Thesis advisor: Marc-Jan Gubbels / The Apicomplexa comprise a group of obligate intracellular parasites some of which cause severe diseases in humans with malaria the most notorious representative. Toxoplasma gondii infection is the most widespread apicomplexan infection, which is mostly symptomless in healthy people but is associated with a variety of birth defects upon congenital infection and can become life threatening in immunocompromised patients. In addition, T. gondii has been established as a model for the study of intracellular parasitism by Apicomplexa. The lytic destruction of host cells underlies the pathogenesis of all apicomplexan diseases. The T. gondii lytic cycle involves host cell invasion, several rounds of intracellular replication, and is followed by egress of motile parasites in order to infect neighboring host cells. Egress is an increasingly more appreciated aspect of the lytic cycle for which three physiological triggers have been identified. All three triggers converge on the release of Ca2+ stores within the parasite. Large sections of the signaling pathways and molecular players associated with egress and intracellular calcium release remain unknown. The objective of this thesis was to develop and employ a novel enrichment screening procedure that would efficiently isolate egress mutants in response to pharmaceutically induced egress. The biggest caveat to such a screen is the ability to separate intracellular from extracellular parasites, which is hampered by the stickiness of parasites to host cells as well as their fast reinvasion capacity. This hurdle was overcome by saturating the parasite's surface receptors with the glycan heparin to prevent attachment to the host cell. Simultaneously, the oxidizing agent pyrrolidine dithiocarbamate (PDTC) was applied to specifically kill extracellular parasites. The enrichment power of the screen was assessed by diluting a previously identified temperature-sensitive egress mutant called F-P2 in wild type parasites. The screen's enrichment power was assessed by flow cytometry and a 1000-fold enrichment capacity to a 100% F-P2 population could routinely be achieved. Subsequently the screen was applied to generate mutants with defects in the poorly understood NTPase mediated egress-trigger pathway. Chemical mutagenesis as well as insertional mutagenesis was applied and dithiotreitol (DTT) that artificially creates the reducing environment triggering egress was used to screen mutants. Three chemically induced constitutive egress mutants and one insertional mutant were isolated. As expected, all mutants displayed resistance to DTT induced egress. In addition, cross resistance to two other egress inducers upstream of Ca2+ release was observed, however all mutants egressed upon calcium ionophore treatment. Taken together, the developed enrichment procedure will enable the isolation of constitutive as well as conditional egress mutants. Future cosmid complementation will help to fill in important blanks in the egress mechanisms and will ultimately lead to a better understanding of intracellular parasitism. This gained understanding will potentially lead to therapies to combat the destructive effects of apicomplexan parasites. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Düsseldorf, Universiẗat, Diss., 2003.

Düsseldorf, Universiẗat, Diss., 2004.

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.

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.

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

Ng Wai-yan. / Thesis submitted in: September 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 200-225). / Abstracts in English and Chinese. / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.v / Abstract --- p.vii / Abstract (Chinese version) --- p.ix / Table of contents --- p.xi / List of Figure --- p.xx / List of Table --- p.xxiii / Chapter Chapter 1 --- : General Introduction --- p.1 / Chapter 1.1 --- Toxoplasma gondii and Toxoplasmosis --- p.1 / Chapter 1.1.1 --- Biology and life cycle of Toxoplasma gondii --- p.2 / Chapter 1.2 --- Treatment of Toxoplasmosis --- p.4 / Chapter 1.2.1 --- Chemotherapy --- p.4 / Chapter (a) --- Pyrimethamine and sulfadiazine --- p.4 / Chapter (b) --- Clindamycin and Spiramycin --- p.4 / Chapter (c) --- Hydroxynaphthoquinones and new Macrolides --- p.5 / Chapter 1.2.2 --- Toxoplamsa Vaccine --- p.5 / Chapter (a) --- Vaccine using mutant strain of T. gondii --- p.6 / Chapter (b) --- Subunit vaccine --- p.6 / Chapter 1.3 --- The Immune Responses --- p.8 / Chapter 1.3.1 --- Protective immune responses --- p.8 / Chapter (a) --- Cytokines involved in the immunity against T. gondii --- p.10 / IFN-γ: Role in resistance to infection --- p.10 / TNF-α: Synergetic Role with IFN-γ --- p.10 / IL-12: Role in T cell differentiation --- p.11 / IL-10: Role in immuno-regulation --- p.12 / Other cytokines --- p.12 / Chapter (b) --- Immunomodulatory role of Nitric oxide (NO) --- p.13 / Chapter (c) --- Humoral immunity against T. gondii --- p.13 / Chapter 1.3.2 --- Toxoplasma proteins known to elicit T cell-dependent immunity --- p.14 / Chapter (a) --- Surface antigen 1 (SAG1/ P30) --- p.16 / Chapter (b) --- Dense granules protein 2 (GRA2/ P28) --- p.17 / Chapter (c) --- Rhoptry protein 2 (ROP2/ P54) --- p.18 / Chapter 1.3.3 --- Appropriate vaccine design --- p.18 / Chapter 1.4 --- Aim of the study --- p.23 / Chapter Chapter 2 ： --- Expression of the Synthetic Gene Encoding the Multi-epitopic Antigen (MEA) for Toxoplasma gondii in Escherichia coli --- p.25 / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.1.1 --- Multi-epitopic antigen for T. gondii as vaccine --- p.26 / Chapter a) --- "Epitopes from Toxoplasma antigens, P30, GRA2 and ROP2" --- p.26 / "T-cell epitopes from P30 (aa 138-154, aa 191-209)" --- p.26 / T-cell epitope from ROP2 (aa 197-216) --- p.27 / T-and B-cell epitope from GRA2 (aa 171-185) --- p.27 / Chapter b) --- Helper T-cell epitope from tetanus toxin --- p.27 / Chapter c) --- Linker --- p.28 / Chapter 2.1.2 --- Escherichia coli Expression System --- p.31 / Chapter (a) --- The T7 RNA polymerase and T7 lac Promoter --- p.31 / Chapter (b) --- Other translational elements --- p.32 / Chapter (c) --- Fusion partner --- p.33 / Chapter (d) --- Choice of expression host strain --- p.33 / Chapter 2.2 --- Materials --- p.35 / Chapter 2.2.1 --- Bacterial strains --- p.35 / Chapter 2.2.2 --- Mouse strains --- p.35 / Chapter 2.2.3 --- Chemicals --- p.35 / Chapter 2.2.4 --- Nucleic acids --- p.36 / Chapter 2.2.5 --- Kits and reagents --- p.37 / Chapter 2.2.6 --- Antibodies --- p.37 / Chapter 2.2.7 --- Solutions --- p.38 / Chapter 2.2.8 --- Enzymes --- p.40 / Chapter 2.2.9 --- Primers --- p.40 / Chapter 2.3 --- Methods --- p.41 / Chapter 2.3.1 --- Design and synthesis of the synthetic gene encoding the multi-epitopic antigen for Toxoplasma gondii --- p.41 / Chapter 2.3.2 --- Cloning of the MEA into the E. coli expression vector --- p.41 / Chapter (a) --- Preparation of plasmid : pCRII-TOPO-MEA and pET30a+ --- p.43 / Chapter (b) --- PCR amplification of MEA from pCRII-TOPO-MEA --- p.44 / Chapter (c) --- Digestion and purification of the E. coli expression vector pET30a+ with Ncol --- p.44 / Chapter (d) --- Fill-in Ncol cut pET30a+ --- p.44 / Chapter (e) --- Purification of DNA fragment from agarose gel --- p.45 / Chapter (f) --- "Ligation of MEA fragment and Ncol cut, fill-in pET30a+" --- p.45 / Chapter (g) --- Preparation of DH5a competent cells --- p.46 / Chapter (h) --- Transformation of recombinant pET30a+-MEA --- p.46 / Chapter (i) --- PCR screening and plasmid preparation for the putative pET30a+- HisMEA --- p.46 / Chapter (j) --- Cycle sequencing reaction on putative plasmid pET30a+-MEA --- p.47 / Chapter 2.3.3 --- Expression and purification of his-tag MEA --- p.48 / Chapter (a) --- Expression profile of His-tag MEA production by IPTG induction --- p.48 / Chapter (b) --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.49 / Chapter (c) --- Estimation of His-tag MEA production in induced bacterial lysate --- p.50 / Chapter (d) --- Purification of His-tag MEA --- p.50 / Chapter (e) --- Braford Protein Microassay (Bio-rad) --- p.50 / Chapter 2.3.4 --- Characterization of his-tag MEA --- p.51 / Chapter (a) --- Western blot of induced bacteriallysate by monoclonal anti-his-tag antibody --- p.52 / Chapter (b) --- N'-terminal amino acid sequencing of His-tag MEA --- p.52 / Chapter (c) --- Western blot of His-tag MEA with seropositive serum of mice and human --- p.53 / Chapter (d) --- Preparation of Toxoplasma gondii lysate --- p.54 / Chapter (e) --- Western blot of purified his-tag MEA and T. gondii Lysate by anti- serum of recombinant his-tag MEA --- p.54 / Chapter 2.4 --- Results --- p.56 / Chapter 2.4.1 --- Design and synthesis of the synthetic gene encoding the multi- epitopic antigen for Toxoplasma gondii --- p.56 / Chapter 2.4.2 --- Cloning of MEA into E. coli expression vector --- p.59 / Chapter 2.4.3 --- Expression and purification of His-tag MEA --- p.61 / Chapter 2.4.4 --- Charterization of His-tag MEA --- p.67 / Chapter 2.5 --- Discussion --- p.73 / Chapter 2.5.1 --- Design and synthesis of the synthetic gene encoding the multi-epitopic antigen for Toxoplasma gondii --- p.73 / Chapter 2.5.2 --- Cloning of MEA into E. coli expression vector --- p.77 / Chapter 2.5.3 --- Expression and purification of His-tag MEA --- p.77 / Chapter 2.5.3 --- Characterization of His-tag MEA --- p.78 / Chapter Chapter 3: --- Immunological Studies of the Multi-epitopic Antigen (MEA) for Toxoplasma gondii in Mouse Models --- p.82 / Chapter 3.1 --- Introduction --- p.82 / Chapter 3.1.1 --- "Expression of P30, GRA2 and ROP2 in E. coli expression system" --- p.82 / Chapter (a) --- Expression of P30 --- p.82 / Chapter (b) --- Expression of ROP2 --- p.83 / Chapter (c) --- Expression of GRA2 --- p.84 / Chapter 3.1.2 --- Immunizations --- p.84 / Chapter (a) --- Choices of animals --- p.84 / Chapter (b) --- Adjuvants --- p.85 / Chapter 3.1.3 --- Measurements of cellular immune responses in vaccinated mice --- p.86 / Chapter 3.2 --- Materials --- p.87 / Chapter 3.2.1 --- Mouse strains --- p.87 / Chapter 3.2.2 --- Chemicals --- p.87 / Chapter 3.2.3 --- "Culture Medium, buffer and other solutions" --- p.87 / Chapter 3.2.4 --- Nucleic acids --- p.87 / Chapter 3.2.5 --- Kits and reagents --- p.88 / Chapter 3.2.6 --- Solutions --- p.88 / Chapter 3.2.7 --- Enzymes --- p.88 / Chapter 3.2.8 --- Primers --- p.89 / Chapter 3.3 --- Methods --- p.90 / Chapter 3.3.1 --- "Construction of pET-P30, pET-GRA2 and pET-ROP2" --- p.90 / Chapter (a) --- Construction of pET-GRA2 --- p.93 / Chapter (i) --- Preparation of total RNA from tachyzoite of T. gondii (RH strain) --- p.93 / Chapter (ii) --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.93 / Chapter (iii) --- Cloning of pET-GRA2 --- p.94 / Chapter (b) --- Construction of pET-ROP2 --- p.95 / Chapter 3.3.2 --- "Expression and characterization of his-tag P30, his-tag GRA2 and his-tag ROP2" --- p.95 / Chapter (a) --- "Expression profile of his-tag P30, his-tag ROP2 and his-tag GRA2" --- p.96 / Chapter (b) --- Western blot of induced bacterial lysate with mono-clonal anti-his-tag antibody --- p.96 / Chapter (c) --- Western blot of induced bacterial lysate with anti-serum against his-tag MEA --- p.97 / Chapter (d) --- "Purification of his-tag P30, his-tag GRA2 and his-tag ROP2 from induced bacterial lysate" --- p.97 / Chapter (e) --- "Western blot of his-tag P30, his-tag GRA2 and his-tag ROP2 with sero- positive serum of mice and human" --- p.98 / Chapter (f) --- "Western blot of T. gondii lysate by anti-serum against his-tag GRA2, his-tag P30 and his-tag ROP2" --- p.99 / Chapter 3.3.3 --- Measurement of cellular immune responses in his-tag MEA vaccinated mice --- p.99 / Chapter (a) --- Isolation of spleenocytes from vaccinated mice --- p.99 / Chapter (b) --- T-cell proliferation assay --- p.100 / Chapter (c) --- Determination of Thl/ Th2 cytokines expression profile by RT-PCR --- p.100 / Chapter 3.3.4 --- Protection efficacy of different vaccine constructs against lethal challenge --- p.102 / Chapter 3.4 --- Results --- p.102 / Chapter 3.4.1 --- Construction of pET-GRA2 and pET-ROP2 --- p.102 / Chapter 3.4.2 --- "Expression and characterization of his-tag P30, his-tag GRA2 and his-tag ROP2" --- p.106 / Chapter 3.4.3 --- Measurement of cellular immune responses in his-tag MEA vaccinated mice --- p.116 / Chapter 3.4.4 --- Protection efficacy of different vaccine constructs against lethal challenge --- p.119 / Chapter 3.5 --- Discussion --- p.121 / Chapter 3.5.1 --- Construction of pET-GRA2 and pET-ROP2 --- p.121 / Chapter 3.5.2 --- "Expression and characterization of his-tag P30, his-tag GRA2 and his-tag ROP2" --- p.121 / Chapter 3.5.3 --- Measurement of cellular immune responses in his-tag MEA vaccinated mice --- p.124 / Chapter 3.5.4 --- Protection efficacy of different vaccine constructs against lethal challenge --- p.126 / Chapter Chapter 4 --- : Expression of the multi-epitopic antigen in Arabidopsis thaliana --- p.129 / Chapter 4.1 --- INTRODUCTION --- p.129 / Chapter 4.1.1 --- Transgenic plants as vaccine production systems --- p.129 / Chapter (a) --- Advantages of using plants as bioreactor --- p.130 / Chapter (b) --- Transgenic plants for vaccine production --- p.131 / Chapter (c) --- Limitations --- p.135 / Chapter (i) --- Low yields --- p.135 / Chapter (ii) --- Plant-specific glycans --- p.135 / Chapter 4.1.2 --- Model plant - Arabidopsis thaliana --- p.136 / Chapter 4.1.3 --- Strategies for expressing the transgene (MEA) in plant --- p.137 / Chapter (a) --- Seed-specific phaseolin promoter --- p.138 / Chapter (b) --- Lysine-rich protein (LRP) as fusion partner --- p.138 / Chapter (c) --- Fusion protein with membrane anchor for targeting to specific vacuolar compartments --- p.139 / Chapter 4.2 --- MATERIALS --- p.141 / Chapter 4.2.1 --- Bacterial strains --- p.141 / Chapter 4.2.2 --- Arabidopsis strains --- p.141 / Chapter 4.2.3 --- Chemicals --- p.141 / Chapter 4.2.4 --- Antibiotics --- p.141 / Chapter 4.2.5 --- Nucleic acids --- p.141 / Chapter 4.2.6 --- Solutions --- p.142 / Chapter 4.2.7 --- Enzymes and buffers --- p.142 / Chapter 4.2.8 --- Primers --- p.143 / Chapter 4.3 --- METHODS --- p.144 / Chapter 4.3.1 --- Construction of plant expression vectors --- p.144 / Chapter 4.3.1.1 --- Strategy 1: Lysine-rich protein (LRP) fusion --- p.144 / Chapter a) --- Construction of pBI121/ Phaseolin Promoter/ LRP1/ MEA/ his-tag/ phaseolin terminator --- p.149 / Chapter b) --- Construction of pBI121/ Phaseolin Promoter/ LRPIIA/ MEA/ phaseolin terminator --- p.150 / Chapter 4.3.1.2 --- Strategy 2: Fusion proteins with membrane anchor for vesicle targeting to specific vesicle compartments --- p.151 / Chapter ai) --- Construction of pBI121/Phaseolin Promoter/ SP/ MEA/ 491/NOS terminator --- p.156 / Chapter aii) --- Construction of pBI121/Phaseolin Promoter/ SP/ MEA(J)/ 526/ NOS terminator --- p.157 / Chapter 4.3.1.3 --- Construction of transgenic control pBI121/phaseolin promoter/ MEA/ phaseolin terminator --- p.157 / Chapter 4.3.2 --- Agrobacterium-mediated transformation of Arabidopsis thaliana by vaccum infiltration --- p.160 / Chapter (a) --- Preparation of competent cell for Agrobacterium GV3101 --- p.160 / Chapter (b) --- Transformation of electro-competent agrobacterium with plant expression vector by electroporation --- p.160 / Chapter (c) --- PCR screening for agrobacterium carrying the desirable plant expression vector --- p.161 / Chapter (d) --- Vacuum infiltration --- p.161 / Chapter 4.3.3 --- Screening of homozygous transgenic plants with single insertion of transgene --- p.162 / Chapter 4.3.4 --- Detection of MEA or MEA fusion protein in transgenic plants --- p.163 / Chapter (a) --- Extraction of seed protein (Soluble and membrane fractions) --- p.163 / Chapter (b) --- Western blot of seed protein by anti-serum against HisMEA --- p.163 / Chapter 4.4 --- RESULTS --- p.164 / Chapter 4.4.1 --- Construction of plant expression vectors and transgenic A. thaliana --- p.164 / Chapter (a) --- Construction of pBI121/ phaseolin promoter/ LRPI or LRP II/ MEA/ Phaseolin terminator --- p.164 / Chapter (b) --- Construction of pBI121/ PP/ SP/ MEA(J)/ 491 or 526/ PT --- p.165 / Chapter (c) --- Construction of transgenic control pBI121/ PP/ MEA/ PT --- p.166 / Chapter (d) --- Agrobacterium-mediated transformation of A. thaliana --- p.167 / Chapter 4.4.2 --- Screening of homozygous transgenic plant with single insertion of transgene --- p.172 / Chapter 4.4.3 --- Molecular analysis of the MEA/ MEA fusion in transgenic plants --- p.181 / Chapter 4.5 --- DISCUSSION --- p.188 / Chapter 4.5.1 --- Construction of plant expression vector --- p.188 / Chapter 4.5.2 --- Screening of homozygous transgenic plant with single insertion of transgene --- p.189 / Chapter 4.5.3 --- Molecular analysis of transgenic MEA/ MEA fusion in transgenic plants --- p.190 / Chapter Chapter 5 --- : General Discussion --- p.193 / Chapter 5.1 --- Development of a vaccine for toxoplasmosis: current status --- p.193 / Chapter 5.1.1 --- Current status --- p.193 / Chapter (a) --- Vaccine for agricultural animals --- p.193 / Chapter (b) --- Other vaccine candidates and the protective response induced --- p.194 / Chapter 5.1.2 --- Appropriate vaccine design - the multi-epitopic antigen (MEA) --- p.194 / Chapter 5.2 --- "Expression, Characterization and Immunological studies of the MEA expressed in prokaryotic system" --- p.195 / Chapter 5.2.1 --- Expression and characterization of E. coli- expressed his-tag MEA --- p.195 / Chapter 5.2.2 --- Immunological studies on the E. coli- expressed his-tag MEA --- p.196 / Chapter 5.3 --- Expression of MEA in transgenic plants --- p.197 / Chapter 5.4 --- Preclinical Safety Assessment: Considerations in Vaccine Development --- p.198 / References --- p.200

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.