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Toxoplasma gondii, the etiological agent of toxoplasmosis, was first discovered in Ctenodactylus gondi, an small North African rodent, by Nicolle and Manceaux in 1908. Later the same year Splendore observed the organism in a laboratory rabbit. Human infection was first reported by Janku in 1923. Since its discovery, many investigations have shown the organism to be truly unique in the protozoan world. There appears to be only one species and only one serotype. Serologic and microscopic studies reveal Toxoplasma to have a vast host range. At least one representative of virtually every mammalian species tested harbored the parasite or showed the presence of demonstrable antibody. Studies of toxoplasmal infection among mammalian populations show that the prevalence of infection usually increases: 1 ) in warm, moist climates and 2) with age. Sex, however, appears to have little influence on the prevalence of the parasite. One most unusual feature of the organism is its lack of target cell specificity: Toxoplasma is an obligate, intracellular parasite capable of living in any cell except non-nucleated erythrocytes. Infection by Toxoplasma is most often confirmed serologically. Many procedures have been described; however, the Sabin-Feldman dye test, the indirect hemagglutinstion test, and the indirect fluorescent antibody test appear to be superior methods for antibody detection. The indirect fluorescent antibody test was selected for this study because: 1) it does not require the use of live organisms, 2) it is relatively easy to perform, and 3) it correlates well with the standard Sabin-Feldman dye test. This study was undertaken to determine the role of various factors on toxoplasmal prevalence in the Greater Orlando area. Age, sex, and race were considered for each human sample tested. Age, sex, and relative degree of outdoor exposure were considered in the study of each canine and feline sample.

O gênero Sarcocystis é constituído por várias espécies que se diferenciam pelas características morfológicas, biológicas e moleculares. Foram relatadas mais de 196 espécies encontradas em mamíferos, aves e répteis e somente 26 dessas espécies possuem o ciclo completo conhecido. Toxoplasma gondii é um parasito intracelular obrigatório, com distribuição geográfica cosmopolita, capaz de infectar uma ampla variedade de mamíferos e aves, inclusive o homem, caracterizando seu potencial zoonótico. Nas últimas décadas, a quantidade de pinguins vindos da Patagônia argentina e chilena, região de nascimento dessas aves, para o litoral brasileiro, onde muitos encalham e são resgatados, tem aumentado significativamente. Pouco se sabe sobre as doenças causadas por protozoários nessas aves. O presente estudo teve como objetivo conhecer aspectos epidemiológicos da infecção por coccídios da família Sarcocystidae em pinguins-de-magalhães (Spheniscus magellanicus), através de análises moleculares e sorológicas. Foram realizadas duas campanhas, uma em 2014 e outra em 2015, com a finalidade de obter amostras de sangue e tecidos dos pinguins que vieram a óbito durante reabilitação no Instituto de Pesquisa e Reabilitação de Animais Marinhos (IPRAM) localizada em Cariacica, Espírito Santo. Foram colhidas 514 amostras de tecidos (músculo=342, coração=86, cérebro=86) de 310 indivíduos. Dos tecidos de 54 pinguins foi realizado o bioensaio em camundongos para o isolamento de T. gondii, mas nenhum isolado foi obtido. Amostras de 310 indivíduos tiveram o DNA extraído para a pesquisa de coccídios da família Sarcocystidae utilizando-se os marcadores 18S rDNA, espaçador interno transcrito 1 (ITS1), codificador de proteínas de superfície (SAG)2, SAG3 e SAG4, subunidade beta da RNA polimerase (RPOB) e citocromo B (CytB). Destas, 16 (3.0%) amostras de músculo peitoral foram positivas para o gênero Sarcocystis spp., quando analisadas pelo marcador 18S, e todas com resultados idênticos. Com o ITS1, RPOB e Ctv. foram confirmadas as espécies de Sarcocystis em 12 amostras, todas idênticas a S. falcatula-like. Com os marcadores SAGs foi possível observar que as sequências não tinham variabilidade genética. Das 145 amostras de soro avaliadas para a presença de anticorpos anti-T. gondii, pelo Teste de Aglutinação Modificado (MAT ≥20), 18 aves foram positivas com títulos de: 20 (7 aves), 40 (9 aves) e 80 (2 aves). Este é o primeiro relato de S. falcatula-like e de anticorpos anti - T. gondii em pinguins-de-magalhães de vida livre. / The genus Sarcocystis is composed of several species that are differentiated by the morphological, biological and molecular characteristics. More than 196 species found in mammals, birds and reptiles have been reported, and only 26 of these species have the complete known cycle. Toxoplasma gondii is an obligate intracellular parasite with cosmopolitan geographic distribution, capable of infecting a wide variety of mammals and birds, including man, characterizing its zoonotic potential. In recent decades, the number of penguins that have come from Argentine and Chilean Patagonia, the region of birth of these birds, to the Brazilian coast, where many of them are stranded and rescued, has increased significantly. Little is known about the diseases caused by protozoa in these birds. The present study had as objective to study epidemiological aspects of coccidia infection of the family Sarcocystidae in Magellanic Penguins (Spheniscus magellanicus), through molecular and serological analyzes. Two campaigns were carried out, one in 2014 and another in 2015, in order to obtain blood and tissue samples from penguins who died during rehabilitation at the Institute of Research and Rehabilitation of Marine Animals (IPRAM) in the municipality of Cariacica, Espírito Santo. Tissue samples (total = 514: muscle = 342, heart = 86, brain = 86) were collected from 310 birds. From the tissues of 54 penguins the mouse bioassay was performed for the isolation of T. gondii, but no isolates were obtained. Samples of 310 individuals had DNA extracted for coccidia from the Sarcocystidae family using the 18S rDNA, Transcribed internal spacer 1(ITS1), surface protein encoder (SAG)2, SAG3, SAG4, beta subunit of RNA polymerase (RPOB) and citocrome B (CytB) markers. Of these, 16 (3.0%) samples, of pectoral muscle, were positive and all were identical to Sarcocystis spp. when analyzed by the 18S marker. With ITS1, RPOB and CytB the Sarcocystis species were confirmed in 12 samples, all identical to S. falcatula-like. With the SAGs it was possible to observe that the sequences had no genetic variability. Of the 145 serum samples evaluated for the presence of anti-T. gondii antibodies by Modified Agglutination Test (MAT ≥20), 18 were positive with titers of: 20 (7 birds), 40 (9 birds) and 80 (2 birds). This is the first report of S. falcatula-like and the presence of antibodies to T. gondii in free-living magellanic penguins.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 3 uppsatser.

Orientador: Helio Langoni / Resumo: Com relação ao desenvolvimento do quadro clínico e a transmissão transplacentária, a toxoplasmose em humanos e ratos é similar, e a infecção em ratos pode servir como um modelo para a enfermidade humana. Foram inoculados, pela via oral, sete grupos de ratos com 105 (Grupo 1), 104 (Grupo 2) e 103 (Grupo 3) bradizoítos de Toxoplasma gondii cepa BTU-2 (altamente virulenta para camundongos), 105 (Grupo 4), 104 (Grupo 5) e 103 (Grupo 6) bradizoítos de T.gondii cepa ME-49 (pouco virulenta para camundongos) e solução salina estéril (Grupo 7 - Controle). Os animais foram observados durante 84 dias, com colheita semanal de sangue para obtenção de soro e realização das provas sorológicas pelos métodos de aglutinação direta (MAD) e imunofluorescência indireta (RIFI) para anticorpos das classes IgG e IgM. Ao final do período os animais foram sacrificados e fragmentos de tecidos colhidos para reisolamento do parasita em camundongos e para detecção de DNA do T.gondii pela reação em cadeia pela polimerase (PCR), utilizando-se oligonucleotídeos dirigidos para a detecção dos genes SAG- 1, B1, rDNA e para uma seqüência repetida não codificadora (REP). Foi realizada a tipagem da cepa BTU-2 pela avaliação do polimorfismo do gene SAG-2. Todos os ratos desenvolveram títulos de anticorpos detectáveis pelas diferentes técnicas, havendo, independente da cepa inoculada, diferenças mais frequentes entre os grupos que receberam 105 e 103 bradizoítos. Para IgM a partir do 35o DPI e para a RIFI-IgG e o MAD, a cepa BTU-2 induziu títulos mais elevados que a cepa ME-49. A cepa BTU-2 foi isolada de todas as amostras de cérebro e musculatura estudadas, independente da dose utilizada na infecção dos animais, enquanto que para a cepa ME-49, o parasita foi isolado mais frequentemente do cérebro do que da musculatura, com influência da dose infectante... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Referring to the symptoms and transplacentary transmission, toxoplasmosis in humans and rats is similar and rats can be used as a model for the disease in humans. 7 groups of rats were inoculated, by oral administration, using 105 (Group 1), 104 (Group 2) and 103 (Group 3) Toxoplasma gondii bradyzoits from strain BTU-2, which is highly infective to mice and with 105 (Group 4), 104 (group 5) and 103 (Group 6) T.gondii bradyzoits of strain ME-49, which is less infective to mice and, finally, sterile saline solution (Group 7) as a control one. The animals were observed during 84 days, with weekly blood samples, in order to obtain serum and then submitted to serological tests by modified agglutination test (MAT) and imunofluorecent antibody test (IFAT) to antibodies from subtypes IgG and IgM. At the end of time of observation, the animals were killed and samples of their tissues were obtained in order to reisolate the parasite and to detect the T.gondii DNA by polymerase chain reaction (PCR), using specific oligonucleotids to detect the genes SAG-1, B1, rDNA and to no coding repetitive sequence (REP). The BTU-2 strain was classified by the SAG-2 gene polymorphism. All the rats developed detectable antibodies titers, no matter which strain was used between the techniques. The most frequent differences in the serological results were among the groups that received 105 and those who received 103 bradizoits, no matter the strain used. The BTU-2 strain induced higher titles to IgM since 35 DPI and to IFAT-IgG and MAT than the ME-49 strain. The BTU-2 strain also was isolated from all the brain and muscles samples, no matter the dosage used to infect the animals. When the ME- 49 strain was used, the parasite was isolated more frequently... (Complete abstract, click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:31:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T18:42:11Z : No. of bitstreams: 1 silva_av_dr_botfm_prot.pdf: 636648 bytes, checksum: 53bbd69b49e6f85da36a6c425cb8447c (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Com relação ao desenvolvimento do quadro clínico e a transmissão transplacentária, a toxoplasmose em humanos e ratos é similar, e a infecção em ratos pode servir como um modelo para a enfermidade humana. Foram inoculados, pela via oral, sete grupos de ratos com 105 (Grupo 1), 104 (Grupo 2) e 103 (Grupo 3) bradizoítos de Toxoplasma gondii cepa BTU-2 (altamente virulenta para camundongos), 105 (Grupo 4), 104 (Grupo 5) e 103 (Grupo 6) bradizoítos de T.gondii cepa ME-49 (pouco virulenta para camundongos) e solução salina estéril (Grupo 7 – Controle). Os animais foram observados durante 84 dias, com colheita semanal de sangue para obtenção de soro e realização das provas sorológicas pelos métodos de aglutinação direta (MAD) e imunofluorescência indireta (RIFI) para anticorpos das classes IgG e IgM. Ao final do período os animais foram sacrificados e fragmentos de tecidos colhidos para reisolamento do parasita em camundongos e para detecção de DNA do T.gondii pela reação em cadeia pela polimerase (PCR), utilizando-se oligonucleotídeos dirigidos para a detecção dos genes SAG- 1, B1, rDNA e para uma seqüência repetida não codificadora (REP). Foi realizada a tipagem da cepa BTU-2 pela avaliação do polimorfismo do gene SAG-2. Todos os ratos desenvolveram títulos de anticorpos detectáveis pelas diferentes técnicas, havendo, independente da cepa inoculada, diferenças mais frequentes entre os grupos que receberam 105 e 103 bradizoítos. Para IgM a partir do 35o DPI e para a RIFI-IgG e o MAD, a cepa BTU-2 induziu títulos mais elevados que a cepa ME-49. A cepa BTU-2 foi isolada de todas as amostras de cérebro e musculatura estudadas, independente da dose utilizada na infecção dos animais, enquanto que para a cepa ME-49, o parasita foi isolado mais frequentemente do cérebro do que da musculatura, com influência da dose infectante... / Referring to the symptoms and transplacentary transmission, toxoplasmosis in humans and rats is similar and rats can be used as a model for the disease in humans. 7 groups of rats were inoculated, by oral administration, using 105 (Group 1), 104 (Group 2) and 103 (Group 3) Toxoplasma gondii bradyzoits from strain BTU-2, which is highly infective to mice and with 105 (Group 4), 104 (group 5) and 103 (Group 6) T.gondii bradyzoits of strain ME-49, which is less infective to mice and, finally, sterile saline solution (Group 7) as a control one. The animals were observed during 84 days, with weekly blood samples, in order to obtain serum and then submitted to serological tests by modified agglutination test (MAT) and imunofluorecent antibody test (IFAT) to antibodies from subtypes IgG and IgM. At the end of time of observation, the animals were killed and samples of their tissues were obtained in order to reisolate the parasite and to detect the T.gondii DNA by polymerase chain reaction (PCR), using specific oligonucleotids to detect the genes SAG-1, B1, rDNA and to no coding repetitive sequence (REP). The BTU-2 strain was classified by the SAG-2 gene polymorphism. All the rats developed detectable antibodies titers, no matter which strain was used between the techniques. The most frequent differences in the serological results were among the groups that received 105 and those who received 103 bradizoits, no matter the strain used. The BTU-2 strain induced higher titles to IgM since 35 DPI and to IFAT-IgG and MAT than the ME-49 strain. The BTU-2 strain also was isolated from all the brain and muscles samples, no matter the dosage used to infect the animals. When the ME- 49 strain was used, the parasite was isolated more frequently... (Complete abstract, click electronic access below)

O gênero Sarcocystis é constituído por várias espécies que se diferenciam pelas características morfológicas, biológicas e moleculares. Foram relatadas mais de 196 espécies encontradas em mamíferos, aves e répteis e somente 26 dessas espécies possuem o ciclo completo conhecido. Toxoplasma gondii é um parasito intracelular obrigatório, com distribuição geográfica cosmopolita, capaz de infectar uma ampla variedade de mamíferos e aves, inclusive o homem, caracterizando seu potencial zoonótico. Nas últimas décadas, a quantidade de pinguins vindos da Patagônia argentina e chilena, região de nascimento dessas aves, para o litoral brasileiro, onde muitos encalham e são resgatados, tem aumentado significativamente. Pouco se sabe sobre as doenças causadas por protozoários nessas aves. O presente estudo teve como objetivo conhecer aspectos epidemiológicos da infecção por coccídios da família Sarcocystidae em pinguins-de-magalhães (Spheniscus magellanicus), através de análises moleculares e sorológicas. Foram realizadas duas campanhas, uma em 2014 e outra em 2015, com a finalidade de obter amostras de sangue e tecidos dos pinguins que vieram a óbito durante reabilitação no Instituto de Pesquisa e Reabilitação de Animais Marinhos (IPRAM) localizada em Cariacica, Espírito Santo. Foram colhidas 514 amostras de tecidos (músculo=342, coração=86, cérebro=86) de 310 indivíduos. Dos tecidos de 54 pinguins foi realizado o bioensaio em camundongos para o isolamento de T. gondii, mas nenhum isolado foi obtido. Amostras de 310 indivíduos tiveram o DNA extraído para a pesquisa de coccídios da família Sarcocystidae utilizando-se os marcadores 18S rDNA, espaçador interno transcrito 1 (ITS1), codificador de proteínas de superfície (SAG)2, SAG3 e SAG4, subunidade beta da RNA polimerase (RPOB) e citocromo B (CytB). Destas, 16 (3.0%) amostras de músculo peitoral foram positivas para o gênero Sarcocystis spp., quando analisadas pelo marcador 18S, e todas com resultados idênticos. Com o ITS1, RPOB e Ctv. foram confirmadas as espécies de Sarcocystis em 12 amostras, todas idênticas a S. falcatula-like. Com os marcadores SAGs foi possível observar que as sequências não tinham variabilidade genética. Das 145 amostras de soro avaliadas para a presença de anticorpos anti-T. gondii, pelo Teste de Aglutinação Modificado (MAT ≥20), 18 aves foram positivas com títulos de: 20 (7 aves), 40 (9 aves) e 80 (2 aves). Este é o primeiro relato de S. falcatula-like e de anticorpos anti - T. gondii em pinguins-de-magalhães de vida livre. / The genus Sarcocystis is composed of several species that are differentiated by the morphological, biological and molecular characteristics. More than 196 species found in mammals, birds and reptiles have been reported, and only 26 of these species have the complete known cycle. Toxoplasma gondii is an obligate intracellular parasite with cosmopolitan geographic distribution, capable of infecting a wide variety of mammals and birds, including man, characterizing its zoonotic potential. In recent decades, the number of penguins that have come from Argentine and Chilean Patagonia, the region of birth of these birds, to the Brazilian coast, where many of them are stranded and rescued, has increased significantly. Little is known about the diseases caused by protozoa in these birds. The present study had as objective to study epidemiological aspects of coccidia infection of the family Sarcocystidae in Magellanic Penguins (Spheniscus magellanicus), through molecular and serological analyzes. Two campaigns were carried out, one in 2014 and another in 2015, in order to obtain blood and tissue samples from penguins who died during rehabilitation at the Institute of Research and Rehabilitation of Marine Animals (IPRAM) in the municipality of Cariacica, Espírito Santo. Tissue samples (total = 514: muscle = 342, heart = 86, brain = 86) were collected from 310 birds. From the tissues of 54 penguins the mouse bioassay was performed for the isolation of T. gondii, but no isolates were obtained. Samples of 310 individuals had DNA extracted for coccidia from the Sarcocystidae family using the 18S rDNA, Transcribed internal spacer 1(ITS1), surface protein encoder (SAG)2, SAG3, SAG4, beta subunit of RNA polymerase (RPOB) and citocrome B (CytB) markers. Of these, 16 (3.0%) samples, of pectoral muscle, were positive and all were identical to Sarcocystis spp. when analyzed by the 18S marker. With ITS1, RPOB and CytB the Sarcocystis species were confirmed in 12 samples, all identical to S. falcatula-like. With the SAGs it was possible to observe that the sequences had no genetic variability. Of the 145 serum samples evaluated for the presence of anti-T. gondii antibodies by Modified Agglutination Test (MAT ≥20), 18 were positive with titers of: 20 (7 birds), 40 (9 birds) and 80 (2 birds). This is the first report of S. falcatula-like and the presence of antibodies to T. gondii in free-living magellanic penguins.

Toxoplasma gondii is an obligate intracellular parasite that can cause lifethreatening disease in immunocompromised individuals. Host cell invasion is therefore central to the pathology of the disease and parasite survival. Unlike many intracellular pathogens, T. gondii does not enter cells by manipulating the host’s phagocytic machinery; instead, the parasite enters the cell by a process of active penetration. Gliding motility and active penetration are driven by a complex of proteins termed the glideosome. The glideosome consists of four major proteins: TgMyoA, an unconventional myosin XIV, myosin light chain (TgMLC1) and glideosome-associated proteins 45 and 50 (TgGAP45, TgGAP50). TgMyoA has been shown to be essential for parasite motility, but the role of TgMLC1 in regulating myosin function remains unknown. Our lab has identified an inhibitor of T. gondii motility and invasion that results in a post-translational modification (PTM) to TgMLC1. Using molecular genetic and mass spectrometry methods we have shown cysteine 53 and cysteine 58 of TgMLC1 are essential for the modification to occur. To determine if the TgMLC1 PTM alters TgMyoA activity, glideosomes were isolated from DMSO- and 115556-treated parasites. Using an in vitro motility assay we have shown that the TgMyoA actin filament displacement velocities are decreased after 115556 treatment. This is the first evidence that TgMLC1 plays a role in regulating TgMyoA activity. The TgMLC1 PTM is responsible, at least in part, for the invasion and motility defects seen in the parasite after compound treatment. During the course of our investigations we have shown that TgMLC1 is dimethylated on lysine 95. This is an unusual modification for cytosolic proteins and has not been previously described for MLCs. Experiments using parasites expressing a non-methylatable form of TgMLC1 (TgMLC1-K95A) show that dimethylation is not necessary for TgMLC1 peripheral localization, TgMLC1 protein-protein interactions and is not required for TgMyoA activity in vitro. However, TgMLC1-K95A does not appear to be phosphoryalted indicating that TgMLC1 dimethylation is necessary for efficient phosphorylation of TgMLC1. These experiments will provide new insight into the ways in which TgMLC1 regulates this unconventional myosin motor complex.

Thèse de doctorat : Sciences du Vivant. Aspects moléculaires et cellulaires de la Biologie : Strasbourg 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr. 22 p.

Diagnostic tools were developed and utilised to detect Toxoplasma gondii infection in a range of Australian marsupial species and identify epidemiological trends. An ELISA was developed to detect anti-T. gondii IgG in macropod marsupials. When compared with the commercially available MAT (modified agglutination test), the ELISA was in high agreement and yielded a ê coefficient of 0.96. Of 18 western grey kangaroos (Macropus fuliginosus) tested for the presence of T. gondii DNA by PCR, the 9 ELISA positive kangaroos tested PCR positive and the 9 ELISA negative kangaroos tested PCR negative indicating that the ELISA protocol was both highly specific and sensitive and correlated 100% with the more labour intensive PCR assay. A T. gondii seroprevalence study was undertaken on free ranging Australian marsupials. There was a T. gondii seroprevalence of 15.5% (95%CI: 10.7-20.3) in western grey kangaroos located in the Perth metropolitan area. The T. gondii seroprevalence in male western grey kangaroos was significantly less than their female counterparts (p=0.038), which may be related to behavioural differences causing differences in exposure to oocysts or recrudescence of T. gondii infection in pregnant females. Marsupial populations located in islands free from felids had a low overall T. gondii seroprevalence. A case control study determined that marsupials located in areas where felids may roam are 14.20 (95%CI: 1.94-103.66) times more likely to be T. gondii seropositive than marsupials located on felid-free islands. PCR, immunohistochemistry and serological techniques were used to detect T. gondii infection in marsupial dams and their offspring. T. gondii DNA was detected in the pouch young of chronically infected western grey kangaroos and a woylie (Bettongia penicillata). T. gondii DNA was also identified in the mammary gland of the woylie dam suggesting that infection of the woylie pouch young was from suckling milk from the mammary gland. Results of the study demonstrate that vertical transmission of T. gondii occurs in Australian marsupials and may be of importance in the maintenance of T. gondii infection in Australian marsupial populations. Animal tissue and meat from Australia, predominately from Australian marsupials, were screened for T. gondii DNA using PCR primers for the multi-copy, T. gondii specific B1 gene. Sequencing of the B1 gene revealed atypical genotypes in 7 out of 13 samples from Australia. These 7 isolates contained single nucleotide polymorphisms (SNPs) in the B1 gene that could not be matched with known sequences from strains I, II, III and X. Six unique genotypes were identified out of the 7 atypical isolates; two out of the 7 isolates had the same unique sequence at the B1 gene whereas the other 5 isolates each had different combinations of SNPs at the B1 gene. A majority of T. gondii isolates sampled from native Australian marsupials were of an atypical genotype. The discovery of atypical strains of T. gondii in Australia leads to further questions regarding the origin and transmission of these atypical strains. Additional studies linking atypical strains with their clinical manifestation are also warranted.