Made available in DSpace on 2016-01-26T12:51:36Z (GMT). No. of bitstreams: 1 cinaradecassiabrandaodemattos_tese.pdf: 2369764 bytes, checksum: d694ab0e38db337b769ea1ace729b6e3 (MD5) Previous issue date: 2012-04-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / T. gondii is an obligate intracellular parasite and cosmopolitan, whose infection congenital or acquired, results in different forms of toxoplasmosis. The clinical manifestations of this disease are nonspecific and variable. Its diagnosis essentially laboratory is aimed at pregnant women, neonates, patients with immunodeficiencies, patients with eye injury and even normal individuals. Serological methods are often used in the characterization of IgM and IgG anti-T.gondii, but those of nature of molecular favoring genetic characterization of the strains isolated from biological samples. Objectives: The overall objective of this thesis was to investigate the infection by T.gondii in the northwestern region of São Paulo state. Its specific objectives were: 1. to characterize infection in pregnant women, newborns and people with eye diseases 2. to evaluate the applicability of the method of PCR inperipheral blood sample in patients with eye diseases 3. to identify the strains from peripheral blood samples. Casuisitc and Methods: epidemiological data and samples of peripheral blood from patients treated in Outpatient Clinic of High Risk Pregnancy and Retinopathy were collected and analyzed for infection by T. gondii using serological methods (ELISA) and molecular (cnPCR and qPCR). Results: among 574 women with mean age equal to 27.2 ± 6.5, 62% (345/556) showed positive for IgG and 3.4% (n = 19/556) for IgM. In 87 mother-newborn pairs, 64.4% (n = 58) were reactive for anti-T. gondii and 2.3% (n = 2), IgM, 92.9% (52/56) had IgG avidity greater than or equal to 30%. Among 184 patients with different eye diseases, 26% (n = 49) had ocular toxoplasmosis, all reagents for IgG. The PCR (qPCR and cnPCR) applied to the analysis of peripheral blood showed sensitivity and specificity equal to 40.8% and 100%, respectively. Five patients with ocular toxoplasmosis were shown to be infected by the strain toxoDB # 65. Conclusion: The results show that the prevalence of infection with T. gondii in pregnant women, neonates and patients with eye diseases is high in the northwertern region of São Paulo state, and to estimate the rates of congenital infection in the region. In addition to describing the first time the ocular toxoplasmosis in the region, this study reinforces the importance of cnPCR and qPCR methods for the characterization of infection and laboratory strains toxoDB # 65 from peripheral blood samples of patients with chronic infection. / T. gondii é um parasito intracelular obrigatório e cosmopolita, cuja infecção de natureza congênita ou adquirida, resulta nas diferentes formas de toxoplasmose. A manifestação clínica desta doença é inespecífica e variável. Seu diagnóstico, essencialmente laboratorial, é direcionado a gestantes, neonatos, portadores de imunodeficiências, portadores de lesão ocular e mesmo indivíduos normais. Métodos sorológicos são frequentemente utilizados na caracterização de anticorpos IgM e IgG anti-T. gondii, porém aqueles de natureza molecular favorecem a caracterização gênica das cepas em isolados de amostras biológicas. Objetivos: O objetivo geral desta tese foi investigar a infecção por T. gondii na região Noroeste Paulista. Seus objetivos específicos foram: 1. caracterizar a infecção em gestantes, neonatos e indivíduos com doenças oculares; 2. avaliar a aplicabilidade do método de PCR em amostra de sangue periférico em pacientes com doenças oculares; 3. identificar as cepas a partir de amostras de sangue periférico. Casuísitca e Métodos: dados epidemiológicos e amostras de sangue periférico de pacientes atendidos em Ambulatório de Gestação de Alto Risco e Retinopatia foram coletadas e analisadas quanto à infecção por T. gondii por métodos sorológicos (ELISA) e moleculares (cnPCR e qPCR). Resultados: entre 574 gestantes com média de idade igual a 27,2±6,5, 62% (345/556) mostraram-se reagentes para IgG e 3,4% (n=19/556), para IgM. Em 87 pares mãe-bebê, 64,4% (n=58) foram reagentes para anti-T. gondii e 2,3% (n=2), para IgM; 92,9% (52/56) apresentaram IgG com avidez maior ou igual a 30%. Dentre 184 pacientes com diferentes doenças oculares, 26% (n=49) apresentaram toxoplasmose ocular, todos reagentes para IgG. O método PCR (cnPCR e qPCR) aplicado à análise do sangue periférico apresentou sensibilidade e especificidade iguais a 40,8% e 100%, respectivamente. Cinco pacientes com toxoplasmose ocular mostraram-se infectados pela cepa toxoDB#65. Conclusão: Os resultados demonstram que a prevalência de infecção por T. gondii em gestantes, neonatos e pacientes com doenças oculares é elevada na região Noroeste paulista, e permitem estimar os índices de infecção congênita na região. Além de descrever pela primeira vez a toxoplasmose ocular na região, este estudo reforça a importância dos métodos cnPCR e qPCR na caracterização laboratorial da infecção e da cepas toxoDB #65 a partir de amostras de sangue periférico de pacientes com infecção crônica.

Schizophrenia is a devastating mental disorder that affects around 1% of the world’s population, characterized by the presence of positive symptoms including hallucinations and delusions, negative symptoms including depression and anxiety, and cognitive impairment including deficits in speech and memory. The complete etiology of schizophrenia is not yet understood, though it is known that both genetics and environmental factors play a role. One environmental factor, a chronic cerebral infection by the parasite Toxoplasma gondii, has one of the highest correlations with schizophrenia of any environmental factor, and may play a role in the pathology of the disease. This is especially true in the case of Type I toxoplasma, which is the most virulent of the three common strains of the parasite. Toxoplasmosis causes an increase in dopamine levels in the striatum and substantia nigra through the production of two enzymes that mimic the rate limiting enzyme in dopamine synthesis, tyrosine hydroxylase. Increased dopamine concentrations in these areas are experimentally correlated with positive schizophrenia symptoms. In addition, toxoplasmosis causes chronic upregulation of the kynurenine pathway via INF- release, leading to chronically elevated kynurenic acid levels. This leads to dysfunction of the glutamatergic system via (1) the binding and inhibition ofα7- nicotinic receptors, leading to decreased GABAergic inhibitory activity in the hippocampus and decreased glutamate release in the prefrontal cortex, and (2) NMDA and AMPA receptor hypofunction, causing decreased inhibitory signaling by GABAergic neurons leaving glutamatergic neurons in a hyper-excitable state. These mechanisms, compounded by commonly identified mutations in the genes of schizophrenic individuals affecting the dopaminergic system, the kynurenine pathway,α7-nicotinic receptors, and the glutamatergic system, create a viable theory as to how the interplay between genetics and toxoplasmosis could cause schizophrenia.

Estandariza y evalúa una prueba ELISA casera para la detección de anticuerpos IgG anti T. gondii en población peruana. Se sometió a evaluación mediante un estuche referencial de ELISA mexicano, un total de 178 sueros de personas de Perú, los cuales fueron catalogados como positivos o negativos a la presencia de anticuerpos IgG anti T. gondii. Para la determinación del punto de corte del ELISA casero se emplearon curvas ROC y se determinaron los parámetros diagnósticos de la técnica; así como la correlación inter e intra ensayo. / Tesis

T.gondii is a model organism of the phylum Apicomplexa that infects one third of the human population. While the majority of infections are asymptomatic or manifest with mild flu-like symptoms, toxoplasmosis can be fatal in immunocompromised individuals and in the developing fetus. The lytic cycle of tachyzoite-stage parasites causes damage to the host by repeated rounds of host cell invasion, intracellular replication and lysis of the host cell upon egress. Invasion is a key step for the parasite to maintain its intracellular lifestyle. Apical Membrane Antigen 1 (AMA1) is an adhesin released from a unique set of secretory organelles called micronemes. AMA1 plays a central role in the initial stages of host cell invasion. Although parasites without AMA1 are viable in culture, virulence in an animal model of infection is completely attenuated, highlighting AMA1's functional importance. AMA1 is a type I transmembrane protein with a large ectodomain and a short cytoplasmic tail. The ectodomain of AMA1 interacts with domain 3 (D3) of rhoptry neck protein 2 (RON2), which in turn complexes with RONs 4, 5, and 8 in the host cell. Together, this complex of proteins forms the moving junction, through which the parasite pushes itself during invasion. Rhomboid proteases on the parasite surface cleave AMA1 within its transmembrane domain and parasites expressing a non-cleavable form of AMA1 show reduced invasion of host cells and a growth defect. While much is known about the ectodomain of T. gondii AMA1 (TgAMA1), the fate of the TgAMA1 cytoplasmic tail after cleavage remains unclear, its interacting partners remain unidentified, and its role in invasion or thereafter remains a mystery. To address these questions, we: (a) explored the consequences of TgAMA1-TgRON2 interaction during invasion and (b) generated allelic replacement (AR) parasites with point mutations across the tail of TgAMA1 to determine the effect of these mutations on the parasite's ability to invade host cells. Quantitative proteomic techniques were used to analyze the proteins that bind to the tail of TgAMA1 under these different experimental conditions. The results from this work highlight the importance of TgAMA1 post-translational modifications, and potentially TgAMA1-binding proteins, in regulating invasion-related processes in T. gondii.

La toxoplasmosis es una zoonosis de amplia distribución mundial, causante de problemas de abortos y reabsorción embrionaria. En las explotaciones ovinas es importante desde el punto de vista económico, por las pérdidas que conlleva, tiene manifestaciones clínicas diversas e inespecíficas, por lo tanto es difícil poder precisar el diagnóstico con certeza, el diagnóstico debe contar con el apoyo de las pruebas inmunológicas adecuadas, actualmente existen dos pruebas la hemaglutinación indirecta (HAI) y la inmunofluorescencia indirecta (IFI) utilizadas con frecuencia. Es por esto que el objetivo del presente estudio fue medir el grado de concordancia entre las técnicas de HAI e IFI para la detección del Toxoplasma gondii en ovinos. Se colectaron 310 sueros de ovinos hembras de diferentes edades del Fundo Santa María, Distrito de Nuñoa, Provincia de Melgar, Departamento de Puno, los cuales fueron almacenados a -20° C para su posterior análisis serológico. La muestras fueron analizadas en el Laboratorio de Parasitología de la Facultad de Medicina Veterinaria de la Universidad Nacional Mayor de San Marcos. Al evaluar el grado de concordancia entre ambas tecnicas se halló un valor de Kappa (K) igual a 24% indicando que el grado de asociación entre ambas tecnicas fue del tipo regular y mediante la prueba de Mc Nemar se encontró que existen diferencias significativas entre ambas, no siendo reemplazable una por la otra. Adicionalmente se halló una seroprevalencia para Toxoplasma gondii de 50.3 ± 5.56 % por el método de HAI y 88.1 ± 3.6 % por el método de IFI. La frecuencia por edades en animales menores de 1 año, mayores de 1 año y menores de 2 años, mayores de 2 años y menores de 3 años y mayores de 3 años fue de 23.2 %, 33.3 %, 52.1 % y 58.7 % respectivamente por el método de HAI y de 72.01 %, 90,0 %, 91.2 % y 90.5 % por el método de IFI. Estos resultados confirmarían que la concordancia entre las pruebas de HAI e IFI en el diagnóstico de la toxoplasmosis ovina fue de tipo regular no siendo reemplazables entre ellas. / --- The toxoplasmosis is a zoonosis of worldwide distribution, which causes problems as abortions and embryonic reabsorption. In the sheep developments, it is very important from the economic point of view for the losses that it bears. The Toxoplasma gondii infection has clinical diverse and unspecific declarations, therefore it is hard to certainly precise the diagnosis, that's why the diagnosis must count on the support of the suitable immunological tests, at the moment two test exist indirect hemagglutination (HAI) and indirect inmunofluorescencia (IFI) wich are used frequently. The present work takes as a target measuring the grade of congruity between the skills of immunofluorescence innuendo and hemagglutination innuendo for the detection of the Toxoplasma gondii in sheep. For the study there were collected 310 wheys of sheep females of different ages from the farm Santa Maria, District of Nuñoa, County of Melgar, and Department of Puno. After collected the wheys were stored to 20° C for his later serologic analysis. The samples were analyzed in the laboratory of parasitology of the faculty of veterinary medicine of the University of San Marcos. The results were submitted to statistical analysis, being a grade of congruity between these two tests determined by the value of Kappa (K) equal to 0.24 what corresponds to a grade of affiliation of regular type and by means of the test of Mc Nemar it was found that significant differences between both exist not being replaceable one for other one, also the study found a seroprevalence for Toxoplasma gondii of 50.3±5.6% for the method of HAI and 88.1±3.6% for the method of IFI. The seroprevalence for ages which was categorized for practical ends in animals of until one year, up to two, up to three and bigger than 3, it was of 23.3, 33.3, 52.1 and 58.7 respectively for the method of HAI and of 72.1, 90,1, 91.7 and 90.5 for the method of IFI. These results confirm that the congruity between the tests of HAI and IFI in the diagnosis of the sheep toxoplasmosis is of regular type and both tests are not replaceable.

El Objetivo del presente estudio fue determinar la seroprevalencia de Toxoplasma gondii en búfalos del distrito de Jenaro Herrera, ubicado en la región Loreto. En Agosto del 2008 se recolectaron 70 muestras de sangre de búfalos hembra para la detección de anticuerpos contra Toxoplasma gondii mediante dos técnicas, hemaglutinación indirecta (HAI) e inmunofluorescencia indirecta (IFI). Los resultados indicaron que el 35.7 ± 11.2% de las muestras fueron reactivas a la prueba de HAI, y el 17.14 ± 8.8% fueron reactivas a la prueba IFI. Presentando una relación directa entre el aumento de la prevalencia y la edad de los animales. La prevalencia encontrada en el presente estudio fue moderada. -- Palabras clave: toxoplasmosis, búfalos, HAI, IFI. / -- The aim of this study was to determine the seroprevalence of Toxoplasma gondii in bufalos in the Jenaro Herrera district, located at Loreto Department. A total of 70 blood samples were collected in female bufalos for the detection of antibodies against Toxoplasma gondii by 2 different tecniques, the Indirect Hemaglutination Test (IHA), and the Indirect Inmunofluorescence Test (IFI). The results indicated that 35.71 ± 11.2% of the samples were reactive to the Indirect Hemaglutination Test, and 17.14 ± 8.8% were reactive to IFI. Showing a direct relationship between the increase of seroprevalence and the age of the animals. The prevalence found an the present study was considerated moderate. --Key Words: toxoplasmosis, buffalos, IHA, IFI.

Orientador: Katia Denise Saraiva Bresciani / Banca: Marcelo Vasconcelos Meireles / Banca: Solange Maria Gennari / Resumo: Doze gatas, sorologicamente negativas para Toxoplasma gondii, foram distribuídas em três grupos de quatro animais cada. Os animais dos grupos 1 e 2 foram infectados com cistos teciduais de T. gondii, por via oral, no terço médio de gestação, sendo: grupo 1 - isolado tipo I altamente virulento para camundongos; grupo 2 - isolado tipo III não virulento para camundongos; e grupo 3 - gatas controle não infectadas. Antes da infecção e durante todo o período experimental foram efetuadas avaliações clínicas. Amostras sangüíneas foram colhidas nos dias 0 (antes da inoculação), 15 e 30 dias pós-infecão para a realização de hemogramas e pesquisa de anticorpos contra T. gondii pela reação de imunofluorescência indireta (RIFI 16). A infecção com o isolado de T. gondii virulento (tipo I) ocasionou diarréia, vômito, secreções ocular e vaginal e aborto de uma cria. Nas gatas inoculadas com o isolado tipo III não patogênico, foram observados diarréia, vômito, hiporexia, secreções nasal e ocular purulentas e um parto prematuro com natimortos. Todas as oito gatas infectadas com cistos teciduais de T. gondii apresentaram anticorpos contra T. gondii pela RIFI. Em dois filhotes da mesma ninhada, pertencentes ao grupo 1, foram detectados títulos de 16 e 64 aos 10 dias de vida. No grupo 2, títulos de 16, 32 e 32 foram detectados em três filhotes da mesma ninhada. Os resultados deste estudo demonstram que cistos teciduais de T. gondii de genótipos dos tipos I e III (pelo marcador SAG 2) provocaram transtornos reprodutivos semelhantes em gatas gestantes primoinfectadas. / Abstract: Twelve pregnant queens, serologically negative to Toxoplasma gondii, were distributed into three groups of four animals each. Animals were infected orally with tissue cysts of T. gondii, in the meddle third of their pregnancies as follows: group 1- was infected with isolate type I highly virulent for mice; group 2 - was infected with isolate type III not virulent for mice; group 3 - no infected control queens. Clinical profiles of the animals were determined before infection and during the whole experimental period. Blood samples of the queens were taken in the following days: 0 (before the inoculation), 15 and 30 days pos infection for blood count and for the antibodies detection against T. gondii by the indirect immunofluorescent antibody test (IFAT 16). Virulent T. gondii (type I) infection caused vomiting, diarrhea, purulent eye secretion, haemorrhagic vaginal secretion and abortion of one pregnancy. Queens infected with non pathogenic type III isolate developed diarrhea, emesis, hyporexia, purulent nose and ocular secretions and one premature stillbirth. In all eight infected queens with tissue cysts T. gondii antibody were detectable by IFAT. In two kittens of the same litter, from group 1, had been detected titers of 16 and 64 at 10 days of age. In group 2, titers of 16, 32 and 32 were detected in 3 kittens of the same litter. Infection with tissue cysts of types I and III (SAG 2 marker) of T. gondii isolates developed similar clinical and reproductive disturbance in primary infected pregnant queens. / Mestre

Toxoplasma gondii is an obligate intracellular parasite, that is able to infect any nucleated cell. An important global pathogen, T. gondii can cycle between primary and secondary hosts, thus enabling widespread penetrance. Within its intracellular niche – a membrane-bound parasitophorous vacuole – T. gondii is nevertheless able to subvert a variety of host cell processes to allow its continued survival and replication. This includes modulation of host signalling processes as well as the scavenging of nutrient macromolecules. In recent years, microRNAs have emerged as important regulators of cellular processes including inflammation, tumorigenesis and metabolism, as well as development. It has become increasingly clear that this species of non-coding RNA is of great importance in ‘fine tuning’ many cellular responses. I hypothesise in this work that host cell miRNAs may be yet another means by which T. gondii manipulates its host upon infection. Using high-throughput-sequencing, I examine host cell transcriptional responses to infection both at the mRNA and microRNA level, using two strains of T. gondii at a variety of Multiplicities of Infection over a time course of 43 hours. Through these transcriptional analyses I identify a number of dysregulated pathways common in tumorigenesis, and contemplate the hypothesis that T. gondii may be behaving as an ‘intracellular tumour’, subverting host cell metabolic processes to mimic a long-known feature of cancer metabolism – that of aerobic glycolysis (the Warburg effect) – in order to satisfy its own energetic and metabolic needs.

La toxoplasmosis es una zoonosis que afecta a una gran variedad de animales, incluyendo al hombre. La ingesta de ooquistes excretados por el hospedero definitivo, el gato, constituye una vía de transmisión importante. El objetivo del presente estudio fue evaluar la seroprevalencia y el estado de infección por Toxoplasma gondii en gatos domésticos de Lima Metropolitana (Perú) y la relación que guardan con el tipo de crianza del animal como posibles factores de riesgo. Se colectaron 118 muestras de suero, de las cuales 112 pertenecían a gatos de 22 diferentes distritos de la capital peruana atendidos en clínicas veterinarias y 6 provenientes de un albergue situado en el distrito de Cieneguilla. Se determinó la frecuencia de anticuerpos IgG y el estado de infección mediante las técnicas de ELISA y ELISA de avidez, respectivamente. Los factores de riesgo analizados fueron los siguientes: sexo, grupo etario, raza (pura, mestiza), alimentación (comercial, mixta), acceso a la calle y hábitos de caza. Se detectaron anticuerpos IgG anti T. gondii en 29 de 118 gatos (24.6%), los que a su vez evidenciaron una alta avidez (29/29), correspondiendo a infecciones tardías. Se encontró asociación significativa (p p<0.05) en los factores edad (p=0.02), alimentación (p=0.04), acceso a la calle (p=0.02) y hábitos de caza (p=0.05). Los factores sexo y raza no mostraron asociación significativa. La frecuencia y Odds ratio de seropositivos aumentó en gatos de alimentación mixta (38.2%, OR=2.63), con acceso a la calle (41.9%, OR=3.56), cazadores (31.6%, OR=1.96) y mayores de un año (30.5%, OR=3.51), donde dentro esta categoría, la más alta prevalencia se encontró en mayores de siete años (8/18, 44.4%). Se reporta un aumento de la seroprevalencia de T. gondii en gatos en comparación a estudios previos en Lima, asociada a sus hábitos de crianza. Asimismo, la prueba de ELISA resulta una herramienta útil para este tipo de estudios, debiendo ser implementada en el Perú. / Tesis

A Toxoplasmose é uma zoonose cosmopolita que ocorre principalmente em indivíduos imunodeprimidos, gestantes e em pacientes em terapia anti-câncer. Na Medicina Veterinária, animais como ovelhas, cabras e porcos são seriamente afetados podendo ocorrer abortos e com isto perdas significativas em termos econômicos. Atualmente não se dispõem de vacinas contra a toxoplasmose, mas tem-se desenvolvido modelos animais em ratas e camundongos para o ensaio imunológico contra a enfermidade. Os modelos atualmente utilizados, possuem limitações, por exemplo, não é possível ensaiar no modelo rata um imunógeno de Toxoplasma gondii, uma vez que todas as linhagens de ratas disponíveis são resistentes. Portanto não se pode ensaiar desafios vacinais altamente exigentes neste modelo. Por isso é aconselhável contar com experimentos efetuados em diversas espécies animais. Está sendo proposto testar o Hamster (Mesocricetus auratus), com o intuito de estabelecer se é ou não um modelo que se aproxima das respostas esperadas nos humanos.Antes de se comprometer a custos muito elevados com ensaios finais de vacinações, o aconselhável é contar com ensaios em diversas espécies, pois os animais respondem de formas dIferentes aos mesmos imunógenos. Na transmissão congênita crônica dez Hamsters foram inoculados com 40 oocistos por via oral da cepa Me-49 do T.gondii e após sessenta dias foram postas para cruzarem com os machos. Obteve-se resultados promissores uma vez que a transmissão congênita aos seus filhotes nesta fase crônica da infecção foi nula. Na transmissão congênita aguda da infecção, obteve-se 42,9% de taxa de transmissão de T.gondii. Não foi observada a transmissão lactogênica, do toxoplasma, na fase aguda da infecção. / Taxoplasmosis is a cosmopolitan disease that causes a lot of damage to immunocompromised people, pregnant women, patients who have had transplants and people undergoing anticancer therapy. Animals like sheep, goats and pigs are seriously affected causing abortions and consequently significant economic losses. This is why, the importance of studing the transmission, the mechanisms of transmission and the forms of infection. Currently, vaccines against toxoplasmosis are not available, but animals’ models, in rats and mice, have been developed to an immune assay against this disease. These models considered to the study have some limitations, however it is not possible to apply a toxoplasma immune assay on a rat model, because it is a susceptible race and it has already been proven that rats are resistant to toxoplasma. We proposed to test the hamster (Mesocrisetua auratus) with the intention of establishing if it is or not one model that approaches the same results expected on human beings, therefore before compromising high costs with final assays of vaccinations, the advisable thing to do is to turn to assays on diverse species, because animals have different answers to the same immune. We intended to test hamster (Mesocrisetus auratus), with the intention of establishing if it is or no a model that approaches the expected ansewer from diferential ways to the same strains. In the chronic congenital transmit, in Hamsters was obtained promising results because yours creates didn’t present title when their organs (liver and lung), they were bioassayed in mice and twenty five days after they were bled and their serum tested with direct agglutination of Desmonts and Remington (1980), confirming that the passage wasn’t made during the gestation of females infected before the conception. In the sharp congenital transmission of the infection, it was obtained 42,9% that passed to their descendants antibodies of T. gondii. In the lactogenic transmission, wasn’t have title for T.gondii