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Prolonged in vitro cultivation of Leishmania mexicana venezuelensis
TRANSACTIONS OF THE ROYALSOCIETY OF TROPICALMEDICINEAND HYGIENE(1986) 80, CORRESPONDENCE Rocha e Silva, E. 0. (1975). Ciclo evolutivo do Hepatozoon rriatomae (Sporozoa, haemogregarinidae) parasita de triatomineos. Revista de Saude Publica, Sao Paulo, 9, 383-391. Schofield, C. J., Apt, W. & Miles, M. A. (1982). The ecology of Chagas’ diseasein Chile. Ecology of Disease,1, 117-129. Accepted for publication
15th November, 1985.
Prolonged in vitro cultivation
of Leishmania mexicana
venezuelensis BONFANTE-GARRIDO (1980, 1983) described a new sub-species of the Leishmania mexicana complex, L. m. venezuelensis(which he isolated from the cutaneous lesion of a patient in the State of Lara, Venezuela. The parasite grows quickly in hamster skin and produces the large lesions characteristic of organisms of the L. mexicana group (LAINSON, 1983). Inoculation of amastigote-infected hamster skin into conventional blood agar type media used for the in vitro isolation of Leishmania spp. produces heavy growth of leishmanial promastigotes. Sub-cultivation of the promastigotes for more than one or two passageshas always been unsuccessful, the organisms rapidly dying out (LAINSON, 1983; Dr. J. J. Shaw and Dr. G. Grimaldi, personal communications). In 1984 we received from Professor Ralph Lainson a primary culture of L. m. venezuelensis MHOMNEi 74/PM-H3 originally isolated by Professor Rafael Bonfante-Garrido from a 60-year-old male patient in Lara State, Venezuela. This organism had been deep-frozen in liquid nitrogen and had had several passagesin hamsters since its initial isolation in 1974. We successfully inoculated hamsters from the primary culture, but like many others before us, failed to maintain the organisms in vitro. Sevenmonths ago we re-isolated the parasite into culture from an infected hamster. The initial isolation was into Evans’ modified Tobie’s medium (EVANSet al.. 1984). where the parasite grew well. Subsequent sub-cultures were made into modified Tobie’s medium, where the parasites gradually declined and died, and also into a semi-solid blood aear medium “sloo~v Evans” (EVANSet al., 1984): In the semi-solid*l;lkdium the organisms declined in number but did not die out. After three weeks the culture was sub-inoculated into fresh semi-solid medium and this time the organisms began to grow. A further sub-inoculation was made into semi-solid medium after 10 days, and within one week the culture was full of active promastigotes. Sub-cultures were made into modified Tobie’s medium and into MEM-FCS-EBLB medium (EVANS et al., 1984). The sub-cultures all thrived and the organism has been maintained by serial passagetwice weekly for six months in MEM-FCS-EBLB medium. The incubation temperature was 22°C. Isoenzyme analysis of promastigotes maintained in serial subculture for this period retain the differences from other members of the L. mexicana group in ALAT, PGM, GPI, G6PDH, MDH and MPI reported by MILES (1983). DAVID A. EVANS VALERIE SMITH Dept. of Medical Protozoology, London School of Hygiene and Tropical Medicine, Keppel St., London WCIE 7HT
493
References
Bonfante-Garrido, R. (1980). New subspecies of leishmaniasis isolated in Venezuela. Xth Inmrnatkmal Congress on Tropical Medicine and Malaria,
Manilla,
PhilippTnes,
1980, p. 203. Bonfante-Garrido, R. (1983). Observaciones sobre Leishmania mexicana vene.zuelensis. Proceedings of the 3rd Venezwksn Congress of Microbiology and Symposium on Leishmaniasis. Barouisimeto. Nov. 6-12. 1982.
Evans, D. A., Lanham’, S. M.,‘Baldwin, C.‘I. & Peters, W. (1984). The isolation and isoenzyme characterization of Leishmania braziliensis subsp.. from patients with cutaneous leishmaniasis acquired in Belize. Transactions of the Royal Society of Tropical Medicine and Hygiene, 78, 35-42. Lainson, R. (1983). The American leishmaniases: some observations on their ecology and epidemiology. Transactions of the Royal Societyof Tropical Medicine and Hygiene, 77, 569-596. Miles, M. A. (1983). Biochemical identification of leishmanias. Proceedings of the 3rd Venezuelan Congress of Microbiology and Symposium on Leishmaniasis, quisimeto, November 6-12th, 1982.
Bar-
Accepted for publication 29th November, 1985.
Anti-tubulin
antibodies
in visceral
leishmaniasis
Elevated levels of anti-tubulin antibodies have been reported in Grave’s disease, Hashimoto’s thyroiditis, demyelinating disease, infectious mononucleosis and alcoholic liver disease and taken to indicate autoimmune pathology or polyclonal activation of the immune system (VAINIO et al., 1983; KURKI & VIRTANEN, 1984; NEWCOMBEet al., 1985). Antitubulin antibodies have been described in sera from dogs infected with Leishmania donovani (PATERAKIet al., 1983), the causative agent of visceral leishmaniasis in man, and in a single infected human patient (BORDIER et al., 1982). Whilst preparing mouse monoclonal antibodies to L. donovani we noticed that a large number of anti-tubulin antibodies were produced of both IgGl and IgM subclasses(Howard, Usha Datta & Miles, unpublished). We believed that the high tubulin content of L. donovani might have been responsible for inducing anti-tubulin antibodies. We used an enzyme-linked immunosorbent assay (ELISA) (VOLLER et al., 1979) to examine serum ‘from 49 patients with visceral leishmaniasis from ‘localities in South America, Africa and India for the presenceof antibodies to purified sheepbrain tubulin. The mean optical density, with serum dilutions of 1:SO.was 0.36 (+0*32) for the 49 natients and was 0*09’(+0.03) for the control group (24 individuals). 32 (65%) of the patients with visceral leishmaniasis had antibody levels higher (optical density range 0.2 to 1.8) than the normal
mean plus three standard
deviations. Antibodies were rarely detected at dilutions of sera greater than 1:1000, which corresponds with the range of anti-tubulin tnres that have been induced in animals (KARSENTIet al., 1977). Elevated anti-tubulin antibodies were also frequently found in sera of patients with the helminth infections onchocerciasis and schistosomiasis. In contrast antitubulin antibodies were not increased in patients with malaria (Plasmodium vivax) and tuberculosis. Caution is required in interpreting the significance of these autoantibodies (KHOURY et al., 1983; KIERS-
15th November, 1985.
Prolonged in vitro cultivation
of Leishmania mexicana
venezuelensis BONFANTE-GARRIDO (1980, 1983) described a new sub-species of the Leishmania mexicana complex, L. m. venezuelensis(which he isolated from the cutaneous lesion of a patient in the State of Lara, Venezuela. The parasite grows quickly in hamster skin and produces the large lesions characteristic of organisms of the L. mexicana group (LAINSON, 1983). Inoculation of amastigote-infected hamster skin into conventional blood agar type media used for the in vitro isolation of Leishmania spp. produces heavy growth of leishmanial promastigotes. Sub-cultivation of the promastigotes for more than one or two passageshas always been unsuccessful, the organisms rapidly dying out (LAINSON, 1983; Dr. J. J. Shaw and Dr. G. Grimaldi, personal communications). In 1984 we received from Professor Ralph Lainson a primary culture of L. m. venezuelensis MHOMNEi 74/PM-H3 originally isolated by Professor Rafael Bonfante-Garrido from a 60-year-old male patient in Lara State, Venezuela. This organism had been deep-frozen in liquid nitrogen and had had several passagesin hamsters since its initial isolation in 1974. We successfully inoculated hamsters from the primary culture, but like many others before us, failed to maintain the organisms in vitro. Sevenmonths ago we re-isolated the parasite into culture from an infected hamster. The initial isolation was into Evans’ modified Tobie’s medium (EVANSet al.. 1984). where the parasite grew well. Subsequent sub-cultures were made into modified Tobie’s medium, where the parasites gradually declined and died, and also into a semi-solid blood aear medium “sloo~v Evans” (EVANSet al., 1984): In the semi-solid*l;lkdium the organisms declined in number but did not die out. After three weeks the culture was sub-inoculated into fresh semi-solid medium and this time the organisms began to grow. A further sub-inoculation was made into semi-solid medium after 10 days, and within one week the culture was full of active promastigotes. Sub-cultures were made into modified Tobie’s medium and into MEM-FCS-EBLB medium (EVANS et al., 1984). The sub-cultures all thrived and the organism has been maintained by serial passagetwice weekly for six months in MEM-FCS-EBLB medium. The incubation temperature was 22°C. Isoenzyme analysis of promastigotes maintained in serial subculture for this period retain the differences from other members of the L. mexicana group in ALAT, PGM, GPI, G6PDH, MDH and MPI reported by MILES (1983). DAVID A. EVANS VALERIE SMITH Dept. of Medical Protozoology, London School of Hygiene and Tropical Medicine, Keppel St., London WCIE 7HT
493
References
Bonfante-Garrido, R. (1980). New subspecies of leishmaniasis isolated in Venezuela. Xth Inmrnatkmal Congress on Tropical Medicine and Malaria,
Manilla,
PhilippTnes,
1980, p. 203. Bonfante-Garrido, R. (1983). Observaciones sobre Leishmania mexicana vene.zuelensis. Proceedings of the 3rd Venezwksn Congress of Microbiology and Symposium on Leishmaniasis. Barouisimeto. Nov. 6-12. 1982.
Evans, D. A., Lanham’, S. M.,‘Baldwin, C.‘I. & Peters, W. (1984). The isolation and isoenzyme characterization of Leishmania braziliensis subsp.. from patients with cutaneous leishmaniasis acquired in Belize. Transactions of the Royal Society of Tropical Medicine and Hygiene, 78, 35-42. Lainson, R. (1983). The American leishmaniases: some observations on their ecology and epidemiology. Transactions of the Royal Societyof Tropical Medicine and Hygiene, 77, 569-596. Miles, M. A. (1983). Biochemical identification of leishmanias. Proceedings of the 3rd Venezuelan Congress of Microbiology and Symposium on Leishmaniasis, quisimeto, November 6-12th, 1982.
Bar-
Accepted for publication 29th November, 1985.
Anti-tubulin
antibodies
in visceral
leishmaniasis
Elevated levels of anti-tubulin antibodies have been reported in Grave’s disease, Hashimoto’s thyroiditis, demyelinating disease, infectious mononucleosis and alcoholic liver disease and taken to indicate autoimmune pathology or polyclonal activation of the immune system (VAINIO et al., 1983; KURKI & VIRTANEN, 1984; NEWCOMBEet al., 1985). Antitubulin antibodies have been described in sera from dogs infected with Leishmania donovani (PATERAKIet al., 1983), the causative agent of visceral leishmaniasis in man, and in a single infected human patient (BORDIER et al., 1982). Whilst preparing mouse monoclonal antibodies to L. donovani we noticed that a large number of anti-tubulin antibodies were produced of both IgGl and IgM subclasses(Howard, Usha Datta & Miles, unpublished). We believed that the high tubulin content of L. donovani might have been responsible for inducing anti-tubulin antibodies. We used an enzyme-linked immunosorbent assay (ELISA) (VOLLER et al., 1979) to examine serum ‘from 49 patients with visceral leishmaniasis from ‘localities in South America, Africa and India for the presenceof antibodies to purified sheepbrain tubulin. The mean optical density, with serum dilutions of 1:SO.was 0.36 (+0*32) for the 49 natients and was 0*09’(+0.03) for the control group (24 individuals). 32 (65%) of the patients with visceral leishmaniasis had antibody levels higher (optical density range 0.2 to 1.8) than the normal
mean plus three standard
deviations. Antibodies were rarely detected at dilutions of sera greater than 1:1000, which corresponds with the range of anti-tubulin tnres that have been induced in animals (KARSENTIet al., 1977). Elevated anti-tubulin antibodies were also frequently found in sera of patients with the helminth infections onchocerciasis and schistosomiasis. In contrast antitubulin antibodies were not increased in patients with malaria (Plasmodium vivax) and tuberculosis. Caution is required in interpreting the significance of these autoantibodies (KHOURY et al., 1983; KIERS-