MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 92(1) Jan/Feb 1997
PAGES: 109-114 DOI: Full paper
Comparison of Some Molecular-genetic Techniques for Identification of Leishmania Circulating in Natural Foci of Zoonotic Cutaneous Leishmaniasis in the Central Asia Region

Margarita V Strelkova, Raquel S Pacheco* +, Sergey A BulatII, Tatjana A RakitskayaIII

Martsinovsky Institute of Medical Parasitology and Tropical Medicine, Moscow, Russia
IDepartamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
IIInstitute of Nuclear Physics, St. Petersburg, Russia
IIIVavilov Institute of General Genetics, Moscow, Russia

Abstract

Different molecular-genetic methods were used to identify a cohort of Leishmania strains from natural foci of zoonotic cutaneous leishmaniasis located in Central Asia, on the former USSR territory. The results obtained using isoenzymes, PCR, restriction fragment length polymorphisms of kDNA and molecular hybridization techniques are discussed in terms of their applicability, discrimination power and feasibility for answering questions related to molecular epidemiological research and for detecting mixed Leishmania infections.

The natural foci of Leishmania major infection comprise the vast territory of the Turan plain and coincide with the distribution of the reservoir host Rhombomys opimus. According to Dubrovsky (1978), very active leishmaniasis foci are found in irrigation oases in Turkmenistan, Uzbekistan and Kazakhstan (former USSR). It has, until recently, been assumed that leishmanial parasites circulating in the R. opimus population are considered to be L. major, displaying a great range of virulence and infectivity for humans. However, the introduction of new techniques for Leishmania characterization indicated that the parasites formerly identified as L. major in these areas belong to a polytype group comprising three independent species namely L. major sensu stricto, L. turanica (Strelkova et al. 1990b) and L. gerbilli. In fact, the three species are able to coexist in a single gerbil (R. opimus). L. major and L. turanica are generally found together even in the same skin lesion. Each species has its own relatively limited range of virulence; while L. major is found to be pathogenic for humans, L. turanica and L. gerbilli are not (Strelkova et al. 1990a, Strelkova 1990).

All the above mentioned stress the necessity to look for feasible methods for Leishmania identification both in a single or in mixed infections. The present paper attempts to evaluate some phenotypic and genotypic techniques for this purpose and discusses their relationship to epidemiological studies.

 

MATERIALS AND METHODS

Parasite strains - The strains studied are listed in Table. The isolates were obtained from patients with cutaneous leishmaniasis, from great gerbils (R. opimus) and from a sandfly (Phlebotomus papatasi) in Turkmenistan, Uzbekistan and Kazakhstan.

Isolation of the parasites - Parasite isolations were made by injecting as much sterile saline as possible containing penicillin (250 U/ml) and streptomycin (250 mg/ml) intradermally into gerbils' affected ears. The saline was aspirated and inoculated into tubes containing NNN medium. Fragments of human cutaneous lesions were obtained using a sterile scarifier or a scalpel and transferred into tubes of NNN medium containing antibiotics as above.

The tubes were incubated at 22-24oC and examined weekly for the presence of promastigotes for up to six weeks. The positive cultures were cryopreserved in liquid nitrogen.

Cloning - Clones were made using a Fonbrune micromanipulator (Alekseev & Safjanova 1977). With this micromanipulator a single promastigote can be aspirated and released into a separate drop of medium under microscopic control.

Development in experimental animals - The pathogenicity of the Leishmania isolates was assessed in hamsters (Kelina 1982). The inoculum consisted of 7.5 x 104 promastigotes from early stationary phase cultures, injected intradermally into the ears. The hamsters were examined weekly during the first month and every two weeks during the five subsequent months with registration of clinical signs of leishmaniasis. Parasite cultures which produced progressive ulcerative lesions in the hamsters within six weeks of inoculation were classified as highly virulent and those which produced only infiltrative lesions within six months were classified as of low virulence. Some parasite cultures produced abortive ulcerative lesions during five or six months of observation after inoculation and were considered as of moderate virulence. On the other hand, some cultures were found to be avirulent causing no infection in hamsters.

Isoenzyme electrophoresis - Promastigotes were harvested from seven-day cultures in MEM:EBLB:FCS medium (Evans 1987), washed three times in saline solution and centrifuged at 4oC, 2.700 g for 10 min. Two volumes of 0.01 M aminocaproic acid were added to the sediment after the last washing and the cells were lysed by temperature shock. The lysates were centrifuged at 12,000 g for 20 min and the clear supernatants were analyzed by eletrophoresis. Leishmania isolates were identified by using the following marker strains: L. major(MRHO/SU/59/NEALP); L. turanica (MRHO/SU/83/KD51); L. gerbilli (MRHO/CN/60/GERBILLI). The electrophoresis analyses were carried out in polyacrylamide gels and a total of nine enzymes were used (PGI, PGM, 6PGD, MDH, ME, G6PD, ALAT, ASAT, Est.D). Conditions for electrophoresis and histochemical staining have been previously described (Harris & Hopkinson 1976, Altychov et al. 1981).

DNA isolation and PCR amplification - The DNA was obtained according to Sambrook et al. (1989). Promastigotes (104 - 105) were suspended in 200 µl of buffer containing 50 mM Tris-HCL pH 7.8, 50 mM EDTA, 100 mM NaCL, 1% Triton X-100, 1 µg/ml Proteinase K and incubated at 65oC for 30 min. The cell residues were sedimented by centrifugation at 10,000 g for 2 min. PCR amplification was carried out according to Saiki et al. (1985) in 30 µl of the reaction buffer (20 mM Tris-HCL pH 8.8, 10 mM (NH4)2 SO4, 8 mM MgCL2, 100 µg/ml BSA) containing 0.25 mM of each dNTP, 2 units of Taq polymerase and 40-80 ng of primer. The PCR procedure was carried out in a thermocycler developed at St. Petersburg Institute of Nuclear Physics using 32 cycles regime: denaturation condition 92oC for 50 sec, primer hybridization condition 55oC for 4 sec and DNA extension 69oC for 50 sec. PCR products were separated on 1.8% agarose gels. The gels were stained by ethidium bromide and photographed under an ultraviolet transiluminator. The oligonucleotide primer with the following sequence: 5' TAAGGGCGGTGCCAGT was used (Bulat & Mironenko 1992).

kDNA extraction and digestion with restriction enzymes - The technique of extraction and analysis of kDNA restriction profiles (schizodeme analysis) has been previously described (Pacheco et al. 1986). Parasites were lysed using a strong detergent (sarkosyl) and digested by pronase at 60oC. The kDNA networks were collected by centrifugation at 33,000 rpm for 2 hr, extracted by phenol-chloroform followed by ethanol precipitation. Two micrograms of purified kDNA preparations were digested with the restriction enzymes Hae III and Alu I in the appropriate buffers according to the manufacturer's instructions. Maxi and minicircle kDNA fragments were separated in 5%-10% linear polyacrylamide gradient gels and photographed under ultraviolet light.

Dot blot hybridization - Purified kDNAs (20 ng/dot) were spotted onto nylon membranes (Zetaprobe, Biorad) after alkaline denaturation in 0.4 N NaOH using a microfiltration apparatus connected to a vacuum pump. After blotting the membranes were rinsed in 2X SSC (1X SSC is 150 mM sodium chloride/15 mM sodium citrate) and the DNA immobilized by ultraviolet light cross-link. Total kDNAs from L. major and L. turanica were radiolabelled with alfa 32P dATP by the technique of Random Primer (Feinberg & Volgelstein 1983) modified according to the protocol described by Pacheco et al. (1994) and used as probes. Hybridizations were carried out overnight at 65oC and the membranes were washed at 65oC in 0.1X SSC/0.5% SDS, 3 times 30 min each. Blots were exposed to Kodak X-OMAT X ray films with an intensifying screen overnight.

 

RESULTS

The isoenzyme analysis allowed us to separate the strains into four groups (Fig. 1). The first group was composed by the strains KD-704, NARK and M-97 of L. major, the second by the strains CL3720 and BK-7 of L. turanica. L. gerbilli (E-18) belonged to the third group and the last one was composed by a mixture of L. major and L. turanica (DA-11). After cloning procedures, 24 and 18 clones have been respectively obtained from strains BK-7 and DA-11. The isoenzyme analysis enabled us to identify 1 clone from the strain BK-7 as L. major and the remaining 23 clones as L. turanica, while among clones isolated from the strain DA-11, L. turanica (12 clones) and L. major (6 clones) have been identified (not shown).

Analysis of PCR patterns (Fig. 2) showed that the strains identified by isoenzymes as L. major and L. turanica displayed significant differences in their amplification products. Although, the sample composed by an artificial mixture of L. major and L. turanica promastigotes (1:1) was similar to L. major. The low molecular weight amplification product (320 bp) probably belong to L. turanica.

Results of schizodeme analysis showed that NARK and KD-704 strains characterized as L. major displayed the same kDNA restriction profile, while the other strains analyzed each had a different genotypic profile. Strain DA-11 which is, according to the isoenzyme analysis, composed of a mixture of L. turanica and L. major showed a profile very closed to L. major (NARK and KD-704), although some slight microheterogeneities were observed in the kDNA minicircle regions. The isolate BK-7 displayed a more complex restriction profile; additional minicircle bands were observed when compared with the other isolates. On the other hand, the isolate KD-51 was found to possess a very distinct profile. The results were confirmed by using a second enzyme, Alu I, which did not cut kDNA minicircles of this isolate suggesting that it is, in fact, a different parasite.

Molecular hybridization experiments have confirmed, in some aspects, the results of schizodeme analysis. By this approach, strains DA-11 and BK-7 are closely related to L. major. The radioactive labelled kDNA of L. major used as probe showed strong hybridization signals with the homologous kDNAs (NARK and KD-704) indicating a high degree of sequence homology and also with the isolates DA-11 and BK-7, although in different degrees. In addition, faint hybridization signals were detected with L. turanica (KD-51) and with L. gerbilli (E-18). Results of both methodologies are shown in Figs 3 and 4. By comparing the results of filter 1 with that of filter 3 one can observe that there is no strong cross-homology between L. turanica (KD-51) kDNA probe and kDNAs from L. major strains. These results indicate that strain KD-51 is indeed less closely related to L. major. No strong DNA homology was detected between the isolates KD-51 and DA-11 in cross-hybridization analysis. According to these results it is reasonable to establish the existence of a more closely genotypic relationship between L. turanica KD-51 and BK-7 than with the isolate DA-11 (Fig. 4).

 

DISCUSSION

Until the 1970s, studies on natural leishmaniasis foci were based on methods used in clinical epidemiology and population ecology. Theses approaches allowed researchers to identify natural hosts and vectors of Leishmania, to analyse their biological and ecological features and to use this knowledge for initiating a series of programs for control of human leishmaniasis.

However, extensive data accumulated during the last two decades showed that these methods have incontestable limitations, particularly with respect to the identification of Leishmania species. At the same time, new molecular-genetic methods have been developed and subsequent progress in studies of Leishmania was associated with their introduction into laboratory routine. On the other hand, these methods proved to be not always adequated to the problems faced by epidemiologists and parasitologists.

The major goal of this report was to justify the systems that have been used for Leishmania identification by evaluating the potential of each technique as a discriminatory tool. Isoenzyme electrophoresis is considered a very discriminatory system for Leishmania identification (Le Blancq & Peters 1986, Le Blancq et al. 1986). Some authors use izoenzyme analysis to investigate variations at intraspecies level in Leishmania from different areas and hosts. By using nine enzymatic loci we were able to detect L. major and L. turanica as well as mixtures of both species in some isolates from endemic areas (for example DA-11). But, we were unable to distinguish in others as for example the isolate BK-7. Polymerase chain reaction, on the other hand, is a very promising technique made available for a rapid parasite identification. The results of PCR reaction in the present study suggest significant differentiation among L. major, L. turanica and L. gerbilli genomes, but mixture of L. major/L. turanica could hardly be identified. Such results encourage us to consider PCR amplification as a step for future development of specific primers for detecting sympatric disease agents which are relatively difficult to discriminate. Restriction fragment length polymorphisms of kDNA analyses also showed the feasibility of distinguishing among L. major, L. turanica and L. gerbilli. In addition, the technique revealed the presence of the same schizodeme of L. major circulating in two different hosts in the same geographic area, suggesting the wide spreading of this genotypic profile in humans and gerbils, as well as confirming the role of R. opimus as a reservoir host. Restriction endonuclease analyses showed genetic differences or similarities among the isolates emphasizing that is a valuable tool in epidemiological surveys. Mixed infections and/or clonal heterogeneity can be detected by schizodeme analysis (Pacheco et al. 1990, 1995) but the L. major/L. turanica infection in the present study was not recognized. Hybridization experiments gave an estimation of genetic proximity or distance of the strains.

In selecting suitable methods for a particular study, their properties and limitations need to be considered in relationship to the scope and purpose of the study. In endemic areas where distinct Leishmania species are concurrently transmitted, the possibility of mixed infections should not be discarded. In this work, we compared different methods of Leishmania identification and found that isoenzyme, PRC and restriction fragments of kDNA analyses are equally useful for discriminating single infections. However, in studies of mixed Leishmaniainfection or when difficulties in the taxonomic classification of an isolate occur these integrated methodologies in conjunction with biological data can produce reliable results. Nevertheless, in some particular cases, the analysis should be performed in several stages (identification - cloning - identification), which makes it much more labor-consuming.

 

REFERENCES

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Altykhov JU, Khilchevskaya RI, Shurkhal AV 1981. Level of polymorphism and heterozigosis of Moscow Russian population: data on 22 gene loci specifying blood proteins. Genetics 17: 548-555.

Bulat SA, Mironenko NV 1992. Polymorphism of yest-like fungus Aureobasidium pullulans (De Bary) revealed by universally primed polymerase chain reaction: species divergence state. Genetics 4: 19-30 (in Russian).

Dubrovsky YA 1978. The gerbil and natural focality of cutaneous leishmaniasis. Nauka, Moscow, 137 pp.

Evans DA 1987. Leishmania, p. 52-75. In In vitro methods for parasite cultivation. Acad. Press Ltd, London.

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Pacheco RS, Lopes UG, Morel CM, Grimaldi Jr G, Momen H 1986. Schizodeme analysis of Leishmania isolates and comparison with some phenotypic techniques, p. 57-65. In JA Rioux, Leishmania Taxonomy et Phylogenese. Applications Eco-Epidemiologiques. IMEEE, Montpellier.

Pacheco RS, Grimaldi Jr G, Momen H, Morel CM 1990. Population heterogeneity among clones of New World Leishmania species. Parasitology 100: 393-398.

Pacheco RS, Brandão AA, Sibajev A, Cupolillo E, Momen H, Degrave W 1994. The genus Crithidia: genotypic diversity among species. J Protozool Res 4: 71-82.

Pacheco RS, Martinez JE, Valderrama L, Momen H, Saravia NG 1995. Genotypic polymorphisms in experimental metastatic dermal leishmaniasis. Mol Biochem Parasitol 69: 197-209.

Saiki RK, Sharf S, Falcona F, Mullis K, Horn G, Erlich H, Arnhein N 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354.

Sambrook J, Fritsch EF, Maniatis T 1989. In Molecular cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York.

Strelkova MV 1990. Isoenzyme identification and pathogenic characteristics of clones of Leishmania major, L.spn and L. gerbilliMed Parasit i Parasit Bolezni 6: 9-13 (in Russian).

Strelkova MV, Shurkhal AV, Eliseev LN, Kelina OI, Rakitskaya TA, Zvagintseva TV, Peters W, Evans DA 1990a. Isoenzyme identification and pathogenic characteristics of Leishmania isolated from natural foci of cutaneous leishmaniasis in the USSR. Med Parasit i Parasit Bolezni 5: 43-48 (in Russian).

Strelkova AV, Shurkhal AV, Kelina OI, Eliseev IN, Evans DA, Peters W, Chapman CJ, Le Blancq SM, Van-Eys GJJ 1990b. A new species of Leishmania isolated from the great gerbil Rhombomys opimus. Parasitology 101: 327-335.

+Corresponding author. Fax: +55-21-590.3495
Received 22 May 1996
Accepted 6 August 1996

This work was supported by FAPERJ and CNPq (Brazil) and UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases

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