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Understanding disease aetiology and pathologic mechanisms is essential for fish health evaluation. Carp edema virus (CEV) is the causative agent of a disease (CEVD) responsible for high mortality rates in both wild and cultured common carp Cyprinus carpio. Inspection of two carp specimens from a pond with high fish mortality revealed CEV infection in both the host and its ectoparasite (Argulus foliaceus). In addition to flavobacteria, well known to be associated with gill lesions, we found that free-living eukaryotes (amoebae and ciliates) and a temporary parasite (Ichthyobodo spp.) colonizing the gills may also contribute to alterations in gill structure and/or function, either directly, through firm (Ichthyobodo) or weak (amoebae) attachment of trophozoites to the gill epithelium, or indirectly, through carriage of pathogenic bacteria. Bacterial assemblages rich in families and genera, with predominance of Cetobacterium spp. in low-intensity alteration of the gill tissue and of Flavobacterium spp. in gills with extensive necrotic lesions, were detected in gills and within the cytoplasm of associated amoebae using high-throughput sequencing. Quantitative PCR indicated F. swingsii as the prevailing flavobacterial species within amoebae from less affected gills and F. psychrophilum within amoebae from extensively affected gills. This case study suggests that eukaryotic organisms as part of the gill pathobiome may also contribute to irreversible gill lesions seen in CEVD. Emphasizing the complexity of mutual relationships between bacterial assemblages and eukaryotic co-pathogens, further studies regarding factors that trigger pathology and influence severity in the CEV-positive carp are needed.

Aquatic pathogens are a major concern for fish hatchery production, fisheries management, and conservation, and disease control needs to be addressed. Two important salmonid pathogens are Myxobolus cerebralis and Flavobacterium psychrophilum that cause whirling disease and bacterial coldwater disease (BCWD), respectively. Innate disease resistance is a potential option for reducing disease-related mortality in hatchery-reared rainbow trout (Oncorhynchus mykiss, Walbaum). Two experiments were conducted to assess pathogen resistance of first-generation (F1) rainbow trout created by crossing M. cerebralis- and F. psychrophilum-resistant strains. In the first experiment, we exposed two rainbow trout strains and one F1 cross to six treatments: control (no exposure), mock injection, F. psychrophilum only, M. cerebralis only, F. psychrophilum then M. cerebralis, and M. cerebralis then F. psychrophilum. Results indicated that the F1 cross was not resistant to either pathogen. In the second experiment, we exposed five rainbow trout strains and four rainbow trout crosses to F. psychrophilum. The second experiment indicated that at least one rainbow trout cross was F. psychrophilum-resistant. Achieving dual resistance may be possible using selective breeding but only some multigenerational strains are suitable candidates for further evaluation.

Intensive aquaculture conditions expose fish to bacterial infections, leading to significant financial losses, extensive antibiotic use and risk of antibiotic resistance in target bacteria. Flavobacterium columnare causes columnaris disease in aquaculture worldwide. To develop a bacteriophage-based control of columnaris disease, we isolated and characterized 126 F. columnare strains and 63 phages against F. columnare from Finland and Sweden in 2017. Bacterial isolates were virulent on rainbow trout (Oncorhynchus mykiss) and fell into four previously described genetic groups A, C, E and G, with genetic groups C and E being the most virulent. Phage host range studied against a collection of 227 bacterial isolates (from 2013 to 2017) demonstrated modular infection patterns based on host genetic group. Phages infected contemporary and previously isolated bacterial hosts, but bacteria isolated most recently were generally resistant to previously isolated phages. Despite large differences in geographical origin, isolation year or host range of the phages, whole-genome sequencing of 56 phages showed high level of genetic similarity to previously isolated F. columnare phages (Ficleduovirus, Myoviridae). Altogether, this phage collection demonstrates a potential for use in phage therapy.

Previously, rainbow trout fed deoxynivalenol (DON) or partially fed (pair-fed) for 4 weeks before and during experimental infection with Flavobacterium psychrophilum had significantly decreased mortality rates. Similar results were obtained in the present study after 12 days, but not after 6 days, feeding 5 ppm DON or pair-fed before infection. Furthermore, feeding 250 ppm chloroquine (CQ) also reduced mortality (p = .052) compared with controls and may have promise for treatment of some fish disease. Parallel groups of fish were maintained on the respective treatments for 15 days, with an additional group that was fasted, but were not infected to monitor autophagy. Fish that were fasted or fed DON had significantly increased LC3II in the liver and fasted fish had significantly decreased LC3II in muscle compared with controls using western blot. There was no difference in LC3II signal in the spleen of any treatment group. Fish that were fasted or pair-fed had significant up-regulation of the Atg genes atg4, atg7, lc3, gabarap and atg12 in muscle using quantitative PCR. Less alteration of Atg expression was seen in liver. Fish treated with CQ had significantly increased expression of atg4, becn1, lc3 and atg12 in the liver. Fish fed DON for 15 days had few alterations of Atg genes in either the liver or muscle. It is still not clear if autophagy is responsible for the resistance of rainbow trout fed DON, CQ or pair-fed before F. psychrophilum infection.

Phage predation constitutes a major mortality factor for bacteria in aquatic ecosystems, and thus, directly impacts nutrient cycling and microbial community dynamics. Yet, the population dynamics of specific phages across time scales from days to months remain largely unexplored, which limits our understanding of their influence on microbial succession. To investigate temporal changes in diversity and abundance of phages infecting particular host strains, we isolated 121 phage strains that infected three bacterial hosts during a Baltic Sea mesocosm experiment. Genome analysis revealed a novel Flavobacterium phage genus harboring gene sets putatively coding for synthesis of modified nucleotides and glycosylation of bacterial cell surface components. Another novel phage genus revealed a microdiversity of phage species that was largely maintained during the experiment and across mesocosms amended with different nutrients. In contrast to the newly described Flavobacterium phages, phages isolated from a Rheinheimera strain were highly similar to previously isolated genotypes, pointing to genomic consistency in this population. In the mesocosm experiment, the investigated phages were mainly detected after a phytoplankton bloom peak. This concurred with recurrent detection of the phages in the Baltic Proper during summer months, suggesting an influence on the succession of heterotrophic bacteria associated with phytoplankton blooms.

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

Iron is essential for growth and virulence in most pathogenic bacterial strains. In some cases, the hosts for these pathogenic bacteria develop specialized strategies to sequester iron and limit infectivity. This in turn may result in the invading pathogens utilizing high-affinity iron transport mechanisms, such as the use of iron-chelating siderophores, to extend beyond the host defences. Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD) in salmonids, relies on iron metabolism for infectivity, and the genome of the model CSF-259-93 strain has recently been made available. Further, this strain serves as a parent strain for a live-attenuated vaccine strain, B.17, which has been shown to provide rainbow trout with protection against BCWD. To elucidate specific gene expression responses to iron metabolism and compare strain differences, both F. psychrophilum strains were grown under iron-limiting conditions and 26 genes related to iron metabolism were mapped for 96 hr in culture via qPCR analyses. Results indicate increased production of the ferrous iron transport protein B (FITB; p =.008), and ferric receptor CfrA (FR 1; p =.012) in the wild-type CSF-259-93 strain at 72 hr and 96 hr post-exposure to iron-limiting media. In the B.17 vaccine strain, siderophore synthase (SS) expression was found to be downregulated at 72 hr, in comparison with 0h (p =.018). When strains were compared, FITB (p =.021), FR1 (p =.009) and SS (p =.016) were also elevated in B.17 at 0 hr and TonB outer protein membrane receptor 1 (TBomr1; p =.005) had a lower expression at 96 hr. Overall, this study demonstrated strain-related gene expression changes in only a fraction of the iron metabolism genes tested; however, results provide insight on potential virulence mechanisms and clarification on iron-related gene expression for F. psychrophilum.

Background Industrial Fuling Zhacai is pickled by a method summarized as 'three times pickled and pressed', in which raw mustard tubers are subjected to three stages of pickling in different salt concentrations, with a pressing operation at the end of each stage to remove brine. This study used Illumina MiSeq technology and multivariate statistical analyses to investigate microbial community succession during the pickling process and its correlation with Zhacai biochemical indices. Results A total of 19 phyla, 208 genera, and 295 species of bacteria were identified. Lactobacillus was the dominant genus of bacteria in all three stages and Lactobacillus sakei was the dominant species in the first and second stages. A total of six phyla, 200 genera and 301 species of fungi were also identified. According to a PICRUSt2 prediction, the main functions of the bacterial and fungal communities were carbohydrate and protein metabolism, while alcohol metabolism was also a function of fungi. Nine bacterial genera closely correlated with Zhacai biochemical indices: Acinetobacter, Pseudomonas, Pedobacter, Erwinia, Lactobacillus, Chryseobacterium, Flavobacterium, Duganella, and Paenarthrobacter. Six genera of fungi correlated closely: Penicillium, Cystobasidium, Cladosporium, Plenodomus, Aspergillus, and Simplicillium. All these genera probably originated from the surface microorganisms of raw mustard tuber. Conclusion This study reveals the succession patterns of microbial community structures during the pickling process of industrial Zhacai and infers the core functional flora, providing reference data for Zhacai pickling process control. © 2020 Society of Chemical Industry.

Columnaris disease generates substantial losses of many freshwater fish species; one is the hybrid striped bass. The ubiquitous aquatic bacterium Flavobacterium columnare can be highly effective in biofilm formation on fish skin and gills. Previous research showed a difference between columnaris disease susceptibility of hybrid striped bass (Morone saxatilis × M. chrysops) and white bass (M. chrysops). To understand these differential susceptibilities and possible mucosal relationship, we assessed total bacterial growth and biofilm formation with mucus derived from each moronid parental species: white bass and striped bass (M. saxatilis). Differential susceptibility was confirmed of the other parent species, the striped bass (M. saxatilis). In addition to intraspecies investigations, individual hybrid striped bass mucosal affects were also studied for deferential responses to bacterial growth and biofilm formation. Species- and concentration-dependent differences were detected in the total growth of the bacteria to host mucus. Our data suggest that bass mucus can significantly affect biofilm formation with the F. columnare isolate tested. There appears to be a correlation between the bacteria's response of growth and biofilms and bass species susceptibility. This study provides insight into our understanding of the host-pathogen interaction between F. columnare and moronids.

Flavobacteria are among the most important pathogens in freshwater salmonid aquaculture worldwide. Due to concerns regarding development of antibiotic resistance, phage therapy has been proposed as a solution to decrease pathogen load. However, application of phages is challenged by the development of phage resistance, and knowledge of the mechanisms and implications of phage resistance is therefore required. To study this, twenty-seven phage-resistant isolates of F. psychrophilum were genome sequenced and characterized to identify genetic modifications and evaluate changes in phenotypic traits, including virulence against rainbow trout. Phage-resistant isolates showed reduction or loss of gliding motility, proteolytic activity, and adhesion to surfaces, and most isolates were completely non-virulent against rainbow trout fry. Genomic analysis revealed that most phage resistant isolates had mutations in genes associated with gliding motility and virulence. Reversal of these mutations in a sub-set of isolates led to regained motility, proteolytic activity, virulence, and phage susceptibility. Although costly, the fast generation of phage resistance driven by single, reversible mutations likely represents a flexible and efficient phage defence mechanism in F. psychrophilum. The results further suggest that phage administration in aquaculture systems to prevent F. psychrophilum outbreaks selects for non-virulent phage resistant phenotypes.

The flavobacterium Chryseobacterium polytrichastri was investigated for its volatile profile by use of a closed‐loop stripping apparatus (CLSA) and subsequent GC‐MS analysis. The analyses revealed a rich headspace extract with 71 identified compounds. Compound identification was based on a comparison to library mass spectra for known compounds and on a synthesis of authentic standards for unknowns. Important classes were phenylethyl amides and a series of corresponding imines and pyrroles., Over 70 volatile organic compounds have been identified in a headspace extract from C. polytrichastri. The extract was particularly rich in nitrogen‐containing natural products, many of which were structurally derived from 2‐phenylethylamine. Several compounds are new natural products, their suggested structures are based on the observed mass spectral fragmentation, confirmed by synthesis of reference substances.

Burbot (Lota lota maculosa) are a potential new species for commercial aquaculture. As burbot culture expands, there is a need to further define pathogen susceptibility and characterize aspects of the burbot immune response in an effort to assess fish health. A recent clinical diagnostic case from juvenile burbot reared at a commercial production facility resulted in the isolation and identification of Flavobacterium columnare along with several Aeromonas spp. The F. columnare isolate was assigned to genetic group 1 via multiplex PCR, a genetic group commonly associated with columnaris disease cases in rainbow trout (Oncorhynchus mykiss). Virulence of the F. columnare isolate was assessed in vivo in both juvenile burbot and rainbow trout. Additionally, several of the Aeromonas sp. case isolates were identified via sequencing (16S rRNA, gyrB and rpoD) and a putative A. sobria isolate (BI-3) was used to challenge burbot, along with a known virulent Aeromonas sp. (A141), but BI-3 was not found to be virulent. Burbot were refractory to F. columnare when challenged by immersion, and it is likely that this is a secondary pathogen for burbot. Although refractory in burbot, the identified F. columnare isolate (BI-1) was found to be virulent in rainbow trout.

Marine flavobacteria are specialists for polysaccharide degradation. They dominate in habitats enriched with polysaccharides, but are also prevalent in pelagic environments where polysaccharides are less available. These niches are likely occupied by distinct lineages, but evolutionary processes underlying their niche differentiation remain elusive. Here, genomic analyses and physiological assays indicate that the sister flavobacteria lineages Leeuwenhoekiella and Nonlabens likely explore polysaccharide-rich macroalgae and polysaccharide-poor pelagic niches respectively. Phylogenomic analyses inferred that the niche separation likely occurred anciently and coincided with increased sequence evolutionary rate in Nonlabens compared with Leeuwenhoekiella. Further analyses ruled out the known mechanisms likely driving evolutionary rate acceleration, including reduced selection efficiency, decreased generation time and increased mutation rate. In particular, the mutation rates were determined using an unbiased experimental method, which measures the present-day populations and may not reflect ancestral populations. These data collectively lead to a new hypothesis that an ancestral and transient mutation rate increase resulted in evolutionary rate increase in Nonlabens. This hypothesis was supported by inferring that gains and losses of genes involved in SOS response, a mechanism known to drive transiently increased mutation rate, coincided with evolutionary rate acceleration. Our analyses highlight the evolutionary mechanisms underlying niche differentiation of flavobacteria lineages.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout syndrome in freshwater salmonid fish worldwide, generating injuries and high mortality rates. Despite several studies on this bacterium, the infection mechanism remains unknown due to limitations in the employed animal models. In this work, we propose using zebrafish (Danio rerio) as a model for studying bacterial pathogenicity. To substantiate this proposal, zebrafish infection by F. psychrophilum strain JIP 02/86 was characterized. Zebrafish larvae were infected using the bath method, and morphological changes and innate immune system activation were monitored using transgenic fish. Salmonid-like infection phenotypes were observed in 4.74% of treated larvae, as manifested by fin, muscle and caudal peduncle damage. Symptomatic and dead larvae accounted for 1.35% of all challenged larvae. Interestingly, infected larvae with no infection phenotypes showed stronger innate immune system activation than specimens with phenotypes. A failure of function assay for myeloid factor pu.1 resulted in more infected larvae (up to 43.5%), suggesting that low infection rates by F. psychrophilum would be due to the protective actions of the innate immune system against this bacterium in zebrafish larvae. Our results support the use of zebrafish as an infection model for studying F. psychrophilum. Furthermore, the percentage of infected fish can be modulated by disturbing, to varying extents, the differentiation of myeloid cells. Using this evidence as a starting point, different aspects of the infection mechanism of F. psychrophilum could be studied in vivo.

Columnaris disease is responsible for substantial losses throughout the production of many freshwater fish species. One of the ways in which the bacterium Flavobacterium columnare is so effective in initiating disease is through the formation of biofilms on fish skin and gills. To further explore the interaction between host factors and bacterial cells, we assayed the ability of vertebrate mucus to enhance F. columnare biofilm development. Different concentrations of catfish, tilapia and pig mucus (5-60 µg/ml) increased biofilm growth at varying degrees among F. columnare isolates. Our data suggest that vertebrate mucus acts as a signalling molecule for the development of F. columnare biofilms; however, there are clear disparities in how individual isolates respond to different mucus fractions to stimulate biofilms. The expression of iron acquisition genes among two genomovar II isolates showed that ferroxidase, TonB receptor and the siderophore synthetase gene were all significantly upregulated among F. columnare biofilms. Interestingly, the siderophore acetyltransferase gene was only shown to be significantly upregulated in one of the genomovar II isolates. This work provides insight into our understanding of the interaction between F. columnare and vertebrate mucus, which likely contributes to the growth of planktonic cells and the transition into biofilms.

In late April 2015, the River Dee Trust informed Marine Scotland Science, Fish Health Inspectorate (FHI), that there had been observations of dead and moribund European eels on the River Dee. Later in May, the Spey Fishery Board also reported a number of moribund European eels in a rotary screw smolt trap on the River Spey. In total, 10 cases involving moribund eels were investigated in 2015 and one case in 2016. In addition, a health screen was conducted to investigate the potential presence of Flavobacterium psychrophilum in healthy eels and Atlantic salmon from the River Dee in 2015. Externally, the diseased eels demonstrated white patches in different locations of the body. In all cases, F. psychrophilum was detected by bacterial isolation and/or molecular methods. Three isolates were further characterized by whole-genome sequencing (WGS) as belonging to sequence type 15 (ST15). Histological examination of diseased European eels revealed lesions at the level of the integument. The pathogen screen for F. psychrophilum in wild healthy fish tested negative by PCR. Further investigation is required to understand the pathogenicity of this bacterium on the health of eels and the potential impact on the wild salmonid population.

International audience; In rainbow trout farming, Flavobacterium psychrophilum, the causative agent of bacterial cold water disease, is responsible for important economic losses. Resistance to F. psychrophilum is heritable, and several quantitative trait loci (QTL) with moderate effects have been detected, opening up promising perspectives for the genetic improvement of resistance. In most studies however, resistance to F. psychrophilum was assessed in experimental infectious challenges using injection as the infection route, which is not representative of natural infection. Indeed, injection bypasses external barriers, such as mucus and skin, that likely play a protective role against the infection. In this study, we aimed at describing the genetic architecture of the resistance to F. psychrophilum after a natural disease outbreak. In a 2000-fish cohort, reared on a French farm, 720 fish were sampled and genotyped using the medium-throughput Axiom (TM) Trout Genotyping Array. Overall mortality at the end of the outbreak was 25%. Genome-wide association studies were performed under two different models for time to death measured on 706 fish with validated genotypes for 30 060 SNPs. This study confirms the polygenic inheritance of resistance to F. psychrophilum with a few QTL with moderate effects and a large polygenic background, the heritability of the trait being estimated at 0.34. Two new chromosome-wide significant QTL and three suggestive QTL were detected, each of them explaining between 1% and 4% of genetic variance.

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country-wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin-inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin-killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate-buffered saline or vaccine formulated with Montanide by intra-peritoneal (i.p.) injection and challenged by intra-muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post-vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un-adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.

In recent years, several studies have examined the gut microbiome of lepidopteran larvae and how factors such as host plant affect it, and in turn, how gut bacteria affect host plant responses to herbivory. In addition, other studies have detailed how secretions of the labial (salivary) glands can alter host plant defense responses. We examined the gut microbiome of the cabbage looper (Trichoplusia ni) feeding on collards (Brassica oleracea) and separately analyzed the microbiomes of various organs that open directly into the alimentary canal, including the labial glands, mandibular glands, and the Malpighian tubules. In this study, the gut microbiome of T. ni was found to be generally consistent with those of other lepidopteran larvae in prior studies. The greatest diversity of bacteria appeared in the Firmicutes, Actinobacteria, Proteobacteria, and Bacteriodetes. Well‐represented genera included Staphylococcus, Streptococcus, Corynebacterium, Pseudomonas, Diaphorobacter, Methylobacterium, Flavobacterium, and Cloacibacterium. Across all organs, two amplicon sequence variants (ASVs) associated with the genera Diaphorobacter and Cloacibacterium appeared to be most abundant. In terms of the most prevalent ASVs, the alimentary canal, Malpighian tubules, and mandibular glands appeared to have similar complements of bacteria, with relatively few significant differences evident. However, aside from the Diaphorobacter and Cloacibacterium ASVs common to all the organs, the labial glands appeared to possess a distinctive complement of bacteria which was absent or poorly represented in the other organs. Among these were representatives of the Pseudomonas, Flavobacterium, Caulobacterium, Anaerococcus, and Methylobacterium. These results suggest that the labial glands present bacteria with different selective pressures than those occurring in the mandibular gland, Malpighian tubules and the alimentary canal. Given the documented effects that labial gland secretions and the gut microbiome can exert on host plant defenses, the effects exerted by the bacteria inhabiting the labial glands themselves deserve further study., Labial gland secretions and the gut microbiota of insects affect defense responses of plants to herbivory. However, the microbiome of the labial gland has not been investigated. This work using cabbage looper, a serious pest of cole crops, demonstrates that the labial glands possess a community of bacteria that is distinct from the gut and other associated organs. This discovery warrants additional research to determine what role the labial gland microbiota has on the plant response to insect pests.

Bacterial diseases cause major financial damage to the producers of Channel Catfish Ictalurus punctatus in the southeastern USA. The two most common bacterial diseases among pond-raised Channel Catfish are enteric septicemia of catfish, caused by the gram-negative bacterium Edwardsiella ictaluri, and columnaris disease, caused by the rod-shaped, gram-negative bacterium Flavobacterium columnare. Streptococcosis is another, less-common bacterial disease in catfish and is caused by the gram-positive coccus Streptococcus iniae. Catfish farmers typically rely on commercial antibiotics and other chemicals to prevent the economic damage from these diseases. Environmentally benign and efficacious alternatives to the currently used antibiotics and chemicals will tremendously help the catfish aquaculture industry. As part of our ongoing efforts in the search for such novel compounds, we investigated ethyl acetate and methanol extracts of mangosteen Garcinia mangostana fruit pericarp via bioassay-guided fractionation. Gamma-mangostin (γ-mangostin) was isolated and identified as the most promising active metabolite against F. columnare. One of the constituents in the mangosteen fruit pericarp, alpha-mangostin (α-mangostin), is the major xanthone; α-mangostin was found to be 10-fold less active than γ-mangostin when minimum inhibitory concentration values were compared.

Flavobacterial diseases are significant impediments to hatchery-based fishery conservation and aquaculture productivity worldwide. Recent studies revealed a multitude of novel flavobacteria within the reproductive fluids and unfertilized eggs of feral Chinook salmon Oncorhynchus tshawytscha broodstock, some of which were associated with systemic disease. Herein, embryonated eggs/fry from these broodstock were assayed for flavobacteria while in incubator stacks in three hatcheries over 2 years, as was the water entering hatchery incubators. Overall, >65% of sampled eggs and 38% of fry were colonized by flavobacteria. One hundred and ninety-one egg and fry-associated flavobacterial isolates were characterized phenotypically and via 16S rRNA gene sequencing and phylogenetic analyses, revealing that the majority fell into 22 clades (i.e., 15 Flavobacterium spp. groups and seven Chryseobacterium spp. groups) that varied in presence by facility. Although some matched previously described fish-pathogenic species, the majority were distinct from all described flavobacteria and likely represent novel species. Of concern, iodophor disinfection at the commonly utilized dose/duration for egg-surface disinfection did not eliminate flavobacteria. Results also implicated maternal routes of infection and source water for some flavobacteria. In total, study findings underscore the complexity of flavobacterial ecology within hatchery environments and highlight the need for improved hatchery biosecurity practices.

Algal polysaccharides of diverse structures are one of the most abundant carbon resources for heterotrophic, marine bacteria with coevolved digestive enzymes. A putative sulfo-mannan polysaccharide utilization locus, which is conserved in marine flavobacteria, contains an unusual GH99-like protein that lacks the conserved catalytic residues of glycoside hydrolase family 99. Using X-ray crystallography, we structurally characterized this protein from the marine flavobacterium Ochrovirga pacifica to help elucidate its molecular function. The structure reveals the absence of potential catalytic residues for polysaccharide hydrolysis, which-together with additional structural features-suggests this protein may be noncatalytic and involved in carbohydrate binding.

Columnaris disease is a major cause of mortality in tilapia hatcheries and commonly occurs during the summer season in Thailand. One way of reducing the problem is by selective breeding for increased disease resistance. The objective of this study was to estimate quantitative genetic parameters for resistance against columnaris in the Chitralada 4 strain of Nile tilapia. Data from 43 full-sib families (2,580 records) of fry (age = 32 ± 4 days post-hatch) were used in the analyses. Initially, fry were subjected to bath challenge with Flavobacterium columnare (LD50 concentration = 1.2 × 106 CFU/ml) for 14 days. Disease resistance was defined as the number of days from challenge until death (DD) or as a binary trait (dead/alive) on day 14. Linear animal and sire-dam models were used for DD, while threshold animal, threshold sire-dam, binary linear animal and binary linear sire-dam models were used for binary outcomes. Covariate effect of age, fixed effect of challenge day and random effects of the individual animals or sires and dams were included in the models. Mean survival was 32.4 ± 11.6%, and survival rates of the best and poorest families were 70% and 8%, respectively. The highest estimate of heritability (0.30 ± 0.025) was obtained under the threshold sire-dam model. Heritability estimates for DD (0.16 ± 0.034 and 0.17 ± 0.046) were comparable to those obtained from the threshold animal (0.15 ± 0.031) and the binary linear (0.14 ± 0.045 and 0.15 ± 0.044) models. The linear animal and sire-dam models for DD and the threshold sire-dam models performed equally with similar values of rEBV (0.629, 0.628 and 0.627) and accuracy of selection (0.793, 0.793 and 0.791). This study reveals the potential of selective breeding to increase disease resistance to F. columnare in the studied population of Nile tilapia.

Flavobacterium columnare, the causative agent of columnaris disease, causes substantial mortality worldwide in numerous freshwater finfish species. Due to its global significance and impact on the aquaculture industry continual efforts to better understand basic mechanisms that contribute to disease are urgently needed. The current work sought to evaluate the effect of L-rhamnose on the growth characteristics of F. columnare. While we initially did not observe any key changes during the total growth of F. columnare isolates tested when treated with L-rhamnose, it soon became apparent that the difference lies in the ability of this carbohydrate to facilitate the formation of biofilms. The addition of different concentrations of L-rhamnose consistently promoted the development of biofilms among different F. columnare isolates; however, it does not appear to be sufficient as a sole carbon source for biofilm growth. Our data also suggest that iron acquisition machinery is required for biofilm development. Finally, the addition of different concentrations of L-rhamnose to F. columnare prior to a laboratory challenge increased mortality rates in channel catfish (Ictalurus punctatus) as compared to controls. These results provide further evidence that biofilm formation is an integral virulence factor in the initiation of disease in fish.

Arsenotrophic bacteria contribute to the nutrient cycling in arsenic (As) affected groundwater. This study employed a culture-independent and -dependent investigation of arsenotrophic microbiomes in As affected groundwater samples collected from Madhabpur, Sonatengra, and Union Porishod in Singair Upazila, Manikganj, Bangladesh. Total As contents, detected by Atomic Absorption Spectrophotometry (AAS) of the samples, were 47 µg/L (Madhabpur, SNGW-1), 53 µg/L (Sonatengra, SNGW-2), and 12 µg/L (Union porishod, SNGW-3), whereas the control well (SNGW-4; depths >150 m) showed As content of 6 µg/L. Denaturing Gradient Gel Electrophoresis (DGGE) analysis of the amplified 16S rRNA gene from As-affected groundwater samples revealed the dominance of aerobic bacteria Pseudomonas within heterogeneous bacterial populations. DGGE of heterotrophic enrichments supplemented with arsenite [As (III)] for 4 weeks showed the dominance of Chryseobacterium, Flavobacterium, and Aquabacterium, whereas the dominant genera in that of autotrophic enrichments were Aeromonas, Acinetobacter, and Pseudomonas. Cultured bacteria retrieved from both autotrophic and heterotrophic enrichments were distinguished into nine genotypes belonging to Chryseobacterium, Acinetobacter, Escherichia, Pseudomonas, Stenotrophomonas, Janibacter, Staphylococcus, and Bacillus. They exhibited varying range of As(III) tolerance from 4 to 27 mM. As(III) transformation potential was confirmed within the isolates with oxidation rate as high as 0.143 mM/h for Pseudomonas sp. Sn 28. The arsenotrophic microbiome specifies their potential role in groundwater As-cycling and their genetic information provide the scientific basis for As-bioremediation.

The objective of this study was to determine whether common bacterial catfish pathogens could attach and colonize surfaces commonly found in aquaculture facilities. In addition, we evaluated the role of calcium in biofilm formation. Attachment to polystyrene plates was used to quantify biofilm formation by five bacterial pathogens (i.e., Flavobacterium columnare, Aeromonas hydrophila, Edwardsiella ictaluri, E. tarda, and E. piscicida). Flavobacterium columnare and A. hydrophila formed thick biofilms that were enhanced by calcium supplementation. Biofilm formation was significantly lower in all Edwardsiella species tested and calcium had little to no effect on Edwardsiella biofilm formation. Attachment to natural and artificial surfaces was quantified by a standard plate count method. Scanning electron microscopy (SEM) was used to confirm biofilm formation on the substrates. Flavobacterium columnare formed biofilm on the liner, flexible PVC, and nets. Bamboo prevented F. columnare attachment and inhibited cell growth. Aeromonas hydrophila and E. ictaluri formed biofilm on all materials tested, although significant differences were found among substrates. While E. ictaluri failed to form biofilm on microtiter polystyrene plates, it was able to colonize and multiply on all aquaculture materials tested. Our results demonstrated that common bacterial pathogens had the potential of colonizing surfaces and may use biofilm as reservoirs in fish farms. Received July 19, 2016; accepted January 19, 2017.

PROBLEM Intrauterine microbial colonization and its association with the pathogenesis of endometriosis via an innate immune cascade have been reported. As a potential source of microbial transmission, information on microbial colonization in cervical mucus is unknown. We investigated pattern of microbiota in the cervical mucus collected from women with and without endometriosis using next-generation sequencing (NGS) technology. METHOD OF STUDY Cervical mucus samples were collected from women with (n = 30) and without (n = 39) endometriosis. The communities of microbiota in cervical mucus in the endometriosis group and the control group were examined by Gram staining and NGS targeting the V5-V6 region of 16S ribosomal RNA gene. Copy number of some target bacteria was detected by real-time PCR. RESULTS We confirmed visual presence of bacteria in cervical mucus by Gram staining. NGS analysis showed that distribution of microbiota was similar in cervical mucus of women with and without endometriosis regardless of the phases of the menstrual cycle. In addition to predominant Lactobacilli spp., the populations of Corynebacterium, Enterobacteriaceae, Flavobacterium, Pseudomonas, and Streptococcus were increased in the endometriosis group. Of them, Enterobacteriaceae and Streptococcus were identified as the more significant candidates in the endometriosis group than in controls by real-time PCR (P

Co-infection of rainbow trout with infections haematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum is known to occur, and it has been speculated that a combined infection can result in dramatic losses. Both pathogens can persist in fish in an asymptomatic carrier state, but the impact of co-infection has not been well characterized or documented. In this study, it was hypothesized that fish co-infected with F. psychrophilum and IHNV would exhibit greater mortality than fish infected with either pathogen alone. To test this, juvenile rainbow trout were co-infected with low doses of either IHNV or F. psychrophilum, and at 2 days post-initial challenge, they were given a low dose of the reciprocal pathogen. This combined infection caused high mortality (76.2%-100%), while mortality from a single pathogen infection with the same respective dose was low (5%-20%). The onset of mortality was earlier in the co-infected group (3-4 days) when compared with fish infected with F. psychrophilum alone (6 days) or IHNV (5 days), confirming the synergistic interaction between both pathogens. Co-infection led to a significant increase in the number of F. psychrophilum colony-forming units and IHNV plaque-forming units within tissues. This finding confirms that when present together in co-infected fish, both pathogens are more efficiently recovered from tissues. Furthermore, pathogen genes were significantly increased in co-infected groups, which parallel the findings of increased systemic pathogen load. Extensive tissue necrosis and abundant pathogen present intracellularly and extracellularly in haematopoietic tissue. This was pronounced in co-infected fish and likely contributed to the exacerbated clinical signs and higher mortality. This study provides novel insight into host-pathogen interactions related to co-infection by aquatic bacterial and viral pathogens and supports our hypothesis. Such findings confirm that mortality in fish exposed to both pathogens is greatly elevated compared to a single pathogen infection.

Drought stress adversely affects the growth and yield of wheat. The present study was planned to investigate the effect of inoculation of plant-growth promoting rhizobacteria (PGPR) strains IG 3 (Klebsiella sp.), IG 10 (Enterobacter ludwigii) and IG 15 (Flavobacterium sp.) in improving drought tolerance in wheat. These PGPR strains were screened for drought tolerance in nutrient broth supplemented with different concentrations (0-25%) of polyethylene glycol (PEG6000). Effect of PGPR inoculation on various physiological, biochemical parameters and gene expression of stress responsive genes were studied under drought stress. Root colonization at the surface and interiors of roots was demonstrated using scanning electron microscopy (SEM) and tetrazolium staining, respectively. Drought stress significantly affected various growth parameters, water status, membrane integrity, osmolyte accumulation and stress-responsive gene expressions, which were positively altered by PGPR-inoculation in wheat. Quantitative real-time (qRT)-PCR analysis revealed the up regulation of some stress-related genes (DREB2A and CAT1) in un-inoculated wheat plants exposed to drought stress. PGPR-inoculated plants showed attenuated transcript levels suggesting improved drought tolerance due to interaction of PGPRs. The PGPR strain IG 3 was found to be the best in terms of influencing biochemical and physiological status of the seedlings under drought stress. Our report demonstrates the role of PGPRs Enterobacter ludwigii and Flavobacterium sp. in plant growth promotion of wheat plants under drought stress. The study reports the potential of PGPR in alleviating drought stress in wheat which could be used as potent biofertilizers.

The gliding aquatic bacterium Flavobacterium columnare causes columnaris disease, a common problem for wild and farmed freshwater fish worldwide. Recently, a broth microdilution method was standardized to test the susceptibility of F. columnare against antimicrobials commonly used in aquaculture. We used this new method to measure the minimal inhibitory concentrations (MICs) of ten antimicrobials against 120 F. columnare isolates. The resulting MIC frequency distributions for each antimicrobial (1 MIC/isolate) were used to estimate epidemiological cut-off values (ECVs) which separate isolates with typical wild-type (WT) susceptibility from isolates with decreased non-wild-type (NWT) susceptibility. We identified 22 NWT isolates with elevated MICs relative to the ECV that covered 99.9% of the MIC distribution against one or more of the antimicrobials: ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, oxolinic acid or oxytetracycline. Ten of the NWT isolates had decreased susceptibility to a single antimicrobial class, six isolates to two antimicrobial classes and six isolates to three or more antimicrobial classes. The MIC frequency distributions and provisional cut-off values provide data needed to set epidemiological cut-off values to monitor for the development of antimicrobial resistance among F. columnare.

In inflammatory bowel diseases (IBD), microbial communities often become imbalanced suggesting abnormal microbial-gut interactions. In this study, we analysed the mucosa-attached gut microbiota from 26 Crohn's disease (CD) patients using 16S rRNA gene amplicon sequencing. The samples were stratified according to their disease activity (Crohn's disease activity index, CDAI). The different disease activity categories had a comparable bacterial richness. Bacterial communities of patients in remission and intermediate CDAI (0-220) were relatively similar and dominated by the genus Bacteroides (>40%). The bacterial composition of patients assigned to a high CDAI category was dominated by Pelomonas (25%) and Flavobacterium (13%) but had a low relative abundance of Bacteroidetes (4%). This indicates the presence of specific abundant bacterial taxa at different CDAI levels. In addition, bacterial communities were also significantly influenced when a tumour necrosis factor (TNF)-α inhibitor was applied or by the local mucosal inflammation level. As a consequence, a shift of the microbial composition may also indicate a change of the disease activity in CD patients.

Flavobacterium columnare, the aetiological agent of columnaris disease, causes significant losses in fish worldwide. In this study, the prevalence of F. columnare infection was assessed in representative Great Lakes fish species. Over 2000 wild, feral and hatchery-propagated salmonids, percids, centrarchids, esocids and cyprinids were examined for systemic F. columnare infections. Logistic regression analyses showed that the prevalence of F. columnare infection varied temporally and by the sex of the fish, whereby females had significantly higher prevalence of infection. A total of 305 isolates of F. columnare were recovered. Amplification of the near complete 16S rRNA gene from 34 representative isolates and subsequent restriction fragment length polymorphism analyses demonstrated that all belonged to F. columnare genomovar I. Phylogenetic analysis of near complete 16S rRNA gene sequences also placed the isolates in genomovar I, but revealed some intragenomovar heterogeneity. Together, these results suggest that F. columnare genomovar I is widespread in the Great Lakes Basin, where its presence may lead to mortality.

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016.

Bacterial counts of 170 radish sprout (Raphanus sativus) retailed in Japan between June 2012 and February 2013 was examined. Aerobic plate counts and coliform counts exhibited seasonal variations. No Shiga toxin-producing Escherichia coli or Salmonella spp. were detected from them. 16S rRNA pyrosequencing analysis of 16 representative samples from one farm (farm D) revealed a predominance of Pseudomonas spp. throughout seasons, and summer samples exhibited an increase in Enterobacteriaceae including Escherichia spp. and decreases in Oxalobacteraceae (primarily Duganella and Janthinobacterium spp.) and Flavobacteriaceae (Flavobacterium spp.) compared with winter samples. Quantitative-PCR analysis of 40 samples from the farm D confirmed the seasonal dynamics of E. coli and Flavobacterium spp. Radish seeds increased proportion of Pseudomonas spp. after sprouting, compared with pre-sprouted seeds, suggesting that increased proportion of Pseudomonas spp. in retailed samples might be sourced from their seeds. Our data thus provides that atmospheric control at postsprouting stage might be hygienic practical point to secure microbial safety. Practical Applications Fresh sprouts are common vehicles of foodborne pathogens that have been responsible for much numbers of outbreaks worldwide. Through the use of indicator bacterial counts in combination with metagenomic approaches, we confirmed the seasonal and growth-dependent variation of bacterial community structure in retailed radish sprouts. Information and relief provided to manufacturers/consumers could help to secure microbial safety associated with fresh sprouts.

A survey of kidney bacteria from female landlocked fall Chinook Salmon Oncorhynchus tshawytscha broodstock from Lake Oahe, South Dakota, was conducted to investigate possible links to subsequent egg survival. Bacteria were isolated from 22 spent females. The kidneys of two females contained no bacteria, and no eggs survived from the spawn of these two fish. All kidneys sampled appeared normal with no gross abnormalities. Twelve bacteria genera were isolated and identified to the genus level: Acinetobacter, Aeromonas, Brevundimonas, Chryseobacterium, Flavobacterium, Acidovorax, Arthrobacter, Deinococcus, Enterococcus, Leucobacter, Micrococcus, and Oerskovia. Screening for Renibacterium salmoninarum was not conducted. Colony forming units (CFU) per gram ranged from 0 to 10,542. There were no significant correlations between egg survival and CFU from samples incubated on agar containing either R2A or Coomassie Brilliant Blue. In addition, no relationship was observed between any of the bacterial spec...

Fish diseases caused by multiple genera within the family Flavobacteriaceae (Phylum Bacteroidetes) are a major impediment to farmed teleosts. At least one fish-pathogenic flavobacterial species (e.g., Flavobacterium psychrophilum) is transmitted from parents to progeny via infected sexual products, but the majority of research has focused on the role that ovarian fluid and eggs play in bacterial transmission. However, a handful of studies have revealed that milt can also harbor F. psychrophilum. Herein, we report on the isolation and molecular identification of multiple Flavobacterium and Chryseobacterium spp. from the milt of feral Great Lakes Chinook Salmon Oncorhynchus tshawytscha. Some of the milt-associated flavobacteria were identified as well-known fish pathogens (e.g., F. columnare, etiological agent of columnaris disease), whereas others were most similar to emerging fish pathogens (e.g., C. piscium) or were distinct from all our described Flavobacterium and Chryseobacterium spp., accordi...

The aims of this study are the description of diversity for proteorhodopsin (PR)‐containing flavobacteria in marine environments, the finding of novel photoreceptive membrane proteins, and the elucidation of the effect of light on the growth of three rhodopsin genes containing flavobacterium. We investigated novel sodium ion rhodopsin (NaR) and halorhodopsin (HR) genes from PR‐containing flavobacteria that were previously isolated from diverse aquatic sites, mainly from tidal flat sediment (62.5%). In 16 PR‐containing isolates, three new types of genes were found. Among these three isolates, one (Nonlabens sp. YIK11 isolated from sediment) contained both the NaR and chloride ion rhodopsin (ClR) ‐ HR type of gene. The sequences showed that the DTE (proton pump), NDQ (sodium ion pump) and NTQ (chloride ion pump) motifs corresponding to the D85, T89, and D96 positions in bacteriorhodopsin (BR) were well conserved. Phylogenetic analysis indicated that three NaR and one ClR grouped within the same clade, as previously reported. Illumination of cell suspensions showed the change in proton pump activity, supporting that one or more rhodopsins are functional. The qRT‐PCR study revealed that three rhodopsin genes, especially NaR, are highly induced when they are incubated in the presence of light or in the absence of sufficient nutrients. The expression levels of the DTE, NDQ, and NTQ motif‐containing rhodopsin genes in YIK11 correlate positively with illumination, but negatively with nutrient levels. Based on those results, we concluded that light has a positive impact on the relative expression levels of the three rhodopsin genes in the flavobacterium, Nonlabens sp. YIK11, but with no apparent positive impact on growth. Consequently, light did not stimulate the growth of YIK11 as determined by cell numbers in a nutrient‐limited or ‐enriched medium, although it contains and induces three rhodopsins.

The freshwater trematode Nanophyetus salmincola has been demonstrated to impair salmonid immune function and resistance to the marine pathogen Vibrio anguillarum, potentially resulting in ocean mortality. We examined whether infection by the parasite N. salmincola similarly increases mortality of juvenile Chinook Salmon Oncorhynchus tshawytscha when they are exposed to the freshwater pathogens Flavobacterium columnare or Aeromonas salmonicida, two bacteria that juvenile salmonids might encounter during their migration to the marine environment. We used a two-part experimental design where juvenile Chinook Salmon were first infected with N. salmincola through cohabitation with infected freshwater snails, Juga spp., and then challenged with either F. columnare or A. salmonicida. Cumulative percent mortality from F. columnare infection was higher in N. salmincola-parasitized fish than in nonparasitized fish. In contrast, cumulative percent mortality from A. salmonicida infection did not differ between N. salmincola-parasitized and nonparasitized groups. No mortalities were observed in the N. salmincola-parasitized-only and control groups from either challenge. Our study demonstrates that a relatively high mean intensity (>200 metacercariae per posterior kidney) of encysted N. salmincola metacercariae can alter the outcomes of bacterial infection in juvenile Chinook Salmon, which might have implications for disease in wild fish populations.

Bacterial coldwater disease (BCWD), caused by Flavobacterium psychrophilum, threatens wild and propagated salmonids worldwide and leads to substantial economic losses. In addition to being horizontally transmitted, F. psychrophilum can be passed from infected parents to their progeny, furthering the negative impacts of this pathogen. In Michigan, both feral and captive salmonid broodstocks are the gamete sources used in fishery propagation efforts. A 5-year study was initiated to follow the prevalence of systemic F. psychrophilum infections in feral broodstocks of four species (steelhead Oncorhynchus mykiss [potadromous Rainbow Trout]; Coho Salmon O. kisutch; Chinook Salmon O. tshawytscha; and Atlantic Salmon Salmo salar) residing in three Great Lakes watersheds. Additionally, captive broodstocks of four species (Rainbow Trout, Brown Trout Salmo trutta, Lake Trout Salvelinus namaycush, and Brook Trout Salvelinus fontinalis) maintained at two facilities were assessed for the presence of F. psychrophilum. The resultant offspring from each broodstock population were sampled for F. psychrophilum infections multiple times throughout hatchery residency. Using selective flavobacterial culture and PCR confirmation, F. psychrophilum was detected in all broodstocks except the captive Lake Trout and Brook Trout. Logistic regression analysis demonstrated that among the infected feral broodstocks, Chinook Salmon from the Lake Michigan watershed had the highest prevalence of systemic F. psychrophilum infection (mean = 63.2%). Among the captive broodstocks, the Gilchrist Creek strain of Brown Trout had the highest infection prevalence (mean = 5%). Collectively, the captive broodstocks were found to have significantly lower infection prevalence than the feral broodstocks. Despite the high prevalence of systemic F. psychrophilum infections in many broodstock populations, the bacterium was rarely detected in their progeny during hatchery rearing. However, heavy losses associated with clinical BCWD outbreaks did occur. Collectively, our results reinforce that BCWD continues to threaten Great Lakes basin salmonids.

Columnaris disease, caused by the bacterium Flavobacterium columnare, is currently the most frequently reported bacterial disease affecting farm-raised channel catfish in the USA. Common treatments against the disease include the use of medicated feed that has led to emergent antibiotic resistant strains of F. columnare. Nigella sativa (Black cumin) is a medicinal herb commonly used by many cultures as a natural remedy for numerous disorders. Recently, we have discovered the antibacterial activity of N. sativa and its oil extract against F. columnare. In this study, we showed N. sativa oil (NSO) strongly inhibited the growth of all of the strains of F. columnare tested and yielded significantly larger zones of inhibition than those produced by oxytetracyclin. We tested the protective effect against columnaris disease in vivo by incorporating NSO (5%) or N. sativa seeds (NSS) (5%) into fish feeds. Fishes (Ictalurus punctatus and Danio rerio) fed amended diets displayed significantly lower mortality than those fed control diets. Per cent mortalities in control groups ranged from 77% to 44% and from 70% to 18% in zebrafish and channel catfish, respectively. A dose study using different NSS concentrations showed that 5% NSS offered the most protection against columnaris disease in channel catfish.

Numerous isolates of Flavobacterium columnare were previously recovered from red tilapia, Oreochromis sp., exhibiting columnaris-like disease in Thai farms, and the phenotypic and genetic characteristics were described. The objective of this study was to determine the virulence of two morphotypes (rhizoid and non-rhizoid colonies) of F. columnare and to determine their ability to adhere to and persist in red tilapia fry. The results showed that the typical rhizoid isolate (CUVET1214) was a highly virulent isolate and caused 100% mortality within 24 h following bath challenge of red tilapia with three different doses. The non-rhizoid isolate (CUVET1201) was avirulent to red tilapia fry. Both morphotypes adhered to and persisted in tilapia similarly at 0.5 and 6 h post-challenge as determined by whole fish bacterial loads. At 24 and 48 h post-challenge, fry challenged with the rhizoid morphotype exhibited significantly higher bacterial loads than the non-rhizoid morphotype. The results suggested that an inability of the non-rhizoid morphotype to persist in tilapia fry may explain lack of virulence.

In our on-going pursuit to discover natural products and natural product-based compounds to control the bacterial species Flavobacterium columnare, which causes columnaris disease in channel catfish (Ictalurus punctatus), we synthesized flavone and chalcone analogs, and evaluated these compounds, along with flavonoids from natural sources, for their antibacterial activities against two isolates of F. columnare (ALM-00-173 and BioMed) using a rapid bioassay. The flavonoids chrysin (1a), 5,7-dihydroxy-4'-methoxyflavone (11), isorhamnetin (26), luteolin (27), and biochanin A (29), and chalcone derivative 8b showed strong antibacterial activities against F. columnare ALM-00-173 based on minimum inhibition concentration (MIC) results. Flavonoids 1a, 8, 11, 13 (5,4'-dihydroxy-7-methoxyflavone), 26, and 29 exhibited strong antibacterial activities against F. columnare BioMed based upon MIC results. The 24-h 50% inhibition concentration (IC50 ) results revealed that 27 and 29 were the most active compounds against F. columnare ALM-00-173 (IC50 of 7.5 and 8.5 mg/l, resp.), while 26 and 29 were the most toxic compound against F. columnare BioMed (IC50 of 9.2 and 3.5 mg/l, resp.). These IC50 results were lower than those obtained for wogonin against F. columnare ALM-00-173 and F. columnare BioMed (28.4 and 5.4 mg/l, resp.). However, based on MIC results, none of the compounds evaluated in this study were as active as wogonin (MIC 0.3 mg/l for each F. columnare isolate). Further modification of the wogonin structure to enhance antibacterial is of interest.

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, has lead to the loss of significant numbers of hatchery-reared salmonids. The bacteria can be spread from parent to progeny within contaminated sperm and ovarian fluid and can enter the egg during fertilization. The addition of antibiotics to diluents and water-hardening solutions could prevent the spread of the disease. In separate trials, a mixture of 0.197 mg/mL penicillin plus 0.313 mg/mL streptomycin was added to both a 0.5% sodium chloride fertilization diluent and hatchery well water during hardening. Tests showed that the addition of the antibiotics to the diluent and during up to 60 min of water hardening had no effect on the eye-up, hatch and deformity rates of Rainbow Trout Oncorhynchus mykiss eggs compared with the nonantibiotic-treated controls. Also, significant reductions in the prevalence of F. psychrophilum on the surface and inside eggs were observed when compared with controls. These results indicate that the addition of penicillin and streptomycin to diluents and during water hardening can prevent the vertical transmission of bacterial coldwater disease.

Summary Lettuce is one of the most common raw foods worldwide, but occasionally also involved in pathogen outbreaks. To understand the correlative structure of the bacterial community as a network, we studied root microbiota of eight ancient and modern Lactuca sativa cultivars and the wild ancestor Lactuca serriola by pyrosequencing of 16S rRNA gene amplicon libraries. The lettuce microbiota was dominated by Proteobacteria and Bacteriodetes, as well as abundant Chloroflexi and Actinobacteria. Cultivar specificity comprised 12.5% of the species. Diversity indices were not different between lettuce cultivar groups but higher than in L. serriola, suggesting that domestication lead to bacterial diversification in lettuce root system. Spearman correlations between operational taxonomic units (OTUs) showed that co-occurrence prevailed over co-exclusion, and complementary fluorescence in situ hybridization-confocal laser scanning microscopy (FISH-CLSM) analyses revealed that this pattern results from both potential interactions and habitat sharing. Predominant taxa, such as Pseudomonas, Flavobacterium and Sphingomonadaceae rather suggested interactions, even though these are not necessarily part of significant modules in the co-occurrence networks. Without any need for complex interactions, single organisms are able to invade into this microbial network and to colonize lettuce plants, a fact that can influence the susceptibility to pathogens. The approach to combine co-occurrence analysis and FISH-CLSM allows reliably reconstructing and interpreting microbial interaction networks.