research article leishmania infantum genetic diversity and

Research ArticleLeishmania infantum Genetic Diversity and Lutzomyialongipalpis Mitochondrial Haplotypes in Brazil

Paulo Eduardo Martins Ribolla1 Letiacutecia Tsieme Gushi1

Maria do Socorro Pires e Cruz2 Carlos Henrique Nery Costa3

Dorcas Lamounier Costa3 Manoel Sebastiatildeo da Costa Lima Juacutenior4

Maria Elizabeth Moraes Cavalheiros Dorval4 Alessandra Gutierrez de Oliveira5

Mirella Ferreira da Cunha Santos5 Vera Luacutecia Fonseca Camargo-Neves6

Carlos Magno Castello Branco Fortaleza7 and Diego Peres Alonso1

1Departamento de Parasitologia Universidade Estadual Paulista ldquoJulio de Mesquita Filhordquo (UNESP)Instituto de Biotecnologia de Botucatu (IBTEC) 18607-440 Botucatu SP Brazil2Departamento de Morfofisiologia Veterinaria Centro de Ciencias Agrarias Universidade Federal do Piauı64049-550 Teresina PI Brazil3Laboratorio de Pesquisa em Leishmaniose Visceral Instituto de Doencas Tropicais Natan Portella 64001-450 Teresina PI Brazil4Centro de Ciencias Biologicas e da Saude Universidade Federal do Mato Grosso do Sul (UFMS)79070-900 Campo Grande MS Brazil5Programa de Pos-Graduacao em Doencas Infecciosas e Parasitarias Faculdade de Medicina (FAMED)Universidade Federal de Mato Grosso do Sul (UFMS) 79070-900 Campo Grande MS Brazil6Superintendencia de Controle de Endemias (SUCEN) 01027-000 Sao Paulo SP Brazil7Departamento de Doencas Tropicais Faculdade de Medicina de BotucatuUniversidade Estadual Paulista ldquoJulio de Mesquita Filhordquo (UNESP) 18618-687 Botucatu SP Brazil

Correspondence should be addressed to Diego Peres Alonso alonsoibbunespbr

Received 6 November 2015 Revised 22 February 2016 Accepted 7 March 2016

Academic Editor Amogh A Sahasrabuddhe

Copyright copy 2016 Paulo Eduardo Martins Ribolla et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Leishmania infantum is the etiological agent of visceral leishmaniasis (VL) in the Americas with domestic dogs being its majorreservoir hosts The main VL vector is the sandfly Lutzomyia longipalpis while other Lutzomyia species may play a role in diseasetransmission Although the genetic structure of L infantum populations has been widely evaluated only a few studies haveaddressed this subject coupled to the genetic structure of the respective sandfly vectors In this study we analyzed the populationstructure of L infantum in threemajorVL endemic areas in Brazil and associated it withLutzomyia longipalpis geographic structure

1 Introduction

Leishmaniases are parasitic diseases caused by protozoansfrom the genus Leishmania which are transmitted by the biteof female sandflies from the family Psychodidae The clini-cal manifestations of leishmaniases are particularly diverseand present different characteristics visceral leishmaniasis(VL) the most severe one mucocutaneous leishmaniasis

characterized as a mutilating disease diffuse cutaneous leish-maniasis caused by a deficient cellular immune responseand cutaneous leishmaniasis which causes single or multiplelesions on the skin The epidemiology of leishmaniasis ishighly complex there are 20 known species of Leishmaniapathogenic to humans and at least 30 species of sandfliesvectors Furthermore this disease can be designated as azoonosis which involves animals as the reservoir hosts or

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 9249217 11 pageshttpdxdoiorg10115520169249217

2 BioMed Research International

as an anthroponosis when humans are the only source ofparasites for sandflies Leishmaniasis is widely spread in 98countries and 3 territories from which more than 70 aredeveloping countries and 13 are among the least developedones [1]

Visceral leishmaniasis can be either an anthroponosis(eg in the Indian subcontinent) or a zoonosis (eg in theMediterranean or in the Americas) and it is characterizedby chronic evolution and systemic involvement which ifuntreated may result in death In the Americas Leishmaniainfantum is the etiological agent of the disease and Brazilaccounts for over 90 of the cases in the continent [12] Domestic dogs are the proven reservoir hosts in ruraland urban areas while the role of naturally infected wildmammals (canids and marsupials) as L infantum reservoirhosts is still controversial [3] The main sandfly vector isLutzomyia longipalpis but other Lutzomyia species mightplay a role in disease transmission for example in CorumbaMato Grosso do Sul naturally Leishmania-infected Lu cruzihave been discovered and because there is still no evidenceof Lu longipalpis in this region that sandfly is considered themain vector [4 5]

In Brazil VL typically occurred in rural settings butsince 1980 its incidence has been changing due to widespreadurban outbreaks The first major VL urban epidemic inthe country happened in Teresina Piauı State Since thenepidemics occurred in Natal (Rio Grande do Norte) andSao Luıs (Maranhao) and the disease subsequently spreadto other regions of the country Autochthonous cases wererecently described for the first time in the southernmost Stateof RioGrande do SulThe current epidemiological scenario ofVL leaves no doubt regarding the severity of the situation andthe unchecked geographic spread of the disease In the 1990sonly 10 of the cases occurred outside the Northeast Regionbut in 2007 the proportion reached 50 of cases From 2006to 2008 autochthonous transmission of VL was reported inmore than 1200 municipalities in 21 states [6]

The broad spectrum of leishmaniasis-associated symp-toms coupled with the wide diversity of vertebrate andinvertebrate host species suggests that both parasitesrsquo andhostsrsquo genetic backgrounds determine the patterns of thedisease [7] On the other hand clonal diversity and geneticheterogeneity which can cause variability in parasite viru-lence are quite common in Leishmania [8]

Several studies showed that genetic variability of Linfantum in Brazil is low with restricted diversity and limitedpopulation clustering In a recent work assessing parasitepopulations distributed over 18 states three major clusteredpopulations could be inferred using microsatellite typingWhen the analysis is performed in parasites from closelyrelated geographic regions the overall diversity is even lower[9ndash11]

When we look at sandfly genetic variability there iscompelling evidence that the Lutzomyia population structurein Brazil is complex with different genotypes identifieddepending on the geographic region assessed and also thespecies involved in parasite transmission [12ndash14]

Based on these studies it is logical to hypothesize that theinteractions of L infantum genotypic variants with different

hosts and vector populations may ultimately influence thetransmission dynamics and severity of eventual outbreaksHence assessing the genetic structure of both vector popula-tions and parasitesmay help us to understand the dynamics ofvector-parasite interactions and the epidemiological aspectsof American visceral leishmaniasis Here we used PCR-RFLP of kinetoplast minicircle DNA (kDNA) to identify Linfantum genotypic variants from three VL endemic areasin Brazil Teresina in Piauı State Campo Grande in MatoGrosso do Sul State and Bauru in Sao Paulo State kDNA-RFLP analysis when compared to microsatellite genotypinghas proven to be more sensitive to examine genetic data ofclosely related sympatric L infantum strains [15] In additionin order to identify different haplotypes of Lu longipalpisand Lu cruzi sandflies from those three VL endemic areaswe used mitochondrial 12S rDNA sequencing As a maternalinheritance rapidly evolving nonrecombinant and haploidmolecular marker 12S rDNA is suitable to trace genealogyand evolutionary history To our knowledge this is the firststudy that seeks to compare genetic variability of Leishmaniainfantum parasites to the genetic structure of its vectors inBrazil

2 Methods

21 Ethics Statement For insect collections in Mato Grossodo Sul State we obtained a permanent license for collectingand transporting zoological material N∘ 25592-1 on behalf ofDr Alessandra Gutierrez de Oliveira issued by the Systemof Authorization and Information on Biodiversity of theBrazilian Institute of Environment and Renewable NaturalResources (SisbioIBAMA) For insect collections in SaoPaulo State and Piauı State no specific permissions wererequired since the specimens were kindly provided by theCenter for the Control of Endemic Diseases (SUCEN) andFederal Piauı State University respectively The collectionswere performed at private residences whose owners person-ally granted permission to enter their backyards to capture thesandflies All of these residences were located in urban areasand no endangered or protected species were collected in thisstudy

22 Sandfly Collections Sandflies were captured by bothmanual collection and electric traps Manual collection wasperformed with electric aspirators restricting the use of aCastro catcher to locations where aspiration could not beusedThe selected collection points were preassessed in orderto establish the best capturing location in the peridomicile Ateach selected point modified CDC light traps were installedfrom 6 pm to 6 am

The collections took place in different areas in Braziland were performed by the respective local teams Sao Paulo(SP) State performed by Center for the Control of EndemicDiseases (SUCEN) Piauı (PI) State performed by PiauıFederal University and Mato Grosso do Sul (MS) Statecarried out by Mato Grosso do Sul Federal University

Lu cruzi was collected in Corumba (MS) and Lu longi-palpis in all other places Campo Grande (MS) Teresina

BioMed Research International 3

Table 1 Haplotype diversity analysis of the six sandfly populations assessed

Populationssampled

Number of individualssampled (119873) Number of haplotypes Haplotype

diversity (Hd)Variance of haplotype

diversity

Standard deviationof haplotypediversity

Andradina 30 1 0 0 0Aracatuba 30 6 0545 001027 0101Birigui 30 1 0 0 0CampoGrande 14 2 0143 001412 0119

Corumba 7 2 0286 003856 0196Teresina 29 7 0672 000346 0059

(PI) Andradina (SP) Aracatuba (SP) and Birigui (SP) Allidentified insects were kept in 70 ethanol until use

23 Sandfly Genomic DNA Isolation The field-derived sand-flies were grinded with the help of a plastic pestle in 15mLtubes containing 300 120583L of 5 Chelex (Bio-Rad) Thesolution was then vortexed for 15 s centrifuged at 11000 gfor 20 s and incubated at 80∘C for 30min after whichthe procedure was repeated The supernatant was finallyremoved transferred to another 15mLmicrocentrifuge tubeand stored at minus20∘C We had an average of 45 ng of DNA persandflymeasuredwithNanoDrop 1000 (ThermoScientific)

24 Sandfly Mitochondrial 12S rDNA Amplification andSequencing PCR amplification of the Lutzomyia sp 12SrDNAmitochondrial region was performed with the primersT1B (51015840-AAACTAGGATTAGATACCT-31015840) and T2B (51015840-AATGAGAGCGACGGGCGATG-31015840) according to Beati etal [16] Reactions of 25 120583L were set up as follows 137 120583L ofultrapure water 25120583L of 10x Platinum buffer (Life Technolo-gies) 10 120583L MgCl2 (50mM) 05 120583L dNTPs (01mM) 10 120583Lof each oligonucleotide (10 pmol120583L) 03 120583L of PlatinumTaq(Life Technologies 5 U120583L) and 10 ng of genomic DNA Thereactionwas carried out in a thermal cycler as follows 5 cyclesof 94∘C for 15 s 51∘C for 30 s and 68∘C for 30 s followed by25 cycles of 94∘C for 15 s 53∘C for 30 s and 70∘C for 30 sand a final extension step of 70∘C for 5min The amplifiedDNA fragments were UV visualized after electrophoresis on1 agarose gel stained with ethidium bromide

The resulting DNA fragments were purified withExoSAP-IT kit (GE Healthcare) according to the manufac-turerrsquos protocol The 20120583L sequencing reactions consistedof 2120583L of BigDye Terminator (Life Technologies) 60 120583L ofBigDyeTerminator 5x SequencingBuffer (Life Technologies)32 120583L of the primers (1 pmol120583L) 48 120583L of ultrapure waterand 200 ng of DNAmeasured with NanoDrop 1000 (ThermoScientific) All reactions were carried out in a thermal cyclerwith 35 cycles of 95∘C for 20 s 50∘C for 15 s and 60∘C for2minThe amplifiedDNAwas precipitatedwith 80120583L of 65isopropanol washed with 200120583L of 70 ethanol and air-dried for 5min Before injection samples were resuspendedin 10 120583L of HI-DI formamide (Life Technologies) and heatedat 95∘C for 3min for DNA denaturation and immediately

cooled on ice Sample processing occurred in an ABI377automatic sequencer

25 Sequencing Analysis The forward and reverse 12S rDNAsequences were manually checked for quality and thepolymorphisms confirmed and then matched using theonline EMBOSS GUI tool package (httpimedmeducmescgi-binembosspl action=inputamp app=merger)The obtainedconsensus sequences were aligned using Clustal X2 softwarePolymorphisms in each sequence were identified and ahaplotypic diversity test (Table 1) was performed with theDnaSP 510 software Haplotype diagram generation wasperformed by TCS phylogenetic network using statisticalestimation parsimony software

26 Parasite Samples and DNA Isolation Parasites used inthis study were collected between 2007 and 2009 (Table 2)The DNA from the promastigotes (from all cultured sam-ples used and for the two parasite samples obtained fromsandflies) was isolated with Chelex (Bio-Rad) Briefly 1mLaliquots of the cultures were transferred to 15mL centrifugetubes and spun down for 1min at 10000 g The supernatantwas discarded and the pellet resuspended in 300120583L of 10Chelex (wv) Following the samples were incubated for15min at 95∘C and then centrifuged again for 1min at10000 g The supernatant containing the DNA was thencarefully recovered and stored in a new tube at minus20∘C Forthe two parasite samples isolated from sandflies the wholeinsect was grinded in 300120583L of 10 Chelex with the help ofa motorized tissue grinder following the same steps aboveWe had an average of 200 ng of DNA per culture sampledand 20 ng per sample for the two sandfly-derived parasitesmeasured with NanoDrop 1000 (Thermo Scientific)

The DNA of L infantum amastigotes was extractedfollowing two different approaches For dog bone marrowaspirates we used the Illustra Blood GenomicPrep Mini Spinkit (GE Healthcare) according to the manufacturerrsquos recom-mendations For slide-fixed humanbonemarrow aspiratesweused the same protocol after scraping the contents of eachslide into a 15mL tube as previously described [17] We hadan average of 100 ng per dog bone marrow sample and 25 ngof DNA per slide measured with NanoDrop 1000 (ThermoScientific)


Table 2 Parasite samples genotyped in the study

Laboratory code WHO code Life stage Type of sample Host Year of isolation LocationTER1 MCANBR2007TER1 Amastigotes Fresh blood marrow aspirates Dog 2007 Teresina PITER2 MCANBR2007TER2 Amastigotes Fresh blood marrow aspirates Dog 2007 Teresina PITER3 MCANBR2007TER3 Amastigotes Fresh blood marrow aspirates Dog 2007 Teresina PITER4 MCANBR2007TER4 Amastigotes Fresh blood marrow aspirates Dog 2007 Teresina PITER5 MCANBR2007TER5 Amastigotes Fresh blood marrow aspirates Dog 2007 Teresina PITER6 MCANBR2007TER6 Amastigotes Fresh blood marrow aspirates Dog 2007 Teresina PITER7 MCANBR2007TER7 Amastigotes Fresh blood marrow aspirates Dog 2007 Teresina PITER8 MCANBR2007TER8 Amastigotes Fresh blood marrow aspirates Dog 2007 Teresina PITER9 MCANBR2008TER9 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER10 MCANBR2008TER10 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER11 MCANBR2008TER11 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER12 MCANBR2008TER12 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER13 MCANBR2008TER13 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER14 MCANBR2008TER14 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER15 MCANBR2008TER15 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER16 MCANBR2008TER16 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER17 MCANBR2008TER17 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER18 MCANBR2008TER18 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER19 MCANBR2008TER19 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER20 MCANBR2008TER20 Amastigotes Fresh blood marrow aspirates Dog 2008 Teresina PITER21 MCANBR2009TER21 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER22 MCANBR2009TER22 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER23 MCANBR2009TER23 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER24 MCANBR2009TER24 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER25 MCANBR2009TER25 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER26 MCANBR2009TER26 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER27 MCANBR2009TER27 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER28 MCANBR2009TER28 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER29 MCANBR2009TER29 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER30 MCANBR2009TER30 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER31 MCANBR2009TER31 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER32 MCANBR2009TER32 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER33 MCANBR2009TER33 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER34 MCANBR2009TER34 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER35 MCANBR2009TER35 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER36 MCANBR2009TER36 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER37 MCANBR2009TER37 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER38 MCANBR2009TER38 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER39 MCANBR2009TER39 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER40 MCANBR2009TER40 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER41 MCANBR2009TER41 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER42 MCANBR2009TER42 Amastigotes Fresh blood marrow aspirates Dog 2009 Teresina PITER43 ILONBR2009TER43 Promastigotes Cultured parasites Sandfly 2009 Teresina PITER44 ILONBR2009TER44 Promastigotes Cultured parasites Sandfly 2009 Teresina PITER45 MHOMBR2009TER45 Promastigotes Cultured parasites Human 2009 Teresina PITER46 MHOMBR2008TER46 Promastigotes Cultured parasites Human 2008 Teresina PITER47 MHOMBR2008TER47 Promastigotes Cultured parasites Human 2008 Teresina PITER48 MHOMBR2008TER48 Promastigotes Cultured parasites Human 2008 Teresina PITER49 MHOMBR2008TER49 Promastigotes Cultured parasites Human 2008 Teresina PITER50 MHOMBR2008TER50 Promastigotes Cultured parasites Human 2008 Teresina PI


Table 2 Continued

Laboratory code WHO code Life stage Type of sample Host Year of isolation LocationTER51 MHOMBR2008TER51 Promastigotes Cultured parasites Human 2008 Teresina PITER52 MHOMBR2007TER52 Promastigotes Cultured parasites Human 2007 Teresina PITER53 MHOMBR2007TER53 Promastigotes Cultured parasites Human 2007 Teresina PITER54 MHOMBR2007TER54 Promastigotes Cultured parasites Human 2007 Teresina PITER55 MHOMBR2007TER55 Promastigotes Cultured parasites Human 2007 Teresina PITER56 MHOMBR2007TER56 Promastigotes Cultured parasites Human 2007 Teresina PITER57 MHOMBR2007TER57 Promastigotes Cultured parasites Human 2007 Teresina PITER58 MHOMBR2007TER58 Promastigotes Cultured parasites Human 2007 Teresina PITER59 MHOMBR2007TER59 Promastigotes Cultured parasites Human 2007 Teresina PITER60 MHOMBR2007TER60 Promastigotes Cultured parasites Human 2007 Teresina PITER61 MHOMBR2007TER61 Promastigotes Cultured parasites Human 2007 Teresina PITER62 MHOMBR2007TER62 Promastigotes Cultured parasites Human 2007 Teresina PITER63 MHOMBR2007TER63 Promastigotes Cultured parasites Human 2007 Teresina PITER64 MHOMBR2007TER64 Promastigotes Cultured parasites Human 2007 Teresina PITER65 MHOMBR2007TER65 Promastigotes Cultured parasites Human 2007 Teresina PITER66 MHOMBR2009TER66 Promastigotes Cultured parasites Human 2009 Teresina PITER67 MHOMBR2009TER67 Promastigotes Cultured parasites Human 2009 Teresina PITER68 MHOMBR2009TER68 Promastigotes Cultured parasites Human 2009 Teresina PITER69 MHOMBR2009TER69 Promastigotes Cultured parasites Human 2009 Teresina PITER70 MHOMBR2009TER70 Promastigotes Cultured parasites Human 2009 Teresina PITER71 MHOMBR2009TER71 Promastigotes Cultured parasites Human 2009 Teresina PITER72 MHOMBR2009TER72 Promastigotes Cultured parasites Human 2009 Teresina PITER73 MHOMBR2009TER73 Promastigotes Cultured parasites Human 2009 Teresina PITER74 MHOMBR2009TER74 Promastigotes Cultured parasites Human 2009 Teresina PITER75 MHOMBR2009TER75 Promastigotes Cultured parasites Human 2009 Teresina PITER76 MHOMBR2009TER76 Promastigotes Cultured parasites Human 2009 Teresina PITER77 MHOMBR2009TER77 Promastigotes Cultured parasites Human 2009 Teresina PITER78 MHOMBR2009TER78 Promastigotes Cultured parasites Human 2009 Teresina PITER79 MHOMBR2009TER79 Promastigotes Cultured parasites Human 2009 Teresina PITER80 MHOMBR2009TER80 Promastigotes Cultured parasites Human 2009 Teresina PITER81 MHOMBR2008TER81 Promastigotes Cultured parasites Human 2008 Teresina PITER82 MHOMBR2008TER82 Promastigotes Cultured parasites Human 2008 Teresina PITER83 MHOMBR2008TER83 Promastigotes Cultured parasites Human 2008 Teresina PITER84 MHOMBR2008TER84 Promastigotes Cultured parasites Human 2008 Teresina PITER85 MHOMBR2008TER85 Promastigotes Cultured parasites Human 2008 Teresina PITER86 MHOMBR2008TER86 Promastigotes Cultured parasites Human 2008 Teresina PITER87 MHOMBR2008TER87 Promastigotes Cultured parasites Human 2008 Teresina PITER88 MHOMBR2009TER88 Promastigotes Cultured parasites Human 2009 Teresina PICGR89 MHOMBR2009CGR89 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR90 MHOMBR2009CGR90 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR91 MHOMBR2009CGR91 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR92 MHOMBR2009CGR92 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR93 MHOMBR2009CGR93 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR94 MHOMBR2009CGR94 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR95 MHOMBR2009CGR95 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR96 MHOMBR2009CGR96 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR97 MHOMBR2009CGR97 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR98 MHOMBR2009CGR98 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR99 MHOMBR2009CGR99 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR100 MHOMBR2009CGR100 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR101 MHOMBR2009CGR101 Promastigotes Cultured parasites Human 2009 Campo Grande MS


Table 2 Continued

Laboratory code WHO code Life stage Type of sample Host Year of isolation LocationCGR102 MHOMBR2009CGR102 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR103 MHOMBR2009CGR103 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR104 MHOMBR2009CGR104 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR105 MHOMBR2009CGR105 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR106 MHOMBR2009CGR106 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR107 MHOMBR2009CGR107 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR108 MHOMBR2009CGR108 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR109 MHOMBR2009CGR109 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR110 MHOMBR2009CGR110 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR111 MHOMBR2008CGR111 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR112 MHOMBR2008CGR112 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR113 MHOMBR2008CGR113 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR114 MHOMBR2008CGR114 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR115 MHOMBR2008CGR115 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR116 MHOMBR2008CGR116 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR117 MHOMBR2008CGR117 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR118 MHOMBR2008CGR118 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR119 MHOMBR2008CGR119 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR120 MHOMBR2008CGR120 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR121 MHOMBR2008CGR121 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR122 MHOMBR2008CGR122 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR123 MHOMBR2008CGR123 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR124 MHOMBR2008CGR124 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR125 MHOMBR2008CGR125 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR126 MHOMBR2008CGR126 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR127 MHOMBR2008CGR127 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR128 MHOMBR2008CGR128 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR129 MHOMBR2008CGR129 Promastigotes Cultured parasites Human 2008 Campo Grande MSCGR130 MHOMBR2009CGR130 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR131 MHOMBR2009CGR131 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR132 MHOMBR2009CGR132 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR133 MHOMBR2009CGR133 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR134 MHOMBR2009CGR134 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR135 MHOMBR2009CGR135 Promastigotes Cultured parasites Human 2009 Campo Grande MSCGR136 MHOMBR2007CGR136 Promastigotes Cultured parasites Human 2007 Campo Grande MSCGR137 MHOMBR2007CGR137 Promastigotes Cultured parasites Human 2007 Campo Grande MSCGR138 MHOMBR2007CGR138 Promastigotes Cultured parasites Human 2007 Campo Grande MSCGR139 MHOMBR2007CGR139 Promastigotes Cultured parasites Human 2007 Campo Grande MSCGR140 MHOMBR2007CGR140 Promastigotes Cultured parasites Human 2007 Campo Grande MSCGR141 MHOMBR2007CGR141 Promastigotes Cultured parasites Human 2007 Campo Grande MSCGR142 MHOMBR2007CGR142 Promastigotes Cultured parasites Human 2007 Campo Grande MSBAU143 MHOMBR2007BAU143 Amastigotes Bone marrow aspirates slides Human 2007 Bauru SPBAU144 MHOMBR2007BAU144 Amastigotes Bone marrow aspirates slides Human 2007 Bauru SPBAU145 MHOMBR2007BAU145 Amastigotes Bone marrow aspirates slides Human 2007 Bauru SPBAU146 MHOMBR2008BAU146 Amastigotes Bone marrow aspirates slides Human 2008 Bauru SPBAU147 MHOMBR2008BAU147 Amastigotes Bone marrow aspirates slides Human 2008 Bauru SPBAU148 MHOMBR2008BAU148 Amastigotes Bone marrow aspirates slides Human 2008 Bauru SPBAU149 MHOMBR2008BAU149 Amastigotes Bone marrow aspirates slides Human 2008 Bauru SPBAU150 MHOMBR2008BAU150 Amastigotes Bone marrow aspirates slides Human 2009 Bauru SPBAU151 MHOMBR2007BAU151 Amastigotes Bone marrow aspirates slides Human 2007 Bauru SPBAU152 MHOMBR2009BAU152 Amastigotes Bone marrow aspirates slides Human 2009 Bauru SP


Table 2 ContinuedLaboratory code WHO code Life stage Type of sample Host Year of isolation LocationBAU153 MHOMBR2009BAU153 Amastigotes Bone marrow aspirates slides Human 2009 Bauru SPBAU154 MHOMBR2009BAU154 Amastigotes Bone marrow aspirates slides Human 2009 Bauru SPBAU155 MHOMBR2009BAU155 Amastigotes Bone marrow aspirates slides Human 2009 Bauru SPBAU156 MHOMBR2009BAU156 Amastigotes Bone marrow aspirates slides Human 2009 Bauru SPBAU157 MHOMBR2009BAU157 Amastigotes Bone marrow aspirates slides Human 2009 Bauru SP

27 PCR-RFLP of Kinetoplast DNA (kDNA) and RFLP Anal-ysis We had initially started our analysis using a panel of7 microsatellite markers (Li22-35 Li23-41 Li45-24 Li71-33 Lm2TG Lm4TA and TubCA) [10] However only onemarker (Li45-24) was polymorphic and due to its lowvariability only two alleles could be identified For thisreason we decided to perform only PCR-RFLP of kinetoplastDNA (kDNA) and RFLP analysis

For the analysis of the kinetoplast minicircle DNA 157L infantum isolates were used (Table 2) 98 cultured sam-ples initially isolated from human patients by sternal bonemarrow aspiration (44 from Teresina and 54 from CampoGrande) 42 samples from dog bone marrow aspirates fromTeresina 2 samples from sandflies blood-fed on L infantum-infected dogs from this same study in Teresina and 15 slide-derived samples originated from bone marrow aspirates ofhumanpatients in Bauru Sao Paulo State PCR reactionswereperformedwith primers LINR4 and LIN19 [18] and generateda 720 bp amplicon which covers almost the entire minicircleThe 50120583L reactions contained 1mMMgCl2 10mM Tris-HCl (pH 83) 03 pmol of each oligonucleotide 01mMdNTPs 1 unit of Taq polymerase (GE Healthcare) and5 120583L of sample DNA The amplification conditions were asfollows 3min at 94∘C 33 cycles of 30 s at 95∘C 30 s at 58∘Cand 1min at 72∘C followed by a final extension step of 10minutes at 72∘C The PCR products were then precipitatedwith ethanol resuspended in water and digested with therestriction enzymes RsaI and HpaII (Promega) as previouslydescribed [15] Approximately 1 120583g of each PCR product wasused per digestion in order to ensure that all reactions hadthe same initial amount of DNA Since the products smallerthan 100 bp can be confused with primer dimers and theones larger than 700 bp can be misidentified as undigestedproducts only the fragments within this range were used inour RFLP analysis

Data analysis was performed using R software environ-ment A binary matrix was constructed based on the profileof fragments generated by each digestion where 1 representsthe presence of a fragment and 0 represents its absenceThis matrix was converted into a similarity matrix using thepackage ldquoproxyrdquo and used for cluster analysis After119870-meanspartitioning method was used to infer the number of clustersusing the package ldquo119896-meansrdquo andAgglomerativeHierarchicalClustering dendrogram was built using the binary distancemethod and ward cluster method with the package ldquohclustrdquo

3 Results

31 Sandflies Genetic Analysis DNA was extracted froma total of 140 individuals as follows 30 individuals from

PI

MS

SP

Teresina

CampoAndradina

BiriguiBauru

Sandflies and parasitesSandflies onlyParasites only

Araccedilatuba

Corumb a Grande

Figure 1 Map of Brazil with emphasis on the states of MatoGrosso do Sul (MS) Sao Paulo (SP) and Piauı (PI) The positionof each studied locality in the states where samples were collected isdepicted

Andradina (SP) Aracatuba (SP) and Birigui (SP) 29 individ-uals from Teresina (PI) 14 individuals from Campo Grande(MS) 7 individuals from Corumba (MS) classified mor-phologically as Lu cruzi (Figure 1) PCR reactions generateda mitochondrial 12S ribosomal DNA fragment of approxi-mately 360 bp as previously described [19] which was thenpartially sequenced (263 bp) Sequences were screened forsignificant polymorphisms and 10 variable sites were found(Table 3) When polymorphisms were assessed with DnaSP510 program 13 haplotypes were generated six haplotypes(H8 H9 H10 H11 H12 andH13) containing only individualsfrom Teresina (PI) five haplotypes (H3 H4 H5 H6 and H7)containing only Aracatuba individuals (SP) one haplotype(H1) containing one individual from Corumba and oneindividual from Campo Grande (MS) and one haplotype


Table 3 Variable sites per haplotype of 12S mitochondrial DNA inLutzomyia sp

Haplotypes SNPsH1 C T C C C T G T A TH2 sdot C sdot sdot sdot sdot sdot sdot sdot sdotH3 T C sdot sdot sdot sdot sdot sdot sdot sdotH4 sdot C sdot sdot sdot G sdot sdot sdot GH5 sdot C sdot sdot sdot G sdot sdot sdot sdot

H6 T C sdot sdot sdot sdot sdot sdot sdot GH7 T C sdot T sdot sdot sdot sdot sdot GH8 sdot C sdot sdot sdot sdot sdot C sdot sdot

H9 sdot C sdot sdot sdot sdot sdot C G sdot

H10 sdot C sdot sdot T sdot sdot C sdot sdot

H11 sdot C T T sdot sdot sdot C sdot sdot

H12 sdot C sdot sdot sdot sdot A sdot sdot sdot

H13 sdot C sdot T T sdot sdot C sdot sdot

SNPs positiona 36 71 80 84 107 178 194 243 244 257aSNPs positions are given in relation to the beginning of 12S rDNA sequencedeposited as KF485516 in GenBank

(H2) covering most of the sequences (111 individuals) Dataare represented in a diagram of haplotypes (Figure 2) Thehaplotypic diversity test showed that Teresina presented thehighest diversity (0672) followed by Aracatuba (0545)Corumba (0286) and Campo Grande (0143) Andradinaand Birigui presented no haplotypic diversity at all (Table 1)

32 Parasites RFLPAnalysis ThekDNA fragments of interestwere successfully amplified from the LinR4 and Lin19 oligosused in this study RFLP analysis of kinetoplast minicirclesDNA was also efficient in detecting restriction patternsbetween different samples From the 157 tested samples wecould observe 55 unique genotypes in the cluster analysisdendrogram illustrated in Figure 3 119870-means partitioningidentified 6 major clusters there was a clear distinctionbetween samples fromTeresina which grouped in two almostexclusive clusters and all other samples an exclusive Baurucluster was also found Two clusters presented with Teresinaand Campo Grande samples and one cluster presented withBauru and Campo Grande samples It is noteworthy thatCampo Grande is distributed over 3 major clusters one thatgroups together with one Teresinamajor cluster and the othertwo that are closer toBauru clusters in a separate branch of thedendrogram There was no clustering differentiation relatedto the years of collection

4 Discussion

During the past 20 years the epidemiology of VL hasbeen constantly changing due to a continuous urbaniza-tion process an increasing incidence of HIVLeishmaniacoinfections and syringe sharing by intravenous drug users[20] and the identification of novel L infantum mammalianhostsreservoirs [21]This highlights the necessity of molecu-larly tracking the geographic distribution of different parasiteand vector populations in order to enhance the knowledgeon basic epidemiological aspects of the disease such as itsnatural history and transmission

H2

H8

H9

H10

H11

H12

H13

H5

H6

H3

H7

H4

H1

110

1341

Teresina

AndradinaAraccedilatubaBirigui

Campo GrandeCorumbaacute

Figure 2 The diagram of 12S mitochondrial haplotypes generatedfor Lutzomyia sp Haplotypes found after the analysis of a 263 bpfragment of 12S mitochondrial rRNA The diameter of the circles isrelated to the numbers of individuals foundwith the samehaplotypeThe connections between haplotypes are of the same size in relationto the center of each circle The black dots represent the number ofsteps (SNPs) between the haplotypes

Several molecular approaches have been used in thecharacterization of genetic variants in the genus Leishmaniaamplified polymorphic DNA (RAPD) markers [22] analysisby size polymorphism of restriction fragments (RFLP) ofthe ITS regions ribosomal DNA [23] and kinetoplast DNA[24] analysis confirmed sequence amplified regions [25]and analysis of regions of DNA with microsatellite markers[19 26ndash29] We then decided to proceed with PCR-RFLPanalysis of minicircle kDNA because it has a higher resolvingpower when applied to population genetics studies involvingeither genetically or geographically closely related strains [2430 31] Our data revealed a clear distinction between sam-ples from Teresina which grouped in two almost exclusiveclusters and all other samples an exclusive Bauru cluster wasalso found Two clusters presented with Teresina and CampoGrande samples and one cluster presented with Bauru andCampo Grande samples These results allowed us to draw arelationship between genetic distance and geographic originInterestingly geographic origin related to diverse geneticbackground was also found for L infantum parasites in Brazilin the study performed by Segatto et al [10]

Our data is partially in accordance with a previousmicrosatellite based genotyping study performed with par-asite populations from all 5 Brazilian regions In the studythree well-defined populations could be identified one that


TeresinaCampo GrandeBauru

25

20

15

10

05

00

TER8

3TE

R80

TER3

3

TER2

5TE

R39

TER8

4

CGR9

1TE

R07

TER3

5

TER2

2TE

R10

TER0

1

TER1

2TE

R04

TER2

5

TER8

8TE

R06

TER0

9

TER8

5TE

R86

TER8

7

TER7

6TE

R77

TER8

1

TER7

3TE

R74

TER7

5

TER7

0TE

R71

TER7

2

TER6

7TE

R68

TER6

9

TER6

4TE

R65

TER6

6

TER6

1TE

R62

TER6

3

TER5

9TE

R58

TER6

0

TER5

5TE

R56

TER5

7

TER5

2TE

R53

TER5

4

TER4

9TE

R50

TER5

1

TER4

6TE

R47

TER4

8

TER4

3TE

R44

TER4

5

TER1

4TE

R15

TER2

4

TER0

2TE

R11

TER1

6

TER2

1TE

R30

TER0

3

TER3

6TE

R08

CGR8

9

TER2

6TE

R32

TER3

1

TER3

4TE

R27

TER3

7TE

R41

TER3

8

TER0

5TE

R29

TER4

2

TER1

7TE

R13

TER1

9

TER4

0TE

R28

TER2

0

CGR1

09CG

R138

CGR9

0

TER8

2TE

R79

CGR1

20

CGR1

25CG

R100

CGR9

7

CGR1

06CG

R123

CGR1

35

CGR1

37CG

R136

CGR1

17

BAU

144

BAU

145

BAU

146

CGR9

3CG

R92

BAU

143

CGR9

6CG

R95

CGR9

4

CGR1

07TE

R16

TER7

8

CGR1

01CG

R102

CGR1

14

BAU

155

BAU

156

CGR1

11

BAU

149

BAU

151

BAU

152

CGR1

31BA

U14

7BA

U14

8

CGR1

21CG

R113

CGR1

22

CGR1

18CG

R130

CGR1

04

CGR1

05CG

R103

CGR1

16

CGR1

24CG

R127

CGR1

15

CGR1

41CG

R140

CGR1

26

CGR9

9CG

R96

CGR1

10

CGR1

34CG

R142

CGR1

19

CGR1

12CG

R133

CGR1

32

BAU

154

BAU

157

CGR1

08

CGR1

39BA

U15

0BA

U15

3

CGR1

28CG

R129

Figure 3 Cluster analysis generated from PCR-RFLP data for Leishmania infantum Hierarchical Agglomerative Clustering for 157 samplesof Leishmania infantum parasites assessed in the study119870-means partitioning identified six major clusters which are depicted with pie chartscontaining the proportions of parasites from each geographic area assessed

was presentmostly inNortheast region (including Piauı Statethat was sampled in our study) and the other two presentin Midwest region (including Mato Grosso and Mato Grossodo Sul States that were sampled in our study) On the otherhand parasites typed in Southeast region (including SaoPaulo State that was sampled in our study) are closely relatedto northeastern parasites while in our study they are closelyrelated toMidwestern parasites [9] Our findings corroboratethe use of this technique in Leishmania genotyping studiesand reinforce the idea that in some cases especially whenanalyzing strains of very close geographical origin it isthe only molecular marker capable of producing detectablepatterns of polymorphism [24 32]

All these genotyping studies on L infantum suggest thatthe nuclear genomic variability of this species is likely tobe low Our hypothesis is that the kinetoplast genome canserve as a source of genetic variability for these parasitesThe kDNA minicircles are essential for the function of thetrypanosomatidrsquos mitochondrial genes as minicircles codefor guide RNAs which play an essential role in editingmessenger RNA (mRNA) from the maxicircles that containgenes for essential mitochondrial proteins [33] ThereforethisDNA ismore prone to a rapid response to diverse ambient

conditions and stress situations and parasite fitness confer-ring different selective advantages might depend on whichminicircle classes prevail in different Leishmania strains

A similar phenomenon known as transkinetoplastidyhas been described in Leishmania and is responsible forchanges in minicircles classes when the parasites are chal-lenged with increasing concentrations of drugs that arenormally lethal This will in turn cause a dramatic changein the abundance of certain minicircles classes which duringtranskinetoplastidy will be increased or reduced and replacedby a previously less frequent class [34]

When we look at sandfly genetic analysis we can clearlyobserve a main haplotype (H2) comprising all individualsfrom Andradina and Birigui 13 out of 14 individuals fromCampo Grande 6 out of 7 individuals from Corumba 20 outof 30 individuals from Aracatuba and 11 out 30 individualsfrom TeresinaThere is also a major haplotype (H8) compris-ing only individuals from Teresina (13 out of 29) and minorhaplotypes from Aracatuba From the 12S rDNA sequencingdata it was not possible to differentiate Lu longipalpis fromLu cruzi (Corumba) since there was no haplotype clusteringamong Corumba sandflies This may suggest that the processof speciation is recent or still occurring A microsatellite


based study assessing the genetic variability of Lu longipalpisand Lu cruzi populations inMatoGrosso do Sul State showedevidence of introgression and limited gene flow between thetwo species corroborating our findings [12]

In general we can summarize the data obtained fromhaplotyping as follows a major haplotype composed of 111individuals (comprising 89 of SP 90 of MS and 38 ofPI individuals) a main haplotype composed of 13 individualsexclusively from Teresina and giving rise to other 4 Teresinaexclusive haplotypes (62 of individuals from Teresina withexclusive haplotypes) minor haplotypes comprising onlyindividuals from SP (11 total) and from the same locality(Aracatuba)

When we compare data from parasite genotyping withsandfly 12S rDNA sequencing the correlation of the twodatasets is remarkable Both show most samples from PIclearly separated from the MS and SP ones which are inturn much more related to each other when compared toPI that presented the highest haplotypic diversity (Table 1)The exception comes from the minor vector haplotypesonly found in Aracatuba samples Aracatuba represents animportant landmark in the natural history of VL in SP giventhe fact that the first VL outbreak registered in the stateoccurred in this location [35 36] This could be a possibleexplanation to its greater number of unique haplotypes as onecan assume that coevolution between parasites and vectorshappens for a longer time in this area this is supportedby the high haplotypic diversity found for this population(Table 1) Taken together these data corroborate that thesandfly vector probably plays an important role in shapingthe genetic structure of L infantum in Brazil as described byFerreira et al [9]

This work presents new insights towards the under-standing of the population structure of L infantum and Lulongipalpis fromVL endemic areas in Brazil Further analyseswill be needed to elucidate how different vector populationsshape the genetic variability of L infantum

5 Conclusions

Taken together our data indicate that the sandfly vectormight play a role in selecting specific parasite strains ata regional level and therefore contributing to the geneticstructure of L infantum in Brazil Assessing the geneticstructure of both vector and parasite populations may helpus to understand the evolution process surrounding vector-parasite interactions and shed light on a fundamental aspectof the ecoepidemiology of American visceral leishmaniasis

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors would like to acknowledge the Fundacao deAmparo a Pesquisa do Estado de Sao Paulo (Research Grant

200910030-9 to Paulo Eduardo Martins Ribolla and PhDFellowship 200661151-2 to Diego Peres Alonso)

References

[1] P Desjeux ldquoLeishmaniasis current situation and new perspec-tivesrdquo Comparative Immunology Microbiology and InfectiousDiseases vol 27 no 5 pp 305ndash318 2004

[2] J Alvar I D Velez C Bern et al ldquoLeishmaniasis worldwideand global estimates of its incidencerdquo PLoS ONE vol 7 no 5Article ID e35671 2012

[3] M E C Dorval E T Oshiro E Cupollilo A C C Castro andT P Alves ldquoOcorrencia de leishmaniose tegumentar americanano Estado do Mato Grosso do Sul associada a infeccao porLeishmania (Leishmania) amazonensisrdquo Revista da SociedadeBrasileira de Medicina Tropical vol 39 no 1 pp 43ndash46 2006

[4] O Mangabeira Filho ldquoSobre a sistematica e biologia dosPhlebotomus do Cearardquo Revista Brasileira de Malariologia eDoencas Tropicais vol 21 pp 3ndash26 1969

[5] R D Ward A L Ribeiro P D Ready and A MurtaghldquoReproductive isolation between different forms of Lutzomyialongipalpis (Lutz amp Neiva) (Diptera Psychodidae) the vectorof Leishmania donovani chagasi Cunha amp Chagas and its signif-icance to kala-azar distribution in South Americardquo Memoriasdo Instituto Oswaldo Cruz vol 78 no 3 pp 269ndash280 1983

[6] G L Werneck ldquoGeographic spread of visceral leishmaniasis inBrazilrdquo Cadernos de Saude Publica vol 26 no 4 pp 644ndash6452010

[7] M E JWoolhouse L H Taylor and D T Haydon ldquoPopulationbiology of multihost pathogensrdquo Science vol 292 no 5519 pp1109ndash1112 2001

[8] Y J-F Garin A Sulahian F Pratlong et al ldquoVirulence ofLeishmania infantum is expressed as a clonal and dominantphenotype in experimental infectionsrdquo Infection and Immunityvol 69 no 12 pp 7365ndash7373 2001

[9] G E M Ferreira B N dos Santos M E C Dorval et alldquoThe genetic structure of leishmania infantum populations inBrazil and its possible association with the transmission cycleof visceral leishmaniasisrdquo PLoS ONE vol 7 no 5 Article IDe36242 2012

[10] M Segatto L S Ribeiro D L Costa et al ldquoGenetic diversity ofLeishmania infantum field populations from BrazilrdquoMemoriasdo Instituto Oswaldo Cruz vol 107 no 1 pp 39ndash47 2012

[11] L F D S Batista M Segatto C E S Guedes et al ldquoAnassessment of the genetic diversity of Leishmania infantumisolates from infected dogs in Brazilrdquo American Journal ofTropical Medicine and Hygiene vol 86 no 5 pp 799ndash806 2012

[12] M F C Santos P E M Ribolla D P Alonso et al ldquoGeneticstructure of lutzomyia longipalpis populations in Mato Grossodo Sul Brazil based on microsatellite markersrdquo PLoS ONE vol8 no 9 Article ID e74268 2013

[13] A S Araki F M Vigoder L G Bauzer et al ldquoMolecularand behavioral differentiation among Brazilian populations ofLutzomyia longipalpis (Diptera Psychodidae Phlebotominae)rdquoPLoS Neglected Tropical Diseases vol 3 no 1 article e365 2009

[14] V de Queiroz Balbino I V Coutinho-Abreu I V Sonodaet al ldquoGenetic structure of natural populations of the sandfly Lutzomyia longipalpis (Diptera Psychodidae) from theBrazilian northeastern regionrdquo Acta Tropica vol 98 no 1 pp15ndash24 2006


[15] D P Alonso D L Costa I L De Mendonca C H NCosta and P E M Ribolla ldquoShort report heterogeneity ofLeishmania infantum chagasi kinetoplast DNA in Teresina(Brazil)rdquo American Journal of Tropical Medicine and Hygienevol 82 no 5 pp 819ndash821 2010

[16] L Beati A G Caceres J A Lee and L E MunstermannldquoSystematic relationships among Lutzomyia sand flies (DipteraPsychodidae) of Peru and Colombia based on the analysis of12S and 28S ribosomal DNA sequencesrdquo International Journalfor Parasitology vol 34 no 2 pp 225ndash234 2004

[17] H Motazedian M Karamian H A Noyes and S Arde-hali ldquoDNA extraction and amplification of Leishmania fromarchived Giemsa-stained slides for the diagnosis of cutaneousleishmaniasis by PCRrdquo Annals of Tropical Medicine and Para-sitology vol 96 no 1 pp 31ndash34 2002

[18] A M Aransay E Scoulica and Y Tselentis ldquoDetection andidentification of Leishmania DNA within naturally infectedsand flies by seminested PCR onminicircle kinetoplastic DNArdquoApplied and Environmental Microbiology vol 66 no 5 pp1933ndash1938 2000

[19] N Kebede S Oghumu A Worku A Hailu S Varikutiand A R Satoskar ldquoMultilocus microsatellite signature andidentification of specific molecular markers for Leishmaniaaethiopicardquo Parasites and Vectors vol 6 article 160 2013

[20] J Alvar P Aparicio A Aseffa et al ldquoThe relationship betweenleishmaniasis and AIDS the second 10 yearsrdquo Clinical Microbi-ology Reviews vol 21 no 2 pp 334ndash359 2008

[21] M Gramiccia and L Gradoni ldquoThe current status of zoonoticleishmaniases and approaches to disease controlrdquo InternationalJournal for Parasitology vol 35 no 11-12 pp 1169ndash1180 2005

[22] A Toledo J Martın-Sanchez B Pesson C Sanchiz-Marın andF Morillas-Marquez ldquoGenetic variability within the speciesLeishmania infantum by RAPD A lack of correlation withzymodeme structurerdquo Molecular and Biochemical Parasitologyvol 119 no 2 pp 257ndash264 2002

[23] E Cupolillo L R Brahim C B Toaldo et al ldquoGenetic polymor-phism and molecular epidemiology of Leishmania (Viannia)braziliensis from different hosts and geographic areas in BrazilrdquoJournal of Clinical Microbiology vol 41 no 7 pp 3126ndash31322003

[24] T Laurent S Rijal V Yardley et al ldquoEpidemiological dynamicsof antimonial resistance in Leishmania donovani genotypingreveals a polyclonal population structure among naturally-resistant clinical isolates from Nepalrdquo Infection Genetics andEvolution vol 7 no 2 pp 206ndash212 2007

[25] J-P Gangneux J Menotti F Lorenzo et al ldquoProspective valueof PCR amplification and sequencing for diagnosis and typingof Old World Leishmania infections in an area of nonendemic-ityrdquo Journal of ClinicalMicrobiology vol 41 no 4 pp 1419ndash14222003

[26] M B Jamjoom R W Ashford P A Bates S J Kemp and HA Noyes ldquoTowards a standard battery ofmicrosatellite markersfor the analysis of the Leishmania donovani complexrdquo Annals ofTropical Medicine and Parasitology vol 96 no 3 pp 265ndash2702002

[27] B Bulle L Millon J-M Bart et al ldquoPractical approachfor typing strains of Leishmania infantum by microsatelliteanalysisrdquo Journal of Clinical Microbiology vol 40 no 9 pp3391ndash3397 2002

[28] S Ochsenreither K Kuhls M Schaar W Presber and GSchonian ldquoMultilocus microsatellite typing as a new tool for

discrimination of Leishmania infantumMON-1 strainsrdquo Journalof Clinical Microbiology vol 44 no 2 pp 495ndash503 2006

[29] L Montoya M Gallego B Gavignet et al ldquoApplication ofmicrosatellite genotyping to the study of a restricted Leishmaniainfantum focus different genotype compositions in isolatesfrom dogs and sand fliesrdquo The American Journal of TropicalMedicine and Hygiene vol 76 no 5 pp 888ndash895 2007

[30] A Nasereddin K Azmi C L Jaffe et al ldquoKinetoplast DNAheterogeneity among Leishmania infantum strains in centralIsrael and Palestinerdquo Veterinary Parasitology vol 161 no 1-2pp 126ndash130 2009

[31] Y Botilde T Laurent W Q Tintaya et al ldquoComparison ofmolecular markers for strain typing of Leishmania infantumrdquoInfection Genetics and Evolution vol 6 no 6 pp 440ndash4462006

[32] G Van der Auwera N R Bhattarai S Odiwuor and MVuylsteke ldquoRemarks on identification of amplified fragmentlength polymorphisms linked to SAG resistance in LeishmaniardquoActa Tropica vol 113 no 1 pp 92ndash93 2010

[33] B Liu Y Liu S A Motyka E E C Agbo and P T EnglundldquoFellowship of the rings the replication of kinetoplast DNArdquoTrends in Parasitology vol 21 no 8 pp 363ndash369 2005

[34] T A Shapiro and P T Englund ldquoThe structure and replicationof kinetoplast DNArdquoAnnual Review ofMicrobiology vol 49 pp117ndash143 1995

[35] V L F de Camargo-Neves and G Katz ldquoLeishmaniose visceralamericana no Estado de Sao Paulordquo Revista da SociedadeBrasileira de Medicina Tropical vol 32 supplement 2 pp 63ndash64 1999

[36] M Z Galimberti G Katz V L F de Camargo-Neves et alldquoLeishmaniose visceral americana no Estado de Sao PaulordquoRevista da Sociedade Brasileira de Medicina Tropical vol 32supplement 1 pp 217ndash218 1999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


as an anthroponosis when humans are the only source ofparasites for sandflies Leishmaniasis is widely spread in 98countries and 3 territories from which more than 70 aredeveloping countries and 13 are among the least developedones [1]

Visceral leishmaniasis can be either an anthroponosis(eg in the Indian subcontinent) or a zoonosis (eg in theMediterranean or in the Americas) and it is characterizedby chronic evolution and systemic involvement which ifuntreated may result in death In the Americas Leishmaniainfantum is the etiological agent of the disease and Brazilaccounts for over 90 of the cases in the continent [12] Domestic dogs are the proven reservoir hosts in ruraland urban areas while the role of naturally infected wildmammals (canids and marsupials) as L infantum reservoirhosts is still controversial [3] The main sandfly vector isLutzomyia longipalpis but other Lutzomyia species mightplay a role in disease transmission for example in CorumbaMato Grosso do Sul naturally Leishmania-infected Lu cruzihave been discovered and because there is still no evidenceof Lu longipalpis in this region that sandfly is considered themain vector [4 5]

In Brazil VL typically occurred in rural settings butsince 1980 its incidence has been changing due to widespreadurban outbreaks The first major VL urban epidemic inthe country happened in Teresina Piauı State Since thenepidemics occurred in Natal (Rio Grande do Norte) andSao Luıs (Maranhao) and the disease subsequently spreadto other regions of the country Autochthonous cases wererecently described for the first time in the southernmost Stateof RioGrande do SulThe current epidemiological scenario ofVL leaves no doubt regarding the severity of the situation andthe unchecked geographic spread of the disease In the 1990sonly 10 of the cases occurred outside the Northeast Regionbut in 2007 the proportion reached 50 of cases From 2006to 2008 autochthonous transmission of VL was reported inmore than 1200 municipalities in 21 states [6]

The broad spectrum of leishmaniasis-associated symp-toms coupled with the wide diversity of vertebrate andinvertebrate host species suggests that both parasitesrsquo andhostsrsquo genetic backgrounds determine the patterns of thedisease [7] On the other hand clonal diversity and geneticheterogeneity which can cause variability in parasite viru-lence are quite common in Leishmania [8]

Several studies showed that genetic variability of Linfantum in Brazil is low with restricted diversity and limitedpopulation clustering In a recent work assessing parasitepopulations distributed over 18 states three major clusteredpopulations could be inferred using microsatellite typingWhen the analysis is performed in parasites from closelyrelated geographic regions the overall diversity is even lower[9ndash11]

When we look at sandfly genetic variability there iscompelling evidence that the Lutzomyia population structurein Brazil is complex with different genotypes identifieddepending on the geographic region assessed and also thespecies involved in parasite transmission [12ndash14]

Based on these studies it is logical to hypothesize that theinteractions of L infantum genotypic variants with different

hosts and vector populations may ultimately influence thetransmission dynamics and severity of eventual outbreaksHence assessing the genetic structure of both vector popula-tions and parasitesmay help us to understand the dynamics ofvector-parasite interactions and the epidemiological aspectsof American visceral leishmaniasis Here we used PCR-RFLP of kinetoplast minicircle DNA (kDNA) to identify Linfantum genotypic variants from three VL endemic areasin Brazil Teresina in Piauı State Campo Grande in MatoGrosso do Sul State and Bauru in Sao Paulo State kDNA-RFLP analysis when compared to microsatellite genotypinghas proven to be more sensitive to examine genetic data ofclosely related sympatric L infantum strains [15] In additionin order to identify different haplotypes of Lu longipalpisand Lu cruzi sandflies from those three VL endemic areaswe used mitochondrial 12S rDNA sequencing As a maternalinheritance rapidly evolving nonrecombinant and haploidmolecular marker 12S rDNA is suitable to trace genealogyand evolutionary history To our knowledge this is the firststudy that seeks to compare genetic variability of Leishmaniainfantum parasites to the genetic structure of its vectors inBrazil

2 Methods

21 Ethics Statement For insect collections in Mato Grossodo Sul State we obtained a permanent license for collectingand transporting zoological material N∘ 25592-1 on behalf ofDr Alessandra Gutierrez de Oliveira issued by the Systemof Authorization and Information on Biodiversity of theBrazilian Institute of Environment and Renewable NaturalResources (SisbioIBAMA) For insect collections in SaoPaulo State and Piauı State no specific permissions wererequired since the specimens were kindly provided by theCenter for the Control of Endemic Diseases (SUCEN) andFederal Piauı State University respectively The collectionswere performed at private residences whose owners person-ally granted permission to enter their backyards to capture thesandflies All of these residences were located in urban areasand no endangered or protected species were collected in thisstudy

22 Sandfly Collections Sandflies were captured by bothmanual collection and electric traps Manual collection wasperformed with electric aspirators restricting the use of aCastro catcher to locations where aspiration could not beusedThe selected collection points were preassessed in orderto establish the best capturing location in the peridomicile Ateach selected point modified CDC light traps were installedfrom 6 pm to 6 am

The collections took place in different areas in Braziland were performed by the respective local teams Sao Paulo(SP) State performed by Center for the Control of EndemicDiseases (SUCEN) Piauı (PI) State performed by PiauıFederal University and Mato Grosso do Sul (MS) Statecarried out by Mato Grosso do Sul Federal University

Lu cruzi was collected in Corumba (MS) and Lu longi-palpis in all other places Campo Grande (MS) Teresina



Populationssampled



diversity
















Table 2 Continued



Table 2 Continued







3 Results


PI

MS

SP

Teresina

CampoAndradina

BiriguiBauru


Araccedilatuba

Corumb a Grande















4 Discussion


H2

H8

H9

H10

H11

H12

H13

H5

H6

H3

H7

H4

H1

110

1341

Teresina








25

20

15

10

05

00

TER8

3TE

R80

TER3

3

TER2

5TE

R39

TER8

4

CGR9

1TE

R07

TER3

5

TER2

2TE

R10

TER0

1

TER1

2TE

R04

TER2

5

TER8

8TE

R06

TER0

9

TER8

5TE

R86

TER8

7

TER7

6TE

R77

TER8

1

TER7

3TE

R74

TER7

5

TER7

0TE

R71

TER7

2

TER6

7TE

R68

TER6

9

TER6

4TE

R65

TER6

6

TER6

1TE

R62

TER6

3

TER5

9TE

R58

TER6

0

TER5

5TE

R56

TER5

7

TER5

2TE

R53

TER5

4

TER4

9TE

R50

TER5

1

TER4

6TE

R47

TER4

8

TER4

3TE

R44

TER4

5

TER1

4TE

R15

TER2

4

TER0

2TE

R11

TER1

6

TER2

1TE

R30

TER0

3

TER3

6TE

R08

CGR8

9

TER2

6TE

R32

TER3

1

TER3

4TE

R27

TER3

7TE

R41

TER3

8

TER0

5TE

R29

TER4

2

TER1

7TE

R13

TER1

9

TER4

0TE

R28

TER2

0

CGR1

09CG

R138

CGR9

0

TER8

2TE

R79

CGR1

20

CGR1

25CG

R100

CGR9

7

CGR1

06CG

R123

CGR1

35

CGR1

37CG

R136

CGR1

17

BAU

144

BAU

145

BAU

146

CGR9

3CG

R92

BAU

143

CGR9

6CG

R95

CGR9

4

CGR1

07TE

R16

TER7

8

CGR1

01CG

R102

CGR1

14

BAU

155

BAU

156

CGR1

11

BAU

149

BAU

151

BAU

152

CGR1

31BA

U14

7BA

U14

8

CGR1

21CG

R113

CGR1

22

CGR1

18CG

R130

CGR1

04

CGR1

05CG

R103

CGR1

16

CGR1

24CG

R127

CGR1

15

CGR1

41CG

R140

CGR1

26

CGR9

9CG

R96

CGR1

10

CGR1

34CG

R142

CGR1

19

CGR1

12CG

R133

CGR1

32

BAU

154

BAU

157

CGR1

08

CGR1

39BA

U15

0BA

U15

3

CGR1

28CG

R129












5 Conclusions


Competing Interests


Acknowledgments



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Populationssampled



diversity
















Table 2 Continued



Table 2 Continued







3 Results


PI

MS

SP

Teresina

CampoAndradina

BiriguiBauru


Araccedilatuba

Corumb a Grande















4 Discussion


H2

H8

H9

H10

H11

H12

H13

H5

H6

H3

H7

H4

H1

110

1341

Teresina








25

20

15

10

05

00

TER8

3TE

R80

TER3

3

TER2

5TE

R39

TER8

4

CGR9

1TE

R07

TER3

5

TER2

2TE

R10

TER0

1

TER1

2TE

R04

TER2

5

TER8

8TE

R06

TER0

9

TER8

5TE

R86

TER8

7

TER7

6TE

R77

TER8

1

TER7

3TE

R74

TER7

5

TER7

0TE

R71

TER7

2

TER6

7TE

R68

TER6

9

TER6

4TE

R65

TER6

6

TER6

1TE

R62

TER6

3

TER5

9TE

R58

TER6

0

TER5

5TE

R56

TER5

7

TER5

2TE

R53

TER5

4

TER4

9TE

R50

TER5

1

TER4

6TE

R47

TER4

8

TER4

3TE

R44

TER4

5

TER1

4TE

R15

TER2

4

TER0

2TE

R11

TER1

6

TER2

1TE

R30

TER0

3

TER3

6TE

R08

CGR8

9

TER2

6TE

R32

TER3

1

TER3

4TE

R27

TER3

7TE

R41

TER3

8

TER0

5TE

R29

TER4

2

TER1

7TE

R13

TER1

9

TER4

0TE

R28

TER2

0

CGR1

09CG

R138

CGR9

0

TER8

2TE

R79

CGR1

20

CGR1

25CG

R100

CGR9

7

CGR1

06CG

R123

CGR1

35

CGR1

37CG

R136

CGR1

17

BAU

144

BAU

145

BAU

146

CGR9

3CG

R92

BAU

143

CGR9

6CG

R95

CGR9

4

CGR1

07TE

R16

TER7

8

CGR1

01CG

R102

CGR1

14

BAU

155

BAU

156

CGR1

11

BAU

149

BAU

151

BAU

152

CGR1

31BA

U14

7BA

U14

8

CGR1

21CG

R113

CGR1

22

CGR1

18CG

R130

CGR1

04

CGR1

05CG

R103

CGR1

16

CGR1

24CG

R127

CGR1

15

CGR1

41CG

R140

CGR1

26

CGR9

9CG

R96

CGR1

10

CGR1

34CG

R142

CGR1

19

CGR1

12CG

R133

CGR1

32

BAU

154

BAU

157

CGR1

08

CGR1

39BA

U15

0BA

U15

3

CGR1

28CG

R129












5 Conclusions


Competing Interests


Acknowledgments



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Table 2 Continued



Table 2 Continued







3 Results


PI

MS

SP

Teresina

CampoAndradina

BiriguiBauru


Araccedilatuba

Corumb a Grande















4 Discussion


H2

H8

H9

H10

H11

H12

H13

H5

H6

H3

H7

H4

H1

110

1341

Teresina








25

20

15

10

05

00

TER8

3TE

R80

TER3

3

TER2

5TE

R39

TER8

4

CGR9

1TE

R07

TER3

5

TER2

2TE

R10

TER0

1

TER1

2TE

R04

TER2

5

TER8

8TE

R06

TER0

9

TER8

5TE

R86

TER8

7

TER7

6TE

R77

TER8

1

TER7

3TE

R74

TER7

5

TER7

0TE

R71

TER7

2

TER6

7TE

R68

TER6

9

TER6

4TE

R65

TER6

6

TER6

1TE

R62

TER6

3

TER5

9TE

R58

TER6

0

TER5

5TE

R56

TER5

7

TER5

2TE

R53

TER5

4

TER4

9TE

R50

TER5

1

TER4

6TE

R47

TER4

8

TER4

3TE

R44

TER4

5

TER1

4TE

R15

TER2

4

TER0

2TE

R11

TER1

6

TER2

1TE

R30

TER0

3

TER3

6TE

R08

CGR8

9

TER2

6TE

R32

TER3

1

TER3

4TE

R27

TER3

7TE

R41

TER3

8

TER0

5TE

R29

TER4

2

TER1

7TE

R13

TER1

9

TER4

0TE

R28

TER2

0

CGR1

09CG

R138

CGR9

0

TER8

2TE

R79

CGR1

20

CGR1

25CG

R100

CGR9

7

CGR1

06CG

R123

CGR1

35

CGR1

37CG

R136

CGR1

17

BAU

144

BAU

145

BAU

146

CGR9

3CG

R92

BAU

143

CGR9

6CG

R95

CGR9

4

CGR1

07TE

R16

TER7

8

CGR1

01CG

R102

CGR1

14

BAU

155

BAU

156

CGR1

11

BAU

149

BAU

151

BAU

152

CGR1

31BA

U14

7BA

U14

8

CGR1

21CG

R113

CGR1

22

CGR1

18CG

R130

CGR1

04

CGR1

05CG

R103

CGR1

16

CGR1

24CG

R127

CGR1

15

CGR1

41CG

R140

CGR1

26

CGR9

9CG

R96

CGR1

10

CGR1

34CG

R142

CGR1

19

CGR1

12CG

R133

CGR1

32

BAU

154

BAU

157

CGR1

08

CGR1

39BA

U15

0BA

U15

3

CGR1

28CG

R129












5 Conclusions


Competing Interests


Acknowledgments



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 2 Continued







3 Results


PI

MS

SP

Teresina

CampoAndradina

BiriguiBauru


Araccedilatuba

Corumb a Grande















4 Discussion


H2

H8

H9

H10

H11

H12

H13

H5

H6

H3

H7

H4

H1

110

1341

Teresina








25

20

15

10

05

00

TER8

3TE

R80

TER3

3

TER2

5TE

R39

TER8

4

CGR9

1TE

R07

TER3

5

TER2

2TE

R10

TER0

1

TER1

2TE

R04

TER2

5

TER8

8TE

R06

TER0

9

TER8

5TE

R86

TER8

7

TER7

6TE

R77

TER8

1

TER7

3TE

R74

TER7

5

TER7

0TE

R71

TER7

2

TER6

7TE

R68

TER6

9

TER6

4TE

R65

TER6

6

TER6

1TE

R62

TER6

3

TER5

9TE

R58

TER6

0

TER5

5TE

R56

TER5

7

TER5

2TE

R53

TER5

4

TER4

9TE

R50

TER5

1

TER4

6TE

R47

TER4

8

TER4

3TE

R44

TER4

5

TER1

4TE

R15

TER2

4

TER0

2TE

R11

TER1

6

TER2

1TE

R30

TER0

3

TER3

6TE

R08

CGR8

9

TER2

6TE

R32

TER3

1

TER3

4TE

R27

TER3

7TE

R41

TER3

8

TER0

5TE

R29

TER4

2

TER1

7TE

R13

TER1

9

TER4

0TE

R28

TER2

0

CGR1

09CG

R138

CGR9

0

TER8

2TE

R79

CGR1

20

CGR1

25CG

R100

CGR9

7

CGR1

06CG

R123

CGR1

35

CGR1

37CG

R136

CGR1

17

BAU

144

BAU

145

BAU

146

CGR9

3CG

R92

BAU

143

CGR9

6CG

R95

CGR9

4

CGR1

07TE

R16

TER7

8

CGR1

01CG

R102

CGR1

14

BAU

155

BAU

156

CGR1

11

BAU

149

BAU

151

BAU

152

CGR1

31BA

U14

7BA

U14

8

CGR1

21CG

R113

CGR1

22

CGR1

18CG

R130

CGR1

04

CGR1

05CG

R103

CGR1

16

CGR1

24CG

R127

CGR1

15

CGR1

41CG

R140

CGR1

26

CGR9

9CG

R96

CGR1

10

CGR1

34CG

R142

CGR1

19

CGR1

12CG

R133

CGR1

32

BAU

154

BAU

157

CGR1

08

CGR1

39BA

U15

0BA

U15

3

CGR1

28CG

R129












5 Conclusions


Competing Interests


Acknowledgments



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






3 Results


PI

MS

SP

Teresina

CampoAndradina

BiriguiBauru


Araccedilatuba

Corumb a Grande















4 Discussion


H2

H8

H9

H10

H11

H12

H13

H5

H6

H3

H7

H4

H1

110

1341

Teresina








25

20

15

10

05

00

TER8

3TE

R80

TER3

3

TER2

5TE

R39

TER8

4

CGR9

1TE

R07

TER3

5

TER2

2TE

R10

TER0

1

TER1

2TE

R04

TER2

5

TER8

8TE

R06

TER0

9

TER8

5TE

R86

TER8

7

TER7

6TE

R77

TER8

1

TER7

3TE

R74

TER7

5

TER7

0TE

R71

TER7

2

TER6

7TE

R68

TER6

9

TER6

4TE

R65

TER6

6

TER6

1TE

R62

TER6

3

TER5

9TE

R58

TER6

0

TER5

5TE

R56

TER5

7

TER5

2TE

R53

TER5

4

TER4

9TE

R50

TER5

1

TER4

6TE

R47

TER4

8

TER4

3TE

R44

TER4

5

TER1

4TE

R15

TER2

4

TER0

2TE

R11

TER1

6

TER2

1TE

R30

TER0

3

TER3

6TE

R08

CGR8

9

TER2

6TE

R32

TER3

1

TER3

4TE

R27

TER3

7TE

R41

TER3

8

TER0

5TE

R29

TER4

2

TER1

7TE

R13

TER1

9

TER4

0TE

R28

TER2

0

CGR1

09CG

R138

CGR9

0

TER8

2TE

R79

CGR1

20

CGR1

25CG

R100

CGR9

7

CGR1

06CG

R123

CGR1

35

CGR1

37CG

R136

CGR1

17

BAU

144

BAU

145

BAU

146

CGR9

3CG

R92

BAU

143

CGR9

6CG

R95

CGR9

4

CGR1

07TE

R16

TER7

8

CGR1

01CG

R102

CGR1

14

BAU

155

BAU

156

CGR1

11

BAU

149

BAU

151

BAU

152

CGR1

31BA

U14

7BA

U14

8

CGR1

21CG

R113

CGR1

22

CGR1

18CG

R130

CGR1

04

CGR1

05CG

R103

CGR1

16

CGR1

24CG

R127

CGR1

15

CGR1

41CG

R140

CGR1

26

CGR9

9CG

R96

CGR1

10

CGR1

34CG

R142

CGR1

19

CGR1

12CG

R133

CGR1

32

BAU

154

BAU

157

CGR1

08

CGR1

39BA

U15

0BA

U15

3

CGR1

28CG

R129












5 Conclusions


Competing Interests


Acknowledgments



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













4 Discussion


H2

H8

H9

H10

H11

H12

H13

H5

H6

H3

H7

H4

H1

110

1341

Teresina








25

20

15

10

05

00

TER8

3TE

R80

TER3

3

TER2

5TE

R39

TER8

4

CGR9

1TE

R07

TER3

5

TER2

2TE

R10

TER0

1

TER1

2TE

R04

TER2

5

TER8

8TE

R06

TER0

9

TER8

5TE

R86

TER8

7

TER7

6TE

R77

TER8

1

TER7

3TE

R74

TER7

5

TER7

0TE

R71

TER7

2

TER6

7TE

R68

TER6

9

TER6

4TE

R65

TER6

6

TER6

1TE

R62

TER6

3

TER5

9TE

R58

TER6

0

TER5

5TE

R56

TER5

7

TER5

2TE

R53

TER5

4

TER4

9TE

R50

TER5

1

TER4

6TE

R47

TER4

8

TER4

3TE

R44

TER4

5

TER1

4TE

R15

TER2

4

TER0

2TE

R11

TER1

6

TER2

1TE

R30

TER0

3

TER3

6TE

R08

CGR8

9

TER2

6TE

R32

TER3

1

TER3

4TE

R27

TER3

7TE

R41

TER3

8

TER0

5TE

R29

TER4

2

TER1

7TE

R13

TER1

9

TER4

0TE

R28

TER2

0

CGR1

09CG

R138

CGR9

0

TER8

2TE

R79

CGR1

20

CGR1

25CG

R100

CGR9

7

CGR1

06CG

R123

CGR1

35

CGR1

37CG

R136

CGR1

17

BAU

144

BAU

145

BAU

146

CGR9

3CG

R92

BAU

143

CGR9

6CG

R95

CGR9

4

CGR1

07TE

R16

TER7

8

CGR1

01CG

R102

CGR1

14

BAU

155

BAU

156

CGR1

11

BAU

149

BAU

151

BAU

152

CGR1

31BA

U14

7BA

U14

8

CGR1

21CG

R113

CGR1

22

CGR1

18CG

R130

CGR1

04

CGR1

05CG

R103

CGR1

16

CGR1

24CG

R127

CGR1

15

CGR1

41CG

R140

CGR1

26

CGR9

9CG

R96

CGR1

10

CGR1

34CG

R142

CGR1

19

CGR1

12CG

R133

CGR1

32

BAU

154

BAU

157

CGR1

08

CGR1

39BA

U15

0BA

U15

3

CGR1

28CG

R129












5 Conclusions


Competing Interests


Acknowledgments



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



25

20

15

10

05

00

TER8

3TE

R80

TER3

3

TER2

5TE

R39

TER8

4

CGR9

1TE

R07

TER3

5

TER2

2TE

R10

TER0

1

TER1

2TE

R04

TER2

5

TER8

8TE

R06

TER0

9

TER8

5TE

R86

TER8

7

TER7

6TE

R77

TER8

1

TER7

3TE

R74

TER7

5

TER7

0TE

R71

TER7

2

TER6

7TE

R68

TER6

9

TER6

4TE

R65

TER6

6

TER6

1TE

R62

TER6

3

TER5

9TE

R58

TER6

0

TER5

5TE

R56

TER5

7

TER5

2TE

R53

TER5

4

TER4

9TE

R50

TER5

1

TER4

6TE

R47

TER4

8

TER4

3TE

R44

TER4

5

TER1

4TE

R15

TER2

4

TER0

2TE

R11

TER1

6

TER2

1TE

R30

TER0

3

TER3

6TE

R08

CGR8

9

TER2

6TE

R32

TER3

1

TER3

4TE

R27

TER3

7TE

R41

TER3

8

TER0

5TE

R29

TER4

2

TER1

7TE

R13

TER1

9

TER4

0TE

R28

TER2

0

CGR1

09CG

R138

CGR9

0

TER8

2TE

R79

CGR1

20

CGR1

25CG

R100

CGR9

7

CGR1

06CG

R123

CGR1

35

CGR1

37CG

R136

CGR1

17

BAU

144

BAU

145

BAU

146

CGR9

3CG

R92

BAU

143

CGR9

6CG

R95

CGR9

4

CGR1

07TE

R16

TER7

8

CGR1

01CG

R102

CGR1

14

BAU

155

BAU

156

CGR1

11

BAU

149

BAU

151

BAU

152

CGR1

31BA

U14

7BA

U14

8

CGR1

21CG

R113

CGR1

22

CGR1

18CG

R130

CGR1

04

CGR1

05CG

R103

CGR1

16

CGR1

24CG

R127

CGR1

15

CGR1

41CG

R140

CGR1

26

CGR9

9CG

R96

CGR1

10

CGR1

34CG

R142

CGR1

19

CGR1

12CG

R133

CGR1

32

BAU

154

BAU

157

CGR1

08

CGR1

39BA

U15

0BA

U15

3

CGR1

28CG

R129












5 Conclusions


Competing Interests


Acknowledgments



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






5 Conclusions


Competing Interests


Acknowledgments



References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article leishmania infantum genetic diversity and

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