Amebas de vida livre (AVL) são protozoários amplamente distribuídos na natureza e em ambientes artificiais. Alguns gêneros são oportunistas ou patogênicos, com característica anfizóica, são esses: Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., e recentemente Vermamoeba spp. AVL podem servirem de reservatórios de várias bactérias patogênicas, uma vez que se alimentam dessas no ambiente por fagocitose, algumas espécies de bactérias após internalizadas escapam da via de degradação sobrevivem e multiplicam-se no interior das amebas. Face ao exposto, o presente trabalho objetivou isolar e identificar AVL e as bactérias associadas presentes em águas de torres de resfriamento, cuja temperatura da água varia entre 25 a 30°C (ideal para o desenvolvimento de diversos microrganismos). Amebas foram isoladas em cultivo monoxênico com Escherichia coli inativadas pelo calor. Das amostras positivas, foram realizadas Reações em Cadeia de Polimerase (PCR), utilizando oligonucleotídeos específicos para cada gênero. Os produtos das PCR foram sequenciados e as sequências geradas foram comparadas com sequências parciais de cada gene alvo depositadas no GenBank. Das 36 amostras, 33 (91,66%) foram positivas para presença de AVL, destas 15 (45,4%) foram positivas para Acanthamoeba spp., 12 (36,3%) para Naegleria spp., 6 (18,2%) para Vermamoeba spp. Dos endossimbiontes pesquisados, somente Pseudomonas spp. foram identificadas em 13 (39,3%) isolados de AVL. Os resultados obtidos no presente estudo confirmam a presença de amebas de vida livre potencialmente patogênicas e carreadoras de bactérias patogênicas oportunistas que podem representar um risco à saúde humana. / Free-living amoebae (FLA) are protozoa widely distributed in natural and artificial environments. Some genus are opportunistic or pathogenic, with anfizoic feature : Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., and recently Vermamoeba spp. AVL may serve as reservoirs for various pathogenic bacteria, since they feed by phagocytosis in the environment, some species of bacteria after internalized escape to the pathway of degradation surviving and multiplying within the amoebae. The aim of this study was to isolate and identify FLA and their associated bacteria present in water from cooling tower, whose temperature water varies between 25 to 30 °C (ideal for the development of different organisms). Amoebae were isolated in monoxenic cultivation with Escherichia coli inactivated by heat. Of the positive samples Polymerase Chain Reactions (PCR) were performed using specific primers for each genus. The PCR products were sequenced and the sequences were generated compared with partial sequences of each gene target deposited in GenBank. From the 36 samples, 33 (91.66%) were positive for the presence of FLA. Of these, 15 (45.4%) were positive for Acanthamoeba spp., 12 (36.3%) for Naegleria spp. and 6 (18.2 %) for Vermamoeba spp. Of the amoeba isolated, 13 (39.3%) were associated to bacteria wich belong to Pseudomonas genus (100%). Another bacteria investigated were absent. The results of this study confirm the presence of potentially pathogenic free-living amoebae as vehicles of opportunistic/pathogenic bacteria that can pose a risk to the human health.

Os Passeriformes compõem a maior ordem de aves do mundo. No Brasil, representam 55,52%, sendo os animais mais envolvidos no tráfico da fauna silvestre. Só no município de São Paulo são registradas aproximadamente 150 espécies. Embora componham um grupo importante e bem representativo, pouco se sabe sobre as causas de morbidade e mortalidade de Passeriformes de vida livre da fauna nacional. O presente estudo teve como objetivo identificar, caracterizar e comparar as causas de morte de Passeriformes de vida livre residentes na Região Metropolitana de São Paulo e Passeriformes oriundos do tráfico apreendidos na mesma localidade. Foram estudadas 149 aves, sendo 42,3% de vida livre e 57,7% oriundas do tráfico. As causas de morte nas aves provenientes de tráfico e de vida livre foram respectivamente iguais a 51,17% e 24,42% decorrentes a processos infecciosos; 34,92% e 42,86% a processos não infecciosos; 10,46% e 12,70% à suspeita de processos infecciosos e 13,95% e 9,52% à causas indeterminadas. Óbitos devido a processos infecciosos foram os mais predominantes entre os animais de tráfico, caracterizados principalmente por infecções mistas por agentes bacterianos e fúngicos, enquanto os processos não infecciosos, principalmente traumatismos, se destacaram entre aqueles de vida livre. Os resultados obtidos permitem atuar como importante ferramenta auxiliando e direcionando o atendimento clínico, diagnóstico e tratamento, além de gerar informações que contribuam com os programas de conservação in situ, como as solturas, hoje amplamente realizadas e ainda muito controversas. / Passeriformes are the largest and most diverse avian order in the world. In Brazil, they are around 55,52% and most aimed in the illegal wildlife trade. There are 150 species in São Paulo city and surroundings. Although passerines are an important and very representative group, morbidity and mortality of brazilian free-living birds are unknown. The goal of this work was to identify, characterize and compare the causes of death of free-living Passeriformes in the São Paulo Metropolitan area and those from illegal wildlife trade in the same area. In the total of 149 birds studied, 42,3% was free-living and 57,7% from illegal trade. The causes of death of passerines from illegal wildlife trade and free-living are: infectious diseases (respectively, 51,17% and 24,42%); non-infectious diseases (34,92% and 42,86%); suspicious infeccious diseases (10,46% and 12,70%) and indeterminate causes (13,95% and 9,52%). Deaths due to infectious diseases were the majority among birds of illegal trade, mainly concomitant bacterial and fungic infections, while non-infectious diseases, specially trauma, were more predominant among the free-living ones. Obtained results play as an important tool helping and guiding clinic assistance, diagnosis and treatment, besides to contribute with conservation programs in situ, like releases, that are very frequent nowdays, but they are still controversial.

Os Passeriformes compõem a maior ordem de aves do mundo. No Brasil, representam 55,52%, sendo os animais mais envolvidos no tráfico da fauna silvestre. Só no município de São Paulo são registradas aproximadamente 150 espécies. Embora componham um grupo importante e bem representativo, pouco se sabe sobre as causas de morbidade e mortalidade de Passeriformes de vida livre da fauna nacional. O presente estudo teve como objetivo identificar, caracterizar e comparar as causas de morte de Passeriformes de vida livre residentes na Região Metropolitana de São Paulo e Passeriformes oriundos do tráfico apreendidos na mesma localidade. Foram estudadas 149 aves, sendo 42,3% de vida livre e 57,7% oriundas do tráfico. As causas de morte nas aves provenientes de tráfico e de vida livre foram respectivamente iguais a 51,17% e 24,42% decorrentes a processos infecciosos; 34,92% e 42,86% a processos não infecciosos; 10,46% e 12,70% à suspeita de processos infecciosos e 13,95% e 9,52% à causas indeterminadas. Óbitos devido a processos infecciosos foram os mais predominantes entre os animais de tráfico, caracterizados principalmente por infecções mistas por agentes bacterianos e fúngicos, enquanto os processos não infecciosos, principalmente traumatismos, se destacaram entre aqueles de vida livre. Os resultados obtidos permitem atuar como importante ferramenta auxiliando e direcionando o atendimento clínico, diagnóstico e tratamento, além de gerar informações que contribuam com os programas de conservação in situ, como as solturas, hoje amplamente realizadas e ainda muito controversas. / Passeriformes are the largest and most diverse avian order in the world. In Brazil, they are around 55,52% and most aimed in the illegal wildlife trade. There are 150 species in São Paulo city and surroundings. Although passerines are an important and very representative group, morbidity and mortality of brazilian free-living birds are unknown. The goal of this work was to identify, characterize and compare the causes of death of free-living Passeriformes in the São Paulo Metropolitan area and those from illegal wildlife trade in the same area. In the total of 149 birds studied, 42,3% was free-living and 57,7% from illegal trade. The causes of death of passerines from illegal wildlife trade and free-living are: infectious diseases (respectively, 51,17% and 24,42%); non-infectious diseases (34,92% and 42,86%); suspicious infeccious diseases (10,46% and 12,70%) and indeterminate causes (13,95% and 9,52%). Deaths due to infectious diseases were the majority among birds of illegal trade, mainly concomitant bacterial and fungic infections, while non-infectious diseases, specially trauma, were more predominant among the free-living ones. Obtained results play as an important tool helping and guiding clinic assistance, diagnosis and treatment, besides to contribute with conservation programs in situ, like releases, that are very frequent nowdays, but they are still controversial.

As sequencing technologies advance genome studies are becoming a basic tool for studying an organism, and with more genomes available comparative genomics is maturing into a powerful tool for biological research. This thesis demonstrates the strength of a comparative genomics approach on a group of understudied eukaryotes, the diplomonads. Diplomonads are a group of single cell eukaryotic flagellates living in oxygen-poor environments. Most diplomonads are intestinal parasites, like the well-studied human parasite Giardia intestinalis. There are seven different G. intestinalis assemblages (genotypes) affecting different hosts, and it’s under debate whether these are one species. A genome-wide study of three G. intestinalis genomes from different assemblages reveals little inter-assemblage sexual recombination, supporting that the different G. intestinalis assemblages are genetically isolated and thus different species. A genomic comparison between the fish parasite S. salmonicida and G. intestinalis reveals genetic differences reflecting differences in their parasitic lifestyles. There is a tighter transcriptional regulation and a larger metabolic reservoir in S. salmonicida, likely adaptations to the fluctuating environments it encounters during its systemic infection compared to G. intestinalis which is a strict intestinal parasite. The S. salmonicida genome analysis also discovers genes involved in energy metabolism. Some of these are experimentally shown to localize to mitochondrion-related organelles in S. salmonicida, indicating that they possess energy-producing organelles that should be classified as hydrogenosomes, as opposed to the mitosomes in G. intestinalis. A transcriptome analysis of the free-living Trepomonas is compared with genomic data from the two parasitic diplomonads. The majority of the genes associated with a free-living lifestyle, like phagocytosis and a larger metabolic capacity, are of prokaryotic origin. This suggests that the ancestor of the free-living diplomonad was likely host-associated and that the free-living lifestyle is a secondary adaptation acquired through horizontal gene transfers.  In conclusion, this thesis uses different comparative genomics approaches to broaden the knowledge on diplomonad diversity and to provide more insight into how the lifestyle differences are reflected on the genetic level. The bioinformatics pipelines and expertise gained in these studies will be useful in other projects in diplomonads and other organismal groups.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 5 uppsatser.

Amebas de vida livre (AVL) são protozoários amplamente distribuídos na natureza e em ambientes artificiais. Alguns gêneros são oportunistas ou patogênicos, com característica anfizóica, são esses: Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., e recentemente Vermamoeba spp. AVL podem servirem de reservatórios de várias bactérias patogênicas, uma vez que se alimentam dessas no ambiente por fagocitose, algumas espécies de bactérias após internalizadas escapam da via de degradação sobrevivem e multiplicam-se no interior das amebas. Face ao exposto, o presente trabalho objetivou isolar e identificar AVL e as bactérias associadas presentes em águas de torres de resfriamento, cuja temperatura da água varia entre 25 a 30°C (ideal para o desenvolvimento de diversos microrganismos). Amebas foram isoladas em cultivo monoxênico com Escherichia coli inativadas pelo calor. Das amostras positivas, foram realizadas Reações em Cadeia de Polimerase (PCR), utilizando oligonucleotídeos específicos para cada gênero. Os produtos das PCR foram sequenciados e as sequências geradas foram comparadas com sequências parciais de cada gene alvo depositadas no GenBank. Das 36 amostras, 33 (91,66%) foram positivas para presença de AVL, destas 15 (45,4%) foram positivas para Acanthamoeba spp., 12 (36,3%) para Naegleria spp., 6 (18,2%) para Vermamoeba spp. Dos endossimbiontes pesquisados, somente Pseudomonas spp. foram identificadas em 13 (39,3%) isolados de AVL. Os resultados obtidos no presente estudo confirmam a presença de amebas de vida livre potencialmente patogênicas e carreadoras de bactérias patogênicas oportunistas que podem representar um risco à saúde humana. / Free-living amoebae (FLA) are protozoa widely distributed in natural and artificial environments. Some genus are opportunistic or pathogenic, with anfizoic feature : Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., and recently Vermamoeba spp. AVL may serve as reservoirs for various pathogenic bacteria, since they feed by phagocytosis in the environment, some species of bacteria after internalized escape to the pathway of degradation surviving and multiplying within the amoebae. The aim of this study was to isolate and identify FLA and their associated bacteria present in water from cooling tower, whose temperature water varies between 25 to 30 °C (ideal for the development of different organisms). Amoebae were isolated in monoxenic cultivation with Escherichia coli inactivated by heat. Of the positive samples Polymerase Chain Reactions (PCR) were performed using specific primers for each genus. The PCR products were sequenced and the sequences were generated compared with partial sequences of each gene target deposited in GenBank. From the 36 samples, 33 (91.66%) were positive for the presence of FLA. Of these, 15 (45.4%) were positive for Acanthamoeba spp., 12 (36.3%) for Naegleria spp. and 6 (18.2 %) for Vermamoeba spp. Of the amoeba isolated, 13 (39.3%) were associated to bacteria wich belong to Pseudomonas genus (100%). Another bacteria investigated were absent. The results of this study confirm the presence of potentially pathogenic free-living amoebae as vehicles of opportunistic/pathogenic bacteria that can pose a risk to the human health.

Amebas de vida livre (AVL) são protozoários amplamente distribuídos na natureza e em ambientes artificiais. Alguns gêneros são oportunistas ou patogênicos, com característica anfizóica, são esses: Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., e recentemente Vermamoeba spp. AVL podem servirem de reservatórios de várias bactérias patogênicas, uma vez que se alimentam dessas no ambiente por fagocitose, algumas espécies de bactérias após internalizadas escapam da via de degradação sobrevivem e multiplicam-se no interior das amebas. Face ao exposto, o presente trabalho objetivou isolar e identificar AVL e as bactérias associadas presentes em águas de torres de resfriamento, cuja temperatura da água varia entre 25 a 30°C (ideal para o desenvolvimento de diversos microrganismos). Amebas foram isoladas em cultivo monoxênico com Escherichia coli inativadas pelo calor. Das amostras positivas, foram realizadas Reações em Cadeia de Polimerase (PCR), utilizando oligonucleotídeos específicos para cada gênero. Os produtos das PCR foram sequenciados e as sequências geradas foram comparadas com sequências parciais de cada gene alvo depositadas no GenBank. Das 36 amostras, 33 (91,66%) foram positivas para presença de AVL, destas 15 (45,4%) foram positivas para Acanthamoeba spp., 12 (36,3%) para Naegleria spp., 6 (18,2%) para Vermamoeba spp. Dos endossimbiontes pesquisados, somente Pseudomonas spp. foram identificadas em 13 (39,3%) isolados de AVL. Os resultados obtidos no presente estudo confirmam a presença de amebas de vida livre potencialmente patogênicas e carreadoras de bactérias patogênicas oportunistas que podem representar um risco à saúde humana. / Free-living amoebae (FLA) are protozoa widely distributed in natural and artificial environments. Some genus are opportunistic or pathogenic, with anfizoic feature : Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., and recently Vermamoeba spp. AVL may serve as reservoirs for various pathogenic bacteria, since they feed by phagocytosis in the environment, some species of bacteria after internalized escape to the pathway of degradation surviving and multiplying within the amoebae. The aim of this study was to isolate and identify FLA and their associated bacteria present in water from cooling tower, whose temperature water varies between 25 to 30 °C (ideal for the development of different organisms). Amoebae were isolated in monoxenic cultivation with Escherichia coli inactivated by heat. Of the positive samples Polymerase Chain Reactions (PCR) were performed using specific primers for each genus. The PCR products were sequenced and the sequences were generated compared with partial sequences of each gene target deposited in GenBank. From the 36 samples, 33 (91.66%) were positive for the presence of FLA. Of these, 15 (45.4%) were positive for Acanthamoeba spp., 12 (36.3%) for Naegleria spp. and 6 (18.2 %) for Vermamoeba spp. Of the amoeba isolated, 13 (39.3%) were associated to bacteria wich belong to Pseudomonas genus (100%). Another bacteria investigated were absent. The results of this study confirm the presence of potentially pathogenic free-living amoebae as vehicles of opportunistic/pathogenic bacteria that can pose a risk to the human health.

Il est aujourd’hui bien établi que les amibes libres jouent le rôle de vecteurs à la bactérie pathogène Legionella pneumophila favorisant ainsi son développement et sa propagation dans l’environnement. Ainsi, et jusqu’à maintenant, toutes les espèces d’amibes libres étudiées ont démontré une capacité à soutenir et favoriser la croissance de la bactérie responsable des légionelloses. Toutefois, l’ensemble des études a porté sur un nombre restreint d’espèces amibiennes, et leurs capacités relatives à soutenir la croissance bactérienne n’ont que très peu été abordées. Nous avons comparé la capacité de différentes espèces amibiennes à soutenir la prolifération de différentes souches de L. pneumophila du sérogroupe 1. Ces études ont mis en évidence les propriétés particulières d’une souche d’amibe appartenant à l’espèce Willaertia magna. Cette souche présente, au contraire de toutes les autres espèces, la capacité à inhiber et diminuer la prolifération de certaines souches de L. pneumophila. Nous avons par ailleurs pu démontrer l’existence d’une phagocytose interamibienne entre différentes espèces d’amibes, un phénomène jamais mis en évidence à notre connaissance. Les conséquences de cette phagocytose interamibienne sur la croissance et la prolifération de L. pneumophila sont aussi rapportées. La microscopie électronique suggère fortement que la bactérie L. pneumophila ne peut inhiber la fusion phagolysosomale chez W. magna à l’inverse du phénomène observé chez les espèces amibiennes permissives. Ces observations démontrent l’importance de phénomènes membranaires dans la capacité des bactéries à parasiter leur hôte amibien. L’analyse comparée de la composition lipidique des membranes de différentes espèces amibiennes montre chez W. magna une expression élevée de phosphatidylcholine. L’inhibition de la voie de biosynthèse de ce phospholipide par méthylation de phosphatidyléthanolamine résulte en une forte diminution de la croissance amibienne, suggérant que cette voie métabolique joue un rôle important dans les capacités de résistance de W. magna / Free living amoeba is a known vector of L. pneumophila in the environment and it has been shown to favour bacterial growth. Until now, all studied amoeba species showed a capacity to support the growth of the bacterium responsible for the Legionnaire’s disorder. However, these studies were related to a restricted number of amoebic species, and their relative capacity to support the bacterial growth. We compared the capacity of various amoebic species to support the proliferation of various strains of L. pneumophila. These studies highlighted the particular properties of a strain belonging to the Willaertia magna species. This amoeba has, contrary to all the other species, the capacity to inhibit and decrease L. pneumophila proliferation. We also demonstrate the existence of an inter-amoebic phagocytosis between various species of amoebas, a phenomenon never studied to our knowledge. The consequences of this inter-amoebic phagocytosis on the growth and proliferation of L. pneumophila are also reported. Electron microscopy strongly suggests that the bacterium cannot inhibit the phagolysosomal fusion in W. magna contrary to permissive amoebic species. These observations suggest the importance of membrane phenomena in the capacity of the bacteria to infest their amoebic host. Compared analysis of the lipidic composition of various amoebic species shows in W. magna a high expression of phosphatidylcholine the major phospholipid. The inhibition of the PE N-methyltransferase biosynthesis pathway of this phospholipid results in a strong reduction of the amoebic growth, suggesting that this metabolic pathway plays an important role in the resistance capacity of W. magna to L.pneumophila.

No description available.

Trichomonads (Parabasalia) are anaerobic microeukaryotes classified in the supergroup Excavata. Inclusion of parabasalids within Excavata is exclusively based on the molecular- phylogenetic evidence. Over 400 species of parabasalids have been described so far, and the vast majority of them are endobiotic. In contrast, only few species of free-living parabasalids forming four independent lineages have been described (Pseudotrichomonas keilini, Ditrichomonas honigbergii, Monotrichomonas carabina, Honigbergiella sp., Tetratrichomonas undula, and Lacusteria cypriaca). Lacusteria cypriaca is a new species and genus described in our recent paper. In this paper we published the first two sequences of SSU rDNA from Pseudotrichomonas keilini as well. All of these lineages are likely secondarily free-living, and they developed from endobiotic ancestors. In addition to the already published Lacusteria cypriaca and Pseudotrichomonas keilini strains, we have recently obtained seven another isolates of free-living trichomonads (LAGOS2D, E2NT, CK, LAGOS2M, GR8, GOU23 LIVADIAN, and VAV1A1); from all of these isolates we sequenced SSU rDNA and performed phylogenetic analyses. These isolates split into four independent evolutionary lineages, which indicate that free-living parabasalids are more diversed and...