The immune response and PBMC subsets in

canine visceral leishmaniasis before,

and after, chemotherapy

Javier Morenoa, Javier Nietoa, Cristina Chamizoa,Fernando GonzaÂlezb, Fernando Blancoc,

Douglas C. Barkerd, Jorge Alvara,*

aWHO Collaborating Centre for Leishmaniasis, Research Unit for Tropical Diseases and International Health.

Centro Nacional de MicrobiologõÂa, Instituto de Carlos III, Ctra. Majadahonda-Pozuelo,

Km 2.2, 28220- Majadahonda, Madrid, SpainbDpto. de ToxicologõÂa y FarmacologõÂa, Facultad de Veterinaria, Universidad Complutense,

28040-Madrid, SpaincServicios MeÂdicos de IBERIA, Madrid, Spain

dPathology Department, University of Cambridge, Cambridge CB2 1TS, UK

Received 19 February 1999; received in revised form 16 June 1999; accepted 24 June 1999

Abstract

Peripheral blood mononuclear cell subsets, in vitro lymphoproliferative response to leishmanial

antigen, and Leishmania-specific serum antibody levels were examined in 11 dogs, naturally

infected with L. infantum, and 9 healthy control dogs. A decrease in the percentage of CD4� T-cells

and an increase in the proportion of gd T-cells and sIgG� B-cells were observed during canine

visceral leishmaniasis (CVL). These changes may be responsible for the marked humoral response

and the absence of in vitro lymphoproliferation to mitogen and specific parasite antigens. This

possibility was supported by the analysis of these subsets after treatment with amphotericin B. One

month after therapy, a significant increase in the percentage of CD4� T-cells and a decrease of gdT-cells and sIgG� B-cells were observed. At the same time, the lymphocyte blastogenesis assay

with leishmanial antigen was positive and the levels of specific antibodies to Leishmania were

significantly lower than before the treatment. Five months after therapy, lymphocyte proliferative

response to LSA disappeared, antibody and lymphocyte subsets levels returned to those observed

during CVL. Therapeutic failure in CVL is associated with the inability of antileishmanial drugs to

Veterinary Immunology and Immunopathology

71 (1999) 181±195

* Corresponding author. Tel.: �34-1-509-79-78; fax: �34-1-509-70-34

E-mail address: jalvar@isciii.es (J. Alvar)

0165-2427/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 5 - 2 4 2 7 ( 9 9 ) 0 0 0 9 6 - 3

completely revert the profound immunodepression induced by the infection and prevent relapse.

# 1999 Elsevier Science B.V. All rights reserved.

Keywords: Canine visceral leishmaniasis; Immune response; Leishmania infantum; Chemotherapy; Lymphoid

subsets

1. Introduction

Zoonotic visceral leishmaniasis, caused by both, Leishmania infantum and L. chagasi,

represents 20% of human visceral leishmaniasis in the world (100 000 cases annually)

and its incidence is growing in urban and periurban areas of the tropics (Dye, 1996). The

dog constitutes the main domestic reservoir of these parasites and plays a central role in

the transmission cycle of the parasite to humans by phlebotomine sandflies. Current

strategies to control zoonotic visceral leishmaniasis have proved ineffective; the killing of

seropositive dogs is expensive, socially not acceptable (Tesh, 1995; Ashford, 1996), and

its impact on human disease has been partial (Ashford et al., 1998) or null (Dietze et al.,

1997). Antileishmanial drugs are generally expensive and ineffective in the case of dogs

(Ashford, 1996) and only a temporary remission of the clinical symptoms out

parasitological clearance is obtained (Alvar et al., 1994; Oliva et al., 1995). Therapeutic

failure has important epidemiological implications, as we have previously shown dogs

remain asymptomatic, but become infective to sandflies early after treatment (Alvar et al.,

1994). Furthermore, successive courses of treatment after relapses could induce resistant

strains of Leishmania, which would represent a clear risk for human health (Gramiccia et

al., 1992).

Human visceral leishmaniasis is characterised by a depression of cell-mediated

immunity to Leishmania spp. and a marked humoral response. Patients have negative

delayed-type hypersensitivity skin test (Manson-Bahr, 1961), no response in lymphocyte

proliferation assay in vitro (Carvalho et al., 1981) and decreased IL-2 and IFN-gproduction when cultured with parasite antigens (Carvalho et al., 1985). Restoration of

cell-mediated immunity to the parasite is necessary for an effective pentavalent

antimonial therapy (Carvalho et al., 1981; Murray et al., 1989).

In murine visceral leishmaniasis, susceptibility to L. donovani infection is associated

with the loss of capacity of spleen cells to produce IFN-g in vitro (Kaye et al., 1991).

Posterior development of parasite-specific cell-mediated response requires both, CD4�and CD8� T-cells (Stern et al., 1988), and reduces the parasite burden by the production

of endogenous IFN-g and TNF-a, macrophages activation and the formation of hepatic

granulomas (Kaye et al., 1991; Murray et al., 1987; Tumang et al., 1994).

In the case of CVL, the cellular basis of immunosupression remains unknown.

Although both, naturally and experimentally infected dogs are able to present

lymphoproliferative responses to leishmanial antigens, these observations were related

to the resistant status of the dog (Abranches et al., 1991a; Cabral et al., 1992; Pinelli et

al., 1994). Studies on the restoration in sick dogs of cell-mediated immunity after

treatments are lacking.

182 J. Moreno et al. / Veterinary Immunology and Immunopathology 71 (1999) 181±195

In contrast to the abundant data existing about experimental murine leishmaniasis

(Liew and O'Donnell, 1993), little information is available on the immunological basis of

visceral leishmaniasis in dogs and on canine immunology in general, mainly due to the

lack of specific markers and reagents. The recent development of monoclonal antibodies

against canine homologues of human CD antigens (Cobbold and Metcalfe, 1994) has

provided the opportunity to define more precisely lymphoid subpopulations and to

analyse different functions related to them in both, normal and pathological conditions

(Cobbold et al., 1994).

The present work describes the changes in peripheral blood mononuclear cell

subpopulations and in the specific cell-mediated immunity to the parasite occurring in

naturally infected dogs before, and after, treatment with amphotericin B. Knowledge of

the host response and factors that may more precisely define the pathogenesis of the

disease and acquired resistance in the natural reservoirs host should contribute to the

rational development of the effective treatment of infected dogs.

2. Materials and methods

2.1. Dogs

Eleven naturally infected dogs of various breeds, 2±6 years old, were included in the

study. CVL diagnosis was confirmed, both by serological test (IFAT and ELISA) and

isolation of the parasite in NNN medium by culture of bone marrow aspirates, skin

biopsies or lymph node biopsies. The positive dogs were grouped according to clinical

pattern (Abranches et al., 1991b) as: asymptomatic, when there were no external signs

compatible with leishmaniasis; oligosymptomatic, when popliteal lymph node swelling

and/or furfuraceous eczema around the eyes and/or snout were present; or polysympto-

matic, when signs as for oligosymptomatic cases plus other signs compatible with

leishmaniasis were associated.

A group of nine healthy dogs with no symptoms of CVL, negative serology and no

isolation of parasites in NNN medium culture served as controls.

2.2. Treatment

Four infected dogs (cases 2, 3, 6 and 7): were treated, following the experimental

protocols: 25 mg/m2 of amphotericin B (Fungizona1, Bristol-Myers-Squibb, UK) diluted

in 35 ml of Intralipid1 (Pharmacia, Uppsala, Sweden) administered by intravenous

infusion (1 h) 3 days a week during 2 weeks.

2.3. Leishmania antigen preparation

Leishmania infantum zymodeme 1 (MHOM/FR/78/LEM 75) promastigotes were

grown at 288C in RPMI 1640 (Gibco, Paisley, UK), supplemented with 100 IU/ml

of penicillin, 100 mg/ml of streptomycin, 2 mM L-glutamine and 10% heat-inactivated

foetal calf serum (Boehringer Mannheim, Mannheim, Germany). After reaching

J. Moreno et al. / Veterinary Immunology and Immunopathology 71 (1999) 181±195 183

the stationary phase, the parasites were harvested, washed in PBS and used to prepare

soluble leishmanial antigen as previously described (Scott et al., 1987). Protein

concentrations were determined using the bicinchoninic acid protein assay (Pierce,

Rockford, IL).

2.4. Cell-proliferation assay

PBMCs were cultured at a density of 1 � 106 cells/ml in 0.2 ml of RPMI 1640 (Gibco,

Paisley, UK), supplemented with 100 UI/ml of penicillin, 100 mg/ml of streptomycin,

2 mM L-glutamine, 5 � 10ÿ5 M 2-mercaptoethanol and 10% heat-inactivated foetal calf

serum (Biological Industries, Israel). Assays were conducted in flat-bottomed 96-well

microtiter plates incubated (378C, 5% CO2) for 5 days with either 5 mg/ml PHA, 10 mg/

ml soluble leishmanial antigen (SLA) or without antigen (blank) and pulsed during the

last 24 h with 10 mM BrdU. BrdU incorporation was determined using a specific ELISA

system (BIOTRAK, Amersham, Buckinghamshire, UK) The results are expressed as the

mean increase of optical density with respect to blank, in triplicate cultures.

2.5. Indirect immunofluorescence antibody test (IFAT)

Stationary phase promastigotes were washed thrice in phosphate-buffered saline (PBS)

and 10 ml of a 2 � 107 parasites/ml suspension were dispensed in 15-well immuno-

fluorescence slides. Slides were air dried for 1 h at 378C and fixed with cold acetone

(ÿ208C) for 5 min. Sera from infected and control dogs were assayed in serial twofold

dilutions from 1/10 to 1/640 and incubated with parasites for 30 min at 378C. After three

washes in PBS, antibody fixation was revealed with FITC-conjugated sheep anti-dog IgG

(ICN, Aurora, OH), and diluted at 1/150 in 0.01% Evans blue for counterstaining. The

slides were then incubated for 30 min at 378C, washed and examined using a Zeiss

fluorescence microscope. The IFAT result was regarded as positive when a 1/80 dilution

of the serum gave fluorescence.

2.6. rK39 Antigen enzyme-linked immunosorbent assay (ELISA-rK39)

Infected and control dogs sera described above were tested by ELISA for antibody

levels to rK39 antigen as previously described (Burns et al., 1993). Sera were tested at

1 : 100 dilution. Each serum was assayed in duplicate. The optical density was read at

405 nm.

2.7. Flow cytometry

Heparinized blood samples were collected from the jugular vein and fractionated by

centrifugation (30 min at 800 � g) over lymphocyte separation medium (Rafer, Madrid,

Spain) at room temperature. PBMCs were obtained and were stored frozen in liquid

nitrogen until use.

For one colour analysis, 0.5 � 106 cells were incubated for 30 min. with the specific

monoclonal antibody (Table 1) and, after PBS washing, with FITC-conjugated rabbit F

184 J. Moreno et al. / Veterinary Immunology and Immunopathology 71 (1999) 181±195

(ab0)2 anti-rat Igs (Southern Biotechnology, Birmingham, AL) or FITC-conjugated goat

anti-mouse IgG (Tago, Burlingame, CA), depending on the specific mAb.

For two-colour analysis, cells were incubated with a specific monoclonal antibody

made in rat, followed by incubation with FITC-conjugated rabbit F(ab0)2 anti-rat Igs, then

incubated with a specific monoclonal antibody made in mouse (IgG1 isotype) and,

finally, with PE-conjugated goat anti-mouse IgG1 Igs (Southern Biotechnology,

Birmingham, AL).

Cells were fixed with 1% formaldehyde in PBS and relative immunofluorescence

intensities were measured by flow cytometry (EPICs 751, Coulter) after gating for

lymphoid cells.

2.8. Statistics

The mean value � SE of percentages of positive cells were calculated and differences

were evaluated by Student's t test.

3. Results

Sex, breed, clinical, serological and parasitological data of dogs included in this study

are shown in Table 2. Infected dogs showed clinical symptoms in a varied pattern, but all

presented high levels of specific serum antibodies to Leishmania detected by IFAT and by

rK39-ELISA. In all CVL cases, infection was confirmed by parasite isolation from

several biopsies (bone marrow, lymph node, skin and PBMCs) cultured in NNN medium.

Dogs from the control group did not present significant levels of specific serum

antibodies to L. infantum or the recombinant protein rK-39, and no parasites were isolated

from cultured biopsies.

Table 1

Monoclonal antibodies used in this study

Antigenic specificity Clone Dilution Source

Canine

CD3 CA17.2A12 1 : 10 Dr. Peter F. Moore

CD8 b CA15.4G2 1 : 10 University of California

CD11b CA16.3E10 1 : 10 USA

CD45RA CA4.ID3 1 : 10

TCR ab CA15.8G7 1 : 10

TCR gd CA20.6A3 1 : 10

CD4 YKIX 302.9 1 : 10 Serotec (England)

CD5 YKIX 322.3 1 : 10

CD8 a YCATE 55.9 1 : 300

MHC/Class II YKIX 334.2 1 : 10

IgG (H�L) Policlonal 1 : 80 ICN Pharmaceuticals (USA)

Human

CD14 TUK4 1 : 50 Dako (Denmark)

J. Moreno et al. / Veterinary Immunology and Immunopathology 71 (1999) 181±195 185

3.1. In vitro proliferative response to SLA and mitogens

No Leishmania specific proliferative response was observed in CVL dogs and the

response to PHA was low or absent in most of the infected dogs (Fig. 1). PBMCs from

controls showed high levels of lymphoproliferation when cultured in presence of PHA,

and no response to SLA (Fig. 1).

3.2. PBMCs populations in canine visceral leishmaniasis

The percentage of total T-cells identified by staining of PBMCs with both, CD3- and

CD5-specific antibodies showed a decreased of this population during CVL. This decline

was the result of the sharp decrease in the percentage of CD4� cells, specifically the

CD4�TCRab� cell subset. Within this T-helper population, the naive/memory state of

the cells (indicated by the expression of CD45RA molecules) also presented variation.

While 82% of the CD4� cells expressed CD45RA in controls, only 50% of the CD4�cells expressed CD45RA in CVL cases (Fig. 2).

The percentage of CD8� cells presented an elevation in dogs with L. infantum

infection. This increase corresponds, in part, to cells expressing the ab T-cell receptor,

Table 2

Profile, clinical, serological and parasitological characteristics of Leishmania-infected and control dogs

Group Dog Sexa Breed Clinical Serology Parasite

No. patternb

IFAT rK-39

ELISA

isolationc

Infected 1 M German shepherd Asymptom. 1/640 0.743 BM, LN

2 M German shepherd Asymptom. 1/160 1.321 BM, LN

3 M German shepherd Asymptom. 1/640 1.198 BM, LN

4 F Beagle Asymptom. 1/640 0.812 BM, LN, S

5 M Rottweiler Oligosympt. 1/640 2.179 LN

6 M Labrador retriever Oligosympt. 1/640 2.046 BM, LN

7 M Spaniel cocker Oligosympt. 1/640 2.227 BM, LN, Mon

8 F Boxer Oligosympt. 1/320 0.995 BM, LN

9 F Epagneul breton Polysympt. 1/320 0.767 LN, S

10 M Beagle Polysympt. 1/640 2.272 BM, LN, S

11 M Fox terrier Polysympt. 1/640 1.167 BM, LN, S

Control 12 M Beagle Healthy <1/40 0.028

13 F Beagle Healthy <1/40 0.010

14 M German shepherd Healthy <1/40 0.017

15 M German shepherd Healthy 1/40 0.028

16 M Spaniel cocker Healthy 1/40 0.022

17 F German shepherd Healthy <1/40 0.010

18 F Beagle Healthy <1/40 0.037

19 M German shepherd Healthy <1/40 0.059

20 M German shepherd Healthy <1/40 0.032

a M, male; F, female.b Asymptom, asymptomatic; Oligosympt, oligosymptomatic; Polysympt, polysymptomatic.c BM, Bone marrow; LN, lymph node; S, skin; Mon, peripheral blood mononuclear cells.

186 J. Moreno et al. / Veterinary Immunology and Immunopathology 71 (1999) 181±195

Fig. 1. Proliferative response of PBMCs from Leishmania-infected (CVL), and healthy control, dogs (Control)

stimulated in vitro with phytohemaglutinin (PHA) or Leishmania infantum soluble antigen (SLA). Each point

represents proliferative response of individual animals in O.D.450. PBMCs from dogs number 9 (CVL group) and

20 (control group) were not assayed. Bars represent arithmetic mean of O.D.450.

Fig. 2. Percentages of different lymphoid subsets in healthy controls and Leishmania-infected animals (CVL

cases). In controls, and dogs with CVL, mean percentage of CD4, CD8-negative (DN) TCR ab positive cells in

both groups are also shown. The results are expressed as the mean percentage � SE. Statistically significant

differences are marked as follows: ***(p � 0.01), **(p � 0.05), or *(p � 0.1).

J. Moreno et al. / Veterinary Immunology and Immunopathology 71 (1999) 181±195 187

and in other part to CD8� cells expressing the aa homodimer, as staining of PBMCs

with the anti-CD8 b-chain monoclonal antibody indicated. No variation of CD8� cells

expressing CD45RA between control and infected animals was observed (Fig. 2)

The proportion of cells bearing the gd T-cell receptor increased with the disease as did

the B-cell population (determined by IgG expression). On the contrary, there were no

changes on the CD4±CD8±TCRab� cell proportion and the expression of MHC/Class II

molecules in peripheral blood lymphocytes of CVL cases, when compared to healthy

animals (Fig. 2).

The relative number of monocytes (CD14�) in the peripheral blood of dogs did not

change with L. infantum infection, being the mean percentage of CD14� cells similar in

CVL cases and controls.

3.3. Changes after treatment

Drug treatment resulted in an improvement of the external signs of disease in all CVL

cases and dogs remained asymptomatic for several months. This clinical improvement did

not correlate with the serological and parasitological findings after treatment. One month

after therapy, lower levels of antibodies were recorded and isolation of parasites from

cultured biopsies was negative in most of the dogs. Leishmania-specific antibody levels

determined by IFAT returned to pre-treatment levels 5 months after administration of the

drugs, while rK-39-specific antibody levels remained lower despite positive. Dogs

remained with no external signs of leishmaniasis during the period of study (Table 3).

Chemotherapeutic regimen was able to restore a proliferative response in PBMCs

when cultured in presence of Leishmania antigen or mitogen. Treatment with

amphotericin B resulted in the recovery in all dogs of proliferative responses to PHA,

similar to that observed in controls (1.712 � 0.21 O.D.450), although only one of them

remained responsive at month 5. Dog Nos. 6 and 7 also showed a positive response when

cultured with SLA (1.450 and 1.266 O.D.450, respectively), although they all became

unresponsive at month 5 (0.175 � 0.10 O.D.450) (Fig. 3).

Changes in humoral and in vitro proliferative responses obtained were related to those

alterations observed in PBMCs subsets after treatment. Treated dogs showed a

Table 3

Serological and parasitological characteristic of Leishmania-infected dogs before, and after, treatment with

amphotericin B

Dog No. Before treatment After treatment

1 month 5 months

IFAT rK-39

ELISA

parasite

isolationa

IFAT rK-39

ELISA

parasite

isolationa

IFAT rK-39

ELISA

parasite

isolationa

2 1/160 1.321 BM, LN 1/160 0.367 Neg 1/640 0.309 LN

3 1/640 1.198 BM, LN 1/640 0.580 Neg 1/640 0.440 Neg

6 1/640 2.046 BM, LN 1/40 0.632 Neg 1/320 0.756 LN

7 1/640 2.224 BM, LN, Mon 1/320 0.800 BM 1/640 0.967 BM, LN, S

a BM, Bone marrow; LN, lymph node; S, skin; Mon, peripheral blood mononuclear cells; Neg, negative; n.t.non tested.

188 J. Moreno et al. / Veterinary Immunology and Immunopathology 71 (1999) 181±195

statistically significant elevation of the CD4�TCRab� cells percentage 1 month after

treatment while those of B-cell and TCRgd� populations were significantly reduced. All

subsets studied reached pre-treatment levels in the fifth month after treatment. The

Fig. 3. In vitro proliferative response of PBMCs from infected dogs before treatment (B.T.), 1 month (1 m), and

5 months (5 m) after amphotericin B chemotherapy, stimulated with phytohemaglutinin (PHA) or Leishmania

infantum soluble antigen (SLA). Each point represents proliferative response of individual animals in O.D.450.

Fig. 4. Percentage of CD4/ TCR ab, CD8/ TCR ab, TCR gd, and sIgG subsets before, 1 month and 5 months

after treatment with amphotericin B. (a) Bars represent mean percentage � SE. p � 0.05 significant differences

with mean percentage before treatment; and (b) p � 0.05 significant differences with mean percentage 5 months

after treatment.

J. Moreno et al. / Veterinary Immunology and Immunopathology 71 (1999) 181±195 189

percentage of CD8�TCRab� cells remained unaltered in this group during the period of

study (Fig. 4).

4. Discussion

CVL is characterised by a marked humoral response and lack of in vitro response to

Leishmania antigens (Abranches et al., 1991a), immunological manifestations which

have been confirmed by the clinical, parasitological, serological and immunological data

obtained in our work. The present study demonstrates that active CVL induce changes in

lymphoid subpopulations that are responsible for the severe depression of T-cell function

and the high levels of Leishmania specific serum antibodies observed in infected dogs.

The lack or low response to PHA mitogen also reflects the severity of this

immunodepression. Effectiveness of treatment in dogs depends on the recovery of these

lymphoid subpopulations to control values, which are accompanied by the appearance of

a specific T-cell response to Leishmania antigens.

Immunosupressive mechanisms underlying unresponsiveness are likely to be complex.

In human visceral leishmaniasis, it has been proposed that a serum factor (Barral et al.,

1986) and cell-mediated suppression (Carvalho et al., 1989) are involved in addition to

the loss of responsive T-cells (Carvalho et al., 1985; Rohtagi et al., 1996). The main

change confirmed by our results is the dramatic decrease of CD4� T-cells in CVL. The

same observation was made in human visceral leishmaniasis (Cenini et al., 1993) and,

recently, in L. infantum infected dogs (Bourdoiseau et al., 1997a), and it can be

considered an important cause of the diminished cellular immunity. We have also

observed in our laboratory (data not shown) that, in experimentally infected dogs, the loss

of T-helper cells occurs very early after infection, during the prepatent period, when no

other signs of the disease are evident. This decrease in T-cells could account for the

severity of the anergic state in infected dogs, reflected not only by the lack of specific

response to SLA but also by the absence in most CVL cases of proliferation in the

presence of mitogens, as previously reported in naturally infected dogs by Cabral et al.

(1992).

The mechanism responsible for the loss of CD4� T-cells is unknown. In experimental

murine leishmaniasis, it has been demonstrated that Leishmania-parasiteze macrophages

have decreased capability to express MHC Class II molecules (Reiner et al., 1988), and

are unable to deliver costimulatory signals to T-helper cells (Saha et al., 1995). A

decreased expression of costimulatory molecules has also been observed in Leishmania-

infected canine macrophages (Pinelli et al., 1999). Hence, it is conceivable that in

infected dogs, defective antigen presentation by Leishmania infected macrophages to T-

helper cells could induce anergy or programmed cell death of responding cells.

Increase of CD8� cells in peripheral blood has been reported in visceral leishmaniasis

patients (Cillari et al., 1988), although other authors have not found changes (Cenini et

al., 1993). Bourdoiseau et al. (1997a) have reported an increased proportion of CD8�cells in dogs with active visceral leishmaniasis. Nevertheless, the CD8� cell population is

a heterogeneous group of cell types. Therefore, the interpretation of the results is not

clear. CD8�TCRab� cell levels were increased in CVL, as well as the percentage of

190 J. Moreno et al. / Veterinary Immunology and Immunopathology 71 (1999) 181±195

CD8b chain expressing cells. However, this increase was less than of the total CD8�population. A more detailed and discriminatory characterisation of CD8� cell subsets in

dogs will be necessary to the understanding of their role in CVL. The percentage of

monocytes presented no differences between infected and control animals, findings that

agree with the in vitro observation that depletion of CD8� and adherent cells do not

restore the response to Leishmania antigens (Sacks et al., 1987; Carvalho et al., 1989).

The differences in the decrease of CD45RA molecule expression between CD4� T

cells and CD8� T cells indicate that T-helper cells are preferentially primed during CVL.

In contrast to previous report in human visceral leishmaniasis (Cenini et al., 1993; Cillari

et al., 1995), we have not observed an increase in the percentage of cells expressing

MHC/Class II molecules due to activation of T-cells. Although it is probably because the

expression of MHC/Class II molecules is constitutive on canine lymphocytes (Cobbold

and Metcalfe, 1994).

Elevation of TCR gd� cells has been demonstrated in human visceral leishmaniasis

(Cenini et al., 1993; Raziuddin et al., 1992; Russo et al., 1993), but this is the first time

this finding is reported in CVL. The role of TCR gd� cells in visceral leishmaniasis

remains unknown; however, these cells have been associated with the B-cell induced

humoral immune response including the secretion of high levels of BCGF and BCDF

involved in B-cell growth and differentiation (Raziuddin et al., 1992). In line with the

previous data we have also found an increased percentage of sIgG� B-cells consistent

with the high levels of Leishmania-specific serum antibodies detected. An elevation in the

proportion of B-cells has been previously described in CVL (Bourdoiseau et al., 1997a)

CVL is normally treated with antimonial drugs, that produce clinical improvement

although do not prevent relapses (Mancianti et al., 1988). Amphotericin B has been

previously used in the treatment of canine leishmaniasis with good results (Lamothe,

1997). In our case, treatment with amphotericin B included the administration of the drug

with a lipid vehicle in order to achieve lower toxicity, better drug distribution to infected

tissues, more specific targeting to macrophages and prolonged availability of the drugs in

the organism (Caillot et al., 1993). Experimental treatment regimen utilised in the present

study was designed to evaluate alternative chemotherapies against CVL. Specifications of

treatment as well as pharmacological data will be reported independently.

Dogs treated with amphotericin B showed good clinical recovery (i.e. disappearance of

external signs of leishmaniasis), as previously seen by Lamothe (1997). Reduction of

serum antibody levels, parasite load and lymphoproliferative responses with Leishmania

antigen were transitory and returned to pre-treatment status several months later, as

observed in previous studies (Oliva et al., 1995). Nevertheless, changes induced by the

treatment confirm the association between the lack of response and the loss of T-helper

cells. In fact, those dogs treated with amphotericin B presented a significant increase of

CD4� T-cells in peripheral blood which did correlate with the restoration of the

mitogenic response in all the cases and the appearance of lymphoproliferative response in

presence of SLA in two cases. Recovery of the T-helper subset in Leishmania-infected

dogs after treatment with meglumine antimoniate has also been observed by Bourdoiseau

et al. (1997b), who considered it a restoration of the cell-mediated immunity.

In a similar way, variations of Leishmania-specific serum antibody levels tends to

occur in parallel with the significant decrease of both, TCR gd� cells and sIgG� B-cells,

J. Moreno et al. / Veterinary Immunology and Immunopathology 71 (1999) 181±195 191

1 month after treatment and the posterior return to levels observed during active CVL.

Although the group studied is small and more research needs to be done, these findings

could corroborate the role of these cell subsets in the humoral response to Leishmania

infection in canine leishmaniasis, as seen in human visceral leishmaniasis (Raziuddin et

al., 1992).

Based on the present results, we conclude that immunosupression induced in the dog

during CVL is largely due to the loss of T-helper cells which, in turn, prevents adequate

T-cell response to the parasite. Other mechanisms might also contribute to the diminished

responsiveness; the presence of inhibitory Th2 cytokines, mainly IL-4 and IL-10, have

been demonstrated in human visceral leishmaniasis (Ghalib et al., 1993; Holaday et al.,

1993; Zwingenberger et al., 1990), but have yet to be tested in CVL. This possibility

would be examined when specific reagents are available.

The poor response to therapy observed in infected dogs may be due to the grade of

immunosupression induced by CVL, which is not completely restored by chemotherapy.

Nabors and Farrel (1996) have shown that in Leishmania major-infected BALB/c mice

pre-treated with anti-IFN-g to develop a polarised Th2 response, sodium stibogluconate

therapy was ineffective. Considering this, we postulate that patent cases of CVL present

an exaggerated Th2 response, which may explain the refractoriness of these animals to

treatment in the same way that diffuse cutaneous leishmaniasis patients (WHO, 1990), or

HIV/Leishmania coinfected patients (Alvar et al., 1997; Laguna et al., 1999), are difficult

to treat successfully. In such cases, dogs could be then suitable candidates for combined

therapy with anti-leishmanial drugs and immunomodulatory cytokines, such as IL-12 and

IFN-g to shift the cell response towards a Th1 protective response, although the

expensiveness of this therapy would not make it a practical epidemiological control

measure.

Acknowledgements

This work was supported by an FIS grant Ref. 96/0302 from the Ministerio de Sanidad

y Consumo and a INCO-DC research project Ref. IC18*CT970213 from the European

Commission. J. Moreno was supported by a fellowship from the `̀ Instituto de Salud

Carlos III''. C. Chamizo was supported by an FPI fellowship from the Ministerio de

EducacioÂn y Cultura, Spain. J. Alvar was granted by the Fando de Investigaciones

Sanitarias (BAE nuu 99/5038) and Christ's College, University of Cambridge, UK. The

authors are grateful to Dr. Nancy G. Saravia for critical reading and comments on the

manuscript.

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