An appraisal of the taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and Endotrypanum.

We propose a taxonomic revision of the dixenous trypanosomatids currently classified as Endotrypanum and Leishmania, including parasites that do not fall within the subgenera L. (Leishmania) and L. (Viannia) related to human leishmaniasis or L. (Sauroleishmania) formed by leishmanias of lizards: L. colombiensis, L. equatorensis, L. herreri, L. hertigi, L. deanei, L. enriettii and L. martiniquensis. The comparison of these species with newly characterized isolates from sloths, porcupines and phlebotomines from central and South America unveiled new genera and subgenera supported by past (RNA PolII gene) and present (V7V8 SSU rRNA, Hsp70 and gGAPDH) phylogenetic analyses of the organisms. The genus Endotrypanum is restricted to Central and South America, comprising isolates from sloths and transmitted by phlebotomines that sporadically infect humans. This genus is the closest to the new genus Porcisia proposed to accommodate the Neotropical porcupine parasites originally described as L. hertigi and L. deanei. A new subgenus Leishmania (Mundinia) is created for the L. enriettii complex that includes L. martiniquensis. The new genus Zelonia harbours trypanosomatids from Neotropical hemipterans placed at the edge of the Leishmania-Endotrypanum-Porcisia clade. Finally, attention is drawn to the status of L. siamensis and L. australiensis as nomem nudums.

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.

Infection rates and genotypes of Trypanosoma rangeli and T. cruzi infecting free-ranging Saguinus bicolor (Callitrichidae), a critically endangered primate of the Amazon Rainforest.

Parasites of wild primates are important for conservation biology and human health due to their high potential to infect humans. In the Amazon region, non-human primates are commonly infected by Trypanosoma cruzi and T. rangeli, which are also infective to man and several mammals. This is the first survey of trypanosomiasis in a critically endangered species of tamarin, Saguinus bicolor (Callitrichidae), from the Brazilian Amazon Rainforest. Of the 96 free-ranging specimens of S. bicolor examined 45 (46.8%) yielded blood smears positive for trypanosomes. T. rangeli was detected in blood smears of 38 monkeys (39.6%) whereas T. cruzi was never detected. Seven animals (7.3%) presented trypanosomes of the subgenus Megatrypanum. Hemocultures detected 84 positive tamarins (87.5%). Seventy-two of 84 (85.7%) were morphologically diagnosed as T. rangeli and 3 (3.1%) as T. cruzi. Nine tamarins (9.4%) yielded mixed cultures of these two species, which after successive passages generated six cultures exclusively of T. cruzi and two of T. rangeli, with only one culture remaining mixed. Of the 72 cultures positive for T. rangeli, 62 remained as established cultures and were genotyped: 8 were assigned to phylogenetic lineage A (12.9%) and 54 to lineage B (87.1%). Ten established cultures of T. cruzi were genotyped as TCI lineage (100%). Transmission of both trypanosome species, their potential risk to this endangered species and the role of wild primates as reservoirs for trypanosomes infective to humans are discussed.

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.

Polyamines and ornithine decarboxylase activity during growth and differentiation in Blastocladiella emersonii.

The activity of ornithine decarboxylase (EC 4.1.1.17, L-ornithine carboxy-lyase) was determined during the life cycle of Blastocladiella emersonii. The specific activity of the enzyme was found to be low in the zoospores, to rise 20-fold during germination and early growth, to fall during growth and to rise again during sporulation. This rise in enzyme activity was shown to be dependent on protein synthesis. Putrescine levels, on a per mg of protein basis, paralleled the fluctuation found in ornithine decarboxylase activity. Putrescine and spermidine were the only polyamines found in extracts of B. emersonii.

Phytomonas and other trypanosomatid parasites of plants and fruit.

Trypanosomatid parasites are fairly common in the latex, phloem, fruit sap, seed albumen, and even in the nectar, of many plant families. They are transmitted to the plants in the saliva of phytophagous hemipterous bugs (Insecta). Morphologically, plant trypanosomatids have no special characteristic, except perhaps a very twisted cell body. Most occur in plants as promastigotes and a few as choanomastigotes. It is still controversial whether or not they are pathogenic in lactiferous plants or fruit, but it is certain that the phloem parasites are pathogenic in coconut palms and coffee bushes. In these plants, they cause lethal diseases responsible for the destruction of many plantations in Central and South America, but fortunately nowhere else in the world. Probably more than one genus of Trypanosomatidae is represented among the plant parasites. The most important is certainly Phytomonas, but Leptomonas, Crithidia and Herpetomonas may also be present. The distinction between them is difficult and only recently have molecular markers become available to help in their identification. At present, Phytomonas can be identified by DNA hybridization with a specific probe (SL3') complementary to a sequence of the mini-exon or spliced leader gene. The development of a polymerase chain reaction coupled to SL3' hybridization has facilitated the detection of Phytomonas in plants. The phylogeny of Phytomonas is still being worked out. For the moment it can only be said that the genus is very close to Herpetomonas.

Possible artifacts in the radioiodination of surface proteins of trypanosomatids.

Three different methods for the radioiodination of surface proteins in trypanosomatids have been comparatively analyzed. Data interpretation in the lactoperoxidase-mediated method was impaired by autoiodination of the enzyme and its tight retention by the cells. In the enzymobeads method, iodinated enzymes were released from the beads upon solubilization of the preparation with SDS, thus mixing with iodinated proteins of the trypanosomatids. Therefore, upon separation by polyacrylamide gel electrophoresis of the cell lysates, both methods yielded autoradiographic patterns in which prominent bands did not represent labeled cell surface components. In contrast, the autoradiographic patterns obtained by use of the Iodo-Gen method were apparently free of artifacts.

Leishmania tarentolae taxonomic relatedness inferred from phylogenetic analysis of the small subunit ribosomal RNA gene.

The sequence of the Leishmania tarentolae SSU rRNA (small subunit or 18S rRNA) gene was completely determined from 2 different strains and used to determine phylogenetic relationships between this organism and other trypanosomatids. Extensive structural similarities were observed between L. tarentolae and mammalian leishmanias the SSU rRNA. Phylogenetic reconstructions, using distance matrix or parsimony methods, showed large evolutionary distances between trypanosomes, either African and American, and L. tarentolae. Further analysis using intergenic rDNA spacer (IGS) sequences as probes in dot blot experiments confirmed the results obtained with the SSU rDNA comparisons. The data presented here clearly indicate that L. tarentolae is closely related to the mammalian parasite Leishmania donovani and highly divergent from trypanosomes.

Restriction fragment length polymorphisms in the ribosomal gene spacers of Trypanosoma cruzi and Trypanosoma conorhini.

The ribosomal RNA genes of two species of Trypanosoma, Trypanosoma cruzi, the etiological agent of Chagas' disease, and Trypanosoma conorhini, a non-pathogenic rodent trypanosome, were cloned and partially characterized. The physical maps derived for their rRNA genes were similar throughout the region that encompasses the SSU-and LSU-rRNA coding sequences. However, the non-transcribed spacer DNA of both T. cruzi and T. conorhini was found to be polymorphic for several restriction enzyme sites. We show that strains of T. cruzi can be typed according to the characteristic restriction fragment length polymorphism of their NTS DNAs.

Cross-reactivity between Trypanosoma cruzi and insect trypanosomatids as a basis for the diagnosos of Chagas' disease.

Immunological cross-reactivity between Trypanosoma cruzi and insect trypanosomatids was demonstrated by immunofluorescence and confirmed by complement fixation, direct agglutination and cross-absorption experiments. As antigens, the following organisms were surveyed: Crithidia deanei, Crithidia fasciculata, Crithidia luciliae, Herpetomonas samuelpessoai, Herpetomonas megaseliae, Herpetomonas muscarum muscarum, Leptomonas seymouri and Blastocrithidia culicis. Sera from patients with Chagas' disease or sera from rabbits immunized against various trypanosomatids were used as sources of antibodies. The demonstration of cross-reactivity was followed by a survey of 500 human sera (from normal persons or Chagas' disease patients) by immunofluorescence using insect trypanosomatids (H. muscarum muscarum, C. fasciculata and L. seymouri) as antigens. With H. muscarum muscarum 98.7% coincident positive results and 100% of coincident negative results were obtained. These findings may validate the use of insect trypanosomatids as an alternative source of antigen in the serodiagnosis of Chagas' disease by indirect immunofluorescence.

Unstable hypoendemic malaria in Rondonia (western Amazon region, Brazil): epidemic outbreaks and work-associated incidence in an agro-industrial rural settlement.

A longitudinal study was conducted from January 1991 to January 1992 on the Urupa farm, a rural agro-industrial forestry settlement in Rondonia state (Western Amazon Region, Brazil) to define the parasitologic and clinical profile of malaria. Three cross-sectional, parasitologic, and clinical surveys were performed. In the intervals between surveys, malaria cases were monitored by twice a week medical visits to the farm and permanent local surveillance. The population of residents was approximately 170 and was characterized by high mobility. The slide positive rates found in the cross-sectional surveys were 0.5, 4.2 and 2.1, respectively, for the total population (Plasmodium vivax plus P. falciparum). Spleen rate values in children 2-9 years old were always less than 1%. However, this basically hypoendemic malaria situation was unstable, with occurrence of a typical epidemic outbreak at the end of the dry season. The total number of malaria cases recorded from January to December 1991 was 163, giving an annual parasite index of 970 per 1,000 inhabitants. However, sex and age distribution of cases showed rare incidence of malaria in infants and low incidence in children less than the age of 10. Male adults 16-40 years of age represented the main risk group. The observed clustering of cases allowed us to identify the place of work as a factor responsible for high incidence of malaria among adults. The general epidemiologic profile indicated that indoors transmission of malaria by the local Anopheles vector was low or absent.

Hypoendemic malaria in Rondonia (Brazil, western Amazon region): seasonal variation and risk groups in an urban locality.

A longitudinal epidemiologic survey (1989-1991) plus a cross-sectional parasitologic, clinical, and sociodemographic survey (July-October 1990) were conducted in Candeias do Jamary, a village with approximately 7,000 inhabitants in Rondonia, Brazil. Analysis of the results revealed hypoendemic malaria with a complex epidemiology. Plasmodium vivax predominated over P. falciparum infections while infections with P. malariae were absent. Malaria is present throughout the year but was clearly seasonal with epidemic outbreaks in the dry season from June to August. Malaria prevalence was lower in children less than 10 years of age and significantly higher in young adult males, which represent the high-risk group. The incidence of locally acquired infections (autochthonous cases) was significantly lower in the rainy season as compared with the dry season. This is not true with respect to heterotochthonous (imported) malaria cases, that is, malaria acquired elsewhere by Candeias residents, most of whom are male adults working outside the town. In both cases, however, the age and sex distribution of prevalence and its relationship with occupational activities indicate a predominance of outdoor transmission. The results of the cross-sectional survey are in agreement with those of the longitudinal passive survey and, in addition, disclose the absence of asymptomatic infection.

PCR amplification of the spliced leader gene for the diagnosis of trypanosomatid parasites of plants and insects in methanol-fixed smears.

A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.

Staphylococci adherence to trypanosomes exposed to immune sera as a method for the diagnosis of Chagas' disease.

A new method, the staphylococci adherence test (SAT), for the serological diagnosis of Chagas' disease is described; it is based on the affinity of staphylococcal Protein A for IgG globulins and uses epimastigotes of Trypanosoma cruzi which are fixed on to glass slides and incubated stepwise with probing sera and a Staphylococcus aureus A suspension. After Giemsa staining, epimastigote preparations which have been incubated with positive sera appear covered by cocci. Results using indirect immunofluorescence, complement fixation and SAT were in agreement in 98.37% of 860 human sera. A slightly modified SAT (SAT') may also be utilized for the diagnosis of acute Chagas' disease. The simplicity of the method may allow its adaptation for field work. Other possible uses were investigated.

Prevalence and risk factors associated with Helicobacter pylori infection in native populations from Brazilian Western Amazon.

We evaluated the prevalence of, and factors associated with, Helicobacter pylori infection in 222 subjects from 3 distinct communities of native populations (Uru-Eu-Wau-Wau Indians and 2 riverine communities living on the banks of the Machado river and in Portuchuelo) living in isolation in the rainforest of Brazilian Western Amazon. The overall prevalence was 78.8% (95% CI 72.7-83.9). The prevalence was higher in the Machado river community compared with Portuchuelo (chi2 = 3.84, P = 0.05), but no significant difference was observed between the Machado river community and the Uru-Eu-Wau-Wau Indians. Logistic regression showed that residential crowding and age were factors associated with the presence of H. pylori infection. Acquisition of the bacterium started early in life and by the age of 2 years 50% of children were infected. The prevalence increased with age, reaching near universal levels during adulthood (97.9%). Residential crowding was high with a global index of 3.3 persons/room (SD = 1.8), varying significantly between the 3 communities (P = 0.001). These data provide further evidence supporting direct person-to-person spread of the bacterium.

Humoral immune response to the 72 kDa heat shock protein from Plasmodium falciparum in populations at hypoendemic areas of malaria in western Brazilian Amazon.

The heat-shock protein Pf72/Hsp70-1 from the human malaria parasite Plasmodium falciparum has been suggested as a potential candidate antigen for a multivalent vaccine. We have investigated the prevalence and levels of IgG antibodies to the recombinant protein PfR44, derived from Pf72/Hsp70-1, in individuals from different age groups living in Candeias do Jamari, an Amazonian town characterized by unstable and hypoendemic malaria transmission. Blood were collected from a household-based random sample comprising 241 people and the sera were comparatively tested against recombinant antigen PfR44 and a detergent-soluble extract of P. falciparum (PfAg-T). The prevalence and levels of IgG antibodies to both recombinant and total P. falciparum antigens were positively correlated with cumulative exposure to malaria, as estimated by the age of the individuals and the duration of their stay in the study area. Nevertheless, correlations between antibody responses to Pf72/Hsp70-1 and the acquisition of protective anti-malarial immunity could not be derived from our data.

The epidemiology of malaria in Rondonia (Western Amazon region, Brazil): study of a riverine population.

We report on a longitudinal study concerning the incidence of malaria in a riverine population (Portuchuelo) settled on the riverbanks of Rio Madeira, in the State of Rondonia, Brazil. We found the incidence of malaria to be seasonal, prevailing in the dry months of June and July. The Annual Parasite Index (API) was 292/1000 inhabitants, almost three times that of the state of Rondonia for the same period. In contrast with other studied Rondonian populations, malaria in Portuchuelo was more prevalent in youngsters < 16 years old, particularly in the 0-1 year age group. Adults were relatively spared, particularly those over 50 years. Besides being indicative of indoor transmission, these facts may suggest the existence of a certain degree of acquired resistance to infection and/or of lessened symptoms in older people. Riverine populations are spread over the entire Amazon region where most of its members were born. Due to the permanent presence of malaria among riverine populations, we are proposing that they may act as perennial reserves of malaria and, therefore, as sources of infection for migrants or eventual settlers at their vicinity. To date, the opposite view has been generally held. Anopheles darlingi, the main vector species in the area, is essentially sylvatic, which contributes to make the control of malaria highly problematic. The only hopes for control rest on permanent surveillance and the prompt treatment of patients, which are also problematic considering the vastness of the Amazon region and the remoteness of some of its riverine settlements.

Intracytoplasmic flagellum in trypanosomatids.

Electron microscopy of thin sections and detergent extracted preparations of certain trypanosomatids of the genus Phytomonas revealed the unusual presence in some flagellates of an intracytoplasmic flagellum which appears as a naked axoneme encircling the nucleus before emerging at the anterior end of the cell. Another feature of these atypical flagellates is the absence of the flagellar pocket normally present in trypanosomatids at the emergence of the flagellum.

Gynandromorphism in Amblyomma cajennense and Rhipicephalus sanguineus (Acari: Ixodidae).

During a survey of ticks on horses in November 2000, at the University of São Paulo farm in Pirassununga county, São Paulo, Brazil, 1 gynandromorph of Amblyomma cajennense was collected from a naturally infested horse. In another survey on dogs in March 2001, in the urban area of Monte Negro, Rondônia, Brazil, a gynandromorph of Rhipicephalus sanguineus was collected from a naturally infested dog. Both specimens are described and classified as perfect bipartite protogynanders.