237

Genomic diversity in the Leishmania donovani complex

I. L. M A U R I C IO*, M. K. H O W A R D†, J. R. S T O T H A RD‡ and M. A. M I L ES

Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of
Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK

(Received 25 November 1998 ; revised 9 April 1999 ; accepted 9 April 1999)


The Leishmania donovani complex is considered to be composed of 3 species ; L. donovani, L. infantum and L. chagasi,
although this classification has been challenged. Genotypic relationships within the complex were evaluated at different
levels by : binding of the probe Lmet9, specific for L. chagasi and Old World Leishmania spp. ; partial sequencing of a
constitutive major surface protease single gene (mspC) and random amplification of polymorphic DNA (RAPD). The Old
World Leishmania spp. and the L. donovani complex have a monophyletic origin. Leishmania chagasi clearly belongs to
the L. donovani complex but it is indistinguishable from L. infantum, which suggests introduction of L. chagasi into the
New World in recent history. Leishmania infantum\L. chagasi was identified as a monophyletic group within the L.
donovani complex but L. donovani may be paraphyletic. Diversity within L. donovani is substantial and phylogeographical
patterns of association were found.

Key words : Leishmania donovani complex, Leishmania infantum, Leishmania chagasi, visceral leishmaniasis, RAPD
analysis.

studies of the L. donovani complex were based on


Visceral leishmaniasis or kala-azar is a potentially
fatal human disease, the aetiological agents of which
are species of the Leishmania donovani complex
(Lainson & Shaw, 1987) : L. donovani (Laveran &
Mesnil, 1903) Ross, 1903 (Old World), L. infantum
Nicolle, 1908 (Old World) and L. chagasi Cunha &
Chagas, 1937 (New World). Taxonomy within this
complex is controversial. Leishmania donovani may
include African L. donovani-like parasites (Rioux et
al. 1990) but these are also referred to as L. donovani
sensu lato (Le Blancq & Peters, 1986 ; Ashford &
Bettini, 1987 ; Ellis & Crampton, 1991 ; Mebrahtu et
al. 1992). Leishmania infantum is usually considered
a distinct species (Shaw, 1994), included in the L.
donovani complex or forming its own complex
(Rioux et al. 1990). It has also, however, been
considered as a subspecies, L. donovani infantum
(Ashford & Bettini, 1987). Inclusion of L. chagasi in
the L. donovani complex is widely accepted, yet its
taxonomic affinities and origin are still a matter of
controversy (Shaw, 1994). Synonymy of L. chagasi
with L. infantum is strongly supported (Momen,
Grimaldi & Deane, 1987 ; Rioux et al. 1990 ;
Grimaldi & Tesh, 1993). Most previous taxonomic
* Corresponding author : Tel : j44 171 927 2399. Fax :
j44 171 636 8739. E-mail : i.mauricio!Ishtm.ac.uk
† Present address : Cantab Pharmaceutical Research Ltd,
184 Cambridge Science Park, Milton Rd, Cambridge CB4
4GN.
‡ Present address : Biomedical Parasitology, Department
of Zoology, Natural History Museum, Cromwell Road,
London SW7 5BD, UK.
isoenzyme electrophoresis, restriction fragment
length polymorphisms and monoclonal antibodies,
with random amplification of polymorphic DNA
(RAPD) also used, although not systematically.
We chose to evaluate the taxonomy of the L.
donovani complex by (1) hybridization of parasite
total DNA to a probe Lmet9, (2) sequencing of a
Leishmania major surface glycoprotein (gp63) gene
and (3) RAPD. Simultaneously with the Lmet2
probe (Howard et al. 1991), diagnostic for the L.
donovani complex, another probe (Lmet9), was
developed, that is specific for Old World (OW)
Leishmania species (L. donovani complex, L.
aethiopica, L. major, L. arabica, L. tropica) and the
New World (NW) L. chagasi. Lmet9 was used here
to establish the general geographical origin of L.
chagasi. The major surface protease (msp) gp63, a
surface glycosyl phosphatidylinositol (GPI)-
anchored zinc protease of 63 kDa, present in all
Leishmania species (Chakrabarty et al. 1996 ; Mauel,
1996), is encoded by multigene families in
Leishmania. In L. chagasi 3 gp63 families have been
described (Ramamoorthy et al. 1992) in a tandem
array (Roberts et al. 1993) : mspL, mspS and mspC
(single gene), which can be distinguished by the 3h
end half of the coding region and the translated
noncoding 3h end. Gp63 genes have been studied in
L. donovani and L. infantum (Maingon et al. 1990 ;
Webb, Button & McMaster, 1991 ; Morales et al.
1997) but comparisons with those described for L.
chagasi are not clear. The more variable 3h end of
mspC was chosen here for DNA sequence com-
parisons, as this region is discriminatory for mspC

Parasitology (1999), 119, 237–246. Printed in the United Kingdom # 1999 Cambridge University Press
I. L. Mauricio and others
and is not known to contain functional regions,
except for a Zn binding site. Leishmania isolates were
also screened with RAPD (see Williams et al. 1990),
recognized to be a useful taxonomic method for
Leishmania (Tibayrenc et al. 1993 ; Motazedian,
Noyes & Maingon, 1996).
The objective of this study was to provide genetic
data at different levels of resolution and to clarify
phylogeny within the L. donovani complex.
  
Leishmania strains
Genomic DNA was extracted by phenol\chloroform
according to the method described by Kelly (1993)
from diverse Leishmania strains with a wide range of
geographical origins (Table 1).
Hybridization with the Lmet9 probe
The Lmet9 probe was isolated from a L. donovani
HU3 cDNA library in the bacteriophage vector
λgt10, simultaneously to Lmet2 (Howard et al.
1991). Samples of 10& whole cells of 16 WHO
reference strains of Leishmania spp. and 10' cells of
12 L. chagasi and L. infantum strains were dot-
blotted and hybridized to the Lmet9 and, as control,
to the $#P-labelled CTI (hsc70, MacFarlane et al.
1990) probes, made by primer extension of single-
stranded recombinant M13 DNA (Howard et al.
1991). Stringency washes were in 0n25iSSC at
55 mC for the reference strains and at 60 mC for the
L. infantum and L. chagasi strains. Autoradiographs
were exposed overnight at k70 mC.
Direct sequencing of the 3h end of the mspC gene
A total of 1083 bases of the 3h coding end of mspC
(mspC3h), between bases 924 and 2238 (GenBank
accession number M80671), were sequenced for
comparison. Three pairs of primers were designed
from an alignment of L. chagasi mspC, mspL and
mspS1 and L. donovani gp63 genes (GenBank
accession numbers, respectively, M80671, M80672,
M80669 and M60048). The pairs of primers were (5h
to 3h) : A-C4F (tgt aaa acg acg gcc agt CAC GTC
GGC TTC AGT GGA)jC6R (cag gaa aca gct atg
acc AAA AAC CCT GCT GCC AAC) ; B-C6F
(CCA GTC GTC TGA TGG TCG)jC7R (CTG
CTG GAG CTG TCG GAG) ; C-C11F (GCG
CGG CAG TAT GGA CTA)jC8R (TGG ACC
GGA GGA GAC GAG). Primers C4F and C6R
had, respectively, -21M13 and M13Rev primers
added (lowercase). Each 100 µl of amplification
reaction contained : 125 ng genomic DNA, 50 pmol
each primer, 0n2 m dNTPs, 2 m MgCl , 5 %
#
dimethyl sulfoxide (B and C only) and 5U Taq
238
Polymerase (Bioline), in the buffer recommended by
the manufacturer. The amplification thermal profiles
were : 94 mC for 1 min ; 30 cycles at 94 mC, then at
55 mC for A and at 65 mC for B and C, followed by, in
all cases, 72 mC for 1 min ; and 72 mC for 10 min. The
PCR products from the 3 regions were purified using
a QIAEX II4 gel purification Kit (Qiagen), from a
0n8 % agarose gel in TAE buffer.
Direct sequencing was done with ABI PRISM4
Dye Terminator Cycle Sequencing Ready Reaction
kits (Perkin-Elmer, UK) or AmpliTaq2 DNA
Polymerase, FS Thermo-sequenase Dye Terminator
Cycle Sequencing Premix kits (Amersham, Life
Science) according to the manufacturers’ instruc-
tions, using the PCR primers. The thermal profile
was, 25 cycles at 96 mC for 30 s, 50 mC for 15 s, 60 mC
for 240 s. The sequencing products were purified by
ethanol precipitation, and separated in an ABI
Prism4 377 DNA Sequencer (Perkin-Elmer, UK).
Consensus sequences were obtained from forward
and reverse reactions aligned in ABI Prism Sequence
Navigator4 Version 1.0.1 (Perkin-Elmer, UK) and
using Clustal V (Higgins, Bleasby & Fuchs, 1992).
Eight (the L. infantum and L. chagasi WHO
reference strains, L. chagasi strain WR285 and the L.
donovani strains) of the full mspC3h sequences
obtained, were aligned by Clustal V (Higgins et al.
1992) and were used to infer phylogenies using
programs within the PHYLIP package (Felsenstein,
1993). Putative homologues of L. chagasi mspC,
L. major gp63-6 and L. mexicana gp63-C1 (GenBank
accession numbers, respectively, AF039721 and
X64394), were used as outgroups. Dendrograms
were produced from sequence alignment data using
maximum parsimony and maximum likelihood
methods, and from a distance matrix (corrected
nucleotide divergence using the Kimura ‘ 2-
parameter ’ model) using neighbour joining and
Fitch-Margoliash-least squares methods. Bootstrap
analyses were based upon 1000 replicate data
sets, except for the Fitch-Margoliash-least squares
method, where 100 replicates were used and
the L. chagasi PP75 sequence (identical to the
L. infantum) was excluded, and for the maximum
likelihood method owing to the long computing
times necessary.
Random amplified polymorphic DNA
Ten decameric primers (5h-3h : A2-GAAACGGG-
TG, A4-AATCGGGCTG, A5-CTCACGTA GG,
A6-CTGATCGCAG, D3-TTCCGAACCC, D8-
AGCCAGCGAA, D10-GTTGCGATC C, H1-
CGCGCCCGCT, H4-TGCCGAGCTG, L2-
CGGACGTCGC) were used upon 33 Leishmania
isolates (Table 1). Each 20 µl of RAPD reaction
contained : 25 ng genomic DNA, 2n5 m MgCl ,
#
0n2 m dNTPs, 20 pmol primer, 1U Taq poly-
merase (Bioline) in the buffer recommended by the
 Diversity in the Leishmania donovani complex
Table 1. Leishmania spp. strains and analyses performed

International Code Analysis† International Code Analysis† International Code Analysis† International Code Analysis†

L. aethiopica MCAN\BR\89\DOG136 3 MCOE\PA\65\C8* 1 IARI\PT\89\IMT170 2a

MHOM\ET\72\L100* 1 MCER\BR\89\M12084 3 L. infantum IARI\PT\89\IMT171 2
MHOM\ET\70\L96 3 MCER\BR\89\M12085 3 MHOM\CN\80\STRAIN A 2, 3 IARI\PT\89\IMT172 2a
L. arabica MHOM\BR\84\M8270 1, 3 MHOM\CY\63\L53 2a, 3 MCAN\PT\93\IMT193 3
MPSM\SA\83\JISH220 1 MHOM\BR\85\M9702 1, 3 MHOM\FR\78\LEM75 1, 2a, 3 MHOM\ES\87\LOMBARDI 1, 2a, 3
L. brasiliensis MHOM\PA\78\WR285 1, 2, 3 MCAN\FR\82\PHAROAH 1, 2a, 3 MHOM\TU\80\IPT-1* 1, 2, 3
MOM\BR\76\LTB300* 1 MHOM\PA\80\WR341 1 MHOM\IT\81\ALESSANDRO 2a L. major
L. guyanensis L. donovani MCAN\PT\81\L82 2a, 3 MHOM\SU\73\5ASKH* 1, 3
MHOM\BR\75\M4147* 1 MHOM\ET\67\HU3(LV9)* 1, 2, 3 MCAN\PT\81\IMT89 3 L. amazonensis
L. panamensis MHOM\IN\80\DD8* 1, 2, 3 MHOM\PT\82\IMT104 1, 3 MHOM\BR\67\PH8* 1
MHOM\PA\71\LS94* 1, 2, 3 MHOM\IN\82\PATNA1 2, 3 MCAN\PT\84\IMT124 3 L. mexicana
L. chagasi 2 MHOM\KE\67\MRC(L)3 2, 3 MVUL\PT\82\IMT108 3 MHOM\BZ\82\BEL21* 1
MHOM\BR\74\PP75* 3 MHOM\KE\73\MRC74 2, 3 MVUL\PT\84\IMPT128 1 L. pifanoi
MCER\BR\81\M6445 1, 2a, 3 L. gerbilli MCAN\PT\87\IMT150 3 MHOM\VE\57\LL1* 1
MCER\BR\83\M7633 3 MRHO\CN\60\GERBILLI 1 MCAN\PT\87\IMT152 3 L. tropica
MCAN\BR\84\C0910 2, 3 L. hertigi MCAN\PT\88\REBELO2 1, 2a MHOM\SU\74\K27* 1, 3
MCAN\BR\89\DOG118
MCAN\BR\89\DOG124

* WHO reference strains. Key for countries : BR : Brazil ; BZ : Belize ; CN : China ; CY : Cyprus ; ET : Ethiopia ; FR : France ; HN : Honduras ; IN : India ; IT : Italy ; KE : Kenya ;
PA : Panama ; PT : Portugal ; ES : Spain ; SU : former Soviet Union ; TU : Tunisia ; VE : Venezuela.
† 1 – Lmet9 ; 2 – mspC3h end sequence (2a – incomplete sequences) ; 3 – RAPD.

239
I. L. Mauricio and others
Fig. 1. Hybridization of Lmet9 to Leishmania dot blots, with CTI probe as positive control. (A) WHO reference
strains for the named species (L. donovani ss is DD8 and L. donovani sl is HU3). (B) L. infantum and L. chagasi
strains. The Lmet9 probe binds specifically to all Old World Leishmania spp. and Leishmania chagasi.
Fig. 2. Potential diagnostic sites for Leishmania
infantum\L. chagasi in the mspC3h sequence alignment in
the L. donovani complex. Numbers above refer to
position in the alignment, and before each line is the
base number in each EMLB database sequence where
the alignment started (nS). The sites between 371 and
385 are shown as an interval. Note site 582, the unique
polymorphic site found within the sequenced L.
infantum\L. chagasi. The upper 3 strains are L.
infantum\L. chagasi and the lower 5 are L. donovani
strains.
manufacturer. Amplifications were carried out using
1 cycle of : 94 mC for 5 min, 37 mC for 1 min, 72 mC for
1 min ; 37 cycles of : 94 mC, 37 mC, 72 mC for 1 min
each ; and 1 cycle of : 72 mC for 10 min. RAPD
products were separated on 1n2 % agarose gels in
TAE buffer, with 1 µg\ml ethidium bromide, at
50 V for 4 h.
RAPD profiles were scored as presence or absence
data. Dendrograms were built from a distance matrix
[Jaccard index : 1-a\(ajbjc)] using single linkage
and unweighted pair group method with arithmetic
averages (UPGMA) clustering algorithms. In ad-
dition a principal coordinates analysis was performed
with a superimposed minimum spanning tree.
Computer analyses used program SYNTAX-pc 5n0
(Podani, 1993).
240

The Lmet9 probe is specific for the Old World
Leishmania spp.
The Lmet9 probe is 670 kb in length, it is located in
a 700 kbp chromosome in L. donovani and belongs to
a multigene family, although not in tandem repeats
(data not shown). The analysis with the Fickett’s
Testcode programme indicated that Lmet9 is un-
likely to represent a coding region but the pro-
gramme Staden Analysec identified 2 regions within
the probe that are compatible with tRNA like
structures (data not shown).
The Lmet9 probe was able to recognize all WHO
reference strains of Old World Leishmania species
and also L. chagasi but failed to recognize any other
New World Leishmania WHO reference strain (Fig.
1 A). In an extended survey of L. infantum and L.
chagasi, all strains tested positive with probe Lmet9
(Fig. 1 B).
Sequence analysis of mspC3h
Full 1083 bp mspC3h sequences from L. infantum
and L. chagasi strains were identical (1PT-1, PP75,
DOG124, M6445, STRAIN A, IMT171 ; EMBL
accession numbers AJ010234, AJ00908, AJ009911,
AJ010240-2 respectively), except for a single base
pair in strain WR285 (at nucleotide 582 ; EMBL
accession number AJ009909), but variation was
found among L. donovani strains : strains DD8,
PATNA1, HU3, MRC(L)3 and MRC74 (EMBL
accession numbers AJ010235-9). Incomplete mspC3h
sequences from other L. infantum and L. chagasi
strains were also identical to the complete sets :
strains PHAROAH, LOMBARDI, LEM75,
ALESSANDRO (599–625 nt, EMBL accession
numbers AJ010248-51), and L82, REBEL02, L53,
Diversity in the Leishmania donovani complex 241

Table 2. Distance and similarity matrices for mspC3h sequence data of the Leishmania donovani complex
(Corrected nucleotide divergence (Kimura ‘ 2-parameter ’) is shown in the lower triangular matrix and pairwise similarity
is shown in the upper matrix. Maximum and minimum values for each measure within the L. donovani complex are
indicated in bold.)

L. infantum\L. chagasi L. donovani

L. mexicana L. major PP75 WR285 IPT-1 DD8 HU3 MRC(L)3 MRC74 PATNA1

L. mexicana — 0n848 0n121 0n122 0n121 0n126 0n132 0n128 0n130 0n127
L. major 0n176 — 0n114 0n115 0n114 0n123 0n125 0n124 0n124 0n123
PP75 0n135 0n127 — 0n999 1n000 0n972 0n965 0n971 0n984 0n976
WR285 0n136 0n128 0n009 — 0n999 0n978 0n964 0n970 0n969 0n975
IPT-1 0n135 0n127 0n000 0n009 — 0n972 0n965 0n971 0n984 0n976
DD8 0n141 0n137 0n028 0n029 0n028 — 0n989 0n992 0n989 0n995
HU3 0n148 0n140 0n035 0n036 0n035 0n008 — 0n984 0n980 0n988
MRC(L)3 0n144 0n138 0n029 0n030 0n029 0n008 0n013 — 0n994 0n989
MRC74 0n148 0n138 0n031 0n032 0n031 0n012 0n019 0n006 — 0n985
PATNA1 0n141 0n138 0n028 0n029 0n028 0n002 0n008 0n008 0n012 —

Fig. 3. A neighbour-joining dendrogram built from the

distances from the mspC3h sequence in the Leishmania
donovani complex, rooted to L. major and L. mexicana.
Neighbour-joining bootstrap values of more than 90 %
from 1000 trees are indicated at nodes. Maximum
parsimony and Fitch-Margoliash-least squares bootstrap
values were equivalent.

IMT170, IMT172, C0910 (784–840 nt, EMBL

accession numbers AJ010243-47, AJ009910). In
total 47 nucleotide positions (4n3 %) were found to be
polymorphic within the L. donovani complex, with
22 amino acid alterations, including in one of the
Zn binding sites in the L. donovani strains
(Ramamoorthy et al. 1992). A number of poly-
morphisms could be specific for L. infantum\L.
chagasi (Fig. 2).
All sequences (including those from L. donovani
strains) possessed highest similarity to L. chagasi
mspC on a BLAST search (Experimental WU-
BLAST server from the Bioinformatics Group of
the Swiss Institute for Experimental Cancer Re-
search – ISREC), confirmed by visual inspection,
but diverged from it by 3 extra base pairs in the
coding region.
In the sequence analysis L. infantum and L.
chagasi WHO reference strains were identical and
strains within L. donovani were variable (Table 2).
In the neighbour joining dendrogram, rooted to L.
Fig. 4. RAPD amplification patterns of strains of the
Leishmania donovani complex with primers (A) H1 and
(B) A5. Note the polymorphic band patterns produced
by primer A5 and the differentiating bands for
Leishmania donovani in primer H1, indicated by arrows.
L. chagasi : 1-DOG 118 ; 2-M7633 ; 3-M12085 ; 4-
M12084. L. donovani : 5-HU3 ; 6-MRC(L)3 ; 7-MRC74 ;
8-PATNA1. L. infantum : 9-IMPT104 ; 10-IMPT128 ;
11-IMT150 ; 12-IMT152 ; 13-IMT89 ; 14-IMT124 ; 15-
IMT108 ; 16-IMT193.
major and L. mexicana (Fig. 3), the L. donovani
complex was monophyletic and divided into L.
infantum\L. chagasi and L. donovani. The latter
group had a higher degree of diversity and the
Kenyan strains were a distinct branch. In all
clustering methods tested, the topologies were
identical. The bootstrap values were higher than
88 % for the above mentioned branches (Fig. 3). The
L. infantum\L. chagasi group was closer to the root
of the tree and formed a very homogeneous group.
 I. L. Mauricio and others
Table 3. Pairwise distance (Jaccard index) and band sharing among Leishmania donovani complex strains for RAPD data
(The 3 groups of strains are, from top : outgroup, L. infantum\L. chagasi and L. donovani. In the upper triangular matrix are shared over total number of bands. In the lower matrix
are Jaccard distances and in the diagonal the number of bands for each strain. Underlined are maximum and minimum distances within the L. donovani complex.)

5-ASKH 82 20\156 24\181 24\185 23\178 25\188 22\169 24\180 23\184 24\189 23\185 24\188 23\182 24\189 22\185 24\181 25\183 23\181 23\183 23\184 22\180 22\171 24\188 24\184 23\182 25\189 22\181 24\185 22\184 24\180 26\187 22\183 26\188
L96 0n853 74 44\173 21\177 20\170 22\180 19\161 21\172 21\176 21\181 21\177 21\180 21\174 21\181 20\177 21\173 22\175 21\173 20\175 20\176 20\172 18\163 21\180 22\176 19\174 23\181 19\173 21\177 20\176 22\172 21\179 20\175 21\180
K27 0n847 0n659 99 25\202 25\195 24\205 19\186 25\197 24\201 24\206 24\202 24\205 24\199 25\206 24\202 24\198 25\200 25\198 24\200 25\201 25\197 20\188 24\205 24\201 24\199 26\206 24\198 24\202 25\201 25\197 24\204 23\200 25\205

IPT-1 0n851 0n865 0n859 103 94\199 97\209 84\190 94\201 96\205 99\210 96\206 97\209 93\203 99\210 95\206 92\202 92\204 95\202 94\204 96\205 94\201 83\192 97\209 94\205 94\203 96\210 93\202 96\206 86\205 83\201 83\208 78\204 83\209
L82 0n852 0n867 0n853 0n105 96 93\202 84\183 93\194 93\198 93\203 92\199 93\202 90\196 94\203 91\199 88\195 89\197 90\195 92\197 94\198 92\194 84\185 93\202 90\198 91\196 92\203 88\195 92\199 81\198 82\194 78\201 74\197 77\202
Pharoah 0n847 0n861 0n867 0n134 0n147 106 85\193 96\204 101\208 104\213 100\209 102\212 98\206 104\213 98\209 97\205 98\207 97\205 95\207 96\208 95\204 87\195 102\212 97\208 96\206 101\213 94\205 99\209 87\208 87\204 84\211 80\207 86\212
Lombardi 0n850 0n866 0n866 0n208 0n152 0n213 87 84\185 83\189 84\194 82\190 83\193 81\187 84\194 81\190 82\186 80\188 81\186 86\188 83\185 81\185 79\176 83\193 81\189 81\187 82\194 78\186 82\190 72\189 75\185 71\192 69\188 70\193
LEM75 0n846 0n861 0n855 0n121 0n079 0n111 0n168 98 96\200 96\205 95\201 96\204 93\198 96\205 93\201 91\197 92\199 91\197 93\199 94\200 91\196 83\187 96\204 93\200 93\198 95\205 89\197 94\201 82\200 86\196 80\203 76\199 81\204
IMPT104 0n857 0n865 0n864 0n119 0n114 0n056 0n217 0n077 102 102\209 101\205 101\208 99\202 100\209 98\205 95\201 96\203 95\201 93\203 96\204 93\200 83\191 100\208 97\204 96\202 99\209 94\201 98\205 86\204 85\200 83\207 79\203 84\208
IMT150 0n855 0n869 0n868 0n108 0n155 0n046 0n236 0n119 0n047 107 102\210 102\213 99\207 103\214 99\210 96\206 98\208 97\206 94\208 96\209 95\205 85\196 103\213 97\209 96\207 102\214 95\206 99\210 88\209 86\205 84\212 80\208 87\213
IMT152 0n858 0n865 0n865 0n127 0n140 0n083 0n241 0n104 0n029 0n056 103 102\209 100\203 99\210 99\206 96\202 96\204 95\202 92\204 95\205 92\201 82\192 100\209 98\205 97\203 99\210 96\202 99\206 87\205 84\201 84\208 80\204 86\209
IMT89 0n854 0n868 0n867 0n134 0n147 0n073 0n245 0n111 0n056 0n081 0n047 106 100\206 103\213 102\209 96\205 97\207 95\205 94\207 97\208 92\204 83\195 101\212 100\208 99\206 100\213 98\205 103\209 87\208 86\204 86\211 81\207 88\212
IMT124 0n855 0n863 0n863 0n155 0n151 0n093 0n236 0n114 0n039 0n083 0n029 0n057 100 97\207 97\203 95\199 95\201 94\199 90\201 93\202 90\198 82\189 97\206 98\202 95\200 96\207 94\199 97\203 85\202 84\198 83\205 80\201 84\206
IMT108 0n855 0n869 0n862 0n108 0n138 0n046 0n236 0n119 0n083 0n072 0n108 0n064 0n118 107 100\210 96\206 98\208 98\206 96\208 98\209 96\205 86\196 102\213 97\209 97\207 101\214 97\206 100\210 89\209 87\205 83\212 78\208 88\213
IMT193 0n865 0n873 0n865 0n144 0n157 0n117 0n257 0n139 0n084 0n108 0n075 0n047 0n085 0n091 103 93\202 93\204 92\202 92\204 95\205 90\201 80\192 97\209 96\205 96\203 96\210 95\202 99\206 86\205 82\201 83\208 80\204 84\209
Strain A 0n847 0n862 0n862 0n164 0n178 0n102 0n212 0n142 0n104 0n127 0n094 0n119 0n087 0n127 0n147 99 94\200 94\198 90\200 91\201 88\197 82\188 95\205 94\201 92\199 94\206 92\198 93\202 84\201 84\197 82\204 80\200 83\205
L53 0n842 0n856 0n857 0n179 0n176 0n101 0n259 0n140 0n103 0n109 0n111 0n118 0n104 0n109 0n162 0n113 101 94\200 90\202 93\203 90\199 83\190 98\207 95\203 92\201 97\208 92\200 94\204 85\203 86\199 80\206 77\202 86\207
PP75 0n854 0n862 0n855 0n112 0n143 0n102 0n229 0n142 0n104 0n110 0n112 0n136 0n105 0n093 0n164 0n096 0n113 99 91\200 94\201 92\197 84\188 98\205 95\201 93\199 97\206 93\198 94\202 84\201 83\197 80\204 77\200 82\205
WR285 0n856 0n871 0n864 0n145 0n124 0n152 0n157 0n123 0n155 0n175 0n179 0n168 0n189 0n143 0n179 0n182 0n196 0n165 101 96\203 93\199 83\190 95\207 91\203 92\201 94\208 89\200 93\204 82\203 83\199 83\206 77\202 82\207
CO910 0n857 0n872 0n858 0n119 0n096 0n143 0n217 0n113 0n111 0n150 0n136 0n126 0n147 0n117 0n136 0n173 0n155 0n121 0n103 102 94\200 83\191 97\200 94\204 95\202 96\209 92\201 96\205 84\204 83\200 82\207 76\203 82\208
M9702 0n861 0n868 0n855 0n121 0n098 0n128 0n221 0n133 0n131 0n136 0n156 0n179 0n167 0n119 0n189 0n193 0n174 0n124 0n123 0n113 98 85\187 95\204 90\200 91\198 94\205 89\197 91\201 82\200 81\196 78\203 74\199 80\204
M8270 0n852 0n876 0n881 0n239 0n168 0n194 0n186 0n202 0n231 0n234 0n255 0n259 0n234 0n218 0n286 0n226 0n224 0n192 0n224 0n231 0n167 89 86\195 83\191 82\189 85\196 80\188 82\192 73\191 75\187 70\194 67\190 72\195
M12727 0n854 0n868 0n867 0n134 0n147 0n073 0n245 0n111 0n074 0n064 0n083 0n090 0n110 0n081 0n134 0n136 0n101 0n084 0n152 0n126 0n128 0n211 106 100\208 99\206 104\213 96\205 100\209 86\208 87\204 84\211 80\207 86\212
M12734 0n850 0n857 0n864 0n170 0n167 0n126 0n250 0n131 0n093 0n134 0n084 0n074 0n058 0n134 0n119 0n121 0n120 0n104 0n188 0n145 0n182 0n231 0n074 102 98\202 98\209 95\201 99\205 83\204 85\200 84\207 81\203 84\208
M12337 0n855 0n877 0n863 0n138 0n133 0n127 0n236 0n114 0n094 0n135 0n085 0n075 0n095 0n118 0n103 0n140 0n156 0n123 0n156 0n112 0n150 0n234 0n075 0n058 100 98\207 95\199 99\203 83\202 83\198 83\205 79\201 82\206
M7633 0n848 0n854 0n856 0n158 0n171 0n098 0n268 0n136 0n100 0n089 0n108 0n115 0n135 0n106 0n158 0n161 0n126 0n110 0n175 0n150 0n153 0n234 0n046 0n117 0n101 107 96\206 100\210 85\209 86\205 84\212 81\208 85\213
M12085 0n862 0n877 0n862 0n147 0n178 0n153 0n278 0n176 0n121 0n144 0n094 0n084 0n105 0n110 0n112 0n132 0n148 0n114 0n198 0n156 0n176 0n259 0n119 0n104 0n087 0n127 99 97\202 84\201 80\197 81\204 76\200 86\205
M12084 0n851 0n865 0n865 0n127 0n140 0n100 0n241 0n121 0n084 0n108 0n075 0n028 0n085 0n091 0n075 0n147 0n145 0n130 0n162 0n119 0n173 0n255 0n083 0n066 0n048 0n091 0n076 103 84\205 84\201 85\208 80\204 85\209

DD8 0n864 0n872 0n858 0n277 0n308 0n281 0n385 0n305 0n271 0n273 0n263 0n281 0n274 0n258 0n277 0n282 0n280 0n282 0n322 0n300 0n305 0n381 0n295 0n314 0n303 0n315 0n282 0n306 102 83\200 83\207 78\203 91\208
HU3 0n846 0n853 0n855 0n297 0n268 0n256 0n318 0n218 0n261 0n277 0n282 0n271 0n263 0n263 0n311 0n257 0n239 0n272 0n284 0n291 0n296 0n330 0n256 0n261 0n278 0n277 0n316 0n282 0n291 98 83\203 80\199 84\204
MRC(L)3 0n839 0n867 0n867 0n336 0n366 0n339 0n413 0n350 0n331 0n344 0n323 0n312 0n320 0n357 0n336 0n328 0n365 0n355 0n325 0n344 0n376 0n435 0n339 0n317 0n320 0n344 0n341 0n309 0n331 0n308 105 93\206 87\211
MRC74 0n863 0n871 0n870 0n381 0n398 0n370 0n420 0n382 0n363 0n375 0n355 0n357 0n339 0n400 0n355 0n333 0n384 0n374 0n384 0n402 0n408 0n455 0n370 0n336 0n352 0n362 0n387 0n355 0n376 0n328 0n177 101 78\207
Patna1 0n840 0n868 0n861 0n341 0n384 0n317 0n431 0n341 0n323 0n310 0n301 0n290 0n311 0n296 0n328 0n320 0n289 0n333 0n344 0n349 0n355 0n415 0n317 0n323 0n339 0n336 0n277 0n315 0n222 0n300 0n298 0n395 106

242
Diversity in the Leishmania donovani complex
Fig. 5. A single linkage dendrogram built from Jaccard
distances recorded for RAPD data in strains of the
Leishmania donovani complex, outgrouped by L. tropica
(T, L96), L. aethiopica (A, K27) and L. major (M,
5ASKH) strains. Cophenetic correlation with distance
matrix is 0n9941. L. infantum strains are mixed with L.
chagasi. The strains of L. infantum\L. chagasi are
individualized from L. donovani and the latter show
geographical patterns. L. donovani (1–5) : 1 – MRC(L)3 ;
2 – MRC74 ; 3 – DD8 ; 4 – PATNA1 ; 5 – HU3, K –
Kenyan ; I – Indian. L.infantum\L. chagasi (6-33) : 6 –
M8270 ; 7 – LOMBARDI ; 8 – IPT-1 ; 9 – WR285 ; 10 –
L53 ; 11 – M9702 ; 12 – CO910 ; 13 – STRAIN A ; 14 –
PP75 ; 15 – M12085 ; 16 – DOG 124 ; 17 – M7633 ; 18 –
DOG 136 ; 19 – DOG 118 ; 20 – IMT193 ; 21 – M12084 ;
22 – IMT89 ; 23 – IMPT104 ; 24 – IMT152 ; 25 –
IMT124 ; 26 – IMT108 ; 27 – IMT150 ; 28 –
PHAROAH ; 29 – LEM75 ; 30 – L82.
Random amplification of polymorphic DNA
RAPDs produced very distinct profiles between the
L. donovani complex, and L. aethiopica, L. major and
L. tropica, with all tested primers. Primer H1
separated the L. chagasi and L. infantum strains from
the L. donovani strains (Fig. 4 A). Variation was
identified within the L. donovani complex and even
within L. infantum and L. chagasi (Fig. 4 B). In both
single linkage and UPGMA dendrograms, built
from a Jaccard distance matrix (Table 3), L. infantum
and L. chagasi were resolved from the L. donovani
strains but not from each other (Fig. 5), forming a
243
Fig. 6. A principal coordinates analysis with a
superimposed minimum spanning tree depicting the first
(29n48 %), second (10n69 %) and fourth axes as
coordinates (cumulative percentage : 47n25 %), from
RAPD data. Outgrouped by Leishmania aethiopica (A),
L. major (M) and L. tropica (T) strains. Note that
strains of L. infantum\L. chagasi are individualized from
L. donovani and the latter show geographical patterns.
L. donovani strains are : Kenyan, 1 – HU3, 2 – MRC(L)3 ;
Indian, 3 – PATNA1, 4 – DD8 and MRC74.
branch within L. donovani. The L. donovani strains
of the same geographical origin (Indian or Kenyan)
were grouped. The dendrograms were rooted to the
outgroups (L. aethiopica, L. major, L. tropica)
between the L. donovani Kenyan strains and the
remainder, contrasting with the mspC3h findings.
Other distance coefficients, like simple matching
[1-(ajd)\n], Yule [1-(a.dkb.c)\(a.djb.c)] and
Euclidean distance [N(bjc)] produced similar
topologies by single linkage and UPGMA.
Cophenetic correlation was high using either method
(0n9941 for single linkage and 0n9958 for UPGMA),
suggesting that the dendrograms were good represen-
tations of the patristic distances, however, it was not
possible to measure robustness of the nodes by
bootstrap analysis. A 3-dimensional principal
coordinates analysis, plotted with a superimposed
minimum spanning tree (Fig. 6), illustrated again
that L. infantum\L. chagasi were indistinguishable,
and that L. donovani strains grouped according to
geographical origin.

Evaluation of techniques
Three approaches were used which provided genetic
information from Leishmania spp. at different levels
of resolution : between genera and complexes
(Lmet9) and between species or groups of strains
I. L. Mauricio and others
Fig. 7. Approximate levels of resolution for each of the
techniques employed in this work shown on a
hypothetical evolutionary tree of Leishmania spp. Not
drawn to scale. M, A, T are respectively L. major, L.
aethiopica and L. tropica complexes of species.
(mspC3h sequencing and RAPD) – Fig. 7. The
combined use of these different types of molecular
characters provides a reliable basis for taxonomy
(Shaw, 1994 ; Tibayrenc, 1998). Lmet9 was specific
for all OW Leishmania and L. chagasi and is therefore
a useful identification tool for L. chagasi in the NW.
The mspC3h sequence detected genetic diversity
between L. donovani strains, but not L. infantum\L.
chagasi, and high bootstrap values strongly support
the main branches. As far as we are aware, this is the
first time that the sequence of a gp63 gene has been
shown to be of value for inference of phylogenies in
Leishmania.
The nature of variation detected with RAPDs and
ease of its use make it suitable to investigate closely
related species. Variation detected within RAPDs
often requires further support from other techniques
though it has seldom been used for Leishmania
taxonomy (Tibayrenc et al. 1993 ; Andresen et al.
1996 ; Motazedian et al. 1996 ; Noyes, Belli &
Maingon, 1996). In our hands, RAPDs distinguished
between species complexes and resolved variation
within the L. donovani complex. RAPD data was,
however, in conflict with the mspC3h data on the
relative positioning of L. infantum to L. donovani.
Common origin and phylogeny of Old World
Leishmania spp.
All OW Leishmania species were recognized by the
probe Lmet9. This suggests a common genetic
background of the OW Leishmania species, which is
consistent with monophyletism as previously
proposed (Thomaz-Soccol et al. 1993 ; Croan,
Morrison & Ellis, 1997 ; Noyes et al. 1997). The fact
that L. chagasi was the only NW species to be
recognized by the probe Lmet9 suggests that it is
244
more related to OW Leishmania spp. than other NW
species. The L. donovani complex was confirmed to
be monophyletic (Thomaz-Soccol et al. 1993 ; Croan
et al. 1997) by both RAPD and mspC sequence.
Thus, the L. donovani complex is a valid ‘ discrete
typing unit ’ (Tibayrenc, 1998).
Leishmania chagasi and Leishmania infantum are
indistinguishable and form a monophyletic group
Upon mspC3h sequence analysis, L. infantum and L.
chagasi strains were indistinguishable and although
some strain variability was found by RAPD analysis,
it was not possible to discriminate L. chagasi from L.
infantum. The close relationship between L. infantum
and L. chagasi contrasts with a higher level of
variability of L. donovani. Although a few genetic
and phenetic methods were reported to distinguish
between L. infantum and L. chagasi (Decker-Jackson
& Tang, 1982 ; Santoro et al. 1984 ; Palatnik et al.
1990 ; Ellis & Crampton, 1991), all L. chagasi have
isoenzyme profiles similar to reference L. infantum
strain IPT-1 (Moreno et al. 1984 ; Momen et al.
1987), the most common L. infantum zymodeme in
Europe, and L. chagasi could not be distinguished by
most genetic methods from L. infantum (Beverley,
Ismach & McMahon-Pratt, 1987 ; van Eys et al.
1991 ; Cupolillo, Grimaldi & Momen, 1994 ;
Schonian et al. 1996). The impossibility of dis-
tinguishing here between L. infantum and L. chagasi
strains, with similar intraspecific variability within
each, gives further support to the hypothesis that
these species are synonymous (Momen et al. 1987).
The low genetic variability of L. chagasi is consistent
with recent importation into the NW. Leishmania
infantum (L. chagasi) formed a single branch in both
mspC3h and RAPD dendrograms showing that L.
infantum is a well-individualized, monophyletic
group.
Diversity of L. donovani
A substantial amount of diversity within L. donovani
was observed in both mspC3h sequence and RAPD
dendrograms. From the RAPD dendrogram, the L.
donovani complex was rooted within L. donovani
strains, with L. infantum depicted as a recent branch
of L. donovani, suggestive of L. donovani as a
paraphyletic group, contrasting with earlier findings
(Moreno et al. 1984 ; Thomaz-Soccol et al. 1993),
but confirming others (Mebrahtu et al. 1992). The
sequence data, on the contrary, suggest mono-
phyletism of L. donovani. An extended analysis of L.
donovani, however, is in progress and may help
clarify this question.
Presently, the taxonomic status of African L.
donovani and L. infantum is unclear with some
strains included in the L. donovani complex
recognized as different from L. donovani sensu stricto
Diversity in the Leishmania donovani complex
(Lainson & Shaw, 1987) but not assigned a species
name and often designated as L. donovani s.l. or
‘ Leishmania sp [place of isolation] ’. The L. donovani
s.l. characterized here seem at least as divergent as L.
infantum (L. chagasi) is from L. donovani s.s. and
both mspC3h sequence and RAPD grouped L.
donovani strains according to geographical origins,
specifically Indian and Kenyan, showing a sub-
stantial divergence within the taxon. However,
Ashford et al. (1992) found a seemingly single
population with the putative original L. donovani
and L. infantum zymodemes, with minimal iso-
enzyme differences (Rioux et al. 1990), and thus
proposed that there was no justification for divisions
within the L. donovani complex. Contradictory
findings by other authors and in this report on the
phylogeny of L. donovani are further indictments for
a systematic reevaluation of the L. donovani complex,
with more exhaustive data sets that include
epidemiological and clinical aspects (Tibayrenc,
1998). Specific diagnostic methods and epidemio-
logical markers are clearly warranted, but would first
require a stable definition of groups within the L.
donovani complex, though this may be complicated
by genetic exchange (Rioux et al. 1990 ; Shaw, 1994).
I. L. M. acknowledges personal financial support from
Praxis XXI (Portugal-EU) ; we also acknowledge funding
from European contract No. ERBTS3*CT920113 and the
Wellcome Trust. Professor J. J. Shaw (Instituto Evandro
Chagas, Brazil) kindly provided Brazilian L. chagasi
strains, and Professor P. Abranches (Instituto de Higiene
e Medicina Tropical, Lisbon, Portugal) some Portuguese
L. infantum strains. Rachel Gregory and Margaret Long
operated the automated ABI 377 sequencer.

, ., , . ., , . . 
, . (1996). Random amplified polymorphic
DNA for the differentiation of Leishmania donovani
isolates from Sudan. Transactions of the Royal Society
of Tropical Medicine and Hygiene 90, 204–205.
, . .  , . (1987). Ecology and
Epidemiology : Old World. In The Leishmaniasis in
Biology and Medicine. Vol. I Biology and Epidemiology
(ed. Peters, W. & Killick-Kendrick, R.), pp. 366–424.
Orlando Academic Press, Orlando, USA.
, . ., , ., , .  ,
. (1992). Epidemic visceral leishmaniasis in Southern
Sudan : identity and systematic position of the
parasites from patients and vectors. Transactions of the
Royal Society of Hygiene and Tropical Medicine 86,
379–380.
, . ., , . .   , .
(1987). Evolution of the genus Leishmania as revealed
by comparisons of nuclear DNA restriction fragment
patterns. Proceedings of the National Academy of
Sciences, USA 84, 484–488.
, ., , ., , .-., ,
. ., , .-.  , . . (1996). Kinetics of
entry of virulent and avirulent strains of Leishmania
245
donovani into macrophages : a possible role of
virulence molecules (gp63 and LPG). Journal of
Parasitology 82, 632–635.
, . ., , . .  , . . (1997).
Evolution of the genus Leishmania revealed by
comparison of DNA and RNA polymerase gene
sequences. Molecular and Biochemical Parasitology 89,
149–159.
, .,  .   , . (1994). A
general classification of New World Leishmania using
numerical zymotaxonomy. American Journal of
Tropical Medicine and Hygiene 50, 296–311.
-, . .  , . . (1982). Identification
of Leishmania spp. by radiorespirometry II. A
statistical method of data analysis to evaluate the
reproducibility and sensitivity of the technique. In
Biochemical Characterization of Leishmania.
Proceedings of a Workshop held at the Pan American
Health Organization, 9–11 December, 1980 (ed.
Chance, M. C. & Walton, B. C.), pp. 205–245.
UNDP\World Bank\WHO Special Programme for
Research and Training in Tropical Diseases, Geneva.
, . .  , . . (1991). Differences
between Leishmania (Leishmania) chagasi, Leishmania
(L.) infantum and Leishmania (L.) donovani as shown
by DNA fingerprinting. MemoT rias do Instituto
Oswaldo Cruz 86, 479–481.
, . (1993). PHYLIP (Phylogeny Inference
Package) Version 3.5c. Distributed by the author.
Department of Genetics, University of Washington,
Seattle.
, .   , . . (1993). Leishmaniasis of
the New World : current concepts and implications for
future research. Clinical Microbiology Reviews 6,
230–250.
, . ., , . .  , . (1992). Clustal
V : improved software for multiple alignment.
Computer Applications in the BioSciences 8, 189–191.
, . ., , . ., , . .  , . .
(1991). A sensitive repetitive DNA probe that is
specific for the Leishmania donovani complex and its
use as an epidemiological and diagnostic reagent.
Molecular and Biochemical Parasitology 44, 63–72.
, . . (1993). Isolation of RNA and DNA from
Leishmania. In Methods in Molecular Biology, Vol. 21.
(ed. Hyde, J. E.), pp. 123–131. Humana Press, New
Jersey, USA.
, .  , . . (1987). Evolution, classification
and geographical distribution. In The Leishmaniasis in
Biology and Medicine. Vol. I Biology and Epidemiology
(ed. Peters, W. & Killick-Kendrick, R.), pp. 1–120.
Orlando Academic Press, Orlando, USA.
 , . .  , . (1986). Leishmania in the
Old World : 4. The distribution of L. donovani sensu
lato zymodemes. Transactions of the Royal Society of
Tropical Medicine and Hygiene 80, 367–377.
, ., , . ., , . ., ,
. .  , . . (1990). Identification and
characterization of a Leishmania donovani antigen
belonging to the 70 kDa heat shock protein family.
European Journal of Biochemistry 190, 377–384.
, . . ., , ., , ., ,
. .  , . . (1990). Multiple distinct gp63-
like genes in amastigotes of Leishmania donovani. In
I. L. Mauricio and others
Parasites : Molecular Biology, Drug and Vaccine
Design, pp. 191–204. Wiley-Liss, Inc., New York,
USA.
, . (1996). Intracellular survival of protozoan
parasites with special reference to Leishmania spp.,
Toxoplasma gondii and Trypanosoma cruzi. Advances
in Parasitology 38, 1–51.
, . ., , . ., , ., , .,
, . ., , . ., , . .  ,
. . (1992). Biochemical characterization and
zymodeme classification of Leishmania isolates from
patients, vectors, and reservoir hosts in Kenya.
American Journal of Tropical Medicine and Hygiene
47, 852–892.
, ., , .   , . . (1987).
Leishmania infantum, the aetiological agent of
American Visceral Leishmaniasis (AVL) ? MemoT rias
do Instituto Oswaldo Cruz 82, 447–448.
, ., , ., , . ., , .,
, . ., , . .  , . (1997).
Mapping of the antigenic determinants of the
Leishmania infantum gp63 protein recognized by
antibodies elicited during canine visceral
leishmaniasis. Parasitology 114, 507–516.
, ., , . ., , ., , . 
, . (1984). Le complexe Leishmania donovani
s.l. Analyse enzymatique et traitement numerique.
Individualization du complexe Leishmania infantum.
Corollaires biogeographiques et phyletiques. A propos
de 146 souches originaires de l’Ancien et du Nouveau
Monde. In Leishmania. Taxonomie et PhylogeneZ se.
Applications ET co-eT pidemiologiques (ed. Rioux, J. A.),
pp. 105–107. IMEEE, Montpellier, France.
, ., , .  , . (1996).
Leishmania and Sauroleishmania : the use of random
amplified polymorphic DNA for the identification of
parasites from vertebrates and invertebrates.
Experimental Parasitology 83, 150–154.
, . ., , . .  , . (1996).
Appraisal of various random amplified polymorphic
DNA-polymerase chain reaction primers for
Leishmania identification. American Journal of
Tropical Medicine and Hygiene 55, 98–105.
, . ., , . ., , . .  , .
(1997). The Leishmania hertigi (Kinetoplastida ;
Trypanosomatidae) complex and the lizard
Leishmania : their classification and evidence for a
neotropical origin of the Leishmania – Endotrypanum
clade. Journal of Eukaryotic Microbiology 44, 511–517.
, . ., , . ., : -, .
 , . (1990). A new approach to the
phylogeny of Leishmania : species specificity of
glycoconjugate ligands for promastigote internalization
into murine macrophages. Parasitology Research 76,
289–293.
, . (1993). SYN-TAX pc Computer Programs for
Multivariate Data Analysis in Ecology and Systematics,
Version 5.0. Sciencia Publishing, Budapest.
, ., , . ., , . .,
, ., , . .  , . . (1992).
246
Three distinct RNAs for the surface protease gp63 are
differentially expressed during development of
Leishmania donovani chagasi promastigotes to an
infectious form. Journal of Biological Chemistry 267,
1888–1895.
, . ., , ., , ., , .,
, .  , . (1990). Taxonomy of
Leishmania. Use of isoenzymes. Suggestions for a new
classification. Annales de Parasitologie Humaine et
CompareT e 65, 111–125.
, . ., , . ., , . .,
, ., , . .  , . .
(1993). Sequence diversity and organization of the msp
gene family encoding gp63 of Leishmania chagasi.
Molecular and Biochemical Parasitology 62, 157–172.
, ., , . ., , . ., , . 
, . (1984). Spe! cificite! au niveau des
prote! ines de surface des promastigotes de Leishmania
donovani (Laveran et Mesnil, 1903), Leishmania
infantum Nicolle, 1908 et Leishmania chagasi Cunha
et Chagas, 1937. In Leishmania. Taxonomie et
PhylogeneZ se. Applications ET co-eT pidemiologiques (ed.
Rioux, J. A.), pp. 71–75. IMEEE, Montpellier,
France.
, ., , ., , ., , .,
, ., , .  , . (1996).
Identification and determination of the relationships
of species and strains within the genus Leishmania
using single primers in the polymerase chain reaction.
Molecular and Biochemical Parasitology 77, 19–29.
, . . (1994). Taxonomy of the genus Leishmania :
present and future trends and their implications.
MemoT rias do Instituto Oswaldo Cruz 89, 471–478.
-, ., , ., , . ., ,
., -, .  , . (1993).
Monophyletic origin of the genus Leishmania Ross,
1903. Annales de Parasitologie Humaine et CompareT e
68, 107–108.
, ., , ., , ., , .,
, ., , . . (1993). Genetic
characterization of six parasitic protozoa : parity
between random-primer DNA typing and multilocus
enzyme electrophoresis. Proceedings of the National
Academy of Sciences, USA 90, 1335–1339.
, . (1998). Genetic epidemiology of parasitic
protozoa and other infectious agents : the need for an
integrated approach. International Journal for
Parasitology 28, 85–104.
 , . . . ., , ., , . . 
, . (1991). A nuclear DNA probe for the
identification of strains within the Leishmania
donovani complex. Experimental Parasitology 72,
459–463.
, . ., , . .  , . (1991).
Heterogeneity of the genes encoding the major surface
glycoprotein of Leishmania donovani. Molecular and
Biochemical Parasitology 48, 173–184.
, . . ., , . ., , . ., ,
. .  , . . (1990). DNA polymorphisms
amplified by arbitrary primers are useful as genetic
markers. Nucleic Acids Research 18, 6531–6535.