Evolutionary history of trypanosomes from South American caiman (Caiman yacare) and African crocodiles inferred by phylogenetic analyses using SSU rDNA and gGAPDH genes.

In this study, using a combined data set of SSU rDNA and gGAPDH gene sequences, we provide phylogenetic evidence that supports clustering of crocodilian trypanosomes from the Brazilian Caiman yacare (Alligatoridae) and Trypanosoma grayi, a species that circulates between African crocodiles (Crocodilydae) and tsetse flies. In a survey of trypanosomes in Caiman yacare from the Brazilian Pantanal, the prevalence of trypanosome infection was 35% as determined by microhaematocrit and haemoculture, and 9 cultures were obtained. The morphology of trypomastigotes from caiman blood and tissue imprints was compared with those described for other crocodilian trypanosomes. Differences in morphology and growth behaviour of caiman trypanosomes were corroborated by molecular polymorphism that revealed 2 genotypes. Eight isolates were ascribed to genotype Cay01 and 1 to genotype Cay02. Phylogenetic inferences based on concatenated SSU rDNA and gGAPDH sequences showed that caiman isolates are closely related to T. grayi, constituting a well-supported monophyletic assemblage (clade T. grayi). Divergence time estimates based on clade composition, and biogeographical and geological events were used to discuss the relationships between the evolutionary histories of crocodilian trypanosomes and their hosts.

Phylogeny of snake trypanosomes inferred by SSU rDNA sequences, their possible transmission by phlebotomines, and taxonomic appraisal by molecular, cross-infection and morphological analysis.

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 2.4% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannamyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.

Morphological and molecular diversity and phylogenetic relationships among anuran trypanosomes from the Amazonia, Atlantic Forest and Pantanal biomes in Brazil.

We examined for the presence of trypanosomes in blood samples from 259 anurans (47 species from 8 families), the majority of which were from the Brazilian Amazonia, Atlantic Forest and Pantanal biomes. Trypanosomes were detected by a combination of microhaematocrit and haemoculture methods in 45% of the anurans, and 87 cultures were obtained: 44 from Hylidae, 22 from Leptodactylidae, 15 from Bufonidae, 5 from Leiuperidae and 1 from an unidentified anuran. High morphological diversity (11 morphotypes) was observed among blood trypanosomes from anurans of different species and of the same species as well as among trypanosomes from the same individual. Conversely, morphologically similar trypanosomes were found in anurans from distinct species and biomes. ITS and SSU rDNA polymorphisms revealed high diversity among the 82 isolates examined. Twenty-nine genotypes could be distinguished, the majority distributed in 11 groups. Phylogenetic relationships based on rDNA sequences indicated that isolates from more phylogenetically related anurans are more closely related. Comparison of anuran trypanosomes from Brazil and other countries revealed several new species among the isolates examined in this study. Phylogenetic relationships suggest that host restriction, host switching and overall ecogeographical structure may have played a role in the evolution of the anuran trypanosomes.

Randomly amplified polymorphic DNA analysis of Trypanosoma rangeli and allied species from human, monkeys and other sylvatic mammals of the Brazilian Amazon disclosed a new group and a species-specific marker.

We characterized 14 trypanosome isolates from sylvatic mammals (9 from primates, 1 from sloth, 2 from anteaters and 2 from opossum) plus 2 human isolates of Brazilian Amazon. These isolates were proven to be Trypanosoma rangeli by detection of metacyclic trypomastigotes in the salivary glands of triatomines and by a specific PCR assay. Polymorphism determined by randomly amplified polymorphic DNA (RAPD) revealed that most (12) of the Brazilian T. rangeli isolates from the Amazon differed from those of other geographical regions, thus constituting a new group of T. rangeli. Four Brazilian isolates clustered together with a previously described group (A) that was described as being composed of isolates from Colombia and Venezuela. Isolates from Panama and El Salvador form another group. The isolate from Southern Brazil did not cluster to any of the above-mentioned groups. This is the first study that assesses the genetic relationship of a large number of isolates from wild mammals, especially from non-human primates. A randomly-amplified DNA fragment (Tra625) exclusive to T. rangeli was used to develop a PCR assay able to detect all T. rangeli groups.

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.

Morphological, biochemical and molecular characterization of Herpetomonas samuelpessoai camargoi n. subsp., a trypanosomatid isolated from the flower of the squash Cucurbita moschata.

We report the morphological, biochemical and molecular characteristics of a trypanosomatid isolated from the flower of Cucurbita moschata. Although the trypanosomatid was isolated from a plant, the lack of recognition of Phytomonas-specific molecular markers based on spliced-leader and ribosomal genes as well as by monoclonal antibodies specific for Phytomonas argues against assigning it to this genus. Because the isolate displayed typical opisthomastigote forms in culture, it is assigned to the genus Herpetomonas. Analysis of randomly amplified polymorphic DNA (RAPD) patterns and characterization of ribosomal SSU and ITS markers suggest that it is more closely related to H. samuelpessoai than to any other species. However, the presence of spined flagellates in culture (displaying lateral expansions of the plasma membrane originating near the flagellar pocket) and isolate-specific RAPD fingerprints argue strongly that the trypanosomatid belongs to a new subspecies, for which the name Herpetomonas samuelpessoai camargoi n. subsp. is proposed.

Molecular and morphological studies of Brazilian Trypanosoma evansi stocks: the total absence of kDNA in trypanosomes from both laboratory stocks and naturally infected domestic and wild mammals.

The kinetoplast DNA (kDNA) minicircle molecules of 14 Brazilian stocks of Trypanosoma evansi were studied by morphological approaches (Giemsa and 4'-6'-diamidino-2-phenylindole staining and transmission electron microscopy) and molecular approaches (probing with an oligonucleotide complementary to the minicircle origin of replication and polymerase chain reaction amplification of a minicircle sequence). All methods indicated the absence of both a typical kinetoplast and kDNA minicircles, even in a very small number of parasites of a single stock or in small numbers of copies of molecules per cell. We did not detect any altered kDNA molecules. There were no kDNA molecules in either old or new stocks of T. evansi maintained by successive passages in mice. Similarly, no kDNA minicircles were detected in trypanosomes in blood smears from naturally infected domestic and wild animals. Thus, the total absence of kDNA in Brazilian stocks of T. evansi from both domestic and wild mammals is probably the natural state of Brazilian T. evansi.

Ribosomal and kDNA markers distinguish two subgroups of Herpetomonas among old species and new trypanosomatids isolated from flies.

We examined files of various families for the presence of trypanosomatids. Of 592 insects, 113 (19%) were positive. From these insects, we obtained 42 cultures and selected 14 for further analysis. Seven of the cultures had the characteristics of Herpetomonas species, they displayed typical opisthomastigotes, lacked arginase, possessed a Pvu II restriction site at 360 bp from the 5' end of the small subunit ribosomal gene, and did not possess a Hin dIII site at 1,500 bp from the ribosomal alpha-large subunit 5' end. Hybridization with synthetic oligonucleotides complementary to the sequences flanking the Pvu II site in Herpetomonas samuelpessoai and Herpetomonas muscarum, permitted the distribution of the old species and the 7 isolates into 2 subgroups of Herpetomonas spp. Of the remaining 7 cultures, 4 were probably Leptomonas spp., whereas the other 3, together with Herpetomonas roitmani, seem to constitute a novel group with morphological and molecular characteristics quite distinct from those of Herpetomonas spp. or any other genera of Trypanosomatidae. We also studied some trypanosomatid species of questionable taxonomic status, Leptomonas samueli and Phytomonas davidi yielded results identical to those of Herpetomonas spp., thus confirming their already suspected affiliation to this genus. On the other hand, Herpetomonas anglusteri, Herpetomonas dedonderi, and Herpetomonas mcgheei displayed morphological and molecular characteristics incompatible with their placement in the genus Herpetomonas.

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.

[Susceptibility of Vero cell line to vaccine strains of the measles virus]. / Suscetibilidade da linhagem de células Vero a cepas vacinais do vírus do sarampo.

Vero cells used by distinct measles vaccine control laboratories had their susceptibility to Moraten, Schwarz and Biken CAM-70 vaccine strains assayed. Of a total of 72 lots of measles vaccine whose potency was titrated by microtechnique in two Vero cell samples (Vero IB and Vero INCQS), 25 had been produced with Moraten strain, 24 with Schwarz and 23 with Biken CAM-70. The statistical analysis of the results demonstrated that both Vero cells assayed presented comparable susceptibility to Moraten and Biken CAM-70 strains. As to the Schwarz strain, Vero IB cells were more susceptible than the other cell sample tested, thus confirming the existence of different sensitivities of Vero cells to some measles vaccine strains, or even to viruses derived from the same strain but with different passage histories. An altered cell susceptibility to virus replication may significantly alter the results in potency testing. Such alteration may be caused not only by the adoption of distinct protocols for the maintenance of cell cultures by different control laboratories but also by the methodology followed in the vaccine titration. In order to minimize the differences existing among the results obtained in the potency testing, it is suggested that all control laboratories should use the same protocols for cell culture maintenance as well as for vaccine potency testing.

[Photosensitivity and stability of freeze-dried and/or reconstituted measles vaccines (Biken CAM-70 strain)]. / Fotossensibilidade e termoestabilidade de vacinas contra o sarampo (cepa Biken CAM-70) liofilizadas e/ou reconstituídas para administração.

Three different lots of measles vaccines produced with the Biken CAM-70 virus strain were requested from the central cold store of the Public Health Department of the State of S. Paulo, Brazil, and assays on photosensitivity at 2-8 degrees C, and on stability at 28, 36.5 and 45 degrees C were carried out to find out for how long these vaccines would maintain their minimum potency, established as being 3.70 log10 or 5000 TCID50 (50% tissue culture infective dose) per human dose. The analysis of the adjusted straight regression lines indicated that, with the passage of time, the potency of lyophilized or reconstituted vaccines, as well as of vaccines exposed to or protected from light decreased. Light-exposed vaccines, however, became less potent than vaccines protected from the light. None of the vaccine lots studied, reconstituted and stored at 2-8 degrees C, exhibited homogeneity as to sensitivity to light. When freeze-dried vaccines had their photosensitivity studied at 2-8 degrees C, lots 1 and 2 presented greater thermal degradation when exposed to light than when protected from it. However, in both instances, it was found that potency fell below that taken as minimum for the Biken CAM-70 virus strain. At all other temperatures considered, even when protected from light, lots 1 and 2 did not retain the minimum potency. Lot 3 kept the expected stability for 60 days at 2-8 degrees C when protected from light and for 40 days when unprotected, but its thermal degradation at other temperatures was more intense (28 degrees C: 5 days; 36.5 degrees C: 2 days; 45 degrees C: 0.5 day).(ABSTRACT TRUNCATED AT 250 WORDS)

Sorbitol-gelatin and glutamic acid-lactose solutions for stabilization of reference preparations of measles virus.

The work reported here sought to assess the protection afforded by two stabilizing solutions (sorbitol-gelatin and glutamic acid-lactose) in preserving the potency of freeze-dried Schwarz strain measles virus during storage with a view to the production of reference preparations and working lots of virus suspensions. Stabilized virus suspensions and control suspensions were stored at -70 degrees C or were freeze-dried and stored at -20 degrees C, and their potency was determined over a storage period of 21 months. It was found that the sorbitol-gelatin imparted more satisfactory stability (r = +0.18) to the freeze-dried virus suspensions than did the glutamic acid-lactose. The results also indicate that sorbitol-gelatin, used under the conditions of this study, is an effective stabilizer in the preparation of freeze-dried suspensions of Schwarz strain measles virus employed as reference preparation working lots.