H. MOTAZEDIAN,*,1 H. NOYES,* AND R. MAINGON

EXPERIMENTAL PARASITOLOGY 83, 150 154 (1996) ARTICLE NO. 0059 RESEARCH BRIEF Leishmania and Sauroleishmania: The Use of Random Amplified Polymorphic DNA for the Identification of Parasites from Vertebrates and Invertebrates H. MOTAZEDIAN,*,1 H. NOYES,* AND R. MAINGON *Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA United Kingdom; and Department of Biological Sciences, Centre for Applied Parasitology and Entomology, Keele University, Staffordshire, United Kingdom MOTAZEDIAN, H., NOYES, H., AND MAINGON, R. 1996. Leishmania and Sauroleishmania: The use of random amplified polymorphic DNA for the identification of parasites from vertebrates and invertebrates. Experimental Parasitology 83, 150 154. We used the RAPD PCR method for distinguishing the main Old World Leishmania parasites Leishmania tropica, L. major, and L. infantum and applied it to Sauroleishmania species from Iran. Twelve out of 21 tested primers were suitable for identification of these parasites. The Jaccard similarity index was 0.30 for L. tropica and L. infantum as well as for L. major and L. infantum. The similarity coefficient for L. major and L. infantum was 0.22 and for L. tropica and L. major it was 0.26. These data agree well with established phylogenetic/ taxonomic classification. The index between different isolates derived from various hosts and sandfly vectors for the same Leishmania species was 1, indicating that this method is suitable for epidemiological analysis. 1996 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Leishmania; L. infantum; L. major; L. tropica; Sergentomyia; random amplified polymorphic DNA; Sauroleishmania; S. gymnodactyli; S. tarentolae; Tatera; trypanosomatidae DNA, deoxyribonucleic acid; OTU, operational taxonomic unit; RAPD, random amplified polymorphic DNA; PCR, polymerase chain reaction; SSU rrna, Small subunit ribosomal ribonucleic acid. The RAPD PCR method was used to distinguish between the main Old World Leishmania parasites Leishmania tropica, L. major, and L. infantum and it was applied to Sauroleishmania species from Iran. Twenty-five primers were tested, 13 of which were suitable for identification of these parasites as they generated near identical fingerprints from strains of the same species isolated from various countries. The Jaccard similarity indices for these species, which may be used for the classification of Leishmania, agreed well with the same measure calculated from isoenzyme data. The epidemiology of Leishmania is complex and not fully understood; for the detailed studies required to elucidate the remaining problems, it is important to identify large numbers of isolates from a wide range of potential hosts and vectors across the full geographic range of the parasite. A number of biochemical methods are available for the identification and classification of Leishmania but none of them are ideal. Isoenzyme analysis has become the standard technique; however, it requires bulk in vitro culture of parasites and a relatively large range of reagents (Abderrazak et al. 1 Present address: Department of Microbiology, University of Medical Sciences, Shiraz, Iran. 1993). Monoclonal antibodies for the identification of Leishmania are not available for the majority of Old World species. As DNA is more chemically stable than both isoenzymes and antigens/antibodies, it has been the subject of intensive research for genetic markers suitable for Leishmania identification. DNA probes derived from the various DNA molecules have been developed (Bozza et al. 1995; Ellis and Crampton 1988; Guevara et al. 1992; Qiao et al. 1995; Ready et al. 1988), but are available for only a few species. The PCR offers very high sensitivity but resolution is lower than with isoenzymes, and PCR primers have been published only for L. major and the L. donovani complex of the Old World species (Ibrahim et al. 1994; Smyth et al. 1992). Recently the RAPD PCR method has attracted a great deal of interest (Caetano-Anolles, 1994; Tibayrenc et al. 1993; Waitumbi and Murphy 1993; Williams et al. 1990). It is relatively simple to perform, requires no prior knowledge of the parasite genome, and requires a minimal number of parasites. Furthermore it may be manipulated experimentally to yield information at the species or subspecies level. We have previously shown that RAPD can be useful for identifying members of a sandfly species complex (Adamson et al. 1993) and that it is useful for identifying and classifying members of the L. braziliensis complex (Noyes et al. 1995). We show here the results of a prelimi- 0014-4894/96 $18.00 Copyright 1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 150

MOTAZEDIAN, NOYES, AND MAINGON 151 TABLE I Parasite Stocks Species Strain number Identification 1 L. tropica MHOM/IR/60/LV357 a 2 L. tropica MHOM/IR/66/LV556 a 3 L. tropica MHOM/IR/89/ARD22 a 4 L. major MHOM/ET/XX/LV305 a 5 L. major MHOM/IR/59/LV 39 a 6 L. major MHOM/IR/XX/LV114 a 7 S. tarentolae RTAR/IT/XX/LV 35 a 8 S. gymnodactyli RAGE/SU/XX/LV 247 a 9 L. infantum MHOM/BE/67/ITMA263 a 10 L. infantum MHOM/FR/80/LEM 188 a 11 Unknown IPAP/IR/94/LMIRP1 L. major 12 Unknown MTAT/IR/94/LMIRR2 L. major 13 Unknown MHOM/SD/95/LMIRP3 L. major 14 Unknown MHOM/IR/94/LMIRP4 L. infantum 15 Unknown ISER/IR/94/SLIRP5 S. gymnodactyli 16 Unknown RLIZ/IR/94/SLIRP6 S. gymnodactyli nary study to investigate the application of RAPD to the problem of identifying the Leishmania and Sauroleishmania species found in Iran. Reference strains and recent isolates of L. tropica, L. major, L. infantum, Sauroleishmania gymnodactyli, and S. tarentolae used in this study are shown in Table I. DNA was isolated from the promastigote stage which had been in vitro cultured at 25 C in Schneider s insect medium (Sigma). Parasites (5 10 6 ) were harvested by centrifugation (1000g, 10 min) and washed once with Locke s solution (150 mm NaCl, 6 mm KCl, 4 mm CaCl 2,2mMNaHCO 3,and5mM glucose). The pellet was resuspended in 100 l lysis buffer (150 mm Tris HCl ph 7.6, EDTA (ph 8), 0.5% v/v Tween 20, and 200 g/ml proteinase K) and incubated at 55 C for 1 hr. The lysate was extracted once with equal volumes of 1:1 (v/v) phenol:chloroform and once with 24:1 (v/v) chloroform:isoamylalcohol and precipitated by ethanol. The DNA was resuspended in 100 lof10mmtris HCl, 1 mm EDTA, and a 1:10 dilution was prepared in PCR grade water. Amplification reactions were done in a total volume of 25 l containing 20 mm (NH 4 ) 2 SO4, 75 mm Tris HCl, ph9, 0.01% (w/v) Tween 20, 2 mm MgCl 2, 200 M each deoxynucleotide triphosphate, 1 mm primer and 1 unit of Taq polymerase. One microliter of diluted DNA (approximately 5 ng) was added by centrifugation through the mineral oil overlay and the reaction was carried out in a thermocycler (Perkin Elmer or Omnigene, Hybaid) programmed at one cycle of 94 C, 2 min, followed by 30 cycles of 94 C, 30 sec; 36 C, 1 min; 72 C, 2 min. Twelve microliters of each reaction was run on 1.5% agarose gels and visualised under UV light with ethidium bromide. Primers used in this study are listed in Table II. Fingerprint patterns were compared by the Jaccard similarity coefficient which was calculated as described by Cibulskis et al. (1986). Twenty-five primers were tested for the amplification of reproducible species specific products. Thirteen of those primers generated at least five reproducible products each that could be visualised on an ethidium bromide stained gel (Table I). Figure 1 shows the fingerprints produced by primer M13( 40) with eight reference strains: two L. infantum isolated in Belgium France in 1967 and 1980, three L. major isolated from Ethiopia and Iran, and three L. tropica isolated Primer TABLE II PCR Primers Sequence M13( 40) a GTTTTCCCAGTCACGAC M13 a GTAAAACGACGGCCAGT A1 CAGGCCCTTC A4 GAAACGGGTG A8 GTGACGTAGG AB1-01 GTTTCGCTCC AB1-07 GGTGACGCAG AB1-09 TGGGGGACTC AB1-12 CCTTGACGCA AB1-14 TTCCCCCGCT AB1-15 GGAGGGTGTT AB1-18 CCACAGCAGT 3301 TCGTAGCCAA Note. Table II Primers that reliably amplified Leishmania and Sauroleishmania. Amplification was 1 cycle of 94 C, 2 min, followed by 30 cycles of 94 C, 30 sec; 36 C, 1 min; 72 C, 2 min, except for primers marked with an a, which were amplified as follows: 2 cycles of 94 C, 2 min; 40 C, 5 min; 72 C, 5 min, followed by 35 cycles of 94 C, 30 sec; 60 C, 1 min; 72 C, 2 min.

JOBNAME: JEP 83#1 96 PAGE: 3 SESS: 12 OUTPUT: Thu Jun 20 15:41:00 1996 /xypage/worksmart/tsp000/70152f/6 152 RAPD FOR THE IDENTIFICATION OF Leishmania AND Sauroleischmania FIG. 1. RAPD products of primer M13 ( 40) with Leishmania reference strains separated on a 1.5% agarose gel. L. inf L. infantum; L. maj L. major, L. trop L. tropica. in Iran between 1960 and 1980. All strains produced species-specific patterns despite large separations in time and distance. With primer M13( 40) L. tropica shares a strong band at 900 bp with L. major and a band at 400 bp with L. infantum; however, all three species are readily distinguishable. The primers that produced fingerprints with the greatest difference between L. tropica and L. major were OP-A8 and AB1-12. Primers AB1-18 and AB1-14 gave the greatest difference between L. tropica and L. infantum. Four primers (AB1-12, AB1-14, AB1-18, and OP-A1) gave fingerprints with little similarity between L. major and L. infantum. Unknown parasite isolates were identified with primer AB1-18 as follows: LMIRP2 from the gerbil Tatera from Iran and LMIRP3 from a child from the Sudan were both L. major. LMIRP4 from a human case in Iran was L. infantum (Fig. 2). Although fingerprints were generally very reproducible, the range of product sizes did vary as can be seen from the L. major LV305 in Fig. 1, lane 5, in which the higher molecular weight bands (above 1000 bp) have not amplified. On other occasions products below 600 bp did not amplify. However, in a given run only higher or lower bands were lost but not both, so there was always a region in which comparison could be made with confidence. FIG. 2. RAPD products of primer AB1-18 with Leishmania reference strains and parasites isolated from humans, Sergentomyia and a lizard. L. inf, L. infantum; L. maj L. major; S. tar, Sauroleishmania tarentolae; S. gym Sauroleishmania gymnodactyli. All of the primers tested distinguished between any of the Leishmania from Iran and Sauroleishmania. which is found in sandflies in the same area. S. tarentolae and S. gymnodactyli were easily differentiated from each other using a variety of primers, as shown for the primer AB1-18 (Fig. 2). Two unknown flagellates isolated from Sergentomyia spp. (IRP5) and from a lizard (IRP6) were identified as S. gymnodactyli. L. tropica and S. tarentolae generated very similar fingerprint patterns (Fig. 2) emphasizing the intermediate nature of the latter lizard parasite which has been also described as L. tarentolae (Simpson and Simpson 1978). RAPD has not been found to be suitable for the comparison of distantly related species so this apparent similarity may not be an accurate representation of the relationship between the two genomes (Black 1993). There is very little comparative data on Sauroleishmania and Leishmania, but the two genera cannot be satisfactorily resolved using the sequence of the 18S SSU rrna gene (Briones et al. 1992; Marché et al. 1995). The genus Sauroleishmania was created on the basis of host and vector specificity and some biological characteristics at a time when very little comparative biochemical molecular biological data were available (Killick-Kendrick et al., 1986). We are currently examining a larger group of isolates to investigate the relationship between these genera (Noyes and Maingon, in preparation). The 13 primers listed in Table II were used to calculate

MOTAZEDIAN, NOYES, AND MAINGON 153 similarity coefficients (Table III). The Jaccard coefficient has values between 1, for identical OTUs, and 0, for OTUs with no similarity. Although a relatively limited number of isolates have been tested in this study, the values for the three pairs of Leishmania species studied ranged between 0.22 and 0.30, showing that RAPD PCR clearly differentiated between these species. The results obtained from reference strains are encouraging for the general applicability of the RAPD PCR as parasites of a given species collected in widely different places at different times gave consistently similar patterns. Although RAPD fingerprints can only be obtained from cultured parasites, sufficient parasites are available in the supernatant of the biphasic media that are routinely used for parasite isolation. As a single primer is sufficient for identification purposes, RAPD has considerable potential for epidemiological surveys in which it is necessary to combine identification to the species level with the capacity to process large numbers of samples. As RAPD can be used to identify any species for which reference stains are available without prior development work, it is valuable for the identification of lesser known species such as the Sauroleishmania shown here. Isoenzymes have been used to identify S. tarentolae but this requires bulk culture of parasites (Pozio et al. 1986). Since it has been reported that Sauroleishmania can cross react with Leishmania in the Montenegro skin test for at least 2 years after inoculation and may give some cross protection, the Sauroleishmania may have some epidemiological significance (Wilson and Southgate 1979). As stocks isolated from both invertebrate and vertebrate hosts appeared identical, RAPD could provide a useful tool for the further study of the ecology of these lesser known trypanosomatidae. RAPD must be used with caution for the classification of even closely related taxa, as some products of equal size may not be homologous. Furthermore the mix of conserved and variable sequences represented is not known and may vary between taxa (Black 1993). Nevertheless the values of the Jaccard coefficient found from the RAPD fingerprints compare well with others produced from isoenzyme data. Cibulskis et al. (1986) examined 280 Old World stocks with 13 enzymes; values of the Jaccard coefficient for the same pairs of species as examined in the present study were interpolated from their dendrograms. The values ranged between 0.24 and 0.31, compared with the values between 0.22 and 0.30 found in the present study. The same authors found the most closely related species to be either L. infantum and L. major or L. tropica and L. major, according to TABLE III Jaccard Similarity Coefficients between Leishmania Species Found in Iran L. tropica v. L. major 0.26 L. tropica v. L. infantum 0.30 L. major v. L. infantum 0.22 whether the data were analyzed by the average linkage or single linkage methods. In the present study L. infantum and L. tropica were most closely related, however, Lanotte et al. (1986) also found L. infantum and L. tropica to be the most closely related using three enzymes and 202 stocks. There are no reliable methods for determining the precise relationships between taxa that are almost equidistant from each other (Felsenstein 1988), so these discrepancies are not significant. RAPD has already proved useful in a study of the ecoepidemiology of L. braziliensis in Brazil (Gomez et al. 1995). 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