scholarly journals Molecular characterization of a re-emergent Brugia malayi parasite in Sri Lanka, suggestive of a novel strain with close nucleotide homology to Brugia pahangi

2020 ◽  
Author(s):  
Chandana Harendra Mallawarachchi ◽  
Nilmini T. G. A. N Chandrasena ◽  
Ranjan Premarathna ◽  
S.M.N.S.M. Maleesha Mallawarachchi ◽  
Nilmini Y. I. S. Gunawardane ◽  
...  
Keyword(s):  
Sri Lanka ◽  
Its2 Region ◽  
Species Identity ◽  
Internal Transcribed Spacer Region ◽  
Region Gene ◽  
Specific Pcr ◽  
Baited Traps ◽  
Human Blood Samples ◽  
Polymerase Chain

Abstract Background Brugian filariasis has re-emerged in Sri Lanka after four decades of quiescence. As microscopy alone was insufficient for ascertaining the species identity of the re-emerged sub-periodic Brugia spp. parasite, molecular speciation was performed. The transmission dynamics of the parasite was studied by entomological procedures.Methods Human blood samples positive for Brugia spp. microfilariae (MF) (n=8) were collected and DNA extracted using ReliaPrep™ Blood DNA Miniprep System (modified). Polymerase chain reaction (PCR) was performed with pan-filarial primers specific for the internal transcribed spacer region 2 (ITS2) of the ribosomal DNA (rDNA) of MF. Results Of those tested, seven (87.5%) yielded a band at 615bp establishing the species identity of the re-emerged filarial parasite as B. malayi. Comparison of the ITS2 region gene sequences of B. malayi MF isolated from humans (n=2), dogs (n=3) and cats (n=6) with GenBank sequences revealed a higher sequence homology with B. pahangi than B. malayi, but phylogeny was closer to B. malayi. A total of 82 mosquitoes of genus Mansonia comprising of M. annulifera (65), M. uniformis (14) and M. indiana (3) were collected by cattle-baited traps. Mosquito dissections identified 17 infected mosquitoes: one M. uniformis (7.14%) and 16 M. annulifera (24.6%). The DNA extracts of all infected Mansonia mosquitoes elicited the 615bp band on pan-filarial primer specific PCR. Conclusions The re-emergent B. malayi is a genetic variant or a novel species closely related to B. malayi and B. pahangi. Mansonia spp. mosquitoes were vectors of this zoonotic variant B. malayi circulating among cats, dogs and humans in Sri Lanka.

scholarly journals Molecular Characterization of a Re-Emergent Brugia Malayi Parasite in Sri Lanka, Suggestive of a Novel Strain With Close Nucleotide Homology to Brugia Pahangi

2020 ◽  
Author(s):  
Chandana Harendra Mallawarachchi ◽  
Nilmini T. G. A. N Chandrasena ◽  
Ranjan Premarathna ◽  
S.M.N.S.M. Maleesha Mallawarachchi ◽  
Nilmini Y. I. S. Gunawardane ◽  
...  
Keyword(s):  
Sri Lanka ◽  
Its2 Region ◽  
Species Identity ◽  
Internal Transcribed Spacer Region ◽  
Region Gene ◽  
Specific Pcr ◽  
Baited Traps ◽  
Human Blood Samples ◽  
Polymerase Chain

Abstract Background Brugian filariasis has re-emerged in Sri Lanka after four decades of quiescence. As microscopy alone was insufficient for ascertaining the species identity of the re-emerged sub-periodic Brugia spp. parasite, molecular speciation was performed. The transmission dynamics of the parasite was studied by entomological procedures.Methods Human blood samples positive for Brugia spp. microfilariae (MF) (n=8) were collected and DNA extracted using ReliaPrep™ Blood DNA Miniprep System (modified). Polymerase chain reaction (PCR) was performed with pan-filarial primers specific for the internal transcribed spacer region 2 (ITS2) of the ribosomal DNA (rDNA) of MF. Results Of those tested, seven (87.5%) yielded a band at 615bp establishing the species identity of the re-emerged filarial parasite as B. malayi. Comparison of the ITS2 region gene sequences of B. malayi MF isolated from humans (n=2), dogs (n=3) and cats (n=6) with GenBank sequences revealed a higher sequence homology with B. pahangi than B. malayi, but phylogeny was closer to B. malayi. A total of 82 mosquitoes of genus Mansonia comprising of M. annulifera (65), M. uniformis (14) and M. indiana (3) were collected by cattle-baited traps. Mosquito dissections identified 17 infected mosquitoes: one M. uniformis (7.14%) and 16 M. annulifera (24.6%). The DNA extracts of all infected Mansonia mosquitoes elicited the 615bp band on pan-filarial primer specific PCR. Conclusions The re-emergent B. malayi is a genetic variant or a novel species closely related to B. malayi and B. pahangi. Mansonia spp. mosquitoes were vectors of this zoonotic variant B. malayi circulating among cats, dogs and humans in Sri Lanka.

scholarly journals Molecular Characterization of a Reemergent Brugia malayi Parasite in Sri Lanka, Suggestive of a Novel Strain

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
C. H. Mallawarachchi ◽  
T. G. A. N. Chandrasena ◽  
G. P. Withanage ◽  
R. Premarathna ◽  
S. M. N. S. M. Mallawarachchi ◽  
...  
Keyword(s):  
Sri Lanka ◽  
Brugia Malayi ◽  
Public Health Issue ◽  
Hybrid Strain ◽  
Species Identity ◽  
Internal Transcribed Spacer Region ◽  
Pairwise Divergence ◽  
The Mean ◽  
Polymerase Chain

Sri Lanka achieved elimination status for lymphatic filariasis in 2016; still, the disease remains a potential public health issue. The present study is aimed at identifying a subperiodic Brugia sp. parasite which has reemerged in Sri Lanka after four decades via molecular-based analysis. Polymerase chain reaction performed with pan-filarial primers specific for the internal transcribed spacer region-2 (ITS-2) of the rDNA of Brugia filarial parasites isolated from human, canine, and feline blood samples yielded a 615 bp band establishing the species identity as Brugia malayi. Comparison of the ITS2 sequences of the reemerged B. malayi isolates with GenBank sequences revealed a higher sequence homology with B. pahangi than B. malayi with similar phylogenetic evidence. However, the mean interspecies Kimura-2-parameter pairwise divergence between the generated Brugia sequences with B. malayi and B. pahangi was less than 3%. During the analysis of parsimony sites of the new ITS2 sequences, substitutions at A36T, A296G, T373A, and G482A made the sequences different from both B. pahangi and B. malayi suggesting the possibility of a new genetic variant or a hybrid strain of B. malayi and B. pahangi. Mosquito dissections and xenomonitoring identified M. uniformis and M. annulifera as vectors of this novel strain of B. malayi circulating among cats, dogs, and humans in Sri Lanka.

scholarly journals Characterization of a New Alloantigen (SH) on the Human Neutrophil Fcγreceptor IIIb

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1027-1034 ◽  
Author(s):  
Juergen Bux ◽  
Ernst-Ludwig Stein ◽  
Philippe Bierling ◽  
Patricia Fromont ◽  
Mary Clay ◽  
...  
Keyword(s):  
Nucleotide Sequence Analysis ◽  
Polymorphism Analysis ◽  
Mutational Event ◽  
Coding Region ◽  
Specific Pcr ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Antigen Frequency ◽  
Neonatal Neutropenia

Abstract Polymorphic structures of the neutrophil Fcγreceptor IIIb (FcγRIIIb) result in alloantibody formation that causes alloimmune neonatal neutropenia and transfusion reactions. Alloantigens located on FcγRIIIb include the antigens NA1 and NA2. In four cases of alloimmune neonatal neutropenia, granulocyte-specific alloantibodies directed against a thus far unknown antigen were detected by granulocyte agglutination and immunofluorescence tests in the maternal sera. By the use of the monoclonal antibody–specific immobilization of granulocyte antigens (MAIGA) assay, the new antigen, termed SH, was located on the FcγRIIIb. Nucleotide sequence analysis of the FcγRIIIb coding region from a SH(+) individual showed a single-base C→A mutation at position 266, which results in an Ala78Asp amino acid substitution. A family study confirmed that this nucleotide difference is inherited, and corresponds to the SH phenotype. Serologic typing of 309 randomly selected individuals showed an antigen frequency of 5% in the white population. The same frequency was found by genotyping, for which a technique based on polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP) was developed. Typing of all SH(+) individuals for NA1 and NA2, and PCR-restriction fragment length polymorphism analysis of the NA-specific PCR products from five SH(+) individuals using the SH-specific endonuclease SfaN I showed that SH antigen is very probably the result of an additional mutational event in the NA2 form of the FcγRIIIB gene. Immunochemical studies also demonstrated that the SH determinants reside on the 65- to 80-kD NA2 isoform of the FcγRIIIb. Our findings show the existence of an additional polymorphism of the FcγRIIIb, which can result in alloantibody formation causing alloimmune neonatal neutropenia.

scholarly journals Characterization of Verticillium dahliae and V. tricorpus Isolates from Lettuce and Artichoke

Plant Disease ◽  
2008 ◽  
Vol 92 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Qing-Ming Qin ◽  
Gary E. Vallad ◽  
Krishna V. Subbarao
Keyword(s):  
Sequence Information ◽  
Root Weight ◽  
Internal Transcribed Spacer Region ◽  
Early Introduction ◽  
Reduced Height ◽  
Verticillium Tricorpus ◽  
Pathogenicity Tests ◽  
Plausible Mechanism ◽  
Polymerase Chain

Verticillium isolates collected from lettuce and artichoke were characterized for morphology, growth and pathogenicity. Several isolates were identified as Verticillium tricorpus based on morphological and cultural characteristics, including the production of dark resting mycelia, chlamydospores, microsclerotia, and yellow to orange pigmentation in culture. Compared with isolates of V. dahliae, these isolates also produced microsclerotia and conidia that were significantly larger and exhibited a distinct growth pattern at varying temperatures. Using database sequence information, primers were developed from the internal transcribed spacer region to produce a diagnostic 337-bp product specific to V. tricorpus and used to confirm the identification of isolates. Pathogenicity tests indicated that isolates of V. tricorpus were weak pathogens, causing a median disease severity (DS) of <1 (0-to-5 scale) on lettuce and artichoke. In contrast, isolates of V. dahliae consistently caused severe wilt with a median DS of >3.5 on lettuce and 5.0 on artichoke. Although lettuce and artichoke inoculated with isolates of V. tricorpus exhibited reduced height and fresh foliar and root weight, the reductions were not statistically significant, unlike in plants inoculated with isolates of V. dahliae. Lettuce co-inoculated with isolates of V. tricorpus and V. dahliae exhibited reduced symptoms of Verticillium wilt and improved growth relative to those inoculated with V. dahliae alone. The early introduction of V. tricorpus in soil-drench inoculations appeared to provide better relief from subsequent V. dahliae inoculation than when the two species were co-inoculated simultaneously using the root-dip method, suggesting competitive exclusion as a plausible mechanism. A spore-polymerase chain reaction assay developed using cultured spores directly as template and primers specific to V. tricorpus confirmed the presence of V. tricorpus on inoculated roots. This work demonstrates the potential use of V. tricorpus to directly reduce the effect of V. dahliae on lettuce and artichoke and, to our knowledge, is the first reported characterization of V. tricorpus isolates collected from lettuce and artichoke.

scholarly journals Genetic and Morphological Characterization of Colletotrichum acutatum Causing Anthracnose of Lupins

2002 ◽  
Vol 92 (9) ◽  
pp. 986-996 ◽  
Author(s):  
Pedro Talhinhas ◽  
S. Sreenivasaprasad ◽  
João Neves-Martins ◽  
Helena Oliveira
Keyword(s):  
Morphological Characters ◽  
Morphological Characterization ◽  
Colletotrichum Acutatum ◽  
Distinct Subgroup ◽  
Specific Pcr ◽  
Pathogen Diversity ◽  
Polymerase Chain ◽  
Arbitrarily Primed Pcr ◽  
Pathogen Diagnosis

Anthracnose, caused by Colletotrichum sp., is a serious problem of lupins (Lupinus spp.) worldwide. Morphological characters and molecular markers were used to characterize 43 Colletotrichum isolates from lupins, 8 isolates from other hosts, and 18 reference isolates representing related Colletotrichum spp., to assess the pathogen diversity and resolve its taxonomy. All lupin Colletotrichum isolates tested positive with C. acutatum-specific polymerase chain reaction (PCR) and did not test positive with C. gloeosporioides-specific PCR. Spore shape and colony diameter as well as insensitivity to benomyl grouped the lupin anthracnose isolates closer to C. acutatum than to C. gloeosporioides. Analysis of internal transcribed spacer (ITS) sequences of 57 Colletotrichum isolates grouped all lupin isolates with C. acutatum and distinct from C. gloeosporioides. Further, tub2 and his4 sequences revealed groups concordant with ITS, reducing the excessive dependence on the latter. Arbitrarily primed-PCR and amplified fragment length polymorphism analyses revealed intraspecific subgroups, but neither was useful to decipher species level relationships. ITS, tub2, and his4 results strongly support designating lupin anthracnose pathogen as C. acutatum or its subspecies. Most Colletotrichum isolates from lupins from worldwide locations are genetically homogeneous and form a distinct subgroup within C. acutatum. Present results also underline the potential of the C. acutatum-specific PCR for routine pathogen diagnosis.

Design of plant-specific PCR primers for the ETS region with enhanced specificity for tribe Bromeae and their application to other grasses (Poaceae)

Botany ◽  
2014 ◽  
Vol 92 (10) ◽  
pp. 693-699 ◽  
Author(s):  
Alicia Alonso ◽  
Roger D. Bull ◽  
Carmen Acedo ◽  
Lynn J. Gillespie
Keyword(s):  
Repeat Unit ◽  
Pcr Primers ◽  
Nuclear Ribosomal Dna ◽  
Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Specific Pcr ◽  
External Transcribed Spacer ◽  
Preliminary Study ◽  
Polymerase Chain ◽  
Ribosomal Repeat

Selective primers were developed for the amplification via polymerase chain reaction (PCR) of the external transcribed spacer (ETS) region in the nuclear ribosomal repeat unit. These primers were intended to be specific to the Poaceae tribe Bromeae but were found to function broadly across subfamily Pooideae. ETS primers previously developed for grasses were unable to amplify tribe Bromeae taxa because of ETS variability and several subrepeats. After detailed analysis, a region was chosen and four candidate primers were designed to amplify and sequence the ETS fragment. This fragment of approximately 800–900 bp is located in the 3′ region of the ETS of 18S–26S nuclear ribosomal DNA. A preliminary study with these primers was conducted in eight samples of Bromus. The best two primers showed strong amplification and successful sequencing for all samples tested. We also tested the specificity of these two primers in samples belonging to all large taxa of Bromeae, including all three genera and five subgenera and samples of eight tribes and 15 subtribes of the subfamily Pooideae, and both worked for most of the taxa tested. Our results demonstrate that the ETS region is more informative for resolving relationships at different taxonomic levels than internal transcribed spacer region. Also, these results indicate the utility of these new primers for studying the ETS region in the tribe Bromeae as well as across the subfamily Pooideae.

Genetic variability and molecular identification of Brazilian Biomphalaria species (Mollusca: Planorbidae)

Parasitology ◽  
2001 ◽  
Vol 123 (7) ◽  
pp. 197-209 ◽  
Author(s):  
O. S. CARVALHO ◽  
R. L. CALDEIRA ◽  
A. J. G. SIMPSON ◽  
T. H. D. A. VIDIGAL
Keyword(s):  
Polymerase Chain Reaction ◽  
Morphological Characteristics ◽  
Neotropical Region ◽  
Snail Host ◽  
Molecular Techniques ◽  
Its2 Region ◽  
Intermediate Hosts ◽  
Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain

Freshwater snails belonging to the genus Biomphalaria are intermediate hosts of the trematode Schistosoma mansoni in the Neotropical region and Africa. In Brazil, one subspecies and ten species of Biomphalaria have been identified: B. glabrata, B. tenagophila, B. straminea, B. occidentalis, B. peregrina, B. kuhniana, B. schrammi, B. amazonica, B. oligoza, B. intermedia and B.t. guaibensis. However, only the first three species are found naturally infected with S. mansoni. The classical identification of these planorbids is based on comparison of morphological characteristics of the shell and male and female reproductive organs, which is greatly complicated by the extensive intra-specific variation. Several molecular techniques have been used in studies on the identification, genetic structure as well as phylogenetic relationships between these groups of organisms. Using the randomly amplified polymorphic DNAs (RAPD) analysis we demonstrated that B. glabrata exhibits a remarkable degree of intra-specific polymorphism. Thus, the genetics of the snail host may be more important to the epidemiology of schistosomiasis than those of the parasite itself. Using the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) in intra-populational and intra-specific studies we have demonstrated that snails belonging to the B. straminea complex (B. straminea, B. kuhniana and B. intermedia) clearly presented higher heterogeneity. Using the low stringency polymerase chain reaction (LS-PCR) technique we were able to separate B. glabrata from B. tenagophila and B. tenagophila from B. occidentalis. To separate all Brazilian Biomphalaria species we used the restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer region (ITS) of the DNA gene. The method also proved to be efficient for the specific identification of DNA extracted from snail eggs. Recently we have sequenced the ITS2 region for phylogenetic studies of all Biomphalaria snails from Brazil.

scholarly journals Characterization of Colletotrichum Isolates from Tamarillo, Passiflora, and Mango in Colombia and Identification of a Unique Species from the Genus

2003 ◽  
Vol 93 (5) ◽  
pp. 579-587 ◽  
Author(s):  
Lucia Afanador-Kafuri ◽  
Dror Minz ◽  
Marcel Maymon ◽  
Stanley Freeman
Keyword(s):  
Morphological Characterization ◽  
Its2 Region ◽  
Specific Primers ◽  
Molecular Analyses ◽  
Rapd Pcr ◽  
Morphological Criteria ◽  
Polymerase Chain ◽  
Species Specific ◽  
Unique Species

This study was conducted to identify the species of Colletotrichum infecting tamarillo, mango, and passiflora in Colombia and to assess whether cross-infection between host species is occurring. Isolates of Colletotrichum spp. from tamarillo (n = 54), passiflora (n = 26), and mango (n = 15) were characterized by various molecular methods and by morphological criteria. Morphological characterization grouped the tamarillo isolates as C. acutatum and the passiflora and mango isolates as C. gloeosporioides. Species-specific primer analysis was reliable and confirmed grouping of the tamarillo isolates (besides Tom-6) as C. acutatum and the mango isolates (besides Man-76) as C. gloeosporioides. However, DNA of the passiflora isolates was not amplified by either C. acutatum- or C. gloeosporioides-specific primers, but reacted with a new primer, Col1, designed according to the internal transcribed spacer (ITS) 1 region of these isolates. Isolates Tom-6 and Man-76 also reacted positively with the Col1 primer. All the isolates reacting with the C. acutatum- and C. gloeosporioides-specific primers failed to react with primer Col1. Isolate Pass-35 from passiflora did not react with any of the taxon-specific primers. Arbitrarily primed polymerase chain reaction (ap-PCR), random amplified polymerase DNA (RAPD)-PCR, and A+T-rich DNA analyses delineated representative isolates into subgroups within the designated species. Molecular analyses indicated that the C. acutatum tamarillo isolates were uniform or clonal, whereas the C. gloeosporioides mango isolates and Colletotrichum passiflora isolates were heterogeneous. Likewise, sequence analysis of the complete ITS (ITS1-5.8S-ITS2) region identified certain isolates to their respective species: tamarillo isolates as C. acutatum; mango isolates as C. gloeosporioides; passiflora, Tom-6, and Man-76 isolates as a Colletotrichum sp. as yet undefined; and the Pass-35 isolate as an additional undefined Colletot-richum sp. Molecular analyses of the population of Colletotrichum isolates from passiflora, Tom-6 from tamarillo, and Man-76 from mango indicate that this population may not be host specific.

scholarly journals Characterization of the Colletotrichum Species Causing Anthracnose in Andean Blackberry in Colombia

Plant Disease ◽  
2014 ◽  
Vol 98 (11) ◽  
pp. 1503-1513 ◽  
Author(s):  
Lucía Afanador-Kafuri ◽  
Alonso González ◽  
Lederson Gañán ◽  
Juan Fernando Mejía ◽  
Nadya Cardona ◽  
...  
Keyword(s):  
Colletotrichum Gloeosporioides ◽  
Morphological Characteristics ◽  
High Sensitivity ◽  
Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Colletotrichum Spp ◽  
Polymerase Chain ◽  
Polymerase Chain Reaction Primers ◽  
Multilocus Phylogeny

Since 1992, anthracnose of Andean blackberry (Rubus glaucus) has generated losses as high as 40% for farmers in Colombia. In this study, our goal was to characterize 240 Colletotrichum isolates from Andean blackberry in eight areas of Colombia. These isolates were evaluated according to morphological characteristics, sensitivity to benomyl, pathogenicity, and genetic variability. Identification of the genus Colletotrichum was achieved by using species complex-specific polymerase chain reaction primers. A multilocus phylogeny approach was used to identify isolates to the species level with sequences from the ribosomal internal transcribed spacer region and partial sequences of the actin, β-tubulin 2, calmodulin, chitin synthase 1, glutamine synthetase, and glyceraldehyde-3-phosphate dehydrogenase genes. Most of the isolates were identified as Colletotrichum gloeosporioides sensu lato, were associated with the Castilla ecotype, showed high sensitivity to benomyl, and were highly aggressive. Isolates identified as C. acutatum sensu lato were found mainly on the Thornless ecotype, were highly resistant to benomyl, and showed intermediate aggressiveness. Only three isolates were identified as C. boninense sensu lato. The species identified included C. fructicola, C. kahawae subsp. ciggaro, C. godetiae, C. karstii, C. brassicicola, and undetermined Colletotrichum spp. This study is the first report of these species associated with anthracnose in Andean blackberry.

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