PCR primers sequences, reaction and cycling conditions used.

<p>PCR primers sequences, reaction and cycling conditions used.</p

Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia

<div><p>A gp63PCR method was evaluated for the detection and characterization of <i>Leishmania (Leishmania)</i> (<i>L.</i>) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a <i>L. infantum</i> specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different <i>L. infantum</i> endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to <i>L. infantum</i> species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of <i>L. infantum</i> infections in dogs in Tunisia.</p></div

Results of parasitology, serology and PCR investigations on Tunisian and control dogs.

<p>Results of parasitology, serology and PCR investigations on Tunisian and control dogs.</p

Gp63PCR-RFLP patterns of <i>Leishmania</i> parasites obtained from dog biopsies.

<p>A: digestion with <i>Msc</i>I restriction enzyme; B: digestion with <i>Sal</i>I restriction enzyme. 1: <i>L. donovani</i>, 2: <i>L. infantum</i>, 3: LN112, 4: LN129, 5: LN26, 6: LN11, 7: LN80, 8: LN2, 9: LN39, 10: LN77, 11: LN102, 12: LN110, 13: J1, 14: J3, 15: J5, 16: J6, 17: J7. All sizes are indicated in bp.</p

Pair wise concordance values calculated using the kappa coefficient for parasitology, serology and PCR investigations.

<p>Pair wise concordance values calculated using the kappa coefficient for parasitology, serology and PCR investigations.</p

Kitch dendrogram constructed using Nei-modified distances calculated from gp63PCR-RFLP results obtained with lymph node biopsies of dogs from Tunisia.

<p>Branches corresponding to Old World representative <i>Leishmania</i> strains are indicated.</p

Screening and Characterization of RAPD Markers in Viscerotropic <i>Leishmania</i> Parasites

<div><p>Visceral leishmaniasis (VL) is mainly due to the <i>Leishmania donovani</i> 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 <i>Leishmania</i> 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 <i>L. donovani</i>, <i>L. archibaldi</i> and <i>L. infantum</i> 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 <i>L. infantum</i> and <i>L. donovani</i>. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents.</p></div

RAPD profiles obtained with primers OP-O7 (A) and OP-O13 (B) to illustrate examples of monomorphic and polymorphic profiles.

<p>Laboratory codes of the strains are indicated for each lane. SD: Sudan; TN: Tunisia; M: 100 bp DNA size marker. Indicated sizes are in bp.</p

Panel of <i>Leishmania</i> strains used for screening of RAPD markers.

<p>WHO that summarizes Host, geographical origin, year of isolation and laboratory code is presented together with pathology and zymodeme code whenever available. MON– corresponds to zymodeme code attributed by the reference center in Montpellier. The table also gathers study codes assigned to some of the isolates in other studies: D21, D28, D29, D31 and D32: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Mauricio1" target="_blank">[20]</a>; DON-39 and ARC-43: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Kuhls1" target="_blank">[21]</a>; Devi, H9, LRC-L57, ADDIS 164: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Jamjoom1" target="_blank">[18]</a>; Devi, GEBRE1 and KA-Jeddah: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Thiel1" target="_blank">[53]</a>; DON-81and ARC-43 (LG11): strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Kuhls2" target="_blank">[28]</a>; DON-09, DON-31, DON-39 and ARC-11: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Chocholov1" target="_blank">[25]</a>. Country abbreviations are shown as specified by WHO recommendations (SD: Sudan; TN: Tunisia; ET: Ethiopia; SA: Saudi Arabia; KE: Kenya; IN: India). ND: Not Determined; CL: cutaneous leishmaniasis; VL: visceral leishmaniasis; PKDL: Post Kala azar Dermal Leishmaniasis.</p><p>Panel of <i>Leishmania</i> strains used for screening of RAPD markers.</p

Comparative inter-species analysis of mutations within the RAPD priming sites.

<p>Comparative inter-species analysis of mutations within the RAPD priming sites.</p