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This study aimed to investigate the effects of different concentrations of fructooligosaccharide (FOS) instead of glucose (GLU) as the carbon source in the biofloc system. GLU was used as the carbon source in the control group, and 2.5%, 5.0%, 7.5%, and 10.0% FOS were used instead of GLU as the carbon source in experimental groups. Ammonium chloride was used the nitrogen source with a C/N ratio of 15. Bacillus subtilis was supplemented to promote biofloc formation. The results showed that the concentrations of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and total nitrogen were reduced effectively in all groups. High-throughput 16S rRNA sequencing revealed that the abundance of Flavobacterium was significantly increased in the 2.5% FOS group (P < 0.05), while Acinetobacter was significantly increased in the 10.0% FOS group on the 14th day (P < 0.05), the abundance of Aeromonadaceae was significantly reduced in all experimental groups (P < 0.05). Compared with the control group, the abundance of Aeromonadaceae was significantly decreased in 2.5% FOS on the 28th day (P < 0.05). Correlation analysis between microbial community composition and environmental factors indicated Flavobacterium and norank_f__norank_o__Saccharimonadales were negatively associated with three forms of nitrogen. In summary, the substitution of GLU with 2.5% FOS as the carbon source in the biofloc system significantly increased the abundance of core bacteria (Flavobacterium and Acinetobacter) and significantly reduced harmful bacteria (Aeromonadaceae), thereby stabilizing bacterial communities within the aquatic environment. The positive effects exhibited by 10.0% FOS were similar to 2.5% FOS. Based on experimental results and cost, the recommended proportion of FOS as a substitute for GLU is 2.5%. This research provides a foundation for the application of FOS as a carbon source in biofloc system and serves as a reference for future studies in this field. [ABSTRACT FROM AUTHOR]

The intricate relationship between regeneration and microbiota has recently gained attention, spanning diverse model organisms. Axolotl (Ambystoma mexicanum) is a critically endangered salamander species and a model organism for regenerative and developmental biology. Despite its significance, a noticeable gap exists in understanding the interplay between axolotl regeneration and its microbiome. Here, we analyse in depth bacterial 16S rRNA amplicon dataset that we reported before as data resource and profile fungal community by sequencing ITS amplicons at the critical stages of limb regeneration (0–1–4–7–30–60 days post amputation, 'dpa'). Results reveal a decline in richness and evenness in the course of limb regeneration, with bacterial community richness recovering beyond 30 dpa unlike fungi community. Beta diversity analysis reveals precise restructuring of the bacterial community along the three phases of limb regeneration, contrasting with less congruent changes in the fungal community. Temporal dynamics of the bacterial community highlight prevalent anaerobic bacteria in initiation phase and Flavobacterium bloom in the early phase correlating with limb blastema proliferation. Predicted functional analysis mirrors these shifts, emphasising a transition from amino acid metabolism to lipid metabolism control. Fungal communities shift from Blastomycota to Ascomycota dominance in the late regeneration stage. Our findings provide ecologically relevant insights into stage specific role of microbiome contributions to axolotl limb regeneration. [ABSTRACT FROM AUTHOR]

ABSTRACT Edwardsiella ictaluri and Flavobacterium covae are two bacteria species that cause diseases in farm‐raised channel catfish (Ictalurus punctatus) that cause heavy economic damage to the aquaculture industry, particularly to the channel catfish farming. In search for environmentally benign antibacterial compounds active against E. ictaluri and F. covae, we investigated the constituents isolated from Brazilian red, brown and green propolis. We have also synthetically modified active constituents to see if lipophilicity plays a role in enhancing antibacterial activities. Vestitol, neovestitol and methylvestitol were found to be the active constituents with minimum inhibitory concentration (MIC) relative to drug control florfenicol (RDCF) values (MIC−RDCF) of 7.6, 7.6 and 7.9 mg/L, respectively, against F. covae. The activity against E. ictaluri was not significant. [ABSTRACT FROM AUTHOR]

ABSTRACT Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD), is one of the leading pathogens in rainbow trout (Oncorhynchus mykiss) aquaculture. To date, there is little knowledge of the transmission kinetics of F. psychrophilum over the course of infection. In particular, how transmission is affected by host genotype and pathogen exposure dosage are not well studied. In order to fill in these knowledge gaps, we exposed two divergently selected lines of rainbow trout (ARS‐Fp‐R and ARS‐Fp‐S) to a range of dosages of F. psychrophilum (strain CSF117‐10). We then measured mortality and bacterial shedding to estimate transmission risk at multiple time points since initial infection. As dosage increased, the number of fish shedding and the amount of bacteria shed increased ranging from 0% to 100% and 103 to 108 cells fish−1 h−1, respectively. In addition, we found that disease resistance (survival) was not correlated with transmission risk blocking, in that 67% of fish which shed bacteria experienced no clinical disease. In general, fish mortality began on Day 3, peaked between Days 5–7 and was higher in the ARS‐Fp‐R line. Results from this study could be used to develop epidemiological models and improve disease management, particularly in the context of aquaculture and selective breeding. [ABSTRACT FROM AUTHOR]

Flavobacterium is a genus of microorganisms living in a variety of hosts and habitats across the globe. Some species are found in fish organs, and only a few, such as Flavobacterium psychrophilum and Flavobacterium columnare, cause severe disease and losses in fish farms. The evolution of flavobacteria that are pathogenic to fish is unknown, and the protein changes accountable for the selection of their colonization to fish have yet to be determined. A phylogenetic tree was constructed with the complete genomic sequences of 208 species of the Flavobacterium genus using 861 softcore genes. This phylogenetic analysis revealed clade CII comprising nine species, including five pathogenic species, and containing the most species that colonize fish. Thirteen specific amino acid changes were found to be conserved across 11 proteins within the CII clade compared with other clades, and these proteins were enriched in functions related to replication, recombination, and repair. Several of these proteins are known to be involved in pathogenicity and fitness adaptation in other bacteria. Some of the observed amino acid changes can be explained by preferential selection for certain codons and tRNA frequency. These results could help explain how species belonging to the CII clade adapt to fish environments. [ABSTRACT FROM AUTHOR]

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Abstract The generic term “Gill disease” refers to a wide range of disorders that affect the gills and severely impact salmonid aquaculture systems worldwide. In rainbow trout freshwater aquaculture, various etiological agents causing gill diseases have been described, particularly Flavobacterium and Amoeba species, but research studies suggest a more complex and multifactorial aetiology. Here, a cohort of rainbow trout affected by gill disease is monitored both through standard laboratory techniques and 16S rRNA Next-Generation Sequencing (NGS) analysis during a natural disease outbreak and subsequent antibiotic treatment with Oxytetracycline. NGS results show a clear clustering of the samples between pre- and post-treatment based on the microbial community of the gills. Interestingly, the three main pathogenic bacteria species in rainbow trout (Yersinia ruckeri, Flavobacterium psychrophilum, and Flavobacterium branchiophilum) appear to be weak descriptors of the diversity between pre-treatment and post-treatment groups. In this study, the dynamics of the gill microbiome during the outbreak and subsequent treatment are far more complex than previously reported in the literature, and environmental factors seem of the utmost importance in determining gill disease. These findings present a potential novel perspective on the diagnosis and management of gill diseases, showing the limitations of conventional laboratory methodologies in elucidating the complexity of this disease in rainbow trout. To the authors’ knowledge, this work is the first to describe the microbiome of rainbow trout gills during a natural outbreak and subsequent antibiotic treatment. The results of this study suggest that NGS can play a critical role in the analysis and comprehension of gill pathology. Using NGS in future research is highly recommended to gain deeper insights into such diseases correlating gill’s microbiome with other possible cofactors and establish strong prevention guidelines.

A novel amylase AmyFlA from Flavobacterium sp. NAU1659, AmyFlA, was cloned and expressed in Esherichia coli. Based on phylogenetic and functional analysis, it was identified as a novel member of the subfamily GH13_46, sharing high sequence identity. The protein was predicted to consist of 620 amino acids, with a putative signal peptide of 25 amino acids. The enzyme was able to hydrolyze soluble starch with a specific activity of 352.97 U/mg at 50 °C in 50 mM phosphate buffer (pH 6.0). The Km and Vmax values of AmyFlA were respectively 3.15 mg/ml and 566.36 µmol·ml−1·min−1 under optimal conditions. Its activity towards starch was enhanced by 63% in the presence of 1 mM Ca2+, indicating that AmyFlA was a Ca2+-dependent amylase. Compared to the reported maltogenic amylases, AmyFlA produced a lower variety of intermediate oligosaccharides at the start of the reaction so that the product mixture contained a higher proportion of maltose. These results indicate that AmyFlA may be potential application value in the production of high-maltose syrup. [ABSTRACT FROM AUTHOR]

β-1,6-Glucan plays a crucial role in fungal cell walls by linking the outer layer of mannoproteins and the inner layer of β-1,3-glucan, contributing significantly to the maintenance of cell wall rigidity. Therefore, the hydrolysis of β-1,6-glucan by β-1,6-glucanase directly leads to the disintegration of the fungal cell wall. Here, a novel β-1,6-glucanase FlGlu30 was identified from the endophytic Flavobacterium sp. NAU1659 and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of purified FlGlu30 were 50℃ and pH 6.0, resulting in a specific activity of 173.1 U/mg using pustulan as the substrate. The hydrolyzed products of FlGlu30 to pustulan were mainly gentianose within 1 h of reaction. With the extension of reaction time, gentianose was gradually hydrolyzed to glucose, indicating that FlGlu30 is an endo-β-1,6-glucanase. The germination of Magnaporthe oryzae Guy11 spores could not be inhibited by FlGlu30, but the appressorium formation of spores was completely inhibited under the concentration of 250.0 U/mL FlGlu30. The disruptions of cell wall and accumulation of intracellular reactive oxide species (ROS) were observed in FlGlu30-treated M. oryzae Guy11 cells, suggesting the significant importance of β-1,6-glucan as a potential antifungal target and the potential application of FlGlu30. Key points: • β-1,6-Glucan is a key component maintaining the rigid structure of fungal cell wall. • β-1,6-Glucanase is an antifungal protein with significant potential applications. • FlGlu30 is the first reported β-1, 6-glucanase derived from Flavobacterium. [ABSTRACT FROM AUTHOR]

Petroleum hydrocarbons (PHCs) can be biodegraded into CO2, and PHC‐contaminated aquifers are always deemed as carbon sources. Fortunately, some carbon fixation microorganisms have been found in PHC‐contaminated sites. However, most of the studies are related to volatile short‐chain PHC, and few studies focus on long‐chain PHC‐contaminated sites. To reveal the carbon fixation microorganisms in these sites, in the study, a long‐chain PHC polluted site in North China was selected. Through hydrochemical and metagenomics analysis, the structure and capacity of carbon fixing microorganisms in the site were revealed. Results showed that there were many kinds of carbon fixed microorganisms that were identified such as Flavobacterium, Pseudomonas. HP/4HB, rTCA, and DC/4HB cycles were dominated carbon fixation pathways. The long‐chain PHC were weakly correlated with carbon fixation microorganisms, but it may stimulate the growth of some carbon fixation microorganisms, such as microorganisms involved in rTCA cycle. Practitioner Points: The microorganisms with carbon fixation gene exist in the aquifer contaminated by long‐chain petroleum hydrocarbon.Microorganisms that have the ability to degrade petroleum also have the ability to carbon fixation.Long‐chain petroleum hydrocarbon may promote the growth of carbon fixation microorganisms. [ABSTRACT FROM AUTHOR]

The α-1,3-glucanase Agl-EK14 from Flavobacterium sp. EK-14 comprises a signal peptide (SP), a catalytic domain (CAT), a first immunoglobulin-like domain (Ig1), a second immunoglobulin-like domain (Ig2), a ricin B-like lectin domain (RicinB), and a carboxy-terminal domain (CTD). SP and CTD are predicted to be involved in extracellular secretion, while the roles of Ig1, Ig2, and RicinB are unclear. To clarify their roles, domain deletion enzymes Agl-EK14ΔRicinB, Agl-EK14ΔIg2RicinB, and Agl-EK14ΔIg1Ig2RicinB were constructed. The insoluble α-1,3-glucan hydrolytic, α-1,3-glucan binding, and fungal cell wall hydrolytic activities of the deletion enzymes were almost the same and lower than those of Agl-EK14. Kinetic analysis revealed that the K m values of the deletion enzymes were similar and uniformly higher than those of Agl-EK14. These results suggest that the deletion of RicinB causes a decline in binding and hydrolytic activity and increases the K m value. To confirm the role of RicinB, Ig1, Ig2, and RicinB were fused with green fluorescent protein (GFP). As a result, RicinB-fused GFP (GFP-RicinB) showed binding to insoluble α-1,3-glucan and Aspergillus oryzae cell walls, whereas Ig1- and Ig2-fused GFP did not. These results indicated that RicinB is involved in α-1,3-glucan binding. The fusion protein GFP-Ig1Ig2RicinB was also constructed and GFP-Ig1Ig2RicinB showed strong binding to the cell wall of A. oryzae compared to GFP-RicinB. Gel filtration column chromatography suggested that the strong binding was due to GFP-Ig1Ig2RicinB loosely associated with itself. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 97 (GH97) comprises enzymes like anomer‐inverting α‐glucoside hydrolases (i.e., glucoamylase) and anomer‐retaining α‐galactosidases. In a soil bacterium, Flavobacterium johnsoniae, we previously identified a GH97 enzyme (FjGH97A) within the branched dextran utilization locus. It functions as an α‐glucoside hydrolase, targeting α‐(1→6)‐glucosidic linkages in dextran and isomaltooligosaccharides (i.e., glucodextranase). FjGH97A exhibits a preference for α‐(1→6)‐glucoside linkages over α‐(1→4)‐linkages, while Bacteroides thetaiotaomicron glucoamylase SusB (with 69% sequence identity), which is involved in the starch utilization system, exhibits the highest specificity for α‐(1→4)‐glucosidic linkages. Here, we examined the crystal structures of FjGH97A in complexes with glucose, panose, or isomaltotriose, and analyzed the substrate preferences of its mutants to identify the amino acid residues that determine the substrate specificity for α‐(1→4)‐ and α‐(1→6)‐glucosidic linkages. The overall structure of FjGH97A resembles other GH97 enzymes, with conserved catalytic residues similar to anomer‐inverting GH97 enzymes. A comparison of active sites between FjGH97A and SusB revealed differences in amino acid residues at subsites +1 and +2 (specifically Ala195 and Ile378 in FjGH97A). Among the three mutants (A195S, I378F, and A195S‐I378F), A195S and A195S‐I378F exhibited increased activity toward α‐(1→4)‐glucoside bonds compared to α‐(1→6)‐glucoside bonds. This suggests that Ala195, located on the Gly184‐Thr203 loop (named loop‐N) conserved within the GH97 subgroup, including FjGH97A and SusB, holds significance in determining linkage specificity. The conservation of alanine in the active site of the GH97 enzymes, within the same gene cluster as the putative dextranase, indicates its crucial role in determining the specificity for α‐(1→6)‐glucoside linkage. [ABSTRACT FROM AUTHOR]

The recent development of single cell sequencing technologies has revolutionized the state-of-art of cell biology, allowing the simultaneous measurement of thousands of genes in single cells. This technology has been applied to study the transcriptome of single cells in homeostasis and also in response to pathogenic exposure, greatly increasing our knowledge of the immune response to infectious agents. Yet the number of these studies performed in aquacultured fishspecies is still very limited. Thus, in the current study, we have used the 10x Genomics single cell RNA sequencing technology to study the response of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs) to infectious pancreatic necrosis virus (IPNV), an important trout pathogen. The study allowed us to obtain a transcriptomic profile of 12 transcriptionally distinct leukocyte cell subpopulations that included four different subsets of B cells, T cells, monocytes, two populations of dendritic-like cells (DCs), hematopoietic progenitor cells, non-specific cytotoxic cells (NCC), neutrophils and thrombocytes. The transcriptional pattern of these leukocyte subpopulations was compared in PBL cultures that had been exposed in vitro to IPNV for 24 h and mock-infected cultures. Our results revealed that monocytes and neutrophils showed the highest number of upregulated protein-coding genes in response to IPNV. Interestingly, IgM+IgD+ and IgT+ B cells also upregulated an important number of genes to the virus, but amuch fainter response was observed in ccl4+ or plasma-like cells (irf4+ cells). A substantial number of protein-coding genes and genes coding for ribosomal proteins were also transcriptionally upregulated in response to IPNV in T cells and thrombocytes. Interestingly, although genes coding for ribosomal proteins were regulated in all affected PBL subpopulations, the number of such genes transcriptionally regulated was higher in IgM+IgD+ and IgT+ B cells. A further analysis dissected which of the regulated genes were common and which were specific to the different cell clusters, identifying eight genes that were transcriptionally upregulated in all the affected groups. The data provided constitutes a comprehensive transcriptional perspective of how the different leukocyte populations present in blood respond to an early viral encounter in fish. [ABSTRACT FROM AUTHOR]

Both above‐ and below‐ground parts of plants are constantly challenged with microbes and interact closely with them. Many plant‐growth‐promoting rhizobacteria, mostly interacting with the plant's root system, enhance the immunity of plants in a process described as induced systemic resistance (ISR). Here, we characterized local induced resistance (IR) triggered by the model PGPR Pseudomonas simiae WCS417r (WCS417) in Arabidopsis thaliana. Hydroponic application of WCS417 to Arabidopsis roots resulted in propagation of WCS417 in/on leaves and the establishment of local IR. WCS417‐triggered local IR was dependent on salicylic acid (SA) biosynthesis and signalling and on functional biosynthesis of pipecolic acid and monoterpenes, which are classically associated with systemic acquired resistance (SAR). WCS417‐triggered local IR was further associated with a priming of gene expression changes related to SA signalling and SAR. A metabarcoding approach applied to the leaf microbiome revealed a significant local IR‐associated enrichment of Flavobacterium sp.. Co‐inoculation experiments using WCS417 and At‐LSPHERE Flavobacterium sp. Leaf82 suggest that the proliferation of these bacteria is influenced by both microbial and immunity‐related, plant‐derived factors. Furthermore, application of Flavobacterium Leaf82 to Arabidopsis leaves induced SAR in an NPR1‐dependent manner, suggesting that recruitment of this bacterium to the phyllosphere resulted in propagation of IR. Together, the data highlight the importance of plant‐microbe‐microbe interactions in the phyllosphere and reveal Flavobacterium sp. Leaf82 as a new beneficial promoter of plant health. [ABSTRACT FROM AUTHOR]

Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products. [ABSTRACT FROM AUTHOR]

Flavobacterial diseases, caused by bacteria in the order Flavobacteriales, are responsible for devastating losses in farmed and wild fish populations worldwide. The genera Flavobacterium (Family Flavobacteriaceae) and Chryseobacterium (Weeksellaceae) encompass the most well-known agents of fish disease in the order, but the full extent of piscine-pathogenic species within these diverse groups is unresolved, and likely underappreciated. To identify emerging agents of flavobacterial disease in US aquaculture, 183 presumptive Flavobacterium and Chryseobacterium isolates were collected from clinically affected fish representing 19 host types, from across six western states. Isolates were characterized by 16S rRNA gene sequencing and phylogenetic analysis using the gyrB gene. Antimicrobial susceptibility profiles were compared between representatives from each major phylogenetic clade. Of the isolates, 52 were identified as Chryseobacterium species and 131 as Flavobacterium. The majority of Chryseobacterium isolates fell into six clades (A-F) consisting of ≥ 5 fish isolates with ≥ 70% bootstrap support, and Flavobacterium into nine (A-I). Phylogenetic clades showed distinct patterns in antimicrobial susceptibility. Two Chryseobacterium clades (F & G), and four Flavobacterium clades (B, G-I) had comparably high minimal inhibitory concentrations (MICs) for 11/18 antimicrobials tested. Multiple clades in both genera exhibited MICs surpassing the established F. psychrophilum breakpoints for oxytetracycline and florfenicol, indicating potential resistance to two of the three antimicrobials approved for use in finfish aquaculture. Further work to investigate the virulence and antigenic diversity of these genetic groups will improve our understanding of flavobacterial disease, with applications for treatment and vaccination strategies.

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease, is a devastating, worldwide distributed, fish pathogen causing significant economic loss in inland fish farms. Previous epidemiological studies showed that prevalent clonal complexes (CC) differ in fish species affected with disease such as rainbow trout, coho salmon and ayu, indicating significant associations between particular F. psychrophilum genotypes and host species. Yet, whether the population structure is driven by the trade of fish and eggs or by host-specific pathogenicity is uncertain. Notably, all F. psychrophilum isolates retrieved from ayu belong to Type-3 O antigen (O-Ag) whereas only very few strains retrieved from other fish species possess this O-Ag, suggesting a role in outbreaks affecting ayu. Thus, we investigated the links between genotype and pathogenicity by conducting comparative bath infection challenges in two fish hosts, ayu and rainbow trout, for a collection of isolates representing different MLST genotypes and O-Ag. Highly virulent strains in one host species exhibited low to no virulence in the other. F. psychrophilum strains associated with ayu and possessing Type-3 O-Ag demonstrated significant variability in pathogenicity in ayu, ranging from avirulent to highly virulent. Strikingly, F. psychrophilum strains retrieved from rainbow trout and possessing the Type-3 O-Ag were virulent for rainbow trout but not for ayu, indicating that Type-3 O-Ag alone is not sufficient for pathogenicity in ayu, nor does it prevent pathogenicity in rainbow trout. This study revealed that the association between a particular CC and host species partly depends on the pathogen's adaptation to specific host species. [ABSTRACT FROM AUTHOR]

The rainbow trout (Oncorhynchus mykiss) is an important commercial fish. Studies have shown that probiotics can promote the healthy growth of fish. In this study, we assessed the impact of an endogenous Bacillus subtilis strain (RT-BS07) on the growth, survival and immunological response of juvenile rainbow trout. Additionally, the morphology of rainbow trout's intestinal tissue was measured. Control (CT) and experimental groups (B) were fed with conventional and probiotic-supplemented diets (1.0 × 108 CFU per gram) for 42 days, respectively. Following feeding, a challenge experiment was carried out with Aeromonas hydrophila at 1.0 × 107 CFU/mL to determine the experimental fish's tolerance to the bacteria. The results showed that the weight gain rate (WGR) in group B was 118.2%, which was significantly higher than that in group CT (54.86%) after 42 days. The height of intestinal villi and the number of goblet cells increased in group B, thus increasing the resistance of rainbow trout to pathogenic bacteria. The alkaline phosphatase (AKP) activity (U/mg prot) in the liver, spleen and intestinal tissues of group B was significantly higher than that in the CT group. The expression of interleukin-1β (IL-1β) in the intestinal tissue of group B was 6-fold higher than that in group CT. Furthermore, the expression of growth hormone-2 (GH-2) in the spleen was 3-fold higher than that in group CT. After 12 days of A. hydrophila injection, the survival rate was 40% and 70% in group CT and group B, respectively. Collectively, the present study demonstrated that the addition of endogenous B. subtilis strain (1.0 × 108 CFU per gram) to the feed can effectively promote the growth of rainbow trout and enhance immunity, which is beneficial to rainbow trout culture. [ABSTRACT FROM AUTHOR]

Flavobacterium psychrophilum (Fp) causes Bacterial Cold Water Disease in salmonids. During host‐pathogen interactions, gram‐negative bacteria, such as Fp, release external membrane vesicles (OMVs) harbouring cargos, such as DNA, RNA and virulence factors. This study aimed to characterise the potential role of the OMVs' small RNAs (sRNAs) in the Fp‐rainbow trout host‐pathogen interactions. sRNAs carried within OMVs were isolated from Fp. RNA‐Seq datasets from whole‐cell Fp and their isolated OMVs indicated substantial enrichment of specific sRNAs in the OMVs compared to the parent cell. Many of the OMV‐packaged sRNAs were located in the pathogenicity islands of Fp. Conservation of sRNAs in 65 strains with variable degrees of virulence was reported. Dual RNA‐Seq of host and pathogen transcriptomes on day 5 post‐infection of Fp ‐resistant and ‐susceptible rainbow trout genetic lines revealed correlated expression of OMV‐packaged sRNAs and their predicted host's immune gene targets. In vitro, treatment of the rainbow trout epithelial cell line RTgill‐W1 with OMVs showed signs of cytotoxicity accompanied by dynamic changes in the expression of host genes when profiled 24 h following treatment. The OMV‐treated cells, similar to the Fp ‐resistant fish, showed downregulated expression of the suppressor of cytokine signalling 1 (SOCS1) gene, suggesting induction of phagosomal maturation. Other signs of modulating the host gene expression following OMV‐treatment include favouring elements from the phagocytic, endocytic and antigen presentation pathways in addition to HSP70, HSP90 and cochaperone proteins, which provide evidence for a potential role of OMVs in boosting the host immune response. In conclusion, the study identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections. [ABSTRACT FROM AUTHOR]

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February–April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non‐nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi‐aetiological origin. [ABSTRACT FROM AUTHOR]

Fruit shape is an important character of watermelon. And the compositions of rhizospheric and endophytic microorganisms of watermelon with different fruit shape also remains unclear. To elucidate the biological mechanism of watermelon fruit shape formations, the rhizospheric and endophytic microbial community compositions between oval (OW) and circular watermelons (CW) were analyzed. The results showed that except of the rhizospheric bacterial richness (P < 0.05), the rhizospheric and endophytic microbial (bacterial and fungal) diversity were not statistically significant between OW and CW (P > 0.05). However, the endophytic microbial (bacterial and fungal) compositions were significantly different. Firstly, Bacillus, Rhodanobacter, Cupriavidus, Luteimonas, and Devosia were the unique soil dominant bacterial genera in rhizospheres of circular watermelon (CW); In contrast, Nocardioides, Ensifer, and Saccharomonospora were the special soil dominant bacterial genera in rhizospheres of oval watermelons (OW); Meanwhile, Cephalotrichum, Neocosmospora, Phialosimplex, and Papulaspora were the unique soil dominant fungal genera in rhizospheres of circular watermelon (CW); By contrast, Acremonium, Cladosporium, Cryptococcus_f__Tremellaceae, Sodiomyces, Microascus, Conocybe, Sporidiobolus, and Acremonium were the unique soil dominant fungal genera in rhizospheres of oval watermelons (OW). Additionally, Lechevalieria, Pseudorhodoferax, Pseudomonas, Massilia, Flavobacterium, Aeromicrobium, Stenotrophomonas, Pseudonocardia, Novosphingobium, Melittangium, and Herpetosiphon were the unique dominant endophytic bacterial genera in stems of CW; In contrast, Falsirhodobacter, Kocuria, and Kineosporia were the special dominant endophytic genera in stems of OW; Moreover, Lectera and Fusarium were the unique dominant endophytic fungal genera in stems of CW; By contrast, Cercospora only was the special dominant endophytic fungal genus in stems of OW. All above results suggested that watermelons with different fruit shapes exactly recruited various microorganisms in rhizospheres and stems. Meanwhile, the enrichments of the different rhizosphric and endophytic microorganisms could be speculated in relating to watermelon fruit shapes formation. [ABSTRACT FROM AUTHOR]

Background: Francisella tularensis, the bacterium that causes tularemia, has been a persistent and widespread pathogen in various regions of the world for centuries. Francisella tularensis can affect humans and various domestic and wild animals. The current study aimed to determine the epidemiological status of tularemia in countries of the WHO Eastern Mediterranean Region (EMRO) through a systematic review and meta-analysis. Methods: All included studies were identified through a systematic search of online databases, including Scopus, PubMed, Web of Science, and EMBASE, through July 26, 2022, using keywords and suitable combinations. We focused on cross-sectional studies investigating the prevalence of F. tularensis. The weighted pooled prevalence was calculated using a random-effects model. Results: A total of 206 studies were identified, of which 20 were finally included in the analysis. The human seroprevalence of tularemia in WHO-EMRO countries was 6.2% (95% CI, 4.2 9.2). In the subgroup analysis, anti-F. tularensis antibodies were found in 6.92% and 5.5% of the high-risk individuals and Iran, respectively. The pooled prevalence of F. tularensis in environmental samples (water and soil) from the WHO-EMRO countries was 5.8% (9.4% by PCR and 0.5% by culture). In addition, 2.5% (95% CI, 0.2 0.22.7) of ticks in WHO-EMRO countries were positive for F. tularensis. The pooled prevalence of F. tularensis in rodents is 2.0% (1.1% by PCR and 3.7% by serology). In addition, 0.6% of domestic ruminants (0.4% by PCR and 2.4% by serology) were positive for F. tularensis in WHO-EMRO countries. Conclusion: According to the results of the present study, tularemia is an endemic but neglected disease in the WHO-EMRO region. However, most studies on tularemia are limited to a few countries in this region. Studies on tularemia in human populations, reservoirs, and vectors have been conducted in all countries in the WHO-EMRO region to obtain more detailed information about the epidemiology of tularemia in these regions. Author summary: Tularemia is a bacterial zoonotic disease caused by Francisella tularensis. This bacterium can infect animals (domestic and wild vertebrates), invertebrates, and humans. Francisella tularensis is transferred to humans through contact with infected wild animals (, inhalation of infected aerosols, arthropod bites, consumption of contaminated water or contaminated food, and swimming and contact in polluted hydro-telluric environments. The mortality rate associated with this disease ranges from 2 to 60% in the absence of proper antibiotic treatment. The prevalence of F. tularensis varies significantly between countries, as these pathogens can infect a wide range of hosts and have multiple vectors. However, there needs to be complete data on the geographical distribution of different species of F. tularensis, as well as the status of reservoirs, vectors, and human cases in most parts of the world. Therefore, this study aimed to determine F. tularensis prevalence in the countries of the WHO Eastern Mediterranean Region (WHO-EMRO). Based on our study, the human seroprevalence of tularemia in WHO-EMRO countries was 6.2%. The pooled prevalence of F. tularensis in environmental samples (water and soil), ticks, rodents, and domestic ruminants were 5.8%, 2.5%, 2.0%, and 0.6% in the WHO-EMRO countries, respectively. [ABSTRACT FROM AUTHOR]

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

Aphanizomenon flos-aquae (AFA) is the dominant filamentous cyanobacterium that develops into blooms in Upper Klamath Lake, Oregon, each year. During AFA bloom and collapse, ecosystem conditions for endangered Lost River and shortnose suckers deteriorate, thus motivating the need to identify processes that limit AFA abundance and decline. Here, we investigate the relations between AFA and other members of the microbial community (photosynthetic and nonphotosynthetic bacteria and archaea), how those relations impact abundance and collapse of AFA, and the types of microbial conditions that suppress AFA. We found significant spatial variation in AFA relative abundance during the 2016 bloom period using 16S rRNA sequencing. The Pelican Marina site had the lowest AFA relative abundance, and this was coincident with increased relative abundance of Candidatus Sericytochromatia, Flavobacterium , and Rheinheimera , some of which are known AFA antagonists. The AFA collapse coincided with phosphorus limitation relative to nitrogen and the increased relative abundance of Cyanobium and Candidatus Sericytochromatia, which outcompete AFA when dissolved inorganic nitrogen is available. The data collected in this study indicate the importance of dissolved inorganic nitrogen combined with microbial community structure in suppressing AFA abundance. [ABSTRACT FROM AUTHOR]

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

Background: Annual or seasonal killifishes (Cyprinodontiformes: Nothobranchiidae) are unique among fish in their ability to enter into developmental arrests (diapauses: DI, DII, and DIII). They have a short lifespan and their embryos are exceptionally tolerant to a variety of environmental stresses. These traits make them a popular model for studying vertebrate diapause, aging, stress tolerance, genome adaptation, and evolution. In such issues, in a comparative evolutionary framework, Fundulopanchax gardneri, a popular aquarium fish from Africa, is commonly used as a representative non‐annual model though its development is not studied in detail and whether it includes diapauses remains uncertain. Results: We described in detail for the first time embryonic development of F. gardneri and revealed it to resemble that in the undoubtedly annual Austrofundulus limnaeus killifish in displaying two developmental depressions. However, if compared with A. limnaeus, these developmental states look like "less intense" versions of DII and DIII rather than true diapauses. Conclusions: To determine whether developmental depressions in F. gardneri represent "true" diapauses or only their functional equivalents, detailed studies of embryonic development of different killifish both annual and non‐annual are needed. Before that, acceptance of F. gardneri as a representative non‐annual fish seems premature. Key Findings: Aplocheloid annual killifish (Cyprinodontiformes) are unique among fish in the ability to arrest development at different embryonic stages and prolong it for months and years. These and other traits make them a popular model in studying of aging, stress tolerance etc. In these studies, in a comparative framework, aplocheloid Fundulopanchax gardneri is usually used as a representative non‐annual fish though its developmental life‐history remains obscure.This study provides detailed description of F. gardneri development from early embryonic stages till sexual maturation with focus on developmental arrests, divergent developmental trajectories, duration of embryonic period, etc.The data provided in this study indicate F. gardneri to display developmental traits characteristic for annual killifish but in the "less expressed" form. Acceptance of F. gardneri as annual vs non‐annual is premature until the detailed study of embryonic development in other killifish both annual and non‐annual. [ABSTRACT FROM AUTHOR]

The seed‐endophytic bacterial community is a potentially beneficial and heritable fraction of the plant microbiome. Its utilization as a sustainable crop improvement strategy could be especially valuable for species such as hemp, where production is being scaled up and new challenges will be faced in managing crop productivity and health. However, little is known about the makeup and variation of the hemp seed microbiome. This study profiled the endophytic bacterial communities harboured by 16 hemp cultivars sourced from commercial suppliers in Europe. A 16S rDNA amplicon sequencing approach identified 917 amplicon sequence variants across samples. Taxonomic classification of sequences revealed 4 phyla and 87 genera to be represented in the dataset. Several genera were widespread while some were specific to one or a few cultivars. Flavobacterium, Pseudomonas, and Pantoea were notable in their high overall abundance and prevalence, but community composition was variable and no one taxon was universally abundant, suggesting a high degree of flexibility in community assembly. Taxonomic composition and alpha diversity differed among cultivars, though further work is required to understand the relative influence of hemp genetic factors on community structure. The taxonomic profiles presented here can be used to inform further work investigating the functional characteristics and potential plant‐growth‐promoting traits of seed‐borne bacteria in hemp. [ABSTRACT FROM AUTHOR]

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]

Francisella tularensis secretes tubular outer membrane vesicles (OMVs) that contain a number of immunoreactive proteins as well as virulence factors. We have reported previously that isolated Francisella OMVs enter macrophages, cumulate inside, and induce a strong pro-inflammatory response. In the current article, we present that OMVs treatment of macrophages also enhances phagocytosis of the bacteria and suppresses their intracellular replication. On the other hand, the subsequent infection with Francisella is able to revert to some extent the strong pro-inflammatory effect induced by OMVs in macrophages. Being derived from the bacterial surface, isolated OMVs may be considered a "non-viable mixture of Francisella antigens" and as such, they present a promising protective material. Immunization of mice with OMVs isolated from a virulent F. tularensis subsp. holarctica strain FSC200 prolonged the survival time but did not fully protect against the infection with a lethal dose of the parent strain. However, the sera of the immunized animals revealed unambiguous cytokine and antibody responses and proved to recognize a set of well-known Francisella immunoreactive proteins. For these reasons, Francisella OMVs present an interesting material for future protective studies. [ABSTRACT FROM AUTHOR]

Aims Stem rot caused by Fusarium concentricum is a new disease of Paris polyphylla reported by our research group. The present study investigates the growth inhibitory and apoptotic effects of Bacillus velezensis FJAT-54560 lipopeptide against F. concentricum. Methods and Results HPLC preparation and LC-MS analysis results show that the crude lipopeptides secreted by Bacillus velezensis FJAT-54560 isolated from Jasminum sambac consist of C14–17 iturin A, C14 fengycin B, C16 fengycin A/A2, C18 fengycin A, C20 fengycin B2, C21 fengycin A2, C22–23 fengycin A, C12–16 surfactin A, and C15 surfactin A derivatives. The mass ratios (g/g) of iturin, fengycin, and surfactin in lipopeptides are 2.40, 67.51, and 30.08%, respectively. Through inhibition zone and inhibition rate experiments, we found that crude lipopeptides and purified fengycin exhibit strong antifungal activity against F. concentricum , including accumulation of reactive oxygen species, loss of mitochondrial membrane potential, DNA fragmentation, Ca2+ accumulation, chromatin condensation, and phosphatidylserine externalization. Transcriptomic analysis indicates that crude lipopeptide-induced apoptosis in F. concentricum cells may be mediated by apoptosis-inducing factors and apoptosis mediators and can serve as a metacaspase-independent model. Conclusion Lipopeptides from Bacillus velezensis FJAT-54560 can control the pathogenic fungus F. concentricum by inducing apoptosis. [ABSTRACT FROM AUTHOR]

Infectious hematopoietic necrosis (IHN) is a common disease in the intensive production of salmonids. The IHN virus (IHNV) was isolated for the first time in North Macedonia in 2018. This study aimed to investigate the distribution and genotype of IHN in farmed rainbow trout and autochthonous salmonid fish in North Macedonia following the first detection. The samples were collected from 47 trout farms. Trout fry with or without clinical signs of IHN were selected as individual samples. Kidney, spleen, and heart were taken from each fish during the dissection. Three pooled samples were collected from each farm. A total of 141 pooled samples were collected: Rainbow trout (Oncorhynchus mykiss) n=127, Macedonian trout (Salmo macedonicus) n=11, and Ohrid trout (Salmo letnica) n=3. The virus was detected in 43 samples (30.50%): rainbow trout (n=40), Macedonian trout (n=2), and Ohrid trout (n=1). There were 18 (38.30%) positive fish farms. The MAKIHNV1 isolate from 2018 (MN641902) and the newly isolated virus shared a similarity of >99 and were placed in clade E-1 of European genogroup E. The IHN has spread throughout the country and is also present in the autochthonous salmonids. [ABSTRACT FROM AUTHOR]

Interleukin-21 (IL-21) is produced mainly by activated CD4+ T cells and promotes the proliferation, survival and differentiation of immune cells. This study characterised a high affinity binding receptor of IL-21 (IL-21 Ra) from grass carp (Ctenopharyngodon idella, Ci) and analyzed the expression of CiIL-21 and CiIL-21 Ra. It has been shown that they were modulated by PAMPs and during infection with bacterial and viral pathogens. Recombinant CiIL-21 protein produced in bacteria was active in inducing the expression of genes involved in Th responses. A monoclonal CiIL-21 antibody was generated against the rCiIL-21 and showed good reactive specificity with the rCiIL-21 proteins expressed in E. coli cells, and HEK293 T cells and the native protein from PHA-stimulated kidney leukocytes. In addition, the IL-21-producing cells detected by the CiIL-21 monoclonal antibody were found to be increased to in the kidney, spleen and hindgut after infection with Flavobacterium columnare. Our data indicate that IL-21 is involved in the regulation of immune response to bacterial and viral infection. [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]

Fusarium verticillioides is one of the most important fungal pathogens causing maize ear and stalk rots, thereby undermining global food security. Infected seeds are usually unhealthy for consumption due to contamination with fumonisin B1 (FB1) mycotoxin produced by the fungus as a virulence factor. Unveiling the molecular factors that determine fungal development and pathogenesis will help in the control and management of the diseases. Kex2 is a kexin-like Golgi-resident proprotein convertase that is involved in the activation of some important proproteins. Herein, we identified and functionally characterized FvKex2 in relation to F. verticillioides development and virulence by bioinformatics and functional genomics approaches. We found that FvKex2 is required for the fungal normal vegetative growth, because the growth of the ∆Fvkex2 mutant was significantly reduced on culture media compared to the wild-type and complemented strains. The mutant also produced very few conidia with morphologically abnormal shapes when compared with those from the wild type. However, the kexin-like protein was dispensable for the male role in sexual reproduction in F. verticillioides. In contrast, pathogenicity was nearly abolished on wounded maize stalks and sugarcane leaves in the absence of FvKEX2 gene, suggesting an essential role of Fvkex2 in the virulence of F. verticillioides. Furthermore, high-performance liquid chromatography analysis revealed that the ∆Fvkex2 mutant produced a significantly lower level of FB1 mycotoxin compared to the wild-type and complemented strains, consistent with the loss of virulence observed in the mutant. Taken together, our results indicate that FvKex2 is critical for vegetative growth, FB1 biosynthesis, and virulence, but dispensable for sexual reproduction in F. verticillioides. The study presents the kexin-like protein as a potential drug target for the management of the devastating maize ear and stalk rot diseases. Further studies should aim at uncovering the link between FvKex2 activity and FB1 biosynthesis genes. Key points: •The kexin-like protein FvKex2 contributes significantly to the vegetative growth of Fusarium verticillioides. •The conserved protein is required for fungal conidiation and conidial morphology, but dispensable for sexual reproduction. •Deletion of FvKEX2 greatly attenuates the virulence and mycotoxin production potential of F. verticillioides. [ABSTRACT FROM AUTHOR]

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

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

In this study, 2 yeasts, Candida zeylanoides isolate Y12‐3 (MN073455.1) and Hyphopichia pseudoburtonii isolate Y12‐1 (MN073489.1), were isolated from rainbow trout hindguts. Their identity was confirmed by amplification of internal transcribed spacer (ITS) gene regions. Their in vitro probiotic properties showed that they were hydrophobic and did not inhibit bacterial growth. However, they displayed positive co‐aggregation with several bacterial pathogens. It was interesting that Y12‐1 and Y12‐3 isolates survived and grew well in bile salt concentrations ranging from 0.6% to 1.5% and at low pH conditions. After a 35‐day feeding trial, the results of dietary incorporation of the yeast isolates showed that serum myeloperoxidase activity was unchanged in the treated and control groups. Meanwhile, serum lysozyme activity in yeast groups was decreased significantly compared to the control. However, fish fed with Y12‐1 had higher total protein, albumin, and globulin than other groups. Total cholesterol levels were decreased significantly in yeast groups compared to the control. Interestingly, the Y12‐1 and Y12‐3 groups showed higher survival (69.05% and 80.95%) after the challenge with Lactococcus garvieae compared to the control (45.24%). In brief, the aforementioned findings showed that Y12‐1 and Y12‐3 strains could be potential probiotic candidates for use in rainbow trout farming. [ABSTRACT FROM AUTHOR]

BACKGROUND: Evolving climatic conditions impact the behavior of microorganisms. The lack of efficiency of beneficial microorganisms against pathogens can be due to these evolving abiotic factors more favorable to the development and adaptation of pathogens. It is therefore of great interest to understand their impact (especially temperature increase and relative humidity (RH) variation) on pathogenic and non‐pathogenic microorganisms. This work aimed to examine the possible effects of increasing temperature (20, 25, 30 and 33 °C) and RH (40%, 50%, 60% and 80%) on the growth and mycotoxin production (deoxynivalenol (DON) and zearalenone (ZEN)) of Fusarium graminearum, on the growth of three commercial biocontrol agents (BCAs; Mycostop®, Xedavir® and Polyversum®) and on the pathogen–BCA interaction. RESULTS: Results demonstrated that BCAs have contrasting impacts on the growth and mycotoxinogenesis of F. graminearum depending on abiotic factors. At 25 °C and regardless of RH, commercial BCAs limit DON production by F. graminearum, but at 30 °C and intermediate RH, Xedavir® is no longer effective. The ability of Xedavir® to control the production of ZEN production by F. graminearum is also affected by abiotic factors. However, increasing temperature has an opposite effect on its ability to control the accumulation of ZEN. Polyversum® oomycete is the BCA with the most resilient efficacy against F. graminearum toxinogenesis under the different abiotic factors. CONCLUSION: This work provides new knowledge of the effect of these abiotic parameters on the interaction between BCA and F. graminearum, especially on the production of mycotoxins. It paves the way for the development of efficient and resilient mycotoxin biocontrol strategies using beneficial microorganisms against F. graminearum, thus contributing to global food security. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: Tea is one of the most widely consumed beverages in the world, with significant economic and cultural value. However, tea production faces many challenges due to various biotic and abiotic stresses, among which fungal diseases are particularly devastating. Results: To understand the identity and pathogenicity of isolates recovered from tea plants with symptoms of wilt, phylogenetic analyses and pathogenicity assays were conducted. Isolates were characterized to the species level by sequencing the ITS, tef-1α, tub2 and rpb2 sequences and morphology. Four Fusarium species were identified: Fusarium fujikuroi, Fusarium solani, Fusarium oxysporum, and Fusarium concentricum. The pathogenicity of the Fusarium isolates was evaluated on 1-year-old tea plants, whereby F. fujikuroi OS3 and OS4 strains were found to be the most virulent on tea. Conclusions: To the best of our knowledge, this is the first report of tea rot caused by F. fujikuroi in the world. This provides the foundation for the identification and control of wilt disease in tea plants. [ABSTRACT FROM AUTHOR]

Aim: Fusobacterium nucleatum has gained attention in the endodontic literature as it is a predominant microorganism in primary, secondary, and persistent root canal infections. Current irrigants have lot of drawbacks and are unlikely to predictably achieve a bacteria-free root canal system, which can lead to these failures. Herbal alternatives are now gaining popularity in the field of endodontics, paving a new way in the disinfection of root canals. The aim of this study is to evaluate the antimicrobial efficacy of green tea extract and Triphala as root canal irrigants against F. nucleatum using real-time Polymerase chain reaction (PCR). Methods: Extracted teeth were taken and inoculated with F. nucleatum, which was grown in a Mueller-Hinton agar plate under anaerobic conditions. The samples were cleaned and shaped and irrigated with green tea extract, Triphala extract, and dimethyl sulfoxide, which were compared to the widely used irrigant, sodium hypochlorite. Cycle threshold value was used to calculate the efficacy of the irrigant against F. nucleatum after which the samples were subjected to reverse transcriptase PCR. The data were analyzed and the data between the group were compared using the one-way ANOVA followed by a post hoc Tukey's test. Results: Triphala extract demonstrated high antimicrobial activity against F. nucleatum. It revealed statistically significant differences between Triphala extract and sodium hypochlorite (NaOCL) (P = 0.001). Green tea extract also displayed antimicrobial activity. Both the Triphala and green tea extracts showed a significant difference (P < 0.001). Conclusion: Triphala extract showed better antibacterial efficacy when compared to 3% NaOCl and green tea extract against F. nucleatum. [ABSTRACT FROM AUTHOR]

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

The eye of vertebrates is constantly faced with numerous challenges from aquatic or airborne pathogens. As a crucial first line of defense, the ocular mucosa (OM) protects the visual organ from external threats in vertebrates such as birds and mammals. However, the understanding of ocular mucosal immunity in early vertebrates, such as teleost fish, remains limited, particularly concerning their resistance to bacterial infections. To gain insights into the pivotal role of the OM in antibacterial immunity among teleost fish, we developed a bacterial infection model using Flavobacterium columnare in rainbow trout (Oncorhynchus mykiss). Here the qPCR and immunofluorescence results showed that F. columnare could invade trout OM, suggesting that the OM could be a primary target and barrier for the bacteria. Moreover, immune-related genes (il-6, il-8, il-11, cxcl10, nod1, il1-b, igm, igt, etc.) were upregulated in the OM of trout following F. columnare infection, as confirmed by qPCR, which was further proved through RNA-seq. The results of transcriptome analyses showed that bacterial infection critically triggers a robust immune response, including innate, and adaptive immune-related signaling pathways such as Toll-like, NOD-like, and C-type lectin receptor signaling pathway and immune network for IgA production, which underscores the immune role of the OM in bacterial infection. Interestingly, a substantial reduction in the expression of genes associated with visual function was observed after infection, indicating that bacterial infection could impact ocular function. Overall, our findings have unveiled a robust mucosal immune response to bacterial infection in the teleost OM for the first time, providing valuable insights for future research into the mechanisms and functions of ocular mucosal immunity in early vertebrate species. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase (GH) 87 α-1,3-glucanase (Agl-EK14) gene was cloned from the genomic DNA of the gram-negative bacterium Flavobacterium sp. EK14. The gene consisted of 2940 nucleotides and encoded 980 amino acid residues. The deduced amino acid sequence of Agl-EK14 included a signal peptide, a catalytic domain, a first immunoglobulin-like domain, a second immunoglobulin-like domain, a ricin B-like lectin domain, and a carboxyl-terminal domain (CTD) involved in extracellular secretion. Phylogenetic analysis of the catalytic domain of GH87 enzymes suggested that Agl-EK14 is distinct from known clusters, such as clusters composed of α-1,3-glucanases from bacilli and mycodextranases from actinomycetes. Agl-EK14 without the signal peptide and CTD hydrolyzed α-1,3-glucan, and the reaction residues from 1 and 2% substrates were almost negligible after 1440 min reaction. Agl-EK14 hydrolyzed the cell wall preparation of Aspergillus oryzae and released glucose, nigerose, and nigero-triose from the cell wall preparation. After treatment of A. oryzae live mycelia with Agl-EK14 (at least 0.5 nmol/ml), mycelia were no longer stained by red fluorescent protein-fused α-1,3-glucan binding domains of α-1,3-glucanase Agl-KA from Bacillus circulans KA-304. Results suggested that Agl-EK14 can be applied to a fungal cell wall lytic enzyme. [ABSTRACT FROM AUTHOR]

Aquatic pathogens contribute to the most severe economic loss in fishes. Nanoparticles are being developed as potent antimicrobial agents against various pathogens. The bacteria Aeromonas caviae is one of the most common aquatic pathogens that infects the majority of economically important fishes causing significant losses to the aquaculture industry. This study involved synthesizing and characterizing of a novel chitosan‐selenium nanoflower employing multiple spectroscopic and microscopic approaches. The UV‐vis spectra obtained at 265 nm indicated the formation of the Chitosan‐selenium nanoflower. The particle size analysis revealed the size of the nanoflowers to be 186.3 nm. The transmission electron micrographs revealed a unique nanoflower-like morphology. XRD spectrum revealed amorphous nature. The Raman spectrum showed a strong resonance peak at 254 cm−1 which is a characteristic absorption band for monoclinic Se and α‐Se. Cytotoxicity analysis of the synthesized nanoflowers against isolated fish pathogen Aeromonas caviae showed increasing toxicity in a dose‐dependent manner with maximum cytotoxicity of 75.06% at 1000 µg/mL. The DCFDA assay was conducted to estimate the increase in reactive oxygen species (ROS) production, and the highest percentage increase in ROS of 96.02% was observed at 1000 µg/mL. The lipid peroxidation assay was performed by quantification of the lipid oxidation product malondialdehyde (MDA). The highest percentage of lipid peroxidation was found to occur at a dose of 500 µg/mL. As a result, the synthesized chitosan‐selenium nanoflowers can be exploited as a promising antibacterial treatment against the fish pathogen Aeromonas caviae. [ABSTRACT FROM AUTHOR]

Maize grain production in the tropics is affected by Fusarium verticillioides, leading to significant losses in yield and quality. Fungicides are commonly used to control foliar diseases and increase grain yield, but have limited efficacy against F. verticillioides and may even stimulate the fungus to produce mycotoxins. Biocontrol can reduce F. verticillioides populations and fumonisin, but little is known about the resulting microbial interactions on the phylloplane and their relationship with F. verticillioides. This study aimed at evaluating the impact of different foliar treatments on the cultivable phylloplane community of fungi and bacteria and their potential in controlling F. verticillioides in maize. Results showed that two sprays of Bacillus subtilis BIOUFLA2 increased the percentage of native phylloplane antagonist bacteria and fungi against F. verticillioides by 25.0% and 27.3%, respectively, compared with the water control. However, two sprays with a triazole + strobilurin fungicide at different phenological stages in maize reduced such antagonistic communities. From field trials, 34 bacteria and 13 fungi from the phylloplane were selected for their inhibitory activity against F. verticillioides, with most coming from the exclusive treatment with BIOUFLA2. The study also revealed that the biocontrol agent‐recruited phylloplane indigenous lineages act through antibiosis, competition, and parasitism. Therefore, the adoption of biocontrol to limit F. verticillioides population build‐up not only has a direct activity on the pathogen but also recruits fungal and bacterial antagonists that may act additively or synergistically with the applied biocontrol agent. [ABSTRACT FROM AUTHOR]

The upsurge in havoc being wreaked by antibiotic-resistant bacteria has led to an urgent need for efficacious alternatives to antibiotics. This study assessed the antibacterial efficacy of two isobutyl cyanoacrylate nanoparticles (iBCA-NPs), D6O and NP30, against major bacterial pathogens of fish. In vivo tests on rainbow trout were preceded by in vitro tests of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). NP30 exhibited higher efficacy than D60, but both iBCA-NPs demonstrated dose-dependent and species-specific in vitro antibacterial properties against the bacterial isolates. Generally, Gram-negative bacteria were more resistant to the iBCA-NPs. Streptococcus iniae, Tenacibaculum maritimum, and Photobacterium damselae were particularly sensitive to both iBCA-NPs. Administered to rainbow trout at 3571.4 mg (iBCA-NP)/kg feed, the iBCA-NPs produced a relative gain rate and survival rates comparable to the control (p > 0.05). The condition factor and the hepatosomatic and viscerosomatic indices of fish were indifferentiable (p > 0.05) between the iBCA-NP groups and the control. The iBCA-NPs caused no alteration in stress, oxidative stress (superoxide dismutase, SOD), plasma complement titer, or lysozyme activity. This study presents the first report of antibacterial activity of iBCA-NPs against Gram-negative bacteria. The results of this study suggest that D60 and NP30 may contribute to reducing the amounts of antibiotics and chemotherapeutic agents used in aquaculture. [ABSTRACT FROM AUTHOR]

Bacterial coldwater disease (BCWD), caused by Flavobacterium psychrophilum, results in significant losses among multiple salmonid (family Salmonidae) species. Molecular epidemiology and serotyping studies have suggested that some variants are host specific; however, these associations have not been evaluated by cross‐challenging fish species with putatively host‐associated F. psychrophilum isolates via more natural (i.e. immersion) exposure routes. To this end, F. psychrophilum isolates US19‐COS, US62‐ATS and US87‐RBT, each originally recovered from diseased coho salmon (Oncorhynchus kisutch), Atlantic salmon (Salmo salar) or rainbow trout (O. mykiss), and belonging to a host‐associated multilocus sequence typing clonal complex (e.g. CC‐ST9, CC‐ST232 or CC‐ST10), were PCR‐serotyped, evaluated for proteolytic activity, and used to challenge adipose fin‐clipped 4‐month old Atlantic salmon, coho salmon and rainbow trout via immersion. Findings showed US87‐RBT caused disease and mortality only in rainbow trout (e.g. 56.7% survival probability). US19‐COS and US62‐ATS caused more mortality in coho salmon and Atlantic salmon but also caused disease in both other host species, albeit to a lesser extent. Observed survival differences may be due to variant antigenic/virulence determinants as differences in serotype and proteolytic activity were discovered. Collectively, results highlight the intricacies of F. psychrophilum‐host interactions and provide further in vivo evidence that some F. psychrophilum MLST variants are host specific, which may have implications for the development of BCWD prevention and control strategies. [ABSTRACT FROM AUTHOR]

Background: Columnaris disease, instigated by Gram-negative Flavobacterium covae (F. covae), is a dreadful disease of many different genera of freshwater fish with alarmingly high mortality rates. Ictalurus punctatus (I. punctatus) is extremely prone to this disease. Despite the terrible implications of this disease, very little is known about how the host and the bacterium's virulence factor interact. Secreted extracellular proteins (SEPs) are key bacterial virulence agents that frequently provide immunogenicity. In a previous study, the major secreted extracellular proteins of F. covae were identified and their protective efficacy was verified in I. punctatus, and the results were published. In the current study, an endeavor has been made to examine the progressive shifts in I. punctatus immunity following immunization with F. covae SEPs in order to more thoroughly comprehend the host's intrinsic and acquired immune responses against F. covae infection. An enzyme-linked immunosorbent test (ELISA) is operated to analyze specific humoral immune responses against SEPs of F. covae. Moreover, the expressions of immune-associated genes that are related to inherent and acquired immunity were monitored in the anterior kidneys and spleens of the vaccinated I. punctatus. Results: Marked rise in immunoglobulin M (IgM) levels were detected in the serum of I. punctatus vaccinated with different SEP formulations, including crude SEPs, heat-deactivated SEPs, and SEPs combined with adjuvant, at 14 and 21 days post-vaccination. At the same time points, a marked increase in the expression levels of clusters of differentiation (CD4-1 and CD8-β) and major histocompatibility complex (MHC-II) genes was detected in the vaccinated fish's anterior kidneys and spleens, while the highest levels of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) up-regulations were detected in the immunized fish's anterior kidneys 14 days following vaccination. Conclusions: This work revealed that SEPs are an effective basis for vaccination against F. covae infection, eliciting humoral and inherent immune responses of I. punctatus after SEPs immunization. Improvements are needed to further enhance the I. punctatus immunological responses to SEPs of F. covae since these antigens remain a prospective candidate for future optimization and clinical trials in aquaculture settings. [ABSTRACT FROM AUTHOR]

• A rare bacterium was isolated from synovial fluids of a patient with prosthesis. • Sequence analyses of the bacterial 16S ribosomal RNA gene and whole genome were performed. • The isolate was identified as Falsarthrobacter (Arthrobacter) nasiphocae. • The isolate had a genome size of 2.4 Mb and G+C content of 70.4 mol%. • This is the first report of F. nasiphocae periprosthetic joint infection. We report the isolation of a rare Gram-positive coccobacillary bacterium from synovial fluids of a patient with periprosthetic joint infection on three occasions over an 8-month period. As routine microbiological methods were not able to identify the isolate definitely, sequence analyses of the bacterial 16S ribosomal RNA gene and whole genome were performed. Analysis of the bacterial 16S ribosomal RNA gene showed the highest similarity (98.1%) with that of Falsarthrobacter (previously known as Arthrobacter) nasiphocae , which was first isolated from the nasal cavities of common seals (Phoca vitulina). The genome size of the strain (designated as UM1) is 2.4 Mb. With a high G+C content (70.4 mol%), strain UM1 is phylogenetically most closely related to F. nasiphocae based on whole genome analysis. Strain UM1 was susceptible to vancomycin, linezolid, trimethoprim-sulfamethoxazole, doxycycline, and intermediate to penicillin and ciprofloxacin. Ceftriaxone resistance was noted. The patient who was also on hemodialysis for his end stage kidney disease died approximately 3 weeks following implant removal and fusion with an external fixator. This study describes the first isolation of F. nasiphocae from human clinical samples. The use of emerging technologies has supported more definitive etiological diagnosis associated with rarely encountered organisms in periprosthetic joint infection. [ABSTRACT FROM AUTHOR]