LeishmaniaandSauroleishmania:The Use of Random Amplified Polymorphic DNA for the Identification of Parasites from Vertebrates and Invertebrates

Motazedian, H; Noyes, Harry; Maingón, R.

doi:10.1006/expr.1996.0059

Cited by 29 publications

(18 citation statements)

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Section: Methodsmentioning

confidence: 99%

“…The geographical origins of L. major isolates studied for heterogeneity and their generic code are shown in Table 2. Genomic DNA was extracted from promastigote stages according to the procedure described (Motazedian et al 1996;Ishikawa et al 2002) with some modification. Briefly, 2 · 10 8 parasites were harvested, washed with PBS and the pellet was re-suspended in 100 ll lysis buffer containing 50 lg ⁄ ml proteinase K and incubated at 42°C for 2 h. DNA was extracted by phenol ⁄ chloroform ⁄ isoamyl alcohol (25:24:1) and precipitated by 1 ⁄ 10 of its volume using 3 m NaOAc solution pH 7.0 and 2.5 volumes of ethanol and kept at )20°C for 8 h.…”

Section: Testing Of Isolates For Polymorphismmentioning

confidence: 99%

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Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran

Mahmoudzadeh-Niknam

Ajdary

Riazi-Rad

et al. 2012

Tropical Med Int Health

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Abstractobjective To determine the geographical distribution of Leishmania species causing cutaneous leishmaniasis (CL) and to study the genetic heterogeneity of Leishmania major isolates from different endemic areas of Iran.methods A total of 341 isolates from lesions of patients living in 11 provinces of Iran were grown in culture medium and inoculated to BALB ⁄ c mice to detect possible visceralisation. The species were identified by isoenzyme analysis using a battery of six enzymes and kinetoplast (k) DNA-PCR technique. Genetic variation among L. major isolates was analysed by random amplified polymorphic DNA (RAPD) technique.results Of the total 341 isolates, 283 isolates were L. major and 58 isolates were Leishmania tropica.In rural areas, the causative agent of CL was mainly L. major (95% L. major vs. 5% L. tropica), in urban areas it was L. tropica (65% L. tropica vs. 35% L. major). All isolates of L. major and 8.6% of L. tropica isolates showed visceralisation in BALB ⁄ c mice. There is considerable genetic diversity between L. major strains from different endemic areas and even between some isolates of the same endemic area.conclusion Leishmania major is the most frequent species in the endemic areas of CL in eleven provinces of Iran, and genetic diversity is a common feature of L. major in the country.

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Section: Methodsmentioning

confidence: 99%

Section: Testing Of Isolates For Polymorphismmentioning

confidence: 99%

Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran

Mahmoudzadeh-Niknam

Ajdary

Riazi-Rad

et al. 2012

Tropical Med Int Health

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“…More than 80% of the strains of L. infantum isolated thus far belong to the predominant zymodeme, 42). The alternative methods for species and/or strain discrimination include analysis with monoclonal antibodies (17); molecular karyotyping (7, 18); PCR fingerprinting-random amplified polymorphic DNA analysis (1,19,36,51,60); and PCR amplification of distinct nuclear multicopy target sequences or kinetoplast DNA, followed by an analysis of the amplificates by sequencing, fragment length polymorphism analysis, restriction fragment length polymorphism analysis (6,29,30,32,35,44), and singlestrand conformation polymorphism analysis (10). These methods are all limited in the intrinsic level of polymorphism that they can detect.…”

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confidence: 99%

Multilocus Microsatellite Typing as a New Tool for Discrimination of Leishmania infantum MON-1 Strains

et al. 2006

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The Leishmania donovani complex, which consists of L. donovani, L. infantum-L. chagasi, and L. archibaldi, is responsible for visceral manifestations of leishmaniasis. Multilocus enzyme electrophoresis is the standard method for the characterization and identification of strains of Leishmania. For L. infantum, the predominance of zymodeme MON-1 significantly reduces the discriminative power of this approach. In the present study, we developed 17 independent polymorphic microsatellite markers for the typing of strains of L. infantum, with the main emphasis on zymodeme MON-1. The discriminative powers of 11 markers selected from among these markers were tested by using a panel of 63 isolates of the L. donovani complex. Unique multilocus genotypes were observed for the strains analyzed, with only three exceptions. Model-based and distance-based analyses of the data set showed comparable results. It was possible to discriminate between L. donovani sensu stricto, a non-MON-1 group of L. infantum isolates, and a MON-1 group of L. infantum isolates. Within MON-1, three clusters with geographical correlations became apparent. The frequency of heterozygosity in the alleles analyzed varied extremely between the different groups of isolates. The main clusters described are not consistent with species definitions based on isoenzyme analysis but confirm the results of former PCR-based investigations.Leishmania is a genus of protozoan flagellates that cause a broad spectrum of diseases, ranging from self-limiting localized cutaneous lesions to visceral leishmaniasis with fatal spontaneous evolution (2). The majority of visceral manifestations are caused by parasites of the Leishmania donovani complex (26), which consists of L. donovani Ross, 1903; L. infantum Nicolle, 1908-L. chagasi Cunha and Chagas, 1937; and L. archibaldi Castellani and Chalmers, 1919. Currently, multilocus enzyme electrophoresis (MLEE) is the generally accepted "gold standard" for the identification and classification of isolates of Leishmania. By this method, strains are divided into groups with identical enzyme patterns, called "zymodemes." The main criticism of this approach is that genotypes are assayed indirectly, with the consequence that nucleotide substitutions may not be observed in synonymous sites or in nonsynonymous sites, if it is assumed that subsequent changes in the amino acid composition do not lead to different electrophoretic mobilities. In contrast, posttranslational modifications may change the electrophoretic mobilities, despite identical genotypes. Furthermore, the method is quite slow, laborious, and costly. Growth in vitro is inevitable, and the data sets of the few laboratories in which these analyses are performed are difficult to compare.Unlike, e.g., L. donovani or L. tropica, which present extended genetic and enzymatic polymorphisms (27, 53), L. infantum is a relatively uniform species (46). More than 80% of the strains of L. infantum isolated thus far belong to the predominant zymodeme, 42). The alternative methods for ...

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“…The genomic sites selected for analysis often include variable, but redundant sequences, such as kDNA minicircles (Simpson 1987; Stuart 1983) and the intergenic or intragenic regions of the multi‐copied nuclear genes (Luis et al 1998; Mendoza‐Leon, Havercroft, and Barker 1995; Victoir et al 1998). The sensitivity of these approaches is enhanced by PCR amplification of relevant sequences using specific (de Brujin et al 1993; Rodgers, Popper, and Wirth 1990) or random primers (Motazedian et al 1996; Pogue et al 1995; Tibayrenc et al 1993). Regardless of the methodology used, the differences detected presumably reflect the genetic heterogeneity of these parasites.…”

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confidence: 99%

Multi‐Site DNA Polymorphism Analyses of Leishmania Isolates Define their Genotypes Predicting Clinical Epidemiology of Leishmaniasis in a Specific Region

Akman

Aksu

Wang

et al. 2000

J Eukaryotic Microbiology

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Leishmania isolates from 57 cases of human cutaneous (CL), human visceral (VL), and canine visceral (CVL) leishmaniasis in Turkey were grouped by multi-site DNA polymorphism analyses into five genotypes. The initial grouping was based on DNA heterogeneity of the faster-evolving mitochondrion (kinetoplast) minicircles and the intergenic regions of two nuclear repetitive genes. Taxonomic affiliation and phylogenetic relationships of the five genotypes were inferred by comparing them with reference species for sequence heterogeneity in a approximately 1.4 kb conserved single-copy gene, encoding N-acetylglucosamine-1-phosphate transferase (NAGT). Alignment of the available sequences revealed no gap, but up to 7% scattered base substitutions, suggesting that this functionally important gene is a suitable marker. Three genotypes are completely identical to the NAGTs of the reference species, identifying them as L. infantum, L. tropica. and L. major, respectively. The remaining two are recognized as L. major NAGT variants with one and four base substitutions, respectively. As expected, Maximum Likelihood analysis of the NAGT sequences separates them into three clades, corresponding to the three species. The majority of the isolates obtained are L. infantum and L. tropica, which have been known to cause infantile VL and anthroponotic CL in western and southeastern Turkey, respectively. Unexpected is the finding of Leishmania major variants and their dispersal, possibly as previously unrecognized clinico-epidemiologic entities of CL and VL.

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LeishmaniaandSauroleishmania:The Use of Random Amplified Polymorphic DNA for the Identification of Parasites from Vertebrates and Invertebrates

Cited by 29 publications

References 0 publications

Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran

Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran

Multilocus Microsatellite Typing as a New Tool for Discrimination of Leishmania infantum MON-1 Strains

Multi‐Site DNA Polymorphism Analyses of Leishmania Isolates Define their Genotypes Predicting Clinical Epidemiology of Leishmaniasis in a Specific Region

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