Parasitol Res (2012) 110:1277–1285 DOI 10.1007/s00436-011-2628-5
ORIGINAL PAPER
Evaluation of parasitological and immunological parameters of Leishmania chagasi infection in BALB/c mice using different doses and routes of inoculation of parasites Dulcilene M. Oliveira & Mariana Amália F. Costa & Miguel A. Chavez-Fumagalli & Diogo G. Valadares & Mariana C. Duarte & Lourena E. Costa & Vivian T. Martins & Rosângela F. Gomes & Maria N. Melo & Manuel Soto & Carlos Alberto P. Tavares & Eduardo Antonio F. Coelho
Received: 3 September 2010 / Accepted: 29 August 2011 / Published online: 14 September 2011 # Springer-Verlag 2011
Abstract Experimental vaccines to protect against visceral leishmaniasis (VL) have been developed by using BALB/c mice infected with a large (107 to 108) inoculum of parasites. Remarkably, prior literature has reported that the poor protection observed is mainly due to the high susceptibility of this strain. To determine factors inherent to mice that might abrogate vaccine-induced efficacy, the present research sought to investigate the impact of the administration of different infective inoculums of Leishmania chagasi (syn. L. infantum) in BALB/c mice, evaluating subcutaneous and intravenous routes of administration as well as parasitological and immunological parameters over different periods of time. This study shows that the injection of a highly infective inoculum in mice, through both subcutaneous and intrave-
nous routes, results in a sustained infection. The mice developed a high parasite load in the liver; however, these values diminished over time. This result did not corroborate with the parasite load in the bone marrow and brain and proved to be expressively different in the spleen and draining lymph nodes, where the values increased over time. Mice infected with a low dose of parasites (103) showed a certain resistance against infection, based mainly on the IFN-γ and oxide nitric production. Considering all the elements, it could be concluded that the models employing high doses (107) of L. chagasi in BALB/c mice can bring about an imbalance in the animals’ immune response, thus allowing for the development of the disease at the expense of efficacy within the vaccine candidates.
D. M. Oliveira Programa de Pós-Graduação em Neurociências, Universidade Federal de Minas Gerais, 31.270-901( Belo Horizonte, Minas Gerais, Brazil
R. F. Gomes : M. N. Melo Departamento de Parasitologia, Universidade Federal de Minas Gerais, 31.270-901( Belo Horizonte, Minas Gerais, Brazil
M. A. F. Costa : D. G. Valadares : V. T. Martins : C. A. P. Tavares Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, 31.270-901( Belo Horizonte, Minas Gerais, Brazil M. A. Chavez-Fumagalli Programa de Pós-Graduação em Medicina Molecular, Universidade Federal de Minas Gerais, 31.270-901( Belo Horizonte, Minas Gerais, Brazil M. C. Duarte : L. E. Costa : E. A. F. Coelho Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, 31.270-901( Belo Horizonte, Minas Gerais, Brazil
M. Soto Centro de Biología Molecular Severo Ochoa, CSIC, UAM, Departamento de Biología Molecular, Universidad Autónoma de Madrid, 28049 Madrid, Spain E. A. F. Coelho (*) Laboratório de Biotecnologia Aplicada ao Estudo das Leishmanioses, Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Campus Pampulha, Avenida Antônio Carlos, 6627, 31.270-901( Belo Horizonte, Minas Gerais, Brazil e-mail:
[email protected]
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Introduction Visceral leishmaniasis (VL), a severe disease that is responsible for a high degree of morbidity and mortality in mammal hosts, is characterized by the infiltration of amastigote forms in different organs such as the liver, spleen, lymph node (LN), bone marrow (BM), and brain of the hosts. Several experimental animal models of VL have been developed over the years (Abreu-Silva et al. 2003; Carrión et al. 2006; Garcia-Alonso et al. 1996; Keenan et al. 1984; Muigai et al. 1983; Nieto et al. 1996; Prasad and Sen 1996; Ramos et al. 1994; Viñuelas et al. 2001). Of these, the hamster model most commonly mimics the human disease, given that these animals follow a progressively fatal path following visceral infection (Melby et al. 2001a). By contrast, Leishmania donovani infection in mice has induced variable outcomes, depending on the genetic background of the mouse strain; the route of inoculation; as well as the parasite stage (promastigote or amastigote), dose, and infectivity of the challenge organism (Mukherjee et al. 2003). The development of a vaccine against leishmaniasis is a long-term goal in both human and veterinary medicine. This situation unquestionably demands the use of a proper screening model for vaccine candidates. To substantiate the results of primary screening, therapeutic trials are performed under controlled conditions on laboratory animals. If the efficacy is established and a dose relationship does in fact exist, the results of the secondary screening would aid in predicting the necessary dose to protect humans against the parasite (Garg and Dube 2006). Murine models have been used widely for the development of vaccines against VL (Afrin et al. 2000; Carrión et al. 2006; Zanin et al. 2007). Studies employing the mouse model have led to the characterization of the immune mechanisms needed to develop organ-specific immune responses, which would clear the parasites from the liver but not from the spleen (Kaye et al. 1995; Carrión et al. 2006). BALB/c mice infected with L. donovani or L. chagasi is the most widely studied model of VL, but it is also considered to be rather vulnerable, given that the infection progresses during the first two weeks. However, after this time, it can be controlled by the host immune response, depending on the infective inoculum inoculated into the animals (Murray et al. 1987). An intradermal murine model of VL has been explored in an attempt to establish a chronic infection pattern in susceptible BALB/c mice, which resembles a course of disease similar to that of human VL (Ahmed et al. 2003). Resistance and susceptibility are closely related to the development of T-cell responses of Th1 or Th2, respectively. In the murine VL, C57BL/6 mice mount an early Th1 immune response,
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which prevents the further growth of the parasite, and in turn produce a self-healing phenotype (Lehmann et al. 2000), whereas susceptible BALB/c mice mount early Th2 response, which results in a non-healing lesion and an exaggeration of the disease (Himmelrich et al. 2000). In addition, the induction of parasite specific immune suppressive IL-10-mediated responses has proven to be linked to disease progression in the experimentally infected mice (Stager et al. 2010). Finally, respective resistance and susceptibility of C57BL/6 and BALB/c strains depend not only on the Th1 and Th2 types of immune responses from CD4+ T cells but also on the genetic background of the host (Afonso and Scott 1993; Barral et al. 1991; Coelho et al. 2003). In this manner, the immune responses, following the infection of inbred mouse strains with viscerotropic Leishmania species such as L. donovani and L. infantum, can be considered to be similar to those observed in the L. major mouse model. Recent reports of a low dose infection by Leishmania, which generates a Th1 response and immunity and in turn furthering infection, have revived the interest in this field (Bretscher et al. 1992; Kaur et al. 2008). In studies with L. chagasi, most vaccine candidates evaluated in the experimental protocols are commonly challenged with high concentrations (about 107 to 108) of parasites, which may well be responsible for or bring about an imbalance in the immune response of the host, thus allowing for the development of the disease at the expense of efficacy within the vaccine candidates due to the unfavorable conditions of the experimental infection (Sacks and Melby 2001; Ahmed et al. 2003; Malafaia et al. 2009; Peters and Sacks 2006; Yazdanbakhsh and Sacks 2010). Since the dose and route of administration of parasites can influence the development of the T helper response, the present study was carried out to investigate the impact of the inoculation of different parasite dose combinations (low, medium and high) and routes of inoculation (subcutaneous and intravenous) to evaluate the development of the disease in BALB/c mice after infection with L. chagasi. The parasite burden in the liver, spleen, BM, brain, draining popliteal LN, cytokine (IFN-γ, IL-4, and IL-10) levels, oxide nitric production, as well as humoral response were investigated at 15, 30, 45, and 60 days after infection in the infected mice.
Material and methods Mice Female BALB/c mice (6–8 weeks of age) were obtained from the breeding facilities of the Department of Biochemistry and Immunology, Institute of Biological Sciences,
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Federal University of Minas Gerais (UFMG) and were maintained under specific pathogen-free conditions. The Committee on the Ethical Handling of Research Animals from UFMG approved all the animal handling methods and procedures. Parasites All experiments were carried out using the L. chagasi (MOM/BR/1970/BH46) strain. Parasites were grown at 24°C in Schneider’s medium (Sigma, St. Louis, MO, USA) supplemented with 20% heat-inactivated fetal bovine serum (FBS, Sigma), 20 mM L-glutamine, 200 U/mL penicillin, 100 μg/mL streptomycin, and 50 μg/mL gentamicin at pH 7.2. Soluble L. chagasi antigenic (SLA) extract was prepared from stationary cultures, as previously described (Coelho et al. 2003).
Experimental infection Mice (n=12 per group) were infected subcutaneously with 103, 105, or 107 stationary promastigotes of L. chagasi injected into their right hind footpad. An additional group (n=12) was challenged with 107 stationary promastigotes by intravenous route in the tail vein. As a control, mice (n= 12) received only sterile phosphate buffer saline (PBS) at pH 7.4. At 15, 30, 45, and 60 days after infection, mice (n= 3 per group) were sacrificed and the liver, spleen, BM, brain, draining LN, and serum were collected for parasitological and immunological assays. Estimation of parasite load The liver, spleen, BM, brain, and draining LN were collected for parasite quantification, following a technical protocol from Vieira et al. (1996) modified by Coelho et al. (2003). Briefly, total organs were collected, weighed, and homogenized using a glass tissue grinder in sterile PBS (1×). Tissue debris was removed by centrifugation at 150×g, and cells were concentrated by centrifugation at 2,000×g. Pellets were resuspended in 1 mL of Schneider’s insect medium supplemented with 20% FBS. Two hundred and twenty microliters were plated onto 96-well flat-bottom microtiter plates (Nunc, Nunclon®) and diluted in log-fold serial dilutions in supplemented Schneider’s culture medium with a 10-1 to 10-24 dilution. Each sample was plated in triplicate and read 10 days after the beginning of the culture at 24°C. Pipette tips were discarded after each dilution to avoid carrying adhered parasites from one well to another. Results are expressed as the negative log of the titer (i.e., the dilution corresponding to the last positive well) adjusted per microgram of tissue.
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Cytokine analysis To measure IFN-γ, IL-4, and IL-10 in culture supernatants, spleen cells were prepared and incubated, in duplicate, for 48 h at 37°C in 5% CO2, in 24-well round-bottomed plates (Nunc, Nunclon®) using 5×106 cells/well, with or without SLA extract (50 μg/well), in a total volume of 1 mL. Following incubation, the concentrations of IFN-γ, IL-4, and IL-10 were determined in the culture supernatants by enzyme-linked immunosorbent assay (ELISA), according manufacturer instructions (BD Biosciences). Nitrite production Oxide nitric production by spleen cells was determined as previously described (Carrión et al. 2006). Briefly, 100 μL of supernatant cultures were mixed with an equal volume of Griess reagent (Sigma). After an incubation of 30 min at room temperature, absorbances were measured in an ELISA reader (LAB-660 model, LGC Biotechnology) at 540 nm, and the nitrite concentrations were calculated from a standard curve constructed using the NaNO2 standard. Data are expressed as μM per 5×106 cells. Humoral response Parasite-specific IgG, IgG1, and IgG2a antibodies were measured by ELISA, as described by Coelho et al. (2003). Briefly, 96-well plates (Falcon) were sensitized with SLA (1 μg/well) for 18 h at 4°C. Next, the plates were washed five times with PBS 1×/Tween 20 0.05%, and the wells were blocked with a solution of PBS 1×/bovine serum albumin 10%/Tween 20 0.05% for 2 h at 37°C. The plates were then washed five more times under the same conditions, and serum samples (1:100 diluted) were added, in duplicate, for 1 h at 37°C. After that, the plates were washed 7 times, and specific peroxidase-labeled antibodies for mouse IgG, IgG1, and IgG2a isotypes (Sigma) were added separately (1:5,000 diluted). In addition, incubation was performed for 1 h at 37°C, when H2O2 and ophenylenediamine were added for the development of reactions. Optical densities were read in an ELISA reader at 492 nm. Statistical analysis Comparisons among the groups were carried out by twoway ANOVA and Bonferroni’s post-test. Differences were considered significant when P
