Contrasting Disease Progression, Microglia Reactivity, Tolerance, and Resistance to Toxoplasma gondii Infection in Two Mouse Strains.

Our study investigated the innate immune response to Toxoplasma gondii infection by assessing microglial phenotypic changes and sickness behavior as inflammatory response markers post-ocular tachyzoite instillation. Disease progression in Swiss albino mice was compared with the previously documented outcomes in BALB/c mice using an identical ocular route and parasite burden (2 × 105 tachyzoites), with saline as the control. Contrary to expectations, the Swiss albino mice displayed rapid, lethal disease progression, marked by pronounced sickness behaviors and mortality within 11-12 days post-infection, while the survivors exhibited no apparent signs of infection. Comparative analysis revealed the T. gondii-infected BALB/c mice exhibited reduced avoidance of feline odors, while the infected Swiss albino mice showed enhanced avoidance responses. There was an important increase in microglial cells in the dentate gyrus molecular layer of the infected Swiss albino mice compared to the BALB/c mice and their respective controls. Hierarchical cluster and discriminant analyses identified three microglial morphological clusters, differentially affected by T. gondii infection across strains. The BALB/c mice exhibited increased microglial branching and complexity, while the Swiss albino mice showed reduced shrunken microglial arbors, diminishing their morphological complexity. These findings highlight strain-specific differences in disease progression and inflammatory regulation, indicating lineage-specific mechanisms in inflammatory responses, tolerance, and resistance. Understanding these elements is critical in devising control measures for toxoplasmosis.

Three-dimensional architecture of Cyrilia lignieresi gametocyte-stage development inside red blood cells.

The Haemogregarinidae family (Apicomplexa: Adeleina) comprises hemoprotozoa that infect mammals, birds, amphibians, fish, and reptiles. Some morphological characteristics of the Cyrilia lignieresi have been described previously, but the parasite-erythrocyte relationship is still poorly understood. In order to understand the structural architecture of C. lignieresi-infected red blood cells, electron microscopy-based three-dimensional reconstruction was carried out using TEM as well as FIB-SEM tomography. Results showed that development of the macrogametocyte-stage inside the red blood cell is related to an increase in cleft-like structures in the host cell cytoplasm. Furthermore, other aspects related to parasite intraerythrocytic development were explored by 3D visualization techniques. We observed the invagination of a large extension of the Inner Membrane Complex (IMC) on the parasite body, which results from or induces a folding of the posterior end of the parasite. Small tubular structures were seen associated with areas related to IMC folding. Taken together, results provide new information on the remodeling of erythrocytes induced by the protozoan C. lignieresi.

Characterization in vivo and in vitro of a strain of Leishmania (Viannia) shawi from the Amazon Region.

The present study analyses complement resistance, cell surface carbohydrates expression, lipidic composition and morphology in vivo and in vitro, of Leishmania (Viannia) shawi, a parasite identified in the Amazon region, Pará state, in 1989. We demonstrated that promastigotes in the stationary (STAT) growth phase are more resistant to complement lysis than in the logarithmic (LOG) growth phase. Ultrastructural analyses and imidazol technique showed accumulation of lipids in STAT growth phase promastigotes, which was confirmed by biochemical approach. Light and electron microscopy of skin lesion in hamster footpads caused by promastigotes in STAT growth phase, 90 days post inoculation, showed amastigotes inside of macrophage and free in the tissue surrounded by collagen fibers as well as extensive inflammatory reaction with tissue destruction. We also demonstrated, using lectins by agglutination assays and flow cytometry, the presence of fucose, mannose and/or glucose carbohydrate residues on the surface of LOG and STAT promastigotes. The results constitute the first characterization essay combining biochemical and morphological approaches dedicated to LOG and STAT growth phase promastigotes of L. (V) shawi contributing for a better knowledge of this poorly studied species of the New World.

Cutaneous localized annular chromoblastomycosis.

Chromoblastomycosis (CBM) is a difficult-to-treat dermal mycosis characterized by the presence of round, pigmented, sclerotic bodies formed by black fungi found in polymorphic lesions. According to the morphology of a lesion, different clinical types of the disease have been described. We present three patients who each developed a single, 10-cm diameter, 8 to 15-year-old, well-circumscribed, slow-growing, annular, papulosquamous or papulosquamous-verrucous lesion, with no regression despite the use of topical antifungals. Skin scrapings and biopsies confirmed CBM and microculture defined the agent as Fonsecaea pedrosoi. The patients were treated with 200 mg/day of itraconazole for 6-9 months and were discharged after complete regression of the lesions. All were examined after the first and second year of the end of treatment and there were no signs of recurrence. A new clinical type of CBM is described, and itraconazole appears to be effective and safe in curing these patients after no more than 9 months of therapy.

Characterization of Minaçu virus (Reoviridae: Orbivirus) and pathological changes in experimentally infected newborn mice.

Minaçu virus was isolated from Ochlerotatus scapularis (Diptera: Culicidae) in Minaçu, Goiás State, Brazil, in 1996. In attempting characterization of virus serological (hemagluttination inhibition, HI; indirect immunofluorescence assay, IFA), physicochemical [test for deoxycholate acid (DCA) sensitivity; polyacrylamide gel electrophoresis (PAGE)] tests and ultrastructural studies were made. Virus was also assayed in suckling mice after intracerebral inoculation of 0.02 ml and in VERO and C6/36 cells with 0.1 ml of viral suspension containing 10(5) LD50/ml. Inoculated and control systems were observed daily. Every 24 h, one control and two inoculated animals were killed for tissue testing, including histopathological changes by haematoxylin and eosin (HE)-stained sections, which were semi-quantified. Research into viral antigen in the tissues of mice [central nervous system (CNS), liver, heart, lungs, spleen and kidneys] was carried out by the immunohistochemical technique using the peroxidase system. The virus only replicated in VERO cells, with antigen positive by IFA. Positive complement fixation tests were only obtained using antiserum of Minaçu virus. Minaçu virus is DCA resistant; haemagglutinating activity was negative. By electronic microscopy non-enveloped virus particles were 75 nm in diameter. PAGE analysis showed Minaçu virus genome profile with 10 RNA segments. Infected, non-killed animals died 7 days after inoculation. Tissue lesions were observed in all organs, except the lungs. Intense lesions were observed in the CNS and the heart, where neurone and cardiocyte necroses, respectively, were noted. The liver, spleen and kidneys had moderate tissue changes. Viral antigens were more abundant in the CNS and the heart, and absent in the lungs. In conclusion, Minaçu virus belongs to the family Reoviridae, genus Orbivirus.

Blood and inflammatory cells of the lungfish Lepidosiren paradoxa.

A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In summary, this lungfish appears to possess the typical inflammatory granulocytes of teleosts, however, further functional studies are necessary to better understand the polymorphonuclear agranulocyte.

West Nile virus infection of primary mouse neuronal and neuroglial cells: the role of astrocytes in chronic infection.

Primary cultures of embryonic murine neurons and newborn mouse astrocytes were inoculated with West Nile virus (WNV) strain NY385-99 to compare the pathogenesis of WNV infection in these types of CNS cells. Two different outcomes were observed. WNV infection in the neurons was rapidly progressive and destructive; within 5 days, all of the neurons were destroyed through apoptosis. WNV infection in the astrocytes evolved more slowly and did not seem to be highly lethal to the cells. The infected astrocytes continued to produce infectious virus (10(4.6)-10(6.5) PFU/mL) for 114 days, in a permissive, persistent infection. During this period, WNV antigen could be shown in the cytoplasm of the infected astrocytes by immunocytochemical assay, transmission electron microscopy of ultrathin sections, and in the cell culture medium by complement fixation test. Our results with this in vitro experimental murine cell model indicate that astrocytes can develop chronic or persistent infection with WNV, suggesting that these cells may play a role in the maintenance of WNV in the CNS.

Araguari virus, a new member of the family Orthomyxoviridae: serologic, ultrastructural, and molecular characterization.

This paper reports the results of serologic, structural, biochemical, and genetic studies indicating that Araguari virus, a previously unassigned viral agent, is a member of the family Orthomyxoviridae and genus Thogotovirus. Araguari virus has six RNA fragments; biologically, it shares several properties with other viruses in the family Orthomyxoviridae. Nucleotide sequencing of the RNA segments 4 (glycoprotein) and 5 (nucleoprotein) of Araguari virus aligned with the orthomyxoviruses, showing the closest relationship with Thogoto virus (sequence similarity = 61.9% and 69.1%, respectively, for glycoprotein and nucleoprotein), but also sharing a more distant similarity with Dhori and Influenza C viruses, especially for the glycoprotein gene. Based on these results, we propose that Araguari virus should be assigned as a new member of the family Orthomyxoviridae and genus Thogotovirus.

Cytochemical and functional characterization of blood and inflammatory cells from the lizard Ameiva ameiva.

The fine structure and differential cell count of blood and coelomic exudate leukocytes were studied with the aim to identify granulocytes from Ameiva ameiva, a lizard distributed in the tropical regions of the Americas. Blood leukocytes were separated with a Percoll cushion and coelomic exudate cells were obtained 24 h after intracoelomic thioglycollate injection. In the blood, erythrocytes, monocytes, thrombocytes, lymphocytes, plasma cells and four types of granulocytes were identified based on their morphology and cytochemistry. Types I and III granulocytes had round intracytoplasmic granules with the same basic morphology; however, type III granulocyte had a bilobued nucleus and higher amounts of heterochromatin suggesting an advance stage of maturation. Type II granulocytes had fusiformic granules and more mitochondria. Type IV granulocytes were classified as the basophil mammalian counterpart based on their morphology and relative number. Macrophages and granulocytes type III were found in the normal coelomic cavity. However, after the thioglycollate injection the number of type III granulocyte increased. Granulocytes found in the coelomic cavity were related to type III blood granulocyte based on the morphology and cytochemical localization of alkaline phosphatase and basic proteins in their intracytoplasmic granules. Differential blood leukocyte counts showed a predominance of type III granulocyte followed by lymphocyte, type I granulocyte, type II granulocyte, monocyte and type IV granulocyte. Taken together, these results indicate that types I and III granulocytes correspond to the mammalian neutrophils/heterophils and type II to the eosinophil granulocytes.

Ultrastructural, antigenic and physicochemical characterization of the Mojuí dos Campos (Bunyavirus) isolated from bat in the Brazilian Amazon region.

The Mojuí dos Campos virus (MDCV) was isolated from the blood of an unidentified bat (Chiroptera) captured in Mojuí dos Campos, Santarém, State of Pará, Brazil, in 1975 and considerated to be antigenically different from other 102 arboviruses belonging to several antigenic groups isolated in the Amazon region or another region by complement fixation tests. The objective of this work was to develop a morphologic, an antigenic and physicochemical characterization of this virus. MDCV produces cytopathic effect in Vero cells, 24 h post-infection (p.i), and the degree of cellular destruction increases after a few hours. Negative staining electron microscopy of the supernatant of Vero cell cultures showed the presence of coated viral particles with a diameter of around 98 nm. Ultrathin sections of Vero cells, and brain and liver of newborn mice infected with MDCV showed an assembly of the viral particles into the Golgi vesicles. The synthesis kinetics of the proteins for MDCV were similar to that observed for other bunyaviruses, and viral proteins could be detected as early as 6 h p.i. Our results reinforce the original studies which had classified MDCV in the family Bunyaviridae, genus Bunyavirus as an ungrouped virus, and it may represent the prototype of a new serogroup.

Comparison of Fonsecaea pedrosoi sclerotic cells obtained in vivo and in vitro: ultrastructure and antigenicity.

The parasitic form of Fonsecaea pedrosoi from the hyperkeratotic layer of the skin was obtained from four patients with chromoblastomycosis. Primary cultures containing hyphae and conidia were successfully converted into sclerotic cells in the presence of 800 microM propranolol and low pH as described before. The morphology of sclerotic cells of F. pedrosoi obtained in vivo and in vitro was analyzed by light and electron microscopy. Their antigenicity was also compared by immunofluorescence microscopy and ELISA assays, using serum samples from untreated patients infected with F. pedrosoi. Due to the similarity of the sclerotic cells obtained in vivo and in vitro, the latter can be more adequately in studies of host-parasite interactions in chromoblastomycosis.

Comparison of Fonsecaea pedrosoi sclerotic cells obtained in vivo and in vitro: ultrastructure and antigenicity

The parasitic form of Fonsecaea pedrosoi from the hyperkeratotic layer of the skin was obtained from four patients with chromoblastomycosis. Primary cultures containing hyphae and conidia were successfully converted into sclerotic cells in the presence of 800 microM propranolol and low pH as described before. The morphology of sclerotic cells of F. pedrosoi obtained in vivo and in vitro was analyzed by light and electron microscopy. Their antigenicity was also compared by immunofluorescence microscopy and ELISA assays, using serum samples from untreated patients infected with F. pedrosoi. Due to the similarity of the sclerotic cells obtained in vivo and in vitro, the latter can be more adequately in studies of host-parasite interactions in chromoblastomycosis