Leishmania donovani: cellular and humoral immune responses in Indian langur monkeys, Presbytis entellus

Acta Tropica 73 (1999) 37 – 48

Leishmania dono6ani: cellular and humoral immune responses in Indian langur monkeys, Presbytis entellus Anuradha Dube a,*, J.K. Srivastava a, P. Sharma a, A. Chaturvedi a, J.C. Katiyar a, S. Naik b b

a Di6ision of Parasitology, Central Drug Research Institute, Lucknow 226001, India Department of Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226014, India

Received 17 December 1997; received in revised form 3 December 1998; accepted 18 December 1998

Abstract We have previously reported that disease mimicking human visceral leishmaniasis can be established in Presbytis entellus, the Indian langur monkey, following a single intravenous challenge of 108 Leishmania dono6ani amastigotes. In the present report, infection was assessed in monkeys infected intravenously with a single dose of 108 amastigotes (HDA group), three weekly doses of 107 amastigotes (LDA group) and three weekly doses of 5×107 promastigotes (HDP group). Typical clinical infection was established in all three groups with significant parasite load. There was a gradual and sustained rise in anti-leishmania specific immunoglobulin G response, and a severe fall in the lymphoproliferative response to the T cell mitogens PHA and Con A by day 80 post infection (p.i.). The antibody level remained elevated until death in monkeys of the HDA and HDP groups; the T-cell responses showed a recovery prior to death. T-cell responses to leishmania antigen, however, could not be demonstrated in any of these monkeys prior to death. One monkey of the LDA group survived for 155 days and two monkeys spontaneously eradicated the infection. Surprisingly, one monkey of the HDA group also achieved spontaneous cure. In the three monkeys which eradicated infection spontaneously, the antibody level declined to baseline

* Corresponding author. Tel.: +91-522-212411-18, ext. 4398; fax: + 91-522-223405. E-mail address: [email protected] (A. Dube) 0001-706X/99/$ - see front matter © 1997 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 9 9 ) 0 0 0 0 7 - 8

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levels on day 180 p.i. with a well demonstrable antigen specific lymphoproliferative response; no parasites could be demonstrated in splenic aspirates by direct examination of culture. These data demonstrate that disease severity may be the function of the total inoculum dose rather than the stage of the parasite and that the immunological responses in the Indian langur model parallel the reported changes in human visceral leishmaniasis. This makes the langur a potentially useful model for the evaluation of candidate anti-leishmanial drugs and vaccines. © 1997 Elsevier Science B.V. All rights reserved. Keywords: Leishmania dono6ani; Presbytis entellus; Langur monkeys; Enzyme linked immunosorbent assay; Lymphocyte proliferation assay

1. Introduction Visceral leishmaniasis (V.L.) caused by Leishmania dono6ani is endemic in many parts of the world and affects an estimated 15 million persons worldwide (Ashford et al., 1992). Immunological studies in experimental models for V.L. (hamsters and mice) have contributed greatly to our understanding of the disease (Neal and Croft, 1984; Nickol and Bonventre, 1985). However, since these animals differ from man and from each other in their physiology, metabolism, handling of drugs and immunological responses, there is a need for availability of suitable non-human primate model(s) for V.L. Such models could be used for studying several as yet poorly understood aspects of pathogenesis of the disease, and for testing new drugs and vaccines. Although, the susceptibility of various monkeys to Leishmania infections has been established, these animals have limitations and have not been widely used to study chemotherapeutic and immunological responses (Hommel et al., 1995). We have established experimental infection closely resembling human V.L. in Indian langur monkeys, Presbytis entellus, with a single intravenous inoculation of 108 L. dono6ani amastigotes (Anuradha et al., 1990, 1992). This dose resulted in progressive infection which reached a peak of 130–195 L. dono6ani bodies per 500 cell nuclei at the time of death by day 110–120 post infection (p.i.). The infected monkeys lost body weight, developed severe anemia, lymphocytosis, hyperproteinemia, hypergammaglobulinemia and an increase in serum alkaline phosphatase and alanine aminotransferase. Resistance against Leishmania infection is associated with development of cell mediated immunity (Liew, 1990; Curry et al., 1994); antibodies are considered to play a less important role (Olobo et al., 1980; Howard et al., 1984). In fact, a TH2-type response with high anti-leishmanial antibody levels is seen in susceptible mice strains which develop disseminated disease following Leishmania tropica infection (Kaye et al., 1991; Bretscher et al., 1992). The present study was designed to compare cellular and humoral immune responses in the langur monkey model for visceral leishmaniasis that we have previously described, both during clinical and subclinical infections with L. dono6ani.

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2. Materials and methods Young male langur monkeys (P. entellus) caught in the wild and weighing approximately 3 – 5 kg were quarantined for 45 days prior to use. During this period, they were tested for Mycobacterium tuberculosis infection as well as gastrointestinal and parasitic infections. The langurs were fed on commercial primate feed pellets (Lipton India) supplemented with seasonal fruits and vegetables and water was provided ad libitum. L. dono6ani (Man/IN/80/Dd8) obtained from Imperial College, London, was maintained in the laboratory and used for inoculation of monkeys. The parasites were maintained in male golden hamsters (Mesocricetus auratus) of 40–45 g body weight through serial passages (amastigote to amastigote). Promastigotes were cultured in NNN medium with RPMI 1640 medium as an overlay. Stationary phase promastigotes were harvested by centrifugation at 2000 rpm for 15 min at 4°C. The pellets were washed three times in sterile phosphate buffered saline (PBS) by centrifugation and appropriate concentrations were prepared for inoculation. Twelve monkeys were divided into four groups of three. Three monkeys received an intravenous inoculation of 108 amastigotes of L. dono6ani (high dose amastigotes (HDA)); this was the dose that we had been previously reported (Anuradha et al., 1990, 1992) to establish a V.L.-like disease and has been used as a reference group. A second group of three monkeys received three intravenous doses of 107 amastigotes at 1-week intervals with the aim of inducing a subclinical infection (low dose amastigotes (LDA)). The third group of three animals received three weekly intravenous inoculations of 5× 107 promastigtes (high dose promastigotes (HDP)). Control monkeys did not receive any parasite and were followed up with the experimental animals. The animals were kept under constant observation for their general condition during the study period. The monkeys were bled at the time of parasite challenge (day 0) and on days 26, 33, 45, 60, 80, 100 and 105 p.i. for assessing the cellular and humoral parameters. The protocols including the number of animals were approved by the Animal Ethics Committee of Central Drug Research Institute (CDRI) and number of animals in each group was decided by them. The intensity of infection was assessed by splenic biopsy on days 40 and 180 p.i. Animals were immobilized by an intramuscular injection of Ketamine HCl at a dose of 10 mg/kg of body weight. Laparotomy was performed through a 2 cm incision on the left of the lower abdomen, the spleen exposed and a biopsy piece of 3 mm ×3 mm was taken. Tissue was subjected to contact smears and inoculation into NNN culture medium with RPMI 1640 as an overlay. The incision was sutured with absorbable surgical sutures and adequate amount of antibiotic powder and/or ointment was applied locally. In addition, Terramycin/tetracycline (50 mg/animal) was injected intramuscularly once daily for 5 days. The contact smears were fixed in methanol and stained with 10% Giemsa. The number of amastigotes per 500 macrophage nuclei was counted. The presence of parasites was confirmed by growth of amastigotes in the cultures after 7–10 days of incubation at 269 1°C.

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Antigen was prepared by the method described previously (Anuradha et al., 1990, 1992). In brief, the harvested metacyclic promastigotes were disrupted by sonication (Soniprep 150 model) at 20kHz for three cycles of 1 min each separated by intervals of 2 min. The sonicated material was rapidly frozen in liquid nitrogen and thawed four times and left overnight at 4°C. The supernatant was centrifuged at 40 000 rpm for 30 min and its protein content was assessed by the method of Lowry et al. (1951): preparations were stored at − 70°C until use. Enzyme linked immunosorbent assay (ELISA) was standardised according to the procedure described by Engvall and Perlmann (1972). Different dilutions of promastigote antigen (1 – 20 mg/ml), pool positive and pool negative sera (1:25 to 1:1600) and conjugate (1:500 to 1:2000) were simultaneously evaluated. Maximum difference in OD between pool positive and pool negative sera was observed at 1:100 sera dilution with the antigen and conjugate concentration at 10 mg/ml and 1:1000, respectively. Using these optimum dilutions of reagents, the cut-off value for positivity was assessed employing 10 pool negative (normal) sera which was calculated as the mean OD value 9 3×SD. Thus, the OD value obtained for the cut-off point for positivity was 0.22. For lymphocyte proliferation assays, heparinized blood was collected from the femoral vein of monkeys. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation (Boyum, 1968) and washed three times with RPMI 1640. The cells were adjusted to a final concentration of 1 × 106 cells/ml in complete RPMI (cRPMI) supplemented with 10% fetal calf serum (Biological Industries, Israel), 25 mM Hepes buffer (Sigma), 2 mM L-glutamine (Sigma), 50 × 10 − 6 M 2-mercaptoethanol (Sigma), 1 mM sodium pyruvate (Gibco), 100 U/ml of penicillin and 100 mg/ml of streptomycin. Cultures were set up in triplicate with 100 ml of cell suspension and 100 ml of leishmania antigen or mitogens, phytohemagglutinin (PHA) or concavalin A (Con A), to give a final concentration of 5 mg/ml. This dose was selected based on standardization experiments using normal monkeys (data not shown). The plates were incubated at 37°C in a CO2 incubator (Hereaus) with 5% CO2 for 3 days in the case of mitogens and 6 days in the case of antigens. Eighteen hours prior to termination of cultures, 0.5 mCi of 3H thymidine (BARC, Bombay; specific activity 6 mCi/mM) was added to each well. The cells were harvested onto glass fiber mats and counted in a liquid scintillation counter. The counts were expressed as counts per minute (cpm) and means of the triplicate wells used for analysis. Results were expressed as stimulation index (SI) which was calculated as mean cpm of stimulated cultures/mean cpm of control cultures. The SI of more than 2.5 was taken as a positive response for antigen stimulated cultures.

3. Results All the animals of HDA and HDP groups became listless and weak within 60 days p.i. There was progressive deterioration in their condition, with loss of body weight of 12.3 and 14.3%, respectively, in these two groups (Table 1). Discoloration

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of facial skin being more prominent around the eyes, was observed by day 75– 90 p.i. The face had a puffy appearance and there was intermittent rise in body temperature by 2 – 4°C. Similar but milder alterations were observed in the LDA group animals which showed a marginal loss of body weight (4.6%). These changes became evident only around 120 days p.i. and gradually subsided in the three monkeys (one of HDA and two of LDA groups) who lived beyond 180 days p.i. All the monkeys had established infection by day 40 p.i. and amastigotes could be quantitated in splenic imprints. The average splenic parasitic loads (amastigotes/500 macrophage nuclei) were as follows: HAD, 21; LDA, 12; and HDP, 25. The parasite counts reached a peak at the time of death (107–133 p.i.) in the HDA and HDP groups, and mean parasite counts were 199 and 158, respectively. Monkeys of the LDA group had a longer survival and only one monkey of this group died on day 153 p.i. with a parasite load of 34/500 cell nuclei. One monkey of the HDA group and two from the LDA group who survived until day 180 p.i. had no parasites in the splenic aspirate on repeat biopsy. The splenic tissue of all the infected monkeys biopsied on day 40 p.i. and cultured in vitro developed into promastigotes, while the tissue from the three surviving monkeys obtained on day 180 p.i. failed to grow the parasite. In all infected monkeys, there was a progressive rise in immunoglobulin G (IgG) class specific antileishmanial antibodies from day 30 p.i., which attained a peak on day 60 p.i. in the HDA group of 0.84 and fell to 0.58 on day 105 p.i. (Fig. 1). The mean OD in the LDA group on day 60 p.i. was 0.51 and peaked on day 105 p.i. at 0.73. The antibody level in the HDP group rose until day 105 p.i. with an O.D. value of 0.81. In the three long survivors, a gradual but appreciable fall in antibody levels to the pre-infection levels was seen on day 180 p.i. (Table 2). The mean proliferative response to 5 mg/ml of PHA of all the three groups was comparable to that of the control group prior to parasite inoculation. On day 60 p.i., the LDA and HDP groups showed a decreased response which further declined by day 80 p.i. The response of the HDA group was normal on day 60 p.i. and was depressed on day 80 p.i. These remained lower than the control group even on day 100 p.i. for the HDA group. However, in all groups at day 100 p.i., there was some recovery of proliferation towards levels close to normal values and in the HDP group, proliferation actually surpassed the normal values. The responses to 5 mg/ml of Con A in all the groups was comparable to controls prior to inoculation and also on day 60 p.i. On day 80 p.i., the proliferative responses were considerably lower than pre-infection values and the day 60 p.i. values in the LDA and HDP groups. The responses of the HDA group did not show as marked a depression as that of the other groups. On day 100 p.i., the responses had shown some recovery in the HDA and LDA groups, while responses in the HDP group showed a marked recovery (Figs. 2 and 3). The mean cpm values of control cultures without any stimulant was 10979 1187. None of these trends could be statistically evaluated as the groups were

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Groups

HDA LDA HDP Controls a

Inoculum size

Amastigotes 108, single Amastigotes 107×3, weekly Promastigotes 5×107×3, weekly Saline

Number of amastigotes/500 cell nuclei. Monkeys died on days 108 and 118 p.i. c Monkey died on day 153 p.i. d Monkeys died on days 107, 111, and 133 p.i. b

% Decrease in body weight

12.3 4.6 14.3 2.0

Parasite Number in spleena Day 40 p.i.

At autopsy/biopsy

21 (17,19,28) 12 (05,13,19) 25 (15,25,35) –

199 (0,212,386) 11 (0,0,34) 158 (70,195,210) –

Survival \180 days

1/3b 2/3c 0/3d 3/3

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Table 1 The parasite load and survival of monkeys given intravenous infection with L. dono6ani amastigotes and promastigotes (mean of the values of three monkeys in each group is given, with individual parasite burden in parentheses)

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Fig. 1. Sequential anti-leishmanial IgG levels (mean O.D. 9 SD of three monkeys at each time point) of the experimental groups. The cut-off for pooled normal monkeys samples is 0.22 9 0.073.

too small and only one normal monkey was available for simultaneous evaluation. The values of the normal monkey have been shown only to indicate that the experimental conditions were satisfactory. No monkey in any group showed antigen specific lymphoproliferative responses up to the time of death except the three monkeys who survived longer and showed spontaneous cure. These three monkeys (one of the HDA group and two of the LDA group) were tested on days 60, 80, 100, 120, 150 and 180 p.i. Significant responses to L. dono6ani antigen at 180 days p.i. was observed at which time there was also a sharp decrease in their antibody levels reaching the baseline levels (Table 2). Table 2 Immunological parameters at day 180 p.i. in the three animals with prolonged survival (\180 days) Groups

HDA LDA HDA

Proliferation response (mean cpm with SI)a PHA (5 mg/ml)

Con A (5 mg/ml) Antigen (5 mg/ml)

34,793 (25.3) 75,073 (46.2) 23,863 (17.8)

27107 (27.5) 6243 (5.7) 38,854 (11.6)

11731 (23.7) 8732 (25.6) 8384 (16.1)

Antibody levelb (O.D. value)

0.24 0.28 0.18

a Mean SI ( 9 SEM) in normal monkey is 34.4( 9 5.5) for PHA (5 mg/ml), 36.2( 9 12.1) for Con A (5 mg/ml) and B2.5 for antigen (5 mg/ml). b Cut-off point for pooled negative samples is 0.22 9 0.73 and for pooled positive samples is 1.7849 0.26 at the sera concentration of 1:100.

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Fig. 2. The lymphoproliferative responses of control and experimental groups to PHA (5 mg/ml). Values are expressed as the mean cpm 9SEM of three monkeys in each group.*, SEM bar is not shown since bar represents value of only one monkey.

4. Discussion We have revalidated our earlier findings (Anuradha et al., 1992) that P. entellus (Indian langur monkey) can serve as a good non-human primate model for human visceral leishmaniasis. Additionally, we have shown that this model enables us to study the evolution of immune responses during the course of visceral disease. We believe this to be the first report of serial immunological studies in this monkey model through the evolution of disease. Six of the nine monkeys died by day 133 p.i. irrespective of dose and stage of parasite inoculum (median survival, 111 days). One monkey from the HDA group and all three from the LDA group had longer survival, with three of these four animals achieving self cure. Establishment of disseminated disease with promastigotes has been reported in vervets for the L. major parasite (Githure et al., 1986). It seems that V.L.-like disease can be established with either promastigotes or amastigotes if an adequate number of parasites is given intravenously. Our findings suggest that the inoculating dose is critical for achieving adequate infection, since lower doses resulted in milder disease and self cure. It has been shown in a number of models for infectious diseases that the infecting dose is an important factor in the type of disease that will develop. The influence of parasite dose on the development of Leishmania infection has been demonstrated in the murine model for L. major (Bretscher et al., 1992). The leishmania specific IgG response gradually increased following infection, plateaued by day 60 p.i., and was maintained up to day 105 p.i. Antibody levels were comparable in the three groups of animals and also in the three animals who

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had spontaneous cure. In V.L., there is an exponential increase in antibodies probably due to polyclonal activation of B cells resulting in the marked elevation of IgG and IgM levels against various proteins and haptens. These antibodies are non-protective (Rezai et al., 1978). An elevated antibody response has also been reported in the vervet model and African green monkeys infected with L. major and L. dono6ani, respectively (Olobo et al., 1992; Binhazim et al., 1993). In human V.L., no correlation has been found between disease type or severity and the antibody response. All the monkeys showed a good lymphoproliferative response to the common T-cell mitogens, PHA and Con A prior to infection, but the responses showed a dramatic fall on day 80 p.i. (Figs. 2 and 3) that recovered on day 100 p.i. The values of PHA and Con A stimulation of the normal monkey were also low on days 60 and 80 p.i.; however, it is difficult to comment on the results of one animal. The responses were decreased to the same extent in the animals that progressed towards self cure. A depressed response to PHA was reported in squirrel monkeys infected with L dono6ani by the intravenous route (Dennis et al., 1986) but these authors found no alteration in the response to Con A, in sharp contrast to our finding of parallel depression to both mitogens. The findings reported by Dennis et al. (1986) are rather surprising since both PHA and Con A are known to stimulate the same population of T cells by similar mechanisms of cell activation. Using a protocol similar to ours, Binhazim et al. (1993) found very little alteration in the Con A responses of African green monkeys infected with L. dono6ani amastigotes; they also reported that at 12 weeks, the antibody responses peaked and the Con A

Fig. 3. The lymphoproliferative responses of control and experimental groups to Con A (5 mg/ml). Values are expressed as mean cpm 9SEM of the three monkeys in each group.*, SEM bar is not shown since bar represents value of only one monkey.

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responses showed a mild decrease coinciding with the peak parasite burden. It appears that the pattern of disease is very similar in the two monkey models. The antibody levels in the African green monkey model doubled compared with a 3.5 times rise in our monkeys; the T cell responses were also milder in their model. Marked depression of T-cell lymphoproliferative responses to mitogens has also been reported in human V.L. The present observations suggest that our langur monkeys develop a disease that is more akin to human disease. None of the monkeys in the three groups showed any lymphoproliferative response to L dono6ani antigen prior to infection or up to 135 days p.i., including those who died by 120 days p.i. Lymphoproliferative responses were only seen on day 184 p.i. in the three monkeys who survived. These monkeys had not shown any response when they were tested on days 60, 80, and 100 p.i. Thus, it appears that antigen specific responses are mounted late and were only demonstrable in monkeys which survived for adequate time. These animals had no parasites in their splenic aspirates on day 175 p.i. The antibody responses in these three monkeys also fell from the mean level of 0.659 0.21 on day 105 p.i. to 0.24, 0.28 and 0.18 on day 180 p.i., which was close to the cut-off value for normal monkeys (0.22). An inverse relationship between DTH responses which reflect cell mediated immunity and antibody levels has been demonstrated in human V.L. during evolution of the disease (Turk and Bryceson, 1971). Similar evidence has also been put forward in studies with BALB/c mice (Sacks et al., 1984) and in vervet monkeys (Gicheru et al., 1985). While we realize that our observations are based on small number of animals and we would not like to draw any definitive conclusions, the data suggest that development of antigen specific lymphoproliferative responses along with loss of the antibody response make this model closely resemble human V.L. We are aware that prolonged observation of the infected animals up to their death/recovery, could inflict suffering to the animals. However, we felt that these experiments were justified for validation of the animal model, especially in view of the fact that progressive untreated disease in man is also fatal. The Institutional Ethics Committee accepted the justification and suggested for judicious planning of future experiments. The immunological findings from the present study, in the light of previous studies, suggest that the langur model is very akin to the human situation. Studies on the type of helper T lymphocyte profile of cytokines, which has been shown to be closely associated with the clinical outcome of infection, would further strengthen the potentiality of langur as a model for V.L.

Acknowledgements The technical assistance by S.C. Bhar is gratefully acknowledged. JKS, PS and AC were receipients of the Scientist Pool Scheme (Government of India), Research Associateship (CSIR, New Delhi) and Junior Research Fellowhip (WHO, Geneva), respectively. The Ivestigation received financial support from the UNDP/World

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Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR). This is CDRI Communication No. 5576.

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