full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (2) 1183-1192 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1184expanding. The fact that there are manyasymptomatic cases and systematic underreportingfurther complicates the possibilities ofdisease control (1, 2).In Panama, although the main manifestationare the cutaneous and mucocutaneous forms, thedisease is considered a major health problem,accounting for about 3000 new cases every yearand a possible under report of about 50% (3).Although several Leishmania species have beensporadically reported in Panama, including thepotentially pathogens for humans Leishmaniamexicana, Leishmania amazonensis, andLeishmania colombiensis, the most common isLeishmania (Viannia) panamensis, being themajor causative agent of cutaneous andmucocutaneous leishmaniasis in the country (3,4, 5, 6). The mucocutaneous form accounts forabout 5% of the patients and has the worstprognosis, frequently producing deforming lesions.Recently, the application of molecular detectionmethods has allowed some evidence of previouslyunreported species for the country, Leishmanianaiffi, in Lutzomya vectors in the Panama Canalarea (7).The study of the genetic variability ofLeishmania parasites is considered a topic of highrelevance to many aspects of the natural history,ecology and epidemiology of the organism. Thecurrent methods for exploring the genetic diversityin Leishmania have included the use of multilocusenzyme electrophoresis [8, 9], PCR-RFLPs [10],RAPDs (11), multilocus DNA sequencing (12)and microsatellites (13).The Amplified Fragment LengthPolymorphisms (AFLP) is a technique developedto explore genetic diversity of organisms whenno DNA sequence information is available (14).This technique has been extremely useful to assessthe degree of genetic variability of many biologicalgroups, showing high reproducibility androbustness. Several authors have successfullytested AFLP to assess genetic diversity inprotozoa species such as Plasmodium chabaudi(15), Plasmodium falciparum (16),Trypanosoma brucei (17, 18) and Eimeriatenella (19). Although the AFLP is a techniquewidely employed for that purpose in many othertaxa, its use for studying Leishmania parasites isscarce. Here we optimize experimental conditionsfor the application of AFLP to Leishmaniapanamensis parasites and show that thistechnique has a great potential for examining bothinter and intraspecific genetic variability.Materials and MethodsStrains Culture Conditions and DNAextractions : Several field isolates of Leishmaniapanamensis were obtained from the Institute ofScientific Research and High Technology Servicesof Panama (INDICASAT-AIP), InstitutoConmemorativo Gorgas de Estudios de la Salud(ICGES) and Center for Research and Diagnosisof Parasitic Diseases, CIDEP, Faculty ofMedicine, University of Panama. All strains used(indicated here as P2, P5, P6, P7, P8 and P9)were isolated from patients of cutaneousleishmaniasis from endemic areas in centralPanama, and were previously characterized usingmultilocus isoenzyme typing or kinetoplast PCR-RFLP DNA typing [20]. Additionally, a referencestrain of Leishmania guyanensis (MHOM/BR/1975/M4147) was included for comparison.Promastigotes were cultured at 25ºC, in tissueculture flasks (Nunc, USA), using Schneidermedium (Sigma, USA) with 0.4 g/L de NaHCO 3,20% FCS (Invitrogen, USA) and 50 µg/mlgentamycin (Invitrogen, USA). After reachinghigh density, 5 ml cultures were centrifuged at3000 rpm for 5 min, pellets washed in samevolume of PBS 1X, and DNA immediatelyextracted using a commercial kit (Wizard ®Genomic DNA purification kit, Promega, USA).DNA quality, concentration and digestibility wereCarlos M. Restrepo et al