Isoenzyme characterization of 112 Leishmania isolates from French Guiana

610 TRANSACTIONS OFTHE ROYALSOCIETY OPTROPICALMHDIUNEAND HYGIENE

lsoenzyme

characterization

P. Desjeux” and J. P. Dede? Pasteur de Guyane Frangaise

of 112 Leishmania ‘IBBA,

(1989) 83, 610-612

isolates from French Guiana

Embajada de Francis, Casilla 824, La Paz, Bolivia; ‘Institut

Abstract 112Leishmunia isolates, obtained in French Guiana from human lesions, phlebotomine sandflies and wild mammals, were characterized by isoenxyme electrophoresis. Leishmania braziknsis guyanensisand L. mexicana amazonensiswere found parasitizing different natural hosts. L.b. gzganensiswas the dominant species (103 isolates) responsible for most of the human lesions (96.7%). Based on variations observed in 2 enzymes, 3 distinct xymodemes were distinguished within the L.b. guyanensis taxon. Introduction Between 1981 and 1987 we carried out an epidemiological investigation of cutaneous leishmaniasis in French Guiana. We have already published several aspects of this research dealing with natural hosts (DEDET et al., 1985), sandfly taxonomy and ecology (PAJOT et al., 1986; GEOFFROYet al., 1986; LEBBEet al., 1987), the condition of transmission and control (ESTERREet al., 1986; DEDETet al., 1987; ALEXANDREet al., 1987) and, more recently, aspects of the human disease (DEDET et al., 1989). During this investigation, we obtained numerous isolates from sandB.ies, wild mammal and human lesions; 112 of these were characterized by isoenxyme electrophoresis, and the results of this characterization are presented here.

rogenaseEC 1.4.1.2 (GDH NAD), glutamate dehydrogenase EC 1.4.1.2 (GDH NADP+), glutamate oxaloacetate transaminase EC 2.6.1.1 (GOT), phosphoglucomutase EC 2.7.5.1 (PGM), peptidase EC 3.4.11.11 (PEP), aconitate hydrolase EC 4.2.1.3 (ACON), mannose phosphate isomerase EC 5.3.1.8 iM$) and glucose phosphate isomerase EC 5.3.1.9 The electrophoretic procedures were adapted from those of LANHAM et al. (1981) and have been described by TIBAYRENC & LE RAY (1984). Cellulose acetate plates and reagents were purchased from Helena Laboratories. The following six reference strains were used as standards: Leishmunia braziliensis guyanensisMHOM/ BR/75/M4147, L.b. bradiensis MHOM/BR/75/ M2904, L.b. panumensisMHOM/PA/75/M4037, L. mexicana mexicana MNYWBZl62iM379, L.m. amazonettsisIFLAiBR/67/PH8 and L.m. pganoi MHOM/ VEi77iL20. Results Between.1981 and 1987, 1% isolates were obtained from human lesions, sandflies and wild mammals. of which 120 were maintained in culture and c;opreserved in liquid nitrogen, and 112 characterized. Isoenzyme characterization showed that 103 isolates were related to L.b. guyanensisand 7 to L.m. amuzonensis.Two isolates obtained from the sandfly Lutwmyia umbratilis differed from the L.b. guyanensis reference strain in the electrophoretic pattern of 5 enzymes. They also differed from the other reference strains of the L. brazihensis and L. mexicana complexes and from 2 Endonypanum strains, but appeared to be identical to the isolates obtained from Lu.

Materials and Methods Isolates were obtained from the digestive tract of phlebotomine sandflies, from the shin and internal organs (spleen, liver, blood and bone-marrow) of wild mammals, and from human cutaneous lesions. The conditions under which the parasites were isolated have been fully documented in previous papers (DEDET et al., 1982, 1985). Promastigotes of the different isolates were cultivated in RPM1 medium supplemented with 15% foetal calf serum. The cultures were collected at the end of the log phaseand washed 3 times in saline. The final pellet was preserved at -20°C until use. Just before use, an equal volume of hypotonic enzyme stabilizer was added to the pellets, and the promastigotes were lysed by 3 consecutive freeze-thaw cycles. The following 13 enzymes were used: malate dehydrogenase EC 1.1.1.37 (MDH), malic enzyme EC 1.1.1.40 (ME), isocitrate dehydrogenase EC 1.1.1.42 (ICD), 6-phosphogluconate dehydrogenase EC 1.1.1.44 (6PGDH), glucosed-phosphate dehydrogenase EC 1.1.1.49 (G6PDH), glutamate dehyd-

M4147

‘Present address: Parasitic Disease Programme, Ttypanosomiasis/Leishmaniasis, World Health Organization, 1211 Geneva 27, Switzerland. **Present address: IBBA, Embajada de Francis, Casilla 824, La Paz, Bolivia.

Fig. 1. Electrophoreticpatternsof G6PDH showinga slightly more isolates(atrows)obtained rapid migrationof two L.m. (I~~SOMIL(L( from Proechimys cuvieriin French Guiana,than that of the L.m. umazonen5is (L.m.a.) referencestraio PHS. L.m.p.=L. m. pif&, L.b.g.=L.b. guycmensis.

L 20 L.m.p.

MPROIGF182 MPRO/GF

L.b.g. @’

IA35 /83/A94

I

611 umbrutilis by ARIASet al. (1985) in the Brazilian state of Rondonia. Among the L. amazonensis isolates, 3 were obtained from normal skin of the rodent Proechimyscuvieri, 1 from the sandfly Lu. jlkiscutellata and 3 from human lesions. Thev were all identical to each other havine G6PDH with more rapid migration than that of the Ly amuacmensisreference strain PH8 (Fig. 1). 103 strains were closely related to the L. guyunensis reference strain M4147, with variation in 2 enzymes: MDH and ME. In this respect, we distinguished 3 different zymodemes: (i) zymodeme 1 with more rapid MDH and slower ME than those of the reference strain, (ii) zymodeme 2 with more rapid MDH and ME identical to those of the reference strain, and (iii) zymodeme 3 with normal MDH and ME slower than that of the reference strain (Fig. 2). Zymodeme 1 was the most common, both in mammals and in sandflies (Table). The following 6 isolates belonged to this group: 2 isolates from the skin and internal organs of the two-toed sloth, Choloepus diductylus, the principal natural reservoir of L. guyunensis;2 isolates from the marsupial Didelphis marsupialis, which is also a host of T. cruzi in French Guiana; and 2 isolates obtained from normal skin of the rodent P. cur&i. Zymodeme 1 also contained most of the isolates from Lu. umbrutilis and 55 isolates from human lesions. Zymodeme 2 contained one strain from C. diductylus and 33 human isolates. A single isolate from the digestive tract of Lu. umbratilis constituted the third zymodeme. MDH

ME

ml 12345676

12345676

Table. Distribution of the various isolates tested within thee L.6. zymodemes

Zymodemes Isolates Mammals Cholcwpuc didaclylus Did&his marsupialis

Proechimys Human lesions Lutzomyia

Total

umbratilis

23

The existence of different zymodemeswithin recognized species of Leishmania is well known. In the speciesL. guyanensis and L. amazonensis it is generally agreed that the level of infraspecific variation is low, as was also found in this study. Isoenzvmatic variation of G6PDH in L. amazonensishas already been reported by MILES et al. (1979) in Brazilian isolates. The slight variation observed in L. myanetasis from French Guiana corroborates the unpublished observations of several Brazilian authors who tirst noticed it in isolates from other geographical regions (Mohmen, personal communication). From an epidemiological point of view, our results demonstrate the coexistencein the tropical rain forest of French Guiana of at least 2 different species of Leishmania with distinct life-cycles. L. b. guyanensis was found in various wild mammals: in the arboreal edentate C. diductylus as well as in the marsupial D. marsupialis and the rodent P. cuvieri. both of which are terrestrial animals. Until now the only vector of this specieshad appearedto be Lu. (Nyssumyiu)umbrutilis, a speciesmore commonly found in the canopy (LE PONT & PAJOT, 1980), but also encountered at ground level all the year round (PAJOT et al., 1986). L.m. amazonensis in French Guiana appeared to be restricted to the P. cuoieri in contrast to the situation in Para State of Brazil. where it was isolated from 11 different species of wild rodents and marsupials (LAINSON ef al., 1981). L.b. guyanensii was responsible for the vast majority (96.7%) of the human lesions, which were limited to the skin without any mucous involvement (DEDET et al., 1989). Not one isolate of L. b. braziliensis was observed in’our sample, while 9 enzymesout of the 13 used discriminated between L.b. &yanensis and L.b. braziliensis. This finding is in oooosition to the work of COURTOIS er 01. (198?i), who’rkported 2 isolates of L.b. bruziliensis among 10 obtained from soldiers assumed to have been infected in French Guiana. Acknowledgements

Fig. 2. Electrophoretic patterns of tnalic enzyme (ME) and malate dehydrogenase(MDH), showing the profiles of the three zymodemes occurring in French Guiana and those of reference sttains. 1, M 4147 L.6. guyan& reference strain; 2, M 4037L.b. panamen.Greference strain; 3, M 2904L.6. guywmi~; 4, IUMBIGF/82/P30 L.6. guyawnti zymodeme 3 isolate from French Guiana; 5, MCHO/GF/83/A116 L.b. guyanenti zymodeme 2 isolate from French Guiana; 6, MHOM/GF/82&i40 L.b. gnyanenrirzymodeme 1 isolate from French Guiana; 7, PH8 L.m. amuaonen.tisreference strain; 8, M379 L.m. mexicana reference strain.

guyanensis

Discussion

Total

Zl

z2

2 2

1

2

I

I

3 2 2

55

33

-

88

7

-

1

8

68

34

1

103

This study received financial support from the French Ministry of Research and Technology. We thank Drs F. X. Pajot,, J. P. Chippaux and B. Geoffroy for collection and idennfication of the sand&es, Mr G. Chatenay for collection of the mammals, and MS F. Gay for the cultivation of Leishmania isolates. References Alexandre, D. Y., Dedet, 1. P. 81 Esterre. P. (1987). La leishmaniose en Guy&e francake. 7.-Caracte&ques structurales de quelques sites de contamination humaine en for&. Cahiers ORSTOM, Skrie Entomologie Midicale et Parasitologic, 25, 101-109. Arias, J. R., Miles, M. A., Naiff, R. D., Povoa, M. M., Freitas, R. A., Biancardi. C. B. & Castellon. E. G. (1985): Flagellate infections of Brazilian sandflies (Diptera, Psychodidae): isolation in vitro and biochemical identification of Endotypanum and Leishmania. American 3oumal of Tropical Medicine and Hygiene, 34, 1098-l 108. Courtois, D., Rioux, J. A., Pratlong, F., Moreno, G., Lanotte, G. & Courrier, P. L. (1986). Leishmania brasiliensis Vianna, 1911 s.st., en Guyane fran9aise. t;cfle;

de Parasitologic

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Dedet, J. P:, Safjanova, V., Desjeux, P., Emelyanova, L. P., Schnur, L. F., & Chance, M. L. (1982). Ecologic d’un foyer de leishmaniose cutanee dans la region de

612 Thits (Senegal, Afrique de I’Ouest). 6.-Caracterisation et typage des souchesde Leiskmania isoRes. Bulletin de la So&% de Patho&ie Exotique, 75, 155-168. Dedet, J. P., Pajot, F. X., Desjeux, P., Goyot, P., Chippaux., J. P. & Geoffroy, B. (1985). Natural hosts of Leishmanta mexicana amaaonensis Lainson and Shaw, 1972 (Rinetoplastida: Trypanosomatidae) in French Guiana. Transactions of the Royal Society of Tropical Medicine and Hygiene, 79, 302-305.

Dedet, J. P., Esterre, P. & Pradinaud, R. (1987). Individual clothing prophylaxis of cutaneous leishmaniasis in the Amazonian region. Transactions of the Royal Society of Tropical &edicine and Hygiene, 81, 748. Dedet, J. P., Pradinaud, R. & Gay, F. (1989). Epidemiological aspectsof human cutaneousleishmaniasisin French Guiana. Transactions of the Royal Society of Tropical Medicine and Hygiene, in press. Esterre, P., Chippaux, J. P., Lefait, J. F. & Dedet, J. P. (1986). Evaluation d’un programme de lutte contre la leishm.aniosecuts&e dam un village forestier de Guyane FS~T~F.

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tions Institut Pasteur de Guyane francaise: 165~~. + disquette. Le Pont, F. & Pajot F. X. (1980). La leishmaniose en Guyane francaise. 1. Etude de l’ecologie et du taux d’infection naturelle du vecteur Lutzomyia (Nysscmyiu) umbratilis Ward et Fraiha, 1977 en saison s&he. Consid&ations epidtmiologiques. Cahiers ORSTOM, S&e Entomologie Medicale et Parasitologie,

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30 Januaty 1989

1989; accepted

Belge de

for publication

28