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Previous issue date: 2015-04-23 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Nas Am?ricas, a infec??o por Leishmania tem como principal agente etiol?gico Leishmania infantum. Nos ?ltimos 30 anos o padr?o de distribui??o das leishmanioses tem mudado substancialmente e a doen?a tem apresentado um perfil emergente na periferia dos grandes centros urbanos. A infec??o por Leishmania pode evoluir com um amplo espectro cl?nico desde o acometimento da pele, mucosas e v?sceras. Dos indiv?duos infectados por L. infantum apenas 10% desenvolvem a doen?a, sabe-se que 90% da infec??o humana ? assintom?tica e diversos fatores est?o envolvidos no curso da infec??o. Os principais fatores envolvidos no desenvolvimento da doen?a s?o a resposta imune do hospedeiro, a esp?cie e o conte?do g?nico do parasita. O sequenciamento dos isolados de Leishmania poderia aumentar a compreens?o acerca da sintomatologia dos indiv?duos. Dessa forma, o objetivo desse estudo foi avaliar a diversidade gen?tica de cepas de Leishmania circulantes entre humanos, sintom?ticos e assintom?ticos e c?es de ?reas end?micas do Rio Grande do Norte. A variabilidade gen?tica de um grupo de amostras de humanos e caninos com a doen?a visceral, doen?a cut?nea e infec??o assintom?tica foi avaliado com os marcadores moleculares hsp70 e ITS1. O genoma completo de 20 isolados de Leishmania oriundos de humanos, sintom?ticos e assintom?ticos, e c?es foram sequenciados para avaliar a diversidade dessas amostras. Os fragmentos amplificados de hsp70 e ITS1 das amostras e foram analisados e montadas utilizando o pacote Phred/Phrap. Os dendogramas foram constru?dos aplicando o m?todo neighbor joining com 500 bootstraps, seguido das infer?ncias sobre as rela??es entre as variantes de Leishmania. As sequ?ncias dos 20 isolados brasileiros foram mapeadas com o genoma de refer?ncia Leishmania infantum JPCM5, usando o programa Bowtie2, com identifica??o de 36 contigs. As informa??es dos SNPs v?lidos foram utilizadas na PCA. Os SNPs foram visualizados pelo Geneious 7.1 e IGV. As anota??es do genoma foram ent?o transferidas para seus respectivos cromossomos e visualizadas utilizando o Geneious. As sequ?ncias consenso de todos os cromossomos (com m?nimo de 75% das reads com a mesma base) foram alinhadas usando Mauve. As ?rvores filogen?ticas foram calculadas de acordo com c?lculos de m?xima verossimilhan?a e modelos JTT. Como resultados obtivemos que hsp70 e ITS1 n?o foram capazes de definir variabilidade gen?tica entre os isolados de humanos e c?es; nem para os isolados de cultura e de sangue perif?rico, oriundos de um mesmo paciente.O sequenciamento gen?mico dos 20 isolados brasileiros revelou uma forte rela??o entre as cepas de Leishmania circulantes em no Rio Grande do Norte. Os isolados da Grande Natal de humanos e caninos permaneceram agrupados em todas as an?lises, sugerindo que existe proximidade genot?pica e geogr?fica entre os isolados. As amostras isoladas na d?cada de 1990 apresentaram uma maior diversidade genot?pica quando comparadas as amostras recentemente isoladas. De forma geral, n?o encontramos correla??o entre as formas cl?nicas sintom?ticas e assintom?ticas e o conte?do g?nico dos isolados brasileiros de Leishmania. / Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the
New World. The pattern of distribution of leishmaniasis has changed
substantially and has presented an emerging profile within the periphery of the
Large Urban Centers. Leishmania infection can compromise skin, mucosa and
viscera. Only 10% of the individuals infected develop the disease and 90% of
human infection is asymptomatic. The main factors involved in the development
of the disease are the host immune response, the vector?s species and the
parasite?s genetic content. The sequencing of Leishmania isolated seeks to
increase the understanding of the symptoms of individuals. The aim of this
study was to evaluate the genetic diversity of circulating Leishmania strains
among humans, and symptomatic and asymptomatic, and dogs from endemic
areas of Rio Grande do Norte State and analyze sandflies from endemic areas
for cutaneous and visceral disease. The genetic variability was evaluated by the
use of markers hsp70 , ITS1 and a whole genome sequencing was also carried
out. The amplified hsp70 and ITS1 of samples were analyzed and assembled
using a Phred / Phrap package. The dendograms were constructed using the
same methodology, but adding 500 bootstraps, followed by inferences on the
relationships between Leishmania variants. The sequences of the 20 Brazilian
isolates were mapped to the reference genome L. infantum JPCM5, using the
Bowtie2 program and the identification of 36 contigs. The information of the
valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and
IGV. The genome annotations were transferred to their respective
chromosomes and displayed on Geneious. The matching sequences of all
chromosomes were aligned using Mauve. The phylogenetic trees were
calculated according to maximum likelihood and JTT models. Sandflies were
analyzed by PCR for the identification of Leishmania infection, a blood meal
source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of
identifying genetic variability among human isolates from symptomatic and
asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates
revealed a strong similarity between the circulating Leishmania strains in Rio
Grande do Norte. The isolates collected in the city of Natal from humans and
canines remained grouped in all analyzes, suggesting that there is genotypic
and geographic proximity among the isolates. The isolated samples in the
1990s had a higher genotypic diversity when compared to freshly isolated
samples. All isolates presented 36 chromosomes with variable ploidy among
them, no correlation was found between the number of amastina genes copies,
gp63, A2 and SSG with such clinic forms. In general, we did not find correlation
between symptomatic and asymptomatic clinical forms and the gene content of
the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the
upper west region were L. longipalpis and the main sources of blood meal were
humans, dogs and chickens.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/21157 |
| Date | 23 April 2015 |
| Creators | Silva, Virg?nia Pen?llope Macedo e |
| Contributors | 20007256434, http://lattes.cnpq.br/4231925509338101, Medeiros, Iara Marques de, 36970360415, http://lattes.cnpq.br/0702456215392682, Carvalho, Lucas Pedreira de, 88323145504, http://lattes.cnpq.br/7308383096084856, Moreira, Paula Viviane de Souza Queiroz, 02498237488, Salha, Daniella Regina Arantes Martins, Ximenes, Maria de F?tima Freire de Melo |
| Publisher | Universidade Federal do Rio Grande do Norte, PROGRAMA DE P?S-GRADUA??O EM BIOQU?MICA, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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