Brazilian isolates of Trypanosoma (Megatrypanum) theileri: diagnosis and differentiation of isolates from cattle and water buffalo based on biological characteristics and randomly amplified DNA sequences.

We detected and cultivated isolates of Trypanosoma (Megatrypanum) theileri from cattle and water buffaloes in São Paulo state, southeastern Brazil, which were characterized by comparing morphological, growth and molecular features. Although isolates from cattle and water buffalo were morphologically indistinguishable, they differed in their growth characteristics. A dendrogram based on randomly amplified polymorphic DNA (RAPD) patterns indicated close-genetic relationships among all isolates from both species, which were all tightly clustered together and distant from Megatrypanum species from wild mammals. In addition, isolates within the T. theileri-cluster were clearly segregated into two host-associated groups. The sequence of a synapomorphic RAPD-derived DNA fragment (Tth625), which was shared by all T. theileri trypanosomes from cattle and buffalo but not detected in any of 13 other trypanosome species, was used as target for a conventional T. theileri-specific PCR assay. We also defined RAPD fragments (Tthc606 and Tthb606) that distinguished cattle from buffalo isolates. Thus, distinct growth features and genetic variability distinguished between isolates from cattle and water buffaloes of the same geographic origin, suggesting an association of these isolates with their host species. The trypanosomes from water buffalo reported here are the first T. theileri-like isolates from the Asiatic buffalo (Bubalus bubalis) to be continuously cultured and compared with cattle isolates using biological and molecular methods.

A giant phosphoprotein localized at the spongiome region of Crithidia luciliae thermophila.

A giant protein with an apparent molecular mass of 2,300-kDa was identified in the Triton X-100 soluble fraction of Crithidia luciliae thermophila. Polyclonal antibody raised against this protein reacted by immunoblot analysis with proteins of similar molecular mass in Crithidia fasciculata and Crithidia oncopelti. In addition, the antibody immunoprecipitates the protein either after in vivo phosphorylation with [32P]orthophosphoric acid or after metabolically labeling with [35S]methionine. Indirect immunofluorescence microscopy analysis performed either with fixed or with live parasites showed a single fluorescent spot at the level of the flagellar pocket region. Immunogold electron microscopy of thin sections of the parasite revealed that the antigen is localized at a restricted area of the spongiome, between the contractile vacuole and the flagellar pocket. Furthermore, Triton X-114 phase separation of whole cell membrane proteins, metabolically labeled with [35S]methionine, demonstrated that the giant protein remains in the aqueous phase. These results indicate that this phosphoprotein behaves as a peripheral membrane protein localized at the spongiome region, suggesting that it might be involved in the osmoregulatory process.

A giant protein associated with the anterior pole of a trypanosomatid cell body skeleton.

A megadalton protein was found to be a cytoskeleton component of the promastigote forms of the flagellate Phytomonas serpens. This protein migrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis as a doublet of polypeptides with a molecular mass similar to muscle beta-connectin (titin) 2500-3000 kDa. A polyclonal antibody raised against this protein reacts, by immunoblot analysis, with Phytomonas serpens and two others Phytomonas species. In addition, the Phytomonas serpens protein was immunoprecipitated after being metabolically labeled with [35S]methionine. This antibody did not cross-react with the cytoskeletal proteins of Trypanosoma cruzi, Crithidia luciliae thermophila, Crithidia fasciculata and Leptomonas samueli or with beta-connectin (titin). Indirect immunofluorescence microscopy analysis revealed a punctate fluorescence staining at the anterior region of the parasite's body skeleton. Moreover, immunogold electron microscopy of cytoskeletal preparations and of thin sections of whole cells indicates that the giant protein appears to cap the anterior end of the cell body microtubules at the level of the junctional complex. We suggest that this giant protein may serve as a linker between the cell body skeleton and the flagellum membrane.

Heat shock induction of apoptosis in promastigotes of the unicellular organism Leishmania (Leishmania) amazonensis.

Apoptosis and/or programmed cell death have been described in examples ranging from fungi to man as gene-regulated processes with roles in cell and tissue physiopathology. These processes require the operation of an intercellular communicating network able to deliver alternative signals for cells with different fates and is thus considered a prerogative of multicellular organisms. Promastigotes from Leishmania (Leishmania) amazonensis, when shifted from their optimal in vitro growth temperature (22 degrees C) to the temperature of the mammalian host (37 degrees C), die by a calcium-modulated mechanism. More parasites die in the presence of this ion than in its absence, as detected by a colorimetric assay based on the activity of mitochondrial and cytoplasmic dehydrogenases which measures cell death, independently of the process by which it occurs. A heat shock, unable to induce detectable parasite death (34 degrees C for 1 h), is able to significantly raise the concentration of intracellular free calcium in these cells. Heat-shocked parasites present ultrastructural and molecular features characteristic of cells dying by apoptosis. Morphological changes, observed only in the presence of calcium, are mainly nuclear. Cytoplasmic organelles are preserved. Heat shock is also able to induce DNA cleavage into an oligonucleosomal ladder detected in agarose gels by ethidium bromide staining and autoradiography of [alpha 32P]ddATP-labeled fragments. These results indicate that death by apoptosis is not exclusive of multicellular organisms.

Naturally acquired infections with Leishmania enriettii Muniz and Medina 1948 in guinea-pigs from São Paulo, Brazil.

Two domestic guinea-pigs (Cavia porcellus), bought in Pinheros, São Paulo State, Brazil, were taken by their owners to a farm in the rural district of Capão Bonito, close to the Atlantic Forest, São Paulo, where they both developed tumour-like and ulcerating lesions on the ears. The causative agent was identified as Leishmania (L.) enriettii, based on biological characters and isoenzyme profiles. Sources of the parasite in wild mammals, and the possible sandfly vector species are discussed.

Ultrastructural analysis of the interaction between host cells and Trypanosoma cruzi in experimental chagomas.

The initial interaction between infective forms of Trypanosoma cruzi and host cells in vivo was studied at the ultrastructural level. In order to follow these events, T. cruzi bloodstream forms were inoculated into the cheek-pouch of hamsters--a peculiar region devoid of lymphatic vessels. This region was chosen as injection site because, unlike other regions, trypanosomes remained there and multiplied locally up to 15 days after inoculation. Parasites were detected initially outside cells or inside neutrophils. Only after the first week following inoculation were developing and multiplying trypanosomes seen inside macrophages or other resident cells. Parasites persisted until 15-20 days after inoculation, but by about the 28th day they were no longer seen.

Trypanosoma cruzi amastigotes: development in vitro and infectivity in vivo of the forms isolated from spleen and liver.

Trypanosoma cruzi amastigotes were isolated from liver and spleen of previously infected mice and purified in discontinuous gradients of Metrizamide and Percoll. The amastigotes were well preserved as judged by electron microscopy. The amastigotes were readily interiorized by macrophages and multiplied actively within these cells in vitro. However, their capacity of differentiation was hampered as estimated by the absence of trypomastigotes until day 6 of cultivation. The purified amastigotes were infective for mice but the onset of parasitemia was somewhat delayed and less intense when compared to mice infected with trypomastigotes.

The influence of lymphatic drainage in experimental Trypanosoma cruzi infection.

The rapid disappearance of infective forms of Trypanosoma cruzi from the site of inoculation as well as the initial phase of infection produced by the parasite are not yet fully understood. To investigate this problem we used the hamster as an animal model considering the existence of the cheek pouch--a peculiar region devoid of lymphatic vessels. T. cruzi trypomastigotes were inoculated into the cheek pouch or into the footpad of animals previously infected or not with the same parasite. The results were followed from 3 up to 21 days postinoculation, by histological examination. In the cheek pouch of normal animals a large number of parasites could be seen up to 15 days post-inoculation and the inflammatory infiltrate had a focal distribution. Conversely, in the footpad the infiltrate was diffuse and no parasites could be detected. These observations indicate that the lymphatic system is the main route of T. cruzi dissemination from the site or inoculation. When hamsters were first inoculated in the footpad and 7 days later in the pouch, the inflammatory infiltrate at this point was less aggressive and no parasites could be detected.

Trypanosoma cruzi interaction with macrophages: differences between tissue culture and blodstream forms.

Macrofagos obtidos do peritoneo de camundongos apos estimulo, com peptona, foram cultivados em laminulas, infectados com tripomastigotas das cepas F y Y de T. cruzi, obtidos de cultivo de tecidos ou do sangue de camundongos infectados. Os parasitas, obtidos de cultivo de tecidos, tanto da cepa Y como os da cepa F, sao interiorizados por macrofagos em proporcao muito mais elevada do que os sanguicolas.Parasitas de cultivo de tecidos incubados com soro de camundongos normais, ou soro hiperimune especifico em diluicao subaglutinante, comportam-se esencialmente como parasitas nao opsonizados. Foram observadas diferencas a nivel ultraestrutural na fase inicial de interacao entre macrofagos e tripomastigotas das duas origens. Apos 30 minutos, tripomastigotas de cultivo de tecidos localizam-se em agrupamentos na area de contato com os macrofagos. Enquanto os tripomastigotas sanguicolas estao na maioria das vezes no interior de vacuolos fagociticos largos, apos 3 horas de interacao os tripomastigotas de cultura situam-se em um unico vacuolo estreito. Tanto as formas de cultivo de tecidos quanto os tripomastigotas sanguicolas de cepa Y multiplicam-se em macrofagos; os tripomastigotas sanguicolas da cepa F sao destruidos no interior da celula hospedeira, enquanto os tripomastigotas de cultivo de tecidos desta cepa sao capazes de multiplicar-se

Identification of Leishmania mexicana mexicana in the State of Sao Paulo, Brazil.

Foi identificado pela primeira vez a presenca de L. mexicana mexicana no Estado de Sao Paulo - Vale do Ribeira, a partir de casos humanos e de cao domestico, atraves da caracterizacao biologica (in vivo e in vitro) e bioquimica (enzimas)

Macrophage-trypanosome infection. Dead and live cell scoring.

Descreve-se uma tecnica, usando azul de tripano e posterior fixacao, que permite visualizacao perfeita de T. cruzi no interior de macrofagos e ao mesmo tempo a distincao entre celulas hospedeiras vivas e mortas