Microbiome and network analysis reveal potential mechanisms underlying Carassius auratus diseases: The interactions between critical environmental and microbial factors.

Despite the rapid development of research on aquatic environment microbiota, limited attention has been paid to exploring the complex interactions between microbial communities and aquatic environments. Particularly, the mechanisms underlying fish diseases based on such dynamic interactions remain unknown. This study aimed to address the gap by conducting microbiome and co-occurrence network analyses on the typical freshwater aquaculture systems. High-throughput 16S rRNA gene sequencing results revealed significant differences in the microbiota between the disease and healthy groups. Notably, disease mortality varied consistently with the gradient of relative abundance of Proteobacteria (intestine, R2 = 0.46, p < 0.05) and Cyanobacteria (gill, R2 = 0.67, p < 0.01), indicating their potential use as diagnostic criteria. Furthermore, the elevated hepatosomatic index, NO3-N, COD and TC (sediment) were directly correlated with diseases (r > 0.54, p < 0.01). Mean concentrations of NO3-N, COD and TC were elevated by 78.87%, 25.63% and 44.2%, respectively, in ponds where diseases occurred. Quantitative analysis (qPCR) revealed that Aeromonas sobria infected hosts through a potential pathway of "sediment (4.4 × 105 copy number/g)-water (1.1 × 103 copy number/mL)-intestine (1.2 × 106 copy number/g)". Similarly, the potential route for Aeromonas veronii was sediment (4.9 × 106 copy number/g) to gill (5.1 × 105 copy number/g). Additionally, the complexity of microbial networks in the intestine, water, and sediment was significantly lower in the disease group, although no similar phenomenon was observed in the gill microbial network. In summary, these findings reveal that elevated concentrations of crucial environmental factors disrupt the linkages within microbiota, fostering the growth of opportunistic bacteria capable of colonizing fish gut or gills. This offers new insights into potential mechanisms by which environmental factors cause disease in fish.

Deltamethrin increased susceptibility to Aeromonas hydrophila in crucian carp through compromising gill barrier.

Pyrethroids serve as a significant method for managing and preventing parasitic diseases in fish. Among these, deltamethrin (DEL) is used extensively in aquatic environments. Our previous work has been confirmed that DEL exposure can induce oxidative stress and immunosuppression on the gill mucosal barrier of crucian carp (Carassius auratus). However, it is not clear whether DEL affects the susceptibility of farmed fish to bacterial infection. In this study, fish was pre-exposed to different DEL concentration (0, 0.3 and 0.6 µg L-1) and then challenged by immersion with Aeromonas hydrophila (1.0 × 10^8 CFU mL-1). After immersion challenge, fish pre-exposed to DEL developed prominent lipopolysaccharides level in gill and serum and had a significantly lower survival rate compared to the control group. In DEL pre-exposure fish after immersion, the gill apoptosis levels were significantly higher and disrupted the tight junction barrier by downregulating the zo1 and claudin12. Furthermore, fish pre-exposed to DEL exhibited increased activities of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malonaldehyde (MDA) levels in the early stage after immersion but experiencing decreased activities of glutathione peroxidase (GPx) and lysozyme (LZM) in the later stage after immersion. And this process was regulated by the NRF2 pathway. Additionally, fish pre-exposed to DEL after immersion had significantly lower mRNA levels of immune-related genes tlr4, myd88, tnfα, and il-1ß. Overall, these findings indicate that DEL damaged the gill barrier, weakened the immune response, raised LPS levels, and heightened vulnerability to A. hydrophila infection in crucian carp, resulting in mortality. Thus, this work will help social groups and aquaculture workers to understand the potential risk of DEL exposure for bacterial secondary infection in cultured fish.

Potential biocontrol for bacterial and viral disease treatment in aquaculture: a minireview.

Aquaculture is part of the crucial industry that supplies food, especially for the global human population that is gradually increasing annually. Innovations of culture techniques have been improved throughout the years but aquaculture is regularly susceptible to bacterial and viral diseases. Numerous factors could contribute to occurrence of disease and usually they are from environmental or human stressors on the cultured animals. Synthetic chemicals in commercial treatments may yield fast results however, the side effects are usually unknown until it has taken effect. Therefore, biological control methods to treat diseases in aquaculture are preferred. This mini review provides an overview of different potential biocontrol practices for treatment of bacterial and viral diseases. Bacteriophage causes death of pathogenic bacteria by killing the cell and continue to multiply until all targeted pathogenic bacteria are eliminated. Probiotic, prebiotic, synbiotic, biofloc, and immunostimulants are beneficial products from the respective organisms that are effective in inhibiting pathogens. Vaccines introduce inactivated pathogen into the body to stimulate the immune system, while genetic modifications involve alteration and selection of disease resistant genetics.

Applying the FAO surveillance evaluation tool (SET) to assess the fish farming disease surveillance system in Spain.

EU Member States should ensure that they implement adequate health surveillance schemes in all aquaculture farming areas, as appropriate for the type of production. This study presents the results of applying the FAO's Surveillance Evaluation Tool (SET) to assess the Spanish disease surveillance system for farmed fish species, which although applied previously in livestock production, is applied here to aquaculture for the first time. Overall, there were important score differences between trout and marine fish (seabass and seabream) surveillance, which were higher for trout in the following areas: Institutional (70.8% versus 50.0%), Laboratory (91.7% versus 47.2%), and Surveillance activities (75.3% versus 61.3%). For other categories, the values were lower and no significant differences were found. However, most surveillance efforts focused only on trout, for which there are EU and WOAH listed (notifiable) diseases. In contrast, for seabream and seabass, for which there are no listed diseases, it was considered that surveillance efforts should, nevertheless, be in place and should focus on the identification of abnormal mortalities and emerging diseases, for which there are as yet no standardized harmonised methodologies.

Pathogenesis of Experimental Infection of Nile Tilapia (Oreochromis niloticus) with Nucleospora Braziliensis Pathology and Proteomic of Microsporidia.

The recent discovery of disease caused by Nucleospora braziliensis in Nile tilapia (Oreochromis niloticus) is important as it has highlighted the high prevalence of infection and associated mortality in cultured fish. Thus, this study conducted an experimental infection of this microsporidium to evaluate pathological alterations and conduct proteomic analysis. For pathological observation, samples of brain, eyes, gall bladder, gut, heart, kidney, liver, muscle, skin, spleen, and stomach tissue, were collected, and liquid chromatography-mass spectrometry (LC-MS/MS) was performed for proteomic analysis. The most prevalent lesions were brownish color of the liver, gill filament fusion, gut ischemia, hemorrhage of the lips and fins, hepatomegaly, spleen atrophy, splenomegaly, and stomach congestion. The most common microscopic lesions were degeneration, hemorrhage, and inflammation in the brain, gills, gut, kidney, liver, muscle, spleen, and stomach. The digested peptides were identified by LC-MS/MS and the intersection of each group showed that in the spleen there were 121 exclusive proteins in the infected sample and 252 in the control, while in the kidney, 129 proteins were identified in the infected specimen compared to 83 in the control. In conclusion, this study demonstrates the proteome profile of O. niloticus kidney and spleen tissue in response to infection with N. braziliensis.

Computational design of novel chimeric multiepitope vaccine against bacterial and viral disease in tilapia (Oreochromis sp.).

Regarding several infectious diseases in fish, multiple vaccinations are not favorable. The chimeric multiepitope vaccine (CMEV) harboring several antigens for multi-disease prevention would enhance vaccine efficiency in terms of multiple disease prevention. Herein, the immunogens of tilapia's seven pathogens including E. tarda, F. columnare, F. noatunensis, S. iniae, S. agalactiae, A. hydrophila, and TiLV were used for CMEV design. After shuffling and annotating the B-cell epitopes, 5,040 CMEV primary protein structures were obtained. Secondary and tertiary protein structures were predicted by AlphaFold2 creating 25,200 CMEV. Proper amino acid alignment in the secondary structures was achieved by the Ramachandran plot. In silico determination of physiochemical and other properties including allergenicity, antigenicity, glycosylation, and conformational B-cell epitopes were determined. The selected CMEV (OSLM0467, OSLM2629, and OSLM4294) showed a predicted molecular weight (MW) of 70 kDa, with feasible sites of N- and O-glycosylation, and a number of potentially conformational B-cell epitope residues. Molecular docking, codon optimization, and in-silico cloning were tested to evaluate the possibility of protein expression. Those CMEVs will further elucidate in vitro and in vivo to evaluate the efficacy and specific immune response. This research will highlight the new era of vaccines designed based on in silico structural vaccine design.

Proteomic analysis of Neobenedenia sp. and Rhabdosynochus viridisi (Monogenea, Monopisthocotylea): Insights into potential vaccine targets and diagnostic markers for finfish aquaculture.

Monogeneans are parasitic flatworms that represent a significant threat to the aquaculture industry. Species like Neobenedenia melleni (Capsalidae) and Rhabdosynochus viridisi (Diplectanidae) have been identified as causing diseases in farmed fish. In the past years, molecular research on monogeneans of the subclass Monopisthocotylea has focused on the generation of genomic and transcriptomic information and the identification in silico of some protein families of veterinary interest. Proteomic analysis has been suggested as a powerful tool to investigate proteins in parasites and identify potential targets for vaccine development and diagnosis. To date, the proteomic dataset for monogeneans has been restricted to a species of the subclass Polyopisthocotylea, while in monopisthocotyleans there is no proteomic data. In this study, we present the first proteomic data on two monopisthocotylean species, Neobenedenia sp. and R. viridisi, obtained from three distinct sample types: tissue, excretory-secretory products (ESPs), and eggs. A total of 1691 and 1846 expressed proteins were identified in Neobenedenia sp. and R. viridisi, respectively. The actin family was the largest protein family, followed by the tubulin family and the heat shock protein 70 (HSP70) family. We focused mainly on ESPs because they are important to modulate the host immune system. We identified proteins of the actin, tubulin, HSP70 and HSP90 families in both tissue and ESPs, which have been recognized for their antigenic activities in parasitic flatworms. Furthermore, our study uncovered the presence of proteins within ESPs, such as annexin, calcium-binding protein, fructose bisphosphate aldolase, glutamate dehydrogenase, myoferlin, and paramyosin, that are targets for immunodiagnostic and vaccine development and hold paramount relevance in veterinary medicine. This study expands our knowledge of monogeneans and identified proteins that, in other platyhelminths are potential targets for vaccines and drug discovery.

Association of ectoparasite Lepeophtheirus salmonis counts on farmed Atlantic salmon and wild sea trout in Scotland.

Parasitic sea lice (Copepoda: Caligidae) colonising marine salmonid (Salmoniformes: Salmonidae) aquaculture production facilities have been implicated as a possible pressure on wild salmon and sea trout populations. This investigation uses monitoring data from the mainland west coast and Western Isles of Scotland to estimate the association of the abundance of adult female Lepeophtheirus salmonis (Krøyer) colonising farmed Atlantic salmon Salmo salar L. with the occurrence of juvenile and mobile L. salmonis on wild sea trout, anadromous S. trutta L. The associations were evaluated using generalised linear mixed models incorporating farmed adult female salmon louse abundances which are temporally lagged relative to dependent wild trout values. The pattern of lags, which is consistent with time for L. salmonis development between egg and infective stage, was evaluated using model deviances. A significant positive association is identified between adult female L. salmonis abundance on farms and juvenile L. salmonis on wild trout. This association is consistent with a causal relationship in which increases in the number of L. salmonis copepodids originating from lice colonising farmed Atlantic salmon cause an increase of L. salmonis abundance on wild sea trout.

Vibrio infection in freshwater fish in Poland.

Vibrio species are common inhabitants of aquatic environments and have been described in connection with fish and human diseases. Six Vibrio species were isolated from diseased freshwater and ornamental fish in Poland. The strains were identified based on morphological and biochemical characteristics and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as V. albensis (n=3) from Gymnocephalus cernua, Sander lucioperca, Paracheirodon innesi, and Xiphophorus hellerii; V. mimicus (n=1) from Xiphophorus maculatus; and V. vulnificus (n=1) from Nematobrycon palmeri. This is the first time that Vibrio species have been isolated and described from ornamental fish in Poland. The isolates were resistant to ampicillin (83.3%), gentamicin (16.6%), ciprofloxacin (16.6%), sulfamethoxazole-trimethoprim (16.6%), and chloramphenicol (16.6%). The multiple antibiotic resistance (MAR) index was 0.00-0.08 for V. albensis, 0.17 for V. mimicus, and 0.33 for V. vulnificus. Our study confirmed the presence of potentially pathogenic Vibrio species in freshwater and ornamental fish. Therefore, further monitoring of the presence of Vibrio species, mainly in ornamental fish, is necessary.

Determination of Raman spectrum under different culture conditions: preliminary research on bacterial fish pathogens.

The intensive labour and time required for conventional methods to identify bacterial fish pathogens have revealed the need to develop alternative methods. Raman spectroscopy has been used in the rapid optical identification of bacterial pathogens in recent years as an alternative method in microbiology. Strains of bacterial fish pathogens (Vibrio anguillarum, Lactococcus garvieae and Yersinia ruckeri) that often cause infectious diseases in fish were here identified and analyzed in terms of their biochemical structures in different media and at different incubation times, and the data were specified by using Raman spectroscopy. The results demonstrated that Raman spectroscopy presents species-specific Raman spectra of each disease-causing bacteria and that it would be more appropriate to choose general microbiological media over selective media for routine studies. Additionally, it was found that species-specific band regions did not differ in 24- and 48-hour cultures, but there could be a difference in peak intensity which may lead to difficult characterization of spectrum. The current study, conducted for the first time with bacterial fish pathogens under different incubation conditions, is believed to provide a basis for the routine use of Raman spectroscopy for quick pathogen identification and the precise determination of the methodology for further research.

Riding the wave of innovation: immunoinformatics in fish disease control.

The spread of infectious illnesses has been a significant factor restricting aquaculture production. To maximise aquatic animal health, vaccination tactics are very successful and cost-efficient for protecting fish and aquaculture animals against many disease pathogens. However, due to the increasing number of immunological cases and their complexity, it is impossible to manage, analyse, visualise, and interpret such data without the assistance of advanced computational techniques. Hence, the use of immunoinformatics tools is crucial, as they not only facilitate the management of massive amounts of data but also greatly contribute to the creation of fresh hypotheses regarding immune responses. In recent years, advances in biotechnology and immunoinformatics have opened up new research avenues for generating novel vaccines and enhancing existing vaccinations against outbreaks of infectious illnesses, thereby reducing aquaculture losses. This review focuses on understanding in silico epitope-based vaccine design, the creation of multi-epitope vaccines, the molecular interaction of immunogenic vaccines, and the application of immunoinformatics in fish disease based on the frequency of their application and reliable results. It is believed that it can bridge the gap between experimental and computational approaches and reduce the need for experimental research, so that only wet laboratory testing integrated with in silico techniques may yield highly promising results and be useful for the development of vaccines for fish.

The effects of two dietary synbiotics on growth performance, haematological parameters, and non-specific immune responses in Japanese eel (Anguilla japonica).

Feed additives have attracted increased attention in aquaculture due to their ability to modulate fish gut microbiota, resulting in improved fish growth and immunity. This study assessed two synbiotics' effects in Japanese eels: Bacillus subtilis with mannooligosaccharide (MOS) and Enterococcus faecium with fructooligosaccharide (FOS). Six diets, including a control (CON), oxytetracycline (OTC), and four synbiotic diets - B.subtilis at 1 × 106 and 107 CFU/g with 5 g/kg MOS (BS6MO and BS7MO) and E. faecium at 1 × 106 and 107 CFU/g with 5 g/kg FOS (EF6FO and EF7FO) - were fed to triplicate groups of 20 fish averaging 6.00 ± 0.07g for eight weeks. Fish fed the BSMOS diets showed significantly higher weight gain (WG), specific growth rate (SGR), and feed efficiency (FE) compared to the CON and OTC diets (P < 0.05), but not significantly different from those fed the EFFOS diets. Weight gain, SGR of fish fed EFFOS were not significantly different from those fed all other diets (P > 0.05). Fish fed the OTC diet showed a higher mean aspartate aminotransferase (AST) level, though the difference was not statistically significant. The myeloperoxidase (MPO) activity of fish fed the BS7MO diet was significantly higher than in all other diets, and the superoxide dismutase (SOD) activity of fish fed the BS7MO diet was also significantly higher than in the EF7FO diet. Overall, the BSMOS synbiotic diets were significantly more effective than the CON diet in enhancing fish survival against Vibrio anguillarum. Our findings suggest that synbiotics can be a preferable alternative to antibiotics in aquaculture.

Community structure, seasonal dynamics, and impact of some biological parameters of the African catfish Clarias gariepinus on the infection level of the helminth parasites.

Fish parasitic diseases impose a major economic concern on aquaculture. Identified parasites of Clarias gariepinus include one monogenean, Macrogyrodactylus clarii (gills), three digeneans Orientocreadium batrachoides, Eumasenia bangweulensis and Sanguinicola sp. (intestine), two cestodes Tetracampose ciliotheca and Monobothrioides chalmersius (intestine) and two nematodes Paracamallanus cyathopharynx and Procamallanus pseudolaeviconchus (intestine and stomach). Most nematodes, digeneans and cestodes occurred in all months of the study period. However, M. clarii and Sanguinicola sp. disappeared for 6 and 8 months of the year, respectively. The digenean group was the most dominant followed by the cestode and nematode groups, respectively. The nematodes attained the highest infection rate over the digeneans and cestodes while the monogenean M. clarii recorded the lowest infection rate. The infection level of examined parasites varied seasonally, but no overall significant pattern was detected. E. bangweulensis showed a highly significant difference for all parameters seasonally. A higher prevalence was obvious in males than females for most parasites, and the opposite for the mean intensity except for P. pseudolaeviconchus which was significantly different between females and males in the mean abundance. There were variations in the relationship between the host condition factor and helminth parasite infection levels. O. batrachoides, E. bangweulensis and P. cyathopharynx recorded the highest infection level in class II. The mean prevalence was highly significantly different between host classes for T. ciliotheca, M. chalmersius and P. pseudolaeviconchus.

Fish-associated Streptococcus agalactiae ST283: first human cases reported from Malaysia.

In South East Asia, Streptococcus agalactiae ST283 causes sepsis in healthy adults. Raw freshwater fish consumption is the only known risk factor. These two case reports are the first from Malaysia. Although they cluster with Singapore ST283, the epidemiology is complicated by the flow of people and fish across borders.

High immunogenicity of virus-like particles (VLPs) decorated with Aeromonas salmonicida VapA antigen in rainbow trout.

The Gram-negative bacterium A. salmonicida is the causal agent of furunculosis and used to be one of the most loss-causing bacterial infections in the salmonid aquaculture industry with a mortality rate of about 90% until the 1990s, when an inactivated vaccine with mineral oil as adjuvant was successfully implemented to control the disease. However, the use of this vaccine is associated with inflammatory side effects in the peritoneal cavity as well as autoimmune reactions in Atlantic salmon, and incomplete protection has been reported in rainbow trout. We here aimed at developing and testing a recombinant alternative vaccine based on virus-like particles (VLPs) decorated with VapA, the key structural surface protein in the outer A-layer of A. salmonicida. The VLP carrier was based on either the capsid protein of a fish nodavirus, namely red grouper nervous necrotic virus (RGNNV) or the capsid protein of Acinetobacter phage AP205. The VapA and capsid proteins were expressed individually in E. coli and VapA was fused to auto-assembled VLPs using the SpyTag/SpyCatcher technology. Rainbow trout were vaccinated/immunized with the VapA-VLP vaccines by intraperitoneal injection and were challenged with A. salmonicida 7 weeks later. The VLP vaccines provided protection comparable to that of a bacterin-based vaccine and antibody response analysis demonstrated that vaccinated fish mounted a strong VapA-specific antibody response. To our knowledge, this is the first demonstration of the potential use of antigen-decorated VLPs for vaccination against a bacterial disease in salmonids.

Effect of dietary celery (Apium graveolens) on the growth performance, immune responses, and bacterial resistance against Vibrio anguillarum of European seabass (Dicentrarchus labrax).

In this study, we evaluated to reveal the effects of aqueous methanolic extract of celery (Apium graveolens) on the growth performance, immune responses, and resistance against Vibrio anguillarum in European seabass (Dicentrarchus labrax). For this purpose, twenty fish (initial mean weight of 4.80 ± 0.06 g) were placed into twelve tanks (400 L) in triplicate and fish were fed with control (C) and three different levels (0.01, 0.05, and 0.1 g/kg) of A. graveolens (AG) extract-containing diets (AG0.01, AG0.05, and AG0.1) for 30 days. Blood and tissue (kidney, spleen, and intestine) samples were taken from the fish every 10 days during the study to determine the immune responses of the fish. Respiratory burst activity (RBA) was significantly decreased in the AG0.1 group compared to all other groups on the 10th day of the study (P < 0.05). Significance was noticed in the RBA of fish in all AG groups compared to the C group (P < 0.05) on the 30th day of the experiment Lysozyme activity (LYS) was raised on the 10th day of the study in all celery groups compared to the C group (P < 0.05). No differences in the myeloperoxidase activity (MPO) were observed among the experimental groups (P > 0.05). The final mean weight (FMW) was not affected in any experimental groups (P > 0.05). However, in the AG0.05 group, the specific growth rate (SGR) increased, and the feed conversion ratio (FCR) decreased compared to other groups (P < 0.05). IL-1ß in the kidney was highly elevated in the AG0.01 group on the 20th day of the study (P < 0.05). Similar results were observed on IL-6, IL-8, and TNF-α expression in the kidney (P < 0.05). Anti-inflammatory responses (IL-10 and TGF-ß) also increased in all experimental groups and tissues compared to the C group (P < 0.05). COX-2 was upregulated on the 20th day of the study in all tissues (P < 0.05). At the end of the feeding trial, the survival rate of the AG0.1 group in fish infected with Vibrio anguillarum infection was higher than the C group. Dietary celery extract did not affect growth performance directly but increased innate immune responses and a high survival rate. Overall, compared to the control group, the growth, immunity, and resistance of European seabass fed with a diet containing 0.05 g/kg celery aqueous methanolic extract has been improved, and this could be used as an immunostimulant feed additive.

First preliminary study on identification of bacterial fish pathogens with Raman spectroscopy.

Accurate and rapid determination of bacterial disease agents of fish is an important step for sustainable and efficient aquaculture production. In general, biochemical and molecular methods are used for pathogen detection but they are usually time-consuming and required qualified personnel. Recently spectroscopic methods are preferred in clinical and food microbiology and declared as a promising alternative method for pathogens diagnosis with many advantages. In this study, the significant spectra of three important bacterial fish pathogens (Lactococcus garvieae, Vibrio anguillarum and Yersinia ruckeri) were determined by Raman spectroscopy. The first data of the pathogens were obtained and the distinctive differences in polysaccharides, nucleic acids, fatty acids and amino acids were identified. This preliminary study aimed to be pioneer for further studies in aquaculture and veterinary microbiology toward developing an alternative method for routine identification.

The first outbreak of citrobacteriosis caused by Citrobacter gillenii in reared Russian sturgeon (Acipenser gueldenstaedtii) in Turkiye.

Russian sturgeon (Acipenser gueldenstaedtii) is an endangered fish species and also an important resource for the sturgeon aquaculture industry in Turkiye. Recently, a fatal and persistent bacterial disease occurred in the reared sturgeon kept in a trout farm in Turkiye. The disease outbreak has been with notable external signs including petechial hemorrhages and systemic anemia. This outbreak lasted for six weeks, and cumulative mortality reached around 35.00 - 40.00%. In this study, no parasitic and viral agents were observed in the sturgeons. Citrobacter gillenii was isolated from the diseased fish and identified by biochemical and molecular methods including API 20E and 20NE and 16S rRNA gene region sequencing, respectively. As a result, C. gillenii was identified for the first time in Russian sturgeon in Turkiye. The sequence was also deposited under the Genbank with MW057770 accession number. According to the result of disc diffusion method, bacteria were sensitive to enrofloxacin, streptomycin, amoxicillin and oxytetracycline and resistant to penicillin, trimethoprim/sulfamethoxazole, florfenicol and erythromycin. Also, ampC, sul1 and floR resistance genes were detected in the isolated bacteria. The results of this study provide important information for the diagnosis and treatment of this newly emerged disease of Russian sturgeon.

Identification of Key Functions Required for Production and Utilization of the Siderophore Piscibactin Encoded by the High-Pathogenicity Island irp-HPI in Vibrionaceae.

Piscibactin is a widespread siderophore system present in many different bacteria, especially within the Vibrionaceae family. Previous works showed that most functions required for biosynthesis and transport of this siderophore are encoded by the high-pathogenicity island irp-HPI. In the present work, using Vibrio anguillarum as a model, we could identify additional key functions encoded by irp-HPI that are necessary for piscibactin production and transport and that have remained unknown. Allelic exchange mutagenesis, combined with cross-feeding bioassays and LC-MS analysis, were used to demonstrate that Irp4 protein is an essential component for piscibactin synthesis since it is the thioesterase required for nascent piscibactin be released from the NRPS Irp1. We also show that Irp8 is a MFS-type protein essential for piscibactin secretion. In addition, after passage through the outer membrane transporter FrpA, the completion of ferri-piscibactin internalization through the inner membrane would be achieved by the ABC-type transporter FrpBC. The expression of this transporter is coordinated with the expression of FrpA and with the genes encoding biosynthetic functions. Since piscibactin is a major virulence factor of some pathogenic vibrios, the elements of biosynthesis and transport described here could be additional interesting targets for the design of novel antimicrobials against these bacterial pathogens.

Therapeutic Intervention with Dietary Chitosan Nanoparticles Alleviates Fish Pathological and Molecular Systemic Inflammatory Responses against Infections.

Marine bio-sourced chitosan nanoparticles (CSNP) are antimicrobial and immunomodulatory agents beneficial for fish medicine. Herein, dietary CSNP was investigated for the amelioration of the systemic inflammatory responses of an induced fish model. One hundred and forty-four rainbow trout were assigned to one pathogen-free and non-supplemented group (negative control), and three challenged groups: non-supplemented (positive control), CSNP-preventive, and CSNP-therapeutic. After a feeding experiment extended for 21 days, the organosomatic indices (OSI) and molecular aspects were assessed. After a challenge experiment extended for further 28 days, CSNP-therapeutic intervention was assessed on fish survival and systemic inflammatory responses on pathology, histo-morphology, and molecular aspects. With CSNP administration, OSI nonsignificantly decreased and the relative expression of targeted inflammatory-mediator genes was significantly increased. The CSNP-therapeutic fish showed an RPS of 80% as compared to the positive control group, and CSNP-therapeutic administration retained the highest gene expression augmentation up to 28 days after the challenge. Notably, the splenic reticulin fibers framework of the CSNP-therapeutic group retained the highest integrity among the groups during the infection. After recovery, reticulin fibers density in the CSNP-therapeutic samples was significantly higher than in the negative control group, which indicates high innate immunity. Thus, CSNP showed promising biotherapeutic features enhancing fish resistance against infections.