9 research outputs found
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
Selected features characterizing the cloned RAPD markers.
a<p>: Non coding Sequence;</p>b<p>: Overlap with a coding sequence;</p>c<p>: Matching with a coding sequence;</p>d<p>: Minicircle sequence;</p><p>* Imperfect Microsatellite: one mutation in one repeat;</p><p>**Imperfect Microsatellite: one mutation in two repeats;</p><p>*** Imperfect Microsatellite: one mutation in three repeats; (d): a 58 bp deletion associated to the microsatellite; NA: not applicable; −: no microsatellite observed or no mutations at priming site; +: presence of mismatch at priming site.</p><p>Selected features characterizing the cloned 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
Temporal distribution of RABV subtypes in Bangui as determined by the MCC analysis (number of specimens positive for rabies by months from January 2003 to March 2012 and same color code as in Fig 2).
<p>Black arrows indicates RABV introduction events in the city (first introduction of a new subtype circulating in Bangui). Grey areas indicated periods without any reported cases.</p
Sampling of RABV samples.
<p>Map of the Central African Republic and neighbouring countries (a) showing the location (red points and squares) of the 162 isolates of RABV analyzed in this study. The cities in red indicate the presence of RABV belonging to the Africa 1 clade. Cities in blue represent those where RABV belonging to the Africa 2 clade were found. In Bangui, both clades were identified. Detailed map of Bangui (b), showing the location of the 122 samples analyzed in the context of the landscape. Number of specimens positive for rabies (c) by trimester during sentinel surveillance, January 1993 to March 2012.</p
Estimation of the rate of introduction of rabid dogs in Bangui, <i>λ</i><sub><i>t</i></sub> (in rabid dogs imported to Bangui per week), the instantaneous effective reproduction number, <i>R</i><sub><i>t</i></sub>, and observed number of rabid dogs infected locally (red bar) or from outside the city (blue bar) and simulated number of rabid dogs from the model (black line: posterior median; grey area: 95% credible interval of the posterior distribution).
<p>If <i>R</i><sub><i>t</i></sub>, is above 1, a local self-sustaining epidemic in dogs may occur in the city.</p
MCC tree of 162 sequences from the Central African Republic and other locations in Africa estimated from 5000 nt of dog RABV genome.
<p>Tips representing isolates from Bangui are coloured according to the selected subtypes of RABV; other tips are colored by location. Tip times are scaled to the date of sampling (years) and branches are estimated in time units as indicated by the time bar.</p