Exploitation of parasite-derived antigen in therapeutic success against canine visceral leishmaniosis

veterinary parasitology ELSEVIER

Veterinary Parasitology 54 (1994) 367-373

Exploitation of parasite-derived antigen in therapeutic success against canine visceral leishmaniosis Asit B. Neogy a, Ioannis Vouldoukis a'b, Jose M. da Costa a, Loic Monjour *'a, The Veterinary Group of Lupino c aLaboratoire de Parasitologie, FacultOde MOdecinePitid-Salpdtrikre, 91 Boulevard de l'Hdpital, Paris, France bDepartment of Geographical Medicine and Microbiology, Faculty of Medicine, Iraklion, Crete, Greece cClinique VOtOrinairede Lupino, Bastia, Corse, France (Accepted 26 October 1993)

Abstract

In an attempt to obtain therapeutic success against canine visceral leishmaniosis, the potential of LiF2 antigen (Leishmania infantum-derived Fraction 2, 94-67 kDa), given alone or in combination with the chemotherapeutic agent N-methylglucamine antimonate, was compared with conventional chemotherapy with that drug. Absence of any parasite in direct microscopic examination of bone-marrow aspirates in treated dogs was considered a parasitological cure, i.e. therapeutic success. Results showed that the disappearance of clinical symptoms did not always indicate parasitological healing in dogs. The parasitological healing rates with chemotherapy and immunotherapy alone were 37.5% and 25% respectively, in contrast to the 100% cure rate observed with chemotherapy combined with immunotherapy. The development of a protective response in dogs, as measured by the in vitro leishmanicidal activity of monocyte-derived macrophages in the presence of autologous lymphocytes, was found to correlate well with the success of therapy. The overall findings of this study give an important insight into the immunotherapeutic strategy by which therapeutic success can be achieved in canine visceral leishmaniosis. Keywords: Leishmania infantum; Dog; Control methods-Trematoda

*Corresponding author. SSDI0304-4017(93)00613-4

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1. Introduction

In recent years, the consistent increase in the incidence of canine and human visceral leishmaniosis (VL) caused by Leishmania infantum in some endemic areas of the Mediterranean basin (Micelli and Mansueto, 1987; Quilici et al., 1987) has given rise to grave concern. It has been suggested that eradication of infected dogs is necessary to control VL in the area (World Health Organization, 1984). Since killing infected domestic dogs often meets with emotional and ethical resistance, control of disease largely depends on the success of treatment. However, drugs currently in use for canine VL are only partially effective; relapses are the rule (Slappendel, 1988 ) and the presence of parasites in clinically cured dogs is common (I. Vouldoukis and L. Monjour, unpublished observations, 1993). Leishmania is an obligatory intracellular parasite and successful host resistance is T-cell-dependent and mediated by macrophages activated by Tcell-derived lymphokines (Murray, 1988). In a more acceptable approach, various immunopotentiators (biological and immunological) have been used in recent years to achieve therapeutic success against leishmanial infection, largely through their ability to activate macrophages non-specifically (reviewed by Alexander and Russel, 1992). This preliminary study investigates the potential of L. infantum promastigote-derived Fraction 2 (94-67 kDa), known to be mitogenic (Ogunkolade, 1987), given separately or in combination with conventional chemotherapy to bring about parasitological healing, i.e. therapeutic success, in canine VL. Along with clinical and parasitological evaluation, the protective response in dogs, as expressed by in vitro leishmanicidal activity of monocyte-derived macrophages in the presence of autologous lymphocytes, was assessed throughout the study period in an attempt to give an insight into the process operating in the therapeutic success.

2. Materials and methods

2.1. Dogs Twenty-four VL-infected dogs of various breeds and 3-7 years old, originating from different areas of Bastia, Corsica, where canine VL is endemic, were used in this study. They were housed in a dog kennel under close clinical observation of veterinarians of the Lupino Clinic. Informed consent of the dog owners was obtained. All the dogs had typical clinical signs of canine VL, i.e. lymphadenopathy, splenomegaly, anaemia, depilation, onychogryphosis, presence of cutaneous lesions, emaciation, conjunctivitis and weight loss. Leishmania parasites were identified, by direct microscopical examination, in all Giemsa-stained bonemarrow smears and parasite load was graded according to the method described by Chulay and Bryceson (1983). In contrast, no amastigote was detected in peripheral blood leucocytes or in fresh bone marrow from healthy control dogs.

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2.2. Therapeutic approaches The dogs were randomly allocated to three groups each of eight animals. Groups I, II and III were subjected to chemotherapy, immunotherapy, and chemotherapy followed by immunotherapy (combined therapy), respectively. For chemotherapy, N-methylglucamine antimonate (Glucantime ®; Rhone M6deux, France ) was used; 20 intramuscular injections, each containing 300 mg per kg body weight, were given at 2 day intervals. Partially purified antigenic Fraction 2, derived from L. infantum promastigotes using SDS-PAGE (LiF2, 94-67 kDa) (Monjour et al., 1988), was used as the immunotherapeutic agent. Dogs were injected intramuscularly three times with 50 #g (protein equivalent) of LiF2 in normal saline at 7 days apart. For the combined therapy, immunotherapy was started at 15 days after completion of chemotherapy. In addition to the evaluation of the general clinical improvement of the dogs, the decrease in their parasite load was quantitated to assess the therapeutic response 3 and 6 months after commencement of treatment. Absence of any parasite in direct examination of bone-marrow aspirate was always confirmed by culture before considering the dog as parasitologically cured.

2.3. Isolation and infection of dog macrophages For macrophage leishmanicidal assay, monocytes and lymphocytes from dogs' venous blood were isolated before and after treatment (see Table 2 ) by two-step centrifugation in Percoll (Pharmacia Fine Chemicals, France) as described by Pertoft et al. (1980). Monocytes were seeded at 2 × 105 cells per dish into 35 mm diameter plastic Petri dishes (Falcon) and cultured for 6 days in complete medium (RPMI 1640 containing 25 mM Hepes, 20% heat-inactivated fetal calf serum, L-glutamine, penicillin and streptomycin) at 37°C in an atmosphere of 5% CO2 in air. After 6 days of culture, monocyte-derived macrophages were washed, overlaid with fresh medium and exposed to stationary phase L. infantum promastigotes at a ratio of 1:5 for 24 h (see Table 2) at 37°C. Culture in some Petri dishes was terminated and the time point was taken as 0 h. 2.4. A ntileishmanial activity induced by macrophages in the presence of

lymphocytes In the remaining Petri dishes, macrophages were washed to remove non-phagocytosed parasites, fresh medium was added and cells were incubated either alone or in the presence of autologous lymphocytes at 37°C in 5% CO2. Lymphocytes were incubated separately for 6 days in complete medium, then washed, counted and added at a concentration of 1 × 105 cells per dish to macrophages. At 0, 24 and 48 h of co-culture, the cells were washed in phosphate-buffered saline (pH 7.2), fixed in 1% glutaraldehyde and Giemsa-stained. Leishmanicidal activity of macrophages was estimated microscopically by determining the number of intact parasites per 100 cells in two separate culture dishes. Parasite killing was further

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investigated by the transfer and incubation ofmacrophages in an axenic medium. Kept at 26 ° C, killed amastigotes from treated macrophages will not be able to differentiate into promastigotes over a 6 day period (Ogunkolade et al., 1988 ).

3. Results

The results of the therapeutic responses in the three groups of dogs are summarized in Table 1. Although the degree of clinical signs and parasite load in the 24 dogs in the three groups were similar before the treatment was started, the rates of cure and clinical improvement were different in response to the different therapeutic measures. The rate ofparasitological cure in Group III dogs given the combined therapy was 100% as recorded 3 months after commencement of treatment. Clinical improvement was prompt in this group. On the other hand, dogs treated with either Glucantime (Group I ) or LiF2 (Group II ) alone showed parasitological cure rates of 37.5% and 25%, respectively, after 3 months, and the values remained the same even after 6 months. Surprisingly, the remaining dogs in both groups that were parasitologically positive (2 + ) at the 3 month observation period became clinically cured after 6 months, although the clinical improvement in Group II was slower than that of Group I. Leishmanicidal activity of monocyte-derived macrophages in dogs is presented in Table 2. Resistance of macrophages against infection, which is inversely proportional to the number of parasites per 100 cells at 0 h, was significantly (P< 0.01 ) higher in parasitologically cured dogs than in infected, normal and clinically cured parasite-positive ones. In the absence of leishmanicidal activity, uncontrolled multiplication of intracellular parasites inside the macrophages of infected as well as normal dogs was recorded after 24 and 48 h of culture, and autologous lymphocytes had shown Table 1 Parasitological a n d clinical observations in canine VL in response to therapy Pretreatment

After starting treatment 3 months

Group (No. of dogs )

Therapeutic agent

1 (8) II(8) III (8)

Glucantime ® LiF2 Glucantime ® + LiF2

DE + dogs

6 months

DE + dogs

D E - dogs

DE + dogs

DE

dogs

No.

p.l.

Clinical No. sign

p.1. Clinical No. Clinical No. p.l. sign sign

Clinical No. sign

Clinical sign

8 8 8

4+ 4+ 4+

++ ++ ++

2+ 2+

-

-

5 6 0

_+ +

3 2 8

-

5 6

2+ 2+ 0

3 2 8

DE +, direct e x a m i n a t i o n (positive) of Giemsa-stained b o n e - m a r r o w smear; p.l., parasite load. Index: 4 + , 3 + , 2 + a n d 1 + denote the presence o f 1-10 amastigotes per 1, 10, 100 a n d 1000 oil immersion ( X 100) microscopic fields, respectively; D E - , absence o f any amastignte in 1000 oil immersion fields. Degree of clinical sign ( + + , prominent; + , mild; _+, disappearing; - , disappeared) was assessed conventionally by veterinarians, a n d represents the average trend o f clinical i m p r o v e m e n t groupwise.

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Table 2 In vitro leishmanicidal activity of monocyte-derived macrophages in canine VL Dogsa

No. of L. infantum per 100 macrophages (mean _+SD) 0 hb

24 h

48 h With Witout lymphocytes lymphocytes

With lymphocytes

472_+67

lll0_+ 160

1040± 172

d

21±7

20+8

e

90_+18

71_+21

63+ 12

392-+89

900_+130

830+ 115

Without lymphocytes Before treatment (n=24) Parasite load: 4 + Clinicially and parasitologically cured 3 months after treatment (n=13) Parasite load: Clinically cured 6 months after treatment (n=ll) Parasite load: 2 + Healthy controls f (n=6)

310+_42 520+74

70+11 c

45-+9

105-+22 112-+25 217+_57

470-+71

d

4+3

an=24 (Group I, 8; Group II, 8; Group III, 8); n= 13 (Group I, 3; Group II, 2; Group III, 8); n= 11 (Group I, 5; Group II, 6). Groupwise treatment and index of parasite load are described in Table 1. bAfter 24 h exposure of macrophages to L. infantum promastigotes at a ratio of 1:5 as detailed in the text. q'he value is significantly different (P<0.01, Student's t-test, unpaired) from other values of the column vertically. dDifference between the values is significant (P< 0.01, Student's t-test, paired). eDifference between the values is insignificant (P> 0.05, Student's t-test, paired). fDogs with no history or symptoms of leishmaniosis and a negative serological test.

hardly any effect in the event. In clinically cured parasite-positive dogs, the leishm a n i c i d a l activity o f m a c r o p h a g e s in the presence o f l y m p h o c y t e s was d e m o n strated by a strong reduction o f parasite load, yet the intracellular amastigotes m a i n t a i n e d their viability a n d capacity to differentiate a n d grow at 26 ° C. It was only in parasitologically cured dogs that leishmanicidal activity o f m a c r o p h a g e s was clearly evident at 24 a n d 48 h o f culture a n d greatly e n h a n c e d ( P < 0.01 ) in the presence o f l y m p h o c y t e s . Only a limited n u m b e r o f intracellular amastigotes could be seen; f u r t h e r m o r e , w h e n transferred at 26°C, these amastigotes were unable to c o m p l e t e their cycle a n d t r a n s f o r m into promastigotes. As neither m o n o c l o n a l antibodies against cell-surface antigens n o r r e c o m b i n a n t cytokines were available, we were not able to identify the l y m p h o c y t e subsets a n d their p r o d u c t s responsible for inducing leishmanicidal activity in m a c rophages in cured dogs.

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4. Discussion

It is evident from this study that clinical recovery in canine VL does not always reflect parasitological cure. A major proportion of treated dogs (62.5% undergoing chemotherapy and 75% undergoing immunotherapy), despite having a parasite load score of 2 +, became clinically normal. It seems that when the parasite load decreases to a certain level (2 + ), clinical signs disappear in the majority of infected dogs. Return of overt clinical disease to normality in treated but parasitologically positive dogs has also been recorded earlier (Mansour et al., 1970). However, the present findings indicate that, in canine VL, chemotherapy followed by immunotherapy is highly effective (100%) in completely eliminating the parasites from the host body, whereas immunotherapy or chemotherapy alone is largely ineffective. Despite chemotherapy, which may induce temporary remissions, dog mortality can exceed 90% (Ogunkolade et al., 1988). We have provided evidence that protection against infection, as indicated by in vitro leishmanicidal activity of macrophages in the presence of autologous lymphocytes, develops at an appreciable level only when the dogs become absolutely free of parasites. Although antigen-specific lymphocyte responses in the active phase of canine VL was shown to be depressed similarly to that in human disease (Abranches et al., 1991 ), almost nothing is known concerning the manipulation of T-cell-mediated responses during the course of treatment and after cure. As seen from experimental and human studies, the high efficacy of our combined therapy is possibly because, as a consequence of drug-induced reduction in parasite load, sensitized T-lymphocytes become stimulated by specific mitogenic antigen leading to the production of lymphokines which induce the activation of macrophages for killing residual parasites in dogs. However, how LiF2 alone brought about a parasitological cure with development of a protective response in two of the eight infected dogs, and clinical cure in others, is difficult to explain. Notable success has also been recorded in the treatment of human cutaneous leishmaniosis with BCG (bacille Calmette Gurrin) plus killed promastigotes (Convit et al., 1987 ), and with semipurified parasite-derived antigen (Monjour et al., 1991 ). The role of an immunomodulator in modifying the course of infection in VL, largely through the activation of macrophages, is obviously a complex phenomenon and awaits further clarification. Finally, we believe that parasitological cure of infected dogs is of utmost importance to control canine VL effectively in an endemic area; the presence of even a trace of parasites in clinically cured dogs may constitute a disease reservoir and may cause a relapse, particularly in a situation where immune status of the host is severely compromised by other factors. Since no new drug options are available, our combined therapy might help to obtain a radical cure for canine VL, especially in rural endemic areas where posttreatment surveillance is difficult. A study is now in progress to suitably modify this therapeutic approach as a simple and practical method requiring a short duration of treatment.

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Acknowledgements The authors wish to thank O.A. Silva, C. Alfred-Morin and I. Malet for their assistance. This work was supported by the European Economic Community, the Conseil Scientifique-Universit6 Paris VI and Laboratoire Virbac, Carros, France. A.B. Neogy was a recipient of a WHO Research Training Grant under the Special Programme for Research and Training in Tropical Diseases.

References Abranches, P., Santos-Gomes, G., Rachamin, N., Campino, L., Schnur, L.F. and Jaffe, C.L., 1991. An experimental model for canine visceral leishmaniasis. Parasite Immunol., 13: 537-550. Alexander, J.A. and Russel, D.G., 1992. The interaction of Leishmania species with macrophages. In: J.R. Baker and R. Muller (Editors), Advances in Parasitology, Vol. 31. Academic Press, London, pp. 175-253. Chulay, J.D. and Bryceson, A.D., 1983. Quantification of amastigotes of Leishmania donovani in smears of splenic aspirates from patients with visceral leishmaniasis. Am. J. Trop. Med. Hyg., 32: 475-479. Convit, J., Castellanos, P.L., Rondon, A., Pinardi, M.E., Ulrich, M., Cast6s, M., Bloom, B. and Garcia, L., 1987. Immunotherapy versus chemotherapy in localised cutaneous leishmaniasis. Lancet, 1: 401-405. Mansour, N.S., Stauber, L.A. and McCoy, J.R., 1970. Leishmaniasis in the Sudan Republic. 29. Comparison and epidemiological implications of experimental canine infections with Sudanese, Mediterranean, and Kenyan strains ofLeishmania donovani. J. Parasitol., 56: 468-472. Micelli, D.M. and Mansueto, S., 1987. Canine leishmaniasis in Western Sicily. Trans. R. Soc. Trop. Med. Hyg., 81: 175. Monjour, L., Vouldoukis, I., Ogunkolade, B.W., Hetzel, M., Ichen, M. and Frommel, D., 1988. Immunoprophylaxis against Leishmania infections with a semipurified vaccine. Trans. R. Soc. Trop. Med. Hyg., 88: 412-415. Monjour, L., Mansouri, P., Cuba, A.C., Rolland-Burger, L., Barzegari, M. and Dowlati, Y., 1991. Immunotherapy as treatment of cutaneous leishmaniasis. J. Infect. Dis., 164:1244-1245. Murray, H.W., 1988. Interferon-gamma, the activated macrophage and host defence against microbial challenge. Ann. Intern. Med., 108: 595-608. Ogunkolade, B.W., 1987. Immunoprophylaxie anti-leishmanienne:nouvelles appr6ches. D.Sc. Thesis, University of Paris VII, pp. 123-126. Ogunkolade, B.W., Vouldoukis, I., Frommel, D., Davoust, B., Feullette, A.R. and Monjour, L., 1988. Immunizationof dogs with Leishmania infantum-derived vaccine. Vet. Parasitol., 28:33-41. Pertoft, H., Johnsson, A., Warmegard, B. and Seljelid, R., 1980. Separation of human monocytes on density gradients of Percoll. J. Immunol. Methods, 33:221-229. Quilici, M., Dunan, S., Dumon, H., Franck, J., Gambarelli, F. and Toga, I., 1987. La leishmaniose visc6rale humaine en provence. M6d. Chir. Digest., 16:511-514. Slappendel, R.J., 1988. Canine leishmaniasis. A review based on 95 cases in the Netherlands. Tijdschr. Diergeneeskd., 113: 3-18. World Health Organization, 1984. Leishmaniasis. Technical Report Series No. 701. World Health Organization, Geneva.