The synergistic effect of dielectric barrier discharge plasma and phycocyanin on shelf life of Oncorhynchus mykiss rainbow fillets.

This study aimed to evaluate the efficacy of dielectric barrier discharge treatment (DBD) combined with phycocyanin pigment (PC) in extending the shelf life of Oncorhynchus mykiss rainbow fillets stored at 4 ± 0.1 °C. Microbiological, physicochemical, sensory and antioxidant properties were assessed over an 18-day storage period. The combined DBD and PC treatment significantly inhibited total viable counts and Psychrotrophic bacteria counts compared to the rest of the samples throughout storage. While Total Volatile Nitrogen concentrations remained below international standard until day 18, they exceeded this threshold in control sample by day 9. DBD treatment notably reduced Trimethylamine levels compared to controls (p < 0.05). PC and DBD combined inhibited DPPH and ABTS radical scavenging capacities by 80% and 85%, respectively, while demonstrating heightened iron-reducing antioxidant activity compared to controls. Analysis of 24 fatty acids indicated that PC mitigated DBD's adverse effects, yielding superior outcomes compared to controls. The ratio of n-3 to n-6 fatty acids in all samples met or fell below international standard. Thus, the combined use of DBD and PC shows promise in extending fillet shelf life by over 15 days at 4 °C.

Antibiotic susceptibility and resistance genes profiles of Vagococcus salmoninarum in a rainbow trout (Oncorhyncus mykiss, Walbaum) farm.

Disease outbreaks negatively affect fish production. Antimicrobial agents used in the treatment of diseases become ineffective over time because of antibiotic resistance developed by bacteria distributed in the aquaculture environment. This study was conducted for 4 months (cold period) in a fish farm to detect the fish disease, cold water streptococcosis. In the study, four brood stock showing disease signs were detected. Bacteria isolates were obtained and identified as Vagococcus salmoninarum. Antimicrobial susceptibility of V. salmoninarum was tested and antibiotic resistance gene profiles of V. salmoninarum isolates were screened. The phylogenetic relation of the isolates with the previously reported strains was evaluated. Antibiotic resistance developed by pathogenic bacteria is distributed in the aquaculture environment. The transfer of resistance genes from one bacterium to another is very common. This situation causes the antimicrobial agents used in the treatment of diseases to become ineffective over time. The disc diffusion test showed that all four isolates developed resistance to 13 (FFC30, AX25, C30, E15, CF30, L2, OX1, S10, T30, CRO30, CC2, PT15 and TY15) of the evaluated antibiotics and were about to develop resistance to six others (AM 10, FM 300, CFP75, SXT25, APR15 and TE30). Furthermore, antibiotic resistance genes tetA, sul1, sul2, sul3, dhfr1, ereB and floR were detected in the isolated strain. Moreover, the phylogenetic analysis showed that isolated V. salmoninarum strain (ESN1) was closely related to the bacterial strains isolated from USA and Jura.

The effects of Juniperi fructus essential oil and vacuum packing on the shelf life of rainbow trout fillets during storage at 2±1°C.

We hypothesized that the combined effect of vacuum packaging and Juniperi fructus essential oil addition would increase shelf life. Six different treatments were tested. The effects of the different concentrations of J. fructus essential oil (0%, 0.3% and 0.6%) and packing method (non-vacuum and vacuum) on the fish (Oncorhynchus mykiss) fillets of stored 4±1 °C were investigated in terms of its microbiological (mesophilic aerobic bacteria and yeast-mold), chemical (pH,  total volatile alkaline nitrogen (TVB-N), thiobarbituric acid (TBA value)) and sensory quality. The results showed that J. fructus essential oil had a positive significant effect on quality parameters (p<0.05). In conclusion, based primarily on sensory, TVB-N and mesophilic bacteria data the shelf-life of fresh rainbow trout was 4 days (non-vacuum packaged), 13 days (vacuum packaged), 19 and 28 days treated with J. fructus oil (0.3 and 0.6%, v/w) under vacuum packaged, respectively. J. fructus essential oil application and vacuum packaging; extended the shelf life of fish fillets by an average of 15 days. The combined use of J. fructus essential oil and packaging techniques could form the basis for new studies.

Pharmacokinetic behaviour and pharmacokinetic-pharmacodynamic integration of doxycycline in rainbow trout (Oncorhynchus mykiss) after intravascular, intramuscular and oral administrations.

OBJECTIVE: Doxycycline (DO) has been used in fish for a long time, but there are some factors that have not yet been clarified regarding its pharmacokinetic (PK) and pharmacodynamic (PD) properties. Therefore, the aim of this study was to investigate the PK and PK/PD targets of DO after 20 mg/kg intravascular (IV), intramuscular (IM) and oral (OR) gavage administration in rainbow trout (Oncorhynchus mykiss). METHODS: Plasma samples were collected at specific time points and subsequently analysed by HPLC-ultraviolet. The PK/PD indices were calculated based on the MIC90 (Aeromonas hydrophila and Aeromonas sobria) values obtained for the respective bacteria and the PK parameters obtained for DO following both IM and OR administration. RESULTS: After IV administration, the elimination half-life (t1/2 ʎz), area under the concentration vs. time curve (AUC), apparent volume of distribution at steady-state and total body clearance of DO were 34.81 h, 723.82 h µg/mL, 1.24 L/kg and 0.03 L/kg/h, respectively. The t1/2λz of the DO was found to be 37.39 and 39.78 h after IM, and OR administration, respectively. The bioavailability was calculated 57.02% and 32.29%, respectively, after IM and OR administration. The MIC90 of DO against A. hydrophila and A. sobria was 4 µg/mL. The PK/PD integration showed that DO (20 mg/kg dose) for A. hydrophila and A. sobria with MIC90 ≤4 µg/mL achieved target AUC/MIC value after IM administration. CONCLUSIONS: These results suggest that when rainbow trout was treated with 20 mg/kg IV and IM administered DO, therapeutically effective concentrations were reached in the control of infections caused by A. hydrophila and A. sobria.

Nutrient enrichment increases virulence in an opportunistic environmental pathogen, with greater effect at low bacterial doses.

Eutrophication of aquatic ecosystems is associated with an increased risk of pathogen infection via increased pathogen growth and host exposure via increased pathogen doses. Here, we studied the effect of nutrients on the virulence of an opportunistic bacterial pathogen of fish, Flavobacterium columnare, in challenge experiments with rainbow trout fingerlings. We hypothesized that removing all nutrients by washing the bacteria would reduce virulence as compared to unwashed bacteria, but adding nutrients to the tank water would increase the virulence of the bacterium. Nutrient addition and increase in bacterial dose increased virulence for both unwashed and washed bacteria. For unwashed bacteria, the addition of nutrients reduced the survival probability of fish challenged with low bacterial doses more than for fish challenged with higher bacterial doses, suggesting activation of bacterial virulence factors. Washing and centrifugation reduced viable bacterial counts, and the addition of washed bacteria alone did not lead to fish mortality. However, a small addition of nutrient medium, 0.05% of the total water volume, added separately to the fish container, restored the virulence of the washed bacteria. Our results show that human-induced eutrophication could trigger epidemics of aquatic pathogens at the limits of their survival and affect their ecology and evolution by altering the dynamics between strains that differ in their growth characteristics.

Complete genome sequence and potential pathogenic assessment of Flavobacterium plurextorum RSG-18 isolated from the gut of Schlegel's black rockfish, Sebastes schlegelii.

Flavobacterium plurextorum is a potential fish pathogen of interest, previously isolated from diseased rainbow trout (Oncorhynchus mykiss) and oomycete-infected chum salmon (Oncorhynchus keta) eggs. We report here the first complete genome sequence of F. plurextorum RSG-18 isolated from the gut of Schlegel's black rockfish (Sebastes schlegelii). The genome of RSG-18 consists of a circular chromosome of 5,610,911 bp with a 33.57% GC content, containing 4858 protein-coding genes, 18 rRNAs, 63 tRNAs and 1 tmRNA. A comparative analysis was conducted on 11 Flavobacterium species previously reported as pathogens or isolated from diseased fish to confirm the potential pathogenicity of RSG-18. In the SEED classification, RSG-18 was found to have 36 genes categorized in 'Virulence, Disease and Defense'. Across all Flavobacterium species, a total of 16 antibiotic resistance genes and 61 putative virulence factors were identified. All species had at least one phage region and type I, III and IX secretion systems. In pan-genomic analysis, core genes consist of genes linked to phages, integrases and matrix-tolerated elements associated with pathology. The complete genome sequence of F. plurextorum RSG-18 will serve as a foundation for future research, enhancing our understanding of Flavobacterium pathogenicity in fish and contributing to the development of effective prevention strategies.

Persistence of heterologous Flavobacterium psychrophilum genetic variants in microcosms simulating fish farm and hatchery environments.

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, causes substantial economic losses in salmonid farms and hatcheries. Some multilocus sequence types (ST) of F. psychrophilum are more likely to be associated with fish farms and hatcheries, but it is unclear if these patterns of association represent genetic lineages that are more adapted to aquaculture environments. Towards elucidating the disease ecology of F. psychrophilum, the culturability of 10 distinct F. psychrophilum STs was evaluated for 13 weeks in three microcosms including sterilized well water, sterilized well water with commercial trout feed, or sterilized well water with raceway detritus. All STs remained culturable in each of the microcosms for at least 8 weeks, with bacterial concentrations often highest in the presence of raceway detritus. In addition, most (e.g., 90%) STs remained culturable for at least 13-weeks. Significant differences in log10 cfus were observed among STs, both within and between microcosms, suggesting potential variability in environmental persistence capacity among specific variants. Collectively, results highlight the ability of F. psychrophilum to not only persist for weeks under nutrient-limited conditions but also thrive in the presence of organic substrates common in fish farms and hatchery-rearing units.

Studying the impact of phycoerythrin on antioxidant and antimicrobial activity of the fresh rainbow trout fillets.

Marine cyanobacteria present a significant potential source of new bioactive compounds with vast structural diversity and relevant antimicrobial and antioxidant activities. Phycobiliproteins (PBPs) like phycocyanin (PC), phycoerythrin (PE), and water-soluble cyanobacterial photosynthetic pigments, have exhibited strong pharmacological activities and been used as natural food additives. In this study, phycoerythrin (PE) isolated from a marine strain of cyanobacterium Nostoc sp. Ft salt, was applied for the first time as a natural antimicrobial as well as an antioxidant to increase the shelf life of fresh rainbow trout i.e., (Oncorhynchus mykiss) fillets. Fresh trout fillets were marinated in analytical grade PE (3.9 µg/mL) prepared in citric acid (4 mg/mL), and stored at 4 °C and 8 °C for 21 days. Microbiological analysis, antioxidant activity and organoleptic evaluation of both control and treated fish fillets were then statistically compared. The results demonstrated noticeable (P < 0.05) differences in the microbial counts, antioxidant activity, and organoleptic characteristic values between PE-treated and non-treated groups. In addition, we observed that treating fresh fish fillets with a PE solution leads to a significant increase in shelf life by at least 14 days. Consequently, PE could be an alternative to synthetic chemical additives since it does not contain the potentially dangerous residues of the synthetic chemical additives and is thus healthier to the consumers.

Preparation of multifunctional hydrogels based on co-pigment-polysaccharide complexes and establishment of a machine learning monitoring platform.

Economic loss due to fish spoilage exceeds 25 billion euros every year. Accurate and real-time monitoring of the freshness of fish can effectively cut down economic loss and food wastage. In this study, a dual-functional hydrogel based on sodium alginate-co-pigment complex with volatile antibacterial and intelligent indication was prepared and characterized. The characterization results indicated that the sodium alginate-co-pigment complex successfully improved the stability and color development ability of blueberry anthocyanins and bilberry anthocyanins at different temperatures and pH. The double cross-linking network inside the hydrogel conferred it with excellent mechanical properties. During rainbow trout storage, the hydrogel indicated a color difference of 73.55 on the last day and successfully extended the shelf-life of rainbow trout by 2 days (4 °C). Additionally, four dual-channel monitoring models were constructed using machine learning. The validation error of the genetic algorithm back propagation model (GA-BP) was only 5.6e-3, indicating that GA-BP can accurately monitor the freshness of rainbow trout. The rainbow trout real-time monitoring platform built based on GA-BP model can monitor the freshness of rainbow trout in real time through the images uploaded by users. The results of this study have broad applicability in the food industry, environmental conservation, and economic sustainability.

Phenotypic and genomic characterization of a non-pathogenic Epilithonimonas ginsengisoli isolated from diseased farmed rainbow trout (Oncorhynchus mykiss) in Chile.

Flavobacterial infection associated with diseased fish is caused by multiple bacterial species within the family Flavobacteriaceae. In the present study, the Chilean isolate FP99, from the gills of a diseased, farmed rainbow trout (Oncorhynchus mykiss), was characterized using phenotypic and genomic analyses. Additionally assessed was pathogenic activity. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that isolate FP99 belonged to the genus Epilithonimonas, an average nucleotide identity value of 100% was detected with the Chilean isolate identified as Epilithonimonas sp. FP211-J200. In silico genome analysis, mechanisms for toxins production, and superantigens, adhesion, or other genes associated with virulence were not detected. However, genes encoding proteins for antibiotic resistance were found, including the chrA gene and the nucleotide sequence that encodes for multiple antibiotic resistance MarC proteins. Furthermore, the blaESP-1 gene (87.85% aminoacidic sequence identity), encoding an extended-spectrum subclass B3 metallo-ß-lactamase and conferring carbapenem-hydrolysing activity, and the tet(X) gene, which encodes a monooxygenase that catalyses the degradation of tetracycline-class antimicrobials were carried by this isolate. Phenotyping analyses also supported assignment as E. ginsengisoli. Challenge trials against healthy rainbow trout resulted in no observed pathogenic effect. Our findings identify for the first time the species E. ginsengisoli as associated with fish farming, suggesting that this isolate may be a component of the microbiota of the freshwater system. Notwithstanding, poor environmental conditions and any stressors associated with aquaculture situations or lesions caused by other pathogenic bacteria, such as F. psychrophilum, could favour the entry of E. ginsengisoli into rainbow trout.

Varying Flavobacterium psychrophilum shedding dynamics in three bacterial coldwater disease-susceptible salmonid (Family Salmonidae) species.

Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and is responsible for substantial losses in farm and hatchery-reared salmonids (Family Salmonidae). Although F. psychrophilum infects multiple economically important salmonids and is transmitted horizontally, the extent of knowledge regarding F. psychrophilum shedding rates and duration is limited to rainbow trout (Oncorhynchus mykiss). Concurrently, hundreds of F. psychrophilum sequence types (STs) have been described using multilocus sequence typing (MLST), and evidence suggests that some variants have distinct phenotypes, including differences in host associations. Whether shedding dynamics differ among F. psychrophilum variants and/or salmonids remains unknown. Thus, three F. psychrophilum isolates (e.g., US19, US62, and US87) in three MLST STs (e.g., ST13, ST277, and ST275) with apparent host associations for coho salmon (O. kisutch), Atlantic salmon (Salmo salar), or rainbow trout were intramuscularly injected into each respective fish species. Shedding rates of live and dead fish were determined by quantifying F. psychrophilum loads in water via quantitative PCR. Both live and dead Atlantic and coho salmon shed F. psychrophilum, as did live and dead rainbow trout. Regardless of salmonid species, dead fish shed F. psychrophilum at higher rates (e.g., up to ~108-1010 cells/fish/hour) compared to live fish (up to ~107-109 cells/fish/hour) and for a longer duration (5-35 days vs 98 days); however, shedding dynamics varied by F. psychrophilum variant and/or host species, a matter that may complicate BCWD management. Findings herein expand knowledge on F. psychrophilum shedding dynamics across multiple salmonid species and can be used to inform future BCWD management strategies.IMPORTANCEFlavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, both of which cause substantial losses in farmed and hatchery-reared salmon and trout populations worldwide. This study provides insight into F. psychrophilum shedding dynamics in rainbow trout (Oncorhynchus mykiss) and, for the first time, coho salmon (O. kisutch) and Atlantic salmon (Salmo salar). Findings revealed that live and dead fish of all fish species shed the bacterium. However, dead fish shed F. psychrophilum at higher rates than living fish, emphasizing the importance of removing dead fish in farms and hatcheries. Furthermore, shedding dynamics may differ according to F. psychrophilum genetic variant and/or fish species, a matter that may complicate BCWD management. Overall, study results provide deeper insight into F. psychrophilum shedding dynamics and will guide future BCWD management strategies.

Comprehensive Analysis of YTH Domain-Containing Genes, Encoding m6A Reader and Their Response to Temperature Stresses and Yersinia ruckeri Infection in Rainbow Trout (Oncorhynchus mykiss).

YTH domain-containing genes are important readers of N6-methyladenosine (m6A) modifications with ability to directly affect the fates of distinct RNAs in organisms. Despite their importance, little is known about YTH domain-containing genes in teleosts until now. In the present study, a total of 10 YTH domain-containing genes have been systematically identified and functionally characterized in rainbow trout (Oncorhynchus mykiss). According to the phylogenetic tree, gene structure and syntenic analysis, these YTH domain-containing genes could be classified into three evolutionary subclades, including YTHDF, YTHDC1 and YTHDC2. Of them, the copy number of OmDF1, OmDF2, OmDF3, and OmDC1 were duplicated or even triplicated in rainbow trout due to the salmonid-specific whole-genome duplication event. The three-dimensional protein structure analysis revealed that there were similar structures and the same amino acid residues that were associated with cage formation between humans and rainbow trout, implying their similar manners in binding to m6A modification. Additionally, the results of qPCR experiment indicated that the expression patterns of a few YTH domain-containing genes, especially OmDF1b, OmDF3a and OmDF3b, were significantly different in liver tissue of rainbow trout under four different temperatures (7 °C, 11 °C, 15 °C, and 19 °C). The expression levels of OmDF1a, OmDF1b and OmDC1a were obviously repressed in spleen tissue of rainbow trout at 24 h after Yersinia ruckeri infection, while increased expression was detected in OmDF3b. This study provides a systemic overview of YTH domain-containing genes in rainbow trout and reveals their biological roles in responses to temperature stress and bacterial infection.

Co-occurrence of heterogeneous Flavobacterium psychrophilum isolates within the same Chilean farm and during the same infectious outbreak.

Flavobacterium psychrophilum is a pathogenic bacterium affecting Chilean salmonid farms. High antigenic and genetic diversity exists among Chilean F. psychrophilum isolates, but the distribution thereof among farms is poorly understood. These epidemiological data are key for developing isolate-specific vaccines. The present study isolated F. psychrophilum in diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) from five freshwater farms between 2018 and 2019. Each farm only raised one salmonid species and was geographically separated from and did not share culturing water with the other farms. Antigenic and genetic analyses were conducted to shed light on the possibility of isolates coexisting within the same farm during outbreaks. A total of 68 Chilean F. psychrophilum isolates were recovered from skin lesions, gills, fins, kidney and spleen of moribund and live fish. Among the 68 Chilean isolates, mPCR-serotyping indicated three major serotypes (i.e. 23.5% type 0; 47.1% type 2; and 26.5% type 4) and, to a lesser degree, serotype 1 (2.9%). Sixteen antigenic groups were detected by slide agglutination. Genetic characterizations by 16S rRNA alleles identified 71% of the isolates as the virulent genogroup CSF259-93 allele. A predominant serotype was associated with each farm, with types 0 and 4 related to Atlantic salmon and types 1 and 2 to rainbow trout. Notwithstanding, several antigenic groups coexisted within some farms. Likewise, the experimental intramuscular challenges (n = 20) demonstrated that the type-2 isolates from rainbow trout were the most pathogenic among isolates recovered from infectious outbreaks in Atlantic salmon, especially as compared to those from types-0 and -4. These results allow us to suggest that prevention measures, specifically vaccines, should be developed according to dominant isolates and with specificity to each farm, that is the use of autogenous or site-specific vaccines.

Immunomodulatory effects of bacteriocinogenic and non-bacteriocinogenic Lactococcus cremoris of aquatic origin on rainbow trout (Oncorhynchus mykiss, Walbaum).

Lactic Acid Bacteria (LAB) are a group of bacteria frequently proposed as probiotics in aquaculture, as their administration has shown to confer positive effects on the growth, survival rate to pathogens and immunological status of the fish. In this respect, the production of antimicrobial peptides (referred to as bacteriocins) by LAB is a common trait thoroughly documented, being regarded as a key probiotic antimicrobial strategy. Although some studies have pointed to the direct immunomodulatory effects of these bacteriocins in mammals, this has been largely unexplored in fish. To this aim, in the current study, we have investigated the immunomodulatory effects of bacteriocins, by comparing the effects of a wild type nisin Z-expressing Lactococcus cremoris strain of aquatic origin to those exerted by a non-bacteriocinogenic isogenic mutant and a recombinant nisin Z, garvicin A and Q-producer multi-bacteriocinogenic strain. The transcriptional response elicited by the different strains in the rainbow trout intestinal epithelial cell line (RTgutGC) and in splenic leukocytes showed significant differences. Yet the adherence capacity to RTgutGC was similar for all strains. In splenocyte cultures, we also determined the effects of the different strains on the proliferation and survival of IgM+ B cells. Finally, while the different LAB elicited respiratory burst activity similarly, the bacteriocinogenic strains showed an increased ability to induce the production of nitric oxide (NO). The results obtained reveal a superior capacity of the bacteriocinogenic strains to modulate different immune functions, pointing to a direct immunomodulatory role of the bacteriocins, mainly nisin Z.

First detection of Flavobacterium psychrophilum in juvenile Siberian sturgeons (Acipenser baerii) and description of the pathological findings.

Flavobacterium psychrophilum affects many cultured fish species and is considered one of the most important bacterial pathogens causing substantial economic losses in salmonid aquaculture worldwide. Here, F. psychrophilum was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nested PCR as the aetiological agent causing mortality in diseased juvenile Siberian sturgeons (Acipenser baerii) reared on a freshwater fish farm. Diseased sturgeons were lethargic and displayed dark skin pigmentation, increased mucus production and the presence of skin ulcerations and haemorrhages specially on the ventral side and the base of fins. The histological examination of fish revealed proliferative branchitis, ulcerative and necrotizing dermatitis and myositis, lymphoid tissue atrophy, liver and kidney degeneration and thrombosis. To the best of our knowledge, this is the first report describing the infection of Siberian sturgeons by F. psychrophilum. The detection of F. psychrophilum in diseased Siberian sturgeons and the description of the pathological findings observed during the outbreak may contribute to a better understanding of the bacterium pathogenicity and the range of fish species susceptible to infection.

Interplay between a bacterial pathogen and its host in rainbow trout isogenic lines with contrasted susceptibility to cold water disease.

Infectious diseases are a major constraint on aquaculture. Genetic lines with different susceptibilities to diseases are useful models to identify resistance mechanisms to pathogens and to improve prophylaxis. Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum represents a major threat for freshwater salmonid farming worldwide. A collection of rainbow trout (Oncorhynchus mykiss) isogenic lines was previously produced from a French domestic population. Here, we compared BCWD resistance phenotypes using a subset of isogenic lines chosen for their contrasted susceptibilities to F. psychrophilum. We applied individual monitoring to document the infection process, including time-course quantification of bacteremia and innate immune response. Strikingly, BCWD resistance was correlated with a lower bacterial growth rate in blood. Several immune genes were expressed at higher levels in resistant fish regardless of infection: the Type II arginase (arg2), a marker for M2 macrophages involved in anti-inflammatory responses and tissue repair, and two Toll-like receptors (tlr2/tlr7), responsible for pathogen detection and inflammatory responses. This study highlights the importance of innate and intrinsic defense mechanisms in determining the outcome of F. psychrophilum infections, and illustrates that non-lethal time-course blood sampling for individual monitoring of bacteremia is a powerful tool to resolve within-host pathogen behavior in bacterial fish diseases.

Molecular mechanisms of the virulence and efficacy of a highly virulent Vibrio anguillarum strain and its formalin-inactivated vaccine in rainbow trout.

In this study, we have isolated four strains of Vibrio anguillarum, revealing that they share the same serotype of O1, biochemical characteristics and virulence factor genes. However, there were differences in haemolytic activity among the bacterial strains; a strain with lower pathogenicity showed γ-haemolytic activity, whereas other virulent strains showed α-haemolytic activity on blood agar and higher empA gene expression in RTG-2 cell line. The most virulent strain was V. anguillarum RTBHR from diseased masu salmon (Oncorhynchus masou), which resulted in mortality of 100% and 93.3% when injected intraperitoneally at concentrations of 9 × 105 and 6.3 × 105 colony-forming units/fish in rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), respectively. A formalin-inactivated vaccine of V. anguillarum RTBHR induced a protective and specific immunity in rainbow trout as the vaccinated fish exhibited low cumulative mortality in a challenge test and a high specific antibody response in enzyme-linked immunosorbent assay at 8 weeks post-vaccination. The produced antibody was bound to bacterial proteins of 30-37 kDa in size. This adaptive immune response was detected as early as day 1, with quantitative polymerase chain reaction analysis revealing the upregulated expression of genes encoding for TCRα, T-bet, mIgM and sIgM in rainbow trout. This suggested that the vaccine induced T (probably a more dominant Th1 response) and B cell responses. In conclusion, the vaccine successfully protected fish from V. anguillarum infection by eliciting cellular and humoral immune responses.

Investigation of the Genus Flavobacterium as a Reservoir for Fish-Pathogenic Bacterial Species: the Case of Flavobacterium collinsii.

Bacteria of the genus Flavobacterium are recovered from a large variety of environments. Among the described species, Flavobacterium psychrophilum and Flavobacterium columnare cause considerable losses in fish farms. Alongside these well-known fish-pathogenic species, isolates belonging to the same genus recovered from diseased or apparently healthy wild, feral, and farmed fish have been suspected to be pathogenic. Here, we report the identification and genomic characterization of a Flavobacterium collinsii isolate (TRV642) retrieved from rainbow trout spleen. A phylogenetic tree of the genus built by aligning the core genome of 195 Flavobacterium species revealed that F. collinsii stands within a cluster of species associated with diseased fish, the closest one being F. tructae, which was recently confirmed as pathogenic. We evaluated the pathogenicity of F. collinsii TRV642 as well as of Flavobacterium bernardetii F-372T, another recently described species reported as a possible emerging pathogen. Following intramuscular injection challenges in rainbow trout, no clinical signs or mortalities were observed with F. bernardetii. F. collinsii showed very low virulence but was isolated from the internal organs of survivors, indicating that the bacterium is able to survive inside the host and may provoke disease in fish under compromised conditions such as stress and/or wounds. Our results suggest that members of a phylogenetic cluster of fish-associated Flavobacterium species may be opportunistic fish pathogens causing disease under specific circumstances. IMPORTANCE Aquaculture has expanded significantly worldwide in the last decades and accounts for half of human fish consumption. However, infectious fish diseases are a major bottleneck for its sustainable development, and an increasing number of bacterial species from diseased fish raise a great concern. The current study revealed phylogenetic associations with ecological niches among the Flavobacterium species. We also focused on Flavobacterium collinsii, which belongs to a group of putative pathogenic species. The genome contents revealed a versatile metabolic repertoire suggesting the use of diverse nutrient sources, a characteristic of saprophytic or commensal bacteria. In a rainbow trout experimental challenge, the bacterium survived inside the host, likely escaping clearance by the immune system but without provoking massive mortality, suggesting opportunistic pathogenic behavior. This study highlights the importance of experimentally evaluating the pathogenicity of the numerous bacterial species retrieved from diseased fish.

Immune profiling of rainbow trout (Oncorhynchus mykiss) exposed to Lactococcus garvieae: Evidence in asymptomatic versus symptomatic or vaccinated fish.

Lactococcosis, caused by the Gram-positive bacterium Lactococcus garvieae, is a major concern in rainbow trout (Oncorhynchus mykiss) farms, which are regularly affected by outbreaks especially during the summer/fall months. In these farms, unvaccinated healthy and symptomatic fish can coexist with vaccinated fish. In the present study, innate (leukogram, serum lysozyme activity, peroxidase activity, antiprotease activity, bactericidal activity, total IgM and total proteins), and specific immune parameters (serum antibodies to L. garvieae) were assessed in unvaccinated adult rainbow trout naturally exposed to the pathogen, with or without evidence of clinical signs, or subjected to vaccination. Blood was drawn from all three groups, and blood smears were prepared. Bacteria were found in the blood smears of 70% of the symptomatic fish but not in any of the asymptomatic fish. Symptomatic fish showed lower blood lymphocytes and higher thrombocytes than asymptomatic fish (p ≤ .05). Serum lysozyme and bactericidal activity did not vary substantially among groups; however, serum antiprotease and peroxidase activity were significantly lower in the unvaccinated symptomatic group than in the unvaccinated and vaccinated asymptomatic groups (p ≤ .05). Serum total proteins and total immunoglobulin (IgM) levels in vaccinated asymptomatic rainbow trout were significantly higher than in unvaccinated asymptomatic and symptomatic groups (p ≤ .05). Similarly, vaccinated asymptomatic fish produced more specific IgM against L. garvieae than unvaccinated asymptomatic and symptomatic fish (p ≤ .05). This preliminary study provides basic knowledge on the immunological relationship occurring between the rainbow trout and L. garvieae, potentially predicting health outcomes. The approach we proposed could facilitate infield diagnostics, and several non-specific immunological markers could serve as reliable indicators of the trout's innate ability to fight infection.

Efficacy of polyvalent vaccine on immune response and disease resistance against streptococcosis/lactococcosis and yersiniosis in rainbow trout (Oncorhynchus mykiss).

Diseases are the most significant challenge in the development and stability of aquaculture. In this study, the immunogenic efficiency of polyvalent streptococcosis/lactococcosis and yersiniosis vaccines was evaluated by injection and immersion methods in rainbow trout.. The 450 fish with an average weight of 50 ± 5 g were divided into three treatments and three replications as follows: injection vaccine treatment, immersion vaccine treatment and control group without vaccine administration. Fish were kept for 74 days and sampling was done on days 20, 40 and 60. Then, from the 60th to the 74th day, the immunized groups were challenged with three bacteria Streptococcus iniae (S. iniae), Lactococcus garvieae (L. garvieae) and Yersinia ruckeri (Y. ruckeri) separately. A significant difference was observed in the weight gained (WG) in the immunized groups compared to the control group (P < 0.05). The relative survival percentage (RPS) after 14 days of challenge with S. iniae, L. garvieae and Y. ruckeri in the injection group compared to the control group increased respectively (60%, 60% and 70%), (P < 0.05). Also, RPS in the immersion group had an increase respectively (30%, 40% and 50%) after the challenge with S. iniae, L.garvieae and Y. ruckeri compared to the control group. Immune indicators such as antibody titer, complement and lysozyme activity significantly increased in comparison to the control group (P < 0.05). In general, it can be concluded that applying three vaccines by injection and immersion method has significant effects on immune protection and survival rate. However, the injection method is more effective and more suitable than the immersion method.