Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The equine protozoal mieloencefalite (EPM) is caused mainly by Sarcocystis neurona. It s definitive host is the opossum (Didelphis spp.), which becomes infected by ingesting sporocysts from tissues of intermediate hosts, that belong to different species. Clinical manifestations occur with neurologic signs, which vary according to the area of the nervous system affected. Clinical disease is not common, however cases of immunosuppression as senility, stress, use of corticosteroids may lead to development of clinical signs. From an epidemiological standpoint, it is interesting to determine the distribution of the protozoa in order to know the areas where animals were exposed to S. neurona through serological tests, in order to consider EPM in differential diagnosis of neurological diseases and guide the treatment. In the U.S. it is estimated that half of the horses are reagents for S. neurona, in Brazil there are not many reports about prevalence of seropositive horses. The transplacental infection has been described for other species of Sarcocystis, however there is no evidence of intrauterine infection by S. neurona, only some studies suggesting the vertical transmission as another way of agent maintenance, which could be confirmed by detecting antibodies in sera from neonates before colostrum ingestion. While S. neurona is the primary agent of equine mieloencefalite, S. cruzi is related to losses in cattle. Although parasites present similar life cycle, but with different definitive hosts, horses were exposed to both species of Sarcocystis, and these two species may infect horses and spread concurrently in herds. Therefore, this study aimed to detect the occurrence of transplacental transmission by S. neurona in horses and compare the prevalence of flocks against S. neurona and S. cruzi. Results were arranged in two chapters. In chapter one, we investigated the occurrence of transplacental infection of S. neurona in horses. For this, blood was collected from mares and their newborns for antibodies against S. neurona. In the second chapter, we compared, by indirect immunofluorescence, serum samples from 189 mares for the presence of antibodies against Sarcocystis spp. using as antigen S. neurona and S. cruzi. The analysis of the results revealed the majority of animals responding to antigens of S. cruzi and a third of seropositive animals reacted to antigens of both species. / A mieloencefalite equina por protozoário (MEP) é causada principalmente por Sarcocystis neurona. O hospedeiro definitivo de S. neurona é o gambá (Didelphis spp.), que se infecta ingerindo esporocistos dos tecidos dos hospedeiros intermediários, que pertencem a diversas espécies. Os sinais clínicos cursam com sinais neurológicos, que variam conforme a área do sistema nervoso afetada. A doença clínica não é comum, porém casos de imunossupressão como senilidade, estresse, uso de corticoides podem levar ao desenvolvimento dos sinais clínicos. Do ponto de vista epidemiológico, é interessante determinar a distribuição geográfica do protozoário a fim de se conhecer as áreas onde os animais foram expostos a S. neurona por meio de testes sorológicos, a fim considerar a MEP no diagnóstico diferencial de doenças neurológicas e direcionar o tratamento. Nos EUA estima-se que metade dos equinos seja reagentes para S. neurona, no Brasil não há muitos relatos sobre prevalência de equinos soropositivos. A infecção transplacentária já foi descrita para outras espécies de Sarcocystis, porem ainda não há evidências da infecção intrauterina pelo S. neurona, somente alguns estudos que sugerem a transmissão vertical como outra forma de manutenção do agente, o que poderia ser confirmada pela detecção de anticorpos no soro do neonato antes da ingestão do colostro. Enquanto o S. neurona é o principal agente da mieloencefalite equina, a infecção por S. cruzi está relacionada a prejuízos em bovinos. Apesar dos parasitas apresentarem ciclo de vida distintos, com hospedeiros definitivos diferentes, equinos estão expostos a ambas espécies de Sarcocystis, sendo que as duas espécies podem infectar equinos e se disseminar concomitantemente nos planteis. Assim sendo, este trabalho teve por objetivo detectar a ocorrência da transmissão transplacentária por S. neurona em equinos e comparar a soroprevalência dos plantéis frente a S. neurona e S. cruzi. Os resultados encontrados foram dispostos em dois capítulos. No capítulo um, investigou-se a ocorrência de infecção transplacentária de S. neurona em éguas. Para tal foi coletado sangue desses animais e seus neonatos para a pesquisa de anticorpos contra S. neurona. No segundo capítulo, comparou-se, por imunofluorescência indireta, amostras sorológicas de 189 éguas quanto a presença de anticorpos contra Sarcocystis spp. utilizando como antígeno S. neurona e S. cruzi. Na análise dos resultados revelou a maioria dos animais reagindo a antígenos de S. cruzi e um terço dos animais soropositivos reagiu a antígenos das duas espécies.

Equine protozoal myeloencephalitis (EPM), caused by the protozoan parasite Sarcocystis neurona, is one of the most important neurological diseases of horses in the Americas. While seroprevalence of S. neurona in horses is high, clinical manifestation of EPM occurs in less than 1% of infected horses. Factors governing the occurrence and severity of EPM are largely unknown, although horse immunity might play an important role in clinical outcome. We hypothesize that EPM occurs due to an aberrant immune response, which will be discernable in the equine IgG subisotypes a, b, and (T) that recognize S. neurona in infected diseased horses versus infected but clinically healthy horses. Based on previously-established serum antibody concentrations for IgG subisotypes in healthy horses, standard curves were generated and served to establish the concentration of antigen-specific IgG subisotypes in equine serum and CSF in infected diseased and infected normal horses. The subisotype concentrations and ratios between subisotypes were analyzed to assess whether neurological disease is associated with detectable differences in the antibody response elicited by infection. Results indicate a type I biased immune response in infected diseased horses, implicating the role of immunity in the development of EPM.

Orientador: Rogerio Martins Amorim / Resumo: Estudos com células estromais mesenquimais multipotentes (MSCs) estão em crescente progresso devido às suas propriedades imunomoduladoras, antiinflamatórias, antiapoptóticas e de regeneração tecidual, tornando essa modalidade de terapia celular promissora no tratamento de diversas doenças. Devido à limitada capacidade regenerativa do sistema nervoso central (CNS), causando sequelas funcionais, as MSCs estão sendo investigadas como uma alternativa terapêutica para condições neurológicas inflamatórias, vasculares, traumáticas e degenerativas em diversas espécies animais. A Mieloencefalite protozoária equina (EPM) causada por ambos os protozoários do filo Apicomplexa, Sarcocystis neurona e Neospora hughesi, permanece como uma importante doença neurológica dos equinos nas Américas, embora a maioria dos casos seja devida à infecção por S. neurona. A aplicação da proteômica com sua gama de ferramentas na clínica de equinos pode contribuir significativamente para o entendimento de processos patológicos e facilitar a descoberta de novos alvos terapêuticos ou marcadores diagnósticos. Neste contexto, os objetivos deste estudo foram avaliar o perfil proteômico do líquido cefalorraquidiano (CSF) antes e após múltiplos transplantes intratecal de MSCs em equinos hígidos e o perfil proteômico do CSF de equinos cronicamente afetados pela EPM. Doze cavalos adultos clinicamente saudáveis foram divididos aleatoriamente em três grupos experimentais: grupo DPBS (DPBS ou control; n = 4) onde a sol... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Multipotent mesenchymal stromal cell (MSCs) studies are under increasing progress because of their immunomodulatory, anti-inflammatory, antiapoptotic and tissue regeneration properties, making this modality of cell therapy promising in the treatment of various diseases. Due to the limited regenerative capacity of the central nervous system (CNS), causing functional sequelae, MSCs are being investigated as a therapeutic alternative for inflammatory, vascular, traumatic and degenerative neurological conditions in various animal species. Equine protozoal myeloencephalitis (EPM) caused by both protozoa of the Apicomplexa phylum, Sarcocystis neurona and Neospora hughesi, remains an important neurological disease in horses in the Americas, although most cases are due to S. neurona infection. The application of proteomics with its range of tools in the equine clinic can contribute significantly to the understanding of pathological processes and facilitate the discovery of new therapeutic targets or diagnostic markers. In this context, the objectives of this study were to evaluate the proteomic profiling of cerebrospinal fluid (CSF) before and after multiple intrathecal transplantations of MSCs in healthy horses and the CSF proteomic profiling of horses chronically affected by EPM. Twelve clinically healthy adult horses were randomly divided into three experimental groups: DPBS (DPBS or control; n = 4), in which intrathecal "transplants" with Dulbecco's phosphate buffered saline (DPB... (Complete abstract click electronic access below) / Doutor

Sarcocystis neurona is a protozoan parasite that causes the serious neurologic disease equine protozoal myeloencephalitis (EPM). The life cycle of S. neurona progresses through multiple developmental stages that differ morphologically and molecularly. The S. neurona merozoite surface is covered by multiple related proteins, which are orthologous to the surface antigen (SAG) gene family of Toxoplasma gondii. The SAG surface antigens in T. gondii and another related parasite Neospora caninum are life cycle stage-specific and seem necessary for parasite transmission and persistence of infection. The present research was conducted to explore the gene family of SnSAGs in S. neurona. Specifically, the project identified new SnSAGs in the draft genome sequence of S. neurona and examined the stage-specific expression and potential function of these surface antigens. For the first part of the study, expression of the S. neurona merozoite surface antigens was evaluated in the sporozoite and bradyzoite stages. The studies revealed that SnSAG2, SnSAG3 and SnSAG4 are expressed by sporozoites, while SnSAG5 appeared to be downregulated in this life cycle stage. In S. neurona bradyzoites, SnSAG2, SnSAG3, SnSAG4 and SnSAG5 were either absent or expression was greatly reduced. For the second part of the study, the draft sequence of the S. neurona genome was searched for potential new SnSAGs. Multiple searches revealed sixteen potential new SnSAG genes, and bioinformatic analyses of the sequences revealed characteristics consistent with the SAG gene family. Two of the new SnSAGs, designated SnSAG7 and SnSAG8, have been characterized in detail. The studies showed that SnSAG7 is expressed by the merozoite stage, while SnSAG8 is expressed by the bradyzoite stage. The third part of the study assessed the role of SnSAGs in host cell attachment and/or invasion by S. neurona. Serum neutralization assays using polyclonal serum raised against SnSAG1, SnSAG2, SnSAG3, and SnSAG4 suggested that SnSAG1 and SnSAG4 play a role in host cell attachment and/or invasion; treatment with antibodies against SnSAG2 and SnSAG3 were inconclusive. The information acquired about the stage-specific expression of the SnSAGs, identification of new SnSAG paralogues, and their functional characterization will help to understand the importance of the SnSAG proteins for parasite survival and could lead to improved methods for EPM prevention and/or treatment.

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Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-03T13:51:17Z No. of bitstreams: 1 elisabethcamposdeandrade.pdf: 1614173 bytes, checksum: 02bbfb073bbecc90f3e7f67d2c386c90 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-03T19:01:36Z (GMT) No. of bitstreams: 1 elisabethcamposdeandrade.pdf: 1614173 bytes, checksum: 02bbfb073bbecc90f3e7f67d2c386c90 (MD5) / Made available in DSpace on 2017-04-03T19:01:36Z (GMT). No. of bitstreams: 1 elisabethcamposdeandrade.pdf: 1614173 bytes, checksum: 02bbfb073bbecc90f3e7f67d2c386c90 (MD5) Previous issue date: 2009-03-26 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / As parasitoses intestinais são um importante problema de saúde pública principalmente nos países subdesenvolvidos ou em desenvolvimento. Embora o parasitismo intestinal seja amplamente reconhecido como relevante no contexto epidemiológico de diversas comunidades, os estudos sobre o assunto são ainda insuficientes, principalmente no Brasil. Em vista da dificuldade de diagnóstico específico das parasitoses, muitas vezes são realizados tratamentos empíricos com mais de uma droga. O objetivo principal do presente estudo foi avaliar a efetividade e segurança do uso de nitazoxanida no tratamento de poliparasitoses comparado à terapêutica tradicional ofertada pelo serviço público. Além deste objetivo, foi possível investigar a prevalência e os fatores associados às parasitoses intestinais na população de Colônia do Paiol, uma comunidade quilombola, na Zona da Mata, Minas Gerais. Na comunidade analisada, procedeuse um estudo transversal por censo, sendo que dos 425 moradores, 391 (92%) foram avaliados através de questionário estruturado e exame coproparasitológico. Os resultados mostraram uma alta positividade (63,8%), sendo as espécies patogênicas mais freqüentes Ascaris lumbricoides (22,4%) e Trichuris trichiura (17,9%). O poliparasitismo ocorreu em 36,5% dos investigados. O ensaio clínico, controlado, duplo cego, randomizado avaliou 65 indivíduos em dois grupos de tratamento. A taxa de cura foi de 32,4% e 38,7% com a nitazoxanida e com o tratamento convencional, respectivamente, mas esta diferença não foi estatisticamente significativa (p=0,599). A ocorrência de vômito (p= 0,031) esteve associada ao tratamento convencional e de urina esverdeada ao uso de nitazoxanida (p=0,002). Os outros efeitos adversos foram independentes da droga. Houve diferença estatisticamente significativa em relação à cor da pele e a taxa de cura para ambos os tratamentos. A menor eficácia efetividade foi apresentada pelos indivíduos de cor preta. São necessários outros estudos para esclarecer a baixa efetividade nos casos de poliparasitismo, assim como, reavaliar as práticas preventivas e terapêuticas, com o uso de novas drogas e de agentes de largo espectro, podendo a nitazoxanida ser uma droga alternativa neste contexto. Agrega-se às novas possibilidades terapêuticas, a necessidade de políticas públicas que garantam qualidade de vida, através de saneamento básico, educação para saúde e acesso ao sistema público de saúde, minimizando as iniqüidades na sociedade. / Intestinal parasitism is an important public health concern, chiefly in underdeveloped or developing countries. Although widely recognized as a relevant community epidemiological issue, intestinal parasitism has not been sufficiently studied in Brazil. Because specific diagnosis is difficult and generally cumbersome, empiric treatment, sometimes with more than one drug, is frequently employed. The main aim of this study was the assessment of the effectiveness and safety of nitazoxanide for the treatment of intestinal polyparasitism, as compared to traditional therapy provided by the public service. The study also investigated the prevalence of and factors associated with intestinal parasitism in the population of Colônia do Paiol, a quilombola community from the Zona da Mata, Minas Gerais, Brazil. A census-based cross-sectional study used a structured questionnaire and stool examination to assess 391 people (92%) of the 425 inhabitants of the community. The frequency of intestinal parasitism was as high as 63.8%, with predominance of Ascaris lumbricoides (22.4%) and Trichuris trichiura (17.9%). Polyparasitism occurred in 36.5% of those investigated. A double-blind randomized controlled trial assessed 65 individuals in two treatment groups. Cure rates were 32.4% and 38.7% with nitazoxanide and conventional treatment, respectively, but the difference was not statistically significant (p = 0.599). Vomiting (p = 0.031) was associated with conventional treatment and greenish urine with nitazoxanide use (p = 0.002). Other untoward effects were independent of which drug was used. There was a statistically significant difference concerning skin color and cure rates for both treatments. Dark-skinned subjects had lower cure rates. Further studies are necessary to clarify the reasons for the low effectiveness found in these cases of polyparasitism, and to reevaluate preventive and therapeutic approaches with new and broad-spectrum drugs, nitazoxanide being an option in this context. In addition to new therapeutic approaches, there is a clear need to develop public policies that, through the provision of basic sanitation, health education and access to the public health system, assure quality of life and minimize inequity.