Caractérisation moléculaire et fonctionnelle de la jonction mobile contrôlant l'invasion de la cellule hôte par Toxoplasma gondii. Les apicomplexes sont des parasites eucaryotes responsables d'infections humaines et animales, dont le paludisme et la toxoplasmose. La plupart sont des parasites intracellulaires obligatoires ; l'entrée dans la cellule hôte est donc un évènement crucial dans leur cycle de développement. Ce processus, conservé au sein du phylum, implique la sécrétion séquentielle du contenu de deux organites : les micronèmes et les rhoptries. Lors de l'invasion, le parasite établit un contact étroit entre son extrêmité apicale et la membrane plasmique de la cellule hôte, appelé la jonction mobile (JM). La JM est un point d'ancrage à la cellule hôte qui est initié chez Toxoplasma par la sécrétion de protéines du col des rhoptries appelées TgRON2/RON4/RON5/RON8 (complexe de RONs). Ces protéines sont sécrétées dans la cellule hôte et TgRON2 est insérée dans la membrane de la cellule hôte. TgRON2 peut servir de récepteur à la protéine TgAMA1 (Apical Membrane Antigen 1) qui est une protéine de micronèmes sécrétée à la surface du parasite durant l'invasion. L'interaction AMA1-RON2 est également conservée chez Plasmodium, mais il n'existe pas de réactivité croisée entre espèces d'apicomplexes. La résolution de la structure de la protéine recombinante TgAMA1 en complexe avec un peptide TgRON2 nous a permis de déterminer des résidus critiques à l'interaction entre ces deux protéines in vitro et à l'invasion du parasite in vivo, et de définir les bases structurales de la spécificité intra-espèce de l'interaction AMA1-RON2. Par l'obtention d'une souche dépourvue de TgAMA1, nous montrons qu'AMA1 n'est pas essentielle à la survie du toxoplasme, comme il avait été supposé depuis longtemps. Nous confirmons le rôle clé de cette protéine dans l'invasion et la formation de la JM. Les mutants dépourvus d'AMA1 sont capables d'insérer le complexe de RONs dans la cellule hôte mais se détachent plus fréquemment, entrainant des invasions abortives. L'invasion résiduelle observée en absence d'AMA1 pourrait impliquer des protéines homologues à TgAMA1, TgRON2 et TgRON4, dont nous avons entamé la caractérisation moléculaire et fonctionnelle.Mot-clés : Apicomplexes, Toxoplasma gondii, invasion, jonction mobile, micronèmes, rhoptries / Molecular and functional characterisation of the moving junction controlling host cell invasion by Toxoplasma gondiiAbstract:Apicomplexa are eukaryotic parasites responsible for a variety of human and animal diseases, including malaria or toxoplasmosis. Most of them have an obligatory intracellular stage; thus, the invasive process is a crucial step in their developmental cycle. It implies the sequential secretion of two organelles: micronemes and rhoptries. During invasion, the parasite establishes a structure called the moving junction (MJ), which is a close apposition between the apical end and the plasma membrane of host cell. The MJ is an anchoring point for invasion that is initiated in Toxoplasma by the secretion of rhoptry neck proteins named TgRON2/RON4/RON5/RON8 (the RONs complex). These proteins are exported to the host cell cytoplasm and TgRON2 spans the host cell membrane. There, TgRON2 will function as a receptor to Apical Membrane antigen 1 (TgAMA1), which is a micronemal protein displayed on the surface of the parasite during the invasion process. The AMA1-RON2 interaction is conserved in Plasmodium but there is no interspecies cross-binding.We have determined the structure of a TgAMA1 recombinant protein in complex with a TgRON2 peptide, which allowed us to determine which residues are critical for the interaction between both proteins in vitro and for parasite invasion in vivo. Moreover, the co-structure explains at the structural level the evolutionary constraint of the AMA1-RON2 interaction. By generating an AMA1 null strain in T. gondii, we demonstrate that TgAMA1 is not an essential gene, as claimed before. We confirm the importance of AMA1 in invasion and its key role in MJ formation. AMA1 null parasites insert the RON complex into the host cell but are more frequently detached from it, causing abortive invasions. The residual invasion might involve proteins homologous to TgAMA1, TgRON2 and TgRON4, for which the molecular and functional characterization is undertaken.Keywords: Apicomplexes, Toxoplasma gondii, invasion, moving junction, micronemes, rhoptries

M.Sc. / Research into blood protozoans (haematozoans) infecting African tortoises is scanty with only a few records published, many during the early part of the last century. Little research had been done on the blood parasites of tortoises examined in this study namely, Kinixys lobatsiana, K. belliana belliana, K. natalensis, Geochelone pardalis pardalis, G. pardalis babcocki and Chersina angulata. The study therefore aimed to: 1) examine apicomplexan haematozoan parasites infecting several of South Africa’s indigenous tortoises and compare them with published species descriptions, especially from neighbouring Mozambique; 2) provide host details (identity, ectoparasites, host weight and gender, effects of blood parasites on host cells) and locality records in different seasons for described and new apicomplexan species; 3) describe new and recorded parasites using morphometrics and, if possible, ultrastructural characteristics 4) attempt apicomplexan DNA extraction, amplification and, if feasible, purification; and 5) establish a basis for future research as a result of the acquired knowledge. During the current study, 154 tortoises of six species in three genera, both captive and wild, and from four South African provinces (Gauteng, North West, Kwazulu-Natal and Western Cape) were sampled. Giemsa stained blood smears and use of image analysis enabled morphometric analysis of the apicomplexans and their effects on host cells, while some blood preserved in Karnovsky’s and Todd’s fixatives received detailed examination by transmission electron microscopy. Lastly, blood preserved in lysis buffer during collection, and with the highest parasitaemias, was subjected to parasite DNA extraction and amplification. Comparisons between a published account of apicomplexans recorded from K. b. belliana in Mozambique, and those found in the current study, identified two haemogregarine species. In the present research, Haemogregarina fitzsimonsi Dias, 1953 infected 2/27 (7%) wild North West K. lobatsiana, 2/3 (66%) captive Kwazulu-Natal K. natalensis, 7/14 (50%) captive Kwazulu- Natal K. b. belliana, 3/6 (50%) captive Kwazulu-Natal G. p. pardalis, 2/41 (5%) wild G. p. babcocki and 13/37 (35%) captive Gauteng G. pardalis. In addition, Haemogregarina parvula Dias, 1953, infected 2/14 (14%) captive K. b. belliana and 1/10 (10%) captive G. p. pardalis. An unknown species of haemogregarine, possibly also H. fitzsimonsi occurred in 6/16 (38%) Chersina angulata from the Western Cape. As well as haemogregarines, two haemoproteids were identified: Haemoproteus balazuci Dias, 1953 infected 2/27 (7%) wild North West K. lobatsiana, 2/2 (100%) captive Gauteng K. lobatsiana and 1/41 (2%) wild North West G. p. babcocki; Haemoproteus sp., a likely new species, was found in 1/3 (33%) captive K. natalensis. Infections with Haemogregarina and Haemoproteus were not concurrent in this study, but were found to occur concurrently in Dias (1953) findings, and only the two Haemogregarina spp. occurred together in captive Kwazulu-Natal G. p. pardalis tortoises, which do not occur naturally in the region. Haemogregarina fitzsimonsi did not appear region or host specific, since it infected 5/6 species of tortoises from all provinces sampled. Haemogregarina parvula apparently existed only in tortoises from Kwazulu-Natal. Furthermore, captive Gauteng female tortoises were found to have a higher rate of infection than males and heavier tortoises showed a lower intensity infection than lighter and younger tortoises. On average season appeared to have a slight affect on parasite prevalence, with a higher prevalence during the summer rather than the winter, possibly a result of the activity of the assumed vector, which may be the tick species Amblyomma marmoreum (found on G. pardalis) and/or Amblyomma hebraeum (found on C. angulata). For the new Haemoproteus sp., the small sample size meant that meaningful data on host-specificity and range was not gathered, but Hp. balazuci occurred in K. lobatsiana in the drier regions of the North West and Gauteng. Although DNA extraction was possible for H. fitzsimonsi, the technique requires further refinement and samples with greater parasitemias before it can be used with additional material, and sequencing can be attempted. Thus, new localities, hosts, host data and possible vectors (ticks) were recorded for the apicomplexan species identified by Dias (1953) and they were re-described using modern techniques. Also, possibly new Haemogregarina and Haemoproteus spp. were recorded, but their identity requires confirmation by DNA analysis. It is anticipated that these, and future results, will increase the knowledge of the ecology and biodiversity of apicomplexan haematozoans parasitising chelonian hosts in South Africa, with possible application to the conservation of these and other tortoise species around the world.

A research report submitted to the faculty of Medicine, University of the Witwatersrand, Johannesburg, in part fulfillment of the requirements for the degree of Master of Medicine (in the branch of Haematology) Johannesburg 1992. / The observation by Haldane in 1949 that the distribution of malaria and certain thalassaemias were similar and that the former disease must be a selective force tor the continued existence of the latter by preservation of the heterozygotes. This theory which later became known as lithe malaria hypothesis" has been applied to other inherited conditions such as G6PD deficiency, membranopathies, certain blood group polymorphisms, other heamoglobinopathies such as sickle cell disease, blood group polymorphisms and more recently HLA phenotypes. It has been shown that the Duffy blood group antigens are the receptors for. Plasmodium vivax and since these antigens are lacking in most black Africans this species of malaria is virtually absent in Africa. It has also been shown that the glycophorins are at least in part the receptors for Pfalciparum. Several variants of the glycophorins exist and the biochemistry and, where known, the molecular mechanisms by which these arise is reviewed. Experimental work is carried out to establish the growth characteristics of Pfalciparum in an in vitro culture system using cells with glycophorin variants on their membranes. Three such variants were compared to normal cells and two (S~s-U-and Dantu) were found to be partially resistant to invasion by Pfalciparum merozoites whereas the third (Henshaw) was found to be no different to controls. / MT2018

A resposta imune ao P. chabaudi se caracteriza pela ativação policlonal de linfócitos B na fase aguda da doença, que resulta em uma intensa produção de anticorpos IgM e IgG de baixa afinidade e autoanticorpos. Apesar de esses anticorpos contribuírem para o controle da infecção, pode retardar a geração de anticorpos de alta afinidade que garantem a destruição dos parasitas e a proteção contra infecções subseqüentes. O nosso objetivo foi caracterizar os mecanismos envolvidos na ativação policlonal dos linfócitos B na infecção pelo P. chabaudi AS. Os nossos resultados mostraram que a cooperação T-B e importante nessa fase da infecção. Os ensaios in vitro mostraram que os linfócitos T da fase aguda da infecção são capazes de estimular linfócitos B naives, aumentando a expressão do CD69 e induzindo proliferação e produção de anticorpos. A molécula de classe II do MHC é importante na ativação policlonal dos linfócitos B, aumentando a expressão do CD69 e induzindo a proliferação de anticorpos, devido ao seu envolvimento na ativação dos linfócitos T. Assim, moléculas como CD28, CD40L e ICOS são importantes na ativação, proliferação e produção de anticorpos. / Polyclonal B cell activation is a feature of the early spleen B cell response to human and murine malaria, which results in intense production of parasite low-affinity IgM and IgG and autoantibodies. Although this response may contribute for the initial parasite control, it may also hinder the generation of high-affinity IgG that guarantees parasite elimination and protection against secondary infections. To characterize the mechanism involved in the polyclonal B cell response to P. chabaudi AS, we have analyzed in vivo and in vitro the role of several molecules known to be involved in antibody production. According to our in vivo results, this response depends on the cooperation of CD4+ T cells with B cells. The in vitro assays shows that the expression of MHC class II molecules is crucial for B cell activation in terms of CD69 expression, proliferation and antibody production, apparently due to their requirement for T cell activation. Signaling pathways mediated by the co-stimulatory receptor CD28, CD40L and ICOS has a central role for B cell proliferative response and antibody production. This study may help to clarify the molecular basis of polyclonal B cell activation induced during acute malaria.

As doenças causadas por patógenos emergentes e oportunistas são uma grande preocupação para os profissionais de Saúde Pública. A criptosporidiose é uma doença cosmopolita, cujo agente etiológico é o protozoário coccídia Cryptosporidium. Este parasita emergiu como um importante patógeno de veiculação hídrica, responsável por surtos em diferentes países e atualmente é considerado um problema para indivíduos imunocomprometidos e imunocompetentes. A taxonomia do gênero, baseada em aspectos morfológicos, é de difícil realização devido ao reduzido tamanho dos oocistos e também devido à perda de suas características morfológicas, principalmente em amostras ambientais. Essa limitação estimulou a aplicação de métodos moleculares na identificação das espécies destes microrganismos. Neste estudo, amostras de águas superficiais foram submetidas à ested-PCR para detecção de Cryptosporidium. No total 30 amostras de águas de 10 pontos de coleta situados no estado de São Paulo foram analisadas. Cryptosporidium foi detectado em 30% das amostras analisadas e a genotipagem permitiu a identificação de C. hominis, C. meleagridis e C. andersoni. Embora a identificação de Cryptosporidium em amostras ambientais seja uma tarefa complexa, os métodos moleculares são essenciais para determinação de espécies, ajudando a elucidar a epidemiologia deste protozoário em nosso país. / The illnesses caused by emerging and opportunist pathogens are a great concern for Public Health professionals. Criptosporidiosis is a cosmopolitan disease, whose etiologic agent is the coccidian protozoan Cryptosporidium. This parasite has emerged as an important waterborne pathogen, responsible for outbreaks in different countries, and it is currently considered a problem for immunocompromised and immunocompetent patients. The taxonomy of the genus, based on morphologic aspects, is of difficult accomplishment due to the small size of the oocysts, and also due to loss of its characteristics, mainly in environmental samples. This limitation has estimulated the application of molecular methods for species identification of these microorganisms. In this study, superficial water samples were submitted to nested-PCR to detect the presence of Cryptosporidium. A total of 30 water samples, from 10 points located in the state of São Paulo, were analyzed. Cryptosporidium was detected in 30% of the studied samples and genotyping allowed the identification of C. hominis, C. meleagridis and C. andersoni. Although the identification of Cryptosporidium in environmental samples is a complex task, molecular methods are essential for the species determination, helping to elucidate the epidemiology of this protozoan in our country.

As doenças causadas por patógenos emergentes e oportunistas são uma grande preocupação para os profissionais de Saúde Pública. A criptosporidiose é uma doença cosmopolita, cujo agente etiológico é o protozoário coccídia Cryptosporidium. Este parasita emergiu como um importante patógeno de veiculação hídrica, responsável por surtos em diferentes países e atualmente é considerado um problema para indivíduos imunocomprometidos e imunocompetentes. A taxonomia do gênero, baseada em aspectos morfológicos, é de difícil realização devido ao reduzido tamanho dos oocistos e também devido à perda de suas características morfológicas, principalmente em amostras ambientais. Essa limitação estimulou a aplicação de métodos moleculares na identificação das espécies destes microrganismos. Neste estudo, amostras de águas superficiais foram submetidas à ested-PCR para detecção de Cryptosporidium. No total 30 amostras de águas de 10 pontos de coleta situados no estado de São Paulo foram analisadas. Cryptosporidium foi detectado em 30% das amostras analisadas e a genotipagem permitiu a identificação de C. hominis, C. meleagridis e C. andersoni. Embora a identificação de Cryptosporidium em amostras ambientais seja uma tarefa complexa, os métodos moleculares são essenciais para determinação de espécies, ajudando a elucidar a epidemiologia deste protozoário em nosso país. / The illnesses caused by emerging and opportunist pathogens are a great concern for Public Health professionals. Criptosporidiosis is a cosmopolitan disease, whose etiologic agent is the coccidian protozoan Cryptosporidium. This parasite has emerged as an important waterborne pathogen, responsible for outbreaks in different countries, and it is currently considered a problem for immunocompromised and immunocompetent patients. The taxonomy of the genus, based on morphologic aspects, is of difficult accomplishment due to the small size of the oocysts, and also due to loss of its characteristics, mainly in environmental samples. This limitation has estimulated the application of molecular methods for species identification of these microorganisms. In this study, superficial water samples were submitted to nested-PCR to detect the presence of Cryptosporidium. A total of 30 water samples, from 10 points located in the state of São Paulo, were analyzed. Cryptosporidium was detected in 30% of the studied samples and genotyping allowed the identification of C. hominis, C. meleagridis and C. andersoni. Although the identification of Cryptosporidium in environmental samples is a complex task, molecular methods are essential for the species determination, helping to elucidate the epidemiology of this protozoan in our country.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 129-143).

The concern of a Cryptosporidium or Giardia waterborne outbreak due to treated wastewater has had water treatment utilities using some of the highest water cleansing technologies available. Cryptosporidiosis and Giardiasis are severe diarrheal diseases which can lead to death, thus it is important that appropriate steps are taken to assure these parasites are not present in the effluent of treated wastewater. This study examined the results of 863 assays for Giardia and Cryptosporidium on the effluent of wastewater treatment facilities and found that county of collection, watershed of collection, and laboratory analyzing the sample have the most significant impact on the detection of Cryptosporidium oocysts and Giardia cysts in wastewater effluent and that there were minimal but significant differences in method of treatment and method of filtration. To date no other comprehensive analysis of this data has been done.

Theileria parva is a complex protozoan parasite causing East Coast fever in Eastern and Central Africa. Vaccination using live parasites is an effective control measure and has been used in Zambia based on locally isolated and introduced T. parva stocks. Diversity among T. parva populations was investigated in parasites from two Zambian provinces with different disease epidemiologies and control histories. Isolates from the pre-vaccination era, local and exotic stocks used for vaccination, and one recent field isolate were cloned and passaged in vitro to study genomic stability over time. The results of the data from three genome-wide probes indicate a marked homogeneity and stability among the Zambian isolates in contrast to East African isolates. Results from Southern blot profiles and the polymorphic immunodominant molecule (PIM) sequence analysis suggest a common origin for the Zambian isolates from the pre-vaccination era, except for one isolate (Zam5) from Southern Province. This isolate showed characteristics suggesting a buffalo origin. Assays for genotype characterisation were developed using five allelic markers. Multilocus characterisation revealed identical profiles in a recent Zambian isolate from Southern Province and two components of an exotic cocktail vaccine, indicating the escape of one of the vaccine stocks in the field. Characterisation of T. parva field populations by RFLP-PCR assays after immunisation revealed the presence of dominant genotypes from those that had been used for vaccination. Circumstantial evidence for the involvement of one of the exotic vaccine parasites in epidemics in Southern Province is presented and a hypothesis formulated for the rapid spread of this genotype. Analysis of the characterisation data suggested the existence of two groups of T. parva parasites of different origin. The classic T. parva group, characterised by a dimorphism of the p150, p104 and p32 loci and the absence of a p67 insert and a buffalo-derived group which showed a polymorphism of p150, p104 and p32 and the presence of a p67 insert. There is evidence that recombination occurs, resulting in parasites that have characteristics of both groups. The relevance of these recombinant parasites in the epidemiology of the disease seems low. Characterisation of larger samples from areas of regular buffalo-cattle contact is necessary to clarify this. Sequence analysis of the most discriminative locus (PIM) was undertaken and gene conversion could be the main mechanism generating diversity. A more appropriate nomenclature for T. parva is proposed based on the growing evidence of molecular differences among isolates and stocks.

The withdrawal of water sources concluded the reuse of treated wastewaters, especially for non-potable purposes. Agricultural use of the reclaimed wastewaters is one of the reuse options. However health considerations of the reuse of reclaimed wastewaters for public related purposes are underestimated, since wastewaters contain a variety of microbial pathogens, which may be transmitted to workers and consumers through the crops irrigated. Of these, parasitic eggs have a special place, as they are capable of surviving in the soil for months or even years, depending on environmental conditions. There is insufficient accumulated information on the health related criteria for the reuse of treated wastewaters in Turkey. The aim of this study was therefore to determine the helminthic eggs in raw sewage and in effluents of ASKi municipal wastewater treatment plant in Ankara. The study involved examining to decide whether these organisms exist in the wastewaters at all, and if so in what concentrations. Modified Bailenger&rsquo / s method, which published in the &ldquo / WHO Laboratory Manual of Parasitological and Bacteriological Techniques&rdquo / and &ldquo / U.S.EPA ICR Microbial Laboratory Manual&rdquo / were used in developing the specific methods used in this study.