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Leishmania (Viannia) lainsoni occupies a unique niche within the subgenus Viannia
231
TRWSACTIONS OF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE (1995) 89,231-236
Leishmania Viannia
(Viannia)
lainsoni
occupies
a unique
niche within
the subgenus
Salih Eresh, Maarten H. L. de Bruijn, J. Alexis Mendoza-Le6n and Douglas C. Barker* Medical Research Council Outstation of the National Institute of Medical Research, Molten0 Laboratories, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 IQI’, UK Abstract Leishmania Zuinsonihas recently been recognized as a new peripylarian species belonging to the subgenus Viannia and the L. bruziliensis complex. It has been isolated from its sandfly vector., reservoir host and cuta-
neous lesions of human patients. Microscopical examination has shown characteristics which are different from those of other members of the L. bruziliensis complex. Nuclear deoxyribonucleic acid (DNA) hybridization patterns with a B tubulin probe and kinetoplast DNA buoyant density measurements show close similarities with other speciesof the L. braziZiensis complex. However, kinetoplast DNA restriction enzyme fragment patterns of L. (V.) lainsoni isolates show similarities to L. mexicanacomplex speciesaswell asweak cross hybridization. L. (V.) Zuinsoni is also amplified with L. braziliensis complex specific polymerase chain reaction (PCR) primers but it requires a lower annealing temperature and gives a 300 base pair PCR product. A possible model for the binding of PCR primers to the L. (V.) Iuinsoni kinetoplast DNA minicircle is proposed. Keywords:
Leishmanialainsoni, characterization,nuclear DNA, kDNA, PCR
Introduction
Parasites of the genus Leishmania can be classified as either peripylarian, with development in the hindgut of the sandfly vector, or suprapylarian, with no such development (LAINSON & SHAW, 1979). These groups correspond with the subgenera Viannia and Leishmania (see LAINSON & SHAW, 1987). The subgenus Leishmania encompassesL. (L.) mexicana, L. CL.1 amazonensis and L. (L. ) chagusi from the ‘New World’ as well as all known @d W&ld’ Leishmania species. The subgenus Viannia contains the speciesbelonging to the L. braziliensis complex [L. (V.) bruziliensis, L. (V.) guyanensis, L. (V.) panamensisand L. (V.) peruviana], all of which infect humans, together with recently discovered species. The first of these newly recognized specieswas L. (V.) luinsoni SILVEIRAet al., 1987, which was isolated from the single, cutaneous lesions of 6 patients in the state of Par& Brazil. Although clearly peripylarian, these parasites differ from speciesin the L. bruziliensis complex by their typically elongated amastigotes, their voluminous kinetoplast and their abundance in lesions. The parasites can be further distinguished from the other silvatic svecies. L. IL. j amazonensis. L. IV. j braziliensis and L. (i/., guyanensis; by isoenzyme promes and by speciesspecific monoclonal antibodies. The sandfly Lutzomyia ubiquitalis and the rodent Agouti paca have recently been implicated as the first known natural vector and wild animal reservoir, respectively (SILVEIRA et al., 1991a, 1991b; LAINSON et al., 1992). Two additional Amazonian peripylarian species have been described. L. iv.) naiffi LAINSON & SHAW. 1989 and L. (V.j sha& L~I~soN~& al., 1989. As with i. (V.) luinsoni, they can be distinguished from each other and from other specieswithin the subgenus by a combination of biological, biochemical and serological criteria, including morphology, vector and host range, isoenzyme profiles and monoclonal antibodies. L. (V.j naifj? has been identified in 3 anthropophilic sandfly species(Psychodopygus paraensis, I’. ayrozai and P. squamiventris), the nine-banded armadillo (Dasypus novemcinctus), and humans (LAINSON & SHAW,1989; LAINSONet al., 1990; NAIFF et al., 1991). L. (V.) shawi occurs in the sandfly Lu. (Nyssomyiaj whirmani and in a variety of wild mammals, including monkeys and sloths (LAINSON et al., 19891.It has also been found to infect humans (SHAWer al., 1991). The most recently reported addition to the L. bruziliensis comvlex is L. iv. ) colombiensis. isolated from human cutaneous leishmaniasis cases,sandflies (Lu. hartmanni) and sloths (Choloepus hoffmani) ( KREUTZER et al., 1991). Based on isoenzyme and morphological data, L. ‘Author for correspondence.
(V.) colombiensis was judged to be closely related to L. (V.) luinsoni.
We have recently reported a new diagnostic method for leishmaniasis which specifically detects speciesof the L. bruziliensis comnlex bv amolilication in vitro of kinetoplast deoxyribonucleic acid *(kDNA) minicircles using the polymerase chain reaction (PCR) (DE BRUIJN, 1988; DE BRUIJN & BARKER, 1992; DE BRUIJN et al., 1993). The method is suitable for direct identification in crudelv prepared samples from patients and animals and is capable of detecting a single parasite. Since L. (V.) luinsoni, L. (V.) shawi and L. (V.) naiffi are pathogenic to humans, extention of the diagnostic method to these 3 specieswould be desirable. Here we present a molecular analysis at the level of nuclear and kDNA which further defines the phylogenetic status of the 3 new speciesof Vianniu and indicates that L. (V.) luinsoni is substantially different from the other speciesin the subgenus. The data further indicate that our PCR-based diagnostic method is specific for all recognized specieswithin the subgenus.
Table 1. Details the study
of Leishmania
isolates used during
IFLAIBR’67IPH8CS L. [L.) amazonensis MHOMlBR73lM2269 L. (L. j amazonensis MHOMiPEi86iLC53 L (V.) braziliensis MHOMiBR84iLTB300 L . (V. ) braziliensis MHOMIBRi75iM2903 L. (V.) braziliensis MHOMIBRi74iPP75 L. (L.) chug& MHOMIIN180IDD8 L. CL.1 donovani MHOMiBR75iM4147 L. iv.j guyanensis MHOM/FR8O/LEM75 L. (L.) infantum CCAN/TN/87/LEM78 L. (L.) infantum MDIDIBR71IM1597 L. (V.) lainsoni MHOMIBR8 l/M6424 L. [V.) lainsoni MHOMiBR82iM6887 L. (V.) Zuinsoni IUBI/BR/83/M7556 L. iv. 1 lainsoni L. iv.j lainsoni MAGUiBR84iM8617 MRHOiSU/59/P-strain L. (L.) major MHOMIBZ182IBEL2 1 L. (L.) mexicana MNYCIBZi62iM379 L. (L.) mexicana MDASlBIU79iM5533 L. (V.) naiffi MHOMIPAI71ILS94 L. (V.) panamensis MHOMiPEi84iLC26 L. (V.) peruviana MHOMIVEI57ILL 1 L. (L.) pifanoi MCEBiBRi84iM8408 L. (L.) shawi “In the text and Table 2, isolates are referred to by the last component of the code; e.g., PH8C5, M2269, etc.
232 Materials
and Methods
Leishmania speciesand culture conditions All Leishmania species/strains were obtained from either Winches Farm Laboratories (London School of Hygiene and Tropical Medicine, UK) or Instituto Evandro Chagas, Belem, Brazil and cultured in MEM-EBLB medium (EVANS, 1989). The isolates used in this study are shown in Table 1. Isolation of kinetoplast and nuclear DNA
Parasites were cultured to log phase and kDNA was isolated as described by KENNEDY (1984). Purified kDNA was digested with the restriction enzyme HaeIII.
1
2
3
4
5
6
7
Nuclear DNA (nDNA) was isolated and purified as described by ERESHet al. (1993). The nDNA sampleswere digested with BamHI. Hybridization conditions
Digested DNA was electrophoresed on a 1% agarose gel in 1X TBE, Southern blotted and hybridized with probes which were labelled with [a-QP]dATP using the Amersham Multiprime@, DNA labelling kit. After hybridization, the digest was washed under medium stringency conditions (4 washes, 15 min each, with 2xSSC (NaCl 0.3M+trisodium citrate @03M) containing 0.1% sodium dodecyl sulphate (SDS).
8 9 10 11 12 13 14 15
16 17 18 19 20 21
kb 23.13
-
9.42 6.68
-
4.36
-
2.32
-
2.03
-
0.560
-
Fig. 1. Autoradiograph of nuclear DNA digested with the restriction enzyme BarnHI, fractionated on a 1% agarose gel, Southern blotted to a nitrocellulose membrane and hybridized to a radiolabelled clone of the L. (V.) guyanensis fi-tubulin gene. Lane 1, L. (L.)plfanoi LLl. Lane 2, L. CL.) mewicann M379. Lane 3, L. (L.) mexicana BEL21. Lane 4, L. (L.) amazonensir PHSCS. Lane 5, L. (V.) braziliensis LTB300. Lane 6, L. (V.) braziliensis M2903. Lane 7, t. (V.) braziliensis LC53. Lane 8, L. (V .) guyanensis M4147. Lane 9, L. (V.)pannmensis LS94. Lane 10, L. (V.) penrviana LC26. Lane 11, L. (V.) lainsoni M6426. Lane 12, L. (L.) mnjc~r P-strain. Lane 13, L. CL.) donovani DD8. Lane 14, L. (L.) infanrum LEM75. Lane 15, Typan~xom~ brucet. Lane 16, prelabelled DNA marker h/HindIII. Lanes 17-21, L. (V.) lainsoni isolates: lane 17, M6462; lane 18, M1597; lane 19, M8617; lane 20, M6887; lane 21, M7556; kb=kilobases.
233 Dot blot hybridizations
Parasites growing in log phase were washed in NET 100 buffer (100 mM NaClilOO mM ethylenediaminetetraacetic acid (EDTA)/lO mM Tris) and 105parasiteswere spotted on nitrocellulose membranes.After air drying, the filters were processedby placing, twice for 5 min, on to Whatmann 3MM paper soaked in denaturing solution (0.5 M NaOHI1.5 M NaCl) followed by 2 incubations of 5 min each in neutralizing solution (1.5 M NaCIIO.5M TrisHCl, pH 8.0). After hybridization with radioactively labelled probes the filters were washed at 60°C as follows: 2 x 30 min with 2 X SSC/O.l% SDS; 1x 30 min with 1x SSC/O.l% SDS; 1X 15 min with2x SSC/O.l% SDS.
bp 1353 1076 872
Polymerase chain reaction amplification
Crude samples of parasites were prepared by centrifuging 200 uL of a logarithmically growing culture at 13 600 g for 10 min at room temperature. The pellet was resuspended in 20 uL of sterile distilled water and the parasites were allowed to lyse at room temperature for 10 min. These lysates were then used directly for PCR or stored at -20°C. Unless stated otherwise, 2 uL of culture lysate was amplified in a total volume of 100 uL containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3)? 1.5 mM MgCl2, 0.01% gelatin, 0.2 mM of each deoxyrtbonucleotide, 100 pmol of each primer (Bl and B2), and 2.5 units of Taq DNA polymerase (Perkin Elmer Cents@or Cambio@).Primers Bl (21-mer) and B2 (19-mer) have already been shown to be specific for the amplification of L. braziliensis complex species (DE BRUIJN & BARKER, 1992). Samples were overlaid with light mineral oil (Sigma) and initially denatured at 96°C for 6 min before the polymerasewas added. Cycles consisted of annealing at 67.5”C or 60.5”C for 1 min, extension at 72°C for 1 min, and denaturation at 93°C for 0.5 min. Programmes were run for 35 cycles on a Cambio@ Intelligent Heating Block (Genesys Instruments, Cambridge, UK). Amplification products (15 uL) were analysed by electrophoresis on 1% agarosegels in Tris-borate-EDTA buffer. Results Comparisons at the level of nDNA
Nuclear DNA from a variety of ‘New’ and ‘Old World’ Leishmania that had been digested with BamH I was subsequently hybridized to a cloned and radiolabelled fragment of the L. (V.) guyanensis @-tubulin gene (pLgl34; unpublished data) (Fig. 1). Variable patterns of fragments with sequence homology to the labelled probe were obtained, the complexity of which was due to restriction recognition site polymorphism within the corre-
Table 2. Buoyant density measurements of kinetoplast DNA of the species belonging to the L. braziliensis and L. mexicana complexes
Species L. L. L. L. L. L. L. L. L. L. L. L.
(V.) (V.) (V.) (V.) (V.) (V.) (V. ) (If.) (V.) (V.) (L.) (L.)
Code” lainsoni lainsoni lainsoni lainsoni naiffi shawi braziliensis guyanensis
panamensis
peruviana amazonensis
mexicana
Ml597 M6887 M7556 M8617 M5533 M8408 M2903 M4147 LS94 SL5 PH8 M379
Buoyant density (dmL) 1.692 1.693 1.693 1.694 1.696b 1.692’ 1.692 1.692 1.695 1.693 1.699 1.700
Yee Table 1. bL~~~~~~ & SHAW(1989); the value given in that paper for the kinetoolast DNA of L. W. ) lainsoni was the value for nuclear DNA. ‘LAINSON et al. (1989).
603
310
Fig. 2. Restriction enzyme patterns of kinetoplast DNA from isolates of the L. braziliensisand L. mexicanacomplexes, digested with HaeIII. Lane I, L. (V.) guyanensis M4147. Lane 2, L. (If.1 braziliensisM2903. Lane 3, L. (V.) panamensis LS94. Lanes 4-8, L. (V.) lainroni isolates: Lane 4, M6426; lane 5, M1597; lane 6, M6887; lane 7, M7556; lane 8, M8617; lane 9, L. (L.) amaaonensisPHICS. Lane 10, L. (L.) mexicana M379. Lane 11, DNA marker r~X174IHaeIH; bp=base pairs.
sponding l)-tubulin genes. It is clear, however, that the hybridization pattern for specieswithin the L. mexicana complex (lanes l-4) and for ‘Old World’ Leishmania species (lanes 12-14) was substantially different from that observed for the L. braziliensis complex species (lanes 5-10). It was equally evident that the hybridization pattern of L. (V.) lainsoni (lanes 11 and 17-21) closely conformed to that of speciesof the L. braziliensis complex. The apparently different patterns in lanes 8,20 and 2 1 were due to variation in the amount of DNA analysed. The hybridization results thus confirmed at the molecular level that L. lainsoni belongs to the subgenus Viannia Comparisons at the level of kDNA
Buoyant density measurementsof kDNA from L. (V.) lainsoni isolates fell within the range observed for the L. braziliensis complex and not within that of the L. mexicana complex (Table 2; BARKER & BUTCHER, 1983). Differences between L. (V.) lainsoni and members of the L. braziliensis complex were nevertheless also apparent at the kDNA level. Upon digestion with Hae III, 1
2
3
I 5 6 I s 9
Fig. 3. Dot blots of whole parasites, representing the L. braailiensisand L. mextcunacomplexes, hybridized to radiolabelled kinetoplast DNA of (A) L. iv.) lainroni M6426, (B) L. (L.) amazonensis PH8C5, and (C)L. iv.) brazrlumu M2903. (1) L. iv.1 lainsoni M6426; (2) L. (V.) miff2 M5533; (3) L. CV.) shawt M8408; (4) L. (V.) braailrensrsM2903; (5) L. iv.) lamron~ M6426; (6) L. (L.) amuazonensir M2269; (7) L. (L.) amamwm PHBCS; (8) L. (L.) mexrcanaM379; (9) L. (V.) naiffi M5533. (10’ parasiteswere blotted for nos. l-3 and 10sparasites for nos. 4-9).
234
kDNA of L. (V.) lainsoni isolates gave a pattern of multiple fragments (Fig. 2; lanes 4-8) that was different from that of L. (V.)guyanensis, L. (V.) braziliensis and L. (V.) panamensis (Fig. 2; lanes 1, 2 and 3, respectively). The latter 3 species and L. (V.) peruviana (data not shown) gave a single product slightly smaller than a whole linearized minicircle (about 750 base pairs (bp); DE BRUIJN & BARKER, 1992), due to the close proximity of two Hae III recognition sites. The L. (V.) Zainsoni pattern of 3 major bands as well as at least 3 other bands was similar to, but distinct from, that produced by kDNA of L. (L.) amazonensis and L. (L.) mexicana (Fig. 2; lanes 9 and 10). Previous studies (WIRTH & PRATT, 1982; BARKER& BUTCHER, 1983; BARKER, 1987) have conclusively shown that virtually no cross-homology exists between kDNAs of any member of the L. braziliensis complex and any member of the L. mexicana complex. To investigate whether or not this applied to the subgenus Viannia as a whole, and to L. (V.) Zainsoni in particular, we have extended these studies. Dot blot hybridizations of L. (V.) Zainsoni and other species of the subgenus Viannia, as well as speciesof the L. mexicana complex, are shown in Fig. 3. Under conditions of high stringency, radiolabelled kDNA of L. (V.) braziliensis strongly crosshybridized with lysed parasites of L. (V.) na#i and L. (V.) shawi but only weakly with those of L. (V.) lainsoni (Fig. 3, C). Labelled kDNA of L. (V.) lainsoni, however, not only cross-hybridized strongly with L. (V.) braziliensis, L. (V.) naiffi and L. (V.) shawi, but also with L. (L.) amazonensis, and weakly with L. (L.) mexicana (Fig. 3, A). Labelled kDNA of L. (L.) amazonensis showed no cross-hybridization with speciesnot belonging to the L. mexicana complex (Fig. 3, B). The differences in the hybridization were probably due to the different copy number of the homologous sequencesin the target DNA. Analysis of the new speciesof the subgenus Viannia by PCR
We have previously shown that parasites belonging to the L. braziliensis complex can be unambiguously and positively identified by their PCR product (DE BRUIJN& BARKER,1992). This identification was basedon the specific recognition of 2 adjacent and highly conserved sites 1
2
3
4
5
6
7
0
9
10
11
12
13
A
bp 1353872603310-
6
bp 1353872am310-
Fig. 4. Polymerasechainreactionamplificationof kinetoplastDNA using lysed parasitesat the annealingtemperaturesof (A) 67.X and (B) 60~5°C.Arrows showthe diagnosticband of about 750basepairs (bp). Lanes1 and 13,L. (V.)pemviana LC26, Lanes2-6, L. iv.) lainsoniisolates:lane2, M6426;lane3, M6887;lane4, M7556;lane5, M8617;lane 6, M1597. Lanes 7-9: L. (V.) naiffi isolates:lane 7, M5533;lane 8, M52101;lane 9, M11712.Lanes 10-12:L. iv.) shawi isolates: lane 10, M8408;lanell,M11044;lane12,M7731.
on the kDNA minicircles of those parasites by synthetic oligonucleotides with a complementary sequence. At primer annealing temperatures above 60°C only L. braziZiensiscomplex-specific products are amplified and none from human-infecting species of the subgenus Leishmania, Tvpanosoma cruzi, and a number of other human pathogens (DE BRUIJN et al., 1993). The PCR products of kDNA isolated from the complex representative L. (V.) peruviana, at annealing temperatures of 67.5”C and 60.5”C respectively, are shown in lanes 1 and 13 of Fig. 4, A and B. The diagnostic and smallest product at both temperatures corresponded in size to a whole linearized kDNA minicircle (about 750 bp, indicated by the arrows in Fig. 4). Five isolates of L. (V.) lainsoni, 3 of L. (V.) naiffi and 3 of L. (V.) shawi, including the reference strain of each species, were similarly analysed. Parasites from all isolates of L. (V.) naiffi (Fig. 4, lanes 7-9) and L. (V.) shawi (lanes 10-12) produced the diagnostic band upon amplification irrespective of the annealing temperature used. Thev were therefore indistinguishable bv PCR from species of the L. braziliensis complex (lanes 1 and 13). None of the 5 isolates of L . (V. ) lainsoni gave any amplification product at 67.5”C (Fig. 4, A, lanes 2-6), but they did give a very distinct product of about 300 bp at the less stringent annealing temperature of 60.5”C (Fig. 4, B, lanes 2-6). In this, L. (V.) Zainsoni is dissimilar from both the L. braziliensis and L. mexicana complexes. No amplification product was observed for L. (V.) Zainsoni isolates when tested with L. mexicana complex-specific PCR primers (ERESHet al., 1994). Discussion
The comparative data obtained with nDNA and kDNA present a slightly confusing picture of the molecular relationships between L. (V.) Zainsoni, L. (V.) naiffi, L. (V.) shawi and the established speciesof the L. braziliensis complex. With L. (V.) luinsoni, the nDNA hybridization patterns with the B-tubulin probe and the kDNA buoyant density measurementsfor 5 isolates suggest close molecular similarities with species of the L. braziliensis comnlex and dissimilarities with members of the subgenus Leishmania. Homogeneity at the B-tubulin locus has also been found for Peruvian isolates, irrespective of whether they originated from patients with cutaneous or mucocutaneous leishmaniasis (REINER et al., 1989). Previous studies have shown that molecular karyotypes hybridized to a radiolabelled telomeric probe derived from Trypanosoma brucei (see VAN DER PLOEG et al., 1984) gave a strikinglv non-stochiometrical hvbridization signal with the -3- smallest chromosome-bands visible in L. IV.) lainsoni. in contrast to L. IV.) guyanensis, L . @. ) panamens& and L . (Y.) braziliensis‘, all the chromosome bands of which hybridized strongly to the probe (ERESH et al., 1993). This could be explained if there were a low copy number of the telomeric sequence represented by the T. brucei probe in these particular chromosomes and/or by existence of a telomeric sequence dissimilar to that of the probe. The 300 bp PCR product obtained with crude lysates of L. (V.) lainsoni confirmed both its inclusion in the subgenus Viannia and its status as a separate species, since at an annealing temperature of 60.5”C our primers do not give amplification products from parasites belonging to the subgenus Leishmania, and the L. (V.) Zainsoni product differed from the 750 bp product generated by the other parasites of the subgenus Viannia. This latter finding also suggests that 15. (V.) Zainsoni stands alone within the subgenus, since all other specieshave a minicircle-size PCR product in common. The presence of the 300 bp PCR product has been used for the identification and diagnosis of L. (V. ) lainsoni in isolates from areasof the Peruvian and Brazilian Amazon basin. This will be the subject of a separatecommunication, which will describe the kDNA sequenceof a major class of minicircle from Brazilian and Peruvian isolates of L. (V.) lainsoni.
235 cal comments. We thank Roger Williams buoyant density analysis and photography.
750 bp/Obp
VA,
3oo bp
Fig. 5. A possible model for the arrangement of polymerase chain reaction (PCR) primers Bl and B2 around an L. (V.) lainsoni kinetoplast DNA minicircle. The positions of Bl and B2 can be interchanged without affecting the end result. The arrows indicate the direction of the PCR DNA synthesis. (Not drawn to scale; bp=base pairs).
The digestion products in Fig. 2 suggest that L. (V.) Zuinsoniminicircles are about the same size as those of other Viunniu species. The L. (V.) lainsoni PCR product of 300 bp is almost half the size of a minicircle. This could imply that these minicircles are made up of 2 repeat units, as in T. lewisi (seePONZI et al., 1984), similarly to the 4 repeat units in a T. crtzi minicircle (MACINA et al., 1986). At low annealing temperatures bur primers pioduce a ‘ladder’ of PCR products with T. cruzi, corresponding to one, 2, 3 and 4 repeat units respectively (MACINA et al., 1986)(result not shown). A possible model for the annealing sites of the PCR primers (Bl/B2) around the L. (V.) l&soni kDNA
minicircle
is shown
in Fig. 5. It is proposed
that
there are 2 annealing sites for each primer and when, all the sites are occupied, only a product of 300 bp is amplified. However, if the-starting-concentration of kDNA is increased. DNA bands of annroximatelv 600 bo and 750 bp are obtained as well as %e 300 bp brands(&.ults not shown). This could be explained by the fact that, as the target kDNA concentration is increased to excess,not all the annealing sites are occupied by the primers, which permits the amplification of the larger DNA fragments. The PCR primers are derived from a region of the minicircle which is highly conserved (DE BRUIJN& BARKER, 1992), and correspond to the ‘stem’ and ‘loop’ sequences, respectively, of what is considered to be the origin of minicircle replication (NTAMBI & ENGLUND, 1985; BARKER et al., 1986). The ‘stem’ sequence IS large1 conserved within the Trypanosomatidae and is thererore probably also conserved in L. (V.) luinsoni. The ‘loop’ sequence is conserved only in the subgenus Viannia (see BARKER et al., 1986; BARKER, 1987), and is therefore likely to be less conserved in i. (V.) kzinsoni. This mav also be the exnlanation for the amolification of L. (V.) iainsoni kDNA*at 60.5”C rather thin at 67.5”C, the temperature at which all the other speciesof the L. braziliensis complex are amplified. Since there appearsto be selection pressure to maintain this ‘loop’ sequence within the subgenus, L. (V.) lainsoni must have evolved either faster than other speciesof Viannia or for a greater length of time. Acknowledgements We are most grateful to Professor R. Lainson and Dr J. J. Shaw (Instituto Evandro Chagas, BelCm, Par& Brazil) for provision of isolates, for their interest in our work, and for their criti-
for performing
the
References Barker, D. C. (1987). DNA diagnosis of human leishmaniasis. Parasitolo Today, 3,177-184. Barker, D. t? & Butcher, J. (1983). The use of DNA probes in the identification of leishmaniasis: discrimination between isolates of the Leishmania mexicana and L. bra&lie& complexes. Transactions of the Royal Society of Tropical Medicine and Hygiene, 7?., 285-297. Barker, D. C., Gibson, L. J., Kennedy, W. I’. K., Nasser, A. A. A. A. & Williams, R. H. (1986). The potential of using recombinant DNA species-specific probes for the identification of tropical Leishmania. Parasitology, 91, S139-S174. De Bruijn? M. H. L. (1988). Diagnostic DNA amplification. No respite for the elusive parasite. Parasitology Today, 4,293295. De Bruijn, M. H. L. & Barker, D. C. (1992). Diagnosis of New World leishmaniasis: specific detection of species of the Leishmania braziliensis cbmplex by amplificatibn of kinetoolast DNA. Acta Trobica. 52.45-58. De’ Bruijn, M. H. L., iab;ad& L. A., Smyth, A. J., Santrich, C. & Barker, D. C. (1993). A comparative study of diagnosis by the polymerase chain reaction and by current clinical methods using biopsies from Colombian patients with suspected leishmaniasis. Tropical Medicine and Parasitology, 44, 201-207. Eresh, S., Mendoza-Leon, A. & Barker, D. C. (1993). A small chromosome of Leishmania (Viannia) braziliensis contains multicopy sequences which are complex specific. Acta Tropica, 55,33-46. Eresh, S., MacCallum, S. M. & Barker, D. C. (1994). Identificanon and diagnosis of Leishmania mexicana complex isolates by polymerase chain reaction. Parasitology, 109,423-433. Evans, D. (1989). Handbook on Isolation, Characterisation and Ctyopreservation of Leishmania. Geneva: World Health Organization, pp. 10-15. Kennedy, W. P. K. (1984). Novel identification of differences in the kinetoplast DNA of Leishmania isolates by recombinant DNA techniques and in situ hybridisation. Molecular and Biochemical Parasitology, 12,3 13-325. Kreutzer, R. D., Corredor, A., Grimaldi, G., jr, Grog& M., Rowton, E. D., Young, D. G., Morales, A., McMahon-Pratt, D.? Guzman, H. & Tesh, R. B. (1991). Characterization of Lelshmania colombiensis sp. n. (Kinetoplastida: Trypanosomatidae), a new parasite infecting humans, animals and phlebotomine sandflies in Colombia and Panama. American Journal of Tropical Medicine and Hygiene, 44,662-675. Lainson, R. & Shaw, J. J. (1979). The role of animals in the epidemiology of South American leishmaniasis. In: Biology of the Kinetoplastida, vol. 2, Lumsden, W. H. R. & Evans, D. A. (editors). London: Academic Press, pp. 1-116. Lainson, R. & Shaw? J. J. (1987). Evolution, classification and geographical distribution. In: The Leishmaniases in Biology and Medicine, vol. 1, Peters W. & Killick-Kendrick, R. (editors). London: Academic Press, pp. l-120. Lainson, R. & Shaw, J. J. (1989). Leishmania (Vianniaj naiffi sp. n., a parasite of the armadillo, Dasypus novemcinctus (L.) in Amazonian Brazil. Annales de Parasitoloaie Humaine et Comparle, 64,3-9. Lainson, R., Braga, R. R., De Souza, A. A. A., PBvoa, M. M., Ishikawa, E. A. Y. & Silveira, F. T. (1989). Leishmania (Viannia) shawi sp. n., a parasite of monkeys, sloths and procyonids in Amazonian Brazil. Annales de Parasitologic Humaine et Cornparke, 64,200-207. Lainson, R., Shaw, J. J., Silveira, F. T., Braga, R. R & Ishikawa, E. A. Y. (1990). Cutaneous leishmaniasis of man due to Leishmania (Viannia) naiffi Lainson and Shaw, 1989. Annales de Parasitologic Humaine et Cornparke, 65,282-284. Lainson, R., Shaw, J. J., Souza, A. A. A., Silveira, F. T. & Falqueto, A. (1992). Further observations on Lutzomyia ubiquitalis (Psychodidae: l’hlebotominae), the sandfly vector of Leishmania (Viannia) lainsoni. Memorias do Institute Oswald0 Cruz, 87,437-439. Macina, R. A., Sanchez, D. O., Gluschankof, D. A., Burrone, 0. R. & Frasch. A. C. C. (19861. Seauence diversitv in the kinetoplast DNA minicircles of ftypariosoma cruzi. tiolecular and Biochemical Parasitology, 21,25-32. Naiff, R. D., Freitas, R. A., Naiff, M. F., Arias, J. R., Barrett, T. V., Momen, H. & Grimaldi, G., jr (1991). Epidemiologlcal and nosological aspects of Leishmania naiffi Lainson and Shaw, 1989. Memorias do Instituto Oswald0 Cruz, 86,317-321. Ntambi, J. M. & Englund, I’. T. (1985). A gap at a uni ue location m newly replicated kinetoplast DNA miniclrc.4 es from
236 Typanosoma equiperdum. Journal of Biological Chemistry, 260, 55745579. Ponzi, M., Birago, C. & Battaglia, I’. A. (1984). Two identical symmetrical regions in the minicircle structure of Typanosomalewisi kinetoplast DNA. Molecular and Biochemical Parasitology, 13, 11l-l 19. Reiner, N. E., Lo, R., Llanos-Cuentas, A., Guerra, H., Button, L. L. & McMaster, W. R. (1989). Genetic heterogeneity in Peruvian Leishmania isolates. American Journal of Tropical Medicine and Hygiene, 41,416421. Shaw, J. J., Ishikawa, E. A., Lainson, R., Braga, R. R. & Silveira, F. T. (1991). Cutaneous leishmaniasis of man due to Leishmania (Viannia) shawi Lainson, de Souza, Pbvoa,, Ishikawa & Silveira, in Para State, Brazil. Annales de Parasttologie Humaine et Cornpuree, 66, 243-246.
Silveira, F. T., Shaw, J. J., Braga, R. R. & Ishikawa, E. (1987). Dermal leishmaniasis in the Amazonian region of Brazil: Leishmania (Viannia) lainsoni sp. n., a new parasite from the state of Para. Memorias do Institute Oswald0 Cruz, 82, 289292. Silveira, F. T., Souza, A. A. A., Lainson, R., Shaw, J. J., Braga, R. R. & Ishikawa, E. E. A. (1991a). Cutaneous leish-
1Announcements
maniasis in the Amazon region: natural infection of the sandfly Lutzomyia ubiquitalis (Psychodidae: Phlebotominae) by Leishmania (Viannia) lainsoni in Para State, Brazil. Memorias do Instituto Oswald0 Cruz, 86,127-130.
Silveira, F. T., Lainson, R., Shaw, J. J., Braga, R. R., Ishikawa, E. E. A. & Souza, A. A. A. (1991b). Leishmaniose cutlnea na Amazonia: isolamento de Leishmania (Viunnia) luinsoni do roedor Agouti paca (Rodentia, Dasyproctidae), no estado do Para, Brasil. Revista da Institute de Medicina Tropical de Sbo Paula, 33, 18-22. Van der Ploeg, L. H. T., Liu, H. Y. & Borst, P. (1984). Structure of the growing telomeres of trypanosomes. Cell, 36,459468. Wirth, D. F. & Pratt, D. M. (1982). Rapid identification of Leishmania species by specific hybridization of kinetoplast DNA in cutaneous leisions. Proceedings of the National Academy of Sciencesof the USA, 79,6999-7003. Received 22 July 1994; revised 26 September accepted for publication 27 September 1994
1 Diagnostic Parasitology Course 24 July-4 August 1995 Uniformed Services University of the Health Sciences Bethesda, Maryland 208144799, USA
Registration fee: US$ 1000 (reduced rate for US government and military personnel). Intending participants should register as soon as possible. Further information can be obtained from Dr John H. Cross (phone + 1 (301) 2953139), Dr Edward H. Michelson (+ 1 (301) 2953138), or MS Ellen Goldman (+ 1 (301) 2953129).
Eighth International Training Course on Identification of Helminth Parasites of Economic Importance d July-I 6 August 1996; St Albans, UK Further details can be obtained from: Dr L. M. Gibbons, International field Road, St Albans, Hertfordshire, AL4 OXU, UK.
Institute of Parasitology, 395A Hat-
L
The University of Edinburgh Training for the Tropics 1995-1996
I
Courses will be held throughout this period on the following broad topics: Agriculture, Animal production, Veterinary medicine, Management skills, Media techniques, Forestry, and Geoscience. Further information can be obtained from Catherine Bancroft, The University of Edinburgh, UnivEd Technologies Ltd, 16 Buccleuch Place, Edinburgh, EH8 9LN, Scotland, UK; telephone +44 (0)131 650 3475; fax +44 (0)131650 3474; telex 727442 (UNIVED G).
1994;
TRWSACTIONS OF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE (1995) 89,231-236
Leishmania Viannia
(Viannia)
lainsoni
occupies
a unique
niche within
the subgenus
Salih Eresh, Maarten H. L. de Bruijn, J. Alexis Mendoza-Le6n and Douglas C. Barker* Medical Research Council Outstation of the National Institute of Medical Research, Molten0 Laboratories, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 IQI’, UK Abstract Leishmania Zuinsonihas recently been recognized as a new peripylarian species belonging to the subgenus Viannia and the L. bruziliensis complex. It has been isolated from its sandfly vector., reservoir host and cuta-
neous lesions of human patients. Microscopical examination has shown characteristics which are different from those of other members of the L. bruziliensis complex. Nuclear deoxyribonucleic acid (DNA) hybridization patterns with a B tubulin probe and kinetoplast DNA buoyant density measurements show close similarities with other speciesof the L. braziZiensis complex. However, kinetoplast DNA restriction enzyme fragment patterns of L. (V.) lainsoni isolates show similarities to L. mexicanacomplex speciesaswell asweak cross hybridization. L. (V.) Zuinsoni is also amplified with L. braziliensis complex specific polymerase chain reaction (PCR) primers but it requires a lower annealing temperature and gives a 300 base pair PCR product. A possible model for the binding of PCR primers to the L. (V.) Iuinsoni kinetoplast DNA minicircle is proposed. Keywords:
Leishmanialainsoni, characterization,nuclear DNA, kDNA, PCR
Introduction
Parasites of the genus Leishmania can be classified as either peripylarian, with development in the hindgut of the sandfly vector, or suprapylarian, with no such development (LAINSON & SHAW, 1979). These groups correspond with the subgenera Viannia and Leishmania (see LAINSON & SHAW, 1987). The subgenus Leishmania encompassesL. (L.) mexicana, L. CL.1 amazonensis and L. (L. ) chagusi from the ‘New World’ as well as all known @d W&ld’ Leishmania species. The subgenus Viannia contains the speciesbelonging to the L. braziliensis complex [L. (V.) bruziliensis, L. (V.) guyanensis, L. (V.) panamensisand L. (V.) peruviana], all of which infect humans, together with recently discovered species. The first of these newly recognized specieswas L. (V.) luinsoni SILVEIRAet al., 1987, which was isolated from the single, cutaneous lesions of 6 patients in the state of Par& Brazil. Although clearly peripylarian, these parasites differ from speciesin the L. bruziliensis complex by their typically elongated amastigotes, their voluminous kinetoplast and their abundance in lesions. The parasites can be further distinguished from the other silvatic svecies. L. IL. j amazonensis. L. IV. j braziliensis and L. (i/., guyanensis; by isoenzyme promes and by speciesspecific monoclonal antibodies. The sandfly Lutzomyia ubiquitalis and the rodent Agouti paca have recently been implicated as the first known natural vector and wild animal reservoir, respectively (SILVEIRA et al., 1991a, 1991b; LAINSON et al., 1992). Two additional Amazonian peripylarian species have been described. L. iv.) naiffi LAINSON & SHAW. 1989 and L. (V.j sha& L~I~soN~& al., 1989. As with i. (V.) luinsoni, they can be distinguished from each other and from other specieswithin the subgenus by a combination of biological, biochemical and serological criteria, including morphology, vector and host range, isoenzyme profiles and monoclonal antibodies. L. (V.j naifj? has been identified in 3 anthropophilic sandfly species(Psychodopygus paraensis, I’. ayrozai and P. squamiventris), the nine-banded armadillo (Dasypus novemcinctus), and humans (LAINSON & SHAW,1989; LAINSONet al., 1990; NAIFF et al., 1991). L. (V.) shawi occurs in the sandfly Lu. (Nyssomyiaj whirmani and in a variety of wild mammals, including monkeys and sloths (LAINSON et al., 19891.It has also been found to infect humans (SHAWer al., 1991). The most recently reported addition to the L. bruziliensis comvlex is L. iv. ) colombiensis. isolated from human cutaneous leishmaniasis cases,sandflies (Lu. hartmanni) and sloths (Choloepus hoffmani) ( KREUTZER et al., 1991). Based on isoenzyme and morphological data, L. ‘Author for correspondence.
(V.) colombiensis was judged to be closely related to L. (V.) luinsoni.
We have recently reported a new diagnostic method for leishmaniasis which specifically detects speciesof the L. bruziliensis comnlex bv amolilication in vitro of kinetoplast deoxyribonucleic acid *(kDNA) minicircles using the polymerase chain reaction (PCR) (DE BRUIJN, 1988; DE BRUIJN & BARKER, 1992; DE BRUIJN et al., 1993). The method is suitable for direct identification in crudelv prepared samples from patients and animals and is capable of detecting a single parasite. Since L. (V.) luinsoni, L. (V.) shawi and L. (V.) naiffi are pathogenic to humans, extention of the diagnostic method to these 3 specieswould be desirable. Here we present a molecular analysis at the level of nuclear and kDNA which further defines the phylogenetic status of the 3 new speciesof Vianniu and indicates that L. (V.) luinsoni is substantially different from the other speciesin the subgenus. The data further indicate that our PCR-based diagnostic method is specific for all recognized specieswithin the subgenus.
Table 1. Details the study
of Leishmania
isolates used during
IFLAIBR’67IPH8CS L. [L.) amazonensis MHOMlBR73lM2269 L. (L. j amazonensis MHOMiPEi86iLC53 L (V.) braziliensis MHOMiBR84iLTB300 L . (V. ) braziliensis MHOMIBRi75iM2903 L. (V.) braziliensis MHOMIBRi74iPP75 L. (L.) chug& MHOMIIN180IDD8 L. CL.1 donovani MHOMiBR75iM4147 L. iv.j guyanensis MHOM/FR8O/LEM75 L. (L.) infantum CCAN/TN/87/LEM78 L. (L.) infantum MDIDIBR71IM1597 L. (V.) lainsoni MHOMIBR8 l/M6424 L. [V.) lainsoni MHOMiBR82iM6887 L. (V.) Zuinsoni IUBI/BR/83/M7556 L. iv. 1 lainsoni L. iv.j lainsoni MAGUiBR84iM8617 MRHOiSU/59/P-strain L. (L.) major MHOMIBZ182IBEL2 1 L. (L.) mexicana MNYCIBZi62iM379 L. (L.) mexicana MDASlBIU79iM5533 L. (V.) naiffi MHOMIPAI71ILS94 L. (V.) panamensis MHOMiPEi84iLC26 L. (V.) peruviana MHOMIVEI57ILL 1 L. (L.) pifanoi MCEBiBRi84iM8408 L. (L.) shawi “In the text and Table 2, isolates are referred to by the last component of the code; e.g., PH8C5, M2269, etc.
232 Materials
and Methods
Leishmania speciesand culture conditions All Leishmania species/strains were obtained from either Winches Farm Laboratories (London School of Hygiene and Tropical Medicine, UK) or Instituto Evandro Chagas, Belem, Brazil and cultured in MEM-EBLB medium (EVANS, 1989). The isolates used in this study are shown in Table 1. Isolation of kinetoplast and nuclear DNA
Parasites were cultured to log phase and kDNA was isolated as described by KENNEDY (1984). Purified kDNA was digested with the restriction enzyme HaeIII.
1
2
3
4
5
6
7
Nuclear DNA (nDNA) was isolated and purified as described by ERESHet al. (1993). The nDNA sampleswere digested with BamHI. Hybridization conditions
Digested DNA was electrophoresed on a 1% agarose gel in 1X TBE, Southern blotted and hybridized with probes which were labelled with [a-QP]dATP using the Amersham Multiprime@, DNA labelling kit. After hybridization, the digest was washed under medium stringency conditions (4 washes, 15 min each, with 2xSSC (NaCl 0.3M+trisodium citrate @03M) containing 0.1% sodium dodecyl sulphate (SDS).
8 9 10 11 12 13 14 15
16 17 18 19 20 21
kb 23.13
-
9.42 6.68
-
4.36
-
2.32
-
2.03
-
0.560
-
Fig. 1. Autoradiograph of nuclear DNA digested with the restriction enzyme BarnHI, fractionated on a 1% agarose gel, Southern blotted to a nitrocellulose membrane and hybridized to a radiolabelled clone of the L. (V.) guyanensis fi-tubulin gene. Lane 1, L. (L.)plfanoi LLl. Lane 2, L. CL.) mewicann M379. Lane 3, L. (L.) mexicana BEL21. Lane 4, L. (L.) amazonensir PHSCS. Lane 5, L. (V.) braziliensis LTB300. Lane 6, L. (V.) braziliensis M2903. Lane 7, t. (V.) braziliensis LC53. Lane 8, L. (V .) guyanensis M4147. Lane 9, L. (V.)pannmensis LS94. Lane 10, L. (V.) penrviana LC26. Lane 11, L. (V.) lainsoni M6426. Lane 12, L. (L.) mnjc~r P-strain. Lane 13, L. CL.) donovani DD8. Lane 14, L. (L.) infanrum LEM75. Lane 15, Typan~xom~ brucet. Lane 16, prelabelled DNA marker h/HindIII. Lanes 17-21, L. (V.) lainsoni isolates: lane 17, M6462; lane 18, M1597; lane 19, M8617; lane 20, M6887; lane 21, M7556; kb=kilobases.
233 Dot blot hybridizations
Parasites growing in log phase were washed in NET 100 buffer (100 mM NaClilOO mM ethylenediaminetetraacetic acid (EDTA)/lO mM Tris) and 105parasiteswere spotted on nitrocellulose membranes.After air drying, the filters were processedby placing, twice for 5 min, on to Whatmann 3MM paper soaked in denaturing solution (0.5 M NaOHI1.5 M NaCl) followed by 2 incubations of 5 min each in neutralizing solution (1.5 M NaCIIO.5M TrisHCl, pH 8.0). After hybridization with radioactively labelled probes the filters were washed at 60°C as follows: 2 x 30 min with 2 X SSC/O.l% SDS; 1x 30 min with 1x SSC/O.l% SDS; 1X 15 min with2x SSC/O.l% SDS.
bp 1353 1076 872
Polymerase chain reaction amplification
Crude samples of parasites were prepared by centrifuging 200 uL of a logarithmically growing culture at 13 600 g for 10 min at room temperature. The pellet was resuspended in 20 uL of sterile distilled water and the parasites were allowed to lyse at room temperature for 10 min. These lysates were then used directly for PCR or stored at -20°C. Unless stated otherwise, 2 uL of culture lysate was amplified in a total volume of 100 uL containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3)? 1.5 mM MgCl2, 0.01% gelatin, 0.2 mM of each deoxyrtbonucleotide, 100 pmol of each primer (Bl and B2), and 2.5 units of Taq DNA polymerase (Perkin Elmer Cents@or Cambio@).Primers Bl (21-mer) and B2 (19-mer) have already been shown to be specific for the amplification of L. braziliensis complex species (DE BRUIJN & BARKER, 1992). Samples were overlaid with light mineral oil (Sigma) and initially denatured at 96°C for 6 min before the polymerasewas added. Cycles consisted of annealing at 67.5”C or 60.5”C for 1 min, extension at 72°C for 1 min, and denaturation at 93°C for 0.5 min. Programmes were run for 35 cycles on a Cambio@ Intelligent Heating Block (Genesys Instruments, Cambridge, UK). Amplification products (15 uL) were analysed by electrophoresis on 1% agarosegels in Tris-borate-EDTA buffer. Results Comparisons at the level of nDNA
Nuclear DNA from a variety of ‘New’ and ‘Old World’ Leishmania that had been digested with BamH I was subsequently hybridized to a cloned and radiolabelled fragment of the L. (V.) guyanensis @-tubulin gene (pLgl34; unpublished data) (Fig. 1). Variable patterns of fragments with sequence homology to the labelled probe were obtained, the complexity of which was due to restriction recognition site polymorphism within the corre-
Table 2. Buoyant density measurements of kinetoplast DNA of the species belonging to the L. braziliensis and L. mexicana complexes
Species L. L. L. L. L. L. L. L. L. L. L. L.
(V.) (V.) (V.) (V.) (V.) (V.) (V. ) (If.) (V.) (V.) (L.) (L.)
Code” lainsoni lainsoni lainsoni lainsoni naiffi shawi braziliensis guyanensis
panamensis
peruviana amazonensis
mexicana
Ml597 M6887 M7556 M8617 M5533 M8408 M2903 M4147 LS94 SL5 PH8 M379
Buoyant density (dmL) 1.692 1.693 1.693 1.694 1.696b 1.692’ 1.692 1.692 1.695 1.693 1.699 1.700
Yee Table 1. bL~~~~~~ & SHAW(1989); the value given in that paper for the kinetoolast DNA of L. W. ) lainsoni was the value for nuclear DNA. ‘LAINSON et al. (1989).
603
310
Fig. 2. Restriction enzyme patterns of kinetoplast DNA from isolates of the L. braziliensisand L. mexicanacomplexes, digested with HaeIII. Lane I, L. (V.) guyanensis M4147. Lane 2, L. (If.1 braziliensisM2903. Lane 3, L. (V.) panamensis LS94. Lanes 4-8, L. (V.) lainroni isolates: Lane 4, M6426; lane 5, M1597; lane 6, M6887; lane 7, M7556; lane 8, M8617; lane 9, L. (L.) amaaonensisPHICS. Lane 10, L. (L.) mexicana M379. Lane 11, DNA marker r~X174IHaeIH; bp=base pairs.
sponding l)-tubulin genes. It is clear, however, that the hybridization pattern for specieswithin the L. mexicana complex (lanes l-4) and for ‘Old World’ Leishmania species (lanes 12-14) was substantially different from that observed for the L. braziliensis complex species (lanes 5-10). It was equally evident that the hybridization pattern of L. (V.) lainsoni (lanes 11 and 17-21) closely conformed to that of speciesof the L. braziliensis complex. The apparently different patterns in lanes 8,20 and 2 1 were due to variation in the amount of DNA analysed. The hybridization results thus confirmed at the molecular level that L. lainsoni belongs to the subgenus Viannia Comparisons at the level of kDNA
Buoyant density measurementsof kDNA from L. (V.) lainsoni isolates fell within the range observed for the L. braziliensis complex and not within that of the L. mexicana complex (Table 2; BARKER & BUTCHER, 1983). Differences between L. (V.) lainsoni and members of the L. braziliensis complex were nevertheless also apparent at the kDNA level. Upon digestion with Hae III, 1
2
3
I 5 6 I s 9
Fig. 3. Dot blots of whole parasites, representing the L. braailiensisand L. mextcunacomplexes, hybridized to radiolabelled kinetoplast DNA of (A) L. iv.) lainroni M6426, (B) L. (L.) amazonensis PH8C5, and (C)L. iv.) brazrlumu M2903. (1) L. iv.1 lainsoni M6426; (2) L. (V.) miff2 M5533; (3) L. CV.) shawt M8408; (4) L. (V.) braailrensrsM2903; (5) L. iv.) lamron~ M6426; (6) L. (L.) amuazonensir M2269; (7) L. (L.) amamwm PHBCS; (8) L. (L.) mexrcanaM379; (9) L. (V.) naiffi M5533. (10’ parasiteswere blotted for nos. l-3 and 10sparasites for nos. 4-9).
234
kDNA of L. (V.) lainsoni isolates gave a pattern of multiple fragments (Fig. 2; lanes 4-8) that was different from that of L. (V.)guyanensis, L. (V.) braziliensis and L. (V.) panamensis (Fig. 2; lanes 1, 2 and 3, respectively). The latter 3 species and L. (V.) peruviana (data not shown) gave a single product slightly smaller than a whole linearized minicircle (about 750 base pairs (bp); DE BRUIJN & BARKER, 1992), due to the close proximity of two Hae III recognition sites. The L. (V.) Zainsoni pattern of 3 major bands as well as at least 3 other bands was similar to, but distinct from, that produced by kDNA of L. (L.) amazonensis and L. (L.) mexicana (Fig. 2; lanes 9 and 10). Previous studies (WIRTH & PRATT, 1982; BARKER& BUTCHER, 1983; BARKER, 1987) have conclusively shown that virtually no cross-homology exists between kDNAs of any member of the L. braziliensis complex and any member of the L. mexicana complex. To investigate whether or not this applied to the subgenus Viannia as a whole, and to L. (V.) Zainsoni in particular, we have extended these studies. Dot blot hybridizations of L. (V.) Zainsoni and other species of the subgenus Viannia, as well as speciesof the L. mexicana complex, are shown in Fig. 3. Under conditions of high stringency, radiolabelled kDNA of L. (V.) braziliensis strongly crosshybridized with lysed parasites of L. (V.) na#i and L. (V.) shawi but only weakly with those of L. (V.) lainsoni (Fig. 3, C). Labelled kDNA of L. (V.) lainsoni, however, not only cross-hybridized strongly with L. (V.) braziliensis, L. (V.) naiffi and L. (V.) shawi, but also with L. (L.) amazonensis, and weakly with L. (L.) mexicana (Fig. 3, A). Labelled kDNA of L. (L.) amazonensis showed no cross-hybridization with speciesnot belonging to the L. mexicana complex (Fig. 3, B). The differences in the hybridization were probably due to the different copy number of the homologous sequencesin the target DNA. Analysis of the new speciesof the subgenus Viannia by PCR
We have previously shown that parasites belonging to the L. braziliensis complex can be unambiguously and positively identified by their PCR product (DE BRUIJN& BARKER,1992). This identification was basedon the specific recognition of 2 adjacent and highly conserved sites 1
2
3
4
5
6
7
0
9
10
11
12
13
A
bp 1353872603310-
6
bp 1353872am310-
Fig. 4. Polymerasechainreactionamplificationof kinetoplastDNA using lysed parasitesat the annealingtemperaturesof (A) 67.X and (B) 60~5°C.Arrows showthe diagnosticband of about 750basepairs (bp). Lanes1 and 13,L. (V.)pemviana LC26, Lanes2-6, L. iv.) lainsoniisolates:lane2, M6426;lane3, M6887;lane4, M7556;lane5, M8617;lane 6, M1597. Lanes 7-9: L. (V.) naiffi isolates:lane 7, M5533;lane 8, M52101;lane 9, M11712.Lanes 10-12:L. iv.) shawi isolates: lane 10, M8408;lanell,M11044;lane12,M7731.
on the kDNA minicircles of those parasites by synthetic oligonucleotides with a complementary sequence. At primer annealing temperatures above 60°C only L. braziZiensiscomplex-specific products are amplified and none from human-infecting species of the subgenus Leishmania, Tvpanosoma cruzi, and a number of other human pathogens (DE BRUIJN et al., 1993). The PCR products of kDNA isolated from the complex representative L. (V.) peruviana, at annealing temperatures of 67.5”C and 60.5”C respectively, are shown in lanes 1 and 13 of Fig. 4, A and B. The diagnostic and smallest product at both temperatures corresponded in size to a whole linearized kDNA minicircle (about 750 bp, indicated by the arrows in Fig. 4). Five isolates of L. (V.) lainsoni, 3 of L. (V.) naiffi and 3 of L. (V.) shawi, including the reference strain of each species, were similarly analysed. Parasites from all isolates of L. (V.) naiffi (Fig. 4, lanes 7-9) and L. (V.) shawi (lanes 10-12) produced the diagnostic band upon amplification irrespective of the annealing temperature used. Thev were therefore indistinguishable bv PCR from species of the L. braziliensis complex (lanes 1 and 13). None of the 5 isolates of L . (V. ) lainsoni gave any amplification product at 67.5”C (Fig. 4, A, lanes 2-6), but they did give a very distinct product of about 300 bp at the less stringent annealing temperature of 60.5”C (Fig. 4, B, lanes 2-6). In this, L. (V.) Zainsoni is dissimilar from both the L. braziliensis and L. mexicana complexes. No amplification product was observed for L. (V.) Zainsoni isolates when tested with L. mexicana complex-specific PCR primers (ERESHet al., 1994). Discussion
The comparative data obtained with nDNA and kDNA present a slightly confusing picture of the molecular relationships between L. (V.) Zainsoni, L. (V.) naiffi, L. (V.) shawi and the established speciesof the L. braziliensis complex. With L. (V.) luinsoni, the nDNA hybridization patterns with the B-tubulin probe and the kDNA buoyant density measurementsfor 5 isolates suggest close molecular similarities with species of the L. braziliensis comnlex and dissimilarities with members of the subgenus Leishmania. Homogeneity at the B-tubulin locus has also been found for Peruvian isolates, irrespective of whether they originated from patients with cutaneous or mucocutaneous leishmaniasis (REINER et al., 1989). Previous studies have shown that molecular karyotypes hybridized to a radiolabelled telomeric probe derived from Trypanosoma brucei (see VAN DER PLOEG et al., 1984) gave a strikinglv non-stochiometrical hvbridization signal with the -3- smallest chromosome-bands visible in L. IV.) lainsoni. in contrast to L. IV.) guyanensis, L . @. ) panamens& and L . (Y.) braziliensis‘, all the chromosome bands of which hybridized strongly to the probe (ERESH et al., 1993). This could be explained if there were a low copy number of the telomeric sequence represented by the T. brucei probe in these particular chromosomes and/or by existence of a telomeric sequence dissimilar to that of the probe. The 300 bp PCR product obtained with crude lysates of L. (V.) lainsoni confirmed both its inclusion in the subgenus Viannia and its status as a separate species, since at an annealing temperature of 60.5”C our primers do not give amplification products from parasites belonging to the subgenus Leishmania, and the L. (V.) Zainsoni product differed from the 750 bp product generated by the other parasites of the subgenus Viannia. This latter finding also suggests that 15. (V.) Zainsoni stands alone within the subgenus, since all other specieshave a minicircle-size PCR product in common. The presence of the 300 bp PCR product has been used for the identification and diagnosis of L. (V. ) lainsoni in isolates from areasof the Peruvian and Brazilian Amazon basin. This will be the subject of a separatecommunication, which will describe the kDNA sequenceof a major class of minicircle from Brazilian and Peruvian isolates of L. (V.) lainsoni.
235 cal comments. We thank Roger Williams buoyant density analysis and photography.
750 bp/Obp
VA,
3oo bp
Fig. 5. A possible model for the arrangement of polymerase chain reaction (PCR) primers Bl and B2 around an L. (V.) lainsoni kinetoplast DNA minicircle. The positions of Bl and B2 can be interchanged without affecting the end result. The arrows indicate the direction of the PCR DNA synthesis. (Not drawn to scale; bp=base pairs).
The digestion products in Fig. 2 suggest that L. (V.) Zuinsoniminicircles are about the same size as those of other Viunniu species. The L. (V.) lainsoni PCR product of 300 bp is almost half the size of a minicircle. This could imply that these minicircles are made up of 2 repeat units, as in T. lewisi (seePONZI et al., 1984), similarly to the 4 repeat units in a T. crtzi minicircle (MACINA et al., 1986). At low annealing temperatures bur primers pioduce a ‘ladder’ of PCR products with T. cruzi, corresponding to one, 2, 3 and 4 repeat units respectively (MACINA et al., 1986)(result not shown). A possible model for the annealing sites of the PCR primers (Bl/B2) around the L. (V.) l&soni kDNA
minicircle
is shown
in Fig. 5. It is proposed
that
there are 2 annealing sites for each primer and when, all the sites are occupied, only a product of 300 bp is amplified. However, if the-starting-concentration of kDNA is increased. DNA bands of annroximatelv 600 bo and 750 bp are obtained as well as %e 300 bp brands(&.ults not shown). This could be explained by the fact that, as the target kDNA concentration is increased to excess,not all the annealing sites are occupied by the primers, which permits the amplification of the larger DNA fragments. The PCR primers are derived from a region of the minicircle which is highly conserved (DE BRUIJN& BARKER, 1992), and correspond to the ‘stem’ and ‘loop’ sequences, respectively, of what is considered to be the origin of minicircle replication (NTAMBI & ENGLUND, 1985; BARKER et al., 1986). The ‘stem’ sequence IS large1 conserved within the Trypanosomatidae and is thererore probably also conserved in L. (V.) luinsoni. The ‘loop’ sequence is conserved only in the subgenus Viannia (see BARKER et al., 1986; BARKER, 1987), and is therefore likely to be less conserved in i. (V.) kzinsoni. This mav also be the exnlanation for the amolification of L. (V.) iainsoni kDNA*at 60.5”C rather thin at 67.5”C, the temperature at which all the other speciesof the L. braziliensis complex are amplified. Since there appearsto be selection pressure to maintain this ‘loop’ sequence within the subgenus, L. (V.) lainsoni must have evolved either faster than other speciesof Viannia or for a greater length of time. Acknowledgements We are most grateful to Professor R. Lainson and Dr J. J. Shaw (Instituto Evandro Chagas, BelCm, Par& Brazil) for provision of isolates, for their interest in our work, and for their criti-
for performing
the
References Barker, D. C. (1987). DNA diagnosis of human leishmaniasis. Parasitolo Today, 3,177-184. Barker, D. t? & Butcher, J. (1983). The use of DNA probes in the identification of leishmaniasis: discrimination between isolates of the Leishmania mexicana and L. bra&lie& complexes. Transactions of the Royal Society of Tropical Medicine and Hygiene, 7?., 285-297. Barker, D. C., Gibson, L. J., Kennedy, W. I’. K., Nasser, A. A. A. A. & Williams, R. H. (1986). The potential of using recombinant DNA species-specific probes for the identification of tropical Leishmania. Parasitology, 91, S139-S174. De Bruijn? M. H. L. (1988). Diagnostic DNA amplification. No respite for the elusive parasite. Parasitology Today, 4,293295. De Bruijn, M. H. L. & Barker, D. C. (1992). Diagnosis of New World leishmaniasis: specific detection of species of the Leishmania braziliensis cbmplex by amplificatibn of kinetoolast DNA. Acta Trobica. 52.45-58. De’ Bruijn, M. H. L., iab;ad& L. A., Smyth, A. J., Santrich, C. & Barker, D. C. (1993). A comparative study of diagnosis by the polymerase chain reaction and by current clinical methods using biopsies from Colombian patients with suspected leishmaniasis. Tropical Medicine and Parasitology, 44, 201-207. Eresh, S., Mendoza-Leon, A. & Barker, D. C. (1993). A small chromosome of Leishmania (Viannia) braziliensis contains multicopy sequences which are complex specific. Acta Tropica, 55,33-46. Eresh, S., MacCallum, S. M. & Barker, D. C. (1994). Identificanon and diagnosis of Leishmania mexicana complex isolates by polymerase chain reaction. Parasitology, 109,423-433. Evans, D. (1989). Handbook on Isolation, Characterisation and Ctyopreservation of Leishmania. Geneva: World Health Organization, pp. 10-15. Kennedy, W. P. K. (1984). Novel identification of differences in the kinetoplast DNA of Leishmania isolates by recombinant DNA techniques and in situ hybridisation. Molecular and Biochemical Parasitology, 12,3 13-325. Kreutzer, R. D., Corredor, A., Grimaldi, G., jr, Grog& M., Rowton, E. D., Young, D. G., Morales, A., McMahon-Pratt, D.? Guzman, H. & Tesh, R. B. (1991). Characterization of Lelshmania colombiensis sp. n. (Kinetoplastida: Trypanosomatidae), a new parasite infecting humans, animals and phlebotomine sandflies in Colombia and Panama. American Journal of Tropical Medicine and Hygiene, 44,662-675. Lainson, R. & Shaw, J. J. (1979). The role of animals in the epidemiology of South American leishmaniasis. In: Biology of the Kinetoplastida, vol. 2, Lumsden, W. H. R. & Evans, D. A. (editors). London: Academic Press, pp. 1-116. Lainson, R. & Shaw? J. J. (1987). Evolution, classification and geographical distribution. In: The Leishmaniases in Biology and Medicine, vol. 1, Peters W. & Killick-Kendrick, R. (editors). London: Academic Press, pp. l-120. Lainson, R. & Shaw, J. J. (1989). Leishmania (Vianniaj naiffi sp. n., a parasite of the armadillo, Dasypus novemcinctus (L.) in Amazonian Brazil. Annales de Parasitoloaie Humaine et Comparle, 64,3-9. Lainson, R., Braga, R. R., De Souza, A. A. A., PBvoa, M. M., Ishikawa, E. A. Y. & Silveira, F. T. (1989). Leishmania (Viannia) shawi sp. n., a parasite of monkeys, sloths and procyonids in Amazonian Brazil. Annales de Parasitologic Humaine et Cornparke, 64,200-207. Lainson, R., Shaw, J. J., Silveira, F. T., Braga, R. R & Ishikawa, E. A. Y. (1990). Cutaneous leishmaniasis of man due to Leishmania (Viannia) naiffi Lainson and Shaw, 1989. Annales de Parasitologic Humaine et Cornparke, 65,282-284. Lainson, R., Shaw, J. J., Souza, A. A. A., Silveira, F. T. & Falqueto, A. (1992). Further observations on Lutzomyia ubiquitalis (Psychodidae: l’hlebotominae), the sandfly vector of Leishmania (Viannia) lainsoni. Memorias do Institute Oswald0 Cruz, 87,437-439. Macina, R. A., Sanchez, D. O., Gluschankof, D. A., Burrone, 0. R. & Frasch. A. C. C. (19861. Seauence diversitv in the kinetoplast DNA minicircles of ftypariosoma cruzi. tiolecular and Biochemical Parasitology, 21,25-32. Naiff, R. D., Freitas, R. A., Naiff, M. F., Arias, J. R., Barrett, T. V., Momen, H. & Grimaldi, G., jr (1991). Epidemiologlcal and nosological aspects of Leishmania naiffi Lainson and Shaw, 1989. Memorias do Instituto Oswald0 Cruz, 86,317-321. Ntambi, J. M. & Englund, I’. T. (1985). A gap at a uni ue location m newly replicated kinetoplast DNA miniclrc.4 es from
236 Typanosoma equiperdum. Journal of Biological Chemistry, 260, 55745579. Ponzi, M., Birago, C. & Battaglia, I’. A. (1984). Two identical symmetrical regions in the minicircle structure of Typanosomalewisi kinetoplast DNA. Molecular and Biochemical Parasitology, 13, 11l-l 19. Reiner, N. E., Lo, R., Llanos-Cuentas, A., Guerra, H., Button, L. L. & McMaster, W. R. (1989). Genetic heterogeneity in Peruvian Leishmania isolates. American Journal of Tropical Medicine and Hygiene, 41,416421. Shaw, J. J., Ishikawa, E. A., Lainson, R., Braga, R. R. & Silveira, F. T. (1991). Cutaneous leishmaniasis of man due to Leishmania (Viannia) shawi Lainson, de Souza, Pbvoa,, Ishikawa & Silveira, in Para State, Brazil. Annales de Parasttologie Humaine et Cornpuree, 66, 243-246.
Silveira, F. T., Shaw, J. J., Braga, R. R. & Ishikawa, E. (1987). Dermal leishmaniasis in the Amazonian region of Brazil: Leishmania (Viannia) lainsoni sp. n., a new parasite from the state of Para. Memorias do Institute Oswald0 Cruz, 82, 289292. Silveira, F. T., Souza, A. A. A., Lainson, R., Shaw, J. J., Braga, R. R. & Ishikawa, E. E. A. (1991a). Cutaneous leish-
1Announcements
maniasis in the Amazon region: natural infection of the sandfly Lutzomyia ubiquitalis (Psychodidae: Phlebotominae) by Leishmania (Viannia) lainsoni in Para State, Brazil. Memorias do Instituto Oswald0 Cruz, 86,127-130.
Silveira, F. T., Lainson, R., Shaw, J. J., Braga, R. R., Ishikawa, E. E. A. & Souza, A. A. A. (1991b). Leishmaniose cutlnea na Amazonia: isolamento de Leishmania (Viunnia) luinsoni do roedor Agouti paca (Rodentia, Dasyproctidae), no estado do Para, Brasil. Revista da Institute de Medicina Tropical de Sbo Paula, 33, 18-22. Van der Ploeg, L. H. T., Liu, H. Y. & Borst, P. (1984). Structure of the growing telomeres of trypanosomes. Cell, 36,459468. Wirth, D. F. & Pratt, D. M. (1982). Rapid identification of Leishmania species by specific hybridization of kinetoplast DNA in cutaneous leisions. Proceedings of the National Academy of Sciencesof the USA, 79,6999-7003. Received 22 July 1994; revised 26 September accepted for publication 27 September 1994
1 Diagnostic Parasitology Course 24 July-4 August 1995 Uniformed Services University of the Health Sciences Bethesda, Maryland 208144799, USA
Registration fee: US$ 1000 (reduced rate for US government and military personnel). Intending participants should register as soon as possible. Further information can be obtained from Dr John H. Cross (phone + 1 (301) 2953139), Dr Edward H. Michelson (+ 1 (301) 2953138), or MS Ellen Goldman (+ 1 (301) 2953129).
Eighth International Training Course on Identification of Helminth Parasites of Economic Importance d July-I 6 August 1996; St Albans, UK Further details can be obtained from: Dr L. M. Gibbons, International field Road, St Albans, Hertfordshire, AL4 OXU, UK.
Institute of Parasitology, 395A Hat-
L
The University of Edinburgh Training for the Tropics 1995-1996
I
Courses will be held throughout this period on the following broad topics: Agriculture, Animal production, Veterinary medicine, Management skills, Media techniques, Forestry, and Geoscience. Further information can be obtained from Catherine Bancroft, The University of Edinburgh, UnivEd Technologies Ltd, 16 Buccleuch Place, Edinburgh, EH8 9LN, Scotland, UK; telephone +44 (0)131 650 3475; fax +44 (0)131650 3474; telex 727442 (UNIVED G).
1994;