El objetivo del presente estudio fue determinar la seroprevalencia de Toxoplasma gondii en cerdos procedentes de granjas tecnificadas y no tecnificadas de Lima-Perú e identificar los factores asociados a su transmisión. El trabajo se desarrolló en 407 animales provenientes de 7 granjas con crianzas porcinas tecnificadas y 10 no tecnificadas ubicadas en la franja costera del departamento de Lima. Se aplicaron encuestas epidemiológicas en las granjas para identificar los potenciales factores asociados a la transmisión de T. gondiien porcinos. Posteriormente se colectaron las muestras de sangre de cerdos en la fase de acabado procedentes de granjas tecnificadas (264) y crianzas no tecnificadas (143), las muestras de suero fueron conservadas en congelación (-70ºC) hasta su procesamiento en el Laboratorio de Parasitología de la Facultad de Medicina Veterinaria de la UNMSM. Para el diagnóstico de Toxoplasmosis porcina se utilizó la técnica de ELISA indirecta. La asociación entre la seroprevalencia a T. gondii y cada una de las variables evaluadas (sexo, procedencia, densidad animal, fuente de agua, tipo de alojamiento, presencia de felinos y control de roedores) fueron analizadas mediantes múltiples modelos de regresión logística. La seroprevalencia de T.gondii en cerdos de granjas tecnificadas y no tecnificadas de Lima-Perú es de 4.5 y 33.6% respectivamente. Los factores asociados a la transmision de T. gondii en porcinos son la procedencia (OR: 10.61), fuente de agua (OR: 6.44), tipo de alojamiento: mixto (OR: 6.14) y no estabulado (OR: 13.59);la presencia de felinos: de 1 a 3 (OR: 5.29) y ≥4 (OR: 16.02); y el control de roedores (OR: 7.81). Palabras clave: porcinos, Toxoplasma gondii, ELISA indirecto, regresión logística / --- The study objective was to determine the seroprevalence of Toxoplasma gondii in pigs from well managed and poor managed farms of the Lima, Peru and identify factors associated with transmission. The work was done in 407 animals from 7 farms whit pig well managed and 10 poor managed located in the coastline of the department of Lima. Epidemiological surveys were applied on farms to identify potential factors associated with the transmission of T. gondii in pigs. Subsequently blood samples were collected from pigs in the finishing phase from well managed (264) and poor managed (143), Serum samples were preserved frozen (-70 ° C) until processing in the Laboratory of Parasitology, Faculty of Veterinary Medicine of UNMSM. To diagnose of swine toxoplasmosis was used the indirect technique ELISA. The association between seroprevalence of T. gondii and each of the evaluated variables (sex, provenance, animal density, water source, type of accommodation, presence of cats and rodent control) were analyzed mediantes multiple models of logistic regression. The seroprevalence of T. gondii in pig of the well managed farms and poor managed farms from Lima-Peru is 4.5 and 33.6% respectively. Factors associated with the transmission of T. gondii in pigs are the provenance (OR: 12.31), water source (OR: 6.44), type of accommodation: mixed (OR: 6.14) and not stabled (OR: 13.59); the presence of felines: 1 to 3 (OR: 5.29) and ≥4 (OR: 16.02); and the rodent control (OR: 7.81). KEYWORDS: swine, Toxoplasma gondii, indirect ELISA, logistic regression

Thesis advisor: Marc-Jan Gubbels / The obligate intracellular parasite Toxoplasma gondii exhibits closed mitosis, as chromosome segregation occurs with the confines of the nuclear envelope. Distinct structural changes are absent during mitosis, as the nucleolus is maintained and condensation of chromosomes is largely restricted. Moreover, the centromeres are clustered and remain persistently associated with the centrocone (spindle pole). To elucidate the process of chromosome segregation during mitosis in the parasite, the role of kinetochores and microtubules was examined. Localization studies of the functionally conserved kinetochore proteins TgNuf2 and TgNdc80 revealed that clustered kinetochores colocalize with clustered centromeres at the centrocone throughout the cell cycle. Pharmacological disruption of microtubules resulted in partial loss of clustering, which indicates spindle microtubules are necessary, but not strictly required for this process. Furthermore, the generation of a conditional TgNuf2 knockdown revealed this kinetochore protein is essential for chromosome segregation but dispensable for clustering of centromeres, which remain associated with the centrocone. Moreover, in the absence of TgNuf2 the centrosome behaves normally, but looses its association with the centrocone. Further analysis of this phenotype revealed that the centrocone is devoid of spindle microtubules following depletion of this essential kinetochore protein. Examination of tubulin localization dynamics through parasite development showed that the initiation of spindle microtubules occurs at the basal region of the nucleus prior to centrosome duplication. Furthermore, acetylation of α-tubulin, a posttranslational modification associated with microtubule stability, was confirmed to be specifically associated with stabilization of the spindle microtubules following comigration of the centrocone and centrosome to the apical end of the nucleus. Collectively, these data demonstrate that the persistent association of clustered centromeres with the centrocone is independent of spindle microtubules. These discoveries are contributing unprecedented details to chromosome anchoring and segregation during the cell cycle in this protozoan parasite. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

IV ­^¤åºK­n SAG1 and SAG2 are important surface molecules of T. gondii for the invasion of tachyzoites into host-cells. Previous studies have been demonstrated they are good candidates for development of vaccines against toxoplasmosis. Therefore, we used SAG1 and SAG2 to generate subunit vaccines and evaluated the protective capacity in BALB/c mice. Anti-idiotype IgG (aId-IgG) with the SAG2 internal image was prepared from anti-SAG2 monoclonal antibody in accordance with the network theory. Lymphocytes from mice immunized subcutaneously twice with 2, 4 or 8 µg aId-IgG showed great proliferations and secreted high level of IFN-£^, which is an important cytokines secreted by Th1 cells. After challenged subcutaneously with 1¡Ñ103 tachyzoites, the highest survival rate reached 88%. Further, SAG1 and SAG2 genes were respectively cloned and recombinant proteins rSAG1 and rSAG2 were prepared. Lymphocytes from mice immunized intraperitoneally twice with rSAG1 or rSAG2 displayed apparently Th1-associated responses, while lymphocytes from mice immunized subcutaneously twice with rSAG1 or rSAG2 did not. Mice immunized intraperitoneally twice with rSAG1 or rSAG2 had a survival rate of 64% which was higher than those mice immunized subcutaneously with rSAG1 or rSAG2. When mice immunized intraperitoneally twice with rSAG1 mixed with rSAG2, survival rate was even higher (71%). Therefore, mixed antigens induced higher protection. Subsequently, SAG1 gene was fused with SAG2 gene and a recombinant protein rSAG1/2 was expressed from the fused gene. Th1-associated responses were detected from lymphocytes of mice immunized intraperitoneally twice with 10 µg rSAG1/2. Interestingly, 80% rSAG1/2-immunized mice survived and it was extremely higher V than rSAG1- or rSAG2-immunized mice (64%). In an attempt to stimulate immune responses against T. gondii infections in the mucosal system, we chose heat-labile enterotoxin (LT) secreted from toxigenic E. coli as the stimulator. LTA2B and LTB genes were respectively cloned and then rLTA2B and rLTB were obtained. Moreover, LTA2B gene or LTB gene fused with SAG1 and SAG2 genes was performed and then recombinant proteins rLTA2B-SAG2/1 and rLTB-SAG1/2 were prepared. Subsequently, mice were immunized intranasally twice with rLTA2B-SAG2/1, rLTB-SAG1/2, rLTA2B mixed with rSAG1/2, or rLTB mixed with rSAG1/2. A strong protection (67%) was shown by the group of mice immunized intranasally with 10 µg rLTB-SAG1/2 or 4 µg rLTB mixed with 10 µg rSAG1/2. rLTB, rather than rLTA2B, will be a better candidate for stimulation the mucosal system. In summary, different survival rates caused by antigens prepared in the study may be attributed to many factors such as the treatment, the preparation and the dose of antigen. The highest survival rate is caused by aId-IgG (88%); the second is shown by rSAG1/2 (80%); the third is resulted from rSAG1mixed rSAG2 (71%). Although the survival rate raised by rLTB-SAG1/2 or rLTB mixed rSAG1/2 is slightly low (67%), these data demonstrate stimulators such as LT could induce anti-Toxoplasma immune response of the mucosal system. Antigens capable of inducing higher survival rates in mice should be worthy of further investigation for searching better vaccine candidates.

El objetivo del presente trabajo fue determinar la seroprevalencia del Toxoplasma gondii en llamas hembras adultas de dos puntas de parición de la Unidad de producción Alianza-Antacalla en la Empresa de propiedad social “Rural Alianza” en la provincia de Melgar-Puno, mediante la prueba de Inmunofluorescencia indirecta; para lo cual se obtuvieron 157 muestras sanguíneas de llamas hembras adultas, de ellas 112 provenían de la punta de parición de Alianza y 45 de la punta de Rio grande. Se encontró que el 10.19 + 4.7% (16/157) del total de llamas hembras adultas presentaron anticuerpos contra T. gondii ; la seroprevalencias halladas en las puntas de Río grande y Alianza fueron de 13.33 + 9.8% (6/45) y 8.93 + 5.3% (10/112) respectivamente, no encontrándose diferencia estadística significativa entre ellas. Las seroprevalencias de T.gondii según rangos de edades de 2 a 3 , 4 a 5 y mayor o igual a 6 años, fueron de 9.09 + 8.5, 15.38 + 13.87 y 9.19 + 6.07 respectivamente; no presentando diferencia estadística significativa entre grupos etarios debido posiblemente a que estos animales eran adultos. Los resultados de este estudio muestran una seroprevalencia a T.gondii relativamente baja en esta empresa, en relación a otros estudios similares en camélidos; además se determinó que las variables edad y punta de parición no representaron factores de riesgo para la infección de T. gondii en llamas hembras adultas. / The objective of the present work was to determine the seroprevalence of Toxoplasma gondii in llamas adult females of two ends of parturition of the Unit of production Alianza-Antacalla in the Company of social property Rural Alliance in the province of Melgar-Puno, by means of the test of indirect inmunofluorescence; for which 157 of blood samples of llamas were obtained adult females, of them 112 proven of the end of parturition of alliance and 45 of the end of Río Grande. Found that the 10.19± 4.7% (16/157) of the total of llamas adult females they presented antibodies against T. gondii; the seroprevalences found in the ends of Río Grande and Alianza were of 13.33± 9.8% (6/45) and 8.93± 5.3% (10/112) respectively, not found significant statistic difference among them. The seroprevalences of T. gondii for ranks of ages from 2 to 3, 4 to 5 and greater or equal to 6 ages, were of 9.09± 8.5, 15.38± 13.87 and 9.19± 6.07 respectively; not presenting significant statistic difference between ages groups had possibly to that these animals were adult. The results of this study show a seroprevalence of T. gondii relatively low in this company, in relation to other camelids in similar studies; moreover we determ that the variables age and end of parturition did not represent factors of risk for infection of T. gondii in llamas adult females.

La toxoplasmosis es una zoonosis parasitaria ampliamente difundida en la naturaleza y causante de cuantiosas pérdidas económicas en la producción ovina y caprina. Esto sirve de referencia para determinar su posible papel en la presentación de problemas reproductivos en Camélidos Sudamericanos, motivo por el cual, el objetivo del estudio fue cuantificar la seroprevalencia de Toxoplasma gondii en alpacas de diversas comunidades alpaqueras pertenecientes a los distritos de Marangani, Pitumarca, Checacupe y San Pablo, localizados en la provincia de Canchis, departamento de Cusco. Se recolectaron 272 muestras de sangre, en marzo del 2003, para la detección de anticuerpos anti - T. gondii. La prueba empleada fue la técnica de Inmunofluorescencia Indirecta (IFI), encontrando una seroprevalencia moderada de 35.7 ± 5.7 % (97/272). No se encontró asociación entre las variables: distrito, sexo, raza y la respuesta a la prueba de IFI. Sin embargo, si se encontró asociación entre la edad y la respuesta a la prueba, incrementándose el porcentaje de alpacas serorreactoras a medida que aumentaba la edad de las mismas. Así, animales de 2 años presentaron un 20.0 + 12.4 % (8/40), animales de 4 años un 33.8 + 11.5 % (22/65) y animales de 6 años a más un 40.1 + 7.4 % (67/167). Los resultados obtenidos en alpacas de comunidades concuerdan con otros ya encontrados en Camélidos Sudamericanos en otras zonas del sur del Perú. / Toxoplasmosis is a parasitic zoonosis widely spread in nature and causes of large economic losses in sheep and goat production. This is used as a reference to determine its possible role in the presentation of reproductive problems in South-American Camelids. The objective of the present study was to quantify the seroprevalence of Toxoplasma gondii in alpacas of diverse communities belonging to the districts of Marangani, Pitumarca, Checacupe and San Pablo, located in the province of Canchis, department of Cusco. A total of 272 blood samples were collected, in march 2003, for the detection of antibodies anti - T. gondii. The used proof was the indirect immunofluorescence test (IFAT), finding a moderate seroprevalence of 35.7 ± 5.7 % (97/272). There were no association among the following variables: district, sex, breed and IFAT response. However it was association between the age and the IFAT response and the percentage of reagent alpacas was increased of the animal matures. Animals of 2 years of age where presented 20.0 ± 12.4 % (8/40), animals of 4 years of age 33.8 ± 11.5 % (22/65) and animals of 6 years or older 40.1 ± 7.4 % (67/167). The results obtained in alpacas of communities agree with other studies already found in South- American Camelids in different zones of the south of Peru.

Thèse de doctorat : Sciences de la vie et de la santé : Tours : 2008. / Titre provenant de l'écran-titre.

Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2008. / Titre provenant de l'écran titre. Bibliogr. p. 107-113.

Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2009. / Titre provenant de l'écran titre. Bibliogr. p. 59-64.

Thesis (Ph. D., Microbiology, Molecular Biology, and Biochemistry)--University of Idaho, March 2009. / Major professor: Gustavo A. Arrizabalaga. Includes bibliographical references. Also available online (PDF file) by subscription or by purchasing the individual file.

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