Made available in DSpace on 2018-08-24T22:53:02Z (GMT). No. of bitstreams: 1 tese_3674_Marcus Ocorrencia de oocistos de Cryptosporidium e cistos de Giardia em sistemas de abastecimento de agu.pdf: 2228950 bytes, checksum: d34171fd8e0650c28dbd8418a531464d (MD5) Previous issue date: 2009-07-17 / Os protozoários Cryptosporidium parvum e Giardia lamblia têm emergido como principais causadores de doenças veiculadas à água, tanto em países desenvolvidos quanto naqueles em desenvolvimento. A presença destes parasitos em água tem chamado a atenção das organizações internacionais de saúde para uma intervenção nos padrões de qualidade da água para abastecimento público. Os cistos de Giardia e oocistos de Cryptosporidium são resistentes às pressões ambientais, podendo sobreviver por vários meses no ambiente aquático, além resistir ao cloro no processo convencional de desinfecção. A pesquisa foi realizada no período de abril de 2008 a março de 2009, e teve como objetivo avaliar a ocorrência de Giardia e Cryptosporidium em dois sistemas de tratamento de água da região da Grande Vitória, no Estado do Espírito Santo. O sistema Carapina (A) tem duas opções de tratamento na mesma planta, filtração direta e flotofiltração, sendo esta última operada em situações de turbidez > 30 uT na água bruta. O sistema Vale Esperança (B) possui duas linhas independentes de tratamento, uma por filtração direta e outra por tratamento convencional. As técnicas de concentração de protozoários por floculação com CaCO3 e filtração em membranas foram avaliadas através de testes de eficiência relativa em diferentes matrizes (água bruta, filtrada e destilada), para definir qual técnica apresenta melhor desempenho na recuperação dos protozoários. A floculação foi melhor para Giardia (48,30%), mas semelhante à filtração na recuperação de Cryptosporidium (p > 0,05), sugerindo que em pesquisas dos dois protozoários na mesma amostra deve-se utilizar a floculação com CaCO3. Os cistos e oocistos foram visualizados por imunofluorescência direta e contraste de fase. Os dois mananciais estudados não apresentaram diferenças no nível de contaminação dos protozoários (p > 0,05). O tratamento convencional teve o melhor desempenho na remoção dos protozoários, pois não foi detectada a presença dos parasitos no seu efluente filtrado. Na água filtrada da flotofiltração identificou-se a maior freqüência de cistos (50,00%) e oocistos (66,67%). No efluente da filtração direta do sistema B, Giardia e Cryptosporidium foram detectados em 16,67% e 41,67% das amostras, respectivamente. A flotofiltração e a filtração direta do sistema B apresentaram turbidez > 0,5 uT na água filtrada em 100% e 58,33% das amostras, respectivamente. No efluente da filtração direta do sistema A não foram identificados oocistos de Cryptosporidium, somente cistos de Giardia em 16,67% das amostras. Na água tratada dos reservatórios não se detectou os parasitos, porém os resultados podem estar subestimados devido às limitações das técnicas de detecção. A remoção dos protozoários por flotofiltração variou de 1,00 a 2,36 log e na filtração direta nos sistemas A e B ficou entre 0,39 e 1,91 log sendo que na maioria dos meses não foi possível enquadrar a eficiência de remoção na recomendação feita pela USEPA (3,0 log para Giardia e 2,0 log para Cryptosporidium) devido à concentração encontrada em água bruta e o limite de detecção dos métodos. Correlações significativas foram observadas na água bruta, entre os protozoários e os parâmetros turbidez, coliformes totais, E.coli, cor verdadeira e aparente. O presente trabalho foi importante para mostrar a real situação dos sistemas de tratamento de água na região da Grande Vitória, ES, quanto à remoção de protozoários patogênicos, evidenciando a importância do monitoramento de Giardia e Cryptosporidium em águas destinadas ao abastecimento público.

The protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis) can cause diarrhea in humans, cats, dogs and other animals. Giardia duodenalis consists of eight assemblages (A-H) that are morphologically identical but genetically distinct. Assemblages C-H are generally species-specific, while A and B infect people and animals and are considered potentially zoonotic. Most canine and feline isolates belong to their respective species-specific assemblages, but isolates of assemblages A and B (predominantly found in humans) have also been recovered from dogs and cats. Diagnosis of infection has historically been by morphologic techniques (observing trophozoites on direct fecal smears or cysts on centrifugal zinc sulfate fecal flotations), and it is currently recommended to use morphologic techniques in conjunction with a sensitive and specific antigen test. Diagnosis is important for management of clinical giardiasis in cats and dogs and also to identify the assemblage present to determine its zoonotic potential. In my dissertation research I evaluated diagnostic techniques in use for companion animals, including centrifugal zinc sulfate fecal flotation, antigen tests optimized for use in dogs and cats, direct immunofluorescent assay (IFA), and Polymerase Chain Reaction (PCR). I showed that when compared to the reference IFA the veterinary optimized antigen tests performed similarly and had no statistically significant differences in sensitivity or specificity when combined with a centrifugal zinc sulfate fecal flotation test. Sensitivity and specificity by comparison to IFA was ≥ 82% and ≥ 90%, respectively, for all diagnostic tests evaluated in dogs and cats. When analyzed via Bayesian analysis sensitivity and specificity for all diagnostic tests was ≥83% and ≥95%, respectively. The Bayesian analysis also showed that using the direct immunofluorescent assay (IFA) as the reference test was supported. I also evaluated PCR as a molecular diagnostic technique to detect Giardia infections in dogs with soft stool or diarrhea (mimicking clinical signs of infection). I utilized both conventional and real time PCR assays and compared the results to the recommended method of diagnosis, the zinc sulfate fecal flotation combined with an immunoassay test. I found that agreement between PCR and microscopy combined with an immunoassay was poor to fair and varied depending on the molecular parameters and size of the DNA target underscoring the complexity of test evaluation and molecular diagnostics for Giardia. I also evaluated cats from a varied population (owned, shelter, feral) in Virginia to determine to what extent (if any) they were infected with potentially zoonotic assemblages of Giardia. The species-specific assemblage F was detected in 57% of the samples and assemblage A, which is considered potentially zoonotic, was recovered from 32% of the sampleI. In 11% both assemblages F and A were detected. We showed for the first time that cats in Virginia are infected with potentially zoonotic assemblages of Giardia. / PHD

El objetivo del estudio fue determinar la prevalencia de Giardia sp. en caninos y niños de comunidades campesinas de los distritos de Ajoyani, Palca y Santa Lucía en Puno. Se recolectaron 130 muestras fecales tanto de niños como de canes. Las muestras fueron conservadas en formol al 10%, siendo luego enviadas al Laboratorio de Parasitología de la FMV-Lima para su procesamiento. Para el diagnóstico de Giardia sp. cada muestra fue analizada mediante las técnicas de Sedimentación Espontánea y Sheather. Considerando como positivo el hallazgo del parásito en una de las dos técnicas usadas, se obtuvo una prevalencia global de 14.6+6.1% y 28.5+7.8% en caninos y niños respectivamente. En caninos se hallaron prevalencias de 31.8%, 18.2% y 9.3% en los distritos de Ajoyani, Palca y Santa Lucía, respectivamente; las prevalencias en machos y hembras fueron de 14.6% y 17.4% respectivamente y según los grupos de edad de 0-6 meses, >6-12 meses, >12-72 meses y >72 meses fueron de 7.7%, 21.7%, 11.4% y 16.0% respectivamente. En niños se obtuvieron prevalencias de 36.4%, 13.6% y 30.2% en Ajoyani, Palca y Santa Lucia, respectivamente; los niños presentaron prevalencias de 14.6% y las niñas 17.4%; según los grupos etarios de 0-3años, >3-7años, >7-12 años fueron de 33.3%, 29.7%, 25.9% respectivamente. Para el análisis estadístico se empleó la prueba de chi cuadrado, con un nivel de significancia de 0,05%. No se encontró asociación estadística significativa (p>0,05) entre la presencia de Giardia sp. y las variables estudiadas. Además la técnica de sedimentación espontánea demostró ser la técnica más eficaz para el diagnóstico del parásito. Las prevalencias halladas en caninos y niños sugieren infecciones independientes. Sin embargo, sólo se esclarecería con futuros estudios moleculares, para descartar posible riesgo zoonótico. Palabras clave: Giardia sp., Puno, Sedimentación espontánea, Sheather, zoonosis / --- The aim of this study was to determine the prevalence of Giardia sp. in dogs and children in rural communities of the districts Ajoyani, Palca and Santa Lucía in Puno. We collected 130 fecal samples from both children and dogs. The samples were preserved in formaldehyde at 10%, then sent to the Laboratory of Parasitology of the FMV-Lima for processing. For the diagnosis of Giardia sp. each sample was analyzed by spontaneous sedimentation techniques and Sheather. Considering as positive the parasite founding in one of two techniques used, we obtained an overall prevalence of 14.6+6.1% and 28.5+7.8% in dogs and children respectively. In dogs, were found prevalences of 31.8%, 18.2% and 9.3% in the districts of Ajoyani, Palca, and Santa Lucía, respectively; the prevalences in males and females were 14.6% and 17.4% respectively and according to age groups from 0-6 months,> 6-12 months, > 12-72 months and > 72 months were 7%, 21.7%, 11.4% and 16.0% respectively. In children, were obtained 36.4%, 13.6% and 30.2% prevalences in Ajoyani, Palca and Santa Lucía, respectively; boys had prevalences of 14.6% and girls had 17.4%, according to the age groups from 0-3years,> 3-7years, > 7-12 years were 33.3%, 29.7%, 25.9%, respectively. For statistical analysis we used the chi square test with a significance level of 0.05%. There was not significant association (p> 0.05) between the presence of Giardia sp. and the variables studied. Furthermore, the spontaneous sedimentation technique proved to be the most effective technique for the diagnosis of the parasite. The prevalence found in dogs and children suggest independent infections. However, only molecular studies would clarify in future to rule out possible zoonotic risk. Keywords: Giardia sp., Puno, spontaneous sedimentation, sheather, zoonosis

The unicellular parasite Giardia intestinalis is one of the organisms carrying mitochondrion-related organelle known as mitosome, which is adapted to the microaerobic lifestyle. The only known fuction of the mitosome is the synthesis of the iron-sulphur clusters. The research of the mitosomal proteome provides new information on the biogenesis and function of this unusual organelle. One of the means of the mitosome research is the analysis of the interactome of the known mitosomal proteins. The state-of- the-art method of the interactome approach is the use of the chemical crosslinking and the subsequent immunoaffinity isolation of the complexes, containing the protein of interest. In this thesis, the interactomes of GiTom40 and GiMOMP35 were characterized with the bioinformatic tools. The cellular localization of four of the chosen proteins was determined by the fluorescent microscopy. One of the proteins, the predicted dynein intermediate chain DIC6939, was phylogenetically classified as an axonemal dynein. The superresolution microsopy was utilized to observe the possible colocalization of DIC6939 with the mitosomes and blue native PAGE led to the visualization of its native complexes. In this work, the optimal conditions for DIC6939 interactome isolation were succesfully determined. The outcome...

Memoria para optar al Título Profesional de Médico Veterinario / El presente estudio tuvo como objetivo pesquisar Cryptosporidium spp. y Giardia spp. en mamíferos terrestres del Buin Zoo muestreados durante el período 2013-2014. Se realizó un muestreo censal estacional de recintos de ungulados, carnívoros y primates, obteniéndose un total de 204 muestras fecales, cada una correspondiente a un pool de heces obtenidas de tres días alternos; las cuales fueron procesadas en el Laboratorio de Parasitología de la Facultad de Ciencias Veterinarias y Pecuarias de la Universidad de Chile, mediante tinción de Ziehl-Neelsen modificado y Telemann modificado. 47 muestras fecales resultaron positivas a algún parásito (23,04%), siendo los carnívoros principalmente infectados (51,06%); y Giardia spp. mayormente encontrado (significativo en invierno y verano). Se encontró monoparasitismo en 91,49% de las muestras positivas y aquellas con ambos parásitos fueron sólo de carnívoros. Las variaciones estacionales encontradas para cada parásito no fueron estadísticamente significativas, pero al analizarse sin corrección de continuidad, se obtuvo como resultado que las muestras de ungulados y primates positivas a Giardia spp. fueron menos abundantes durante las estaciones frías que en verano. Esta es la primera descripción de Cryptosporidium spp. en leones africanos y tigres de bengala, así como también de Giardia spp. en pudúes, nyalas, jirafas, suricatas y monos tití cabeza algodón. Se concluye que hubo presencia de estos agentes parasitarios en el zoológico durante todo el periodo 2013-2014, lo que es relevante debido al posible riesgo zoonótico que conlleva, recomendándose así la implementación de un programa de control y prevención, con énfasis en vías de transmisión parasitaria

Recombinant giardial arginine deiminase (rADI) was characterized. The enzyme was found to have a specific activity of 12 U (mg protein)-1under at pH 7.4 and 1 mM arginine. The maximum velocity was 14.75 U (mg protein-1) and the KM was 0.167 mM. The specific activity and maximum velocity values are significantly lower than the values reported previously for giardial rADI, while the KM value is quite similar. The optimum pH for the giardial rADI was 6-9, a broad range compared to other arginine deiminases. Recombinant ADI is very specific in its binding specificity, with canavanine (KI 2.4 mM) and ornithine (KI 2.1 mM) being the only substrate analogues giving significant inhibition from the wide variety of analogues tested. None of the analogues could be shown to act as alternative substrates. The contribution of conserved, catalytic and C-terminal residues proposed by previous research towards ADI activity was investigated by site-directed mutagenesis. Mutations of catalytic site residues Asp175, Glu226, His280 and Cys424 decreased the rADI activity to below 2%. Conservative mutations showed significant activity for Asp175 to Glu175 (DE175) and Glu226 to Asp226 (ED226). Site directed mutagenesis on the conserved non-catalytic site Leu46 showed activities below 15%, with the highest activity observed for the mutation to Val46 (LV46), which differs in one CH2 to Leu46 on its side chain. The KM of the mutant LV46 was 3.64 mM while for LA46 (Leu to Ala mutation) was 1.33 mM. Excising 5, 13, 16 amino acids from the C-terminal residues resulted in activity decreasing to 0.8% of the wild type, while excising 54 amino acids caused the rADI to be insoluble. Sequence alignment of Giardia and Dictyostelium suggests a homologous area within the C-terminal extension. Site directed mutagenesis on the Tyr567 residue in this region resulted in a decrease in activity, with the highest activity observed for a Tyr to Phe mutation. Studies using specific cysteine protease inhibitors demonstrated partial protection against proteolysis of ADI against giardial proteases. Degradation of ADI by giardial proteases was more rapid at pH 6 than at pH 7.4.

Bibliography: leaves 183-228

El objetivo del estudio fue determinar la prevalencia de Giardia sp. en caninos y niños de comunidades campesinas de los distritos de Ajoyani, Palca y Santa Lucía en Puno. Se recolectaron 130 muestras fecales tanto de niños como de canes. Las muestras fueron conservadas en formol al 10%, siendo luego enviadas al Laboratorio de Parasitología de la FMV-Lima para su procesamiento. Para el diagnóstico de Giardia sp. cada muestra fue analizada mediante las técnicas de Sedimentación Espontánea y Sheather. Considerando como positivo el hallazgo del parásito en una de las dos técnicas usadas, se obtuvo una prevalencia global de 14.6+6.1% y 28.5+7.8% en caninos y niños respectivamente. En caninos se hallaron prevalencias de 31.8%, 18.2% y 9.3% en los distritos de Ajoyani, Palca y Santa Lucía, respectivamente; las prevalencias en machos y hembras fueron de 14.6% y 17.4% respectivamente y según los grupos de edad de 0-6 meses, >6-12 meses, >12-72 meses y >72 meses fueron de 7.7%, 21.7%, 11.4% y 16.0% respectivamente. En niños se obtuvieron prevalencias de 36.4%, 13.6% y 30.2% en Ajoyani, Palca y Santa Lucia, respectivamente; los niños presentaron prevalencias de 14.6% y las niñas 17.4%; según los grupos etarios de 0-3años, >3-7años, >7-12 años fueron de 33.3%, 29.7%, 25.9% respectivamente. Para el análisis estadístico se empleó la prueba de chi cuadrado, con un nivel de significancia de 0,05%. No se encontró asociación estadística significativa (p>0,05) entre la presencia de Giardia sp. y las variables estudiadas. Además la técnica de sedimentación espontánea demostró ser la técnica más eficaz para el diagnóstico del parásito. Las prevalencias halladas en caninos y niños sugieren infecciones independientes. Sin embargo, sólo se esclarecería con futuros estudios moleculares, para descartar posible riesgo zoonótico. Palabras clave: Giardia sp., Puno, Sedimentación espontánea, Sheather, zoonosis / The aim of this study was to determine the prevalence of Giardia sp. in dogs and children in rural communities of the districts Ajoyani, Palca and Santa Lucía in Puno. We collected 130 fecal samples from both children and dogs. The samples were preserved in formaldehyde at 10%, then sent to the Laboratory of Parasitology of the FMV-Lima for processing. For the diagnosis of Giardia sp. each sample was analyzed by spontaneous sedimentation techniques and Sheather. Considering as positive the parasite founding in one of two techniques used, we obtained an overall prevalence of 14.6+6.1% and 28.5+7.8% in dogs and children respectively. In dogs, were found prevalences of 31.8%, 18.2% and 9.3% in the districts of Ajoyani, Palca, and Santa Lucía, respectively; the prevalences in males and females were 14.6% and 17.4% respectively and according to age groups from 0-6 months,> 6-12 months, > 12-72 months and > 72 months were 7%, 21.7%, 11.4% and 16.0% respectively. In children, were obtained 36.4%, 13.6% and 30.2% prevalences in Ajoyani, Palca and Santa Lucía, respectively; boys had prevalences of 14.6% and girls had 17.4%, according to the age groups from 0-3years,> 3-7years, > 7-12 years were 33.3%, 29.7%, 25.9%, respectively. For statistical analysis we used the chi square test with a significance level of 0.05%. There was not significant association (p> 0.05) between the presence of Giardia sp. and the variables studied. Furthermore, the spontaneous sedimentation technique proved to be the most effective technique for the diagnosis of the parasite. The prevalence found in dogs and children suggest independent infections. However, only molecular studies would clarify in future to rule out possible zoonotic risk. Keywords: Giardia sp., Puno, spontaneous sedimentation, sheather, zoonosis

Gerbils, experimentally infected with Giardia lamblia trophozoites, had trophozoites and encysting trophozoites in all 3 equal sections of the small intestine and in the colon at necropsy. Cysts were found in the second and third sections of the small intestine and in the colon. WB strain derived trophozoites (WB, D1, WB-C6, and V1) differed in levels of encystation in vitro but not in gerbils. Passage in gerbils increased the in vitro encystation levels of WB and D1 but decreased that of WB-C6 and V1. No differences were found in the total protein profiles or isoenzyme patterns of these G. lamblia populations. Immunization of mice with in vitro cysts produced monoclonal antibodies (mAbs) recognizing cyst protein antigens. In Immunofluorescence (IFA), mAb 5A4.G6 recognized cyst walls. This mAb reacted with a 38 kD band on Western blots. IFA results showed that mAb 8C5.C11 reacts with vesicles in encysting trophozoites and with cyst walls. It recognized 26, 28, 42 and 46 kD bands in Western blots. When mAb 8C5.C11 and Guinea pig complement were added to 0-9 hour encysting cultures, the numbers of cysts produced were significantly reduced compared to control. MAb 5A4.G6 did not affect in vitro encystation.

No description available.