Toxoplasma gondii is a protozoan pathogen of birds and mammals, including humans. The infective stage, the bradyzoite, lives within cysts, which occur predominantly in cells of the central nervous system and skeletal and cardiac muscles, characterizing the chronic phase of toxoplasmosis. In the present study, we employed for the first time primary mouse culture of skeletal muscle cells (SkMC) infected with bradyzoites, as a cellular model for cystogenesis. The interconversion of bradyzoite and tachyzoite was analyzed by immunofluorescence using 2 stage-specific antibodies, i.e., anti-bradyzoite (anti-BAG1) and anti-tachyzoite (anti-SAG1). After 24 hr of interaction only bradyzoites were multiplying, as revealed by anti-BAG1 incubation; interconversion to tachyzoites was not observed. After 48 hr of infection, 2 types of vacuoles were seen, i.e., BAG1+ and SAG1+, indicating the presence of bradyzoites as well as their interconversion to tachyzoites. After 96 hr of infection, BAG1+ vacuoles presented a higher number of parasites when compared to 48 hr, indicating multiplication of bradyzoites without interconversion. Using ultrastructural analysis, bradyzoites were found to adhere to the cell membranes via both the apical and posterior regions or were associated with SkMC membrane expansions. During bradyzoite invasion of SkMC, migration of the rough endoplasmic reticulum (RER) profiles to the parasite invasion site was observed. Later, RER profiles were localized between the mitochondria and parasitophorous vacuole membrane (PVM) that contained the parasite. After 31 days of parasite-host cell infection, RER profiles and mitochondria were not observed in association with the cyst wall. Alterations of the PVM, including increased thickness and electrondensity gain on its inner membrane face, were observed 48 hr after infection. Cystogenesis was complete 96 hr after infection, resulting in the formation of the cyst wall, which displayed numerous membrane invaginations. In addition, an electron-dense granular region enriched with vesicles and tubules was present, as well as numerous intracystic bradyzoites. These results show that the in vitro T. gondii model and SkMC are potential tools for both the study of cystogenesis using molecular approaches and the drug screening action on tissue cysts and bradyzoites.

Angiostrongylus felineus n. sp. (Nematoda, Metastrongyloidea), parasitic in Puma (Herpailurus) yagouaroundi (É. Geoffroy, 1803) (Carnivora, Felidae) from the municipality of Juiz de Fora, Minas Gerais state, Brazil, is described and illustrated herein. Angiostrongylus felineus n. sp. differs from all congeneric species by having the anterior extremity with accentuated cuticular expansion and by smaller size of spicules. This study describes for the first time a species of Angiostrongylus in a wild Felidae in Brazil.

No abstract

Olfactory ensheathing cells (OECs) are a type of specialized glial cell currently considered as having a double function in the nervous system: one regenerative, and another immune. Streptococcus pneumoniae is a major agent of severe infections in humans, including meningitis. It is commonly found in the nasopharynx of asymptomatic carriers, and, under certain still unknown conditions, can invade the brain. We evaluated whether pneumococcal cells recovered from lysed OECs and microglia are able to survive by manipulating the host cell activation. An intracellular-survival assay of S. pneumoniae in OECs showed a significant number of bacterial CFU recovered after 3 h of infection. In contrast, microglia assays resulted in a reduced number of CFU. Electron-microscopy analysis revealed a large number of pneumococci with apparently intact morphology. However, microglia cells showed endocytic vesicles containing only bacterial cell debris. Infection of OEC cultures resulted in continuous NF-κB activation. The IFN-γ-induced increase of iNOS expression was reversed in infected OECs. OECs are susceptible to S. pneumoniae infection, which can suppress their cytotoxic mechanisms in order to survive. We suggest that, in contrast to microglia, OECs might serve as safe targets for pneumococci, providing a more stable environment for evasion of the immune system.

BackgroundToxoplasma gondii belongs to a large and diverse group of obligate intracellular parasitic protozoa. Primary culture of mice skeletal muscle cells (SkMC) was employed as a model for experimental toxoplasmosis studies. The myogenesis of SkMC was reproduced in vitro and the ability of T. gondii tachyzoite forms to infect myoblasts and myotubes and its influence on SkMC myogenesis were analyzed.ResultsIn this study we show that, after 24 h of interaction, myoblasts (61%) were more infected with T. gondii than myotubes (38%) and inhibition of myogenesis was about 75%. The role of adhesion molecules such as cadherin in this event was investigated. First, we demonstrate that cadherin localization was restricted to the contact areas between myocytes/myocytes and myocytes/myotubes during the myogenesis process. Immunofluorescence and immunoblotting analysis of parasite-host cell interaction showed a 54% reduction in cadherin expression at 24 h of infection. Concomitantly, a reduction in M-cadherin mRNA levels was observed after 3 and 24 h of T. gondii-host cell interaction.ConclusionsThese data suggest that T. gondii is able to down regulate M-cadherin expression, leading to molecular modifications in the host cell surface that interfere with membrane fusion and consequently affect the myogenesis process.

Infection by the protozoan parasite

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells

Tachyzoites of Toxoplasma gondii were located inside the nucleus of both skeletal muscle cells infected in vitro and peritoneal exudate cells collected from infected mouse in vivo. Ultrastructural analysis demonstrated that T. gondii invades the nucleus of host cells by the parasite apical region and with constriction of its body. We noted that the rhoptry, a secretory organelle of the parasite that is involved in the host cell invasion mechanism, was empty in the intranuclear T. gondii. The parasites were found in the nuclear matrix without evidence of the vacuolar membrane. Frequently, new parasites invaded host cell nucleus, which was already infected. The significance of this nuclear invasion could reflect an alternative route of T. gondii for its transitory survival or an escape mechanism from the host immune response during the in vivo infection (or both).