Multiplex droplet digital PCR assay for detection of Flavobacterium psychrophilum and Yersinia ruckeri in Norwegian aquaculture

Lewin, Anna; Haugen, Tone; Netzer, Roman; Tøndervik, Anne; Dahle, Stine Wiborg; Hageskal, Gunhild

doi:10.1016/j.mimet.2020.106044

Cited by 17 publications

(14 citation statements)

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“…Digital droplet PCR has demonstrated to be an important new tool in clinical microbiology to quantify pathogens [67]. Assays based on ddPCR technology can be more sensitive as a screening tool of species-specific parasites in marine environments, since the end-point quantification method is less affected by inhibitors in water samples compared to conventional quantitative PCR [68][69][70]. Adopting eDNA quantification based on a ddPCR assays in combination with models of physical-chemical and environmental parameters together with the marine bacterial composition could provide important insights about aquatic animal health and microbial management for the aquaculture industry.…”

Section: Discussionmentioning

confidence: 99%

Digital Droplet PCR-Based Environmental DNA Tool for Monitoring Cryptocaryon irritans in a Marine Fish Farm from Hong Kong

et al. 2021

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The adoption of new investigative strategies based on environmental DNA (eDNA) can be used to monitor parasites, associated bacterial microbiomes, and physical-chemical parameters in fish farms. In this study, we used the economically important and globally distributed fish ciliate parasite Cryptocaryon irritans as a model to understand the parasite abundance and potential drivers of its presence in marine fish farms. Environmental (rainfall) and physical-chemical (temperature, oxygen, salinity, pH) data collected from a marine fish farm in Hong Kong were analyzed together with the eDNA approach targeting C. irritans abundance based on digital droplet PCR and 16S metagenomics to determine associations and triggers between parasites and specific bacterial groups. Rainfall and temperature demonstrated positive associations with high abundance of C. irritans (eDNA) at the studied marine fish cage farm. However, rainfall was the only parameter tested that demonstrated a significant association with parasite eDNA, indicating that the raining season is a risky period for fish farmers in Hong Kong. Coraliomargarita was the bacterial genus with the most significant relationship with low abundance of C. irritans in water. Understanding the environmental triggers of ciliate parasites propagation and associated bacterial microbiome could elucidate new insights into environmental control, microbial management, and promote the reduction of chemical use in marine fish farms.

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Section: Discussionmentioning

confidence: 99%

Digital Droplet PCR-Based Environmental DNA Tool for Monitoring Cryptocaryon irritans in a Marine Fish Farm from Hong Kong

et al. 2021

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“…Lewin et al. (2020) have recently developed a multiplex droplet PCR assay for detecting F. psychrophilum in Norwegian Atlantic salmon RAS culture, which points to potential future concerns. Avendaño‐Herrera et al.…”

Section: Discussionmentioning

confidence: 99%

Assessment of Flavobacterium psychrophilum‐associated mortality in Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis)

Bruce

Jones

et al. 2021

Journal of Fish Diseases

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Salmonid diseases caused by infections of Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, remain difficult to manage as novel, pathogenic strains continue to emerge in aquaculture settings globally. To date, much of the research regarding treatment options and vaccine development has focused on rainbow trout (Oncorhynchus mykiss), but other inland‐reared salmonids are also impacted by this Gram‐negative bacterium. As such, Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis) were injection‐challenged with a variety of previously reported F. psychrophilum strains isolated from disease diagnostic cases in salmonids, as well as a standard and well‐studied F. psychrophilum strain (CSF 259–93) known to be virulent in rainbow trout. In three separate virulence assessments (Trials A, B and C), strains US063 (isolated from lake trout; Salvelinus namaycush) and US149 (isolated from Atlantic salmon) caused a significantly higher cumulative per cent mortality (CPM) relative to other strains in Atlantic salmon (p <.001 for all trials), with US149 causing significantly greater mortality than US063 in Trials A (CPM 97% vs. 65%, p =.008) and B (CPM 96% ± 2.3% vs. 81.33% ± 4.8%, p =.014). Trial C used a lower dose (1.86 × 108 CFU/mL) for US149, resulting in a lower mortality (78.67% ± 9.33%) relative to Trials A and B. CSF259‐93 did not cause significant mortality in any trials. In brook trout, the strain 03–179 (originally isolated from steelhead trout; Oncorhynchus mykiss) was significantly more virulent than any other (CPM 100% ± 0%, p <.001), followed by US063 (73% ± 3.8%) and US149 (40% ± 6.1%,) respectively. Again, CSF259‐93 did not cause significant mortality relative to a mock challenge treatment. Results provide information about the applicability of strain selection in F. psychrophilum virulence testing in Atlantic salmon and brook trout, demonstrating the high virulence of US063 and US149 for these salmonid species. This information is applicable for the development of therapeutics and vaccines against F. psychrophilum infections and demonstrates the reproducibility of the experimental challenge model.

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“…Another bacterial pathogen of concern in New Zealand O. tshawytscha farms is an endemic strain of Y. ruckeri (serotype O1b), which causes enteric red-mouth disease. This bacterium originates in freshwater hatcheries but can persist in fish following transfer to marine farms ( Tobback et al, 2007 ; Chapela et al, 2018 ; Lewin et al, 2020 ). Collectively, these four bacteria cause disease responsible for multi-billion dollar losses globally ( Assefa and Abunna, 2018 ); therefore, cost-effective diagnostics tools are required to enable early detection and appropriate management responses to outbreaks ( Brosnahan, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

“…Novel molecular-based assays using droplet digital Polymerase Chain Reaction (ddPCR) can produce immediate results and high sample throughput without compromising detection sensitivity ( Miotke et al, 2014 ; Cao et al, 2020 ). While traditional PCR technologies are currently applied by New Zealand’s Ministry for Primary Industries (MPI) for salmon aquaculture surveillance ( Brosnahan, 2020 ), the advantages of ddPCR are now fully acknowledged and targeted assays are being used for routine monitoring and commercial applications across medicine to biosecurity ( Rowlands et al, 2019 ; Wood et al, 2019 ; Kiefer et al, 2020 ; Lewin et al, 2020 ; Netzer et al, 2021 ; Orioles et al, 2022 ). The digital droplet PCR system QX100/QX200 (Bio-Rad, California, United States) is based on partitioning each sample (e.g., extracted DNA from fish tissue) into approximately 20,000 individual droplets, with each small reaction volume containing a single target DNA fragment, which minimizes inhibition ( Mazaika and Homsy, 2014 ; Nathan et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, partitioning into droplets enables absolute quantification of the targeted gene fragments to be conducted through direct measurement of DNA copy numbers, removing the need for replicates and standard curve extrapolation ( Hindson et al, 2011 ). Finally, the possibility to multiplex several target genes into a single ddPCR reaction through two optical channels and adjusting the fluorescence signal of the different targets allows for significant time and cost savings when large datasets are being processed ( Hughesman et al, 2016 ; Lewin et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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An Efficient Tetraplex Surveillance Tool for Salmonid Pathogens

et al. 2022

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Fish disease surveillance methods can be complicated and time consuming, which limits their value for timely intervention strategies on aquaculture farms. Novel molecular-based assays using droplet digital Polymerase Chain Reaction (ddPCR) can produce immediate results and enable high sample throughput with the ability to multiplex several targets using different fluorescent dyes. A ddPCR tetraplex assay was developed for priority salmon diseases for farmers in New Zealand including New Zealand Rickettsia-like organism 1 (NZ-RLO1), NZ-RLO2, Tenacibaculum maritimum, and Yersinia ruckeri. The limit of detection in singleplex and tetraplex assays was reached for most targets at 10−9 ng/μl with, respectively, NZ-RLO1 = 0.931 and 0.14 copies/μl, NZ-RLO2 = 0.162 and 0.21 copies/μl, T. maritimum = 0.345 and 0.93 copies/μl, while the limit of detection for Y. ruckeri was 10−8 with 1.0 copies/μl and 0.7 copies/μl. While specificity of primers was demonstrated in previous studies, we detected cross-reactivity of T. maritimum with some strains of Tenacibaculum dicentrarchi and Y. ruckeri with Serratia liquefaciens, respectively. The tetraplex assay was applied as part of a commercial fish disease surveillance program in New Zealand for 1 year to demonstrate the applicability of tetraplex tools for the salmonid aquaculture industry.

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Multiplex droplet digital PCR assay for detection of Flavobacterium psychrophilum and Yersinia ruckeri in Norwegian aquaculture

Cited by 17 publications

References 18 publications

Digital Droplet PCR-Based Environmental DNA Tool for Monitoring Cryptocaryon irritans in a Marine Fish Farm from Hong Kong

Digital Droplet PCR-Based Environmental DNA Tool for Monitoring Cryptocaryon irritans in a Marine Fish Farm from Hong Kong

Assessment of Flavobacterium psychrophilum‐associated mortality in Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis)

An Efficient Tetraplex Surveillance Tool for Salmonid Pathogens

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