A Revised Classification for Leishmania and Endotrypanum

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A Revised Classification for Leishmania and Endotrypanum E. Cupolillo, E. Medina-Acosta, H. Noyes, H. Momen and G. Grimaldi Jr Leishmania and Endotrypanum are two genera of digenetic parasites belonging to the family Trypanosomatidae; this family also includes the genera Trypanosoma, and at least four genera of parasites that only infect invertebrates. Within the Trypanosomatidae, Leishmania and Endotrypanum, both transmitted by phlebotomine sandfly vectors, are the two most closely related genera1. However, while Endotrypanum is restricted to Neotropical tree sloths (Xenarthra; genera Choloepus and Bradypus), parasites of the genus Leishmania can infect reptiles and several orders of mammals, and have a worldwide distribution in tropical and subtropical areas. The reptilian Leishmania have been classified in a separate genus – Sauroleishmania – although this division has been questioned by several authors. As the classification of several species isolated from lizards is currently the subject of a separate study, we will not discuss Leishmania of reptiles here. At present, about 30 species of Leishmania that infect mammals have been described, and these are divided into species that (1) develop within the midgut and foregut of the sandfly host – classified as the subgenera L. (Leishmania), and (2) undergo an additional developmental phase within the hindgut – classified as the subgenera L. (Viannia)2. Species of both subgenera can be human pathogens and produce a range of clinical conditions3. Parasites of the genus Endotrypanum are unique among the Trypanosomatidae in that they infect the erythrocytes of the mammalian host4; however, these forms are rarely seen in naturally infected sloths. Diagnosis usually relies on the examination of parasites from cultures or from sandflies, and these forms of the parasite are morphologically indistinguishable from the Leishmania promastigotes5,6. Currently, there are only two named species of Endotrypanum: E. schaudinni7 and E. monterogeii8. The intraerythrocytic forms of these species are trypomastigotes and epimastigotes, respectively. Recent Studies Recently, molecular studies have shown that several species of Leishmania are more closely related to Endotrypanum than to the other species of Leishmania9–12. The correct classification of these parasites 142

has important epidemiological significance besides assisting in the understanding of the evolution of the trypanosomatids. A variety of molecular techniques, including multilocus enzyme electrophoresis (MLEE) and analysis of the rRNA gene cluster by restriction fragment length polymorphism (RFLP) of the intergenic transcribed spacers (ITSrRNA)12, measurement of sialidase activity13 and primary DNA sequencing of the small subunit (SSU) rRNA gene9 (all previously shown to be useful in the classification of Leishmania strains) have been used to re-examine representative strains and species of Leishmania and Endotrypanum. On the basis of these results, and using these techniques with additional species14, we propose a revised classification for the genera Leishmania and Endotrypanum. Two Lineages The results of these studies (Fig. 1) suggest the presence of two major phylogenetic lineages within Leishmania that are clearly divergent. We propose that these two lineages be called ‘Sections’, by analogy to the Sections within the genus Trypanosoma. One Section (which we named Euleishmania; Eu 5 well, true, easily compared) includes species classified within the subgenera L. (Leishmania) and L. (Viannia)2. The other Section (which we named Paraleishmania; Para 5 beside, closely related) comprises several Leishmania species, the previous classification of which had been uncertain. It includes L. herreri isolated from sloths, L. equatorensis, a recently described parasite infecting arboreal mammals (sloth and squirrel), L. deanei and L. hertigi from porcupines (Coendu spp), and L. colombiensis, which has been isolated from a variety of hosts, including humans. It also includes species and strains currently classified within the genus Endotrypanum9–12. This division is corroborated by the different techniques. For example, the data from the SSU rRNA sequences and the ITSrRNA analysis give dendrograms (not shown) with branching similar to that in the MLEE phenogram in Fig. 1. The presence of sialidase activity in the Paraleishmania species and the absence of activity in Euleishmania further confirm the separation. This is a specific case of the ‘concordance principle where

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independent gene phylogenies (or phylogenies from independent genetic loci) provide concordant support for the identified assemblages of organisms. The enzyme glucose-6-phosphate dehydrogenase (G6PDH; E.C.1.1.1.49) always produced a single band in Paraleishmania species and a triplet-banding pattern in Euleishmania. Another enzyme that varied between the two Sections was the malic enzyme (ME; E.C. 1.1.1.40). In Euleishmania species, this enzyme had only one locus while in Paraleishmania it had two loci, except for L. hertigi and L. deanei. In fact, these two species were the most divergent members of the Paraleishmania, not only in the MLEE analysis, but also in the analysis of the ITSrRNA and the sequence of the SSU rRNA. This divergence is sufficient to consider these parasites as a separate group within the Paraleishmania. Although our results indicate that the two Sections are clearly divergent [Nei’s genetic distance (D) 5 1.04; E. Cupolillo, unpublished], there are certain similarities between the Paraleishmania and the subgenera L. (Viannia) and L. (Leishmania). For example, a kDNA probe shows crosshybridization between Endotrypanum and L. braziliensis16, while the GP46/M-2 genes occur in L. (Leishmania) but not in L. (Viannia), nor in those of the Paraleishmania that have been studied (Ref. 17 and E. Cupolillo unpublished). Moreover, some of the Leishmania species included in the Paraleishmania were previously described as belonging to L. (Viannia) and others to L. (Leishmania), according to their biological, biochemical or molecular characteristics2,18,19. The Real Endotrypanum? Intraerythrocytic forms of Endotrypanum have been seen only in fresh blood preparations from captured wild sloths, and these forms have never been identified inside erythrocytes in experimental infections. Sloths are reservoirs of many Leishmania species3 as well as Endotrypanum4, and current laboratory strains of Endotrypanum generally grow easily in standard culture media. Therefore, these strains of Endotrypanum might be Paraleishmania parasites present in the original isolates that, during in vitro culture, have grown preferentially over the intraerythrocytic Parasitology Today, vol. 16, no. 4, 2000

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Fig. 1. Dendrogram of MLEE (multilocus enzyme electrophoresis) data of Leishmania species and Endotrypanum. The similarity among the species/zymodemes was calculated by the presence/absence of bands (electromorphs) using the Jaccard’s coefficient, and the dendrogram constructed by the UPGMA algorithm. Results from the other techniques have been added to this phenogram, according to the clusters formation, to summarize the relationships among the species. The two principal clusters, named here as Sections Euleishmania and Paraleishmania, are also distinguished by the presence/absence of sialidase activity, glucose-6-phosphate dehydrogenase (G6PDH) enzyme structure and the SSU rRNA sequencing analysis (a and a9 ). [NB The cladogram produced by the analysis of the SSU rRNA sequence has the same topology as that shown by Noyes et al.5 The species not analyzed in this paper5, L. colombiensis (GenBank accession number AF133836) and L. equatorensis (GenBank accession number AF133837), cluster together with L. herreri and E. monterogeii.] The clusters b, c, d and e represent the groups observed by the analysis of the restriction fragment length polymorphism of the intergenic transcribed spacers of the rRNA gene (ITSrRNA). The dashed lines represent zymodemes of strains currently classified as Endotrypanum23. These zymodemes are related to different Paraleishmania species. EZ12 is closest to L. hertigi/L. deanei and is almost identical to these two Leishmania species by ITSrRNA analysis. EZ01 is very similar to L. herreri and L. colombiensis by MLEE and ITSrRNA analysis.

parasite. Current laboratory strains of Endotrypanum are polyphyletic in origin (Fig. 1). If current isolates of Endotrypanum are, in fact, Paraleishmania, then the intraerythrocytic trypomastigote and epimastigote forms seen in vivo could be developmental stages of an as yet uncultured trypanosomatid parasite of sloths. The alternative possibility of transferring L. hertigi, L. deanei, L. herreri, L. colombiensis and L. equatorensis to a revised genus Endotrypanum is not favoured as it is impossible to classify these Leishmania species within the genus Endotrypanum under the existing definitions for this genus. If species of a revised Endotrypanum genus do develop as amastigotes in their hosts, it would not be possible to discriminate between Leishmania and Endotrypanum on the basis of their morphological development in the vertebrate host. This classification would require a radical re-description of Endotrypanum, including an extension of its host range to include rodents and humans. In fact L. colombiensis can cause both cutaneous and visceral leishmaniasis12,18, and its classification in the Paraleishmania is a further reason for maintaining these parasites as a Section within Leishmania and not transferring them to Endotrypanum. Parasitology Today, vol. 16, no. 4, 2000

Many aspects of the natural history of Endotrypanum and the Paraleishmania are still unknown4,20; therefore, on the basis of available molecular data9,12–14, we propose placing the Paraleishmania Section as a polyphyletic clade within the genus Leishmania and, provisionally classifying all current laboratory strains of Endotrypanum as Paraleishmania. All species of this Section are Neotropical, indicating a New World origin for the Paraleishmania. By definition, Leishmania are digenetic parasites with two distinct stages in their life cycle: a motile flagellated promastigote stage that lives extracellularly within the alimentary tract of the sandfly vectors21, and a nonmotile amastigote stage that resides within macrophages of the vertebrate host22. Endotrypanum are also digenetic parasites, but these flagellates assume an epimastigote or trypomastigote form inside the erythrocytes of sloths, while in the sandfly the parasite assumes promastigote morphology4. We therefore reserve the genus Endotrypanum for the description of the intraerythrocytic trypanosomatid parasite of sloths. The re-description of this genus will require further studies on fresh isolates from sloths, which can be clearly demonstrated to have an intraerythrocytic cycle.

Acknowledgements We sincerely thank Stephen Beverley, Toby Barrett and Michael Miles for valuable discussions. References 1 Fernandes, A.P. et al. (1993) Evolution of nuclear ribosomal RNAs in kinetoplastid protozoa: perspectives on the age and origins of parasitism. Proc. Natl. Acad. Sci. U. S. A. 90, 11608–11612 2 Lainson, R. and Shaw, J.J. (1987) Evolution, classification and geographical distribution, in The Leishmaniases in Biology and Epidemiology Vol. 1 (Peters, W. and Killick-Kendrick, R., eds), pp 1–120, Academic Press 3 Grimaldi, G., Jr and Tesh, R.B. (1993) Leishmaniases of the New World: current concepts and implications for future research. Clin. Microbiol. Rev. 6, 230–250 4 Shaw, J.J. (1992) Endotrypanum, a unique intraerythrocytic flagellate of New World tree sloths. An evolutionary link or an evolutionary backwater? Ciên. Cult. 44, 107–116 5 Croft, S.L. et al. (1980) Ultrastructural and biochemical characterization of stocks of Endotrypanum. Ann. Trop. Med. Parasitol. 74, 585–589 6 Soares, M.J. et al. (1990) Ultrastructural and stereological analysis of trypanosomatids of the genus Endotrypanum. Mem. Inst. Oswaldo Cruz 86, 175–180 7 Mesnil, F. and Brimont, E. (1908) Sur un hematozoaire nouveau (Endotrypanum n.gen.) d’un edente de la Guyane. C. R. Soc. Biol. 65, 581 8 Shaw, J.J. (1969) The Haemoflagellates of Sloths (London School of Hygiene and Tropical Medicine Memoir No.13), Lewis, H.K. & Co. Ltd

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Comment 9 Noyes, H.A. et al. (1997) The Leishmania hertigi (Kinetoplastida; Trypanosomatidae) complex and the lizard Leishmania: their classification and evidence for a neotropical origin of the Leishmania-Endotrypanum clade. J. Euk. Microbiol. 44, 511–517 10 Croan, D.G. et al. (1997) Evolution of the genus Leishmania revealed by comparison of DNA and RNA polymerase gene sequences. Mol. Biochem. Parasitol. 89, 149–159 11 Noyes, H.A. et al. (1996) Leishmania herreri (Kinetoplastida; Trypanosomatidae) is more closely related to Endotrypanum (Kinetoplastida; Trypanosomatidae) than to Leishmania. Mol. Biochem. Parasitol. 80, 119–123 12 Cupolillo, E. et al. (1998) Genetic data showing evolutionary links between Leishmania and Endotrypanum. Mem. Inst. Oswaldo Cruz 93, 677–683 13 Medina-Acosta, E. et al. (1994) Trans-sialidase and sialidase activities discriminate between morphologically indistinguishable trypanosomatids. Eur. J. Biochem. 225, 333–339 14 Cupolillo, E. et al. (1998) Genetic relationships and evolution of new world Leishmania: suggestion of evolutive link with Endotrypanum. Mem. Inst. Oswaldo Cruz 93, 186

15 Avise, J.C. and Ball, R.M. (1990) Principles of genealogical concordance in species concepts and biological taxonomy. Oxford Surv. Evol. Biol. 76, 45–67 16 Pacheco, R.S. et al. (1990) kDNA crosshybridization between Endotrypanum and peripylarian Leishmania. Trans. R. Soc. Trop. Med. Hyg. 84, 531 17 McMahon-Pratt, D. et al. (1992) Loss of the GP46/M-2 surface membrane glycoprotein gene family in the Leishmania braziliensis complex. Mol. Biochem. Parasitol. 50, 151–160 18 Kreutzer, R.D. et al. (1991) Characterization of Leishmania colombiensis sp.n. (Kinetoplastida: Trypanosomatidae), a new parasite infecting humans, animals, and phlebotomine sand flies in Colombia and Panama. Am. J. Trop. Med. Hyg. 44, 662–675 19 Grimaldi, G., Jr et al. (1992) Description of Leishmania equatorensis sp.n. (Kinetoplastida: Trypanosomatidae), a new parasite infecting arboreal mammals in Ecuador. Mem. Inst. Oswaldo Cruz 87, 221–228 20 Lainson, R. (1997) On Leishmania enriettii and other enigmatic Leishmania species of the neotropics. Mem. Inst. Oswaldo Cruz 92, 377–387

21 Walters, L.L. (1993) Leishmania differentiation in natural and unnatural sand fly hosts. J. Euk. Microbiol. 40, 196–206 22 Chang, K-P. et al. (1985) Biology of Leishmania and leishmaniasis. In Leishmaniasis (Chang, K-P. and Bray, R.S., eds), pp 1–30, Elsevier 23 Franco, A.M.R. et al. (1996) Enzyme polymorphism in Endotrypanum and numerical analysis of isoenzyme data. Parasitology 11, 39–48

Elisa Cupolillo and Gabriel Grimaldi Jr are at the Laboratório de Leishmaniose, Department of Immunology, Instituto Oswaldo Cruz, FIOCRUZ, Av Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, Brazil. Enrique Medina-Acosta is at the Universidade Estadual do Norte Fluminense, Campos, RJ, Brazil. Harry Noyes is at the Department of Genetics, University of Liverpool, Liverpool, UK L3 SQA. Hooman Momen is at the Department of Biochemistry and Molecular Biology, Instituto Oswaldo Cruz, FIOCRUZ, RJ, Brazil. Tel: +55 21 280 1486, Fax: +55 21 280 1589, e-mail: [email protected]

ParaSite Fungi, Lynx and Gills on the Web Parasites in general Pathogens of pathogens? Russ Farris wanted examples, but particularly of bacteria attacking helminths. While Tom McCloud knew of ‘fungi which trap (and eat) nematodes’, Alan Trudgett, an immunologist from Queens University, Belfast, Northern Ireland, was interested in viruses. He knew of ‘virus-like particles’ – ‘they looked to me a little like herpes viruses but I’m not an electron microscopist.’ He thought, however, that the rather scattered distribution of helminths makes transmission to a fresh individual difficult, so a helminth virus must either integrate into the genetic material of the host (eg. like retroviruses) or form latent infections, as do herpesviruses. ‘Perhaps it’s time for a bit of blue skies research looking for herpes transcripts in helminth neurons?’ François Dreyer (Western Cape Provincial Veterinary Laboratory, South Africa) had seen an EM of ‘‘viruslike’? structures attached to especially the rectum and genital pore area of the nematode’, and he too mentioned nematophagous fungi, especially Duddingtonia flagrans, a fungus now being sought in South Africa. John T. Jones (University of Dundee, UK) offered Pasteuria, bacteria that attack nematodes that infect plants, and which also could be developed into biological control agents, especially of root-knot nematodes. He wondered too about Wolbachia, found in filarial and plant parasitic nematodes, ‘but whether these are symbionts or parasites is something I’m not sure about.’ [He should have read M.J. Taylor and A. Hoerauf, Wolbachia bacteria of filarial nematodes, Parasitol. Today 15, 437–442, 1999, and C. Bandi et al., Wolbachia genomes and the many faces of symbiosis. Parasitol. Today 15, 428–429; see also ParaSite, Parasitol. Today 14, 259–260, 1998 for an earlier internet discussion of viruses of parasites.] Finally, Rob Knell (Queen Mary & Westfield College, London, UK) added that there are numerous examples among parasitoid wasps, ‘where one species lays an egg in 144

a host (say a caterpillar) and then another species comes along and oviposits in the same host, killing the initial parasitoid and taking the host over.’ [Nematophagous fungi may be grouped by how they trap the vermiform stage of the plant parasitic nematodes (eg. by adhesive knobs, networks or conidia, or by rings or constricting rings); some of them parasitize cyst nematodes, consuming everything inside, including eggs. For dramatic pictures, also of Pasteuria spp, see http://sacs.cpes.peachnet. edu/nemabc sponsored by the Society of Nematologists.] Trichinellosis in Switzerland – in lynx The ProMED-mail post is more usually concerned with virus infections than with worms, but there was an exception in December when Bruno Gottstein (University of Berne, Switzerland) described work done there on the corpses of lynx. Five out of 19 were found to harbour Trichinella musclestage larvae, identified by PCR as Trichinella britovi, a species known to infect the red fox. (Swiss pigs are free of Trichinella spp and the red fox was the only Trichinella-affected host there.) Apparently, the lynx was re-introduced into Switzerland about ten years ago and the population has increased and spread. The nematode is believed to be transmitted directly, by lynx eating dead foxes. Hamburger gill disease This infection of catfish, formally known as proliferative gill disease (PGD), causes the gills to swell, bleed easily and become necrotic, and the fish to gasp for air. Farm-raised catfish are the 5th most popular food fish in the USA, worth about US$592 million per year, and the disease costs the industry US$50–100 million a year. It featured in the ProMED-mail post when Gary Burtle (University of Georgia, USA) was reported by Associated Press as having demonstrated that the flathead minnow Pimphales promelas might offer protection by eating

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