Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing
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Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing Yutaka Asato a, * , Minoru Oshiro b , Chomar Kaung Myint a , Yu-ichi Yamamoto a , Hirotomo Kato c , Jorge Diego Marco d , Tatsuyuki Mimori e , Eduardo A.L. Gomez f , Yoshihisa Hashiguchi d , Hiroshi Uezato a a Division of Dermatology, Unit of Organ-oriented Medicine, University of the Ryukyu, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan b Division of Cell Biology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan c Department of Veterinary Hygiene, Faculty of Agriculture, University of Yamaguchi, Yamaguchi 753-8515, Japan d Department of Parasitology, Kochi Medical School, Kochi University, Kochi 783-8505, Japan e Division of Tumor Genetics and Biology, Department of Developmental and Reconstructive Medical Science, Unit of Advanced Biochemical Science, University School of the Kumamoto, Kumamoto 860-8555, Japan f Department of Epidemiology and Parasitology, National Institute of Health and Tropical Medicine and Department of Tropical Medicine, Catholic University, Guayaquil, Ecuador article info Article history: Received 13 May 2008 Received in revised form 17 December 2008 Accepted 18 December 2008 Available online 25 December 2008 Keywords: Leishmania Endotrypanum PCR Cytochrome b Phylogeny abstract In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human path- ogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylo- genic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classifi- cation proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclu- sion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L. tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction Leishmaniasis is an infectious disease caused by the genus Leish- mania transmitted via sandfly, which belongs to Trypanosomatidae. Leishmaniases are now endemic in 88 countries on five continents, Africa, Asia, Europe, and North and South Americas with a total of 350 million people at risk. The disease presents in humans in four different forms with a broad range of clinical manifestations; vis- ceral, mucocutaneous, diffuse cutaneous and cutaneous leishmani- asis. These different symptoms mainly depend on the species of Leishmania, with more than 20 species identified to date, as well as the host immune system (Herwaldt, 1999; WHO, 2000). The original definition and classification of Leishmania species and subspecies was established by Lainson and Shaw (1987). There are several techniques for identifying species utilizing isoenzyme electrophoresis, monoclonal antibodies and kinetoplast DNA and genomic restriction analysis, but these methods are time consum- ing and require various materials (Kreutzer and Chiristensen, 1980; Lopes et al., 1984; Grimaldi et al., 1987). Recently, molecular biological techniques, such as DNA-based analysis, has replaced traditional methods, as they tend to be more specific and stable. In general, DNA analysis requires repeated copying of the genome, and the levels of inter- and intra-species variability has to be taken into consideration. Thus, genes such as 18S ribosomal RNA (18S-rRNA), gp63 and cytochrome b (Cyt b) have been used as targets for taxonomy of Leishmania (Uliana et al., 1991; Mauricio et al., 1999; Luyo-Acero et al., 2004). The mitochondrial genome is particularly valuable for understanding evolutionary relationships among individuals, populations and species of different organisms. Cyt b is the central redox catalytic subunit of quinol, which belongs to the mitochondrial genome and is useful for phylogenic study (Howell and Gilbert, 1988; Degli-Esposti et al., 1993). As each species tested previously has a 22–24bp region that is very similar to the RNA-editing region in L. tarentolae, which 0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.12.013 * Corresponding author. E-mail address: [email protected] (Y. Asato). Experimental Parasitology 121 (2009) 352–361 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Transcript of Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing
Experimental Parasitology 121 (2009) 352–361
Contents lists available at ScienceDirect
Experimental Parasitology
journal homepage: www.elsevier .com/ locate /yexpr
Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing
Yutaka Asato a,*, Minoru Oshiro b, Chomar Kaung Myint a, Yu-ichi Yamamoto a, Hirotomo Kato c,Jorge Diego Marco d, Tatsuyuki Mimori e, Eduardo A.L. Gomez f, Yoshihisa Hashiguchi d, Hiroshi Uezato a
a Division of Dermatology, Unit of Organ-oriented Medicine, University of the Ryukyu, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japanb Division of Cell Biology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japanc Department of Veterinary Hygiene, Faculty of Agriculture, University of Yamaguchi, Yamaguchi 753-8515, Japand Department of Parasitology, Kochi Medical School, Kochi University, Kochi 783-8505, Japane Division of Tumor Genetics and Biology, Department of Developmental and Reconstructive Medical Science, Unit of Advanced Biochemical Science,University School of the Kumamoto, Kumamoto 860-8555, Japanf Department of Epidemiology and Parasitology, National Institute of Health and Tropical Medicine and Department of Tropical Medicine, Catholic University, Guayaquil, Ecuador
a r t i c l e i n f o a b s t r a c t
Article history:Received 13 May 2008Received in revised form 17 December 2008Accepted 18 December 2008Available online 25 December 2008
Keywords:LeishmaniaEndotrypanumPCRCytochrome bPhylogeny
0014-4894/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.exppara.2008.12.013
* Corresponding author.E-mail address: [email protected] (Y. A
In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis isan effective method for classifying several isolates of the genus Leishmania; hence, we have furtherapplied this method to other Leishmania species in an effort to enhance the accuracy of the procedureand to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHOreference strains, clinical isolates from our patients assigned to 28 strains (human and non-human path-ogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each samplewas amplified by PCR, and was then sequenced by several primers, as reported previously. The phylo-genic tree was constructed based on the results obtained by the computer software MEGA v3.1 andPAUP� v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classifi-cation proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclu-sion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L. tarentolae in the genusSauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L.(L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L.(L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis isthus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separatingnon-human pathogenic species from human pathogenic species.
� 2008 Elsevier Inc. All rights reserved.
1. Introduction
Leishmaniasis is an infectious disease caused by the genus Leish-mania transmitted via sandfly, which belongs to Trypanosomatidae.Leishmaniases are now endemic in 88 countries on five continents,Africa, Asia, Europe, and North and South Americas with a total of350 million people at risk. The disease presents in humans in fourdifferent forms with a broad range of clinical manifestations; vis-ceral, mucocutaneous, diffuse cutaneous and cutaneous leishmani-asis. These different symptoms mainly depend on the species ofLeishmania, with more than 20 species identified to date, as wellas the host immune system (Herwaldt, 1999; WHO, 2000).
The original definition and classification of Leishmania speciesand subspecies was established by Lainson and Shaw (1987). Thereare several techniques for identifying species utilizing isoenzymeelectrophoresis, monoclonal antibodies and kinetoplast DNA and
ll rights reserved.
sato).
genomic restriction analysis, but these methods are time consum-ing and require various materials (Kreutzer and Chiristensen,1980; Lopes et al., 1984; Grimaldi et al., 1987).
Recently, molecular biological techniques, such as DNA-basedanalysis, has replaced traditional methods, as they tend to be morespecific and stable. In general, DNA analysis requires repeatedcopying of the genome, and the levels of inter- and intra-speciesvariability has to be taken into consideration. Thus, genes such as18S ribosomal RNA (18S-rRNA), gp63 and cytochrome b (Cyt b)have been used as targets for taxonomy of Leishmania (Ulianaet al., 1991; Mauricio et al., 1999; Luyo-Acero et al., 2004). Themitochondrial genome is particularly valuable for understandingevolutionary relationships among individuals, populations andspecies of different organisms. Cyt b is the central redox catalyticsubunit of quinol, which belongs to the mitochondrial genomeand is useful for phylogenic study (Howell and Gilbert, 1988;Degli-Esposti et al., 1993).
As each species tested previously has a 22–24bp region that isvery similar to the RNA-editing region in L. tarentolae, which
Y. Asato et al. / Experimental Parasitology 121 (2009) 352–361 353
undergoes insertion of 39 U residues at 15 sites (Feagin et al.,1988), we postulated that the Cyt b gene in the genus Leishmaniaconstitutes two regions, the edited region (50 region of 22–24 bp),which undergoes RNA editing, and the non-edited region (30 regionof 1056 bp), and that the editing region (22–24 bp) may be cor-rected by the RNA-editing process, possibly by adding zero, one,or two U residues downstream of position 22 in the edited region(Luyo-Acero et al., 2004).
We thus established a method for identifying species bysequencing the Cyt b gene and have since analyzed the phylogenyof Leishmania (Luyo-Acero et al., 2004). In this study, we analyzed24 Leishmania species (28 strains), including human and non-hu-man pathogenic species, using Cyt b gene analysis and constructeda novel phylogenic tree based on the results obtained.
2. Materials and methods
2.1. Parasites
The Leishmania strains examined were WHO reference strainsand clinical isolates from patients. Parasites of the genus Endotry-panum (E. schaudinni and E. monterogeii) were also chosen for com-parison with genus Leishmania; some of these were kindlyprovided by Dr. M. Hide (IRD de Montpellier, Laboratory CEPMUMR CNRS/IRD 9926, Cedex 5, France).
2.2. Cell culture
Leishmania promastigotes were grown at 26 �C in RPMI 1640medium (Sigma, Ronkonkoma, NY, USA) supplemented with 10%heat-inactivated fetal bovine serum (Bio Whittaker, Walkersville,MD, USA), 50 U/ml penicillin and 50 lg/ml streptomycin. Prom-astigotes were harvested at the stationary phase of growth by cen-trifugation at 2000g for 10 min. Genomic DNA was extracted frompromastigote pellets using GenomicPrepTM Cell and a Tissue DNAIsolation Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA)according to the manufacturer’s instructions.
2.3. Design of primer and PCR conditions
Oligonucleotide primers were designed based on the consensussequence found in the Cyt b gene and adjacent COIII and MURF4genes conserved among Kinetoplastida. Polymerase chain reaction(PCR) was performed in a total volume of 50 ll. Each reaction mix-ture contained 400 ng of DNA template, 0.25 lM each primer,0.2 mM each dNTP, 1.25 U of EX Taq polymerase, 10 mM Tris–HCl, pH 8.3, 50 mM KCl and 1.5 mM MgCl2 (Takara, Tokyo, Japan).DNA was amplified using an Gene Amp� PCR SYSTEM 9700 (Ap-plied Biosystems, Foster City, CA, USA) with initial denaturationat 94 �C for 1 min, followed by 40 cycles of denaturation at 94 �Cfor 1 min, annealing at 50 �C for 1 min and extension at 72 �C for1 min, and a final extension step at 74 �C for 5 min. In some exper-iments, Phusion polymerase (Finzyme, Espo, Finland) was used.
Each PCR product was run on a 0.7% agarose gel, stained withethidium bromide, and visualized by UV illumination. Approxi-mately 1080-bp PCR products were purified from the gels using aQIAquick Gel Extracion Kit (Qiagen, Germantown, MD, USA).
2.4. DNA sequencing
PCR products were analyzed directly on ABI PRISMTM 310 auto-mated sequencer (Applied Biosystem, USA) using the Big Dye ter-minator cycle sequencing ready reaction kit (Applied Biosystem,USA). DNA sequencing was performed with four gene-specificprimers and 10 internal primers (COIIIF, MURF4RLCB1F, LCBR2,
LCBF2, LCBF3, LCBF30, LCBF4, LCBF5, LCBR1, LCBR3, LCBR30, LCBR4and LCBR5), as reported previously (Luyo-Acero et al., 2004).
2.5. Phylogenic tree analysis
The sequencing results were processed using the software Gen-etyx Mac. ver 11.0.0 (Software Development Co. Ltd, Japan) andMolecular Evolutionary Genetic Analysis (MEGA) version 3.1(Kumar et al., 2001). The neighbor joining (NJ) method and Maxi-mum-likelihood estimation (MLE) were used to construct phylo-genic trees with MEGA v3.1 and Phylogenic Analysis UsingParsimony (PAUP�) version 4.0 Beta (Swofford, 1998).
3. Results
3.1. Sequence analysis of Leishmania Cyt b genes
As reported previously (Luyo-Acero et al., 2004), the first nucle-otide position of the edited region is regarded as the first nucleo-tide position of the Cyt b gene, and the translation terminationcodon (UAA or UAG) in the last part of the non-edited region is re-garded as the end of the Cyt b gene. The total length of the Cyt bgene ranges from 1078 to 1080 bp (edited region: 22–24 bp, non-edited region: 1056 bp). The frequency of base A ranges from0.248 to 0.281, that of base T ranges from 0.488 to 0.512, that ofbase G ranges from 0.153 to 0.175, and that of base C ranges from0.060 to 0.087. The Cyt b gene of Leishmania is thus A + T rich (seeTable 1).
The nucleotide sequences of the Cyt b gene obtained in thisstudy were compared with sequences from L. tarentolae in homol-ogy searches. The range of homology for each strain was 86.7% (L.(L.) deanei) to 90.8% (L. (V.) braziliensis INH-03). As the positions ofL. (L.) hertigi complex and L. (V.) equatorensis (recently removed tothe genus Endotrypanum) are near the genus Endotrypanum in thepresent tree, L. (L.) hertigi was compared with E. monterogeii and E.schaudinni. The homology between L. (L.) hertigi and E. monterogeiiwas 90.1%, and that between L. (L.) hertigi and E. schaudinni wasalso 90.1%. With regard to L. (V.) equatorensis, the homology was100%, showing that the species could not be distinguished fromthe genus Endotrypanum in Cyt b gene analysis.
All Cyt b gene sequences from 28 strains of Leishmania werealigned. A total of 333 nucleotide polymorphisms were observed,of which 59 were singleton and 274 were parsimony-informative.Two nucleotide positions involving insertion or deletion were ob-served in the boundaries between the edited and non-edited re-gions. Alignment of the amino acid sequence corresponding tothe non-edited region revealed amino acid substitutions at 27 posi-tions: 12 Val-Ile substitutions, 3 Leu-Ile substitutions, 2 Met-Leusubstitutions, 2 Phe-Leu substitutions, 1 Cys-Ser substitution, 1Ser-Thr substitution, 1 Val-Thr substitution, 1 Met-Lys substitu-tion, 1 Ala-Val-Leu substitution, 1 Val-Ile-Ala substitution, 1 Val-Ile-Ala substitution, and 1 Tyr-Phe-Cys substitution.
The nucleotide sequence data reported in this paper have beensubmitted to the DDBJ/EMBL/GenBank nucleotide sequence dat-abases with the Accession Nos. AB434674–AB434689 (Table 2).
3.2. Phylogenic analysis of Leishmania Cyt b genes
3.2.1. Human pathogenic LeishmaniaIn the NJ and MLE trees, human pathogenic Leishmania was clas-
sified into 5 clades. Clade 1 (L. (L.) major and L. (L.) major-like) andClade 2 (L. (L.) tropica, L. (L.) killiki and L. (L.) aethiopica) corre-sponded to L. (L.) tropica complex, Clade 3 (L. (L.) donovani, L. (L.)archibaldi, L. (L.) infantum and L. (L.) chagasi = L. (L.) infantum) cor-responded to L. (L.) donovani complex, Clade 4 (L. (L.) amazonensis,
Table 1Leishmania strains used in this study, gene size, A + T and G + C contents, and frequencies of Cyt b gene.
Species International code Size of Cyt b (bp) A + T content (Frequency) G + C content (Frequency)
L. (L.) donovani MHOM/SD/62/2S-25M 1079 296 + 527(0.274 + 0.488) 178 + 78(0.164 + 0.072)L. (L.) chagasi MHOM/BR/74/PP/75 1080 291 + 531(0.269 + 0.491) 180 + 78(0.167 + 0.072)L. (L.) infantum MHOM/TN/80/IPTI 1079 295 + 529(0.273 + 0.490) 179 + 76(0.166 + 0.070)L. (L.) archibaldi MHOM/ET/72/GEBBRE1 1080 296 + 527(0.274 + 0.488) 178 + 78(0.165 + 0.072)L. (L.) hertigi MCOP/PA/65/C8 1080 271 + 535(0.251 + 0.495) 183 + 91(0.169 + 0.084)L. (L.) deanei MCOE/BR/74/M2674 1080 268 + 531(0.248 + 0.491) 186 + 94(0.172 + 0.087)L. (V.) equatorensis MCOH/EC/82/LSP-1 1080 289 + 545(0.268 + 0.505) 176 + 70(0.163 + 0.065)L. (V.) equatorensis MSCI/EC/82/LSP-1 1080 289 + 545(0.268 + 0.505) 176 + 70(0.163 + 0.065)L. (L.) major MHOM/AU/73/5ASKH 1080 275 + 540(0.254 + 0.5) 189 + 76(0.175 + 0.070)L. (L.) major like MHOM/EC/88/PT-115 1080 275 + 541(0.254 + 0.501) 189 + 75(0.175 + 0.069)L. (L.) aethiopica MHOM/ET/72/L100 1080 288 + 531(0.267 + 0.492) 180 + 81(0.167 + 0.075)L. (L.) amazonesis MHOM/BR/75/M2904 1078 285 + 551(0.264 + 0.511) 173 + 69(0.160 + 0.064)L. (L.) garnhami MHOM/VE/76/JAP78 1079 286 + 552(0.265 + 0.512) 172 + 69(0.159 + 0.064)L. (L.) mexicana MHYC/BZ/62/M379 1079 284 + 546(0.263 + 0.506) 175 + 74(0.162 + 0.068)L. (L.) tropica MHOM/SU/58/StrainOD 1080 284 + 537(0.262 + 0.497) 183 + 76(0.169 + 0.070)L. (L.) killiki MHOM/TN/86/LEM163 1080 287 + 532(0.265 + 0.492) 180 + 81(0.167 + 0.075)L. (L.) aristidesi MORY/PA/69/GML 1078 295 + 537(0.273 + 0.498) 169 + 77(0.156 + 0.071)L. (L.) pifanoi MHOM/VE/57/LL1 1079 284 + 546(0.263 + 0.506) 175 + 74(0.162 + 0.069)L. (L.) enrietti MCAV/BR/45/L88 1080 297 + 530(0.275 + 0.491) 173 + 80(0.160 + 0.074)L. (L.) braziliensis MHOM/BR/75/M2904 1078 299 + 548(0.277 + 0.508) 166 + 65(0.154 + 0.060)L. (L.) braziliensis MHOM/BR/75/M2903 1078 298 + 549(0.276 + 0.509) 166 + 65(0.154 + 0.060)L. (L.) braziliensis MHOM/BR/00/LTB300 1078 298 + 549(0.276 + 0.509) 166 + 65(0.154 + 0.060)L. (L.) braziliensis MHOM/EC/88/INH-03 1078 298 + 548(0.276 + 0.508) 167 + 65(0.155 + 0.060)L. (L.) guyanensis MHOM/BR/79/M4147 1078 303 + 544(0.281 + 0.504) 165 + 66(0.153 + 0.061)L. (L.) panamensis MHOM/BR/71/LS94 1078 298 + 546(0.276 + 0.506) 168 + 66(0.155 + 0.061)L. (L.) shawi MHOM/BR/79/M15065 1078 300 + 551(0.278 + 0.511) 165 + 62(0.153 + 0.058)L. (L.) turanica MRHO/SU/80/CLONE3720 1080 277 + 527(0.256 + 0.488) 183 + 93(0.169 + 0.086)L. (V.) arabica MPSA/SA/83/JISH220 1080 278 + 523(0.257 + 0.484) 189 + 90(0.175 + 0.083)
354 Y. Asato et al. / Experimental Parasitology 121 (2009) 352–361
L. (L.) garnhami, L. (L.) mexicana and L. (L.) pifanoi) corresponded to L.(L.) mexicana complex, Clade 5 (L. ((V.) braziliensis, L. (V.) guyanensis,L. (V.) panamensis and L. (V.) shawi) corresponded to L. (V.) brazili-ensis complex, as described by Lainson and Shaw (1987) (see Figs.1 and 2).
Table 2Sequencing results for Cyt b gene (br:braziliensis; eq:equatorensis).
3.2.2. Non-human pathogenic LeishmaniaThe clade of L. (L.) hertigi and L. (L.) deanei (Clade I), which is
defined as the L. (L.) hertigi complex by Lainson and Shaw(1987), is close to the genus Endotrypanum. The alignment ofboth strains (LSP-1 and LSP-2) of L. (V.) equantorensis was the
Table 2 (continued)
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Y. Asato et al. / Experimental Parasitology 121 (2009) 352–361 355
same as the genus Endotrypanum (E. schaudinni and E. montero-geii). L. (L.) turanica and L. (L.) arabica, non-human pathogenicLeishmania, form another clade (Clade II). L. (L.) aristidesi, whichis not yet known to be a human pathogenic species and is con-sidered to be a member of L. (L.) mexicana complex by Lainsonand Shaw (1987), was actually placed in L. (L.) mexicana complexin the present tree. However, it diverged from the human path-ogenic group in the clade. Although L. (L.) enrietti was also de-fined as the L. (L.) mexicana complex, it was placed near thegenus Endotrypanum and L. (L.) hertigi complex in the presenttree (see Figs. 1 and 2).
4. Discussion
In a previous report (Luyo-Acero et al., 2004), we demon-strated that phylogenic analysis based on the Cyt b genesfrom Leishmania species/subspecies was sufficient to distin-guish them, and allowed us to explore their phylogenic rela-tionships. Thus, we attempted to examine more preciserelationships among species/subspecies using additional mate-rials (isolates) by the same method. The results obtainedclearly demonstrate that the method is useful for such phylo-genic analyses.
Table 2 (continued)
356 Y. Asato et al. / Experimental Parasitology 121 (2009) 352–361
Although the phylogenic tree corresponded to the classificationof Leishmania proposed by Lainson and Shaw (1987), our previousreport included two suggestions: (1) the exclusion of L. (L.) majorfrom within the L. (L.) tropica complex, because of its notable ear-lier divergence from the L. (L.) tropica/L. (L.) aethiopica clade (clade2); and (2) the placement of L. tarentolae. In the NJ tree, L. tarento-lae, which was placed in another genus (Sauroleishmania) on thebasis that L. tarentolae infects lizards but not mammals, placed
near the L. (V.) braziliensis complex (Luyo-Acero et al., 2004). Inaddition to these findings, the current investigation led to morequestions.
The first issue is the placement of L. (L.) deanei and L. (L.) hertigi;these two species are classified as the L. (L.) hertigi complex (Lain-son and Shaw, 1987). However, this complex is located near thegenus Endotrypanum in both the NJ and MLE trees. It remains con-troversial whether these species should be included in the genus
Table 2 (continued)
(continued on next page)
Y. Asato et al. / Experimental Parasitology 121 (2009) 352–361 357
Leishmania (Lainson, 1997). In fact, several reports indicate thatthese two species are different from all the known species of Leish-mania (Croft et al., 1978; Miles et al., 1980; Cupolillo et al., 2000).Noyes et al. (1997), who performed RFLP (restriction fragmentlength polymorphisms) of SSUr (sequence of small subunitribosomal) RNA and hybridization studies of kinetoplast DNA ofthe L. (L.) hertigi complex and Endotrypanum parasites, found thatthe L. (L.) hertigi complex was more closely related to the genusEndotrypanum than the genus Leishmania. In addition, Cupolilloet al. (2000), who used MLEE (multilocus enzyme electrophoresis),
RFLP of ITS-rRNA (the intergenic transcribed spacer), SSU rRNAgene sequencing and measurement of sialidase activity, also foundthat L. (L.) hertigi complex, L. (L.) herreri isolated from sloths, L (V.)equatorensis infecting arboreal mammals (sloths and squirrels) andL. (V.) colombiensis isolated from a variety of host were classifiedwithin the genus Endotrypanum (Cupolillo et al., 2000). Our resultscorresponded with these reports.
In the present study, we could not distinguish L. (V.) equatorensisfrom the genus Endotrypanum in our study. This suggests that L. (V.)equatorensis is genetically approximate to the genus Endotrypanum,
able 2 (continued)
358 Y. Asato et al. / Experimental Parasitology 121 (2009) 352–361
T
and Katakura et al. (2003), who performed PCR amplification andsequencing of the Mini-Exon gene for characterization andidentification of Endotrypanum species, mentioned that L. (V.)equatorensis parasites were phylogenically related to the genusEndotrypanum. As only two species of the genus Endotrypanum,E. schaudinni and E. monterogiil, have been reported to date, someresearchers believe that this genus should be included in the genusLeishmania. Interestingly, Cupolillo et al. (2000) suggested that thismay be due to the possibility that, during in vitro culture, Paraleish-mania parasites present in the original host were cultured insteadof Endotrypanum organisms, and they pointed out the necessity offurther precise studies on the genus Endotrypanum. Therefore, withregard to this genus, further research is needed.
The second issue is the position of L. (L.) enrietti. This species issupposed to belong to the L. (L.) mexicana complex (Lainson andShaw, 1987). L. (L.) enrietti remains mysterious, due to the obscurityof its wild mammalian host and sandfly vector. The domestic guineapig, Cavia porcellus, is considered to be its sole mammalian host, andno human cases have been reported to date. In the NJ tree, L. (L.)enrietti shows early divergence from other groups. It is placed nearthe genus Endotrypanum in the MLE tree, which shows that it maynot be part of the genus Leishmania (Lainson, 1997).
The third issue is placement of L. (L.) turanica and L. (L.) arab-ica. These two species form another clade in trees, and this iscompletely different from the 5 clades reported previously(Luyo-Acero et al., 2004), while the results of the NJ and MLEtrees imply that these species may be a subgroup of L. (L.) tropicacomplex. L. (L.) turanica was isolated from the auricular tissue ofgreat gerbils in Uzbekistan by Kellina and Passova (1985) and wasnamed by Strelkova et al (1990). L. (L.) arabica was found in therodent Psammomys obeseu from the eastern province of SaudiArabia (Peters et al., 1986). The fact that the two species werefound around the area in which L. (L.) major was distributedmay be consistent with the divergence from the L. (L.) major cladein the NJ and MLE trees. L. (L.) turanica and L. (L.) arabica wereregarded as non-human pathogenic species. No human cases in-fected with these two species have been observed, although thepathogenicity of L. (L.) turanica in humans based on artificial (orexperimental) infection has been reported. Its earlier divergencefrom L. (L.) tropica clade in our trees will be a useful landmarkfor differential diagnosis between human and non-human patho-genic Leishmania species.
The same concept will be applied to the position of L.(L.) aristidesi.This species was isolated from the rodents Proechimys semispinosus,
L.(L.)arabica
L.(L.)turanica L.(L.)major
L.(L.)major-like
L.(L.)aethiopica
L.(L.)killicki L.(L.)tropica
L.(L.)chagasi
L.(L.)infantum
L.(L.)archibaldi
L.(L.)donovani
L.(L.)aristidesiL.(L.)amazonensis
L.(L.)garnhami
L.(L.)mexicana L.(L.)pifanoi
L.tarentolae
L.(V.)shawi
L.(V.)panamensis
L.(V.)guyanensis L.(V.)braziliensis INH-03
L.(V.)braziliensis M2903
L.(V.)braziliensis LTB300
L.(V.)braziliensis M2904
L.(L.)enriettii
L.(L.)deanei
L.(L.)hertigi
L.(V.)equatorensis LSP2
L.(V.)equatorensis LSP1
E.monterogeii
E.schaudinni
100
100
100
100
100
100
9883
100
60
100
99
87
58
83
95
91
90
76
85
100
50
56
99
71
0.01
L.tropicacomplex
L.donovanicomplex
L.mexicana complex
L.braziliensiscomplex
L.hertigicomplex
Clade 1
Clade 2
Clade 3
Clade 4
Clade 5
Clade I
Clade II
Fig. 1. Phlogeny of Leishmania according to the Neighbour Joining (NJ) method. Brackets and dotted lines indicate Leishmania complexes as described by Lainson and Shaw(1987). Arrows indicate clades formed based on Cyt b gene. Clades labeled with Arabic numbers are human pathogenic Leishmania. Clades labeled with Roman numerals arenon-human pathogenic Leishmania. Numbers in the branches correspond to the bootstrap values based on 1000 replicates. The NJ tree was constructed using theTamura and Nei (1993) distance.
Y. Asato et al. / Experimental Parasitology 121 (2009) 352–361 359
Orysomys capito and Agouti paca, and the marsupial Marmosa rob-insoni in the Sasardi forest, San Blas Territory, eastern Panama(Herrer, 1971). Although L. (L.) aristidesi was defined as a memberof the L. (L.) mexicana complex by Lainson and Shaw (1987), itspathogenicity in humans is uncertain. Uliana et al. (2000) re-ported that the L. (L.) mexicana complex was divided into twosubgroups according to their PCR hybridization assay derivedfrom SSU rRNA sequences, and the patterns of SSU rRNA inL. (L.) aristidesi are more similar to L. (L.) amazonensis (Ulianaet al., 2000). Our trees revealed that L. (L.) mexicana complexhad two subgroups, and proved that L. (L.) aristidesi belongs tothe L. (L.) mexicana clade. However, its position is completely
separated from L. (L.) amazonensis and L. (L.) garnham in our trees.Lainson (1997) speculated that the habit of the suspected vector,Lutzomyia olmeca bicolor, which bites humans on rare occasions,may be a main factor for the absence of reported human cases(Lainson, 1997). We believe that the parasite itself may affectthe pathogenicity of Leishmania parasites, as in the present phy-logeny, L. (L.) aristidesi diverged early from L. (L.) mexicana, L. (L.)amazonensis and L. (L.) garnhami (these show pathogenicity inhumans). This suggests that genetic factors are involved in theseparation from human pathogenic species. Thus, we need toinvestigate other non-human pathogenic species in order to eval-uate this hypothesis.
0.8
L.(V.)guyanensis
L.(L.)killicki
L.(L.)arabica
L.(V.)braziliensis M2904
L.(V.)braziliensis INH03
L.(L.)aethiopica
L.(L.)major
L.(V.)panamensis
L.(L.)major-like
L.tarentolae
L.(V.)shawi
L.(L.)deanei
L.(L.)infantum
L.(L.)aristidesi
L.(V.)equatorensis LSP2
L.(L.)pifanoi
E.schaudinni
L.(L.)enriettii
L.(L.)turanica
L.(L.)amazonensis
L.(L.)hertigi
L.(L.)chagasi
E.monterogeii
L.(L.)tropica
L.(L.)donovaniL.(L.)archibaldi
L.(V.)braziliensis LTB300L.(V.)braziliensis M2903
L.(L.)mexicana
L.(L.)garnhami
L.(V.)equatorensis LSP1
87
10
10
99
80
98
80
98
8498
100
99
100
10075
85
5298
100
100
59
9962
L.hertigicomplex
Clade I
Clade II
L.tropica complex
Clade 1
Clade 2
L.donovanicomplex
Clade 3
L.mexicana complexClade 4
Clade 5
L.braziliensiscomplex
Fig. 2. Phlogeny of Leishmania according to Maximum-likelihood estimation (MLE). lnL = �3357.76042. Brackets and dotted lines indicate Leishmania complexes as describedby Lainson and Shaw (1987). Arrows indicate clades formed based on Cyt b gene. Clades labeled with Arabic numbers are human pathogenic Leishmania. Clades labeled withRoman numerals are non-human pathogenic Leishmania. Numbers in the branches correspond to the bootstrap values based on 1000 replicates. The HKY85 model(Hasegawa-Kishino-Yano 1985) of evolution was used.
360 Y. Asato et al. / Experimental Parasitology 121 (2009) 352–361
On the whole, our classification was almost identical to thetraditional method of classification proposed by Lainson and Shaw(1987). However, their classification could not distinguish humanLeishmania from non-human Leishmania, on the other hand ourclassification could do it. Furthermore, our classification could de-tect small subgroups of Leishmania as described previously. Inthese respects, we could mention that our classification would beimproved, compared to traditional classification.
Although our study could not discriminate the precise relation-ship between Leishmania and Endotrypanum, the present Cyt b geneanalysis is applicable to phylogeny of the genus Leishmania. As allnon-human pathogenic species diverge early from human patho-genic species, the present method may be useful for separatingthe non-human pathogenic species from the pathogenic species.Several other species of Leishmania still require precisestudy, and such studies are sure to contribute to knowledge con-cerning the clinical diagnosis and epidemiological research ofleishmaniasis.
Acknowledgments
We thank Dr. Mallorie Hide for providing L. (L.) chagasi (= L. (L.)infantum), and Dr. Nonaka for his support in the initial phases of
the study. This study was financially supported by the Ministryof Education, Science, Culture, Sports and Technology of Japan(Grant Nos. 14256002, 15590371 and 18256004).
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