scholarly journals Free-Living Amoebae in Soil Samples from Santiago Island, Cape Verde

2021 ◽  
Vol 9 (7) ◽  
pp. 1460
Author(s):  
Djeniffer Sousa-Ramos ◽  
María Reyes-Batlle ◽  
Natália K. Bellini ◽  
Rubén L. Rodríguez-Expósito ◽  
José E. Piñero ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
18S Rrna Gene ◽  
Soil Samples ◽  
Cape Verde ◽  
Beach Sand ◽  
Rrna Gene ◽  
Pathogenic Microorganisms ◽  
Free Living ◽  
First Report ◽  
Santiago Island

Free-Living Amoebae (FLA) are widely distributed protozoa, which contain some groups considered as pathogenic microorganisms. These members are able to produce several opportunistic diseases including epithelial disorders, such as keratitis and fatal encephalitis. Even though they have been reported in numerous sources, such as soils, dust and water, there is no legislation related to the presence of these protozoa in soil-related environments worldwide. Therefore, there are no established prevention or disinfection protocols to advise the population regarding FLA infections or eliminate these microorganisms from human-related environments to date. Acanthamoeba spp. are the most common FLA isolated in soil samples, which is also the most common genera found in clinical cases. Thus, the aim of the present study was to evaluate the presence of potentially pathogenic FLA in human-related soil samples of Santiago Island, Cabo Verde. A total of 26 soil samples were seeded in non-nutrient agar plates (2%), incubated at 26 °C, and monitored daily to evaluate the presence of FLA. DNA was extracted from those plates on which there was suspected FLA growth, and PCR amplification of the 18S rRNA gene was carried out. A total of 17 from the 26 analysed samples were positive for FLA, where Acanthamoeba is the most abundant isolated genus (14/17; 82,4%), with the T4 genotype being the most common (13/14; 92,9%), followed by the T5 genotype, A. lenticulata (1/14; 7,1%). Moreover, Vermamoeba vermiformis, Stenamoeba dejonckheerei and Vannella pentlandi were isolated in three other samples. To the best of our knowledge, this is the first report of FLA presence in Cape Verde and the first report of V. vermiformis in beach sand worldwide.

scholarly journals Detection of Protozoan Hosts for Legionella pneumophila in Engineered Water Systems by Using a Biofilm Batch Test

2010 ◽  
Vol 76 (21) ◽  
pp. 7144-7153 ◽  
Author(s):  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Dick van der Kooij
Keyword(s):  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Batch Test ◽  
Free Living ◽  
Water Types ◽  
Operational Taxonomic Units ◽  
Hartmannella Vermiformis

ABSTRACT Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.

scholarly journals First Report of Root-Knot Nematode Meloidogyne enterolobii on Poinsettia ‘Luv U Pink’ in Taiwan

Plant Disease ◽  
2021 ◽  
Author(s):  
Che-Chang Liang ◽  
P. Janet Chen
Keyword(s):  
United States ◽  
18S Rrna ◽  
The United States ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
First Report ◽  
Meloidogyne Enterolobii ◽  
Mock Control ◽  
Haplotype 1 ◽  
Coii Region

Poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch.), originated in southern Mexico and northern Guatemala, is the most valuable potted flowering plant in the spurge family (Euphorbiaceae). The European Union and the United States are two biggest poinsettia markets (Taylor et al. 2011), with a wholesale value of $153 million in the United States in 2019. Root knot galls of poinsettia ‘Luv U Pink’ were collected from a production greenhouse located in Nantou County, Taiwan in March 2021. No aboveground symptoms were observed. A nematode population was established from a single female and used for identification and the Koch’s postulate. The perineal patterns of randomly picked 5 females are round or ovoid with moderate to high dorsal arches, but no distinct lateral lines, ventral striae are fine and smooth. The Morphometric characters of second-stage juvenile include: a vermiform body shape, tail narrow and tapering with rounded tail tips, and a distinct hyaline tail end. Measurements of 20 J2 are as follows: body length, 430 (398 - 473) μm; body width, 15.4 (13.4 - 17.8) μm; stylet length,13.4 (13.0 - 14.0) μm; dorsal esophageal gland orifice to basal knob, 3.4 (2.8 - 3.9) μm; tail length, 52.9 (47.6 - 62.2) μm. All morphometric data were consistent with the original description of Meloidogyne enterolobii (Yang and Eisenback 1983). Nematode DNA was extracted using GeneMark Tissue & Cell Genomic DNA Purification Kit (GeneMark, Taiwan) from approximately 1500 J2 and used for amplification of 18S rRNA gene, a D2-D3 region of 28S rRNA gene, and a mtDNA COII region with primer sets 1A/MelR, D2A/D3B, and C2F3/1108, respectively (Power and Harris 1993, Subbotin et al. 2006, Tigano et al. 2005). The sequence of 18S rRNA gene (accession no. MZ948800 haplotype 1 and MZ955998 haplotype 2), haplotype 1 shared 100% identity with that of M. enterolobii from the United States (KP901058) and China (MN832688); haplotype 2 shared 99.8% identity with that of KP901058 and MN832688. The sequence of the D2-D3 region (MZ955995) shared 99% identity with that M. enterolobii from the United States (KP901079). Sequence of the COII region (MZ964625) also shared 99% identity with that of M. enterolobii from the United States (AY446975) and China (MN840970). Phylogenetic trees of the three gene sequences plotted as described by Ye et al. (2021) revealed that the newly described nematode was grouped with M. enterolobii. Sequence analysis of two fragments: 236 bp and 520 bp amplified with gene specific primers Me-F/R and MK7F/R, respectively (Long et al. 2006, Tigano et al. 2010) also confirmed the identity of M. enterolobii. To measure the reproductive factor (Rf), the Poinsettia ‘Luv U Pink’ seedlings with eight true leaves were transplanted into three 12-cm diameter pots each containing 6000 eggs or water (mock control). Forty-five days after inoculation, the average Rf value of three inoculated plants was 6, and no galls were observed on mock control plant roots, confirming that poinsettia is the host of M. enterolobii. M. enterolobii has been reported in several Euphorbia species, including E. heterophylla, E. prostrata, E. punicea and E. tirucalli (Han et al. 2012, Rich et al. 2009). To the best of our knowledge, this is the first report of M. enterolobii infecting E. pulcherrima ‘Luv U Pink’. 

scholarly journals Legionella Species Diversity in an Acidic Biofilm Community in Yellowstone National Park

2005 ◽  
Vol 71 (1) ◽  
pp. 507-511 ◽  
Author(s):  
Kathy B. Sheehan ◽  
Joan M. Henson ◽  
Michael J. Ferris
Keyword(s):  
Yellowstone National Park ◽  
National Park ◽  
Pcr Amplification ◽  
18S Rrna Gene ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Thermal Gradients ◽  
Legionella Species ◽  
Legionella Micdadei

ABSTRACT Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30�C) and higher (35 to 38�C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.

Identification of Acanthamoeba genotype T4 and Paravahlkampfia sp. from two clinical samples

2008 ◽  
Vol 57 (3) ◽  
pp. 392-396 ◽  
Author(s):  
Soykan Ozkoc ◽  
Sema Tuncay ◽  
Songul Bayram Delibas ◽  
Ciler Akisu ◽  
Zeynep Ozbek ◽  
...  
Keyword(s):  
18S Rrna ◽  
Single Agent ◽  
18S Rrna Gene ◽  
Maximum Temperature ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Free Living ◽  
History Of ◽  
Simplex Virus ◽  
Corneal Trauma

In this study, two free-living amoebae strains, Acanthamoeba genotype T4 and Paravahlkampfia sp., which were isolated from keratitis cases are presented. While the Acanthamoeba strain was isolated as a single agent, the Paravahlkampfia strain was found together with herpes simplex virus. Neither of the patients were contact lens wearers, but they did have a history of minor corneal trauma. Amoebae were detected on non-nutrient agar covered with Escherichia coli. Based on PCR-amplified 18S rRNA-gene analysis the first isolate was identified as Acanthamoeba genotype T4 and the second as Paravahlkampfia sp. In thermotolerance tests, the maximum temperature at which trophozoites continued to divide was determined as 37 °C for this Acanthamoeba strain and 35 °C for the Paravahlkampfia strain. To the best of our knowledge, the Acanthamoeba strain described herein is the second molecularly identified Acanthamoeba strain in an Acanthamoeba keratitis patient in Turkey. However, the Paravahlkampfia isolate is believed to be the first strain that has been isolated from a keratitis patient and has been molecularly differentiated from Vahlkampfia.

scholarly journals Failure To Differentiate Cryptosporidium parvum from C. meleagridis Based on PCR Amplification of Eight DNA Sequences

1998 ◽  
Vol 64 (4) ◽  
pp. 1454-1458 ◽  
Author(s):  
Dominique Champliaud ◽  
Philippe Gobet ◽  
Muriel Naciri ◽  
Odile Vagner ◽  
José Lopez ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Dna Sequences ◽  
Target Genes ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Restriction Enzyme Digestion ◽  
Human Pathogens

ABSTRACT In order to determine the specificities of PCR-based assays used for detecting Cryptosporidium parvum DNA, eight pairs of previously described PCR primers targeting six distinct regions of theCryptosporidium genome were evaluated for the detection ofC. parvum, the agent of human cryptosporidiosis, andC. muris, C. baileyi, and C. meleagridis, three Cryptosporidium species that infect birds or mammals but are not considered to be human pathogens. The four Cryptosporidium species were divided into two groups: C. parvum and C. meleagridis, which gave the same-sized fragments with all the reactions, and C. muris and C. baileyi, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification, C. parvum and C. meleagridiscouldn’t be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen C. parvum.

scholarly journals Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies, Identified by Phylogenic Analysis of 18S rRNA Gene Sequences

2009 ◽  
Vol 75 (14) ◽  
pp. 4736-4746 ◽  
Author(s):  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Geo Bakker ◽  
Hauke Smidt ◽  
Dick van der Kooij
Keyword(s):  
Drinking Water ◽  
Legionella Pneumophila ◽  
Distribution System ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Free Living ◽  
Water Supplies ◽  
Molecular Approaches ◽  
High Level

ABSTRACT Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa.

scholarly journals Microscopic and Molecular Studies of the Diversity of Free-Living Protozoa in Meat-Cutting Plants

2008 ◽  
Vol 74 (18) ◽  
pp. 5741-5749 ◽  
Author(s):  
Mario J. M. Vaerewijck ◽  
Koen Sabbe ◽  
Julie Baré ◽  
Kurt Houf
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Screening Method ◽  
18S Rrna Gene ◽  
Hot Water ◽  
Rrna Gene ◽  
Heterotrophic Flagellates ◽  
Free Living ◽  
Protozoan Community ◽  
Food Borne ◽  
Gradient Gel Electrophoresis

ABSTRACT The diversity of free-living protozoa in five meat-cutting plants was determined. Light microscopy after enrichment culturing was combined with sequencing of PCR-amplified, denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments, which was used as a fast screening method. The general results of the survey showed that a protozoan community of amoebae, ciliates, and flagellates was present in all of the plants. Protozoa were detected mainly in floor drains, in standing water on the floor, on soiled bars of cutting tables, on plastic pallets, and in out-of-use hot water knife sanitizers, but they were also detected on surfaces which come into direct contact with meat, such as conveyer belts, working surfaces of cutting tables, and needles of a meat tenderizer. After 7 days of incubation at refrigerator temperature, protozoa were detected in about one-half of the enrichment cultures. Based on microscopic observations, 61 morphospecies were found, and Bodo saltans, Bodo spp., Epistylis spp., Glaucoma scintillans, Petalomonas spp., Prodiscophrya collini, and Vannella sp. were the most frequently encountered identified organisms. Sequencing of DGGE bands resulted in identification of a total of 49 phylotypes, including representatives of the Amoebozoa, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. Sequences of small heterotrophic flagellates were affiliated mainly with the Alveolata (Apicomplexa), Stramenopiles (Chrysophyceae), and Rhizaria (Cercozoa). This survey showed that there is high protozoan species richness in meat-cutting plants and that the species included species related to known hosts of food-borne pathogens.

scholarly journals MORPHOLOGICAL AND MOLECULAR VARIATION OF FUSARIUM OXYSPORUM F.SP LYCOPERSICI ISOLATES CAUSING FUSARIUM WILT IN TOMATO

2020 ◽  
Vol 21 (supplement 1) ◽  
Author(s):  
K. Vignesh ◽  
K. Rajamohan ◽  
R. Anandan ◽  
R. Udhayakumar
Keyword(s):  
Fusarium Oxysporum ◽  
Fusarium Wilt ◽  
18S Rrna ◽  
Sequence Similarity ◽  
Fusarium Species ◽  
Critical Role ◽  
Pcr Amplification ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Molecular Variation

Tomato (Solanum lycopersicum L.) is one of the most important, commercial and widely grown vegetable crop in the world. Tomato plays a critical role in nutritional food requirements, income and employment opportunities for the people. However, its production is threatened by the Fusarium wilt caused by Fusarium oxysporum f.sp. lycopersici and productionlossesbetween30%to40%. In the present investigation an attempt has been made to study the morphological and molecular variation of Fusarium oxysporum f.sp lycopersici isolates. Usual identification of Fusarium species based on their micro and macroscopic features and morphological characters alone may lead to incorrect designation. In order to identify the correct species, we amplified the 18S rRNA gene region by PCR, sequenced and analyzed for sequence similarity among the NCBI data through BLAST. Further, PCR amplification of ITS regions was performed using ITS primers. The amplified product of 18S rRNA gene was sequenced and deposited to Gen Bank with the accession numbers.

Molecular characterization of Theileria spp. in livestock and the first report on the occurrence of Theileria sp. OT3 in Iran

2018 ◽  
Vol 63 (3) ◽  
pp. 515-521 ◽  
Author(s):  
Seyyed Javad Seyyed Tabaei ◽  
Adel Spotin ◽  
Ramin Pouriran ◽  
Abbas Shahbazi ◽  
Amirreza Javadi Mamaghani
Keyword(s):  
Distance Matrix ◽  
18S Rrna Gene ◽  
Pairwise Distance ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Nested Polymerase Chain Reaction ◽  
Blood Samples ◽  
First Report ◽  
Theileria Lestoquardi ◽  
Causative Agents

Abstract Theileria lestoquardi, T. ovis, and T. annulata are recognized as major causative agents for ovine and cattle theileriosis in Iran, respectively. Recently, there have been reports on the presence of Theileria spp. (Theileria sp. OT1, Theileria sp. OT3, and Theileria sp.). In this study, 37 blood samples were collected from sheep and cattle with clinically suspected Theileria infection in the Northwest of Iran. The samples were analyzed using a light microscope. DNA samples were amplified via nested-polymerase chain reaction (PCR) of 18S rRNA gene. The amplicons were digested with HpaII, following restriction fragment length polymorphism (RFLP) and sequenced to reconfirm Theileria species. The microscopic examination indicated that 4 out of 37 (10.8%) blood samples were infected with Theileria spp. Based on the nested PCR-RFLP and sequencing data, 5.4%, 13.5%, and 27% of blood samples were infected with Theileria sp. OT3, T. ovis, and T. annulata, respectively. The pairwise distance matrix of Theileria sp. OT3 showed 99.8–100% identity and 0–0.2% divergence in comparison with the registered sequences. The present study is the first report of Theileria sp. OT3 in Iran. To the evaluate evolution of Theileria spp. and providing resultant genetic data, further research with a larger sample is necessary in the region.

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