Eukaryote symbionts of animals are major drivers of ecosystems not only because of their diversity and host interactions from variable pathogenicity but also through different key roles such as commensalism and to different types of interdependence. However, molecular investigations of metazoan eukaryomes require minimising co-amplification of homologous host genes. In this study we 1) identified a previously published ‘anti-metazoan’ reverse primer to theoretically enable amplification of a wider range of microeukaryotic symbionts, including more evolutionarily divergent sequence types, 2) evaluated in silico several anti-metazoan primer combinations, and 3) optimised the application of the best performing primer pair for high throughput sequencing (HTS) by comparing one-step and two-step PCR amplification approaches, testing different annealing temperatures and evaluating the taxonomic profiles produced by HTS and data analysis. The primer combination 574*F – UNonMet_DB tested in silico showed the largest diversity of non-metazoan sequence types in the SILVA database and was also the shortest available primer combination for broadly-targeting anti-metazoan amplification across the 18S rRNA gene V4 region. We demonstrate that the one-step PCR approach used for library preparation produces significantly lower proportions of metazoan reads, and a more comprehensive coverage of host-associated microeukaryote reads than the two-step approach. Using higher PCR annealing temperatures further increased the proportion of non-metazoan reads in all sample types tested. The resulting V4 region amplicons were taxonomically informative even when only the forward read is analysed. This region also revealed a diversity of known and putatively parasitic lineages and a wider diversity of host-associated eukaryotes.

The World Health Organization considers antimicrobial resistance as one of the most pressing global issues which poses a fundamental threat to human health, development, and security. Due to demographic and environmental factors, the marine environment of the Gulf Cooperation Council (GCC) region may be particularly susceptible to the threat of antimicrobial resistance. However, there is currently little information on the presence of AMR in the GCC marine environment to inform the design of appropriate targeted surveillance activities. The objective of this study was to develop, implement and conduct a rapid regional baseline monitoring survey of the presence of AMR in the GCC marine environment, through the analysis of seawater collected from high-risk areas across four GCC states: (Bahrain, Oman, Kuwait, and the United Arab Emirates). 560 Escherichia coli strains were analysed as part of this monitoring programme between December 2018 and May 2019. Multi-drug resistance (resistance to three or more structural classes of antimicrobials) was observed in 32.5% of tested isolates. High levels of reduced susceptibility to ampicillin (29.6%), nalidixic acid (27.9%), tetracycline (27.5%), sulfamethoxazole (22.5%) and trimethoprim (22.5%) were observed. Reduced susceptibility to the high priority critically important antimicrobials: azithromycin (9.3%), ceftazidime (12.7%), cefotaxime (12.7%), ciprofloxacin (44.6%), gentamicin (2.7%) and tigecycline (0.5%), was also noted. A subset of 173 isolates was whole genome sequenced, and high carriage rates of qnrS1 (60/173) and bla_CTX-M-15 (45/173) were observed, correlating with reduced susceptibility to the fluoroquinolones and third generation cephalosporins, respectively. This study is important because of the resistance patterns observed, the demonstrated utility in applying genomic-based approaches to routine microbiological monitoring, and the overall establishment of a transnational AMR surveillance framework focussed on coastal and marine environments.

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.

Diseases of bivalve molluscs caused by paramyxid parasites of the genus Marteilia have been linked to mass mortalities and the collapse of commercially important shellfish populations. Until recently, no Marteilia spp. have been detected in common cockle (Cerastoderma edule) populations in the British Isles. Molecular screening of cockles from ten sites on the Welsh coast indicates that a Marteilia parasite is widespread in Welsh C. edule populations, including major fisheries. Phylogenetic analysis of ribosomal DNA (rDNA) gene sequences from this parasite indicates that it is a closely related but different species to Marteilia cochillia, a parasite linked to mass mortality of C. edule fisheries in Spain, and that both are related to Marteilia octospora, for which we provide new rDNA sequence data. Preliminary light and transmission electron microscope (TEM) observations support this conclusion, indicating that the parasite from Wales is located primarily within areas of inflammation in the gills and the connective tissue of the digestive gland, whereas M. cochillia is found mainly within the epithelium of the digestive gland. The impact of infection by the new species, here described as Marteilia cocosarum n. sp., upon Welsh fisheries is currently unknown.

While both virulent and putatively avirulent Yersinia ruckeri strains exist in aquaculture environments, the relationship between the distribution of virulence-associated factors and de facto pathogenicity in fish remains poorly understood. Pan-genome analysis of 18 complete genomes, representing established virulent and putatively avirulent lineages of Y. ruckeri, revealed the presence of a number of accessory genetic determinants. Further investigation of 68 draft genome assemblies revealed that the distribution of certain putative virulence factors correlated well with virulence and host-specificity. The inverse-autotransporter invasin locus yrIlm was, however, the only gene present in all virulent strains, while absent in lineages regarded as avirulent. Strains known to be associated with significant mortalities in salmonid aquaculture display a combination of serotype O1-LPS and yrIlm, with the well-documented highly virulent lineages, represented by MLVA clonal complexes 1 and 2, displaying duplication of the yrIlm locus. Duplication of the yrIlm locus was further found to have evolved over time in clonal complex 1, where some modern, highly virulent isolates display up to three copies.

Infectious disease causes significant mortality in wild and farmed systems, threatening biodiversity, conservation and animal welfare, as well as food security. To mitigate impacts and inform policy, tools such as mathematical models and computer simulations are valuable for predicting the potential spread and impact of disease. This paper describes the development of the Aquaculture Disease Network Model, AquaNet-Mod, and demonstrates its application to evaluating disease epidemics and the efficacy of control, using a Viral Haemorrhagic Septicaemia (VHS) case study. AquaNet-Mod is a data-driven, stochastic, state-transition model. Disease spread can occur via four different mechanisms, i) live fish movement, ii) river based, iii) short distance mechanical and iv) distance independent mechanical. Sites transit between three disease states: susceptible, clinically infected and subclinically infected. Disease spread can be interrupted by the application of disease mitigation measures and controls such as contact tracing, culling, fallowing and surveillance. Results from a VHS case study highlight the potential for VHS to spread to 96% of sites over a 10 year time horizon if no disease controls are applied. Epidemiological impact is significantly reduced when live fish movement restrictions are placed on the most connected sites and further still, when disease controls, representative of current disease control policy in England and Wales, are applied. The importance of specific disease control measures, particularly contact tracing and disease detection rate, are also highlighted. The merit of this model for evaluation of disease spread and the efficacy of controls, in the context of policy, along with potential for further application and development of the model, for example to include economic parameters, is discussed.