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This study reports the localization and partial characterization of thioredoxin from the parasitic trematode Fasciola hepatica. Snails (Pseudosuccinia columella) were raised in culture and infected with F. hepatica so that Western blotting and immunohistochemical techniques could be utilized to determine the presence of thioredoxin in different stages of the parasite’s development. The results of these experiments showed that thioredoxin was present in the tegument, gut epithelium, excretory canal epithelium and sperm, of the adult parasite as well as in the tegument and gut of the redia and cercaria intermediate stages. In situ hybridization was used to determine the localization and possible differential mRNA expression of two different F. hepatica thioredoxin isotypes (Fh2020.A and Fh2020.SL) in the adult parasite. The in situ hybridization results showed that both isotypes are expressed in the tegument and gut epithelium. Fh2020.A stains with a greater intensity possibly demonstrating a difference in the amount of expression between the two isotypes. Recombinant F. hepatica thioredoxin expressed in bacteria using the pMAL™ Protein Fusion and Expression System was used to test its affects on the production of super oxide anion by murine peritoneal macrophages, bovine monocyte-derived macrophages and bovine whole blood neutrophils, and nitric oxide production by mouse peritoneal macrophages and bovine monocyte-derived macrophages. The results of the cellular assays were not definitive due to the fact that the maltose binding protein (MBP) moiety of the recombinant thioredoxin, when tested alone, increased production of nitric oxide by bovine monocyte-derived macrophages. Consequently, since the MBP could not be effectively separated from the thioredoxin portion of the recombinant, allowing the thioredoxin affects to be tested independently, no true conclusions regarding its affects on the host immune cells tested could be drawn. This is the first report of the localization of thioredoxin in both the adult F. hepatica as well as in specific intermediate stages of the parasite. These studies demonstrate the possible affects that a protein tag can have on experimental results and demonstrate how such data may be interpreted when a non-cleaved recombinant protein is used in cellular or other assays when compared to native or cleaved recombinant proteins.

Here we report the expression and purification of two recombinant Fasciola hepatica enzymes, catL2 and catL5 which were used to perform cleavage studies with substrates potentially encountered by the parasite in vivo; BSA, hIgG3K and the important T cell marker, CD4. We examined the digestion products generated by the cleavage of human CD4 with catL5 using mass spectrometry and predicted candidate cleavage sites by performing a theoretical digest of the protein. / Ovine CD4 is also of interest to us as a target of F. hepatica cathepsin L activity. Here we confirm a recently reported ovine CD4 cDNA sequence and the existence of a single nucleotide polymorphism (T/C) within this sequence. The polymorphism translates to a serine-proline switch near the hinge region of the protein. Additionally, we have found that this polymorphism is also present in genomic DNA, suggesting that two alleles of CD4 exist in the ovine genome.

As proteínas ligadoras de ácidos graxos (Fatty Acid Binding Proteins, FABPs) de parasitos têm um papel importante no processo de infecção por estes organismos. Por este motivo, estas proteínas são antígenos candidatos para vacina contra a infecção por Schistosoma mansoni e Fasciola hepatica. No presente trabalho foram caracterizadas FABPs de F. hepatica e comparadas com a proteína Sm14 de S. mansoni, a FABP de parasito melhor caracterizada, mediante análise de sequências e estruturas modeladas. Também foram clonadas, expressas e purificadas as FABPs tipo 1 e tipo 3 de F. hepatica. Os resultados do presente estudo indicam que a FABP tipo 3 de F. hepatica é relacionada estrutural, imunológica e funcionalmente com a Sm14, um candidato vacinal amplamente estudado. Devido à importância da Sm14 como alvo para o desenvolvimento de vacina para a esquistossomose, as características apresentadas pela FhFABP3 de F. hepatica apontam esta proteína como um candidato importante também para o desenvolvimento de uma vacina contra a fasciolose / Parasites fatty acid binding proteins (FABPs) have an important role in infection process by these organisms. For this reason, these proteins are candidates as vaccine antigens against Schistosoma mansoni and Fasciola hepatica infection. In the present study, FABPs from F. hepatica were characterized and compared with Sm14 protein from S. mansoni, the best characterized parasite FABP, by sequence analysis and modeled structure. Type 1 and type 3 FABPs from F. hepatica were also cloned, expressed and purified. The results of this study indicated that type 3 FABP from F. hepatica is structural, immunological and functionally related with Sm14, a vaccine candidate widely studied. Due to the importance of Sm14 as a target for vaccine development for schistosomiasis, the characteristics presented by the FhFABP3 from F. hepatica suggest this protein as a candidate also important for the development of a vaccine against fasciolosis

Publicación a texto completo no autorizada por el autor / Estandariza dos pruebas de ELISA indirecto para la detección de anticuerpos IgM e IgG4 anti Fasciola hepatica y aplica las pruebas en sueros de pobladores de la comunidad andina de Pamparomás, Ancash. Desarrolla un estudio observacional, descriptivo y transversal. Las principales medidas de resultados son anticuerpos contra F. hepatica detectados mediante las pruebas de ELISA IgM e IgG4 anti F. hepatica. Encuentra que las condiciones de reacción de la prueba de ELISA IgM fueron concentración de antígeno excretado-secretado (E/S) de F. hepatica 5 µg/100 µL y dilución del suero 1/10, dilución del conjugado anti IgM 1/500. Las condiciones de reacción de la prueba de ELISA IgG4 anti F. hepatica fueron concentración de antígeno E/S de F. hepatica 2,5 µg/100 µL, dilución del suero 1/200, dilución del conjugado anti IgG4 biotinilado 1/2 000 y conjugado estreptavidina-peroxidasa 1/1 000. La prueba de ELISA IgG4 anti F. hepatica determinó una frecuencia de 14,5% (16/110) de reactividad en sueros humanos. La sensibilidad y especificidad para la prueba de ELISA IgG4 anti F. hepatica fue de 100% y 93,1%. Los valores de sensibilidad y especificidad de la prueba de ELISA IgM anti F. hepatica resultaron no concluyentes. Determina que la prueba de ELISA IgG4 anti F. hepatica es útil como prueba de tamizaje para el diagnóstico de la fasciolosis en pobladores infectados de zonas endémicas. / Tesis

Objetivo general: Estimar la prevalencia y factores asociados a la infección de Fasciola hepatica por PCR en muestras de heces recogidas de niños escolares del distrito de San Pablo, Cajamarca, Perú. Objetivos específicos: - Estimar la prevalencia de infección por Fasciola hepatica por PCR en niños escolares del distrito de San Pablo, según edad, sexo y contacto con ganados. - Establecer los factores asociados a la infección por Fasciola hepatica por PCR en niños escolares del distrito de San Pablo. Métodos: Se realizó un estudio transversal prospectivo de abril a junio de 2015 en niños de 5 a 12 años de la Institución Educativa Estatal del nivel primario de Educación Básica Regular del distrito de San Pablo provincia de Cajamarca, Perú. La muestra estuvo constituida por un total de 150 niños de los cuales fueron obtenidas las muestras de heces y se analizaron por reacción en cadena de la polimerasa (PCR) para determinar la infección por Fasciola hepatica. Resultados: Se detectó ADN de Fasciola hepatica en el 10.67% de muestras de heces, con una prevalencia en niños de 11,54% y niñas de 9,72%. El grupo de edad de 5-8 años (13,64%) mostró un ligero mayor porcentaje de infectados con respecto al grupo edad de 9-12 años (8,33%). No se encontró ninguna diferencia estadística entre un animal específico y los pacientes con muestras positivas para Fasciola hepatica. Los factores asociados a la infección fueron; el consumo de agua de acequia y el consumo de menestra presentando una asociación positiva y negativa, respectivamente. Conclusiones: La prevalencia de infección por Fasciola hepática en niños escolares hallada en el distrito de San Pablo fue de 10,67%. Con respecto a la presencia de fasciolasis, no se encontró diferencia de acuerdo al género, grupo de edad y contacto con animal. Se determinó que el consumo de agua de acequia predispone a la infección por Fasciola hepática, y que la proporción de niños con esta infección fue menor con el consumo de menestras.

Evalúa el efecto de la dosificación con Triclabendazol+Fenbendazol y Albendazol sobre los parámetros productivos y la frecuencia de lesiones y hallazgos macroscópicos en hígados en bovinos de engorde procedentes de una zona endémica a Fasciola hepatica. Este estudio utilizó 90 bovinos de 2 a 4 años de edad, procedentes del distrito de Cora Cora, provincia de Ayacucho. Los animales fueron transportados a un centro de engorde en el distrito de Lurín, Lima. Posteriormente, estos fueron distribuidos en los siguientes tratamientos: [T1] control no tratado, [T2] Albendazol a dosis única oral y [T3] Triclabendazol+Fenbendazol a dosis única oral. Los tratamientos se realizaron a la semana de arribo de los animales y fueron mantenidos en engorde por 60 días. Los pesos se registraron a los días 0, 30 y 60, así como el consumo de alimento acumulado. Finalmente, los bovinos se sacrificaron al día 60 y se evaluaron las lesiones macroscópicas en hígados. Los resultados demostraron valores más altos de peso corporal, ganancias de peso y mejor conversión alimenticia en los bovinos tratados con Triclabendazol+Fenbendazol en comparación a los bovinos tratados con Albendazol y los bovinos control [p<0.05]. Lesiones y hallazgos macroscópicos no se observaron en bovinos tratados con Triclabendazol+Fenbendazol en comparación a los bovinos control y tratados con Albendazol [p<0.05]. Estos resultados demuestran que la combinación Triclabendazol+Fenbendazol a dosis única oral es adecuada y óptima para ser aplicada en sistemas de engorde intensivo ya que la productividad y apariencia de hígados mejoró en forma significativa. / Tesis

The liver fluke, Fasciola hepatica, is one of the most important parasites of livestock, and it also infects humans. The proteolytic system of trematodes is critical for their interaction with the host and is a potential target for the development of novel vaccines. This work is focused on proteases secreted by F. hepatica adults and on FheCy2, a new protease inhibitor from the cystatin family. The proteolytic activity of the secreted proteases was analyzed using: (a) chromogenic protein substrates and fluorogenic peptide substrates, (b) selective protease inhibitors, and (c) a fluorescent activity-based probe for visualization of proteases. The results showed that the secreted proteases are cysteine proteases of papain family belonging to cathepsins L and B. These proteases were effectivelly inhibited by FheCy2 as demonstrated by enzymological analysis. It can be assumed that FheCy2 participates in the physiological regulation of endogenous proteases secreted by F. hepatica adults, which makes it attractive candidate protein for vaccination studies. Key words: Fasciola hepatica, cathepsins, proteolytic activity, substrate specificity, protease inhibitors (In Czech)

Fasciola cause considerable monetary loss in the agriculture industry, while parasitism of humans is an emerging disease. Fasciola cathepsin L-like proteases are believed to aid parasite invasion and survival through a range of functions including feeding, immune evasion and modulation, tissue migration, egg production and excystment. As such these proteases are considered good targets for chemotherapies and vaccine development. Fasciola cathepsins are evolutionarily divided into clades that reflect function and life stage of expression. Analysis of F. gigantica genomic DNA and mRNA identified novel cathepsin L-like sequences which are incorporated into a phylogenetic analysis of the complete Fasciola cathepsin L-like protease family. Analysis of mRNA transcripts isolated in this study also points to trans-splicing occurring amongst cathepsin transcripts, the first time this has been identified in Fasciola species. S2 subsite specificity is important in determining substrate interactions with cathepsin L-like proteases. Previous work has shown that amino acid substitutions at this site can dramatically influence substrate specificity. A number of substitutions, specifically those that have been observed, or predicted to occur during the evolution of Fasciola cathepsins L-like proteases, were introduced into the S2 subsite of FhCatL5 at aa69 to determine their influence. The introduction of L69C and L69S substitutions resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants amongst fluke. The L69F variant showed an increase in the ability to cleave substrates with P2 proline, indicating F69 variants expressed by fluke are also likely to have this ability, similar to that shown with L69Y and FhCatL2. The introduction of a L69W substitution leads to increased cleavage of substrates with P2 proline, along with a decrease in cleavage of substrates with P2 phenylalanine. FgCatL1G transcripts were isolated from F. gigantica metacercariae. This contrasts with FhCatL5 and FhCatL2 which have been isolated in adult F. hepatica. These cathepsins differ at aa69, possessing tryptophan, leucine and tyrosine respectively. The processing and substrate specificities of each recombinant enzyme was analysed and compared. While FhCatL5 and FhCatL2 process in vitro in a manner similar to that reported for FhCatL1, FgCatL1G requires different processing conditions, including neutral pH. Combined with FgCatL1G possessing increased stability at acidic pH, this reflects the different environment into which FgCatL1G is expressed by immature compared to the adult flukes. The substrate specificity of FgCatL1G also differed from previously reported cathepsins, with a preference for P2 proline and low activity against substrates with P2 phenylalanine. This is the first time recombinant expression and purification of a cathepsin L-like protease specific to the immature life stages of Fasciola has been undertaken and had enzyme specificity analysed. This work has expanded knowledge of the repertoire of cathepsin proteases expressed at various life-stages of the liver fluke. Vaccination and/or drug inhibition studies may in the future be targeted towards cathepsins that are expressed in either the adult or immature stage, or perhaps both in a multi-targeted approach. The knowledge gained in this study may allow such targets to be chosen.