Short communication

First evidence of transmission of Leishmania (Viannia) lainsoniin a Sub Andean region of Bolivia

B. Bastrenta a,*, R. Buitrago b, F. Vargas b, F. Le Pont a, M. Torrez b,M. Flores b, N. Mita b, S.F. Breniere c

a Institut de recherche pour le Developpement (IRD), UMR CNRS/IRD no. 9926, ‘Genetique Moleculaire des Parasites et des Vecteurs’,

CP 9214 La Paz, Boliviab Instituto Boliviano de Biologıa de Altura, (IBBA), CP 641 La Paz, Bolivia

c Institut de Recherche pour le Developpement (IRD), UR 008, Pathogenie des Trypanosomatidae, BP 5045, 34032 Montpellier, France

Received 3 October 2000; received in revised form 22 February 2002; accepted 15 April 2002

Abstract

Using ubiquitous primers which amplify the variable parts of kDNA minicircle of all Leishmania spp, we obtained

for Leishmania (viannia) lainsoni a major band of 605 bp (band 1) shared with L. V. braziliensis and a minor 524 bp

band (band 2) specific of L. V. lainsoni . The specificity of the two bands was examined through Southern blot

hybridization of kDNA PCR obtained from reference strains belonging to L. braziliensis , L. mexicana , L. donovani

complexes with L. V. lainsoni species. Band 1 was not specific of L. V. lainsoni since it hybridized with some isolates

belonging to L. braziliensis complex. In contrast, band 2 was L. V. lainsoni specific. PCR-based detection followed by

hybridization with the new L. V. lainsoni probe (Band 2) and L. V. braziliensis probe (564 bp), was assayed using

sample from a pool of 25 females of Lutzomiya nuneztovari anglesi , blood, skin and liver samples of 18 mammals, spinal

cords of four mammals and blood and cutaneous ulcers aspirates from 95 patents from Sub Andean region of La Paz,

Bolivia. We observed a ositive hybridization of four patients lesions and the pool of L. nuneztovari anglesi with the L.

V. lainsoni probe. It is the first time that L. V. lainsoni is observed in a cycle of transmission in Bolivia. PCR products of

three patients lesions and the pool of L. nuneztovari anglesi were also hybridized with the specific probe of L. V.

braziliensis suggesting mixed infection in this focus. # 2002 Elsevier Science B.V. All rights reserved.

Keywords: Leishmania (Viannia ) lainsoni ; Bolivia; Transmission; PCR-based diagnosis; Specific probe; Kinetoplast

We report in this study the first evidence of the

presence of Leishmania Viannia lainsoni in vector

and patients from the Yungas valleys situated in

the Sub Andean region of ‘La Paz’ department,

Bolivia. This region is highlly endemic for cuta-

neous and muco-cutaneous leishmaniasis due to L.

V. braziliensis, the consequences being severe

mutilations mostly on the patients’ faces (Torres

Espejo et al., 1989; David et al., 1993; Dedet et al.,

1995). Moreover, a recent study showed a new

focus in the same region due to L. Leishmania

* Corresponding author. Tel.: �/591-2-278-2969; fax: �/591-

2-278-2944

E-mail address: bastrenta@mail.megalink.com (B.

Bastrenta).

Acta Tropica 83 (2002) 249�/253

www.parasitology-online.com

0001-706X/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 0 0 1 - 7 0 6 X ( 0 2 ) 0 0 1 2 9 - 8

amazonensis (Martinez et al., 1998) and some cases

of visceral leishmaniasis have been described

previously (Desjeux et al., 1983). L. V. lainsoni

was a newly recognized species which was first

identified in infected humans in the state of Para,

Brazil (Silveira et al., 1987) and was also isolated

from patients in the Sub Andean region in Peru

(Lucas et al., 1994). This species can be distin-

guished from L. V. braziliensis by typically

elongated amastigotes and their voluminous kine-

toplast and from L. L. amazonensis and L. V.

braziliensis by isoenzyme profiles (Guerrini, 1993;

Lucas et al., 1994; Eresh et al., 1995). To

investigate the presence of L. V. lainsoni in the

Yungas valleys, we used in this study the ubiqui-

tous primers defined by Breniere et al., (1999)

which amplify the variable parts of kDNA mini-

circles of all Leishmania sp. and other Kinetoplas-

tidae. These primers generated high

polymorphism, which correlate with isoenzyme

analysis (Breniere et al., 1999). Therefore, three

complex-specific kDNA probes have been pro-

Fig. 1. Fig, 1 (a): Ethidium bromide stained 1.5% agarose gel containing kDNA PCR products from references strains. (b,c) High

stringency hybridization of Southern blotted PCR products with L. V. lainsoni probe (M6426, band 2: 524 bp) and L. V. braziliensis

probe (CG), respectively. L. V. guyanensis : lane 1: MHOM/GF/85/Lem 669; lane 2: MHOM/BR/78/M5378; L. V. braziliensis : lane 3;

MHOM/BR/84/LTB 300; lane 4: MHOM/CO/83/lem 469; lane 5: MHOM/BO/90/CG; lane 6: MHOM/BO/90/JP; lane 7: MHOM/BO/

90/JM; lane 8: MHOM/BO/90/AM; Lane 9: MHOM/BO/90/EL; lane 10: MHOM/BO/90/CS; lane 11: MHOM/BR/75/M2904; lane 12:

MHOM/PE/90/LH 1016; lane 13: MHOM/BO/84/LPZ 595; L. V. peruviana: lane 14: MHOM/PE/90/HB44; L. V. lainsoni (Banuls,

1998): lane 15: MHOM/BR/81/M6426; lane 18: MHOM/PE/91/LC2288; lane 19: MHOM/PE/91/LH1154; lane 20: MHOM/PE/00/

LH762; lane 21: L. L. mexicana : MINYC/BZ/62/M379; L. L. tropica : lane 16: MHOM/SU/74/k-27; lane 17, 22; Puc 19/Ra sI.

B. Bastrenta et al. / Acta Tropica 83 (2002) 249�/253250

duced from major PCR bands of reference stocks

belonging to L. V. braziliensis , (MHOM/BO/90/

CG), L. L. mexicana (MINYC/BZ/62/M379) and

L. L. chagasi (MHOM/BR/74/PP75). The applica-

tion of the PCR-based diagnosis, followed by

hybridization with these specific probes allowed

determination of the putative reservoirs of L. L.

amazonensis (Telleria et al., 1999) and the reser-

voir of L. V. braziliensis (study in process) in the

Yungas valleys.

Similarly, the specificity of two kDNA PCR

bands obtained from L. V. lainsoni (MHOM/BR/

81/M6426) were tested: the major band of 605 bp

(band 1) shared with L. V. braziliensis and the

minor 524 bp band (band 2) which seemed to be

specific of L. V. lainsoni (Figs. 1a and 2a). The two

probes were labeled using the enhanced chemilu-

minescence gene detection system (ECL). The

specificity of the probes (bands 1 and 2) was

examined by Southern blot hybridization tokDNA PCR products obtained from reference

strains belonging to L. braziliensis , L. mexicana ,

L. donovani complexes and L. V. lainsoni species.

Hybridization was performed at 42 8C overnight

in a rotating oven (Appligen, Illkirch, France).

The membranes were washed twice under highly

stringent conditions (6 M urea, 0.1�/SSC at

42 8C during 10 min), and then twice in 2�/SSCat room temperature. Two exposures were per-

formed (1 and 30 min) on autoradiography film

(HyperfilmTM-MP, Amersham, Buckingham-

shire, UK).

As expected, the band 1 probe was not specific

of L. V. lainsoni since it hybridized also with some

isolates belonging to L. braziliensis complex (data

not shown). In contrast, band 2 was L. V. lainsoni

specific (five strains tested: MHOM/BR/81/

M6426, MHOM/PE/91/LC2288, MHOM/PE/91/

LH1154, MHOM/PE/00/LH762, MHOM/BR/81/

LH619 (Figs. 1b and 2b), whereas no signal could

be observed among a large set of L. V. braziliensis

reference strains, L. L. mexicana : MNYC/BZ/62/

M379, L. major : MHOM/SU/73/5Askh. More-

over, no hybridization of band 2 was observedwhen using additional strains belonging to L.

donovani complex: MHOM/BR/74/PP75,

MHOM/BR/79/L101, MHOM/IN(–)/61/L13, L.

mexicana complex, IFLA/BR/67/PH8, MHOM/

BR/76/LTB012, MHOM/FG/84/H142, MORY/

PA/68/GML3, MHOM/VE/76/JAP78, MHOM/

VE/57/LV135, L. tarentolae : RTAR/SN/67/G10,

and L. V. panamensis : MHOM/CO/83/REST417(data not shown).

These results support the hypothesis that L. V.

lainsoni species is substantially different from the

other species of the braziliensis complex (Eresh et

al., 1995). However the phylogenetic analysis

based on kDNA-PCR polymorphism, showed

that it is more related to the braziliensis complex

than to others (Breniere et al., 1999).In order to evaluate the effectiveness of band 2

as a diagnosis marker of L. V. lainsoni infection,

the PCR-based detection followed by hybridiza-

tion with the new L. V. lainsoni probe, was assayed

using sample from vectors and mammals, includ-

ing humans. Ninety-five patients were received in

the hospital of Chulumani (South Yungas pro-

Fig. 2. (a): Ethidium bromide stained 1.5% agarose gel contain-

ing kDNA PCR products. (b,c) Hybridization of Southern

blotted PCR products with L. V. lainsoni probe (band 2: 524

bp) and L. V. braziliensis probe (CG), respectively. Lanes 1, 2,

4, 5: spinal cord of mammal; lane 3: blood of mammal; lanes 6�/

12: lesion of patients; lane 13: vector: Lutzomyia nuneztovari

anglesi ; L. V. lainsoni: lane 14: MHOM/BR/81/M6426; lane 15:

MHOM/BR/81/LH619; L. V. braziliensis : lane 16: MHOM/BO/

84/LPZ 595; L. L. mexicana : lane 17: MNYC/BZ/62/M379.

B. Bastrenta et al. / Acta Tropica 83 (2002) 249�/253 251

vince, La Paz department, 1800 m a.s.l.) for aleishmaniasis diagnosis, first based on clinical

observation. For each patient, 2 ml of blood

were mixed with an equal volume of 6 M

guanidine HCL/200 mM EDTA, pH 8 (Avila et

al., 1991) and lesion aspirates were taken from

cutaneous ulcers with 3 ml syringes containing 0.5

ml of sterile normal saline solution (NaCl 0.9%).

DNAs were extracted by phenol-chloroform andprecipitated with ethanol (Wincker et al., 1997).

The pellet was mixed in 50 ml of distilled water and

stored at �/20 8C. A pool of 25 females of

Lutzomyia nuneztovari anglesi , vector that showed

in a previous study a natural infection by L. V.

braziliensis (Torrez et al., 1998) was immersed in

50 ml of lysis buffer containing proteinase K,

incubated at 42 8C for 30 min, and then at95 8C for 30 min. Blood, skin and liver samples

of 18 mammals and spinal cords of four mammals,

were also DNA extracted according to Telleria et

al. (1999). Five microliters of DNA extracts were

used to the amplification kDNA-PCR procedures,

then, the products were Southern blotted and

hybridized. The conditions were according to

Breniere et al. (1999).Fig. 2 illustrates the electrophoretic patterns of

kDNA PCR products obtained from patients,

vector, mammals samples and reference strains

and the hybridization results with (b) L. V.

lainsoni probe (band 2: 524 bp), (c) L. V.

braziliensis probe (Breniere et al., 1999). Among

all the samples tested, we observed a positive

hybridization of four patients lesions and thepool of L. nuneztovari anglesi with the L. V.

lainsoni probe. PCR products of three patients

lesions and the pool of L. nuneztovari anglesi were

also hybridized with the specific probe of L. V.

braziliensis , suggesting mixed infection in this

focus. It is the first time that L. V. lainsoni is

observed in a cycle of transmission in Bolivia. This

result suggests that L. V. lainsoni is probably morewidespread in the Sub Andean region. L. nunez-

tovari anglesi is the candidate vector of L. V.

braziliensis and L. L.mexicana in this area (Le

Pont et al., 1989; Telleria et al., 1999) and should

be also the vector of L. V. lainsoni . Further studies

based on the same PCR/hybridization procedure

will confirm that L. nuneztovari anglesi is the main

vector of various Leishmania species in this region.Extensive studies should provide information

critical to the development of strategies aimed at

the control of leishmaniasis.

Acknowledgements

This work received financial support from,

UNDP/World Bank Special Program for research

and Training in Tropical Diseases (Tegumentary

Leishmaniasis: risk factors and self-protection no.

940902) and from IRD (l’Institut de Recherchepour le Developpement, France). We thank Pro-

fessor Jorge Arevalo (Instituto de Medecina Tro-

pical Alexander Von Humbolt, Universidad

Peruana Cayetano Heredia from Lima) for the

L. V. Lainsoni reference stocks used in this study.

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