Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.

Tissue damage and infection are deemed likewise triggers of innate immune responses. But whereas neutrophil responses to microbes are generally protective, neutrophil recruitment into damaged tissues without infection is deleterious. Why neutrophils respond to tissue damage and not just to microbes is unknown. Is it a flaw of the innate immune system that persists because evolution did not select against it, or does it provide a selective advantage? Here we dissect the contribution of tissue damage signaling to antimicrobial immune responses in a live vertebrate. By intravital imaging of zebrafish larvae, a powerful model for innate immunity, we show that prevention of tissue damage signaling upon microbial ear infection abrogates leukocyte chemotaxis and reduces animal survival, at least in part, through suppression of cytosolic phospholipase A2 (cPla2), which integrates tissue damage- and microbe-derived cues. Thus, microbial cues are insufficient, and damage signaling is essential for antimicrobial neutrophil responses in zebrafish.

Microbe transmission from pet shop to lab-reared zebrafish reveals a pathogenic birnavirus.

Zebrafish are popular research organisms selected for laboratory use due in part to widespread availability from the pet trade. Many contemporary colonies of laboratory zebrafish are maintained in aquaculture facilities that monitor and aim to curb infections that can negatively affect colony health and confound experiments. The impact of laboratory control on the microbial constituents associated with zebrafish in research environments compared to the pet trade are unclear. Diseases of unknown causes are common in both environments. We conducted a metatranscriptomic survey to broadly compare the zebrafish-associated microbes in pet trade and laboratory environments. We detected many microbes in animals from the pet trade that were not found in laboratory animals. Cohousing experiments revealed several transmissible microbes including a newly described non-enveloped, double-stranded RNA virus in the Birnaviridae family we name Rocky Mountain birnavirus (RMBV). Infections were detected in asymptomatic animals from the pet trade, but when transmitted to laboratory animals RMBV was associated with pronounced antiviral responses and hemorrhagic disease. These experiments highlight the pet trade as a distinct source of diverse microbes that associate with zebrafish and establish a paradigm for the discovery of newly described pathogenic viruses and other infectious microbes that can be developed for study in the laboratory.

Gliding motility proteins GldJ and SprB contribute to Flavobacterium columnare virulence.

Flavobacterium columnare causes columnaris disease in fish. Columnaris disease is incompletely understood, and adequate control measures are lacking. The type IX secretion system (T9SS) is required for F. columnare gliding motility and virulence. The T9SS and gliding motility machineries share some, but not all, components. GldN (required for gliding and for secretion) and PorV (involved in secretion but not required for gliding) are both needed for virulence, implicating T9SS-mediated secretion in virulence. The role of motility in virulence is uncertain. We constructed and analyzed sprB, sprF, and gldJ mutants that were defective for motility but that maintained T9SS function to understand the role of motility in virulence. Wild-type cells moved rapidly and formed spreading colonies. In contrast, sprB and sprF deletion mutants were partially defective in gliding and formed nonspreading colonies. Both mutants exhibited reduced virulence in rainbow trout fry. A gldJ deletion mutant was nonmotile, secretion deficient, and avirulent in rainbow trout fry. To separate the roles of GldJ in secretion and in motility, we generated gldJ truncation mutants that produce nearly full-length GldJ. Mutant gldJ563, which produces GldJ truncated at amino acid 563, was defective for gliding but was competent for secretion as measured by extracellular proteolytic activity. This mutant displayed reduced virulence in rainbow trout fry, suggesting that motility contributes to virulence. Fish that survived exposure to the sprB deletion mutant or the gldJ563 mutant exhibited partial resistance to later challenge with wild-type cells. The results aid our understanding of columnaris disease and may suggest control strategies.IMPORTANCEFlavobacterium columnare causes columnaris disease in many species of freshwater fish in the wild and in aquaculture systems. Fish mortalities resulting from columnaris disease are a major problem for aquaculture. F. columnare virulence is incompletely understood, and control measures are inadequate. Gliding motility and protein secretion have been suggested to contribute to columnaris disease, but evidence directly linking motility to disease was lacking. We isolated and analyzed mutants that were competent for secretion but defective for motility. Some of these mutants exhibited decreased virulence. Fish that had been exposed to these mutants were partially protected from later exposure to the wild type. The results contribute to our understanding of columnaris disease and may aid development of control strategies.

Edwardsiella tarda Attenuates Virulence upon Oxytetracycline Resistance.

The relationship between antibiotic resistance and bacterial virulence has not yet been fully explored. Here, we use Edwardsiella tarda as the research model to investigate the proteomic change upon oxytetracycline resistance (LTB4-ROTC). Compared to oxytetracycline-sensitive E. tarda (LTB4-S), LTB4-ROTC has 234 differentially expressed proteins, of which the abundance of 84 proteins is downregulated and 15 proteins are enriched to the Type III secretion system, Type VI secretion system, and flagellum pathways. Functional analysis confirms virulent phenotypes, including autoaggregation, biofilm formation, hemolysis, swimming, and swarming, are impaired in LTB4-ROTC. Furthermore, the in vivo bacterial challenge in both tilapia and zebrafish infection models suggests that the virulence of LTB4-ROTC is attenuated. Analysis of immune gene expression shows that LTB4-ROTC induces a stronger immune response in the spleen but a weaker response in the head kidney than that induced by LTB4-S, suggesting it's a potential vaccine candidate. Zebrafish and tilapia were challenged with a sublethal dose of LTB4-ROTC as a live vaccine followed by LTB4-S challenge. The relative percentage of survival of zebrafish is 60% and that of tilapia is 75% after vaccination. Thus, our study suggests that bacteria that acquire antibiotic resistance may attenuate virulence, which can be explored as a potential live vaccine to tackle bacterial infection in aquaculture.

Role of cold shock proteins B and D in Aeromonas salmonicida subsp. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus).

Cold shock proteins (Csp) are pivotal nucleic acid binding proteins known for their crucial roles in the physiology and virulence of various bacterial pathogens affecting plant, insect, and mammalian hosts. However, their significance in bacterial pathogens of teleost fish remains unexplored. Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is a psychrotrophic pathogen and the causative agent of furunculosis in marine and freshwater fish. Four csp genes (cspB, cspD, cspA, and cspC) have been identified in the genome of A. salmonicida J223 (wild type). Here, we evaluated the role of DNA binding proteins, CspB and CspD, in A. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus). A. salmonicida ΔcspB, ΔcspD, and the double ΔcspBΔcspD mutants were constructed and characterized. A. salmonicida ΔcspB and ΔcspBΔcspD mutants showed a faster growth at 28°C, and reduced virulence in lumpfish. A. salmonicida ΔcspD showed a slower growth at 28°C, biofilm formation, lower survival in low temperatures and freezing conditions (-20°C, 0°C, and 4°C), deficient in lipopolysaccharide synthesis, and low virulence in lumpfish. Additionally, ΔcspBΔcspD mutants showed less survival in the presence of bile compared to the wild type. Transcriptome analysis revealed that 200, 37, and 921 genes were differentially expressed in ΔcspB, ΔcspD, and ΔcspBΔcspD, respectively. In ΔcspB and ΔcspBΔcspD virulence genes in the chromosome and virulence plasmid were downregulated. Our analysis indicates that CspB and CspD mostly act as a transcriptional activator, influencing cell division (e.g., treB), virulence factors (e.g., aexT), and ultimately virulence.

Protective effect of fructooligosaccharide against oxidative stress and apoptosis induced by Aeromonas hydrophila in Megalobrama amblycephala.

This research aimed to investigate the effects of dietary fructooligosaccharides (FOS) on attenuating the Aeromonas hydrophila (A. hydrophila)-induced oxidative stress and apoptosis in blunt snout bream Megalobrama amblycephala. Fish were divided into three groups as follows: C1 (Control), T1 (A. hydrophila), and T2 (A. hydrophila + 4 g/kg FOS). The results showed that the activities of antioxidant enzymes increased, the liver morphology had disorderly arrangement, and extensive cell necrosis occurred because of A. hydrophila-infection. While the dietary FOS improved the above-mentioned liver damage. Additionaly, FOS elevated mRNA levels of pro-apoptotic molecules, including caspase-8 and 9, and down-regulated mRNA levels of the anti-apoptotic molecule Bcl-2, which is triggered by A. hydrophila-infection. The transcriptome analysis showed that the oxidative stress-related DEGs pathways were activated in intestine of blunt snout bream by A. hydrophila-infection. The FOS-added group led to the enrichment of more pathways to health. Further WGCNA co-expression network analysis showed that the screened single genes were clustered into 49 modules. The two modules with the highest association to the five traits (10 hub genes) were chosen to build the network by combining the physiological and biochemical characteristic. In summary, this research offers a foundation for the exploring of A. hydrophila-restoration genes in dietary FOS, and also lays a theoretical foundation for aquaculture in the future.

Yersinia ruckeri Infection and Enteric Redmouth Disease among Endangered Chinese Sturgeons, China, 2022.

During October 2022, enteric redmouth disease (ERM) affected Chinese sturgeons at a farm in Hubei, China, causing mass mortality. Affected fish exhibited characteristic red mouth and intestinal inflammation. Investigation led to isolation of a prominent bacterial strain, zhx1, from the internal organs and intestines of affected fish. Artificial infection experiments confirmed the role of zhx1 as the pathogen responsible for the deaths. The primary pathologic manifestations consisted of degeneration, necrosis, and inflammatory reactions, resulting in multiple organ dysfunction and death. Whole-genome sequencing of the bacteria identified zhx1 as Yersinia ruckeri, which possesses 135 drug-resistance genes and 443 virulence factor-related genes. Drug-susceptibility testing of zhx1 demonstrated high sensitivity to chloramphenicol and florfenicol but varying degrees of resistance to 18 other antimicrobial drugs. Identifying the pathogenic bacteria associated with ERM in Chinese sturgeons establishes a theoretical foundation for the effective prevention and control of this disease.

Persistence of heterologous Flavobacterium psychrophilum genetic variants in microcosms simulating fish farm and hatchery environments.

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

Antagonistic activity of Phaeobacter piscinae against the emerging fish pathogen Vibrio crassostreae in aquaculture feed algae.

Aquaculture provides a rich resource of high-quality protein; however, the production is challenged by emerging pathogens such as Vibrio crassostreae. While probiotic bacteria have been proposed as a sustainable solution to reduce pathogen load in aquaculture, their application requires a comprehensive assessment across the aquaculture food chain. The purpose of this study was to determine the antagonistic effect of the potential probiotic bacterium Phaeobacter piscinae against the emerging fish pathogen V. crassostreae in aquaculture feed algae that can be an entry point for pathogens in fish and shellfish aquaculture. P. piscinae strain S26 produces the antibacterial compound tropodithietic acid (TDA). In a plate-based assay, P. piscinae S26 was equally to more effective than the well-studied Phaeobacter inhibens DSM17395 in its inhibition of the fish pathogens Vibrio anguillarum 90-11-286 and V. crassostreae DMC-1. When co-cultured with the microalgae Tetraselmis suecica and Isochrysis galbana, P. piscinae S26 reduced the maximum cell density of V. crassostreae DMC-1 by 2 log and 3-4 log fold, respectively. A TDA-deficient mutant of P. piscinae S26 inhibited V. crassostreae DMC-1 to a lesser extent than the wild type, suggesting that the antagonistic effect involves TDA and other factors. TDA is the prime antagonistic agent of the inhibition of V. anguillarum 90-11-286. Comparative genomics of V. anguillarum 90-11-286 and V. crassostreae DMC-1 revealed that V. crassostreae DMC-1 carries a greater arsenal of antibiotic resistance genes potentially contributing to the reduced effect of TDA. In conclusion, P. piscinae S26 is a promising new candidate for inhibition of emerging pathogens such as V. crassostreae DMC-1 in algal feed systems and could contribute to a more sustainable aquaculture industry.IMPORTANCEThe globally important production of fish and shellfish in aquaculture is challenged by disease outbreaks caused by pathogens such as Vibrio crassostreae. These outbreaks not only lead to substantial economic loss and environmental damage, but treatment with antibiotics can also lead to antibiotic resistance affecting human health. Here, we evaluated the potential of probiotic bacteria, specifically the newly identified strain Phaeobacter piscinae S26, to counteract these threats in a sustainable manner. Through a systematic assessment of the antagonistic effect of P. piscinae S26 against V. crassostreae DMC-1, particularly within the context of algal feed systems, the study demonstrates the effectiveness of P. piscinae S26 as probiotic and thereby provides a strategic pathway for addressing disease outbreaks in aquaculture. This finding has the potential of significantly contributing to the long-term stability of the industry, highlighting the potential of probiotics as an efficient and environmentally conscious approach to safeguarding aquaculture productivity against the adverse impact of pathogens.

Redefining piscine lactococcosis.

Piscine lactococcosis is a significant threat to cultured and wild fish populations worldwide. The disease typically presents as a per-acute to acute hemorrhagic septicemia causing high morbidity and mortality, recalcitrant to antimicrobial treatment or management interventions. Historically, the disease was attributed to the gram-positive pathogen Lactococcus garvieae. However, recent work has revealed three distinct lactococcosis-causing bacteria (LCB)-L. garvieae, L. petauri, and L. formosensis-which are phenotypically and genetically similar, leading to widespread misidentification. An update on our understanding of lactococcosis and improved methods for identification are urgently needed. To this end, we used representative isolates from each of the three LCB species to compare currently available and recently developed molecular and phenotypic typing assays, including whole-genome sequencing (WGS), end-point and quantitative PCR (qPCR) assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), API 20 Strep and Biolog systems, fatty acid methyl ester analysis (FAME), and Sensititre antimicrobial profiling. Apart from WGS, sequencing of the gyrB gene was the only method capable of consistent and accurate identification to the species and strain level. A qPCR assay based on a putative glycosyltransferase gene was also able to distinguish L. petauri from L. garvieae/formosensis. Biochemical tests and MALDI-TOF MS showed some species-specific patterns in sugar and fatty acid metabolism or protein profiles but should be complemented by additional analyses. The LCB demonstrated overlap in host and geographic range, but there were relevant differences in host specificity, regional prevalence, and antimicrobial susceptibility impacting disease treatment and prevention. IMPORTANCE: Lactococcosis affects a broad range of host species, including fish from cold, temperate, and warm freshwater or marine environments, as well as several terrestrial animals, including humans. As such, lactococcosis is a disease of concern for animal and ecosystem health. The disease is endemic in European and Asian aquaculture but is rapidly encroaching on ecologically and economically important fish populations across the Americas. Piscine lactococcosis is difficult to manage, with issues of vaccine escape, ineffective antimicrobial treatment, and the development of carrier fish or biofilms leading to recurrent outbreaks. Our understanding of the disease is also widely outdated. The accepted etiologic agent of lactococcosis is Lactococcus garvieae. However, historical misidentification has masked contributions from two additional species, L. petauri and L. formosensis, which are indistinguishable from L. garvieae by common diagnostic methods. This work is the first comprehensive characterization of all three agents and provides direct recommendations for species-specific diagnosis and management.

Genetic and phenotypic diversity of Flavobacterium psychrophilum isolates from Czech salmonid fish farms.

BACKGROUND: The salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database (n = 187). RESULTS: The Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint. CONCLUSION: The prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.

Susceptibility of the cultured Amazonian fish, Colossoma macropomum, to experimental infection with Aeromonas species from ornamental fish.

The global ornamental fish trade carries important risk factors for spreading pathogens between different countries and regions, not only for ornamental fish but also for cultured fish and even other animal species. In the current study, we reported the capacity of Aeromonas veronii and A. hydrophila isolated from ornamental fish to experimentally infect the reared Amazonian fish Colossoma macropomum. For this, those bacteria were identified, and a primary characterization was performed. Fish were inoculated with 0.1 mL of increasing concentrations of A. hydrophila or A. veronii (C1 = 1 × 102; C2 = 1.8 × 104; C3 = 2.1 × 106; C4 = 2.4 × 108 bacterial cells per mL) in the coelomic cavity. In the control group, fish received the same volume of sterile saline solution (0.9 %). Fish presented petechiae, skin suffusions, and mortality rates up to 100 % according to the inoculum concentration. Histopathologically, fish presented necrosis with karyolysis, loss of the cytoplasmic delimitation of cells of the renal tubules and hepatocytes, hemorrhage, cellular edema, and the presence of bacterial cells. The LD50-96h of A. veronii on C. macropomum was estimated at 2.4 × 106 CFU mL-1 and of A. hydrophila at 1.408 × 105 CFU mL-1. The results demonstrated that it is possible that Aeromonas species isolated from ornamental fish affect C. macropomum, causing similar clinical signs and lesions. This shows the importance of promoting risk control measures worldwide regarding the trade of ornamental fish.

RNA-seq analysis revealed the pathogenicity of Vibrio vulnificus to American eel (Anguilla rostrata) and the strategy of host anti-V. vulnificus infection.

Vibrio vulnificus is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-V. vulnificus infection remains uncertain. In this study, American eels were infected with different dose of V. vulnificus to determine the LD50. Then, bacterial load in the liver and kidney histopathology were assessed post the LD50 of V. vulnificus infection. Additionally, gene expressions of 18 immune related genes in the liver, spleen and kidney were detected. Furthermore, transcriptome sequencing and enrichment of differentially expressed genes (DEGs) were analyzed in the eel spleens between pre-infection (Con_0), post-36 h (Vv_36), and post-60 h (Vv_60) infection. The results showed that LD50 of V. vulnificus to American eels was determined to be 5.0 × 105 cfu/g body weight, and the bacterial load peaked at 24 and 12 h post the infection (hpi) in the kidney and liver, respectively. The histopathology was highlighted by necrotic hepatocytes and splenic cells, congestion blood vessels in liver and spleen, atrophied glomeruli and vacuolization of renal tubular epithelial cells. The results of RT-PCR revealed that 18 host immune-related genes showed significantly up or downregulated expression post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 16 DEGs play essential role to the immunosuppression in American eels, and the protein-protein interactions shed light on the widespread upregulation GEGs related to metabolism and immune response maintained the host cell homeostasis post the V. vulnificus infection, shedding new light on our understanding of the V. vulnificus pathogenesis towards understudied American eel and the host anti-V. vulnificus infection strategies in gene transcript.

Deletion of speA and aroC genes impacts the pathogenicity of Vibrio anguillarum in spotted sea bass.

Vibrio anguillarum is one of the major pathogens responsible for bacterial infections in marine environments, causing significant impacts on the aquaculture industry. The misuse of antibiotics leads to bacteria developing multiple drug resistances, which is detrimental to the development of the fisheries industry. In contrast, live attenuated vaccines are gradually gaining acceptance and widespread recognition. In this study, we constructed a double-knockout attenuated strain, V. anguillarum ΔspeA-aroC, to assess its potential for preparing a live attenuated vaccine. The research results indicate a significant downregulation of virulence-related genes, including Type VI secretion system, Type II secretion system, biofilm synthesis, iron uptake system, and other related genes, in the mutant strain. Furthermore, the strain lacking the genes exhibited a 67.47% reduction in biofilm formation ability and increased sensitivity to antibiotics. The mutant strain exhibited significantly reduced capability in evading host immune system defenses and causing in vivo infections in spotted sea bass (Lateolabrax maculatus), with an LD50 that was 13.93 times higher than that of the wild-type V. anguillarum. Additionally, RT-qPCR analysis of immune-related gene expression in spotted sea bass head kidney and spleen showed a weakened immune response triggered by the knockout strain. Compared to the wild-type V. anguillarum, the mutant strain caused reduced levels of tissue damage. The results demonstrate that the deletion of speA and aroC significantly reduces the biosynthesis of biofilms in V. anguillarum, leading to a decrease in its pathogenicity. This suggests a crucial role of biofilms in the survival and invasive capabilities of V. anguillarum.

Impact of Chaetomorpha aerea-enriched diet on growth, feed utilization, and haemato-immunological responses in Clarias batrachus challenged with Aeromonas hydrophila.

The bacteria Aeromonas hydrophila, which causes motile Aeromonas septicemia (MAS), is dangerous to aquaculture because it affects the fish's well-being and production. As the aquaculture industry seeks sustainable and effective methods to enhance fish immunity and growth, natural supplements such as marine algae have gained attention. This study explored the potential benefits of incorporating the green marine algae Chaetomorpha aerea into the fish diet, focusing on disease resistance, growth, feed utilization, and hematological and immunological responses. Five diets were prepared, varying concentrations of C. aerea (0 control, T1: 1 g/kg; T2: 2 g/kg: T3: 5 g/kg: and T4: 10 g/kg) and administered to fish over 30 days. Following the feeding trial, the fish were exposed to A. hydrophila, and their survival rates were observed for the next 14 days. The findings demonstrated that the final weight, weight gain, relative growth rate, specific growth rate, and daily growth rate were all positively impacted by a diet containing 5 g/kg of C. aerea. Additionally, fish in the 5 g/kg C. aerea group demonstrated improved feed conversion efficiency compared to the control group. While there were no significant changes in red and white blood cell counts on the initial day, serum lysozyme activity and overall resistance to infection were enhanced in fish receiving C. aerea at 2 and 5 g/kg. These results imply that C. aerea supplementation with fish supplements may be a useful immunostimulant, boosting improved health and growth in sustainable aquaculture practices.

Gill lesions are the main cause of death in yellowfin seabream (Acanthopagrus latus) following infection with Amyloodinium ocellatum.

Amyloodiniosis, caused by the ectoparasite Amyloodinium ocellatum, affects the healthy development of mariculture. This study used a local infection method to identify the pathogenic target organ responsible for the death of infected fish. Comparing the relationship between the abundance of trophonts in gills and skin with the mortality of infected fish using local infection showed that severe gill infections cause the mortality of infected fish. At the 40 % survival rate of infected fish, the parasite abundance in the gill was 14,167 ± 4371. The gill filaments of the infected fish were structurally disordered, with pronounced lesions associated with the presence of trophonts, such as epithelial cell degeneration and massive lymphocytic infiltration. However, the skin showed no obvious pathological changes. The TUNEL assay showed a significant presence of apoptotic cells concentrated in the area of A. ocellatum infection. The trophonts on the gills developed faster than those parasitising the skin and fins. Microbiome analysis revealed that at the phylum level, Proteobacteria, Bacteroidota, and Firmicutes are abundant in the skin, while Verrucomicrobiota, Bacteroidota, and Proteobacteria are abundant in the gills of A. latus. Furthermore, A. ocellatum infection significantly reduced (p < 0.05) the richness and diversity of the gill microbial community of A. latus. Infection by A. ocellatum increased the relative abundance of several putative pathogenic bacteria (Flavobacterium and Nocardia) in the gill and skin of A. latus, possibly increasing the likelihood of disease in the host. In conclusion, these results evidenced that severe gill infections by A. ocellatum cause mortality in infected fish, which clarifies the direction for exploring the pathogenesis of amyloodiniosis.

Br - nanoconjugate enhances the antibacterial efficacy of nimboloide against Flavobacterium columnare infection in Labeo rohita: A nanoinformatics approach.

BACKGROUND: The bacterial pathogen, Flavobacterium columnare causes columnaris disease in Labeo rohita globally. Major effects of this bacterial infection include skin rashes and gill necrosis. Nimbolide, the key ingredient of the leaf extract of Azadirachta indica possesses anti-bacterial properties effective against many microorganisms. Nano-informatics plays a promising role in drug development and its delivery against infections caused by multi-drug-resistant bacteria. Currently, studies in the disciplines of dentistry, food safety, bacteriology, mycology, virology, and parasitology are being conducted to learn more about the wide anti-virulence activity of nimbolide. METHODS: The toxicity of nimbolide was predicted to determine its dosage for treating bacterial infection in Labeo rohita. Further, comparative 3-D structure prediction and docking studies are done for nimbolide conjugated nanoparticles with several key target receptors to determine better natural ligands against columnaris disease. The nanoparticle conjugates are being designed using in-silico approaches to study molecular docking interactions with the target receptor. RESULTS: Bromine conjugated nimbolide shows the best molecular interaction with the target receptors of selected species ie L rohita. Nimbolide comes under the class III level of toxic compound so, attempts are made to reduce the dosage of the compound without compromising its efficiency. Further, bromine is also used as a common surfactant and can eliminate heavy metals from wastewater. CONCLUSION: The dosage of bromine-conjugated nimbolide can be reduced to a non-toxic level and thus the efficiency of the Nimbolide can be increased. Moreover, it can be used to synthesize nanoparticle composites which have potent antibacterial activity towards both gram-positive and gram-negative bacteria. This material also forms a good coating on the surface and kills both airborne and waterborne bacteria.

The current status and development forecasts of vaccines for aquaculture and its effects on bacterial and viral diseases.

The aquaculture sector predicts protein-rich meals by 2040 and has experienced significant economic shifts since 2000. However, challenges emanating from disease control measures, brood stock improvement, feed advancements, hatchery technology, and water quality management due to environmental fluctuations have been taken as major causative agents for hindering the sector's growth. For the past years, aquatic disease prevention and control have principally depended on the use of various antibiotics, ecologically integrated control, other immunoprophylaxis mechanisms, and chemical drugs, but the long-term use of chemicals such as antibiotics not only escalates antibiotic-resistant bacteria and genes but also harms the fish and the environments, resulting in drug residues in aquatic products, severely obstructing the growth of the aquaculture sector. The field of science has opened new avenues in basic and applied research for creating and producing innovative and effective vaccines and the enhancement of current vaccines to protect against numerous infectious diseases. Recent advances in vaccines and vaccinology could lead to novel vaccine candidates that can tackle fish diseases, including parasitic organism agents, for which the current vaccinations are inadequate. In this review, we study and evaluate the growing aquaculture production by focusing on the current knowledge, recent progress, and prospects related to vaccinations and immunizations in the aquaculture industry and their effects on treating bacterial and viral diseases. The subject matter covers a variety of vaccines, such as conventional inactivated and attenuated vaccines as well as advanced vaccines, and examines their importance in real-world aquaculture scenarios. To encourage enhanced importation of vaccines for aquaculture sustainability and profitability and also help in dealing with challenges emanating from diseases, national and international scientific and policy initiatives need to be informed about the fundamental understanding of vaccines.

Evaluation of virulence of Aeromonas veronii strain GZ21-2 and development of a highly effective vaccine for grass carp with the potential for industrial application.

Bacterial septicemia represents a significant disease affecting cultured grass carp culture, with the primary etiological agent identified as the Gram-negative bacterium Aeromonas veronii. In response to an outbreak of septicemia in Guangzhou, we developed a formaldehyde-inactivated vaccine against an A. veronii strain designated AV-GZ21-2. This strain exhibited high pathogenicity in experimental infections across at all developmental stages of grass carp. Mortality rates for grass carp weighing 15 ± 5 g ranged from 16 % to 92 % at exposure temperatures of 19 °C-34 °C, respectively. The median lethal dose (LD50) for grass carp groups weighing 15 ± 5 g, 60 ± 10 g, 150 ± 30 g and 500 ± 50 g were determined to be 1.43, 2.52, 4.65 and 7.12 × 107(CFU/mL), respectively. We investigated the inactivated vaccine in conbination with aluminum hydroxide gel (AV-AHG), Montanide ISA201VG (AV-201VG), and white oil (AV-WO) adjuvants. This study aimed to optimize inactivation conditions and identify the adjuvant that elicits the most robust immune response. The AV-GZ21-2 inactivated bacterial solution (AV),when combined with various adjuvants, was capable of inducing a strong specific immune response in grass carp. The relative percent survival (RPS) following a lethal challenge with AV-GZ21-2 were 94 % for AV-AHG, 88 % for AV-201VG, 84 % for AV-WO and 78 % for AV alone. The minimum immunization dose of the AV-AHG vaccine was determined to be 6.0 × 107 CFU per fish, providing immunity for a duration of six months with an immune protection level exceeding 75 %. Furthermore, the AV-AHG vaccine demonstrated significant protective efficacy against various epidemic isolates of A. veronii. Consequently, we developed an inactivated vaccine targeting a highly pathogenic strain of A. veronii, incorporating an aluminum hydroxide gel adjuvant, which resulted in high immune protection and a duration of immunity exceeding six months. These findings suggest that the AV-AHG vaccine holds substantial potential for industrial application.

Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus).

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1ß, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila, the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection.