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. Brenie`re 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 Biologa 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
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: email@example.com (B.
Acta Tropica 83 (2002) 249/253
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 Brenie`re 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 (Brenie`re 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
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 overnightin a rotating oven (Appligen, Illkirch, France).
The membranes were washed twice under highly
stringent conditions (6 M urea, 0.1/SSC at42 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-
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. lainsonispecific (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 (Brenie`re 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 andstored at /20 8C. A pool of 25 females ofLutzomyia 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 samplesof 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
Brenie`re 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 (Brenie`re 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.
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 (lInstitut 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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