Rapid detection and quantification of Neoparamoeba perurans in the marine environment

Bridle, Andrew R.; Crosbie, Philip; Cadoret, K; Nowak, Barbara F.

doi:10.1016/j.aquaculture.2010.09.018

Cited by 60 publications

(66 citation statements)

References 31 publications

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“…Previously Bridle et al (2010) reported excellent agreement between AGD gross gill scores (Powell et al, 2001) and molecular results (gill scores 1 through to 4, no gill score 0), though the gill area sampled, fish size and season are not specified. The results of the current field study showed lower agreement, which may reflect the wider gill score range used in this study (from 0 to 5).…”

Section: Discussionmentioning

confidence: 99%

“…Each assay was designed to amplify specific regions of the 18S rRNA gene, which is generally chosen due to its high copy number, thus potentially increasing sensitivity. The 18S rRNA gene is an established marker for microbial identification, used in numerous studies, with a large database of species specific sequences (Bridle et al, 2010). Another reason for this choice is that multiple copies of this gene are encoded within the eukaryotic genome (Long and Dawid, 1980;Young et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

“…Protocol 4 was a SYBR R Green protocol targeting the 18S rRNA gene sequence of N. perurans generating an amplicon of 146 bp (Bridle et al, 2010). Each qPCR reaction contained 0.4 µM of each primer, 2x SensiFAST SYBR R Lo-ROX Master Mix (Bioline) and nuclease-free water.…”

Section: Protocolmentioning

confidence: 99%

“…The identification of N. perurans as the causal agent of AGD (Young et al, 2007) has allowed the development of specific DNA based molecular diagnostic assays for the detection of the amoeba. Currently there are two conventional polymerase chain-reaction (PCR) assays published for the detection of N. perurans, (Young et al, 2008;Rozas et al, 2011), while three real-time PCR assays were developed based on SYBR R green (Bridle et al, 2010) and TaqMan R chemistries (Fringuelli et al, 2012;Downes et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of Non-destructive Molecular Diagnostics for the Detection of Neoparamoeba perurans

et al. 2017

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Amoebic gill disease (AGD) caused by Neoparamoeba perurans, has emerged in Europe as a significant problem for the Atlantic salmon farming industry. Gross gill score is the most widely used and practical method for determining AGD severity on farms and informing management decisions on disease mitigation strategies. As molecular diagnosis of AGD remains a high priority for much of the international salmon farming industry, there is a need to evaluate the suitability of currently available molecular assays in conjunction with the most appropriate non-destructive sampling methodology. The aims of this study were to assess a non-destructive sampling methodology (gill swabs) and to compare a range of currently available real-time polymerase chain-reaction (PCR) assays for the detection of N. perurans. Furthermore a comparison of the nondestructive molecular diagnostics with traditional screening methods of gill scoring and histopathology was also undertaken. The study found that all molecular protocols assessed performed well in cases of clinical AGD with high gill scores. A TaqMan based assay (protocol 1) was the optimal assay based on a range of parameters including % positive samples from a field trial performed on fish with gill scores ranging from 0 to 5. A higher proportion of gill swab samples tested positive by all protocols than gill filament biopsies and there was a strong correlation between gill swabs tested by protocol 1 and gross gill score and histology scores. Screening for N. perurans using protocol 1 in conjunction with non-destructive gill swab samples was shown to give the best results.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Protocolmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of Non-destructive Molecular Diagnostics for the Detection of Neoparamoeba perurans

et al. 2017

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“…The nucleic acid pellet was rinsed twice with 70% ethanol and resuspended in 50 µl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8). Real-time PCR assays for Neoparamoeba perurans were conducted to quantify amoeba abundance for each sample using a CFX Connect PCR Detection System (Bio-Rad) to detect an N. perurans-specific 18S rRNA sequence as described by Bridle et al (2010), but with modifications (Bridle et al 2015). ).…”

Section: Amoeba Quantificationmentioning

confidence: 99%

Depth distribution of the amoebic gill disease agent, Neoparamoeba perurans, in salmon sea-cages

Wright¹,

Nowak²,

Oppedal³

et al. 2015

Aquacult. Environ. Interact.

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Identifying where and when parasites occur in farming environments is vital to understand transmission dynamics and develop preventative measures that reduce host−parasite encounters. A major parasite concern for Atlantic salmon farming is Neoparamoeba perurans, a marine amoeba that causes the potentially fatal amoebic gill disease (AGD), for which few control options exist. We explored whether free-living N. perurans abundance differs among depths in commercial Atlantic salmon Salmo salar sea-cages. Water samples collected from the surface to 10 m depth at multiple cage sites and times, and subsequently subjected to qPCR analysis, revealed that N. perurans abundance was influenced by depth at the time of year when amoeba numbers were highest, with more amoebae in surface waters. No distinct depth patterns were observed when amoebae were in low abundance. Across all times, temperature and salinity were largely homogeneous throughout cage depths. Possible factors explaining the presence of amoebae at the surface are discussed. Our results suggest that excluding caged salmon from upper cage depths where N. perurans is more abundant could be an effective management strategy to reduce the speed at which initial infections occur and delay the development of AGD outbreaks.

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Rapid Detection and Monitoring of Flavobacterium psychrophilum in Water by Using a Handheld, Field‐Portable Quantitative PCR System

et al. 2018

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Advances in technology are making it easier for rapid field detection of microbes in aquaculture. Specifically, real-time quantitative PCR (qPCR) analysis, which has traditionally been confined to laboratory-based protocols, is now available in a handheld, field-portable system. The feasibility of using the Biomeme handheld qPCR system for rapid (<50 min) on-site detection and monitoring of Flavobacterium psychrophilum from filtered water samples was evaluated. Paired water samples were collected over a 23-d period from microcosm tanks that housed fish injected with known levels of F. psychrophilum. Water samples were filtered through 0.45-μm nitrocellulose filters and were analyzed with both the Biomeme qPCR platform and a traditional bench qPCR protocol. The two methods identified similar fluctuations in F. psychrophilum DNA throughout the study. Standard curves relating quantification cycles to the number of F. psychrophilum colonyforming units (CFU) were constructed and analyzed; results indicated that CFU increased rapidly between days 6 and 8 of the trial and then progressively decreased during the remaining 15 d. Average calculated log 10 (CFU/mL) values were significantly correlated between the two platforms. Rapid, field-based qPCR can be incorporated into daily water quality monitoring protocols to help detect and monitor microbes in aquaculture systems.In aquaculture, the health status of farmed stocks is often directly linked to the microbe composition within the aquatic environment (Bentzon-Tilia et al. 2016).

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Rapid detection and quantification of Neoparamoeba perurans in the marine environment

Cited by 60 publications

References 31 publications

Evaluation of Non-destructive Molecular Diagnostics for the Detection of Neoparamoeba perurans

Evaluation of Non-destructive Molecular Diagnostics for the Detection of Neoparamoeba perurans

Depth distribution of the amoebic gill disease agent, Neoparamoeba perurans, in salmon sea-cages

Rapid Detection and Monitoring of Flavobacterium psychrophilum in Water by Using a Handheld, Field‐Portable Quantitative PCR System

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