The humoral response of humans, calves and horses to Cryptosporidium sporozoite antigens was evaluated using a western blot technique. Sera from calves, humans and horses were obtained at various times following the detection of infection. Sera were reacted with detergent-solubilized, sporozoite antigens form sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The number of antigens recognized by immune sera from humans and animals increased with time post infection (P.I.). A 20 kDa antigen appeared to be a major sporozoite surface determinant since it was labelled via membrane protein biotinylation and recognized by mouse monoclonal antibodies using indirect immunofluorescence and western blotting. Detectable recognition of the 20 kDa band occurred in 3 week post infection (P.I.) sera from all species tested. Sera reactivity to the 20 kDa band diminished significantly within 5 months P.I. when infected humans had no further recurrence of cryptosporidial diarrhea. In contrast, 12 month P.I. sera from an individual constantly exposed to oocysts under working conditions was as strongly reactive as the 3 week convalescent sera. Therefore, reactivity to the 20 kDa antigen appeared to be a good indicator of exposure to Cryptosporidium. Anti-sporozoite indirect immunofluorescent titers decrease in reactivity from convalescent to post convalescent sera which correlated with western blot results. Chromosomal DNA of five Cryptosporidium parvum isolates and one Cryptosporidium baileyi isolate were compared by field inversion gel electrophoresis (FIGE). FIGE analyses of parasite DNA prepared from purified sporozoites versus intact oocysts showed no observable differences. Chromosomal DNA migration patterns of the five Cryptosporidium parvum isolates were indistinguishable. Distinct differences in chromosomal DNA were evident between the Cryptosporidium baileyi and Cryptosporidium parvum isolates, yet the overall pattern was similar. Five C. parvum isolates were also compared using two dimensional electrophoretic analyses. Silver stained patterns of sporozoite proteins showed a shift in a 106 kDa protein in three of the isolates. One isolate (Mexico) showed a complete absence of this protein (106 kDa) and the presence of an additional 40 kDa protein not found in any other isolate.

Thesis (Ph. D.)--University of Adelaide, 1984. / Some ill. mounted. Includes bibliographical references (leaves [473]-504).

Thesis (M.Sc.) -- Memorial University of Newfoundland, 1989. / Typescript. Bibliography: leaves 53-57. Also available online.

Thesis (M.A.)--Indiana University, 2007. / Title from screen (viewed on May 23, 2007) Department of Pharmacology & Toxicology, Indiana University-Purdue University Indianapolis (IUPUI) Includes vita. Includes bibliographical references (leaves 204-226)

Orientador: Nelson da Silva Cordeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-25T05:22:08Z (GMT). No. of bitstreams: 1 Adriano_EdsonAparecido_M.pdf: 4529408 bytes, checksum: 15cb3c4cf8fdb72903e217116508c6b5 (MD5) Previous issue date: 1999 / Resumo: Entre janeiro e dezembro de 1998 foram examinados 450 exemplares de três espécies de aves da família Columbidae capturados no município de Junqueirópolis, região oeste do estado de São Pal!lo, Brasil. O presente estudo avaliou nessas aves, a ocorrência de protozoários"Apicomplexa (Haemoproteidae e Eimeriidae), parasitos de sangue e de intestino, dando ênfase para os aspectos taxonômicos dos parasitos, bem como o estudo da prevalência. A determinação específica dos parasitos encontrados nas aves foi realizada mediante comparações morfológicas com as espécies descritas na literatura. As aves foram capturadas quinzenalmente com o auxílio de armadilha-telada, os esfregaços foram confeccionados com sangue obtido mediante picada na artéria braquial, fixados com álcool metílico, corados com Giemsa e examinados em microscópio óptico. Apenas Haemoproteus columbae Kruse, 1890 foi encontrado no sangue das aves: a prevalência do parasito e a intensidade do parasitismo apresentaram variações entre Zenaida auriculata Des Murs, 1847, Columbina talpacoti Temmink, 1810 e Scardafella squammata Lesson, 1831. Para obter as fezes visando o estudo dos coccídios, as aves foram isoladas em gaiolas por um período de 2 horas, posteriormente anilhadas e liberadas. Amostras fecais foram diluídas em solução aquosa de dicromato de potássio e após concentração foram examinadas ao microscópio óptico. Em 23,9% dos exemplares de Z auriculata foram encontrados oocistos de protozoários do gênero Eimeria Schneider, 1875, com características diferentes das espécies previamente descritas em Columbiformes. Ooocistos encontrados em 17,4% dos exemplares de C. talpacoti e em 12,8% dos exemplares de S. squammata foram diferentes daqueles registrados infectando outros columbídeos e daqueles encontrados em Z. auriculata. Os oocistos encontrados nos exemplares de Z. auriculata, e os encontrados simultaneamente em C. talpacoti e S. squammata foram considerados como duas novas espécies de Eimeria / Abstract: During the period between january and december of 1998, were examined 450 specimens of 3 species of birds from the Columbidae family captured in the minicipality of Junqueirópol_, West Regio"n of São Paulo State, Brazil. The present study evaluated in this doves, the occurrence of protozoan Apicomplexa (Haemoproteidae and Eimeriidae), parasites of blood and of intestine, emphasizing the aspects taxonomics of the parasite and the study of the prevalence. The specific determination of the parasites found in the doves was made by morphological camparation with those parasites reported in literature The doves were captured fortnighthy by gauze-trap, and the blood smears were made from blood obtained from the brachial artery, air-dried, fixed in 100% metanol, stained with Giemsa and examined in light microscope. Only Haemoproteus columbae kruse (1890), was reported in blood of the doves examined; the prevalence of the parasite and intensity of the parasitism, revealed variant among Zenaida auriculata Des Murs (1847),62 of Columbina talpacotiTemmink (1810) and 57 of Scardafella squammata Lesson (1831). For obtain the faecal samples, the doves were individually isoled in cage for a period of 2 hours, afterwards tagged and liberated. Individual faecal samples were placed in a solution of 2.5% potassium dicromate and examined microscopically after flotation, using Sheather's sugar solution. In 23.9% of the specimens of Z. auriculata, were found oocysts of protozoan of the genus Eimeria Schneider (1881), than appears aspects than distinguished its of the species of Eimeria previously described of Columbiformes. Oocysts found in 17.4% of the specimens of the C. talpacoti and 12.8% of the specimens of the S. squammata, were distinguished from those oocysts of the species of Eimeria reported infecting columbforms hosts, as well as from those found in Z. auriculata. Oocysts found in Z. a uriculata , and those found in both, C. talpacoti and S. squammata, were considered to be oocysts of two news species of Eimeria / Mestrado / Parasitologia / Mestre em Ciências Biológicas

Neospora caninum é um protozoário pertencente ao filo Apicomplexa que atinge, dentre diversos hospedeiros intermediários, principalmente bovinos e tem emergido como um importante causador de problemas reprodutivos e abortos em rebanhos de corte e leiteiro. Organismos do filo Apicomplexa são parasitas intracelulares obrigatórios que, para locomoverem-se e realizarem a invasão das células hospedeiras, utilizam um mecanismo próprio de locomoção ativa impulsionada pelo motor actina/miosina denominado motilidade por deslizamento (gliding motility), cujo complexo motor localiza-se entre a membrana plasmática e o complexo de membrana interno do parasita. A investigação a respeito do funcionamento desse mecanismo de locomoção e invasão vem sendo realizada principalmente em Toxoplasma gondii e Plasmodium spp., entretanto não há nenhuma publicação envolvendo actina em N. caninum. Esse trabalho envolveu a clonagem e expressão da sequência NcAct201-310 e deu início a caracterização da actina de N. caninum (NcAct). A sequência NcAct foi obtida em banco de dados ToxoDB, e uma comparação por alinhamento entre as actinas de Apicomplexas relacionados revelou que NcAct é idêntica à TgACT1 (100% identidade). Com outras espécies, a NcAct tem maior identidade/similaridade com a actina de Eimeria tenella (97%/99%), seguida da actina de Plasmodium falciparum PfACT1 (93%/97%), da actina de Babesia bovis (86%/94%) e PfACT2 (80%/92%). Quando localizada com anticorpo anti-?-actina C4, NcAct apresenta-se em duas bandas de 43 e 45 kDa em gel de acrilamida 1D e em nove isoformas em gel de acrilamida 2D. Todas as identidades das bandas e spots foram confirmados por espectrometria de massas (MS/MS). Além disso, NcAct localiza-se, em sua maioria, na região periférica do taquizoíta de N. caninum e sua distribuição é alterada após incubação dos taquizoítas com 5 ?M de jasplakinolida (JAS) ou 2 ?M de citocalasina D (CytD). Por fim, por meio de ensaio de fracionamento de actina monomérica (actina-G) e filamentosa (actina-F), demonstramos que a JAS é capaz de aumentar a quantidade de actina-F em taquizoítas de N. caninum. / Neospora caninum is an Apicomplexan protozoan that infects, among a whole range of intermediate hosts, bovine where it is emerging as a relevant cause of reproductive problems and abortion in dairy and beef cattle. As obligatory intracellular organisms, parasites from Apicomplexa Phylum use their own active locomotion system to move and invade host cells. This mechanism is driven by the actin/myosin motor known as gliding motility, localized between the plasma and the inner membrane complex. Studies involving this locomotion and invasion system have been conducted mainly in Toxoplasma gondii and Plasmodium spp. To our knowledge there is no publication involving actin in N. caninum, so this work was outlined and involved the cloning and expression of the sequence NcAct201-310, initiating the characterization of actin of N. caninum (NcAct). The sequence NcAct was obtained from the Database ToxoDB, and a comparison of actins from Apicomplexa-related revealed total identity of NcAct with TgACT1 (100% identity). With other species, NcAct has higher identity/similarity with Eimeria tenella actin (97%/99%), followed by Plasmodium falciparum actin PfACT1 (93%/97%), Babesia bovis actin (86%/94%) and PfACT2 (80%/92%). When localized with the antibody anti-?-actin C4, NcAct is presented as two bands of 43 and 45 kDa in 1D acrylamide gel and as nine isoforms in 2D acrylamide gel. All these findings were confirmed by mass spectrometry (MS/MS). Moreover, NcAct localizes predominantly in the peripheric region of N. caninum tachyzoites. This distribution is altered after incubation of the tachyzoites with 5 ?M of jasplakinolide (JAS) or 2 ?M of cytochalasin D (CytD). Finally through fractionating assay of monomeric (actin-G) and filamentous (actin-F), we demonstrated that JAS is capable of increasing the quantity of actin-F in N. caninum tachyzoites.

Neospora caninum é um protozoário pertencente ao filo Apicomplexa que atinge, dentre diversos hospedeiros intermediários, principalmente bovinos e tem emergido como um importante causador de problemas reprodutivos e abortos em rebanhos de corte e leiteiro. Organismos do filo Apicomplexa são parasitas intracelulares obrigatórios que, para locomoverem-se e realizarem a invasão das células hospedeiras, utilizam um mecanismo próprio de locomoção ativa impulsionada pelo motor actina/miosina denominado motilidade por deslizamento (gliding motility), cujo complexo motor localiza-se entre a membrana plasmática e o complexo de membrana interno do parasita. A investigação a respeito do funcionamento desse mecanismo de locomoção e invasão vem sendo realizada principalmente em Toxoplasma gondii e Plasmodium spp., entretanto não há nenhuma publicação envolvendo actina em N. caninum. Esse trabalho envolveu a clonagem e expressão da sequência NcAct201-310 e deu início a caracterização da actina de N. caninum (NcAct). A sequência NcAct foi obtida em banco de dados ToxoDB, e uma comparação por alinhamento entre as actinas de Apicomplexas relacionados revelou que NcAct é idêntica à TgACT1 (100% identidade). Com outras espécies, a NcAct tem maior identidade/similaridade com a actina de Eimeria tenella (97%/99%), seguida da actina de Plasmodium falciparum PfACT1 (93%/97%), da actina de Babesia bovis (86%/94%) e PfACT2 (80%/92%). Quando localizada com anticorpo anti-?-actina C4, NcAct apresenta-se em duas bandas de 43 e 45 kDa em gel de acrilamida 1D e em nove isoformas em gel de acrilamida 2D. Todas as identidades das bandas e spots foram confirmados por espectrometria de massas (MS/MS). Além disso, NcAct localiza-se, em sua maioria, na região periférica do taquizoíta de N. caninum e sua distribuição é alterada após incubação dos taquizoítas com 5 ?M de jasplakinolida (JAS) ou 2 ?M de citocalasina D (CytD). Por fim, por meio de ensaio de fracionamento de actina monomérica (actina-G) e filamentosa (actina-F), demonstramos que a JAS é capaz de aumentar a quantidade de actina-F em taquizoítas de N. caninum. / Neospora caninum is an Apicomplexan protozoan that infects, among a whole range of intermediate hosts, bovine where it is emerging as a relevant cause of reproductive problems and abortion in dairy and beef cattle. As obligatory intracellular organisms, parasites from Apicomplexa Phylum use their own active locomotion system to move and invade host cells. This mechanism is driven by the actin/myosin motor known as gliding motility, localized between the plasma and the inner membrane complex. Studies involving this locomotion and invasion system have been conducted mainly in Toxoplasma gondii and Plasmodium spp. To our knowledge there is no publication involving actin in N. caninum, so this work was outlined and involved the cloning and expression of the sequence NcAct201-310, initiating the characterization of actin of N. caninum (NcAct). The sequence NcAct was obtained from the Database ToxoDB, and a comparison of actins from Apicomplexa-related revealed total identity of NcAct with TgACT1 (100% identity). With other species, NcAct has higher identity/similarity with Eimeria tenella actin (97%/99%), followed by Plasmodium falciparum actin PfACT1 (93%/97%), Babesia bovis actin (86%/94%) and PfACT2 (80%/92%). When localized with the antibody anti-?-actin C4, NcAct is presented as two bands of 43 and 45 kDa in 1D acrylamide gel and as nine isoforms in 2D acrylamide gel. All these findings were confirmed by mass spectrometry (MS/MS). Moreover, NcAct localizes predominantly in the peripheric region of N. caninum tachyzoites. This distribution is altered after incubation of the tachyzoites with 5 ?M of jasplakinolide (JAS) or 2 ?M of cytochalasin D (CytD). Finally through fractionating assay of monomeric (actin-G) and filamentous (actin-F), we demonstrated that JAS is capable of increasing the quantity of actin-F in N. caninum tachyzoites.

Orientador: Ana Maria Aparecida Guaraldo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T14:53:24Z (GMT). No. of bitstreams: 1 Hofstatter_PauloGonzalez_M.pdf: 2633496 bytes, checksum: 19c74e9814c573f8ebf8f7645de76a37 (MD5) Previous issue date: 2011 / Resumo: Psittacidae são aves de estimação bem conhecidas e comuns em zoológicos, parques e criatórios particulares. Têm uma ampla distribuição mundial, principalmente em regiões tropicais. Apesar de sua popularidade, pouco se sabe a respeito de seus parasitas, principalmente coccídios. O filo Apicomplexa é um grupo de protozoários predominantemente parasíticos de imensa importância médica e veterinária, o qual apresenta afinidades com Dinozoa, Ciliophora e Heterokonta. Apesar da presença de Apicomplexa em psitacídeos ter sido relatada diversas vezes, poucas espécies de Eimeria (6) e Isospora (2) foram descritas até o momento. O objetivo deste estudo foi investigar, identificar e descrever os parasitas coccidianos encontrados em aves psitaciformes. Para isto, fezes de aves cativas originárias de zoológicos e criadores particulares foram coletadas entre 2009 e 2010 e submetidas a técnicas de flutuação a fim de recuperar oocistos de coccidios e ovos de nematódeos. 43 das 237 amostras estavam positivas (18,1%). Oocistos típicos de Eimeria foram observados em Amazona aestiva, Ara rubrogenis e Platycercus sp. e uma nova espécie de Eimeria foi descrita em A. aestiva. Nenhum oocisto de Isospora foi observado. Além disso, tivemos a oportunidade de observar rica variedade de ovos de nematódeos, possivelmente pertencendo a Capillaria plagiaticia, Ascaridia sp., Heterakis sp. e outro material não identificado. Observou-se também que espécimes de zoológicos foram significativamente mais parasitados do que aves oriundas de criatórios particulares. A prevalência de nematódeos (16,9%) foi muito maior que de protozoários (2,1%) / Abstract: Psittacidae are well-known companion birds and common as zoo specimens, in parks and breeding facilities. They have a wide distribution around the world, mainly in tropical regions. Despite their popularity, not much is known about the parasites associated to them, mainly coccidia. The phylum Apicomplexa is a predominantly parasitic protozoan group of huge medical and veterinary importance, which bears evolutionary affinities to Dinozoa, Ciliophora and Heterokonta. Although the presence of apicomplexan parasites in psittacine birds was reported several times, only a handful of Eimeria (6) and Isospora (2) species has been described until now. The aim of this study was to investigate, identify and describe the coccidian parasites found in psittacine birds. For this, feces of captive birds originating from zoos and private breeders were collected from 2009 to 2010 and they were submitted to flotation techniques in order to recover coccidian oocysts and nematode eggs. 43 of 237 samples were positive (18,1%). Typical eimerian oocysts were observed in Amazona aestiva, Ara rubrogenis and Platycercus sp. and a new Eimeria species was described in A. aestiva. No isosporan oocysts were detected. Besides, we were able to observe a rich variety of nematode eggs, possibly belonging to Capillaria plagiaticia, Ascaridia sp., Heterakis sp. and another non-identified material. Zoo specimens were significantly more heavily parasitized than birds from private breeding facilities. Nematode prevalence was 16,9%, which was much more prevalent than that of coccidia, at about 2,1% / Mestrado / Parasitologia / Mestre em Parasitologia

Excystation of coccidian oocysts is accomplished in vitro by altering wall permeability with CO2 and a reducing agent, then activating the enclosed sporozoites with a solution of trypsin and bile. Elucidation of the mechanism of action of the CO2-reducing agent treatment was the basic intent of this study. Oocysts of Eimeria stiedae (rabbit) and E. tenella (chicken) were tested for the presence of an excystation-associated enzyme by incubating sporulated oocysts in fluids extracted from variously treated oocysts, and by carbon-14 labelling; the effect of CO2 -reducing agent treatment on oocyst walls was investigated by titration with acid or base for secondary bonding groups, or with dithionitrobenzoate (DTNB) for sulfhydryl groups. Electron microscopy was used to observe some effects of the excystation treatment. An excystation-associated enzyme was not found. Sulfhydryl groups appear to be present in the walls of both species. The DTNB titration indicated an increase in such groups with E. stiedae after outer wall removal and/or exposure to CO2-reducing agent treatment. A similar increase was not seen with E. tenella. Carbon dioxide probably acts as an allosteric affector, enabling the reducing agent to attack wall-stabilizing disulfide bonds in oocyst walls, thereby altering the permeability of the walls by reducing such bonds to sulfhydryl groups.

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