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Revista do Instituto de Medicina Tropical de São Paulo

On-line version ISSN 1678-9946

Rev. Inst. Med. trop. S. Paulo vol.32 no.1 São Paulo Jan./Feb. 1990 



Vaccination of dogs against Leishmania (Viannia) braziliensis



Canine cutaneous leishmaniasis was first recorded in Brazil in the second decade of this century3,17 from the State of São Paulo. Epidemiological studies in the Rio Doce Valley of the State of Minas Gerais7,8 have shown that about 3 per cent of the dog population have active leishmanial lesions. In this focus of human American cutaneous leishmaniasis, both dogs and man are probably accidental hosts of Leishmania (Viannia) braziliensis. However, in a rural area in the municipality of Viana, State of Espírito Santo, 25 per cent of the dogs were found to be infected with L. braziliensis9 and a close correlation between prevalence and distribution of human and canine infections was established10. A similar high level of canine infections has been reported from the municipality of Nova Iguaçu, State of Rio de Janeiro1, where 20-25 per cent of the dog population are infected with this parasite. The prevalence of canine cutaneous leishmaniasis in the Viana and Nova Iguaçu foci is much higher than the 10 per cent infection rate amongst domestic dogs living on the western slopes of the Peruviana Andes12, where the infective agent is L. (V.) peruviana. It is usually considered that canine infections constitute the major source of human uta peruana. The development of a prophyllactic vaccine against canine cutaneous leishmaniasis would be of value, at least in certain areas, in controlling human cutaneous and muco-cutaneous leishmaniasis. The vaccine developed for use in man14, with or without Corynebacterium parvum as adjuvant, proved to be inadequate15. After receiving three weekly doses of vaccine, experimental dogs showed increases in IgG titres but developed skin lesions when challenged with promastigotes of L. braziliensis. The immunotherapeutic methods developed in Venezuela for the treatment of leprosy5 and later modified to treat cases of human American cutaneous leishmaniasis6 suggested us to use it as a means for protecting dogs against L. braziliensis. Various other vaccination schemes (Route, dose, interval between doses, booster, addition of adjuvants) were tested and these are the first results that suggest the possibility of success. The preliminary results obtained in the use of this new vaccine are recorded herein. Eight 4-month-old mongrel puppies were used in the experiments. Before vaccination, the puppies were treated with anthelminthics, vaccinated against parvovirus, leptospirosis and distemper, and tested with Montenegro antigen containing 200µg/0,1 ml of protein nitrogen2,11,13. All animals had negative Montenegro reactions before being vaccinated. The vaccine was prepared from promastigotes of two WHO reference stocks of Leishmania: MHOM/BR/75/M2903 (L. braziliensis) and IFLA/BR/67/PH8 (L. amazonensis). The organisms were grown in LIT medium4 and harvested in the stationary phase. Harvested promastigotes were autoclaved for 15 minutes at 120 b/in2. Autoclaved promastigotes were suspended in buffered saline to give a concentration of 600µg of nitrogen/ml. The eight puppies were divided into three experimental groups: I) Four animals received a single intradermic injection of the vaccine, containing 600 µg of nitrogen/ml of autoclaved promastigotes (300µg of nitrogen of each slock) plus 500µg of viable, lyophilized BCG; II) Two animals were given a single intradermal injection of 500 UG of BCG; III) Two animals served as unvaccinated controls, and were given a single intradermic injection of buffered saline. Thirty days later, the animals were again tested with Montenegro antigen, and the results were read after 72 hours15. 2/4 in group I, 1/2 in group II, and 2/2 in group III had developed positive reactions to the antigen. These results suggest that it is necessary to use other methods to assess cellular hypersensitivity in dogs. In our observations (in press) on the use of Montenegro tests as indicators of cells producing delayed hypersensitivity in vaccinated individuals, histological studies showed that a single dose of Montenegro antigen induces sensibility, without inducing nodule formation in persons receiving placebos. Immediately after reading responses to Montenegro antigen, the animals were challenged with 1 x 105 LIT cultured promastigotes of strain MCAN/BR/82/BH348 (L. braziliensis isolated from a dog living in the Rio Doce Valley), administered intradermally on the inner surface of the ear. Two months after challenge, four of the dogs developed leishmanial lesions at the sites of inoculation. These included both animals in group III, one of group II and one in group I. The dog belonging to group I that developed a lesion had given a positive Montenegro reaction before being challenged. The dogs were kept under observation for a further eight months and none of the remaining four developed lesions during this period. The negative animals included the two dogs belonging to Group I with an unchanged (negative) response to Montenegro antigen after immunization with the Leishmania/BCG vaccine and a dog of Group II that became Montenegro positive after receiving only BCG. Although no conclusions can be drawn from these preliminary observations, the results obtained justify further investigations on the utility of the Leishmania/BCG vaccine as a means of protecting dogs against infection by L. braziliensis. The results also suggest the need to develop a more sensitive Montenegro antigen to skin test dogs.


Departamento de Parasitologia do
Instituto de Ciências Biológicas da
Universidade Federal de Minas Gerais
Caixa Postal 2486
31270 Belo Horizonte, Minas Gerais
Magno DIAS
Carlos Alberto da COSTA
Marilene Suzan Marques MICHALICK
Maria Norma MELO
Roberto Teodoro da COSTA



1. AGUILAR, C.M.; RANGEL, E.F.; GRIMALDI Fº, G. & MOMEN, H. - Alta freqüência de leishmaniose tegumentar canina em um foco endêmico do Estado do Rio de Janeiro, Brasil. Mem. Inst. Oswaldo Cruz, 82(Suppl. 1): 56, 1987.

2. BARBOSA-SANTOS, E.G.O.; MARZOCHI, M.C.A.; SILVA, V.L.; CONCEIÇÃO, N.F.; SILVA, P.C.T. & SANTOS, C. - Elicitation of the delayed hypersensitivity reaction in canine muco-cutaneous leishmaniasis. Mem. Inst. Oswaldo Cruz, 81(Suppl.): 146, 1986.

3. BRUMPT, E. & PEDROSA, A.M. - Pesquisas epidemiológicas sobre a leishmaniose americana das florestas no Estado de São Paulo. An. paul. Med. Cirurg., 1: 97-136, 1913.

4. CAMARGO, E.P. - Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclie trypanosomes in liquid media. Rev. Inst. Med. Trop. S. Paulo, 6: 93-100, 1964

5. CONVIT, J.; ARANZAZU, N.; ULRICH, M.; PINARDI, M.E.; REYES, O. & ALVARADO, J. - Immunotherapy with a mixture of Mycobacterium leprae and BCG in different forms of leprosy and in Mitsuda-negative contacts. Int. J. Leprosy, 50: 415-424, 1982.

6. CONVIT, J.; RONDON, A.; ULRICH, M.; BLOOM, B.; CASTELLANOS, P.L.; PINARDI, M.E.; CASTES, M. & GARCIA. L. - Immunotherapy versus chemotherapy in localised cutaneous leishmaniasis. Lancet, 1: 401-405, 1987.

7. DIAS, M. - Leishmaniose tegumentar americana na zona do Rio Doce, Minas Gerais. Aspectos da doença no homem e estudo de reservatórios. Belo Horizonte, 1982. [Tese de Doutoramento - Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais].

8. DIAS, M.; MAYRINK, W.; DEANE, L.M.; COSTA, C.A. da; MAGALHÃES, P.A.; MELO, M.N.; BATISTA, S.M.; ARAUJO, F.G.; COELHO, M.V. & WILLIAMS, P. - Epidemiologia da leishmaniose tegumentar americana. I. Estudo de reservatórios em área endêmica no Estado de Minas Gerais. Rev. Inst. Med. trop. S. Paulo, 19: 403-410, 1977.

9. FALQUETO, P. - Leishmaniose tegumentar em Viana, estado do Espírito Santo. Investigação sobre a infecção natural em animais e sua relação com a ocorrência da doença humana. Rio de Janeiro, 1984. [Dissertação de Mestrado - Faculdade de Medicina da Universidade Federal do Rio de Janeiro].

10. FALQUETO, A.; COURA, J.R.; BARROS, G.C.; GRIMALDI Fº, G.; SESSA, P.A.; CARIAS, V.R.D.; JESUS, A.C. de & ALENCAR, J.T.A. - Participação do cão no ciclo de transmissão de leishmaniose tegumentar no município de Viana, estado do Espírito Santo, Brasil. Mem. Inst. Oswaldo Cruz, 81: 155-163, 1986.

11. GENARO, O.; MAYRINK, W.; RASO, P.; DIAS, M.; PINTO, F.M.; COSTA, C.A. da; WILLIAMS, P.; MICHALICK, M.S.M.; MELO, M.N.; MAGALHÃES, P.A.; COSTA, R.T. da & OLIVEIRA LIMA, A. - Kinectics of Montenegro skin test in dogs infected with Leishmania (Viannia) braziliensis. Mem. Inst. Oswaldo Cruz, 83(Suppl. 1): 163, 1988.

12. HERRER, A. - Estudios sobre leishmaniasis tegumentaria en el Peru. Leishmaniasis natural en perros procedentes de localidades utogenas. Rev. Med. esp. (Lima), 8: 87-118, 1949/1951.

13. MARZOCHI, M.C.A.; BARBOSA-SANTOS, E.G.O.; CONCEIÇÃO, N.F. & SILVA, V.L. - Epidemiological survey of canine cutaneous leishmaniasis by intradermal reaction in an endemic area of Rio de Janeiro, Brazil. Mem. Inst. Oswaldo Cruz, 82(Suppl. 1): 163, 1987.

14. MAYRINK, W.; COSTA, C.A. da; MAGALHÃES, P.A.; MELO, M.N.; DIAS, M.; OLIVEIRA LIMA, A.; MICHALICK, M.S.M. & WILLIAMS, P.- A field trial of a vaccine against American dermal leishmaniasis. Trans. roy. Soc. trop. Med. Hyg., 73: 385-387, 1979.

15. MAYRINK, W.; GENARO, O.; DIAS, M.; COSTA, C.A. da; MICHALICK, M.S.M.; MELO, M.N.; WILLAMS, P; COSTA, R.T. da; NASCIMENTO, E. & OLIVIEIRA LIMA, A. - Experimental vaccination of dogs against Leishmania (Viannia) braziliensis (Pilot trial). Mem. Inst. Oswaldo Cruz, 83:(Suppl. 1): 159, 1988.

16. MICHALICK, M.S.M.; COSTA, C.A. da; COSTA, R.T. da; MELO, M.N.; DIAS, M.; GENARO, O.; MAYRINK, W.; LIMA, A.O. & WILLIAMS, P. - An experimental vaccination against canine cutaneous leishmaniasis. Mem. Inst. Oswaldo Cruz, 82(Suppl. 1): 153, 1987.

17. PEDROSO, A.M. - Leishmaniose local de cão. An. paul. Med. Cirurg., 1: 33-40, 1913.



Recebido para publicação cm 20/02/1989.
Work financed by FINEP, UFOP UFMG and W.H.O.

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