Open Access

Evolutionary history of Leishmania killicki (synonymous Leishmania tropica) and taxonomic implications

  • Dhekra Chaara1, 2, 3Email author,
  • Christophe Ravel2, 3,
  • Anne- Laure Bañuls3,
  • Najoua Haouas1,
  • Patrick Lami2, 3,
  • Loïc Talignani2, 3,
  • Fouad El Baidouri2, 3, 4,
  • Kaouther Jaouadi1,
  • Zoubir Harrat5,
  • Jean-Pierre Dedet2, 3,
  • Hamouda Babba1 and
  • Francine Pratlong2, 3
Parasites & Vectors20158:198

https://doi.org/10.1186/s13071-015-0821-6

Received: 18 September 2014

Accepted: 21 March 2015

Published: 1 April 2015

Abstract

Background

The taxonomic status of Leishmania (L.) killicki, a parasite that causes chronic cutaneous leishmaniasis, is not well defined yet. Indeed, some researchers suggested that this taxon could be included in the L. tropica complex, whereas others considered it as a distinct phylogenetic complex. To try to solve this taxonomic issue we carried out a detailed study on the evolutionary history of L. killicki relative to L. tropica.

Methods

Thirty-five L. killicki and 25 L. tropica strains isolated from humans and originating from several countries were characterized using the MultiLocus Enzyme Electrophoresis (MLEE) and the MultiLocus Sequence Typing (MLST) approaches.

Results

The results of the genetic and phylogenetic analyses strongly support the hypothesis that L. killicki belongs to the L. tropica complex. Our data suggest that L. killicki emerged from a single founder event and that it evolved independently from L. tropica. However, they do not validate the hypothesis that L. killicki is a distinct complex. Therefore, we suggest naming this taxon L. killicki (synonymous L. tropica) until further epidemiological and phylogenetic studies justify the L. killicki denomination.

Conclusions

This study provides taxonomic and phylogenetic information on L. killicki and improves our knowledge on the evolutionary history of this taxon.

Keywords

Leishmania killicki Leishmania tropica Evolutionary history Phylogeny Isoenzymatic polymorphism

Background

Leishmaniases are neglected tropical diseases caused by Leishmania parasites and transmitted to mammals through bites by infected Phlebotomine sandflies of the genus Phlebotomus [1]. In humans, these diseases can have cutaneous (CL), muco-cutaneous (MCL) or visceral (VL) clinical manifestations.

Since the first description of the genus Leishmania Ross, 1903, the classification methods have considerably evolved. Indeed, between 1916 and 1987, Leishmania taxonomy followed the Linnaean classification system, mainly based on extrinsic features, such as clinical manifestations, geographical distribution, epidemiological cycles and behaviour in sandfly vectors. This method has led to the subdivision of the Leishmania genus in the two sub-genera Leishmania and Viannia [2,3].

In the eighties, the biochemical classification based on the study of the parasite isoenzymatic patterns started to be developed. This approach has evolved from the classical Adansonian to the numerical cladistic classification method that uses isoenzymes as evolutionary markers [4-8]. The description of several Leishmania complexes in the Old and New World is based on these analyses. Specifically, by using numerical phenetic and phylogenetic approaches, Rioux et al. [9] identified four main Leishmania groups in the Old World, while Thomaz et al. and Cupolillo et al. [10,11] defined eight complexes and two Leishmania groups in the New World.

Currently, the numerical taxonomic approach based on isoenzyme analysis is considered as the gold standard for the classification of the genus Leishmania and is routinely used for classification updates and for epidemiological studies [12,13]. The drawbacks of this approach are the need of bulk cultures of Leishmania parasites and its relatively poor discriminatory power. It is also time-consuming. Therefore, DNA-based techniques represent valuable alternatives for the identification and the classification of these parasites.

Since the nineties, several DNA-based approaches that target nuclear and/or kinetoplastid markers have been used for phenetic and phylogenetic studies of Leishmania, including sequencing of PCR-generated fragments (PCR-sequencing) [14], nested PCR [15], random amplified polymorphic DNA (RAPD) [16,17], single strand conformation polymorphism (SSCP) analysis [18,19], multilocus sequence typing (MLST) [20], multilocus microsatellite typing (MLMT) [21,22], restriction fragment length polymorphism analysis of PCR-amplified fragments (PCR-RFLP) [23,24], high-resolution melting (HRM) [25] and amplified fragment length polymorphism (AFLP) [26]. These techniques have improved our epidemiological knowledge and consequently also leishmaniasis control and treatment. Due to the wealth of new taxonomic data generated by these DNA-based approaches, it is currently debated whether the genus Leishmania classification should be revised [27,28].

MLST is one of the most appropriate approaches for taxonomic studies because it provides data on the genetic variations of housekeeping genes. This approach has been increasingly used for phylogenetic investigations to understand the epidemiological and transmission features of many Leishmania complexes [20,29-33]. However, because of the complexity of this genus and the lack of studies, several taxa need to be detailed further [34].

Leishmania killicki is a recently described taxon that causes CL in Tunisia [35], Libya [36] and Algeria [37]. L. killicki taxonomic status and evolutionary history relative to L. tropica are based on very few studies and samples. The numerical taxonomic analysis using the Multilocus Enzyme Electrophoresis (MLEE) approach first included this parasite in the L. tropica complex [9,38]. However, after the revision of the Leishmania genus classification, it was considered as a separate phylogenetic complex [39]. Recently, an update study by Pratlong et al. [12] confirmed the inclusion of L. killicki within the L. tropica complex. Phenetic and phylogenetic studies using MLMT [40], PCR-sequencing [41] and MLST [31] also classified L. killicki within the L. tropica complex and suggested a closer genetic link with L. tropica from Morocco. However, these data were obtained using only seven L. killicki strains: two strains were analyzed by Schwenkenbecher et al. [40], two by Chaouch et al. [41] and three by El Baidouri et al. [31]. Therefore, the present study wanted to analyze by MLST a large number of L. killicki and L. tropica strains in order to precisely determine the evolutionary history and the taxonomic status of L. killicki.

Methods

Origin of strains

For this study, strains of L. killicki (n = 35), L. tropica (n = 25), L. major (n = 1) and L. infantum (n = 1) from different geographic areas and with various zymodeme patterns were included (total = 62 strains). These strains were from human cutaneous lesions, except the L. infantum strain that was isolated from a patient with VL. Most strains (n = 53) were selected from the Cryobank of the Centre National de Référence des Leishmanioses (CNRL) (Montpellier, France) and nine L. killicki strains were collected by the team of the Laboratoire de Parasitologie - Mycologie Médicale et Moléculaire (Monastir, Tunisia) during epidemiological investigations.

Forty-eight strains, among which 34 L. killicki strains (six from Algeria, one from Libya and 27 from Tunisia) and 14 L. tropica strains from Morocco were analyzed by MLST for the first time during this study. The eleven remaining L. tropica strains were from several countries (one from Egypt, one from Greece, two from Israel, two from Jordan, three from Kenya and two from Yemen) and were previously typed by MLST. Their sequences were published in Genbank under the following accession numbers: KC158621, KC158637, KC158643, KC158677, KC158682, KC158683, KC158690, KC158696, KC158711, KC158722 and KC158761 (see [31]). One L. killicki strain (LEM163) MHOM/TN/80/LEM163 had also already been analyzed by MLST (Genbank accession number KC158820 (see [31]).

The L. major (LEM62) MHOM/YE/76/LEM62 and L. infantum (LEM75) MHOM/FR/78/LEM75 strains, previously typed by MLST, were used as outgroups [31].

Isoenzymatic identification

All studied strains were identified by MLEE, according to Rioux et al. [9], using 15 enzymatic systems: malate dehydrogenase (MDH, EC 1.1.1.37), malic enzyme (ME, EC 1.1.1.40), isocitrate dehydrogenase (ICD, EC 1.1.1.42), phosphogluconate dehydrogenase (PGD, EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), glutamate dehydrogenase (GLUD, EC 1.4.1.3), NADH diaphorase (DIA, EC 1.6.2.2), nucleoside purine phosphorylase 1 and 2 (NP1, EC 2.4.2.1 and NP2, EC 2.4.2*), glutamate oxaloacetate transaminase 1 and 2 (GOT1 and GOT2, EC 2.6.1.1), phosphoglucomutase (PGM, EC 5.4.2.2), fumarate hydratase (FH, EC 4.2.1.2), mannose phosphate isomerase (MPI, EC 5.3.1.8) and glucose phosphate isomerase (GPI, EC 5.3.1.9).

DNA extraction

Genomic DNA from cultured parasites was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer’s recommendations and eluted in 150 μl.

Analysis by Multilocus sequence typing (MLST)

The L. killicki (n = 34) and L. tropica (n = 14) strains that had not been previously assessed by MLST were typed using the MLST approach based on the analysis of seven loci coding for single-copy housekeeping genes that was developed and optimized by El Baidouri et al. [31]. Genomic DNA was amplified by real-time PCR using the SYBR Green method (Light cycler 480 II, Roche). The amplified products were sequenced on both strands (Eurofins MWG Operon, Germany) and the obtained sequences were aligned and checked in both directions using the CodonCode Aligner software, v.4.0.1 (Codon Code Co., USA). For each strain, polymorphic sites (PS) and ambiguous positions corresponding to heterozygous sites (HS) were identified in each locus using the same software. The DnaSP software v.5 [42] was used to calculate the number of haplotypes from the concatenated sequences.

Phylogenetic relationships were inferred using a Bayesian approach implemented with the MrBayes software v. 3.2.3 [43]. The concatenated duplicated sequence alignments of the seven loci for the 32 Leishmania strains representing all the identified haplotypes and the two outgroup strains (n = 34 in total) were used to run two independent chains for 10,000,000 generations each and trees sampled every 1000 generations. The burn-in period was set to 200,000 generations to fit the first 20% of the analyses. Analyses were conducted using the General time reversible model of substitution with a proportion of invariable sites and gamma distribution estimated by the program (GTR + G + I).

The chain convergence was assessed using the average standard deviation of split frequencies (ASDSF). If two runs converge onto the stationary distribution, the ASDSF is expected to approach zero, reflecting the fact that the two tree samples become increasingly similar. An average standard deviation below 0.01 is thought to be a very good indication of convergence (below 0.004 in our analysis). The consensus tree was constructed using 1000 trees sampled from the stationary phase. The MEGA 5.10 software [44] was used to identify amino acid variations between L. killicki and L. tropica.

Results

Isoenzymatic identification of Leishmania strains

Among the 62 strains under study, 53 had been previously characterized by MLEE at the Centre National de Référence des Leishmanioses. The nine strains collected by the team of the Laboratoire de Parasitologie - Mycologie Médicale et Moléculaire (Monastir, Tunisia) were identified for the first time in this study using the same technique [12,35-38,45-47]. Nevertheless, all the strains were analyzed again by MLEE at the Centre National de Référence des Leishmanioses (Montpellier, France).

Seventeen zymodemes were identified: three for L. killicki, 12 for L. tropica and a single zymodeme for each L. major and L. infantum strain (Table 1). For L. killicki, besides the two known zymodemes MON-8 (n = 28) and MON-301 (n = 6), a new zymodeme (MON-317) was characterized in a single Tunisian strain (LEM6173: MHOM/TN/2010/MET300). The zymodeme MON-317 differed from MON-8 by only a single enzyme (FH) [35]. On the other hand, the MDH, ME, GOT1, GOT2 and FH profiles were different in the zymodemes MON-317 and MON-301 [37], and the MDH, GOT1, GOT2 and FH profiles allowed discriminating between MON-317 and MON-306 (a zymodeme described in Algeria, but not included in our sample collection) [48] (Table 2). For L. tropica, all the identified zymodemes were already known [12,45]: MON-54 (n = 1), MON-71 (n = 2), MON-102 (n = 5), MON-109 (n = 3), MON-112 (n = 2), MON-113(n = 3), MON-114 (n = 1), MON-119 (n = 3), MON-137 (n = 2), MON-200 (n = 1), MON-264 (n = 1) and MON-265 (n = 1) (Table 1).
Table 1

Details about the origin, taxon and zymodeme of the 62 strains under study

CNRL code

WHO code

Origin

Taxon

Zymodeme

LEM95

MHOM/TU/79/LEM95

Tunisia

L. killicki

MON-8

LEM160

MHOM/TN/80/LEM160

Tunisia

L. killicki

MON-8

LEM163

MHOM/TN/80/LEM163

Tunisia

L. killicki

MON-8

LEM174

MHOM/TN/80/LEM174

Tunisia

L. killicki

MON-8

LEM177

MHOM/TN/80/LEM177

Tunisia

L. killicki

MON-8

LEM179

MHOM/TN/80/LEM179

Tunisia

L. killicki

MON-8

LEM180

MHOM/TN/80/LEM180

Tunisia

L. killicki

MON-8

LEM181

MHOM/TN/80/LEM181

Tunisia

L. killicki

MON-8

LEM182

MHOM/TN/80/LEM182

Tunisia

L. killicki

MON-8

LEM183

MHOM/TN/80/LEM183

Tunisia

L. killicki

MON-8

LEM184

MHOM/TN/80/LEM184

Tunisia

L. killicki

MON-8

LEM185

MHOM/TN/80/LEM185

Tunisia

L. killicki

MON-8

LEM186

MHOM/TN/80/LEM186

Tunisia

L. killicki

MON-8

LEM193

MHOM/TN/80/LEM193

Tunisia

L. killicki

MON-8

LEM194

MHOM/TN/80/LEM194

Tunisia

L. killicki

MON-8

LEM904

MHOM/TN/80/LEM904

Tunisia

L. killicki

MON-8

LEM1013

MHOM/TN/80/LEM1013

Tunisia

L. killicki

MON-8

LEM4390

MHOM/TN/2002/LSL65

Tunisia

L. killicki

MON-8

LEM4741

MHOM/TN/2004/CRE139

Tunisia

L. killicki

MON-8

LEM5420

MHOM/TN/2007/LPN306

Tunisia

L. killicki

MON-8

LEM6175

MHOM/TN/2010/MET315

Tunisia

L. killicki

MON-8

LEM6226

MHOM/TN/2004/PLC3

Tunisia

L. killicki

MON-8

LEM6228

MHOM/TN/2003/LC39

Tunisia

L. killicki

MON-8

LEM6229

MHOM/TN/2006/SSC36

Tunisia

L. killicki

MON-8

LEM6230

MHOM/TN/2006/SSC37

Tunisia

L. killicki

MON-8

LEM6231

MHOM/TN/2005/LC24 bras

Tunisia

L. killicki

MON-8

LEM6423

MHOM/TN/2012/NAS12

Tunisia

L. killicki

MON-8

LEM6173

MHOM/TN/2010/MET300

Tunisia

L. killicki

MON-317

LEM6227

MHOM MN/2005/PLC5

Libya

L. killicki

MON-8

LEM4995

MHOM/DZ/2005/LIPA07

Algeria

L. killicki

MON-301

LEM6404

MHOM/DZ/2005/LIPA11

Algeria

L. killicki

MON-301

LEM6416

MHOM/DZ/2011/LIPA283

Algeria

L. killicki

MON-301

LEM6418

MHOM/DZ/2005/LIPA14

Algeria

L. killicki

MON-301

LEM6420

MHOM/DZ/2011/LIPA281

Algeria

L. killicki

MON-301

LEM6421

MHOM/DZ/2011/LIPA282

Algeria

L. killicki

MON-301

LEM1623

MHOM/MA/89/LEM1623

Morocco

L. tropica

MON-102

LEM1663

MHOM/MA/89/LEM1663

Morocco

L. tropica

MON-102

LEM2017

MHOM/MA/90/LEM2017

Morocco

L. tropica

MON-102

LEM5276

MHOM/MA/2000/INHW02

Morocco

L. tropica

MON-102

LEM5506

MHOM/MA/2007/INHS10

Morocco

L. tropica

MON-102

LEM1591

MHOM/MA/89/LEM1591

Morocco

L. tropica

MON-109

LEM1880

MHOM/MA/90/LEM 1880

Morocco

L. tropica

MON-109

LEM1922

MHOM/MA/89/LEM 1922

Morocco

L. tropica

MON-109

LEM1879

MHOM/MA/89/LEM 1879

Morocco

L. tropica

MON-112

LEM1918

MHOM/MA/89/LEM 1918

Morocco

L. tropica

MON-112

LEM1778

MHOM/MA/89/LEM1778

Morocco

L. tropica

MON-113

LEM5283

MHOM/MA/2000/INHW19

Morocco

L. tropica

MON-113

LEM5295

MHOM/MA/2000/INHW20

Morocco

L. tropica

MON-113

LEM3015

MHOM/MA/95/LEM3015

Morocco

L. tropica

MON-264

LEM0617

MHOM/IL/80/SINGER 

Israel

L. tropica

MON-54

LEM955

MHOM/YE/86/LEM955 

Yemen

L. tropica

MON-71

LEM1015

MHOM/YE/86/LEM1015 

Yemen

L. tropica

MON-71

LEM1904

MHOM/GR/88/LA615 

Greece

L. tropica

MON-114

LEM1824

MHOM/KE/86/EB103 

Kenya

L. tropica

MON-119

LEM2313

IGUG/KE/91/000 

Kenya

L. tropica

MON-119

LEM2454

MHOM/KE/92/EB000 

Kenya

L. tropica

MON-119

LEM2001

MHOM/EG/90/LPN65 

Egypt

L. tropica

MON-137

LEM3956

MHOM/IL/96/LRC-L691 

Israel

L. tropica

MON-137

LEM2869

MHOM/JO/93/JH67 

Jordan

L. tropica

MON-200

LEM3322

MHOM/JO/96/JH-88 

Jordan

L. tropica

MON-265

LEM62

MHOM/YE/76/LEM62

Yemen

L. major

MON-26

LEM75

MHOM/FR/78/LEM75

France

L. infantum

MON-1

Table 2

Isoenzyme patterns for the 15 enzyme systems of the four Leishmania killicki zymodemes

Taxon

Zymodeme

Enzyme profiles

MDH

ME

ICD

PGD

G6PD

GLUD

DIA

NP1

NP2

GOT1

GOT2

PGM

FH

MPI

GPI

L. killicki

MON-317

100

100

100

93

82

110

100

300

100

127

90

100

110

110

76

 

MON-8

100

100

100

93

82

110

100

300

100

127

90

100

100

110

76

 

MON-301

112

93, 66

100

93

82

110

100

300

100

140

85

100

100

110

76

 

MON-306

112

100

100

93

82

110

100

300

100

140

85

100

100

110

76

Sequence analysis

The sequences of the L. killicki (n = 34) and L. tropica (n = 14) strains were submitted to GenBank (accession numbers from KM085998 to KM086333). The sizes of the seven loci under study were identical to those reported by El Baidouri et al. [31], except for locus 12.0010 (only 579 pb instead of 714 pb), leading to a total length of 4542 pb for the concatenated sequences (Table 3). All chromatograms were clearly readable. Polymorphic sites (PS) and heterozygous sites (HS), which corresponded to ambiguous positions with two peaks, were easily identified. No tri-allelic site was detected.
Table 3

Genetic diversity indices calculated from the MLST data considering the seven loci and all the Leishmania killicki (n = 35) and Leishmania tropica (n = 25) strains

Locus

Length (bp)

No of PS (% of length)

No of HS (% of length)

03.0980

678

18 (2,65%)

11 (1,62%)

04.0580

711

15 (2,1%)

9 (1,26%)

10.0560

636

12 (1,88%)

5 (0,78%)

12.0010

579

12 (2,07%)

4 (0.69%)

14.0130

642

11 (1,71%)

8 (1,24%)

31.0280

810

21 (2,6%)

16 (1,97%)

31.2610

486

6 (1,23%)

6 (1,23%)

Concatenated

4542

95 (2,09%)

59 (1,3%)

PS: polymorphic sites, HS: heterozygous sites.

Genetic polymorphisms in L. killicki and in L. tropica

Ninety-five (2.09%) PS of which 59 (1.3%) were heterozygous positions (HS) were identified in the 60 L. killicki and L. tropica strains. The number of PS varied from six (1.23%) for locus 31.2610 to 21 (2.6%) for locus 31.0280 (Table 3).

In the L. killicki strains, 11 (0.24%) PS were identified and they corresponded only to HS. Locus 31.2610 was the most polymorphic with three (0.61%) PS, whereas locus 12.0010 had none. In the L. tropica strains, 87 (1.91%) PS among which 48 (1.06%) HS were found. The number of PS varied from six (1.23%) for locus 31.2610 to 19 (2.36%) for locus 31.0280 (Table 4).
Table 4

Comparison of the MLST data for the Leishmania killicki (n = 35) and Leishmania tropica (n = 25) strains at the seven loci under study

Locus

Length (bp)

No of PS (% of length)

No of HS (% of length)

  

L. killicki*

L. tropica

L. tropica

03.0980

678

2 (0,29%)

16 (2,36%)

9 (1,33%)

04.0580

711

2 (0,29%)

13 (1,81%)

7 (0,98%)

10.0560

636

1 (0,16%)

11 (1,72%)

4 (0,63%)

12.0010

579

0

12 (2,07%)

4 (0,69%)

14.0130

642

1 (0,15%)

10 (1,56%)

7 (1,09%)

31.0280

810

2 (0,24%)

19 (2,36%)

14 (1,73%)

31.2610

486

3 (0,61%)

6 (1,23%)

3 (0,36%)

Concatenated

4542

11 (0,24%)

87 (1,91%)

48 (1,06%)

S: polymorphic sites, HS: heterozygous sites.

*For L. killicki PS=HS.

Assessment of the presence of mutations in the seven loci under study in the L. killicki and L. tropica (heterozygous mutations were excluded from the analysis) identified 55 mutations of which 29 were silent substitutions and 26 resulted in altered amino acid residues (Table 5). All L. killicki mutations corresponded to a single amino acid change. Conversely, in the L. tropica strains, mutations could lead to more than one amino acid change.
Table 5

Amino acid variations in Leishmania killicki and Leishmania tropica sequences at the seven loci assessed by MLST

Locus

Position

Amino acid

 
  

L. killicki

L. tropica (number of strains/total number of strains)

03.0980

24

G

G

 

231

L

L

 

623

V

V (0.2), A(0.8)

 

630

S

S

04.0580

6

V

V

 

36

H

H (0.96), R (0.04)

 

258

I

I (0.96), V (0.04)

 

261

V

V (0.92), A(0.08)

 

421

V

V (0.44), I (0.56)

 

463

L

L

 

711

R

R

10.0560

24

F

F

 

42

G

G

 

51

V

V

 

54

A

A

 

61

A

S (0.6), A (0.4)

 

496

D

D (0.96), N (0.04)

 

535

Y

Y (0.88), N (0.12)

 

545

I

I (0.96), V (0.04)

 

619

T

T (0.64), A (0.36)

 

633

E

E

12.0010

72

D

D

 

75

G

G

 

120

S

S

 

156

K

K

 

249

R

R

 

261

S

S

 

344

A

A (0.56), V (0.44)

 

438

N

N (0.96), K (0.04)

 

507

G

G

14.0130

77

L

L (0.88), Q (0.12)

 

85

S

P (0.52), S (0.48)

 

246

E

E

 

303

T

T

 

364

M

V (0.52), M (0.48)

 

367

R

R (0.36), H (0.48), C (0.16)

 

470

Q

R (0.52), Q (0.48)

31.0280

7

L

L (0.52), I (0.48)

 

107

N

N (0.48), S (0.52)

 

110

I

I (0.48) ,S (0.52)

 

120

A

A

 

187

I

I (0.36),V (0.64)

 

216

A

A

 

229

T

T (0.88), A (0.12)

 

239

D

D (0.92), G (0.08)

 

417

E

E

 

553

Q

Q (0.92), K (0.08)

 

649

F

F (0.92), L (0.08)

 

717

S

S

 

807

V

V

31.2610

133

L

L

 

162

A

A

 

210

A

A

 

327

I

I

 

368

L

P (0.68), L (0.32)

Phylogenetic analysis of L. killicki

In total, 32 different haplotypes were identified: 10 for the 35 L. killicki strains and 22 for the 25 L. tropica strains. Twenty-six haplotypes were unique (eight for L. killicki and 18 for L. tropica) and the two taxa did not share any haplotype. The L. killicki MON-317 (strain LEM6173) had its own haplotype (Table 6). The Bayesian consensus tree using 32 strains representing all the identified haplotypes was constructed based on the concatenated sequences and duplicated nucleotide sites to avoid the loss of genetic information in ambiguous positions (Figure 1). The phylogenetic tree showed that L. killicki formed a separate group, although it belonged to the L. tropica complex. The L. killicki cluster showed low structuring and low polymorphism (see Figure 1). In contrast, L. tropica was highly polymorphic with strong structuring supported by high bootstrap values and some links with the country of origin, especially for strains from Kenya and Yemen. The larger and main clade was composed by all the Moroccan strains with the addition of other strains from other countries.
Table 6

Haplotypes of Leishmania killicki and Leishmania tropica strains based on the concatenated sequences of the seven loci used for the MLST analysis

Taxon

Haplotype

Number of strains

CNRL code

L. killicki

1

24

LEM95, LEM160, LEM163, LEM174, LEM179, LEM183, LEM184, LEM186

   

LEM194, LEM904, LEM1013, LEM4390, LEM4741, LEM4995, LEM6226, LEM6229

   

LEM6230, LEM6231, LEM6404, LEM6416, LEM6418, LEM6420, LEM6421, LEM6423

 

2

3

LEM0177, LEM0182, LEM5420

 

3

1

LEM180

 

4

1

LEM181

 

5

1

LEM185

 

6

1

LEM193

 

7

1

LEM6173

 

8

1

LEM6175

 

9

1

LEM6227

 

10

1

LEM6228

L. tropica

11

2

LEM1591, LEM1922

 

12

1

LEM1623

 

13

2

LEM1663, LEM5506

 

14

1

LEM1778

 

15

2

LEM1879, LEM1918

 

16

1

LEM1880

 

17

2

LEM2017

 

18

1

LEM3015

 

19

1

LEM5276

 

20

1

LEM5295

 

21

1

LEM5283

 

22

1

LEM617

 

23

1

LEM2869

 

24

1

LEM1904

 

25

1

LEM0955

 

26

1

LEM1015

 

27

1

LEM3322

 

28

1

LEM2001

 

29

1

LEM3956

 

30

1

LEM1824

 

31

1

LEM2313

 

32

1

LEM2454

Figure 1

Bayesian consensus tree of the seven concatenated loci from the 32 Leishmania strains. L. infantum (LEM75) and L. major (LEM62) were used as outgroups. Posterior probabilities (>0.9) are shown in black at nodes. The coloured boxes on the tree define the two Leishmania groups: L. killicki (green) and L. tropica (pink).

Discussion

Previous studies using a small number of strains and different molecular tools and analytic methods [9,12,31,38,40] included L. killicki in the L. tropica complex, except the study by Rioux and Lanotte [39] in which it was considered as a separate phylogenetic complex. The present study wanted to improve the knowledge on L. killicki phylogeny and its evolutionary history relative to L. tropica by using a larger sample of L. killicki strains from different countries.

The phylogenetic analyses performed in this study confirm the position of this taxon within L. tropica in agreement with the previous biochemical and genetic findings. The close phylogenetic relationships between these taxa were also confirmed by the low number of polymorphic sites compared to those found between various Leishmania species [30,31]. The phylogenetic tree shows that L. killicki creates an independent group within L. tropica with high bootstrap value and no common haplotypes between them. Nevertheless, this taxon is included in the L. tropica complex and our data indicate that the species status of L. killicki is not justified. Furthermore, based on the L. tropica complex diversity and the multiple monophyletic branches in this complex, if L. killicki were to be considered as a species, the L. tropica complex would be composed of many species. Therefore, we suggest calling this taxon L. killicki (synonymous L. tropica) as it was previously done before for L. chagasi (synonymous L. infantum) [9,11,49,50]. Further epidemiological and clinical studies in the different countries where this taxon has been reported will say whether the L. killicki denomination should be maintained.

From an evolutionary point of view, these data strongly suggest that L. killicki descends from L. tropica following only one founder event. This hypothesis is supported by the structure of the phylogenetic tree and by biochemical and genetic data. Indeed, the isoenzymatic characterization showed a low number of L. killicki zymodemes compared to those of L. tropica. This low polymorphism in L. killicki was confirmed by the low numbers of PS, HS and haplotypes and amino acid variations in the sequence of the different strains. The analysis of the phylogenetic tree suggests that L. killicki could have originated from an L. tropica ancestor from the Middle East. This ancestor would have separated into L. tropica in Morocco and other countries and into L. killicki in several other countries.

Finally, the lack of shared haplotypes and the identification of the new zymodeme MON-317 and its own haplotype suggest that L. killicki is now evolving independently from L. tropica, probably due to their different transmission cycles (zoonotic for L. killicki [51,52] and both anthroponotic and zoonotic for L. tropica [45,53,54]).

As the L. killicki strains showed low structuring and low polymorphism, we could not determine the precise evolutionary history of this taxon and particularly the country in which it emerged for the first time. Based on the epidemiological data, the higher genetic diversity and especially the relatively high number of described cases in Tunisia compared to the other countries [35,55-57], it is likely that this taxon has emerged for the first time in Tunisia and then has spread in other North-African countries. Nevertheless, this should be further investigated.

Conclusion

The present work brings new insights into the evolutionary history of L. killicki and its taxonomic classification relative to L. tropica. However, more investigations need to be carried out on this model and particularly a detailed population genetics analysis to better understand the epidemiology and population dynamics of this parasite in comparison to L. tropica.

Declarations

Acknowledgements

This study was funded by the Institut Français de Coopération de Tunisie fellowship for PhD students. We thank Elisabetta Andermarcher for editing the English.

Authors’ Affiliations

(1)
Département de Biologie Clinique B, Laboratoire de Parasitologie-Mycologie Médicale et Moléculaire (code LR12ES08), Faculté de Pharmacie, Université de Monastir
(2)
Département de Parasitologie-Mycologie, Centre National de Référence des Leishmanioses, CHRU de Montpellier, Université de Montpellier, France
(3)
UMR MIVEGEC (CNRS 5290-IRD 224-Université de Montpellier)
(4)
School of Life Sciences University of Lincoln, Joseph Banks Laboratories
(5)
Laboratoire d’éco-épidémiologie Parasitaire et Génétique des Populations, Institut Pasteur d’Algérie

References

  1. WHO: Control of the leishmaniases: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases. World Health Organ Tech Rep; 2010: Ser 949, Geneva.Google Scholar
  2. Safjanova VM, Sér. Protozoologie. Le problème de taxonomie chez les Leishmania. In: Les Leishmanies. Léningrad: Académie Soviétique des Sciences. Science; 1982. p. 5–109.Google Scholar
  3. Lainson R, Shaw JJ. Evolution, classification and geographical distribution. In: Peters W, Killicki- Kendrick R, editors. The Leishmaniases Biology and medicine. London: Academie Press; 1987. p. 1–120.Google Scholar
  4. Lanotte G, Rioux JA, Maazoun R, Pasteur N, Pratlong F, Lepart J. Application de la méthode numérique à la taxonomie du genre Leishmania Ross, 1903. A propos de 146 souches originaires de l’Ancien Monde. Utilisation des Allozymes. Corollaires épidémiologiques et phylétiques. Annals Parasitol Hum Comp. 1981;56:575–92.Google Scholar
  5. Lanotte G, Rioux JA, Lepart J, Maazoun R, Pasteur N, Pratlong F. Contribution de la cladistique numérique à la phylétique de genre Leishmania Ross, 1903 (Kinetoplastida- Trypanosomatidae). Utilisation des caractères enzymatiques. C R Acad Sci, Paris. 1984;299:769–72.Google Scholar
  6. Lanotte G, Rioux JA, Serres E. Approche cladistique du genre Leishmania Ross, 1903. A propos de 192 souches originaires de l’Ancien Monde. Analyse numérique de 50 zymodèmes identifiés par 15 enzymes et 96 isoenzymes. In: Rioux JA, editor. Leishmania, Taxonomie et Phylogénèse. Montpellier: IMEE/CNRS/INSERM, Montpellier; 1986. p. 269–88.Google Scholar
  7. Le Blancq SM, Cibulkis RE, Peters W. Leishmania in the Old World. 5. Numerical analysis of isoenzyme data. Trans R Soc Trop Med Hyg. 1986;80:517–24.View ArticlePubMedGoogle Scholar
  8. Moreno G, Pratlong F, Velez ID, Restrepo M, Rioux JA. Individualisation du complexe Leishmania guyanensis. A propos de l’analyse enzymatique de sept zymodèmes. In: Rioux JA, editor. Leishmania, Taxonomie et phylogénèse. Montpellier: IMEEE/CNRS/INSERM; 1986. p. 165–72.Google Scholar
  9. Rioux JA, Lanotte G, Pratlong F, Bastien P, Perieres J. Taxonomy of Leishmania. Use of isoenzymes. Suggestions for a new classification. Ann Parasitol Hum Comp. 1990;65:111–5.PubMedGoogle Scholar
  10. Thomaz- Soccol V, Lanotte G, Rioux JA, Pratlong F, Martini- Dumas A, Serres E. Phylogenetic taxonomy of New Word Leishmania. Annals Parasitol Hum Comp. 1993;68:104–6.Google Scholar
  11. Cupolillo E, Grimaldi GJ, Momen H. A general classification of New World Leishmania using numerical zymotaxonomy. Am J Trop Med Hyg. 1994;50:296–311.PubMedGoogle Scholar
  12. Pratlong F, Dereure J, Ravel C, Lami P, Balard Y, Serres G, et al. Geographical distribution and epidemiological features of Old World cutaneous leishmaniasis foci, based on the isoenzyme analysis of 1048 strains. Trop Med Int Health. 2009;14:1071–85.View ArticlePubMedGoogle Scholar
  13. Pratlong F, Lami P, Ravel C, Balard Y, Dereure J, Serres G, et al. Geographical distribution and epidemiological features of Old World Leishmania infantum and Leishmania donovani foci, based on the isoenzyme analysis of 2277 strains. Parasitology. 2013;140:423–34.View ArticlePubMedGoogle Scholar
  14. Van Eys GJ, Schoone GJ, Kroon NC, Ebeling SB. Sequence analysis of small subunit ribosomal RNA genes and its use for detection and identification of Leishmania parasites. Mol Biochem Parasitol. 1992;51:133–42.View ArticlePubMedGoogle Scholar
  15. Noyes HA, Reyburn H, Bailey JW, Smith D. A nested-PCR based schizodeme method for identifying Leishmania kinetoplast minicercle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan. J Clin Microbiol. 1998;36:2877–81.PubMed CentralPubMedGoogle Scholar
  16. Bañuls AL, Jonquieres R, Guerrini F, Le Pont F, Barrera C, Espinel I, et al. Genetic analysis of Leishmania parasites in Ecuador: are Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis distinct taxa? Am J Trop Med Hyg. 1999;61:838–45.PubMedGoogle Scholar
  17. Hide M, Bañuls AL, Tibayrenc M. Genetic heterogeneity and phylogenetic status of Leishmania (Leishmania) infantum zymodeme MON-1: epidemiological implications. Parasitology. 2001;123:425–32.View ArticlePubMedGoogle Scholar
  18. Schönian G, Schnur L, El Fari M, Oskam L, Kolesnikov AA, Sokolowska- Köhler W, et al. Genetic heterogeneity in the species Leishmania tropica revealed by different PCR- based methods. Trans R Soc Trop Med Hyg. 2001;95:217–24.View ArticlePubMedGoogle Scholar
  19. Spanakos G, Piperaki ET, Menounos PG, Tegos N, Flemetakis A, Vakalis NC. Detection and species identification of Old World Leishmania in clinical samples using a PCR-based method. Trans R Soc Trop Med Hyg. 2008;102:46–53.View ArticlePubMedGoogle Scholar
  20. Mauricio IL, Yeo M, Baghaei M, Doto D, Pratlong F, Zemanova E, et al. Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD). Int J Parasitol. 2006;36:575–69.View ArticleGoogle Scholar
  21. Oddone R, Schweynoch C, Schönian G, de Sousa CS, Cupolillo E, Espinosa D, et al. Development of a multilocus microsatellite typing approach for discriminating strains of Leishmania (Viannia) species. J Clin Microbiol. 2009;47:2818–25.View ArticlePubMed CentralPubMedGoogle Scholar
  22. Gouzelou E, Haralambous C, Antoniou M, Christodoulou V, Martinković F, Živičnjak T, et al. Genetic diversity and structure in Leishmania infantum populations from southeastern Europe revealed by microsatellite analysis. Parasit Vect. 2013;6:342.View ArticleGoogle Scholar
  23. Montalvo AM, Fraga J, Monzote L, Montano I, De Doncker S, Dujardin JC, et al. Heat-shick protein 70 PCR-RFLP: a universal simple tool for Leishmania species discrimination in the New and Old World. Parasitology. 2010;137:1159–68.View ArticlePubMedGoogle Scholar
  24. Haouas N, Garrab S, Gorcii M, Khorchani H, Chargui N, Ravel C, et al. Development of a polymerase chain reaction-restriction fragment length polymorphism assay for Leishmania major/Leishmania killicki/Leishmania infantum discrimination from clinical samples, application in a Tunisian focus. Diagn Microbiol Infect Dis. 2010;68:152–8.View ArticlePubMedGoogle Scholar
  25. Talmi-Frank D, Abedelmajeed N, Schnur LF, Schönian G, Toz SO, Jaffe CL, et al. Detection and Identification of Old World Leishmania by High Resolution Melt Analysis. PLoS Negl Trop Dis. 2010;4:e581.View ArticlePubMed CentralPubMedGoogle Scholar
  26. Odiwuor S, Vuylsteke M, De Doncker S, Maes I, Mbuchi M, Dujardin JC, et al. Leishmania AFLP: paving the way towards improved molecular assays and markers of diversity. Infect Genet Evol. 2011;11:960–7.View ArticlePubMedGoogle Scholar
  27. Schönian G, Mauricio I, Cupolillo E. Is it time to revise the nomenclature of Leishmania? Trends Parasitol. 2010;26:466–9.View ArticlePubMedGoogle Scholar
  28. Van der Auwera G, Fraga J, Montalvo AM, Dujardin JC. Leishmania taxonomy up for promotion. Trends Parasitol. 2011;27:49–50.View ArticlePubMedGoogle Scholar
  29. Zemanova E, Jirku M, Mauricio IL, Horak A, Miles MA, Lukes J. The Leishmania donovani complex: genotypes of five metabolic enzymes (ICD, ME, MPI, G6PDH, and FH), new targets for multilocus sequence typing. Int J Parasitol. 2007;37:149–60.View ArticlePubMedGoogle Scholar
  30. Boité MC, Mauricio IL, Miles MA, Cupolillo E. New insights on taxonomy, phylogeny and population genetics of Leishmania (Viannia) parasites based on multilocus sequence analysis. Plos Negl Trop Dis. 2012;6:e1888. doi:10.1371/journal.pntd.0001888.View ArticlePubMed CentralPubMedGoogle Scholar
  31. El Baidouri F, Diancourt L, Berry V, Chevenet F, Pratlong F, Marty P, et al. Genetic structure and evolution of the Leishmania genus in Africa and Eurasia: What does MLST tell us? Plos Negl Trop Dis. 2013;7:e2255. doi:10.1371/journal.pntd.0002255.View ArticlePubMed CentralPubMedGoogle Scholar
  32. Zhang CY, Lu XJ, Du XQ, Jian J, Shu L, Ma Y. Phylogenetic and evolutionary analysis of Chinese Leishmania isolates based on multilocus sequence typing. Plos one. 2013;8:e63124. doi:10.1371/journal.pone.0063124.View ArticlePubMed CentralPubMedGoogle Scholar
  33. Marlow MA, Boité MC, Ferreira GEM, Steindel M, Cupolillo E. Multilocus sequence analysis for Leishmania braziliensis outbreak investigation. Plos Negl Trop Dis. 2014;8(2):e2695. doi:10.1371/journal.pntd.0002695.View ArticlePubMed CentralPubMedGoogle Scholar
  34. Bañuls AL, Hide M, Prugnolle F. Leishmania and the leishmaniases: A parasite genetic updates and advances in taxonomy, epidemiology and pathogeniecity in Humans. Adv Parasitol. 2007;64:1–109.View ArticlePubMedGoogle Scholar
  35. Rioux JA, Lanotte G, Pratlong F. Leishmania killicki n.sp (Kinetoplastida: Trypanosomatidae) Leishmania. Taxonomie et phylogenèse. In: Application éco-épidémiologique (Coll. Int. CNRS/INSERM, 1984). Montpellier: IMEEE; 1986. p. 139–42.Google Scholar
  36. Aoun K, Bousslimi N, Haouas N, Babba H, El-Buni A, Bouratbine A. First report of Leishmania (L.) killicki Rioux, Lanotte and Pratlong, 1986 in Libya. Parasite. 2006;13:87–8.PubMedGoogle Scholar
  37. Harrat Z, Boubidi SC, Pratlong F, Benikhlef R, Selt B, Dedet JP, et al. Description of a dermotropic Leishmania close to L. killicki (Rioux, Lanotte & Pratlong 1986) in Algeria. Trans R Soc Trop Med Hyg. 2009;103:716–20.View ArticlePubMedGoogle Scholar
  38. Pratlong F, Lanotte G, Ashford RW, Rioux JA. Le complexe Leishmania tropica. A propos de l’analyse numérique de 29 souches identifiées par la méthode enzymatique. Leishmania. Taxonomie et phylogenèse. In: Applications éco-épidémiologiques (Coll. Int. CNRS/INSERM, 1984). Montpellier: IMEEE; 1986. p. 129–37.Google Scholar
  39. Rioux JA, Lanotte G. Apport de la cladistique à l’analyse du genre Leishmania Ross, 1903 (Kinetoplastida: Trypanosomatidae). Corollaires épidémiologiques. Biosystema. 1993;8:79–80.Google Scholar
  40. Schwenkenbecher JM, Wirth T, Schnur LF, Jaffe CL, Schallig H, Al-Jawabreh A, et al. Microsatellite analysis reveals genetic structure of Leishmania tropica. Int J Parasitol. 2006;36:237–46.View ArticlePubMedGoogle Scholar
  41. Chaouch M, Fathallah-Mili A, Driss M, Lahmadi R, Ayari C, Guizani I, et al. Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis. Acta Trop. 2013;125:357–65.View ArticlePubMedGoogle Scholar
  42. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25:1451–2.View ArticlePubMedGoogle Scholar
  43. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012;61:539–42.View ArticlePubMed CentralPubMedGoogle Scholar
  44. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9.View ArticlePubMed CentralPubMedGoogle Scholar
  45. Pratlong F, Rioux JA, Dereure J, Mahjour J, Gallego M, Guilvard E, et al. Leishmania tropica in Morocco. IV-Intrafocal enzyme diversity. Ann Parasitol Hum Comp. 1991;66:100–4.PubMedGoogle Scholar
  46. Haouas N, Chargui N, Chaker E, Ben Said M, Babba H, Belhadj S, et al. Anthroponotic cutaneous leishmanisis in Tunisia: Presence of Leishmania killicki outside its original focus of Tataouine. Trans R Soc Trop Med Hyg. 2005;99:499–501.View ArticlePubMedGoogle Scholar
  47. Haouas N, Chaker E, Chargui N, Gorcii M, Belhadj S, Kallel K, et al. Geographical distribution updating of Tunisian leishmaniasis foci: About the isoenzymatic analysis of 694 strains. Acta Trop. 2012;124:221–8.View ArticlePubMedGoogle Scholar
  48. Mansouri R, Pratlong F, Bachi F, Hamrioui B, Dedet JP. The first isoenzymatic characterizations of the Leishmania strains responsible for cutaneous leishmaniasis in the Area of Annaba (Eastern Algeria). Open Conf proceed J. 2012;3(Suppl 2-M2):6–11.View ArticleGoogle Scholar
  49. Momen H, Gabriel Grimaldi JR, Leonidas MD. Leishmania infantum, the aetiological agent of American visceral leismaniasis (AVL)? Mem Inst Oswaldo Cruz. 1987;82:447–8.View ArticlePubMedGoogle Scholar
  50. Mauricio IL, Stothard JR, Miles MA. The strange case of Leishmania chagasi. Parasitol Today. 2000;16:188–9.View ArticlePubMedGoogle Scholar
  51. Jaouadi K, Haouas N, Chaara D, Gorcii M, Chargui N, Augot D, et al. First detection of Leishmania killicki (Kinetoplastida, Trypanosomatidae) in Ctenodactylus gundi (Rodentia, Ctenodactylidae), a possible reservoir of human cutaneous leishmaniasis in Tunisia. Parasite Vector. 2011;11:159.View ArticleGoogle Scholar
  52. Bousslimi N, Ben-Ayed S, Ben-Abda I, Aoun K, Bouratbine A. Natural infection of North African gundi (Ctenodactylus gundi) by Leishmania tropica in the focus of cutaneous leishmaniasis, Southeast Tunisia. Am J Trop Med Hyg. 2012;86:962–5.View ArticlePubMed CentralPubMedGoogle Scholar
  53. Sang DK, Njeru WK, Ashford RW. A zoonotic focus of cutaneous leishmaniasis due to Leishmania tropica at Utut, Rift Valley Province Kenya. Trans R Soc Trop Med Hyg. 1994;88:35–7.View ArticlePubMedGoogle Scholar
  54. Svobodova M, Votypka J, Peckova J, Dvorak V, Nasereddin A, Baneth G, et al. Distinct transmission cycles of Leismania tropica in 2 adacent foci, Northern Israel. Emerg Infect Dis. 2006;12:1860–8.View ArticlePubMed CentralPubMedGoogle Scholar
  55. Chaffai M, Ben Rachid MS, Ben Ismail R. Formes clinico-épidémiologiques des Leishmanioses cutanées en Tunisie. Ann Dermatol Venereol. 1988;115:1255–60.PubMedGoogle Scholar
  56. Aoun K, Ben Abda I, Bousslimi N, Bettaieb J, Siala E, Ben Abdallah R, et al. Caractérisation comparative des trois formes de leishmaniose cutanée endémique en Tunisie. Ann Dermatol Venereol. 2012;139:452–8.View ArticlePubMedGoogle Scholar
  57. Jaouadi K, Depaquit J, Haouas N, Chaara D, Gorcii M, Chargui N, et al. Twenty-four new human cases of cutaneous leishmaniasis due to Leishmania killicki in Metlaoui, southwestern Tunisia: probable role of Phlebotomus sergenti in the transmission. Acta Trop. 2012;122:276–83.View ArticlePubMedGoogle Scholar

Copyright

© Chaara et al.; licensee BioMed Central. 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Advertisement