isolar e caracterizar essas comunidades, sementes de 12 cultivares de soja foram amostradas, de onde 3504 isolados bacterianos foram obtidos. Os isolados foram agrupados morfologicamente de acordo com a coloração e taxa de crescimento das colônias, sendo representantes de cada grupo morfológico (no total 176 isolados) agrupados pela técnica de ARDRA (Análise de Restrição de DNA Ribossomal Amplificado). Um total de 12 ribotipos foi observado compondo a comunidade cultivada de bactérias endofíticas de sementes de soja. Representantes destes ribotipos tiveram seus genes 16S rDNA parcialmente sequenciados, identificando os integrantes desta comunidade como: Acinetobacter sp., Bacillus sp., Brevibacterium sp., Chryseobacterium sp., Citrobacter sp., Curtobacterium sp., Enterobacter sp., Methylobacterium sp., Microbacterium sp., Micromonospora sp., Pantoea sp., Paenibacillus sp., Pseudomonas sp., Ochrobactrum sp., Streptomyces sp. e Tsukamurella sp. A comunidade bacteriana endofítica de sementes de soja provenientes de plantas genticamente modificadas apresentou uma diversidade maior comparada à comunidade bacteriana de plantas convencionais. Em relação ao potencial biotecnológico desta comunidade, os resultados demonstraram que os isolados foram capazes de controlar o crescimento de fungos fitopatogênicos por antagonismo (18%), sintetizar AIA (100%), solubilizar fosfato (39%) e fixar nitrogênio (18%). Os isolados com os melhores resultados nas análises in vitro foram inoculados em sementes de soja e avaliados em casa de vegetação quanto à habilidade de promover o crescimento das plantas. As plantas apresentaram diferentes respostas à inoculação das bactérias. A maior parte dos tratamentos mostrou influência negativa das bactéria nas plantas, enquanto que um isolado de Enterobacter sp. aumentou a massa da matéria seca de raiz. Mesmo não diferindo estatisticamente, alguns isolados mostraram tendência de aumento e outros de diminuição de biomassa da planta. / Plant tissues, including seeds, are inhabited by microorganisms called endophytes, whose interaction with the plant can offer advantages to the host. It is known that plants growth promotion is induced by indole acetic acid (IAA) synthesis, phosphate solubilization and nitrogen fixation, among others. The control of phytopathogenic fungi is also related to a good plant development. Before commercialization, the seed storage conditions can restrict the development of microorganism, due to the low temperature and humidity. This fact leads to the interest of exploring resistant microorganisms to those conditions. The study and the characterization of these communities are of great agronomic and biotechnological interest, being possible its application onto seeds, introducing in the field plants with a greater production potential. In this context, the aim of this study was to isolate and identify the endophytic bacteria community in soybean seeds and study the capacity of these isolates to promote growth in the host plant, including: phosphate solubilization, nitrogen fixation, IAA synthesize and antagonism against phytopathogenic fungi. From seeds of 12 cultivars, 3504 bacteria were isolated. The isolates were morphologically grouped according to the coloration and growth rate of the colony. Representatives of each morph group, totalizing 176, were analyzed using the Amplified Ribosomal Restriction Analysis (ARDRA) technique. A total of 12 ARDRA ribotypes were observed in the cultivable endophytic community of soybean seeds. Representatives of each ribotype had their 16S rDNA gene partially sequenced, allowing to the identification of the members of this community as: Acinetobacter sp., Bacillus sp., Brevibacterium sp., Chryseobacterium sp., Citrobacter sp., Curtobacterium sp., Enterobacter sp., Methylobacterium sp., Microbacterium sp., Micromonospora sp., Pantoea sp., Paenibacillus sp., Pseudomonas sp., Ochrobactrum sp., Streptomyces sp. and Tsukamurella sp. The endophytic bacterial community of soybean seeds from genetically modified plants showed a greater diversity compared to the bacterial community of conventional seeds. In relation to the biotechnological potential of the community, the outcomes demonstrate that the isolates were able to antagonist phytopathogenic fungi (18%), synthesize IAA (100%), solubilize phosphate (39%) and fix nitrogen (18%). The isolates with best in vitro outcomes were inoculated onto seeds and tested in greenhouse for their ability to promote growth in soybean. The plants answered differently to the inoculation of each bacterial isolate. The major part of the treatments demonstrated a negative influence of bacteria onto plants, while one Enterobacter sp. isolate increased the dry mass weight of roots. Even not differing statistically, some isolates showed a tendency to increase, meanwhile others to decrease the biomass of the plant.

Vias redox são importantes reguladores da homeostase e sinalização celular, mas o entendimento dos mecanismos desses processos é incompleto. Tiol-proteínas como a dissulfeto isomerase proteica (PDI) podem ser moduladores dessas vias. A PDI(PDIA1) é o protótipo da família das PDIs, cuja função canônica é o enovelamento redox de proteínas no retículo endoplasmático. Além disso, PDI exerce regulação de NADPH oxidases, as principais fontes de oxidantes celulares, e é necessária para ativação de RhoGTPases, organização do citoesqueleto e migração de células vasculares. No estudo de mecanismos pelos quais a PDI regula RhoGTPases, mostramos, em redes computacionais e em experimentos de co-imunoprecitação, associação entre PDIA1 e o regulador de RhoGTPases RhoGDIalfa. Além disso, identificamos forte proximidade entre os genes codificando estas proteínas. Neste estudo, caracterizamos o perfil e implicações desta sintenia gênica.A análise bioinformática pelos programs Ensembl, NCBI e UCSC evidencia um padrão de sintenia entre diferentes isoformas destas duas famílias: PDIA1 (P4HB), PDIA2 (PDIP) e PDIA8 (Erp27) são vizinhos, respectivamente, a RhoGDIbeta, RhoGDIy e RHOGDIalfa, com correspondentes regiões intergênicas de 7.1, 2.9 e 0.14 kb em distintos cromossomos em H. sapiens. O padrão dessa sintenia foi fortemente conservado emC. elegans, alguns peixes e uniformemente em anfíbios, répteis, aves e mamíferos. Leveduras expressam no mesmo cromossomo , porém em locais distantes (i.emacrossintenia) ortólogos da PDIA1 e RhoGDI?, mas não expressam outras PDIs e RhoGDIssintênicasnos eucariotos complexos. No entanto, sintenia entre PDI e RhoGDI foi também observada na planta A. thaliana, sem evidência de um ancestral comum. Os pares sintênicos associam-se a blocos vizinhos conservados, porém diversos para cada par, enquanto cada bloco contem um gene codificando um distinto regulador da PP1 (fosfatase proteica-1). Análise filogenética mostrou topologia semelhante entre as duas famílias.Análise dos dados do estudo ENCODE e predição pelo Softberry identificou sítios de ligação a fatores de transcrição comuns entre os distintos pares, cuja ontologia indicou principalmente desenvolvimento, processos metabólicos e resposta imune. O estudo de possíveis implicações funcionais dessa sintenia mostrou que manipulações da expressão proteica de PDIA1 não promovem mudança consistente na expressão proteica de RhoGDIalfa, tanto in vitro (silenciamento da PDI por siRNA e superexpressão por vetor lentiviral induzível) como in vivo (camundongo transgênico com superexpressão constitutiva da PDIA1). No entanto, as mudanças da expressãogênica de ambos os genes na camada íntima de artérias carótidas de camundongo durante remodelamento induzido por fluxo foram fortemente correlacionadas. Experimentos de coimunoprecipitação e co-localização à microscopia confocal sugeriram interação física entre PDIA1 e RhoGDIAalfa. Deste modo, estes dados mostram um intrigante padrão de conservação evolutiva da proximidade gênica entre PDIs e RhoGDIs, não usual em eucariotos. Genes sintênicos frequentemente codificam proteínas que tendem a interagir física e/ou funcionalmente. Com efeito, nosso dados sugerem co-regulação e interação física entre PDIA1 e RhoGDIAalfa, corroborando a convergência entre essas proteínas como possível mecanismo envolvido na regulação redox do citoesqueleto pela PDIA1 / Redox pathways are important regulators of homeostasis and cell signaling, but the understanding of the mechanisms of these processes is incomplete. Thiol proteins such as protein disulfide isomerase (PDI) can be modulators of these pathways. PDI (PDIA1) is the prototype of the family of PDIs whose canonical function is a redox protein folding in the endoplasmic reticulum. In addition, PDI exerts regulatory NADPH oxidase, the main sources of cellular oxidant, and is required for activation RhoGTPases, cytoskeletal organization and migration of vascular cells. In the study of mechanisms by which regulates PDI RhoGTPases, we showed in computer networks and co-imunoprecitation experiments association between PDIA1 and the regulator of RhoGTPases, RhoGDI?. In addition, we identified strong proximity of the genes encoding these proteins. In this study, we characterize the profile and implications of this synteny. .A bioinformatic analysis by programs Ensembl, NCBI and UCSC shows a pattern of synteny between different isoforms of these two families: PDIA1 (P4HB), PDIA2 (PDIP) and PDIA8 (Erp27) are neighbors , respectively RhoGDIalfa, and RhoGDIy RHOGDIbeta with corresponding intergenic regions 7.1, 2.9 and 0:14 kb in different chromosomes of H. sapiens. The pattern of this synteny was strongly maintained in C. elegans, some fish and evenly amphibians, reptiles, birds and mammals. Yeasts express on the same chromosome, but in distant places (i.e macrosintenia) orthologs of PDIA1 and RhoGDI?, but do not express other syntenics PDIs and RhoGDIs in complex eukaryotes. However, synteny between PDI and RhoGDI was also observed in the plant A. thaliana, no evidence of a common ancestor. The syntenic pairs are associated with the stored neighboring blocks, but different for each pair, while each block contains a gene encoding a regulator of distinct PP1 (protein phosphatase-1). Phylogenetic analysis showed similar topology between the two famílias. The identified binding sites common transcription factors between different pairs, which mainly indicated ontology development, metabolic and immune response. The study of possible functional implications of synteny showed that manipulations of PDIA1 protein expression do not promote consistent change in protein expression RhoGDI, both in vitro (silencing of PDI by siRNA and overexpression of inducible lentiviral vector) and in vivo (transgenic mice overexpressing constitutive of PDIA1). The study of possible functional implications of synteny showed that manipulations of PDIA1 protein expression do not promote consistent change in protein expression RhoGDIalfa, both in vitro (silencing of PDI by siRNA and overexpression of inducible lentiviral vector) and in vivo (transgenic mice overexpressing constitutive of PDIA1). However, changes of gene expression of both genes in the intima of mouse carotid arteries during remodeling induced by flow were strongly correlated. Immunoprecipitation experiments and co-location to confocal microscopy suggested physical interaction between PDIA1 and RhoGDIAalfa. Thus, these data show an intriguing pattern of evolutionary conservation of gene proximity between POIs and RhoGDIs not common in eukaryotes. sintênicos genes often encode proteins that tend to interact physically and / or functionally. Indeed, our data suggest co-regulation and physical interaction between PDIA1 and RhoGDIAalfa, supporting the convergence of these proteins as a possible mechanism involved in redox regulation of cytoskeleton by PDIA1

This study was conducted to measure the reduction in polycyclic aromatic hydrocarbons (PAHs) content in oil sludge by co-composting the sludge with pig, cow, horse and poultry manures under laboratory conditions. Four kilograms of soil spiked with 800g of oil sludge was co-composted differently with each manure in a ratio of 2:1 (w/w) spiked soil: manure and wood-chips in a ratio of 2:1 (w/v) spiked soil: wood-chips. Control was set up similar as the one above but without manure. Mixtures were incubated for 10 months at room temperature. Compost piles were turned weekly and moisture level was maintained at between 50% and 70%. Moisture level, pH, temperature, CO2 evolution and oxygen consumption were measured monthly and the ash content at the end of experimentation. Bacteria capable of utilizing PAHs were isolated, purified and characterized by molecular techniques using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), amplification of the 16S rDNA gene using the specific primers (16S-P1 PCR and 16S-P2 PCR) and the amplicons were sequenced. Extent of reduction of PAHs was measured using automated soxhlet extractor with Dichloromethane as the extraction solvent coupled with gas chromatography/mass spectrometry (GC/MS). Temperature did not exceed 27.5OC in all compost heaps, pH ranged from 5.5 to 7.8 and CO2 evolution was highest in poultry manure at 18.78μg/dwt/day. Microbial growth and activities were enhanced. Bacteria identified were Bacillus, Arthrobacter and Staphylococcus species. Results from PAH measurements showed reduction between 77 and 99%. The results from the control experiments may be because it was invaded by fungi. Co-composting of spiked soils with animal manures enhanced the reduction in PAHs. Interestingly, all bacteria isolated and identified in this study were present in all treatments, including the control. / Environmental Sciences / M.Sc. (Environmental Sciences)

[ES] De entre los patógenos emergentes presentes en agua, las bacterias del género Helicobacter son de las más alarmantes, ya que se encuentran directamente relacionadas con el cáncer gástrico y hepatobiliar y su epidemiología aún no está clara. Se ha planteado que H. pylori puede ser adquirida por diferentes vías de transmisión, entre las que se destaca el agua. Se ha demostrado su capacidad de supervivencia frente a los tratamientos comunes de desinfección de aguas. Por todo ello, en esta tesis se ha investigado la presencia de células viables, y por tanto infectivas, de H. pylori en aguas potables y de riego, mediante la mejora y la optimización de técnicas de cultivo y moleculares. Este trabajo se inició con la búsqueda de un medio de cultivo óptimo. Se obtuvieron resultados muy positivos con el medio de cultivo Agar Dent con sulfato de polimixina B, independientemente del origen de la muestra. Seguidamente, se desarrolló un método de pre-tratamiento con Propidium Monoazide y PEMAXTM para la detección y cuantificación de células de H. pylori viables, por PCR. Se confirmó que el PMA a una concentración de 50 µM y un periodo de incubación de 5 minutos sería la metodología óptima de tratamiento antes del análisis mediante qPCR. A continuación, se analizaron 20 muestras de agua residual. Mediante la técnica de cultivo, fue posible la detección de 4 colonias sospechosas de Helicobacter spp. y H. pylori, cuya identificación fue confirmada mediante amplificación y posterior secuenciación. La técnica DVC-FISH indico la presencia de células viables de Helicobacter spp. en 15 (75%) de las muestras. Respecto a la detección de células de H. pylori, mediante DVC-FISH y FISH, estos microorganismos se observaron en 10 (50%) y 11 (55%) de las 20 muestras analizadas, respectivamente. La técnica qPCR determino la presencia de H. pylori en las muestras, con un porcentaje de detección del 60%. Finalmente, mediante metagenómica de secuenciación dirigida, se analizó el microbioma de 16 muestras de aguas residuales. Los filos dominantes de las muestras analizadas fueron Proteobacteria, seguido de Bacteroidetes y Firmicutes. H. pylori se detectó en 6 muestras de aguas residuales. Además, se detectaron otras especies de Helicobacter spp., como H. hepaticus, H. pullorum y H. suis. Igualmente, mediante PCR se identificó el gen cagA en 5 muestras de agua residual y una de agua potable. Respecto el genotipo vacAs1, se observó en 4 muestras de agua residual; el genotipo vacAm1, se identificó en una muestra de agua potable y 2 de agua de riego. En las biopelículas analizadas, 2 fueron positivas para el tipo vacAm1, y otros dos para el gen de resistencia pbp1A. Del mismo modo se analizaron 45 muestras de heces. No se observaron colonias sospechosas en Agar Dent selectivo. Mediante qPCR se evidenció H. pylori en 41 muestras (45,56%). Fue posible cuantificar 10 muestras directas y 18 enriquecidas, con concentraciones entre 3,39*103 y 2,61*103 UG/mL, las restantes presentaban niveles superiores al umbral de fiabilidad (>35 ciclos). Por DVC-FISH se observaron células viables de H. pylori en 26 (57,78%) de las 45 muestras directas. La identificación de mutaciones en el 23S rDNA, de la resistencia a la claritromicina, mostro que el 37,79% de las muestras de heces presentaban células de H. pylori potencialmente resistentes. Mediante secuenciación dirigida y posterior análisis bioinformático se identificó H. pylori en 13 muestras directas y 13 muestras enriquecidas, y otras especies como H. hepaticus y H. pullorum. Por último, se evaluó la capacidad de H. pylori para formar biopelículas y su resistencia frente a los tratamientos comunes de desinfección. Se analizaron 27 biopelículas procedentes del sistema de distribución de agua potable para detectar la presencia de H. pylori mediante qPCR. El porcentaje de detección fue del 23%, siendo posible la cuantificación en 5 muestras, con concentraciones entre 7,32*101 y 1,16*101 unidades genómicas/mL. Los resultados obtenidos en esta Tesis confirman la existencia de células viables de H. pylori y otros Helicobacter spp. en aguas residuales tras su tratamiento, lo que significa un riesgo potencial para la Salud Pública. De igual forma se demuesta su presencia en muestras de heces, proporcionando un punto de partida para el estudio del riesgo que puede suponer para el ser humano la trasmisión fecal-oral de estas especies. Este trabajo también demuestra la capacidad de H. pylori de formar biopelículas y su resistencia frente a tratamientos comunes de desinfección y confirma su existencia en sistemas de distribución de agua potable. / [CAT] D'entre tots els patògens emergents presents en aigua, els bacteris del gènere Helicobacter són dels més alarmants, ja que es troben directament relacionats amb el càncer gàstric i hepatobiliar i la seua epidemiologia encara no és clara. S'ha plantejat que H. pylori es pot transmetre per diferents vies de transmissió, entre les quals destaca l¿aigua. S'ha demostrat la seua capacitat de supervivència enfront dels tractaments comuns de desinfecció d'aigües. Per tant, en aquesta tesi s'ha investigat la presència de cèl·lules viables, i per tant infectives, d' H. pylori en aigües potables i de reg, mitjançant la millora i l'optimització de tècniques de cultiu i moleculars. Aquest treball es va iniciar amb la cerca d'un cultiu òptim. Es van obtenir resultats molt positius amb el mitjà de cultiu Agar Dent amb sulfat de polimixina B, independentment de l'origen de la mostra. Seguidament, es va desenvolupar un mètode de pretractament amb Propidium Monoazide i PEMAXTM per a la detecció i quantificació exclusiva de cèl·lules d' H. pylori viables per PCR. Es va confirmar que el PMA a una concentració de 50 µM i un període d'incubació de 5 minuts seria la metodologia òptima de tractament abans de l'anàlisi mitjançant qPCR. A continuació, es van analitzar 20 mostres d'aigua residual. Mitjançant la tècnica de cultiu, va ser possible la detecció de 4 colònies sospitoses d' Helicobacter spp. i H. pylori, la identificació de la qual va ser confirmada mitjançant amplificació i posterior seqüenciació. La tècnica DVC-FISH va demostrar la presència de cèl·lules viables d' Helicobacter spp. en 15 (75%) de les mostres, sense necessitat d'un pas previ d'enriquiment. Respecte a la detecció de cèl·lules d' H. pylori, mitjançant DVC-FISH i FISH, aquests microorganismes es van observar en 10 (50%) i 11 (55%) de les 20 mostres analitzades, respectivament. La tècnica qPCR determinà la presència d' H. pylori a les mostres amb un percentatge de detecció del 60%. Finalment, mitjançant metagenòmica de seqüenciació dirigida, es va analitzar el microbioma de 16 mostres d'aigües residuals. Els talls dominants de les mostres analitzades van ser Proteobacteria, seguit de Bacteroidetes i Firmicutes. H. pylori es va detectar mitjançant aquesta tècnica en 6 mostres d'aigües residuals. A més, es van detectar altres espècies d' Helicobacter spp., com H. hepaticus, H. pullorum i H. suis. Igualment mediant PCR es va identificar el gen cagA en 5 mostres d'aigua residual i una d'aigua potable. Respecte al genotip vacAs1, es va observar en 4 mostres d'aigua residual; el genotip vacAm1, es va identificar en una mostra d'aigua potable i 2 d'aigua de reg. En les biopel·lícules analitzades, 2 van ser positives per al tipus vacAm1, i altres dos per al gen de resistència pbp1A. De la mateixa manera es van analitzar 45 mostres de femta. No es van observar colònies sospitoses en Agar Dent selectiu. Mitjançant la tècnica qPCR es va demostrar la presència d'H. pylori en 41 mostres (45,56%). Va ser possible quantificar 10 mostres directes i 18 enriquides, amb concentracions d'entre 3,39*103 i 2,61*103 UG/ml, les restants presentaven nivells per damunt del llindar de fiabilitat (>35 cicles). Mitjançant DVC-FISH es van observar cèl·lules viables d' H. pylori en 26 (57,78%) de les 45 mostres directes. La detecció de mutacions en el 23S rDNA, específiques de la resistència a la claritromicina, va indicar que el 37,79% de les mostres de femta presentaven cèl·lules d' H. pylori potencialment resistents. Mitjançant seqüenciació dirigida i posterior anàlisi bioinformàtica es va identificar H. pylori en 13 mostres directes i 13 mostres enriquides, i altres espècies com H. hepaticus i H. pullorum. Finalment, es va avaluar la capacitat d' H. pylori per a formar biopel·lícules i la seua resistència enfront dels tractaments comuns de desinfecció. Es van analitzar 27 biopel·lícules procedents del sistema de distribució d'aigua potable per a detectar la presència d' H. pylori mitjançant qPCR. El percentatge de detecció va ser del 23%, sent possible la quantificació en 5 mostres corresponents a concentracions d’entre 7,32*101 i 1,16*101 unitats genòmiques/ml. Els resultats obtinguts en aquesta Tesi confirmen la presència de cèl·lules viables d' H. pylori i altres Helicobacter spp. en aigües residuals després del seu tractament, la qual cosa suposa un risc potencial per a la Salut Pública. D'igual forma, s'evidencia la seua presència en mostres de femta, proporcionant un punt de partida per a l'estudi del risc que la transmissió fecal-oral d'aquestes espècies pugui suposar per als humans. Aquest treball també demostra la capacitat d' H. pylori de formar biopel·lícules i la seua resistència enfront als tractaments comuns de desinfecció, i confirma la seua presència en sistemes de distribució d'aigua potable. / [EN] Among all the emergent pathogens in water, bacteria of the Helicobacter genus are among the most disturbing, as they are directly related to gastric and hepatobiliary cancer and their epidemiology is still unclear. It has been suggested that H. pylori can be acquired through different transmission routes, among which water stands out. Its ability to survive against common water disinfection treatments has been demonstrated. In addition, H. pylori can form insoluble biofilms, which favors its resistance to the different disinfection and potabilization treatments. Therefore, in this thesis has investigated the presence of viable cells, and therefore potentially infective, of H. pylori in drinking and irrigation waters, through the improvement and optimization of culture and molecular techniques. This work began with the development of an optimal culture medium. Positive results were obtained with the culture medium Agar Dent with polymyxin B sulfate. Subsequently, a pretreatment method with Propidium Monoazide and PEMAXTM was developed for the exclusive detection and quantification of viable H. pylori cells by PCR. It was confirmed that PMA at a concentration of 50 µM and an incubation period of 5 minutes, would be the optimal treatment methodology before analysis by qPCR. A total of 20 wastewater samples, aseptically collected at the outlet of the biological reactor and after disinfection treatment, were then analyzed. Using the culture technique, it was possible to detect 4 suspicious colonies of Helicobacter spp. and H. pylori, whose identification was confirmed by amplification and subsequent sequencing. DVC-FISH technique demonstrated the presence of viable Helicobacter spp. cells in 15 (75%) of the samples. Regarding the detection of H. pylori cells, by DVC-FISH and FISH, these microorganisms were observed in 10 (50%) and 11 (55%) of the 20 samples analyzed, respectively. The qPCR technique determined the presence of H. pylori in the samples with a detection rate of 60%. Finally, using deep-amplicon sequencing, the microbiome of 16 wastewater samples was analyzed. The dominant phylum in the samples analyzed were Proteobacteria, followed by Bacteroidetes and Firmicutes. H. pylori was detected by this technique in 6 wastewater samples. In addition, others Helicobacter spp., such as H. hepaticus, H. pullorum and H. suis were detected. PCR technique was used to identify the cagA gene in 5 wastewater samples and one drinking water sample. Regarding the vacAs1 genotype, it was observed in 4 samples of wastewater; the vacAm1 genotype, was identified in one drinking water sample and 2 irrigation water samples. In the biofilms analyzed, 2 were positive for the vacAm1 type, and two for the resistance gene pbp1A. Likewise, 45 stool samples were analyzed. No suspicious colonies were observed on selective Dent Agar. The qPCR technique demonstrated the presence of H. pylori in 41 samples (45.56%). It was possible to quantify 10 direct samples and 18 enriched samples, with concentrations between 3.39*103 and 2.61*103 GU/mL, the remaining samples had levels above the reliability threshold (>35 cycles). DVC-FISH showed viable H. pylori cells in 26 (57.78%) of the 45 direct samples. Detection of 23S rDNA mutations specific for clarithromycin resistance indicated that 37.79% of stool samples had potentially resistant H. pylori cells. Through Deep-amplicon sequencing and subsequent bioinformatics analysis, H. pylori was identified in 13 direct samples and 13 enriched samples, as well as other species such as H. hepaticus and H. pullorum. Finally, the ability of H. pylori to form biofilms and their resistance to common disinfection treatments was evaluated. Twenty-seven biofilms from the drinking water distribution system were also tested for the presence of H. pylori by qPCR. The detection rate was 23%, being possible the quantification in 5 samples corresponding to concentrations between 7.32*101 and 1.16*101 GU/mL. / Esta Tesis Doctoral ha sido posible gracias a las ayudas de carácter predoctoral: “Ayuda de conselleria para la contratación de personal investigador en formación -Irene Hortelano Martin (determinación del riesgo para el consumidor de la presencia de H. pylori y otros helicobacters patógenos en aguas de consumo mediante técnicas moleculares y metagenómica) (ACIF/2016/150) Generalitat Valenciana (2016-2019). Y a la financiación de los proyectos: “Helicobacter pylori y otros helicobacters patógenos en aguas y alimento: Desarrollo y aplicación de herramientas moleculares dirigidas a la evaluación del riesgo para el consumidor (REF. AGL2014-53875-R)”. Ministerio de Economía y Competitividad, España. “Determinación del riesgo para la salud pública debido a la presencia de H. pylori en agua y alimentos: Detección (AICO/2018/273)”. Generalitat Valenciana (2018-2020). / Hortelano Martín, I. (2021). Determinación del riesgo para el consumidor de la presencia de H. pylori y otros Helicobacter spp. patógenos en aguas de consumo mediante técnicas moleculares y metagenómica [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/178942

Nesta pesquisa, calculou-se, por meio da modelagem molecular, parâmetros físico-químicos importantes à capacidade anti-radicalar de compostos fenólicos extraídos de plantas da flora nacional, Chimarrhis turbinata e Arrabidaea samydoides. As propriedades eletrônicas também podem ser analisadas por meio de superfícies representadas por legendas de cores no campo 3D. Mapa de potencial eletrostático, distribuição orbitalar de HOMO e de LUMO e densidade de spin foram superfícies avaliadas neste trabalho. Em adição, estudos de QSAR (Quantitative Structure-Activity Relationships), cálculos de descritores moleculares holísticos por meio dos programas DRAGON e VOLSURF, cálculos estatísticos incluindo algoritmo genético e PLS (Partial Least Squares), demonstraram a influência de determinadas características moleculares como fundamentais à atividade biológica. A pesquisa concluiu que o grupo farmacofórico favorável à atividade antioxidante é estrutura que apresenta predominantemente características hidrofílicas, grupos hidroxila como substituintes, características eletrônicas favoráveis à doação de elétron e à estabilização do radical fenóxi formado, além de reduzido comprometimento estérico. Consideramos que os métodos empregados no trabalho podem ser considerados como abordagem inovadora para a Ciência Cosmética, indicando potencial ação antioxidante, que poderá ser utilizada em formulações antienvelhecimento. / In this research, the calculated physico-chemical parameters, by molecular modelling, have been reported in the literature for supplying important information about the antiradicalar behavior of phenolic compounds, as the studied herein from Chimarrhis turbinata sp. and Arrabidaea samydoides sp. The electronic properties also can be analyzed by means of surfaces represented by legends of colors in the 3D field. Map of electrostatic potential, HOMO and LUMO distribution orbitalar and spin density have been used in this work. In addition, QSAR studies (Quantitative Structure-Activity Relationships), calculations of holistic molecular descriptors by softwares DRAGON and VOLSURF, statistical analysis including genetic algorithm and PLS (Partial Least Squares), demonstrate the influence of the molecular structure in the biological activity. Therefore, pharmacofor favorable to the antioxidant activity structure that presents predominantly characteristic hydrophilic, groups hydroxyl as substituintes, electronic characteristics favorable to the donation of electron and the stabilization of the radical formed, besides reduced inibition esteric. These recent methods can be considered as an innovative approach for Cosmetic Science toward antioxidant action that could be used in antiaging products.

O Huanglongbing (HLB) é uma das mais temidas doenças da citricultura mundial, associada a bactérias do gênero Candidatus Liberibacter, que foram detectadas no Brasil em 2004. Em 2008, detectou-se outra bactéria associada a sintomas de HLB no Estado de São Paulo, que foi caracterizada como sendo um fitoplasma do grupo 16 SrIX. Fitoplasmas são molicutes fitopatógenos restritos ao floema de plantas, disseminados por insetos vetores. O presente trabalho buscou identificar cigarrinhas potencialmente vetoras do fitoplasma associado ao HLB e plantas hospedeiras desses insetos, bem como desenvolver uma técnica de quantificação de DNA desse patógeno em insetos e plantas. Amostragens de cigarrinhas foram realizadas quinzenalmente por 12 meses em dois pomares de laranja com histórico de ocorrência do fitoplasma 16SrIX na região norte do Estado de São Paulo, usando-se armadilhas adesivas amarelas em duas alturas (0,3 e 1,5 m) da copa de árvores cítricas, e rede de varredura na vegetação espontânea. Dados faunísticos identificaram uma espécie de Agalliinae (Agallia albidula Uhler) e três de Deltocephalinae, [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stål) e Scaphytopius (Convelinus) marginelineatus (Stål)], como os cicadelídeos (Hemiptera: Cicadellidae) mais abundantes e frequentes nas áreas estudadas. Essas espécies predominaram na amostragem com rede de varredura e na menor altura de coleta com armadilhas adesivas, indicando comportamento de alimentação em vegetação rasteira. Com observações visuais, verificou-se associação das espécies com certas plantas invasoras, e influência da composição florística da vegetação rasteira sobre a abundância das cigarrinhas. S. marginelineatus e P. flavicosta ocorreram com maior frequência em Sida rhombifolia L. e Althernantera tenella Colla, respectivamente, enquanto que A. albidula foi predominante em Conyza bonariensis (L.) Cronq., e B. hebe ocorreu exclusivamente em gramíneas, principalmente Panicum maximum Jacq.. Plantas invasoras amostradas nas áreas foram testadas para a presença do fitoplasma 16SrIX, porém sem resultados positivos. No entanto, amostras de campo da cigarrinha S. marginelineatus foram positivas por PCR e sequenciamento para o referido fitoplasma. Indivíduos de S. marginelineatus criados em laboratório e mantidos por um período de acesso à aquisição de 72 h em citros infectado com o fitoplasma 16SrIX, foram capazes de transmití-lo para citros, após 21 dias de latência, porém com baixa eficiência (0,5%). Por meio de PCR quantitativo desenvolvido para esse fitoplasma, verificou-se baixo título do patógeno tanto em S. marginelineatus, quanto em plantas cítricas infectadas, o que pode explicar, pelo menos em parte, a baixa eficiência de transmissão pelo inseto tendo citros como fonte. Isto sugere a existência de outros hospedeiros mais adequados como fontes de inóculo para aquisição do fitoplasma por S. marginelineatus ou outro vetor ainda desconhecido. / Huanglongbing (HLB) is a severe citrus disease associated to phloemlimited bacteria in the genus Candidatus Liberibacter, which were detected in Brazil in 2004. In 2008, another bacterium was found in association with HLB symptom in the São Paulo State, and characterized as a phytoplasma belonging to 16SrIX group. Phytoplasmas are vector-borne phytopathogenic mollicutes that inhabit plant sieve elements. The goals of this study were to identify potential leafhopper vectors of the HLB-associated phytoplasma and their host plants, as well as to establish a real-time PCR procedure for pathogen quantification in vectors and plants. Leafhoppers were sampled fortnightly for 12 months by yellow sticky cards placed at two heights (0.3 and 1.5 m) on the citrus tree canopy and by sweep net in the ground vegetation of two sweet orange groves with history of infection by the 16SrIX phytoplasma, in the northern region of São Paulo State. Faunistic analyses indicated 1 Agalliinae (Agallia albidula Uhler) and 3 Deltocephalinae [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stål) e Scaphytopius (Convelinus) marginelineatus (Stål)] species that were the most abundant and frequent leafhoppers (Hemiptera: Cicadellidae) in the experimental areas. These species predominated in sweep net and in sticky traps catches at 0.3 m above soil, showing that they inhabit the ground vegetation. Visual observations indicated a strong association of leafhopper species with some weeds and the influence of weed species composition on leafhopper abundance in the ground vegetation. S. marginelineatus and P. flavicosta were more frequent on Sida rhombifolia L. and Althernantera tenella Colla, respectively, while A. albidula was observed more often on Conyza bonariensis (L.) Cronq., and B. hebe occurred solely on grasses, more abundantly on Panicum maximum Jacq. Fourteen weed species sampled in the area were PCR tested for infection by the 16SrIX phytoplasma, but none was found infected. Nevertheless, 3 out of 30 field-collected samples (10 adults per sample) of S. marginelineatus tested positive for this phytoplasma by PCR and sequencing. Healthy lab-reared adults of S. marginelineatus were able to transmit inefficiently (0,5%) the 16SrIX phytoplasma to healthy citrus after a 72-h acquisition access period on infected citrus plants followed by a 21-day latent period on S. rhombifolia plants. By using the qPCR method developed for this phytoplasma, a very low pathogen titer was found both in S. marginelineatus and in infected citrus plants, which may explain, at least partially, the low transmission efficiency by this vector using citrus as a source plant. It also suggests that existence of alternative hosts that might be more adequate as inoculum sources for phytoplasma acquisition and spread by S. marginelineatus or another vector yet to be discovered.

O Huanglongbing (HLB) é uma das mais temidas doenças da citricultura mundial, associada a bactérias do gênero Candidatus Liberibacter, que foram detectadas no Brasil em 2004. Em 2008, detectou-se outra bactéria associada a sintomas de HLB no Estado de São Paulo, que foi caracterizada como sendo um fitoplasma do grupo 16 SrIX. Fitoplasmas são molicutes fitopatógenos restritos ao floema de plantas, disseminados por insetos vetores. O presente trabalho buscou identificar cigarrinhas potencialmente vetoras do fitoplasma associado ao HLB e plantas hospedeiras desses insetos, bem como desenvolver uma técnica de quantificação de DNA desse patógeno em insetos e plantas. Amostragens de cigarrinhas foram realizadas quinzenalmente por 12 meses em dois pomares de laranja com histórico de ocorrência do fitoplasma 16SrIX na região norte do Estado de São Paulo, usando-se armadilhas adesivas amarelas em duas alturas (0,3 e 1,5 m) da copa de árvores cítricas, e rede de varredura na vegetação espontânea. Dados faunísticos identificaram uma espécie de Agalliinae (Agallia albidula Uhler) e três de Deltocephalinae, [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stål) e Scaphytopius (Convelinus) marginelineatus (Stål)], como os cicadelídeos (Hemiptera: Cicadellidae) mais abundantes e frequentes nas áreas estudadas. Essas espécies predominaram na amostragem com rede de varredura e na menor altura de coleta com armadilhas adesivas, indicando comportamento de alimentação em vegetação rasteira. Com observações visuais, verificou-se associação das espécies com certas plantas invasoras, e influência da composição florística da vegetação rasteira sobre a abundância das cigarrinhas. S. marginelineatus e P. flavicosta ocorreram com maior frequência em Sida rhombifolia L. e Althernantera tenella Colla, respectivamente, enquanto que A. albidula foi predominante em Conyza bonariensis (L.) Cronq., e B. hebe ocorreu exclusivamente em gramíneas, principalmente Panicum maximum Jacq.. Plantas invasoras amostradas nas áreas foram testadas para a presença do fitoplasma 16SrIX, porém sem resultados positivos. No entanto, amostras de campo da cigarrinha S. marginelineatus foram positivas por PCR e sequenciamento para o referido fitoplasma. Indivíduos de S. marginelineatus criados em laboratório e mantidos por um período de acesso à aquisição de 72 h em citros infectado com o fitoplasma 16SrIX, foram capazes de transmití-lo para citros, após 21 dias de latência, porém com baixa eficiência (0,5%). Por meio de PCR quantitativo desenvolvido para esse fitoplasma, verificou-se baixo título do patógeno tanto em S. marginelineatus, quanto em plantas cítricas infectadas, o que pode explicar, pelo menos em parte, a baixa eficiência de transmissão pelo inseto tendo citros como fonte. Isto sugere a existência de outros hospedeiros mais adequados como fontes de inóculo para aquisição do fitoplasma por S. marginelineatus ou outro vetor ainda desconhecido. / Huanglongbing (HLB) is a severe citrus disease associated to phloemlimited bacteria in the genus Candidatus Liberibacter, which were detected in Brazil in 2004. In 2008, another bacterium was found in association with HLB symptom in the São Paulo State, and characterized as a phytoplasma belonging to 16SrIX group. Phytoplasmas are vector-borne phytopathogenic mollicutes that inhabit plant sieve elements. The goals of this study were to identify potential leafhopper vectors of the HLB-associated phytoplasma and their host plants, as well as to establish a real-time PCR procedure for pathogen quantification in vectors and plants. Leafhoppers were sampled fortnightly for 12 months by yellow sticky cards placed at two heights (0.3 and 1.5 m) on the citrus tree canopy and by sweep net in the ground vegetation of two sweet orange groves with history of infection by the 16SrIX phytoplasma, in the northern region of São Paulo State. Faunistic analyses indicated 1 Agalliinae (Agallia albidula Uhler) and 3 Deltocephalinae [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stål) e Scaphytopius (Convelinus) marginelineatus (Stål)] species that were the most abundant and frequent leafhoppers (Hemiptera: Cicadellidae) in the experimental areas. These species predominated in sweep net and in sticky traps catches at 0.3 m above soil, showing that they inhabit the ground vegetation. Visual observations indicated a strong association of leafhopper species with some weeds and the influence of weed species composition on leafhopper abundance in the ground vegetation. S. marginelineatus and P. flavicosta were more frequent on Sida rhombifolia L. and Althernantera tenella Colla, respectively, while A. albidula was observed more often on Conyza bonariensis (L.) Cronq., and B. hebe occurred solely on grasses, more abundantly on Panicum maximum Jacq. Fourteen weed species sampled in the area were PCR tested for infection by the 16SrIX phytoplasma, but none was found infected. Nevertheless, 3 out of 30 field-collected samples (10 adults per sample) of S. marginelineatus tested positive for this phytoplasma by PCR and sequencing. Healthy lab-reared adults of S. marginelineatus were able to transmit inefficiently (0,5%) the 16SrIX phytoplasma to healthy citrus after a 72-h acquisition access period on infected citrus plants followed by a 21-day latent period on S. rhombifolia plants. By using the qPCR method developed for this phytoplasma, a very low pathogen titer was found both in S. marginelineatus and in infected citrus plants, which may explain, at least partially, the low transmission efficiency by this vector using citrus as a source plant. It also suggests that existence of alternative hosts that might be more adequate as inoculum sources for phytoplasma acquisition and spread by S. marginelineatus or another vector yet to be discovered.

Nesta pesquisa, calculou-se, por meio da modelagem molecular, parâmetros físico-químicos importantes à capacidade anti-radicalar de compostos fenólicos extraídos de plantas da flora nacional, Chimarrhis turbinata e Arrabidaea samydoides. As propriedades eletrônicas também podem ser analisadas por meio de superfícies representadas por legendas de cores no campo 3D. Mapa de potencial eletrostático, distribuição orbitalar de HOMO e de LUMO e densidade de spin foram superfícies avaliadas neste trabalho. Em adição, estudos de QSAR (Quantitative Structure-Activity Relationships), cálculos de descritores moleculares holísticos por meio dos programas DRAGON e VOLSURF, cálculos estatísticos incluindo algoritmo genético e PLS (Partial Least Squares), demonstraram a influência de determinadas características moleculares como fundamentais à atividade biológica. A pesquisa concluiu que o grupo farmacofórico favorável à atividade antioxidante é estrutura que apresenta predominantemente características hidrofílicas, grupos hidroxila como substituintes, características eletrônicas favoráveis à doação de elétron e à estabilização do radical fenóxi formado, além de reduzido comprometimento estérico. Consideramos que os métodos empregados no trabalho podem ser considerados como abordagem inovadora para a Ciência Cosmética, indicando potencial ação antioxidante, que poderá ser utilizada em formulações antienvelhecimento. / In this research, the calculated physico-chemical parameters, by molecular modelling, have been reported in the literature for supplying important information about the antiradicalar behavior of phenolic compounds, as the studied herein from Chimarrhis turbinata sp. and Arrabidaea samydoides sp. The electronic properties also can be analyzed by means of surfaces represented by legends of colors in the 3D field. Map of electrostatic potential, HOMO and LUMO distribution orbitalar and spin density have been used in this work. In addition, QSAR studies (Quantitative Structure-Activity Relationships), calculations of holistic molecular descriptors by softwares DRAGON and VOLSURF, statistical analysis including genetic algorithm and PLS (Partial Least Squares), demonstrate the influence of the molecular structure in the biological activity. Therefore, pharmacofor favorable to the antioxidant activity structure that presents predominantly characteristic hydrophilic, groups hydroxyl as substituintes, electronic characteristics favorable to the donation of electron and the stabilization of the radical formed, besides reduced inibition esteric. These recent methods can be considered as an innovative approach for Cosmetic Science toward antioxidant action that could be used in antiaging products.

Einleitung: Eimeria (E.) spp. und Toxoplasma (T.) gondii sind intrazelluläre Protozoen aus dem Phylum Apicomplexa und stellen bedeutende Pathogene dar. Infektionen mit Eimeria spp. sind Auslöser der Kokzidiose, welche eine der ökonomisch bedeutsamsten Erkrankungen in der Geflügelproduktion darstellt. Die Toxoplasmose des Huhnes hingegen verläuft in der Regel subklinisch. Da beide Parasiten, Eimeria spp. und Toxoplasma gondii, weltweit verbreitet sind, können natürliche Koinfektionen auftreten. Prävalenzstudien mit Hilfe molekularer und serologischer Untersuchungsmethoden sind geeignet, um die Vorkommen, Verbreitung, und Speziesidentität von einzelnen Apicomplexa zu untersuchen, was zu besserer Diagnose, Kontrolle, und Bestandsüberwachung beiträgt. Trotz vorliegender Hinweise, dass bei Koinfektionen zu einer Interaktion zwischen Eimeria und Toxoplasma kommt, existieren Erkenntnislücken hinsichtlich der Häufigkeit und der Bedeutung von natürlichen Koinfektionen des Huhnes unter Feldbedingungen, insbesondere mit Augenmerk auf verschiedene Nutzungstypen, Produktions- und Haltungsbedingungen. Ziele der Untersuchungen: Die Ziele der Untersuchungen waren die Erhebung der Prävalenzen von verschiedenen Eimeria spp. und von T. gondii bei Hühnern, sowie die Häufigkeit ihres Auftretens als Einzel- und Koinfektionen unter Feldbedingungen unter Berücksichtigung verschiedener Nutzungstypen und Haltungsbedingungen in Griechenland. Tiere, Material und Methoden: Die Auswahl der für diese Feldstudie untersuchten Hühnerbestände erfolgte auf Basis der Anzahl von kommerziellen Hühnerhaltungen in drei Hauptregionen Griechenlands. Nach offiziellen Angaben des griechischen Ministeriums für Ländliche Entwickung und Lebensmittel konzentriert sich die griechische Geflügelindustrie hauptsächlich auf Epirus (im Nordwesten), Zentralmazedonien, und Zentralgriechenland. Die Probenentnahme erfolgte in kommerziellen und Hinterhofhaltungen, wobei die Probenanzahl proportional zur Anzahl entsprechender Haltungsformen in der jeweiligen Region gewählt wurde (n = 50). Es wurden Legehennen (n = 21), langsam wachsende Broiler (n = 15) und Hinterhofhühner beprobt (n = 14), welche verschiedenen Produktions- und Haltungsbedingungen zugeordnet waren (Haltung in ausgestalteten Käfigen, Bodenhaltung mit Einstreu und Biohaltung). Konventionelle intensive Broileraufzuchten wurden nicht berücksichtigt. Für die Erhebung der Häufigkeiten von Eimeria spp.-Infektionen wurden aus allen ausgewählten Beständen Einstreuproben gesammelt (n = 756). Die Broilerherden wurden zu einem Zeitpunkt bei der Schlachtung erneut beprobt, indem die Därme der Einzeltiere gesammelt wurden (n = 162). Die Einstreu-/Darminhaltsproben wurden quantitativ mittels McMaster-Verfahrens auf den Eimeria spp.-Oozystengehalt (als Oozysten pro Gramm Kot, OPG) untersucht. Die Eimeria-Oozysten aus positiven Proben wurden für weitere molekulare Analysen aufgereinigt und aufbewahrt. Die Eimeria-Speziesbestimmung erfolgte durch spezies-spezifische DNA-Nachweise, und die Quantifizierung der einzelnen Arten erfolgte semiquantitativ. Um die Häufigkeit von T. gondii-Infektionen zu bestimmen, wurden Blutproben (n = 1,021) von Einzeltieren zur serologischen Untersuchung mittels TgSAG1 ELISA entnommen, zeitgleich zur o. g. Beprobung der Bestände für die Einstreuproben. Für Hinterhofhaltungen mit Tieren variablen Alters wurden ältere Tiere bevorzugt beprobt, und in kommerziellen Legehennenhaltungen wurden die Tiere im Alter von ca. 10 Monaten beprobt. In beiden Fällen wurden von T. gondii-seropositiven Tieren bei der Schlachtung die Herzen gewonnen (n = 322). In Broilerhaltungen wurden ebenfalls Blutproben und das Herz während des Schlachtungsprozesses zum T. gondii-Nachweis entnommen. Das Herzgewebe wurde in einer magnetic capture Polymerasekettenreaktion (mc-PCR) sowie einem Mausbioassay auf Präsenz und Genotyp einzelner Isolate untersucht. Parallel zu den Laboruntersuchungen wurden Daten zu Haltungsbedingungen, Biosicherheitsmaβnahmen, Lage, Bestandsgröβe, Krankheitsgeschichte etc. mittels standardisierter Fragebögen erfasst, um potenzielle Risikofaktoren für Eimerien- und/oder T. gondii-Infektionen zu bestimmen. Die Datenanalyse erfolgte durch Multilevel-Modelling (generalized linear mixed modelling fit by maximum likelihood (Laplace approximation)) mittels dem R-Programm (https://www.r-project.org, Version 4.0.2, Paket lme4), dem Kruskal-Wallis-Test und bivariaten Spearman-Korrelationen im PSPP-Prgramm (GNU PSPP 1.3.0). Ergebnisse: Insgesamt lag die Nachweisrate für Eimeria spp.-Infektionen bei 85,7 %. Alle sieben Hühnereimerienspezies wurden identifiziert, wobei E. acervulina (79,3 %) und E. tenella (65,5 %) die höchste Prävalenz aufwiesen. Infektionen mit mehreren Eimeria-Arten (79,3 %) waren deutlich häufiger anzutreffen als Einzelinfektionen (20,7 %). Als Risikofaktoren wurden Herdengröβe, Art des Auslaufs und Produktionssystem identifiziert. Zwischen respiratorischen Erkrankungen und mittlerer OPG wurde in Broilerhaltungen eine sehr starke Korrelation beobachtet (P < 0.001). Biohaltungen zeigten eine höhere Prävalenz von E. tenella (P = 0,023). Nutzung einer bewachsenen Auslauffläche war stark mit der Präsenz von E. brunetti korreliert (P < 0,001). Die T. gondii-Seroprävalenz über alle untersuchten Tiere betrug 9,5 %. Dabei testeten 41,2 % aller Hinterhofhühner seropositive. In 70 % der Bio- und Freilaufhaltungen wurde mindestens ein Tier seropositiv getestet. Es wurden keine T. gondii-seropositiven Broiler gefunden, obwohl mit Hilfe der mc-PCR positive DNA-Nachweise erfolgten. Dies belegt die hohe Sensitivität der mc-PCR und ihre potenzielle Eignung für die Detektion früher Infektionen bei Hühnern. Die T. gondii-Isolate, welche im Mausbioassay gewonnen wurden, wurden als Typ II (ToxoDB#3) genotypisiert, was durch Mikrosatellitenanalyse bestätigt wurde. Für T. gondii-Infektionen wurden Produktionssystem und Futterautomatisierung als Risikofaktoren identifiziert, wobei Auslaufbeweidung die Wahrscheinlichkeit für T. gondii-Infektionen erhöht. Die Gegenwart von Katzen stellte hingegen keinen nachweisbaren Risikofaktor für T. gondii-Seropositivität auf Bestands- oder Einzeltierniveau dar. Koinfektionen mit beiden Protozoen wurden in 87% aller untersuchten Hühnerbestände nachgewiesen, wobei Hinterhof-, Bio-, und Freilandhaltungen am häufigsten betroffen waren. In den moisten Fällen von Koinfektionen wurde E. acervulina nachgewiesen. Schlussfolgerungen: Die Prävalenz sowohl von Eimeria spp. als auch von T. gondii war generell hoch und auf einem vergleichbaren Niveau mit den Ergebnissen früherer Studien in anderen Ländern. Faktoren wie Produktionssystem, Haltungs- und Managementbedingungen sind mit dem Risiko von Mono- oder Koinfektionen verbunden. Die gewonnenen Erkenntisse erlauben die gezielte Planung zukünftiger Studien hinsichtlich sich ändernder Haltungsbedingungen, z. B. dem Trend zur verstärkten Bio- und tierfreundlichen Hühnerhaltung uns dem Einsatz langsam wachsender Rassen. Es besteht auch in Griechenland ein Bedarf an nachhaltiger Kontrolle von Kokzidieninfektionen, einschlieβlich der Minimierung zoonotischer Erreger wie T. gondii in Nutztierbeständen. Die verwendeten serologischen und molekularen Methoden können nach den Studienerkenntnisse ausserdem zur frühzeitigen Überwachung von T. gondii–Infektionen in Hühnerbeständen beitragen.:Chapter 1 - Introduction Chapter 2 - Literature Review 2.1 Eimeria spp. 2.1.1 Life cycle 2.1.2 Diagnosis and control of Coccidiosis in chickens 2.1.3 Economic Impact of Coccidiosis 2.1.4 Prevalence of Eimeria spp. in chicken farms 2.1.5 Data of chicken coccidiosis from Greece 2.2 Toxoplasma gondii 2.2.1 Life cycle 2.2.2 Toxoplasmosis and Zoonotic Importance 2.2.3 Prevalence in poultry and risk factors 2.2.4 T. gondii data from Greece 2.3 Co-existence of Eimeria spp. and Toxoplasma gondii in chickens Chapter 3 - Overview of own scientific work 3.1 Aims 3.2 Presentation of own scientific work: Publications 3.2.1 Publication 1: Prevalence and molecular detection of Eimeria species in different types of poultry in Greece and associated risk factors. 3.2.2 Publication 2: Prevalence and molecular characterization of Toxoplasma gondii in different types of poultry in Greece, associated risk factors and co-existence with Eimeria spp. Chapter 4 - Overreaching Discussion Chapter 5 - Conclusions Chapter 6 - Summary Chapter 7 - Zusammenfassung Chapter 8 - References Acknowledgements / Introduction: Eimeria spp. and Toxoplasma gondii are intracellular Apicomplexan protozoa and represent important pathogens for chickens. Coccidiosis, caused by Eimeria spp. is one of the most notable diseases in chickens having a high economic impact on the poultry industry worldwide, while toxoplasmosis is usually subclinical in these hosts. As both Eimeria spp and Toxoplasma gondii have a broad worldwide distribution, natural co-infections in chickens can occur. Prevalence studies using molecular and serological techniques have proven a very useful approach to study the diversity and distribution of these parasites’ mono-infections, further contributing to more efficient diagnosis, control and monitoring. Despite existing indications that these two parasites interact when the host is infected simultaneously, there are still knowledge gaps regarding the frequency and impact of naturally occurring co-infections under field conditions, particularly in different types of poultry, production and housing systems. Objective: Determination of the level of occurrence of Eimeria spp. and Toxoplasma gondii mono- and co-infections under field conditions, in different types of chickens and farm profiles in Greece. Animals, materials and methods: Selection of poultry operations was based on the number of commercial farms in three major Greek regions, as poultry farms in Greece are highly concentrated in Epirus (in North-Western Greece), Central Macedonia, and Central Greece, based on the data from the Hellenic Ministry of Rural Development and Food. Sampling from both commercial operations and backyard farms was conducted proportionately to their frequency (n=50) and type of flocks included in the study were layers (n=21), slow-growing broilers (n=15) and backyard chickens (n=14), under different production and housing systems (enriched cages, floor-housed in litter, free-range and organic systems). Conventional intensively reared broilers were not included in the study. To record Eimeria spp. occurrence, faecal samples were collected from the litter of the chicken housing (n=756). Broiler flocks were followed up to the slaughterhouse for a second sampling where the whole gut was collected (n=162). Samples were quantitatively examined by a modified McMaster technique to calculate oocysts per gram (OPG) of faeces, followed by collection and purification of the oocysts for further molecular analysis. Species identification was performed by multiple PCR assays using species-specific primers and PCR bands were categorized by intensity semiquantitavely. To record Toxoplasma gondii infections, simultaneously to faecal sampling, blood samples (n=1,021) were also collected from individual animals for serological T. gondii detection via TgSAG1 ELISA. In our sampled broiler flocks, animals were sampled at slaughter, where blood samples and the heart were collected for T. gondii detection. In backyard flocks, blood samples were taken from the older animals and in layers from individual animals (at the age of approximately 10 months). Toxoplasma positive animals were followed up to slaughter to collect heart tissue (n=322), further processed for bioassay in KO mice and mc-PCR in order to characterize Toxoplasma gondii isolates. In parallel, potential risk factors and impact regarding mono- and co-infection of both parasites were investigated through an obtained questionnaire containing additional information about farm management, biosecurity status, location, production rate and diseases history. For the data analysis a multilevel-modelling (generalized linear mixed modelling fit by maximum likelihood (Laplace approximation)) was performed using R (https://www.r-project.org) version 4.0.2, by applying the package lme4, as well as Kruskal-Wallis tests and bivariate correlations in PSPP statistical program (GNU PSPP 1.3.0). Results: Overall Eimeria spp. positivity level was 85.7%. All seven Eimeria species were identified with E. acervulina (79.3%) and E. tenella (65.5%) being the most prevalent ones. Mixed infections (79.3%) were more common than single-species (20.7%) Significant identified risk factors were flock size, type of outdoor area, and production system. A very strong correlation (p < 0.001) was found between the presence of respiratory disease and the average OPG level in broiler farms. Organic flocks showed higher prevalence of E. tenella (p = 0.023), while presence of vegetation at the outdoor area correlated strongly with E. brunetti (p < 0.001). Toxoplasma gondii seroprevalence was 9.5%. 41.2% of the backyard chickens sampled were seropositive and 70% of the organic and free-range layer farms had at least one Toxoplasma gondii seropositive hen. No serologically positive broilers were found, however mc-PCR revealed positive samples, stressing out the high sensitivity and potential contribution of this method in early detection of the parasite. Toxoplasma gondii isolates obtained by mouse bioassay were genotyped and found to belong to type II (ToxoDB#3) as confirmed also by microsatellite typing. The most significant risk factors were production system and feeding system automation, with free-grazing practices increasing the likelihood for Toxoplasma infections. Presence of cats showed no association with Toxoplasma gondii seropositivity on a farm and individual animal level. The two protozoan parasites were found to co-exist in 87% of the studied poultry operations, with backyard, organic and free-range farms showing the highest occurrence. E. acervulina was the species identified in most of the co-existence cases. Conclusions: Prevalence of both Eimeria spp. and Toxoplasma gondii is overall high and comparable with findings from similar studies in other countries. Production system, husbandry and management conditions relate to increased risk of both mono- and co-infections, giving useful insights and indications for future studies, particularly in the light of increasing application of slow-growing, organic and “higher welfare” poultry farming practices. It could be shown that also in Greece there is a need for sustainable coccidiosis control, both in terms of Eimeria spp. infection control and of a minimization of T. gondii introduction to operations. A combination of the serological and molecular methods used in this study can contribute to earlier and more accurate diagnosis of Toxoplasma gondii infections in chickens, which is a crucial and sensitive subject for safeguarding transmission to humans.:Chapter 1 - Introduction Chapter 2 - Literature Review 2.1 Eimeria spp. 2.1.1 Life cycle 2.1.2 Diagnosis and control of Coccidiosis in chickens 2.1.3 Economic Impact of Coccidiosis 2.1.4 Prevalence of Eimeria spp. in chicken farms 2.1.5 Data of chicken coccidiosis from Greece 2.2 Toxoplasma gondii 2.2.1 Life cycle 2.2.2 Toxoplasmosis and Zoonotic Importance 2.2.3 Prevalence in poultry and risk factors 2.2.4 T. gondii data from Greece 2.3 Co-existence of Eimeria spp. and Toxoplasma gondii in chickens Chapter 3 - Overview of own scientific work 3.1 Aims 3.2 Presentation of own scientific work: Publications 3.2.1 Publication 1: Prevalence and molecular detection of Eimeria species in different types of poultry in Greece and associated risk factors. 3.2.2 Publication 2: Prevalence and molecular characterization of Toxoplasma gondii in different types of poultry in Greece, associated risk factors and co-existence with Eimeria spp. Chapter 4 - Overreaching Discussion Chapter 5 - Conclusions Chapter 6 - Summary Chapter 7 - Zusammenfassung Chapter 8 - References Acknowledgements