scholarly journals Identification of Cryptosporidium Species and Genotypes in Scottish Raw and Drinking Waters during a One-Year Monitoring Period

2010 ◽  
Vol 76 (17) ◽  
pp. 5977-5986 ◽  
Author(s):  
R. A. B. Nichols ◽  
L. Connelly ◽  
C. B. Sullivan ◽  
H. V. Smith
Keyword(s):  
Drinking Water ◽  
Fragment Length Polymorphism ◽  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Raw Water ◽  
Pcr Assay ◽  
Drinking Waters ◽  
One Year ◽  
Repetitive Analysis

ABSTRACT We analyzed 1,042 Cryptosporidium oocyst-positive slides (456 from raw waters and 586 from drinking waters) of which 55.7% contained 1 or 2 oocysts, to determine species/genotypes present in Scottish waters. Two nested PCR-restriction fragment length polymorphism (RFLP) assays targeting different loci (1 and 2) of the hypervariable region of the 18S rRNA gene were used for species identification, and 62.4% of samples were amplified with at least one of the PCR assays. More samples (577 slides; 48.7% from raw water and 51.3% from drinking water) were amplified at locus 1 than at locus 2 (419 slides; 50.1% from raw water and 49.9% from drinking water). PCR at loci 1 and 2 amplified 45.4% and 31.7% of samples containing 1 or 2 oocysts, respectively. We detected both human-infectious and non-human-infectious species/genotype oocysts in Scottish raw and drinking waters. C ryptosporidium andersoni, C ryptosporidium parvum, and the Cryptosporidium cervine genotype (now C ryptosporidium ubiquitum) were most commonly detected in both raw and drinking waters, with C. ubiquitum being most common in drinking waters (12.5%) followed by C. parvum (4.2%) and C. andersoni (4.0%). Numerous samples (16.6% total; 18.9% from drinking water) contained mixtures of two or more species/genotypes, and we describe strategies for unraveling their identity. Repetitive analysis for discriminating mixtures proved useful, but both template concentration and PCR assay influenced outcomes. Five novel Cryptosporidium spp. (SW1 to SW5) were identified by RFLP/sequencing, and Cryptosporidium sp. SW1 was the fourth most common contaminant of Scottish drinking water (3%).

scholarly journals Detection and molecular characterization of piroplasms species from naturally infected dogs in southeast Brazil

2012 ◽  
Vol 21 (2) ◽  
pp. 137-142 ◽  
Author(s):  
Tatiana Didonet Lemos ◽  
Aloysio de Mello Figueiredo Cerqueira ◽  
Helena Keiko Toma ◽  
Adrianna Vieira da Silva ◽  
Rafael Gomes Bartolomeu Corrêa ◽  
...  
Keyword(s):  
Rio De Janeiro ◽  
Fragment Length Polymorphism ◽  
Restriction Enzymes ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Pcr Assay ◽  
Babesia Vogeli ◽  
Babesia Spp

Rangelia vitalii is a protozoon described from dogs in the south and southeast regions of Brazil. It is phylogenetically related to Babesia spp. that infects dogs, but data on this enigmatic parasite is still limited. The aim of this work was to detect piroplasm species in dogs in the state of Rio de Janeiro, Brazil, by 18S rRNA gene-based PCR assay, restriction fragment length polymorphism (RFLP) and sequence analyses. Of 103 dogs examined, seven (6.8%) were positive for Babesia spp. by PCR. The amplified products were digested by restriction enzymes to differentiate the Babesia species, and one sample was identified as Babesia vogeli. The pattern observed for the other six amplification products did not match with pattern described for large Babesia infecting dogs. Sequencing analysis confirmed these six samples as R. vitalii, with high homologies (99-100%) with a sequence from south Brazil. This study confirms the presence of Babesia vogeli and Rangelia vitalii circulate in domestic dogs in Teresópolis, Rio de Janeiro, Brazil.

scholarly journals Molecular characterisation of Cryptosporidium spp. in lambs in the South Central region of the State of São Paulo

2015 ◽  
Vol 67 (2) ◽  
pp. 441-446 ◽  
Author(s):  
A.S. Zucatto ◽  
M.C.C. Aquino ◽  
S.V. Inácio ◽  
R.N. Figueiredo ◽  
J.C. Pierucci ◽  
...  
Keyword(s):  
Central Region ◽  
18S Rrna Gene ◽  
Sao Paulo ◽  
The State ◽  
Rrna Gene ◽  
São Paulo ◽  
The South ◽  
Cryptosporidium Spp ◽  
South Central ◽  
One Year

Considering the proximity of sheep farmers to animals that are possibly diseased or releasing fecal oocysts into the environment and the marked pathogenicity in lambs, the aim of this study was to determine the occurrence and to molecularly characterize the infection by Cryptosporidium spp. in lambs in the South Central region of the state of São Paulo, Brazil. A total of 193 fecal samples were collected from sheep of several breeds, males and females, aged up to one year. Polymerase chain reaction (nested-PCR) was used to amplify DNA fragments from the subunit 18S rRNA gene and indicated 15% positivity; sequencing of amplified fragments was possible for 19 samples. Analysis of the obtained sequences showed that the identified species were Cryptosporidium xiaoi for 15 samples, constituting thus the first molecular characterization study of this Cryptosporidium species in Brazil. Cryptosporidium ubiquitum was identified for three samples and Cryptosporidium meleagridis for one sample; the latter two are considered zoonotic species.

scholarly journals MixedTheileriainfections in free-ranging buffalo herds: implications for diagnosingTheileria parvainfections in Cape buffalo (Syncerus caffer)

Parasitology ◽  
2011 ◽  
Vol 138 (7) ◽  
pp. 884-895 ◽  
Author(s):  
RONEL PIENAAR ◽  
FRED T. POTGIETER ◽  
ABDALLA A. LATIF ◽  
ORIEL M. M. THEKISOE ◽  
BEN J. MANS
Keyword(s):  
South Africa ◽  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Control Measures ◽  
Locked Nucleic Acid ◽  
Rrna Gene ◽  
Mixed Infections ◽  
Endemic Region ◽  
Signal Suppression

SUMMARYBuffalo-adaptedTheileria parvacauses Corridor disease in cattle. Strict control measures therefore apply to the movement of buffalo in South Africa and include mandatory testing of buffalo for the presence ofT. parva. The official test is a real-time hybridization PCR assay that amplifies the V4 hypervariable region of the 18S rRNA gene ofT. parva, T.sp. (buffalo) andT.sp. (bougasvlei). The effect that mixedT. parvaandT.sp. (buffalo)-like infections have on accurateT. parvadiagnosis was investigated in this study.In vitromixed infection simulations indicated PCR signal suppression at 100 to 1000-foldT.sp. (buffalo) excess at lowT. parvaparasitaemia. Suppression of PCR signal was found in field buffalo with mixed infections. TheT. parva-positive status of these cases was confirmed by selective suppression ofT.sp. (buffalo) amplification using a locked nucleic acid clamp and independent assays based on the p67, p104 andTprgenes. The incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, while the prevalence in buffalo outside the endemic area is currently low. A predicted increase ofT.sp. (buffalo)-like infections can affect future diagnoses where mixed infections occur, prompting the need for improvements in current diagnostics.

scholarly journals Short-term responses to ocean acidification: effects on relative abundance of eukaryotic plankton from the tropical Timor Sea

2020 ◽  
Author(s):  
Janina Rahlff ◽  
Sahar Khodami ◽  
Lisa Voskuhl ◽  
Matthew P. Humphreys ◽  
Christian Stolle ◽  
...  
Keyword(s):  
Ocean Acidification ◽  
Community Composition ◽  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Marine Biodiversity ◽  
Rrna Gene ◽  
Tropical Regions ◽  
Timor Sea ◽  
Decreasing Ph ◽  
Species Specific

ABSTRACTAnthropogenic carbon dioxide (CO2) emissions drive climate change and pose one of the major challenges of our century. The effects of increased CO2 in the form of ocean acidification (OA) on the communities of marine planktonic eukaryotes in tropical regions such as the Timor Sea are barely understood. Here, we show the effects of high CO2 (pCO2=1823±161 μatm, pHT=7.46±0.05) versus in situ CO2 (pCO2=504±42 μatm, pHT=7.95±0.04) seawater on the community composition of marine planktonic eukaryotes immediately and after 48 hours of treatment exposure in a shipboard microcosm experiment. Illumina sequencing of the V9 hypervariable region of 18S rRNA (gene) was used to study the eukaryotic community composition. Down-regulation of extracellular carbonic anhydrase occurred faster in the high CO2 treatment. Increased CO2 significantly suppressed the relative abundances of different eukaryotic operational taxonomic units (OTUs), including important primary producers. These effects were consistent between abundant (DNA-based) and active (cDNA-based) taxa after 48 hours, e.g., for the diatoms Trieres chinensis and Stephanopyxis turris. Effects were also very species-specific among different diatoms. Planktonic eukaryotes showed adaptation to the CO2 treatment over time, but many OTUs were adversely affected by decreasing pH. OA effects might fundamentally impact the base of marine biodiversity, suggesting profound outcomes for food web functioning in the future ocean.

scholarly journals Species, Sequence Types and Alleles: Dissecting Genetic Variation in Acanthamoeba

Pathogens ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 534
Author(s):  
Paul A. Fuerst ◽  
Gregory C. Booton
Keyword(s):  
Genetic Variation ◽  
18S Rrna ◽  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Sequence Type ◽  
Rrna Gene ◽  
Dna Databases ◽  
Molecular Approaches ◽  
History Of ◽  
Sequence Types

Species designations within Acanthamoeba are problematic because of pleomorphic morphology. Molecular approaches, including DNA sequencing, hinted at a resolution that has yet to be fully achieved. Alternative approaches were required. In 1996, the Byers/Fuerst lab introduced the concept of sequence types. Differences between isolates of Acanthamoeba could be quantitatively assessed by comparing sequences of the nuclear 18S rRNA gene, ultimately producing 22 sequence types, designated T1 through T22. The concept of sequence types helps our understanding of Acanthamoeba evolution. Nevertheless, substantial variation in the 18S rRNA gene differentiates many isolates within each sequence type. Because the majority of isolates with sequences in the international DNA databases have been studied for only a small segment of the gene, designated ASA.S1, genetic variation within this hypervariable region of the 18S rRNA gene has been scrutinized. In 2002, we first categorized variation in this region in a sample of T3 and T4 isolates from Hong Kong, observing ten “alleles” within type T4 and five “alleles” within T3. Subsequently, confusion occurred when different labs applied redundant numerical labels to identify different alleles. A more unified approach was required. We have tabulated alleles occurring in the sequences submitted to the international DNA databases, and determined their frequencies. Over 150 alleles have occurred more than once within 3500+ isolates of sequence type T4. Results from smaller samples of other sequence types (T3, T5, T11 and T15, and supergroup T2/6) have also been obtained. Our results provide new insights into the evolutionary history of Acanthamoeba, further illuminating the degree of genetic separation between significant taxonomic units within the genus, perhaps eventually elucidating what constitutes a species of Acanthamoeba.

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 Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

2016 ◽  
Vol 82 (19) ◽  
pp. 5878-5891 ◽  
Author(s):  
Ian M. Bradley ◽  
Ameet J. Pinto ◽  
Jeremy S. Guest
Keyword(s):  
Community Structure ◽  
18S Rrna ◽  
Target Genes ◽  
Hypervariable Region ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Sequencing Bias ◽  
Primer Sets

ABSTRACTThe use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3′ end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest.IMPORTANCEThe quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.

scholarly journals Molecular characterization of Eurytrema coelomaticum in cattle from Paraná, Brazil

2014 ◽  
Vol 23 (3) ◽  
pp. 383-386 ◽  
Author(s):  
Gustavo Freire Figueira ◽  
Victor Henrique Silva de Oliveira ◽  
Alessandra Taroda ◽  
Amauri Alcindo Alfieri ◽  
Selwyn Arlington Headley
Keyword(s):  
Molecular Characterization ◽  
Gene Sequence ◽  
Domestic Animals ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Sequence Analyses ◽  
Pcr Assay ◽  
Bovine Pancreas

This study investigated the occurrence of Eurytremaspp. in cattle by analysis of the partial 18S rRNA gene sequence. Trematodes from 44 bovine pancreas were collected and classified based on typical morphological features. PCR assay and sequence analyses of amplified products confirmed that the trematodes classified as Eurytrema coelomaticum were phylogenetically distinct from those identified as E. pancreaticum. The results of this study represent the first molecular characterization of E. coelomaticum within the Americas, and provide an efficient method to differentiate digenean trematodes of domestic animals.

scholarly journals Metagenetic Analysis for Microbial Characterization of Focaccia Doughs Obtained by Using Two Different Starters: Traditional Baker’s Yeast and a Selected Leuconostoc citreum Strain

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1189
Author(s):  
Massimo Ferrara ◽  
Angelo Sisto ◽  
Giuseppina Mulè ◽  
Paola Lavermicocca ◽  
Palmira De Bellis
Keyword(s):  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Biochemical Properties ◽  
Baker’S Yeast ◽  
Rrna Gene ◽  
Baker's Yeast ◽  
Hypervariable Regions ◽  
Leuconostoc Citreum ◽  
Significant Difference ◽  
Microbial Environment

Lactic acid bacteria (LAB) decisively influence the technological, nutritional, organoleptic and preservation properties of bakery products. Therefore, their use has long been considered an excellent strategy to improve the characteristics of those goods. The aim of this study was the evaluation of microbial diversity in different doughs used for the production of a typical Apulian flatbread, named focaccia. Leavening of the analyzed doughs was obtained with baker’s yeast or by applying an innovative “yeast-free” protocol based on a liquid sourdough obtained by using Leuconostoc citreum strain C2.27 as a starter. The microbial populations of the doughs were studied by both a culture-dependent approach and metagenetic analyses. The flours used for dough preparation were also subjected to the same analyses. The metagenetic analyses were performed by sequencing the V5–V6 hypervariable regions of the 16S rRNA gene and the V9 hypervariable region of the 18S rRNA gene. The results indicate that these hypervariable regions were suitable for studying the microbiota of doughs, highlighting a significant difference between the microbial community of focaccia dough with baker’s yeast and that of the dough inoculated with the bacterial starter. In particular, the dough made with baker’s yeast contained a microbiota with a high abundance of Proteobacteria (82% of the bacterial population), known to be negatively correlated with the biochemical properties of the doughs, while the Proteobacteria in dough produced with the L. citreum starter were about 43.5% lower than those in flour and dough prepared using baker’s yeast. Moreover, the results show that the L. citreum C2.27 starter was able to dominate the microbial environment and also reveal the absence of the genus Saccharomyces in the dough used for the production of the “yeast-free” focaccia. This result is particularly important because it highlights the suitability of the starter strain for obtaining an innovative “yeast-free” product.

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