El Toxoplasma gondii es un protozoo, que tiene como hospedero definitivo a los felinos y como hospederos intermediarios, a un amplio rango de hospederos entre ovinos, caprinos, porcinos, aves y humanos. Este protozoo, presenta una distribución mundial y es causante de problemas reproductivos en el ganado, principalmente el ovino. Su importancia radica en la implicancia zoonótica. Los estudios realizados en camélidos sudamericanos silvestres como la vicuña son escasos, sobre todo aquellos dirigidos a determinar los problemas de tipos reproductivos y epidemiológicos. El objetivo del presente estudio fue estimar la seroprevalencia de T. gondii en vicuñas de la Reserva Nacional de Pampa Galeras, en el Departamento de Ayacucho, para lo cual se muestrearon 191 vicuñas, entre machos y hembras, a las cuales se les realizó la prueba de Inmunofluorescencia indirecta. La seroprevalencia obtenida fue de 5.8 ± 3.3 %, no observándose diferencias estadísticas significativas en los resultados, al evaluar las variables sexo y grupo etáreo relacionados a los reactores a la prueba serológica. El presente estudio demuestra la infección por T. gondii en vicuñas de la Reserva Nacional de Pampa Galeras-Proyecto San Cristóbal y Aledaños. Palabras Clave: Toxoplasmosis, CSA silvestre, serología, anticuerpos, Ayacucho. / --- Toxoplasma gondii is a protozoan parasite, which definitive host is the feline and has a wide range of species like sheep, goats, pigs, birds and humans as intermediate hosts. This protozoan is worldwide distributed and causes reproductive problems in cattle, specially sheep. Its importance takes root in its zoonótica implication. Studies performed in wild South American camelids like the vicunas are scarce, specially those directed to determine reproductive and epidemiological problems. The objective of this study was to determine the Seroprevalence of T. gondii in vicunas of the National Reserve of Pampa Galeras in the department of Ayacucho. Therefore, 191 serum of vicunas were evaluated, male and female, by using the inmunofluorescent antibody test (IFAT). The Seroprevalence obtained was 5.8 % +/- 3.3. Significant statistical differences were not observed in the results, comparing the variables age and sex. The present study demonstrates that infection of T. gondii in vicunas of the National Reserve of Pampa Galeras occurred. Key Words: Toxoplasmosis, wild SC , serology, antibodies, Ayacucho.

Toxoplasma gondii y Neospora caninum son dos protozoarios apicomplexos de distribución mundial y potenciales causantes de enfermedades neuromusculares en canes. Sin embargo, en el Perú son escasos los estudios sobre la implicancia de estos agentes en la especie canina. Así, el objetivo de este estudio fue determinar la frecuencia de anticuerpos IgG contra Neospora caninum y Toxoplasma gondii en canes con alteraciones neuromusculares y observar el tipo de asociación que existe entre la presencia de anticuerpos y la afección neuromuscular. Para el estudio fueron examinados 96 sueros de canes con signos clínicos de afección neuromuscular y 120 sueros de canes sin presencia de signos neuromusculares. Todas las muestras fueron obtenidas en la Clínica de Animales Menores de la FMV – UNMSM. Luego, el diagnóstico se realizó a través de la prueba de Inmunofluorescencia Indirecta (IFI) para la detección de anticuerpos IgG, considerándose positivos los sueros que presentaron fluorescencia completa del taquizoíto en la dilución 1:50. En los canes con afección neuromuscular, la frecuencia para Toxoplasma gondii fue de 23.96 ± 8.5% (23/96) y para Neospora caninum fue de 5.21 ± 4.4 (5/96), mientras que en los canes sin afección neuromuscular la frecuencia para Toxoplasma gondii fue de 3.34 ± 3.1 (4/120) y para Neospora caninum fue de 1.67 ± 2.5 (2/120). Finalmente, se observó que existe asociación entre la afección neuromuscular y la presencia de anticuerpos anti-Toxoplasma gondii, mientras que no se halló asociación entre tal afección y la presencia de anticuerpos anti-Neospora caninum. / Toxoplasma gondii and Neospora caninum are two apicomplex protozoans that can cause neuromuscular diseases in dogs. In Peru, are only a few studies on the implication of these agents in the canine species. The target of this investigation was to find the association between IgG antibodies and neuromuscular diseases. In this study, 96 sera of dogs with clinical signs of neuromuscular disease and 120 sera of dogs without neuromuscular signs were examinated. All the samples were obtained at the Small Animal Clinic of the FMV – UNMSM. The diagnosis was performed by means of indirect immunofluorescence test (IFI) for the detection of IgG antibodies, considering positive the sera that showed complete fluorescence of the tachyzoite in the dilution 1:50. In the dogs with neuromuscular disease, the frequency for Toxoplasma gondii was 23.96 ± 8.5% (23/96) and for Neospora caninum was 5.21 ± 4.4 (5/96), meanwhile, in the dogs without neuromuscular disease the frequency for Toxoplasma gondii was 3.34 ± 3.1 (4/120) and for Neospora caninum was 1.67 ± 2.5 (2/120). Finally, it was observed association between the neuromuscular disease and the presence of anti-Toxoplasma gondii antibodies, while did not find association between such disease and the presence of anti-Neospora caninum antibodies.

Toxoplasma gondii es un patógeno intracelular obligatorio dentro del Phylum Apicomplexa, con un amplio rango de hospederos incluyendo muchas aves y mamíferos en el mundo. El objetivo del estudio fue determinar la seroprevalencia de anticuerpos anti-Toxoplasma gondii en alpacas de la Unidad de Producción de Cuyo de la SAIS Pachacútec, ubicada en el distrito de Marcapomacocha, Provincia de Yauli, Departamento de Junín. Para tal fin se recolectaron en el mes de enero del 2003, sueros sanguíneos de 258 alpacas hembras para la detección de anticuerpos de Toxoplasma gondii; mediante la técnica de Inmunofluorescencia Indirecta (IFI), encontrándose que el 8.53±3.41% (22/258) de las muestras, presentaban anticuerpos contra el parásito; siendo estos menores en proporción con los hallados en alpacas de otras zonas ganaderas del país, por lo tanto se hace necesario la continuar con estudios similares a fin de determinar el verdadero rol de este parásito con los problemas reproductivos en alpacas. / --- Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa, with a broad host range including many birds and mammals in the world. The objective of this study was determine the seroprevalence of antibodies against Toxoplasma gondii in alpacas in the United of Production of Cuyo at the SAIS Pachacutec, located in the district of Marcapomacocha, Province of Yauli, Departament of Junin. In order to do this, 258 sera from female alpacas were collected in the month of January of 2003. Sera was tested against Toxoplasma gondii antibodies using Indirect Immunoflorescence (IFI), being that 8.53±3.41% (22/258) of the samples showed antibodies against the parasite. These results show a lower proportion compared to those findings in alpacas from other zones of the country; therefore, it is necessary to continue with similar studies, which will determine the real pathological role of this parasite in alpacas.

El Toxoplasma gondii, agente causal de la toxoplasmosis, es un parásito importante en la salud animal, por ser una de las principales infecciones parasitarias que ocasiona problemas reproductivos, y también constituye una zoonosis muy importante alrededor del mundo. El objetivo del presente estudio fue determinar la seroprevalencia de Toxoplasma gondii en alpacas de la Unidad de Producción de Cochas de la SAIS Túpac Amaru, ubicada en el distrito de Canchayllo, Provincia de Jauja, Departamento de Junín. Para tal fin, se recolectaron sueros sanguíneos procedentes de 200 alpacas entre hembras y machos, en el mes de diciembre de año 2000, para la detección de anticuerpos mediante la técnica de Hemaglutinación Indirecta (HAI). Los resultados generales indicaron que el 21 ± 5.64 % (42/200) de las muestras de alpacas presentaron anticuerpos con títulos que variaron desde 1/16 hasta 1/1024. La seroprevalencia hallada en machos 26.47 10.49 % (18/68) fue mayor que en hembras 18.18 6.57 % (24/132), y por rangos de edad desde los 8 a 12 meses, de 12 hasta 36 meses y más de 36 meses las seroprevalencias fueron 33.33 16.08 %, 15.38 9.81, 20 7.31 respectivamente. Al realizar el análisis estadístico respectivo mediante la Prueba de regresión Logística no se encontró relación estadística significativa entre la presencia de anticuerpos contra T. gondii y el sexo de las alpacas, pero si entre la edad del animal y la infección por T. gondii, siendo las alpacas más jóvenes (8 a 12 meses) aproximadamente 2,9 veces más susceptibles que los otros dos grupos etéreos, este resultado podría deberse a la presencia de anticuerpos maternales, manejo de los animales, factores ambientales y frecuencia del hospedero definitivo. La prevalencia encontrada en el presente estudio fue moderada, por lo tanto, se hace necesario la realización de más estudios a fin de determinar el verdadero rol de este parásito en los problemas reproductivos de las alpacas. / --- Toxoplasma gondii, causative agent of toxoplasmosis, is an important parasite in animal health because it is one of the major parasitic infections that produce reproductive problems, and also it is an important zoonosis worldwide. The objective of this study was to determine the seroprevalence of antibodies against Toxoplasma gondii in alpacas from the Unit of Production of Cochas of the SAIS Tupac Amaru, located in the district of Canchayllo, Province of Jauja, Departament of Junin, Perú. To this end, sera from 200 alpacas among females and male were gathered in the month of December of 2000, for the detection of antibodies by using the method of Indirect Hemaglutination (IHA). 21 5.64 % (42/200) of the samples showed antibodies, with titles ranging from 1/16 up to 1/1024. The seroprevalence found in males was 26.47 (10.47% (18/64), and it was greater than in females, 18.18 6.57 % (24/132), and for age groups, from the birth until 1 year old, more than 1 up to 2 years old and more than 3 years old; the seroprevalence found was 33.33 16.08%, 15.38 9.8, 20 7.31 respectively. The analysis by using The Logistic regression found that the genus did not represent a risk factor. The age did represent a risk factor, being younger alpacas (8 to 12 months) more susceptible than other age groups. These results are coherent with other studies obtained in different parts of world, however, it is observed that there are differences with recent studies obtained in Peru; this, this could be due to environmental factors, frequency of the definitive host. Prevalence found in the present study was moderate, should be done further studies to determine the real importance of T. gondii in the occurrence of abortion in alpacas.

Thesis advisor: Marc-Jan Gubbels / The apicomplexan parasite <italic>Toxoplasma gondii</italic> divides rapidly and asexually through a unique process of internal daughter budding. The physical infrastructure for this process is the cytoskeleton, which is composed of subpellicular microtubules, flattened vesicles (alveoli), and a meshwork of intermediate filament-like proteins. This meshwork is composed of a family of 14 inner membrane complex (IMC) proteins that were identified based on the presence of a repeat sequence shared across the Alveolata, the alveolin-repeat. All 14 proteins were cloned as YFP fusions to study their subcellular localization and antibodies were generated against several representative IMC proteins. Each IMC displays unique spatio-temporal dynamics throughout development, but four physically distinct localizations were identified: eight IMCs localize to the alveoli, four IMCs localize to a structure known as the basal complex, IMC11 localizes to the apical cap in mature parasites, and IMC15 localizes primarily to the centrosomes and early buds. IMC15 is of particular interest because its appearance before membrane occupation and recognition nexus 1 (MORN1) in the early bud suggests that it is the first cytoskeletal component to associate with the buds. A conditional knockdown of this protein using the destabilization domain (DD) reveals IMC15 has a strong affinity for the centrosomes that overcomes targeting of the DD fusion protein to the proteasome and the presence of IMC15 in the early bud may not be necessary for the division process. Conditional knockdowns using a tetracycline repressible promoter reveal that a minimal amount of IMC15 is sufficient for parasite survival. In order to further characterize IMC15, dominant negative constructs based on mutating putative palmitoylation sites or overexpression of deletion constructs are being pursued. Collectively, the IMC family is being incorporated into the temporal and spatial dynamics of cytoskeletal development through the creation of a comprehensive timeline of daughter bud assembly. These findings are contributing unprecedented detail to the cell division process. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Marc-Jan Gubbels / Toxoplasma gondii – an obligate intracellular parasite – has a complex multistep mechanism for host cell invasion with a pivotal role played by calcium signaling. However, the coordination amongst the players in all the key steps of this signaling pathway, essential for parasitic life cycle of Toxoplasma, is still not entirely known. Given the evolutionary relationship between ciliates and apicomplexans, this work evaluates the functions of three orthologous proteins in cell signaling of alveolates leading to calcium-dependent exocytosis of vesicles such as trichocysts and micronemes. The proteins investigated are calmodulin (CaM) which is a calcium sensor, calcineurin (CN), a protein phosphatase, and parafusin-related protein 1 (PRP1), the Toxoplasma ortholog of ciliate protein parafusin. In the ciliate, CaM activates CN upon rise in intracellular calcium. Activated CN then leads to the dephosphorylation of the secretory vesicle scaffolding protein called parafusin. Parafusin dephosphorylation dissociates it from the calcium-dependent secretory vesicles, which then discharge their contents. As expected upon conditional depletion, we found CaM to be essential for Toxoplasma tachyzoites and CN to be significant for the lytic cycle. Surprisingly, the microneme secretion remained normal in the CN depleted parasites, although there was significant reduction in the attachment of the parasite to the host cell. We also found that PRP1 is dispensable for Toxoplasma lytic cycle, despite its absence affecting the microneme secretion induced by the calcium ionophore, A23187. However the secretion defect is not uniform and remains comparable to the wild type when it is induced using other pharmacological agents like ethanol, zaprinast and propranolol. Therefore despite phylogenetic conservation of the three proteins in alveolates, this work demonstrates their involvement in distinct functional aspects in Toxoplasma compared to other ciliates. Alongside the limited understanding of the molecular mechanism, our knowledge about the cellular sensors in Toxoplasma is also scarce. Our laboratory has previously identified a double C2 (DOC2) domain containing protein called TgDOC2 and demonstrated its role in overall microneme secretion. However, TgDOC2 does not regulate the dosed microneme release associated with varied steps of the egress-invasion trajectory. In general, DOC2 domain containing proteins work in coordination with each other to execute their cellular function, mostly in calcium-dependent manner. We therefore, wanted to expand our knowledge of this domain containing proteins in the parasite. In this work, we investigated a conserved apicomplexan protein called FER2 containing multiple DOC2 domains. Conditional depletion of FER2 appeared detrimental due to significant loss of invasion and attachment of the parasites. FER2 depleted parasites have normal microneme secretion, which is currently the only known calcium-dependent secretory organelle in Toxoplasma, but abrogated the release of rhoptries, the second secretory organelle to be released during invasion. Altogether this work extends the importance of calcium signaling in Toxoplasma gondii and brings into light the novel aspects of some parasitic proteins which are significant in reconstructing our understanding of the signaling pathway in this parasite. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Marc Jan Gubbels / Thesis advisor: Thomas Chiles / The length of the tachyzoite cell cycle, in particular G1, is an important virulence factor in Toxoplasma gondii. Cdk and Cyclin activities ultimately control the cell cycle; however, the checkpoint control mechanisms diverge from higher eukaryotes and are poorly understood. In order to elucidate these mechanisms, temperature sensitive (ts) mutants were generated by chemical mutagenesis. One of these mutants, called FV-P6, dies within one cell cycle in the G1 phase upon transfer from the permissive (35°C) to the restrictive temperature (40°C). Cosmid complementation identified the gene responsible for this G1 arrest as a `Cactin' ortholog. A single point mutation in this gene that resulted in an amino acid substitution from Tyrosine to Histidine at position 661 in the highly conserved C-terminus was shown to underlay the temperature sensitive effect. Cactin is highly conserved across eukaryotes and plays a role in embryonic development of metazoa although its mechanism of action is poorly understood. In agreement with the predicted nuclear localization signal in the N-terminus, expression of a fluorescent reporter gene fusion resulted in nuclear localization. Genome-wide expression profiling analysis of mutant and wild type at the permissive and restrictive temperatures confirmed the G1 arrest and furthermore demonstrated up-regulation of bradyzoite and Toxoplasma cat life cycle stage genes, hinting at TgCactin's role as a repressor. Since DNA binding domains or enzymatic domains are absent in TgCactin, TgCactin must act in a complex. Native blue gel electrophoresis demonstrated that TgCactin is present in large complexes of 720 and 800 kDa. A yeast two-hybrid screen (YTH) identified 40 potential TgCactin-interacting proteins of which 10 were selected for further validation. Eight out of these ten candidates are involved in DNA/RNA processes pertaining to transcription and translation, respectively. One-on-one YTH interactions between mutated and N-terminal deletion mutants of TgCactin and the above 10 interactors were abolished except for a single RNA helicase. Studies in Toxoplasma of four of these interactors demonstrated that only the RNA helicase localized to the nucleus; however, co-immunoprecipitation experiments to demonstrate that this protein is present in a complex with TgCactin were inconclusive. Furthermore, TgCactin self interactions identified domains necessary for TgCactin-TgCactin binding. Taken together, these findings indicate that TgCactin likely functions as a repressor of gene expression, possibly through an epigenetic mechanism reminiscent of an RNA/DNA helicase- based system in plants. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Orientador: Paulo Francisco Domingues / Banca: Hélio Langoni / Banca: Alexander Welker Biondo / Resumo: A toxoplasmose é uma das mais freqüentes zoonoses parasitárias, de distribuição mundial, cujo agente etiológico é o Toxoplasma gondii. A infecção em suínos tem sido descrita em diversos países, demonstrando seu caráter cosmopolita e a susceptibilidade desta espécie. O T.gondii pode ser transmitido sob várias formas, e com relação à espécie suína, a ingestão de carne crua ou mal cozida e seus derivados são importantes vias de transmissão para o homem. Estudou-se a prevalência de anticorpos anti-Toxoplasma gondii em 550 amostras de soros de suínos de criações de "fundo de quintal" na microrregião de Registro - SP, utilizando como prova sorológica o método de aglutinação direta (MAD). Este mostrou-se uma ferramenta diagnóstica de fácil realização, baixo custo, não necessitando de equipamentos especiais para a leitura e interpretação dos resultados. Das 550 amostras de soro, 111(20,18%) reagiram positivamente com os títulos 64 (15,32%), 256 (27,93%), 1024 (39,64%), 4096 (10,81%), 16384 (4,50%) e 65536 (1,80%). Concluí-se que os resultados obtidos indicam alta prevalência desta enfermidade nas condições e região de origem dos animais. À medida que foram obtidos títulos altos de anticorpos contra o agente, pode-se concluir também que há risco potencial de transmissão da toxoplasmose para o homem, sugerindo que muitos desses animais poderiam apresentar bradizoítos em sua musculatura / Abstract: Toxoplasmosis is one of the most frequent parasitic zoonosis with worldwide distribution whose etiological agent is Toxoplasma gondii. The infection in swine has been described in many countries, showing the cosmopolitan character and susceptibility of this specie. T.gondii can be transmitted by many ways. The ingestion of swine raw or badly cocked meat and derivatives are important pathways of transmission to man. The anti-T.gondii antibodies prevalence was studied in 550 serum samples of swine under rustic condition (animals with no breed, popular named as "backyard breeding") in Registro-SP, using the modified agglutination test (MAT). Such method is verified as an easy-appliance tool, with low costs and non necessity of special equipment in order to achieve interpretation of results. In 550 samples of serum, 111 (20.18%) were positive with titles of 64 (15.32%), 256 (27.93%), 1024 (39.64:%), 4096 (10.81%), 16384 (4.50%), 65536 (1.80%). These results achieved indicate a high prevalence of the disease on the region studied and under the specific conditions described. The high rate obtained as result also leads to the conclusion that there is potential risk of toxoplasmosis transmission to man due to the possibility of the animals to have bradyzoits in their musculature / Mestre

El objetivo del presente trabajo fue determinar la frecuencia de anticuerpos contra Toxoplasma gondii en gatos de Lima Metropolitana. Además, estimar el grado de concordancia entre las técnicas de diagnóstico de inmunofluorescencia indirecta y hemaglutinación indirecta. Se analizaron las muestras de sueros de 178 gatos, obtenidas de diferentes clínicas veterinarias de Lima Metropolitana. Los resultados mostraron una frecuencia de reactores a Toxoplasma gondii de 17.9% con un intervalo de confianza de 95% entre 12.0 y 23.5% para la técnica IFI y 11.2% con un intervalo de confianza de 95% entre 6.6 y 15.8% para la técnica HAI. La evaluación de reactores según edad y sexo, no mostraron diferencia estadística significativa (p>0,05). Por otro lado, al evaluar el grado de concordancia entre ambas pruebas se halló un valor de Kappa (K) igual a 0.73 indicando que el grado de concordancia entre ambas pruebas fue del tipo sustancial; mientras que con la prueba de Mc Nemar se encontró significancia estadística (p menor 0.05), entendiendo que las pruebas no pueden ser reemplazadas mutuamente. / --- The aim of the present work was to estimate the frequency of Toxoplasmosis in cats in the city of Metropolitan Lima. Aside from determining the rate of concordance between the Indirect immunofluorescence (IFI) and Indirect haemaglutination (HAI) Assays, two of the very common diagnostic tests for Toxoplasmosis in our area, a number of 178 cat serum samples, collected in veterinary clinics of this city, were analyzed. The results determined a frequency of reactors to Toxoplasma gondii in 17.90% with a 95% confidence interval (CI) between 12.00% and 23.50% for IFI test and in 11.20% with a 95% confidence interval (CI) between 6.60% and 15.80% for HAI test. The evaluation of reactors, according to age and sex, did not provide statistically significant difference (p> 0, 05). On the other hand, when the rate of concordance among both assays was tested, there was a value of Kappa (K) similar to 0.73 indicating that the concordance is substantial between the two test; while in Mc Nemar’s test, there was statistically significant difference (p less 0.05), indicating that both tests cannot be replaced reciprocally. / Tesis

El objetivo del presente estudio fue determinar la seroprevalencia de T. gondii en mamíferos del Orden Carnivora y Primates mantenidos en cautiverio e identificar las variables epidemiológicas que intervienen en su presentación. El trabajo se desarrolló en el zoológico del Patronato del Parque de las Leyendas, distrito de San Miguel. Se colectaron muestras de sangre a 101 animales silvestres pertenecientes a las órdenes Carnivora (49) y Primates (52); así como de 87 roedores y 18 felinos domésticos obtenidos mediante el uso de trampas de captura viva. Las muestras de suero fueron conservadas en refrigeración y procesadas en el Laboratorio de Parasitología de la FMV-UNMSM. Se realizaron encuestas para identificar los potenciales factores de riesgo de cada Orden muestreada. Asimismo se obtuvo información concerniente de cada roedor (parámetros morfométricos, edad estimada, sexo y peso) y felino doméstico (edad, sexo). Para el diagnóstico de T. gondii se utilizó la técnica de Hemaglutinación Indirecta, preparándose diluciones de 1:16 a 1:2048, considerándose como positivos títulos mayores a 1/16. Se determinó anticuerpos IgM mediante el uso del 2-Mercaptoetanol para determinar una infección aguda. La frecuencia de anticuerpos anti- Toxoplasma gondii en roedores (Rattus sp.) y gatos procedentes de un área de tenencia de animales silvestres fue de 25,3 y 77,8 % respectivamente. La asociación entre la seroprevalencia a T. gondii y cada una de las variables evaluadas (sexo, origen, tiempo en la institución y tipo de alimentación) fue analizada mediante la razón de posibilidades (odds ratio–OR) con su respectivo IC 95%. La seroprevalencia de T. gondii en mamíferos del Orden Carnivora y Primates criados en cautiverio fue de 87,8 y 80,8% respectivamente. Se encontró asociación significativa (p<0.05) entre la seroprevalencia de T. gondii en Primates y el tipo de alimentación, donde el ser omnívoro constituyó un factor de riesgo (OR: 40,9), para la presentación de la infección. No fue encontrada asociación significativa (p>0.05) con el sexo, origen y tiempo en la institución. PALABRAS CLAVES: factor de riesgo, Toxoplasma gondi, seroprevalencia, Carnivora, Primates. / Tesis