Ticks as potential vectors of Mycobacterium leprae: Use of tick cell lines to culture the bacilli and generate transgenic strains.

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation

Genotipagem de isolados de Mycobacterium leprae de pacientes hansenianos do Brasil

Submitted by Anderson Silva ([email protected]) on 2012-10-15T19:20:49Z No. of bitstreams: 1 amanda_n_b_fontes_ioc_bcm_0050_2011.pdf: 3773109 bytes, checksum: 8933f048c4d79d33efa1313c366344a8 (MD5)Made available in DSpace on 2012-10-15T19:20:49Z (GMT). No. of bitstreams: 1 amanda_n_b_fontes_ioc_bcm_0050_2011.pdf: 3773109 bytes, checksum: 8933f048c4d79d33efa1313c366344a8 (MD5) Previous issue date: 2011-05-26Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilA hanseníase é uma doença infecto-contagiosa crônica, causada pelo Mycobacterium leprae. Após o sequenciamento do genoma deste patógeno, inúmeros esforços têm sido feitos na busca por sequências repetitivas com potencial para diferenciar isolados de M. leprae. Atualmente, VNTRs têm sido utilizados com o objetivo de compreender a diversidade genética deste patógeno em áreas com alta prevalência da doença. Além disso, SNPs também têm contribuído para elucidar aspectos referentes à disseminação da hanseníase pelo mundo. Neste estudo, a variabilidade genética de 292 isolados de M. leprae oriundos de amostras clínicas de pacientes hansenianos das regiões norte, nordeste e sudeste do país foi avaliada utilizando 16 VNTRs e três SNPs. O polimorfismo dos diferentes VNTRs foi determinado através de MLVA (Multiple-locus VNTR analysis) utilizando FLA (Fragment lenght analysis), enquanto que os SNPs foram analisados através de PCR-RFLP e/ou sequenciamento. O poder discriminatório dos 16 VNTRs foi de 0.999, sendo que as repetições de dinucleotídeos AT e o trinucleotídeo GAA21 apresentaram os maiores índices de discriminação alélica. Além da genotipagem de isolados de M. leprae de biópsias de pele, material de linfa fixado em lâmina de baciloscopia também foi utilizado como uma fonte alternativa para obtenção de DNA deste bacilo. Com relação à genotipagem por SNP, foi possível observar a predominância do genótipo 3, associado à população européia, em Rondônia, Rio de Janeiro e São Paulo. Já o genótipo 4, oriundo da África Ocidental, foi predominante em Pernambuco e Fortaleza. A presença dos diferentes genótipos e sua predominância em determinadas áreas corroboram com o processo de colonização do país que se reflete na atual composição étnica da população brasileira. Com base nos perfis obtidos através dos VNTRs e SNPs, foram identificados seis grupos geneticamente idênticos: quatro do Ceará, um de Rondônia e outro formado entre amostras de Minas Gerais e São Paulo. Nenhuma associação entre os pacientes enquadrados nos grupos da região norte e sudeste do país foi estabelecida. Todavia, foi possível observar que os grupos foram formados entre indivíduos oriundos de mesmo estado ou região geográfica. Com relação aos grupos formados entre as amostras do Ceará, associações entre o gênero masculino e o local de origem das amostras foram estabelecidas com base nos genótipos de M. leprae, sugerindo que estes seriam fatores de risco para a transmissão da hanseníase. Neste estudo, também foi possível avaliar amostras de pacientes com hanseníase recidiva para quatro VNTRs e os achados sugerem que estes pacientes foram re-infectados ou que houve mudança da população bacteriana durante a recidiva da doença. Os resultados comprovam que a genotipagem de M. leprae é uma ferramenta importante na elucidação de aspectos relativos à transmissão e disseminação da doença pelo mundo.Leprosy is a chronic infectious disease caused by Mycobacterium leprae. After sequencing the genome of this pathogen, numerous efforts have been made to identify repetitive sequences with potential to differentiate isolates of M. leprae. Currently, VNTRs have been used in order to understand the genetic diversity of this pathogen in areas with high prevalence of leprosy. Another form of genetic polymorphism, namely single nucleotide polymorphisms (SNPs), elucidated aspects related to the spread of leprosy in the world. In this study, the genetic variability of 292 M. leprae isolates from clinical leprosy patients from the north, northeast and southeast of the country, were analyzed for composition of 16 VNTRs and three SNPs. The genetic variability of different VNTRs was evaluated by MLVA (Multiple-locus VNTR analysis) using FLA (Fragment length analysis) while the SNPs were analyzed by RFLP-PCR and/or sequencing. The discriminatory power of 16 VNTRs was 0.999 and the AT dinucleotides and the GAA21 trinucleotide demonstrated the higher rates of allelic discrimination. In addition to the genotyping of isolates of M. leprae derived from skin biopsy samples, lymph fixed in ZN slides was also used as an alternative source to obtain DNA. By SNP typing, we observed the high prevalence of genotype 3, previously associated with Europeans, in the states of Rondônia, Rio de Janeiro and São Paulo. On the other hand, genotype 4, originating from Western Africa, was predominant in Pernambuco and Fortaleza. The presence of different genotypes and their prevalence in certain areas of Brazil is in accordance to the country’s history of colonization which reflects in the current ethnic composition of the population. Based on the VNTR and SNP profiles, six identical groups of two isolates each were identified: four from Ceará, one from Rondônia and another composed of samples from Minas Gerais and São Paulo. No association between patients from north and southeast of the country was established, however, most groups were composed by patients from the same state or geographic region. When performing an analysis of genotype similarity with patient data in groups from Ceará, we observed association of male gender and place of origin with M. leprae genotype grouping, suggesting these could be risk factors for transmission of leprosy. In this analysis, patients with leprosy relapse were also analyzed for four VNTRs and the results suggest the occurrence of re-infection or of bacterial population shift during disease relapse. The results of this study demonstrate that genotyping of M. leprae is an important tool to elucidate aspects of transmission and spread of disease in the world

Genetic diversity of Mycobacterium leprae isolates from Brazilian leprosy patients

Submitted by Sandra Infurna ([email protected]) on 2018-09-25T17:17:57Z No. of bitstreams: 1 amanda_fontes_etal_IOC_2009.pdf: 376703 bytes, checksum: ec0c4b7b9b14df59fc4f776733a26280 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-09-25T17:24:17Z (GMT) No. of bitstreams: 1 amanda_fontes_etal_IOC_2009.pdf: 376703 bytes, checksum: ec0c4b7b9b14df59fc4f776733a26280 (MD5)Made available in DSpace on 2018-09-25T17:24:17Z (GMT). No. of bitstreams: 1 amanda_fontes_etal_IOC_2009.pdf: 376703 bytes, checksum: ec0c4b7b9b14df59fc4f776733a26280 (MD5) Previous issue date: 2009Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ. Brasil.Colorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, Colorado, USA.Instituto Lauro de Souza Lima, Bauru. SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.Colorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, Colorado, USA.Colorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, Colorado, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ. Brasil.Introduction Leprosy is a chronic disease caused by infection with Mycobacterium leprae, an obligate intracellular parasite. A problem in studying the transmission of leprosy is the small amount of variation in bacterial genomic DNA. The discovery of variable number of tandem repeats (VNTRs) allowed the detection of strain variation in areas with a high prevalence of leprosy. Four genotypes of M. leprae based on three single-nucleotide polymorphism (SNPs) were also discovered to be useful for analysis of the global spread of leprosy

Genotyping comparison of Mycobacterium leprae

Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil under [grant number CNPq–410573/2006-0].Federal University of Ceará. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal University of Ceará. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brazil.Fundação Osvaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Fundação Osvaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Federal University of Ceará. Departamento de Estatística e Matemática Aplicada. Fortaleza, CE, Brazil.Universidade de Fortaleza. Programa de Pós-Graduação em Saúde Coletiva. Fortaleza, CE, Brazil.Centro de Dermatologia Dona Libânia. Department of the State of Ceará. Fortaleza, CE, Brazil.Centro de Dermatologia Dona Libânia. Department of the State of Ceará. Fortaleza, CE, Brazil.Tulane University. School of Public Health and Tropical Medicine. Department of Global Community Health and Behavioral Sciences. New Orleans , LA , USA.Federal University of Ceará. Faculdade de Medicina. Departamento de Saúde Comunitária. Fortaleza, CE, Brazil.This study analyzed the genetic diversity by MIRU-VNTR of Mycobacterium leprae isolates from nasal cavities and related to epidemiological and clinical data. The sample consisted of 48 newly diagnosed leprosy cases that tested positive for M. leprae PCR in nasal secretion (NS) attending to the National Reference Center of Dermatology Dona Libania (CDERM), Fortaleza, Brazil. Total DNA was extracted from NS of each patient and used for amplification of four M. leprae VNTR loci. Four clusters of M. leprae isolates were formed with identical genotypes. In the spatial analysis, 12 leprosy cases presented similar genotypes organized into 4 clusters. The most common genotypes in the current study was AC8b: 8, AC9: 7, AC8a: 8, GTA9: 10, which may represent a genotype of circulating strains most often in Ceará. A minimum set of four MIRU-VNTR loci was demonstrated to study the genetic diversity of M. leprae isolates from NS

Genotyping comparison of Mycobacterium leprae isolates by VNTR analysis from nasal samples in a Brazilian endemic region

Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil under [grant number CNPq–410573/2006-0].Federal University of Ceará. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal University of Ceará. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brazil.Fundação Osvaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Fundação Osvaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Federal University of Ceará. Departamento de Estatística e Matemática Aplicada. Fortaleza, CE, Brazil.Universidade de Fortaleza. Programa de Pós-Graduação em Saúde Coletiva. Fortaleza, CE, Brazil.Centro de Dermatologia Dona Libânia. Department of the State of Ceará. Fortaleza, CE, Brazil.Centro de Dermatologia Dona Libânia. Department of the State of Ceará. Fortaleza, CE, Brazil.Tulane University. School of Public Health and Tropical Medicine. Department of Global Community Health and Behavioral Sciences. New Orleans , LA , USA.Federal University of Ceará. Faculdade de Medicina. Departamento de Saúde Comunitária. Fortaleza, CE, Brazil.This study analyzed the genetic diversity by MIRU-VNTR of Mycobacterium leprae isolates from nasal cavities and related to epidemiological and clinical data. The sample consisted of 48 newly diagnosed leprosy cases that tested positive for M. leprae PCR in nasal secretion (NS) attending to the National Reference Center of Dermatology Dona Libania (CDERM), Fortaleza, Brazil. Total DNA was extracted from NS of each patient and used for amplification of four M. leprae VNTR loci. Four clusters of M. leprae isolates were formed with identical genotypes. In the spatial analysis, 12 leprosy cases presented similar genotypes organized into 4 clusters. The most common genotypes in the current study was AC8b: 8, AC9: 7, AC8a: 8, GTA9: 10, which may represent a genotype of circulating strains most often in Ceará. A minimum set of four MIRU-VNTR loci was demonstrated to study the genetic diversity of M. leprae isolates from NS

Experimental Infection of Rhodnius prolixus (Hemiptera, Triatominae) with Mycobacterium leprae Indicates Potential for Leprosy Transmission

Submitted by sandra infurna ([email protected]) on 2016-07-05T13:05:51Z No. of bitstreams: 1 arthur_neumann_etal_IOC_2016.pdf: 4675130 bytes, checksum: fad9635aadb783994d9389cd46172170 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-07-05T13:20:09Z (GMT) No. of bitstreams: 1 arthur_neumann_etal_IOC_2016.pdf: 4675130 bytes, checksum: fad9635aadb783994d9389cd46172170 (MD5)Made available in DSpace on 2016-07-05T13:20:09Z (GMT). No. of bitstreams: 1 arthur_neumann_etal_IOC_2016.pdf: 4675130 bytes, checksum: fad9635aadb783994d9389cd46172170 (MD5) Previous issue date: 2016Made available in DSpace on 2016-07-08T12:21:57Z (GMT). No. of bitstreams: 3 arthur_neumann_etal_IOC_2016.pdf.txt: 50556 bytes, checksum: 3a1e7f93efce390704119290369be910 (MD5) arthur_neumann_etal_IOC_2016.pdf: 4675130 bytes, checksum: fad9635aadb783994d9389cd46172170 (MD5) license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Laboratório de Bioquímica de Artrópodes Hematófagos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Instituto Lauro de Sousa Lima. Departmento de Biologia, Bauru, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Laboratório de Bioquímica de Artrópodes Hematófagos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Laboratório de Bioquímica de Artrópodes Hematófagos. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Laboratório de Bioquímica de Artrópodes Hematófagos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Leprosy is a chronic dermato-neurological disease caused by infection with Mycobacterium leprae. In 2013 almost 200,000 new cases of leprosy were detected around the world. Since the first symptoms take from years to decades to appear, the total number of asymptomatic patients is impossible to predict. Although leprosy is one of the oldest records of human disease, the mechanisms involved with its transmission and epidemiology are still not completely understood. In the present work, we experimentally investigated the hypothesis that the mosquitoes Aedes aegypti and Culex quinquefasciatus and the hemiptera Rhodnius prolixus act as leprosy vectors. By means of real-time PCR quantification of M. leprae 16SrRNA, we found that M. leprae remained viable inside the digestive tract of Rhodnius prolixus for 20 days after oral infection. In contrast, in the gut of both mosquito species tested, we were not able to detect M. leprae RNA after a similar period of time. Inside the kissing bug Rhodnius prolixus digestive tract, M. leprae was initially restricted to the anterior midgut, but gradually moved towards the hindgut, in a time course reminiscent of the life cycle of Trypanosoma cruzi, a well-known pathogen transmitted by this insect. The maintenance of M. leprae infectivity inside the digestive tract of this kissing bug is further supported by successful mice footpad inoculation with feces collected 20 days after infection.We conclude that Rhodnius prolixus defecate infective M. leprae, justifying the evaluation of the presence of M. leprae among sylvatic and domestic kissing bugs in countries endemic for leprosy

Serological and molecular detection of infection with Mycobacterium leprae in Brazilian six banded armadillos (Euphractus sexcinctus)

Federal Rural University of Rio de Janeiro. Institute of Veterinary Medicine. Department of Animal Parasitology. Multiuser Molecular Biology Laboratory. Seropédica, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Cellular Microbiology. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Cellular Microbiology. Rio de Janeiro, RJ, Brazil.Federal Rural University of Semi-Árido. Hospital Veterinary. Rio Grande do Norte, RN, Brazil.Federal Rural University of Semi-Árido. Hospital Veterinary. Rio Grande do Norte, RN, Brazil.Federal Rural University of Semi-Árido. Hospital Veterinary. Rio Grande do Norte, RN, Brazil.National Hansen's Disease Program. Healthcare Systems Bureau. Health Resources and Services Administration. Department of Health and Humans Services. Baton Rouge, United States.National Hansen's Disease Program. Healthcare Systems Bureau. Health Resources and Services Administration. Department of Health and Humans Services. Baton Rouge, United States.National Hansen's Disease Program. Healthcare Systems Bureau. Health Resources and Services Administration. Department of Health and Humans Services. Baton Rouge, United States.Infectious Disease Research Institute. Seattle, United States.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Geoprocessamento. Ananindeua, PA, Brasil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Molecular Biology Applied to Mycobacteria. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Molecular Biology Applied to Mycobacteria. Rio de Janeiro, RJ, Brazil.Federal Rural University of Rio de Janeiro. Institute of Veterinary Medicine. Department of Animal Parasitology. Multiuser Molecular Biology Laboratory. Seropédica, RJ, Brazil.Leprosy was recognized as a zoonotic disease, associated with nine-banded armadillos (Dasypus novemcinctus) in the Southern United States of America in 2011. In addition, there is growing evidence to support a role for armadillos in zoonotic leprosy in South America. The current study evaluated twenty specimens of the six-banded armadillo (Euphractus sexcinctus), collected from rural locations in the state of Rio Grande do Norte (RN), Brazil for evidence of infection with Mycobacterium leprae. Serum was examined using two "in-house" enzyme-linked immunosorbent assays (ELISAs) and via two commercially available (ML flow and NDO-LID®) immunochromatographic lateral flow (LF) tests, for detection of the PGL-I and/or LID-1 antigens of the bacterium. The presence of M. leprae DNA in liver tissue was examined using the multi-copy, M. leprae-specific repetitive element (RLEP), as target in conventional and nested PCR assays. Molecular and anti-PGL-I-ELISA data indicated that 20/20 (100 %) of the armadillos were infected with M. leprae. The corresponding detection levels recorded with the LF tests were 17/20 (85 %) and 16/20 (85 %), for the NDO-LID® and ML flow tests, respectively. Our results indicate that, in common with D. novemcinctus, six banded armadillos (a species hunted and reared as a food-source in some regions of Brazil, including RN), represent a potential reservoir of M. leprae and as such, their role in a possible zoonotic cycle of leprosy within Brazil warrants further investigation

Numbers of <i>M</i>. <i>leprae</i> recovered from mice footpads after a six month infection with 10⁴ bacillus isolated from <i>Rhodnius prolixus</i> feces that received an infectious blood meal.

<p>Numbers of <i>M</i>. <i>leprae</i> recovered from mice footpads after a six month infection with 10⁴ bacillus isolated from <i>Rhodnius prolixus</i> feces that received an infectious blood meal.</p

<i>M</i>. <i>leprae</i> reaches <i>Rhodnius prolixus</i> feces 20 days after infection.

<p><i>Rhodnius prolixus</i> were allowed to feed rabbit blood containing (A-B) or not (C-D) 10⁷ PKH26-<i>M</i>. <i>leprae</i> / mL. During the next non-infected blood meal (20 days after infection), feces from groups of ten insects were collected in a sterile tube and analyzed by fluorescence microscopy (B and D). Stained <i>M</i>. <i>leprae</i> was identified only in the infected insects (A-B), allowing its counting. Images are representative of six pools of feces, where at least five fields were analyzed. Scale bar indicates 20μm.</p