The gp63 encoding genes were characterized by PCR-RFLP in 35 isolates representative of the Leishmania donovani complex (L. infantum, L. donovani, L. archibaldi and L. chagasi), with special attention to Mediterranean L. infantum from different geographical origins, and in separate groups from Old World Leishmania (L. major, L. tropica and L. aethiopica). The aim was to evaluate how the possible selective pressure by the host on these important surface proteins would influence structuring of our sample. Comparison was carried out with the structure obtained (i) from reported isoenzyme data, characters supposed to vary neutrally, and (ii) from PCR-RFLP analysis of gp63 inter-genic regions, containing nontranslated spacers and regulatory genes. Polymorphism within the gp63-encoding region, was much higher than in gp63 inter-genic regions. In the gp63 intra-genic dendrogram, the 4 species of L. donovani complex were discriminated and quite distinct from outgroups. Within L. infantum, geographical structuring was observed and did not overlap with the structure built-up from isoenzymes and inter-genic data. These results support the idea of a strong host-selection on gp63, at vector level but most of all at vertebrate (human or dog) immunological level. Furthermore, they illustrate how the nature of genetic characters may influence the perception of population structuring.
Eight patients with cutaneous ulcers were referred to the Institute of Endemic Diseases, Khartoum, Sudan, from June 2000 to March 2002 for the diagnosis of suspected cutaneous leishmaniasis (CL). Diagnosis was confirmed parasitologically by both positive Giemsa-stained smears and successful culture of Leishmania promastigotes in NNN medium. The eight parasite isolates were shown to belong to the Leishmania donovani complex by kDNA PCR. Isoenzyme typing of three isolates revealed that they were identical to the L. donovani MON-82 reference strain, and the gp63 PCR-RFLP profile showed similar patterns to a reference strain of MON-82. CL is endemic in most regions of Sudan and has been reported previously as being caused by L. major MON-74. The results of this study suggest that L. donovani is also a cause of CL in Sudan and that further study of isolates from Sudanese patients with cutaneous ulcers is warranted to ascertain whether L. donovani or L. major is the causative agent.
Cutaneous leishmaniasis (CL) due to Leishmania infantum occurs sporadically in Tunisia where its distribution is confined to the northern parts of the country. However, during the past decade there have been occasional repeated reports of cases from areas in central Tunisia, known to be free of CL. Epidemiological, clinical and parasitological data regarding these patients were collected and analysed. Data were very suggestive of the sporadic form of CL due to L. infantum. The parasites contained within the lesions of some of the patients were characterised by two different previously described PCR assays, each having different resolutive powers. The first assay, which amplified complete kDNA minicircles, showed a fragment size characteristic of the L. donovani complex; whilst the second consisted of a PCR-RFLP analysis targeting the gp63 coding sequences that confirmed assignment of the parasites to L. infantum species while illustrating its differences from the reference isolate. These findings confirm the aetiology of CL in the concerned areas in central Tunisia and suggest that L. infantum CL might be more prevalent and widespread than previously thought, or possibly emerging in these areas.
This paper reviews our exploration of the dynamics of the Leishmania genome and its contribution to epidemiology and diagnosis. We used as a model Peruvian populations of L. (Viannia) braziliensis and L. (V.) peruviana, 2 species very close phylogenetically, but phenotypically very different in biotope and pathology. We initially focused on karyotype analysis. Our data showed that chromosomes were subject to a fast rate of evolution, and were sensitive indicators of genetic drift. Therefore, molecular karyotyping appeared an adequate tool for monitoring (i) emergence of close species, (ii) ecogeographical differentiation at the intraspecific level, and (iii) strain 'fingerprinting'. Chromosome size variation was mostly due to the number of tandemly repeated genes (rDNA, mini-exon, gp63, and cysteine proteinase genes), and could involve the deletion of unique genes (L. (V.) braziliensis-specific gp63 families). Considering the importance of these genes in parasitism, their rearrangement might have functional implications: adaptation to different environments and pleomorphic pathogenicity. Our knowledge of genome structure and dynamics was used to develop new polymerase chain reaction (PCR) techniques. Amplification of gp63 genes followed by cleavage with restriction enzymes and study of restriction fragment length polymorphism (gp63 PCR-RFLP) allowed the discrimination of all species tested, even directly in biopsies with 95% sensitivity (compared with PCR amplification of kinetoplast deoxyribonucleic acid). At the intra-specific level, RFLP was also observed and corresponded to mutations in major immunogen domains of gp63. These seem to be under strong selection pressure, and the technique should facilitate addressing how the host's immune pressure may modulate parasite population structure. Altogether, gp63 PCR-RFLP represents a significant operational improvement over the other techniques for molecular epidemiology and diagnosis: it combines sensitivity, discriminatory power and prognostic value.
Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents.
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