Search Results (15)

As global fish consumption rises, improving fish health through immunomodulatory feed ingredients shows promise while also supporting growth performance. This study investigated the effects of yeast prebiotics, probiotics, a postbiotic (butyrate), and black soldier fly larvae (BSFL) meal on fish immune responses. Zebrafish were fed diets containing these ingredients for 63 days and then exposed to either Pseudomonas aeruginosa lipopolysaccharide (LPS) or live Flavobacterium psychrophilum to assess hepatic candidate gene expression and weight gain. No mortalities were observed post-immune challenges, and weight gains were not significantly different across treatments. Liver samples were collected for mRNA analysis, and real-time qPCR was used to evaluate the expression of immune-related genes such as TNF-α, IL-1β, hepcidin, and NF-κB/p65. NF-κB/p65 was upregulated in response to immune challenges, indicating a reaction to both LPS and pathogen exposure. Fish on the BSFL diet showed decreased NF-κB/p65 expression after the pathogen challenge, while probiotic-fed fish had reduced angiopoietin-like 4 (angptl4) levels following LPS exposure. Butyrate supplementation had the most significant impact, downregulating pro-inflammatory cytokines and other immune-related genes, suggesting a protective effect. These findings support the health benefits of BSFL and sodium butyrate during an immune challenge. Full article

Flavobacterium psychrophilum is currently one of the most important pathogens in aquaculture worldwide, causing high losses to farmed salmonids particularly during early growth stages with significant economic impact. Despite previous attempts, no effective vaccine has been developed, and protection against introduction into farms is difficult due to the ubiquitous occurrence of the pathogen. A better understanding of the mechanism of disease development is essential for targeted therapeutic and preventive measures in farms. Unfortunately, the pathogenesis of diseases caused by F. psychrophilum has not been elucidated yet. Previously, several putative virulence factors have been identified. Some appear to be essential for disease development, while others are probably dispensable. The importance of some factors has not yet been explored. This review focuses on the supposed virulence factors of F. psychrophilum and the current knowledge about their importance in the pathogenesis of the disease. Full article

The objective of this study was to investigate the bactericidal activity of blood plasma from cultured rainbow trout obtained from two different fish farms. Plasma from trout naturally infected with the bacterial pathogen Flavobacterium psychrophilum was found to inhibit the growth of Aeromonas hydrophila in vitro. Incubation of A. hydrophila in bacteriostatic trout plasma resulted in agglutination and growth retardation, without causing massive damage to the cell membrane. The proteome of the plasma with high antimicrobial activity revealed an abundance of high-density apolipoproteins, some isoforms of immunoglobulins, complement components C1q and C4, coagulation factors, lectins, periostin, and hemoglobin. Analysis of trout proteins retained on A. hydrophila cells revealed the presence of fish immunoglobulins, lectins, and complement components on bacteria whose growth was inhibited, although the native membrane attack complex of immunised trout plasma did not assemble effectively, resulting in a weak bactericidal effect. Furthermore, this study examined the bacterial response to trout plasma and suggested that the protein synthesis pathway was the target of antimicrobial proteins from fish blood. Taken together, these findings illustrate the advantages of the affinity approach for understanding the role of plasma proteins in host defence against pathogens. Full article

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease in salmonids and rainbow trout fry syndrome. This pathogen has attained a global presence and can spread both horizontally and vertically. However, it was not documented in Korea before September 2018. In this study, the objectives were to characterize Flavobacterium psychrophilum strain FPRT1, isolated from diseased rainbow trout genotypically and phenotypically. We also conducted various investigations to better understand its impact and assess potential control measures. We acquired fifty rainbow trout (approximately 70 g in weight) and transferred them to a laboratory aquarium. During the initial acclimation period, we observed mortality and examined affected fish for clinical signs. We isolated the bacterium from the spleen of infected rainbow trout using tryptone yeast extract salts agar supplemented with glucose, naming this FPRT1. Antibiotic susceptibility testing was carried out, and from the result, we selected enrofloxacin to administer to the trout orally to reduce mortality. To evaluate pathogenicity, we exposed the trout to FPRT1 at different water temperatures (8, 15, and 22 °C). Genomic analysis was conducted to identify the serotype and relatedness of FPRT1 to European strains. Affected fish displayed clinical signs, such as ulcerative lesions in the mandible, anemia with pale gills, exophthalmia, and increased mucus secretion. Internal symptoms included pale liver and enlarged spleen. FPRT1 was susceptible to erythromycin, enrofloxacin, florfenicol, oxytetracycline, and gentamicin, but resistant to oxolinic acid and sulfamethoxazole/trimethoprim. Oral administration of enrofloxacin resulted in a decrease in mortality from 28% to 6%. Pathogenicity tests revealed varying mortality rates due to FPRT1 at different temperatures. The highest rates were observed at 8 °C (ranging from 43% to 100%) for both intraperitoneal and intramuscular injections, and lower rates occurred at 22 °C (ranging from 0% to 30%), with intramuscular injections displaying higher susceptibility. Genomic analysis identified FPRT1 as serotype 2 and indicated its close genetic relationship with European strains based on the core genome and dispensable genome. The substantial genomic similarity between our strain and European strains suggests the possibility of bacterial spread through the importation of fertilized eggs from Europe. In conclusion, this study highlights the introduction of the previously undocumented pathogen (F. psychrophilum) into Korean rainbow trout populations. The detection of this pathogen and its pathogenicity assessment is not only important for understanding its impact on local aquaculture but also for establishing surveillance and control measures to prevent further transmission and outbreaks in the region. Full article

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a multifunctional neuropeptide that is widely distributed and conserved across species. We have previously shown that in teleost fish, PACAP not only possesses direct antimicrobial properties but also immunomodulatory effects against the bacterial pathogens Flavobacterium psychrophilum and Pseudomonas aeruginosa using in vitro and in vivo experiments. These previous results suggest PACAP can be used as an alternative to antibiotics to prevent and/or treat bacterial infections in the aquaculture industry. To accomplish this goal, more studies are needed to better understand the effect of PACAP on pathogens affecting fish in live infections. In the present study, the transcripts PACAP, PRP/PACAP, and VPAC2 receptor were examined in rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri, which exhibited an increase in their expression in the spleen when compared to healthy fish. Synthetic Clarias gariepinus PACAP-38 has direct antimicrobial activity on Y. ruckeri and inhibits up to 60% of the bacterial growth when the peptide is at concentrations between 50 and 100 µM in TSB. The growth inhibition increased up to 90% in the presence of 12.5 µM of PACAP-38 when salt-free LB broth was used instead of TSB. It was also found to inhibit Y. ruckeri growth in a dose-dependent manner when the rainbow trout monocyte/macrophage-like cell line (RTS11) was pre-treated with lower concentrations of the peptide (0.02 and 0.1 µM) before going through infection. Differential gene expression was analyzed in this in vitro model. Overall, the results revealed new evidence to support the role of PACAP as an antimicrobial and immunomodulatory peptide treatment in teleosts. Full article

The diversity and composition of intestinal microbiota in rainbow trout have been studied using next-generation sequencing (NGS), although few studies have examined the effects of antimicrobials. We evaluated the effect of antibiotics florfenicol and erythromycin and infection with or without Flavobacterium psychrophilum on the intestinal microbiota in rainbow trout juveniles (30–40 g) using NGS. Prophylactic oral antibiotic treatments were administered for 10 days before groups of fish were injected intraperitoneally with virulent F. psychrophilum. Intestinal content (allochthonous bacteria) was collected at day −11, 0, 12, and 24 p.i., and the v3–v4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Before prophylactic treatment, Tenericutes and Proteobacteria were the most abundant phyla identified and Mycoplasma was the most abundant genus. Fish infected with F. psychrophilum had decreased alpha diversity and a high abundance of Mycoplasma. Fish administered florfenicol had increased alpha diversity compared to the control at day 24 p.i., although both florfenicol and erythromycin-treated fish had a higher abundance of potential pathogens, specifically Aeromonas, Pseudomonas, and Acinetobacter. Mycoplasma disappeared after treatment but appeared again after day 24. This study demonstrates that prophylactic oral treatment with antibiotics florfenicol and erythromycin as well as F. psychrophilum infection changed the composition of intestinal microbiota in rainbow trout juveniles that did not recover by day 24 p.i. and further long-term effects on the host need to be investigated. Full article

Flavobacterium psychrophilum (Fp), the causative agent of Bacterial Cold-Water disease in salmonids, causes substantial losses in aquaculture. Bacterial outer membrane vesicles (OMVs) contain several virulence factors, enzymes, toxins, and nucleic acids and are expected to play an essential role in host–pathogen interactions. In this study, we used transcriptome sequencing, RNA-seq, to investigate the expression abundance of the protein-coding genes in the Fp OMVs versus the Fp whole cell. RNA-seq identified 2190 transcripts expressed in the whole cell and 2046 transcripts in OMVs. Of them, 168 transcripts were uniquely identified in OMVs, 312 transcripts were expressed only in the whole cell, and 1878 transcripts were shared in the two sets. Functional annotation analysis of the OMV-abundant transcripts showed an association with the bacterial translation machinery and histone-like DNA-binding proteins. RNA-Seq of the pathogen transcriptome on day 5 post-infection of Fp-resistant versus Fp-susceptible rainbow trout genetic lines revealed differential gene expression of OMV-enriched genes, suggesting a role for the OMVs in shaping the host–microbe interaction. Interestingly, a cell wall-associated hydrolase (CWH) gene was the most highly expressed gene in OMVs and among the top upregulated transcripts in susceptible fish. The CWH sequence was conserved in 51 different strains of Fp. The study provides insights into the potential role of OMVs in host–pathogen interactions and explores microbial genes essential for virulence and pathogenesis. Full article

Background: Although Flavobacterium psychrophilum is recognized as the causative pathogen of rainbow trout fry syndrome (RTFS), often resulting in high fry mortality, it is also responsible for bacterial cold water disease (BCWD) in large and older rainbow trout (Oncorhynchus mykiss). These older fish do not experience high mortality, but sustain, through the shedding of bacteria, a constant infection pressure at farm level, which exposes fry to an unnecessary infection risk. We have produced and assessed the immunogenicity of an experimental injection BCWD vaccine, which may be used to decrease the shedding of bacteria from older fish. Methods: A total of 800 fish were i.p.-injected: 200 fish received the bacterin with adjuvant, 200 fish received the bacterin alone, 200 fish received adjuvant alone and 200 fish were injected with physiological saline. Blood samples were taken at day 0 and at three different time points (4, 8 and 14 weeks) post-vaccination. Plasma antibody levels were measured by ELISA for reactivity against both the homologous F. psychrophilum vaccine strain (serotype Fd) and heterologous strains (serotype Th). Results: Significantly elevated antibody titers were found against all serotypes in vaccinated fish. Welfare parameters associated with the vaccination process were evaluated by analyzing trout plasma samples for six different biochemical parameters, but no adverse effects associated with injection were indicated. Conclusions: The study suggests that an injection vaccine containing formalin-inactivated whole cells of F. psychrophilum (serotype Fd), adjuvanted with FIA, may also induce protection against heterologous strains. We advocate for, as the next step, the performance of field trials evaluating if the vaccination of older rainbow trout will (1) reduce the infection pressure in farms, (2) elevate the general health level in all groups and (3) minimize F. psychrophilum infection in fry at farm level. This may reduce the need for the administration of antibiotics in all age classes. Full article

Previously, we reported an in vitro evaluation regarding antibacterial effects against F. psychrophilum by a new Cu (I) complex, [Cu(NN₁)₂](ClO₄). This study presents the results of an in vivo evaluation of [Cu(NN₁)₂](ClO₄) added as a dietary supplement against F. psychrophilum in rainbow trout. The results showed that the administration of [Cu(NN₁)₂](ClO₄) at 29 and 58 µg/g of fish for 15 days does not affect the growth of rainbow trout. On the other hand, the amount of copper present in the liver, intestine, and muscle of rainbow trout was determined. The results showed that the amount of copper in the liver, when compared between treated fish and control fish, does not change. While, in the intestine, an increase in the fish fed at 58 µg/g of fish was observed. In muscle, a slight decrease at 29 µg/g was obtained. Additionally, copper concentrations in the pond water after 15 days of feeding with the [Cu(NN₁)₂](ClO₄) complex showed the highest levels of copper. Finally, the effect of the administration of [Cu(NN₁)₂](ClO₄) for 15 days at 58 µg/g of fish was evaluated against F. psychrophilum, where a 75% survival was obtained during 20 days of challenge. Full article

Infectious bacterial pathogens are a concern for aquaculture as estimates suggest that billions of US dollars are lost annually in aquaculture due to disease. One of the most prevalent salmonid pathogens is the bacterium Flavobacterium psychrophilum that causes bacterial coldwater disease. We reviewed the published F. psychrophilum literature and conducted a Bayesian analysis to examine large-scale patterns in rainbow trout (Oncorhynchus mykiss) mortality associated with laboratory challenge. We incorporated factors that were common across a majority of the laboratory exposure studies and these included bacterial dose, culture time, exposure method, bacterial isolate, experimental duration, and fish weight. The comparison showed that injection as the exposure method produced higher mortality than bath immersion, bacterial isolates differed in their effect on mortality, and bacterial dose has an interactive effect with fish weight and exposure method. Our comparison allows for inference on factors affecting rainbow trout mortality due to exposure to F. psychrophilum and suggests avenues to further optimize research protocols to better reach study goals. Full article

The fish pathogen Flavobacterium psychrophilum is currently one of the main pathogenic bacteria hampering the productivity of salmonid farming worldwide. Although putative virulence determinants have been identified, the genetic basis for variation in virulence of F. psychrophilum is not fully understood. In this study, we analyzed whole-genome sequences of a collection of 25 F. psychrophilum isolates from Baltic Sea countries and compared genomic information with a previous determination of their virulence in juvenile rainbow trout. The results revealed a conserved population of F. psychrophilum that were consistently present across the Baltic Sea countries, with no clear association between genomic repertoire, phylogenomic, or gene distribution and virulence traits. However, analysis of the entire genome of four F. psychrophilum isolates by hybrid assembly provided an unprecedented resolution for discriminating even highly related isolates. The results showed that isolates with different virulence phenotypes harbored genetic variances on a number of consecutive leucine-rich repeat (LRR) proteins, repetitive motifs in gliding motility-associated protein, and the insertion of transposable elements into intergenic and genic regions. Thus, these findings provide novel insights into the genetic variation of these elements and their putative role in the modulation of F. psychrophilum virulence. Full article

The microbial community surrounding fish eyed eggs can harbor pathogenic bacteria. In this study we focused on rainbow trout (Oncorhynchus mykiss) eyed eggs and the potential of bacteriophages against the pathogenic bacteria Flavobacterium psychrophilum and F. columnare. An infection bath method was first established, and the effects of singular phages on fish eggs was assessed (survival of eyed eggs, interaction of phages with eyed eggs). Subsequently, bacteria-challenged eyed eggs were exposed to phages to evaluate their effects in controlling the bacterial population. Culture-based methods were used to enumerate the number of bacteria and/or phages associated with eyed eggs and in the surrounding environment. The results of the study showed that, with our infection model, it was possible to re-isolate F. psychrophilum associated with eyed eggs after the infection procedure, without affecting the survival of the eggs in the short term. However, this was not possible for F. columnare, as this bacterium grows at higher temperatures than the ones recommended for incubation of rainbow trout eyed eggs. Bacteriophages do not appear to negatively affect the survival of rainbow trout eyed eggs and they do not seem to strongly adhere to the surface of eyed eggs either. Finally, the results demonstrated a strong potential for short term (24 h) phage control of F. psychrophilum. However, further studies are needed to explore if phage control can be maintained for a longer period and to further elucidate the mechanisms of interactions between Flavobacteria and their phages in association with fish eggs. Full article

A new copper (I) complex, [Cu(NN₁)₂](ClO₄)_, was synthesized, where NN₁ was a imine ligand 6-((quinolin-2-ylmethylene)amino)-2H-chromen-2-one obtained by a derivatization of natural compound coumarin. The structural characterization in solution was done by NMR techniques, UV-Vis and cyclic voltammetry. The potential antibacterial effect of [Cu(NN₁)₂](ClO₄)_, was assessed for F. psychrophilum isolated 10094. F. psychrophilum is a Gram-negative bacterium which causes diseases such as bacterial cold-water disease and rainbow trout fry syndrome, causing large economic losses in the freshwater salmonid aquaculture industry. This complex show to have antibacterial activity against F. psychrophilum 10094 at non-cytotoxic concentration in cell line derived from trout (F. psychrophilum 10094 IC₅₀ 16.0 ± 0.9; RT-GUT IC₅₀ 53.0 ± 3.1 µg/mL). Full article

Two new models, based upon the principles promulgated by Baranyi and co-workers are presented and resulting growth functions evaluated based upon their ability to mimic bacterial growth of the fish pathogen Flavobacterium psychrophilum. These growth functions make use of a dampening function to suppress potential growth, represented by a logistic, and are derived from rate:state differential equations. Dampening effects are represented by a rectangular hyperbola or a simple exponential, incorporated into a logistic differential equation and solved analytically resulting in two newly derived growth equations, viz. logistic × hyperbola (log × hyp) and logistic × exponential (log × exp). These characteristics result in flexible and robust growth functions that can be expressed as equations with biologically meaningful parameters. The newly derived functions (log × hyp and log × exp), along with the Baranyi (BAR), simple logistic (LOG) and its modified form (MLOG) were evaluated based upon examination of residuals and measures of goodness-of-fit and cross-validation. Using these criteria, log × hyp, log × exp and BAR performed better than, or at least equally well as, LOG and MLOG. In contrast with log × exp and BAR, log × hyp can be easily manipulated mathematically allowing for simple algebraic expressions for time and microbial biomass at inflexion point, in addition to maximum and scaled maximum growth rates. Full article

Cyclotides are circular peptides found in various plant families. A cyclized backbone, together with multiple disulfide bonds, confers the peptides’ exceptional stability against protease digestion and thermal denaturation. In addition, the features of these antimicrobial molecules make them suitable for use in animal farming, such as aquaculture. Fmoc solid phase peptide synthesis on 2-chlorotrityl chlorine (CTC) resin using the “tea-bag” approach was conducted to generate the VarvA cyclotide identified previously from Viola arvensis. MALDI-TOF mass spectrometry determined the correct peptide amino acid sequence and the cyclization sites-critical in this multicyclic compound. The cyclotide showed antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens present in Chilean aquaculture. The highest antimicrobial activity was found to be against Flavobacterium psychrophilum. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and the Sytox Green permeabilization assay showed the ability to disrupt the bacterial membrane. We postulate that this compound can be proposed for the control of fish farming infections. Full article