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On the identity of the parasite causing diffuse cutaneous leishmaniasis in the Dominican Republic
756 TRANSACTIONS OFTHEROYALSOCIETY OFTROPICAL. MEDICINE ANDHYGIENE, VOL.77, No. 6, 756-762 (1983)
On the identity
of the parasite causing in the Dominican
diffuse cutaneous Republic
leishmaniasis
L. F. SCHNUR’, B. C. WALTON 2* AND H. BOGAERT-DIAZ~ ‘Dept. of Parasitology, Kuvin Centre for Infectious and Tropical Diseases, The Hebrew University Hadassak Medical School, Post Ofice Box 1172, Jerusalem, Israel; 2*Gorgas Memorial Laboratory, Box 6991, Panama 5, Republic of Panama; 31nstitute of Dermatology, Apartado 1090, Santo Domingo, Dominican Republic
Summary Three isolates from disseminated cutaneous leishmaniasis (DCL) patients from an unusual focus in the Dominican Republic were studied and typed according to their behaviour in culture and experimental animals, their malate dehydrogenase(MDH) electrophoretic variant type, and excretory factor (EF) serotype. They were compared with parasites known to causeDCL in other parts of the world. On the basis of these characteristics, this parasite appears to be different from Leishmania aerhiopica which causes DCL in the Old World and from both L. mexicanu p$unoi and L. m. umuzonensis, which are associatedwith DCL in the New World. However, it appearedto have some relationship to the L. mexicunu-complex.
Introduction Diffuse cutaneous leishmaniasis (DCL) is a rare clinico-pathological condition that was first recorded in Venezuela (CONVIT & LAPENTA, 1946). It is also known from the Kenyan and Ethiopian highlands and from scattered casesin the New World from Texas to Central Brazil. More recently it has been encountered in the Caribbean, from the Dominican Republic (BOGAERT-DIAZ et a!., 1975). The condition is characterized by multiple non-ulcerating lesions in the form of nodules and Dlaques, very similar in appearanceto lepromatous &p&y (BRY~ESON,1969, 1970a:CONVIT et al.. 1971). Its definine features are: (i) v&y characteri& l&topatholo& of leproid appearance,showing numerous amastigotespacked in vacuolated macrophages, i.e., “foam cells”, with parasites throughout lesions and in skin of normal appearance; (ii) no evidence of visceralization, other than occasional invasion of lymph glands; (iii) resistance to the chemotherapeutics normally used against leishmaniasis; and (iv) specific anergy to leishmanin skin test antigen. These histopathological and immunological features are considered indicative of immunological abnormality in hosts who suffer this condition. It has been suggested (TURK & BRYCESON,1971; TURK & BELEHU, 1974) that the different clinical forms of leishrndasis, dke those of leprosy, constitute a diseasespectrum related to the immunological competence of infected hosts, whose immunological status is characterized by the histopathology of their lesions and their skin responsiveness. The DCL condition, with its associated anergy, appears at one end of this spectrum, leishrnanias~~;e&livans (LR) (DOSTROVSKY, 1934, 1936), with its associated hypersensitivity, at the other end, and the more usual restricted, self-limiting and self-curing lesions are intermediate. TURK & BELEHU (1974) also sueraest that the particular form the irnmbne response Fgkes probably depends on the way antigen is presented and on the genetically determined ability of a person to respond, which many external factors (e.g., age,
nutrition, intercurrent infections, drug treatment) can influence, thus altering the position on the spectrum. This interpretation of the clinical and immunological effects seenamongst the various types of leishmaniasis casesrecorded stressesthe role played by the host and largely ignores the parasite-determined factors contributing to the condition. When DCL was first seen in Venezuela, it was considered to be caused by an unusual parasite that was consequently regarded a separate species and named L. pifunoi (MEDINA & ROMERO,1959). However, it is now recognized that the type of immunological response and clinical condition seen are related to both the type of leishmanial parasite involved and specific hostdermined factors. The type of parasite is very significant and certain clinical syndromes exist only in relation to ,specific .types of parasite. In the Old World,DCL is associatedonly with L. uethiopicu as defined serologicallv and biochemicallv (SCH~~UR & ZUCKERMAN,1977; CHANCEet al., 1978; CHANCE. 19791.In the New World. DCL is associated with L. mexicana endemic areas and all isolates identified to date have belonged to the L. rnexicunucomplex (CHANCE et al., 1977; Lainson, personal communication). It is also recognized that all DCL patients have an abnormal immune response. In individuals with normal responses, these parasites causeulcerating lesions of the usual tvpes that resolve and lead to serf-cure and, supposediy, immunity to reinfection. Thus. DCL is not oroduced bv all snecies of Leishmuniu, b;t can only be caused by certain types, and appears to result from the combination of particular types of parasite, with special intrinsic characteristics, infecting particular hosts with a specific immunological abnormality. Present knowledge of the causative organism of DCL is basedupon casesreported from one particular area in the Old World, the Kenyan and Ethiopian *Present address: Trypanosomiasesand Leishmaniases Unit, ParasiticDiseasesProgramme,World Health Organization, 1211Geneva27, Switzerland.
L. F. SCHNUR et al.
highlands, where the parasite is L. aethiopica, and principally one area in the New World, Venezuela, where the parasite is L. mexicuna pifunoi (= L. pifanoi). However, it now appearsthat other members of the L. mexicana-complexalso possessthe characteristics that can produce DCL, since cases have occurred at the northernmost limit of the distribution of New World leishmanial parasites, in S.E. Texas and N.E. Mexico, as well as in South America, where it is associatedwith L. m. umuzonensis (CHANCE et al., 1977; MILES et al., 1979). A unique focus of DCL exists in the Dominican Renublic (BOGAERT-DIAZ et al.. 1975). with an unusual number of cases,in the complete’absenceof caseswith ordinary ulcerating lesions (Bogaert-Diaz, unpublished data). This feature, as well as the geographical isolation of this insular focus, provokes special interest regarding the identity of its causative agent. This report describes some of the biological, biochemical and serological charactersof this leishmanial parasite and compares them to those of some leishmanial parasites known to cause DCL elsewhere in the world.
Materials
and Methods
The leishmanial strains and their isolation and culture
Three DCL stocks from the Dominican Republic were studied: LRC-L280 (=AMRU-448), from a 54-year-old female; LRC-L281 (=AMRU-450), from a ICyear-old female,*; and LRC-L285 (=AMRU-452), from a 14-yearold male. These were isolated by needle aspirates from skin nodules seeded into USAMRU blood-agar medium (WALTON et al.,, 1977b). Subsequent passages-weremade into the
samemedrumand into modified Senekjie’sand Schneider’s insect tissue culture medium supplemented with 30% foetal calf serum. The isolates and subcultures were maintained at 25°C. The three stocks were sent to the WHO Reference Centre for Leishmaniasis (LRC) in Jerusalem, where they were maintained on modified Tobie, von Brand, Mehlman medium (TOBIE et al., 1950; EL-ON,, 1969) at 28”~. This is a diphasic, blood-agar medmm, containing 2.5 g agar, 1 g peptone, 0.5 g NaCl, 0.39 g beef extract (Difco, Detroit) and 10% dmbrinated rabbit blood or rabbit blood lysate, with an overlay of 0.85% saline, containing 200 units crystalline penicillin and 200 ug streptomycin per ml. Strains used as markers for typing were obtained from the leishmanial strain culture collection of the WHO-LRC.
of laboratoy animals Promastigotes of the LRCL281 (AMRU-450) stock, harvested from the liquid phase of second and third passage cultures grown on USAMRU blood-agar medium (WALTON et al., 1977b), were counted in a haemocytometer. Two Syrian hamsters, Mesocricetus auratus, and two African white-tailed rats, Mystromys albicaudatus, were each inoculated subcutaneously with approximately 4 X lo6 promastigotes: the former into the nose; the latter into the base of the tail.
Inoculation
Excreted factor serotyping
Excreted factors (EF) were collected and serotypes determined by the method described by SCHNURet al. (1972) and SCHNUR& ZUCKERMAN(1977), employing standard type EFs and antisera. *This isolate was made from one of the patients reported in the study by PETERSENet al., 1982, and presumably should have the same characteristics as their strain “Isabel” (NIH).
757
Malate dehydrogenase enzyme variant determination
The electrophoretic mobility of the malate dehydrogenase (MDH) of the stocks was determined by polyacrylamide gel disc electrophoresis, as described by GARDENER et al. (1974) and CHANCE et al. (1977). emolovine several standard marker strains of known ‘MDH variait type: L. major LRC-L137, type XIII; L. m. amazonensis LRC-L259 (=LV160), type III; L. aethiopica LRCL147, type V; L. dcmovani LRC-L133 (=LV9), type VII, and also a L. mexicana s.1. strain from Belize LRC-L94. that seemsto be another, as yet undefined, variant type. Results
The growth of Dominican Republic DCL artificial media
stocks in
The growth of the three strains in USAMRU diphasic medium was initially very indolent. After three or four transfers the growth rate increased, being essentially equal to that usually seen with L. b. punumensis, but never showed the rapid proliferation seen upon the same medium with a stock (AMRU1156) of L. mexicunu isolated in Texas (WALTON et al., 1977a). Following transfer into modified Tobie, von Brand, Mehhnan medium, they grew well, but neither achieved the luxuriant growth described by LAINSON & SHAW (1972) for L. mexicuna umuzonensis grown in diphasic blood-agar medium, nor were there the abundant and quite massive rosettes so typical of L. mexicunu amuzonensis cultures. L. uethiopicu also grows well in this diphasic blood-agar medium, often with rosettes, but it would be difficult to differentiate it easily from the Dominican Republic strains on this basis. The infectivity and puthogenicity of Dominican Republic DCL stocks
One of the DCL stocks, LRCL250, was infective to both Syrian hamsters and African white-tailed rats, when inoculated subcutaneously. Figs. la and lb show the types of lesions seen after 4.5 months. The growth of this Dominican Republic strain in the nose of the hamsters was relatively indolent and very similar to that observed with L. bruziliensis punamensis and unlike that of L. mexicunu (AMRU-1156) in animals from the same colony, in which massive swelling and multiple metastaseswere produced by this time. Similarly, the number of parasites in tissues was less than with L. mexicana. L. uethiopicu strains, generally, barely infect Syrian hamsters (SCHNUR et al., 1973; SCHNUR & ZLJCKERMAN, 1977) and are very
indolent when they do. The lesions produced in the African white-tailed rat were even more indolent than those produced by L. b. bruziliensis in animals from the same colony and remained as large, hard, and highly elevated papules for over two months without ulcerating. Lesions caused by L. b. punumensis ulcerated much more quickly from a small papule, as described in the same model by MCKINNEY & HENDRICKS (1980). Unfortunately behaviour of L. mexicunu strains in this host has not been recorded so no comparison can be made. The excreted factor (EF) serotvtres of the Dominican Republic DCL stocks ’ AZ J
The EFs of all three Dominican Republic DCL stocks reacted identically with the type antisera, proving to be subserotype AJBJ (Fig. 2), a subserotype hitherto unrecorded.
758
IDENTITY
OF
PARASITE
CAUSING
DCL
IN
DOMINICAN
REPUBLIC
Fig. 1. Lesions caused in (a) the nose of a Syrian hamster and (b) the base of the tail of an African while-tailed rat by the Dominican Republic leishmanial strain LRC-L281 isolated from a human case of diffuse cutaneous leishmaniasis (DCL), 4.5 months after subcutaneous inoculation of culture promastigotes. Dashed lines added in (b) indicate margin of elevated papule which does not show well in the black and white photograph.
L. F. scmxm
et al.
Fig. 2. Gel diffusion of EFs from Dominican Republic DCL strains and EFs of known marker strains: the numbers inscribed in the wells are WHO reference numbers and should be prefixed by LRC-L; h indicates EF homologous to a given antiserum. Strains producing EFs that react with anti-l. fropica LRC-L32 are EF serotype A strains, and tlx various types of reactions seen indicate suhserotypes. The type of reaction given in diffusion plate 1 with EF of L. major LRC-L137 from Israel, indicates subserotype A, strains, that given with EF of L. noprca LRC-L32 from Iraq indicates subserotype A, strains, that given with EF of L. mexicana mexzcana LRC-L94 from Belize indicates subserotype Al strains, and that given with EF of Leishmaniu sp. LRC-L285 from the Dominican Republic indicates subserotype A+ It should be noted that EF from a subserotype A, strain produces two precipitation bands when diffused against anti-LRC-L32, one of which shows complete identity with EF from subserotype A4 strains, indicating shared antigenic components. Strains producing EFs that react with anti-l. dmumzni LRC-L52 and anti-L. in/antum LRC-L47 are EF serotype B strains. Anti-LRC-L52 can only indicate that a strain is, or is not, a B strain. Thus, diffusion plate 2 indicates that the Indian L. donovani strain LRC-L52 and the Ethiopian L. donovani strain LRC-L133 are B strains, as are the Dominican Republic stocks LRC-L280, LRC-L281 and LRC-L285, in addition to their being A strains. The type OFreaction given in diffusion plate 3 with EF of L. aethwpica LRC-L147 from Ethiopia indicates subserotype B, strains and that with EF of L. donooani LRC-L52 from India indicates subserotype Bz strains.
760
IDENTITY OF PARASITE CAUSING DCL IN DOMINICAN REPUBLIC
Fig. 3. Polyacrylamidegelscomparingthe electrophoreticmobility of the malatedehydrogenase (MDH) of the DominicanRepublicstock LRC-L285with that of the IsraeliL. majorstrainLRC-L137:b is the positionof the bromophenolblue marking the front of the run; the arrow indicatesnon-specificstaining.
The malate dehydrogenase (MDH) electrophoretic variant type of the Dominican Republic DCL stocks The MDH electrophoretic variant type was the same for all three DCL stocks, being variant type I (Fig. 3), using the system described by GARDENER et al. (1974), CHANCE et al. (1977) and CHANCE (1979). Discussion On the basis of the characters compared here (summarized in the table), it is apparent that the leishmanial agent causing DCL in the Dominican Republic is neither L. aethiopica, the parasite causing DCL in the East African highlands, nor L. m. amazonensis, the parasite causing DCL in parts of
Brazil. Similarly, it does not seem to be L. m. pifanoi (= L. oifanoi). which is associated with DCL in Venezuela, even though the Dominican Republic strains and L. m. pifunoi produce MDH of the same electrophoretic mobility under the electrophoretic conditions used in this study. Since all other DCL in the New World, from Texas to Brazil, is apparently caused by L. mexicanacomplex parasites, it is somewhat surprising that the isolates from the Dominican Republic do not clearly fall into this group, although at this time they cannot be excluded with certainty. Growth in blood-agar media has been shown to be a variable characteristic in the L. bruziliensis-complex (WALTON et al., 1977a), and similar exceptions to the rule might also occur in the L. mexicana-complex. However, the rapid evolution of lesions in the hamster with widespread metastases that has always been considered distinctive of L. mexicana-complex parasites was also not seen. The apparent similarity of L. m. pifunoi (= L. pifunot) and these strains in sharing MDH of variant type I might indicate true identity, but it also could be a superficial similarity, since similarity of the electrophoretic mobilities of two populations of molecules does not guarantee identical molecular structure. Comparison of the EF subserotype of the Dominican Republic stocks, with those of the other types of DCL strain indicates that there is some sharing of antigenic determinants (Fig. 2 and table), indicating components with identical, or very similar, molecular structure to those of some L. mxicana-complex strains. The absence of a B component in the EF of L. m. pifanoi strains from Venezuela, and of an A comoonent in the EF of L. aethioaica. indicates a subsiantial difference between these ’ two DCLinducing forms and the Dominican Republic strains examined. However, a strain of L. mexicana from Belize, LRC-L94, has a mixed EF serotype, AsBr, showing a shared B component. However, this subspecies has, to our knowledge, never been shown to be associated with DCL. DNA characteristics of two of the Dominican Republic strains have been studied by other researchers. BARKER et al. (1982) measured the nuclear and kinetoplast DNA buoyant densities of the strains LRC-L281 (= AMRU-450 = LUMP 1433) and LRCL285 (= AMRU-452 = LUMP 1434), showing them to be similar to two strains of L. m. amnzonensis and different from a strain of L. m. mexicana. Desnite this they concluded that neither strain could- be positively identified with any of the New World and Old World marker strains that they used, which included L. aethiopica strains. SPITHILL et al. (1983) characterized the strain LRC-L285 bv endonuclease fingerprinting and Southern blot hybridization analysis of its kinetoplast DNA, finding it to be similar to L. major (formerly L. tropicn major) strains and different from them in the size of its EcoRl-cut unit length minicircle. In conclusion, the three DCL isolates from the Dominican Republic studied here appear to be different from the other tvnes of leishmanial oreanisms associated with DCL in the Old World and oiher parts in the New World, but do show some antigenic similarity to some strains grouped in the L. mexicanacomplex.
L.
Table-A comparison leishmaniasis (DCL) Species of Leishmania (Origin) L. aethiopica (Ethiopia) L. m. pifanoi (Venezuela) L. m. amazonensis (Brazil) Leishmania sp. (Dominican Republic)
of some characteristics
F.
SCHNUR
of leishmanial
Development of lesions in hamsters none to minimal rapid growth with metastases rapid growth with metastases slow growth without metastases
761
t?t al.
species associated with disseminated
Growth in blood-agar medium
cutaneous
Excreted factor (EF) subserotype
Malate dehydrogenase we
rapid
Bl
Vi
rapid
Al*
I+
rapid
BX
III
slow to medium
A&*
I
* some of the antigenic components in the EF subserotypes A, and A4 are identical (see Fig. 2 and its legend). + data taken from CHANCE et al., 1977 & CHANCE, 1979.
Acknowledgements This investigation received support from the UNDl’i World Ban&WHO Special Programme for Research and Training in Tropical Diseases. The contribution of one of us (B. C. Walton) was made as a part of Project AMRO-0800 of the Pan American Health Organizations at Gorgas Memorial Laboratory in Panama.
References Barker, D. C., Arnot, D. E. & Butcher, J. (1982). DNA characterization as a taxonomic tool for identification of kinetoplastic flagellate protozoa. In: Biochemical Characterization of Leishmania. M. L. Chance and B. C. Walton (Editors). Geneva: UNDP/World BankWHO Special Programme for Research and Training in Tropical Diseases, pp. 139-180. Bogaert-Diaz, H., Rojas, R. F., De Leon? A., Martinez, D. & Quiriones, M. (1975). Leishmamasis tegumentaria americana: reporte de 10s primeros tres cases. Forma anergica en tres hermanos. Revista Dominicana de Dermatologia, 9, 19-33. Bryceson, A. D. M. (1969). Diffuse cutaneous leishmaniasis in Ethiopia. I. The clinical and histological features of the disease. Transactions of the Roval Sociehr of TroDical Medicine and Hygiene, 6& 708-737. - . _ Bryceson, A. D. M. (1970a). Diffuse cutaneous leishmaniasis in Ethiopia. II. Treatment. Transactions of the Royal Society of Tropical Medicine and Hygiene, 64, 369-379. Bryceson, A. D. M. (1970b). Diffuse cutaneous leishmaniasis in Ethiopia. III. Immunological studies. Transactions of the Royal Society of Tropical Medicine and Hygiene, 64, 380-387. Chance, M. L. (1979). The identification of Leishmania. In: Problems in the Identification of Parasites and their Vectors. Angela E. R. Taylor and R. Muller (Editors). Symposia of the British Society of Parasitology, Vol. 17, Oxford, London, Edinburgh, Melbourne: Blackwell Scientific Publications, pp. 55-74. Chance, M. L., Gardener, P. J. & Peters, W. (1977). Biochemical taxonomy of Leishmania as an ecological tool. In: Colloques Internationaux du Centre National de la Recherche Scientifique, No. 239-Ecologic des Leishmanioses. Montpellier, 18-24 Aofit, 1974. Chance, M. L., Schnur, L. F., Thomas, S. C. & Peters, W. (1978). The biochemical and serological taxonomy of Leishmania from the Aethiopian zoogeographical region of Africa. Annals of Tropical Medicine and Parasitology, 72, 533-542.
Convit, J. & Lapenta, P. (1946). Sobre un case de leishmaniosis tegumentaria de forma disseminada. Revista Policlinica, Caracas, 17, 153-158. Convit, J., Pinardi, M. E. & Rondon, A. J. (1971). Diffuse cutaneous leishmaniasis: a disease due to an immunological defect of the host. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 603-610. Dostrovsky, A. (1934). Leishmania recidiva of the skin. Harefuah, 8, 118-124. [In Hebrew, English summary pp. l-2.1 Dostrovsky, A. (1936). Relapses in cutaneous leishmaniasis. Annals of Tropical Medicine and Parasitology, 3, 267-274. El-On, J, (1969). Comparison of discelectrophoretic profiles of in vivo (trypomastigote) forms and in vitro (epimastigote) forms of trypanosomes. M.Sc. thesis, Hebrew University, Jerusalem, in Hebrew. Gardener, I’. J., Chance, M. L. & Peters, W. (1974). Biochemical taxonomy of Leishmania. II. Electrophoretic variation of malate dehydrogenase. Annals of TropIcal Medicine and Parasitology, 68, 317-325. Lainson, R. & Shaw, J. J. (1972). Leishmania of the New World: taxonomic problems. British Medical Bulletin, 28, 44-48. McKinney, L. A. & Hendricks, L. A. (1980). Experimental infection of Mystromys albicaudatus with Leishmania braziliensis. Pathology. American Journal of Tropical Medicine and Hygiene, 29, 753-760. Medina, R. & Romero, J. (1959). Estudio clinic0 y parasitologic0 de una nueva cepa de Leishmania. Archives Venezolanos de Medicina Tropical y Parasitologia Medica, 3, 298-326. Miles, M. A., Povoa, M. M., De Souza,A., Lainson, R. & Shaw, J. J. (1979). Some methods for the enzymic characterization of Latin-American Leishmania with particular reference to Leishmania mexicana amazonensis and subspecies of Letshmania hertigi. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 243-252. Petersen, E. A., Neva, F. A., Oster, C. N. & BogaertDia?, H. (1982). Specific inhibition of lymphocyteprohferation response by adherent suppressor cells in diffuse cutaneous leishmaniasis. New EnglandJournal of Medicine, 306, 387-392. Schnur, L. F. & Zuckerman, Z. (1977). Leishmanial excreted factor (EF) serotypes in Sudan, Kenva and Ethiopia. Annals of Tropical Medictne and Para&ology, 71, 273-294. Schnur, L. F:, Zuckerman, A. & Greenblatt, C. L. (1972). Leishmamal serotypes as distinguished by the gel diffusion of factors excreted in vitro and in vivo. IsraelJournal of Medical Sciences, 8, 982.942.
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OF
PARASITE
CAUSING
Schnur, L. F., Zuckerman, A. & Montillo, B. (1973). Dissemination of leishmanias to the organs of Syrian hamsters following intrasplenic inoculation of promastigotes. Experimental Parasitology, 34, 432-447. Spithill, T., Grumont., R. & Mitchell, G. F. (1983). Characterization of isolates and clones of Leishmania by analysis of kinetoplast DNA. Abstracts of 12th Annual UCLA Symposia, Journal of Cellular Biochemistry, supplement 7Aj p. 27. Tobie, E. J., Von Brand, T. & Mehlman, B. (1950). Cultural and Phvsioloeical observations on Trvnanosoma rhodesiense and f. gam%iense.Journal of Para&logy, 36, 48-54. Turk, J. L. & Belehu, A. (1974). Immunological spectra in infectious diseases.In: Parasites in the Immunized Host: Mechanisms of suruival. Ciba Foundation Symposium 25 (new series), Amsterdam, London, New York: Associated Scientific Publishers, pp. 101-122.
DCL
IN
DOMINICAN
REPUBLIC
Turk, J. L. & Bryceson, A. D. M. (1971). Immunological phenomena in leprosy and related diseases.In: Advances in Immunology, 13, London, New York: Academic Press, pp. 209-266. Walton, B. C., Intermill~ R. W. & Hajduk, M. E. (1977a). Difference in biological characteristics of three Leishmania isolates from patients with espundia. American Journal of Tropical Medicine and Hygiene, 26, 850-855. Walton, B. C., Shaw, J. J. & Lainson, R. (1977b). Observations on the in vitro cultivation of Leishmania braziliensis. Journal of Parasitology, 63, 1118-l 119.
Accepted for publication 21st January,
1983.
On the identity
of the parasite causing in the Dominican
diffuse cutaneous Republic
leishmaniasis
L. F. SCHNUR’, B. C. WALTON 2* AND H. BOGAERT-DIAZ~ ‘Dept. of Parasitology, Kuvin Centre for Infectious and Tropical Diseases, The Hebrew University Hadassak Medical School, Post Ofice Box 1172, Jerusalem, Israel; 2*Gorgas Memorial Laboratory, Box 6991, Panama 5, Republic of Panama; 31nstitute of Dermatology, Apartado 1090, Santo Domingo, Dominican Republic
Summary Three isolates from disseminated cutaneous leishmaniasis (DCL) patients from an unusual focus in the Dominican Republic were studied and typed according to their behaviour in culture and experimental animals, their malate dehydrogenase(MDH) electrophoretic variant type, and excretory factor (EF) serotype. They were compared with parasites known to causeDCL in other parts of the world. On the basis of these characteristics, this parasite appears to be different from Leishmania aerhiopica which causes DCL in the Old World and from both L. mexicanu p$unoi and L. m. umuzonensis, which are associatedwith DCL in the New World. However, it appearedto have some relationship to the L. mexicunu-complex.
Introduction Diffuse cutaneous leishmaniasis (DCL) is a rare clinico-pathological condition that was first recorded in Venezuela (CONVIT & LAPENTA, 1946). It is also known from the Kenyan and Ethiopian highlands and from scattered casesin the New World from Texas to Central Brazil. More recently it has been encountered in the Caribbean, from the Dominican Republic (BOGAERT-DIAZ et a!., 1975). The condition is characterized by multiple non-ulcerating lesions in the form of nodules and Dlaques, very similar in appearanceto lepromatous &p&y (BRY~ESON,1969, 1970a:CONVIT et al.. 1971). Its definine features are: (i) v&y characteri& l&topatholo& of leproid appearance,showing numerous amastigotespacked in vacuolated macrophages, i.e., “foam cells”, with parasites throughout lesions and in skin of normal appearance; (ii) no evidence of visceralization, other than occasional invasion of lymph glands; (iii) resistance to the chemotherapeutics normally used against leishmaniasis; and (iv) specific anergy to leishmanin skin test antigen. These histopathological and immunological features are considered indicative of immunological abnormality in hosts who suffer this condition. It has been suggested (TURK & BRYCESON,1971; TURK & BELEHU, 1974) that the different clinical forms of leishrndasis, dke those of leprosy, constitute a diseasespectrum related to the immunological competence of infected hosts, whose immunological status is characterized by the histopathology of their lesions and their skin responsiveness. The DCL condition, with its associated anergy, appears at one end of this spectrum, leishrnanias~~;e&livans (LR) (DOSTROVSKY, 1934, 1936), with its associated hypersensitivity, at the other end, and the more usual restricted, self-limiting and self-curing lesions are intermediate. TURK & BELEHU (1974) also sueraest that the particular form the irnmbne response Fgkes probably depends on the way antigen is presented and on the genetically determined ability of a person to respond, which many external factors (e.g., age,
nutrition, intercurrent infections, drug treatment) can influence, thus altering the position on the spectrum. This interpretation of the clinical and immunological effects seenamongst the various types of leishmaniasis casesrecorded stressesthe role played by the host and largely ignores the parasite-determined factors contributing to the condition. When DCL was first seen in Venezuela, it was considered to be caused by an unusual parasite that was consequently regarded a separate species and named L. pifunoi (MEDINA & ROMERO,1959). However, it is now recognized that the type of immunological response and clinical condition seen are related to both the type of leishmanial parasite involved and specific hostdermined factors. The type of parasite is very significant and certain clinical syndromes exist only in relation to ,specific .types of parasite. In the Old World,DCL is associatedonly with L. uethiopicu as defined serologicallv and biochemicallv (SCH~~UR & ZUCKERMAN,1977; CHANCEet al., 1978; CHANCE. 19791.In the New World. DCL is associated with L. mexicana endemic areas and all isolates identified to date have belonged to the L. rnexicunucomplex (CHANCE et al., 1977; Lainson, personal communication). It is also recognized that all DCL patients have an abnormal immune response. In individuals with normal responses, these parasites causeulcerating lesions of the usual tvpes that resolve and lead to serf-cure and, supposediy, immunity to reinfection. Thus. DCL is not oroduced bv all snecies of Leishmuniu, b;t can only be caused by certain types, and appears to result from the combination of particular types of parasite, with special intrinsic characteristics, infecting particular hosts with a specific immunological abnormality. Present knowledge of the causative organism of DCL is basedupon casesreported from one particular area in the Old World, the Kenyan and Ethiopian *Present address: Trypanosomiasesand Leishmaniases Unit, ParasiticDiseasesProgramme,World Health Organization, 1211Geneva27, Switzerland.
L. F. SCHNUR et al.
highlands, where the parasite is L. aethiopica, and principally one area in the New World, Venezuela, where the parasite is L. mexicuna pifunoi (= L. pifanoi). However, it now appearsthat other members of the L. mexicana-complexalso possessthe characteristics that can produce DCL, since cases have occurred at the northernmost limit of the distribution of New World leishmanial parasites, in S.E. Texas and N.E. Mexico, as well as in South America, where it is associatedwith L. m. umuzonensis (CHANCE et al., 1977; MILES et al., 1979). A unique focus of DCL exists in the Dominican Renublic (BOGAERT-DIAZ et al.. 1975). with an unusual number of cases,in the complete’absenceof caseswith ordinary ulcerating lesions (Bogaert-Diaz, unpublished data). This feature, as well as the geographical isolation of this insular focus, provokes special interest regarding the identity of its causative agent. This report describes some of the biological, biochemical and serological charactersof this leishmanial parasite and compares them to those of some leishmanial parasites known to cause DCL elsewhere in the world.
Materials
and Methods
The leishmanial strains and their isolation and culture
Three DCL stocks from the Dominican Republic were studied: LRC-L280 (=AMRU-448), from a 54-year-old female; LRC-L281 (=AMRU-450), from a ICyear-old female,*; and LRC-L285 (=AMRU-452), from a 14-yearold male. These were isolated by needle aspirates from skin nodules seeded into USAMRU blood-agar medium (WALTON et al.,, 1977b). Subsequent passages-weremade into the
samemedrumand into modified Senekjie’sand Schneider’s insect tissue culture medium supplemented with 30% foetal calf serum. The isolates and subcultures were maintained at 25°C. The three stocks were sent to the WHO Reference Centre for Leishmaniasis (LRC) in Jerusalem, where they were maintained on modified Tobie, von Brand, Mehlman medium (TOBIE et al., 1950; EL-ON,, 1969) at 28”~. This is a diphasic, blood-agar medmm, containing 2.5 g agar, 1 g peptone, 0.5 g NaCl, 0.39 g beef extract (Difco, Detroit) and 10% dmbrinated rabbit blood or rabbit blood lysate, with an overlay of 0.85% saline, containing 200 units crystalline penicillin and 200 ug streptomycin per ml. Strains used as markers for typing were obtained from the leishmanial strain culture collection of the WHO-LRC.
of laboratoy animals Promastigotes of the LRCL281 (AMRU-450) stock, harvested from the liquid phase of second and third passage cultures grown on USAMRU blood-agar medium (WALTON et al., 1977b), were counted in a haemocytometer. Two Syrian hamsters, Mesocricetus auratus, and two African white-tailed rats, Mystromys albicaudatus, were each inoculated subcutaneously with approximately 4 X lo6 promastigotes: the former into the nose; the latter into the base of the tail.
Inoculation
Excreted factor serotyping
Excreted factors (EF) were collected and serotypes determined by the method described by SCHNURet al. (1972) and SCHNUR& ZUCKERMAN(1977), employing standard type EFs and antisera. *This isolate was made from one of the patients reported in the study by PETERSENet al., 1982, and presumably should have the same characteristics as their strain “Isabel” (NIH).
757
Malate dehydrogenase enzyme variant determination
The electrophoretic mobility of the malate dehydrogenase (MDH) of the stocks was determined by polyacrylamide gel disc electrophoresis, as described by GARDENER et al. (1974) and CHANCE et al. (1977). emolovine several standard marker strains of known ‘MDH variait type: L. major LRC-L137, type XIII; L. m. amazonensis LRC-L259 (=LV160), type III; L. aethiopica LRCL147, type V; L. dcmovani LRC-L133 (=LV9), type VII, and also a L. mexicana s.1. strain from Belize LRC-L94. that seemsto be another, as yet undefined, variant type. Results
The growth of Dominican Republic DCL artificial media
stocks in
The growth of the three strains in USAMRU diphasic medium was initially very indolent. After three or four transfers the growth rate increased, being essentially equal to that usually seen with L. b. punumensis, but never showed the rapid proliferation seen upon the same medium with a stock (AMRU1156) of L. mexicunu isolated in Texas (WALTON et al., 1977a). Following transfer into modified Tobie, von Brand, Mehhnan medium, they grew well, but neither achieved the luxuriant growth described by LAINSON & SHAW (1972) for L. mexicuna umuzonensis grown in diphasic blood-agar medium, nor were there the abundant and quite massive rosettes so typical of L. mexicunu amuzonensis cultures. L. uethiopicu also grows well in this diphasic blood-agar medium, often with rosettes, but it would be difficult to differentiate it easily from the Dominican Republic strains on this basis. The infectivity and puthogenicity of Dominican Republic DCL stocks
One of the DCL stocks, LRCL250, was infective to both Syrian hamsters and African white-tailed rats, when inoculated subcutaneously. Figs. la and lb show the types of lesions seen after 4.5 months. The growth of this Dominican Republic strain in the nose of the hamsters was relatively indolent and very similar to that observed with L. bruziliensis punamensis and unlike that of L. mexicunu (AMRU-1156) in animals from the same colony, in which massive swelling and multiple metastaseswere produced by this time. Similarly, the number of parasites in tissues was less than with L. mexicana. L. uethiopicu strains, generally, barely infect Syrian hamsters (SCHNUR et al., 1973; SCHNUR & ZLJCKERMAN, 1977) and are very
indolent when they do. The lesions produced in the African white-tailed rat were even more indolent than those produced by L. b. bruziliensis in animals from the same colony and remained as large, hard, and highly elevated papules for over two months without ulcerating. Lesions caused by L. b. punumensis ulcerated much more quickly from a small papule, as described in the same model by MCKINNEY & HENDRICKS (1980). Unfortunately behaviour of L. mexicunu strains in this host has not been recorded so no comparison can be made. The excreted factor (EF) serotvtres of the Dominican Republic DCL stocks ’ AZ J
The EFs of all three Dominican Republic DCL stocks reacted identically with the type antisera, proving to be subserotype AJBJ (Fig. 2), a subserotype hitherto unrecorded.
758
IDENTITY
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DCL
IN
DOMINICAN
REPUBLIC
Fig. 1. Lesions caused in (a) the nose of a Syrian hamster and (b) the base of the tail of an African while-tailed rat by the Dominican Republic leishmanial strain LRC-L281 isolated from a human case of diffuse cutaneous leishmaniasis (DCL), 4.5 months after subcutaneous inoculation of culture promastigotes. Dashed lines added in (b) indicate margin of elevated papule which does not show well in the black and white photograph.
L. F. scmxm
et al.
Fig. 2. Gel diffusion of EFs from Dominican Republic DCL strains and EFs of known marker strains: the numbers inscribed in the wells are WHO reference numbers and should be prefixed by LRC-L; h indicates EF homologous to a given antiserum. Strains producing EFs that react with anti-l. fropica LRC-L32 are EF serotype A strains, and tlx various types of reactions seen indicate suhserotypes. The type of reaction given in diffusion plate 1 with EF of L. major LRC-L137 from Israel, indicates subserotype A, strains, that given with EF of L. noprca LRC-L32 from Iraq indicates subserotype A, strains, that given with EF of L. mexicana mexzcana LRC-L94 from Belize indicates subserotype Al strains, and that given with EF of Leishmaniu sp. LRC-L285 from the Dominican Republic indicates subserotype A+ It should be noted that EF from a subserotype A, strain produces two precipitation bands when diffused against anti-LRC-L32, one of which shows complete identity with EF from subserotype A4 strains, indicating shared antigenic components. Strains producing EFs that react with anti-l. dmumzni LRC-L52 and anti-L. in/antum LRC-L47 are EF serotype B strains. Anti-LRC-L52 can only indicate that a strain is, or is not, a B strain. Thus, diffusion plate 2 indicates that the Indian L. donovani strain LRC-L52 and the Ethiopian L. donovani strain LRC-L133 are B strains, as are the Dominican Republic stocks LRC-L280, LRC-L281 and LRC-L285, in addition to their being A strains. The type OFreaction given in diffusion plate 3 with EF of L. aethwpica LRC-L147 from Ethiopia indicates subserotype B, strains and that with EF of L. donooani LRC-L52 from India indicates subserotype Bz strains.
760
IDENTITY OF PARASITE CAUSING DCL IN DOMINICAN REPUBLIC
Fig. 3. Polyacrylamidegelscomparingthe electrophoreticmobility of the malatedehydrogenase (MDH) of the DominicanRepublicstock LRC-L285with that of the IsraeliL. majorstrainLRC-L137:b is the positionof the bromophenolblue marking the front of the run; the arrow indicatesnon-specificstaining.
The malate dehydrogenase (MDH) electrophoretic variant type of the Dominican Republic DCL stocks The MDH electrophoretic variant type was the same for all three DCL stocks, being variant type I (Fig. 3), using the system described by GARDENER et al. (1974), CHANCE et al. (1977) and CHANCE (1979). Discussion On the basis of the characters compared here (summarized in the table), it is apparent that the leishmanial agent causing DCL in the Dominican Republic is neither L. aethiopica, the parasite causing DCL in the East African highlands, nor L. m. amazonensis, the parasite causing DCL in parts of
Brazil. Similarly, it does not seem to be L. m. pifanoi (= L. oifanoi). which is associated with DCL in Venezuela, even though the Dominican Republic strains and L. m. pifunoi produce MDH of the same electrophoretic mobility under the electrophoretic conditions used in this study. Since all other DCL in the New World, from Texas to Brazil, is apparently caused by L. mexicanacomplex parasites, it is somewhat surprising that the isolates from the Dominican Republic do not clearly fall into this group, although at this time they cannot be excluded with certainty. Growth in blood-agar media has been shown to be a variable characteristic in the L. bruziliensis-complex (WALTON et al., 1977a), and similar exceptions to the rule might also occur in the L. mexicana-complex. However, the rapid evolution of lesions in the hamster with widespread metastases that has always been considered distinctive of L. mexicana-complex parasites was also not seen. The apparent similarity of L. m. pifunoi (= L. pifunot) and these strains in sharing MDH of variant type I might indicate true identity, but it also could be a superficial similarity, since similarity of the electrophoretic mobilities of two populations of molecules does not guarantee identical molecular structure. Comparison of the EF subserotype of the Dominican Republic stocks, with those of the other types of DCL strain indicates that there is some sharing of antigenic determinants (Fig. 2 and table), indicating components with identical, or very similar, molecular structure to those of some L. mxicana-complex strains. The absence of a B component in the EF of L. m. pifanoi strains from Venezuela, and of an A comoonent in the EF of L. aethioaica. indicates a subsiantial difference between these ’ two DCLinducing forms and the Dominican Republic strains examined. However, a strain of L. mexicana from Belize, LRC-L94, has a mixed EF serotype, AsBr, showing a shared B component. However, this subspecies has, to our knowledge, never been shown to be associated with DCL. DNA characteristics of two of the Dominican Republic strains have been studied by other researchers. BARKER et al. (1982) measured the nuclear and kinetoplast DNA buoyant densities of the strains LRC-L281 (= AMRU-450 = LUMP 1433) and LRCL285 (= AMRU-452 = LUMP 1434), showing them to be similar to two strains of L. m. amnzonensis and different from a strain of L. m. mexicana. Desnite this they concluded that neither strain could- be positively identified with any of the New World and Old World marker strains that they used, which included L. aethiopica strains. SPITHILL et al. (1983) characterized the strain LRC-L285 bv endonuclease fingerprinting and Southern blot hybridization analysis of its kinetoplast DNA, finding it to be similar to L. major (formerly L. tropicn major) strains and different from them in the size of its EcoRl-cut unit length minicircle. In conclusion, the three DCL isolates from the Dominican Republic studied here appear to be different from the other tvnes of leishmanial oreanisms associated with DCL in the Old World and oiher parts in the New World, but do show some antigenic similarity to some strains grouped in the L. mexicanacomplex.
L.
Table-A comparison leishmaniasis (DCL) Species of Leishmania (Origin) L. aethiopica (Ethiopia) L. m. pifanoi (Venezuela) L. m. amazonensis (Brazil) Leishmania sp. (Dominican Republic)
of some characteristics
F.
SCHNUR
of leishmanial
Development of lesions in hamsters none to minimal rapid growth with metastases rapid growth with metastases slow growth without metastases
761
t?t al.
species associated with disseminated
Growth in blood-agar medium
cutaneous
Excreted factor (EF) subserotype
Malate dehydrogenase we
rapid
Bl
Vi
rapid
Al*
I+
rapid
BX
III
slow to medium
A&*
I
* some of the antigenic components in the EF subserotypes A, and A4 are identical (see Fig. 2 and its legend). + data taken from CHANCE et al., 1977 & CHANCE, 1979.
Acknowledgements This investigation received support from the UNDl’i World Ban&WHO Special Programme for Research and Training in Tropical Diseases. The contribution of one of us (B. C. Walton) was made as a part of Project AMRO-0800 of the Pan American Health Organizations at Gorgas Memorial Laboratory in Panama.
References Barker, D. C., Arnot, D. E. & Butcher, J. (1982). DNA characterization as a taxonomic tool for identification of kinetoplastic flagellate protozoa. In: Biochemical Characterization of Leishmania. M. L. Chance and B. C. Walton (Editors). Geneva: UNDP/World BankWHO Special Programme for Research and Training in Tropical Diseases, pp. 139-180. Bogaert-Diaz, H., Rojas, R. F., De Leon? A., Martinez, D. & Quiriones, M. (1975). Leishmamasis tegumentaria americana: reporte de 10s primeros tres cases. Forma anergica en tres hermanos. Revista Dominicana de Dermatologia, 9, 19-33. Bryceson, A. D. M. (1969). Diffuse cutaneous leishmaniasis in Ethiopia. I. The clinical and histological features of the disease. Transactions of the Roval Sociehr of TroDical Medicine and Hygiene, 6& 708-737. - . _ Bryceson, A. D. M. (1970a). Diffuse cutaneous leishmaniasis in Ethiopia. II. Treatment. Transactions of the Royal Society of Tropical Medicine and Hygiene, 64, 369-379. Bryceson, A. D. M. (1970b). Diffuse cutaneous leishmaniasis in Ethiopia. III. Immunological studies. Transactions of the Royal Society of Tropical Medicine and Hygiene, 64, 380-387. Chance, M. L. (1979). The identification of Leishmania. In: Problems in the Identification of Parasites and their Vectors. Angela E. R. Taylor and R. Muller (Editors). Symposia of the British Society of Parasitology, Vol. 17, Oxford, London, Edinburgh, Melbourne: Blackwell Scientific Publications, pp. 55-74. Chance, M. L., Gardener, P. J. & Peters, W. (1977). Biochemical taxonomy of Leishmania as an ecological tool. In: Colloques Internationaux du Centre National de la Recherche Scientifique, No. 239-Ecologic des Leishmanioses. Montpellier, 18-24 Aofit, 1974. Chance, M. L., Schnur, L. F., Thomas, S. C. & Peters, W. (1978). The biochemical and serological taxonomy of Leishmania from the Aethiopian zoogeographical region of Africa. Annals of Tropical Medicine and Parasitology, 72, 533-542.
Convit, J. & Lapenta, P. (1946). Sobre un case de leishmaniosis tegumentaria de forma disseminada. Revista Policlinica, Caracas, 17, 153-158. Convit, J., Pinardi, M. E. & Rondon, A. J. (1971). Diffuse cutaneous leishmaniasis: a disease due to an immunological defect of the host. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 603-610. Dostrovsky, A. (1934). Leishmania recidiva of the skin. Harefuah, 8, 118-124. [In Hebrew, English summary pp. l-2.1 Dostrovsky, A. (1936). Relapses in cutaneous leishmaniasis. Annals of Tropical Medicine and Parasitology, 3, 267-274. El-On, J, (1969). Comparison of discelectrophoretic profiles of in vivo (trypomastigote) forms and in vitro (epimastigote) forms of trypanosomes. M.Sc. thesis, Hebrew University, Jerusalem, in Hebrew. Gardener, I’. J., Chance, M. L. & Peters, W. (1974). Biochemical taxonomy of Leishmania. II. Electrophoretic variation of malate dehydrogenase. Annals of TropIcal Medicine and Parasitology, 68, 317-325. Lainson, R. & Shaw, J. J. (1972). Leishmania of the New World: taxonomic problems. British Medical Bulletin, 28, 44-48. McKinney, L. A. & Hendricks, L. A. (1980). Experimental infection of Mystromys albicaudatus with Leishmania braziliensis. Pathology. American Journal of Tropical Medicine and Hygiene, 29, 753-760. Medina, R. & Romero, J. (1959). Estudio clinic0 y parasitologic0 de una nueva cepa de Leishmania. Archives Venezolanos de Medicina Tropical y Parasitologia Medica, 3, 298-326. Miles, M. A., Povoa, M. M., De Souza,A., Lainson, R. & Shaw, J. J. (1979). Some methods for the enzymic characterization of Latin-American Leishmania with particular reference to Leishmania mexicana amazonensis and subspecies of Letshmania hertigi. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 243-252. Petersen, E. A., Neva, F. A., Oster, C. N. & BogaertDia?, H. (1982). Specific inhibition of lymphocyteprohferation response by adherent suppressor cells in diffuse cutaneous leishmaniasis. New EnglandJournal of Medicine, 306, 387-392. Schnur, L. F. & Zuckerman, Z. (1977). Leishmanial excreted factor (EF) serotypes in Sudan, Kenva and Ethiopia. Annals of Tropical Medictne and Para&ology, 71, 273-294. Schnur, L. F:, Zuckerman, A. & Greenblatt, C. L. (1972). Leishmamal serotypes as distinguished by the gel diffusion of factors excreted in vitro and in vivo. IsraelJournal of Medical Sciences, 8, 982.942.
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Schnur, L. F., Zuckerman, A. & Montillo, B. (1973). Dissemination of leishmanias to the organs of Syrian hamsters following intrasplenic inoculation of promastigotes. Experimental Parasitology, 34, 432-447. Spithill, T., Grumont., R. & Mitchell, G. F. (1983). Characterization of isolates and clones of Leishmania by analysis of kinetoplast DNA. Abstracts of 12th Annual UCLA Symposia, Journal of Cellular Biochemistry, supplement 7Aj p. 27. Tobie, E. J., Von Brand, T. & Mehlman, B. (1950). Cultural and Phvsioloeical observations on Trvnanosoma rhodesiense and f. gam%iense.Journal of Para&logy, 36, 48-54. Turk, J. L. & Belehu, A. (1974). Immunological spectra in infectious diseases.In: Parasites in the Immunized Host: Mechanisms of suruival. Ciba Foundation Symposium 25 (new series), Amsterdam, London, New York: Associated Scientific Publishers, pp. 101-122.
DCL
IN
DOMINICAN
REPUBLIC
Turk, J. L. & Bryceson, A. D. M. (1971). Immunological phenomena in leprosy and related diseases.In: Advances in Immunology, 13, London, New York: Academic Press, pp. 209-266. Walton, B. C., Intermill~ R. W. & Hajduk, M. E. (1977a). Difference in biological characteristics of three Leishmania isolates from patients with espundia. American Journal of Tropical Medicine and Hygiene, 26, 850-855. Walton, B. C., Shaw, J. J. & Lainson, R. (1977b). Observations on the in vitro cultivation of Leishmania braziliensis. Journal of Parasitology, 63, 1118-l 119.
Accepted for publication 21st January,
1983.