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Essential oils are products from aromatic plants, which, due to their biological, allelopathic, antioxidant and antimicrobial effects, are important for food preservation. Kajikia audax is a fish of great commercial importance, however, it is highly perishable, requiring strategies to extend its shelf life. In vitro antimicrobial activity of Bursera graveolens essential oil was determined against microorganisms isolated from K. audax. Essential oil was extracted by steam distillation, obtaining a yield of 1.25%, a density of 0.83 g/ml and a refractive index of 1.473°, in addition, it was determined by GC-MS that D-limonene (77.6%) is the majority compound. Antimicrobial tests showed that the minimum inhibitory concentration (MIC) for Aeromonas salmonicida and Pichia kudriavzevii was 1.62 mg/ml and 6.48 mg/ml respectively, and the minimum bactericidal concentration (MBC) was 25.92 mg/ml for both microorganisms. Pseudomonas aeruginosa showed total resistance against the concentrations used. B. graveolens essential oil turned out to be a potential product to control the growth of microorganisms isolated from K. audax, however, it should be tested against species of the genera Vibrio, Flavobacterium, Shewanella, Lactococcus and Streptococcus that cause spoiling of hydrobiological products. [ABSTRACT FROM AUTHOR]

Standard disc diffusion and MIC test procedure were used to investigate the susceptibility of two hundred and fifty‐one isolates collected from infected fish in France to florfenicol, oxolinic acid and tetracycline. The tests were performed at 22 ± 2℃ and for the 177 Yersinia ruckeri they were read after 24–28 hr incubation and for the 74 Aeromonas salmonicida isolates they were read after 44–48 hr. Applying epidemiological cut‐off values to the susceptibility data generated in these tests, the isolates were categorized as wild‐type or non‐wild‐type. The agent‐specific categories into each isolate were placed on the basis of the data generated by the two methods were in agreement in 98% of the determinations made. It is argued that, with respect to categorising isolates, disc diffusion and MIC methods can be considered as equally valid at this temperature and after both periods of incubation. [ABSTRACT FROM AUTHOR]

The bacterium Aeromonas salmonicida is the pathogen responsible for furunculosis, which is a serious disease of salmonids. This disease has a significant economic impact on the economic benefits of the global salmon farming industry. However, the pathogenesis of this disease in fish is still unknown. Members of the aldehyde dehydrogenase gene (ALDH) superfamily play a key role in the enzyme detoxification of endogenous and exogenous aldehydes. In this study, we obtained a recombinant aldehyde dehydrogenase 7A1 (ALDH7A1) protein to find its functions on Atlantic salmon infected by A. salmonicida. The transcriptional response in the liver of Atlantic salmon (Salmo salar) with differing levels of A. salmonicida infection was analysed and compared in order to reveal mechanisms by which ALDH7A1 may confer infection resistance. With the addition of ALDH7A1 protein, it was found that a total of 13,369 genes were annotated with one or more KEGG and localized to 360 KEGG pathways in the high concentration infection group. The differential expression genes were more enriched in immune signalling pathways such as the Toll‐like receptor signalling pathway, NF‐kappa B signalling pathway and TNF signalling pathway. On the other hand, at low concentrations of infection, KEGG enriched a smaller number of differential expression genes. However, these differential genes were more concentrated in immune signalling pathways such as the PI3K‐Akt signalling pathway, JAK‐STAT signalling pathway and complement and coagulation cascades. In addition, several known immune‐related genes including HSP90α, HSP70, DNA damage‐inducible transcript 4, integrin alpha 5 and microtubule‐associated protein 2 were among the differentially expressed transcripts. These data provide the first insights into the host–ALDH7A1 vaccine interactome. The results of this study contribute to identifying the potential resistance mechanisms of Atlantic salmon to A. salmonicida infection and determining future treatment strategies. [ABSTRACT FROM AUTHOR]

The fungal strains Pseudogymnoascus are a kind of psychrophilic pathogenic fungi that are ubiquitously distributed in Antarctica, while the studies of their secondary metabolites are infrequent. Systematic research of the metabolites of the fungus Pseudogymnoascus sp. HSX2#-11 led to the isolation of six new tremulane sesquiterpenoids pseudotremulanes A–F (1 – 6), combined with one known analog 11,12-epoxy-12β-hydroxy-1-tremulen-5-one (7), and five known steroids (8 – 12). The absolute configurations of the new compounds (1 – 6) were elucidated by their ECD spectra and ECD calculations. Compounds 1 – 7 were proved to be isomeride structures with the same chemical formula. Compounds 1 / 2 , 3 / 4 , 1 / 4 , and 2 / 3 were identified as four pairs of epimerides at the locations of C-3, C-3, C-9, and C-9, respectively. Compounds 8 and 9 exhibited cytotoxic activities against human breast cancer (MDA-MB-231), colorectal cancer (HCT116), and hepatoma (HepG2) cell lines. Compounds 9 and 10 also showed antibacterial activities against marine fouling bacteria Aeromonas salmonicida. This is the first time to find terpenoids and steroids in the fungal genus Pseudogymnoascus. [ABSTRACT FROM AUTHOR]

Oral delivery is the most convenient way to vaccinate cultured fish, however it is still problematic, primarily due to a lack of a commercially valid vaccine vehicle to protect the antigen against gastric degradation and ensure its uptake from the intestine. With the goal of advancing the potential to vaccinate orally, this study evaluates a novel silicon nanoparticle-based vehicle (VacSaf carrier). Aeromonas salmonicida antigens were formulated with the VacSaf carrier using different preparation methods to generate dry powder and liquid formulations. Twelve formulations were first subjected to an in vitro evaluation where the A. salmonicida bacterin conjugated to VacSaf carriers were found superior at inducing pro-inflammatory cytokine expression in primary leucocyte cultures and the macrophage/monocyte cell line RTS-11 compared with A. salmonicida bacterin alone. This was especially apparent after exposure to acid conditions to mimic stomach processing. One formulation (FD1) was taken forward to oral delivery using two doses and two administration schedules (5 days vs 10 days, the latter 5 days on, 5 days off, 5 days on), and the transcript changes of immune genes in the intestine (pyloric caeca, midgut and hindgut) and spleen were evaluated by qPCR and serum IgM was measured by ELISA. The VacSaf carrier alone was shown to be safe for use in vivo , in that no side-effects were seen, but it did induce expression of some cytokines, and may have value as an oral adjuvant candidate. The FD1 bacterin formulation was effective at inducing a range of cytokines associated with innate and adaptive immunity, mainly in the pyloric caeca, compared to A. salmonicida bacterin alone (which had almost no effect), and confirms the immune competence of this gut region following appropriate oral vaccination. These results reveal that in vitro screening of formulations for oral delivery has value and can be used to assess the most promising formulations to test further. • VacSaf carriers promote stability of A. salmonicida bacterin in acidic environments. • VacSaf carriers are safe to use for bacterin in vivo delivery to fish. • VacSaf carrier is a potential oral adjuvant, impacting gut cytokine expression. • A. salmonicida bacterin/VacSaf carrier induces key cytokines in gut and spleen. • The gut immune system is resilient to different oral administration regimes. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of three commercial diets, standard, immunostimulant-supplemented (β-glucan, vitamins C and E) and plant-based, on the degree of oxidative stress in tissues of rainbow trout (Oncorhynchus mykiss). Selected immune and haematological indices were measured and challenge with Aeromonas salmonicida was conducted. The plant-based diet systemically modulated all oxidative stress biomarkers (thiobarbituric acid reactive substances; reduced and oxidised glutathione and their ratio) in the intestine. The ratio was elevated in all organs (liver, kidney, muscle and intestine) and an enhancement of respiratory burst and complement activity was observed even in the control fish. With the standard diet, an elevation of thiobarbituric acid reactive substances in the intestine and a decrease in some immune indices appeared after challenge. Less distinctive changes and the lowest mortality rate (the highest being with the plant-based feed) were obtained with immunostimulants. Increased total immunoglobulin, relative lymphocytosis and a decrease in the phagocyte count were observed. This study contributes especially by a thorough examination of oxidative stress indices in different tissues. The exact composition of these commercial diets is a trade secret; however, knowledge of their effects is extremely important for fish farmers using them; therefore, this study has a great practical impact. [ABSTRACT FROM AUTHOR]

The movement of live fish for use as bait in recreational angling has been identified as a high-risk pathway for the spread of aquatic invasive species and disease in the Great Lakes region. To better understand the hazards present in Minnesota's live baitfish pathway, we employed both conventional and advanced diagnostic approaches to detect non-target species and pathogens in golden shiners (Notemigonus crysoleucas), a common baitfish sold in retail bait shops. Golden shiners were purchased from bait shops (n = 34 total; n = 15 sampled once, n = 19 sampled twice) in 2014 and 2015 across Minnesota. Of the 53 lots examined, non-target species were observed in 24/53 lots and included nine different fish species and one frog species, however, none of the non-target species found are listed as invasive in Minnesota. Nine parasite taxa were observed by wet mounts of the gills, epidermal mucus, and fin clips in 27/53 lots. The microsporidian parasite, Ovipleistophora ovariae, was detected in 24/53 lots by qPCR. While many bacterial species were identified by culture dependent and independent methods, two notable species including, Aeromonas salmonicida and Yersinia ruckeri, were confirmed in ten and five lots, respectively. Detection of replicating virus in culture was low, with only 2/53 lots positive for the golden shiner reovirus. No other viruses were detected with the standard culture-based assays; however, a total of eight novel viruses were detected by next-generation sequencing. These findings underscore the need for a proactive surveillance approach that includes advanced diagnostic tools for the detection of emerging aquatic pathogens, to better understand and manage the risks associated with the use of live baitfish. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida (As) is a highly heterogeneous bacterial species, and strains' host specificity has been reported. Ballan wrasse (Labrus bergylta Ascanius, 1767) is susceptible to atypical As (aAs) vapA type V and type VI in Scotland and Norway. Identification of the bacterium is achieved by culture and molecular techniques; however, the available methods used to distinguish the As types are costly and time‐consuming. This paper describes the development of a PCR and a restriction enzyme assay for the detection of aAs vapA type V and type VI in ballan wrasse, respectively. Type V‐specific primers were designed on conserved regions of the vapA gene, and the restriction enzyme assay was performed on the PCR products of the hypervariable region of vapA gene for the detection of type VI isolates. Amplification product was produced for type V (254 bp) and restriction bands (368 and 254 bp) for type VI isolates only. In addition, the assays detected type V and type VI isolates in spiked water samples and type V in diagnostic tissue samples. The assays are fast, specific and cost‐effective and can be used as specific diagnostic tools for cleaner fish, to detect infectious divergence strains, and to manage and mitigate aAs disease outbreaks through vaccine development. [ABSTRACT FROM AUTHOR]

Atypical Aeromonas salmonicida (aAs) is currently one of the most routinely recovered bacterial pathogens isolated during disease outbreaks in farmed cleaner fish, ballan wrasse (Labrus bergylta, Ascanius). Vibrionaceae family bacteria have also been isolated from ballan wrasse in Scotland. This study determined the infectivity, pathogenicity and virulence of aAs and Vibrionaceae isolates in juvenile farmed ballan wrasse (n = 50; approx. 2 g) using a bath challenge, and fish were monitored for a period of 16 days. Atypical As caused significant mortalities in contrast to Vibrionaceae isolates. Notably, differential virulence was observed between two aAs vapA type V strains at similar challenge doses. Diseased fish exhibited a systemic infection where aAs was detected in all analysed tissues (liver, spleen and kidney) by PCR and qPCR. Macroscopically, moribund and survivor fish exhibited hepatomegaly and splenomegaly. In moribund and surviving fish, histopathology showed granulomatous hepatitis with eosinophilic granular cells surrounding bacterial colonies and endocarditis along with splenic histiocytosis. This is the first report of a successful aAs bath challenge model for juvenile ballan wrasse which provides an important tool for future studies on vaccine efficacy and immunocompetence. [ABSTRACT FROM AUTHOR]

Aeromonas is a ubiquitous aquatic bacteria, and it is a significant factor contributing to meat spoilage during processing and consumption. The abilities of Aeromonas salmonicida 29 and 57, which exhibit spoilage heterogeneity, to secrete protease, lipase, hemolysin, gelatinase, amylase, and lecithinase were confirmed by plate method. A total of 3948 proteins were identified by ITRAQ in extracellular secretions of A. salmonicida , and 16 proteases were found to be potentially related to spoilage ability. The complete genome sequence of A. salmonicida 57 consists of one circular chromosome and three plasmids, while A. salmonicida 29 consists of one circular chromosome, without a plasmid. Transcriptomic analysis revealed a significant number of DEGs were up-regulated in A. salmonicida 29, which were mainly enriched in metabolic pathways (e.g., amino acid metabolism, carbohydrate metabolism), indicating that A. salmonicida 29 had better potential to decompose and utilize nutrients in meat. Six protease genes (2 pepB , hap , pepA , ftsI , and pepD) were excavated by combined ITRAQ with transcriptome analysis, which potentially contribute to bacterial spoilage ability and exhibit universality among other dominant spoilage bacteria. This investigation provides new insights and evidence for elucidating metabolic and spoilage phenotypic differences and provides candidate genes and strategies for future prevention and control technology development. • Aeromonas salmonicida is considered a widely spread bacteria in chilled meat. • Salmonicida possesses proteases related to pathogenic and spoilage. • The genome of two strains of A. salmonicida with spoilage heterogeneity was uncovered. • Transcriptomics revealed a number of DEGs that were up-regulated in A. salmonicida 29. • Six genes that contribute to meat spoilage were excavated: 2 pepB , hap , pepA , fts I, and pepD. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida is one of the most harmful pathogens in finfish aquaculture worldwide. Immunostimulants such as β-glucans are used to enhance the immunity of cultured fish. However, their effects on fish physiology are not completely understood. In the present work, we evaluated the effect of a single intraperitoneal (ip) injection of zymosan A on fish survival against A. salmonicida infection. A single administration of this compound protected fish against A. salmonicida challenge and reduce the bacterial load in the head kidney one week after its administration. Transcriptome analyses of head kidney samples revealed several molecular mechanisms involved in the protection conferred by zymosan A and their regulation by long noncoding RNAs. The transcriptome profile of turbot exposed only to zymosan A was practically unaltered one week after ip injection. However, the administration of this immunostimulant induced significant transcriptomic changes once the fish were in contact with the bacteria and increased the survival of the infected turbot. Our results suggest that the restraint of the infection-induced inflammatory response, the management of apoptotic cell death, cell plasticity and cellular processes involving cytoskeleton dynamics support the protective effects of zymosan A. All this information provides insights on the cellular and molecular mechanisms involved in the protective effects of this widely used immunostimulant. • The administration of zymosan A in turbot induces protection against A. salmonicida. • Zymosan A balance the increment of metabolites and inflammation induced by the bacteria. • Cytoskeleton dynamics and cell plasticity seems to be essential for the protection. • LncRNAs modulated by zymosan A participate in the regulation of all of those processes. • LncRNAs are essential for the protective effect of zymosan A against A. salmonicida. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida is ubiquitously distributed in aquatic environments and is one of the major pathogens of freshwater aquaculture fish. Recent studies have demonstrated that A. salmonicida plays a crucial role in fish spoilage. In this study, a novel antibacterial agent, lactobionic acid (LBA), was investigated for its antibacterial effect against A. salmonicida. The results showed that the minimum inhibitory concentration (MIC) of LBA was 16 mg/mL. Bacterial cells dropped below the detection limit within 2 h after exposure to 2 MIC of LBA. The cells treated with LBA became rough and sunken, and LBA caused cellular protein leakage and an increase in conductivity. Transcriptomic analysis exhibited that the expression of 1273 genes in A. salmonicida was altered by LBA, including 700 down-regulated and 573 up-regulated genes. The most representative down-regulated genes were enriched in bacterial chemotaxis, two-component system and ATP-binding cassette transporter pathways. LBA showed an effective inhibitory effect on A. salmonicida in refrigerated grass carp and could prolong the storage time of grass carp. Overall, the intervention of LBA resulted in the decrease of cell chemotaxis and disturbance of cellular metabolic processes, which inhibited A. salmonicida. These results revealed the antibacterial mechanism of LBA against A. salmonicida , which provided a strategy for the control of fish spoilage. • LBA can effectively inhibit the growth of A. salmonicida. • LBA destroyed the integrity of cell membrane and caused protein leakage. • LBA disturbed the chemotaxis, two-component system and quorum sensing pathways. • LBA can prolong the storage time of refrigerated grass carp. [ABSTRACT FROM AUTHOR]

Foodborne spoilage bacteria may pose a serious risk to food quality by forming biofilms that adhere to food processing devices and food surfaces, thus causing cross-contamination and deterioration of food products. In order to effectively control microbial contamination, understanding the bacterial profiles related to biofilm formation in situ is crucial. Pseudomonas , Aeromonas , and Serratia are typically considered as prevalent spoilage organisms in chilled meat, which is particularly susceptible to deterioration during storage and transportation. In this study, four strains (Serratia liquefaciens F5, Aeromonas salmonicida H8, Pseudomonas saponiphila G7, and Aeromonas veronii G8) were cultured in situ in chilled chicken claws as the experimental group and in Tryptic Soy Broth (TSB) as the control group. The results showed that tested strains differed in ability to adhere, with S. liquefaciens F5 significantly exhibiting the highest biofilm formation capacity (P < 0.05). Moreover, it was discovered that higher protein and polysaccharides concentrations enhanced bacterial auto-aggregation and that the protein content of extracellular polymeric substances (EPS) positively correlated with bacterial surface hydrophobicity. On the 4th day, the protein content in EPS of each strain was significantly higher in vitro than in situ (P < 0.05). Furthermore, the rate of increase in auto-aggregation capacity and surface hydrophobicity proved faster in vitro than in situ for each strain due to higher polysaccharides and protein contents. The primary components (proteins, polysaccharides) in the EPS of the four strains increased as the biofilm formation process progressed, further confirmed by Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) spectra. Scanning electron microscopy revealed that A. salmonicida H8 can create biofilms on the surface of chicken claws, which severely disrupts the tissue structure of chilled meat. These observations will offer a theoretical foundation for a thorough investigation of the spoiling outcomes and adhesion characteristics of various meat-derived spoilage bacteria. • In situ , S. liquefaciens F5 had the strongest adhesion and spoilage capacity. • Lesser EPS produced by bacteria in situ compared to those in vitro. • The proteins and polysaccharides in EPS boost bacterial auto-aggregation. • Proteins and polysaccharides accelerated the process of biofilm formation. • Proteins in EPS increased the hydrophobicity of bacterial surfaces. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are part of the innate immune response against bacteria. Liver expressed antimicrobial peptides (LEAPs) are one of the most important antimicrobial peptides in fish and belong to a family of cysteine-rich antimicrobial peptides with antimicrobial activity against a variety of pathogens. In this study, we cloned and identified two liver expressed antimicrobial peptides (TaLEAP-1, TaLEAP-2) from Thymallus arcticus grubei , functional studies were performed and the two synthesized TaLEAPs peptides were examined in vitro bacterial inhibition. The results showed that the open reading frame lengths of TaLEAP-1 and TaLEAP-2 were 267 bp and 258 bp, respectively. TaLEAP-1 is a β-sheet polypeptide containing 89 amino acids, and TaLEAP-2 is an α-helix with β-sheet polypeptide containing 85 amino acids. Tissue distribution showed that both TaLEAPs were widely expressed in ten tissues of T. arcticus grubei , with the highest expression in the liver. During the bacterial challenge with Aeromonas salmonicida and Aeromonas hydrophila , two LEAPs genes were differentially expressed. TaLEAP-1 has broad-spectrum antibacterial activity against a variety of bacteria. TaLEAP-1 damages bacteria by disrupting cell membranes, showing that TaLEAP-1 has potent antibacterial effects. Cytotoxicity assay results showed that TaLEAP-1 was not toxic and TaLEAP-2 was slightly toxic to RTG-2. The results of this study provide strategies for resistance to bacterial and fungal infections in T. arcticus grubei. • Two LEAPs gene (TaLEAP-1 and TaLEAP-2) were identified and characterized in Thymallus arcticus grubei Dybowski. • Two LEAPs were widely expressed in tested tissues and induced significantly after bacterial infection. • LEAP-1 is effective by disrupting the bacterial cell membrane exhibited a wide range of antibacterial activity against Gram-negative bacteria. • TaLEAP-1 was no toxic and TaLEAP-2 was slightly toxic to RTG-2. [ABSTRACT FROM AUTHOR]

In the present study, the effects of feeding ground leonardite (LE), which contained 68% of humic substances, to juvenile Oncorhynchus mykiss were examined on overall growth and production performance, feed nutrient digestibility, gut microbiota, health condition, antioxidant status, immune response, and disease resistance. During two consecutive experiments, fish were fed four experimental diets with different LE supplementary levels: 0, 0.5, 1, and 3% of feed mass (LE0, LE0.5, LE1, and LE3, respectively). Firstly, the fish were tested in a growing experiment (GE, n = 300 fish/group, body weight 15.4 ± 2.5 g) for 56 days. Thereafter, these fish (n = 75 fish/group, body weight 51.9 ± 8.7 g) were exposed to an intraperitoneal infection challenge experiment (IC) with Aeromonas salmonicida subsp. salmonicida for 28 days. In the GE, dietary LE did not affect the growth, production, or health performance of O. mykiss juveniles, although the fish more than tripled their initial body weight. The LE dose had an impact on the digestibility of feed nutrients (dry matter, gross energy, and ash), but not on the protein and lipid digestibility or solid waste outputs. The gut microbial communities were highly diverse across the experimental groups and remained distinct between the control and LE groups. The LE had a dose-dependent effect on the cell composition of the head kidney and peripheral blood leukocytes. The highest LE addition exerted the immune suppressive effect, and a decreased count of immune cells was observed in the LE3. The peritoneal cavity infection in the IC trial induced the rapid recruitment of myeloid cells in all groups shortly after the infection, except for the LE3, which had an apparent delayed response. The cumulative mortality and specific trout antibody levels against A. salmonicida did not significantly vary among tested groups, although their average initial level dropped by half at 3 days post-infection (dpi), and subsequently doubled at 28 dpi. The plasma biochemical parameters (TP, TAG, and CHOL) showed an overall decrease at 0–3 dpi, while increasing back to the initial levels at 3–6 dpi. Only LDH negatively correlated with LE inclusion, this can reflect the oxidative stress in the highest LE inclusion. Overall, our results support that there is a dose-dependent effect that should be optimised for ground LE dietary use in aquaculture. • Dietary leonardite (LE 0–3%) had no negative effects on the growth of O. mykiss. • Intestine microbial community differed between the control and LE groups. • Leonardite had a dose-dependent effect on the count of immune cells. • Immune response to the bacterial infection was delayed in the highest LE inclusion. [ABSTRACT FROM AUTHOR]

Although overfeeding in aquaculture can decrease feed efficiency and increase wastage as well as bacterial loads, there are very few investigations on its effects on fish health. This study investigates the physiological changes in overfeeding-induced fish obesity and whether the feeding regimen increases vulnerability to infection. In total, 105 rainbow trout (average weight = 49.26 ± 2.78 g) were divided into three groups (normal-, intermediate-, and over-fed) and fed about 1.7%, 2.55%, and 3.58% of their body weights per day, respectively, for 8 weeks. Serological parameters and metabolites were analyzed to understand the correlation between obesity and physiological state of the fish. Fish at 8 weeks post-feeding (wpf) were challenged with Aeromonas salmonicida subsp. salmonicida at three concentrations of approximately 103, 104, and 105 CFU/fish by intraperitoneal injection. The results showed that the hepatosomatic index (at 2, 4, and 8 wpf), body mass index, and serum triglyceride values (at 4 and 8 wpf) of overfed fish were significantly higher than those of the other groups, suggesting that obesity was successfully induced. Moreover, overfed fish showed significantly higher concentrations of succinate, valine, and isoleucine in their serum compared to the intermediate and control groups, which are known to be associated with obesity. The levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), which serve as indicators of liver function, were consistently higher between 2 and 8 wpf compared to both the normal and intermediate groups. This suggest that overfeeding has detrimental effects on the liver; thus, these enzymes can serve as early indicators of overfeeding. In the challenge test, the fish in the overfed group showed notably higher mortality than those in the other two groups. Our findings therefore suggest that overfeeding for even short periods of time could harm fish health and increase their vulnerability to infection. • Overfeeding causes obesity in fish, leading to physiological changes and liver problems. • Overfeeding elevates serum ALT, AST, and TG levels in rainbow trout, along with obesity-related metabolites. ,as well asobesity-related metabolites, such as succinate, valine, and isoleucine. • Obesity results in immunophysiological imbalances, increasing susceptible to A. salmonicida infection. [ABSTRACT FROM AUTHOR]

The plastisphere is a newly recognized ecosystem. However, its interaction with early life stages of aquatic vertebrates is a multifaceted issue that requires further research. This study investigated the involvement of bacteria in shaping realistic microplastics hazards in zebrafish Danio rerio embryos. Fish were exposed to bottle micro-fragments (FR) and textile micro-fibers (FI) of polyethylene terephthalate (5–15 μm), concomitant with Aeromonas salmonicida achromogenes challenge from 2h post-fertilization for 3 days. Egg chorion showed affinity for FR and FI, inducing earlier embryo hatching. However, this effect was masked by biofilm invasion. Fragments were more detrimental than fibers on developmental parameters, while bacterial presence compromised body length, eye, and yolk sac surface area. In a further finding, MPs alone increased locomotor activity in zebrafish larvae, without synergistic effect when combined with bacteria. Data showed that realistic MPs had no significant effects except for downregulated sod and cyp1a gene expression, whereas bacterial challenge inhibited larval potency for most of the evaluated mRNA levels (mpx (immune system), apoeb (lipid metabolism), nfkb and tfa (inflammation), cyp and sod (oxidative stress)). This study provides new insights into realistic microplastic effects under relevant conditions when combined with environmental pathogen within the first life stages of aquatic vertebrates. [Display omitted] • The 24 hpf zebrafish embryonic chorion can block 5–15 μm PET fragments and fibers. • Bacteria have a high affinity for embryonic chorion. • Triggered MPs toxicity may stem from bacterial association. • Complex exposure may disrupt the larval antioxidant system. [ABSTRACT FROM AUTHOR]

The caspase, functioning as a proteinase, plays a crucial role in eukaryotic cell apoptosis, regulation of apoptosis, cellular growth, differentiation, and immunity. The identification of caspase gene family in Sebastes schlegelii is of great help to understand its antimicrobial research. In S. schlegelii , we totally identified nine caspase genes, including four apoptosis initiator caspases (caspase 2, caspase 8, caspase 9 and caspase 10), four apoptosis executioners (caspase 3a, caspase 3b, caspase 6 , and caspase 7) and one inflammatory executioner (caspase 1). The duplication of caspase 3 genes on chr3 and chr8 may have been facilitated by whole genome duplication (WGD) events or other complex evolutionary processes. In general, the number of caspase genes relatively conserved in high vertebrates, while exhibiting variation in teleosts. Furthermore, syntenic analysis and phylogenetic relationships analysis supported the correct classification of these caspase gene family in S. schlegelii , especially for genes with duplicated copies. Additionally, the expression patterns of these caspase genes in different tissues of S. schlegelii under healthy conditions were assessed. The results revealed that the expression levels of most caspase genes were significantly elevated in the intestine, spleen, and liver. To further investigate the potential immune functions of these caspase genes in S. schlegelii , we challenged individuals with A. salmonicida and V. anguillarum, respectively. After infection with A. salmonicida , the expression levels of caspase 1 in the liver and spleen of S. schlegelii remained consistently elevated throughout the infection time points. The expression levels of most caspase family members in the intestine exhibited significant divergence following V. anguillarum infection. This study provides a comprehensive understanding of the caspase gene families in S. schlegelii , thereby establishing a solid foundation for further investigations into the functional roles of these caspase genes. [Display omitted] • Nine caspase family members were identified in S. schlegelii. • The caspase genes including 4 executioner genes, 4 initiator genes, and 1 inflammatory gene. • Most caspase genes showed high expression levels in intestine, spleen, and liver. • Most caspase genes in liver and spleen were significantly induced by A. salmonicida infection. • The expression pattern of caspase gene was different after the infection of V. anguillarum. [ABSTRACT FROM AUTHOR]

Biofilm formation causes persistent contamination of spoilage microorganisms, leading to serious economic losses in the aquatic processing industry. The antibacterial and anti-biofilm activity of ten carvacrol, cinnamaldehyde, and derivatives against two fish spoilers, Pseudomonas fluorescens and Aeromonas salmonicida, were evaluated by minimum inhibitory concentration (MIC), crystal violet staining, exopolysaccharide, adhesion, swimming, and swarming. Ten essential oils (EOs) tested showed different antibacterial activity, in which carvacrol and thymol exhibited the lowest MIC. Biofilms and exopolysaccharide production were repressed by all EOs at one-fourth MIC without affecting cell viability, as well as, adhesion. Swimming and swarming of two spoilage strains significantly decreased. Carvacrol and eugenol exerted the best anti-biofilm of P. fluorescens (42.2%) and A. salmonicida (62.9%), respectively. All tested EOs components showed that the decrease of acyl-homoserine lactone (AHL) quorum sensing (QS) signals of two strains were positively correlated with anti-biofilm based on violacein production. Molecular docking revealed that the most active EO, carvacrol, competed with C4-HSL to bind with receptor LuxR and AsaR by its hydroxyl group, resulting in the repression of biofilm formation. These results indicated that efficacy of antibiofilm for ten EOs was associated with the structure of compounds, and carvacrol exerted the strongest anti-biofilm activity mediated by QS against two spoilage strains. [ABSTRACT FROM AUTHOR]

High-throughput genomic sequencing has helped to reveal the plasmidome of Aeromonas salmonicida. This literature review provides an overview of A. salmonicida's rich plasmidome by presenting all the plasmids identified so far, addressing their biological importance and the functional links between them. The plasmids of A. salmonicida, especially those bearing antibiotic resistance genes, can provide clues about interactions of this species with other pathogens (animals and humans), as is the case for pRAS3-3432 and Chlamydia suis or pSN254b and Salmonella enterica. In addition to antibiotic resistance, plasmids play an important role in the virulence of A. salmonicida, particularly for the subspecies salmonicida and the plasmid pAsa5, which carries genes for the type-three secretion system, a virulence factor essential for the bacterium. The A. salmonicida plasmidome also has many cryptic plasmidswith no known biological function, but which can be used for the acquisition of new genetic elements. Striking examples are pAsa7 and pAsaXII that provide, respectively, resistance to chloramphenicol and formaldehyde and are derivatives of cryptic pAsa2. [ABSTRACT FROM AUTHOR]

Aeromonas is included in the Dutch Drinking Water Decree as an indicator for elevated microbial regrowth in nonchlorinated drinking water distribution systems (DWDS). The temporal and spatial diversity of Aeromonas species in 10 DWDS and their planktonic growth characteristics with regard to different carbon sources were investigated. Genotyping of the gyrB gene of isolates showed a nonsystematic temporal and spatial variable prevalence of seven different Aeromonas species in these DWDS and no correlation with the level of assimilable organic carbon determined with Pseudomonas fluorescens strain P17 and Spirillum sp. strain NOX (AOC-P17/NOX concentration) and Aeromonas concentrations. Pure cultures of these seven species showed a high affinity for low concentrations (micrograms per liter) of individual amino acids and fatty acids, compounds associated with biomass. Growth occurred at 0.5 #181;g C/literofan amino acid mixture. Growth of a mixed community of Aeromonas rivuli, Aeromonas salmonicida, Aeromonas sobria,and Aeromonas veronii in drinking water occurred in pasteurized samples; however, either no growth or decay occurred in competition with the autochthonous bacteria (nonpasteurized samples). This community also failed to grow in nonpasteurized distribution samples from a location with a clear increase in planktonic Aeromonas concentrations in the transported drinking water. For competitive planktonic growth of Aeromonas, an amino acid concentration of ≥5 µg C/liter is required. AOC-P17/NOX concentrations showed that such concentrations are not expected in Dutch drinking water. Therefore, we suspect that competitive planktonic growth is not the major cause of the observed noncompliance with the Aeromonas standard in nonchlorinated DWDS. [ABSTRACT FROM AUTHOR]

Genomic islands (Aeromonas salmonicida genomic islands , AsaGEI s) are found worldwide in many isolates of Aeromonas salmonicida subsp. salmonicida , a fish pathogen. To date, five variants of AsaGEI (1a, 1b, 2a, 2b and 2c) have been described. Here, we investigate a sixth AsaGEI , which was identified in France between 2016 and 2019 in 20 A. salmonicida subsp. salmonicida isolates recovered from sick salmon all at the same location. This new AsaGEI shares the same insertion site in the chromosome as the other AsaGEI2 s as they all have a homologous integrase gene. This new AsaGEI was thus named AsaGEI2d , and has five unique genes compared to the other AsaGEI s. The isolates carrying AsaGEI2d also bear the plasmid pAsa7, which was initially found in an isolate from Switzerland. This plasmid provides resistance to chloramphenicol thanks to a cat gene. This study reveals more about the diversity of the AsaGEI s. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida strains cause problematic bacterial infections in the aquaculture industry worldwide. The genus Aeromonas includes both mesophilic and psychrophilic species. Bacteriophages that infect Aeromonas spp. strains are usually specific for mesophilic or psychrophilic species; only a few bacteriophages can infect both types of strains. In this study, we characterized the podophage T7-Ah, which was initially found to infect the Aeromonas salmonicida HER1209 strain. The burst size of T7-Ah against its original host is 72 new virions per infected cell, and its burst time is 30 minutes. It has been found that this phage can lyse both mesophilic and psychrophilic A. salmonicida strains, as well as one strain of Escherichia coli. Its genome comprises 40,153 bp of DNA and does not contain any recognizable toxin or antibiotic resistance genes. The adsorption rate of the phage on highly sensitive bacterial strains was variable and could not be related to the presence or absence of a functional A-layer on the surface of the bacterial strains. The lipopolysaccharide migration patterns of both resistant and sensitive bacterial strains were also studied and compared to investigate the nature of the potential receptor of this phage on the bacterial surface. This study sheds light on the surprising diversity of lifestyles of the bacterial strains sensitive to phage T7-Ah and opens the door to the potential use of this phage against A. salmonicida infections in aquaculture. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida is a fish pathogen that has resulted in significant losses to the aquaculture industry worldwide. In the winter of 2018/2019, a serious outbreak of A. salmonicida infection of grass carp (Ctenopharyngodon idella) occurred in China. Since C. idella do not feed at low water temperatures, antibiotics cannot be delivered orally, thus the use of disinfectants is the only viable option to control the disease in the winter months. Therefore, the aim of this study was to elucidate the inhibitory effects of different disinfectants against A. salmonicida at various temperatures and pH values. The microdilution method was used to determine the minimum inhibitory concentration (MIC) of five disinfectants against A. salmonicida at different temperatures. The results showed that A. salmonicida was highly adaptable to temperatures ranging from 8 to 35 °C and pH values from 5 to 9. Moreover, based on the results, benzalkonium bromide (BB) had the strongest inhibitory effect at every temperature. Since A. salmonicida has strong environment adaptability, BB should be chosen to control the disease at low temperatures. [ABSTRACT FROM AUTHOR]

Atypical Aeromonas salmonicida (a As) and Vibrionaceae related species are bacteria routinely recovered from diseased ballan wrasse used as cleaner fish in the Atlantic salmon farming industry. Autogenous (i.e. farm specific inactivated) multivalent vaccines formulated from these microorganisms are widely used to protect farmed wrasse despite limited experimental proof that they are primary pathogens. In this study, the components of a commercial multivalent injection vaccine containing four strains of Aeromonas salmonicida and one strain of Vibrio splendidus previously isolated from ballan wrasse in Scotland, were tested for infectivity, pathogenicity and virulence via intra peritoneal injection at pre-deployment size (25–50 g) and the efficacy of the vaccine for protection against a As assessed. Injection with 3.5 × 109, 8 × 109 1.8 × 109 and 5 × 109 cfu/fish of Vibrio splendidus , V. ichthyoenteri , Aliivibrio logeii and A. salmonicida, respectively, did not cause significant mortalities, lesions or clinical signs after a period of 14 days. IP injection with both a As and Photobacterium indicum successfully reproduced the clinical signs and internal lesions observed during natural outbreaks of the disease. Differences in virulence (LD 50 at day 8-post infection of 3.6 × 106 cfu/fish and 1.6 × 107 cfu/fish) were observed for two a As vapA type V isolates. In addition, the LD 50 for Photobacterium indicum was 2.2 × 107 cfu/fish. The autogenous vaccine was highly protective against the two a As vapA type V isolates after 700-degree days of immunisation. The RPS FINAL values for the first isolate were 95 and 91% at 1 × 106 cfu/fish and 1 × 107 cfu/fish, respectively, and 79% at 1 × 107 cfu/fish for the second isolate tested. In addition, significantly higher anti a As seral antibodies (IgM), were detected by ELISA in vaccinated fish in contrast with control (mock vaccinated) fish. These results suggest wrasse can be effectively immunised and protected against a As infection by injection with oil adjuvanted vaccines prepared with inactivated homologous isolates. • Intraperitoneal challenge model developed for ballan wrasse against Aeromonas salmonicida vapA type V. • Vaccine provided high protection in ballan wrasse against homologous a As vapA type V strains (RPS 79–95%). • Specific serum antibodies (IgM) were detected in vaccinated fish. [ABSTRACT FROM AUTHOR]

Renibacterium salmoninarum causes bacterial kidney disease mainly in salmonid fish. Oligonucleotide primers incorporating R. salmoninarum unique sequences were designed to amplify a 501 bp region of the gene encoding a 57 kDa soluble extra-cellular protein. The primers did not amplify other wide varieties of aquatic or piscine bacteria Aeromonas salmonicida or Yersinia ruckeri. This assay provides a molecular description and definitive identification of R. salmoninarum in Poland. [ABSTRACT FROM AUTHOR]

The present study was aimed at determining antimicrobial susceptibility by a CLSI standard microdilution testing protocol and detecting the resistance genes of motile Aeromonas species isolated from cultured fish. The importance of the minimum inhibitory concentrations was assessed based on statistically determined epidemiological cut‐off values calculated by normalized resistance analysis. Unfortunately, CLSI epidemiological cut‐off values are available only for Aeromonas salmonicida, and there is no further detailed data on Aeromonas isolated from aquatic animals. The antimicrobial susceptibilities of pre‐identified motile Aeromonas species to florfenicol, tetracycline and sulfamethoxazole were determined by calculating epidemiological cut‐off values with fully automated and freely available Excel spreadsheets, applying the normalized resistance interpretation (NRI) method. Furthermore, the presence of the antimicrobial resistance genes floR, tetA, tetB, tetC, tetD, tetE, tetH, sulI, sulII and sulIII was detected by PCR analysis and confirmed by sequence analysis. The presence of up to six different genes (multiple antimicrobial resistance) was determined in the Aeromonas isolates. Significance and Impact of the Study: Significance and Impact of the Study: In this study, we investigated phenotypic and genotypic antimicrobial resistance characteristics by a novel method based on epidemiological cut‐off values. This is the second comprehensive study on the antimicrobial susceptibility characteristics of Aeromonas species using NRI and epidemiological cut‐off values. The present research is related to our previous researches focussed on the identification of motile Aeromonads, their prevalence in relation to different fish lengths, seasons and regions, and covered the investigation of Lactococcus garvieae, Yersinia ruckeri, Flavobacterium spp., Enterobacter spp. and Citrobacter spp. [ABSTRACT FROM AUTHOR]

Furunculosis caused by Aeromonas salmonicida is a major threat to salmonids throughout all their life stages. Vaccination against A. salmonicida has been shown to provide effective protection in aquaculture. The success of many vaccines relies on their association with the appropriate adjuvant to increase their immunogenicity and ensure long‐term protection. In this study, the effects of vaccines using propolis as adjuvants against furunculosis in rainbow trout (Oncorhynchus mykiss) were evaluated. The parameters evaluated included the relative percent survival (RPS), the specific IgM antibody level and the expression profiles of several immune‐related genes in four tissues following immunization and challenge. The results showed that the experimental vaccines containing propolis as adjuvants (ExpVacPro group) could effectively protect the experimental fish against A. salmonicida infection with an RPS of 89.47%. The gene expression data revealed that ExpVacPro could induce the differential expression of the pro‐inflammatory cytokines, MHC class Ⅰ, T‐cell markers and Ig markers in immunized fish, with higher levels in liver and spleen than in head kidney and gill. ExpVacPro initially increased the expression of MHC I and IL8 genes in related pathways, and then gradually increased the expression of IL‐1β and IgT genes for about 2 weeks. During the post‐vaccination challenge, the expression levels of all detected genes were up‐regulated especially for CD8 and IgT genes. The specific IgM antibody titres were significantly induced following immunization and challenge. In conclusion, the results indicated that propolis could improve potency and efficacy of formalin‐inactivated A. salmonicida vaccine as adjuvants. [ABSTRACT FROM AUTHOR]

Atypical Aeromonas salmonicida causes furunculosis infections of non‐salmonid fish, which requires antibiotic therapy. However, antibiotics may induce biofilm in some bacteria, which protects them against hostile conditions while allowing them to persist on surfaces, thus forming a reservoir for infection. The aim of this study was to determine whether atypical isolates of A. salmonicida increased biofilm in the presence of two antibiotics, florfenicol and oxytetracycline. A microtitre plate assay was used to quantify biofilm in the presence and absence of each antibiotic. Fifteen of 28 isolates formed biofilms under control conditions, while 23 of 28 isolates increased biofilm formation in the presence of at least one concentration of at least one antibiotic. For oxytetracycline, the most effective concentration causing biofilm to increase was one‐quarter of that preventing visible bacterial growth, whereas for florfenicol it was one‐half of this value. This is the first study to demonstrate that a bacterial pathogen of fish increases biofilm in response to antibiotics. Biofilm formation may increase the risk of re‐infection in culture systems and this lifestyle favours the transmission of genetic material, which has implications for the dissemination of antibiotic‐resistance genes and demonstrates the need for enhanced disease prevention measures against atypical A. salmonicida. [ABSTRACT FROM AUTHOR]

Vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) are fat-soluble secosteroid hormones obtained from plant and animal sources, respectively. Fish incorporates vitamin D2 and D3 through the diet. In mammals, vitamin D forms are involved in mineral metabolism, cell growth, tissue differentiation, and antibacterial immune response. Vitamin D is an essential nutrient in aquafeeds for finfish. However, the influence of vitamin D on fish cell immunity has not yet been explored. Here, we examined the effects of vitamin D2 and vitamin D3 on Salmo salar primary macrophage immune response to A. salmonicida subspecies salmonicida infection under in vitro conditions. We determined that high concentrations of vitamin D2 (100,000 ng/ml) and D3 (10,000 ng/ml) affect the growth of A. salmonicida and decrease the viability of S. salar primary macrophages. In addition, we determined that primary macrophages pre-treated with a biologically relevant concentration of vitamin D3 for 24 h showed a decrease of A. salmonicida infection. In contrast, vitamin D2 did not influence the antibacterial activity of the S. salar macrophages infected with A. salmonicida. Vitamin D2 and D3 did not influence the expression of canonical genes related to innate immune response. On the other hand, we found that A. salmonicida up-regulated the expression of several canonical genes and suppressed the expression of leukocyte-derived chemotaxin 2 (lect-2) gene, involved in neutrophil recruitment. Primary macrophages pre-treated for 24 h with vitamin D3 counteracted this immune suppression and up-regulated the transcription of lect-2. Our results suggest that vitamin D3 affects A. salmonicida attachment to the S. salar primary macrophages, and as a consequence, the A. salmonicida invasion decreased. Moreover, our study shows that the positive effects of vitamin D3 on fish cell immunity seem to be related to the lect-2 innate immunity mechanisms. We did not identify positive effects of vitamin D2 on fish cell immunity. In conclusion, we determined that the inactive form of vitamin D3, cholecalciferol, induced anti-bacterial innate immunity pathways in Atlantic salmon primary macrophages, suggesting that its utilization as a component of a healthy aquafeed diet in Atlantic salmon could enhance the immune response against A. salmonicida.

Aeromonas salmonicida is a global distributed Gram-negative teleost pathogen, affecting mainly salmonids in fresh and marine environments. A. salmonicida strains are classified as typical or atypical depending on their origin of isolation and phenotype. Five subspecies have been described, where A. salmonicida subsp. salmonicida is the only typical subspecies, and the subsp. achromogenes, masoucida, smithia, and pectinolytica are considered atypical. Genomic differences between A. salmonicida subsp. salmonicida isolates and their relationship with the current classification have not been explored. Here, we sequenced and compared the complete closed genomes of four virulent strains to elucidate their molecular diversity and pathogenic evolution using the more accurate genomic information so far. Phenotypes, biochemical, and enzymatic profiles were determined. PacBio and MiSeq sequencing platforms were utilized for genome sequencing. Comparative genomics showed that atypical strains belong to the subsp. salmonicida, with 99.55% ± 0.25% identity with each other, and are closely related to typical strains. The typical strain A. salmonicida J223 is closely related to typical strains, with 99.17% identity with the A. salmonicida A449. Genomic differences between atypical and typical strains are strictly related to insertion sequences (ISs) activity. The absence and presence of genes encoding for virulence factors, transcriptional regulators, and non-coding RNAs are the most significant differences between typical and atypical strains that affect their phenotypes. Plasmidome plays an important role in A. salmonicida virulence and genome plasticity. Here, we determined that typical strains harbor a larger number of plasmids and virulence-related genes that contribute to its acute virulence. In contrast, atypical strains harbor a single, large plasmid and a smaller number of virulence genes, reflected by their less acute virulence and chronic infection. The relationship between phenotype and A. salmonicida subspecies’ taxonomy is not evident. Comparative genomic analysis based on completed genomes revealed that the subspecies classification is more of a reflection of the ecological niche occupied by bacteria than their divergences at the genomic level except for their accessory genome.

Rainbow trout (Oncorhynchus mykiss) serves as one of the most important commercial fish with an annual production of around 800,000 tonnes. However, infectious diseases, such as furunculosis caused by Aeromonas salmonicida infection, results in great economic loss in trout culture. The brain and kidney are two important organs associated with “sickness behaviors” and immunomodulation in response to disease. Therefore, we worked with 60 trout and investigated transcriptional responses and enrichment pathways between healthy and infected trout. We observed that furunculosis resulted in the activation of toll-like receptors with neuroinflammation and neural dysfunction in the brain, which might cause the “sickness behaviors” of infected trout including anorexia and lethargy. We also showed the salmonid-specific whole genome duplication contributed to duplicated colony stimulating factor 1 (csf-1) paralogs, which play an important role in modulating brain immunomodulation. Enrichment analyses of kidneys showed up-regulated immunomodulation and down-regulated neural functions, suggesting an immune-neural interaction between the brain and kidney. Moreover, the kidney endocrine network was activated in response to A. salmonicida infection, further convincing the communications between endocrine and immune systems in regulating internal homeostasis. Our study provided a foundation for pathophysiological responses of the brain and kidney in response to furunculosis and potentially offered a reference for generating disease-resistant trout strains.

The bacterium Aeromonas salmonicida has long been known to be one of the most feared pathogens in fish farming. However, the more we discover about this bacterial species, the more we question whether it is really exclusively an aquatic pathogen. In recent years, it has become obvious that this bacterial species includes a myriad of strains with various lifestyle and ecological niches, including the well-known strict psychrophiles, the first bacteria known of the species, and the newly described mesophilic strains. The mesophiles are able to grow at low temperatures, but even better at temperatures of approximately 37 °C, which strict psychrophiles cannot do. In this perspective article, we address some aspects surrounding this dual lifestyle in A. salmonicida, including the impact of mobile genetic elements, and how future research around this bacterial species may focus on the psychrophilic/mesophilic dichotomy, which makes A. salmonicida an increasingly interesting and relevant model for the study of speciation.

Abstract Background High-throughput proteomics was used to determine the role of the fish liver in defense responses to bacterial infection. This was done using a rainbow trout (Oncorhynchus mykiss) model following infection with Aeromonas salmonicida, the causative agent of furunculosis. The vertebrate liver has multifaceted functions in innate immunity, metabolism, and growth; we hypothesize this tissue serves a dual role in supporting host defense in parallel to metabolic adjustments that promote effective immune function. While past studies have reported mRNA responses to A. salmonicida in salmonids, the impact of bacterial infection on the liver proteome remains uncharacterized in fish. Results Rainbow trout were injected with A. salmonicida or PBS (control) and liver extracted 48 h later for analysis on a hybrid quadrupole-Orbitrap mass spectrometer. A label-free method was used for protein abundance profiling, which revealed a strong innate immune response along with evidence to support parallel rewiring of metabolic and growth systems. 3076 proteins were initially identified against all proteins (n = 71,293 RefSeq proteins) annotated in a single high-quality rainbow trout reference genome, of which 2433 were maintained for analysis post-quality filtering. Among the 2433 proteins, 109 showed significant differential abundance following A. salmonicida challenge, including many upregulated complement system and acute phase response proteins, in addition to molecules with putative functions that may support metabolic re-adjustments. We also identified novel expansions in the complement system due to gene and whole genome duplication events in salmonid evolutionary history, including eight C3 proteins showing differential changes in abundance. Conclusions This study provides the first high-throughput proteomic examination of the fish liver in response to bacterial challenge, revealing novel markers for the host defense response, and evidence of metabolic remodeling in conjunction with activation of innate immunity.

Abstract Aeromonas salmonicida was isolated from red spot emperor, king soldier bream, white-spotted rabbit fish and tilapia, and an invertebrate (abalone) in Oman during December 2011–May 2012. The cytotoxic enterotoxin ast gene was found widely distributed among the isolates; aerolysin-like protein (act) and the flagellin structural gene lafA less so; and the nuclease gene (nuc) not at all. However, there was not any evidence of pathogenicity among the isolates when tested in laboratory-based experiments using rainbow trout and Nile tilapia. Therefore, the risk of the pathogen to fish in Oman is unclear.

Abstract Background Mortality episodes have affected young-of-year smallmouth bass (Micropterus dolomieu) in several river systems in Pennsylvania since 2005. A series of laboratory experiments were performed to determine the potential role of largemouth bass virus (Ranavirus, Iridoviridae) in causing these events. Results Juvenile smallmouth bass experimentally infected with the largemouth bass virus exhibited internal and external clinical signs and mortality consistent with those observed during die-offs. Microscopically, infected fish developed multifocal necrosis in the mesenteric fat, liver, spleen and kidneys. Fish challenged by immersion also developed severe ulcerative dermatitis and necrotizing myositis and rarely panuveitis and keratitis. Largemouth bass virus-challenged smallmouth bass experienced greater mortality at 28 °C than at 23 or 11 °C. Co-infection with Flavobacterium columnare at 28 °C resulted in significant increase in mortality of smallmouth bass previously infected with largemouth bass virus. Aeromonas salmonicida seems to be very pathogenic to fish at water temperatures

The teleost gut is a multifunction complex structure that plays a pivotal immunological role in homeostasis and the maintenance of health, in addition to digestion of food and/or nutrient absorption. In vitro examination of the intestine leucocyte repertoire has the potential to aid our understanding of gut immune competence and allows a rapid screen of host-microorganism interactions in different immunological contexts. To explore this possibility, in the present study we investigated the response of isolated gut leucocytes to 4 bacterins of Aeromonas salmonicida , prepared from different strains, combinations and strains grown in different environments, in comparison to a Yersinia ruckeri bacterin for which a commercial/effective oral booster vaccine has been developed. To aid this study we also optimized further our method of GALT cell isolation from rainbow trout, so as to avoid mechanical clearance of the intestine contents. This drastically increased the cell yield from ~12 × 106 to ~210 × 106/fish with no change in the percent cell viability over time or presence of transcripts typical of the key leucocyte types needed for the study of immune modulation (i.e. T- and B-cells, dendritic cells and macrophages). A wide array of immune transcripts were modulated by the bacterins, demonstrating the diversity of GALT cell responses to bacterial stimulation. Indeed, the GALT leucocyte responses were sensitive enough to distinguish the different bacterial species, strains and membrane proteins, as seen by distinct kinetics of immune gene expression. However, the response of the GALT cells was often relatively slow and of a low magnitude compared to those of PBL. These results enhance our knowledge of the gut biocapacity and help validate the use of this model for screening of oral vaccine candidates. • Further optimization of the GALT leucocyte isolation procedure boosted the average cell yield more than 17 fold. • Transcripts for markers of T- and B-lymphocytes, dendritic cells and macrophages were detectable in cells isolated. • The GALT cell response to bacterins is diverse and distinct but relatively slow and of a lower magnitude compared to PBL. • The Y. ruckeri elicited a range of changes in the GALT cells that could be biomarkers to evaluate oral vaccine candidates. [ABSTRACT FROM AUTHOR]

Aims: The aim of the study was to quantify the growth kinetic parameters and spoilage‐associated metabolites of an inoculated strain of Aeromonas salmonicida in pre‐rigor filleted Atlantic salmon (Salmo salar L.) stored in vacuum (VP) or modified atmosphere (MAP 60/40% CO2/N2) at 4 and 8°C. Methods and Results: The maximum growth rate of A. salmonicida in VP salmon stored at 4°C was 0·56 ± 0·04 day−1 with no detectable lag‐phase and the concentration of Aeromonas reached 8·33 log CFU per g after 10 days. The growth rates and maximum population density of Aeromonas in MAP salmon were lower but the applied atmosphere did not inhibit the growth. A selection of metabolites associated with fish spoilage were quantified using 1H nuclear magnetic resonance (NMR) spectroscopy. The concentration of trimethylamine (TMA) was significantly affected by storage time and temperature, packaging atmosphere and inoculation with A. salmonicida (General Linear Model (GLM), P < 0·001 for all factors). Conclusion: The study presents preliminary results on A. salmonicida as a potential spoilage organism in vacuum‐packaged salmon during cold storage. The combination of refrigeration and a packaging atmosphere consisting of 60/40 % CO2/N2 did not completely inhibit the growth but prevented the formation of TMA. Significance and Impact of the Study: Little information is available on the spoilage potential of Aeromonas spp. in minimally processed salmon products under different packaging conditions. The study clearly demonstrates the importance of hurdle technology and provides data to further elucidate the significance of Aeromonas spp. as a spoilage organism. [ABSTRACT FROM AUTHOR]

Aeromonas and Pseudomonas are important bacterial species involved in spoilage of refrigerated freshwater fish. In this study, 10 Aeromonas and seven Pseudomonas bacterial strains were isolated from spoiled grass carp and identified. Twelve of seventeen bacterial strains showed high potential of biofilm formation and 14 of 17 can produce extracellular protease. In order to explore the spoilage capacity of dual‐species, the sterile grass carp fillets were inoculated with mono‐ and dual‐species of Aeromonas salmonicida and Pseudomonas azotoformans strains. The results revealed significantly higher levels of the total viable count and total volatile basic nitrogen in dual‐species as compared to mono‐species from day 6. The higher contents of histamine, cadaverine and serious degradation in muscles tissue were also observed in dual‐species after 10 days of storage. Results of in vitro experiments showed that the co‐culture of A. salmonicida and P. azotoformans significantly increased the bacterial maximum growth rate, promoted the biofilm formation and improved the spoilage capacity of bacterial strains. This study has revealed that the co‐culture of Aeromonas and Pseudomonas bacterial strains accelerated spoilage process of grass carp and increased biofilm formation. It indicates that the mixed‐cultures of spoilage micro‐organisms pose a huge threat to food industry. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida is a gram‐negative bacterium that is the causative agent of furunculosis. An A. salmonicida strain was isolated from diseased turbot (Scophthalmus maximus) with the sign of furunculosis from North China. Based on vapA gene, the strain was further classified as A. salmonicida subsp. masoucida RZ6S‐1. Culturing RZ6S‐1 strain at high temperature (28°C) obtained the virulence attenuated strain RZ6S. Genome sequence comparison between the two strains revealed the loss of the type IV secretion system (T4SS) and type III secretion system (T3SS) from the native plasmid pAsmB‐1 and pAsmC‐1 of wild‐type strain RZ6S‐1, respectively. Further study demonstrated that the wild‐type strain RZ6S‐1, but not its derivative mutant RZ6S, can stimulate apoptosis. Elevated protein level of cleaved caspase‐3 was detected from epithelioma papulosum cyprinid (EPC) cells infected with wild‐type strain RZ6S‐1 as compared with that infected with RZ6S strain. Meanwhile, the invasion of the mutant strain RZ6S was about 17‐fold higher than the wild‐type strain RZ6S‐1, suggesting that some protein(s) from A. salmonicida subsp. masoucida RZ6S‐1 suppress its invasion. The RZ6S mutant strain was attenuated, since its LD50 is over 10,000 times higher compared to the wild‐type strain as revealed in the turbot infection model. [ABSTRACT FROM AUTHOR]

The genes encoding chitin-degrading enzymes in Aeromonas salmonicida SWSY-1.411 were identified and cloned in Escherichia coli. The strain contained two glycoside hydrolase (GH) families 18 chitinases: AsChiA and AsChiB, two GH19 chitinases: AsChiC and AsChiD, and an auxiliary activities family 10 protein, lytic polysaccharide monooxygenase: AsLPMO10A. These enzymes were successfully expressed in E. coli and purified. AsChiB had the highest hydrolytic activity against insoluble chitin. AsChiD had the highest activity against water-soluble chitin. The peroxygenase activity of AsLPMO10A was lower compared to SmLPMO10A from Serratia marcescens. Synergism on powdered chitin degradation was observed when AsChiA and AsLPMO10A were combined with other chitinases of this strain. More than twice the increase of the synergistic effect was observed when powdered chitin was treated by a combination of AsLPMO10A with all chitinases. GH19 chitinases suppressed the hyphal growth of Trichoderma reesei. Synergistic effects of chitinases and LPMO of A. salmonicida on the degradation of powdered chitin, and synergy between chitinases of A. salmonicida and SmChiA or SmChiB of S. marcescens, or BcChiA1 of B. circulans. [ABSTRACT FROM AUTHOR]

Effective vaccine programs against Aeromonas salmonicida have been identified as a high priority area for the sablefish (Anoplopoma fimbria) aquaculture. In this study, we established an A. salmonicida infection model in sablefish to evaluate the efficacy of commercial vaccines and an autogenous vaccine preparation. Groups of 40 fish were intraperitoneally (ip) injected with different doses of A. salmonicida J410 isolated from infected sablefish to calculate the median lethal dose (LD 50). Samples of blood, head kidney, spleen, brain, and liver were also collected at different time points to determine the infection kinetics. The LD 50 was estimated as ~3 × 105 CFU/dose. To evaluate the immune protection provided by an autogenous vaccine and two commercial vaccines in a common garden experimental design, 140 fish were PIT-tagged, vaccinated and distributed equally into 4 tanks (35 fish for each group, including a control group). Blood samples were taken every 2 weeks to evaluate IgM titers. At 10 weeks post-immunization, all groups were ip challenged with 100 times the calculated LD 50 for A. salmonicida J410. A. salmonicida was detected after 5 days post-infection (dpi) in all collected tissues. At 30 days post-challenge the relative percentage survival (RPS) with respect to the control group was calculated for each vaccine. The RPS for the bacterin mix was 65.22%, for Forte Micro 4® vaccine was 56.52% and for Alpha Ject Micro 4® was 30.43%, and these RPS values were reflected by A. salmonicida tissue colonization levels at 10 days post-challenge. Total IgM titers peaked at 6–8 weeks post-immunization, where the autogenous vaccine group showed the highest IgM titers and these values were consistent with the RPS data. Also, we determined that the A. salmonicida A-layer binds to immunoglobulins F(ab)' in a non-specific fashion, interfering with immune assays and potentially vaccine efficacy. Our results indicate that vaccine design influences sablefish immunity and provide a guide for future sablefish vaccine programs. • We developed an infection model for sablefish, including determining the LD 50 dose for atypical A. salmonicida (~3x105 CFU/dose). • Atypical A. salmonicida J410 does causes a chronic type of infection that involves colonization of the brain. • The monovalent autogenous vaccine mix using three A. salmonicida strains provided better protection than two polyvalent commercial vaccines. • Vaccine immune protection was associated with the IgM titers, where the autogenous vaccinated fish had the highest RPS and IgM titers. • The A. salmonicida A-layer binds to immunoglobulins in a non-specific fashion, questioning its utility as an immune protective antigen. [ABSTRACT FROM AUTHOR]

The distribution of salmon roe poses a risk to the salmon industry due to the potential transmission of quarantinable disease-causing pathogens. The curing of commercial salmon roe with saturated saline or soy sauce seasoning is widespread in Japan. The aim of this study was to evaluate the effect of commercial curing processes on quarantinable disease-causing pathogens [Aeromonas salmonicida, Renibacterium salmoninarum, Yersinia ruckeri, infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), Oncorhynchus masou virus (OMV)] of salmon. Curing with saturated saline for 8 min slightly decreased the viability of A. salmonicida, from 6.0 log colony-forming units (CFU)/ml to 5.1 log CFU/ml, but did not affect the viability of R. salmoninarum or Y. ruckeri. Curing with soy sauce seasoning for 90 min decreased the viability of A. salmonicida from 5.5 log CFU/ml to undetectable levels, but did not affect those of R. salmoninarum or Y. ruckeri. Neither saturated saline nor soy sauce seasoning affected the infectivity of IHNV, IPNV or OMV. In conclusion, the efficacy of curing was only demonstrated for A. salmonicida when soy seasoning was employed. This study shows that quarantine measures other than those examined here need to be developed for the safe distribution of salmon roe. [ABSTRACT FROM AUTHOR]

LncRNAs have been demonstrated to play pivotal roles in virous biological processes, especially the gene expression regulation, including transcriptional regulation, posttranscriptional control and epigenetic processes. However, most of the current studies of lncRNAs are still limited in mammalian species, the investigations of functional roles of lncRNAs in teleost species are still lacking. In current study, we identified a novel lncRNA (SETD3-OT) in turbot, with 2,504 bp full-length obtained by 5′ and 3′ RACE, located in turbot chromosome 17, ranged from 20,933,835 to 20,936,302 bp. In addition, 8 neighboring genes of SETD3-OT were identified within 100 kbp in genome location. From the annotation of the neighboring adjacent genes, SETD3-OT might involve in regulation of cell apoptosis and cycle, the immune cell development, and the immune response against infection, and its expression pattern is similar to majority of the neighboring genes following Aeromonas salmonicida challenge. Intriguingly, SETD3-OT showed significant high expression levels in mucosal surfaces (intestine, gill and skin), and was dramatically down-regulated in these mucosal tissues following Vibrio anguillarum challenge, especially in gill and skin. In addition, SETD3-OT was distributed in nucleus, it might regulate the neighboring genes in cis or in trans. Taken together, our results provide insights for lncRNA in fish innate immunity, further studies should be conduct to explore the detailed molecular mechanism of the gene regulation between SETD3-OT and its neighboring genes. • A novel lncRNA (SETD3-OT) was identified in turbot with 2504 bp full-length. • Its expression pattern is similar to majority of the neighboring genes following A. salmonicida challenge. • SETD3-OT showed significant high expression level in turbot mucosal surfaces (intestine, gill and skin). • SETD3-OT was dramatically down-regulated in turbot mucosal tissues following V. anguillarum challenge. • SETD3-OT was distributed in nucleus, it might regulate the neighboring genes in cis or in trans. [ABSTRACT FROM AUTHOR]

Aims: This study aimed to evaluate in vitro efficacy of essential oils (EOs) and their compounds (EOCs) alone or in combination against Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in salmonid fish. Methods and Results: Antimicrobial activity of 13 EOs and 16 EOCs was investigated for four A. salmonicida subsp. salmonicida strains using broth microdilution. The checkerboard assay was used to evaluate a putative synergy between the most efficient EOs and EOCs against the tested strains. Cinnamon bark, oregano, clove, and thyme oils and their major compounds cinnamaldehyde, eugenol, carvacrol and thymol showed the lower minimum inhibitory concentration and minimum bactericidal concentration values. The association of cinnamaldehyde and eugenol (V/V: 30%/70%) showed a synergistic activity against three tested strains. The combinations of cinnamon with oregano, clove or thyme EOs showed a neutral or additive activity against all the tested strains. Conclusions: Cinnamon, oregano, clove and thyme oils and their major phytochemical compounds showed strong activities against A. salmonicida subsp. salmonicida strains. Significance and Impact of the Study: To reduce the use of antibiotics in aquaculture, phytochemicals such as cinnamaldehyde and eugenol can be tested alone or in combination in in vivo studies as functional feed alternatives. [ABSTRACT FROM AUTHOR]

Integrons are prokaryotic genetic elements known to carry and exchange antibiotic resistance gene cassettes through a site-specific recombinase called integrase. In this work, 107 Aeromonas isolates from environmental origin, including fish, water, and sediments, were investigated for the presence of integrons. Using specific primers for Class 1, 2 and 3 integrases, only Class 1 and Class 2 integrons were detected. Detection of Class 2 integrases and their associated variable regions required two rounds of polymerase chain reaction (PCR). Sequencing of the intI2 amplicons confirmed them as integrase-derived products. Class 1 integrons were detected in 26 out of 107 isolates. PCR amplification of the variable regions associated to these integrons revealed an outstanding homogeneity, 25 of them having variable regions with an identical dfrA12-orfF-aadA2 cassette array and one integron carrying only the dfrA16 cassette. To assess clone diversity, chromosomal DNA from isolates was subjected to enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), which discarded clonality in all instances. Class 2 integrons were surprisingly more prevalent than Class1 integrons, being detected in 60 out of 107 isolates. Forty-six of them showed a unique ERIC profile, while the remaining 14 strains displayed profiles that could be grouped in five different patterns. Cassette arrangements of all Class 2 variable regions were those described as the most prevalent (dfrA1-sat2-aadA1). A rather startling result of this work is the sensitivity to trimethoprim, streptomycin, and streptothricin of most strains, despite the presence of the cognate resistance genes. To know the integron distribution in environmental Aeromonas species, a phylogenetic reconstruction was done using rpoD/gyrB or rpoD/gyrA gene sequences. Isolates bearing these elements corresponded to Aeromonas hydrophila, Aeromonas veronii, Aeromonas salmonicida, Aeromonas dhakensis, Aeromonas sanarellii, Aeromonas taiwanensis, Aeromonas media, Aeromonas caviae, Aeromonas jandaei, and Aeromonas sp. This work revealed an unusual high incidence of Class 2 integrons and a low variability of cassette arrangements in environmental Aeromonas species. [ABSTRACT FROM AUTHOR]

Previously, Aeromonas sobria and A. salmonicida were identified to be the most prevalent species in salmonid farms in Korea. In this study, we evaluated the biochemical characteristics, antibiotic susceptibility and pathogenicity of A. salmonicida (3 isolates) and A. sobria (8 isolates) isolated from salmonids, and further investigated efficacy of A. salmonicida vaccine. In antibiotic susceptibility test, all of A. sobria isolates were resistant to amoxicillin and ampicillin. Six A. sobria and two A. salmonicida isolates were resistant to oxytetracycline. In challenge test, A. sobria isolates exhibited low pathogenicity in rainbow trout (Oncorhynchus mykiss) while one A. salmonicida isolate showed high pathogenicity with LD50 of 6.4 × 103 CFU/fish in rainbow trout and coho salmon (Oncorhynchus kisutch). Among virulence factors, secretion apparatus (ascV and ascC) and transcription regulatory protein (exsA) of type 3 secretion system and A‐layer protein genes were differentially detected in DNA or cDNA of A. salmonicida isolates, indicating their contribution to the pathogenicity. A formalin‐killed vaccine of highly pathogenic A. salmonicida isolate exhibited a protective effect with relative survival rate of 81.8% and 82.9% at 8 weeks and 16 weeks post‐vaccination, respectively, in challenge test. [ABSTRACT FROM AUTHOR]

To study the mechanism of β-glucan in immune protection, rainbow trout were fed diets with or without 0.2% β-glucan for 42 days and then infected with Aeromonas salmonicida. After that, spleen tissues were sampled on 4- and 6-days post infection (dpi). Transcriptome analysis was compared between control group (CG, without β-glucan addition) and 0.2% β-glucan group (BG). In CG vs BG, 378 and 406 DEGs were identified on 4 dpi and 6 dpi respectively; furthermore, 46 DEGs were shared on 4 dpi and 6 dpi, enriching in GO terms, such as complement activation, inflammatory response, and metabolic process. KEGG pathway analysis revealed that some DEGs in CG vs BG were involved in immune or metabolic signaling pathways such as complement and coagulation cascades, toll-like receptor signaling pathway, NF-κB signaling pathway, antigen processing and presentation, and platelet activation on 4 or 6 dpi. DEGs, such as fgg , fgb , f5 , c9 , c3 , c5 , tlr5 , and myd88 , were analyzed in CG vs BG on 4 dpi and 6 dpi, implying their potential roles in β-glucan-modulated immunity. These results are beneficial to understand the mechanism of β-glucan in resisting bacteria in fish. • RNA-seq technique was firstly used to study the mechanism of β-glucan in protecting fish from bacterial infection. • A total of 378 and 594 DEGs were identified on 4 dpi and 6 dpi in control group vs β-glucan group respectively. • The complement signaling pathway may serve as a communication bridge to the β-glucan-mediated pathways. • Some metabolism pathways might be related to β-glucan function. [ABSTRACT FROM AUTHOR]

The functional spectrum of the teleostean head kidney covers haematopoietic, immune and endocrine signalling pathways with physiological effects that are likely to conflict if activated at the same time. An in vivo experiment on the salmonid fish maraena whitefish (Coregonus maraena) revealed that the head kidney shows a remarkably strong response after injection of Aeromonas salmonicida within 48 h. In order to investigate the potential influence of endocrine signalling on the initiation of immune responses, we established a primary culture of head-kidney cells of maraena whitefish. For the characterisation of this model system, we used flow cytometry complemented with an extensive panel of immunological/haematological and stress-physiological/neuroendocrinological qPCR assays. More than one third of the cells expressed the characteristic signature of myeloid cells, while more than half of the cells expressed those genes typical for lymphocytes and monocytes. In parallel, we quantified the expression of genes encoding endocrine receptors and identified ADRA2D as by far the most highly expressed adrenergic-receptor gene in head-kidney cells. The stimulation of the head-kidney cells with toll-like receptor ligands induced the expression of typical immune genes (IL1B , CXCL8 , TNF , SAA) after only 1 h. The incubation with the stress hormones cortisol, adrenaline and noradrenaline also had an immune-activating effect, though less pronounced. However, cortisol had the strongest suppressive effect on the stimulation-induced immune response, while adrenaline exerted a comparably weaker effect and noradrenaline was almost ineffective. Moreover, we found that cortisol reduced the expression of genes coding for adrenergic and some glucocorticoid receptors, while noradrenaline increased it. In conclusion, the primary head-kidney cells of maraena whitefish reflect the immunological and neuroendocrinological diversity of the entire organ. This in vitro system allowed thus identifying the correlative changes between the activities of hormones and immune factors in salmonid fish in order to contribute to a better understanding of the regulation circuit between stress and immune defence. Image 1 • The head kidney of maraena whitefish shows a pronounced response to A. salmonicida. • Head-kidney cells express a high density of endocrine receptors. • A cell culture of head kidney reflects well the composition of the entire organ. • Cortisol is more efficient than catecholamines in decreasing cytokine expression. • Noradrenaline increases the expression of hormone receptors. [ABSTRACT FROM AUTHOR]