Harry Noyes

The ancient and divergent origins of the human pathogenic trypanosomes, Trypanosoma brucei and T. cruzi

This study presents new findings concerning the evolution of the human pathogens, Trypanosoma brucei and T. cruzi, which suggest that these parasites have divergent origins and fundamentally different patterns of evolution. Phylogenetic analysis of 18S rRNA sequences places T. brucei in a clade comprising exclusively mammalian trypanosomes of African origin, suggesting an evolutionary history confined to Africa. T. cruzi (from humans and sylvatic mammals) clusters with trypanosomes specific to Old and New World bats, T. rangeli and a trypanosome species isolated from an Australian kangaroo. The origins of parasites within this clade, other than some of those from bats, lie in South America and Australia suggesting an ancient southern super-continent origin for T. cruzi, possibly in marsupials; the only trypanosomes from this clade to have spread to the Old World are those infecting bats, doubtless by virtue of the mobility of their hosts. Viewed in the context of palaeogeographical evidence, the results date the divergence of T. brucei and T. cruzi to the mid-Cretaceous, around 100 million years before present, following the separation of Africa, South America and Euramerica. The inclusion in this study of a broad range of trypanosome species from various different hosts has allowed long phylogenetic branches to be resolved, overcoming the limitations of many previous studies. Moreover, T. brucei and the other mammalian tsetse-transmitted trypanosomes appear, from these data, to be evolving several times faster than T. cruzi and its relatives.

The molecular evolution of trypanosomatidae

In the absence of a fossil record, theories relating to the evolution of protozoa have, for most of the twentieth century, been based on morphological and life cycle data despite their known limitations. However, recent advances in molecular methodology, notably the wide availability of accurate, automated DNA sequencing, have made it possible to deduce the evolutionary relationships of extant species from their genes. This paper focuses on new findings concerning the evolution of the Trypanosomatidae, based on the ever-expanding body of molecular data now available. Classically, the evolution of digenetic parasitism in kinetoplastids has centred around two opposing theories--invertebrate first or vertebrate first--depending on which was the original host of the monogenetic parasite. However, data supporting a close phylogenetic relationship between genera of monogenetic insect parasites and digenetic vertebrate parasites challenge the simplicity of these hypotheses and suggest that the transition may not have been a major evolutionary barrier. The implications of these observations for the evolution of parasitism within the group are discussed. Phylogenetic analysis of a diverse selection of trypanosomatid species suggests that the genus Trypanosoma is monophyletic and that the human parasites, T. brucei, T. cruzi and Leishmania spp., have fundamentally different patterns of evolution. T. brucei clusters with mammalian trypanosomes of African origin, suggesting an evolutionary history confined to Africa. T. cruzi shows association with trypanosomes from bats, T. rangeli, and trypanosomes from a range of South American mammals and an Australian kangaroo. The origins of most parasites within this clade lie in South America and Australia, suggesting an ancient southern super-continent origin for T. cruzi, possibly in marsupials. The divergence between the Leishmania and Trypanosoma lineages is also ancient. The topology of Leishmania phylogenies suggests an independent transition to digenetic parasitism, a neotropical origin and an early tertiary radiation of the parasite.

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.

A Revised Classification for Leishmania and Endotrypanum

We sincerely thank Stephen Beverley, Toby Barrett and Michael Miles for valuable discussions.

DNA EXTRACTION AND AMPLIFICATION OF LEISHMANIA FROM ARCHIVED, GIEMSA-STAINED SLIDES, FOR THE DIAGNOSIS OF CUTANEOUS LEISHMANIASIS BY PCR

Abstract DNA was isolated from 92 Giemsa-stained smears of lesions from suspected cases of cutaneous leishmaniasis and used for PCR-based diagnosis of Leishmania infection. Each smear had been examined under a light microscope at ∈1000 and scored for amastigote numbers. Although the smears had been stored for up to 4 years, all the microscopy-positive slides were also positive by PCR and four of the 14 smears that were negative by microscopy (although of lesions that were clinically consistent with leishmaniasis) were also PCR-positive. PCR-based investigations therefore appear to offer an effective method to confirm suspected cases of cutaneous leishmaniasis using (even archived) samples that have been collected, from humans (and reservoir hosts) in the field, by simple methods.

A systematic strategy for large-scale analysis of genotype phenotype correlations: identification of candidate genes involved in African trypanosomiasis.

It is increasingly common to combine Microarray and Quantitative Trait Loci data to aid the search for candidate genes responsible for phenotypic variation. Workflows provide a means of systematically processing these large datasets and also represent a framework for the re-use and the explicit declaration of experimental methods. In this article, we highlight the issues facing the manual analysis of microarray and QTL data for the discovery of candidate genes underlying complex phenotypes. We show how automated approaches provide a systematic means to investigate genotype–phenotype correlations. This methodology was applied to a use case of resistance to African trypanosomiasis in the mouse. Pathways represented in the results identified Daxx as one of the candidate genes within the Tir1 QTL region. Subsequent re-sequencing in Daxx identified a deletion of an amino acid, identified in susceptible mouse strains, in the Daxx–p53 protein-binding region. This supports recent experimental evidence that apoptosis could be playing a role in the trypanosomiasis resistance phenotype. Workflows developed in this investigation, including a guide to loading and executing them with example data, are available at http://workflows.mygrid.org.uk/repository/myGrid/PaulFisher/.

The Leishmania hertigi (Kinetoplastida; Trypanosomatidae) Complex and the Lizard Leishmania: Their Classification and Evidence for a Neotropical Origin of the Leishmania-Endotrypanum Clade

ABSTRACT. The relationships of the Leishmania hertigi complex and the lizard Leishmania species to the main groups of mammalian Leishmania and Endotrypanum parasites were examined. Restriction fragment length polymorphisms and sequences of small subunit ribosomal RNA genes and hybridization studies of kinetoplast DNA indicated that the L. hertigi complex was more closely related to the genus Endotrypanum than to the genus Leishmania. The lizard Leishmania species were found to be at the crown of the Leishmania tree. The data provides strong evidence for a Neotropical origin of the Endotrypanum/Leishmania clade since the parasites closest to the root of the tree are all found exclusively in the Neotropics. The evolution of the Leishmania/Endotrypanum clade in relation to the evolution of the known hosts of these parasites is discussed.

Leishmania donovani and cutaneous leishmaniasis, Sri Lanka.

To investigate the relationship of cutaneous leishmaniasis isolates from Sri Lanka to known species, we performed DNA sequencing and microsatellite analyses. We identified Leishmania donovani as the agent of Sri Lanka cutaneous leishmaniasis and showed that these parasites are closely related to those causing visceral leishmaniasis in the Indian subcontinent.

Genetic identification of two sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) that produce distinct male sex pheromones in Sobral, Ceará State, Brazil.

Lutzomyia longipalpis, the main sandfly vector for New World visceral leishmaniasis is a complex of an as yet undefined number of sibling species. At present, there is no consensus on the status (single species vs. species complex) of Brazilian populations. We applied five microsatellite loci to test the hypothesis that L. longipalpis occurs as two sympatric cryptic species in Sobral, Ceará State, Brazil as predicted by male sex pheromone chemotypes described previously for field specimens from this site [S‐9‐methyl‐germacrene‐B (9MGB) and a cembrene compound]. Abdominal spot morphology corresponds with pheromone type at this locality (9MGB in ‘1 spot’ males and cembrene in ‘2 spot’ males). Genotype data from 190 wild‐caught L. longipalpis specimens collected in October 1999 and April 2001 were used to estimate genetic differentiation between the two sex pheromone populations and sampling dates. No significant (P > 0.05) genetic differences were found between the 1999 and 2001 9MGB samples (θ = 0.018; RST = −0.005), and genetic differentiation was low between the cembrene collections (θ = 0.037, P < 0.05; RST = −0.043, P > 0.05). By contrast, highly divergent allelic frequencies (largely at two microsatellite loci) corresponded to significant (P > 0.05) genetic differentiation (θ = 0.221; RST = 0.215) for all comparisons between samples with different pheromones. When pheromone samples were pooled across sample date, genetic differentiation was high (θ = 0.229; P < 0.001; Nem = 0.84). The allele frequency distribution at each of the five microsatellite loci was similar for males and females from the two collection years. Two of these loci showed highly divergent allele frequencies in the two sex pheromone populations. This was reflected in the highly significant genetic differentiation obtained from the male genotypes, between populations producing different pheromones (θ = 0.229–0.268; P < 0.0001 for the 2001 and θ = 0.254–0.558; P < 0.0001 for the 1999 collections, respectively). Similar results were obtained when the females, assigned to a pheromone type, were included in the analysis. Both a Bayesian analysis of the data set and a population assignment test provided strong evidence for two distinct populations corresponding to pheromone type. Given its genotype, the probability of assigning a 9MGB male to the original 9MGB population was 100% once the two years’ collections were pooled. For cembrene‐producing ‘2 spot’ males this probability although still high, was lower than for 9MGB males, at 86%. This microsatellite data together with previously reported reproductive isolation between the two Sobral populations confirm that premating barriers are important in speciation of L. longipalpis.

A previously unclassified trypanosomatid responsible for human cutaneous lesions in Martinique (French West Indies) is the most divergent member of the genus Leishmania ss.

Two cases of skin lesions similar to those caused by Leishmania parasites have been reported from Martinique. Parasites isolated from these lesions were unlike Leishmania reference strains by isoenzyme analysis and electron microscopy and were assumed to be monoxenous trypanosomatids which normally only infect invertebrates. Both strains have now been retyped by isoenzyme analysis and found to be identical to each other and distantly related to all other Leishmania species. The sequence of the 18S ribosomal RNA gene and partial sequences of the DNA polymerase alpha and RNA polymerase II largest subunit genes were obtained. These sequences indicated that the Martinique parasites clustered with L. enriettii and were basal to all other euleishmania. However, support for both the position basal to all euleishmania and the clustering with L. enriettii was low. The Martinique parasites may cluster with L. (Leishmania) or L. (Viannia) or form a novel clade within the euleishmania either with or without L. enriettii.