Infectivity of cultured Trypanosoma fallisi (Kinetoplastida) to various anuran species and its evolutionary implications

Trypanosoma fallisi, a hemoflagellate infecting Bufo americanus from Ontario, was grown in vitro, and metatrypanosomes from the primary culture were inoculated into 4 uninfected test groups from 3 anuran families: Bufonidae, Hylidae, and Ranidae. In vitro-cultured T. fallisi was found to infect B. americanus and to induce transient infections in Bufo valliceps and Hyla versicolor. The flagellate was not infective to Rana clamitans. Trypanosoma ranarum was uninfective to the bufonids and hylids tested. These data suggest that the potential for host-switching decreases with increased evolutionary distance of the potential anuran host.     

Allozyme comparison of three Trypanosoma species (Kinetoplastida: Trypanosomatidae) of toads and frogs by starch-gel electrophoresis.

Six metabolic enzymes, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, isocitrate dehydrogenase, malate dehydrogenase, phosphoglucomutase, and purine nucleoside phosphorylase, from clonal isolates of 3 presumptive species of Trypanosoma (T. fallisi, T. ranarum, and T. rotatorium) from 3 anuran hosts (Bufo americanus, Rana clamitans, and Rana catesbeiana) were compared using starch-gel electrophoresis. Although bands were shared among the different zymodemes of isolates of the same host genus, low genetic polymorphism of the enzyme loci was observed with few apparent shared bands between samples isolated from frogs and toads. A distance value calculated between toad and frog trypanosome isolates suggests the likelihood of long-time separation of species. Cluster analysis based on overall similarity distinguished the trypanosomes of toads and frogs as separate taxa, suggesting that host specificity and observed morphological differences are consistent with heritable allozyme differences.     

A light and electron microscopic study of Trypanosoma fallisi N. Sp. in toads (Bufo americanus) from Algonquin Park, Ontario

Trypanosoma fallisi n. sp. is described from Bufo americanus in Ontario. The parasite was observed in 65 of 94 toads examined. The trypanosomes were pleomorphic with respect to the age of infections, being longer and broader in early infections (during spring and summer) and shorter and more slender during late summer and autumn. They ranged in size from 38-76 microns in body length and 3-8 microns in width, with a free flagellum 6-30 microns long. Epizootiological and experimental evidence suggests that this trypanosome is transmitted to the toads by the leech, Batracobdella picta. Trypanosoma fallisi is morphologically similar to T. bufophlebotomi described in Bufo boreas from California, but geographic isolation, host and vector differences as well as slight morphological differences indicate that speciation has occurred. Similar trypanosomes from Bufo americanus (which were identified as T. bufophlebotomi) in Michigan, are probably T. fallisi. This species shares many ultrastructural features with trypanosomes of other lower vertebrates and also of mammals.     

The detection of non-RoTat 1.2 Trypanosoma evansi

The majority of Trypanosoma evansi can be detected using diagnostic tests based on the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2. Exceptions are a number of T. evansi isolated in Kenya. To characterize T. evansi that are undetected by RoTat 1.2, we cloned and sequenced the VSG cDNA from T. evansi JN 2118Hu, an isolate devoid of the RoTat 1.2 VSG gene. A 273 bp DNA segment of the VSG gene was targeted in PCR amplification for the detection of non-RoTat 1.2 T. evansi. Genomic DNA samples from different trypanosomes were tested including 32 T. evansi, 10 Trypanosoma brucei, three Trypanosoma congolense, and one Trypanosoma vivax. Comparison was by PCR amplification of a 488 bp fragment of RoTat1.2 VSG gene. Results showed that the expected 273 bp amplification product was present in all five non-RoTat 1.2 T. evansi tested and was absent in all 27 RoTat 1.2-positive T. evansi tested. It was also absent in all other trypanosomes tested. The PCR test developed in this study is specific for non-RoTat 1.2 T. evansi.     

Use of kidney impressions for the detection of trypanosomes of anura

The sensitivities of three techniques used for detecting infections of Trypanosoma spp. in frogs (Rana spp.) were compared. In total, 52 of 99 frogs had detectable infections of T. rotatorium, T. chattoni, T. pipientis or T. ranarum. Two or more Trypanosoma spp. were detected in 12 frogs. Microscopic examination of stained kidney impressions (KIT) was more sensitive than either hematocrit centrifugation (HCT) or wet-mount examination (WME) in detecting T. rotatorium and T. chattoni. The HCT was more sensitive in detecting T. pipientis and T. ranarum. Four infections of T. rotatorium that were missed using the HCT were detected using the WME; one of these was missed using the KIT. Success of the KIT may be related to size of the trypanosome while success of the HCT may be related to size, motility or specific gravity of the trypanosome.     

The evolution of Trypanosoma cruzi: the 'bat seeding' hypothesis

Recent discussions on the evolution of Trypanosoma cruzi have been dominated by the southern super-continent hypothesis, whereby T. cruzi and related parasites evolved in isolation in the mammals of South America, Antarctica and Australia. Here, we consider recent molecular evidence suggesting that T. cruzi evolved from within a broader clade of bat trypanosomes, and that bat trypanosomes have successfully made the switch into other mammalian hosts in both the New and Old Worlds. Accordingly, we propose an alternative hypothesis--the bat seeding hypothesis--whereby lineages of bat trypanosomes have switched into terrestrial mammals, thereby seeding the terrestrial lineages within the clade. One key implication of this finding is that T. cruzi may have evolved considerably more recently than previously envisaged.     

A Light and Electron Microscopic Study of Trypanosoma fallisi N. Sp. in Toads (Bufo americanus) from Algonquin Park, Ontario

Trypanosoma fallisi n. sp. is described from Bufo americanus in Ontario. The parasite was observed in 65 of 94 toads examined. The trypanosomes were pleomorphic with respect to the age of infections, being longer and broader in early infections (during spring and summer) and shorter and more slender during late summer and autumn. They ranged in size from 38–76 μm in body length and 3–8 μm in width, with a free flagellum 6–30 μm long. Epizootiological and experimental evidence suggests that this trypanosome is transmitted to the toads by the leech, Batracobdella picta. Trypanosoma fallisi is morphologically similar to T. bufophlebotomi described in Bufo boreas from California, but geographic isolation, host and vector differences as well as slight morphological differences indicate that speciation has occurred. Similar trypanosomes from Bufo americanus (which were identified as T. bufophlebotomi) in Michigan, are probably T. fallisi. This species shares many ultrastructual features with trypanosomes of other lower vertebrates and also of mammals.     

Trypanosoma rangeli is phylogenetically closer to Old World trypanosomes than to Trypanosoma cruzi

Trypanosoma rangeli and Trypanosoma cruzi are generalist trypanosomes sharing a wide range of mammalian hosts; they are transmitted by triatomine bugs, and are the only trypanosomes infecting humans in the Neotropics. Their origins, phylogenetic relationships, and emergence as human parasites have long been subjects of interest. In the present study, taxon-rich analyses (20 trypanosome species from bats and terrestrial mammals) using ssrRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH), heat shock protein-70 (HSP70) and Spliced Leader RNA sequences, and multilocus phylogenetic analyses using 11 single copy genes from 15 selected trypanosomes, provide increased resolution of relationships between species and clades, strongly supporting two main sister lineages: lineage Schizotrypanum, comprising T. cruzi and bat-restricted trypanosomes, and Tra[Tve-Tco] formed by T. rangeli, Trypanosoma vespertilionis and Trypanosoma conorhini clades. Tve comprises European T. vespertilionis and African T. vespertilionis-like of bats and bat cimicids characterised in the present study and Trypanosoma sp. Hoch reported in monkeys and herein detected in bats. Tco included the triatomine-transmitted tropicopolitan T. conorhini from rats and the African NanDoum1 trypanosome of civet (carnivore). Consistent with their very close relationships, Tra[Tve-Tco] species shared highly similar Spliced Leader RNA structures that were highly divergent from those of Schizotrypanum. In a plausible evolutionary scenario, a bat trypanosome transmitted by cimicids gave origin to the deeply rooted Tra[Tve-Tco] and Schizotrypanum lineages, and bat trypanosomes of diverse genetic backgrounds jumped to new hosts. A long and independent evolutionary history of T. rangeli more related to Old World trypanosomes from bats, rats, monkeys and civets than to Schizotrypanum spp., and the adaptation of these distantly related trypanosomes to different niches of shared mammals and vectors, is consistent with the marked differences in transmission routes, life-cycles and host-parasite interactions, resulting in T. cruzi (but not T. rangeli) being pathogenic to humans.     

Host specificity and incidence of Trypanosoma in some African rainforest birds: a molecular approach

Studies of host-parasite interactions in birds have contributed greatly to our understanding of the evolution and ecology of disease. Here we employ molecular techniques to determine the incidence and study the host-specificity of parasitic trypanosomes in the African avifauna. We developed a polymerase chain reaction (PCR)-based diagnostic test that amplified the small subunit ribosomal RNA gene (SSU rRNA) of Trypanosoma from avian blood samples. This nested PCR assay complements and corroborates information obtained by the traditional method of blood smear analysis. The test was used to describe the incidence of trypanosomes in 479 host individuals representing 71 rainforest bird species from Cameroon, the Ivory Coast and Equatorial Guinea. Forty-two (59%) of these potential host species harboured trypanosomes and 189 individuals (35%) were infected. To examine host and geographical specificity, we examined the morphology and sequenced a portion of the SSU rRNA gene from representative trypanosomes drawn from different hosts and collecting locations. In traditional blood smear analyses we identified two trypanosome morphospecies, T. avium and T. everetti. Our molecular and morphological results were congruent in that these two morphospecies had highly divergent SSU rRNA sequences, but the molecular assay also identified cryptic variation in T. avium, in which we found seven closely allied haplotypes. The pattern of sequence diversity within T. avium provides evidence for widespread trypanosome mixing across avian host taxa and across geographical locations. For example, T. avium lineages with identical haplotypes infected birds from different families, whereas single host species were infected by T. avium lineages with different haplotypes. Furthermore, some conspecific hosts from geographically distant sampling locations were infected with the same trypanosome lineage, but other individuals from those locations harboured different trypanosome lineages. This apparent lack of host or geographical specificity may have important consequences for the evolutionary and ecological interactions between parasitic trypanosomes and their avian hosts.     

The taxonomic position and evolutionary relationships of Trypanosoma rangeli.

This paper presents a re-evaluation of the taxonomic position and evolutionary relationships of Trypanosoma (Herpetosoma) rangeli based on the phylogenetic analysis of ssrRNA sequences of 64 Trypanosoma species and comparison of mini-exon sequences. All five isolates of T. rangeli grouped together in a clade containing Trypanosoma (Schizotrypanum) cruzi and a range of closely related trypanosome species from bats [Trypanosoma (Schizotrypanum) dionisii, Trypanosoma (Schizotrypanum) vespertilionis] and other South American mammals [Trypanosoma (Herpetosoma) leeuwenhoeki, Trypanosoma (Megatrypanum) minasense, Trypanosoma (Megatrypanum) conorhini] and an as yet unidentified species of trypanosome from an Australian kangaroo. Significantly T. rangeli failed to group with (a) species of subgenus Herpetosoma, other than those which are probably synonyms of T. rangeli, or (b) species transmitted via the salivarian route, although either of these outcomes would have been more consistent with the current taxonomic and biological status of T. rangeli. We propose that use of the names Herpetosoma and Megatrypanum should be discontinued, since these subgenera are clearly polyphyletic and lack evolutionary and taxonomic relevance. We hypothesise that T. rangeli and T. cruzi represent a group of mammalian trypanosomes which completed their early evolution and diversification in South America.     

First record of Trypanosoma chattoni in Brazil and occurrence of other Trypanosoma species in Brazilian frogs (Anura, Leptodactylidae)

The present study provides the first record of Trypanosoma chattoni Mathis and Leger, 1911, in a new host, Leptodactylus fuscus Schneider, 1799 (Anura, Leptodactylidae), and the occurrence of Trypanosoma rotatorium-like species in Leptodactylus chaquensis Cei, 1950. The anurans were captured in the State of Mato Grosso, Brazil. Blood samples were obtained by cardiac puncture, and blood smears were examined for the presence of hemoparasites. The Trypanosoma rotatorium-like species in this study refers to a short-bodied trypomastigote that has a conspicuous undulating membrane but lacks a free flagellum; T. chattoni refers to a monomorphic parasite that has a rounded body, a kinetoplast adjacent to the nucleus, and a short flagellum.     

The inadvertent introduction into Australia of Trypanosoma nabiasi, the trypanosome of the European rabbit (Oryctolagus cuniculus), and its potential for biocontrol

Wild rabbits (Oryctolagus cuniculus) in Australia are the descendents of 24 animals from England released in 1859. We surveyed rabbits and rabbit fleas (Spilopsyllus cuniculi) in Australia for the presence of trypanosomes using parasitological and PCR-based methods. Trypanosomes were detected in blood from the European rabbits by microscopy, and PCR using trypanosome-specific small subunit ribosomal RNA (SSU rRNA) gene primers and those in rabbit fleas by PCR. This is the first record of trypanosomes from rabbits in Australia. We identified these Australian rabbit trypanosomes as Trypanosoma nabiasi, the trypanosome of the European rabbit, by comparison of morphology and SSU rRNA gene sequences of Australian and European rabbit trypanosomes. Phylogenetic analysis places T. nabiasi in a clade with rodent trypanosomes in the subgenus Herpetosoma and their common link appears to be transmission by fleas. Despite the strict host specificity of trypanosomes in this clade, phylogenies presented here suggest that they have not strictly cospeciated with their vertebrate hosts. We suggest that T. nabiasi was inadvertently introduced into Australia in the 1960s in its flea vector Spilopsyllus cuniculi, which was deliberately introduced as a potential vector of the myxoma virus. In view of the environmental and economic damage caused by rabbits in Australia and other islands, the development of a virulent or genetically modified T. nabiasi should be considered to control rabbits.     

Steady-state oxygen kinetics of terminal oxidases in Trypanosoma mega

Steady-state oxygen kinetics of Trypanosoma mega reveal the presence of 3 oxidases. These include an oxidase which is sensitive to salicylhydroxamic acid (SHAM) but insensitive to sodium azide. This oxidase could be the L-alpha glycerophosphate oxidase present in bloodstream trypanosomes. In addition, and oxidase is present wthich is azide-sensitive but SHAM-insensitive. This oxidase is inhibited by CO and is probably cytochrome aa3. A 3rd oxidase is insensitive to both azide and SHAM but is inhibited by CO and is possibly cytochrome o. Reciprocal plots of T. mega reveal the presence of 2 oxidases that are inhibited by CO. These results are discussed in the light of previous evidence suggesting the presence of several oxidases and a branched electron transport system in T. mega.     

Trypanosoma saimirii Rodhain, a junior synonym of Trypanosoma rangeli Tejera

In the present study, we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to compare polypeptides of trypanosomes isolated by hemoculture of squirrel monkeys displaying Trypanosoma saimirii blood trypomastigotes, with other trypanosomes that infect primates to evaluate the validity of T. saimirii. The polypeptide profiles of trypanosomes isolated directly from squirrel monkeys or after their passage in mice were identical to those of 3 standard strains of T. rangeli, but they were distinct from those of T. cruzi, T. conorhini, and T. minasense. These results strengthen previous morphological and biological findings by Rodhain on trypanosomes of the squirrel monkey and lead to the conclusion that T. saimirii is indeed a junior synonym of T. rangeli.     

Programmed cell death in procyclic Trypanosoma brucei rhodesiense is associated with differential expression of mRNAs

Procyclic Trypanosoma brucei rhodesiense have a cell death mechanism which can be activated by an external signal, the lectin ConA, in vitro. ConA has been shown to cause profound changes in cellular morphology and induce fragmentation of nuclear DNA in T.b. rhodesiense which are characteristic of apoptosis, a form of programmed cell death (PCD) in other eukaryotic cells. RNA analysis of trypanosomes induced to undergo PCD revealed that RNA remains intact up to 48 h into the process, a time when nuclear DNA fragmentation has already started. Using the randomly amplified differentially expressed sequences polymerase chain reaction method, ConA-induced cell death in T.b. rhodesiense is shown to be associated with differential expression of mRNAs, including up regulation of mRNAs late in the death process. The results demonstrate that trypanosomes actively participate in their own destruction through a PCD process and confirm that cell death in trypanosomes is associated with de novo gene expression.     

Development of metacyclic forms of Trypanosoma brucei sspp. in cultures containing explants of Phormia regina Meigen

When procyclic trypanosomes of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense were cultivated in Nunclon 25 cm2 flasks at 27 C in a liquid medium containing various tissue explants of Phormia regina Meigen, some of them developed into forms infective for mice. The infective stages were present at various periods of up to 29 days when the cultures were terminated. Larger numbers of explants of head-salivary glands than the other tissues used were required to produce infections. Infectivity titrations on trypanosome suspensions of T. b. brucei TRUM 252 and T. b. rhodesiense TRUM 497 indicated that only a small proportion of the populations was infective. Mice were rarely infected with trypanosomes grown in medium without explants. Only 1 mouse of the 11 inoculated developed a parasitemia from a control culture of T. b. rhodesiense TRUM 545. A few trypanosomes resembling epimastigotes and metacyclic forms were seen in stained samples of infective inocula.