Quantitative PCR assay for the detection of the parasitic ciliate Cryptocaryon irritans

Taniguchi, Akito; Onishi, Hideaki; Eguchi, M.

doi:10.1007/s12562-011-0362-7

Cited by 24 publications

(24 citation statements)

References 20 publications

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“…This number may appear extremely elevated, in comparison to 1–15 copies found in bacteria [29], 30 copies found in marine stramenopiles MAST4 [24] and around 150 copies in Saccharomyces cerevisiae [30]. Yet, much higher numbers were reported in ciliates, ranging from 3415 in parasitic species Cryptocaryon irritans [31] to 160,000 copies in Vorticella sp. [32] and up to 200,000 in macronucleus of Stylonychia lemnae [33].…”

Section: Discussionmentioning

confidence: 88%

Can Abundance of Protists Be Inferred from Sequence Data: A Case Study of Foraminifera

2013

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Protists are key players in microbial communities, yet our understanding of their role in ecosystem functioning is seriously impeded by difficulties in identification of protistan species and their quantification. Current microscopy-based methods used for determining the abundance of protists are tedious and often show a low taxonomic resolution. Recent development of next-generation sequencing technologies offered a very powerful tool for studying the richness of protistan communities. Still, the relationship between abundance of species and number of sequences remains subjected to various technical and biological biases. Here, we test the impact of some of these biological biases on sequence abundance of SSU rRNA gene in foraminifera. First, we quantified the rDNA copy number and rRNA expression level of three species of foraminifera by qPCR. Then, we prepared five mock communities with these species, two in equal proportions and three with one species ten times more abundant. The libraries of rDNA and cDNA of the mock communities were constructed, Sanger sequenced and the sequence abundance was calculated. The initial species proportions were compared to the raw sequence proportions as well as to the sequence abundance normalized by rDNA copy number and rRNA expression level per species. Our results showed that without normalization, all sequence data differed significantly from the initial proportions. After normalization, the congruence between the number of sequences and number of specimens was much better. We conclude that without normalization, species abundance determination based on sequence data was not possible because of the effect of biological biases. Nevertheless, by taking into account the variation of rDNA copy number and rRNA expression level we were able to infer species abundance, suggesting that our approach can be successful in controlled conditions.

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Section: Discussionmentioning

confidence: 88%

Can Abundance of Protists Be Inferred from Sequence Data: A Case Study of Foraminifera

2013

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“…For ciliates, the extremely high copy numbers of the rRNA gene might be one of the essential causes for polymorphism in this fragment (see also Gong et al 2013). Previous studies confirmed that ciliates generally host the highest rRNA gene copy number in a single cell than any other protist or fungi (Yao et al 1974;Prescott 1994;Heyse et al 2010;Taniguchi et al 2011;Gong et al 2013). In Vorticella sp., for example, around 300,000 SSU rRNA gene copies could be found in a single cell (Gong et al 2013).…”

Section: Origin Of Sequence Variationmentioning

confidence: 95%

Effects of intragenomic polymorphism in the SSU rRNA gene on estimating marine microeukaryotic diversity: A test for ciliates using single‐cell high‐throughput DNA sequencing

Zhao

Filker

et al. 2019

Limnology & Ocean Methods

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Diversity assessments using the small subunit (SSU) rRNA gene allow for rapid assessments of microeukaryotic communities. Yet, for microeukaryotes, only limited information is available on the extent to which sequence variations within individual genomes exist at this locus. We investigated 14 marine species belonging to four major ciliate groups by single‐cell high‐throughput DNA sequencing to estimate the extent of intragenomic polymorphism in the V4 region of the SSU rRNA gene. Based on these results, we evaluated the potential effect of polymorphism on metabarcoding studies. The intragenomic variability was generally below 3% among about 1000 unique sequences obtained from each studied cell, except of Protocruzia sp., whose intragenomic variability was as high as 15.6% among about 2000 unique sequences. Intragenomic variations significantly correlated with the number of macronuclei, but not with the biovolume of cell or macronuclei, indicating the occurrence of nonuniform evolution within the macronuclei. The removal of rare unique sequences greatly decreased the number of variations. For diversity studies, we therefore propose to conservatively discard rare amplicons. Considering the intragenomic variation within the V4 region, a sequence similarity cutoff of 3% to delimit marine ciliate operational taxonomic units could be substantiated, as at least this threshold could exclude most effect of intragenomic polymorphisms, although it may be too strict for some particular groups.

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“…The reported PCR (Chen et al 2008)-and qPCR (Taniguchi et al 2011)-based diagnostic methods developed for detection of C. irritans can only screen fish breeding waters suspected to contain C. irritans. When a parasite is absent or exists at low concentrations in the surrounding environment, these parasite-specific DNAbased diagnostic methods would not be able to detect the parasites in the water.…”

Section: Figurementioning

confidence: 99%

“…)‐ and qPCR (Taniguchi et al . )‐based diagnostic methods, which generally have higher sensitivity and specificity compared ELISA methods, have good potential in laboratory‐based epidemiology studies. However, use of PCR‐based methods in the open waters such as in mariculture will be impeded by lack of portable instrumentation and experienced personnel to conduct the test.…”

Section: Introductionmentioning

confidence: 99%

“…Accurate and rapid diagnosis of C. irritans is made difficult by asymptomatic infection, infections only of the gill and existence of other diseases that display similar signs. The development of C. irritans PCR (Chen et al 2008)-and qPCR (Taniguchi et al 2011)-based diagnostic methods, which generally have higher sensitivity and specificity compared ELISA methods, have good potential in laboratory-based epidemiology studies. However, use of PCR-based methods in the open waters such as in mariculture will be impeded by lack of portable instrumentation and experienced personnel to conduct the test.…”

Section: Introductionmentioning

confidence: 99%

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Cryptocaryon irritans recombinant proteins as potential antigens for sero‐surveillance of cryptocaryonosis

Lokanathan

Mohd-Adnan

et al. 2016

Journal of Fish Diseases

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Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens.

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Quantitative PCR assay for the detection of the parasitic ciliate Cryptocaryon irritans

Cited by 24 publications

References 20 publications

Can Abundance of Protists Be Inferred from Sequence Data: A Case Study of Foraminifera

Can Abundance of Protists Be Inferred from Sequence Data: A Case Study of Foraminifera

Effects of intragenomic polymorphism in the SSU rRNA gene on estimating marine microeukaryotic diversity: A test for ciliates using single‐cell high‐throughput DNA sequencing

Cryptocaryon irritans recombinant proteins as potential antigens for sero‐surveillance of cryptocaryonosis

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