Serotyping and genetic evaluation of rainbow trout-derived Flavobacterium columnare isolates

Columnaris disease is a highly contagious infection that affects nearly all freshwater fish species worldwide. Grass carp, one of the most economically significant freshwater fish species in China, is particularly susceptible to the disease, leading to large-scale mortality. Flavobacterium columnare and F. covae are the primary pathogens causing columnaris disease in Chinese grass carp aquaculture. Herein, we compare mortality rates, replication rates of typical columnaris symptoms, histopathological changes, and bacterial content in the tissues of grass carp following infection using four challenge models. The mortality rate in grass carp challenged via intraperitoneal injection was 86.7%. All fish infected via intramuscular and intradermal injections died, while immersion resulted in lower mortality. Gill corrosion rates were 67%, 53%, and 87%, respectively, in the intramuscular injection, intradermal injection, and immersion groups. Correspondingly, skin ulceration rates were 75%, 91%, and 63%. However, surface symptoms in the intraperitoneal injection group were milder. Histopathological analysis revealed similar lesions in grass carp subjected to immersion, intramuscular, and intradermal infection, which differed from carp infected via intraperitoneal injection. The trends in bacterial loads in the gills and skin were similar, although the absolute bacterial content varied between tissues. Bacterial loads in the immersion and intraperitoneal injection groups were lower than those in the other groups. Based on these findings, we determined that the optimal model for simulating columnaris disease in grass carp is the intradermal injection of F. covae in 10–12 cm fish. The infection model generated via intradermal injection resembles natural F. covae infection and can serve as a good tool for evaluating the protective effect of anti-F. covae infection vaccines in grass carp.

Isolation of a highly virulent strain of Flavobacterium columnare Ci2323 and development of its live attenuated vaccine candidate in grass carp (Ctenopharyngodon idella)

Study on the correlation between Flavobacterium columnare infection and gill injury in Largemouth bass (Micropterus salmoides) and the protective effect of Enoxolone.

Genomic localization and immune response of IgT in grass carp (Ctenopharyngodon idellus).

Plastisphere is a hotspot for some potential pathogens. Herein, a sudden incident of gill-rot disease in the Haihe River, China, is speculated to be related to the effects of the plastisphere. Significantly higher levels of microplastics (MPs) were present in the gills of dying carps identified with gill-rot disease, while Flavobacterium columnare was identified as the main pathogenic microorganism. A six-week simulated exposure experiment was conducted to further confirm the relationship among MPs, Flavobacterium columnare and the pathogens of gill-rot disease. At an exposure concentration reflecting environmental abundance, all four MPs─PET and PLA in fiber and granule forms─were found to promote F. columnare-driven gill tissue damage, pathogen enrichment in the gills, and pathogenic metabolism. Compared to granular and nonbiodegradable PET MPs, fibrous PLA, which exhibited higher retention in the gills and greater microbial affinity, resulted in the highest disease incidence. For the first time, the risk of respiratory infectious diseases in aquatic animals caused by the plastisphere on MPs was presented and demonstrated.

Abstract Aquaculture is one of the fastest-growing food production sectors and plays a crucial role in meeting the demand for nutritious and affordable food for billions of people worldwide. Nile tilapia is an economically important fish species that is widely farmed in over 140 countries. However, diseases affecting Nile tilapia significantly hinder the expansion and development of sustainable aquaculture. Recently, the industry has faced serious bacterial infections that have resulted in substantial losses for Nile tilapia farmers. These bacterial infections—including Aeromonas hydrophila, Yersinia ruckeri, Escherichia coli, Streptococcus aureus, Flavobacterium columnare, Pseudomonas aeruginosa, Streptococcus agalactiae, Streptococcus iniae, and Edwardsiella tarda affect various parts of the fish such as the skin, eyes, kidneys, gills, and liver. The absence of effective disease prevention and appropriate biosecurity measures makes these infections a major threat to global Nile tilapia farming. Moreover, factors such as high nutrient levels, elevated water temperatures, and high fish density can promote bacterial growth, including that of potentially pathogenic bacteria. Strategies employed to combat diseases in tilapia include the use of feed supplements, herbal extracts, and vaccines. This review aims to provide an updated overview of the disease risks affecting Nile tilapia production, emphasizing the challenges related to these diseases and their treatment approaches. Moreover, it seeks to enhance understanding of country-specific bacterial diseases and addresses a critical gap in knowledge regarding health and disease pathways for sustainable aquaculture and the control strategies implemented thus far.

Flavobacterium columnare is a common pathogen of Chinook Salmon Oncorhynchus tshawytscha in the Klamath River. Elevated water temperatures invoke congregation behavior within thermal refugia and are associated with columnaris disease. A flowing-water F.columnare challenge system was compared with the standard static-bath challenge as an initial step in simulating a riverine exposure. Juvenile Chinook Salmon were exposed to 103 CFU/mL F. columnare for 20 h either in an aerated static bath or within a recirculation swim chamber set at one body length per second. Fish were held at a constant 20°C or exposed to short-term temperature fluctuations to a maximum of 24°C prior to the challenge. Mucus and gill samples were collected at the end of the 20-h challenge and from fish held up to 96 h postchallenge. Samples were assayed for detection of F. columnare by quantitative PCR and conventional plate culture method. In static-bath challenge groups, F. columnare was detected in asymptomatic (38%) and moribund Chinook Salmon (29%). In contrast, F. columnare was detected in only one asymptomatic (4%) and one moribund (4%) Chinook Salmon in the flowing-water challenge groups. Prechallenge temperature conditions had no effect on infection. Other yellow-pigmented bacteria were isolated from the Chinook Salmon (particularly static-bath challenge) but were not associated with morbidity or amplified in the F. columnare quantitative PCR. Low transmission of F. columnare occurred among juvenile Chinook Salmon under flowing-water conditions simulating a thermal refugia during early summer (20°C, flow of one body length per second, 20-h exposure to 103 CFU/mL). The flowing-water system is sufficient to examine the environmental factors (velocity, temperature, host density, duration, and bacterial concentration of exposure) of riverine exposures on F. columnare transmission to juvenile Chinook Salmon.

Transcriptome analysis of head kidney and liver in grass carp (Ctenopharyngodon idella) symptomatically or asymptomatically infected with Flavobacterium columnare.

Striped catfish (Pangasianodon hypophthalmus) farmers' perspectives on challenges and health management practices in the Mekong Delta, Vietnam: A qualitative study.

Columnaris disease, caused by columnaris-causing bacteria (CCB), affects cultured and wild freshwater fish worldwide. Belonging to the phylum Bacteroidota and the family Flavobacteriaceae, CCB are gram-negative, yellow-pigmented bacteria that are presumed ubiquitous in freshwater habitats. Since the first report of the disease in 1922, growth and survival, pathogenesis, and transmission of these pathogens have been intensely researched, aiding in a greater understanding of host-pathogen interactions. Recent classification of Flavobacterium columnare into four distinct species based on genetic similarity, including F. columnare, F. covae, F. davisii, and F. oreochromis, along with their host associations is critical to understanding disease dynamics and prevention strategies. Outbreaks of the disease typically follow stressful events where CCB colonise external tissues, such as skin and gill. Vaccination against columnaris disease was practiced as early as the 1970's. Since then, many different vaccine types have been under development, including killed, live-attenuated, recombinant, and micro- and nano-particle vaccines. Among the different CCB vaccine types, immune responses and level of protection have been variable. However, some vaccines have been reported to offer moderate-significant protection in some fish species. This review synthesises the current knowledge of CCB taxonomy, genetic diversity, virulence, and immunity in various aquaculture species, with the goal of identifying knowledge gaps to be addressed for future CCB vaccine development. The development of an efficacious columnaris vaccine remains a challenge, with promising results in the laboratory that have yet to be reflected under production settings. This highlights the need for improved strategies tailored to the species of CCB most appropriate for the target host species.

BackgroundColumnaris disease, a prevalent disease among farmed and wild freshwater fish, is caused by the Flavobacterium columnare group, which includes four distinct species: F. columnare, F. oreochromis, F. covae, and F. davisii. Among these, F. oreochromis, F. covae, and F. davisii are particularly prevalent in farmed freshwater fish in Thailand. In this study, a comparative genomic analysis of 22 isolates was conducted to elucidate virulence factors, antibiotic resistance genes (ARGs), genomic islands (GIs), phages, insertion elements (ISs), and clustered regularly interspaced short palindromic repeats (CRISPRs).ResultsA total of 212 putative virulence genes were predicted across three species with F. oreochromis exhibiting the highest number of unique virulence genes, followed by F. davisii, and F. covae. Moreover, 195 genes were predicted as ARGs, with F. oreochromis and F. covae showing an abundance of unique genes associated with resistance to quinolone, fluoroquinolone, and tetracycline antibiotics. Antimicrobial susceptibility testing, assessed with epidemiological cut-off values (ECVs), revealed decreased susceptibility to quinolones, fluoroquinolones and tetracycline in several isolates of F. oreochromis and F. covae. F. oreochromis and F. covae exhibited notable decreased susceptibility to quinolones, with mutations observed in the quinolone resistance-determining regions (QRDRs) of gyrA, including Ser83Phe, Ser83Val, Ser83Ala, and Asp87Tyr, the latter representing a novel mutation among isolates from Thailand. As a result, these findings suggest that gyrA is major target for quinolone in F. oreochromis, F. covae, and F. davisii, while gyrB, parC, parE might be less important to the decreased phenotypic susceptibility to this class of antimicrobials. Moreover, a tetracycline resistance gene (tetA_2) was found in only one F. covae isolate, which exhibited decreased phenotypic susceptibility to this drug, marking the first report of decreased susceptibility in this species.ConclusionsThis study provides insights into the genetic and pathogenic diversity of Flavobacterium species, aiding in the development of strategies to manage columnaris disease in farmed freshwater fish in Thailand.

First report on Flavobacterium columnare causing disease outbreaks in cultured black carp, Mylopharyngodon piceus, in Northern Vietnam.

Regular monitoring of laboratory zebrafish health status is crucial for ensuring both animal welfare and scientific validity in aquatic research. While zebrafish usage in research has increased substantially due to their biological advantages and experimental benefits, including high fecundity and vertebrate similarity, systematic health monitoring remains uncommon in South Korean facilities. This study presents a comprehensive assessment of zebrafish colony health monitoring practices in South Korea, combining comparative survey data from 2018 and 2024 with microbiologic and environmental analyses of 11 facilities. Our survey revealed a trend: despite facility scale expansion (proportion of the large-scale facilities with >200 tanks increasing from 41.7% to 54.5%) and universal adoption of recirculation systems, monitoring efforts have declined. The percentage of facilities without active monitoring increased from 50.0% in 2018% to 81.8% in 2024, while awareness of monitoring necessity decreased from 91.7% to 72.7%. To investigate these issues, we conducted analyses across 11 facilities (6 research institutes and 5 local suppliers). The analysis encompassed multiple parameters: 1) detection of key infectious agents (Mycobacterium spp., Aeromonas hydrophila, Flavobacterium columnare, Pseudocapillaria tomentosa, Pseudoloma neurophilia, Pseudomonas aeruginosa) in sump tank biofilm, zebrafish specimens, and feed samples; and 2) evaluation of water chemistry parameters (pH, nitrate concentration, conductivity) in tank water. Our findings revealed that Mycobacterium spp. were present in biofilm samples from all facilities and in >80% of fish samples from research facilities. Aeromonas hydrophila was detected across all sample types. Both Mycobacterium spp. and A. hydrophila are opportunistic pathogens that necessitate careful consideration in long-term zebrafish experiments. Furthermore, evaluation of water quality analyses indicated widespread deviations from acceptable parameters, particularly in nitrate levels and pH values. Our results underscore the need for implementing standardized monitoring protocols and enhanced water system management to safeguard research integrity, animal health, and occupational safety in zebrafish facilities.

Background: Aquaculture, the fastest-growing food production sector worldwide, faces significant challenges due to disease outbreaks, particularly from bacterial pathogens such as Flavobacterium columnare. These pathogens cause severe economic losses, necessitating the development of effective diagnostic tools and vaccines. Methods: This study aimed to express the chondroitin AC lyase gene from F. columnare, a key virulence factor in prokaryotic expression vector. The gene was PCR-amplified, cloned into the pET-32a vector and expressed in Escherichia coli BL21-D3 cells. Positive clones were confirmed through PCR and restriction enzyme digestion. The expressed protein was analyzed by SDS-PAGE and Western blot, with protein expression tested under varying incubation temperatures. In silico analysis was performed to predict secondary and tertiary structures, including identification of key motifs and antigenic sites. Antigenicity was confirmed through an Agar Gel Immunodiffusion (AGID) assay. Result: The SDS-PAGE and Western blot analysis revealed a prominent band at ~77 kDa, indicating successful expression of the recombinant protein. Expression levels varied with incubation temperature. In silico analysis identified significant antigenic sites and the AGID assay showed strong immune reactions, confirming the protein’s potential as a vaccine component.

Due to the recent taxonomic reclassification of the species Flavobacterium columnare into four new species-Flavobacterium columnare, Flavobacterium davisii, Flavobacterium covae, and Flavobacterium oreochromis-it is necessary to re-evaluate isolates of previous outbreaks to better understand the epidemiology related to this bacterial group. Therefore, the aim of this study was to determine the taxonomic profile of Brazilian isolates of Flavobacterium spp. associated with columnaris disease using available diagnostic methods. Fifty isolates from different outbreaks (17 clinical cases) occurring in five different Brazilian states previously identified as F. columnare were selected and identified by multiplex PCR and MALDI-ToF methods. In addition, at least one isolate from each clinical case was analyzed by 16S rRNA gene sequencing. After inclusion of the MSPs (main spectra profiles), the isolates were identifiable, and when compared with the multiplex PCR results, they showed almost perfect agreement (94.2% Kappa = 0.85). Only F. davisii, F. covae, and F. oreochromis were found among the Brazilian isolates, with these species causing disease in neotropical fish hosts not previously reported (e.g., Siluriformes, Serrasalmidae, and Bryconidae), while F. columnare was not detected. This study provides evidence of Flavobacterium species associated with columnaris disease circulating in various aquaculture facilities across different regions of Brazil. This information is crucial for developing control programs and advancing epidemiologic studies on columnaris disease in Brazilian aquaculture.

Several bacterial pathogens employ haemophores to scavenge haem from host haemoprotein to obtain an iron source. However, no homologues of well-characterized haemophores are found in Riemerella anatipestifer, a bacterium belonging to the order Flavobacteriales that encodes haem uptake systems. Herein, a unique haemophore RhuH is characterized in this bacterium. R. anatipestifer used RhuH to grow when duck hemoglobin serves as the sole iron resource. RhuH is secreted as a component of outer membrane vesicles. Recombinant RhuH exhibited a high binding affinity for haem (Kd of 3.44 × 10-11m) and can extract haem from duck hemoglobin. X-ray crystallography elucidated the 3D structure of RhuH at 2.85 Å resolution, showing a dimeric conformation with each monomer exhibiting a unique structure. Structure modeling of RhuH-haem, coupled with mutagenesis, haemin utilization, and binding affinity assays, show that haem is captured in the β-barrel-like region, displaying the classic iron coordination. The RhuH homologues are predominantly distributed in Weeksellaceae and Flavobacteriaceae. Finally, the homologues of RhuH in Riemerella columbina, Flavobacterium columnare, and Flavobacterium soli are used as a proof of concept, demonstrating that these homologues exhibit conserved structures and functions.

NOD-like receptors are significant contributors to the immune response of fish against different types of pathogen invasion. NOD1 and NOD2 genes of yellow catfish (Tachysurus fulvidraco) were identified and characterized in this study. Yellow catfish NOD1 and NOD2 have open reading frames (ORFs) of 2841 and 2949 bp, encoding 946 and 982 amino acids, respectively. Both NOD1 and NOD2 are intracellular proteins lacking transmembrane regions and signal peptides. Sequence homology analysis revealed that the protein sequences of NOD1 and NOD2 of yellow catfish are highly similar to those of channel catfish. Both NOD1 and NOD2 showed high expression in the head kidney, and spleen. Following challenge with Flavobacterium columnare, NOD1 expression obviously increased in the liver, spleen, midgut, and hindgut, whereas NOD2 clearly increased in head kidney, and gut. Microscopic observation of gill tissues revealed evident epithelial hyperplasia in the secondary gill filaments at 3 and 6 hpi, with a notable decrease in the aspect ratio in comparison with the control group, followed by a return to baseline levels. These findings indicate a potential involvement of NOD1 and NOD2 genes in defense against F. columnare invasion. The findings of this study contribute valuable insights into NOD1 and NOD2's functions in the innate immune response of yellow catfish and other fish species to bacterial infection.

In recent years, outbreaks of mixed bacterial infections, similar to bacterial hemorrhagic septicemia, have become more frequent in the cage trout farms of the Leningrad Region. Previously, a similar course of the infectious process was observed mainly in closed water supply installations, especially for the first time years of their operation. High water temperatures play an important role in the development of the disease process (more than 20 °C), which last for a month or more, as well as high planting densities. In the period from 2021 to 2024, in the summer, a bacterial infection, the causative agents of which are Yersenia ruckeri and Flexibacter columnaris, is dangerous in cages. In May, and later during the summer, the presence of mixed bacterial infections caused by the development of opportunistic microflora was revealed — aeromonas (Aeromonas hydrophila, Aeromonas caviae), pseudomonas (Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas chlororaphis) and myxobacteria (Flexibacter psyhrophila, Flexibacter columnaris). It should be noted the appearance of enterobacteria (Enterobacter spp.) as representatives of the secondary microflora, whose pathogenicity has been confirmed. The complex of aeromonas with flavobacteria is characterized by increased aggressiveness, which contributes to the development of serious septic infection. In one farm, two or three associations of pathogenic bacteria could be noted. A significant difficulty in detecting bacterial hemorrhagic septicemia is the selection of antimicrobial drugs. In this case, it is necessary to take into account the sensitivity to them of the main representatives of the association of pathogens. Otherwise, you can provoke anew outbreak of the disease. It was noted that mixed infections were often found in farms where fish purchased from three to four suppliers were grown on one fish farm. An extremely negative effect was given by stocking in the autumn with juveniles with low fatness and with a small amount of cavitary fat. Timely measures taken, including the normalization of conditions of detention and feeding, as well as medical and preventive measures, made it possible to suppress the development of infections and improve the epizootic state of kindergarten farms. In the future, it is recommended to avoid stocking one site with a large number of fish of different origin, and, accordingly, with different opportunistic microflora, the contact of which with each other leads to a deterioration in the epizootic state of farms. To normalize the in testinal microflora and increase the immune-physiological status, the use of probiotics can be proposed.

The objective of this study was to implement population pharmacokinetic (PPK) of enrofloxacin (EF) in grass carp (Ctenopharyngodon idella) after a single oral administration and a single intravenous administration based on a nonlinear mixed effect model. The plasma samples collected by the sparse sampling method were detected by high-performance liquid chromatography with a fluorescent detector. The initial pharmacokinetic (PK) parameters were evaluated by reference search and the calculation of a naïve pooled method. After oral administration, the concentration-time profile was best described by a one-compartment open model. The absorption rate constant (Ka), apparent distribution volume (V), and systemic clearance (CL) were estimated to be 3.11/h, 4.36 L/kg, and 0.079 L/h/kg, respectively. After intravenous administration, the concentration-time curve was best simulated by a two-compartment open model. The apparent distribution volume of the central compartment (V1), apparent distribution volume of the peripheral compartment (V2), CL, and clearance from the central compartment to the peripheral compartment (CL2) were estimated to be 0.42, 2.05 L/kg, 0.067, and 2.94 L/h/kg, respectively. Finally, the bioavailability was calculated to be 84.81%. The parameter of AUC/minimum inhibitory concentration value was estimated to be more than 506.32 for Aeromonas hydrophila, Aeromonas sobria, and Flavobacterium columnare indicating that EF at 20 mg/kg has high effectiveness for these pathogens. This study supported a concise method for conducting PK study in aquatic animals that facilitated the development of PK methodology in aquaculture.