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Am. J. Trop. Med. Hyg., 72(4), 2005, pp. 415-420
Copyright © 2005 by The American Society of Tropical Medicine and Hygiene

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EVALUATION OF 7SL RNA GENE SEQUENCES FOR THE IDENTIFICATION OF LEISHMANIA SPP.

ADRIAN M. ZELAZNY, DANIEL P. FEDORKO, LI LI, FRANKLIN A. NEVA, AND STEVEN H. FISCHER
Microbiology Service, Department of Laboratory Medicine, Clinical Center, and Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We evaluated the use of 7SL RNA gene sequences for the identification of Leishmania spp. A fragment (~137 basepairs) of the 7SL RNA gene from 13 reference strains and 18 clinical isolates of 11 different Leishmania species was amplified and sequenced using conserved primers. Reference strains from each Leishmania spp. complex showed unique sequences. The nucleotide sequences were compared pairwise and a range of 81.0–99.3% intercomplex similarity was observed. Clinical isolates of the same species had sequences identical to the corresponding reference strains; thus, the intraspecies similarity was 100%. A phylogenetic tree derived from the 7SL RNA gene partial sequences was constructed and is in agreement with accepted phylogenetic schemes.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leishmaniasis is an important public health problem in tropical and subtropical countries worldwide. Symptoms range from self-healing cutaneous lesions (caused by different species) to persistent and disfiguring cutaneous and mucocutaneous manifestations (Leishmania braziliensis complex) and the potentially fatal visceral disease (L. donovani complex). Cutaneous leishmaniasis may exhibit signs that are very similar to other diseases presenting with nodules or ulcers, and thus laboratory confirmation is required.1 Leishmania major, L. tropica, L. aethiopica, and rarely L. donovani infantum cause cutaneous disease in the Old World. New World cutaneous leishmaniasis is caused by members of the L. mexicana, L. braziliensis, and L. guyanensis complexes. It is important to identify the complex and/or species of Leishmania for both clinical and epidemiologic reasons. Identification of Leishmania based on clinical presentations can be problematic, since several complexes and/or species cause both cutaneous, and diffuse cutaneous or mucocutaneous disease (L. aethiopica and L. braziliensis complex), while others cause both visceral and cutaneous disease (L. donovani complex). Leishmania identification by geographic origin is inadequate in areas not endemic for this disease, as well as in disease-endemic regions where multiple complexes and/or species of Leishmania co-exist. Moreover, reports indicate that the response to therapeutic drugs can vary among different species present in the same area.2,3

Traditional methods for diagnosis of leishmaniasis include direct microscopic examination of clinical specimens and culture. However, complex and species-specific diagnosis is not possible by microscopy or culture alone. Isoenzyme analysis4 is the current gold standard for the differentiation of Leishmania species, but it is a lengthy and technically demanding procedure. With the advent of polymerase chain reaction (PCR) technology, several PCR-based assays were developed for direct detection and/or for identification of Leishmania spp. with varying specificities and species discrimination capabilities. Targets for amplification have included the ribosomal RNA (rRNA) gene,5,6 the mini-exon gene,7 repetitive nuclear DNA sequences,8,9 the glucose-6-phosphate dehydrogenase gene,10 internal transcribed spacer (ITS) regions,11,12 and extrachromosomal DNA, such as the kinetoplast DNA.13,14 Although these approaches offer valid taxonomic data for species identification, some markers such as rDNA exhibit a high degree of sequence similarity among different species,5 while others such as ITS regions show a high degree of molecular polymorphism.15 Thus, there is still a need for a simple and comprehensive assay that can be used for the identification of Leishmania isolates at the species or complex level.

In eukaryotes, protein translocation across the endoplasmic reticulum is mediated by the signal recognition particle, an essential ribonucleoprotein complex formed by six proteins and one RNA molecule, the 7SL RNA.16 Work carried out on two trypanosomatids, Trypanosoma brucei17 and Leptomonas collosoma,18 demonstrated in each the presence of an abundant RNA homolog of 273 and 280 nucleotides, respectively, that resembles structurally the mammalian 7SL RNA. A putative 7SL RNA gene has been also identified in L. major (Leishmania major Friedlin Genome Project, http://www.sanger.ac.uk/Projects/L_major/). The purpose of our work was to evaluate the use of 7SL RNA gene sequences for the identification of Leishmania spp. This target was chosen for several reasons: 1) 7SL RNA molecules are abundant in the cell,17,19 2) although both the trypanosomatid and the mammalian 7SL RNA fit a canonical secondary structure, their sequences are not very similar (i.e., only 60% similarity between mammalian and T. brucei 7SL RNA),17 and 3) the flanking domains I and IV of the trypanosomatid 7SL RNA are highly conserved, while the central domain III is divergent.20 These characteristics make 7SL RNA a potentially good target for a sensitive assay designed to detect and differentiate Leishmania spp. Here, we show a simple PCR scheme for the differentiation of Leishmania spp. based on partial sequencing of the 7SL RNA gene. A phylogenetic analysis derived from these sequences is also presented.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reference strains and clinical isolates. The sources of 13 Leishmania reference strains and 18 clinical isolates, two Crithidia spp., and two T. cruzi isolates used in this study are listed in Table 1Go. Reference strains, which were obtained from American Type Culture Collection (Manassas, VA), as well as all clinical isolates, were grown on either NNN medium containing rabbit blood agar overlaid with RPMI 1640 medium21 (Gibco-BRL, Gaithersburg, MD) or complete M199 medium containing 20% fetal calf serum.22 DNA was obtained from 2 mL of stationary phase culture of parasites that had been washed twice in 0.85% NaCl (Sigma, St. Louis, MO) and extracted with a NucliSens kit (bioMerieux Inc., Durham, NC) according to the manufacturers recommendations. Samples obtained from the London School of Hygiene and Tropical Medicine consisted of DNA purified at this reference center by phenol/chloroform extraction and isopropanol precipitation.23 All clinical isolates were identified by isoenzyme typing, which was performed by Richard D. Kreutzer (Youngstown State University, Youngstown, OH), as previously described.24


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TABLE 1
Reference and clinical isolates used in this study
 
Primer design and PCR. Two conserved regions were identified after aligning Leptomonas collosoma (GenBank accession no. AF006750) and T. brucei (GenBank accession no. M80262.1) 7SL RNA gene sequences, and L. major putative 7SL RNA gene sequences (GenBank accession no. AL354512.3, nucleotides 23430-23157). These regions were used to design two primers (TRY7SL.For1 and TRY7SL. Rev1), which correspond to L. major putative 7SL RNA gene positions 12–33 and 171–195, respectively. These primers were tailed with M13 sequencing primer sites to facilitate subsequent sequencing of a ~137-basepair fragment of the 7SL RNA gene. Primers TRY7SL.For1.M13 (5'-GTA AAA CGA CGG CCA GTG CTC TGT AAC CTT CGG GGG CT-3') and TRY7SL.Rev1.M13 (5'-CAG GAA ACA GCT ATG ACG GCT GCT CCG TYN CCG GCC TGA CCC-3') were commercially synthesized (Midland Certified Reagent Company, Midland, TX). The M13 tail sequences are shown in bold.

The PCR amplification with primers TRY7SL.For1.M13 and TRY7SL.Rev1.M13 generated a product of 137, 138, or 139 basepairs (excluding the primers). The PCRs were performed in a PerkinElmer (Wellesley, MA) 9600 Thermocycler with a reaction mixture containing 2.5 mM MgCl2, 1x Light-Cycler-Fast Start DNA Hybridization Probes (Roche, Indianapolis, IN), 1 pmol of forward primer, 1 pmol of reverse primer, 3 µL of extracted DNA, and ultra pure water to give a final volume of 25 µL. The PCR thermocycling program consisted of an initial step at 95°C for 5 minutes, followed by 34 cycles at 95°C for 1 minute, 65°C for 1 minute, and 72°C for 1 minute, and a final incubation at 72°C for 10 minutes. The PCR products were visualized by ultraviolet illumination of an ethidium bromide-stained 1.5% agarose gel following electrophoresis. The purification of the remaining PCR product was achieved with Microcon-100 microconcentrators (Millipore, Billerica, MA) by following the manufacturer’s instructions.

Sequencing of DNA. The ABI Prism BigDye Terminator v1.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) was used for the sequencing of the PCR product. The sequencing reaction mixture contained 4 µL of Big Dye premixture, 0.5x buffer, 3.2 pmol of sequencing primer, and approximately 150 ng of PCR product template in a total volume of 20 µL. The following M13 primers were used for sequencing: M13 Forward, 5'-GTAAAA-CGACGGCCAG-3' and M13 Reverse, 5'-CAGGAAA-CAGCTATGAC-3'. The sequencing reactions and template preparation were performed in accordance with the instructions of the manufacturer. Sequencing products were purified with CleanSEQ Sequencing Reaction Clean-Up system (Agencourt, Beverly, MA) and analyzed using the 3100 Genetic Analyzer (Applied Biosystems), following the manufacturer’s recommendations.

The Lasergene program (version 5.51; DNASTAR Inc., Madison, WI) was used for sequence assembly and alignment. Multiple sequence alignment of 7SL RNA sequences was done by the CLUSTAL W method.25 Phylogenetic analyses were performed with the PHYLIP version 3.5c package.26 Distance matrices based on Kimura’s two-parameter model27 were produced with the DNADIST program, and a neighbor-joining tree constructed with the NEIGHBOR program. Bootstrapping (1,000 replicates) was performed to assess the stability of the grouping using SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE programs. The resulting trees were depicted by using the TreeView version 1.4 package.28

Leishmanial 7SL RNA sequences determined in the present study were deposited in GenBank under accession numbers AY722713 to AY722735 and AY781789 to AY781796. Crithidial 7SL RNA partial sequences were deposited in GenBank under accession numbers AY781797 and AY781798.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Primers TRY7SL.For1.M13 and TRY7SL.Rev1.M13 were used to amplify a fragment of the 7SL RNA gene from 13 reference strains of Leishmania spp., as well as 18 clinical isolates grown mostly from cutaneous samples. The nucleotide sequences of amplified DNA segments were determined and compared pairwise. As shown in Figure 1Go, the amplified products from L. major, L. mexicana, L. tropica, and L aethiopica comprised 137 nucleotides (excluding the primers). Insertions of one and two bases in the sequence were observed in L. donovani complex and the members of the subgenus Viannia, L. (Viannia) braziliensis and L. (V) guyanensis, respectively.



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  		    FIGURE 1. Alignment of 7SL RNA sequences from clinically relevant Leishmania spp. Dots indicate identity with the Leishmania major sequence. Since all isolates belonging to the same species had an identical 7SL RNA sequence, only one sequence for each species is shown. V = Viannia.

 
Primers TRY7SL.For1.M13 and TRY7SL.Rev1.M13 also amplified a 138-basepair fragment of the 7SL RNA gene from reference strains of Crithidia fasciculata and C. luciliae. Although we expected these primers to amplify the same segment of the 7SL RNA gene in T. cruzi, only spurious bands were generated upon performing the assay with two reference strains.

Pairwise comparison of the leishmanial nucleotide sequences by the Clustal W method showed a range of 81.0–99.3% intercomplex similarity. Species belonging to different complexes were easily differentiated from one another; however, species within the L. donovani complex (L. donovani, L. infantum/chagasi) showed identical sequences. Most importantly, all reference strains and clinical isolates belonging to the same species had identical 7SL RNA sequences; thus, the intraspecies similarity was 100%. Identification of the clinical isolates by 7SL RNA sequences was in agreement with the results previously obtained by isoenzyme typing. Members of the subgenus Viannia, L. (V) braziliensis and L. (V) guyanensis, had one base difference that was consistent among all isolates tested. Within the L. mexicana complex, the clinical isolate of L. amazonensis displayed one base difference from the closely related species L. mexicana.

A phylogenetic tree of the reference strains and clinical isolates sequenced was constructed by the neighbor-joining method, using the T. brucei 7SL RNA gene (GenBank accession no. M80262.1) as the outgroup (Figure 2Go). A clear division between the two subgenera Leishmania and Viannia was observed. Within the subgenus Leishmania, the Old World species were separated from the L. mexicana complex that is found only in the New World. The recognized species complexes of the Old World (L. donovani, L. tropica, L. aethiopica, and L. major) were all clearly separated.



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  		    FIGURE 2. Phylogenetic tree of 31 reference and clinical isolates of Leishmania spp. and two reference strains of Crithidia (C.) (spp. (Table 1Go) constructed by the neighbor-joining method, using the Trypanosoma brucei 7SL RNA gene (GenBank accession no. M80262.1) as the outgroup. Numbers on the branches represent the percentage of 1,000 bootstrap samples supporting the branch. Only values > 50% are shown. ci = clinical isolate; ATTC = American Type Culture Collection; V. = Viannia.

 
The non-pathogenic species L. tarentolae emerged as an independent branch separated from the pathogenic Old-World Leishmania spp. and the L. mexicana complex. The non-pathogenic C. fasciculata and C. luciliae clustered together as an independent branch separated from the subgenus Viannia. In addition, the two Crithidia spp. could be differentiated from one another.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Correct identification of the etiologic agent of leishmaniasis is important for clinical and epidemiologic reasons. Unfortunately, species identification by the gold standard method, isoenzyme analysis, is technically challenging and not easy to implement in the clinical laboratory. We propose here a simple assay based on partial sequences (~137 basepairs) of the 7SL RNA gene for the identification of Leishmania at the species or complex level. Primers TRY7SL.For1.M13 and TRY7SL.Rev1.M13 amplified the expected product from all 31 strains of Leishmania tested. No amplification was observed when the assay was performed with a control sample of human genomic DNA or total DNA extracted from samples of human stools. Although the primer sequences were derived from conserved sequences based on data available in GenBank on three members of the family Trypanosomatidae (including T. brucei), no amplification of the 7SL RNA gene was observed with two reference strains of T. cruzi, even after repeated attempts under different cycling conditions.

The partial sequences of the 7SL RNA gene from all 31 Leishmania isolates was determined and compared pairwise for similarity. As shown in Figure 1Go, the amplified 7SL RNA sequence from L. major, L. mexicana, L. tropica, and L aethiopica contained 137 nucleotides (excluding the primers). The L. donovani complex and the members of the subgenus Viannia, L. (V) braziliensis and L. (V) guyanensis, had one and two single base insertions, respectively. One remarkable feature was the presence of identical sequences in all species for which we had multiple isolates (intraspecies similarity = 100%). The absence of intraspecies polymorphisms reported in other targets9,15 makes the identification of clinical isolates by this method straighforward.

Although the 18S gene of rDNA has been widely used to explore sequence phylogenies in the Kinetoplastida,29,30 the small inter-species variability among Leishmania species (> 99% identical bases) does not allow for extensive species discrimination and unambiguous phylogenetic inferences.5 In contrast, comparison of 7SL RNA sequences among different species showed a range of 81.0–100% interspecies similarity, thus allowing better species discrimination.

Alignment of Leishmania 7SL RNA partial sequences showed seven variable regions (Figure 1Go). Assuming a secondary structure similar to that of the trypanosomatid Leptomonas collosoma,20 all variable regions were located at (or next to) putative loops. A particularly hypervariable region of Leishmania 7SL RNA (bases 105–119; Figure 1Go) was deduced to be in domain III. This domain has previously been reported to be divergent also between T. brucei and Leptomonas collosoma.20

A phylogenetic tree of the 7SL RNA partial sequences from the reference strains and clinical isolates was constructed by the neighbor-joining method (Figure 2Go). Despite the small size of the partial sequences (~137 basepairs), the separation between subgenera Leishmania and Viannia and the relative position of each species within the tree was similar to that previously reported with DNA polymerase31 and ITS-5.8S rDNA32 using 924-bp and 1512-bp sequences, respectively. Interestingly, the 7SL RNA partial sequences also allowed the differentiation of the two species of Crithidia tested from one another and from all Leishmania spp. Although this study focused mainly on clinically relevant Leishmania spp., it would be interesting to sequence the 7SL RNA gene from additional members of the family Trypanosomatidae for phylogenetic purposes.

In conclusion, PCR amplification and sequencing of a short fragment of the 7SL RNA gene allowed the unequivocal identification of Leishmania isolates to the species or complex level. Future work will focus on amplification and sequencing of 7SL RNA from other (less common) species of cutaneous Leishmania, and the development and evaluation of a real-time PCR assay for direct testing of clinical samples.

Received August 26, 2004. Accepted for publication November 24, 2004.

Acknowledgments: We thank Dennis M. Dwyer (Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health) for providing reference strains of L. tarentolae, C. fasciculata, and C. luciliae. We also thank Patrick R. Murray (Department of laboratory Medicine, Microbiology Service, Clinical Center, National Institutes of Health) for critically reviewing the manuscript.

Authors’ addresses: Adrian M. Zelazny, Daniel P. Fedorko, Li Li, and Steven H. Fischer, Microbiology Service, Department of Laboratory Medicine, Clinical Center, Building 10, Second Floor, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, Telephone: 301-496-4433, Fax: 301-402-1886, E-mails: azelazny{at}cc.nih.gov, dfedorko{at}cc.nih.gov, lli{at}cc.nih.gov and sfischer{at}cc.nih.gov. Franklin A. Neva, Opportunistic Diseases Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Building 4, Room B1-27, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, Telephone: 301-496-2486, Fax: 301-402-0079, E-mail: fneva{at}niaid.nih.gov.

Reprint requests: Steven H. Fischer, Microbiology Service, Department of Laboratory Medicine, Building 10, Room 2C-381B, National Institutes of Health 10 Center Drive, Bethesda, MD 20892.


REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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