full issue - Association of Biotechnology and Pharmacy

More documents

Recommendations

Info

Current Trends in Biotechnology and PharmacyVol. 5 (2) 1183-1192 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)Amplified Fragment Length Polymorphisms Reveals HighIntraspecific Variability in Field Isolates of LeishmaniapanamensisCarlos M. Restrepo 1 , Efraín Pérez Lao 1 , Carolina De La Guardia 1 , Octavio E. Sousa 2 ,José E. Calzada 3 and Ricardo Lleonart 1*1Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Panamá2Centro de Investigaciones y Diagnóstico de Enfermedades Parasitarias (CIDEP), Facultad de Medicina,Universidad de Panamá, Panamá3Instituto Conmemorativo GORGAS de Estudios de la Salud, Panamá*For Correspondence - rlleonart@indicasat.org.pa1183AbstractLeishmania parasites cause leishmaniasis,a potentially deadly re-emergent disease thataffects millions throughout the world. In Panama,the disease is showing an increasing trend, withestimates of thousands of new cases every year.The main manifestations are the cutaneous andmucocutaneous forms. Genetic variability studiesin Leishmania are extremely important to definekey elements of the eco-epidemiology of thedisease. However, few studies have addressedthis issue in Panama, and these have been basedmainly on kinetoplastid DNA RFLP. The amplifiedfragment length polymorphisms (AFLP) is a veryefficient technique for rapid detection of geneticvariability, particularly useful on organisms withoutsequenced genomes. Although this technique hasbeen used successfully on many species, includingseveral protozoa, its use for studying geneticvariability in Leishmania parasites is just in itsbeginnings. We have optimized and used AFLPto address genetic diversity in Leishmaniapanamensis, a poorly studied member of theViannia subgenus. We have found that thistechnique is able to generate high numbers ofpeaks when low selective EcoRI and MseIprimers were used (+0, +1, +2 series).Additionally, we have found that an importantproportion of those alleles, up to 57% for someprimer combinations, are polymorphic. Some ofthese alleles are potentially useful to rapidlydistinguish L. panamensis and L. guyanensis,the two most genetically similar species of thesubgenus. The AFLP was an efficient techniqueto screen the Leishmania panamensis genomefor polymorphisms, allowing the rapid detectionof hundreds of polymorphic alleles.Keywords: AFLP, Leishmania, GeneticVariability, PolymorphismsIntroductionLeishmania parasites (Kinetoplastida:Trypanosomatidae) are the cause of the neglecteddisease leishmaniasis, accounting for significantmorbidity and lethality at more than 88 countriesaround the world. This disease is zoonotic, causedby several species of parasites and transmittedby sand flies (Diptera: Psychodidae). Estimatesfrom the World Health Organization (WHO) pointto several million people infected and more than350 million at risk. The global incidence ofleishmaniasis is on the rise, and has been classifiedas importance level I by the WHO, meaning thatthe disease is re-emergent, uncontrolled andAFLP analysis of Leishmania panamensis.
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
Page 3 and 4:
ISSN 0973-8916Current Trends inBiot
Page 6 and 7:
Current Trends in Biotechnology and
Page 10 and 11:
Page 12 and 13:
Page 14 and 15:
Page 16 and 17:
Page 18 and 19:
Page 20 and 21:
Page 22 and 23:
Page 24 and 25:
Page 26 and 27:
Page 28 and 29:
Page 30 and 31:
Page 32 and 33:
Page 34 and 35:
Page 37 and 38:
Page 39 and 40:
Page 41 and 42:
Page 43 and 44:
Page 45 and 46:
Page 47 and 48:
Page 49 and 50:
Page 51 and 52:
Page 53 and 54:
Page 55: Current Trends in Biotechnology and
Page 58 and 59: Current Trends in Biotechnology and
Page 133: (December 7-9, 2011)Venue: Karunya
show all

full issue - Association of Biotechnology and Pharmacy

Create successful ePaper yourself

Delete template?

Save as template?