ABSTRACT
Bacteria from the family Vibrionaceae have been implicated in mass mortalities of farmed Pacific oysters (Magallana gigas) in multiple countries, leading to substantial impairment of growth in the sector. In Ireland there has been concern that Vibrio have been involved in serious summer outbreaks. There is evidence that Vibrio aestuarianus is increasingly becoming the main pathogen of concern for the Pacific oyster industry in Ireland. While bacteria belonging to the Vibrio splendidus clade are also detected frequently in mortality episodes, their role in the outbreaks of summer mortality is not well understood. To identify and characterize strains involved in these outbreaks, 43 Vibrio isolates were recovered from Pacific oyster summer mass mortality episodes in Ireland from 2008 to 2015 and these were whole-genome sequenced. Among these, 25 were found to be V. aestuarianus (implicated in disease) and 18 were members of the V. splendidus species complex (role in disease undetermined). Two distinct clades of V. aestuarianus - clade A and clade B - were found that had previously been described as circulating within French oyster culture. The high degree of similarity between the Irish and French V. aestuarianus isolates points to translocation of the pathogen between Europe's two major oyster-producing countries, probably via trade in spat and other age classes. V. splendidus isolates were more diverse, but the data reveal a single clone of this species that has spread across oyster farms in Ireland. This underscores that Vibrio could be transmitted readily across oyster farms. The presence of V. aestuarianus clades A and B in not only France but also Ireland adds weight to growing concern that this pathogen is spreading and impacting Pacific oyster production within Europe.
Subject(s)
Crassostrea , Vibrio , Animals , Ireland/epidemiology , Disease OutbreaksABSTRACT
Harmful algal blooms (HABs) are a naturally occurring global phenomena that have the potential to impact fisheries, leisure and ecosystems, as well as posing a significant hazard to animal and human health. There is significant interest in the development and application of methodologies to study all aspects of the causative organisms and toxins associated with these events. This paper reports the first application of nanopore sequencing technology for the detection of eukaryotic harmful algal bloom organisms. The MinION sequencing platform from Oxford Nanopore technologies provides long read sequencing capabilities in a compact, low cost, and portable format. In this study we used the MinION to sequence long-range PCR amplicons from multiple dinoflagellate species with a focus on the genus Alexandrium. Primers applicable to a wide range of dinoflagellates were selected, meaning that although the study was primarily focused on Alexandrium the applicability to three additional genera of toxic algae, namely; Gonyaulax, Prorocentrum, and Lingulodinium was also demonstrated. The amplicon generated here spanned approximately 3 kb of the rDNA cassette, including most of the 18S, the complete ITS1, 5.8S, ITS2 and regions D1 and D2 of the 28S. The inclusion of barcode genes as well as highly conserved regions resulted in identification of organisms to the species level. The analysis of reference cultures resulted in over 99% of all sequences being attributed to the correct species with an average identity above 95% from a reference list of over 200 species (see Supplementary Material 1). The use of mock community analysis within environmental samples highlighted that complex matrices did not prevent the ability to distinguish between phylogenetically similar species. Successful identification of causative organisms in environmental samples during natural toxic events further highlighted the potential of the assay. This study proves the suitability of nanopore sequencing technology for taxonomic identification of harmful algal bloom organisms and acquisition of data relevant to the World Health Organisations "one health" approach to marine monitoring.
ABSTRACT
The Genus Alexandrium is a widespread dinoflagellate marine phytoplankton that is the primary causative organism causing Paralytic Shellfish Poisoning (PSP) intoxications in European waters. EU food safety directives specify that EU Member States must implement a routine monitoring programme to mitigate risks associated with bio-accumulation of biotoxins by bivalve shellfish, such as those produced by Alexandrium. This strategic drive comprises of both direct testing of bivalve flesh for the presence of regulated toxins and an early warning phytoplankton monitoring programme. In the UK the flesh testing moved away from animal bio-assays to analytical chemistry techniques, whereas phytoplankton monitoring methods have seen little technological advancement since implementation. Methods currently utilize light microscopy and manual enumeration of different algal species. These methods although proven are time consuming, reliant on highly trained staff, have high limits of detection (LOD) with low specificity, unable to reliably identify Alexandrium to species level. The implications of these limitations of the techniques mean that in the case of Alexandrium the LOD is also the action limit and as such it is easy to miss positive samples affecting the efficacy of any early warning strategy. This study outlines the development, preliminary method characterisation, validation and trial implementation of an alternative early warning technique, utilizing quantitative PCR to identify water samples containing Alexandrium cells. The approach outlined in this document, showed an improved correlation with flesh toxicity, improved sensitivity, improved throughput compared to traditional light microscopy methods and there was also good correlation with higher cell abundance samples when compared to the light microscopy results. The application of this approach to routine water samples was explored and was found to demonstrate potential as a corroborative method for use during flesh intoxication episodes. This study offers potential for future improvements in the accuracy and sensitivity of phytoplankton monitoring whilst ensuring continuity of public safety, providing cost savings and offering new research opportunities.
