Dataset Information

Determination of genetic diversity of Leishmania species using mini-circle kDNA, in Iran-Iraq countries border.

ABSTRACT: Background and Objective:Cutaneous leishmaniasis (CL) is one of the most important diseases worldwide, with a different range of prevalence in endemic areas. Anthroponotic and zoonotic CL are two epidemiological forms of CL, in Iran. Although Ilam Province in the west of Iran is one of the main endemic areas of the disease, there is no inclusive study to determine the genetic variations of parasite in these areas. The objective of this study was to determine the genetic diversity of Leishmania species in Ilam Province, using mini-circle kDNA gene. Materials and Methods:Direct smears were taken from skin lesions of 200 suspected cases of CL. Smears were stained, screened under light microscope. Polymerase chain reaction (PCR) was performed, using specific kinetoplast DNA primers. Data were analyzed, using the molecular bio-software. Results:All the samples were positive by direct examination. PCR results showed all cases were positive for Leishmania major. Although all isolated cases belong to a different county of Ilam province, all were positive for L. major with intra-species genetic diversity, divided into four clades in the dendrogram. Interpretation and Conclusion:This variation can affect drug resistance and controlling strategies of parasite. It is possible that different species of sand flies and rodents are the vector and reservoir of parasite, respectively; however, further studies are needed to validate this.

SUBMITTER: Mousavi T

PROVIDER: S-EPMC6329268 | biostudies-literature | 2018 Jul-Dec

REPOSITORIES: biostudies-literature

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Determination of genetic diversity of <i>Leishmania</i> species using mini-circle kDNA, in Iran-Iraq countries border.

Mousavi Tahereh T Shokohi Sahar S Abdi Jahangir J Naserifar Razi R Ahmadi Mahmoud M Mirzaei Asad A

Tropical parasitology 20180701 2

<h4>Background and objective</h4>Cutaneous leishmaniasis (CL) is one of the most important diseases worldwide, with a different range of prevalence in endemic areas. Anthroponotic and zoonotic CL are two epidemiological forms of CL, in Iran. Although Ilam Province in the west of Iran is one of the main endemic areas of the disease, there is no inclusive study to determine the genetic variations of parasite in these areas. The objective of this study was to determine the genetic diversity of <i>L ...[more]

PMID: 30693211

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Project description:BackgroundOur investigation uses nucleotide variations of the genera Phlebotomus and Sergentomyia using the EF-1α and Cyt b genotype regions to describe the sand fly fauna and genetic aspects collected at war-torn sites of the Khuzestan boundary between Iraq and Iran.MethodsAll sand fly species were characterized using molecular genetics. The field work was conducted in six districts including 24 locations in remote areas for three years at the peak of sand fly activity during cutaneous leishmaniasis (CL) transmission seasons. The distribution of CL vectors was determined based on the climatic regionalization using the kriging method in ArcGIS model. DNA of sand fly pools were screened via polymerase chain reaction (PCR) using neutrality (Tajima's D) and neutral allele frequency (Fu's F s) tests to measure the effect of randomly evolving DNA sequence on the genetic diversity of sand fly populations in response to habitat fragmentation and landscape modification.ResultsAmong the 1213 specimens, ten species were identified based on morphology. The non-native species Phlebotomus sergenti was unequivocally found for the first time in the studied regions. Nucleotide substitutions of sand fly sequences varied most in the most disrupted districts (Dashte-Azadegan and Abadan; disparity index test: P < 0.05). The haplotypes of Cyt b from the subgenus Sergentomyia and P. papatasi revealed more heterogeneity (Tajima's D > +2) than P. alexandri (D > +1), which suggests widespread heteroplasmic mitochondrial DNA mutations in the same mtDNA gene among different sand fly species. Subgenus Sintonius exhibited greater fitness (D = 0) and (neutrality test; P > 0.05) no evidence of selection. The sequence of the nuclear gene EF-1α indicated similar nucleotide differences, as observed for the Cyt b gene, in all sand fly species, but lower levels of polymorphisms (D > +1) were observed compared with the mitochondrial Cyt b gene (D > +2) in the subgenus Sergentomyia.ConclusionOur findings describe random nucleotide diversity in the Phlebotomus and Sergentomyia population gene pools due to recent anthropogenic influence. A phylogenetic analysis showed that the closely related species are positioned in monophyletic clades, except for the subgenus Sergentomyia and P. sergenti, and highlights the importance of haplotype variations for the development of adaptability.

Project description:Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area.DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed.The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples.A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.

Project description:BackgroundQuery ID="Q1" Text="Graphical abstract: As per journal requirements, graphical abstract is necessary. Kindly check and provide the same."The magnitude of the health problems caused by leishmaniasis has been a major driving factor behind the development and implementation of leishmaniasis control programs by the national authorities in Iran, with a priority for health and environmental management. Such programs are not achievable unless all of the factors leading to the infection, including the parasite's life-cycle, vectors and reservoirs, are recognized. So far in Iran, humans and rodents have been considered the principal reservoirs of Leishmania tropica and Leishmania major, respectively, both associated with cutaneous leishmaniasis (CL), with domestic dogs considered to be the main reservoir for Leishmania infantum, associated with visceral leishmaniasis (VL). The role of other mammals in maintaining the Leishmania parasite has remained unclear. This study aimed to investigate Leishmania infection among livestock in endemic areas of VL and CL in Fars province, southern Iran, using serological and molecular methods.MethodsBlood samples from 181 clinically healthy livestock, including 49 sheep, 114 goats, 16 cattle and two donkeys, were screened to detect Leishmania DNA and anti-Leishmania antibodies using qPCR (quantitative PCR) and the direct agglutination test (DAT), respectively. Four qPCR-positive samples were amplified using the internal transcribed spacer one (ITS1) primers in conventional PCR and sent for directional sequencing.ResultsOf the 181 livestock tested, 51 (28.2%) were infected with Leishmania, using serological and molecular methods. Anti-Leishmania antibodies were detected in 70 (38.7%) (95% confidence interval [CI]: 31.5-46.2) and Leishmania DNA in 93 (51.4%) (95% CI: 43.9-58.9) livestock. The identified Leishmania spp. were L. infantum and L. major.ConclusionsThe findings of the present study show a relatively high prevalence of Leishmania infection among livestock in endemic areas of the disease, in Fars province, southern Iran. Given the large population of this group of animals and the fact that they live in the vicinity of the main reservoirs of the disease and vectors, it seems that sand flies regularly bite these animals. Further studies are needed to determine the role of livestock in the parasite's life-cycle and the epidemiology of Leishmania infection.

Dataset Information

Determination of genetic diversity of Leishmania species using mini-circle kDNA, in Iran-Iraq countries border.

Publications

Determination of genetic diversity of <i>Leishmania</i> species using mini-circle kDNA, in Iran-Iraq countries border.

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