First detection of Leishmania major in peridomestic Phlebotomus papatasi from Isfahan province, Iran: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA
Introduction
In the central Iranian province of Isfahan, the haematophagous females of Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) have been incriminated as the principal vectors of the parasitic protozoan Leishmania major (Yakimoff and Schokhor) (Kinetoplastida: Trypanosomatidae), the causative agent of zoonotic cutaneous leishmaniasis (ZCL) (Nadim et al., 1968, Yaghoobi-Ershadi et al., 1995, Yaghoobi-Ershadi and Javadian, 1996). Other species of phlebotomine sandflies occur in the burrows of the main reservoir host, the gerbil Rhombomys opimus (Licht.) (Rodentia: Gerbillidae) (Nadim and Faghih, 1968, Yaghoobi-Ershadi et al., 1996), and some of these have been found infected with L. major (Yaghoobi-Ershadi et al., 1994) and unidentified Leishmania-like leptomonads (Nadim et al., 1968, Yaghoobi-Ershadi et al., 1996). Nevertheless, most Leishmania-like infections have been detected in P. papatasi (Yaghoobi-Ershadi et al., 1996), which is likely to be the main vector in Isfahan province because of its anthropophily and predominance in gerbil burrows as well as in peridomestic sites (Yaghoobi-Ershadi et al., 1996, Parvizi et al., 2003). However, no infections have been reported from P. papatasi collected in peridomestic sites in Iran, which is the subject of this report.
Isoenzymes provide the gold-standard characters for identifying species and reference strains of Leishmania (Anon., 1990, Rioux et al., 1990), and this method was used by Yaghoobi-Ershadi et al., 1994, Yaghoobi-Ershadi et al., 1995 to identify single infections of the same strain (zymodeme MON26) of L. major in P. papatasi and P. caucasicus captured in the Borkhar focus of ZCL 30–48 km north of Isfahan city. However, isoenzyme characterization has the disadvantage of requiring the culture of large numbers of parasites, and primary isolates can easily become contaminated or, in mixed infections, yield only the strain that grows fastest in laboratory conditions. Consequently, a wealth of molecular techniques have been developed to permit the identification of small numbers of Leishmania parasites in primary infections (Alvar and Barker, 2002). A few of these techniques have been used to identify Leishmania in sandflies, including hybridization of probes of repetitive DNA to squash-blots of sandflies or dot blots of their DNA (Ready et al., 1988, Gebre-Michael et al., 1993) and the amplification by the polymerase chain reaction (PCR) of fragments of repetitive DNA with diagnostic sizes or sequences (Feliciangli et al., 1994, Aransay et al., 2000, Reithinger and Davies, 2002, Rodriguez et al., 2002).
Based on a literature search, we selected two PCR methods that had shown high sensitivity or specificity for detecting and identifying Leishmania and, in the present report, both methods were assessed for their ability to identify L. major in Iranian P. papatasi. The semi-nested PCR described by Aransay et al. (2000) targets the minicircles of kinetoplast (k) DNA of Leishmania. This test is very sensitive for detecting Leishmania, partly because there can be about 10,000 minicircles per kinetoplast (Barker, 1980), but it should not always provide easily detectable markers for Leishmania strains, because each cell (or clone) can contain different classes or ‘species’ of minicircle kDNA (Reviews: Brewster and Barker, 2002, Lambson and Barker, 2002). In contrast, most Leishmania cells contain only hundreds of tandemly repeated nuclear ribosomal genes (rDNA), which should lessen the sensitivity of PCR detection. However, unlike minicircle kDNA, most rDNA copies are believed to be homogeneous (Hillis et al., 1991), which favours the fixation of strain-specific markers. Within each rDNA repeat there are two internal transcribed spacers (ITS), located between the small subunit (SSU) and large subunit (LSU) rRNA genes. ITS1 and ITS2 are separated by the 5.8S rRNA gene, and both provide species-specific sequence markers that have been most frequently detected by Restriction Fragment Length Polymorphism (RFLP) analysis of one-step PCR products (Cupolillo et al., 1995, Schönian et al., 2001, Mauricio et al., 2004). In the current paper, we used a nested PCR that should improve the sensitivity of amplifying the ITS of rDNA.
Section snippets
Culture of reference strains of Leishmania
Reference strains of four Old World species of the subgenus Leishmania were used: L.major MHOM/SU/73/5ASKH, Leishmania tropica MHOM/SU/74/K27, Leishmania infantum MHOM/TN/80/IPTI and Leishmania donovani MHOM/IN/80/DD8. They were stored in liquid nitrogen and resurrected on biphasic Difco blood-agar medium or in liquid MEM:FCS:EBLB medium (Evans, 1989). Promastigotes were grown at 23 °C, and the cultures were examined daily until they had reached late log phase.
Preparing cell suspensions and extracting DNA from quantified numbers of promastigotes of Leishmania
A Neubauer Chamber (an
Specificity and sensitivity of the semi-nested PCR of minicircle kDNA for identifying cultured L. major
The semi-nested PCR amplifies most of each minicircle kDNA molecule and, as reported (Aransay et al., 2000), the size of the fragment was diagnostic for L.major (ca. 650 bp) compared with the three other species of Leishmania (ca. 720 bp) that most frequently infect humans in Eurasia. Size specificity was 100% for 123 fragments of L.major (Table 1, Table 2), compared with seven fragments of L.donovani, two fragments of L.infantum and three fragments of L.tropica (Table 1). At least two
Relative specificity and sensitivity of the semi-nested PCR and the nested PCR for identifying cultured L. major
Both PCR tests have 100% specificity based on sequencing the amplified DNA fragments (Brewster et al., 1998, Schönian et al., 2001), but the semi-nested PCR of minicircle kDNA has the practical advantage of being diagnostic for the size of the amplified DNA fragment (Aransay et al., 2000).
Using DNA extracted from cultured cells of L.major (Table 2, Table 3), the sensitivity of both PCR tests depended on the method of DNA extraction, being significantly better for DNA extracted by the Leishmania
Acknowledgements
We thank Dr. M. Taghikhni (Director) and Dr. M. Assmar (Head of Parasitology Department) of the Pasteur Institute of Iran for facilitating the collections of sandflies, which were assisted by the Centre of Health Services, Isfahan Province. Julia Llewellyn-Hughes and Johann Testa helped with DNA sequencing. This work was supported by a Ph.D. scholarship awarded to Mr. P. Parvizi by the Ministry of Health & Medical Education, Iran.
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Present address: Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.