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Flavobacterium columnare is a virulent intracellular bacterial pathogen that causes an infection known as columnaris in many species of fish. Some economically important fish species are strongly affected by columnaris, leading to a high mortality rate and significant economic losses. Previous in silico studies have provided various biological insights into F. columnare, including its interaction with MHC class I alleles and the epitopic region within outer membrane proteins. However, the protein-protein interaction networks underlying the growth, defense, and pathogenesis of F. columnare remain largely unknown. This study was conducted to identify the protein-protein interaction (PPI) networks and hub proteins of F. columnare that can be used as drug or vaccine targets. A total of 500 protein sequences were retrieved from UniprotKB in FASTA format and analyzed using VaxiJen, PSORTb, STRING, Cytoscape, and BLASTp programs. The results demonstrated that 60% of F. columnare proteins were predicted as antigenic proteins, most of which were associated with catalytic activity and metabolic processes, identified as cytoplasmic proteins. Ten hub proteins with the highest number of functional interactions were identified, which were also antigenic and non-host homologous. In conclusion, F. columnare hub proteins represent potential therapeutic targets in drug and vaccine development against columnaris infection. [ABSTRACT FROM AUTHOR]

Abstract Tilapia is one of the most important farmed fish in the world and the most cultivated in Brazil. The increase of this farming favors the appearance of diseases, including bacterial diseases. Therefore, the aim of this study was to evaluate the bactericidal activity of copaiba oil, Copaifera duckei, against Streptococcus agalactiae and Flavobacterium columnare and the dietary effect of copaiba oil on zootechnical performance, hematological, biochemical, immunological, and histological analysis before and after an intraperitoneal infection (body cavity) with S. agalactiae in Nile tilapia. For this, fish were randomly distributed into 15 fiber tanks in five treatments (0, 0.25, 0.50, 0.75, and 1.0%) and fed with a commercial diet supplemented with copaiba oil for 30 days. After this period, the fish were randomly redistributed for the experimental challenge with S. agalactiae into six treatments (T0, T1, T2, T3, T4, and T5), the fish were anesthetized, and blood samples were collected to assess hematological, biochemical, immunological, and histological parameters. Copaiba oil showed bactericidal activity against Streptococcus spp. and Flavobacterium spp. in vitro. In addition, concentrations of 0.75 and 1.0% of copaiba oil have an anti-inflammatory effect and improve hematological and immunological parameters, increasing leukocyte numbers, albumin, and serum lytic activity. Furthermore, there is an increase in the intestinal villus length and tissue damage in groups at concentrations of 0.75 and 1.0% of copaiba oil. In conclusion, copaiba oil presented bactericidal activity against Streptococcus spp. and Flavobacterium spp. in vitro, and oral supplementation at concentrations of 0.75 and 1.0% compared to the control group enhanced non-specific immune parameters and digestibility in Nile Tilapia.

Tilapia is one of the most important farmed fish in the world and the most cultivated in Brazil. The increase of this farming favors the appearance of diseases, including bacterial diseases. Therefore, the aim of this study was to evaluate the bactericidal activity of copaiba oil, Copaifera duckei, against Streptococcus agalactiae and Flavobacterium columnare and the dietary effect of copaiba oil on zootechnical performance, hematological, biochemical, immunological, and histological analysis before and after an intraperitoneal infection (body cavity) with S. agalactiae in Nile tilapia. For this, fish were randomly distributed into 15 fiber tanks in five treatments (0, 0.25, 0.50, 0.75, and 1.0%) and fed with a commercial diet supplemented with copaiba oil for 30 days. After this period, the fish were randomly redistributed for the experimental challenge with S. agalactiae into six treatments (T0, T1, T2, T3, T4, and T5), the fish were anesthetized, and blood samples were collected to assess hematological, biochemical, immunological, and histological parameters. Copaiba oil showed bactericidal activity against Streptococcus spp. and Flavobacterium spp. in vitro. In addition, concentrations of 0.75 and 1.0% of copaiba oil have an anti-inflammatory effect and improve hematological and immunological parameters, increasing leukocyte numbers, albumin, and serum lytic activity. Furthermore, there is an increase in the intestinal villus length and tissue damage in groups at concentrations of 0.75 and 1.0% of copaiba oil. In conclusion, copaiba oil presented bactericidal activity against Streptococcus spp. and Flavobacterium spp. in vitro, and oral supplementation at concentrations of 0.75 and 1.0% compared to the control group enhanced non-specific immune parameters and digestibility in Nile Tilapia. [ABSTRACT FROM AUTHOR]

Identifying novel natural sources from the marine environment with unique immunomodulatory and antioxidant efficacies is of interest in intensive fish farming. In this sense, marine sponge extract derived from Smenospongia (SS-extract) was tested for its potential anti-inflammatory, antioxidant and antimicrobial activities during the concurrent infection with Trichodina sp. and Flavobacterium columnare in Nile tilapia. A total of 625 Nile tilapia fingerlings were distributed into five groups: the control group (with no additives) and four groups fed the control diet fortified with SS-extract at 50, 100, 150, and 200 mg/kg, respectively, for 60 days. The parasitic challenge with Trichodina sp was done at the endpoint of the feeding trial and at 7 days post-infection (dpi); the fish were coinfected with F. columnare. A significant improvement in growth-related parameters of Nile tilapia was detected in the groups that received SS-extract at the levels of 150 and 200 mg/kg. The scavenging ability for free radicals (2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH)) was maximized with higher SS-extract supplementation levels. Simultaneously, the antioxidant defense of the Nile tilapia fed 150 and 200 mg/kg was augmented with a decline in oxidation-associated indicators [reactive oxygen species (ROS), hydrogen peroxide (H2O2), and malondialdehyde (MDA)]. The higher concentrations of SS-extract in the feed potentiated the immune response of Nile tilapia before infection and even after coinfection and was paralleled by a subsiding in the exaggerated inflammatory response after concurrent infection. Concurrently, the mRNA expression of autophagy-encountered genes (Atg5 and 12, LC3-II and BCLN1) had its highest expression in the fish fed the 200 mg/kg diet with the lowest expression of the mTOR gene in the same fish. The lowest infection intensity and rate and mucus score were displayed in fish fed 200 mg/kg SS-extract at 20 dpi. Interestingly, higher levels of SS-extract triggered antimicrobial peptides (upregulation of Hepcidin and β-defensin-1). In contrast, excessive expression of endoplasmic reticulum genes (atf4, JAK1, PERK, and eif2α) in the control infected group was downregulated by 200 mg/kg of SS-extract. The quantification of F. columnare indicated that columnaris coinfection severity displayed its lowest rate in the group supplemented with 200 mg/kg of SS-extract. In conclusion, a strategic siting based on fortification of the diet of Nile tilapia with SS-extract was elucidated to reinforce its immune and antioxidant defenses during a concurrent infection.

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. [ABSTRACT FROM AUTHOR]

Objective: Columnaris disease is a leading cause of disease‐related losses in the catfish industry of the southeastern United States. The term "columnaris‐causing bacteria" (CCB) has been coined in reference to the four described species that cause columnaris disease: Flavobacterium columnare, F. covae, F. davisii, and F. oreochromis. Historically, F. columnare, F. covae, and F. davisii have been isolated from columnaris disease cases in the catfish industry; however, there is a lack of knowledge of which CCB species are most prevalent in farm‐raised catfish. The current research objectives were to (1) sample columnaris disease cases from the U.S. catfish industry and identify the species of CCB involved and (2) determine the virulence of the four CCB species in Channel Catfish Ictalurus punctatus in controlled laboratory challenges. Methods: Bacterial isolates or swabs of external lesions from catfish were collected from 259 columnaris disease cases in Mississippi and Alabama during 2015–2019. The DNA extracted from the samples was analyzed using a CCB‐specific multiplex polymerase chain reaction to identify the CCB present in each diagnostic case. Channel Catfish were challenged by immersion with isolates belonging to each CCB species to determine virulence at ~28°C and 20°C. Result: Flavobacterium covae was identified as the predominant CCB species impacting the U.S. catfish industry, as it was present in 94.2% (n = 244) of diagnostic case submissions. Challenge experiments demonstrated that F. covae and F. oreochromis were highly virulent to Channel Catfish, with most isolates resulting in near 100% mortality. In contrast, F. columnare and F. davisii were less virulent, with most isolates resulting in less than 40% mortality. Conclusion: Collectively, these results demonstrate that F. covae is the predominant CCB in the U.S. catfish industry, and research aimed at developing new control and prevention strategies should target this bacterial species. The methods described herein can be used to continue monitoring the prevalence of CCB in the catfish industry and can be easily applied to other industries to identify which Flavobacterium species have the greatest impact. Impact statementColumnaris disease is a leading cause of disease‐related losses in the southeastern U.S. catfish industry. Recently, four species of columnaris‐causing bacteria (CCB) have been described, but their prevalence in the industry was unknown. In this research, Flavobacterium covae was identified as the predominant species of CCB impacting the industry and was demonstrated to be highly virulent in Channel Catfish. Research aimed at developing new control and prevention strategies should target this bacterial species. [ABSTRACT FROM AUTHOR]

Columnaris disease is a prevalent disease in freshwater environments caused by the ubiquitous aquatic pathogen Flavobacterium species. Adhesion to the external mucosal surfaces of fishes is the initial stage of infection, and the gills specifically have been identified as both a primary target and release site for this pathogen. Demonstrated here and in previous research, the hybrid striped bass (Morone chrysops x M. saxatilis), a prominent United States aquaculture product, is more susceptible to infection with F. covae than the maternal white bass (M. chrysops) parental species. To further elucidate the mechanisms underlying differences in resistance between these fish we examined gill gene expression profiles using RNA sequencing at different timepoints after F. covae infection. Patterns of differential gene expression and association with key enrichment terms indicate the effective immune response observed in white bass includes the up-regulation of multiple cytokines (IL-1β, IL-17C, TNF-α, G-CSF, IL-8, CCL16, CXCL9), hepcidins (HAMP and HAMP2), ribosomal subunit components (e.g., RPL13), and, interestingly, the down-regulation of leptin (LEPA) during initial (1 – 4 h) stages of infection. Conversely, up-regulation of the same genes was not detected in hybrid striped bass until 24 h after infection, indicating a delay in immune response mechanisms that is ultimately ineffective in protecting the host, as this was concurrent with the onset of mortality in these fish. Collectively, the presented results include several putative pathways and candidate genes for further investigation toward characterizing immune defense mechanisms underlying the resistance (white bass) and susceptibility (hybrid striped bass) for selective breeding efforts and/or biotechnological intervention.

TonB-dependent siderophore receptors play a critical transport role for Flavobacterium columnare virulence formation and growth, and have become valuable targets for the development of novel antimicrobial agents. Traditional Chinese medicine has demonstrated notable efficacy in the treatment of fish diseases and includes potential antibacterial agents. Herein, we performed molecular docking-based virtual screening to discover novel TonB-dependent siderophore receptor inhibitors from traditional Chinese medicine and provide information for developing novel antibacterial agents. Firstly, we efficiently obtained 11 potential inhibitors with desirable drug-like characteristics from thousands of compounds in the TCM library based on virtual screening and property prediction. The antibacterial activity of Enoxolone, along with its interaction characteristics, were determined via an MIC assay and molecular dynamic simulation. Transcriptional profiling, along with validation experiments, subsequently revealed that an insufficient uptake of iron ions by bacteria upon binding to the TonB-dependent siderophore receptors is the antibacterial mechanism of Enoxolone. Finally, Enoxolone's acceptable toxicity was illustrated through immersion experiments. In summary, we have used virtual screening techniques for the first time in the development of antimicrobial agents in aquaculture. Through this process, we have identified Enoxolone as a promising compound targeting the TonB-dependent siderophore receptor of F. columnare. In addition, our findings will provide new ideas for the advancement of innovative antimicrobial medications in aquaculture.

Recent work has identified pituitary adenylate cyclase activating polypeptide (PACAP) as a potential antimicrobial and immune stimulating agent which may be suitable for use in aquaculture. However, its effects on teleost immunity are not well studied and may be significantly different than what has been observed in mammals. In this study we examined the effects of PACAP on the Atlantic salmon macrophage cell line SHK-1. PACAP was able to increase the expression of LPS-induced il-1β in at concentrations of 1 uM when administered 24h prior to LPS stimulation. Furthermore, concentrations as low as 40nM had an effect when administered both 24h prior and in tandem with LPS. PACAP was also capable of increasing the expression of il-1β and tnf-α in SHK-1 cells challenged with a low dose of heat-killed Flavobacterium columnare. We attempted to get a better understanding of the mechanism underlying this enhancement of il-1β expression by manipulating downstream signaling of PACAP with inhibitors of phosphodiesterase and phospholipase C activity. We found that inducing cAMP accumulation with phosphodiesterase inhibitors failed to recapitulate the effect of PACAP administration on LPS-mediated il-1β expression by PACAP, while use of a phospholipase C inhibitor caused a PACAP-like enhancement in LPS-mediated il-1β expression. Interestingly, the VPAC1 receptor inhibitor PG97-269, but not the PAC1 inhibitor max.d.4, also was capable of causing a PACAP-like enhancement in LPS-mediated il-1β expression. This suggests that fish do not utilize the PACAP receptors in the same manner as mammals, but that it still exerts an immunostimulatory effect that make it a good immunostimulant for use in aquaculture.

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish. Columnaris disease can cause heavy economic losses in aquaculture. In this study, whole-genome sequencing was used to characterize this pathogen. F. columnare isolate AH-01 had a circular chromosome and plasmid that encoded a total of 3,022 genes. Isolate GX-01 only had a circular chromosome and encoded 2,965 genes. Genomic islands, prophage regions, and CRISPR/Cas systems were identified in both genomes. Both genomes presented evidence of gene variation and horizontal transfer, both of which are the essential components of genetic diversity, genome plasticity, and functional evolution. Single-gene phylogeny and comparative genome analyses were performed to investigate the variation and evolution of this pathogen. Genetic analysis of 16S rRNA and housekeeping gene sequences significantly clustered 55 F. columnare isolates into four clades. The intragroup identity of the 16S rRNA gene exceeded 99%, while the intergroup identity was below the species delineation threshold. We discovered significant translocation, inversion, and rearrangement events that influenced local synteny within each group. Notably, the observed alignments varied considerably among all the studied groups. The core genomes of all strains with available sequences comprised 747 genes, corresponding to approximately 25% of the genome. Core genome multilocus sequence typing, genome-wide orthology and phylogenetic analyses, and average nucleotide identity suggested that the currently existing F. columnare was an assemblage of several distinct species, with levels of divergence at least equivalent to those between recognized bacterial species. The present investigation provided genomic evidence of gene variation and horizontal transfer,whichwere the basis of genetic diversity, genome plasticity, and functional evolution. The findings supported a proposed new taxonomic perspective on F. columnare. [ABSTRACT FROM AUTHOR]

Flavobacterium columnare, which causes columnaris disease, is responsible for significant mortality in grass carp. Vaccination is a safe and effective measure to combat this disease, and this study aimed to investigate the immune protective effects of different treatments using an inactivated F. columnare vaccine. The vaccine was prepared by inactivating the bacteria with 0.05% formaldehyde at 4°C for 24 hours. The experiments involving grass carp were divided into two parts. In Experiment 1, the immune effects of two isolates, JX-01 (genomovar I) and MU-04 (genomovar II), were compared, along with the impact of white oil adjuvant and the number of immunizations. The results showed that when the white oil adjuvant was used as a booster, the relative percent survival (RPS) of the JW2 group and MW2 group after 8 weeks of the first immunization was 34% and 61%, respectively. In Experiment 2, only the MU-04 (genomovar II) isolate was used as an antigen, with the white oil adjuvant as a booster. The effects of different doses (CFU=108, 107, and 106 bacteria/mL) on immune responses were compared, and the RPS values in the MW6, MW7, and MW8 groups after 4 weeks of the first immunization were found to be 38%, 57%, and 71%, respectively. Furthermore, in the crossantigen protection experiment, the MW2 group exhibited an RPS of 55% against the JX-01 isolate, which was significantly higher than the control group (33%). These findings suggest that an inactivated vaccine comprising an appropriate antigen isolate when administered with a white oil adjuvant as a booster, can provide effective protection in grass carp. [ABSTRACT FROM AUTHOR]

Flavobacterium columnare represent one of the most important bacterial pathogens of cultured sturgeon. However, at present there are no commercially available vaccines to prevent infection and treatment options are limited. β-glucans have been shown to be potent immunostimulants that can provide fish protection against infectious disease. In this study, the effects of dietary β-glucan supplementation on disease susceptibility were examined by exposing 0.3% β-glucan-fed white sturgeon (Acipenser transmontanus) to Flavobacterium columnare in laboratory-controlled challenges. Morbidity and mortality were monitored for 15 days post-challenge (dpc). Additionally, transcript levels for pro-inflammatory cytokines, regulatory cytokines and acute phase proteins (APP) were investigated in the spleen and gills at different time points post-challenge. No evidence of protection was observed in β-glucan-fed fish challenged with the bacteria. Moreover, significantly greater mortalities were observed in β-glucan-fed fish challenged with F. columnare (p

Considering the increasing adoption of RAS technologies in the global aquaculture industry, we sought to characterize in vitro PAA efficacy against three important bacterial fish pathogens in water with representative chemistry profiles of salmonid RAS production. Keywords: Flavobacterium columnare; peracetic acid; RAS disinfection; Weissella ceti; Yersinia ruckeri EN Flavobacterium columnare peracetic acid RAS disinfection Weissella ceti Yersinia ruckeri 5731 5736 6 10/14/22 20221101 NES 221101 Peracetic acid (PAA) has been identified as an efficacious alternative to common disinfectants used in aquaculture settings to reduce fish pathogen loads (Farmer et al., 2013; Liu et al., 2017; Pedersen et al., 2009; Sudová et al., 2010). The majority of important bacterial fish pathogens are Gram-negative, and as such are likely more susceptible to disinfectants such as PAA; however, further research is required to determine differences in susceptibility between additional important Gram-negative (e.g. I Aeromonas salmonicida i , I Edwardsiella tarda i , etc.) and Gram-positive (e.g. I Streptococcus iniae i , I Renibacterium salmoninarum i , etc.) pathogens of farmed fish. Briefly, each selected concentration of PAA was applied to separate flasks of the prepared pathogen-RAS water suspension described above (with sterile phosphate buffered saline administered for the 0 mg/L PAA controls) in triplicate, with each flask gently inverted several times to ensure sufficient mixing. [Extracted from the article]

Flavobacterium columnare, which causes columnaris disease, is one of the costliest pathogens in the freshwater fish-farming industry. The virulence mechanisms of F. columnare are not well understood and current methods to control columnaris outbreaks are inadequate. Iron is an essential nutrient needed for metabolic processes and is often required for bacterial virulence. F. columnare produces siderophores that bind ferric iron for transport into the cell. The genes needed for siderophore production have been identified, but other components involved in F. columnare iron uptake have not been studied in detail. We identified the genes encoding the predicted secreted hemebinding protein HmuY, the outer membrane iron receptors FhuA, FhuE, and FecA, and components of an ATP binding cassette (ABC) transporter predicted to transport ferric iron across the cytoplasmic membrane. Deletion mutants were constructed and examined for growth defects under iron-limited conditions and for virulence against zebrafish and rainbow trout. Mutants with deletions in genes encoding outer membrane receptors, and ABC transporter components exhibited growth defects under iron-limited conditions. Mutants lacking multiple outer membrane receptors, the ABC transporter, or HmuY retained virulence against zebrafish and rainbow trout mirroring that exhibited by the wild type. Some mutants predicted to be deficient in multiple steps of iron uptake exhibited decreased virulence. Survivors of exposure to such mutants were partially protected against later infection by wild-type F. columnare. [ABSTRACT FROM AUTHOR]

Flavobacterium columnare, the causative agent of columnaris disease in a large variety of freshwater fish, is a major problem in commercial aquaculture. A limited number of antimicrobial therapies are available to control this disease; therefore, these agents must be used judiciously. To facilitate effective monitoring for changes in susceptibility, the Clinical Laboratory Standards Institute (CLSI) has a standard broth microdilution test method specific for F. columnare. However, there are no CLSI-approved criteria (termed epidemiological cutoff values [ECVs]) to interpret results. Nevertheless, researchers have developed provisional ECVs based on testing by one laboratory. To satisfy CLSI data requirements, three laboratories used the standard method to generate additional antimicrobial susceptibility data against ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, oxolinic acid, oxytetracycline, sulfadimethoxine/ormetoprim, and sulfamethoxazole–trimethoprim using 109 F. columnare isolates. The new data combined with previously published data from 120 F. columnare isolates were analyzed and ECVs proposed to CLSI. Of the 10 antimicrobials, ECVs were approved for ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, oxolinic acid, and oxytetracycline, which were published in the 2020 edition of the CLSI document VET04 performance standards. These ECVs will help microbiologists categorize decreased antimicrobial susceptibility among F. columnare and will help in surveillance efforts to ensure judicious antimicrobial use. [ABSTRACT FROM AUTHOR]

Flavobacterium columnare, which causes columnaris disease, is one of the costliest pathogens in the freshwater fish-farming industry. The virulence mechanisms of F. columnare are not well understood and current methods to control columnaris outbreaks are inadequate. Iron is an essential nutrient needed for metabolic processes and is often required for bacterial virulence. F. columnare produces siderophores that bind ferric iron for transport into the cell. The genes needed for siderophore production have been identified, but other components involved in F. columnare iron uptake have not been studied in detail. We identified the genes encoding the predicted secreted heme-binding protein HmuY, the outer membrane iron receptors FhuA, FhuE, and FecA, and components of an ATP binding cassette (ABC) transporter predicted to transport ferric iron across the cytoplasmic membrane. Deletion mutants were constructed and examined for growth defects under iron-limited conditions and for virulence against zebrafish and rainbow trout. Mutants with deletions in genes encoding outer membrane receptors, and ABC transporter components exhibited growth defects under iron-limited conditions. Mutants lacking multiple outer membrane receptors, the ABC transporter, or HmuY retained virulence against zebrafish and rainbow trout mirroring that exhibited by the wild type. Some mutants predicted to be deficient in multiple steps of iron uptake exhibited decreased virulence. Survivors of exposure to such mutants were partially protected against later infection by wild-type F. columnare.

In teleost fish, the nucleotide polymorphisms of histone H2A significantly affect the resistance or susceptibility of zebrafish to Edwardsiella piscicida infection. Whether histone H2A variants can enhance the resistance of grass carp to Flavobacterium columnare infection remains unclear. Here, the effects of 7 previously obtained variants (gcH2A-1~gcH2A-7) and 5 novel histone H2A variants (gcH2A-11, gcH2A-13~gcH2A-16) in response to F. columnare infection were investigated. It was found that these histone H2A variants could be divided into type I and II. Among them, 5 histone H2A variants had no any effects on the F. columnare infection, however 7 histone H2A variants had antibacterial activity against F. columnare infection. The gcH2A-4 and gcH2A-11, whose antibacterial activity was the strongest in type I and II histone H2A variants respectively, were picked out for yeast expression. Transcriptome data for the samples from the intestines of grass carp immunized with the engineered Saccharomyces cerevisiae expressing PYD1, gcH2A-4 or gcH2A-11 revealed that 5 and 12 immune-related signaling pathways were significantly enriched by gcH2A-4 or gcH2A-11, respectively. For the engineered S. cerevisiae expressing gcH2A-4, NOD-like receptor and Toll-like receptor signaling pathways were enriched for up-regulated DEGs. Besides NOD-like receptor and Toll-like receptor signaling pathways, the engineered S. cerevisiae expressing gcH2A-11 also activated Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway and C-type lectin receptor signaling pathway. Furthermore, grass carp were immunized with the engineered S. cerevisiae expressing PYD1, gcH2A-4 or gcH2A-11 for 1 month and challenged with F. columnare. These grass carp immunized with gcH2A-4 or gcH2A-11 showed lower mortality and fewer numbers of F. columnare than did the control group. All these results suggest that gcH2A-4 and gcH2A-11 play important roles in evoking the innate immune responses and enhancing disease resistance of grass carp against F. columnare infection. [ABSTRACT FROM AUTHOR]

The major histocompatibility complex (MHC) is an important component of the immune system of vertebrates, which plays a vital role in presenting extrinsic antigens. In this study, we cloned and characterized the mhc ⅡA and mhc ⅡB genes of yellow catfish Pelteobagrus fulvidraco. The open reading frames (ORFs) of mhc ⅡA and mhc ⅡB genes were 708 bp and 747bp in length, encoding 235 and 248 amino acids, respectively. The structure of mhc ⅡA and mhc ⅡB includes a signal peptide, an α1/β1 domain, an α2/β2 domain, a transmembrane region and a cytoplasmic region. Homologous identity analysis revealed that both mhc ⅡA and mhc ⅡB shared high protein sequence similarity with that of Chinese longsnout catfish Leiocassis longirostris. mhc ⅡA and mhc ⅡB showed similar expression patterns in different tissues, with the higher expression level in spleen, head kidney and gill and lower expression in liver, stomach, gall bladder and heart. The mRNA expression level of mhc ⅡA and mhc ⅡB in different embryonic development stages also showed the similar trends. The higher expression was detected from fertilized egg to 32 cell stage, low expression from multicellular period to 3 days post hatching (dph), and then the expression increased to a higher level from 4 dph to 14 dph. The mRNA expression levels of mhc ⅡA and mhc ⅡB were significantly up-regulated not only in the body kidney and spleen, but also in the midgut, hindgut, liver and gill after challenge of Flavobacterium columnare. The results suggest that Mhc Ⅱ plays an important role in the anti-infection process of yellow catfish P. fulvidraco. • The cDNA and amino acid sequences of mhc ⅡA and mhc ⅡB were characterized. • A NJ phylogenetic tree was constructed based on the deduced amino acid sequences of Mhc ⅡA and Mhc ⅡB. • mhc ⅡA and mhc ⅡB were both highly expressed in spleen, head kidney and gill, and lower expression in liver, stomach, etc. • Both of mhc ⅡA and mhc ⅡB showed similar response to F. columnare infection in digested- and immune-related tissues. [ABSTRACT FROM AUTHOR]

Abstract This study reports the comparative analyses of four Flavobacterium columnare isolates that have different virulence and antimicrobial resistance patterns. The main research goal was to reveal new insights into possible virulence genes by comparing the genomes of bacterial isolates that could induce tissue damage and mortality versus the genome of a non-virulent isolate. The results indicated that only the genomes of the virulent isolates possessed unique genes encoding amongst others a methyl-accepting chemotaxis protein possibly involved in the initial colonization of tissue, and several VgrG proteins engaged in interbacterial competition. Furthermore, comparisons of genes unique for the genomes of the highly virulent (HV) carp and trout isolates versus the, respectively, low and non-virulent carp and trout isolates were performed. An important part of the identified unique virulence genes of the HV-trout isolate was located in one particular gene region identified as a genomic island. This region contained araC and nodT genes, both linked to pathogenic and multidrug-resistance, and a luxR-gene, functional in bacterial cell-to-cell communication. Furthermore, the genome of the HV-trout isolate possessed unique sugar-transferases possibly important in bacterial adhesion. The second research goal was to obtain insights into the genetic basis of acquired antimicrobial resistance. Several point-mutations were discovered in gyrase-genes of an isolate showing phenotypic resistance towards first and second-generation quinolones, which were absent in isolates susceptible to quinolones. Tetracycline-resistance gene tetA was found in an isolate displaying acquired phenotypic resistance towards oxytetracycline. Although not localized on a prophage, several flanking genes were indicative of the gene’s mobile character.

The article presents the results of studies on the treatment scheme efficacy for columnaris in Pterophyllum scalare, common under private aquarium husbandry conditions. To establish the diagnosis, the clinical features of the diseased fish, pathological and anatomical changes and the results of microscopic and microbiological studies were taken into account. Separate chemical and microbiological parameters of aquarium water were also studied. It was established that fish disease developed against the background of adverse changes in the chemical composition and microbiocenosis of aquarium water. High alkalinity and excess of phosphates compared to the norm provoked accumulation of opportunistic microbiota, resulting in a balance disorder in the parasite-host system and development of clinical manifestation of the fish disease. During the disease outbreak, bacteriological indices of water indicated a high level of organic contamination and a low intensity of water self-purification processes. Clinically, the disease was manifested in P. scalare by decrease in appetite and motor activity, onset of ulcerative lesions of various shapes and sizes on the surface of the body and on the gill covers. Selected pure cultures of Flavobacterium columnare showed sensitivity to enrofloxacin (growth retardation zone 31.3 ± 1.0 mm); moderate resistance was found to tylosin. The microorganisms were resistant to amoxicillin, doxycycline, benzylpenicillin and tetracycline. Microscopic studies of intestinal specimens of dead P. scalare revealed numerous motile flagellates. It has been shown that an effective treatment regimen that provides recovery for 70% of diseased P. scalare is the use of enroxil 10% solution for five days, metronidazole three times a day, and “API MelaFix” for seven days. It is proved that the following measures are effective to restore the disrupted hydro-balance: periodic water replacement in the amount of 20% of the total volume, providing the aquarium with active aeration systems, planting slow-growing plants and reducing the amount of fish food provided. The measures developed were efficient, they led to elimination of the outbreak of columnaris in the P. scalare and to restoration of biological equilibrium in a closed aquatic ecosystem.

The polymeric immunoglobulin receptor (pIgR) plays an important role in mediating the transcytosis of polymeric immunoglobulins (pIgs) to protect organisms against pathogen invasion. Here, a polyclonal antibody against grass carp (Ctenopharyngodon idellus) recombinant pIgR was developed by immunizing New Zealand white rabbit, and the responses of pIgR, IgM and IgZ were analyzed after bath immunization and intraperitoneal administration with Flavobacterium columnare. The results showed that pIgR transcription level was similar to IgM and IgZ, but pIgR rose much faster and peaked earlier than IgM and IgZ ; the pIgR mRNA levels were higher in the skin and spleen for both immunized groups, while IgM and IgZ mRNA expression were higher in skin, gills, and intestines in bath immersion group, or spleen and head kidney in intraperitoneal immunization group. ELISA revealed that the IgM, IgZ and pIgR protein levels were up-regulated in skin mucus, gill mucus, gut mucus and bile, reaching a higher peak level earlier in skin mucus and gill mucus in bath immersion group, but a higher peak level in bile in injection group. Moreover, secretory component molecules were detected in grass carp's skin, gill and intestine mucus and bile, but not in serum, which molecular mass was near the theoretical mass obtained from the sequence of grass carp pIgR. These results demonstrated that bath and intraperitoneal immunization up-regulated pIgR and secretory Ig expression in secretions, which provided more insights into the role of pIgR in immunity and offer insight into ways of protecting teleost against pathogen invasion. • The polyclonal antibody specifically reacting with rpIgR was developed. • The pIgR transcription level rose much faster and peaked earlier than IgM and IgZ. • The IgM, IgZ and pIgR protein levels were up-regulated in skin, gill and gut mucus and bile. • SC molecules were detected in mucus and bile but not in serum. [ABSTRACT FROM AUTHOR]

Increasing problems with antibiotic resistance have directed interest toward phage therapy in the aquaculture industry. However, phage resistance evolving in target bacteria is considered a challenge. To investigate how phage resistance influences the fish pathogen Flavobacterium columnare, two wild-type bacterial isolates, FCO-F2 and FCO-F9, were exposed to phages (FCO-F2 to FCOV-F2, FCOV-F5, and FCOV-F25, and FCO-F9 to FCL-2, FCOV-F13, and FCOV-F45), and resulting phenotypic and genetic changes in bacteria were analyzed. Bacterial viability first decreased in the exposure cultures but started to increase after 1 to 2 days, along with a change in colony morphology from original rhizoid to rough, leading to 98% prevalence of the rough morphotype. Twenty-four isolates (including four isolates from no-phage treatments) were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion, and biofilm formation, protease activity, whole-genome sequencing, and virulence in rainbow trout fry. The rough isolates arising in phage exposure were phage resistant with low virulence, whereas rhizoid isolates maintained phage susceptibility and high virulence. Gliding motility and protease activity were also related to the phage susceptibility. Observed mutations in phage-resistant isolates were mostly located in genes encoding the type IX secretion system, a component of the Bacteroidetes gliding motility machinery. However, not all phage-resistant isolates had mutations, indicating that phage resistance in F. columnare is a multifactorial process, including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections since phage resistance is associated with decreases in bacterial virulence. [ABSTRACT FROM AUTHOR]

The bacterial pathogen, Flavobacterium columnare causes columnaris disease in Labeo rohita globally. Major effects of this bacterial infection include skin rashes and gill necrosis. Nimbolide, the key ingredient of the leaf extract of Azadirachta indica possesses anti-bacterial properties effective against many microorganisms. Nano-informatics plays a promising role in drug development and its delivery against infections caused by multi-drug-resistant bacteria. Currently, studies in the disciplines of dentistry, food safety, bacteriology, mycology, virology, and parasitology are being conducted to learn more about the wide anti-virulence activity of nimbolide. The toxicity of nimbolide was predicted to determine its dosage for treating bacterial infection in Labeo rohita. Further, comparative 3-D structure prediction and docking studies are done for nimbolide conjugated nanoparticles with several key target receptors to determine better natural ligands against columnaris disease. The nanoparticle conjugates are being designed using in-silico approaches to study molecular docking interactions with the target receptor. Bromine conjugated nimbolide shows the best molecular interaction with the target receptors of selected species ie L rohita. Nimbolide comes under the class III level of toxic compound so, attempts are made to reduce the dosage of the compound without compromising its efficiency. Further, bromine is also used as a common surfactant and can eliminate heavy metals from wastewater. The dosage of bromine-conjugated nimbolide can be reduced to a non-toxic level and thus the efficiency of the Nimbolide can be increased. Moreover, it can be used to synthesize nanoparticle composites which have potent antibacterial activity towards both gram-positive and gram-negative bacteria. This material also forms a good coating on the surface and kills both airborne and waterborne bacteria. • Columnaris is a common infection occurring in the most consumed fish in India, that is, Labeo rohita. • Both nimbolide and bromine have been exploited for their anti-virulence properties in the past. • The synergistic effect of nimbolide and bromine can help fight Flavobacterium columnare infection better in Labeo rohita. • Bromine-conjugated nimbolide can help overcome the fast-growing antibiotic resistance in microbial species. [ABSTRACT FROM AUTHOR]

Abstract Background Flavobacterium columnare is the causative agent of columnaris disease that affects cultured freshwater fishes worldwide. F. columnare easily colonizes surfaces by forming biofilm, which helps the pathogen resist antibiotic and disinfectant treatments. Previously, we had shown that increasing concentrations of calcium (Ca2+) promoted biofilm formation by F. columnare. The objective of this study was to further characterize the role of Ca2+ on biofilm formation and to compare the transcriptome profiles of planktonic and biofilm cells. Results RNA-Seq analysis was conducted to identify genes that were differentially expressed between the following states: i) planktonic cells in control medium (P), ii) planktonic cells in calcium-enriched medium (P/Ca), and iii) biofilm cells in calcium-enriched medium (B/Ca). Overall, we identified 441 significant (FDR-adjusted p 2) differentially expressed genes (DEGs) between P and B/Ca samples; 112 significant DEGs between P/Ca and B/Ca samples, and 175 significant DEGs between P/Ca and P samples, corresponding to 15.87, 4.03 and 6.30% of the total protein-coding sequences, respectively. The significant DEGs fell into different functional categories including iron acquisition, oxidative stress response, extracellular protein secretion, and respiratory metabolism. Conclusions Our results posit Ca2+ as a critical signal in regulating bacterial surface adhesion and biofilm formation in F. columnare. Living in biofilm elicited a shift in several metabolic pathways that allowed the cells to cope with oxidative stress and nutrient starvation. In addition, Ca2+ supplementation induced the expression of putative virulence factors in F. columnare, such as extracellular protein secretion and iron acquisition.

Flavobacterium columnare (Fc) is the causative agent for columnaris disease (CD) in several fish species and an emerging problem for rainbow trout aquaculture. We characterize the virulence phenotype of two Fc isolates, CSF‐298–10 and MS‐FC‐4, against trout from two sources, NCCCWA and a production stock (PS), at the eyed egg and alevin life stages. Immersion challenges demonstrated that NCCCWA eyed eggs were susceptible to the Fc isolate MS‐FC‐4 (>97% mortality) but no mortality was observed against PS eyed eggs. The CSF‐298–10 had little effect on any eyed eggs tested and was not highly virulent to any alevin till day six post‐hatch, up to 38% for NCCCWA and ~80% PS alevin. The MS‐FC‐4 strain produced ≥80% mortality any day an immersion challenge occurred post‐hatch. Significant difference in CFU counts was recorded between the Fc strains on 2 days post‐hatch immersion challenges. Counts for the NCCCWA alevin were 4.4 × 103 CFU/ml‐1 and 1.8 × 106 CFU/ml‐1 for the CSF‐298–10 strain and MS‐FC‐4 strain, respectively, and for the PS alevin CSF‐298–10 measured 9.9 × 101 CFU/ml‐1 and 3.8 × 105 CFU/ml‐1 for MS‐FC‐4. These two Fc isolates present stark differences in virulence phenotypes to both eyed eggs and alevin and present an interesting model system for virulence kinetics and potentially alternative pathogenic pathways. [ABSTRACT FROM AUTHOR]

Aims: Comparative genomics analyses indicated that the Flavobacterium columnare genome has unique denitrification genes relative to Flavobacterium psychrophilum and Flavobacterium johnsoniae, including nasA (nitrate reductase), nirS (nitrite reductase), norB (nitric oxide reductase) and nosZ (nitrous oxide reductase). The current study determines the roles of nasA, nirS, norB and nosZ in anaerobic growth, nitrate reduction, biofilm formation and virulence. Methods and Results: Four in‐frame deletion mutants in virulent F. columnare strain 94‐081 were constructed by allelic exchange using pCP29 plasmid. Compared with parent strain 94‐081, FcΔnasA,FcΔnirS and FcΔnosZ mutants did not grow as well anaerobically, whereas the growth of FcΔnorB strain was similar to the parent strain (FcWT). Exogenous nitrate was not significantly consumed under anaerobic conditions in FcΔnasA, FcΔnirS and FcΔnosZ compared to parent strain 94‐081. Under anaerobic conditions, Fc∆nasA, Fc∆norB and Fc∆nosZ formed significantly less biofilm than the wild type strain at 24 and 96 h, but FcΔnirS was not significantly affected. The nitrite reductase mutant FcΔnirS was highly attenuated in catfish, whereas FcΔnasA, FcΔnorB and FcΔnosZ had similar virulence to FcWT. Conclusions: These results show, for the first time, that denitrification genes enable F. columnare to grow anaerobically using nitrate as an electron acceptor, and nitrite reductase contributes to F. columnare virulence. Significance and Impact of the Study: These findings indicate potential for F. columnare to grow in nitrate‐rich anaerobic zones in catfish production ponds, and they suggest that a Fc∆nirS strain could be useful as a safe live vaccine if it protects catfish against columnaris disease. [ABSTRACT FROM AUTHOR]

This study reports the comparative analyses of four Flavobacterium columnare isolates that have different virulence and antimicrobial resistance patterns. The main research goal was to reveal new insights into possible virulence genes by comparing the genomes of bacterial isolates that could induce tissue damage and mortality versus the genome of a non-virulent isolate. The results indicated that only the genomes of the virulent isolates possessed unique genes encoding amongst others a methyl-accepting chemotaxis protein possibly involved in the initial colonization of tissue, and several VgrG proteins engaged in interbacterial competition. Furthermore, comparisons of genes unique for the genomes of the highly virulent (HV) carp and trout isolates versus the, respectively, low and non-virulent carp and trout isolates were performed. An important part of the identified unique virulence genes of the HV-trout isolate was located in one particular gene region identified as a genomic island. This region contained araC and nodT genes, both linked to pathogenic and multidrug-resistance, and a luxR-gene, functional in bacterial cell-to-cell communication. Furthermore, the genome of the HV-trout isolate possessed unique sugar-transferases possibly important in bacterial adhesion. The second research goal was to obtain insights into the genetic basis of acquired antimicrobial resistance. Several point-mutations were discovered in gyrase-genes of an isolate showing phenotypic resistance towards first and second-generation quinolones, which were absent in isolates susceptible to quinolones. Tetracycline-resistance gene tetA was found in an isolate displaying acquired phenotypic resistance towards oxytetracycline. Although not localized on a prophage, several flanking genes were indicative of the gene's mobile character. [ABSTRACT FROM AUTHOR]

TonB-dependent siderophore receptors play a critical transport role for Flavobacterium columnare virulence formation and growth, and have become valuable targets for the development of novel antimicrobial agents. Traditional Chinese medicine has demonstrated notable efficacy in the treatment of fish diseases and includes potential antibacterial agents. Herein, we performed molecular docking-based virtual screening to discover novel TonB-dependent siderophore receptor inhibitors from traditional Chinese medicine and provide information for developing novel antibacterial agents. Firstly, we efficiently obtained 11 potential inhibitors with desirable drug-like characteristics from thousands of compounds in the TCM library based on virtual screening and property prediction. The antibacterial activity of Enoxolone, along with its interaction characteristics, were determined via an MIC assay and molecular dynamic simulation. Transcriptional profiling, along with validation experiments, subsequently revealed that an insufficient uptake of iron ions by bacteria upon binding to the TonB-dependent siderophore receptors is the antibacterial mechanism of Enoxolone. Finally, Enoxolone's acceptable toxicity was illustrated through immersion experiments. In summary, we have used virtual screening techniques for the first time in the development of antimicrobial agents in aquaculture. Through this process, we have identified Enoxolone as a promising compound targeting the TonB-dependent siderophore receptor of F. columnare . In addition, our findings will provide new ideas for the advancement of innovative antimicrobial medications in aquaculture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Chen, Xu, Li, Geng, Chen, Ouyang, Huang and Deng.)

Flavobacterium columnare causes columnaris disease in fish. Columnaris disease is incompletely understood, and adequate control measures are lacking. The type IX secretion system (T9SS) is required for F. columnare gliding motility and virulence. The T9SS and gliding motility machineries share some, but not all, components. GldN (required for gliding and for secretion) and PorV (involved in secretion but not required for gliding) are both needed for virulence, implicating T9SS-mediated secretion in virulence. The role of motility in virulence is uncertain. We constructed and analyzed sprB , sprF , and gldJ mutants that were defective for motility but that maintained T9SS function to understand the role of motility in virulence. Wild-type cells moved rapidly and formed spreading colonies. In contrast, sprB and sprF deletion mutants were partially defective in gliding and formed nonspreading colonies. Both mutants exhibited reduced virulence in rainbow trout fry. A gldJ deletion mutant was nonmotile, secretion deficient, and avirulent in rainbow trout fry. To separate the roles of GldJ in secretion and in motility, we generated gldJ truncation mutants that produce nearly full-length GldJ. Mutant gldJ 563 , which produces GldJ truncated at amino acid 563, was defective for gliding but was competent for secretion as measured by extracellular proteolytic activity. This mutant displayed reduced virulence in rainbow trout fry, suggesting that motility contributes to virulence. Fish that survived exposure to the sprB deletion mutant or the gldJ 563 mutant exhibited partial resistance to later challenge with wild-type cells. The results aid our understanding of columnaris disease and may suggest control strategies.IMPORTANCE Flavobacterium columnare causes columnaris disease in many species of freshwater fish in the wild and in aquaculture systems. Fish mortalities resulting from columnaris disease are a major problem for aquaculture. F. columnare virulence is incompletely understood, and control measures are inadequate. Gliding motility and protein secretion have been suggested to contribute to columnaris disease, but evidence directly linking motility to disease was lacking. We isolated and analyzed mutants that were competent for secretion but defective for motility. Some of these mutants exhibited decreased virulence. Fish that had been exposed to these mutants were partially protected from later exposure to the wild type. The results contribute to our understanding of columnaris disease and may aid development of control strategies., Competing Interests: The authors declare no conflict of interest.

Frass, a by-product of the larval meal industry, is heterogeneous and includes larval excrement, exoskeleton sheds and residual feed ingredients along with abundant nutrients, chitin and beneficial microbes. The present study was performed to evaluate the changes in growth, feed utilization, body composition, hematology, serum chemistry, immune responses and disease resistance of hybrid tilapia, Nile x Mozambique (Oreocromis niloticus x O. mozambique) fed diets containing frass from black soldier fly larvae, Hermetia illucens. Five diets containing frass at levels of 0, 5, 10, 20, and 30% as partial replacements of a combination of soybean meal, wheat short and corn meal on an equal protein basis were fed to juvenile hybrid tilapia (2.6 ± 0.035 g) in quadruplicate aquaria to apparent satiation twice a day for the first two weeks and once daily for rest of the feeding trail. Final weight gain was significantly increased in fish fed the diet including the highest level of frass (30%). Fish fed diets containing frass (5% to 30%) had significantly higher protein efficiency than the group fed diet without frass (control diet). Feed intake and feed utilization efficiency were not significantly affected by dietary treatments. Survival during the feeding trail, whole-body composition, hematological parameters, and serum biochemistry were not affected by dietary treatment. Serum complement activity of fish fed 30% dietary frass was significantly higher than that of fish fed other treatments. Fish fed the diets containing frass showed significant dose-dependent trends in survival against both Flavobacterium columnare and Streptococcus iniae challenges. Frass from the larvae of black solder flies fed Distillers’ dried grains with solubles has potential for use as feed ingredient for improving growth of hybrid tilapia. Use of frass in tilapia diets may prove beneficial by improving innate immune components and the resistance of hybrid tilapia against bacterial infection.

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

Southeast (SE) Asia plays an important role in global food security as this region has been regarded as one of the major producers of aquaculture product and, to date, freshwater fish accounted for one-third of the total aquaculture in SE Asia. The intensification of freshwater farming corresponding to increase of consumer demands has inevitably led to the emergence and re-emergence of diseases causing tremendous economic loss in the region. Nile tilapia (Oreochromis niloticus) and striped catfish (Pangasianodon hypophthalmus), the major freshwater fish species of SE Asia, have been reported susceptible to several bacterial pathogens, e.g. Streptococcus agalactiae , Edwardsiella ictalurid and Flavobacterium columnare. Since only a limited number of vaccines being registered and marketed, these pathogenic organisms still represent a severe threat to aquaculture industry in SE Asia. However, there is profound advancement in the understanding of disease epidemiology, pathogenic mechanisms, teleost mucosal immunity and vaccine delivery system over the last few years. This review aimed to summarize those recent findings which hopefully can provide novel insight into the future development of suitable vaccine and vaccination regime against bacterial infection in SE Asia region. • S. agalactiae , E. ictaluri and F. columnare caused major losses in SE Asia. • Registered vaccines are available only in Vietnam and Indonesia. • Whereas, the experimental vaccines were constantly developed. • Up-to-date epidemiological data of these pathogens in SE Asia is demonstrated. • Directional guidance for future development of regional vaccines is described. [ABSTRACT FROM AUTHOR]

Columnaris disease, caused by Flavobacterium columnare, is a common bacterial disease, affecting cultured and wild freshwater fish species around the globe. This bacterium has different virulence factors and prolyl oligopeptidase (POP) is among one of them. Currently, there are no effective vaccine for controlling the F. columnare infection. Thus, the production of appropriate vaccines against this pathogen is an important goal to avoid columnaris. Considering the role of POP antigens in virulence and satisfactory immunogenicity, they can be used as promising vaccine candidates against F. columnare. In the present study, in-silico approach was used to analyze various aspects of the POP protein, including physico-chemical characteristics, the potential epitopes of B-cell, the secondary and tertiary structure, the subcellular localization and the transmembrane domain using several bioinformatics tools to design a proper vaccine against F. columnare. The results revealed that POP protein contained 649 amino acid. The secondary structure of the POP protein comprises 20.34% alphahelix (Hh), 44.53% random coil (Cc) and 25.89% extended strand. Moreover, several potential B-cell epitopes were identified for POP.Based on the results of Ramachandran plot, 83.5% of the amino acid residues were located in the favoured, 0.6% in allowed regions. The QMEAN score obtained for our structure was -3.66, which is less than -4.0, which indicate the model has an acceptable quality. Our findings suggested that structural and functional predictions applied to POP protein using in-silico tools can reduce the failure risk of the laboratory research studies. Further studies are needed on the development of effective vaccines in vivo using POP alone or in combination with other antigenic peptide in the future. [ABSTRACT FROM AUTHOR]

Mucins are large glycoproteins that cover epithelial surfaces of the body and play important roles in prevention of inflammatory and various infectious diseases. In this study, five membrane‐bound and seven secreted mucin genes in the channel catfish were identified. All these identified mucin genes possess at least one PTS, von Willebrand D (VWD) or SEA domains. The expression of the 12 mucin genes in channel catfish was first studied with infection of Edwardsiella ictaluri and Flavobacterium columnare. Expression difference in MUC13a, MUC13, MUC2 and MUC5b was found in the intestine after E. ictaluri infection. Eight mucin gene expressions (except MUC3a, MUC2, MUC4 and MUC5f) were up‐regulated at 4 hr and down‐regulated after 24 hr in the gill with F. columnare infection. Expression level of MUC2 gene was up‐regulated in the intestine with E. ictaluri infection without no significant change in the gill under the F. columnare infection, which indicate that MUC2 is tissue‐specific gene expression and has different immune respond to two bacterial challenge. Taken together, the study showed mucin from the gill by F. columnare challenge induced an obvious response than mucin from the intestine with E. ictaluri infection. [ABSTRACT FROM AUTHOR]

Flavobacterium columnare is the causative agent of columnaris disease in a variety of fish hosts. Using modifications to previously established protocols, a quantitative PCR (qPCR) assay was validated for the detection of 2 predominant F. columnare genomovars. The oligonucleotide primer and probe combination was designed to amplify a 203-bp region of the chondroitin AC lyase gene (GenBank AY912281) of F. columnare. There were no significant differences in amplification between genomovars. Comparable quantities of genomic DNA from 10 F. columnare strains, 5 representatives of each genomovar, produced similar results. Serial dilutions of purified PCR product demonstrated the limit of sensitivity for the assay was ~ 10 copies per reaction. The presence of gill and spleen tissue did not significantly affect the sensitivity of the assay. Comparably, bacterial DNA detected from the liver and kidney was less sensitive than pure bacterial DNA. However, detection from these tissues was within one order of magnitude of other tissues, indicating this reduction may have minimal analytic significance. This validated assay was used to approximate the minimum infectious dose for F. columnare isolate 94-081 in channel catfish and assess bacterial loads in gill and kidney tissues 48 h post-infection. [ABSTRACT FROM AUTHOR]

Columnaris disease, induced by Flavobacterium columnare , seriously affects the health of freshwater fish species and damages the mucosal tissues, such as the fins, skin, and gills. Teleosts represent the first bony vertebrate to contain both innate and adaptive immune responses against pathogens. So far, three immunoglobulin isotypes (IgM, IgD, and IgT/IgZ) have been identified in teleost fish, and IgT in mucosal tissues of teleost fish was reported to perform a similar function to IgA in mammals during parasitic infection. However, very limited information is known about the function of IgT in gill mucosal tissues during bacterial infection. In the present study, rainbow trout (Oncorhynchus mykiss) was infected with F. columnare (Fc) via immersion. After Fc infection, the gill structure of rainbow trout showed serious hyperplasia symptoms on the secondary lamellae at 12 h post infection (hpi). Moreover, the mRNA expression levels of NOS2 and cathelicidin-1 were significantly upregulated immediately at 12 hpi and showed high expression throughout the experiment. IgT and IgM showed much higher mRNA expression levels at 28 days post infection (dpi) and 75 dpi, while IgD only showed high mRNA expression levels at 28 dpi. Importantly, the accumulation of IgT+ B cells and strong bacteria-specific IgT responses were detected in the gill lamellae of both infected fish (28 dpi) and survivor fish (75 dpi). Overall, our results suggest that IgT and IgT+ B cells play a central role in the adaptive immune responses of fish gill mucosa against bacterial infection. • Immune functions of IgT, IgM and IgD in gill of trout were illustrated after challenged with bacterial pathogens. • Strong immune responses elicited in trout gill after Flavobacterium columnare infection. • IgT and IgM played an important role against bacteria in fish gill mucosal and systemic immunity, respectively. [ABSTRACT FROM AUTHOR]

Hepcidin links iron metabolism with innate immunity during the inhibition of bacterial infection. Our previous studies had shown that recombinant hepcidin can significantly reduce the mortality rate of Ctenopharyngodon idella infected with Flavobacterium columnare under laboratory conditions. Here, we studied the preventive and therapeutic effects of feed supplemented with different doses of recombinant hepcidin on F. columnare -challenged C. idella reared in a cage culture environment. The results showed that in the prevention groups, 30 and 90 mg/kg of added purified and unpurified hepcidin respectively resulted in a higher survival rate in the early post-infection period, while 60 mg/kg of purified hepcidin significantly improved the survival rate in the therapy group (all compared to the control group). In the hepatopancreas, the expression of hepcidin and ferritin was significantly up-regulated, and the levels of ferroportin and serum iron were significantly decreased, especially in the therapy group. In addition, the expression of iron-related genes in spleen and intestine exhibited a similar trend to that in hepatopancreas. Meanwhile, immune genes were up-regulated to varying degrees, and the therapy group exhibited a significantly improved expression of pro-inflammatory cytokines and specific immunity. In summary, our study shows that different doses of recombinant hepcidin had protective effects against bacterial infection by regulating the iron distribution and immune gene expression, which provides a strong foundation for the application of recombinant hepcidin in aquaculture. • Dietary recombinant CiHep has protective effect on C. idella challenged with F. columnare under cage aquaculture conditions. • Dietary recombinant CiHep increased combined iron levels in hepatopancreas and spleen, while decreasing the serum iron. • The addition of 60 mg/kg Hepcidin to diet could improve the immune indexes of C. idella after infection with F. columnare. [ABSTRACT FROM AUTHOR]

The susceptibility of fish from different culture environments to bacterial infection is not well known. The susceptibility and pathological changes of conventional (CV) and specific pathogen-free (SPF) rare minnow (Gobiocypris rarus) infected with two gram-negative bacteria, Flavobacterium columnare and Pseudomonas fluorescens are investigated. Rare minnows were intraperitoneally challenged with two bacterial species to first determine semi-lethal doses (LD 50), and then with the LD 50 dose, determine innate immune response. Infected rare minnows developed characteristic red bellies and then died. LD 50 doses of F. columnare and P. fluorescens were 4.586 × 108 cfu/mL and 2.319 × 1010 cfu/mL for CV rare minnow, and 2.575 × 108 cfu/mL and 1.935 × 1010 cfu/mL, respectively, for SPF rare minnow. The results of RT-PCR showed that the highest levels of toll-like receptor 3 (TLR3), interleukin-6 (IL-6), interferon-2 (IFN-2) and rare minnow Z-DNA binding protein kinase (GrPKZ) mRNA were noticed at 6–48 h post-infection (hpi). In addition, TLR3, IL-6 and IFN-2 in F. columnare challenged rare minnow were more highly expressed than those in P. fluorescens challenged rare minnow, whereas as opposed in the expression of GrPKZ mRNA. Stimulation of innate immune responses is closely related to bacterial virulence. SPF rare minnow might be more susceptible to these bacteria than CV rare minnow, possibly due to their clean environment and lack of resistance. We speculate that clean environment renders rare minnow more susceptible to bacterial infections. • First time to study the susceptibility and immune response of specific pathogen-free rare minnow to gram-negative bacteria. • Comparing the sensitivity of conventional and specific pathogen-free rare minnows to gram-negative bacteria. • Studying the innate immune response of conventional and specific pathogen-free rare minnows. [ABSTRACT FROM AUTHOR]

The present study was conducted to evaluate the influence of Glycyrrhiza uralensis (G. uralensis) extracts on the growth performance, histological structure, immune response and disease resistance against Flavobacterium columnare (F. columnare) of yellow catfish. Fish were fed with two different diets, i.e., basal diet as control group (CG) and diet containing G. uralensis extracts as experimental group (GG). After 60 days feeding, growth performance of GG fish was significantly improved, with increased WG and SGR but decreased FCR compared to CG fish. Fish were then challenged with F. columnare for two times, as fish showed rare mortality after the first infection. GG fish showed significantly lower cumulative mortality during F. cloumnare infection than CG fish after 21 days infection (dpi). Epithelial cell exfoliation and obvious cellular vacuolization in the skin and congestion of gill lamellae were detected in CG fish, while GG fish showed increased width of epidermis and mucous cells number in skin, and increased length of secondary lamina in gill. GG fish also exhibited higher enzyme activity of lysozyme in serum and mRNA expression of lysozyme in head kidney than CG fish at most time points post infection. G. uralensis extracts supplementation also induced earlier serum anti-oxidative responses, with increased superoxide dismutase activity and total antioxidant capacity in GG fish at 1 dpi. Compared to CG fish, GG fish showed increased expression level of genes involved in TLRs-NFκB signaling (TLR2, TLR3, TLR5, TLR9, Myd88, and p65NFκB), resulting in higher expression levels of pro-inflammatory cytokines (IL-1β and IL-8) in the head kidney post infection. However, these genes showed deviation in the gill of GG fish, which increased at some time points but decreased at other time points. Moreover, G. uralensis extracts supplementation also significantly unregulated the expression levels of IgM and IgD in head kidney, and the expression levels of IgM in the gill of yellow catfish, suggesting the elevated humoral immune response during F. columnare infection. All these results contributed to the elevated disease resistance ability against F. cloumnare infection of yellow catfish after dietary G. uralensis extracts supplementation. • G. uralensis extracts elevated growth performance and disease resistance in yellow catfish. • G. uralensis extracts protected fish mucosal integrity against F. columnare infection.. • G. uralensis extracts stimulated fish immune response against F. columnare infection. [ABSTRACT FROM AUTHOR]

Introduction: Kjellbergiodendron celebicum (Koord.) Merr. (local name: tombe uwa) is a plant endemic to Sulawesi, Indonesia, and grows around lakes or aquatic environments where fish live. Based on phytochemical screening in previous studies, i.e. methanol extract and ethyl acetate fraction from the leaves of Kjellbergiodendron celebicum (Koord.) Merr., the methanol extract gives positive results containing polyphenol compounds in the flavonoid group which have been known to have strong antioxidant and antibacterial properties. Objective: To test the effectiveness of the comparison of the natural content in the compounds (antibacterial and antioxidant properties) and the total content of phenol in Kjellbergiodendron celebicum (Koord.) Merr., which was extracted using two methods, i.e. maceration and Ultrasonic- Assisted Extraction (UAE), in fish-disease bacteria. Method: The leaves were separated to be extracted with two different methods: maceration and Ultrasound-Assisted Extraction (UAE). Extracts were first screened qualitatively for antioxidant activity and then quantified with respect to in vitro antioxidant activity using the 2.2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and the ferric-reducing antioxidant power (FRAP) assay. Antibacterial activity was determined by the paper disc diffusion method and microdilution. Results: 70% Ethanol in leaves extract of Kjellbergiodendron celebicum (Koord.) Merr. The extract which has the highest activity based on the DPPH test and FRAP test is the extract from UAE extraction with IC50 value of 9.81512 μg/mL and ferrous equivalent antioxidant capacity (FeEAC) value of 1.661.3 μmol/gr. UAE method also has a higher potential in antibacterial activity based on the diffusion method of paper discs and microdilution with the MIC obtained as much as 390.6 μg/mL. Conclusion: the UAE extraction method is better at scanning polyphenol compounds compared to the conventional maceration extraction method. Therefore, the results of the antioxidant and antibacterial activity using the UAE method are better than the maceration method. [ABSTRACT FROM AUTHOR]

IFN-γ is an immunomodulatory factor that has been extensively studied in phenotypes of mammalian macrophages and multifarious inflammatory responses. Usually these studies relied on the classical synergistic activation of IFN-γ with LPS (LipoPolySaccharides). However, non-mammalian vertebrates, and in particular fish, are not very susceptible to LPS, and easily acquire tolerance upon repeated exposure. Therefore, for studies in fish, it is necessary to replace the classical IFN-γ +LPS immune system activation method, and find other pathogen-associated molecular patterns (PAMPs) capable of stimulating the fish immune system. Here we used an important farmed fish species, Ctenopharyngodon idella , to study the effects of CiIFN- γ2 (C. idella IFN-γ2) and chitosan (CS) on its immune responses in vivo and vitro. Our results showed that the combination of CS and CiIFN-γ2 significantly enhanced the activation of macrophages, with an activation intensity even stronger than in CiIFN-γ2 and CiIFN-γ2 +LPS groups. In vivo , injection of CiIFN-γ2 could improve the survival rate of C. idella infected with Flavobacterium columnare , while a combined injection of CiIFN-γ2 +CS only improved protection in the early stages after the challenge. Notably, both injections reduced the bacterial load of viscera and improved the levels of several plasma parameters (TP, T-SOD, LA, and NO). However, a dramatic up-regulation of inflammatory factors, severe inflammatory damage in the intestines and hepatopancreas, and increased mortality in late stages of infection were observed in the CiIFN-γ2 +CS group. Our findings provide new insights into the macrophage activation phenotypes and inflammatory responses in fish. They also demonstrate that CiIFN-γ2 could be used as a potential immunopotentiator, but not in combination with CS. This suggests that selection of immunological adjuvants should be carefully tested experimentally. • Chitosan + IFN-γ2 could enhanced the activation of macrophages, with an activation intensity stronger than with IFN-γ2 + LPS. • In vivo , chitosan + IFN-γ2 caused serious inflammatory damage and increase the mortality of fish after infection. • IFN-γ2 played a critical role in improving the ability of C. idella to resist F. columnare infection in vivo. [ABSTRACT FROM AUTHOR]

Stimulatory effects of dietary supplementation of large brown alga, Laminaria digitata polysaccharides (LDPs) at doses of 0, 100, 200, and 300 mg/kg diets were assessed on growth, hemato-biochemical, and antioxidant-immune variables as well as related genes expression in Indian rohu carp (Labeo rohita) during Flavobacterium columnare infection. Fish growth indices were significantly (P < 0.05) influenced by dietary LDPs and bacterial infection but not their interaction. However, growth indices of rohu carp in normal unchallenged (NuC) increased pointedly at treatments of 200 or 300 mg/kg diets with no significant difference between them. In normal challenged bacterial (NbC) groups, highest fish growth was observed at the treatment of 300 mg LDPs/kg diets. Feed intake and FCR values were significantly (P < 0.05) affected by dietary LDPs, bacterial infection, and their interaction. The highest feed intake accompanied with lowest FCR value was observed in the treatment of 200 mg LDPs/kg diets. No mortality was seen in NuC groups but lowest fish survival was observed in NbC groups fed with 0.0 and 100 mg LDPs/kg diets. Highest values of white blood cell, total protein, albumin, and globulin found in NuC fish groups administrated with 200 or 300 mg/kg diets with no differences between them. Feeding NbC fish groups on 300 mg LDPs/kg diet recovered the adverse effects of bacterial infection. It is noted that levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and reduced glutathione (GSH) were significantly (P < 0.05) affected by dietary LDPs, bacterial infection, and their interaction. The values of antioxidant indices were significantly (P < 0.05) higher in NuC fish groups received 200 or 300 mg LDPs/kg diets with no significant (P > 0.05) differences between them. In NbC fish groups, values of antioxidant indices were significantly (P < 0.05) lower that those in NuC fish groups. Malondialdehyde (MDA) was significantly influenced by dietary LDPs alone and its lowest values were observed in both NuC and NbC fish groups fed with 200 or 300 mg/kg diets with no differences between them. Phagocytic activity, alternate complement pathway, lysozyme, respiratory burst, peroxidase, complement C3, and immunoglobulin M values improved significantly (P < 0.05) in both NuC and NbC fish groups fed with 200 or 300 mg/kg diets with no differences between them. The mRNA manifestation of hepatic SOD , CAT , and GPx genes were significantly (P < 0.05) upregulated in both NuC fish groups fed on 200 and 300 mg LDPs/kg diets but feeding NbC fish on 300 mg LDPs/kg diets enhanced their mRNA expression. The HSP70, HSP90, TNF-α, IL-1β , and IL-8 mRNA expression in the NuC fish groups showed no significant (P > 0.05) differences due to dietary supplementation of LDPs to ruho carp, but their expressions were significantly (P < 0.05) higher in NbC fish groups; particularly at the treatment of 100 mg LDPs/kg diets. The mRNA expression of IL-10, IgM , hepcidin , and Mx1 genes were significantly (P < 0.05) upregulated in NuC fish group fed with 200 or 300 mg LDPs/kg diets. The bacterial infection downregulated their expressions but the dietary LDPs recovered the adverse effects of bacterial infection especially at 200 or 300 mg LDPs/kg diets. These results shown that the use of LDPs (200 or 300 mg/kg diets) has a valuable role in enhancing the growth, antioxidant-immune status, and inflammatory responses in rohu carp during F. columnare infection. • Dietary LDPs enhanced the fish growth but it was retarded by bacterial infection. • Immune, and antioxidant indices in NuC fish were higher in treatments of 200 or 300 mg LDPs/kg diet. • Pro-inflammatory genes were down-regulated in NuC fish but it was higher in NbC fish. • Immune related genes were up-regulated in NuC fish but it was lower in NbC fish. • Feeding NbC fish with 200 or 300 mg LDPs/kg diet recovered their growth and welfare. [ABSTRACT FROM AUTHOR]

Vaccines are widely employed in aquaculture to prevent bacterial infections, but their use by the U.S. catfish industry is very limited. One of the main diseases affecting catfish aquaculture is columnaris disease, caused by the bacterial pathogen Flavobacterium columnare. In 2011, a modified-live vaccine against columnaris disease was developed by selecting mutants that were resistant to rifampin. The previous study has suggested that this vaccine is stable, safe, and effective, but the mechanisms that resulted in attenuation remained uncharacterized. To understand the molecular basis for attenuation, a comparative genomic analysis was conducted to identify specific point mutations. The PacBio RS long-read sequencing platform was used to obtain draft genomes of the mutant attenuated strain (Fc1723) and the parent virulent strain (FcB27). Sequence-based genome comparison identified 16 single nucleotide polymorphisms (SNP) unique to the mutant. Genes that contained mutations were involved in rifampin resistance, gliding motility, DNA transcription, toxin secretion, and extracellular protease synthesis. The results also found that the vaccine strain formed biofilm at a significantly lower rate than the parent strain. These observations suggested that the rifampin-resistant phenotype and the associated attenuation of the vaccine strain result from the altered activity of RNA polymerase (RpoB) and possible disrupted protein secretion systems.

Viruses of bacteria, bacteriophages, specifically infect their bacterial hosts with minimal effects on the surrounding microbiota. They have the potential to be used in the prevention and treatment of bacterial infections, including in the field of food production. In aquaculture settings, disease-causing bacteria are often transmitted through the water body, providing several applications for phage-based targeting of pathogens, in the rearing environment, and in the fish. We tested delivery of phages by different methods (via baths, in phage-coated material, and via oral delivery in feed) to prevent and treat Flavobacterium columnare infections in rainbow trout fry using three phages (FCOV-S1, FCOV-F2, and FCL-2) and their hosts (FCO-S1, FCO-F2, and B185, respectively). Bath treatments given before bacterial infection and at the onset of the disease symptoms were the most efficient way to prevent F. columnare infections in rainbow trout, possibly due to the external nature of the disease. In a flow-through system, the presence of phage-coated plastic sheets delayed the onset of the disease. The oral administration of phages first increased disease progression, although total mortality was lower at the end of the experiment. When analysed for shelf-life, phage titers remained highest when maintained in bacterial culture media and in sterile lake water. Our results show that successful phage therapy treatment in the aquaculture setting requires optimisation of phage delivery methods in vivo.

Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide. Many studies have focused on the identification of protective antigens to aid in the development of novel vaccines against the disease. In this study, an immunoblotting approach was employed to identify immunogenic outer membrane proteins (OMPs) from F. columnare in two-dimensional electrophoresis (2-DE) map gels using antibacterial sera obtained from grass carp (Ctenopharyngodon idella), and anti-grass carp-recombinant Ig (rIg) monoclonal antibodies. Five unique immunogenic proteins, including the gliding motility lipoprotein GldJ (GldJ), hypothetical protein FCOL_13420 (Fco1), lipoprotein (Lip), F0F1 ATP synthase subunit beta (F0f1) and outer membrane efflux protein precursor (Omep), were characterized. Over-expression of these proteins in Escherichia coli DE3, and their immunogenicity and protective efficacy were evaluated in grass carp. The relative percent survival (RPS) of the groups immunized separately with recombinant GldJ, Lip and Omep was 72%, 64% and 68%, respectively when compared to control fish. Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of F. columnare. The results suggest that GldJ, Lip and Omep are major protective antigens and may be considered as novel candidates in the development of vaccines against columnaris disease in fish.

Flavobacterium columnare causes substantial losses among cultured finfish species. The Gram-negative bacterium is an opportunistic pathogen that manifests as biofilms on the host's mucosal surfaces as the disease progresses. We previously demonstrated that the dominant mucosal IgM antibody response to F. columnare is to the chaperone protein DnaK that is found in the extracellular fraction. To establish the efficacy of using recombinant protein technology to develop a new vaccine against columnaris disease, we are reporting on two consecutive years of vaccine trials using a recombinant F. columnare DnaK protein (rDnaK). In year one, three groups of channel catfish (n = 300) were immunized by bath immersion with a live attenuated F. columnare isolate, rDnaK or sham immunized. After 6 weeks, an F. columnare laboratory challenge showed a significant increase in survival (>30%) in both the live attenuated and rDnaK vaccines when compared to the non-immunized control. A rDnaK-specific ELISA revealed significant levels of mucosal IgM antibodies in the skin of catfish immunized with rDnaK at 4- and 6-weeks post immunization. In the second year, three groups of channel catfish (n = 300) were bath immunized with rDnaK alone or with rDnaK after a brief osmotic shock or sham immunized. After 6 weeks a laboratory challenge with F. columnare was conducted and showed a significant increase in survival in the rDnaK (> 25%) and in rDnaK with osmotic shock (>35%) groups when compared to the non-immunized control. The rDnaK-specific ELISA demonstrated significant levels of mucosal IgM antibodies in the skin of catfish groups immunized with rDnaK at 4- and 6-weeks post immunization. To further understand the processes which have conferred immune protection in the rDnaK group, we conducted RNA sequencing of skin samples from the non-immunized (n = 6) and rDnaK treated channel catfish at 1-week (n = 6) and 6 weeks (n = 6) post immunization. Significantly altered gene expression was identified and results will be discussed. Work to further enhance the catfish immune response to F. columnare rDnaK is underway as this protein remains a promising candidate for additional optimization and experimental trials in a production setting.

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.