Molecular detection of Toxoplasma gondii, Neospora caninum and Sarcocystis spp in tissues of Sus scrofa slaughtered in southern Brazil / Detecção molecular de Toxoplasma gondii, Neospora caninum e Sarcocystis spp. em tecidos de Sus scrofa abatidos no Sul do Brasil

The aim of this study was to determine the presence of deoxyribonucleic acid (DNA) from Toxoplasma gondii, Sarcocystis spp. and Neospora caninum, in tissues of wild boars slaughtered in southern Brazil. A total of 156 samples were collected from different organs of 25 wild boars, and DNA from at least one of the protozoa investigated was detected in 79 samples. To differentiate between infectious agents, restriction fragment length polymorphism was performed using the restriction enzymes DdeI and HpaII. For N. caninum, conventional PCR was performed with specific primers. The DNA of at least one of the studied pathogens was detected in each animal: 26.58% for T. gondii, 68.36% for Sarcocystis spp. and 5.06% for N. caninum. Coinfection between T. gondii and Sarcocystis spp. occurred in 14 animals, between T. gondii and N. caninum in only one male animal, between Sarcocystis spp. and N. caninum in a female, while co-infection with the three agents was equally observed in only one male animal. Considering the high frequency of detection and its zoonotic risk, especially T. gondii, it appears that wild boars can be potential sources of transmission of infectious agents and the adoption of monitoring measures in these populations should be prioritized.(AU)

O objetivo deste estudo foi determinar a presença de ácido desoxirribonucléico (DNA) de Toxoplasma gondii, Sarcocystis spp. e Neospora caninum, em tecidos de javalis abatidos no sul do Brasil. Foram coletadas 156 amostras de diferentes órgãos de 25 javalis, sendo detectado o DNA de pelo menos um dos protozoários pesquisados em 79 amostras. Para diferenciar entre os agentes infecciosos, o polimorfismo do comprimento do fragmento de restrição, foi realizado usando-se as enzimas de restrição DdeI e HpaII. Para N. caninum, a PCR convencional foi realizada com "primers" específicos. O DNA de pelo menos um dos patógenos estudados foi detectado em cada animal: 26,58% para T. gondii, 68,36% para Sarcocystis spp. e 5,06% para N. caninum. Coinfecção entre T. gondii e Sarcocystis spp. ocorreu em 14 animais; entre T. gondii e N. caninum em apenas um animal macho; entre Sarcocystis spp. e N. caninum em uma fêmea, enquanto a coinfecção com os três agentes foi observada igualmente em apenas um animal macho. Considerando-se a alta frequência de detecção e seu risco zoonótico, especialmente T. gondii, constata-se que os javalis podem ser potenciais fontes de transmissão de agentes infecciosos, e a adoção de medidas de monitoramento nessas populações devem ser priorizadas.(AU)

Copaifera spp. oleoresins impair Toxoplasma gondii infection in both human trophoblastic cells and human placental explants.

The combination of pyrimethamine and sulfadiazine is the standard care in cases of congenital toxoplasmosis. However, therapy with these drugs is associated with severe and sometimes life-threatening side effects. The investigation of phytotherapeutic alternatives to treat parasitic diseases without acute toxicity is essential for the advancement of current therapeutic practices. The present study investigates the antiparasitic effects of oleoresins from different species of Copaifera genus against T. gondii. Oleoresins from C. reticulata, C. duckei, C. paupera, and C. pubiflora were used to treat human trophoblastic cells (BeWo cells) and human villous explants infected with T. gondii. Our results demonstrated that oleoresins were able to reduce T. gondii intracellular proliferation, adhesion, and invasion. We observed an irreversible concentration-dependent antiparasitic action in infected BeWo cells, as well as parasite cell cycle arrest in the S/M phase. The oleoresins altered the host cell environment by modulation of ROS, IL-6, and MIF production in BeWo cells. Also, Copaifera oleoresins reduced parasite replication and TNF-α release in villous explants. Anti-T. gondii effects triggered by the oleoresins are associated with immunomodulation of the host cells, as well as, direct action on parasites.

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure.

Toxoplasma gondii Hsp90: potential roles in essential cellular processes of the parasite.

Hsp90 is a widely distributed and highly conserved molecular chaperone that is ubiquitously expressed throughout nature, being one of the most abundant proteins within non-stressed cells. This chaperone is up-regulated following stressful events and has been involved in many cellular processes. In Toxoplasma gondii, Hsp90 could be linked with many essential processes of the parasite such as host cell invasion, replication and tachyzoite-bradyzoite interconversion. A Protein-Protein Interaction (PPI) network approach of TgHsp90 has allowed inferring how these processes may be altered. In addition, data mining of T. gondii phosphoproteome and acetylome has allowed the generation of the phosphorylation and acetylation map of TgHsp90. This review focuses on the potential roles of TgHsp90 in parasite biology and the analysis of experimental data in comparison with its counterparts in yeast and humans.

Envolvimento de mitocôndrias e do retículo endoplasmático da célula muscular esquelética na cistogênese de Toxoplasma gondii / Involvement of mitochondria and endoplasmic reticulum in skeletal muscle cell on Toxoplasma gondii cystogenesis

T. gondii é parasito intracelular obrigatório, agente etiológico datoxoplasmose, doença com ampla distribuição mundial. Os transtornos mais severos(fase aguda) acometem pacientes imunocomprometidos. Hospedeirosimunologicamente sadios, uma vez infectados, apresentam cistos teciduais (fasecrônica) de modo perene. Estudos sugerem que por possuírem um eficientemetabolismo energético, os principais tecidos eleitos para a cistogênese do T.gondii, são o nervoso e o muscular esquelético. O presente trabalho dedicou-se aoestudo das associações de mitocôndrias e do retículo endoplasmßtico (RE) àmembrana do vacúolo parasitóforo (MVP) e à parede cística. Para tanto, foramutilizados bradizoítos e taquizoítos da cepa ME49 (tipo II) e culturas primßrias decélula muscular esquelética (CME) e da linhagem C2C12. Nossas estratégiasmetodológicas contemplaram microscopia de fluorescência, microscopia eletrônicade transmissão, respirometria de alta resolução e ensaios de efeito de um inibidor dafosforilação oxidativa (ISA-34) sobre a cistogênese. Os dados obtidos apontam aocorrência de: (i) associações entre mitocôndrias com a parede cística; (ii) aspectospeculiares ultraestruturais decorrentes de associações entre mitocôndrias e RE(rugoso e liso) da CME com a MVP de vacúolos contendo bradizoítos; (iii)manutenção do metabolismo mitocondrial da CME pelo T. gondii, durante a fasecrônica; (iv) efeito inibitório do composto ISA-34 sobre o desenvolvimento de cistosteciduais. Estes resultados, além de iniciarem uma linha de pesquisa inédita arespeito das respostas do metabolismo energético da CME frente à cistogênese deT. gondii, também abrem novas perspectivas para uma terapia alternativa voltadapara a fase crônica da toxoplasmose...

Toxoplasma gondii is an obligatory intracellular parasite, agent oftoxoplasmosis, disease with a worldwide distribution. The most severe disorders(acute phase) affect immunocompromised patients. Immunologically healthyindividuals, once infected, develop tissue cysts (chronic phase) that can persist forthe host life span. Studies suggest that an efficient energetic metabolism, as innervous and skeletal muscle tissues, leads to the development of T. gondiicystogenesis. The present work aims the study of the association of skeletal musclecell (SkMC) mitochondria and endoplasmic reticulum (ER) to the parasitophorousvacuole membrane (PVM) and to the cyst wall (CW). Bradyzoites and tachyzoitesfrom ME49 strain (type II) of T. gondii and SkMC cultures and C2C12 cell line wereused. The methodological strategies employed were fluorescence microscopy,transmission electron microscopy, high-resolution respirometry and assay using ISA-34, an inhibitor of oxidative phosphorylation. Our data point out: (i) associationsbetween mitochondria and CW; (ii) ultrastructural aspects of the association of SkMCmitochondria and ER (rough and smooth) with PVM of bradyzoite-containingvacuoles; (iii) maintenance of SkMC mitochondrial metabolism by T. gondii and, (iv)inhibitory effect of ISA-34 on the tissue cysts development. These results stimulatefurther investigation concerning the response of SkMC energy metabolism duringcystogenesis of T. gondii and also open novel perspectives for an alternative therapyagainst toxoplasmosis chronic phase...

Efeitos in vitro e de 3-bromopiruvato e atovaquona na infecção por Toxoplasma gondii em células LLC-MK2 / Effect in vitro of 3-bromopyruvate and atovaquone on infection of toxoplasma gondii in llc-mk2 cells

Toxoplasma gondii causa uma infecção comumente assintomática, porémpode se apresentar de forma grave durante a gravidez e em pacientesimunocomprometidos. A terapia atual para a toxoplasmose é restrita contrataquizoítos, e possuem pouco ou nenhum efeito sobre bradizoítos, que são mantidosem cistos teciduais como fonte recrudescente da infecção. Com isso, novasalternativas terapêuticas vêm sendo propostas, como o uso da Atovaquona, queapresentou alguma eficácia sobre taquizoítos e bradizoítos em cistos teciduais.Neste trabalho, foi estudado o efeito de 3-BrPA, um composto utilizado em testessobre células cancerígenas, durante a interação de células LLC-MK2 infectadas comtaquizoítos da cepa RH de T. gondii. Quanto à célula hospedeira não se observouefeito do composto sobre a proliferação e viabilidade celular. A avaliação dainterferência de 3-BrPA sobre o crescimento in vitro do T. gondii evidenciou umaredução na proliferação intracelular do parasito de cerca de 55 por cento após 24 h detratamento e 61 por cento após 48 h. O desenvolvimento intracelular do parasito, analisadopor MEV, apresentou características morfológicas comumente encontradas emcistos teciduais. A incubação das culturas com lectina DBA confirmou odesenvolvimento de cistos e por MET se evidenciou a presença de bradizoítos. Alémdisso, foram revelados parasitos degradados e a influência do composto sobre aendodiogenia. Outra abordagem adotada foi o tratamento de culturas infectadas coma combinação de 3-BrPA e Atovaquona, que resultou em uma redução de parasitosintracelulares de 73 por cento após 24 h de tratamento e 71 por cento após 48 h, em comparação aocontrole, além da ausência da formação de parede cística nessas culturas...

Toxoplasma gondii usually causes an asymptomatic infection, but it can present severityduring pregnancy and in immunocompromised patients. Current therapies fortoxoplasmosis are restricted only against tachyzoites, and have little or no effect onbradyzoites, which are kept in tissue cysts like source of the infection recrudescent.Consequently, new therapeutic alternatives have been proposed, as the use ofAtovaquone that showed some efficacy against tachyzoites and bradyzoites in tissuecysts. In this work, we propose to study the effect of 3-BrPA, a compound that is beingtested against cancer cells, on the infection of LLC-MK2 cells with tachyzoites of T.gondii (RH strain). No effect of 3-BrPAon host cell proliferation and viability wasobserved. Evaluation of 3-BrPA interference on in vitro growth of T. gondii showed areduction in intracellular parasite proliferation about 55 por cento after 24 h of treatment, and61 por cento after 48 h. Intracellular development of parasite, analyzed by SEM, showedmorphological characteristics commonly found in tissue cysts. Incubation of cultureswith DBA lectin confirmed the development of cysts and TEM showed the presence ofbradyzoites. Moreover, we revealed the presence of degraded parasites and theinfluence of compound on endodyogeny. Another approach used was the treatment ofinfected cultures with combination of 3-BrPA and Atovaquone. This resulted in thereduction of intracellular parasites of 73 por cento after 24 h of treatment and 71 percent after 48 h,compared to control, besides the absence of cyst wall formation in these cultures...

Toxoplasmosis sistémica en un canguro rojo (Macropus rufus) cautivo / Systemic toxoplamosis in a captive red kangaroo (Macropus rufus)

Los objetivos del estudio fueron presentar y documentar los hallazgos histopatológicos de toxoplasmosis sistémica en un canguro rojo (Macropus rufus) mantenido en cautiverio donde se describen los hallazgos macro y microscópicos encontrados y los análisis adicionales realizados. En el laboratorio de histopatología animal (Universidad de los Llanos) se recibieron muestras de tejidos fijados en formol tamponado, al 10% que procedían de un ejemplar macho de Macropus rufus, de ocho años de edad y 50 kg de peso corporal. Las muestras se procesaron mediante métodos rutinarios para microscopía óptica. Los cortes histológicos de 3-4 mm de grosor se colorearon con Hematoxilina-Eosina (H&E) y se realizó en algunos cortes la tinción de Ácido Periódico Schiff (PAS), PCR e IHQ. Al análisis histopatológico se encontró una toxoplasmosis sistémica asociada a quistes de protozoarios con inmunoreactividad positiva para T. gondii. La detección de T gondii en tejidos en formalina fue hecha usando dos ensayos de PCR que señalaban segmentos de ADN de diferentes secuencias repetitivas encontradas en T gondii y la IHQ confirmo lo hallado por PCR. Histopatológicamente se diagnosticó infección crónica por protozoarios eucoccideos de la familia Sarcocystidae. El diagnóstico etiológico fue de toxoplasmosis.

The objetives of this study were to present and document the hystopathologycal findings of systemic toxoplamosis in a captive red kangaroo (Macropus rufus) which described macro and microscopic findings of the hystopathological analysis. In the laboratory of animal histopathology (Universidad de los Llanos) formalin fixed tissue specimens were received, from a captive male Macropus rufus, who was eight years old and weighed 50 kg. The samples were processed by usual methods for optical microscopy. The histological sections of 3-4 mm thick were colored with Hematoxilin-Eosin (H&E) and then some samples stained with Periodic Acid Schiff (PAS), and processed by PCR and IHQ. Once the histopathological analysis was performed systemic toxoplasmosis was associated to protozoa cysts immunoreactives to T. gondii. The molecular detection of T. gondii in formalin fixed tissues was made using two PCR tests and confirmated by IHQ. Histopathologically a chronic infection by an eucoccideo protozoa from the Sarcocystidae family was diagnosed. The etiologic diagnosis was toxoplasmosis.

Toxoplasma gondii Sis1-like J-domain protein is a cytosolic chaperone associated to HSP90/HSP70 complex.

Toxoplasma gondii is an obligate intracellular protozoan parasite in which 36 predicted Hsp40 family members were identified by searching the T. gondii genome. The predicted protein sequence from the gene ID TGME49_065310 showed an amino acid sequence and domain structure similar to Saccharomyces cerevisiae Sis1. TgSis1 did not show differences in its expression profile during alkaline stress by microarray analysis. Furthermore, TgSis1 showed to be a cytosolic Hsp40 which co-immunoprecipitated with T. gondii Hsp70 and Hsp90. Structural modeling of the TgSis1 peptide binding fragment revealed structural and electrostatic properties different from the experimental model of human Sis1-like protein (Hdj1). Based on these differences; we propose that TgSis1 may be a potentially attractive drug target for developing a novel anti-T. gondii therapy.

Effect of macrophage migration inhibitory factor (MIF) in human placental explants infected with Toxoplasma gondii depends on gestational age.

Because macrophage migration inhibitory factor (MIF) is a key cytokine in pregnancy and has a role in inflammatory response and pathogen defense, the objective of the present study was to investigate the effects of MIF in first- and third-trimester human placental explants infected with Toxoplasma gondii. Explants were treated with recombinant MIF, IL-12, interferon-γ, transforming growth factor-ß1, or IL-10, followed by infection with T. gondii RH strain tachyzoites. Supernatants of cultured explants were assessed for MIF production. Explants were processed for morphologic analysis, immunohistochemistry, and real-time PCR analysis. Comparison of infected and stimulated explants versus noninfected control explants demonstrated a significant increase in MIF release in first-trimester but not third-trimester explants. Tissue parasitism was higher in third- than in first-trimester explants. Moreover, T. gondii DNA content was lower in first-trimester explants treated with MIF compared with untreated explants. However, in third-trimester explants, MIF stimulus decreased T. gondii DNA content only at the highest concentration of the cytokine. In addition, high expression of MIF receptor was observed in first-trimester placental explants, whereas MIF receptor expression was low in third-trimester explants. In conclusion, MIF was up-regulated and demonstrated to be important for control of T. gondii infection in first-trimester explants, whereas lack of MIF up-regulation in third-trimester placentas may be involved in higher susceptibility to infection at this gestational age.

Understanding mechanisms and the role of differentiation in pathogenesis of Toxoplasma gondii: a review.

Parasite differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission of the intracellular protozoan pathogen Toxoplasma gondii. The presence of bradyzoite-containing cysts in human hosts and their subsequent rupture can cause life-threatening recrudescence of acute infection in the immunocompromised and cyst formation in other animals contributes to zoonotic transmission and widespread dissemination of the parasite. In this review, we discuss the evidence showing how the clinically relevant process of bradyzoite differentiation is regulated at both transcriptional and post-transcriptional levels. Specific regulatory factors implicated in modulating bradyzoite differentiation include promoter-based cis-elements, epigenetic modifications and protein translation control through eukaryotic initiation factor -2 (eIF2). In addition to a summary of the current state of knowledge in these areas we discuss the pharmacological ramifications and pose some questions for future research.

Understanding mechanisms and the role of differentiation in pathogenesis of Toxoplasma gondii: a review

Parasite differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission of the intracellular protozoan pathogen Toxoplasma gondii. The presence of bradyzoite-containing cysts in human hosts and their subsequent rupture can cause life-threatening recrudescence of acute infection in the immunocompromised and cyst formation in other animals contributes to zoonotic transmission and widespread dissemination of the parasite. In this review, we discuss the evidence showing how the clinically relevant process of bradyzoite differentiation is regulated at both transcriptional and post-transcriptional levels. Specific regulatory factors implicated in modulating bradyzoite differentiation include promoter-based cis-elements, epigenetic modifications and protein translation control through eukaryotic initiation factor -2 (eIF2). In addition to a summary of the current state of knowledge in these areas we discuss the pharmacological ramifications and pose some questions for future research.

Toxoplasma gondii Hsp20 is a stripe-arranged chaperone-like protein associated with the outer leaflet of the inner membrane complex.

BACKGROUND INFORMATION: Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. T. gondii has five sHsps [small Hsps (heat-shock proteins)] located in different subcellular compartments. Among them, Hsp20 showed to be localized at the periphery of the parasite body. sHsps are widespread, constituting the most poorly conserved family of molecular chaperones. The presence of sHsps in membrane structures is unusual. RESULTS: The localization of Hsp20 was further analysed using high-resolution fluorescent light microscopy as well as electron microscopy, which revealed that Hsp20 is associated with the outer surface of the IMC (inner membrane complex), in a set of discontinuous stripes following the same spiralling trajectories as the subpellicular microtubules. The detergent extraction profile of Hsp20 was similar to that of GAP45 [45 kDa GAP (gliding-associated protein)], a glideosome protein associated with the IMC, but was different from that of IMC1 protein. Although we were unable to detect interacting protein partners of Hsp20 either in normal or stressed tachyzoites, an interaction of Hsp20 with phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate phospholipids could be observed. CONCLUSIONS: Hsp20 was shown to be associated with a specialized membranous structure of the parasite, the IMC. This discontinuous striped-arrangement is unique in T. gondii, indicating that the topology of the outer leaflet of the IMC is not homogeneous.

Antiproliferative activities of two novel quinuclidine inhibitors against Toxoplasma gondii tachyzoites in vitro.

OBJECTIVES: To study the antiproliferative effects of ER119884 and E5700, two quinuclidine-based inhibitors of squalene synthase (SQS), against Toxoplasma gondii tachyzoites in epithelial cells. METHODS: The antiproliferative effects of the quinuclidine derivatives, alone or in combination with epiminolanosterol or antifolates, were analysed, resulting in the construction of isobolograms. The ultrastructure of treated tachyzoites was analysed by transmission electron microscopy. RESULTS: The quinuclidine derivatives demonstrated selective anti-T. gondii activity, arresting parasite growth with IC50 values of 0.66 and 0.23 microM for ER119884 and E5700, respectively, after 24 h of interaction and 0.44 and 0.19 microM after 48 h of interaction. Both compounds induced remarkable alterations in the parasite ultrastructure, such as mitochondrial swelling and the presence of autophagosome-like structures, after 24 h of treatment. Combination of these quinuclidine derivatives with the antifolates sulfadiazine and pyrimethamine produced a synergic effect. When epiminolanosterol was combined with E5700, the effect observed was synergic, whereas the combination with ER119884 produced no interaction. CONCLUSIONS: E5700 and ER119884 demonstrated selective activity against T. gondii tachyzoites and are a possible alternative to be used in association with the current therapy. The ultrastructural alterations observed suggest a possible interference with lipid metabolism.

Differential subcellular localization of members of the Toxoplasma gondii small heat shock protein family.

The results of this study describe the identification and characterization of the Toxoplasma gondii alpha-crystallin/small heat shock protein (sHsp) family. By database (www.toxodb.org) search, five parasite sHsps (Hsp20, Hsp21, Hsp28, Hsp29, and the previously characterized Hsp30/Bag1) were identified. As expected, they share the homologous alpha-crystallin domain, which is the key characteristic of sHsps. However, the N-terminal segment of each protein contains unique characteristics in size and sequence. Most T. gondii sHsps are constitutively expressed in tachyzoites and fully differentiated bradyzoites, with the exception of Hsp30/Bag1. Interestingly, by subcellular localization we observed that T. gondii sHsps are located in different compartments. Hsp20 is located at the apical end of the cell, Hsp28 is located inside the mitochondrion, Hsp29 showed a membrane-associated labeling, and Hsp21 appeared throughout the cytosol of the parasites. These particular differences in the immunostaining patterns suggest that their targets and functions might be different.

Severe acute disseminated toxoplasmosis acquired by an immunocompetent patient in French Guiana.

Disseminated toxoplasmosis during acute infections has rarely been observed in immunocompetent patients. We report a case of severe acute disseminated toxoplasmosis acquired by an immunocompetent patient in French Guiana and review the literature.

Redistribution of parasite and host cell membrane components during Toxoplasma gondii invasion.

The initial association of tachyzoites of Toxoplasma gondii with a host cell induces an endocytic process which leads to the formation of a vacuole known as the parasitophorous vacuole (PV). We analyzed the parasite-host cell interaction process using either parasites or host cells whose membrane was previously labeled with probes specific for proteins, sialoglycoconjugates and lipids, and then allowed to interact for periods varying from 5 minutes to 24 hours. The fate of the fluorescents probes was followed by confocal laser scanning microscopy. In host cells previously labeled with PKH26, FITC-Thiosemicarbazide or DTAF, which label membrane proteins, siloglycoconjugates and lipids, respectively, a uniform labeling of the cell surface was observed before interaction. When allowed to interact with T. gondii, labeling for PKH26 and DTAF, but not for FITC-Thiosemicarbazide, was observed initially at the region of contact between the two cells and subsequently on the membrane lining the PV and the intravacuolar parasites. These observations show that some, but not all, membrane components contribute to the formation of the PV membrane. Previously labeled parasites attach to the host cell surface but lose the fluorescent probes during the invasion process so that no labeled parasites were seen within the PV. These observations point to the existence of a dynamic process of membrane-associated components of the parasite and host cell during the interaction process.

Toxoplasmose ocular: utilizaçäo de reaçäo imuno-histoquimica para diagnóstico / Ocular toxoplasmosis: utilization of immunohistochemistry in the diagnosis of ocular toxoplasmosis

O diagnóstico de toxoplasmose ocular baseia-se geralmente em dados clinicos e laboratoriais bastante conhecidos. Porém, casos com apresentaçäo clinica atipica ocorrem, principalmente em pacientes imunossuprimidos, trazendo entäo ao médico clinico dificuldades diagnósticas. Os autores descrevem o caso de uma paciente do sexo feminino, com uma doenca ocular bilateral, de tres anos de duracao, em evoluçäo e sem diagnóstico etiológico estabelecido. Após exaustiva investigaçäo clinica e laboratorial e utilizaçäo de vários medicamentos, sem sucesso clinico, optou-se pela enucleaçäo de olho esquerdo, ja atrofico...

Kinematic analysis of Toxoplasma gondii motility.

Toxoplasma gondii tachyzoites execute a complex and little understood combination of rapid movements to reach and penetrate human or other animals cells. In the present study, computer-assisted simulation was used to quantitatively analyze the motility of these parasites in three-dimensional space with spatial and temporal resolutions in the micrometer and subsecond ranges. A digital model based on electron-micrographs of a serially sectioned tachyzoite was animated according to a videomicrographed sequence of a characteristic repetitive movement. Keyframe animation defined over 150 frames by a total of 36 kinematic parameters for specific motions, of both the whole model and particular domains, resulted in a real-time life-like simulation of the videorecorded tachyzoite movement. The kinematic values indicate that a full revolution of the model is composed of three half-turns accomplished in nearly 5 s with two phases: a relatively slow 180 degrees tilting with regard to the substratum plane, followed by fast (over 200 degrees/s) spinning almost simultaneous with pivoting around the posterior end, each clockwise and for about 180 degrees. Maximal flexing of the body, as well as bowing and retraction of its anterior end, occur at midway during the tilting phase. An estimated 70 degrees. clockwise torsion of the body seems to precede the spinning-pivoting phase. The results suggest the operation of two basic forces in the motility of T. gondii tachyzoites: (1) a clockwise torque that causes torsion, spinning, and pivoting; and (2) a longitudinal pull that contracts, bends and tilts the parasite. We discuss the possibility that both of these forces might result from the action of an actin-myosin system enveloping the twisted framework of microtubules characteristic of these organisms.

Separation of toxoplasma cysts from brain tissue and liberation of viable bradyzoites.

A method was developed to separate Toxoplasma gondii cysts from mouse brain tissue and to liberate intact bradyzoites from cysts. Brains were blended in a Waring blender with 20% dextran solution and the homogenate was centrifuged at 4,000 g for 10 min. Cysts present in the sediment were digested by adding an equal amount of a solution containing 1 g NaCl, 1.4 ml HCl, and 1 mg pepsin (1:60,000 assay activity) per 100 ml water, and incubated at 37 C for 1 min. This method is harmless for bradyzoites, as tested by bioassays in mice. It compares favorably with published methods for separating cysts that require 3 centrifugations to achieve similar results.