Common Fish Pathogens Research Articles

Fish IL-26 collaborates with IL-10R2 and IL-20R1 to enhance gut mucosal barrier during the antibacterial innate immunity

IL-26 is a cytokine that is crucial for the maintenance and function of the gut mucosal barrier. IL-26 signaling pathway relies on a heterodimeric receptor complex, which is composed of two distinct subunits, IL-10R2 and IL-20R1. However, there are no reports on the antibacterial immunity of IL-26 and its receptors in fish. For this purpose, in this study we identified IL-26 and its receptors IL-10R2 and IL-20R1 in Carassius cuvieri × Carassius auratus red var. (named WR-IL-26, WR-IL10R2 and WR-IL20R1, respectively). Phylogenetic analysis confirmed the conservation of these genes, with shared structural motifs similar to those found in higher vertebrates. Upon exposure to Aeromonas hydrophila, a common fish pathogen, there was a significant upregulation of WR-IL-26, WR-IL10R2 and WR-IL20R1 in the gut, indicating a potential role in the immune response to infection. A co-immunoprecipitation assay revealed that WR-IL-26 formed complexes with WR-IL10R2 and WR-IL20R1. In vivo experiments demonstrated that administration of WR-IL-26 activated the JAK1-STAT3 signaling pathway and protected the gut mucosa barrier from A. hydrophila infection. Conversely, silencing WR-IL10R2 and WR-IL20R1 via RNA interference significantly attenuated the activation of WR-IL-26-mediated JAK1-STAT3 pathway. These results provided new insights into the role of IL-26 and its receptors in the gut mucosa barrier and could offer novel therapeutic strategies for managing bacterial infections in aquaculture.

Evaluation of the antibacterial activity of pineapple (Ananas comosus) (L.) Merr. industrial waste against common fish and shellfish pathogens

Disposal of industrial pineapple (Ananas comosus) wastes is a pressing environmental issue due to pollution risks when accumulated in large quantities. These wastes are susceptible to microbial spoilage, posing serious environmental and health concerns. Therefore, exploring their conversion into valuable products is crucial for effective waste management. In this study, we assessed the antibiotic properties of pineapple processing wastes against prevalent fish pathogens in aquaculture. The wastes underwent various drying methods: sun-drying (SD), oven-drying (OD), and mechanical dehydration (DH). Ethanol extraction was used to isolate bioactive compounds, which were then tested for antibacterial activity at a concentration of 1000 mg/ml using the Agar Well Diffusion technique and Zone of Inhibition (ZOI) assay. Minimum Inhibitory Concentration (MIC) values were determined across six concentrations: 31.25 mg/ml, 62.50 mg/ml, 125 mg/ml, 250 mg/ml, 500 mg/ml, and 1000 mg/ml. Results indicated that all extracts from different drying processes effectively inhibited all tested aquaculture pathogens. DH extracts showed the highest antibacterial activity against Vibrio harveyi and Vibrio parahaemolyticus, with ZOI of 24.67 + 1.25 mm and 21.67 + 0.47 mm, respectively, and a consistent MIC of 250 mg/ml across all pathogens. SD extracts displayed a MIC of 125 mg/ml against Streptococcus agalactiae, while OD extracts showed a MIC of 1000 mg/ml against Edwardsiella tarda, Aeromonas veronii, and S. agalactiae, and 250 mg/ml against both Vibrio species. Comparative analysis with oxytetracycline did not reveal significant differences. These findings suggest that pineapple waste extracts have potential as natural antibacterial agents against common aquaculture pathogens, offering an eco-friendly alternative to commercial antibiotics.

Gut dysbiosis induced by florfenicol increases susceptibility to Aeromonas hydrophila infection in Zebrafish Danio rerio after the recommended withdrawal period.

Florfenicol (FFC) is a broad-spectrum antibiotic approved by the U.S. Food and Drug Administration to treat both systemic and external bacterial infections in food fish. The objective of this study was to evaluate the effect of FFC-medicated feed on the gut microbiota of Zebrafish Danio danio to determine (1) if the therapeutic dose of FFC-medicated feed induces dysbiosis and (2) if fish with altered gut microbiota were more susceptible to subsequent infection by the common opportunistic fish pathogen Aeromonas hydrophila. Zebrafish that were treated with regular and FFC-medicated feeds were artificially challenged with A. hydrophila at the end of the recommended 15-day antibiotic withdrawal period. The gut microbiota of the Zebrafish at different stages was analyzed using 16S ribosomal RNA gene sequencing. Our results found that FFC-medicated feed induced disruption of the gut microbiota. Dysbiosis was observed in all treated groups, with a significant increase in bacterial diversity, and was characterized by a remarkable bloom of Proteobacteria and a drastic decline of Mycoplasma and Cetobacterium in treated animals but without noticeable clinical signs or mortalities. In addition, the increase of Proteobacteria was not significantly reduced after the recommended 15-day withdrawal period, and the Zebrafish treated with FFC-medicated feed exhibited a significantly higher mortality rate when they were subsequently challenged with A. hydrophila compared to the control (regular feed) groups. Interestingly, the most dramatic changes in the gut microbiome composition occurred at the transition time between the late stage of the medicated treatment and the beginning of the withdrawal period instead of the time during the Aeromonas infection. The administration of FFC-medicated feed at the recommended dose induced gut dysbiosis in Zebrafish, and fish did not recover to the baseline after the recommended withdrawal period. Our findings suggest that the use of antibiotics in fish elicits a response similar to those previously described in mammals and possibly makes the host more susceptible to subsequent infections of opportunistic pathogens. This study using a controlled model system suggests that antibiotics in aquaculture may have long-term effects on the general well-being of the fish.

Comparative Study of Green and Chemical Synthesized Selenium Nanoparticles and Its Antibacterial Assay Against Fish Pathogens

Nanometals are widely used in industries like engineering, medicine and the environment; but their synthesis is often chemically prone to environmental contamination, high energy usage, and health issues. Green synthesis, which uses plant extracts instead of chemical agents, addresses these challenges by being more cost-effective, reducing pollution, and protecting both the environment and human health. In this study, two different SeNPs were synthesized where one from sodium selenite using Phyllanthus amarus leaf aqueous extract and another one is via chemical mediated SeNPs. The synthesized SeNPs were characterized by UV-Vis Spectroscopy and exhibited a peak at 264 nm and 265 nm for Gr- SeNPs and Ch-SeNPs; XRD, EDX revealed the atom percentage at 89.07% for Gr-SeNPs and for Ch-SeNPs at 78.45%. The electron microscopy results revealed that synthesized SeNPs morphology was spherical in shape. XRD reports explain the sharp peaks indicate the crystallinity of the nanoparticles the crystalline size of Gr-SeNPs and Ch-SeNPs are 32.13 nm and 33.41 nm. FT-IR spectrum results describe the information about the interaction between functional groups of phytochemicals in the leaf extract and the SeNPs. This study also proved the antibacterial potential of green SeNPs against common fish pathogens comparable to chemical SeNPs. Based on these results, it is confirmed that leaf extract capped SeNPs may have potential bio-medicinal applications when compared to chemical synthesized SeNPs.

LPS-Induced Mortality in Zebrafish: Preliminary Characterisation of Common Fish Pathogens.

Disease outbreaks are a common problem in aquaculture, with serious economic consequences to the sector. Some of the most important bacterial diseases affecting aquaculture are caused by Gram-negative bacteria including Vibrio spp. (vibriosis), Photobacterium damselae (photobacteriosis), Aeromonas spp. (furunculosis; haemorrhagic septicaemia) or Tenacibaculum maritimum (tenacibaculosis). Lipopolysaccharides (LPS) are important components of the outer membrane of Gram-negative bacteria and have been linked to strong immunogenic responses in terrestrial vertebrates, playing a role in disease development. To evaluate LPS effects in fish, we used a hot-phenol procedure to extract LPS from common fish pathogens. A. hydrophila, V. harveyi, T. maritimum and P. damselae purified LPS were tested at different concentrations (50, 100, 250 and 500 µg mL-1) at 3 days post-fertilisation (dpf) Danio rerio larvae, for 5 days. While P. damselae LPS did not cause any mortality under all concentrations tested, A. hydrophila LPS induced 15.5% and V. harveyi LPS induced 58.3% of zebrafish larvae mortality at 500 µg mL-1. LPS from T. maritimum was revealed to be the deadliest, with a zebrafish larvae mortality percentage of 80.6%. Analysis of LPS separated by gel electrophoresis revealed differences in the overall LPS structure between the bacterial species analysed that might be the basis for the different mortalities observed.

Analysis of the ability of Channa striatus gut-derived Lactococcus garvieae bacteria to reach and persist in the gut environment along with extracellular metabolites and its behavior against common fish pathogens

Exploration of gut microbiota is a resource for researchers to study the range and role of micro-organisms. Exploring gut microbiota of fishes that survive in harsh environmental conditions, catches eye that opportunity of finding new strain may be more. The present study was undertaken to explore the gastrointestinal tract of adult Channa striatus collected from Doaba region of Punjab. A total of 34 isolates were isolated and screened for antimicrobial activity against fish pathogens: Aeromonas hydrophila and Pseudomonas aeruginosa. Four of these isolates were Gram positive. Of these four isolates, only one isolate PMS1 showed good antimicrobial activity against the Aeromonas hydrophila. The isolate exhibited high tolerance to low pH and strong adhesion capability. The isolate was found susceptible to all tested antibiotics. Based on in vitro assessment, biochemical and 16srRNA analysis the strain was identified as Lactococcus garvieae. Furthermore, extracellular metabolites were isolated and analyzed through Gas Chromatography-Mass Spectrometry technique. The results revealed the presence of two compounds (1, 3, 5, 7, 9, 11- Hexaethylcyclohexasiloxane and 2, 6 Lutidine 3, 5 dichloro-4- dodecylthio-). This study concludes that the isolate showed positive in vitro results and the metabolites do not confirm the pathogenicity of the strain. The findings suggest that further studies are required to evaluate the effect of Lactococcus garvieae PMS1 in fish.

Origanum onites ve Mentha spicata subs. tomentosa Uçucu Yağ Nanoemülsiyonlarının Bakteriyel Balık Patojenlerine Karşı in Vitro Antibakteriyel Aktivitesi

The current study aimed to determine the antimicrobial activities of two different aromatic plants (Origanum onites, Mentha spicata subs. tomentosa) essential oils, and their nanoemulsion formulations against six common fish pathogens, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Aeromonas veronii, Vibrio alginolyticus, Yersinia ruckeri, and Lactococcus garvieae. The main components of Mentha spicata subs. tomentosa essential oil (MEO) were piperitone (25.01%), eucalyptol (1,8-cineole) (19.53%), pulegone (14.50%) and, Piperitenone (10.98%). The major components of Origanum onites essential oil (OEO) which were carvacrol (46.17%) and, p-cymene (13.05%) were detected. The antibacterial effects of OEO and MEO and their nanoemulsions were determined by using the agar disc diffusion method. The OEO and its nanoemulsions were extremely effective against the Gram‐negative Aeromonas veronii than the positive control (enrofloxacin). In addition, it was observed that OEO nanoemulsion was more effective than OEO in terms of antibacterial activity. MEO and its nanoemulsions were found to have low activity against fish pathogens, however, there was no activity on Aeromonas veronii and Lactococcus garvieae.

A large-scale, multi-year microbial community survey of a freshwater trout aquaculture facility.

Aquaculture is an important tool for solving the growing worldwide food demand, but infectious diseases of farmed animals represent a serious roadblock to continued industry growth. Therefore, it is essential to understand the microbial communities that reside within the built environments of aquaculture facilities to identify reservoirs of bacterial pathogens and potential correlations between commensal species and specific disease agents. Here, we present the results from 3 years of sampling a commercial rainbow trout aquaculture facility. We observed that the microbial communities residing on the abiotic surfaces within the hatchery were distinct from those residing on the surfaces at the facility's water source as well as the production raceways, despite similar communities in the water column at each location. Also, a subset of the water community seeds the biofilm communities. Lastly, we detected a common fish pathogen, Flavobacterium columnare, within the hatchery, including at the source water inlet. Importantly, the relative abundance of this pathogen was correlated with clinical disease. Our results characterized the microbial communities in an aquaculture facility, established that the hatchery environment contains a unique community composition and demonstrated that a specific fish pathogen resides within abiotic surface biofilms and is seeded from the natural water source.

Biochemical Components, Immune Characteristics and Antimicrobial Activity of Epidermal Mucus of the Three Indian Major Carp Species

Fish live in an environment that is full of phylogenetically diverse collections of bacterial pathogens. Fish epidermal mucus is regarded as a barrier between fish and infectious microorganisms in their surroundings protecting it against pathogens. The current study aimed to evaluate the biochemical components of skin mucus, mucosal immunity, and the bactericidal effect of epidermal mucus in three carp species, including Catla catla, Labeo rohita, and Cirrhinus mrigala. Biochemical composition showed the presence of protein, carbohydrate and lipid in the mucus of all three carp species, and concentration varied with respect to species. Protein and lipid were higher in C. catla, followed by L. rohita and C. mrigala; whereas, carbohydrate was high in the mucus of C. mrigala. Mucosal immunity indicating parameters, viz. lysozyme, alkaline phosphatase, myeloperoxidase, and protease activity was observed in the epidermal mucus of these species, showing the role of mucus in immunity. Lysozyme and alkaline phosphatase were high in C. mrigala, and myeloperoxidase and protease were high in C. catla. To understand the bactericidal role of raw and aqueous mucus, extracts of the epidermis of these species were tested against the common fish pathogen Aeromonas hydrophila by a well diffusion assay. The results showed that raw as well as aqueous extracts showed the zone of inhibition (ZOI) against A. hydrophila; however, ZOI was high for raw mucus compared to aqueous extract. The variation in biochemical composition and mucosal immunity parameters may be due to variation in their habitat, ecological niche, and the presence of pathogens in the surrounding medium.

In Vitro Antibacterial Activity of Tropical Plant Extracts Against Fish Pathogens in Vietnam

Crude methanol extracts of 43 Vietnamese plants were screened in vitro for their antibacterial activity against three common freshwater fish pathogens, including Aeromonas hydrophila, Edwardsiella ictaluri and Streptococcus agalactiae. The agar disc diffusion method was used to evaluate the antibacterial activity, followed by minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined using the broth dilution method. Among the 43 plants screened, eight plant extracts (Bouea oppositifolia, Wedelia chinensis, Terminalia catappa, Punica granatum, Sonneratia caseolaris, Sonneratia ovata, Ludwigia hyssopifolia and Phyllanthus urinaria) exhibited wide-spectrum antibacterial activity to all three common freshwater fish pathogens. Six plant extracts (Ageratum conyzoides, Alpinia galanga, Borassus flabellifer, Abutilon indicum, Eupatorium odotatum, and Scoparia dulcis) might be good candidates for the prevention of co-infection of E. ictaluri and S. agalactiae in tilapia whereas Muntingia calabura and Camellia sinensis could be applied to striped catfish to combat E. ictaluri and A. hydrophila. Through MIC and MBC determination, L. hyssopifolia, A. galanga, Ageratum conyzoides extracts showed a bactericidal activity to A. hydrophila, E. ictuluri and S. agalactiae, respectively, while the other extracts could prevent the growth of tested bacteria. The screening results suggested the potential application of plant extracts as alternative therapeutic agents against bacterial infections in aquaculture.

A Teleost CXCL10 Is Both an Immunoregulator and an Antimicrobial

Chemokines are a group of cytokines that play important roles in cell migration, inflammation, and immune defense. In this study, we identified a CXC chemokine, CXCL10, from Japanese flounder Paralichthys olivaceus (named PoCXCL10) and investigated its immune function. Structurally, PoCXCL10 possesses an N-terminal coil, three β-strands, and a C-terminal α-helix with cationic and amphipathic properties. PoCXCL10 expression occurred in multiple tissues and was upregulated by bacterial pathogens. Recombinant PoCXCL10 (rPoCXCL10) promoted the migration, cytokine expression, and phagocytosis of flounder peripheral blood leukocytes (PBLs). rPoCXCL10 bound to and inhibited the growth of a variety of common Gram-negative and Gram-positive fish pathogens. rPoCXCL10 killed the pathogens by causing bacterial membrane permeabilization and structure destruction. When introduced in vivo, rPoCXCL10 significantly inhibited bacterial dissemination in fish tissues. A peptide derived from the C-terminal α-helix exhibited bactericidal activity and competed with rPoCXCL10 for bacterial binding. Deletion of the α-helix affected the in vitro bactericidal activity but not the chemotaxis or in vivo antimicrobial activity of PoCXCL10. Together, these results indicate that PoCXCL10 exerts the role of both an immunoregulator and a bactericide/bacteriostatic via different structural domains. These findings provide new insights into the immune function and working mechanism of fish CXC chemokines.

Development and comparative evaluation of real-time PCR and real-time RPA assays for detection of tilapia lake virus

Tilapia lake virus (TiLV) is a newly emerged pathogen responsible for high mortality and economic losses in the global tilapia industry. Early and accurate diagnosis is an important priority for TiLV disease control. In order to evaluate the methodology in the molecular diagnosis of TiLV, we compared newly developed quantitative real-time PCR (qPCR) and real-time recombinase polymerase amplification (real-time RPA) assays regarding their sensitivities, specificities and detection effect on clinical samples. Real-time RPA amplified the target pathogen in less than 30 min at 39 °C with a detection limit of 620 copies, while qPCR required about 60 min with a detection limit of 62 copies. Both assays were specific for TiLV and there were no cross-reactions observed with other common fish pathogens. The assays were validated using 35 tissue samples from clinically infected and 60 from artificially infected animals. The sensitivities for the real-time RPA and qPCR assays were 93.33 and 100%, respectively, and the specificity was 100% for both. Both methods have their advantages and can play their roles in different situations. The qPCR is more suitable for quantitative analysis and accurate detection of TiLV in a diagnostic laboratory, whereas real-time RPA is more suitable for the diagnosis of clinical diseases and preliminary screening for TiLV infection in poorly equipped laboratories as well as in fish farms.

Prevalence, antimicrobial resistance (AMR) pattern, virulence determinant and AMR genes of emerging multi-drug resistant Edwardsiella tarda in Nile tilapia and African catfish

Edwardsiella tarda is a common fish pathogen, causing septicemic diseases and formidable economic losses in freshwater fish farming. This study aimed to investigate the prevalence, molecular typing, antibiogram pattern, pathogenicity, distribution of virulence determinant genes (edw1, cds1, qseC, and pvsA) and antibiotic resistance genes (blaTEM, blaCTX, sul1, tetA, qnrA, qnrB, qnrS, and aadA1) associated with natural E. tarda infection among Nile tilapia (Oreochromis niloticus) and African catfish (Clarias gariepinus). Thus, 200 fish (100 Nile tilapia and 100 catfish) were collected randomly from different localities in Dakahlia Governorate, Egypt and subsequently processed for clinical and bacteriological examinations. E. tarda was recovered from 24 fish, with the highest prevalence being reported in C. gariepinus (14%) followed by O. niloticus (10%). The liver and kidney were affected mostly with a prevalence of 54.5% and 36.4%, respectively. PCR results revealed that all the tested isolates harbored gyrB conserved gene, edw1 and qseC virulence genes, while 77.2% were positive for cds1gene. Paradoxically, none of the tested strains were positive for pvsA gene. Based on antibiogram and molecular analysis of resistance genes, most of the tested isolates were multi-drug resistance to six antimicrobial classes (penicillins, aminoglycosides, fluoroquinolones, tetracyclines, sulfonamides, and lincosamides) and harbored blaTEM, sul1, tetA, blaCTX-M, aadA1, qnrS, and qnrA resistance genes as well as gryB, edwI, qseC, and cds1 virulence genes. The challenged fish exhibited similar clinical signs and postmortem lesions found in the naturally infected fish. Moreover, the mortality rate was 65% and 40% in C. gariepinus and O. niloticus, respectively. Therefore, the emergence of multi-drug resistant isolates of E. tarda represents a threat to fish farming sector in Egypt. Molecular-based detection is the fundamental tool for precise and early detection of E. tarda and for controlling septicemic diseases in aquaculture.

Transcriptome analysis of immune-related gene expression in Yellow River carp (Cyprinus carpio var.) after challenge with Flavobacterium columnare

Yellow River carp (Cyprinus carpio) is an economically-important freshwater fish. It is the common host of the epizootic bacterium Flavobacterium columnare, a common fish pathogen that causes columnaris disease resulting in aquacultural losses. However, information on the functions and mechanisms of the immune system of Yellow River carp infected with F. columnare is limited. Therefore, the aim of this study is to evaluate the genetic and histopathological effects of an experimentally-induced F. columnare infection in Yellow River carp. Sixty fish were divided into control (CT group) and challenged groups. The gills were collected for histological and transcriptome analysis to understand the host immune response after challenge with F. columnare. The infected fish of the IF group presented typical columnaris disease symptoms and higher mortality, as well as histological changes. However, some challenged fish showed asymptomatic infection (IC group). Additionally, there were 1776 significant differentially-expressed genes (DEGs) between the IC and CT groups, 1853 DEGs between the IF and CT groups, and 1836 DEGs between the IF and IC groups, All the DEGs were classified into three gene ontology categories, which were allocated to 158 KEGG pathways. Moreover, immune-related genes were confirmed by qRT-PCR. we quantified the level of IL-1, IL-6, TNF-α and IL-8 by ELISA. The results showed the highest expression levels of inflammatory cytokines as well as stress proteins and the adhesion molecules in the lF group, which may contribute to severe infection, and a higher case fatality rate, while the high expression of chemokines, costimulatory molecules and the up regulation of antigen presentation function could help the carp resist F. columnare infection.

Antibiotic Resistance and Antibiotic Resistance Genes Among Edwardsiella Tarda Isolated from Fish

Edwardsiella tarda is a common fish pathogen, causing significant septicemic diseases in fish. This study was carried out to investigate prevalence of E. tarda among Tilapia zilli at EL manzla lake, Dakahlia governorate and characterize the isolates phenotypically and genotypically in addition to detection of multi-drug resistance genes (B-lactames genes and plasmid-mediated quinolone resistant genes by PCR assay. Therefore, (100) samples of Tilapia zilli collected from different localities. Fish samples were subjected to clinical and post-mortem examination then bacteriological examination from liver, kidney and spleen. The suspected isolates were characterized by cultural and morphological characters, some conventional biochemical tests and API 20E system then by PCR assay. Eighteen % isolates were characterized as E. tarda. Furthermore, detection of antimicrobial resistance genes by PCR, the recovered isolates harbored blaSHV and blaOXA-1 with a prevalence of 50% and 25%, respectively with no detection of aac (6′)-Ib-cr and qepA genes in examined isolates. The results of antibiotics sensitivity showed resistance to Nalidixic acid, Lincomycin, Amoxicillin and Norfloxacin and most of isolate were found to be sensitive to Florfenicol and Neomycin.55.6% of recovered isolates showed resistance to three antibiotic classes and 27.7% of recovered isolates showed resistance to four antibiotic classes and considered as MDR. The emergence of MDR-strains represents a threat-alarm and PCR is very rapid method for identification of E. tarda isolates which may be helpful in control of Edwardsiellosis.

In vitro antibacterial activity of several plant extracts against fish bacterial pathogens

Crude methanol extract of 9 Vietnamese plants were in vitro screened for their antibacterial activity against three common freshwater fish pathogens including Aeromonas hydrophila, Edwardsiella ictaluri, and Streptococcus agalactiae. Agar disc diffusion method was used to evaluate the antibacterial activity, then solvent extract was performed for the extracts which exhibited the strongest and a broad-spectrum antibacterial activity. Minimal inhibitory concentration (MIC) was conducted for effective plant extracts using broth dilution method. The results indicated that most of the plant extracts exhibited antibacterial propeties to at least one tested bacterium. Headache tree (Premna corymbosa), bushwillows (Combretum quadrangulare) and Celandine spider flower (Cleome chelidonii) showed a broad-spectrum antibacterial activity. The largest inhibitory zones of 35 mm and 21 mm were observed for the extract of Premna corymbosa against E. ictaluri and S. agalactiae, respectively. E. ictaluri was found to be the most susceptible for all of the extracts while A. hydrophila was the most resistant. The MIC of effective plant extracts against tested bacteria ranged between 0.39 mg/mL and 3.125 mg/mL. The result can be considered for further investigation of the development of an alternative therapy against bacterial infection in aquaculture.

Dual RNAseq highlights the kinetics of skin microbiome and fish host responsiveness to bacterial infection

BackgroundTenacibaculum maritimum is a fish pathogen known for causing serious damage to a broad range of wild and farmed marine fish populations worldwide. The recently sequenced genome of T. maritimum strain NCIMB 2154T provided unprecedented information on the possible molecular mechanisms involved in the virulence of this species. However, little is known about the dynamic of infection in vivo, and information is lacking on both the intrinsic host response (gene expression) and its associated microbiota. Here, we applied complementary omic approaches, including dual RNAseq and 16S rRNA gene metabarcoding sequencing using Nanopore and short-read Illumina technologies to unravel the host–pathogen interplay in an experimental infection system using the tropical fish Platax orbicularis as model.ResultsWe showed that the infection of the host is characterised by an enhancement of functions associated with antibiotic and glucans catabolism functions but a reduction of sulfate assimilation process in T. maritimum. The fish host concurrently displays a large panel of immune effectors, notably involving innate response and triggering acute inflammatory response. In addition, our results suggest that fish activate an adaptive immune response visible through the stimulation of T-helper cells, Th17, with congruent reduction of Th2 and T-regulatory cells. Fish were, however, largely sensitive to infection, and less than 25% survived after 96 hpi. These surviving fish showed no evidence of stress (cortisol levels) or significant difference in microbiome diversity compared with controls at the same sampling time. The presence of T. maritimum in resistant fish skin and the total absence of any skin lesions suggest that these fish did not escape contact with the pathogen, but rather that some mechanisms prevented pathogens entry. In resistant individuals, we detected up-regulation of specific immune-related genes differentiating resistant individuals from controls at 96 hpi, which suggests a possible genomic basis of resistance, although no genetic variation in coding regions was found.ConclusionHere we focus in detail on the interplay between common fish pathogens and host immune response during experimental infection. We further highlight key actors of defence response, pathogenicity and possible genomic bases of fish resistance to T. maritimum.

Quantitative PCR for detection and quantification of Veronaea botryosa in fish and environmental samples.

Systemic phaeohyphomycosis, aka 'fluid belly', is one of the most important emergent diseases in sturgeon Acipenser spp. aquaculture. The etiologic agent is the saprobic, dematiaceous fungus Veronaea botryosa. Effective vaccines and chemotherapeutic treatments are currently unavailable. Additionally, the fungus is a slow-growing organism, taking from 10-15 d for colonies to be observed in agar media. To this end, a specific quantitative PCR (qPCR) targeting the V. botryosa β-tubulin gene was developed and validated. The specificity of the assay to V. botryosa was initially confirmed in silico and in vivo against common fungal fish pathogens, including closely related members of the order Chaetothyriales (Exophiala spp.) and other black pigmented fungi (Alternaria spp. and Cladosporium spp.), as well as tissues from uninfected sturgeon. The assay possessed high clinical specificity (100%) and clinical sensitivity (74%) in detecting V. botryosa DNA in splenic tissues from laboratory-infected sturgeon. Using V. botryosa genomic DNA as a template, the limit of detection was equivalent to 10 conidia, and the method was found suitable for the detection of fungal DNA in fresh and formalin-fixed tissues. In addition, the presence of non-target DNA from white sturgeon did not influence assay sensitivity. The developed qPCR assay is a sensitive, specific, and rapid diagnostic method for the detection and quantification of V. botryosa DNA from white sturgeon tissues.

Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection.

The health and environmental risks associated with antibiotic use in aquaculture have promoted bacterial probiotics as an alternative approach to control fish infections in vulnerable larval and juvenile stages. However, evidence-based identification of probiotics is often hindered by the complexity of bacteria-host interactions and host variability in microbiologically uncontrolled conditions. While these difficulties can be partially resolved using gnotobiotic models harboring no or reduced microbiota, most host-microbe interaction studies are carried out in animal models with little relevance for fish farming. Here we studied host-microbiota-pathogen interactions in a germ-free and gnotobiotic model of rainbow trout (Oncorhynchus mykiss), one of the most widely cultured salmonids. We demonstrated that germ-free larvae raised in sterile conditions displayed no significant difference in growth after 35 days compared to conventionally-raised larvae, but were extremely sensitive to infection by Flavobacterium columnare, a common freshwater fish pathogen causing major economic losses worldwide. Furthermore, re-conventionalization with 11 culturable species from the conventional trout microbiota conferred resistance to F. columnare infection. Using mono-re-conventionalized germ-free trout, we identified that this protection is determined by a commensal Flavobacterium strain displaying antibacterial activity against F. columnare. Finally, we demonstrated that use of gnotobiotic trout is a suitable approach for the identification of both endogenous and exogenous probiotic bacterial strains protecting teleostean hosts against F. columnare. This study therefore establishes an ecologically-relevant gnotobiotic model for the study of host-pathogen interactions and colonization resistance in farmed fish.

Monitoring water quality changes and ornamental fish behaviour during commercial transport

Live transport of fishes is recognised as a major source of stress leading to poor welfare and mortality within the ornamental fish industry. While previous studies have aimed to simulate the stressors experienced by fishes during transport in the laboratory, there is little documented evidence of the actual conditions experienced by fishes under commercial conditions. The aim of this study was to monitor water quality and fish health (physiology and behaviour) through a commercial supply chain for three popular freshwater ornamentals (neon tetra, oranda, variatus platy). Water samples were collected at nine stages of the supply chain beginning at arrival of the fishes at a UK wholesaler from Singapore through their recovery at the wholesaler, to subsequent shipment to and recovery at retail stores. Water chemistry was recorded at various points in the transport chain and the water tested for common fish pathogens. Fish health parameters measured included mortality, injuries, waterborne cortisol and behavioural changes. Most water parameters were found to change significantly through the transport chain, including pH, carbonate hardness (KH), chloride (Cl−), nitrate (NO3−), sodium (Na+), magnesium (Mg2+), potassium (K+) and calcium (Ca2+). Pathogens were detected in the water at each stage of transport, but no disease outbreaks were observed. Mortality was low and was not affected by transport stage. Behaviour was found to change the most between different stages of transport. Neon tetras and orandas showed some behavioural changes during transport but the behaviour of variatus platys was more affected by transport stage. The findings of this study highlight the changes in water quality experienced by fishes during commercial transport that are often over-looked in simulated studies and confirm the need for species-specific indicators of welfare during commercial transport.