Infectious Diseases In Aquaculture Research Articles

The function of NLRP3 in anti-infection immunity and inflammasome assembly of common carp (Cyprinus carpio L.)

NLRP3 inflammasome can be activated by a variety of stimuli and plays an important role in protecting host from pathogen invasion and maintaining homeostasis. However, the activation mechanism of NLRP3 inflammasome in fish is still unclear. In the present study, the NLRP3 gene (CcNLRP3) was identified from common carp, which was 3069 bp in length and encoded a protein with five domains. Sequence analysis showed that NLRP3 was evolutionarily conserved, and CcNLRP3 was closely related to that in grass carp and zebrafish. Real-time PCR showed that CcNLRP3 was widely expressed in various immune-related tissues of healthy common carp, and significantly increased after stimulation with E. tarda, A. hydrophila and Cyprinus spring viremia virus (SVCV), suggesting that CcNLRP3 might be involved in the immune defense of common carp. The results of co-IP, spot formation, oligomerization and fluorescence localization showed that CcNLRP3 could interact with CcASC and assemble into inflammasome. The cytotoxicity assays showed that CcNLRP3 inflammasome was involved in the pyroptosis induced by CcGSDME. At the same time, CcNLRP3 could directly interact with CcCaspase-A/B and result in increased Caspase-B enzyme activity and LDH release, indicating that CcNLRP3 could also form inflammasome through ASC-independent pathway. Taken together, the results provide targets and theoretical basis for the prevention and control of infectious diseases in aquaculture.

Chitosan Microparticles Enhance the Intestinal Release and Immune Response of an Immune Stimulant Peptide in Oncorhynchus mykiss.

The aquaculture industry is constantly increasing its fish production to provide enough products to maintain fish consumption worldwide. However, the increased production generates susceptibility to infectious diseases that cause losses of millions of dollars to the industry. Conventional treatments are based on antibiotics and antivirals to reduce the incidence of pathogens, but they have disadvantages, such as antibiotic resistance generation, antibiotic residues in fish, and environmental damage. Instead, functional foods with active compounds, especially antimicrobial peptides that allow the generation of prophylaxis against infections, provide an interesting alternative, but protection against gastric degradation is challenging. In this study, we evaluated a new immunomodulatory recombinant peptide, CATH-FLA, which is encapsulated in chitosan microparticles to avoid gastric degradation. The microparticles were prepared using a spray drying method. The peptide release from the microparticles was evaluated at gastric and intestinal pH, both in vitro and in vivo. Finally, the biological activity of the formulation was evaluated by measuring the expression of il-1β, il-8, ifn-γ, Ifn-α, and mx1 in the head kidney and intestinal tissues of rainbow trout (Oncorhynchus mykiss). The results showed that the chitosan microparticles protect the CATH-FLA recombinant peptide from gastric degradation, allowing its release in the intestinal portion of rainbow trout. The microparticle-protected CATH-FLA recombinant peptide increased the expression of il-1β, il-8, ifn-γ, ifn-α, and mx1 in the head kidney and intestine and improved the antiprotease activity in rainbow trout. These results suggest that the chitosan microparticle/CATH-FLA recombinant peptide could be a potential prophylactic alternative to conventional antibiotics for the treatment of infectious diseases in aquaculture.

An Overview Of Diagnostic And Therapeutic Advances In Aquaculture Disease Control

With the growth of the global economy, aquaculture has become increasingly important. Aquaculture faces the greatest challenge from infectious diseases, as they cause billions of dollars in economic losses every year. Using scientifically proven technologies is the key to reducing these impacts. By applying this knowledge to develop novel diagnostic techniques (molecular diagnostics, biosensors, spectroscopy and nanoparticles) and therapeutic agents (vaccines, gene silencing methods, etc.), advances in science are leading to an improved understanding of infectious diseases. Here, we are going to discuss some current diagnostic methods, preventive measures, and therapeutics used to control infectious diseases in aquaculture.

The combined effect of a novel formula of herbal extracts on bacterial infection and immune response in Micropterus salmoides.

Herbal extracts have been considered as ideal alternative to antibiotics in aquaculture and application of combinatory effective extracts always can exhibit the enhanced bioactivity with high efficiency. In our study, a novel herbal extract combination GF-7, which is composed of Galla Chinensis, Mangosteen Shell extracts as well as the effective parts of Pomegranate peel and Scutellaria baicalensis Georgi extracts, was prepared and applied for the therapy of bacterial infection in aquaculture. The HPLC analysis of GF-7 was also investigated for quality control and chemical identification. In the bioassay, GF-7 had excellent antibacterial activity against various aquatic pathogenic bacteria in vitro, and the related MIC values were between 0.045 and 0.36 mg/mL. After feeding Micropterus salmoide with GF-7 (0.1, 0.3, and 0.6%, respectively) for 28 days, the activities of ACP, AKP, LZM, SOD, and CAT of the liver in each treatment group were significantly increased and the content of MDA was significantly decreased. Meanwhile, the hepatic expression of the immune regulators including IL-1β, TNF-α, and Myd88 at different times was up-regulated in varying degrees. The challenge results exhibited a good dose-dependent protective effect on M. salmoides infected with A. hydrophila, which was further confirmed by liver histopathology. Our results imply that the novel combination GF-7 is a potential natural medicine for the prevention and treatment of numerous aquatic pathogenic infectious diseases in aquaculture.

The effect of temperature and challenge route on in vitro hemocyte phagocytosis activation after experimental challenge of common octopus, Octopus vulgaris (Cuvier, 1797) with either Photobacterium damselae subsp. damselae or Vibrio anguillarum O1.

Infectious diseases in aquaculture could be associated with high mortalities and morbidity rates, resulting in negative impacts to fish farming industry, consumers, and the environment. Octopods are reared near marine fish farming areas, and this may represent a major risk since fish pathogens may cause pathologies to octopods. Up to date cephalopods immune defense and pathologies, are incompletely understood. Therefore, the aim of this study was to determine the effect of water temperature and challenge route on hemocyte phagocytosis in vitro after experimental challenge of common octopus with Photobacterium damselae subsp. damselae or Vibrio anguillarum O1. Hemolymph was withdrawn at various time-points post-challenge and the number of circulating hemocytes, and phagocytosis ability were determined. No mortalities were recorded irrespective of pathogen, route of challenge and temperature employed. Great variation was observed in the number of circulating hemocytes of both control and challenged specimens in both experiments (1.04×10⁵ to 22.33×10⁵ hemocytes/ml for the Photobacterium damselae subsp. damselae challenge and 1.35×105 to 24.63×105 hemocytes/ml for the Vibrio anguillarum O1 and at both studied temperatures). No correlation was found between circulating hemocytes and baseline control specimens body weight. Probably, the number of circulating hemocytes is affected by many extrinsic, and intrinsic factors such as size, age, maturity stage, natural fluctuations and temperature, as indicated in the literature. The hemocyte foreign particles binding ability observed in Photobacterium damselae subsp. damselae experiments, at 21±0.5°C and 24±0.5°C, was (mean±SD) 2.26±2.96 and 11.72±12.36 yeast cells/hemocyte for baseline specimens and 7.84±8.88 and 8.56±9.89 yeast cells/hemocyte for control and challenged specimens, respectively. The corresponding values for Vibrio anguillarum O1 experiments were (mean±SD) 6.68±9.26 and 7.00±8.11 yeast cells/hemocyte for baseline specimens and 8.82±9.75 and 6.04±7.64 yeast cells/hemocyte for control and challenged specimens, respectively. Hemocytes of the Photobacterium damselae subsp. damselae and Vibrio anguillarum O1 challenged specimens, were more activated at lower temperature. Apparently, temperature is an important factor in hemocyte activation. In addition, our results indicated that time post challenge, route of challenge and pathogen may influence phagocytosis ability.

Screening and identification of lactic acid bacteria with antimicrobial abilities for aquaculture pathogens in vitro

This study aimed to evaluate the antibacterial properties of nine lactic acid bacterial strains. The agar diffusion method (Oxford cup method) was used to assess the antimicrobial activity against pathogenic bacteria in aquaculture. The results showed that all selected strains inhibited the growth of Photobacterium damsel, Vibrio alginolyticus, Listonella anguillarum, Edwardsiella ictaluri, Aeromonas hydrophila, V. arahaemolyticus, Edwardsiella tarda and V. harveyi, but did not inhibit the growth of Metschnikowia bicuspidata. Among these strains of lactic acid bacteria, r1 was selected for its obvious antibacterial activity against eight kinds of pathogenic bacteria by 96-well plate method. Its inhibitory rate reached 96%, and it could inhibit the growth of six pathogenic bacteria at 121℃, 20min and pH 3.5-4.0. It was identified as Lactiplantibacillus plantarum by morphological observation and 16S rRNA sequencing analysis. Because strain r1 was isolated from culture ponds and exerted strong inhibitory effects on pathogenic bacteria, it holds potential as an agent to prevent and control infectious diseases in aquaculture. This study provides a foundation for the development and utilization of probiotics in aquaculture.

Applying genetic technologies to combat infectious diseases in aquaculture.

Disease and parasitism cause major welfare, environmental and economic concerns for global aquaculture. In this review, we examine the status and potential of technologies that exploit genetic variation in host resistance to tackle this problem. We argue that there is an urgent need to improve understanding of the genetic mechanisms involved, leading to the development of tools that can be applied to boost host resistance and reduce the disease burden. We draw on two pressing global disease problems as case studies-sea lice infestations in salmonids and white spot syndrome in shrimp. We review how the latest genetic technologies can be capitalised upon to determine the mechanisms underlying inter- and intra-species variation in pathogen/parasite resistance, and how the derived knowledge could be applied to boost disease resistance using selective breeding, gene editing and/or with targeted feed treatments and vaccines. Gene editing brings novel opportunities, but also implementation and dissemination challenges, and necessitates new protocols to integrate the technology into aquaculture breeding programmes. There is also an ongoing need to minimise risks of disease agents evolving to overcome genetic improvements to host resistance, and insights from epidemiological and evolutionary models of pathogen infestation in wild and cultured host populations are explored. Ethical issues around the different approaches for achieving genetic resistance are discussed. Application of genetic technologies and approaches has potential to improve fundamental knowledge of mechanisms affecting genetic resistance and provide effective pathways for implementation that could lead to more resistant aquaculture stocks, transforming global aquaculture.

What Can Genetics Do for the Control of Infectious Diseases in Aquaculture?

Simple SummaryInfectious diseases place an economic burden on aquaculture and a limitation to its growth. This state-of-the-art review describes the application of genetics and genomics as novel tools to control infectious disease in aquaculture.Infectious diseases place an economic burden on aquaculture and a limitation to its growth. An innovative approach to mitigate their impact on production is breeding for disease resistance: selection for domestication, family-based selection, marker-assisted selection, and more recently, genomic selection. Advances in genetics and genomics approaches to the control of infectious diseases are key to increasing aquaculture efficiency, profitability, and sustainability and to reducing its environmental footprint. Interaction and co-evolution between a host and pathogen can, however, turn breeding to boost infectious disease resistance into a potential driver of pathogenic change. Parallel molecular characterization of the pathogen and its virulence and antimicrobial resistance genes is therefore essential to understand pathogen evolution over time in response to host immunity, and to apply appropriate mitigation strategies.

Draft Genome Sequence of Lactococcus lactis Subsp. cremoris WA2-67: A Promising Nisin-Producing Probiotic Strain Isolated from the Rearing Environment of a Spanish Rainbow Trout (Oncorhynchus mykiss, Walbaum) Farm.

Probiotics are a viable alternative to traditional chemotherapy agents to control infectious diseases in aquaculture. In this regard, Lactococcus lactis subsp. cremoris WA2-67 has previously demonstrated several probiotic features, such as a strong antimicrobial activity against ichthyopathogens, survival in freshwater, resistance to fish bile and low pH, and hydrophobicity. The aim of this manuscript is an in silico analysis of the whole-genome sequence (WGS) of this strain to gain deeper insights into its probiotic properties and their genetic basis. Genomic DNA was purified, and libraries prepared for Illumina sequencing. After trimming and assembly, resulting contigs were subjected to bioinformatic analyses. The draft genome of L. cremoris WA2-67 consists of 30 contigs (2,573,139 bp), and a total number of 2493 coding DNA sequences (CDSs). Via in silico analysis, the bacteriocinogenic genetic clusters encoding the lantibiotic nisin Z (NisZ) and two new bacteriocins were identified, in addition to several probiotic traits, such as the production of vitamins, amino acids, adhesion/aggregation, and stress resistance factors, as well as the absence of transferable antibiotic resistance determinants and genes encoding detrimental enzymatic activities and virulence factors. These results unveil diverse beneficial properties that support the use of L. cremoris WA2-67 as a probiotic for aquaculture.

Phenotypic assessment of safety and probiotic potential of native isolates from marine fish Moolgarda seheli towards sustainable aquaculture.

Aquaculture is a highly productive and fast-growing agricultural sector. The occurrence of epidemic or sporadic disease outbreak is a major limiting factor in this sector, thus better alternatives are the need of the hour. Use of indigenous probiotics is a promising strategy to control infectious diseases. Thus, the present study was aimed to screen and characterize potent indigenous probiotics from marine fish, Moolgarda seheli, towards enhancing sustainable aquaculture production. Totally 347 bacterial isolates were obtained from M. seheli gastrointestinal tract, out of these, four isolates (KAF121, 124, 135, 136) were confirmed as potent probiotics in terms of biosafety, highly resistant to acidic pH, gastric juice, bile salt, high hydrophobicity to solvents, auto and co-aggregation potential. These four isolates also exhibited virtuous antioxidant activity. Further the isolates, KAF124 and 135 proved their efficiency in growth and survival of fish after challenged againt Aeromonas hydrophila. The isolates were identified based on their 16S rRNA gene sequence and the data were submitted to Genbank as Pseudomonas aeruginosa KAF121 (MH393516), Bacillus cereus KAF124 (MH393226), Bacillus thuringiensis KAF135 (MH393230), and Pseudomonas otitidis KAF136 (MH393230). The results conclude that two isolates, KAF124 and KAF135 are highly safe and potent probiotics which are first time isolated from the marine fish M. seheli. The two Bacillus strains could be used as better alternatives to antibiotics and other chemical-based drugs to prevent/control infectious diseases in aquaculture.

Virulence, immunogenicity and live vaccine potential of aroA and phoP mutants of Edwardsiella piscicida in zebrafish

Vaccination remains the most effective approach for prevention and control of infectious diseases in aquaculture. Edwardsiella piscicida is a causative agent of edwardsiellosis leading to mass mortality in a variety of fish species, leading to huge economic losses in the aquaculture industry. In this study, we have deleted the aroA and phoP genes in E. piscicida and investigated the phenotype, degrees of attenuation, immunogenicity, and ability to confer immune protection in zebrafish host. Our vaccine strain χ16028 with genotype ΔaroA11 ΔphoP12, showed significantly reduced growth, motility, biofilm formation and intracellular replication compared to the wild-type strain J118. In this regard, χ16028 exhibited retarded colonization and attenuation phenotype in zebrafish. Studies showed that χ16028 induced TLR4 and TLR5 mediated NF-kB pathway and upregulated cytokine gene expression i.e., TNF-α, IL-1β, IL-6, IL-8 and type-I IFN in zebrafish. Zebrafish immunized by intracoelomic injection (i.c.) with χ16028 showed systemic and mucosal IgM responses and protection against the wild-type E. piscicida i.c. injection challenge. However, the protection was only 25% in zebrafish following i.c. challenge. We speculate that our vaccine strain might be very attenuated; a booster dose may trigger better immune response and increase the percentage of survival to a more significant level.

Transcriptomic analysis of the immune response against A. hydrophila infection in striped catfish Pangasianodon hypophthalmus

As an emergent bacterial pathogen in aquaculture, A. hydrophila has caused considerable losses to the striped catfish aquaculture industry. Efficient transcriptome research has been widely used to study the immune response of fish against pathogens. This study firstly reported the effect of A. hydrophila infection at 0, 4, 12, 24, 36, and 48 h on Pangasianodon hypophthalmus at the transcriptome level. The changes in multiple immune indices indicated that the earlier stages, including 4, 12, and 24 h, were key time points during A. hydrophila infection. Accordingly, 6824, 2674, and 1571 differentially expressed genes (DEGs) were identified between each key infection time (4, 12, and 24 h) versus 0 h. Most of the DEGs were enriched in the functional categories of positive regulation of response to stimulus, positive regulation of immune system process, activation of immune response, positive regulation of immune response, and defense response. Further analysis based on the short time-series expression miner methods indicated that the genes significantly fluctuated in response to A. hydrophila infection and were enriched in cytokine-related pathways, including cytokine-cytokine interaction, the interleukin 17 signaling pathway, Th cell differentiation, and the chemokine signaling pathway. Weighted co-expression network analysis was applied to identify 14 hub genes involved in the immune response against A. hydrophila infection, and a corresponding gene regulatory network was constructed. The findings in this study provide further in-depth insights into the immune response of P. hypophthalmus against A. hydrophila infection, which could be implicated in the future prevention and treatment of infectious diseases in aquaculture.

Plant-Produced Vaccines: Future Applications in Aquaculture.

Aquaculture has undergone rapid development in the past decades. It provides a large part of high-quality protein food for humans, and thus, a sustainable aquaculture industry is of great importance for the worldwide food supply and economy. Along with the quick expansion of aquaculture, the high fish densities employed in fish farming increase the risks of outbreaks of a variety of aquatic diseases. Such diseases not only cause huge economic losses, but also lead to ecological hazards in terms of pathogen spread to marine ecosystems causing infection of wild fish and polluting the environment. Thus, fish health is essential for the aquaculture industry to be environmentally sustainable and a prerequisite for intensive aquaculture production globally. The wide use of antibiotics and drug residues has caused intensive pollution along with risks for food safety and increasing antimicrobial resistance. Vaccination is the most effective and environmentally friendly approach to battle infectious diseases in aquaculture with minimal ecological impact and is applicable to most species of farmed fish. However, there are only 34 fish vaccines commercially available globally to date, showing the urgent need for further development of fish vaccines to manage fish health and ensure food safety. Plant genetic engineering has been utilized to produce genetically modified crops with desirable characteristics and has also been used for vaccine production, with several advantages including cost-effectiveness, safety when compared with live virus vaccines, and plants being capable of carrying out posttranslational modifications that are similar to naturally occurring systems. So far, plant-derived vaccines, antibodies, and therapeutic proteins have been produced for human and animal health. However, the development of plant-made vaccines for animals, especially fish, is still lagging behind the development of human vaccines. The present review summarizes the development of fish vaccines currently utilized and the suitability of the plant-production platform for fish vaccine and then addresses considerations regarding fish vaccine production in plants. Developing fish vaccines by way of plant biotechnology are significant for the aquaculture industry, fish health management, food safety, and human health.

Vanillic acid combats Vibrio alginolyticus by cell membrane damage and biofilm reduction.

Antibiotics are the most powerful weapon against bacterial infectious diseases in aquaculture. However, the indiscriminate usage of antibiotics often culminates in the emerging development of antibiotic-resistant bacteria, making it imperative to search for novel types of antimicrobial agents. This study investigated the antibacterial and antivirulence effects of vanillic acid (VA) against the fish pathogen, Vibrio alginolyticus. We showed that VA had a good anti-Vibrio activity with minimal inhibitory concentration (MIC) of 1.0mg/ml. In addition, VA wielded its antibacterial action in a dose-/time-dependent manner by causing cell membrane damage and increasing membrane permeability, which is evidenced by increasing the conductivity and malondialdehyde content in the treated cell cultures and the scanning electron microscopy images. Furthermore, VA significantly reduced the biofilm-forming capability, mobility and exotoxin production (protease and exopolysaccharide) and downregulation of the expression of biofilm- and virulence-associated genes (sypG, fliS, fliK, lafA, lafK, asp and luxR) was seen in the V.alginolyticus that exposed to VA at subinhibitory concentrations. Overall, our findings suggested that VA may be of interest for treating V.alginolyticus-associated infections in aquaculture.

Simultaneous visual diagnosis of acute hepatopancreatic necrosis disease and Enterocytozoon hepatopenaei infection in shrimp with duplex recombinase polymerase amplification.

Shrimp is a globally popular seafood. Shrimp farming has been challenged by various infectious diseases that lead to significant economic losses. The prevention of two important shrimp infectious diseases, the acute hepatopancreatic necrosis disease (AHPND) and the Enterocytozoon hepatopenaei (EHP) infection, is highly dependent on early and accurate diagnostic. On-site monitoring of the two diseases in shrimp farming facilities demands point-of-care-testing (POCT) type of diagnostic assays. This study established a duplex recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) combined assay that could simultaneously diagnose the two diseases. The optimized RPA-LFD assay could finish the diagnostic in 35min with good specificity, and the sensitivity reached 101 and 102 gene copies per reaction for EHP and AHPND, respectively, which were at the same level as the currently available molecular diagnostic assays. Test results of clinical samples showed 100% agreement of this assay with the industrial standard nested polymerase chain reaction (PCR) assays, and samples with both diseases were simultaneously identified. Because of the isothermal 37℃ amplification and the visual reading of the signal on dipsticks, the dependence on equipment is minimal. This duplex RPA-LFD assay is well suited for simultaneous POCT diagnostic of the two important shrimp infectious diseases. Moreover, the principle can be applied to multiplex POCT diagnostic of other infectious diseases in aquaculture.

PH-Controlled Release of Antigens Using Mesoporous Silica Nanoparticles Delivery System for Developing a Fish Oral Vaccine.

The development of effective vaccines and delivery systems in aquaculture is a long-term challenge for controlling emerging and reemerging infections. Cost-efficient and advanced nanoparticle vaccines are of tremendous applicability in prevention of infectious diseases of fish. In this study, dihydrolipoamide dehydrogenase (DLDH) antigens of Vibrio alginolyticus were loaded into mesoporous silica nanoparticles (MSN) to compose the vaccine delivery system. Hydroxypropyl methylcellulose phthalate (HP55) was coated to provide protection of immunogen. The morphology, loading capacity, acid-base triggered release were characterized and the toxicity of nanoparticle vaccine was determined in vitro. Further, the vaccine immune effects were evaluated in large yellow croaker via oral administration. In vitro studies confirmed that the antigen could be stable in enzymes-rich artificial gastric fluid and released under artificial intestinal fluid environment. In vitro cytotoxicity assessment demonstrated the vaccines within 120 μg/ml have good biocompatibility for large yellow croaker kidney cells. Our data confirmed that the nanoparticle vaccine in vivo could elicit innate and adaptive immune response, and provide good protection against Vibrio alginolyticus challenge. The MSN delivery system prepared may be a potential candidate carrier for fish vaccine via oral administration feeding. Further, we provide theoretical basis for developing convenient, high-performance, and cost-efficient vaccine against infectious diseases in aquaculture.

Phytogenics in Aquaculture: A Short Review of Their Effects on Gut Health and Microflora in Fish

Increasing pace in aquaculture development to meet the growing food requirements of the population has greatly compromised the carrying capacity of the culture environment and has placed the aquacultured animals at heightened risk of getting diseases due to pathogens. At present, chemotherapy is widely used as means to prevent or treat infectious diseases in aquaculture; however, the use of these drugs poses multiple negative impacts on fish and human health, as well as the environment. Recently, research initiatives are focused on the use of plant products and their derivatives as a means of controlling diseases in aquaculture. They are regarded as a promising alternative to the use of chemical treatments for infectious diseases in fish. Plant-derived products or phytogenics have been shown to stimulate appetite and promote weight gain in farmed animals, act as immunostimulants, and possess potent anti-pathogenic properties in fish. Their potency is mediated by the presence of bioactive molecules including alkaloids, terpenoids, saponins, and flavonoids, among others. Moreover, nutritional strategies are geared towards the use of these phytogenics in modulating immune and physiological responses, as well as promoting optimum health and microbial community in the gastrointestinal tract of fish. This review synthesizes the current knowledge on the use of phytogenic feed additives in aquaculture by focusing on how these substances act as modulators of health and bacterial community in the gut of fish.

Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes

Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide valuable models to study infectious diseases in salmonids, and genome editing using CRISPR/Cas systems provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas editing has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly used salmonid fish cell lines: Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90% cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.

Lactobacillus paraplantarum L34b-2 derived from fermented food improves the growth, disease resistance and innate immunity in Pangasius bocourti

Probiotics have increasingly gained interest as alternatives to antibiotics in controlling infectious diseases in aquaculture. This research aimed to isolate and evaluate lactic acid bacteria (LAB) for potential use as probiotics for Pangasius bocourti. A total of 656 LAB isolates were obtained from fish gut and fermented beef samples, of which 367 isolates displayed antagonistic activity against Aeromonas hydrophila FW52 and/or Streptococcus agalactiae F3S which were employed as indicator fish pathogens. Among these antagonistic LAB, only 18 isolates produced bacteriocin-like activity. These were further evaluated in vitro for other probiotic properties. After in vitro screening, the isolate L34b-2 obtained from Thai indigenous fermented beef was chosen as a suitable probiotic LAB for in vivo studies based on its remarkable probiotic characteristics. These included broad-spectrum bacteriocin-like activity, tolerance to simulated gastrointestinal tract conditions, mucin adhesion ability, co-aggregation ability with fish pathogens tested, non-blood hemolysis, antibiotic susceptibility and protease enzyme production. Identification of the isolate L34b-2 using conventional methods together with 16S rDNA analysis revealed that it belonged to Lactobacillus paraplantarum. Then, the effects of 60-day-dietary administration of L. paraplantarum L34b-2 on fish growth, innate immune responses and disease resistance were investigated in Pangasius bocourti. In feed-trial studies, after feeding for both 30 and 60 days it was found that the Pangasius fish fed with basal diet containing 107 CFU g−1 L. paraplantarum L34b-2 had significantly (p < 0.05) higher weight gain and specific growth rate, and the lowest feed conversion ratio, compared to the control fish that received only basal diet. For immune response studies, it was found that the fish fed with the probiotic strain L34b-2, when compared to the control fish, had significantly higher lysozyme, alternative complement and bactericidal activities but no significant differences (p > 0.05) in phagocytic and respiratory burst activities. After 60-day feeding, fish were subjected to challenge tests by intraperitoneal injection with 106 CFU of a virulent Aeromonas hydrophila FW52 and rearing was continued for two more weeks. As expected, fish that received L34b-2 had a significantly higher (p < 0.05) post challenge survival rate than the control group. These results clearly show that L. paraplantarum L34b-2 displayed beneficial effects on P. bocourti as a probiotic by which it not only promoted growth but also enhanced innate immunity and disease resistance against A. hydrophila.

Thymol Protects Channel Catfish from Aeromonas hydrophila Infection by Inhibiting Aerolysin Expression and Biofilm Formation.

Aeromonas hydrophila is an opportunistic pathogen responsible for a number of diseases in freshwater farming. Moreover, the bacterium has been identified as a zoonotic pathogen that threatens human health. Antibiotics are widely used for treatments of infectious diseases in aquaculture. However, the abuse of antibiotics has led to the emergence of antimicrobial resistant strains. Thus, novel strategies are required against resistant A. hydrophila strains. The quorum sensing (QS) system, involved in virulence factor production and biofilm formation, is a promising target in identifying novel drugs against A. hydrophila infections. In this study, we found that thymol, at sub-inhibitory concentrations, could significantly reduce the production of aerolysin and biofilm formation by inhibiting the transcription of genes aerA, ahyI, and ahyR. These results indicate that thymol inhibits the quorum sensing system. The protective effects of thymol against A. hydrophila mediated cell injury were determined by live/dead assay and lactate dehydrogenase (LDH) release assay. Moreover, the in vivo study showed that thymol could significantly decrease the mortality of channel catfish infected with A. hydrophila. Taken together, these findings demonstrate that thymol could be chosen as a phytotherapeutic candidate for inhibiting quorum sensing system-mediated aerolysin production and biofilm formation in A. hydrophila.