Tilapia farming is one of the most important sectors in aquaculture worldwide and of major importance to global food security. Infectious spleen and kidney necrosis virus (ISKNV) has been identified as an agent of high morbidity and mortality, threatening tilapia aquaculture. ISKNV was detected in Lake Volta, Ghana, in September 2018 and spread rapidly, with mortality rates between 60 and 90% and losses of more than 10 tonnes of fish per day. Understanding the spread and evolution of viral pathogens is important for control strategies. Here, we developed a tiled-PCR sequencing approach for the whole-genome sequencing of ISKNV, using long read sequencing to enable field-based, real-time genomic surveillance. This work represents the first use of tiled-PCR for whole genome recovery of viruses in aquaculture, with the longest genome target (>110 kb dsDNA) to date. Our protocol was applied to field samples collected from the ISKNV outbreaks from four intensive tilapia cage culture systems across Lake Volta, between October 2018 and May 2022. Despite the low mutation rate of dsDNA viruses, 20 single nucleotide polymorphisms accumulated during the sampling period. Droplet digital PCR identified a minimum requirement of template in a sample to recover 50% of an ISKNV genome at 275 femtograms (2410 viral templates per 5 µL sequencing reaction). Overall, tiled-PCR sequencing of ISKNV provides an informative tool to assist in disease control in aquaculture.

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.

2. Bacteria from the Vibrionaceae family have been implicated in mass mortalities of farmed Pacific oysters ( Crassostrea gigas ) in multiple countries, leading to substantial impairment for 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 are not well understood. To identify and characterise strains involved in these outbreaks, 43 Vibrio isolates were recovered from Pacific oyster summer mass mortality episodes in Ireland from 2008-2015 and these were whole genome sequenced. Among these, 25 were found to be V. aestuarianus (implicated in disease) and 18 V. splendidus sensu lato (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. 3. Outcome: Pacific oyster culture in Ireland has increasingly suffered from summer mass mortality events. Many of these mortalities in recent years have been associated with Vibrio aestuarianus ; the role of another pathogen, Vibrio splendidus has, so far, remained inconclusive. Here we show that two clades of V. aestuarianus are circulating in Ireland, and that these are members of two clades that have previously caused extensive oyster die offs in France. Their discovery in Ireland is consistent with transport of infected oyster stock between the two countries. Although V. splendidus- like strains in Ireland were highly diverse, a small clonal group was detected that appears to have spread rapidly from a single source to disparate locations in Ireland. Combined, these findings highlight the appearance of a highly pathogenic Vibrio in Ireland, and the risk of transmission between interconnected oyster production industries in Europe. 4. Data summary: Sequences generated in this study were deposited on the NCBI. Accession number: PRJNA797364. Publicly accessed genomes are listed in Table S2. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files .