INTRODUCTION
Leishmania infantum (L. infantum) is an obligatory intracellular protozoan and causative of visceral leishmaniasis in humans and dogs. Control of parasites in dogs leads to a reduced prevalence of disease in humans (Palatnik-de-Sousa et al. Reference Palatnik-de-Sousa, dos Santos, Franca-Silva, da Costa, Reis, Palatnik, Mayrink and Genaro2001). No effective and safe vaccine against canine visceral leishmaniasis (CVL) is currently available and control of the disease by chemotherapy is compromised because existing drugs are toxic and drug resistance is prevalent. We previously described the generation of an attenuated line of L. infantum (L. infantum H-line) which was selected by culturing promastigotes in vitro under pressure of gentamicin (Daneshvar et al. Reference Daneshvar, Coombs, Hagan and Phillips2003). Gentamicin, an aminoglycoside antibiotic, is produced by micromonospora (Weinstein et al. Reference Weinstein, Luedemann, Oden, Wagman, Rosselet and Marquez1963a, Reference Weinstein, Luedemann, Odem and Wagmanb). It is widely used for the treatment of bacterial infections and shows a broad antibacterial spectrum of action (Edson and Terrell, Reference Edson and Terrell1999). The precise mechanism of bactericidal activity of aminoglycosides is not fully understood, but hypotheses include disruption of ribosomal activity by breaking up polysomes, misreading of mRNA during protein synthesis and production of abnormal or non-functional proteins (Chambers, Reference Chambers, Hardman and Limbird2001). A quantitative proteomic analysis revealed the expression of a group of proteins which mediate the response to reactive oxygen intermediates in L. infantum H-line which had changed compared with wild-type parasite (Daneshvar et al. Reference Daneshvar, Wyllie, Phillips, Hagan and Burchmore2012). We showed that the attenuated L. infantum is more susceptible to oxidative challenge, a change which may explain the loss of virulence (Daneshvar et al. Reference Daneshvar, Wyllie, Phillips, Hagan and Burchmore2012). Thus L. infantum H-line invaded but was unable to survive within bone marrow-derived macrophages of BALB⁄c mice in vitro, whereas L. infantum wild-type (WT) survived and multiplied within infected macrophages (Daneshvar et al. Reference Daneshvar, Coombs, Hagan and Phillips2003). Moreover, the attenuated line of L. infantum failed to spread to, and within, the visceral organs of BALB⁄c mice (Daneshvar et al. Reference Daneshvar, Molaei, Afshar, Kamiabi, Burchmore, Hagan and Phillips2009). Previously we reported that L. infantum H-line induced a cellular immunity which protected dogs against wild type parasites (Daneshvar et al. Reference Daneshvar, Molaei, Kamiabi, Burchmore, Hagan and Phillips2010). In addition, no clinical signs and histopathological abnormalities were found in the dogs immunized with the L. infantum H-line (Daneshvar et al. Reference Daneshvar, Molaei, Afshar, Kamiabi, Burchmore, Hagan and Phillips2009). Several studies have shown that the absence of an adequate T cell response leads to insufficient control of the parasite and consequent appearance of clinical signs of disease with high parasite burdens in visceral organs of symptomatic dogs. Conversely, asymptomatic dogs are characterized by a self-limiting disease in the presence of protective cellular immunity (Guarga et al. Reference Guarga, Moreno, Lucientes, Gracia, Peribanez, Alvar and Castillo2000; Reis et al. Reference Reis, Martins-Filho, Teixeira-Carvalho, Giunchetti, Carneiro, Mayrink, Tafuri and Correa-Oliveira2009, Reference Reis, Giunchetti, Carrillo, Martins-Filho and Moreno2010). Moreover, low levels of CD4+ and CD8+ T cells, CR2 (CD21+) B cells and CD14+ monocytes in the peripheral blood mononuclear cells (PBMCs), is associated with higher parasite density in the bone marrow as seen in symptomatic dogs (Reis et al. 2006a). CD4+ T cells from symptomatic dogs respond to antigen stimulation during the earlier stages of infection but lose this ability as the dogs progress to clinically disseminated disease (Boggiatto et al. Reference Boggiatto, Ramer-Tait, Metz, Kramer, Gibson-Corley, Mullin, Hostetter, Gallup, Jones and Petersen2010). In contrast, a high level of CD8+ T cells reported in the PBMCs of asymptomatic dogs was associated with control of parasitism (Reis et al. Reference Reis, Teixeira-Carvalho, Giunchetti, Guerra, Carvalho, Mayrink, Genaro, Correa-Oliveira and Martins-Filho2006a).
In the present study, the impact of L. infantum H-line on the immunophenotypic profile of popliteal lymph node (PLN) and PBMCs of dogs was assessed. The proportions of CD4+, CD8+, CD4+ CD25+, CD44+ T cells and CD21+, CD79b+ B cells and MHC-II+ (HLA-DR), CD14+ cells of dogs immunized with the attenuated line were analysed. Moreover, the influence of tissue burden parasitism in the PLN of dogs was investigated.
MATERIALS AND METHODS
Parasite
Promastigotes of L. infantum JPCM5 (MCAN/ES/98/LIM-877), were cultivated in complete haemoflagellate minimal essential medium (HOMEM) (GIBCO) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (HI-FCS) (Labtech International), as described elsewhere (Mallinson and Coombs, Reference Mallinson and Coombs1989). L. infantum H-line was generated in the same medium supplemented with 10% (v/v) HI-FCS and gentamicin (Sigma-Aldrich) at 20 μg/ml (Daneshvar et al. Reference Daneshvar, Coombs, Hagan and Phillips2003). Briefly, amastigotes of L. infantum derived from a spleen of an infected hamster were transferred into complete HOMEM medium and incubated at 25 °C, where they differentiated into promastigotes over 72 h. These were transferred into complete HOMEM with or without gentamicin and were subcultured routinely at 25 °C. Cultures grown in the absence of gentamicin were maintained in parallel to those with the antibiotic, to confirm that attenuation was not simply the result of long-term cultivation. Stationary-phase promastigotes of the attenuated line or L. infantum WT were harvested after a total of 48 sub-passages by centrifugation and washed twice in phosphate-buffered saline (PBS, pH 7·4); the pellet was suspended at 109 cells/ml in PBS.
Dogs
Thirty healthy semi-German shepherd dogs, between 6 months and 1 year old were used in this study. All dogs were checked by a veterinary surgeon for clinical sign of leishmaniasis and were shown to be negative for the presence of anti-leishmanial antibody by immunoflourescence assay (IFA) test, and also negative for Leishmania DNA by PCR of PBMCs. The protocols used for animal handling were approved by the animal care committee of Kerman University of Medical Sciences. The welfare of the dogs was closely monitored by a veterinary surgeon. The dogs had previously been vaccinated against canine parvovirus and rabies and were also treated with the anthelmintic drugs praziquantel and pyrantel. The animals were accommodated in indoor kennels with windows covered with deltamethrin-sprayed, double anti-mosquito nets. The dogs were allocated into 3 Groups in a double-blind randomized fashion. Two Groups (10 dogs per Group) were injected intradermally (i.d.) with 100 μl of the suspension of stationary-phase promastigotes of L. infantum H-line (Group H) or L. infantum WT (Group WT). Control Group (Group C) (10 dogs) was injected with PBS. The experiment was terminated after 12 months infection to ensure that the control dogs did not suffer unnecessarily. The animals were anaesthetized by intravenous injection of thiopental sodium 33% (5 ml/kg) (Tafuri et al. Reference Tafuri, Rosa de Oliveira, Melo and Tafuri2001) at the end of the study. The PLN was collected in aseptic conditions for flow cytometry and immunohistochemical examinations.
Leishmania soluble antigen (LSA)
LSA was prepared from stationary-phase promastigotes of L. infantum WT. Cells were harvested by centrifugation and washed 3 times with ice-cold (PBS). The pellet was re-suspended in PBS (107 cells/ml) and sonicated at 4 °C for 1 min at 12-mm peak amplitude. The suspension was finally centrifuged at 10000 g and 4 °C for 15 min. The protein concentration in the supernatant was determined by the method described by Bradford (Reference Bradford1976) and frozen as 1 mg/ml aliquots at 20 °C until use.
Cell preparation
Heparinized blood samples taken from the peripheral veins of dogs were diluted with an equal volume of PBS and layered on Ficoll-Hypaque (Sigma). After centrifugation at 350 g for 40 min at room temperature, the layer containing PBMC fraction was collected. Fragments of PLNs were excised and then teased to obtain single-cell suspensions. PBMCs or PLN cells were washed twice with PBS and adjusted to 5 × 106 cells/ml in complete RPMI-1640 supplemented with 10% FCS, penicillin G (100 U/ml), streptomycin sulphate (100 μg/ml) (Life Technologies, USA) with LSA (50 μg/ml). The cells were incubated at 37 °C in 5% CO2 95% air for 24 h and then harvested.
Flow cytometric analysis
Cell surface and cytoplasmic antigens were identified by flow cytometric analysis based on the method described by Murphy et al. (Reference Murphy, Shibuya, Hosken, Openshaw, Maino, Davis, Murphy and O'Garra1996). The cells were washed twicw with PBSW (PBS with 0·5% bovine serum albumin (BSA) and 0·1% sodium azide), counted, and then re-suspended at 1 × 106 per microcentrifuge tube. The cells were stained with the monoclonal antibodies (mAbs) listed in Table 1 for 40 min at 4 °C. After washing with 2 ml of PBSW, the cells were incubated with fluorescein isothiocyanate (FITC) or phycoerythin (PE) conjugated to secondary antibodies (Table 1). After 2 washes with PBSW, the cells were fixed in 2·8% formaldehyde-PBS. The cells were analysed by flow cytometery with FluMax software. Negative controls included isotype mAbs substituted for mAbs listed in Table 1 and secondary antibodies used alone without primary antibodies.
Table 1. Monoclonal and polyclonal antibodies used in flow cytometry
Immunohistochemical finding
An immunohistochemical investigation was performed to evaluate the number of CD14+ cells and parasite burden in the PLN of dogs. Briefly, 5 μm sections were cut, deparaffinized slides were hydrated and incubated in 4% hydrogen peroxide (H2O2) (30 v/v) in 0·01 M PBS, pH 7·2. The tissue sections were incubated with goat serum (diluted 1:100) for 30 min and then incubated overnight at 4 °C with the primary monoclonal antibody anti-canine CD14 (VMRD, USA) (15 μg/ml in PBS). After washing, the secondary antibody (diluted with PBS at 1:100) was applied, and incubated for 60 min at room temperature. The slides were visualized by incubation with 3,3′-diaminobenzidine-tetrahydrochloride (DAB) (Sigma, St Louis, MO, USA) and H2O2 in 0·1 M imidazole, pH 7·1 for 5 min. Finally, the slides were dehydrated, cleared, counterstained with haematoxylin and eosin (HE), and mounted under coverslips. CD14+ cells with a positive brown immunocytochemical reaction were counted in 15 high-power fields (ocular grit = 0·0625 mm2) on cross-sections by light microscopy (Ensinger et al. Reference Ensinger, Boekhoff, Carison, Beineke, Rohn, Tipoid and Stein2010). The parasite burden in the PLN of dogs was evaluated as described by Tafuri et al. (Reference Tafuri, Santos, Arantes, Gonc-alves, Melo, Michalik and Tafuri2004) as follows. The slides were incubated with goat serum (diluted 1:100). Serum from a dog infected with L. infantum WT (IFAT titre 1:320), diluted 1 in 100 in PBS, was applied as the primary antibody and incubated for 22 h at 4 °C. The slides were then incubated with biotinylated anti-mouse and anti-rabbit antibody (Dako, LSAB2 kit, USA) and subsequently with the streptavidin peroxidase complex (Dako, LSAB2 Kit, USA) for 20 min at room temperature. The slides were visualized as described above and the number of macrophages containing amastigotes with a visible nucleus and a brown immunocytochemical reaction were counted as positive. A semi-quantitative study was carried out by optical microscopy (110×). Parasite load was measured as: (+) discrete, (++) moderate and (+ + +) intense (Xavier et al. Reference Xavier, de Andrade, Monte, Chiarelli, Lima, Michalick, Tafuri and Tafuri2006).
Statistics analysis
Statistical analysis was performed using SPSS software. Parametric ANOVA and Student's t-test test were used to assess significant differences between flow cytometry and IHC data, respectively. A P-value of 0·05 or less was considered as a significant difference.
RESULTS
Characterization of immune cells
Flow cytometric analysis was carried out to investigate the proportions of CD4+, CD8+, CD4+ CD25+, CD44+ T cells and CD21+, CD79b+ B cells and MHC-II expression in the samples from the dogs. The percentages of CD4+ T cells in the SLA-stimulated PLN cells and PBMCs in dogs of Group H were significantly higher than those in dogs from other groups (Figs 1a and 2a respectively; P = 0). There was no significant difference between the percentages of CD8+ T cells in PLN of dogs of Group H with that of dogs of Group WT or Group C dogs (Fig. 1b). But a significant difference was found between the higher percentage of CD8+ T cells in PBMCs of immunized dogs compared with that in dogs of Group WT and healthy dogs (Fig. 2b; P = 0 and P < 0·003 respectively). The CD4/CD8 ratio in the PLN of Group H dogs was significantly higher than that in the dogs of Group WT (P < 0·005). The percentage of CD44+ cells in PLN of dogs immunized with the attenuated parasite was significantly higher than that in dogs of Group WT and Group C (Fig. 1c; P < 0·01). The percentage of CD4+ CD25+ T cells in PLN of Group H dogs decreased notably compared with that in dogs of Group WT (Fig. 1d; P = 0). The percentages of CD21+ B cells in PLN and PBMCs were significantly higher in the immunized dogs after 12 months post-immunization and re-stimulated with LSA in vitro compared to the dogs infected with WT parasites (Figs 1e and 2c respectively; P = 0). In contrast, the percentage of CD79b+ B cells in PBMCs of dogs of Group H was significantly lower than that in dogs of Group WT and healthy dogs (result not shown; P=0 and P<0·005 respectively). There was no significant difference between the percentage of CD79b+ B cells in the PLN of immunized dogs compared with that of control and dogs infected with WT parasites (Fig. 1f). L. infantum WT caused down-regulation of expression of MHC-II in antigen presenting cells (APCs) in PLN and PBMCs of dogs (Figs 1g and 2d; P < 0·001 and P<0·01 respectively) compared with healthy control dogs. There was no significant change in MHC-II expression in PLN cells and PBMCs between immunized and control healthy dogs. MHC expression on the APCs in PLN of dogs immunized with the attenuated parasite was significantly higher than that in dogs infected with WT parasites (Fig. 1g; P = 0).
Fig. 1. Percentage of immunophenotype cells in the PLN of dogs infected with Leishmania infantum WT or dogs immunized with L. infantum H-line or healthy control dogs. The cells were re-stimulated with LSA in vitro. Asterisk represents significant difference.
Fig. 2. Percentage of immunophenotype of PBMCs of dogs infected with Leishmania infantum WT or dogs immunized with L. infantum H-line or healthy control group. The cells were re-stimulated with LSA in vitro. Asterisk represents significant difference.
Immunohistochemical finding
In the cell culturing process, the CD14+ macrophages within the PLN spread and stuck on the surface of the tissue-culture plate. The number of cells isolated from the plates was low for flow cytometric analysis. Thus, the immunohistochemical method was performed to measure the subpopulation of CD14+ cells and parasite burden in the dogs immunized with L. infantum H-line compared with dogs of Group WT. The mean number of CD14+ cells per field was 4·3 ± 3·2 in dogs of Group WT and 11·2 ± 4·2 in dogs of Group H, indicating that there was significantly lower in dogs of Group WT (Fig. 3; P < 0·01). Moreover, no amastigotes were found in the PLN of immunized dogs 12 months post-immunization (Fig. 4a). In contrast, amastigotes were found in the PLN tissue section of six dogs (60%) of Group WT (Fig. 3b). Parasites were found in 3 dogs as (+), 2 dogs as (++) and 1dog as (+ + +).
Fig. 3. Immunohistochemical labelling of CD14, a marker for macrophages in the PLN of a dog immunized with Leishmania infantum H-line. Avidin–biotin–peroxidase complex method.
Fig. 4. Immunohistochemical labelling of numerous amastigotes within macrophages in the PLN of a dog immunized with Leishmania infantum H-line (a) or a dog infected with L. infantum WT (b). Avidin–biotin–peroxidase complex method.
DISCUSSION
In the present study we characterized the immunophenotypic profile of mononuclear cells, including lymphocyte proportions, in PBMCs and PLN of dogs immunized with the attenuated parasites compared with dogs infected with wild-type parasites or uninfected dogs. The parasite burden was also investigated in PLN tissue sections. We previously described the generation of L. infantum H-line, which was attenuated by culturing of promastigotes in the pressure of gentamicin. In prokaryotic cells, aminoglycosides bind directly to the ribosomal A site and cause misreading of mRNA during protein synthesis that leads to production of abnormal or non-functional proteins (Woodcock et al. Reference Woodcock, Moazed, Cannon, Davies and Noller1991; Yoshizawa et al. Reference Yoshizawa, Fourmy and Puglisi1998; Walter et al. Reference Walter, Vicens and Westhof1999). Aminoglycosides exhibit anti-leishmanial activity, although the mechanism of action is not understood (Tekos et al. Reference Tekos, Tsagla, Stathopoulos and Drainas2000). We reported that thiol-redoxin is significantly lower in L. infantum H-line as compared to L. infantum WT. Thiol-redox is critical for Leishmania parasites which are exposed to an oxidative burst when they encounter their mammalian macrophage host cell (Wilson et al. Reference Wilson, Andersen and Britigan1994; Goyal et al. Reference Goyal, Roy and Rastogi1996; Dumas et al. Reference Dumas, Ouellette, Tovar, Cunningham, Fairlamb, Tamar, Olivier and Papadopoulou1997). We also reported that L. infantum H-line invaded but was unable to survive within bone marrow-derived macrophages of BALB/c mice in vitro, whereas L. infantum WT survived and multiplied within these macrophages (Daneshvar et al. Reference Daneshvar, Coombs, Hagan and Phillips2003). In addition, no parasites were found in the visceral organs of dogs immunized with L. infantum H-line (Daneshvar et al. Reference Daneshvar, Molaei, Afshar, Kamiabi, Burchmore, Hagan and Phillips2009). In order to assess the immunophenotypic profile of PLN cells and PBMCs of the difference dogs Leishnania specific, the cells were exposed to LSA to induce a secondary immune response (Belkaid, Reference Belkaid2003; Araújo et al. Reference Araújo, de Andrade, Sathler-Avelar, Teixeira-Carvalho, Andrade, Viannac, Reis, Malaquiase, Mello and Martins-Filho2009). In the present study, we found that numbers of CD4+ T cells in dogs immunized with the L. infantum H-line were significantly higher than those in dogs infected with wild-type parasites. It has been reported that CD4+ T cells play a key role in controlling parasite growth and susceptibility to infection is associated with loss of L. infantum-specific CD4+ T cell function (Pinelli et al. Reference Pinelli, Killick-Kendrick, Wagenaar, Bernadina, Del Real and Ruitenberg1994; Strauss-Ayali et al. Reference Strauss-Ayali, Baneth, Shor, Okano and Jaffe2005; Boggiatto et al. Reference Boggiatto, Ramer-Tait, Metz, Kramer, Gibson-Corley, Mullin, Hostetter, Gallup, Jones and Petersen2010). Several studies have demonstrated that CD8+ T cells play an important role in the development of a protective immunity and are required for the effective clearance of Leishmania (Stern et al. Reference Stern, Oca, Rubin, Anderson and Murray1988; Pinelli, Reference Pinelli, Schijns and Horzinek1997). In agreement with these studies, our results showed that the population of CD8+ T cells in the dogs immunized with the attenuated parasites was higher than that in the dogs of Group WT. We previously reported that L. infantum H-line inhibited IL-10 production and induced a cellular immunity that protected dogs against wild-type parasites (Daneshvar et al. Reference Daneshvar, Molaei, Kamiabi, Burchmore, Hagan and Phillips2010). In the present study we found that the percentage of CD4+ CD25+ T cells significantly decreased in the PLN of dogs immunized with L. infantum H-line compared with that in dogs infected with wild-type parasites. These results are consistent with other studies that showed that CD4+ CD25+ T cells are capable of producing IL-10 and play an important role during the early phase of visceral leishmaniasis in dogs (Belkaid, Reference Belkaid2003; Rodrigues et al. Reference Rodrigues, Marques, Soares-Clementea, Ferronha and Santos-Gomes2009). It has been also reported that the reduction in CD4+ CD25+ T cells in lymph nodes induced a protection against leishmaniasis (Felix De Lima et al. Reference Felix De Lima, Ikeda, Rossi, Feitosa, Vasconcelos, Nunes and Goto2010). Non-Treg CD4+ T cells are often divided into 2 major subpopulations that can be designated naïve phenotype (CD4+ CD25− CD44−) and memory-phenotye (CD4+ CD25− CD44+) T cells (Surh and Sprent, Reference Surh and Sprent2008; Day, Reference Day and Day2008). Memory CD44+ T cells are important in the development of a protective immune response to Leishmania sp (Sanchez et al. Reference Sanchez, Diaz, Zerpa, Negron, Convit and Tapia2004; Kedzierski et al. Reference Kedzierski, Curtis and Kedzierska2009). This observation is in agreement with our result that the percentages of CD44+ T cells in the PLN and PBMCs of the dogs immunized with L. infantum H-line were significantly higher than those in dogs infected with wild-type parasites.
The present study shows the percentage of CD21+ B lymphocytes in vaccinated dogs was significantly higher than that in dogs infected with L. infantum WT. In canine VL, previous studies revealed a negative correlation between the LN CD21+ B cell frequency and parasite burden in symptomatic dogs (Reis et al. Reference Reis, Teixeira-Carvalho, Giunchetti, Guerra, Carvalho, Mayrink, Genaro, Correa-Oliveira and Martins-Filho2006a, Reference Reis, Martins-Filho, Teixeira-Carvalho, Carvalho, Mayrink, Franca-Silva, Giunchetti, Genaro and Correa-Oliveirab). Cabral et al. (Reference Cabral, Silvestre, Santarem, Tavares, Silva and Cordeiro-da-Silva2008) reported that L. infantum tryparedoxin is a potent modulator of B cell activity and induces IL-10 in VL. A quantitative proteomic analysis revealed that the most prominent changes were in expression of thiol-redox active enzyme tryparedoxin peroxidase in L. infantum H-line compared with wild-type parasite (Daneshvar et al. Reference Daneshvar, Wyllie, Phillips, Hagan and Burchmore2012). Thus, the high percentage of CD21+ B cells observed in the immunized dogs could be related to a reduction in trypanothione reductase activity in the attenuated line of L. infantum. In contrast, the percentage of CD79b+ cells in the dogs immunized with the attenuated parasites was significantly lower than that in dogs of Group WT. Our results are in agreement with this observation that mature B cells with CD21, IgM and cytoplasmic CD79b receptors, differentiate toward plasma cells and lead to the loss CD21 and IgM receptors (Chu and Arber, Reference Chu and Arber2001; Wilkerson et al. Reference Wilkerson, Dolce, Koopman, Shuman, Chun, Garrett, Barber and Avery2005).
One of the immune evasion strategies associated with the Leishmania parasite could be a down-regulation of MHC class II on the APCs (Reiner et al. Reference Reiner, Ng, Ma and McMaster1988; Papadogiannakis et al. Reference Papadogiannakis, Koutinas, Saridomichelakis, Vlemmas, Lekkas, Karameris and Fytianou2005). In the present study, we found expression of MHC-II+ in APCs of dogs infected with wild-type parasites significantly down-regulated compared with healthy control dogs (P < 0·001). Whereas, MHC-II+ expression in the dogs immunized with H-line was higher that in dogs of Group WT (P = 0). Consistent with our results, Reis et al. (Reference Reis, Teixeira-Carvalho, Giunchetti, Guerra, Carvalho, Mayrink, Genaro, Correa-Oliveira and Martins-Filho2006a) have demonstrated that asymptomatic dogs display enhanced expression of MHC-II on circulating lymphocytes besides lower overall tissue parasitism (Reis et al. Reference Reis, Martins-Filho, Teixeira-Carvalho, Carvalho, Mayrink, Franca-Silva, Giunchetti, Genaro and Correa-Oliveira2006b).
In the cell culturing process, macrophages spread and stick on the solid membranes, and on these cells an immunohistochemical investigation was performed to measure the population of CD14+ macrophages in the PLN of the dogs. Our results showed that CD14+ cells in dogs infected with wild-type parasites were significantly less numerous than that in dogs immunized with L. infantum H-line. No parasites were found in the PLN of dogs immunized with L. infantum H-line whereas parasites were found in 60% dogs of Group WT and in one of them this was intense. Several studies showed that the immunohistochemical method is highly efficient at revealing the presence on intact parasites in tissues of dogs (Ferrer et al. Reference Ferrer, Rabanal, Fondevila, Ramos and Domingo1988; Lima et al. Reference Lima, Michalick, Melo, Tafuri and Tafuri2004; Giunchetti et al. Reference Giunchetti, Martins-Filho, Carneiro, Mayrink, Marques, Tafuri, Correa-Oliveira and Reis2008a). Moreira et al. (Reference Moreira, Luvizotto, Garcia, Corbett and Laurenti2007) used IHC in the PLN of symptomatic, oligosymptomatic and asymptomatic dogs with a specificity of 100% and a sensitivity of 92·68%, 60% and 73·91%, respectively. Down-regulation of CD14 and MHC-II in the dogs infected with L. infantum WT was associated with parasitism whereas dogs with a higher expression of MHC-II and CD14 were the immunized dogs without a parasite tissue load. Our results are in agreement with reports of this association between a low parasite burden with an enhanced expression of MHC-II. In contrast, in a high parasite burden, there is a reduced expression of MHC-II (Papadogiannakis et al. Reference Papadogiannakis, Koutinas, Saridomichelakis, Vlemmas, Lekkas, Karameris and Fytianou2005; Giunchetti et al. Reference Giunchetti, Mayrink, Carneiro, Correa-Oliveira, Martins-Filho, Marques, Tafuri and Reis2008b; Saridomichelakis et al. Reference Saridomichelakis2009). Our major findings re-emphasize the role of cellular immunity in the immunized dogs.
