Ex vivo and in vivo biological behavior of Leishmania (Viannia) shawi

Abstract

Since the first description of Leishmania (Viannia) shawi, few studies were performed with this parasite. In the present work, the in vivo and ex vivo behavior of L. (Viannia) shawi infection was studied using murine model. Peritoneal macrophages from BALB/c and C57BL/6 mice were infected with promastigotes
in the stationary phase of growth; after 24h, the infection index and nitric oxide (NO) levels in the supernatant of the
cultures were analyzed. BALB/c and C57BL/6 mice were infected into the hind footpad, and at each 2weeks, mice were sacrificed,
and the histological changes of the skin inoculation site, parasitism, and humoral immune responses were evaluated during
8weeks. Ex vivo experiments showed that macrophages of BALB/c presented higher infection index and lesser NO levels than
macrophages of C57BL/6. In vivo experiments showed that BALB/c presented higher lesion size than C57BL/6 mice; similarly,
the histopathological changes and the parasitism in skin were more exacerbate in BALB/c mice. In draining lymph nodes, the
main change was increase of germinative centers, and parasites were detected from 6weeks pi onwards in both mice strain.
IgG was detected in BALB/c mice from 4weeks, while in C57BL/6, from 6weeks pi onwards. Taken together, these results indicate
that BALB/c showed a classical behavior of susceptibility when compared to C57BL/6 mice.

Full text

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ORIGINAL PAPER
Ex vivo and in vivo biological behavior of Leishmania
(Viannia) shawi
Luiz Felipe Domingues Passero & Juliano V. Sacomori & Thaíse Yumie Tomokane &
Carlos Eduardo Pereira Corbett & Fernando Tobias da Silveira &
Márcia Dalastra Laurenti
Received: 21 June 2009 /Accepted: 24 August 2009 /Published online: 16 September 2009
# Springer-Verlag 2009
Abstract Since the first description of Leishmania (Viannia)
shawi, few studies were performed with this parasite. In
the present work, the in vivo and ex vivo behavior of
L. (Viannia) shawi infection was studied using murine
model. Peritoneal macrophages from BALB/c and C57BL/6
mice were infected with promastigotes in the stationary phase
ofgrowth;after24h,theinfectionindexandnitricoxide (NO)
levels in the supernatant of the cultures were analyzed.
BALB/c and C57BL/6 mice were infected into the hind
footpad, and at each 2 weeks, mice were sacrificed, and the
histological changes of the skin inoculation site, parasitism,
and humoral immune responses were evaluated during
8 weeks. Ex vivo experiments showed that macrophages of
BALB/c presented higher infection index and lesser NO
levels than macrophages of C57BL/6. In vivo experiments
showed that BALB/c presented higher lesion size than
C57BL/6 mice; similarly, the histopathological changes and
the parasitism in skin were more exacerbate in BALB/c
mice. In draining lymph nodes, the main change was
increase of germinative centers, and parasites were detected
from 6 weeks pi onwards in both mice strain. IgG was
detected in BALB/c mice from 4 weeks, while in C57BL/6,
from 6 weeks pi onwards. Taken together, these results
indicate that BALB/c showed a classical behavior of
susceptibility when compared to C57BL/6 mice.
Introduction
Leishmaniasis is a disease caused by parasites of the genus
Leishmania, which are capable to infect a broad range of
mammalian species, including humans. Up to now, there
are 27 species described in the World; among which, some
few studied and others recently characterized, such as
Leishmania (Viannia) shawi (Lainson et al. 1989), Leish-
mania (Viannia) naiffii (Lainson and Shaw 1989), Leish-
mania (Viannia) colombiensis (Kreutzer et al. 1991),
Leishmania (Viannia) lainsoni (Silveira et al. 1987), and
Leishmania (Viannia) lindenbergi (Silveira et al. 2002).
In the Amazon region of Brazil, L. (Viannia) shawi was
described and characterized by Lainson et al. (1989), who
isolated it from Cepus apella and Chiropotes satanas
monkeys, Choloepus didactylus sloth, edentate Bradypus
tridactylus, coatimundis Nasua nasua, and sandfly Lutzomyia
whitmany in the Serra dos Carajás, Pará State, Brazil. Inthese
animals,theparasitehaddifferenttissuetropisms:inthesloths,
it wasfoundin thespleenand liver, andin theother reservoirs,
itwasfoundlimitedtotheskin;onlyinthehamstertheparasite
caused a self-limited infection in the skin with moderate
densities of amastigote forms. This first report on L. (Viannia)
L. F. D. Passero:T. Y. Tomokane:C. E. P. Corbett:
M. D. Laurenti
Laboratório de Patologia de Moléstias Infecciosas (LIM-50),
Depto. de Patologia, Universidade de São Paulo,
São Paulo, SP, Brazil
J. V. Sacomori
Laboratório de Patologia de Moléstias Infecciosas (LIM-50),
Departamento de Patologia, Universidade de São Paulo,
Avenida Dr. Arnaldo, 455,
01246-903 São Paulo, Brazil
F. T. da Silveira
Núcleo de Medicina Tropical da Universidade Federal
do Pará/Laboratório de Leishmanioses
do Instituto Evandro Chagas,
Belém, PA, Brazil
L. F. D. Passero (*)
Department of Pathology, São Paulo University Medical School,
Av. Dr. Arnaldo, 455,
01246-903 São Paulo, SP, Brazil
e-mail: felipepassero@usp.br
Parasitol Res (2009) 105:1741–1747
DOI 10.1007/s00436-009-1614-7

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shawi showed that it is capable of infecting a wide range of
animals and with tissue specificity regarding host species.
Two years afterwards, the same research group found
this species related to the human American Cutaneous
Leishmaniasis (ACL). In most cases, around 60%, the
disease was characterized by single ulcerated skin lesion,
although in some individuals, many lesions were recorded
in the body (Shaw et al. 1991). More recently, however, the
opinion is that L. (Viannia) shawi infection has increased in
its importance as an agent of ACL in the Brazilian Amazon
region, being surpassed only by Leishmania (Viannia)
braziliensis and Leishmania (Viannia) guyanensis (Silveira,
personal communication).
Despite these few studies, information regarding the
interaction between L. (Viannia) shawi and the host is
scarce. Therefore, the aims of this study was evaluate the
infectivity of L. (Viannia) shawi in macrophages from
BALB/c and C57BL/6 mice, as well as in vivo evaluating
the lesion size, histopathological changes, parasite density,
and humoral immune response to better understand the
eventsthattake place inthe courseofinfection, characterizing
the murine model for L. (Viannia) shawi infection.
Materials and methods
Parasites
L. (Viannia) shawi (MHOM/BR/96/M15789 strain) was
isolated from a patient with cutaneous leishmaniasis in the
Buriticupu County, Maranhão State, Brazil. It was classified
by monoclonal antibodies and isoenzymes at the Evandro
Chagas Institute, Belém, Pará State, Brazil. The parasites
maintained in BALB/c mice footpad were isolated and
expanded in RPMI-1640 medium, supplemented with
10% heat-inactivated serum, 0.25 mM 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES), 10 mg/ml genta-
micin, and 100 IU/ml penicillin and maintained at 25°C. On
the sixth day of culture, the promastigote forms were washed
three times with phosphate buffer (PBS) pH7.4, centrifuged
at 1,200 g, 10 min, 4°C, for mice infection and production of
soluble antigen.
Antigen production
Stationary phase promastigotes were harvested from 5 to
6 days of culture and washed three times in cold PBS,
resuspended in sterile distillated water, subjected to frozen
in liquid nitrogen, and thawed at room temperature three
times, and the solution was centrifuged at 10,000 g for
30 min. The supernatant was collected, and protein
concentration was determined using a commercial kit
(Biorad, USA). Aliquots were stored at −80°C.
Experimental animals
Eight-week-old BALB/c and C57BL/6 male mice obtained
from the Animal Facility of the São Paulo University,
Medical School Brazil were maintained in our laboratory
during the experiments according to the guidelines of the
institutional rules regarding the welfare of experimental
animals and with the approval of the Animal Ethics
Committee of São Paulo University.
Ex vivo experiments: macrophage infection
and NO quantification
Approximately 105BALB/c and C57BL/6 mice peritoneal
macrophages were placed in a 24-well plate in RPMI
medium. L. (Viannia) shawi promastigotes in a stationary
phase of culture growth were added to the macrophage
monolayers at a ratio of 5 parasites to 1 macrophage, and
the culture was incubated at 5% CO2at 35°C. After 24 h,
the plate was centrifuged (1,000 g, 10 min, 4°C), and
supernatants were collected and stored at −80°C for nitric
oxide (NO) quantification, according to Green et al. 1990.
The NO levels were estimated using standard curve
constituted of nitrite. The slides were dried at room
temperature, fixed in methanol, and stained by Giemsa.
The infection index was determinate in both macrophages
from BALB/c and C57BL/6 mice.
BALB/c and C57BL/6 mice infection
Stationary phase promastigote forms were washed three
times in PBS, and the parasites concentration was adjusted
to 2×107promastigotes/ml. Fifty microliters was injected
subcutaneously into the hind footpads of the mice (around
106promastigotes/mouse), and only PBS was injected in
control animals. The lesion size was measured weekly with
a dial micrometer and expressed as the difference in size
between the infected and the contralateral uninfected
footpad. At 2, 4, 6, and 8 weeks post-infection (pi), one
group of five infected mice of both mouse strains were
bled, and the sera were collected and stored at −80°C.
Biopsies of the skin inoculation site and lymph nodes were
collected and fixed in buffered 5% formalin for histopath-
ological studies and quantification of amastigote forms.
Imprints of spleen and liver were performed to evaluate the
viscera parasitism. The experiment was performed three
times.
Evaluation of histopathological changes and quantitative
morphometric analysis
The histological changes were evaluated through routine
histological HE-staining of 5 μm sections under the light
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microscope and were further graded as negative, light,
moderate, and intense, according to Giunchetti et al. (2008).
The parasitism in skin and lymph nodes of BALB/c and
C57BL/6 mice was evaluated using quantitative morpho-
metric analysis in sections labeled by immunohistochemistry
to evidence the amastigote forms, according to Passero et al.
(2008).
Evaluation of the humoral immune response
The humoral immune response was measured by enzyme-
linked immunosorbent assay (ELISA). High-binding plates
(Costar, USA) were coated with 1µg/well soluble antigen
from L. (Viannia) shawi promastigote, overnight at 4°C.
Plates were blocked with 10% of non-fat milk in PBS for
2 h at 37°C to prevent nonspecific binding. Sera of all
animal groups (1:50) were added for 1 h at 37°C. Goat anti-
mouse IgG monoclonal antibody conjugated with alkaline
phosphatase (Sigma, USA) in a dilution of 1:1,000 was
added for 1 h, 37°C. The substrate p-nitrophenyl phosphate
(1.0 mg/ml) was added for 30 min, and the absorbance was
read in ELISA reader (Uniscience, USA).
Statistical analysis
The results were expressed as mean±standard deviation of
three independent experiments, and the non-parametric test
of Mann–Whitney was employed to compare infection
index, NO levels, lesion size, parasite density, and humoral
immune response between BALB/c and C57BL/6 mice.
Differences were considered statistically significant when
p<0.05. Statistical analysis were performed using Biostat
5.0 software, using N=3.
Results
Macrophage infection index and NO levels
Macrophages from BALB/c mice presented higher infection
index than those from C57BL/6 with statistical significance
(p<0.05). Moreover, macrophages from BALB/c mice
showed tendency to present diminished levels of NO levels
compared to macrophages from C57BL/6; however, no
statistical significance was observed (Fig. 1). Uninfected
macrophages showed no expressive NO levels (data not
shown).
Lesion size
The infection caused by L. (Viannia) shawi induced an
increase in the hind footpad swelling in BALB/c and
C57BL/6 mice. The lesion size showed no significant
differences at 1, 2, and 3 weeks pi between both mice, but
from 4 weeks pi, the lesion size became significantly higher
in BALB/c mice (p<0.05) compared with C57BL/6 mice
(Fig. 2), and at 8 weeks pi, the footpad of BALB/c and
C57BL/6 mice increased 2.1 and 1.6 times in relation to
both healthy mice, respectively.
Histopathological changes in the skin
Microscopically, a discrete inflammatory infiltrate in the
dermis, consisting of moderated densities of polymorpho-
nuclear, mononuclear cells, and light densities of amastigote
forms inside the macrophages (Figs. 3a and 5a) was observed
in BALB/c mice at 2 weeks pi. At 4 weeks pi, the
inflammatory infiltrate enlarged, showing moderate densities
of polymorphonuclear cells, high densities of infected
Fig. 1 Peritoneal macrophages from BALB/c and C57BL/6 mice
were isolated and infected with a rate of five promastigote/one
macrophages. After 24 h, the production of nitric oxide levels from
both mice macrophages were quantified, and the infection index was
calculated. *p<0.05 indicates statistical significance between infection
index of macrophages from C57BL/6 with macrophage from BALB/c
mice
Fig. 2 Evolution of footpad swelling in BALB/c and C57BL/6 mice
infected with 106promastigotes/animal. Cross: p<0.05 indicates
statistical significance between BALB/c and C57BL/6 infected mice
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mononuclear cell, and moderated density of amastigote
forms (Figs. 3b and 5a). A highly inflammatory infiltrate
diffusely distributed in the dermis with light densities of
lymphocyte, plasma cells, and highly infected macrophages
was observed at 6 weeks pi. Moreover, a moderate presence
of polymorphonuclear cells and focal necrosis areas (Figs. 3c
and 5a) was also verified. At 8 weeks pi, an intense and
diffuse inflammatory infiltrate in the dermis, with light
densities of polymorphonuclear and plasma cells, moderate
densities of lymphocytes, and intense density of heavily
infected macrophages, was observed. The necrosis was
considered moderate (Figs. 3d and 5a). The number of
amastigote forms in the parasite inoculation site of BALB/c
mice increased significantly with the time of the infection
(p<0.05).
Concerning to C57BL/6 mice, they also showed inflam-
matory changes in the skin, but in lesser degree. A light
focal inflammatory infiltrate characterized by reduced
densities of polymorphonuclear and mononuclear cells
and light amounts of amastigote forms (Figs. 4a and 5a)
were observed at 2 weeks pi. At 4 weeks pi, the
inflammatory infiltrate was also light but spread through
the dermis; it was characterized by the presence of a
moderate density of polymorphonuclear and mononuclear
cells, light density of lymphocytes, and amastigote forms
(Figs. 4b, a). A moderate inflammatory infiltrate with a
diffuse distribution, moderate presence of polymorphonuclear
and mononuclear cells, light densities of lymphocyte, and
moderate densities of amastigote forms (Figs. 4c, a) were
observed in the dermis at 6 weeks pi. An intense and diffuse
inflammatory infiltrate represented by intensely parasitized
macrophages (Fig. 4a), moderate densities of lymphocytes,
and few polymorphonuclear cells, was observed in the
dermis at 8 weeks pi, additionally, a light area of necrosis
was also found (Fig. 4d).
Comparatively, BALB/c mice showed higher amastigote
densities than C57BL/6 mice (p<0.05). The densities of
amastigote forms at 4, 6, and 8 weeks pi in C57BL/6 mice
increased in relation to 2 weeks pi with statistical
significance (p<0.05), reinforcing the tendency of disease
progression in this mice.
The lymph nodes of both mice strains showed prominent
germinative centers in lymphoid follicles, which were
mostly pronounced in BALB/c mice. The amastigote forms
were detected at 6 and 8 weeks pi, and their localization
and densities were similar in both mouse strains (Fig. 5b).
The spleen and liver of both mouse strains showed no
histological changes, and parasites were not found.
Humoral immune response
BALB/c mice infected with L. (Viannia) shawi presented
detectable anti-Leishmania IgG from 2 weeks pi onward
compared to uninfected BALB/c mice (p<0.05), while in
Fig. 3 Histopathology of the skin lesion produced in BALB/c mice by Leishmania (Viannia) shawi. a Two weeks pi (×200), (b) 4 weeks pi
(×200), c 6 weeks pi (×200), and d 8 weeks pi (×200); details are immunohistochemistry reactions showing amastigote forms (×400)
1744Parasitol Res (2009) 105:1741–1747

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C57BL/6 mice, it was detected only at 6 and 8 weeks pi
compared to uninfected C57BL/6 mice (p<0.05). More-
over, the IgG levels were higher in BALB/c than C57BL/6
mice (p<0.05; Fig. 6).
Discussion
Studies on experimental models can help in the improvement
of new approaches in drugs and vaccines studies. In the case
of L. (Viannia) shawi parasites, information regarding their
infectious evolution in ex vivo and in vivo is missing.
Studies in vitro or ex vivo have been useful in the
understanding of some biological processes in the interaction
of Leishmania sp. with host cells. The results showed that
L. (Viannia) shawi infection induced higher infection index
in macrophages of BALB/c than C57BL/6 mice, indicating
that BALB/c mice presented high susceptibility to the
parasite infection. On other hand, C57BL/6 mice were not
capable of full elimination of intracellular amastigotes,
demonstrating that they also present intermediate suscepti-
bility to infection. In vitro studies demonstrated the essential
role of NO in elimination of intracellular amastigotes (Liew
et al. 1990a; Campos et al. 2008) and possibly, due to low
levels of NO molecule in both macrophages from BALB/c
and C57BL/6 mice, the infection could not be eliminated.
However, other mechanisms associated to parasite elimina-
tion, such as hydrogen peroxide (Das et al. 2001), superoxide
(Gantt et al. 2001), and hydroxyl radicals (Barr and
Gedamu 2003) may be associated to lesser infection index
observed in macrophages from C57BL/6 mice, but they
were not sufficient for eliminate amastigotes within
C57BL/6 macrophages.
Similarly, in vivo studies demonstrated that BALB/c
suffered higher disease progression than C57BL/6 mice. In
infected BALB/c mice, high lesion size was verified,
associated to intense inflammatory reaction in dermis and
high number of macrophages highly parasitized. C57BL/6
mice also presented increase in the footpad swelling with
the presence of inflammatory cells and amastigote forms
within macrophages, however, with low degree compared
to BALB/c mice. These characteristics indicate that BALB/c
and C57BL/6 mice were permissible to the infection caused
by L. (Viannia) shawi; however, some immunological
characteristic may be associated to lesser pathology and
parasitism in C57BL/6 mice. In Leishmania major, infection
is well defined that BALB/c presents a clear susceptible
profile, while C57BL/6 mice present a resistant profile
(Reiner and Locksley 1995). However, in New World
leishmaniasis, this pattern cannot be the same, since
Leishmania mexicana generated progressive disease and
metastatic lesion in BALB/c mice, while C57BL/6 mice
Fig. 4 Histopathology of the skin lesion produced in C57BL/6 mice by Leishmania (Viannia) shawi. a Two weeks pi (×200), b 4 weeks pi
(×200), c 6 weeks pi (×200), and d 8 weeks pi (×200); details are immunohistochemistry reactions showing amastigote forms
Parasitol Res (2009) 105:1741–1747 1745

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developed measurable lesions in low levels, but they fail to
heal for entire experimental time (Aguilar Torrentera et al.
2002). Studies conducted by Abreu-Silva et al. (2004) and
Maioli et al. (2004) also demonstrated similar profile in both
BALB/candC57BL/6infectedwithLeishmania amazonensis.
Moreover, in the dermis of both mice, the growing
densities of amastigote forms inside macrophages indicate
low capacity of effector cells, such as lymphocytes, to
activate macrophages for kill amastigote forms (Liew et al.
1990b). Moreover, the presence of neutrophil and plasma
cells observed in BALB/c mice skin may be associated to
increasing susceptibility to infection, once these cells could
be coupling to the progression of cutaneous lesions in
experimental leishmaniasis (Wanasen et al. 2008; Tacchini-
Cottier et al. 2000). Qualitative analysis showed light
densities of lymphocytes in the dermis of C57BL/6 mice,
at late stages of infection, indicating that lesser number of
amastigote forms may be a direct consequence of lympho-
cytes presence into the site of infection. Necrosis areas were
higher in BALB/c mice than in C57BL/6 mice and appear
at early phase of infection. It could have a pathogenic role
in leishmaniasis, since de Moura et al. (2005) found high
lesion size associated with necrosis into ear dermis in
susceptible mice infected with Leishmania braziliensis.
Into the draining lymph nodes close to skin infection
site, the immune response against Leishmania sp. is
developed. In L. shawi infection, the main histological
finding in popliteal lymph nodes was increasing in the
germinative center, which was higher in BALB/c than
C57BL/6 mice (data not shown). Moreover, IgG antibodies
were detected early in BALB/c mice; however, C57BL/6
mice also presented detectable IgG antibodies but in lesser
levels and in later stages. Classically, high levels of total
IgG antibodies have been associated to disease progression,
indicating that both mice strain were in the direction of the
disease establishment, but in BALB/c mice, high levels of
IgG indicated a fast disease progression compared to
C57BL/6 mice.
The characterization of biological behavior of new
Leishmania species in experimental models can help in
the understanding of basic events associated to pathological
process. Moreover, experimental models of infection can be
useful in the development of novel molecules with
pharmacological interest as well as promising vaccine
candidates. Studies with L. (Viannia) shawi demonstrated
its potential to infect both BALB/c and C57BL/6 mice,
where BALB/c mice presented classical signs of suscepti-
bility, therefore, this mice strain could be an interesting
target for further studies on host-parasite interplay for
Fig. 6 Humoral response in BALB/c and C57BL/6 mice infected
with Leishmania (Viannia) shawi. (a) anti-Leishmania IgG. single and
double asterisks p<0.05 indicates statistical significance comparing
IgG levels of infected BALB/c and C57BL/6, respectively, with mice
at 2 weeks pi; cross: p<0.05 indicates statistical significance
comparing IgG levels of infected BALB/c mice with C57BL/6 mice
Fig. 5 Parasite density in skin (a) and lymph nodes (b) of BALB/c
and C57BL/6 mice evaluated by image analysis system (AxioVison
5.0) in paraffin section stained by immunohistochemistry reaction.
Each value represents the mean±standard deviation of amastigote/
mm2of tissue of at least ten microscopy field. single and double
asterisks p<0.05 indicates statistical significance comparing amasti-
gote densities of BALB/c and C57BL/6 mice with those from of
2 weeks pi, respectively; cross: p<0.05 indicates statistical signifi-
cance comparing amastigote densities of C57BL/6 with amastigote
densities of BALB/c mice
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understanding of immunopathogenesis of American
Tegumentar Leishmaniasis, caused by this parasite species.
Acknowledgements
Amparo à Pesquisa do Estado de São Paulo (FAPESP) and
HCFMUSP-LIM50. It is part of the doctoral thesis of Luiz Felipe D.
Passero under a FAPESP fellowship. The authors of this paper would
like to thank Profa. Dra. Gabriela Santos-Gomes from Unidade de
Leishmanioses, CMDT-LA, Instituto de Higiene e Medicina Tropical,
Universidade Nova de Lisboa, Portugal, for the critical reading of the
manuscript.
This work was supported by Fundação de
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