Immune response against Leishmania antigens in dogs naturally and experimentally
infected with Leishmania infantum
Abdelkebir Rhalem
a,*, Hamid Sahibi
a, Nezha Guessous-Idrissi
b, Saadia Lasri
a, Amale Natami
a, Myriam Riyad
b, Boumediane Berrag
aaDeÂpartement de Parasitologie et Maladies Parasitaires, Institut Agronomique et Veterinaire Hassan II, Rabat-Instituts, Rabat, BP. 6202, Morocco
bLaboratoire de Parasitologie-Mycologie. Faculte de MeÂdecine et de Pharmacie, 19 Rue Tarik Ibnou Ziad Casablanca, Morocco
Received 25 March 1998; accepted 1 October 1998
Abstract
Cell-mediated and humoral immune response in naturally and experimentally infected dogs was studied using crude and pure antigens. Both types of infections induced severe signs of visceral disease, but the symptoms observed in natural infections were more pronounced than in experimental infections. In addition, asymptomatic infections were not observed in experimentally infected animals. Disease evolution in laboratory infections was rapid and an increase in antibody titer to crude parasite antigen was correlated with the appearance and aggravation of clinical symptoms. Peripheral blood lymphocyte proliferation to crude antigen and pure gp63 was observed early following experimental infection, but was abolished once the infected dogs began to exhibit clinical signs. A similar pattern was observed in naturally infected dogs. Serum from all patent dogs showed high antibody titers to rK39 in enzyme-linked immunosorbent assays (ELISA), and reacted by western blotting with several antigens, 12 to 120 KDa, including gp63 and gp70. In the case of asymptomatic dogs, antibody titers to crude antigen were low and only a few antigens were identified by western blotting. None of the pure proteins examined, gp63, gp70, and rK39 were recognized by western blotting or ELISA. However, asymptomatic dogs exhibited specific lymphocyte proliferation to both crude antigen and the potential vaccine candidate gp63.#1999 Elsevier Science B.V. All rights reserved.
Keywords: Leishmania infantum; Dog; Natural and experimental infection; Humoral and cellular responses;
Crude antigen; Pure proteins
* Corresponding author. Tel.: +212-7-77-1745; fax: +212-7-680424.
0304-4017/99/$ ± see front matter#1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 2 4 0 - 4
1. Introduction
Visceral leishmaniasis (VL) caused by members of theLeishmania donovanicomplex is a fatal disease of children and adults (Pearson et al., 1983), and is characterized by hepatosplenomegaly, anemia, immunosuppression, hypergamma-globulinemia and fever (Abranches et al., 1991b). In the Mediterranean Region, human disease, primarily found in children, is caused byL. infantum. In this region, dogs represent the main reservoir for the parasite (Mansueto et al., 1982; Delmont, 1983; Abranches et al., 1991a) and the prevalence of canine VL varies between 1±37% (Bettini and Gradoni, 1986). With the exception of depilation, onychogryphosis and emaciation which are typical symptoms of canine VL (Adler, 1936), all the other symptoms of canine disease are similar to human VL (Bettini and Gradoni, 1986; Peters and Killick-Kendrick, 1987). Human studies suggest that at least 15% of the population exposed to this parasite develop asymptomatic infections which eventually self-cure without recourse to chemotherapy (Jahn et al., 1986; Badaro et al., 1986). Similar reports have been made for canine VL (Pozio et al., 1981).
At present, there is no evidence that anti-leishmanial antibodies play an important role in resistance to established infection (Mitchell, 1984). While studies using mouse models demonstrated that protective immunity against leishmaniasis is cell-mediated, (Sheppard et al., 1983; Scott et al., 1989; Liew, 1990), information on the role of T cells and cytokines in canine disease is limited. In order to identify the immunological mechanisms underlying the resistance or susceptibility of dogs to leishmanial infections, studies on canine VL need to be conducted. In this study, the immune responses of experimentally and naturally infected dogs are compared and correlated with their clinical status.
Lymphocyte proliferation and antibody reactivity of dogs with asymptomatic and patent infections to crude and pure proteins is examined in an attempt to identify antigens associated with immune protection in resistant dogs.
2. Materials and methods
2.1. Animals
2.1.1. Natural infection
Infected dogs were collected in the Khemisset region where the prevalence of canine VL is 23.6% (Berrag et al., 1996). The dogs used in this study were followed in the field for at least one year prior to sampling. Sixteen seropositive dogs were grouped as Leishmania susceptible or resistant animals on the basis of the clinical and parasite examination. Parasitological examinations were carried out by popliteal lymph node and spleen tissues biopsy. Preparations stained with Giemsa were examined by microscopy and material was cultured for parasites in NNN medium. Parasites isolated from infected sick dogs (two isolates) were typed by isoenzyme electrophoresis on cellulose acetate using ten enzymes and shown to be L. infantum with an isoenzyme pattern indistinguishable from reference strain MHOM/TN/80/IPT1 zymo- deme MON-1.
2.1.2. Experimental infection
Four mixed-breed dogs, approximately 1 year old (12±17 kg) were acquired from Rabat pound and kept under observation for two months. They were tested by enzyme- linked immunosorbent assay (ELISA) prior to experimental infection. Infection was carried out using aL. infantumMHOM/TN/80/IPT1, zymodeme MON-1 strain isolated from naturally infected poly-symptomatic dogs. Healthy dogs were infected by the intravenous route at a dose of 105 amastigotes/ kg body weight, and kept under observation for clinical signs and immunological analysis. Amastigotes used in the infection were produced directly from spleen homogenates of sick dogs.
2.2. ELISA and Western blot analysis 2.2.1. Antigens
L. infantumpromastigotes were cultured in Schneiders Drosophila medium containing 10% fetal calf serum,L-glutamine (2 mM, GIBCO), penicillin (100 U/ml, GIBCO) and streptomycin (100mg/ml, GIBCO). Stationary phase parasites (about 2.108cells) were washed by centrifugation three times with PBS (400 g, 10 min at 48C). The pellet was maintained on ice and resuspended in cold lysis buffer (20 mM Tris-HCl, 40 mM NaCl, pH 7.4, containing the proteolytic inhibitors 2 mM phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, 1 mg/ml leupeptin and 5 mM ethylene diamine tetra acetic acid). Protein concentration was determined according to Bradford (Bradford, 1976). Bovine serum albumin was used as a standard.
2.2.2. ELISA
Microtiter plates were coated with the parasite antigen (crude promastigote membranes, 100mg/ml, or recombinant K39, 500 ng/ml). Assays using crude antigen were carried out as previously described (Jaffe et al., 1988) using the dog sera at 1 : 100 and 1 : 1000 dilutions. For assays using rK39 (Kindly provided by Dr. Steven Reed, Corixa Corporation, Seattle, WA), the coated plates were blocked with phosphate buffered saline, pH 7.2 containing 1.0% Tween-20 (PBS-T 1.0%, 1 h), washed and incubated with dog sera (1 : 100 dilution) for 30 min at room temperature. After washing thrice with PBS-T, the plates were incubated with protein A conjugated to horseradish peroxidase (1 : 8000 dilution, Zymed Laboratories Inc., CA, 30 min at 378C).
The plates were washed several times, the substrate 2,20-azino-bis (3-ethylbenzthiazoline) sulfonate (ABTS; Sigma Chemical Co., MO) added, and the absorbance at 405 nm read after 10±30 minutes. Negative and positive controls were included on each plate. Positive/negative serum ratios3 is considered positive and the negative cut off equal 3.
2.2.3. Western blotting technique
Identification of parasite antigens recognized by the serum antibodies was carried out following the separation of total promastigote antigens by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (Laemmli, 1970;
Towbin et al., 1979). Lysates from promastigotesLeishmania infantum (130mg/gel) as well as the pure proteins purified gp70 (Jaffe and Zalis, 1988a) and gp63 (Bouvier et al.,
1985) at 0.8mg/gel were run on 10% polyacrylamide gel prepared in Tris-HCl buffer.
Following SDS-PAGE, the separated proteins were transferred to nitrocellulose sheets.
The sheets were blocked with 0.3% PBS-T, washed twice and incubated with sera. Two dilutions of sera were used: 1 : 1000 for crude antigen and 1 : 100 for pure proteins. The nitrocellulose paper was then washed and probed with a protein A±horseradish peroxidase conjugate (1 : 8000 dilution in PBS-T 0.5% plus 2% fetal calf serum).
Excess of second antibody was removed by washing and the membranes were incubated with the substrate 3,30diaminobenzidine (30 mg/ml).
2.3. Lymphocyte proliferation
Promastigotes were resuspended directly in RPMI, freeze-thawed three times, centrifuged (8000g, 30 min, 48C) and the resulting supernatents collected. Protein concentration was determined using the Bradford assay.
Peripheral blood mononuclear cells (PBMC) were separated on Ficoll-Hypaque by centrifugation at room temperature for 25 min at 400g. Cells (105 cells/well) were cultured in flat bottom 96-well microtiter plate in RPMI containingL-glutamine (2 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml), HEPES (2 mM), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), 2-mercaptoethanol (510ÿ5M) and 5%
fetal calf serum (complete tissue culture medium).The cells were incubated (378C, 5%
CO2) with or without gp63 or parasite lysate. Antigen concentrations used are given in the results. On day four, 5-bromo-200-deoxy uridine (BrdU) was added for 18 h and cell proliferation determined using a non-radioactive ELISA technique, according to the manufacturers instructions (BrdU, cell proliferation detection Kit III, Boehringer Mannheim, Germany). Mitogenic proliferation was also tested using concanavalin A (Con A). Concentrations used are given in the results. The same procedure was used, except that the BrdU was added overnight on day 3.
2.4. Statistical analysis
Data were analysed by Student's t test.
3. Results
3.1. Susceptible and resistant dogs:
Naturally infected dogs were subdivided into symptomatic (n11) and asymptomatic (n5) groups on the basis of clinical and parasitological examinations. All symptomatic dogs were parasitologically positive at the time of sampling and the asymptomatic dogs were negative. In the experimental infected dogs, initial clinical signs of canine VL were observed by 9 weeks post-infection. Severe symptoms of VL, except for adenopathy, and similar to those observed in field with dogs suffering from natural disease were seen by 3 months. Lack of adenopathy in the experimentally infected dogs may be due to the inoculation route used. By 4 months post-infection, all the experimental dogs could be
considered as oligo-symptomatic (except one dog which was poly-symptomatic) and were included in the susceptible group.
3.2. Antileishmanial antibodies in the susceptible and resistant dogs
Antibodies to total promastigote lysate (crude antigen) and rK39, a cloned antigen ofL.
chagasi recently used for the serodiagnosis of human VL (Burns et al., 1993) were examined.
In an ELISA carried out using crude antigens, asymptomatic dogs (K23, K5, K123, K18, K30, Table 1) were clearly seropositive at a 1 : 100 serum dilution when compared with sera obtained from control animals. However, the absorbance values found for the asymptomatic dogs were generally lower than those observed for symptomatic dogs. At higher serum dilutions (1 : 1000) the asymptomatic dogs gave negative reactions, while both groups of symptomatic dogs remained seropositive. Antibody reactivity in the naturally infected dogs appeared to be slightly (P< 0.05) greater than that for the experimentally infected dogs and decreased less at higher serum dilutions (1 : 1000), suggesting that the antibody titers for the latter dogs is higher.
All of the asymptomatic dogs tested seronegative on rK39 (Table 2). In contrast, the sick dogs were clearly seropositive with rK39. The absorbances ranged from 0.377 to 1.222 for dogs both naturally and experimentally infected. The intensity of reactivity on
Table 1
Absorbance values (OD at 405 nm) with crude antigen for asymptomatic and sick dogs, naturally and experimentally infected
E.I. (sick) N.I. (sick) N.I. (asymptomatic)
Dog Dilution
of sera Dog Dilution
of sera Dog Dilution
of sera
1 : 100 1 : 1000 1 : 100 1 : 1000 1 : 100 1 : 1000
Sa1 0.887 0.418 Kh4 1.106 1.115 K23 0.743 0.107
Ma2 1.027 0.888 Kh3 1.047 0.608 K5 0.574 0.091
Lo3 1.118 1.139 Kh1 1.069 1.103 K123 0.743 1.102
Va4 0.739 0.426 Kh2 1.104 0.690 K18 0.671 0.057
Kh5 1.045 1.056 K30 0.604 0.108
Kh6 1.020 1.012
K12 1.105 1.115
K14 1.069 1.017
K19 1.108 1.118
K21 1.061 1.101
K13 1.055 1.086
Absorbance values of control dogs free of leishmania:
control 1: at 1 : 1000.192; at 1 : 10000.052 control 2: at 1 : 1000.098; at 1 : 10000.048 control 3: at 1 : 1000.100; at 1 : 10000.052 OD: Optical density.
E.I.: Experimentally infected dogs.
N.I.: Naturally infected dogs.
rK39 appeared to be related to the antibody titer against total promastigote antigen, with dogs showing high reactivity on crude antigen reacting stronger with rK39.
3.3. Parasite antigens recognized by serum antibodies from susceptible and resistant dogs
Sera from sick dogs recognized by western blotting several different promastigote antigens ranging in MW from 12 to 116 KDa. There was some variation in the number and size of the antigens recognized by the naturally (Fig. 1) and experimentally (Fig. 2) infected dogs. In general, those animals with high antibody titres by crude and rK39 ELISA recognized more antigens. Profile found for dog Lo3is the same as for naturally infected dogs except for dogs Kh3and Kh2which had the same profile as experimentally infected dogs. All of the symptomatic dogs recognized gp63 and gp70 (Figs. 1 and 2), though some variation in the signal intensities was observed between naturally and experimentally infected dogs. Sera of experimentally infected dogs, except for dog Lo3, and sera of two naturally infected dogs (Kh3and Kh2) reacted weakly with the two pure proteins. Gp63 and gp70 are not recognized by sera from dogs in the prepatent phase of infection. As might be expected, there was a strong positive correlation between the recognition of rK39 by ELISA, and gp63 and gp70 by western blotting. None of the pure proteins reacted with serum from asymptomatic dogs.
Table 2
Absorbance values (OD at 405 nm) with rK39 for asymptomatic and sick dogs, naturally and experimentally infected
E.I. sick
dogs N.I. sick
dogs N.I. asymptomatic dogs
Sa1 0.377 Kh4 1.137 K23 0.183
Ma2 0.486 Kh3 1.441 K5 0.097
Lo3 1.222 Kh1 1.108 K123 1.112
Va4 0.429 Kh2 1.489 K18 0.112
Kh5 1.106 K30 0.114
Kh6 1.009
K12 1.112
K14 1.039
K19 1.111
K21 1.106
K13 1.786
Absorbance values of control dogs free of leishmania Control 1: 0.135
Control 2: 0.130 Control 3: 0.128 OD: Optical density.
EI: Experimentally infected dogs.
NI: Naturally infected dogs
Lo3: Experimentally infected dog which showed the same high absorbance as dogs naturally infected.
Kh3 and Kh2: Two naturally infected dogs which showed a low absorbance comparable to experimentally infected dogs.
Fig. 1. Recognition of leishmanial antigens by antibodies from naturally infected sick dogs. Western blots following SDS-PAGE were probed with naturally sick dogs sera diluted to 1 : 1000 for the crude and 1 : 100 for the pure protein antigens. Either crude L. infantum antigens (130mg/gel): Fig. 1(a), pure gp63 (0.8mg/
gel):Fig. 1(b) or pure gp70 (0.8mg/gel):Fig. 1(c), were used as antigen.
Fig. 2. Recognition of leishmanial antigens by antibodies from experimentally infected dogs. Western blots following SDS-PAGE were probed with experimentally sick dogs sera diluted to 1 : 1000 for the crude and 1 : 100 for the pure protein antigens. Either crudeL.infantumantigens (130mg/gel): Fig. 1(a), pure gp63 (0.8mg/
gel): Fig. 1(b) or pure gp70 (0.8mg/gel): Fig. 1(c), were used as antigen.
Fourteen additional proteins, nine between 31 and 48 KDa and five below 20 KDa also reacted with sera from sick dogs. One antigen, 21 KDa, was recognized by all sera examined including those from asymptomatic dogs. Sera from two naturally infected dogs reacted strongly with a 116 KDa protein. Finally, a total of 18 antigens were recognized by sera of sick dogs. Sera of asymptomatic dogs reacted just with few proteins (Fig. 3).
3.4. Lymphocyte proliferation responses
PBMC from all the dogs, both asymptomatic and symptomatic, proliferated to the mitogenic ConA (Table 3). However, the cells of sick dogs responded in a weaker manner than those from control and asymptomatic dogs. The best proliferation was observed at 2.5mg/ml Con A.
Antigen specific proliferation was examined using several concentration of crude promastigote antigen (1±10mg/ml) or pure gp63 (0.05±2mg/ml). The proliferation of PBMC from naturally and experimentally infected sick dogs to both crude antigen and gp63 was inhibited (Table 3). On the other hand, asymptomatic dogs demonstrated significant (P< 0.05) antigen specific proliferation. Peak PBMC proliferation for these dogs was observed at 1mg/ml crude antigen and at 0.5mg/ml gp63. The latter gave a higher cell stimulation than crude antigen.
Fig. 3. Recognition of leishmanial antigens by antibodies from asymptomatic dogs. Western blots following SDS-PAGE were probed with asymptomatic dogs sera diluted to 1 : 1000 for the crude and 1 : 100 for the pure protein antigens. Either crudeL.infantumantigens (130mg/gel):Fig. 1(a), pure gp63 (0.8mg/gel): Fig. 1(b) or pure gp70 (0.8mg/gel):Fig. 1(c), were used as antigen.
4. Discussion
The pathogenicity ofL. infantumstrain (two isolates) isolated from naturally infected dogs was confirmed under controlled conditions, demonstrating that the intravenous route of injection can successfully mimic natural disease in experimentally infected dogs.
When the immune responses of dogs resistant or susceptible to L. infantum were compared, we observed marked differences in both the humoral and cellular responses of these groups to the parasite.
The differences between these groups were clearly shown by ELISA and western blotting using the proteins rK39, gp63 and gp70. While an ELISA using crude parasite antigen, especially at 1 : 1000 dilution, was useful in distinguishing between asymptomatic and symptomatic dogs, such assays can lack specificity and frequently show false positive reactions at high serum concentrations. The assays using the pure and recombinant leishmanial proteins were both specific and sensitive for canine VL (Badaro et al., 1996). A significant correlation was observed between serum reactivity against rK39, gp63 or gp70, and the appearence of clinical symptoms of acute canine VL.
Likewise, both rK39 (Badaro et al., 1996) and gp70 (Jaffe and Zalis, 1988b) have been useful for the diagnosis of patent human VL.
Table 3
Cellular response of sick and asymptomatic dogs
Proliferative response of PBMC OD at 405 nm (colorimetric method)
Dog's reference Crude antigen (1mg) Gp63 (0.5mg) Con A (2.5mg)
Sa1 0.099 0.130 0.421
Ma2 0.121 0.111 0.589
Lo3 0.120 0087 0.560
Va4 0.112 0.110 0.420
Kh4 O.172 0.091 0.420
Kh3 0.151 0.112 0.300
Kh1 0.120 0.090 0.480
Kh2 0.116 0.112 0.400
Kh5 0.170 0.090 0.400
Kh6 0.085 0.098 0.480
K12 0.092 0.110 0.500
K14 0.100 0.111 0.399
K19 0.082 0.097 0.440
K21 0.120 0.089 0.500
K13 0.090 0.110 0.490
K23 0.670 0.700 0.689
K5 0.580 0.680 0.600
K123 0.630 0.680 0.701
K18 0.680 0.720 0.660
K30 0.630 0.690 0.700
Control 1 0.063 0.097 0.588
Control 2 0.060 0.063 0.600
Control 3 0.062 0.062 0.610
Blank well 0.065 0.065 0.065
Mitogenic proliferation of PBMC from dogs with patent disease, both experimentally and naturally infected, was not abolished. This is similar to results reported in other studies on experimentally infected dogs (Abranches et al., 1991b; Pinelli et al., 1994) and with natural infections in humans (Peters and Killick-Kendrick, 1987). In contrast, PBMC from sick dogs failed to respond in vitro to leishmanial crude antigen or gp63. Antigen specific immunosuppression appears to be an indicator of acute VL and was previously demonstrated in humans (Sacks et al., 1987) and experimental animals models (Scott and Farrell, 1981; Pinelli et al., 1994).
In the asymptomatic dogs, strong proliferation was observed with crude antigen and gp63. Interestingly, the reaction to gp63 was consistently better than with crude antigen.
These results suggest that gp63 is highly antigenic in dogs and that prepatent parasite produce good T-cell responses to specific epitopes on this molecule. Gp63, considered to be a prime vaccine candidate, has been the focus of biochemical and immunological studies. It is expressed on all leishmanial species examined so far and is present on both stages of the parasite (Eisenberger and Jaffe, 1997). T-cell responses to both native and recombinant gp63 have been reported in humans with active or cured cutaneous disease.
Both CD4and CD8T-cells from patients withL. mexicana amazonensisproliferate to gp63 and the cytokine response patterns of these cells were typical of the Th1 subset thought to be important in resistance to leishmanial infection. Furthermore, T-cell epitopes capable of inducing Th1 responses in both mice and human have been identified in the recombinant protein (Eisenberger and Jaffe, 1997).
Antibodies to gp63 are absent in asymptomatic dogs and only appear once clinical symptoms of disease become apparent. Antibody responses are inversely correlated with T-cell responses to the pure protein which disappear following the onset of active disease.
Studies in our laboratory show that clinical recovery by sick dogs following pentamidine treatment is associated with the recovery of T-cell proliferation to gp63 and to crude antigen. Following the successful treatment of canine VL, both antibody and T-cell responses to gp63 are present (Data not published). These results suggest that gp63 may be involved in the protection of dogs againstL. infantumand seem to support the general validity of additional studies using dog models. These and other studies (Pinelli et al., 1994) suggest that resistance or susceptibility inL. infantuminfected dogs, as in mouse models for cutaneous leishmaniasis, may be correlated with two dichotomous lymphocyte functions. A Th1 T-cell response, which produces INF-g, TNF-g and IL-2, is important in protection, or a Th2 T-cell response, which produces IL4 and IL10, results in disease (Locksley et al., 1987; Scott et al., 1988).
Finally, an antigen, 21 KDa, not seen in western blotting with sera from control healthy animals is recognized by sera from experimentally and naturally infected sick dogs, as well as asymptomatic dogs. This parasite antigen induces a rapid, early antibody response (Data not shown) and may be useful for both the early serodiagnosis of canine VL, as well as monitoring exposure to parasite infections.
Acknowledgements
This work was supported by the MERC Program Contracts No. NO1-A045186.We are grateful to Prof. Charle Jaffe from the Hadassah Medical School, Jerusalem, Israel, who
kindly provided the pure proteins for the serological and immunological diagnosis, and also edited this manuscript.
References
Abranches, P., Silva-Pereira, M.C.D., Conceicao-Silva, F.M., Santos-Gomes, G.M., Janz, J.G., 1991a. Canine leishmaniasis: Pathological and ecological factors influencing transmission of infection. J. Parasitol. 77, 557±561.
Abranches, P., Santos-Gomes, G., Rachamim, N., Campino, L., Schnur, L., Jaffe, C.L., 1991b. An experimental model for canine visceral leishmaniasis. Parasite Immunol. 13, 537±550.
Adler, S., 1936. Canine Visceral Leishmaniasis with special reference to its relationship to human visceral leishmaniasis. In 3e congress International de Pathologie CompareÂe, section Medicine humaine, Eleftheroudakis Co, Athens, pp. 1±10.
Badaro, R., Jones, T.C., Carvalho, E.M., Sampaio, D., Reed, S.G., Barral, A., Teixeira, R., Johnson, W.D., 1986.
New perspectives on a subclinical form of visceral leishmaniasis. J. Infect. Dis. 154, 1003±1011.
Badaro, R., Benson, D., Eulalio, M.C., Freire, S., Cunha, S., Netto, E.M., Pedral-Sampaio, D., Madureira, C., Burns, J.M., Houghton, R.L., David, J.R., Reed, S.G., 1996. rK39: A cloned antigen ofLeishmania chagasi that predicts active visceral leishmaniasis. J. Infect. Dis. 173, 758±761.
Bettini, S., Gradoni, L., 1986. Canine leishmaniasis in the Mediterranean area and its implications for human leishmaniasis. Insect Sci. Applic. 7, 241±245.
Berrag, B., Sahibi, H., Guessous-Idrissi, N., Rhalem, A., 1996. The severity ofLeishmania infantumin naturally infected dogs in Morocco. 45th Annual meeting of the Am. Soc. Trop. Med. Hyg. 55, pp. 288±289.
Bouvier, J., Etges, R.J., Bordier, C., 1985. Identification and purification of membrane and soluble forms of the major surface protein ofLeishmania promastigotes. J. Biol. Chem. 260, 15504±15509.
Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248±254.
Burns Jr., J.M., Shreffler, W.G., Benson, D.R., Ghalib, H.W., Badaro, R., Reed, S.G., 1993. Molecular characterization of a kinesin-related antigen ofLeishmania chagasithat detects specific antibody in African and American visceral leishmaniasis. Proc. Natl. Acad.Sci. USA. 90, 775±779.
Delmont, J., 1983. Les leishmanioses. La gazette de l'eÂtudiant. Gaz. Med. de France. 90, 2857±2868.
Eisenberger, C.L., Jaffe, C.L., 1997. Development of vaccines against leishmaniasis. In: Levine, M.M., Woodrow G. (Eds.), New Generation Vaccines, 2nd ed. Marcel Dekker, New York.
Jaffe, C.L., Keren, E., Nahary, O., Rachamim, N., Schnur, L., 1988. Canine visceral leishmaniasis at Wadi Hamam in Israel. Trans. R. Soc. Trop. Med. Hyg. 82, 852±853.
Jaffe, C.L., Zalis, M., 1988a. Purification of twoLeishmania donovanimembrane proteins recognized by sera from patients with visceral leishmaniasis. Mol. Biochem. Parasitol. 27, 53±62.
Jaffe, C.L., Zalis, M., 1988b. Use of purified parasite proteins fromLeishmania donovani for the rapid serodiagnosis of visceral leishmaniasis. J. Infect. Dis. 157, 1212±1219.
Jahn, A., Lelmett, J.M., Diesfeld, H.J., 1986. Seroepidemiological study on kala-azar in Baringo district. Kenyan J. Trop. Med. Hyg. 89, 91±104.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680±685.
Liew, F.Y., 1990. Regulation of cell-mediated immunity in leishmaniasis. Curr. Top. Microbiol. Immunol. 155, 53±64.
Locksley, R.M., Heinzel, F.P., Sadick, M.D., Holaday, B.J., Gaidner Jr., K.D., 1987. Murine cutaneous leishmaniasis: Susceptibility correlates with differential expansion of helper T-cell subsets. Ann. Institut.
Pasteur. Immunol. 138, 744±749.
Mansueto, S., Miceli, M.D., Quartararo, P., 1982. Counter immunoelectrophoresis (CIEP) and ELISA tests in the diagnosis of canine leishmaniasis. Ann. Trop. Med. Parasitol. 76, 229±231.
Mitchell, G.F., 1984. Host-protective immunity and its suppresion a parasitic disease: murine cutaneous leishmaniasis. Immunol. Today, 224±226.
Pearson, R.D., Wheeler, D.A., Harrison, L.H., Kay, H.D., 1983. The immunobiology of leishmaniasis. Rev.
Infect. Dis. 5, 907±927.
Peters, W., Killick-Kendrick, R. (Eds.), 1987. The leishmaniasis in Biology and Medicine. vols I and II Academic Press, New York, p. 941.
Pinelli, E., Killick-Kendrick, R., Wagenaar, J., Bernadina, W., Real, G.D., Ruitenberg, J., 1994. Cellular and humoral immune responses in dogs experimentally and naturally infected with Leishmania infantum.
Infect. Immun. 62, 229±235.
Pozio, E., Gradoni, L., Bettini, S., Gramiccia, M., 1981. Leishmaniasis in Tuscany (Italy): VI. Canine leishmaniasis in the focus of Monte Argentario (Grosseto). Acta Tropica. 38, 383±393.
Sacks, D.L., Lal, S.L., Shrivastava, S.N., Blackwell, J., Neva, F.A., 1987. An analysis of T cell responsiveness in Indian Kala-azar. J. Immunol. 138, 908±913.
Scott, P.A., Pearce, E., Cheever, A.W., Coffmann, R.L., Sher, A., 1989. Role of cytokines and CD4T cell subsets in the regulation of positive immunity and disease. Immunol. Rev. 112, 161±182.
Scott, P.A., Farrell, J.P., 1981. Experimental cutaneous leishmaniasis. I. Non-specific immunodepression in BALB/C mice infected withLeishmania tropica. J. Immunol. 127, 2395±2400.
Scott, P., Natovitz, P., Coffman, R.L., Pearce, E., Sher, A., 1988. Immunoregulation of cutaneous leishmaniasis:
T cell lines that transfer protective immunity or exacerbation belong to different T helper subsets and respond to distinct parasite antigens. J. Exp. Med. 168, 1675±1684.
Sheppard, H.W., Scott, P.A., Dwyer, D.M., 1983. Recognition ofLeishmania donovaniantigens by murine T lymphocyte lines and clones. J. Immunol. 131, 1496±1503.
Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Nat. Acad. Sci. 76, 4350±4354.
