A new lineage of trypanosomes from Australian vertebrates and terrestrial bloodsucking leeches (Haemadipsidae)

Little is known about the trypanosomes of indigenous Australian vertebrates and their vectors. We surveyed a range of vertebrates and blood-feeding invertebrates for trypanosomes by parasitological and PCR-based methods using primers specific to the small subunit ribosomal RNA (SSU rRNA) gene of genus Trypanosoma. Trypanosome isolates were obtained in culture from two common wombats, one swamp wallaby and an Australian bird (Strepera sp.). By PCR, blood samples from three wombats, one brush-tailed wallaby, three platypuses and a frog were positive for trypanosome DNA. All the blood-sucking invertebrates screened were negative for trypanosomes both by microscopy and PCR, except for specimens of terrestrial leeches (Haemadipsidae). Of the latter, two Micobdella sp. specimens from Victoria and 18 Philaemon sp. specimens from Queensland were positive by PCR. Four Haemadipsa zeylanica specimens from Sri Lanka and three Leiobdella jawarerensis specimens from Papua New Guinea were also PCR positive for trypanosome DNA. We sequenced the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes in order to determine the phylogenetic positions of the new vertebrate and terrestrial leech trypanosomes. In trees based on these genes, Australian vertebrate trypanosomes fell in several distinct clades, for the most part being more closely related to trypanosomes outside Australia than to each other. Two previously undescribed wallaby trypanosomes fell in a clade with Trypanosoma theileri, the cosmopolitan bovid trypanosome, and Trypanosoma cyclops from a Malaysian primate. The terrestrial leech trypanosomes were closely related to the wallaby trypanosomes, T. cyclops and a trypanosome from an Australian frog. We suggest that haemadipsid leeches may be significant and widespread vectors of trypanosomes in Australia and Asia.     

Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62 degrees C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was approximately 1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions.     

Trypanosomes of the subgenus Megatrypanum from armadillos (Xenarthra:Dasypodidae)

A new species of trypanosome, Trypanosoma (Megatrypanum) peba, is described from the peripheral blood of the armadillo Euphractus sexcinctus setosus from Bahia State, Brazil. Ten out of 29 specimens of the armadillo Dasypus novemcinctus from Pará State were found to have trypanosomes, including epimastigote forms, in impression smears of subcutaneous lymph nodes. The trypanosomes from D. novemcinctus are illustrated and were identified as belonging to the subgenus Megatrypanum on the basis of their general appearance, although they failed to multiply in blood-agar culture medium and no bloodstream forms were seen. This is the first published record of trypanosomes of this subgenus from armadillos and the first demonstration of epimastigote trypanosomes in the mammalian host other than in the bloodstream, or in the anal glands of opossums.     

Vertebrate hosts and phylogenetic relationships of amphibian trypanosomes from a potential invertebrate vector, Culex territans Walker (Diptera: Culicidae)

The blood meals of field-collected female Culex territans (Diptera: Culicidae) were concurrently assayed for the presence of trypanosomes and for vertebrate host identification. We amplified vertebrate DNA in 42 of 119 females and made positive identification to the host species level in 29 of those samples. Of the 119 field-collected Cx. territans females, 24 were infected with trypanosomes. Phylogenetic analysis placed the trypanosomes in the amphibian portion of the aquatic clade of the Trypanosomatidae. These trypanosomes were isolated from Cx. territans females that had fed on the frog species Rana clamitans, R. catesbeiana, R. virgatipes, and Rana spp. Results support a potential new lineage of dipteran-transmitted amphibian trypanosomes may occur within the aquatic clade. The frequency in which female Cx. territans acquire trypanosomes, through diverse feeding habits, indicates a new relationship between amphibian trypanosomes and mosquitoes that has not been examined previously. Combining Trypanosoma species, invertebrate, and vertebrate hosts to existing phylogenies can elucidate trypanosome and host relationships.     

Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62°C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was ∼1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions.     

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.     

Morphological changes in trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo

The effect of α-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3–4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.     

Prevalence and molecular phylogenetic characterization of Trypanosoma (Megatrypanum) minasense in the peripheral blood of small neotropical primates after a quarantine period

Neotropical primates of the Cebidae and Callitrichidae, in their natural habitats, are frequently infected with a variety of trypanosomes including Trypanosoma cruzi, which causes a serious zoonosis, Chagas' disease. The state of trypanosome infection after a 30-day quarantine period was assessed in 85 squirrel monkeys (Saimiri sciureus) and 15 red-handed tamarins (Saguinus midas), that were wild-caught and exported to Japan as companion animals or laboratory animals, for biomedical research, respectively. In addition to many microfilariae of Mansonella (Tetrapetalonema) mariae at a prevalence of 25.9%, and Dipetalonema caudispina at a prevalence of 3.5%, a few trypomastigotes of Trypanosoma (Megatrypanum) minasense were detected in Giemsa-stained thin films of blood from 20 squirrel monkeys at a prevalence of 23.5%. Although few T. minasense trypomastigotes were found in Giemsa-stained blood films from tamarins, a buffy-coat examination detected trypanosomes in 12 red-handed tamarins (80.0%), and PCR amplification of a highly variable region of the small subunit ribosomal RNA genes (SSU rDNA) for Trypanosoma spp. detected the infection in 14 of the 15 tamarins (93.3%). Nucleotide sequences of the amplicons were identical for trypanosomes from tamarins and squirrel monkeys, indicating a high prevalence but low parasitemia of T. minasense in imported Neotropical nonhuman primates. Based on the SSU rDNA and 5.8S rDNA, the molecular phylogenetic characterization of T. minasense indicated that T. minasense is closely related to trypanosomes with Trypanosoma theileri-like morphology and is distinct from Trypanosoma (Tejeraia) rangeli, as well as from T. cruzi. Using some blood samples from these monkeys, amplification and subsequent sequencing of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene fragments detected 4 trypanosome genotypes, including 2 types of T. cruzi clade, 1 type of T. rangeli clade, and 1 T. rangeli-related type, but failed to indicate its phylogenetic position based on the gGAPDH gene. Furthermore, species ordinarily classified in the Megatrypanum by morphological criteria do not form a clade in any molecular phylogenetic trees based on rDNA or gGAPDH genes.     

Phylogenetic position of the giant anuran trypanosomes Trypanosoma chattoni,Trypanosoma fallisi,Trypanosoma mega,Trypanosoma neveulemairei,and Trypanosoma ranarum inferred from 18S rRNA gene sequences

Phylogenetic relationships within the kinetoplastid flagellates were inferred from comparisons of small-subunit ribosomal RNA gene sequences. These included 5 new gene sequences, Trypanosoma fallisi (2,239 bp), Trypanosoma chattoni (2,180 bp), Trypanosoma mega (2,211 bp), Trypanosoma neveulemairei (2,197 bp), and Trypanosoma ranarum (2,203 bp). Trees produced using maximum-parsimony and distance-matrix methods (least-squares, neighbor-joining, and maximum-likelihood), supported by strong bootstrap and quartet-puzzle analyses, indicated that the trypanosomes are a monophyletic group that divides into 2 major lineages, the salivarian trypanosomes and the nonsalivarian trypanosomes. The nonsalivarian trypanosomes further divide into 2 lineages, 1 containing trypanosomes of birds, mammals, and reptiles and the other containing trypanosomes of fish, reptiles, and anurans. Among the giant trypanosomes, T. chattoni is clearly shown to be distantly related to all the other anuran trypanosome species. Trypanosoma mega is closely associated with T. fallisi and T. ranarum, whereas T. neveulemairei and Trypanosoma rotatorium are sister taxa. The branching order of the anuran trypanosomes suggests that some toad trypanosomes may have evolved by host switching from frogs to toads.     

Hemoflagellates of Oregon marine fishes with the description of new species of Trypanosoma and Trypanoplasma

Of 2,122 marine fishes representing 36 species collected in the northeastern Pacific Ocean in the vicinity of Newport, Oregon from 1971 to 1973, 541 individuals (25.5%) representing 8 species (22.2%) were infected with hemoflagellates. Four morphologically distinct trypanosomes and 3 distinct trypanoplasms were found in fishes collected offshore, but no hemoflagellates were observed in fishes from Yaquina Bay estuary. Trypanosoma pacifica was found in English sole Parophrys vetulus, Pacific sanddab Citharichthys sordidus, and slender sole Lyopsetta exilis, and survived in 5 other species after intraperitoneal injection. Trypanosoma gargantua was found in big skate Raja binoculata, and the leech Orientobdella confluens was able to transmit the trypanosome in experimental conditions. Trypanosoma khani n. sp. occurred in P. vetulus, petrale sole Eopsetta jordani, and Dover sole Microstomus pacificus. Trypanosoma murmanense was found in L. exilis collected from 200 m, but not in L. exilis collected from 80 m. Trypanoplasma beckeri parasitized the cabezon Scorpaenichthys marmoratus. Trypanoplasma bobolsoni n. sp. was found in E. jordani, L. exilis, and P. vetulus, and survived in 2 other species after intraperitoneal injection. A distinct, but unnamed trypanoplasm, was found in P. vetulus.     

Rapid change of the repertoire of variant surface glycoprotein genes in trypanosomes by gene duplication and deletion

To study the evolution of the variant surface glycoprotein (VSG) repertoire of trypanosomes we have analysed the DNA region surrounding the VSG 118 gene in different trypanosome strains. We find a remarkable degree of variation in this area. Downstream from the 118 gene a 5.7 X 10(3) base-pair DNA segment containing a potential VSG gene has been quadruplicated in strain 427 of Trypanosoma brucei, but not in most other strains analysed. The VSG 1.1000 gene, located immediately upstream from the 118 gene in one trypanosome strain, has been cleanly deleted in another. Our results are most easily explained by multiple unequal cross-overs between sister chromatids and are the first indication that sister chromatid exchange occurs in trypanosomes.     

Community ecology and conservation of the fishes of the Okavango Delta,Botswana

Synopsis The Okavango Delta is a large inland swamp in northern Botswana which receives an annual flood from the highlands of southern Angola. There are distinct fish taxocenes in the Okavango which can be separated from each other by the physical characteristics of the different habitat types with which they co-evolved. An account is given of the ecology and conservation of the fishes of the Okavango Delta. Their response to the annual flood regime, and the environmental factors that limit their distribution and abundance, are described. In the northern riverine floodplain and perennial swamp a higher species richness and ichthyomass was recorded than in the seasonal swamp and drainage rivers. Suggestions are made on the conservation of Okavango fishes taking into account the ecological characteristics of the Delta.     

Multiplication of Trypanosoma pacifica (Euglenozoa: Kinetoplastea) in English sole, Parophrys vetulus, from Oregon coastal waters

Multiplication of Trypanosoma pacifica was common in the fish host from observations of live flagellates and Giemsa-stained blood smears. Multiplication began with the elongation of the kinetoplast, thickening of the posterior portion of the body, and appearance of a new flagellum near the kinetoplast. The new flagellum was very rigid when less than 3 microm in length, but it became flexible as it elongated. When the new flagellum was approximately 12 microm in length, cell division began and the kinetoplast also began to divide. The timing of nuclear division was variable. Generally, it did not occur until division of the kinetoplast had begun, but occasionally binucleate individuals were observed before cell or kinetoplast division was apparent. As division continued, 1 nucleus migrated past the dividing kinetoplast into the future daughter trypanosome. Finally, the kinetoplast completed division and the trypanosomes separated. Cell division was unequal, with the daughter trypanosome being smaller than the parent and with a more weakly developed undulating membrane.     

ATP-independent DNA topoisomerase II as potential drug target in trypanosomes

Two categories of trypanosomal type II topoisomerases have been isolated from trypanosomes: one is unique since it is able to realize DNA topoisomerization reactions in the absence of ATP, in contrast to the other enzyme and mammalian topoisomerase II. The biochemical properties of ATP-independent topoisomerase II from Trypanosoma cruzi are described in this report. The enzyme can decatenate trypanosome kinetoplast DNA networks, catenate supercoiled DNA molecules, unknot P4 phage DNA, and cleave double-stranded DNA. The enzyme is inhibited by various classes of drugs and is more sensitive than mammalian topoisomerase II. Therefore, trypanosome ATP-independent topoisomerase II provides a potential target for chemotherapy.     

Trypanosoma vivax: characterization of the spliced-leader gene of a Brazilian stock and species-specific detection by PCR amplification of an intergenic spacer sequence.

The sequence of the spliced-leader gene repeat of a Brazilian Trypanosoma vivax stock from cattle showed high similarity to sequences of West African T. vivax in both intron and intergenic sequences. This is the first evidence based on DNA sequences of close-relatedness between Brazilian and West African T. vivax stocks. A T. vivax-specific diagnostic PCR assay based on spliced-leader gene intergenic sequences was able to amplify DNA from T. vivax stocks from South America (Brazil, Bolivia, and Colombia) and West Africa. Species-specificity of this method was confirmed by results obtained by testing 15 other trypanosomes, including other species and subspecies that can also infect cattle. The PCR assay developed presented high sensitivity, detecting the DNA content of only one parasite and also revealing T. vivax infection in asymptomatic animals without detectable parasitemia by microhematocrit or in Giemsa-stained blood smears. Use of crude preparations from field-blood samples collected on both filter paper and glass slides as DNA template suggested that this method could be useful for the diagnosis of T. vivax in large epidemiological studies.     

Novel mechanism that Trypanosoma cruzi uses to adhere to the extracellular matrix mediated by human galectin-3

Binding of Trypanosoma cruzi trypomastigotes to laminin is enhanced by galectin-3, a beta-galactoside binding lectin. The galectin-3 enhanced binding of trypanosomes to laminin is inhibited by lactose. Co-immunoprecipitations indicate that galectin-3 binds to the 45, 32 and 30 kDa trypanosome surface proteins. Binding of galectin-3 to the 45, 32 and 30 kDa surface proteins is inhibited by lactose. Polyclonal and a monoclonal antibodies to galectin-3 immunoprecipitated a major 64 kDa trypanosome surface protein. T. cruzi monoclonal antibody to mucin recognized the 45 kDa surface protein. The 45, 32 and 30 kDa surface proteins interact with galectin-3 in order to enhance trypanosome adhesion to laminin.     

Antigenic variation in trypansosomes: Secrets surface slowly

Among pathogenic micro-organisms that evade the mammalian immune responses, Trypanosoma brucei has developed the most elaborate capacity for antigenic variation. Trypanosomes branched early during eukaryotic evolution. They are characterized by many aberrations, ranging from the unusual compartmentation of metabolic pathways to the heresy of RNA editing. The ubiquitous phenomenon of glycosylphosphatidylinositol-anchoring of eukaryotic plasma membrane proteins and RNA trans-splicing (trypanosome genes contain no introns), which adds an identical leader sequence to all trypanosome mRNAs, were first defined during studies of antigenic variation. Genetic transformation of trypanosomes and the high efficiency of gene targeting provide new opportunities to investigate the regulation of antigenic variation. There is every reason to expect trypanosomes to provide further surprises and insights into the evolution of genetic regulatory mechanisms.     

A nested PCR for the ssrRNA gene detects Trypanosoma binneyi in the platypus and Trypanosoma sp. in wombats and kangaroos in Australia

Trypanosome infections in their natural hosts are frequently difficult to detect by microscopy, and culture methods are unreliable and not suitable for all species of Trypanosoma. A nested PCR strategy for detecting and identifying Trypanosoma species, suitable for detecting both known and unknown trypanosomes, is presented. Thirty-two blood samples from 23 species of Australian birds and mammals were screened by a nested PCR for the presence of Trypanosoma sp. ssrRNA. Three infections were detected, one in an eastern grey kangaroo (Macropus giganteus), one in a common wombat (Vombatus ursinus) and one in a platypus (Ornithorhynchus anatinus). The kangaroo and wombat are new host records for Trypanosoma sp.; the platypus parasite was Trypanosoma hinneyi. The three parasites could be distinguished by restriction fragment length polymorphisms of the amplified fragment of the ssrRNA gene. The kangaroo and wombat parasites were also isolated in a semi-solid blood agar medium. The culture forms of the kangaroo trypanosome had an expanded flagellar sheath in which structures similar to hemidesmosomes were detected by EM. The nested PCR was at least as sensitive as culture, and analysis of the PCR products gave parasite-specific fingerprints. Therefore this method could be suitable for rapidly screening host animals for the presence of trypanosomes and identifying the infecting strain.