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

The Phylum Apicomplexa contains a number of devastating pathogens responsible for tremendous human suffering and mortality. Among these are Plasmodium, which is the causative agent of malaria, Cryptosporidium, which causes diarrheal illness in children and immuncompromised people, and Toxoplasma gondii, which causes congenital defects in the developing fetus and severe disease in immunocompromised people. T. gondii also serves as a model organism for other members of this phylum due to the relative ease of parasite culture and manipulation. Although effective treatments exist for some diseases caused by these apicomplexan parasites, drug resistance for others is widespread, perhaps most notably in Plasmodium species. Development of new therapeutic agents is needed to combat this resistance and alleviate disease burden. It is important that the drugs target parasitic cell components not conserved in humans in order to minimize side effects and drug toxicity. However, in order to target unique processes, a better understanding of apicomplexan biology must be gained. One approach toward understanding the unique biological processes of apicomplexan parasites is to study proteins conserved among the Phylum Apicomplexa, but not present in other organisms. One such protein, photosensitized INA-labeled protein 1 (TgPhIL1) was identified previously. The work presented in this dissertation describes targeted disruption of this gene in T. gondii, which results in parasites with an altered shape and a fitness defect in both tissue culture and a mouse model of infection. Another approach to understanding the unique processes of apicomplexan parasites is to perturb them using small molecules. By identifying the targets of the small molecules, a more detailed understanding of the process can be gained. To this end, a small molecule screen was performed in T. gondii in order to identify small molecules that inhibit the apicomplexan-specific and essential process of host-cell invasion. In addition to identifying 24 invasion inhibitors, 6 enhancers were also identified. One of these enhancers, compound 112762, was shown to enhance invasion of other apicomplexan parasites as well. Described herein are attempts to identify the target(s) of this compound. A derivative of this compound was linked to an affinity resin, and TgProfilin was identified as a putative target that may interact covalently with 112762. Additionally, affinity chromatography was used to demonstrate non-covalent binding of a T. gondii FK506-binding protein to 112762. Finally, based on a report in the literature of a compound nearly identical to 112762 that inhibits yeast and mammalian protein arginine methyltransferase 1 (PRMT1), it was hypothesized that 112762 might target TgPRMT1 in T. gondii. Supportive of this hypothesis, 112762 was shown to inhibit TgPRMT1 in vitro, to inhibit parasite protein methylation in vivo, and to bind the 112762 affinity resin. TgPRMT1 knockout parasites are being generated in order to determine whether they show resistance to compound 112762. As a result of this work, three potential targets of 112762 in T. gondii have been identified. This work opens the door for future studies aimed at understanding and controlling invasion by apicomplexan parasites and other processes specific to the Phylum Apicomplexa.

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.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Resumo: A toxoplasmose é similar em humanos e em ratos, sendo estes, o modelo experimental, mais utilizado para o estudo da doença. Há poucos relatos da transmissão desta enfermidade pelo leite materno, e desta forma o objetivo do presente estudo foi pesquisar a presença de Toxoplasma gondii no leite de ratas experimentalmente infectadas e a transmissão galactogênica. Utilizaram-se ratas Wistar (Rattus norvegicus), divididas em três grupos: G1, G2 e G3, contendo seis fêmeas cada um. Foram inoculadas via oral com 104 bradizoítos da cepa BTU4, genótipo I, isolada de cão com cinomose. As ratas do G1 e G2 foram infectadas 60 dias antes do acasalamento e as do G3 logo após o parto. Os animais do G2 foram submetidos à imunossupressão pós-parição. Para a detecção do parasito no leite utilizou-se a reação em cadeia pela polimerase (PCR) e para verificar a transmissão para os filhotes pesquisou-se nestes anticorpos anti-T. gondii pela imunofluorescência indireta (RIFI) e método de aglutinação direta (MAD), e a bioprova pela inoculação de camundongos com pool de tecidos (cerebral, pulmonar, hepático, cardíaco, muscular esquelético, língua e diafragma) de cada ninhada, bem como pela PCR nestes tecidos individualmente. A PCR foi positiva em três amostras de leite, duas provenientes da rata 1 do G1, e uma da rata 3 do G3. Os filhotes de todas as ratas do G1 soroconverteram, mas foram negativos na bioprova. Filhotes das ratas 1 e 3 do G3 soroconverteram, e a bioprova foi positiva. Amostras de fígado, musculatura esquelética e pulmão de filhotes do G1 foram positivas na PCR. Desta forma conclui-se que ocorreu a transmissão do T. gondii pelo leite, sugerindo-se novos estudos em lactentes, considerando-se a magnitude da doença em crianças e recém-nascidos. / Abstract: Toxoplasmosis is similar in humans and rats, and the latter constitute the most used experimental model to study this disease. Few reports on toxoplasmosis transmission through maternal milk are available in literature; thus, the aim of the present study was to investigate whether Toxoplasma gondii is present in and transmitted through the milk of experimentally infected rats. Wistar (Rattus norvegicus) female rats were divided into three groups: G1, G2 and G3, with six animals each. They were orally inoculated with 104 bradyzoites of BTU4 strain, genotype I, isolated from a dog with distemper. G1 and G2 rats were infected 60 days before mating and those of G3, soon after the parturition. G2 animals were subjected immunosuppression just after parturition. Polymerase chain reaction (PCR) was used to detect the parasite in the milk. To verify the parasite transmission to the offspring, these anti-T. gondii antibodies were investigated through indirect immunofluorescence assay (IFA) and direct agglutination test (DAT). Bioassay consisted of inoculating mice with a pool of tissues (brain, lungs, liver, heart, skeletal muscle, tongue and diaphragm) from each litter, as well as PCR in these tissues individually. PCR was positive in three milk samples, two from rat 1 (G1) and one from rat 3 (G3). The pups of all G1 rats seroconverted but were negative in the bioassay. The pups of rats 1 and 3 (G3) seroconverted and their bioassay was positive. Liver, skeletal muscle and lung samples were PCR-positive in G1 pups. Thus, we can conclude that T. gondii was transmitted through milk, suggesting the need of new studies in breast-feeding mothers as this disease is highly severe in children and newborns. / Orientador: Helio Langoni / Coorientador: Simone B. Lucheis / Banca: Italmar Teodorico Navarno / Banca: Katia Denise Saraiva Bresciani. / Mestre

Made available in DSpace on 2014-06-11T19:29:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-05-28Bitstream added on 2014-06-13T20:38:48Z : No. of bitstreams: 1 costa_vm_me_botfmvz.pdf: 404473 bytes, checksum: 0e9d9447d55f3ca2e11ba7a40a45a785 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A toxoplasmose é similar em humanos e em ratos, sendo estes, o modelo experimental, mais utilizado para o estudo da doença. Há poucos relatos da transmissão desta enfermidade pelo leite materno, e desta forma o objetivo do presente estudo foi pesquisar a presença de Toxoplasma gondii no leite de ratas experimentalmente infectadas e a transmissão galactogênica. Utilizaram-se ratas Wistar (Rattus norvegicus), divididas em três grupos: G1, G2 e G3, contendo seis fêmeas cada um. Foram inoculadas via oral com 104 bradizoítos da cepa BTU4, genótipo I, isolada de cão com cinomose. As ratas do G1 e G2 foram infectadas 60 dias antes do acasalamento e as do G3 logo após o parto. Os animais do G2 foram submetidos à imunossupressão pós-parição. Para a detecção do parasito no leite utilizou-se a reação em cadeia pela polimerase (PCR) e para verificar a transmissão para os filhotes pesquisou-se nestes anticorpos anti-T. gondii pela imunofluorescência indireta (RIFI) e método de aglutinação direta (MAD), e a bioprova pela inoculação de camundongos com pool de tecidos (cerebral, pulmonar, hepático, cardíaco, muscular esquelético, língua e diafragma) de cada ninhada, bem como pela PCR nestes tecidos individualmente. A PCR foi positiva em três amostras de leite, duas provenientes da rata 1 do G1, e uma da rata 3 do G3. Os filhotes de todas as ratas do G1 soroconverteram, mas foram negativos na bioprova. Filhotes das ratas 1 e 3 do G3 soroconverteram, e a bioprova foi positiva. Amostras de fígado, musculatura esquelética e pulmão de filhotes do G1 foram positivas na PCR. Desta forma conclui-se que ocorreu a transmissão do T. gondii pelo leite, sugerindo-se novos estudos em lactentes, considerando-se a magnitude da doença em crianças e recém-nascidos. / Toxoplasmosis is similar in humans and rats, and the latter constitute the most used experimental model to study this disease. Few reports on toxoplasmosis transmission through maternal milk are available in literature; thus, the aim of the present study was to investigate whether Toxoplasma gondii is present in and transmitted through the milk of experimentally infected rats. Wistar (Rattus norvegicus) female rats were divided into three groups: G1, G2 and G3, with six animals each. They were orally inoculated with 104 bradyzoites of BTU4 strain, genotype I, isolated from a dog with distemper. G1 and G2 rats were infected 60 days before mating and those of G3, soon after the parturition. G2 animals were subjected immunosuppression just after parturition. Polymerase chain reaction (PCR) was used to detect the parasite in the milk. To verify the parasite transmission to the offspring, these anti-T. gondii antibodies were investigated through indirect immunofluorescence assay (IFA) and direct agglutination test (DAT). Bioassay consisted of inoculating mice with a pool of tissues (brain, lungs, liver, heart, skeletal muscle, tongue and diaphragm) from each litter, as well as PCR in these tissues individually. PCR was positive in three milk samples, two from rat 1 (G1) and one from rat 3 (G3). The pups of all G1 rats seroconverted but were negative in the bioassay. The pups of rats 1 and 3 (G3) seroconverted and their bioassay was positive. Liver, skeletal muscle and lung samples were PCR-positive in G1 pups. Thus, we can conclude that T. gondii was transmitted through milk, suggesting the need of new studies in breast-feeding mothers as this disease is highly severe in children and newborns.

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.