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1.
Acta Trop ; 207: 105495, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32305295

RESUMO

The applicability of molecular biology/PCR for canine visceral leishmaniasis diagnosis presents challenges, mainly due to the diversity of targets described. The objectives of this study were to compare the sensitivities and reliability of five targets (kDNA/120, kDNA/145, ITS1, hsp70/234 and hsp70/1300) in four different tissue samples (bone marrow, popliteal lymph node, skin and conjunctival swab). Sixty-five dogs (32 males and 33 females) naturally infected with Leishmania infantum and ten dogs without infection were examined. Dogs were characterized by serological and parasitological methods. The parasitological test was considered the gold standard for analysis. All tests presented high specificity 100% (95% CI 0.72-1), and variable sensitivity. The targets kDNA/145, ITS1, kDNA/120, hsp70/234 and hsp70/1300 detected 100% (65/65), 93.4% (61/65), 92.3% (60/65), 84.61% (55/65) and 72.3% (77/65) of positive animals respectively. The performance of PCR methods was analyzed in two different scenarios. The highest sensitivity value identified in all scenarios studied was kDNA/145. Our results suggest that popliteal lymph node and conjunctival swab samples, besides being less invasive collections, represent a good substratum for PCR-based diagnosis, and the target kDNA/145 is the best choice for detecting L. infantum DNA in naturally infected dogs.


Assuntos
Doenças do Cão/diagnóstico , Cães/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase/métodos , Animais , DNA de Cinetoplasto/genética , Feminino , Leishmania infantum/genética , Masculino
2.
Parasitol Res ; 119(5): 1683-1690, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32285265

RESUMO

The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.


Assuntos
Doenças do Cão/diagnóstico , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Medula Óssea/parasitologia , DNA de Cinetoplasto/genética , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/parasitologia , Linfonodos/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e Especificidade , Baço/parasitologia
3.
PLoS Negl Trop Dis ; 14(1): e0007770, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32004318

RESUMO

BACKGROUND: Genetic exchange in Trypanosoma cruzi is controversial not only in relation to its frequency, but also to its mechanism. Parasexual genetic exchange has been proposed based on laboratory hybrids, but population genomics strongly suggests meiosis in T. cruzi. In addition, mitochondrial introgression has been reported several times in natural isolates although its mechanism is not fully understood yet. Moreover, hybrid T. cruzi DTUs (TcV and TcVI) have inherited at least part of the kinetoplastic DNA (kDNA = mitochondrial DNA) from both parents. METHODOLOGY/PRINCIPAL FINDINGS: In order to address such topics, we sequenced and analyzed fourteen nuclear DNA fragments and three kDNA maxicircle genes in three TcI stocks which are natural clones potentially involved in events of genetic exchange. We also deep-sequenced (a total of 6,146,686 paired-end reads) the minicircle hypervariable region (mHVR) of the kDNA in such three strains. In addition, we analyzed the DNA content by flow cytometry to address cell ploidy. We observed that most polymorphic sites in nuclear loci showed a hybrid pattern in one cloned strain and the other two cloned strains were compatible as parental strains (or nearly related to the true parents). The three clones had almost the same ploidy and the DNA content was similar to the reference strain Sylvio (a nearly diploid strain). Despite maxicircle genes evolve faster than nuclear housekeeping ones, we detected no polymorphisms in the sequence of three maxicircle genes showing mito-nuclear discordance. Lastly, the hybrid stock shared 66% of its mHVR clusters with one putative parent and 47% with the other one; in contrast, the putative parental stocks shared less than 30% of the mHVR clusters between them. CONCLUSIONS/SIGNIFICANCE: The results suggest a reductive division, a natural hybridization, biparental inheritance of the minicircles in the hybrid and maxicircle introgression. The models including such phenomena and explaining the relationships between these three clones are discussed.


Assuntos
DNA de Protozoário/genética , Hibridização Genética , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , DNA de Cinetoplasto/genética , Genes de Protozoários , Sequenciamento de Nucleotídeos em Larga Escala , Ploidias , Análise de Sequência de DNA
4.
PLoS Genet ; 16(2): e1008390, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32084124

RESUMO

Base J, ß-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.


Assuntos
DNA de Cinetoplasto/metabolismo , Proteínas de Protozoários/metabolismo , RNA Polimerase II/metabolismo , Terminação da Transcrição Genética , Trypanosoma brucei brucei/fisiologia , Animais , DNA de Cinetoplasto/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes de Protozoários , Glucosídeos/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Leishmania/fisiologia , Mutação , Proteínas de Protozoários/genética , Interferência de RNA , RNA Polimerase II/genética , Timina/metabolismo , Uracila/análogos & derivados , Uracila/metabolismo
5.
Transbound Emerg Dis ; 67(2): 476-480, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31536676

RESUMO

Visceral leishmaniasis is an endemic zoonotic disease identified especially in developing territories. Brazil's northeast, southeast and midwest have been endemic for several years; currently, the infection is spreading to the south. Dogs are the main reservoirs; however, other mammal species have also been infected. Herein, we have identified the infecting Leishmania species in dogs and horses from the south of Brazil, a new outbreak of the infection. Blood samples were collected in the urban area of Uruguaiana city. DNA was extracted from peripheral blood, kinetoplast DNA (kDNA) and ribosomal DNA (rDNA) fragments were obtained by polymerase chain reaction (PCR) and sequenced. Out of 123 samples, 25 of them (14 dogs and 11 horses) were positive for Leishmania spp. Sequence alignment and phylogenetic analysis revealed that the kDNA in positive samples was similar to four species previously reported: L. infantum/L. chagasi, L. donovani, L. major. Despite kDNA minicircles regions are very useful due to high sensitivity to Leishmania spp. DNA detection, the sequence polymorphism among minicircles can be an obstacle to interspecific differentiation. Our results suggest that these strains are circulating in Brazil south region cross-border and indicate the susceptibility of new outbreak for visceral leishmaniasis infection in horses domiciled in endemic region for canine and human visceral leishmaniasis.


Assuntos
Reservatórios de Doenças/parasitologia , Doenças do Cão/epidemiologia , Doenças dos Cavalos/epidemiologia , Leishmania donovani/genética , Leishmaniose Visceral/veterinária , Zoonoses/epidemiologia , Animais , Brasil/epidemiologia , DNA de Cinetoplasto/genética , Doenças do Cão/parasitologia , Cães , Feminino , Doenças dos Cavalos/parasitologia , Cavalos , Humanos , Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Masculino , Mamíferos , Filogenia , Reação em Cadeia da Polimerase/veterinária , Alinhamento de Sequência/veterinária , Zoonoses/parasitologia
6.
BMC Vet Res ; 15(1): 381, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666069

RESUMO

BACKGROUND: Leishmaniosis, zoonosis that produces significant public health impacts, is caused by Leishmania infantum. Canines are the main domestic reservoir and, besides humans, other species of mammals could be infected when living in endemic areas. In this study, we detected equine Leishmania infantum infections in a canine visceral leishmaniosis transmission area and evaluated the clinical, haematological, biochemical and oxidative stress disorders. This study was conducted in Uruguaiana, Rio Grande do Sul, south of Brazil. Peripheral blood samples were collected from 124 animals (98 horses and 26 dogs) of both genders and several breeds after they underwent general and dermatologic examinations. RESULTS: Twenty five Leishmania infantum infected animals (20.16%), 14 horses and 11 dogs were detected by PCR (Polymerase Chain Reaction) amplification of kinetoplast DNA regions with 96% homology to Leishmania infantum (GenBank Accession No. L 19877.1). The clinical and haematological alterations of infected equines were skin lesions, nodules, lymphadenopathy, decreased levels in red blood cells and haematocrit (p < 0.05) and increase in urea serum concentration (p < 0.05), while CVL presented a decrease in red blood cells counts (p < 0.05), increase in lymphocytes (p < 0.05), and decrease in neutrophil-lymphocyte ratio (p < 0.05). Oxidative stress markers of plasma protein carbonyl and plasma lipid peroxidation were not statistically significant (p > 0.05) in both species. CONCLUSIONS: To our knowledge, this has been the first leishmaniosis equine survey performed in south of Brazil, caused by Leishmania infantum that were able to initially identify haematological and biochemical changes in the species, even in asymptomatic animals. We present evidence supporting those findings of haematological and biochemical changes could be related to infection. Surprisingly, the clinical manifestations of equine infection were similar to those found in canine visceral leishmaniosis. The equine population could be play an important role in the cycle of leishmaniosis in south Brazil and consequently indicates a great risk of public health. This evaluation of infected animals is important to establish the clinical and laboratory parameters involved in the disease progression.


Assuntos
Doenças do Cão/parasitologia , Doenças dos Cavalos/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Animais , Brasil/epidemiologia , DNA de Cinetoplasto/genética , Doenças do Cão/sangue , Doenças do Cão/epidemiologia , Cães , Doenças dos Cavalos/sangue , Doenças dos Cavalos/epidemiologia , Cavalos , Leishmania infantum/genética , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Zoonoses
7.
Nucleic Acids Res ; 47(21): 11304-11325, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31665448

RESUMO

Kinetoplastids are protists defined by one of the most complex mitochondrial genomes in nature, the kinetoplast. In the sleeping sickness parasite Trypanosoma brucei, the kinetoplast is a chain mail-like network of two types of interlocked DNA molecules: a few dozen ∼23-kb maxicircles (homologs of the mitochondrial genome of other eukaryotes) and thousands of ∼1-kb minicircles. Maxicircles encode components of respiratory chain complexes and the mitoribosome. Several maxicircle-encoded mRNAs undergo extensive post-transcriptional RNA editing via addition and deletion of uridines. The process is mediated by hundreds of species of minicircle-encoded guide RNAs (gRNAs), but the precise number of minicircle classes and gRNA genes was unknown. Here we present the first essentially complete assembly and annotation of the kinetoplast genome of T. brucei. We have identified 391 minicircles, encoding not only ∼930 predicted 'canonical' gRNA genes that cover nearly all known editing events (accessible via the web at http://hank.bio.ed.ac.uk), but also ∼370 'non-canonical' gRNA genes of unknown function. Small RNA transcriptome data confirmed expression of the majority of both categories of gRNAs. Finally, we have used our data set to refine definitions for minicircle structure and to explore dynamics of minicircle copy numbers.


Assuntos
Genoma Mitocondrial , Anotação de Sequência Molecular , Análise de Sequência de DNA , Trypanosoma brucei brucei/genética , Animais , Sequência de Bases , Sequência Conservada , DNA Circular/análise , DNA Circular/genética , DNA de Cinetoplasto/genética , Ordem dos Genes , Genoma de Protozoário , RNA Guia/genética , Trypanosoma brucei brucei/ultraestrutura
8.
Genes (Basel) ; 10(10)2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561572

RESUMO

The mitochondrial DNA (mtDNA), which is present in almost all eukaryotic organisms, is a useful marker for phylogenetic studies due to its relative high conservation and its inheritance manner. In Leishmania and other trypanosomatids, the mtDNA (also referred to as kinetoplast DNA or kDNA) is composed of thousands of minicircles and a few maxicircles, catenated together into a complex network. Maxicircles are functionally similar to other eukaryotic mtDNAs, whereas minicircles are involved in RNA editing of some maxicircle-encoded transcripts. Next-generation sequencing (NGS) is increasingly used for assembling nuclear genomes and, currently, a large number of genomic sequences are available. However, most of the time, the mitochondrial genome is ignored in the genome assembly processes. The aim of this study was to develop a pipeline to assemble Leishmania minicircles and maxicircle DNA molecules, exploiting the raw data generated in the NGS projects. As a result, the maxicircle molecules and the plethora of minicircle classes for Leishmania major, Leishmania infantum and Leishmania braziliensis have been characterized. We have observed that whereas the heterogeneity of minicircle sequences existing in a single cell hampers their use for Leishmania typing and classification, maxicircles emerge as an extremely robust genetic marker for taxonomic studies within the clade of kinetoplastids.


Assuntos
DNA de Cinetoplasto/genética , Genoma Mitocondrial , Genoma de Protozoário , Leishmania/genética , Leishmania/classificação , Filogenia
10.
Am J Trop Med Hyg ; 101(3): 590-601, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31333156

RESUMO

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the Leishmania genus. In Australia, leishmaniasis is an imported disease that is presenting itself at increased rates because of international travel, the influx of immigrants, and deployment of military operations to endemic regions. Although Leishmania species are morphologically indistinguishable, there is a strong correlation between some causative species of leishmaniasis and the subsequent response to the treatments available and patient outcome. Consequently, identification of the infective species is imperative as misidentification can result in the administering of an ineffective drug. The aim of this study was to develop a simple diagnostic tool with high sensitivity and specificity, which is capable of detecting the presence of the parasite and accurately differentiating the causative species in question. Using the advantageous properties of the maxi-circle kinetoplast DNA, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) targeting the ND7 gene was developed for the analysis of imported cases of human leishmaniasis in Australia. Designed as a dual analysis, concurrent PCR of Leishmania maxi-circle DNA and digestion with two separate enzymes (NlaIII and HpyCH4IV), this study provides an appraisal on 24 imported cases of leishmaniasis between 2008 and 2017. Five Leishmania species were reported, with members of the Viannia subgenus being the most common. The implementation of novel diagnostic procedures for leishmaniasis such as the one reported here is needed to establish a gold standard practice for the diagnosis and treatment of leishmaniasis.


Assuntos
DNA de Cinetoplasto/genética , Leishmaniose Cutânea/diagnóstico , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Adulto , Idoso , Austrália , Criança , Pré-Escolar , Doenças Transmissíveis Importadas/parasitologia , DNA de Protozoário/genética , Feminino , Humanos , Leishmania/classificação , Leishmaniose Cutânea/parasitologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Filogenia , Proteínas de Protozoários/genética , Sensibilidade e Especificidade , Viagem , Adulto Jovem
11.
PLoS Negl Trop Dis ; 13(6): e0007536, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31247047

RESUMO

BACKGROUND: Trypanosoma cruzi, the protozoan causative of Chagas disease, is classified into six main Discrete Typing Units (DTUs): TcI-TcVI. This parasite has around 105 copies of the minicircle hypervariable region (mHVR) in their kinetoplastic DNA (kDNA). The genetic diversity of the mHVR is virtually unknown. However, cross-hybridization assays using mHVRs showed hybridization only between isolates belonging to the same genetic group. Nowadays there is no methodologic approach with a good sensibility, specificity and reproducibility for direct typing on biological samples. Due to its high copy number and apparently high diversity, mHVR becomes a good target for typing. METHODOLOGY/PRINCIPAL FINDINGS: Around 22 million reads, obtained by amplicon sequencing of the mHVR, were analyzed for nine strains belonging to six T. cruzi DTUs. The number and diversity of mHVR clusters was variable among DTUs and even within a DTU. However, strains of the same DTU shared more mHVR clusters than strains of different DTUs and clustered together. In addition, hybrid DTUs (TcV and TcVI) shared similar percentages (1.9-3.4%) of mHVR clusters with their parentals (TcII and TcIII). Conversely, just 0.2% of clusters were shared between TcII and TcIII suggesting biparental inheritance of the kDNA in hybrids. Sequencing at low depth (20,000-40,000 reads) also revealed 95% of the mHVR clusters for each of the analyzed strains. Finally, the method revealed good correlation in cluster identity and abundance between different replications of the experiment (r = 0.999). CONCLUSIONS/SIGNIFICANCE: Our work sheds light on the sequence diversity of mHVRs at intra and inter-DTU level. The mHVR amplicon sequencing workflow described here is a reproducible technique, that allows multiplexed analysis of hundreds of strains and results promissory for direct typing on biological samples in a future. In addition, such approach may help to gain knowledge on the mechanisms of the minicircle evolution and phylogenetic relationships among strains.


Assuntos
Doença de Chagas/parasitologia , DNA de Cinetoplasto/genética , Variação Genética , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Técnicas de Genotipagem , Humanos , Análise de Sequência de DNA
12.
Exp Parasitol ; 203: 23-29, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31150654

RESUMO

In Brazil, Leishmania amazonensis is one of the etiological agents of tegumentary leishmaniasis and can cause a wide spectrum of diseases in humans, resulting in cutaneous, mucosal, diffuse, and even visceral leishmaniasis. Besides, this species has also been reported to affect dogs, causing typical symptoms of visceral disease. Unfortunately, the diagnostic of the Leishmania species is not routinely performed due to the difficulties of the available methods. In view of this, different molecular methods have been used in an attempt to solve the problem of diagnosis. Loop-mediated isothermal amplification (LAMP) is a relatively new nucleic acid amplification method, which has been successfully applied in the diagnosis of Leishmania spp. infections. However, this is the first work that standardizes a specific LAMP reaction for L. amazonensis. The set of primers selected were designed from the kDNA minicircle sequence of the L. amazonensis (GenBank: U19810.1). The LAMP assay developed in the present study showed 100% specificity and 89% sensitivity when compared with conventional PCR and was more sensitive than qPCR. In addition, the LAMP reaction developed here was able to amplify a qPCR sample with a parasite load of only 28 parasites in 50 ng of DNA. Consequently, considering the LAMP reaction specific to L. amazonensis and several advantages of the method (such as high efficiency, sensitivity and specificity), we believe that this reaction can be used as a promising diagnostic tool in clinical practice, field studies, and research.


Assuntos
Leishmania mexicana/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Pele/parasitologia , Animais , Sequência de Bases , Colorimetria , Cricetinae , DNA de Cinetoplasto/química , DNA de Cinetoplasto/genética , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Leishmania mexicana/genética , Leishmaniose Cutânea/parasitologia , Masculino , Mesocricetus , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Coloração pela Prata
13.
Parasitol Res ; 118(3): 793-805, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30729301

RESUMO

Leishmania is a parasitic protozoan which is transmitted to humans through the bite of an infected female Phlebotomus and Lutzomyia sand flies. Cutaneous leishmaniasis (CL), caused by Leishmania major and L. tropica, is an endemic disease in many areas of Jordan and considered as a major public health problem. The political instability in the Syrian Arab Republic has resulted in the immigration of large number of refugees into Jordan where most of them resided in camps near the Syrian borders. Therefore, the main objective of the present study was to inspect Leishmania species/genotypes which are responsible for CL infections among Syrian refugees and compare them with the recovered species/genotypes isolated from Jordanian patients. Three molecular-based assays (ITS1-PCR-RFLP, Nested ITS1-5.8S rDNA PCR, and Kinetoplast DNA PCR) followed by sequencing and phylogenetic analysis were undertaken and compared for their efficiency to confirm CL diagnosis and genotype the infecting Leishmania species. Thereafter, the evolutionary relationships among various Leishmania isolates from Syrian and Jordanian CL patients were elucidated. Results from the present study indicated that 20 and 9 out of the inspected 66 patients (39 Jordanian and 27 Syrian) were infected with L. major and L. tropica respectively. ITS1-PCR RFLP typing proved to be more sensitive in the detection of Leishmania species (positive in 44% of the isolates) compared to both ITS1-5.8S rDNA gene and Kinetoplast DNA PCR which were successful in identifying Leishmania species only in 23% and 33% of the isolates respectively. Sequencing and phylogenetic analysis of ITS1 and ITS1-5.8S rDNA genes revealed high levels of heterogeneity among the sequenced isolates. One sample typed as L. tropica from Jordanian patient showed high similarity with L. tropica sample isolated from a Syrian patient in a Lebanon refugee camp; therefore, the need for comprehensive studies to confirm if any new L. tropica strains might be introduced to Jordan by Syrian refugees is urgently indicated. These observations highlighted the need for further studies to clarify the risk status of species and strains which might be introduced from Syria to Jordan.


Assuntos
Leishmania major/genética , Leishmania tropica/genética , Leishmaniose Cutânea/parasitologia , Psychodidae/parasitologia , Animais , DNA de Cinetoplasto/química , DNA de Cinetoplasto/genética , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Feminino , Genótipo , Humanos , Jordânia , Leishmania major/isolamento & purificação , Leishmania tropica/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Refugiados , Síria
14.
Infect Genet Evol ; 70: 90-100, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30738194

RESUMO

The mitochondrial DNA (mtDNA) is a potentially valuable phylogenetic marker given its presence across all eukaryotic taxa and its relative conservation in structure and sequence. In trypanosomatids, a homologue of the mtDNA referred to as the maxicircle DNA, is located within a specialised structure in the single mitochondrion of the trypanosomatids called the kinetoplast; a high molecular weight network of DNA composed of thousands of catenated minicircles and a smaller number of larger maxicircles. Unique to the kinetoplastid protists, the maxicircle component of this complex network could represent a desirable target for taxonomic inquiry that may also facilitate exploration of the evolutionary history of this important group of parasites. The aim of this study was to investigate the phylogenetic value of the trypanosomatid maxicircle for these applications. Maxicircle sequences were obtained either by assembling raw sequence data publicly accessible in online databases (i.e., NCBI), or by amplification of novel maxicircle sequences from trypanosomatid DNA using long-range (LR) PCR with subsequent Illumina sequencing. This procedure facilitated the generation of nearly complete maxicircle sequences (i.e., excluding the divergent region) for numerous dixenous and monoxenous trypanosomatid species. Annotation of each maxicircle sequence confirmed that their structure was conserved across all taxa examined. Phylogenetic analyses confirmed that Z. australiensis showed a greater genetic relatedness with the dixenous trypanosomatids of the genera Leishmania and Endotrypanum, as opposed to members of the monoxenous genera Crithidia and Leptomonas. Additionally, molecular clock analysis supported that the dixenous Leishmaniinae appeared approximately 75 million years ago during the breakup of Gondwana. In line with previous studies, our results support the Supercontinents hypothesis regarding the origin of dixenous Leishmaniinae. Ultimately, we demonstrate that the maxicircle represents an excellent phylogenetic marker for studying the evolutionary history of trypanosomatids, resulting in trees with very high bootstrap support values.


Assuntos
DNA de Cinetoplasto/genética , Trypanosomatina/genética , Animais , Evolução Biológica , Crithidia/genética , Crithidia/ultraestrutura , Marcadores Genéticos , Leishmania/genética , Leishmania/ultraestrutura , Filogeografia , Trypanosomatina/ultraestrutura
15.
Trends Parasitol ; 35(2): 119-128, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30638954

RESUMO

Inheritance of the single mitochondrial nucleoid (kinetoplast) in the trypanosome requires numerous proteins, many of whose precise roles are unclear. By considering kinetoplast DNA (kDNA) as a template for cleavage into two equal-size networks, we predicted sets of mutant kinetoplasts associated with defects in each of the five steps in the kinetoplast cycle. Comparison of these kinetoplasts with those obtained after gene knockdowns enabled assignment of proteins to five classes - kDNA synthesis, site of scission selection, scission, separation, and partitioning. These studies highlight how analysis of mutant kinetoplast phenotypes may be used to predict functional categories of proteins involved in the biogenesis of kinetoplasts.


Assuntos
DNA de Cinetoplasto/genética , Trypanosoma/citologia , Trypanosoma/genética , DNA de Cinetoplasto/biossíntese , Mutação , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Terminologia como Assunto
16.
Trends Parasitol ; 35(1): 8-12, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30274697

RESUMO

We propose to integrate the existing and new experimental data with computational tools to model interaction networks for the most prominent kinetoplastid pathogens. These interaction networks will vastly expand the functional annotation of the kinetoplastid genomes, which in turn are critical for identifying new routes of disease intervention.


Assuntos
Biologia Computacional , Infecções por Euglenozoa/parasitologia , Genoma de Protozoário/genética , Kinetoplastida/genética , Animais , DNA de Cinetoplasto/genética , Infecções por Euglenozoa/prevenção & controle , Estudos de Associação Genética , Humanos , Kinetoplastida/fisiologia , Mapas de Interação de Proteínas/genética
17.
Clin Microbiol Infect ; 25(2): 242-247, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29730222

RESUMO

OBJECTIVES: Superficial swab sampling of American tegumentary leishmaniasis (ATL) lesions shows higher amounts of Leishmania than those from biopsy. Subcutaneous involvement is also important in ATL, but parasite quantification according to lesion depth has not been evaluated. We aim to present the best depth at which sampling should be performed for molecular exams of ATL. METHODS: Patients with a clinical presentation compatible with ATL were allocated to ATL and control groups. Qualitative and quantitative qPCR assays were performed using SYBR Green and primers amplifying the kDNA minicircle of Leishmania spp. in different skin layers, including the epidermis, the superior dermis, the inferior dermis, and the hypodermis. RESULTS: Fifty-nine patients were included in this study, including 40 who had been diagnosed with ATL and 19 controls. The number of parasites was greater in samples of the epidermis and superior dermis (159.1 × 106, range 4.0-781.7, and 75.4 × 106, range 8.0-244.5, mean Leishmania parasite equivalents per µg of tissue DNA, respectively) than those in samples of the inferior dermis and hypodermis (54.6, range 8.0-256.6, and 16.8 × 106, range 8.0-24.1, mean Leishmania parasite equivalents per µg of tissue DNA, respectively). The best diagnostic accuracy was achieved in the superior dermis (77.9%) and was significantly greater than that in the hypodermis (63.3%; p 0.039). CONCLUSIONS: We conclude that superficial sampling can retrieve a greater quantity of parasites. Future studies of the role of transepidermal elimination as a mechanism of host defence in ATL must be performed as there is a considerable quantity of Leishmania kDNA in the epidermis.


Assuntos
DNA de Cinetoplasto/genética , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Reação em Cadeia da Polimerase/métodos , Pele/parasitologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
18.
Rev Soc Bras Med Trop ; 51(3): 376-381, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29972573

RESUMO

INTRODUCTION: This study proposes to identify the Leishmania species found in the skin lesions of cutaneous leishmaniasis (CL) patients from Brasiléia municipality (Acre). METHODS: Skin biopsy imprints or biopsy fragments were assayed via kDNA-PCR/RFLP and FRET-real-time PCR. RESULTS: Of individuals with suspected CL, 18 were positive for Leishmania kDNA. Leishmania (Viannia) braziliensis (61.1%) and Leishmania (Viannia) guyanensis (5.5%) were identified in the positive samples. CONCLUSIONS: These results are congruent with the previous reports in Acre and Bolivia, revealing L. braziliensis as the most prevalent species. L. guyanensis identification also corroborates with the epidemiology of the disease in the Amazon Basin.


Assuntos
Leishmania braziliensis/genética , Leishmania guyanensis/genética , Leishmaniose Cutânea/diagnóstico , Adolescente , Adulto , Biópsia , Brasil/epidemiologia , Criança , Pré-Escolar , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Doenças Endêmicas , Feminino , Humanos , Lactente , Recém-Nascido , Leishmaniose Cutânea/epidemiologia , Masculino , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
19.
Vet Parasitol ; 259: 61-67, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30056986

RESUMO

Leishmania infantum infection was investigated in 202 wild carnivores, rodents and lagomorphs in Southeast Spain using a real-time PCR (rtPCR) in skin and organ samples, mostly spleen. Lesions compatible with leishmaniosis were not observed in any of the animals. Prevalence defined as the percentage of rtPCR-positive animals was 32% overall, and 45% in foxes (n = 69), 30% in rabbits (n = 80) and stone martens (n = 10), 19% in wood mice (n = 16), 0% in black rats (n = 10) and ranged between 0% and 100% in other minoritarian species including badgers, wild cats, wolves, raccoons, genets and hares. Most infected rabbits were rtPCR-positive in skin and not in spleen samples and the opposite was the case for foxes (p < 0.05). L. infantum prevalence was lowest in spring following months of non-exposure to phlebotomine sand fly vectors, and spatially matched recently estimated Phlebotomus perniciosus vector abundance and the prevalence of subclinical infection in dogs and humans. Prevalence increased with altitude and was greater in drier and less windy South and West compared to the coastal Southeast of the study area (p < 0.05). Genetic diversity of L. infantum from foxes, investigated by PCR-restriction fragment length polymorphisms of kinetoplast DNA, revealed B genotype in all animals, which is frequent in people and dogs in the Iberian Peninsula and Morocco. The study provides further evidence that subclinical L. infantum infection is widespread in wildlife with prevalence depending on environmental factors and that parasite tissue tropism may vary according to host species. Moreover, it suggests that sylvatic and domestic transmission cycles are closely interconnected.


Assuntos
Animais Selvagens/parasitologia , Insetos Vetores/fisiologia , Leishmania infantum/genética , Leishmaniose Visceral/veterinária , Psychodidae/fisiologia , Distribuição Animal , Animais , Carnívoros/parasitologia , Clima , DNA de Cinetoplasto/genética , Cães/parasitologia , Doenças Endêmicas , Raposas/parasitologia , Variação Genética , Genótipo , Humanos , Insetos Vetores/parasitologia , Lagomorpha/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/transmissão , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Prevalência , Psychodidae/parasitologia , Espanha/epidemiologia
20.
Vet Parasitol ; 259: 80-84, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30056989

RESUMO

Leishmaniases are endemic in Brazil, where Leishmania infantum has been detected in humans, dogs, cats, and phlebotomine vectors. Monitoring synanthropic vector populations is critical for leishmaniasis control-surveillance in such transmission-prone areas. Here, a suite of molecular approaches were used to assess Leishmania infection prevalence and to identify blood-meal sources in a large sample of sand flies collected in anthropic environments of a Leishmania-transmission area in Mato Grosso do Sul State (Rio Verde de Mato Grosso municipality), Central-West Brazil. We sampled sand flies monthly (January-June 2014 and 2016) in one peri-domestic site within each of six neighborhoods with recent records of human visceral and/or tegumentary leishmaniasis. kDNA-qPCR plus rDNA ITS-sequencing were used to detect and identify Leishmania in pooled female sand flies. Individual engorged females (n = 58) were used for blood-meal analyses through High-Resolution Melting (HRM) targeting the mtDNA cytb gene. Overall, 90.5% of 420 CDC trap-nights yielded vectors, for a total catch of 24,989 sand flies. We sub-sampled and identified 3088 sand flies of 12 species, including 2775 Lutzomyia longipalpis (the most abundant species at all sampling sites) and 297 Nyssomyia whitmani. Female sand flies (n = 1261) were grouped in 159 pools, of which 92 Lu. longipalpis (minimum infection rate [MIR] 8%) and 7 Ny. whitmani pools (MIR 7%) were Leishmania kDNA-positive. Most positive Lu. longipalpis were collected in the 2016 rainy season. Sequencing confirmed L. infantum in Lu. longipalpis samples. HRM analyses identified chicken DNA in 57 sand flies (98.3%), 37 of which were Leishmania DNA-positive (64.9%); human blood was found in just one (Leishmania-negative) female. Our data show ongoing risk of L. infantum transmission to humans in the study area, where Leishmania-infected sandfly vectors are common and heavily rely on chicken blood in the peri-domestic environment.


Assuntos
Sangue/parasitologia , DNA de Cinetoplasto/genética , Leishmania infantum/genética , Leishmaniose/veterinária , Psychodidae/parasitologia , Animais , Brasil/epidemiologia , Galinhas/parasitologia , DNA de Cinetoplasto/isolamento & purificação , Doenças Endêmicas/prevenção & controle , Comportamento Alimentar , Feminino , Humanos , Insetos Vetores/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose/epidemiologia , Leishmaniose/parasitologia , Leishmaniose/transmissão , Refeições , Reação em Cadeia da Polimerase , Prevalência , Psychodidae/fisiologia , Estações do Ano , Análise de Sequência de DNA , Temperatura de Transição
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