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Aconitate hydratase (ACON), an enzyme that distinguishes Leishmania of the subgenus Viannia from other trypanosomatids
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL
MEDICINE ANDHYGIENE (1991)
85, 597-598
schaudinni [200] (MCHO/BR67/M595,
Aconitate hydratase (ACON), an enzyme that distinguishes Leishmania of the subgenus Viannia from other trypanosomatids J. J. Shaw, R. R. Braga, R. Lainson and E. A. Y. Ishikawa The Wellcome Parasitology Unit, Parasitology Section, Instituto Evandro Chagas, Caixa Postal 3, Belt%n, Para, 66.001, Brazil
The number of enzymes routinely used to identify
Leishmania isolates varies from laboratory to labora-
tory, but it is normally between 10 and 15. As more species of Leishmania are described it becomes increasingly difficult, especially when dealing with New World snecies. to include all the necessarv standard strains in a single run. With these points in mind, it would be useful if strains could be first sorted into groups on one character which would reduce the number of standards with which thev would subsequently have to be compared. KREUTZER et al. (1987) appreciated the difficulties of using many enzymes to identify a strain and mentioned that data from GPI (glucose phosphate isomerase),MPI (mannose phosphate isomerase),and 6PGDH (glucose-6-phosphate dehydrogenase)would separate most stocks of Leishmania. More recently ARANA et al. (1990) drew attention to the usefulness of MPI as a marker’separating L. (Viannia) peruviana from L. (I/‘.) braziliensis. However. these workers found this enzvme did not separate the 2 speciesif a cellulose acetate supporting matrix was used instead of starch gel. This emphasizes the care needed when comparing results obtained with different methods from different laboratories. MILES et al. (1981) were the first to note that the aconitate hydratase (ACON) of L. (V.) braziliensis (MHOMIBRl75iM2903) and L. (V.) guyanensis (MHOMiBRi75iM4147) in thin-layer starch (TLS) gel electrophoresis did not migrate from the origin. while that of L. CL.) amazonensis. L. (L.) deanei&d L. (L.) hertigi migrated positively. Later; LAINSONet al. (1982) found that the ACON of L. (V.) naiffi again did not migrate from its origin. SILVEIRA et al. (1987) noted that the ACON of L. (V.) lainsoni and an undescribed peripylarian Leishmania migrated negatively, but at different rates. Usins the TLS electronhoretic method, described by MILES et al. (1981), we extended our observations on the electroohoretic mobilitv of ACON to a wider range of Leishmania and trybanosomatids of other genera. The relative electrophoretic mobility (REM) of each band is expressed in the list below in square brackets according to the method suggested by LANOTTE et al. (198l), using IFLAiBRi67iPH8 as the standard strain. L. (L.) amazonensis [IOO] (IFLAiBRi67PH8); L. (L.) chagasi [82] (MHOMIBRi74iPP75); L. (L.) mexicana [77] (MNYCIBZi67iM379); Endotypanum
597
MCHOIBRI 77lM4922, MCHO/BR/79/M5725, MCHO/BR,‘80/ M6159); s. schaudinni [ 1381 (MCHOIPAI79I GML30); E. monterogei [178] (MCHOIClU62IEA9); L. (L.) major [ 1401(MHOMVE/63/L20 (zymodeme LON-1)); Crithidia fasciculata [82] (ATCC12858); Leptomonas seymouri [86] (ATCC30220); Herpetomonas muscarum [126] (ATCC30260); Leishmania (L.) deanei [90] (MCOE/BIU78/M5088); L. (L.) deanei [IOO] (MCOE/BR/75/M4042); L. (L.) hertigi [IOO] (MCOEIPAI65IC8); Typanosoma sp. [184] MCHOI BRi80iML6219); Typanosoma sp. [56] (MCHO/BFU 781M6273). ACON of parasites of the subgenus Viannia does not generally migrate from its origin towards the negative pole. Occasionally, however, in some runs we observed that the ACON of a strain migrated negatively, while in others it had not left the origin. The reasonsfor this are not understood, especially as the electrophoretic conditions were not altered. The inclusion of standard marker strains in all runs serves to control this kind of variabilitv. Because of the general lack of migration we have not given the REM values of the different soecies. Besides the Viannia species mentioned above, ACON of the following marker strains did not migrate or had a marginal negative migration: L. (V.) panamensis (MHOMIPAI 67IBOYNTON); L. (V. ) neruviana (MHOM/PE/84/ L26); L. (V.) shazoi (MCEBIBR/84/M8408); MHOMi BZI82IBELl; MDIDIBRI71IM1597; ITUBIBRI77I M4964: MHOMiBRi73iM2166. The suggestion by KREUTZERet al. (1987) that a combination of about 3 enzymes may be enough to identify neotropical Leishmania, and our finding that non-migratory/cathodic ACON is characteristic of parasites of the subgenus Viannia, reinforce the idea of using a limited number of biochemical characters to identify strains. However, it must be emphasized that such a procedure is feasible only when the Leishmania species of a region are known and have been characterized biochemically. Using, perhaps, 4 key enzymesto identify the speciesof Leishmania encountered in clinical and epidemiological studies is better than examining just a sample of the strains. The latter is sometimes resorted to because of the logistical problems involved in looking at many strains with a large number of enzymes. Before such procedures can be adopted, however, it will be necessarycarefully to compare identifications based on, perhaps, 4 key enzymes and the 10 commonly used in the reference centres. If the results are satisfactory, identifications with a few key enzymes should be a regional typing tool that has been based on enzyme studies in depth. The fmding that ACON was anodic for members of the generaEndotypanum, Leptomonas, Crithidia, Herpetomonas and Typanosoma, only some of which have a peripylarian type of development, suggeststhere is no association between non-migratory or cathodic ACON and the attachment of parasites to the hind gut. The ACON of 38 Leishmania strains, other than the Viannia strains mentioned in previous publications and which were identified with monoclonal antibodies as belongina to the subgenus Viannia. either did not migrate &om the origin or migrated to the negative Dole. Non-miaratorv/cathodic ACON is therefore. a consistent character for all of the 118 peripylarian
598
Eeishmaniu stocks so far examined and, in our opinion, this character can be added to the others that differentiate the subgenera Leishmania and Viannia. Besides this, non-migratory/cathodic ACON has so far not been associatedwith any other trypanosomatid genus. In our laboratory ACON has proved to be extremely useful for distinguishing peripyiarian Leishmania from other trypanosomatids, such as speciesof Trypanosoma, Endotrypanum, Herpetomonas and Crithidia, that may also develop in the hind gut of sandflies, aswell as from suprapylarian Leishmania. It must be emphasized, however, that ACON alone cannot be used to identify a Leishmania species of either the subgeneraLeishmania or Viannia, but it can be used to indicate to which subgenus a strain may belong. Acknowledgements
This work was supported by the Wellcome Trust, London and the Funda@o National de Salide of the Brazilian Ministry of Health, Brasilia. We are grateful to Francisco Gomes, Jose Paulo Cruz, and Raimundo MendonFa for technical help.
References
Arana, M., Evans, D. A., Zolessi, A., Llanos Cuenras, A. & Arevalo, J. (1990). Biochemical characterization of Leiihmaniu (Viannia) braziliensis and Leishmania (Vianniu) peruviana by isoenzyme electrophoresis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 84,
526-529.
1 Announcement
Kreutzer, R. I?., Souraty, N. & Semko, M. E. (1987). Biochemical identities and differences among Leishmaniu species and subspecies. American Journal of Tropical Medicine and Hygiene, 36? 22-32.
Lainson, R., Shaw, J. J., Mdes, M. A. & I%voa, M. M. (1982). Leishmaniasis in Brazil: XVII. Enzymic characterization of a Leishmania from the armadillo, Dusypus novemcinctus (Edentata), from Par6 State. Transactionsof the Royal Sociew of Tropical Medicine and Hygiene, 76,
81&811. Lanotte, G., Rioux, J. A., Maazoun, R., Pasteur, N., Pratlong, F. & Lepart, J. (1981). Application de la methode numtrique 51la taxonomie du genre Leishmania Ross, 1903. A propos de 146 souches originaires de 1’Ancien Monde. Utilisation des allozymes. Corollaires CpidCmiologiqueset phylhtiques. Annales de Parasitologic Humaine et Compake, 56, 575-592. Miles, M. A., Lainson, R., Shaw, J. J., Povoa, M. & Souza, A. A. (1981). Leishmaniasis m Brazil: XV. Biochemical distinction of Leishmania mexicana amozonensis, Leishmania braziliensis braziliensis, and Leishmania braziliensis guyanensis-aetiological agents of cutaneous leishmaniasis in the Amazon Basin of Brazil. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75,
524529. Silveira, F. T., Shaw, J. J., Braga, R. R. & Ishikawa, A. (1987). Dermal leishmaniasis in the Amazon region of Brazil: Leishmania (Viannia) lainsoni sp.n., a new parasite from the state of Par% Memdrias do Znstituio Oswaldo Cruz, 82, 289-292. Received 17 October 1990; revised 24 January accepted fm publication I February 1991
1
Sixth International
Training
Course on Identification of Helminth Parasites of Economic Importance International Institute of Parasitology, St Albans, UK. 93uly-14 August, 1992
The aim of this 6 weeks course is to familiarize participants with methods of identifying hehninth
parasites of economic importance, with special emphasis on those of medical and veterinary
importance. This is the only short course of its kind available and should benefit those engaged in routine identification of helminth parasitesin medical and veterinary laboratories, field workers, and those involved in training and teaching. For further information and application forms please contact Dr L. F. Khalii, International Institute of Parasitology, 395a Hatfield Road, St Albans, Hertfordshire, AL4 OXU, UK (telephone: 0727 833151; fax: 0727 868721; telex: 9312102254 IP G).
1991;
MEDICINE ANDHYGIENE (1991)
85, 597-598
schaudinni [200] (MCHO/BR67/M595,
Aconitate hydratase (ACON), an enzyme that distinguishes Leishmania of the subgenus Viannia from other trypanosomatids J. J. Shaw, R. R. Braga, R. Lainson and E. A. Y. Ishikawa The Wellcome Parasitology Unit, Parasitology Section, Instituto Evandro Chagas, Caixa Postal 3, Belt%n, Para, 66.001, Brazil
The number of enzymes routinely used to identify
Leishmania isolates varies from laboratory to labora-
tory, but it is normally between 10 and 15. As more species of Leishmania are described it becomes increasingly difficult, especially when dealing with New World snecies. to include all the necessarv standard strains in a single run. With these points in mind, it would be useful if strains could be first sorted into groups on one character which would reduce the number of standards with which thev would subsequently have to be compared. KREUTZER et al. (1987) appreciated the difficulties of using many enzymes to identify a strain and mentioned that data from GPI (glucose phosphate isomerase),MPI (mannose phosphate isomerase),and 6PGDH (glucose-6-phosphate dehydrogenase)would separate most stocks of Leishmania. More recently ARANA et al. (1990) drew attention to the usefulness of MPI as a marker’separating L. (Viannia) peruviana from L. (I/‘.) braziliensis. However. these workers found this enzvme did not separate the 2 speciesif a cellulose acetate supporting matrix was used instead of starch gel. This emphasizes the care needed when comparing results obtained with different methods from different laboratories. MILES et al. (1981) were the first to note that the aconitate hydratase (ACON) of L. (V.) braziliensis (MHOMIBRl75iM2903) and L. (V.) guyanensis (MHOMiBRi75iM4147) in thin-layer starch (TLS) gel electrophoresis did not migrate from the origin. while that of L. CL.) amazonensis. L. (L.) deanei&d L. (L.) hertigi migrated positively. Later; LAINSONet al. (1982) found that the ACON of L. (V.) naiffi again did not migrate from its origin. SILVEIRA et al. (1987) noted that the ACON of L. (V.) lainsoni and an undescribed peripylarian Leishmania migrated negatively, but at different rates. Usins the TLS electronhoretic method, described by MILES et al. (1981), we extended our observations on the electroohoretic mobilitv of ACON to a wider range of Leishmania and trybanosomatids of other genera. The relative electrophoretic mobility (REM) of each band is expressed in the list below in square brackets according to the method suggested by LANOTTE et al. (198l), using IFLAiBRi67iPH8 as the standard strain. L. (L.) amazonensis [IOO] (IFLAiBRi67PH8); L. (L.) chagasi [82] (MHOMIBRi74iPP75); L. (L.) mexicana [77] (MNYCIBZi67iM379); Endotypanum
597
MCHOIBRI 77lM4922, MCHO/BR/79/M5725, MCHO/BR,‘80/ M6159); s. schaudinni [ 1381 (MCHOIPAI79I GML30); E. monterogei [178] (MCHOIClU62IEA9); L. (L.) major [ 1401(MHOMVE/63/L20 (zymodeme LON-1)); Crithidia fasciculata [82] (ATCC12858); Leptomonas seymouri [86] (ATCC30220); Herpetomonas muscarum [126] (ATCC30260); Leishmania (L.) deanei [90] (MCOE/BIU78/M5088); L. (L.) deanei [IOO] (MCOE/BR/75/M4042); L. (L.) hertigi [IOO] (MCOEIPAI65IC8); Typanosoma sp. [184] MCHOI BRi80iML6219); Typanosoma sp. [56] (MCHO/BFU 781M6273). ACON of parasites of the subgenus Viannia does not generally migrate from its origin towards the negative pole. Occasionally, however, in some runs we observed that the ACON of a strain migrated negatively, while in others it had not left the origin. The reasonsfor this are not understood, especially as the electrophoretic conditions were not altered. The inclusion of standard marker strains in all runs serves to control this kind of variabilitv. Because of the general lack of migration we have not given the REM values of the different soecies. Besides the Viannia species mentioned above, ACON of the following marker strains did not migrate or had a marginal negative migration: L. (V.) panamensis (MHOMIPAI 67IBOYNTON); L. (V. ) neruviana (MHOM/PE/84/ L26); L. (V.) shazoi (MCEBIBR/84/M8408); MHOMi BZI82IBELl; MDIDIBRI71IM1597; ITUBIBRI77I M4964: MHOMiBRi73iM2166. The suggestion by KREUTZERet al. (1987) that a combination of about 3 enzymes may be enough to identify neotropical Leishmania, and our finding that non-migratory/cathodic ACON is characteristic of parasites of the subgenus Viannia, reinforce the idea of using a limited number of biochemical characters to identify strains. However, it must be emphasized that such a procedure is feasible only when the Leishmania species of a region are known and have been characterized biochemically. Using, perhaps, 4 key enzymesto identify the speciesof Leishmania encountered in clinical and epidemiological studies is better than examining just a sample of the strains. The latter is sometimes resorted to because of the logistical problems involved in looking at many strains with a large number of enzymes. Before such procedures can be adopted, however, it will be necessarycarefully to compare identifications based on, perhaps, 4 key enzymes and the 10 commonly used in the reference centres. If the results are satisfactory, identifications with a few key enzymes should be a regional typing tool that has been based on enzyme studies in depth. The fmding that ACON was anodic for members of the generaEndotypanum, Leptomonas, Crithidia, Herpetomonas and Typanosoma, only some of which have a peripylarian type of development, suggeststhere is no association between non-migratory or cathodic ACON and the attachment of parasites to the hind gut. The ACON of 38 Leishmania strains, other than the Viannia strains mentioned in previous publications and which were identified with monoclonal antibodies as belongina to the subgenus Viannia. either did not migrate &om the origin or migrated to the negative Dole. Non-miaratorv/cathodic ACON is therefore. a consistent character for all of the 118 peripylarian
598
Eeishmaniu stocks so far examined and, in our opinion, this character can be added to the others that differentiate the subgenera Leishmania and Viannia. Besides this, non-migratory/cathodic ACON has so far not been associatedwith any other trypanosomatid genus. In our laboratory ACON has proved to be extremely useful for distinguishing peripyiarian Leishmania from other trypanosomatids, such as speciesof Trypanosoma, Endotrypanum, Herpetomonas and Crithidia, that may also develop in the hind gut of sandflies, aswell as from suprapylarian Leishmania. It must be emphasized, however, that ACON alone cannot be used to identify a Leishmania species of either the subgeneraLeishmania or Viannia, but it can be used to indicate to which subgenus a strain may belong. Acknowledgements
This work was supported by the Wellcome Trust, London and the Funda@o National de Salide of the Brazilian Ministry of Health, Brasilia. We are grateful to Francisco Gomes, Jose Paulo Cruz, and Raimundo MendonFa for technical help.
References
Arana, M., Evans, D. A., Zolessi, A., Llanos Cuenras, A. & Arevalo, J. (1990). Biochemical characterization of Leiihmaniu (Viannia) braziliensis and Leishmania (Vianniu) peruviana by isoenzyme electrophoresis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 84,
526-529.
1 Announcement
Kreutzer, R. I?., Souraty, N. & Semko, M. E. (1987). Biochemical identities and differences among Leishmaniu species and subspecies. American Journal of Tropical Medicine and Hygiene, 36? 22-32.
Lainson, R., Shaw, J. J., Mdes, M. A. & I%voa, M. M. (1982). Leishmaniasis in Brazil: XVII. Enzymic characterization of a Leishmania from the armadillo, Dusypus novemcinctus (Edentata), from Par6 State. Transactionsof the Royal Sociew of Tropical Medicine and Hygiene, 76,
81&811. Lanotte, G., Rioux, J. A., Maazoun, R., Pasteur, N., Pratlong, F. & Lepart, J. (1981). Application de la methode numtrique 51la taxonomie du genre Leishmania Ross, 1903. A propos de 146 souches originaires de 1’Ancien Monde. Utilisation des allozymes. Corollaires CpidCmiologiqueset phylhtiques. Annales de Parasitologic Humaine et Compake, 56, 575-592. Miles, M. A., Lainson, R., Shaw, J. J., Povoa, M. & Souza, A. A. (1981). Leishmaniasis m Brazil: XV. Biochemical distinction of Leishmania mexicana amozonensis, Leishmania braziliensis braziliensis, and Leishmania braziliensis guyanensis-aetiological agents of cutaneous leishmaniasis in the Amazon Basin of Brazil. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75,
524529. Silveira, F. T., Shaw, J. J., Braga, R. R. & Ishikawa, A. (1987). Dermal leishmaniasis in the Amazon region of Brazil: Leishmania (Viannia) lainsoni sp.n., a new parasite from the state of Par% Memdrias do Znstituio Oswaldo Cruz, 82, 289-292. Received 17 October 1990; revised 24 January accepted fm publication I February 1991
1
Sixth International
Training
Course on Identification of Helminth Parasites of Economic Importance International Institute of Parasitology, St Albans, UK. 93uly-14 August, 1992
The aim of this 6 weeks course is to familiarize participants with methods of identifying hehninth
parasites of economic importance, with special emphasis on those of medical and veterinary
importance. This is the only short course of its kind available and should benefit those engaged in routine identification of helminth parasitesin medical and veterinary laboratories, field workers, and those involved in training and teaching. For further information and application forms please contact Dr L. F. Khalii, International Institute of Parasitology, 395a Hatfield Road, St Albans, Hertfordshire, AL4 OXU, UK (telephone: 0727 833151; fax: 0727 868721; telex: 9312102254 IP G).
1991;