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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leishmania tropica is genetically a very heterogeneous species and strains are frequently distinguished using biochemical, antigenic, and polymerase chain reaction (PCR)–based molecular markers.8–11 In the present study, among L. tropica strains isolated from sand flies and human CL cases originating in the same locality, a degree of serologic, biochemical, and DNA sequence heterogeneity is demonstrated.
Phlebotomus (Paraphlebotomus) sergenti is the putative vector of L. tropica throughout the Middle East and is a proven vector in Saudi Arabia,12 Morocco,13 and Afghanistan.14 In the Galilee region of northern Israel, both, Ph. sergenti and Ph. (Adlerius) arabicus have been found infected with L. tropica.7 The present study was designed to improve our understanding of the epidemiology of CL caused by L. tropica in desert habitats in this region. The multifarious characterization of promastigotes isolated from three female Ph. sergenti and their comparison with strains of L. tropica isolated from human CL cases verifies that this species is also the vector of L. tropica in the Judean Desert near Jerusalem.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It is located at an altitude of 350 meters above sea level approximately 5 km east of Jerusalem off the main highway that descends from Jerusalem to Jericho. Twenty CL cases were diagnosed in Kfar Adumim and its vicinity between 1989 and 1994, and at least 10 additional cases have been diagnosed since then.15,16 Strains isolated from two of these cases, L590 from Kfar Adumim and L691 from Anatot, a neighboring village approximately 4 km to the west, were used for comparison in characterizing stocks isolated from Ph. sergenti (Table 1).
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). Sand flies were also collected inside houses and off properly protected humans, using mouth aspirators. For taxonomical identification, heads and genitalia were removed and mounted on microscope slides in Berlese’s fluid and identified using several keys.17,18 Female Ph. (Larroussius) spp., were identified by the structures at the base of the spermathecal ducts. Isolation and maintenance of leishmanial stocks. For parasite isolation, forceps and glassware were sterilized in 70% ethanol. Female flies were washed in 5% detergent solution, rinsed several times in sterile water, and placed in sterile phosphate-buffered saline (PBS). Their guts were dissected on glass slides, covered with coverslips, and examined for the presence of promastigotes by phase-contrast microscopy with a 40x objective. The guts of infected sand flies were seeded into rabbit blood hyphenate agar NNN slants overlaid with Schneider’s Drosophila medium (SDM) supplemented with 10% fetal calf serum (FCS) containing penicillin (200 IU/ mL), streptomycin (200 µg/mL) (Biological Industries, Beit Haemek, Israel) and 5-fluorocytosine (1,500 µg/ml) (Sigma, St. Louis, MO).19 For routine culture, parasites were grown in liquid SDM containing 10% FCS.
Biologic characterization of leishmanial strains. Strain L747 from a Ph. sergenti female and strain L590 from a CL case from Kfar Adumim were separately injected subcutaneously into the dorsal surfaces of the hind paws of three Syrian hamsters. Each paw received approximately 0.5 x 106 stationary phase promastigotes. The animals were examined periodically and the sites of infection were monitored for the development of lesions and the presence of parasites. Smears of skin and, at necropsy, spleen and liver tissue were made, stained with Giemsa, and examined microscopically for amastigotes. Tissue from these organs was also seeded into rabbit blood agar medium and the cultures were later checked for the development and growth of promastigotes.
Serologic characterization of the leishmanial strains.
Excreted factor (EF) serotyping.
Spent growth medium from cultures of local leishmanial isolates containing their EFs were compared with standard reference EFs as previously described.20,21 Standard serotyping sera were used: anti-L. tropica L36, which reacts only with serotype A and permits the designation of A subserotypes; anti-L. donovani donovani LRC-L52, which reacts only with serotype B EFs without designating subserotypes; and anti-L. donovani infantum LRC-L47, which reacts only with serotype B and permits the designation of B subserotypes. The reference EFs were obtained from the following strains: L. tropica L36 for subserotype A2, L. tropica LRC-L682 for subserotype A9, L. d. donovani LRC-L133 for subserotype B2, and L. aethiopica LRC-L495 for subserotype B1 (Table 1).
Species-specific monoclonal antibodies (MAbs).
Four MAbs were used to characterize Leishmania promastigotes using an indirect immunofluorescent test. Cultured promastigotes were washed and placed in multi-well microscope slides. Preparations were fixed briefly with cold acetone, blocked with 5% FCS in PBS for 30 minutes at room temperature, and incubated for one hour with the MAbs (undiluted culture supernatant fluid or ascites diluted 10–3) at 37°C. Fluorescein isothiocyanate–conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) was applied for 40 minutes at 37°C. Slides were washed three times in PBS, mounted in PBS/glycerol with 3% 1,4-diazabicy-clo[2.2.2]octane (DABCO; (Sigma), and observed by fluorescent microscopy (Axiovet; Zeiss, Oberkochen, Germany). The MAbs used were T1 and T3, which were raised against L. major L137 and L251, and T11 and T15, which were raised against L. tropica strain L36 (Table 1). T1 reacts primarily with L. major while T3 cross-reacts with many L. tropica strains.8 Both, T11 and T15 react specifically with surface moieties of L. tropica.22 The sand fly and human strains were compared with one another and with control strains of L. major, L. tropica, and L. infantum.
Isoenzyme electrophoresis. Electrophoresis was performed in starch gels according to Rioux and others10 and 15 enzymes were examined to construct enzyme files: malate dehydrogenase (MDH, EC 1.1.1.37); malic enzyme (ME, EC 1.1.1.40); isocitrate dehydrogenase (ICD, EC 1.1.1.42); 6-phosphogluconate dehydrogenase (PGD, EC 1.1.1.44); glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49); glutamate dehydrogenase (GLUD, EC 1.4.1.3); NADH diaphorase (DIA, EC 1.6.2.2); purine nucleoside phosphorylase (NP1, EC 2.4.2.1); purine nucleoside phosphorylase (NP2, EC 2.4.2*); glutamate-oxaloacetate transaminases (GOT1 and GOT2, EC 2.6.1.1); phosphoglucomutase (PGM, EC 5.4.2.2); fumarate hydratase (FH, EC 4.2.1.2); mannose phosphate isomerase (MPI, EC 5.3.1.8); and glucose phosphate isomerase (GPI, EC 5.3.1.9).
Molecular characterization. Polymerase chain reaction–based analysis of kinetoplast DNA (kDNA). The primer pair Uni21 (5'-GGG GTT GGT GTA AAA TAG GCC-3') and Lmj4 (5'-CTA GTT TCC CGC CTC CGA G-3'), which was based on a minicircle sequence of L. major,23 were used to amplify kDNA from cultured promstigotes, whose PCR products were electrophoresed on agarose gels according to Anders and others.24
The PCR products were also column purified using a High PureTM PCR Product Purification Kit (Boehringer Mannheim Corp., Indianapolis, IN) according to the manufacturer’s recommendations. For restriction fragment length polymorphism (RFLP) analysis, 50-ng samples of PCR product were digested with the following endonucleases (each applied separately): Mbo I and Ban II (Promega, Madison, WI). The RFLP samples were subjected to electrophoresis in 2% Metaphor agarose gels, and the DNA was detected with Gel Star Stain (FMC BioProducts, Rockland, ME).
Permissively primed intergenic polymorphic (PPIP)–PCR. The PCRs were performed as previously described16 with minor changes, i.e., using 20 ng/µl of genomic DNA and amplified with 5 µM of the single leishmanial-specific primer 2B (5'-CAG GAG CGC GCA CAC GCA CAC ACG-3'), and two units of recombinant Taq DNA polymerase (MBI Fermentas, Amherst, NY).
Amplification and digestion of the internal transcribed spacer (ITS) sequence. This was done essentially according to Schonian and others.25 The ITS ribosomal operon between the small subunit and large subunit ribosomal RNA genes was amplified using the primers LITSV (5'-ACA CTC AGG TCT GTA AAC-3') and LITSR (5'-CTG GAT CAT TTT CCG ATG-3'). Aliquots of 17µl of the 900–1000 basepair products were digested for two hours with 1 µl of either Taq I or Cfo I (Hybaid GmbH, Heidelberg, Germany), according to the manufacturer’s recommendations, and the fragments separated by agarose gel electrophoresis.
Single strand conformation polymorphism (SSCP) analysis of the ITS1 sequence. The first part of the ITS region (ITS1) was amplified with the primer pair L5.8S (5'-TGATACCACTTATCGCACTT-3') and LITSR (5'-CTGGATCATTTTCCGATG-3') to produce a PCR product of approximately 300 basepairs. Subsequent SSCP analyses were performed as described by El Tai and others.26
DNA fingerprinting. DNA fingerprinting was done essentially as described by Gomes and others27 using the human multilocus probe 33.15.28 Seven micrograms of leishmanial genomic DNA were digested completely with the restriction enzyme Hae III and subjected to electrophoresis. Following Southern blotting, the membrane was hybridized under low stringency with the digoxigenin (DIG)–labeled probe (probe 33.15, cloned in M13mp8, kindly provided by A. J. Jeffreys, University of Leicester, Leicester, United Kingdom). The DIG-labeled hybrids were detected with an anti-DIG–alkaline phosphatase conjugate and the chemiluminescent substrate CDP-Star (Roche, Penzberg, Germany). The relatedness of the strains was determined by analyzing the resulting banding patterns with RAPDistance Package version 1.04 using Pearson’s Phi coefficient to calculate similarities (http://www.anu.edu.au/BoZo/software).
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RESULTS |
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). Phlebotomus sergenti, the most abundant species in light-trap collections from the caves, was not greatly attracted to humans, as demonstrated by the relatively small number collected off human bait in the same locations. Furthermore, aspirator collections inside houses showed that few Ph. sergenti entered houses. Conversely, Ph. papatasi was highly endophilic and anthropophagic but was not greatly attracted to light traps (Table 2). Very small numbers of three other species were also collected: Ph. (Pa.) alexandri, Ph. (Laroussius) tobbi, and Ph. (L.) syriacus. Four of the 105 female Ph. sergenti caught in light traps in the caves were infected with promastigotes, from which three stocks were isolated for characterization: L747, L757, and L758 (Table 1). None of the more than 200 Ph. papatasi caught inside houses were infected.
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Serologic characterization by EF and species-specific MAbs.
The EF serotypes and MAb specificities of all the strains studied are shown in Table 3. The Iraqi strain L36, a reference strain of the EF subserotype A2, reacted strongly with the MAb T11 and relatively weakly with MAb T15, which were raised against it and are species-specific for L. tropica. However, strain L36 did not react with MAbs T1 and T3 raised against L. major. Strain L22 isolated from a human case of CL acquired in the Negev region and included as an Israeli reference strain was also EF subserotype A2, showing that this subserotype also exists in Israel. The promastigotes of strain L22 were not checked for their MAb-binding specificities with the MAbs T11, T15, T3, and T1. However, its EF did bind strongly to MAb T11 and moderately to MAb T15, but not to the MAbs T1 and T3.21 The test strains were either EF subserotype A9 or the mixed subserotype A9B2, both of which and like the EF subserotype A2, are species-specific for L. tropica. They also all bound MAb T11 strongly. Strains L747, L757, and L758 from locally caught Ph. sergenti were serologically similar in not reacting with MAb T3 and separable from strains L590 and L691 from the local human cases of CL and from strain L775 from Sinai, Egypt, which bound MAb T3 strongly. Only two of the test strains, L590 and L775, bound MAb T1 relatively weakly. None of the test strains reacted with MAb T15 (Table 3).
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, and the 15-enzyme profiles associated with these zymodemes, which give the electrophoretic mobility of each electromorph, are shown in Table 4. Strains L747, L757, and L758 from Ph. sergenti caught at Kfar Adumim and strain L691 from a human case of CL from nearby Anatot shared the same profile, which was characteristic of strains belonging to zymodeme MON-137, the reference strain of which is L775 (Table 1). The enzyme profile of strain L590 was different from all the profiles recorded for the established zymodemes. Thus, strain L590 represents a new zymodeme, MON-275. Its profile was much closer to that of the Iraqi reference strain L36, which belongs to zymodeme MON-006 than it was to that of the strains from Kfar Adumim, which belong to zymodeme MON-137, even though it came from a human case of CL living in the village. The enzyme profiles of strains L590 and L36 shared 10 enzyme electrophoretic mobilities, whereas L590 and the strains from Kfar Adumim belonging to the zymodeme MON-137 shared only four, those of ICD, DIA, NP1, and GPI (Table 4). The Israeli strain L22 also represented a new zymodeme, MON-288. Its enzyme profile was closer to that of the local strain L590 than it was to that of the Iraqi strain L36 (Table 4).
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Figure 2 shows the kDNA-RFLP patterns of reference strains of L. tropica and several test strains following digestion with Mbo I (Figure 2a) and Ban II (Figure 2b). All strains were shown to be L. tropica, but strain L590 from the human case of CL from Kfar Adumim was clearly different from the sand fly strains caught in the same vicinity (Figure 2a). Moreover, among the sand fly strains, L758 and L757 were identical to one another and L747 was somewhat different (Figure 2b).
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. This analysis showed that all the strains were L. tropica. They were also easily distinguished from L. major, L. infantum, and L. killicki. Strains L747, L757, and L758 from Ph. sergenti caught at Kfar Adumim, strain L691 from the case of CL from Anatot, and strain L775 from Sinai, Egypt had the same PPIP-PCR type (LtB1). Strain L590 from the case of CL from Kfar Adumim was different (PPIP-PCR type LtA1) and identical to strains L36 and L22 from an Iraqi and an Israeli human case of CL respectively (Figure 3 and 6).
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. Again, based on the patterns seen, all locally isolated strains were L. tropica and easily distinguished from L. major, L. infantum, and L. killicki. All of these strains of L. tropica displayed identical patterns except for L590 from the case of CL from Kfar Adumim, which was identical to that of strain L43 isolated from a human case of VL from the village Abu Ghosh approximately 23 km west of Kfar Adumim. Thus, the results of the SSCP analysis corresponded well with those of the PPIP-PCR and isoenzyme electrophoresis.
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. This generated identical patterns for strains L590 from the case of CL and L747 from a Ph. sergenti from Kfar Adumim and the reference strain of L. tropica K27. After digestion with the restriction enzyme Cfo I, the restriction patterns of strains L590 and L747 were also identical to one another. Both types of pattern distinguished these strains of L. tropica from the reference strains of L. infantum and L. major. The ITS-RFLP analysis of strains L691 and L775 from human cases of CL from Anatot and Sinai, Egypt, respectively, and strains L757 and L758 from Ph. sergenti produced the same patterns, also confirming their identity as L. tropica of the same type. However, L. tropica L36 from a human case of CL from Iraq had an extra band of approximately 300 basepairs after digestion with Taq I and another band of approximately 250–300 base-pairs after digestion with Cfo I.
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shows the inter-relationship of various strains of L. tropica according to their genomic DNA RFLP patterns after digestion with Hae III and hybridization with the minisatellite probe. All strains of L. tropica, including L36 from Iraq, grouped together and formed a single closely-knit cluster relative to the strain of L. major L777 used as an outgroup. This clustering of the strains of L. tropica was similar to the clustering of the strains based on 1) specific binding of MAb T11, 2) PCR-based kDNA analysis using the primer pair Uni21 and Lmj4, and 3) amplification and restriction of the ITS sequence with either Taq I or Cfo I (Table 3). This main cluster divided into two subclusters (I and II), which was supported by 1) EF serotypes, 2) enzyme profiles, 3) PPIP-PCR types, and 4) ITS1-SSCP types (Table 3). The strains within each subcluster displayed further intraspecific microheterogeneity based on 1) the specific binding of the MAbs T15, T3, and T1, and 2) restriction of amplified kDNA with either Mbo I or Ban II (Table 3). It is noteworthy that the strains of L. tropica in subcluster II belonged to zymodeme MON-137 and EF serotype A9B2, and had the PPIP-PCR pattern LtB1and the ITS1-SSCP pattern B (Figure 6 and Table 3). |
DISCUSSION |
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). The reluctance of Ph. sergenti to enter houses and bite humans probably explains the paucity of CL cases despite the high infection rate among females of this species (4%). Although Ph. papatasi was notably endophilic and constituted an important pest, there is no evidence for its having a role in the transmission of CL in this focus. However, Ph. papatasi is the vector of L. major in the Jordan Valley, which is not far from Kfar Adumim but at a lower altitude, where P. obesus is the animal reservoir host.1,2 The absence of P. obesus from rocky terrain is a reasonable explanation for the lack of locally acquired infections of L. major in residents of Kfar Adumim and Anatot. Female Ph. sergenti from Kfar Adumim were distinguishable from females of the same species from other countries. The most prominent differentiating feature was the more swollen appearance of the distal segment of their spermathecae, which was consistent and easily seen after dissection. This feature is not readily apparent in mounted specimens and appears to have been previously overlooked. The taxonomic relevance of this differentiating morphologic feature remains to be determined.
Before the introduction of more modern serologic, biochemical, and molecular biologic methods for identifying leishmanial strains, their infectivity, virulence, dissemination, and pathogenicity in laboratory animals were used as diagnostic characteristics. The Syrian hamster, despite not being a proven natural host of any species of Leishmania, can harbor infections of most species, including some strains of L. tropica.29 In our experiments, infection and pathogenesis caused by the two chosen strains, L590 from human CL and L747 from Ph. sergenti, was essentially the same. The infections were dermatotropic without involvement of the spleen or the liver. Pathogenesis differed substantially from that caused by L. major.30
The three sand fly and two human strains of Leishmania from the Kfar Adumim focus proved to be L. tropica by all the approaches used. Two serologic tests were used: EF serotyping and analysis with species-specific MAbs. An EF comprises all immunogenic molecules secreted by promastigotes growing in culture. The main components of EF are the naturally released lipophosphoglycans.31 The presence of the antigenic components A2 or A9 in an EF and reactivity with MAb T11 are typical of strains of L. tropica. It was noted that the first three strains listed in Table 3, all from human cases of CL, also reacted strongly with MAb T3, which although raised against L. major, binds to the promastigotes of both L. major and L. tropica, whereas the three strains from the sand flies did not. No explanation is currently available for this finding. Isoenzyme electrophoresis confirmed that most of the locally isolated strains belonged to the previously known zymodeme MON-137 of L. tropica. This included strains from a human case of CL and those from Ph. sergenti. Strain L590 was the single exception. Its enzyme profile was different from the rest and it represented a new zymodeme, MON-275, but was sufficiently similar to those of the reference strains of L. tropica L36 and L22 (Table 4) to confirm its identity as L. tropica. Direct amplification of the kDNA from all the local strains with the primer pair Uni21 and Lmj4 generated a product of approximately 800 basepairs, confirming they were all L. tropica.24 After digestion with Mbo I (Figure 2a), differences were seen between the RFLP pattern of the locally isolated strains, which is discussed later in the context of microheterogeneity of the strains. Furthermore, genomic DNA analyses using PPIP-PCR (Figure 3), ITS1-SSCP (Figure 4), and ITS-RFLP (Figure 5) all generated profiles readily identifying the test strains as L. tropica and separating them from other species of Leishmania.
Leishmania tropica is recognized as a very heterogeneous species of Leishmania and intraspecific microheterogeneity has been readily demonstrated by many investigators.4,9,10,11,16,21 In terms of isoenzyme profile variation, Kreutzer and others used 21 enzyme systems on viscerotropic strains of L. tropica isolated from soldiers returning from Operation Desert Storm.32 In the present study, despite the small number of isolates and the restricted geographic region studied, strains of L. tropica were shown to comprise two different zymodemes. Most of the strains belonged to zymodeme MON-137, the reference strain of which is strain L775 from Sinai (Table 1). Other strains of this zymodeme have been isolated from CL cases in Jordan.33 The only other local strain isolated and characterized was strain L590. Its enzyme profile was very different and it constituted a new zymodeme, MON-275. Of the 15 enzymes tested, the electrophoretic mobilities of four of them corresponded with those in the profile associated with the zymodeme MON-137, while 11 differed. However, L590, the reference strain of the new zymodeme MON-275, was much more similar to the reference strain of L. tropica (L36 from Iraq, zymodeme MON-6), with which it shared 10 and differed in five enzyme electrophoretic mobilities. It was even more similar in its profile to reference strain L22 from Israel that represented the new zymodeme MON-288, where it differed in only three enzyme electrophoretic mobilities, those of MDH, PGD, and FH. Strains L36 and L22 differed between themselves in four enzyme electrophoretic mobilities, those of MDH, PGD, DIA, and PGM. All these similarities and differences are seen and compared in Table 4.
The RFLP analysis of kDNA after digestion with Mbo I and Ban II (Figure 2 and Table 3) showed that the strain L590 from the case of CL from Kfar Adumim was notably different from the strains isolated from Ph, sergenti from the caves below Kfar Adumim, and more similar to strain L36 isolated many years ago from a human of case CL from Baghdad. The RFLP pattern of strain LRC-L747 and that shared by strains L757 and L758 were different, even though these three strains came from sand flies caught in the same focus within one month in 1998. However, the sand fly from which strain L747 was isolated was caught on a separate occasion than the sand flies from which strains L757 and L758 were isolated. The genetic microheterogeneity of kDNA among the various strains of L. tropica studied here is not surprising because microheterogeneity of kDNA has been reported even among clones from the same strain of L. major.34
Microheterogeneity was also disclosed by the genomic DNA analyses. The PPIP-PCR (Figure 3) and ITS-SSCP (Figure 4) patterns of strain L590 from the human case of CL from Kfar Adumim also differentiated it from strain L691 from the human case of CL from Anatot and strains L747, 757, and 758 from the sand flies caught near Kfar Adumim, which were all identical. In addition, strain L590 was very similar to the Iraqi reference strain L36 and, in the case of its PPIP-PCR pattern, the Israeli strain L22. However, ITS-RFLP analyses did not detect differences among all these strains (Figure 5).
DNA fingerprinting using a minisatellite probe placed L590 in one subcluster together with the Iraqi strain L36 and the Israeli strain L22, and separating it from the other local strains from Kfar Adumim and Anatot (Figure 6). The two subclusters of strains of L. tropica in the dendrogram were congruent with results obtained by PPIP-PCR, ITS1 SSCP, isoenzyme electrophoresis, and serologic profiles (Figure 6 and Table 3). In summary, microheterogeneity was evident among the locally isolated strains examined, and strain L590 appeared sufficiently different from the other local strains to suggest that the person involved, although he lived in the village of Kfar Adumim, might have contracted his CL elsewhere.
Table 4, which includes the enzyme profiles encountered, shows that the enzyme profile of zymodeme MON 026 associated with the strain of L. major, which served as an out-group in the dendrogram (Figure 6), was very different from the profiles associated with the zymodemes of L. tropica. These profiles are consistent with the dendrogram based on DNA fingerprinting and with the data generated by the other methods used.
The various methods of characterization used here disclosed different degrees of similarities and differences among the strains studied. However, a direct connection between the DNA profiles obtained by examining kinetoplast and nuclear DNA and the phenotypic characteristics discerned by serotyping and enzyme analysis cannot be made. Nevertheless, it is interesting to note that the genetic and phenotypic characteristics identified here are consistent with one another (Figure 6 and Table 3). The methods employed seem to separate into two types irrespective of exposing either genotypic or phenotypic criteria: those that identify leishmanial parasities at the species level and those that reveal intraspecies varation. All of these methods are valid at whichever level they operate. Some of the methods are more useful as tools in the diagnosis of disease, while others would be more useful in analyzing the genetic composition of leishmanial parasite populations. Table 3 shows a full comparison of the methods and the criteria they reveal and assess, the clustering of the strains of L. tropica by DNA fingerprinting, and how the various methods used support this clustering (see results of DNA fingerprinting).
During the preparation of this report, a new focus of human CL caused by L. tropica was investigated in the Galilee region of northern Israel. The sand fly vector there was identified as Ph. (Adlerius) arabicus and the causative agent as L. tropica, which was different from all the other variants of L. tropica described throughout the entire geographic range of the species.7 Similar to Kfar Adumim, this new focus was also associated with relatively recent development of the area and the establishment of new villages that caused localized ecologic and environmental disruption. It appears that human encroachment on natural zoonotic foci is leading to the emergence of human CL in the entire region.
The infected female sand flies caught in the vicinity of Kfar Adumim were identified as Ph. sergenti and the strains of Leishmania isolated from them and the local human cases of CL were identified as L. tropica. Phlebotomus sergenti has long since been the putative vector of L. tropica in the region encompassed by Israel and the West Bank. The overall result of this comparative study establishes it as an actual vector in this region.
Received July 2, 2003. Accepted for publication October 9, 2003.
Financial support: This research was supported by grant SO 220/5-1 from the Deutsche Forschungsgemeinschaft (DFG): "The German-Israeli-Palestinian Cooperative Project on Leishmaniosis in Israel and The West Bank" by grant number 235/99-16.2 from "The Israel Science Foundation" of the Israeli Academy of Sciences and Humanities, and a grant from the Israeli Ministry for the Environment. The Montpellier Centre National de Reference des Leishmania is supported by the French Ministry of Health.
* These authors contributed equally to this paper. 
Authors’ addresses: Lionel F. Schnur, Abdelmageed Nasereddin, Carol L. Eisenberger, Charles L. Jaffe, Gerlind Anders, Tamar Grossman, Raymond L. Jacobson, and Alon Warburg. Department of Parasitology, The Kuvin Center for the Study of Infectious and Tropical Diseases, Hebrew University–Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel, Telephone: 972-2-675-8081. Fax: 972-2-675-7425, E-mails: schnurl{at}cc.huji.ac.il and warburg{at}cc.huji.ac.il. Mustafa El Fari and Gabrielle Schonian, Institut fur Mikrobiologie und Hygiene, Charite Campus Mitte, von Humboldt Universitaet zu Berlin, Dorotheenstrasse, Berlin, Germany, Telephone: 49-30-2093-4741, Fax: 49-30-2093-4703. Kifayia Azmi, Department of Biochemistry, Al-Quds University, Palestinian Authority. Mireille Killick-Kendrick and Robert Killick-Kendrick, Department of Biological Sciences, Imperial College at Silwood Park, Acsot SL5 7PY, United Kingdom. Jean-Paul Dedet and Francine Pratlong, Laboratoire de Parasitologie and Centre National de Référence des Leish-mania, 163 Rue Auguste Broussonet, 34090 Montpellier, France. Moien Kanaan, Department of Biology, Bethlehem University, Palestinian Authority.
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). Caves and crevices inhabited by rock hyraxes were between 40 and 350 meters from the nearest houses. Several houses where cases of cutaneous leishmaniasis lived are marked (
) on the aerial photograph (bottom panel). The triangle indicates from where the photograph in the top panel was taken and the angle covered.
X174 + Hae III and 
