A new set of primers directed to 18S rRNA gene for molecular identification of Cryptosporidium spp. and their performance in the detection and differentiation of oocysts shed by synanthropic rodents

2013 ◽  
Vol 135 (3) ◽  
pp. 551-557 ◽  
Author(s):  
Sheila O.S. Silva ◽  
Leonardo J. Richtzenhain ◽  
Iracema N. Barros ◽  
Alessandra M.M. C. Gomes ◽  
Aristeu V. Silva ◽  
...  
Keyword(s):  
Molecular Identification ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Cryptosporidium Spp ◽  
Synanthropic Rodents

scholarly journals Cryptosporidium spp. in Domestic Dogs: the “Dog” Genotype

2000 ◽  
Vol 66 (5) ◽  
pp. 2220-2223 ◽  
Author(s):  
Una M. Morgan ◽  
Lihua Xiao ◽  
Paul Monis ◽  
Abbie Fall ◽  
Peter J. Irwin ◽  
...  
Keyword(s):  
18S Rrna ◽  
18S Rrna Gene ◽  
Domestic Dogs ◽  
Heat Shock Gene ◽  
Valid Species ◽  
Rrna Gene ◽  
Phylogenetic Characterization ◽  
Cryptosporidium Spp ◽  
Shock Gene

ABSTRACT Genetic and phylogenetic characterization ofCryptosporidium isolates at two loci (18S rRNA gene and heat shock gene) from both Australian and United States dogs demonstrated that dog-derived Cryptosporidium isolates had a distinct genotype which is conserved across geographic areas. Phylogenetic analysis provided support for the idea that the “dog” genotype is, in fact, a valid species.

scholarly journals Molecular survey of Cryptosporidium spp. in calves from the state of Mato Grosso, Brazil

2020 ◽  
Vol 41 (5supl1) ◽  
pp. 2437-2444
Author(s):  
Thábata dos Anjos Pacheco ◽  
Felippe Danyel Cardoso Martins ◽  
Sayanne Luns Hatum de Almeida ◽  
Thiago Borges Fernandes Semedo ◽  
Michelle Igarashi Watanabe ◽  
...  
Keyword(s):  
18S Rrna ◽  
18S Rrna Gene ◽  
The State ◽  
Severe Illness ◽  
Rrna Gene ◽  
Mato Grosso ◽  
Fecal Samples ◽  
Cryptosporidium Spp ◽  
Gp60 Gene ◽  
Wide Range

Cryptosporidium spp. is a protozoan that infects a wide range of vertebrate hosts; it has been reported to be the cause of severe illness or death in livestock worldwide, which leads to decreased performance and production losses, especially in young animals. This study investigated the presence of Cryptosporidium in calves from beef farms in the state of Mato Grosso, midwestern Brazil. For this purpose, fecal samples from 237 animals aged ? 45 days, raised in 20 rural properties were subjected to DNA extraction and nested polymerase chain reaction (nPCR) targeting 18S ribosomal RNA (18S rRNA) gene followed by sequencing. Additionally, positive samples, previously identified as Cryptosporidium parvum by sequencing and phylogenetic analyses based on 18S rRNA gene, were subsequently analyzed focusing the amplification and sequencing using nPCR of a fragment of the 60 kDa glycoprotein (gp60) gene. Of the 237 fecal samples analyzed by PCR (18S rRNA), 50 (21.1%) fecal samples were positive for Cryptosporidium spp., while 14 (70%) of the 20 properties had at least one positive animal. The following Cryptosporidium species were detected: C. bovis, C. parvum, and C. ryanae. Thereafter, two potentially zoonotic subtypes (IIaA15G2R1 and IIaA16G3R1) of C. parvum were identified based on gp60 gene sequences. This study resulted in the detection of subtype IIaA16G3R1 for the first time in South America and showed a wide distribution of the protozoan in beef farms in the studied area of the State.

scholarly journals The first study of molecular prevalence and species characterization of Cryptosporidium in free-range chicken (Gallus gallus domesticus) from Brazil

2017 ◽  
Vol 26 (4) ◽  
pp. 472-478 ◽  
Author(s):  
Maria Paula de Carvalho Ewald ◽  
Felippe Danyel Cardoso Martins ◽  
Eloiza Teles Caldart ◽  
Fernando Emmanuel Gonçalves Vieira ◽  
Milton Hissashi Yamamura ◽  
...  
Keyword(s):  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Gallus Gallus Domesticus ◽  
Free Range ◽  
Cryptosporidium Spp ◽  
High Prevalence ◽  
Cryptosporidium Oocysts ◽  
Genotype C

Abstract Rearing free-range chicken is based on grazing feeding patterns, and these animals could be potential environmental contaminants of Cryptosporidium oocysts for humans and other animals. Therefore, the present study aimed to evaluate the molecular prevalence of Cryptosporidium spp. in free-range chickens from Brazil. A total of 351 fecal samples from chickens were examined from 20 farms. For detection of Cryptosporidium spp., 18S rRNA gene fragments were amplified using a nested PCR reaction. Positive samples were sent for sequencing. The overall prevalence of Cryptosporidium was 25.6% (95% CI = 21.2% - 30.6%). Sequencing of the amplified fragments allowed for the identification of three species: C. meleagridis in 57 (62.6%), C. baileyi in 15 (16.4%), C. parvum in 3 (3.2%) samples, and a new Cryptosporidium genotype (C. genotype BrPR1) in 3 (3.2%) samples. Cryptosporidium genotype BrPR1 has not yet been classified as a species, and its host spectrum is not known. Cryptosporidium, including zoonotic species, exists at a high prevalence in free-range chickens within the region studied.

scholarly journals Prevalence and species identification of Cryptosporidium spp. in the newborn dairy calves from Muang District, Khon Kaen Province, Thailand

2019 ◽  
Vol 12 (9) ◽  
pp. 1454-1459 ◽  
Author(s):  
Phennarin Doungmala ◽  
Patchara Phuektes ◽  
Weerapol Taweenan ◽  
Somboon Sangmaneedet ◽  
Ornampai Japa
Keyword(s):  
18S Rrna ◽  
Polymerase Chain Reaction Amplification ◽  
18S Rrna Gene ◽  
Dairy Calves ◽  
Rrna Gene ◽  
Nested Polymerase Chain Reaction ◽  
Cryptosporidium Spp ◽  
Cryptosporidium Oocysts ◽  
Khon Kaen ◽  
Acid Fast Staining

Aim: This study aims to determine the prevalence of Cryptosporidium spp. infection and to identify the species of Cryptosporidium spp. in newborn dairy calves between December 2016 and March 2017 in Muang District, Khon Kaen Province, Thailand. Materials and Methods: A total of 200 fecal samples from newborn dairy calves of the ages 1 day up to 28 days were collected and the presence of Cryptosporidium oocysts was examined microscopically using the modified Kinyoun's acid-fast staining technique. Then, Cryptosporidium species were identified using nested polymerase chain reaction amplification of 18S rRNA gene and sequencing. Results: The modified Kinyoun's acid-fast staining revealed the presence of Cryptosporidium oocysts in 51% (102/200). Sequence analysis of the 18S rRNA gene identified two species, namely, Cryptosporidium bovis (n=11) and Cryptosporidium ryanae (n=11) and one isolated strain could not be identified. Conclusion: This study indicated that newborn dairy calves aging up to 4 weeks were highly infected with Cryptosporidium spp., and the infection mostly occurred in diarrheic dairy calves. This is the first report of Cryptosporidium in dairy calves in Khon Kaen Province and the results provide baseline information for further studies and control of Cryptosporidium infection in dairy calves in the study area.

scholarly journals Investigation and molecular identification of Eimeria sp. sampled from captive forest musk deer

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11751
Author(s):  
Ziwei Ren ◽  
Dong Yu ◽  
Wei Zhao ◽  
Yan Luo ◽  
Jianguo Cheng ◽  
...  
Keyword(s):  
Molecular Identification ◽  
18S Rrna ◽  
Pcr Amplification ◽  
Plasmid Vector ◽  
18S Rrna Gene ◽  
Ovis Aries ◽  
Molecular Characteristics ◽  
Rrna Gene ◽  
Musk Deer ◽  
Fecal Samples

Forest musk deer (Moschus berezovskii) is an endangered, protected species in China. Intestinal coccidiosis is a significant problem for captive forest musk deer. However, there are few reports on the prevalence and molecular characteristics of Eimeria sp. in forest musk deer. We sought to investigate the prevalence of Eimeria sp. in forest musk deer in the Sichuan and Shaanxi provinces in China. We also investigated the molecular characteristics of Eimeria sp. by analyzing the 18S rRNA gene. We collected a total of 328 fecal samples from forest musk deer on seven farms throughout the Sichuan and Shaanxi provinces. We extracted this parasite’s DNA and used this as a template for nested PCR amplification. The 18S rRNA gene fragment was associated with the plasmid vector, and these products were introduced into Escherichia coli (DH5α). The cultured bacterial solution was used as a PCR reaction template for identification purposes. We collected 328 fecal samples from forest musk deer in Lixian (n = 54), Maoxian (n = 52), Ma’erkang (n = 49), Dujiangyan (n = 55), Hanyuan (n = 41), Luding (n = 36) and Weinan (n = 41). One hundred ninety-eight (60.37%) fecal samples tested positive for Eimeria sp. . In our analysis of the 18S rRNA gene we found 34 types of Eimeria sp. with a similarity of 90.5–100%. We constructed a phylogenetic tree based on the parasite’s 18S rRNA gene sequence. Our findings indicated that the Eimeria sp. that parasitized the intestinal tract of forest musk deer was closely related to Eimeria alabamensis from Bos taurus and Eimeria ahsata from Ovis aries. To the best of our knowledge, ours was the first investigation and molecular identification of Eimeria sp. sampled from captive forest musk deer in China. Our results provide epidemiological data for the monitoring and prevention of Eimeria sp. in captive forest musk deer.

scholarly journals Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Claire Y. T. Wang ◽  
Emma L. Ballard ◽  
Zuleima Pava ◽  
Louise Marquart ◽  
Jane Gaydon ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Plasmodium Falciparum ◽  
18S Rrna ◽  
Drug Efficacy ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Qpcr Assay ◽  
Rrna Gene ◽  
Analytical Sensitivity ◽  
Blood Samples

Abstract Background Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. Methods A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. Results The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. Conclusions The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.

scholarly journals 16S rRNA gene and 18S rRNA gene diversity in microbial mat communities in meltwater ponds on the McMurdo Ice Shelf, Antarctica

Polar Biology ◽  
2021 ◽  
Author(s):  
Eleanor E. Jackson ◽  
Ian Hawes ◽  
Anne D. Jungblut
Keyword(s):  
16S Rrna ◽  
18S Rrna ◽  
High Throughput Sequencing ◽  
Microbial Mats ◽  
Gene Diversity ◽  
Salinity Gradient ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Ice Shelf ◽  
The Impact

AbstractThe undulating ice of the McMurdo Ice Shelf, Southern Victoria Land, supports one of the largest networks of ice-based, multiyear meltwater pond habitats in Antarctica, where microbial mats are abundant and contribute most of the biomass and biodiversity. We used 16S rRNA and 18S rRNA gene high-throughput sequencing to compare variance of the community structure in microbial mats within and between ponds with different salinities and pH. Proteobacteria and Cyanobacteria were the most abundant phyla, and composition at OTU level was highly specific for the meltwater ponds with strong community sorting along the salinity gradient. Our study provides the first detailed evaluation of eukaryote communities for the McMurdo Ice Shelf using the 18S rRNA gene. They were dominated by Ochrophyta, Chlorophyta and Ciliophora, consistent with previous microscopic analyses, but many OTUs belonging to less well-described heterotrophic protists from Antarctic ice shelves were also identified including Amoebozoa, Rhizaria and Labyrinthulea. Comparison of 16S and 18S rRNA gene communities showed that the Eukaryotes had lower richness and greater similarity between ponds in comparison with Bacteria and Archaea communities on the McMurdo Ice shelf. While there was a weak correlation between community dissimilarity and geographic distance, the congruity of microbial assemblages within ponds, especially for Bacteria and Archaea, implies strong habitat filtering in ice shelf meltwater pond ecosystems, especially due to salinity. These findings help to understand processes that are important in sustaining biodiversity and the impact of climate change on ice-based aquatic habitats in Antarctica.

scholarly journals 18S rRNA gene sequences of leptocephalus gut contents, particulate organic matter, and biological oceanographic conditions in the western North Pacific

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsuyoshi Watanabe ◽  
Satoshi Nagai ◽  
Yoko Kawakami ◽  
Taiga Asakura ◽  
Jun Kikuchi ◽  
...  
Keyword(s):  
Organic Matter ◽  
Particulate Organic Matter ◽  
North Pacific ◽  
Western North Pacific ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Gut Contents ◽  
Rrna Gene ◽  
Marine Snow ◽  
Chlorophyll Maximum

AbstractEel larvae apparently feed on marine snow, but many aspects of their feeding ecology remain unknown. The eukaryotic 18S rRNA gene sequence compositions in the gut contents of four taxa of anguilliform eel larvae were compared with the sequence compositions of vertically sampled seawater particulate organic matter (POM) in the oligotrophic western North Pacific Ocean. Both gut contents and POM were mainly composed of dinoflagellates as well as other phytoplankton (cryptophytes and diatoms) and zooplankton (ciliophoran and copepod) sequences. Gut contents also contained cryptophyte and ciliophoran genera and a few other taxa. Dinoflagellates (family Gymnodiniaceae) may be an important food source and these phytoplankton were predominant in gut contents and POM as evidenced by DNA analysis and phytoplankton cell counting. The compositions of the gut contents were not specific to the species of eel larvae or the different sampling areas, and they were most similar to POM at the chlorophyll maximum in the upper part of the thermocline (mean depth: 112 m). Our results are consistent with eel larvae feeding on marine snow at a low trophic level, and feeding may frequently occur in the chlorophyll maximum in the western North Pacific.

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