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Immune response against Leishmania antigens in dogs naturally and experimentally infected with Leishmania infantum


Academic year: 2021

Partager "Immune response against Leishmania antigens in dogs naturally and experimentally infected with Leishmania infantum"


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Immune response against Leishmania antigens in dogs naturally and experimentally

infected with Leishmania infantum

Abdelkebir Rhalem


, Hamid Sahibi


, Nezha Guessous-Idrissi


, Saadia Lasri


, Amale Natami


, Myriam Riyad


, Boumediane Berrag


aDeÂpartement 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


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 (nˆ11) and asymptomatic (nˆ5) 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 : 100ˆ0.192; at 1 : 1000ˆ0.052 control 2: at 1 : 100ˆ0.098; at 1 : 1000ˆ0.048 control 3: at 1 : 100ˆ0.100; at 1 : 1000ˆ0.052 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 CD4‡and CD8‡T-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.


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.


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In view of the undocumented disease burden of ETEC- associated diarrhea in the Federal Capital Territory (FCT, Abuja, Nigeria), this study aimed to not only determine

The strength of the T cell response against a surrogate tumor antigen (TA) induced by oncolytic vesicular stomatitis virus (VSV) therapy does not correlate with tumor control..

infantum native and recombinant proteins indicate that in a cohort of domestic dogs living in Tunisian endemic areas, the antibody titers of specific Tot IgG, IgG1 and IgG2

Within the emerging focus, where infecting bites are probably fewer than in the old focus, we showed a significant IFN-γ production only in naïve individuals, suggesting that