Multi-agent in situ hybridization confirms Ca. Branchiomonas cysticola as a major contributor in complex gill disease in Atlantic salmon

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qPCR screening for Yersinia ruckeri clonal complex 1 against a background of putatively avirulent strains in Norwegian aquaculture

Although a number of genetically diverse Yersinia ruckeri strains are present in Norwegian aquaculture environments, most if not all outbreaks of yersiniosis in Atlantic salmon in Norway are associated with a single specific genetic lineage of serotype O1, termed clonal complex 1. To investigate the presence and spread of virulent and putatively avirulent strains in Norwegian salmon farms, PCR assays specific for Y. ruckeri (species level) and Y. ruckeri clonal complex 1 were developed. Following extensive screening of water and biofilm, the widespread prevalence of putatively avirulent Y. ruckeri strains was confirmed in freshwater salmon hatcheries, while Y. ruckeri clonal complex 1 was found in fewer farms. The formalin-killed bacterin yersiniosis vaccine was detected in environmental samples by both PCR assays for several weeks post-vaccination. It is thus important to interpret results from recently vaccinated fish with great care. Moreover, field studies and laboratory trials confirmed that stressful management procedures may result in increased shedding of Y. ruckeri by sub-clinically infected fish. Analysis of sea water sampled throughout thermal delousing procedures proved effective for detection of Y. ruckeri in sub-clinically infected populations.publishedVersio

Tenacibaculosis in Norwegian Atlantic salmon (Salmo salar) cage-farmed in cold sea water is primarily associated with Tenacibaculum finnmarkense genomovar finnmarkense

Skin conditions associated with Tenacibaculum spp. constitute a significant threat to the health and welfare of sea-farmed Atlantic salmon (Salmo salar L.) in Norway. Fifteen presumptive tenacibaculosis outbreaks distributed along the Norwegian coast during the late winter and spring of 2018 were investigated. Bacteriological culture confirmed the presence of Tenacibaculum spp. Seventy-six isolates cultured from individual fish were selected and subjected to whole-genome sequencing and MALDI-TOF MS analysis. Average nucleotide identity and MALDI-TOF analyses confirmed the presence of T. finnmarkense and T. dicentrarchi, with further division of T. finnmarkense into genomovars (gv.) finnmarkense and ulcerans. Core genome multilocus sequence typing (cgMLST) and single-nucleotide polymorphism (SNP) analyses identified the presence of a genetically conserved cluster of gv. finnmarkense isolates against a background of relatively genetically diverse gv. finnmarkense and gv. ulcerans isolates in 13 of the 15 studied cases. This clustering strongly suggests a link between T. finnmarkense gv. finnmarkense and development of clinical tenacibaculosis in sea-farmed Norwegian salmon in the late winter and spring. Analysis of 25 Tenacibaculum isolates collected during the spring of 2019 from similar cases identified a similar distribution of genotypes. Low water temperatures were common to all cases, and most incidences involved relatively small fish shortly after sea transfer, suggesting that these fish are particularly predisposed to Tenacibaculum infection.publishedVersio

Phylogeography and host specificity of Pasteurellaceae pathogenic to sea-farmed fish in the north-east Atlantic

The present study was undertaken to address the recent spate of pasteurellosis outbreaks among sea-farmed Atlantic salmon (Salmo salar) in Norway and Scotland, coinciding with sporadic disease episodes in lumpfish (Cyclopterus lumpus) used for delousing purposes in salmon farms. Genome assemblies from 86 bacterial isolates cultured from diseased salmon or lumpfish confirmed them all as bona fide members of the Pasteurellaceae family, with phylogenetic reconstruction dividing them into two distinct branches sharing <88% average nucleotide identity. These branches therefore constitute two separate species, namely Pasteurella skyensis and the as-yet invalidly named “Pasteurella atlantica”. Both species further stratify into multiple discrete genomovars (gv.) and/or lineages, each being nearly or fully exclusive to a particular host, geographic region, and/or time period. Pasteurellosis in lumpfish is, irrespective of spatiotemporal origin, linked almost exclusively to the highly conserved “P. atlantica gv. cyclopteri” (Pac). In contrast, pasteurellosis in Norwegian sea-farmed salmon, dominated since the late-1980s by “P. atlantica gv. salmonicida” (Pas), first saw three specific lineages (Pas-1, -2, and -3) causing separate, geographically restricted, and short-lived outbreaks, before a fourth (Pas-4) emerged recently and became more widely disseminated. A similar situation involving P. skyensis (Ps) has apparently been unfolding in Scottish salmon farming since the mid-1990s, where two historic (Ps-1 and -2) and one contemporary (Ps-3) lineages have been recorded. While the epidemiology underlying all these outbreaks/epizootics remains unclear, repeated detection of 16S rRNA gene amplicons very closely related to P. skyensis and “P. atlantica” from at least five cetacean species worldwide raises the question as to whether marine mammals may play a part, possibly as reservoirs. In fact, the close relationship between the studied isolates and Phocoenobacter uteri associated with harbor porpoise (Phocoena phocoena), and their relatively distant relationship with other members of the genus Pasteurella, suggests that both P. skyensis and “P. atlantica” should be moved to the genus Phocoenobacter

Overview and recommendations for the application of digital PCR

The digital Polymerase Chain Reaction (dPCR), for the detection and absolute quantification of DNA, is a relatively new technique but its application in analytical laboratories is steadily increasing. In contrast to quantitative real-time PCR, DNA (fragments) can be quantified without the need for standard curves. Using dPCR, the PCR mix containing the (target) DNA is partitioned – depending on the device used – currently into a maximum of 10,000,000 small compartments with a volume as low as a few picoliters. These can be either physically distinct compartments on a chip (referred to as chamber-based digital PCR [cdPCR]), or these compartments correspond to water-in-oil droplets (referred to as droplet digital [ddPCR]). Common to both approaches, once PCR has been carried out simultaneously in all compartments/droplets, the number of positive and negative signals for each partition is counted by fluorescence measurement. With this technique, an absolute quantification of DNA copy numbers can be performed with high precision and trueness, even for very low DNA copy numbers. Furthermore, dPCR is considered less susceptible than qPCR to PCR inhibitory substances that can be co-extracted during DNA extraction from different sources. Digital PCR has already been applied in various fields, for example for the detection and quantification of GMOs, species (animals, plants), human diseases, food viruses and bacteria including pathogens. When establishing dPCR in a laboratory, different aspects have to be considered. These include, but are not limited to, the adjustment of the type of the PCR master mix used, optimised primer and probe concentrations and signal separation of positive and negative compartments. This document addresses these and other aspects and provides recommendations for the transfer of existing real-time PCR methods into a dPCR format.JRC.F.5-Food and Feed Complianc

Application of whole genome shotgun sequencing for detection and characterization of genetically modified organisms and derived products

The emergence of high-throughput, massive or next-generation sequencing technologies has created a completely new foundation for molecular analyses. Various selective enrichment processes are commonly applied to facilitate detection of predefined (known) targets. Such approaches, however, inevitably introduce a bias and are prone to miss unknown targets. Here we review the application of high-throughput sequencing technologies and the preparation of fit-for-purpose whole genome shotgun sequencing libraries for the detection and characterization of genetically modified and derived products. The potential impact of these new sequencing technologies for the characterization, breeding selection, risk assessment, and traceability of genetically modified organisms and genetically modified products is yet to be fully acknowledged. The published literature is reviewed, and the prospects for future developments and use of the new sequencing technologies for these purposes are discussed

High Throughput Sequencing for Detection of Foodborne Pathogens

High-throughput sequencing (HTS) is becoming the state-of-the-art technology for typing of microbial isolates, especially in clinical samples. Yet, its application is still in its infancy for monitoring and outbreak investigations of foods. Here we review the published literature, covering not only bacterial but also viral and Eukaryote food pathogens, to assess the status and potential of HTS implementation to inform stakeholders, improve food safety and reduce outbreak impacts. The developments in sequencing technology and bioinformatics have outpaced the capacity to analyze and interpret the sequence data. The influence of sample processing, nucleic acid extraction and purification, harmonized protocols for generation and interpretation of data, and properly annotated and curated reference databases including non-pathogenic “natural” strains are other major obstacles to the realization of the full potential of HTS in analytical food surveillance, epidemiological and outbreak investigations, and in complementing preventive approaches for the control and management of foodborne pathogens. Despite significant obstacles, the achieved progress in capacity and broadening of the application range over the last decade is impressive and unprecedented, as illustrated with the chosen examples from the literature. Large consortia, often with broad international participation, are making coordinated efforts to cope with many of the mentioned obstacles. Further rapid progress can therefore be prospected for the next decade