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Application of riboprinting for the identification of isolates of cutaneous Leishmania spp.
Published online by Cambridge University Press: 09 October 2003
Abstract
Riboprinting is one of several molecular methods that can generate comparative data independently of the complexity of the organism's morphology. Restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Leishmania spp. yields a typical ‘riboprint’ profile that can vary intraspecifically. A selection of 76 stocks of L. major and L. tropica, isolated from patients with cutaneous leishmaniasis, was analysed by riboprinting to assess divergence within and between species. L. major and L. tropica could be easily differentiated from each other. Analysis of PCR–RFLP profiles indicated that stocks of Leishmania spp. could be broadly partitioned into 2 species corresponding to L. major and L. tropica. To test if ribosomal 18S sequences were homogeneous within each species, several isolates of each of the Leishmania spp. were digested. Interpretation of the riboprint profiles of the 18S independently amplified by PCR, there would appear to be one restriction pattern present within each Leishmania spp. Homogeneity within copies of the ribosomal 18S within a single genome has, therefore, been demonstrated. The species designation established by riboprinting results were in agreement with the zymodeme analysis of the same isolates. The restriction patterns produced were simple, reproducible and easy to interpret.
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INTRODUCTION
Cutaneous leishmaniasis prevalent in many countries, is caused by different species of Leishmania. In the Old World, there are 4 Leishmania spp. known to cause the disease (Peters & Killick-Kendrick, 1987). These are L. tropica, L. major, L. aethiopica and L. infantum.
The identification of Leishmania species in an endemic area has a great practical significance since for therapeutic purposes it is important to define the species. However, identification of species and subspecies of Leishmania presents several problems, due to the morphological similarities of the different species (Chance, 1979). The taxonomy and the identification of members of this genus have undergone several modifications (Gardener, 1977). To support current taxonomy, diverse methods of genetic characterization have been applied, such as isoenzyme electrophoresis (Chance, 1979; Chance & Walton, 1982), kDNA analysis (Grimaldi & Tesh, 1993), molecular karyotyping (Dujardin et al. 1993) and other molecular techniques (Clark, 1997; Schallig & Oskam, 2002). The development of simpler molecular techniques such as PCR fingerprinting and randomly amplified polymorphic DNA (RAPD) have enabled the detection of intraspecific variation in different Leishmania species (Andersen et al. 1996; Schonian et al. 1996). Genomic (Lopes et al. 1984) and kDNA restriction analysis (Goncalves, Nehme & Morel, 1984) were developed. Endonuclease digestion of kDNA maxi- and mini-circles was applied to discriminate between different species and subspecies (Barker, Arnot & Butcher, 1982; Barker, 1987; Spithill & Grumont, 1984).
Among several genetic characteristics that can be used for parasite identification are those corresponding to repetitive genes that can be very informative for many diagnostic and evolutionary studies. Repetitive genes are detected more easily than single copy genes. Polymorphism can be observed through either variations in a copy number (Iovannisci & Beverly, 1989) or DNA sequence of the non-transcribed spacers (Fernandes et al. 1994). Furthermore, repeated genes can be used to analyse the genetic bases of important phenotypic properties such as pathogenicity. In this context, the high copy number of the ribosomal genes with about 100 copies in the Leishmania genome (Leon, Fouts & Manning, 1978) makes them an ideal target for genetic characterization. They consist of conserved segments flanked by variable spacer sequences under low selective pressure (Long & Dawid, 1980).
In a previous study of the epidemiology of cutaneous leishmaniasis in Jordan (Nimri et al. 2002), Leishmania species and the zymodemes were identified based on the isoenzyme analysis.
The present work aimed to evaluate the use of a rRNA gene locus for genetic characterization of Leishmania spp. isolated from endemic areas. The results are compared to the isoenzyme characterization (zymodeme analysis) of the same isolates.
MATERIALS AND METHODS
Clinical samples
DNA was prepared from clinical samples collected during a prevalence survey in endemic foci. Skin scrapings from 76 patients were obtained from the edge of lesions consistent with cutaneous leishmaniasis (ulcers or scars). These cases were diagnosed by physicians at several health centers in Jordan. A questionnaire was completed for each patient to record demographic and other relevant information such as the site of ulcer(s) on the body, date and place of acquiring the disease, previous travel history.
Parasitological procedures
The sample was divided into 3 portions; one was immediately cultured in Modified Tobie's Medium with L-proline, foetal calf serum and antibiotics (gentamycin, nystatin or 5-flourocytosin as antifungal agent) (Evans, 1987). Culture media were incubated at 24 °C and examined at regular intervals to monitor the growth of the promastigotes or the presence of contaminants. The contaminated cultures were discarded.
Isoenzyme characterization from cultured promastigotes isolated from specimens was performed (Nimri, Soubani & Gramiccia, 2002).
A second portion was thin smeared on a slide for staining with Giemsa's stain and examined microscopically for amastigotes. The third part, for PCR, was placed directly in 200 μl of lysis buffer (10 mM Tris–HCl, 10 mM EDTA), boiled for 10 min in a water bath, and then stored frozen at −70 °C until used (Belli et al. 1998).
DNA isolation
Genomic DNA was isolated directly from scrapings from cutaneous lesions and from cultures using a DNA detect kit (Clonit S.r.1, Milano, Italy) according to the manufacturer's instructions. L. donovani DNA was provided also with the Kit to be used as a positive control.
The Leishmania reference strains used were L. major 5 ASKH (MHOM/SU/73/5-ASKH), and L. tropica Lv140 (MHOM/IL/75/LV140), obtained from the Leishmania collection of the Royal Tropical Institute in Amsterdam, The Netherlands.
Nested PCR conditions
A nested PCR assay to amplify a 380 bp fragment of the rRNA genes was performed. The 2 sets of primers used in the 2 rounds were provided by Clonit according to 18S rRNA sequence of L. major. The first round primers are S1: 5′-GATCTGGATCTGGTTGATTCTGCCAG-3′ and S3: CCATCACCAGCTGATAGGGC, located at positions 10707–10726 and 11057–11076 respectively. The second round primers are S5: 5′-CCTCAGAATCACTGCATTTG-3′ and S7: 5′-CAGTGAAGGCATTGGTTTTA-3′, located at positions 10764–10783 and 10959–10978 respectively. Amplification reactions were performed in volumes of 50 μl containing 2 U of Taq DNA polymerase (Promega) and 5 μl of the sample DNA. Samples were amplified in a cover-heated thermal cycler (GeneAmp® PCR System 9700, Perkin-Elmer, Emeryville, Calif.). Two rounds of PCR were performed, the cycling conditions were 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min.
Samples of 3 μl of the first-round products were used as template for the second round in a total volume of 50 μl under the same conditions as those for the first round, except that primers S5 and S7 were used. The positive control provided with the Clonit kit and reference strains were also amplified under the same conditions. DNase-free water was used as a negative control with every PCR run to monitor for cross contamination.
Samples of 5 μl of the second round PCR products were loaded onto 2% agarose gels to confirm amplification.
Restriction enzyme analysis
The PCR products of the 76 positive samples in addition to 3 L. donovani were digested with 3 restriction enzymes, Hae III, Msp I, and Taq I (Promega, Madison, WI) in a total volume of 20 μl according to the manufacturer's instructions. The products of the digestion were separated by gel electrophoresis in 2·8% agarose and stained with ethidium bromide.
The digestion reaction was repeated twice for each PCR product.
RESULTS
Parasites that were isolated in this study were previously identified by zymodeme analysis as either L. major or L. tropica.
Nested PCR amplification
The 380 bp fragments that were amplified from the rRNA gene were observed in all the 76 samples (Fig. 1).
Fig. 1. Representative samples of the PCR amplification product of the rRNA gene of 380 bp specific for Leishmania spp. Lanes 1, 2, positive controls; Lanes 3–5, Leishmania major isolates; Lanes 6, 7, L. tropica isolates; Lane M, DNA molecular size marker.
Restriction enzyme analysis
All of the PCR products of the 76 samples were digested with two of the three restriction enzymes used, Msp I, and Taq I. This indicates that all of the DNA fragments amplified had the restriction sites for these two endonucleases. All the samples had simple riboprint patterns containing from 1–3 well-resolved fragments. Faint bands were not taken into account because they were not consistently found in different runs.
Samples digested with Msp I endonuclease showed 2 different restriction patterns, one with L. major isolates and one with L. tropica isolates. Three fragments, 175, 130, and 75 bp, were observed in all L. major isolates. Only the 2 smaller fragments, 75, and 130 bp, were observed in L. tropica isolates (Fig. 2). Few exceptional variations from the species profile were observed, using the endonuclease Msp I. In the profiles of L. major strains, which had been previously typed as being identical by enzyme electrophoresis, 3 of the isolates tested in this study showed slightly different banding profiles with Msp I restriction enzyme (data not shown).
Fig. 2. Representatives of the PCR product of Leishmania spp. digested with Msp I restriction endonuclease, Lane M, molecular size marker; Lanes 1–5, L. major isolates showing 3 fragments of 175, 130, and 75 bp, Lane 6, L. tropica isolate showing the 130, and 75 bp fragments only; Lane 7, positive control for L. major.
Digestion with Taq I endonuclease resulted in 1 pattern in all Leishmania isolates. The 2 fragments, 180 and 200 bp, were observed in isolates of both Leishmania spp. (Fig. 3). No digestion with Hae III endonuclease was observed in any of the DNA samples. L. donovani samples were not digested by Msp I (e.g. uncut band was observed). The same digestion product was generated in the repeated reactions for each DNA, which confirms the reproducibility and the stability of the electrophoretic patterns. The restriction patterns were the same in samples having different zymodemes. Geographical distribution did not affect the ribotype pattern.
Fig. 3. The genus restriction pattern resulted from the digestion of the PCR product with Taq I restriction endonuclease, 2 fragments of 180 and 200 bp appeared in digest of both Leishmania spp. Lane M, molecular size marker; Lanes 1, 2 are L. major, Lanes 5, 6, L. tropica; Lanes 3, 4, positive control for L. major and L. tropica respectively.
DISCUSSION
Classical morphology-based methods of identification or taxonomic and phylogenetic analysis are inadequate in many groups of structurally simple but similar eukaryotes as in the case with Leishmania spp. Riboprinting is one of several molecular methods that can generate comparative data independently of the simplicity or complexity of the organism's morphology (Clark, 1997). The utility of the method is illustrated with examples from several genera of intestinal and blood parasites. Among the applications of riboprinting are the detection of cryptic genetic variations within species, organism misidentifications and the rapid generation of data useful in phylogenetic analyses. (Clark, 1997). Some restriction patterns might support genetic exchanges in members of the genus Leishmania (Minodier et al. 1997).
In the present study, first a fragment of the rRNA gene was amplified by nested PCR followed by restriction fragment length polymorphism analysis (RFLP). The digestion product of the two restriction enzymes Msp I and Taq I gave simple restriction patterns. The Msp I digestion produced 2 different patterns that can easily distinguish L. major from L. tropica. The restriction pattern produced for both Leishmania spp. showed 2 fragments of 75 bp and 130 bp, a third 175 bp fragment was only produced in isolates previously identified by zymodeme analysis as L. major. The restriction patterns of both species could be distinguished by the number and size of the fragments compared with known reference strains for both Leishmania spp. The homogenous riboprints of the samples of both Leishmania species might indicate a single parasite clone that spread in these endemic foci.
The Taq I digestion produced a genus pattern for all Leishmania spp. analysed, showing 2 prominent bands of 180 and 200 bp. A genus-specific restriction pattern was previously reported for Pvu II (Uliana et al. 1991) where a specific site was detected in the 5′ domain of 18S ribosomal DNA of Leishmania (Uliana et al. 1991). The restriction patterns produced in the current study by both endonuclease were fully reproducible on replicate DNA. In addition, the nested PCR used in the present study detected extremely small amounts of Leishmania parasites directly in skin scrapings.
No digestion was observed in the partial or complete digestion with Hae III endonuclease indicating the absence of the restriction site of the enzyme in the rRNA sequence amplified. The Hae III restriction endonuclease analysis of kinetoplast DNA was used to distinguish between morphological forms resembling Leishmania spp. from L. tropica and L. major (Mikhail et al. 1996).
Results from enzyme electrophoresis confirmed the species identification made by restriction enzyme analysis of rRNA. All the Leishmania isolated from different foci of cutaneous leishmaniasis with few exceptions displayed similar patterns. Further experiments were performed using the enzyme Taq I on the same isolates. The results show that there is a single restriction pattern for both Leishmania spp. This indicates that the Taq I restriction sites are located in the conserved region of the rRNA gene in these two close species.
The isolates that gave new variants in the isoenzyme analysis showed same restriction pasterns. This might indicate that the genetic variations to identify new strains are not located in the amplified region.
RFLP analysis of L. major and L. tropica by the endonucleases Msp I proved to be useful in distinguishing between the 3 species of Leishmania that are prevalent in the same endemic foci. Our results are in agreement with a previous study (Lopes et al. 1984), using RFLP–PCR, on closely related parasites belonging to the Leishmania species tested in the current study could be easily identified directly following agarose gel electrophoresis by the simple restriction enzyme patterns of their PCR products. The PCR-fingerprinting using restriction analysis of the ITS region in the ribosomal operon was reported to display the highest discriminatory power over other PCR-based methods in the ability to discriminate strains of L. tropica (Schonian et al. 2001).
In a study of the epidemiology of L. tropica, a nested PCR-based schizodeme method was applied for identifying Leishmania kinetoplast minicircle classes directly from clinical samples (Noyes et al. 1998). The complex fingerprint patterns prepared from the clinical samples were not fully reproducible on replicate DNA preparations from the same clinical sample. If this fingerprinting method is to be used in epidemiological studies, at least 3 replicates from each isolate would be necessary to confirm that the fragments included in the analysis were reproducible. In contrast, the riboprint patterns in our study, produced from the same clinical sample were simple and reproducible.
The use of the 16S rRNA gene as the target for PCR and RFLP-based identification of Leishmania takes advantage of the sequence information on this 16S region readily available in GenBank and other sequence databases. Thus, a comparison of the16S rRNA sequences of unusual patient isolates with the well-characterized genomes included in the databases is possible. The results of this study demonstrate the utility of the riboprinting approach for the characterization and identification of Leishmania species directly from clinical specimens.
The study was financially supported by grant #172/2000 from the Deanship of Research at Jordan University of Science and Technology.
References
REFERENCES
ANDERSEN, K.,
IBRAHIM, M. E.,
THEANDER, T. G. &
KHARAZMI, A.
(1996).
Random amplified polymorphic DNA for thee differentiation of Leishmania donovani isolates from Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene
90, 204–205.CrossRefGoogle Scholar
BARKER, D. C.
(1987).
DNA diagnosis of human leishmaniasis.
Parasitology Today
3, 177–184.CrossRefGoogle Scholar
BARKER, D. C.,
ARNOT, D. E. &
BUTCHER, J.
(1982).
DNA characterization as a taxonomic tool for identification of kinetoplastid flagellate protozoans. In
Biochemical Identification of Leishmania (
ed. Chance, M. L. &
Walton, B. C.),
Proceedings of a Workshop held at the Pan American Health Organization, PAHO, Washington, D.C., 9–11 December 1980,
pp. 139–180.
UNDP/World Bank/WHO Special Programme for Research and Training in Tropical diseases, Geneva, Switzerland.
BELLI, A.,
RODRIGUEZ, B.,
AVILES, H. &
HARRIS, A.
(1998).
Simplified PCR detection of New World Leishmania from clinical specimens.
American Journal of Tropical Medicine and Hygiene
58, 102–109.CrossRefGoogle Scholar
CHANCE, M. L.
(1979).
The identification of Leishmania. In
Problems in the Identification of Parasites and their Vector (
ed. Taylor, A. E. R. &
Muller, R.).
Blackwell Scientific Publications, Oxford.
CHANCE, M. L. &
WALTON, B. C.
(1982).
Biomedical characterization of Leishmania.
Proceedings of a Workshop held at the Pan American Health Organization. Washington, D.C., 9–11 December, 1980, pp. 280. UNDP/World Bank/WHO Special Programme for Research and Training in Tropical diseases, Geneva, Switzerland.
CLARK, C. G.
(1997).
Riboprinting: a tool for the study of genetic diversity in microorganisms.
Journal of Eukaryotic Microbiology
44, 277–283.CrossRefGoogle Scholar
DUJARDIN, J. C.,
LANOS-CUENTAS, A.,
CACERES, A.,
ARANA, M.,
DUJARDIN, J. P.,
GUERRINI, F.,
GOMEZ, J.,
ARROYO, J.,
DE DONCKER, S.,
JACQUET, D.,
HAMERS, R.,
GUERRA, H.,
LE RAY, D. &
AREVALO, J.
(1993).
Molecular karyotype variation of Leishmania (Viannia) peruviana evidences geographical population in Peru along a north-south cline.
Annals of Tropical Medicine and Parasitology
87, 335–347.CrossRefGoogle Scholar
EVANS, D. A.
(1987).
Leishmania. In
In vitro Methods for Parasite Cultivation (
ed. Taylor, A. E. R. &
Baker, J. R.),
pp. 58–59.
Academic Press, London and New York.
FERNANDES, O.,
MURTHY, V.,
KURATH, U.,
DEGRAVE, W. &
CAMPBELL, D.
(1994).
Mini-exon gene variation in human pathogenic Leishmania species.
Molecular and Biochemical Parasitology
66, 261–271.CrossRefGoogle Scholar
GARDENER, P. J.
(1977).
Taxonomy of the genus Leishmania. A review of nomenclature and classification.
Bulletin of Tropical Diseases
74, 1069–1088.Google Scholar
GONCALVES, A. M.,
NEHME, N. S. &
MOREL, C. M.
(1984).
Trypanosomatid characterization by schizodeme analysis. In
Genes and Antigens of Parasites
(ed. Morel, C. M.),
pp. 95–109.
Fundacao Oswaldo Cruz,
Rio de Janeiro.
GRIMALDI, G. Jr. &
TESH, R. B.
(1993).
Leishmaniases of the New World: current concepts and implications for future research.
Clinical Microbiology Reviews
6, 230–250.CrossRefGoogle Scholar
IOVANNISCI, D. M. &
BEVERLY, S. M.
(1989).
Structural alterations of Chromosome 2 in Leishmania major as evidence for diploidy, including spontaneous amplification of mini-exon array.
Molecular and Biochemical Parasitology
34, 177–188.CrossRefGoogle Scholar
LEON, W.,
FOUTS, D. L. &
MANNING, J.
(1978).
Sequence arrangement of the 16S and 26S genes in the pathogenic hemoflagellate Leishmania donovani.
Nucleic Acids Research
5, 491–504.CrossRefGoogle Scholar
LONG, E. O. &
DAWID, I. B.
(1980).
Repeated genes in eukaryotes.
Annual Reviews of Biochemistry
49, 727–764.CrossRefGoogle Scholar
LOPES, U. G.,
MOMEN, H.,
GRIMALDI, G. Jr.,
MARZOCHI, M.,
PACHECO, R. &
MOREL, C. M.
(1984).
Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral and cutaneous leishmaniasis.
Journal of Parasitology
70, 89–98.CrossRefGoogle Scholar
MINODIER, P.,
PIARROUX, R.,
GAMBARELLI, F.,
JOBLET, C. &
DUMON, H.
(1997).
Rapid identification of causative species in patients with Old World leishmaniasis.
Journal of Clinical Microbiology
35, 2551–2555.Google Scholar
MIKHAIL, E. M.,
MANSOUR, N. S.,
MOHAREB, E. W. &
FRANCIES, W. M.
(1996).
Identification of a misleading trypanosomatid parasite from Gerbillus pyramidum and G. andersoni in a Leishmania major endemic area in north Sinai.
Journal of Parasitology
82, 400–404.Google Scholar
NIMRI, L.,
SOUBANI, R. &
GRAMICCIA, M.
(2002).
Leishmania species and zymodemes isolated from endemic areas of cutaneous leishmaniasis in Jordan.
Kinetoplastid Biology and Disease
1, 7.CrossRefGoogle Scholar
NOYES, H.,
REYBURN, H.,
BAILY, J. W. &
SMITH, D.
(1998).
A nested PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan.
Journal of Clinical Microbiology
36, 2877–2881.Google Scholar
PETERS, W. &
KILLICK-KENDRICK, R.
(1987).
The Leishmaniases in Biology and Medicine, Vol. 1.
Academic Press, London and New York.
SCHALLIG, H. D. F. H. &
OSKAM, L.
(2002).
Review: Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification.
Tropical Medicine and International Health
7, 641–651.CrossRefGoogle Scholar
SCHONIAN, G.,
SCWEYNOCH, C.,
ZLATEVA, K.,
OSKAM, L.,
KROON, N.,
GRASER, Y. &
PRESBER, W.
(1996).
Identification and determination of the relationship of species and strains within the genus Leishmania using single primers in the polymerase chain reaction.
Molecular and Biochemical Parasitology
77, 19–29.CrossRefGoogle Scholar
SCHONIAN, G.,
SCHNUR, L.,
EL FARI, M.,
OSKAM, L.,
KOLESNIKOV, A. A.,
SOKOLOWSKA-KOHLER, W. &
PRESBER, W.
(2001).
Genetic heterogeneity in the species of Leishmania tropica revealed by different PCR-based methods.
Transactions of the Royal Society of Tropical Medicine and Hygiene
95, 217–224.CrossRefGoogle Scholar
SPITHILL, T. W. &
GRUMONT, R. J.
(1984).
Identification of species, strains and clones of Leishmania by characterization of kinetoplastid DNA minicircles.
Molecular and Bichemical Parasitology
12, 217–236.CrossRefGoogle Scholar
ULIANA, S. R.,
AFFONSO, M. H.,
CAMARGO, E. P. &
FLOETER-WINTER, L. M.
(1991).
Leishmania: genus identification based on a specific sequence of the 18S ribosomal RNA sequence.
Experimental Parasitology
72, 157–163.CrossRefGoogle Scholar
Fig. 1. Representative samples of the PCR amplification product of the rRNA gene of 380 bp specific for Leishmania spp. Lanes 1, 2, positive controls; Lanes 3–5, Leishmania major isolates; Lanes 6, 7, L. tropica isolates; Lane M, DNA molecular size marker.
Fig. 2. Representatives of the PCR product of Leishmania spp. digested with Msp I restriction endonuclease, Lane M, molecular size marker; Lanes 1–5, L. major isolates showing 3 fragments of 175, 130, and 75 bp, Lane 6, L. tropica isolate showing the 130, and 75 bp fragments only; Lane 7, positive control for L. major.
Fig. 3. The genus restriction pattern resulted from the digestion of the PCR product with Taq I restriction endonuclease, 2 fragments of 180 and 200 bp appeared in digest of both Leishmania spp. Lane M, molecular size marker; Lanes 1, 2 are L. major, Lanes 5, 6, L. tropica; Lanes 3, 4, positive control for L. major and L. tropica respectively.
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