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Infection and Immunity, January 2003, p. 401-410, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.401-410.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Intradermal Infection Model for Pathogenesis and Vaccine Studies of Murine Visceral Leishmaniasis

Saeed Ahmed,1 M. Colmenares,1 L. Soong,2 K. Goldsmith-Pestana,1 L. Munstermann,1 R. Molina,3 and Diane McMahon-Pratt1*

Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 06520-8034,1 Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1019,2 WHO Collaborating Center for Leishmaniasis, Servicio de Parasitologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Spain3

Received 26 March 2002/ Returned for modification 20 May 2002/ Accepted 3 October 2002


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ABSTRACT
 
The levels of protection found in vaccine studies of murine visceral leishmaniasis are significantly lower than for cutaneous leishmaniasis; whether this is due to the high-challenge murine model employed and/or is a consequence of differences required in tissue-specific local immune responses is not understood. Consequently, an intradermal murine model of visceral leishmaniasis has been explored. Intradermal inoculation established a chronic infection in susceptible mice which was associated with a pattern of parasite clearance with time postinfection in the liver and skin; in contrast, parasite persistence and expansion was observed in lymphoid tissue (spleen and draining lymph node). The course of disease found appears to be similar to those reported for subclinical canine and human visceral leishmaniasis. Clearance of parasites from the skin was correlated with an inflammatory response and the infiltration and activation of CD4+ and CD8+ T cells. In contrast, in lymphoid tissue (lymph node or spleen), the production of Th1/Th2 cytokines (interleukin-4 [IL-4], IL-10, and gamma interferon) appeared to correlate with parasite burden and pathogenesis. In vaccination experiments employing the Leishmania infantum D-13 (p80) antigen, significantly higher levels of protection were found with the intradermal murine model (29 to 7,500-fold more than naive controls) than were found with a low-dose intravenous infection model (9 to 173-fold). Thus, this model should prove useful for further investigation of disease pathogenesis as well as vaccine studies of visceral leishmaniasis.


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INTRODUCTION
 
Leishmaniasis is a spectrum of diseases (cutaneous, mucocutaneous, and visceral) caused by parasitic protozoans of the genus Leishmania. The parasite exists in two developmental stages: the flagellated promastigote is transmitted with the bite of the sand fly (insect vector) to the mammalian host where it transforms into the amastigote stage, which replicates and survives within the phagolysosome of the host macrophage. Currently leishmaniasis is endemic in 88 countries, with an estimated total of 350 million people at risk; the World Health Organization estimates that 12 million cases exist worldwide. Visceral leishmaniasis (VL), the most severe form of disease, is caused by members of the Leishmania donovani complex (L. donovani, L. infantum, and L. chagasi). Five hundred thousand new cases of VL occur each year. Further, the presence of subclinical infection in these areas has become evident in patients with AIDS (3, 35).

The murine model has proven useful in our understanding of the mechanisms of pathogenesis as well as the immunological response to infection in VL. In contrast to cutaneous leishmaniasis (CL) caused by L. major, a Th1 versus Th2 dichotomy is not evident in VL (murine model). Noncurative (versus curative) mice fail to develop significant interleukin-4 (IL-4) responses to infection, and treatment with anti-IL-4 has been demonstrated to have no effect on the course of infection (28, 42, 75); further, IL-4 has been shown to be necessary for effective drug treatment of murine VL (2). The cytokines IL-2, gamma interferon (IFN-{gamma}), tumor necrosis factor, and IL-12 are important for the resolution of disease (13, 20, 49-51, 64, 68). IL-12 has been shown to be involved in granuloma formation, regulation of Th1 cytokines, and potentially, NK activation. IL-10 and transforming growth factor ß have been implicated in disease progression in leishmaniasis (5, 14, 18, 21, 26, 27, 29, 41, 75, 76); further, IL-10 is observed in human VL patients' responses to infection and appears to correlate with disease severity (14, 27, 29).

Vaccine studies with the murine model of VL have been less extensive than those of CL. In general, the level of protection found in murine VL vaccine studies (with a high-dose intravenous challenge), with one exception (39), ranges from a 2.5- to a 10-fold reduction in parasite burden in vaccinated mice in comparison to control (nonvaccinated) mice (1, 16, 37, 40, 54, 56, 60, 70, 72). This is in contrast to what is found for CL, where reductions in parasite burdens of 103- to 106-fold are observed (10, 22, 25, 37, 56, 59). At present, it is unclear whether the low level of protection observed in VL vaccine studies is due to: (i) the higher infective dose of parasites (1 x 107 to 2 x 107) generally employed for infection in comparison to that employed for CL (103 to 106); (ii) the intravenous versus subcutaneous or intradermal challenge routes employed; (iii) the requirement for immunological effector control mechanisms that are not adequately induced by vaccination at the specific tissue sites (spleen and liver); or (iv) a combination of the above factors.

The literature suggests that, in the murine model, lower numbers of parasites inoculated intravenously may result in the establishment of infection in susceptible strains of mice (24, 69, 70). Further infection, through the intradermal or subcutaneous route that more closely approximates the natural course of infection due to transmission by the bite of a phlebotomine sand fly, has been achieved in some dogs and in the highly susceptible hamster model (30, 74). However, the establishment of visceral infection through intradermal infection in the mouse model remains controversial (41, 52, 61). The experiments presented in the present study with L. infantum were directed towards characterizing the course of infection with the intradermal route of inoculation and an examination of the utility of this murine model in vaccine studies of VL. Evidence clearly indicates that this model system should provide the basis for future vaccine and pathogenesis studies of VL.


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MATERIALS AND METHODS
 
Parasite strains and in vitro cultivation. L. infantum promastigotes (QQ) were grown at 23°C in Schneider's Drosophila medium supplemented with 20% fetal bovine serum; this strain was originally isolated from a case of VL in Spain and was generously provided by Jorge Alvar (Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Spain). The L. infantum strain was periodically passaged through BALB/c mice to ensure virulence.

Infection and parasite burden analyses. Mice were infected with late-log- and stationary-growth-phase promastigotes of L. infantum. As the presence of dead or dying organisms might contribute to the development of a heightened host immune response, live organisms were separated from cellular debris present in stationary-phase cultures on a step Percoll (Sigma Chemical Co.) gradient. The gradient consisted of 90% Percoll-phosphate-buffered saline (PBS) overlaid with parasites resuspended in 45% Percoll (in PBS); the gradient was overlaid with 25% Percoll (in PBS) and then centrifuged at 4°C for 45 min at 4,300 x g. Live parasites were isolated at the 90%-45% interface. After isolation from the Percoll gradient, parasites were washed three times in PBS and the concentration was adjusted to 109parasites/ml for intradermal infection or 105 to 108 parasites/ml for intravenous infection. Mice were infected intradermally either on the top of one rear hind foot or on the ear pinnae; infection at either site successfully resulted in parasite visceralization.

In general, Percoll-isolated organisms were morphologically heterogeneous and very actively motile. The development of metacyclic organisms was monitored by using P-8 expression with indirect immunofluorescence, as previously described (53); the P-8 antigen is not expressed in log-phase organisms, but it is expressed in metacyclic promastigotes and amastigote organisms (12, 53). The level of P-8 expression in promastigotes (various morphological forms) ranged from 40 to 70% at days 6 to 9 of culture.

Parasite burden analyses for the lymph node, spleen, liver, and cutaneous tissues were performed as previously described for cutaneous or visceral tissues with certain modifications (62, 66). Fivefold serial dilutions, made in Schneider's medium supplemented with 20% heat-inactivated fetal calf serum and 25 µg of gentamicin/ml, were plated into 96-well flat-bottom microtiter plates (48 wells/dilution). After 2 to 2.5 weeks, the plates were scored microscopically for growth. Parasite numbers were determined based upon a plot of log10(% negative wells) versus 1/dilution. Experiments with isolated amastigotes of L. infantum and plating under these conditions indicated that the cloning efficiency for this strain was >=85%. Two to three mice/group were used for each parasite burden determination. The results presented are the averaged values (± standard errors) of the parasite burdens found for each group.

Animals. Mice (BALB/c) were purchased from Jackson Laboratories (Bar Harbor, Maine) or the National Cancer Institute (Frederick, Md.) and housed in the Yale University School of Medicine American Association for Accreditation of Laboratory Animal Care-approved animal facility. Sentinel mice were periodically checked for the presence of viruses in the colony.

Measurement of cytokine production and fluorescence-activated cell sorter (FACS) analyses. Cytokine levels were measured as previously described (59). Briefly, spleen and draining lymph node cells were prepared at the indicated times postinfection in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 5 x 10-5M 2-mercaptoethanol (Sigma Chemical Co.), 100 U of penicillin/ml, and 100 µg of streptomycin sulfate/ml. After washing, cells were plated into 24-well tissue culture plates (2 x 106 cells/well; Corning, Corning, N.Y.) and stimulated with promastigote lysates (equivalent to 106 to 107 parasites, as indicated) or concanavalin A (ConA) (5 µg/ml; Sigma Chemical Co.). Supernatants were harvested after 24 or 72 h of incubation and stored at -70°C until employed to measure the levels of IL-2, IL-4, IL-10, or IFN-{gamma} with specific enzyme-linked immunosorbent assays. The sensitivities of the IL-4, IL-2, IFN-{gamma}, and IL-10 assays were 2 U/ml, 0.5 U/ml, 0.5 U/ml, and 200 pg/ml, respectively. Background cytokine levels were determined by using the supernatants from unstimulated cell populations. Background levels for IL-2 and IL-4 were undetectable while the levels for IL-10 were consistently 500 to 800 pg/ml; background levels of IFN-{gamma} (18 U/ml) were detected only at day 3 postinfection.

For FACS analyses, cells from draining infected lymph nodes were isolated and cultured in the presence of 10 µg of brefeldin A (Golgi Plug; Pharmingen, San Diego, Calif.)/ml for 4 h. Subsequently, cells were stained for CD4, CD8, CD45R/B220, CD11b, or CD11c, fixed, permeabilized, and stained for intracellular cytokines (IFN-{gamma} and IL-10). Antibodies for flow cytometry analysis (FACS) were from Pharmingen and were used at a concentration of 0.2 µg/106 cells. Cell surface determinant and intracellular cytokine data were acquired by using a FACScan flow cytometer; data were analyzed by using FlowJo program software.

Immunohistochemistry. Immunohistochemical staining was performed by following previously described procedures (58). Briefly, foot tissues were fixed in PBS containing 2% paraformaldehyde and 5% sucrose at 4°C overnight and then in 20% sucrose in PBS for 8 h prior to freezing in O.C.T. compound (Miles, Elkhart, Ind.). Frozen sections (5 µm thick) were prepared according to standard procedures and fixed with a graded series of acetone solutions (70 to 90%). After 1 h of blocking with TBS (50 mM Tris-HCl and 0.9% NaCl [pH 7.6]) containing 1% bovine serum albumin, 0.01% Triton X-100, 2.5% Blotto, and 10% goat serum, sections were incubated at 4°C overnight either with anti-CD4, anti-CD8{alpha}, or anti-Mac-1 antibody or with isotype control rat immunoglobulin Gs (1:25 to 1:50 dilution). After washing, tissues were treated with 0.8% H2O2 in methanol and then incubated for 1 h with biotin-labeled goat anti-rat immunoglobulin G (mouse serum absorbed; Kirkegaard & Perry Laboratories, Gaithersburg, Md.). Color development was performed with an avidin-peroxidase conjugate (Vectastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, Calif.) and a 3,3-diaminobenzidine—metal substrate solution (Pierce). The sections were counterstained with 1% methyl green and mounted. No significant staining was observed in tissues stained with control antibody. Representative sections were also stained with hematoxylin and eosin (H&E) for general pathological examination. At least 8 sections per animal and 3 mice per group were evaluated for each primary antibody. Images were viewed and captured with a Zeiss Axioskop microscope equipped with a Sony DXC-970MD video camera (40x objective). Positively stained cells were estimated by using MetaView Imaging System, version 4.0, software (Universal Imaging System, West Chester, Pa.).

Preparation of sand fly salivary gland extracts. Sand fly salivary gland extracts were prepared as previously described (65, 67, 71) with either female Phlebotomus perniciosus or Lutzomyia longipalpis sand flies. The P. perniciosus sand flies were originally colonized from an area of endemicity in Spain (43); the L. longipalpis sand flies were from an area of endemicity of L. chagasi (closely related to L. infantum) in El Callejon, Colombia (46). Salivary glands, sand fly thoraxes (in which the salivary gland is embedded), or abdomens (non-salivary tissue control) were isolated at 4°C; samples were then either stored at -70°C or processed for immediate use. Salivary glands or abdominal tissues were macerated in PBS, and the samples were centrifuged at 18,000 x g for 5 min. Mice were infected with the extract equivalent of 0.4 to 1.0 of a salivary gland together with 107 late-log phase L. infantum promastigotes, as described above.

Vaccination experiments with the D-13 (p80) L. infantum antigen. BALB/c mice were immunized intraperitoneally three times at biweekly intervals with 3 to 5 µg of the D-13 antigen isolated as described from L. infantum promastigote membranes (56, 72) together with 50 or 100 µg of Propiniobacterium acnes or Corynebacterium parvum as an adjuvant. Control groups of mice consisted of those receiving P. acnes or C. parvum alone and/or untreated animals. Six weeks after the final immunization, mice were infected with either 104 (low-dose intravenous infection) or 107 (intradermal infection) purified stationary L. infantum promastigotes. At the times postinfection indicated, parasite burdens (3 mice/group) in the liver and spleen, cutaneous tissue, and/or draining lymph node were determined by using the limiting dilution method described above. The results presented are the averaged values (± standard errors) of the parasite burdens found for each group.


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RESULTS AND DISCUSSION
 
Evaluation of a low-dose intravenous infection model of murine VL. In order to evaluate the feasibility of using a lower intravenous challenge to establish visceral infection, BALB/c mice were infected intravenously either with 104, 105, or 106 late-log- or stationary-phase L. infantum promastigotes. Parasites were isolated after 7 to 9 days in culture from Percoll step gradient and were highly enriched for metacyclic organisms (see Materials and Methods). Parasite burdens (Fig. 1) were evaluated in the liver and spleen at various times postinfection by using limiting dilution analysis (62, 66). In the case of mice (Fig. 1) infected with 106 promastigotes (or 107 organisms; data not shown), the parasite burdens in the liver and the spleen persisted at high levels throughout the time period of the experiment (4 months). The parasite numbers in the liver, however, moderately reduced with time postinfection (3 to 4 months) while the parasite levels in the spleen increased. The liver/spleen parasite burden ratio was 7.9 at 3 weeks postinfection, whereas at 4 months postinfection, the ratio was 0.0044. Therefore at later stages of infection, although substantial parasite numbers were present in the liver, the burden in the spleen exceeded that found in the liver. This tissue-specific pattern of infection was more evident in the course of infection at the lower infective doses (104 or 105) where the parasite burdens appeared to be significantly diminished (to undetectable levels in some cases) in the liver with time postinfection. Parasites were initially present and persisted at low levels in the spleen but then increased significantly with time postinfection. At 4 months postinfection, infection is primarily, if not exclusively, in the splenic tissue.



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  		FIG. 1. Susceptibility of mice to intravenous infection with L. infantum promastigotes. BALB/c mice were infected intravenously as described in Materials and Method with various numbers of L. infantum promastigotes (104 to 106) as indicated. Parasite burdens were determined by using the limiting dilution analysis method at the times postinfection indicated. It should be noted that at 16 weeks postinfection, parasites were found in the livers of mice infected with 104 L. infantum promastigotes; however, the parasite burden was below the minimal quantifiable level (<=170 parasites/liver). Symbols are as follows, with the numbers of promastigotes noted in parentheses: {circ}, liver (106); {square}, spleen (106); {blacktriangleup}, liver (105); {blacktriangledown}, spleen (105); •, liver (104); {blacksquare}, spleen (104).

These data indicate that infections with highly reduced numbers (104) of parasites consistently result in parasite burdens that can be quantified; consequently, lower numbers of parasites might be employed for infection in vaccine studies of VL. These results also suggest that, within the same animal, the effective immune response contributing to parasite clearance varies at different tissue sites. Even though initially the level of infection and/or parasite burden is higher in the liver than in the spleen, parasites are effectively diminished and/or cleared from the liver. These observations are consistent with previously reported studies of murine VL (13, 33) caused by either L. infantum or L. donovani. With time postinfection, parasite burdens in the spleen have been observed to increase while parasite burdens in the liver somewhat diminished. However, in these studies, which employed a high challenge inoculum (107), the liver remained substantially and persistently infected. This differs from what is observed with a low challenge dose in the present study, where the parasite is cleared from the liver in some cases. This difference may be a reflection of the limitation of the immune response in the liver in the initially naive host, which can overcome a relatively low (104 to 105), but not higher (106 to 107), parasite challenge. In addition, the susceptibility of the spleen to infection is particularly evident in the present study; as few as 100 parasites are able to successfully establish progressive infection. Consequently, the lymphoid tissue sites are uniquely susceptible and critical for parasite maintenance. Most vaccine studies to date have primarily examined the parasite burdens in the liver; however, these results and those of previous studies (13, 33) emphasize the susceptibility of the spleen to infection and indicate that vaccines need to target the spleen if persistent parasites are to be eliminated.

Evaluation of the intradermal route of infection to establish murine VL. In order to evaluate the feasibility of using an intradermal or subcutaneous challenge to establish visceral infection, BALB/c mice were infected with107 late-log- or stationary-phase L. infantum promastigotes (isolated as indicated in Materials and Methods). Parasite burden analyses were performed at various times postinfection (Fig. 2) at the cutaneous site of infection, draining lymph node, spleen, and liver tissues. At the site of dermal infection, the parasite burdens decreased with time postinfection (Fig. 2). In contrast, in the draining lymph node, parasites rapidly established themselves; parasite burdens were generally maintained at a level of 104to 105 parasites/lymph node throughout the experimental period of observation (>10 months) (data not shown). Thus, despite the elimination of parasites occurring at the dermal site, L. infantum organisms persisted and maintained levels in the draining lymph node. Parasites were evident in the liver at 1 week postinfection. The parasite burdens in the liver reached a peak at 3 to 5 weeks postinfection, and after this time, the parasite burdens declined. In contrast, the parasite burdens in the spleen were evident (albeit lower) at 1 week postinfection; parasite burdens in the spleen persisted and increased with time postinfection. These results are consistent with what was found with the low-dose (104) intravenous infection and indicate that the spleen, infected with low numbers of parasites, leads to progressive disease and/or parasite growth. Thus, at 3 months postinfection, the primary parasite burden was found in the lymphoid tissues (i.e., lymph node and spleen). The progression of disease found in the intradermal murine model appears to reflect that found in humans and the canine reservoir host, where subclinical infection can persist for long periods of time before developing into severe disease (9, 30, 77).



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  		FIG. 2. Susceptibility of BALB/c mice to intradermal infection with L. infantum promastigotes. Shown are the experimental results for mice infected intradermally with 107 promastigotes of L. infantum. Parasite burdens were determined at the site of infection (skin), draining lymph node, spleen, and liver by limiting dilution analysis. The tissue parasite burdens at various times postinfection are indicated. {diamondsuit}, lymph node; {circ}, cutaneous infection site; •, spleen; {square}, liver.

These data are representative of numerous (>12) experiments conducted over 4 years and indicate that, in the murine model, L. infantum metacyclic promastigotes injected intradermally are capable of establishing visceral infection. These results are consistent with those of a previous study, in which intradermal infection of BALB/c mice with two strains of L. donovani (from cutaneous cases) was demonstrated to result in external signs of pathology and parasites found in the liver and spleen at 13 to 18 months postinfection (52). However, our results differ from those of another study examining the course of intradermal L. donovani infection in BALB/c mice (41). The reason for the lack of visceralization found (41) is, at present, unclear. However, it should be noted that in this study, tissue-isolated amastigotes were employed (41) in contrast to the promastigotes used here and in earlier studies (52) in which visceralization was observed. Further, the parasite burdens found in the draining lymph node were 10- to 102-fold less (41) than in the present study, suggesting a potential difference in parasite virulence may have contributed to the lack of visceralization found.

Given the established ability of sand fly salivary components to enhance the level of cutaneous Leishmania infection (over a wide range of parasite doses [103 to 107]) (6, 36, 45, 78), the effect of salivary gland components in the establishment of VL in the murine intradermal model was investigated. Salivary and abdominal (control) extracts from L. longipalpis and P. pernicious sand flies were employed; these sand flies were originally isolated from areas of VL endemicity (43, 46). However, in three separate experiments, no significant differences in parasite burdens were found (site of cutaneous infection, the draining lymph node, spleen, or liver) between mice receiving parasites together with sand fly salivary gland extracts and control mice. The ratio of parasite burdens of mice infected with abdominal extracts or PBS to those receiving salivary gland extracts ranged from 0.6 to 1.1. The lack of an effect of salivary components on infection may reflect known population variability in the ability of specific sand fly colonies to cause enhancement of infectivity (71). In addition, it is of interest that only a small increase in erythema was noted in the ears of mice injected with salivary components. Erythema and/or vascular permeability and the exacerbation of infection caused by salivary gland components are not associated in murine CL (71). However, it is possible that enhancement of visceral infection might be dependent upon changes in vascular permeability (promoting visceralization) in addition to local or cutaneous suppression of the host immune system (23, 36, 65, 67, 71). Hence, further work (possibly using alternate animal models) may be required to determine if sand fly salivary gland components can enhance the visceralization or establishment of L. infantum infection.

Cutaneous response in mice intradermally infected with L. infantum. As intradermal inoculation led to a persistent, established parasitemia in the lymphoid tissues but clearance at the cutaneous site of infection, it was of interest to compare the ongoing site-specific immune responses with time postinfection. The immune responses at the cutaneous site of parasite inoculation at 1.5, 3, 10, and 28 days postinfection were examined via staining of frozen sections with H&E and monoclonal antibodies specific for CD4+ and CD8+ T lymphocytes (Fig. 3). Parasite-carrying macrophages were readily seen at days 1.5 and 3 (Fig. 3A) but markedly declined by day 28 (data not shown). Inflammatory responses at early stages of infection (days 1.5 and 3), in contrast to later times postinfection, were characterized by massive infiltration of polymorphonuclear leukocytes (PMNs) (Fig. 3B versus C). PMNs are characteristic of the innate response to cutaneous infection by species of Leishmania. PMNs have been demonstrated to be capable of Leishmania destruction as well as the early production of IL-4 and development of a Th2 response (7, 31, 32, 55, 63); consequently, PMNs may be involved in both initial local cutaneous parasite containment and/or subsequent disease exacerbation. However, in spite of the evident early local inflammatory cutaneous response, parasites successfully disseminated (by days 7 to 10 postinfection) to the liver and spleen.



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  		FIG. 3. Skin inflammatory responses of BALB/c mice following infection with L. infantum. Frozen foot sections were collected on the indicated day postinfection with 107 promastigotes of L. infantum. Sections were stained with H&E (A to C) or stained with immunoperoxidase and counterstained with methyl green (D to F) at either 1.5 (A, B, and D) or 28 (C, E, and F) days postinfection. At day 1.5 of infection, tissues were composed of a collection of parasitized macrophages (arrows in panel A) and were characterized with massive infiltration of PMNs (arrows in panel B) and low levels of CD4+ T cells (D). In contrast, parasite-containing macrophages were rare in tissues at 28 days postinfection (panel C versus panel A) while CD4+ (E) and CD8+ (F) T cells were readily detected. All images were taken with 40x magnification.

As shown in a representative section in Fig. 3D, lesions at days 1.5 and 3 displayed scattered staining with CD4 monoclonal antibodies (on average 5 to 11 positive cells/field [magnification, 40x] for both time points) or CD8 monoclonal antibody (on average 3 to 5 positive cells/field [magnification, 40x] for both time points). The number of CD4+ and CD8+ cells became evident at 10 days and was highest at 28 days (41 ± 11 per field [magnification, 40x] and 23 ± 6 per field [magnification, 40x], respectively) (Fig. 3E and F). These histological results correlated with our observations of peak parasite load in the infected foot or ear at 7 to 10 days postinfection and general decrease of parasite load in the skin from 3 to 12 weeks of infection (Fig. 2). The subsequent control of L. infantum replication at the site of inoculation correlated to the infiltration and activation of CD4+ and CD8+ T cells as well as to the production of cytokines in lymph nodes draining the site of infection (Fig. 4). Consequently, the loss of parasites from the dermal site appears to be a result of the innate and acquired immune responses as well as the dissemination of the parasites to the lymph node, spleen, and liver tissues.



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  		FIG. 4. Immune response of BALB/c mice intradermally infected with L. infantum. Cytokine responses were examined in the draining lymph node and splenic tissues of BALB/c mice intradermally infected with 107 L.infantum promastigotes. Cells were stimulated with either L. infantum promastigote homogenate antigen or ConA, and cytokines were measured as described in Materials and Methods at various times postinfection. The sensitivities of the IL-4, IL-2, IFN-{gamma}, and IL-10 assays were 2 U/ml, 0.5 U/ml, 0.5 U/ml, and 200 pg/ml, respectively. Background cytokine levels were determined by using the supernatants from unstimulated cell populations. Background levels for IL-2 and IL-4 were undetectable while the levels for IL-10 were consistently 500 to 800 pg/ml; background levels of IFN-{gamma} (18 U/ml) were detected only at day 3 postinfection.

Pattern of immune responsiveness in lymphoid tissue of mice intradermally infected with L. infantum. The immune response to intradermal infection with L. infantum was further evaluated in the susceptible lymphoid tissue sites (draining lymph node and spleen). Previous studies of murine VL (high intravenous challenge model) have established that production of IL-2, IL-10, IL-4, and IFN-{gamma} are observed (41, 42, 51, 75) in response to infection. Further, in the murine model and in human VL, the lymphocyte response to the mitogen ConA becomes significantly suppressed (15, 17, 18, 27, 29, 34, 38, 75). Therefore, whether similar responses were evident in the intradermal VL model and how these responses developed with increasing parasite burden (time postinfection) and pathogenesis were of interest. Cells were isolated from either the draining lymph node or the spleen at various times postinfection (1.5 days to 1 month). Then the cells were either not stimulated or stimulated with whole sonicated leishmanial antigen or with ConA (5 µg/ml) for 24 to 72 h; supernatants were collected, and cytokine levels (IL-2, IL-4, IL-10, and IFN-{gamma}) were measured. The results from these studies are shown in Fig. 4.

In the draining lymph node, the production of both IL-2 and IFN-{gamma} in response to ConA significantly decreased with time postinfection. Interestingly, less of an effect was observed for the levels of IL-4 or IL-10, which appeared to be maintained at low, relatively consistent levels during the experimental period. In contrast, the ConA response in the spleen showed a consistent level of IFN-{gamma} production with time postinfection (1.5 days to 1 month). However, a reduction in the IL-2 response was observed in the spleen at 28 days postinfection. These results are consistent with observations of immunosuppression (to phytohemagglutinin, ConA, or anti-CD3) reported for human VL patients and also observed in susceptible strains of mice (infected intravenously with 1 x 107 to 2 x 107 organisms) (15, 17, 18, 27, 29, 34, 38, 75). Notably, in the high challenge dose model of murine VL, suppression of the ConA response can be complete by 7 days postinfection (18, 34, 51, 75). The relatively early decrease in ConA-induced IL-2 and IFN-{gamma} responses in the draining lymph node and later effect in the spleen (IL-2) suggest that the reductions in ConA responsiveness correlate with parasite burdens found in these tissues (Fig. 2).

In response to antigenic stimulation, a significant IFN-{gamma} response in the draining lymph node was evident at 1.5 days postinfection and did not appear to increase at later times postinfection. The IL-2 and IL-4 responses in the draining lymph node, in response to antigenic stimulation, appeared to increase moderately with time postinfection. These results are similar to results found for the spleen in the high intravenous challenge model, where production of IL-2, IL-4, and IFN-{gamma} have been observed in response to infection (34, 41, 42, 51, 75). Notably, in the draining lymph node, the IL-10 response appeared to peak at day 3 postinfection. The IL-10 response subsequently decreased but remained significantly above background levels in the draining lymph node throughout the course of infection. FACS analyses (data not shown) of the lymph node cells at 3 days postinfection indicated that T, B, and macrophage cells were producing IL-10; these results are consistent with those of previous studies of murine VL which indicate both that adherent macrophage and lymphocyte populations produce IL-10 in response to infection (75). In the spleen, the cytokine responses to antigen were more moderate and appeared to correspond to the increase in parasite burden found with time postinfection. The levels of IFN-{gamma}, IL-2, and IL-10 increased with time postinfection; however, the levels of IL-4 produced by spleen cells (in contrast to the lymph node cells) were minimally detectable.

It should be noted that the production of IL-10 in the spleen and early IL-10 response in the draining lymph node differs from results reported in studies of mice intradermally infected with L. donovani amastigotes (5 x 106) (41). However, in that case, the intradermal injection of L. donovani (41) failed to result in the visceralization or establishment of L. donovani infection in the spleen. In contrast, intravenous L. donovani infection resulted in chronic disease, suppression of the ConA proliferative response, and progressive up regulation of IL-10 mRNA levels (41). Consequently, the establishment of visceral infection appears to correlate with the production of IL-10.

IL-10 has been observed in human VL and is considered to contribute to the immune down-regulation associated with pathogenesis (15, 27, 29). IL-10 is known to block the development and/or maturation of dendritic cells, important antigen-presenting cells, and immunoregulatory cells (4). In contrast, IL-10 has also been observed to down-regulate the expression of CTLA-4 (11, 19, 57); the down-regulation or blockage of CTLA-4 expression has been shown to enhance resistance to murine VL (17, 18, 47). However, IL-10 is known to down-regulate macrophage production of the superoxide anion and synthesis of IL-12 and tumor necrosis factor (44, 57), which are known to be important in the immunologic containment of Leishmania infection. Further, recent in vivo and in vitro studies (8, 48) indicate that IL-10 contributes to the immune down-regulation critical for the maintenance of L. infantum and L. donovani infection in mice as well as that of L. major and L. amazonensis (5, 21, 26). Thus, the early antigen-specific IL-10 response in the draining lymph node and the progressive response in the spleen are consistent with IL-10 being critical to the developing pathogenesis (VL) caused by intradermal infection.

Hence, VL established by intradermal infection appears to result in progressive disease, where the susceptibility of the lymphoid tissue to persistent, progressive infection is particularly evident. The developing pathology and consequent immunological response are consistent with those previously observed for human VL and in the high-dose murine model of VL. However, the evolution of disease and subsequent immune response is slower and most likely reflects that seen for subclinical infection, which subsequently develops into disease (75).

Effect of intradermal or low intravenous parasite challenge dose on the level of protection found in vaccinated mice. It is evident from murine VL vaccine studies with a high intravenous challenge dose that the level of protection observed is significantly less than those found in vaccine studies of murine CL. In murine CL vaccine studies, reductions of 103- to 106-fold have been observed for vaccinated animals in comparison to nonvaccinated controls (10, 22, 25, 56, 59). The level of protection most commonly observed in vaccine studies of murine VL is of the order of 10-fold (37, 56). It is not clear, however, whether the lower level of protection found in murine VL is a result of the relatively high infection dose employed (1 x 107 to 2 x 107organisms) or of differences in the immune response(s) required for containment of infection (CL versus VL [liver or spleen]). Therefore, the possibility that more-significant protection would be observed in vaccinated mice against a low-inoculum intravenous or intradermal challenge infection was examined.

Mice were immunized with D-13 (p80) antigen isolated from L. infantum (56, 72, 73) with C. parvum (or P. acnes) as an adjuvant. An intravenous infecting dose (104) that provided a course of infection similar to that observed in the intradermal model was employed for these studies. Control groups of mice received C. parvum alone or no treatment. Parasite burdens in the liver and spleen tissues were examined at 6, 12, and 18 weeks postinfection; the results from these studies are presented in Table 1. Two previous independent vaccine studies employing challenge doses of 107 L. donovani promastigotes demonstrated that immunization with the D-13 antigen provided four- to fivefold reductions in parasite burdens in the liver (evaluated at 1 month postinfection) (56, 72). Using the lower challenge inoculum, the reduction in parasite burdens ranged from 9- to 173-fold, with the level of protection increasing with time postinfection.


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  		TABLE 1. Parasite burdens in vaccinated BALB/c mice following intravenous infectiona

These vaccine experiments comparing low-dose intravenous (Table 1) and intradermal challenges (Table 2) indicate that the route of infection does contribute to the level of protection found. This is most clearly observed in the spleen, where the level of protection in the intradermal infected mice at 12 and 18 weeks postinfection was 812- and 4,782-fold, respectively. Mice receiving an intravenous infection had 173- and 45-fold reductions in parasite burdens, respectively, at 12 and 18 weeks postinfection. In the case of the liver, at 12 weeks postinfection the reductions in parasite burdens were comparable in the two groups of mice (29- versus 34.5-fold); at 18 weeks postinfection, the reduction in the intradermal challenge group was approximately 1,000-fold in comparison to the 110-fold reduction in parasite burden found in the intravenously infected mice. These data indicate that the intradermal challenge model results in a higher level of protection than that found for the low-dose intravenous challenge model. Consequently, as this infection route more closely approximates that found in nature, this model may better indicate the potential of vaccine candidate molecules.


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  		TABLE 2. Parasite burdens in vaccinated BALB/c mice following intradermal infectiona

In terms of the prospective of a vaccine for VL, it is encouraging that, in general, protection persists and that the reductions (n-fold) in parasite burdens in vaccinated mice increase with time postinfection. However, it should be noted that, even in intravenous infection with significantly lower numbers of promastigotes, the overall the level (n-fold) of protection still does not reach those found in vaccine studies of murine CL at comparable times postinfection (10, 22, 25, 37, 56, 59). These results are consistent with those of previous studies (56) that demonstrated that mice vaccinated with the D-13 L. donovani antigen were better protected against infection with L. major than against L. donovani infection. Taken together, these results suggest that the immunological mechanisms important in the control of visceral infection are not adequately induced and/or activated by using the vaccination approaches that are effective in inducing protection against cutaneous disease. Hence, further development of vaccination methods directed toward the control of VL need to be considered.

Summary
The intradermal or subcutaneous infection model for VL (which more closely approximates the infection route found in nature) successfully establishes chronic VL infection in susceptible mice. Infection was associated with a pattern of parasite clearance with time postinfection in the liver and skin (intradermal infection) and the persistence of parasites in the spleen and draining lymph node. This course of infection found appears to be representative of subclinical canine and human VL. The tissue site-specific parasite clearance or persistence appears to reflect different ongoing local immune responses. Although the levels of protection observed (induced by vaccination with a single antigen) still appear to be less than those found in vaccine studies of murine CL, these vaccine studies indicate that higher levels of protection are achieved by using the intradermal infection or low-dose intravenous challenge models. Hence, these models should prove useful in future vaccine studies or studies of the pathogenesis of infection caused by members of the L. donovani complex.


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ACKNOWLEDGMENTS
 
We thank Judy F. Anderson (University of Texas) for help in pathology analyses.

This work has been supported through grants from the National Institutes of Health to D.M.-P. (AI-45044) and to L.S. (AI43003).

S.A., M.C., and L.S. contributed equally to this work.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College St., New Haven, CT 06520-8034. Phone: (203) 785-4481. Fax: (203) 737-2921. E-mail: diane.mcmahon-pratt{at}yale.edu. Back

Editor: J. M. Mansfield


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Infection and Immunity, January 2003, p. 401-410, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.401-410.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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