Complete, closed and curated genome sequences of Photobacterium damselae subsp. piscicida isolates from Australia indicate mobilome-driven localized evolution and novel pathogenicity determinants.

Despite the recent advances in sequencing technologies, the complete assembly of multi-chromosome genomes of the Vibrionaceae, often containing several plasmids, remains challenging. Using a combination of Oxford Nanopore MinION long reads and short Illumina reads, we fully sequenced, closed and curated the genomes of two strains of a primary aquatic pathogen Photobacterium damselae subsp. piscicida isolated in Australia. These are also the first genome sequences of P. damselae subsp. piscicida isolated in Oceania and, to our knowledge, in the Southern hemisphere. We also investigated the phylogenetic relationships between Australian and overseas isolates, revealing that Australian P. damselae subsp. piscicida are more closely related to the Asian and American strains rather than to the European ones. We investigated the mobilome and present new evidence showing that a host specialization process and progressive adaptive evolution to fish are ongoing in P. damselae subsp. piscicida, and are largely mediated by transposable elements, predominantly in chromosome 2, and by plasmids. Finally, we identified two novel potential virulence determinants in P. damselae subsp. piscicida - a chorismate mutase gene, which is ubiquitously retained and co-localized with the AIP56 apoptogenic toxin-encoding gene on the pPHDP10 plasmid, and transfer-messenger RNA gene ssrA located on the main chromosome, homologous to a critical-to-virulence determinant in Yersinia pseudotuberculosis. Our study describes, to our knowledge, the only fully closed and manually curated genomes of P. damselae subsp. piscicida available to date, offering new insights into this important fish pathogen and its evolution.

The Inner Workings of the Outer Surface: Skin and Gill Microbiota as Indicators of Changing Gut Health in Yellowtail Kingfish.

The mucosal surfaces and associated microbiota of fish are an important primary barrier and provide the first line of defense against potential pathogens. An understanding of the skin and gill microbial assemblages and the factors which drive their composition may provide useful insights into the broad dynamics of fish host-microbial relationships, and may reveal underlying changes in health status. This is particularly pertinent to cultivated systems whereby various stressors may led to conditions (like enteritis) which impinge on productivity. As an economically important species, we assessed whether the outer-surface bacterial communities reflect a change in gut health status of cultivated Yellowtail Kingfish (Seriola lalandi). Active bacterial assemblages were surveyed from RNA extracts from swabs of the skin and gills by constructing Illumina 16S rRNA gene amplicon libraries. Proteobacteria and Bacteroidetes were predominant in both the skin and gills, with enrichment of key ß-proteobacteria in the gills (Nitrosomonadales and Ferrovales). Fish exhibiting early stage chronic lymphocytic enteritis comprised markedly different global bacterial assemblages compared to those deemed healthy and exhibiting late stages of the disease. This corresponded to an overall loss of diversity and enrichment of Proteobacteria and Actinobacteria, particularly in the gills. In contrast, bacterial assemblages of fish with late stage enteritis were generally similar to those of healthy individuals, though with some distinct taxa. In conclusion, gut health status is an important factor which defines the skin and gill bacterial assemblages of fish and likely reflects changes in immune states and barrier systems during the early onset of conditions like enteritis. This study represents the first to investigate the microbiota of the outer mucosal surfaces of fish in response to underlying chronic gut enteritis, revealing potential biomarkers for assessing fish health in commercial aquaculture systems.

Praziquantel delivery via moist pellets to treat monogenean parasites of yellowtail kingfish Seriola lalandi: efficacy and feed acceptance.

Praziquantel (PZQ) is registered for oral use against Benedenia seriolae infecting Seriola spp. in Japan, but its bitter taste causes poor palatability. Incorporating PZQ in a moist pellet may help mask the flavor to improve intake. Altering delivery, however, may influence efficacy. We assessed the minimum effective concentrations of PZQ in moist pellets delivered by intubation for the monogeneans Zeuxapta seriolae and B. seriolae infecting yellowtail kingfish Seriola lalandi in flow-through tanks. The optimised dose was then evaluated in a sea-cage of S. lalandi to assess feed acceptance and efficacy. During intubation trials, efficacy was assessed as a percent reduction compared to control groups; in the field trial, efficacy was assessed by a percent reduction after treatment. PZQ delivered by intubation at 70 mg kg-1 body weight (BW) for 3 d was 99.7 and 81.7% effective against Z. seriolae and B. seriolae, respectively. Increasing the dose to 120 mg kg-1 BW for 3 d had a similar efficacy against Z. seriolae (98.4%) and increased efficacy against B. seriolae to 89.2%, but partial emesis of the medicated feed was sometimes noted. S. lalandi in a sea-cage at 17°C readily consumed PZQ administered daily in moist pellets at 70 mg kg-1 BW for 3 d (inclusion rate: 5.15 g kg-1), which significantly reduced Z. seriolae and B. seriolae abundance with 99.4 and 81.6% efficacy, respectively. Juvenile B. seriolae were common on the eyes of fish post-treatment which indicates a strategically timed repeat treatment is necessary.

Praziquantel treatment for yellowtail kingfish (Seriola lalandi): dose and duration safety study.

Regulatory approval is being sought to use praziquantel (PZQ) to treat flukes infecting yellowtail kingfish (YTK), but accurate safety data were not available. We investigated the effect of increased doses or prolonged exposure of orally administered PZQ on YTK by assessing changes in haematological and biochemical characteristics, and mortality. Fish were intubated daily for 3 days with 0, 100, 300 and 500 mg PZQ kg(-1) BW day(-1) or once daily for 9 days at 0 and 100 mg PZQ kg(-1) BW day(-1). Blood was taken 24 h after the cessation of treatment. There was no significant difference between any of the haematological or biochemical indices in YTK treated with PZQ and controls, indicating that PZQ is safe for use at 100 mg PZQ kg(-1) BW day(-1) in YTK and that exposure to high doses or prolonged duration does not have negative effects on the YTK haematological or biochemical parameters we measured.

Histological evaluation of sodium percarbonate exposure on the gills of rainbow trout.

Ichthyophthirius multifiliis is a recurring problem in Australian rainbow trout Oncorhynchus mykiss farms and requires strategically timed, repeat treatments for effective management. Sodium percarbonate (SPC) is permitted for use in Australia, with host safety margins based on the toxicity of acute exposures to hydrogen peroxide (HP), the active product released when SPC is added to water. The effects of exposure to HP released by SPC, of repeated doses and of doses exceeding 100 mg l-1 on rainbow trout are unknown. We exposed juvenile rainbow trout (mean weight: 30.5 ± 9 g) to repeated doses of 50, 150 and 250 mg l-1 SPC for 1 h on Days 1, 2, 7 and 8 of a treatment regime. The effect of SPC was assessed by histological evaluation of structural changes in gill tissue. Survival was 100% in all groups, but some fish exposed to 250 mg l-1 SPC displayed impaired swimming performance, and on Day 9 after the final treatment, oedema was present in 9.8% of lamella, which was significantly higher than the mean occurrence of 1.7, 4.2 and 1.3% in fish treated with 0, 50 and 150 mg l-1 SPC, respectively. These changes resolved within 24 h of the cessation of treatment. We conclude that SPC is safe to use on rainbow trout in doses of ≤150 mg l-1 at 17°C, however caution is advised at doses approaching 250 mg l-1. Water temperature, fish age, fish size and maturity, intensity of parasite infection and stocking density could alter the sensitivity of rainbow trout to SPC treatments.

Life cycle and settlement of an Australian isolate of Ichthyophthirius multifiliis Fouquet, 1876 from rainbow trout.

Ichthyophthirius multifiliis Fouquet, 1876, a ciliate parasite, is a cosmopolitan and problematic parasite of cultured freshwater fish. Each geographical isolate of I. multifiliis has variations in life cycle timing under different abiotic water conditions, such as temperature and salinity. We assessed the effects of salinity and temperature on the development and the preferred settlement site of a temperate Australian isolate of I. multifiliis. The time until theront release was significantly different between each temperature; development time was longest at 5 °C with a mean time of 189 h and decreased to a mean time of 11.7 h at 30 °C. At 5 °C our isolate produced a mean of 267 theronts per tomont, which increased to a mean of 493 theronts at 25 °C and reduced to a mean of 288 theronts at 30 °C. Theront length showed an inverse relationship to temperature; mean length was 62 µm at 5 °C and 41 µm at 30 °C. Our isolate reproduced faster at all temperatures and a greater sensitivity to salinity than all reported profiles for temperate isolates. Parasite abundance was highest on the dorsal region of the fish. An accurate understanding of temperature-life cycle information and optimal region to sample for surveillance will aid in the development of specific management plans for the Australian isolate of I. multifiliis, facilitating the strategic timing of treatments.

Minimum effective concentrations of formalin and sodium percarbonate on the free-living stages of an Australian isolate of Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis Fouquet, 1876, a ciliate protozoan, is a common cosmopolitan parasite of freshwater teleosts and is a recurring problem during the summer months on Australian rainbow trout Oncorhynchus mykiss (Walbaum, 1792) farms. Preventative strategies include increasing water flow and filtration, but when an infection is established, chemical intervention is often required. Formalin (FOR) has been traditionally used on Australian trout farms as a treatment for I. multifiliis. Treatment using sodium percarbonate (SPC) that releases hydrogen peroxide when dissolved is being implemented on a number of farms. To assess anecdotal reports of low efficacy we evaluated 1 h exposures of FOR and SPC at 12 °C and 17 °C in both hard and soft water against free-living stages of I. multifiliis. Each free-living stage were exposed to FOR and SPC in vitro; theronts were exposed to 8, 16, 32 or 64 mg/l SPC or FOR every 15 min, for a maximum of 6 h, and the number of live theronts at each time point was recorded. Prototomonts and tomocysts were exposed to 64, 128, 256 and 512 mg/l SPC and 16, 32, 64 and 128 mg/l FOR for 1 h, incubated, with the percentage viability and the number of theronts produced recorded. Theronts were more sensitive to treatment than tomonts, and prototomonts were more sensitive to treatment than tomocysts. FOR and SPC killed all theronts within 15 min at 64 mg/l at both temperatures. FOR was effective against all prototomonts at ≥64 mg/l at both temperatures and was effective against all tomocysts at 128 mg/l at 17 °C but did not achieve complete mortality in any doses tested at 12 °C. SPC was effective against prototomonts and tomocysts at 64 m/l at 17 °C but required ≥256 mg/l at 12 °C. These results can be used to aid development of specific treatment strategies for the management of I. multifiliis on Australian rainbow trout farms.

Evolution of the capsular operon of Streptococcus iniae in response to vaccination.

Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins.