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.

Effect of dietary koumine on the immune and antioxidant status of carp (Cyprinus carpio) after Aeromonas hydrophila infection.

Koumine (KM) has anxiolytic, anti-inflammatory and growth-promoting effects in pigs and sheep. Based on the growth-promoting and immunological effects of koumine, the present study was conducted on Cyprinus carpio (C. carpio) with four KM concentrations: 0 mg/kg, 0.2 mg/kg, 2 mg/kg, and 20 mg/kg for 10 weeks, followed by a 1-week Aeromonas hydrophila (A. hydrophila) infection experiment. The effect of KM on the immunity of A. hydrophila infected carp was analyzed by histopathology, biochemical assay, and qRT-PCR to assess the feasibility of KM in aquaculture. The results showed that the presence of KM alleviated pathogen damage to carp tissues. At 2 mg/kg and 20 mg/kg concentrations of KM successively and significantly elevated (p < 0.05) the SOD activities in the intestinal tract, hepatopancreas and kidney of carp. The expression levels of hepatopancreatic antioxidant genes Nrf2 and IGF-1 were significantly up-regulated in the same group (p < 0.05), while the expression levels of immune genes IL-8 and IL-10 were down-regulated. In summary, KM at concentrations of 2 mg/kg and 20 mg/kg could regulate the expression of antioxidant and immune genes in various tissues in an orderly and rapid manner, and significantly improve the antioxidant and immune abilities of carp, which is conducive to the improvement of the resilience of carp.

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.

Oral feed-based administration of phage cocktail protects rohu fish (Labeo rohita) against Aeromonas hydrophila infection.

Aeromonas hydrophila is one of the major freshwater fish pathogens. In the current study, a cocktail of D6 and CF7 phages was given orally to Labeo rohita to assess phage survival in fish organs as well as to determine the therapeutic efficacy of phage treatment against fish mortality caused by A. hydrophila. In the phage-coated feed, prepared by simple spraying method, phage counts were quite stable for up to 2 months with a decline of ≤ 0.23 log10 and ≤ 1.66 log10 PFU/g feed during 4 oC and room temperature storage. Throughout the experimental period of 7 days, both phages could be detected in the gut of fish fed with phage-coated feed. Besides, both CF7 and D6 phages were also detected in fish kidneys indicating the ability of both the phage to cross the intestinal barrier. During challenge studies with LD50 dose of A. hydrophila, phage cocktail doses of 1 × 106 - 1 × 108 PFU/g feed prevented the mortality in L. rohita with relative percentage survival (RPS) of 8.7-65.2. When challenged with LD90 dose of A. hydrophila, an RPS value of 28.6 was obtained at a phage cocktail dose of 1 × 108 PFU/g feed. The RPS data showed that orally-fed phage cocktail protected the fish against the mortality caused by A. hydrophila in a dose-dependent manner. Simple practical approaches for phage cocktail development, medicated feed preparation and oral administration along with phage survival and protection data make the current study useful for farmer-level application.

Construction and characterization of Aeromonas hydrophila crp and fur deletion mutants and evaluation of its potential as live-attenuated vaccines in crucian carp.

Aeromonas hydrophila (A. hydrophila) is a typical zoonotic pathogenic bacterium that infects humans, animals, and fish. It has been reported that the Fur, a Fe2+ regulatory protein, and the Crp, a cAMP receptor protein, play important roles in bacterial virulence in many bacteria, but no research has been investigated on A. hydrophila. In this study, the Δfur and Δcrp mutant strains were constructed by the suicide plasmid method. These two mutant strains exhibited a slightly diminished bacterial growth and also were observed some alterations in the number of outer membrane proteins, and the disappearance of hemolysis in the Δcrp strain. Animal experiments of crucian carp showed that the Δfur and Δcrp mutant strains significantly decreased virulence compared to the wild-type strain, and both mutant strains were able to induce good immune responses by two kinds of administration routes of intraperitoneal immunization (i.p) and immersion immunization, and the protection rates through intraperitoneal injection of Δfur and Δcrp to crucian carp were as high as 83.3 % and 73.3 %, respectively, and immersion immunization route of Δfur and Δcrp to crucian carp provided protection as high as 40 % and 20 %, respectively. These two mutant strains showed abilities to induce changes in enzymatic activities of the non-specific enzymes SOD, LZM, AKP, and ACP in crucian carp. Together, these results indicated the Δfur and Δcrp mutants were safe and effective candidate vaccine strains, showing good protection against the wild-type A. hydrophila challenge.

Infection dynamics of Shewanella spp. in Nile tilapia under varied water temperatures: A hematological, biochemical, antioxidant-immune analysis, and histopathological alterations.

In aquaculture, fluctuating water temperatures can act as a potent stressor, influencing the virulence and transmission dynamics of pathogenic bacteria, potentially triggering outbreaks and impacting fish health. The purpose of this work was to examine the impact of Shewanella spp. infection on hematological, biochemical, and antioxidant-immune parameters of Nile tilapia (Oreochromis niloticus) under different water temperatures. For this purpose, 180 fish were divided into 6 groups in triplicate (30 fish per group; 10 fish per replicate). Group 1 (G1), G2, and G3 were reared at varying water temperatures (22 °C, 28 °C, and 31 °C, respectively) without infection. While G4, G5, and G6 were IP-injected with 0.2 mL of Shewanella spp. (0.14 × 105) and reared at 22 °C, 28 °C, and 31 °C, respectively. Shewanella spp. infection induced significant lowering (p < 0.05) in hematological parameters (red and white blood cells, hemoglobin, and packed cell volume%) and immune-antioxidant responses (phagocytic activity%, phagocytic index, lysozyme, nitric oxide), total antioxidant capacity, catalase, and reduced glutathione, especially at 22 °C. Moreover, a significant increase (p < 0.05) in the hepato-renal function indicators (alanine aminotransferase, aspartate aminotransferase, urea, and creatinine), stress biomarkers (glucose and cortisol), malondialdehyde, and pro-inflammatory cytokines (interleukin-1ß and tumor necrosis factor-α) were the consequences of the Shewanella spp. infection, especially at 22 °C. The Shewanella spp. infection exhibited marked histopathological changes in the hepatic and renal tissues. Worthily, Shewanella spp. can cause detrimental alterations in Nile tilapia's hematological, biochemical, and antioxidant-immune parameters at various water temperatures, but the major detrimental changes were observed at a water temperature of 22 °C. Consequently, we can conclude that the infection dynamics of Shewanella spp. are exaggerated at 22 °C. These outcomes could help in understanding the nature of such an infection in Nile tilapia.

Meningitis caused by Aeromonas hydrophila in Oreochromis niloticus: Proteomics and druggability of virulence factors.

Meningitis caused by Gram-negative bacteria is a serious public health problem, causing morbidity and mortality in both children and adults. Here, we propose a novel experimental model using Nile tilapia (Oreochromis niloticus) to study neuroinflammation. The fish were infected with Aeromonas hydrophila, and the course of infection was monitored in the peripheral blood. Septicemia was obvious in the blood, while in the brain tissue, infection of the meninges was present. The histopathological examination showed suppurative meningitis, and the cellular immune response in the brain tissue during infection was mediated by microglia. These cells were morphologically characterized and phenotyped by MHC class II markers and CD68. The increased production of TNF-α, IL-1ß and iNOS supported the infiltration of these cells during the neuroinflammatory process. In the proteomic analysis of A. hydrophila isolated from brain tissue, we found chemotactic and transport proteins, proteolytic enzymes and enzymes associated with the dismutation of nitric oxide (NO), as well as motor proteins and those responsible for cell division. After characterizing the most abundant proteins during the course of infection, we investigated the druggability index of these proteins and identified promising peptide sequences as molecular targets that are similar among bacteria. Thus, these findings deepened the understanding of the pathophysiology of meningitis caused by A. hydrophila. Moreover, through the proteomics analysis, important mechanisms and pathways used by the pathogen to subvert the host response were revealed, providing insights for the development of novel antibiotics and vaccines.

Response signatures of intestinal microbiota and gene transcription of yellow catfish (Pelteobagrus fulvidraco) to Aeromonas hydrophila infection.

Bacterial intestinal inflammation is a common disease of yellow catfish (Pelteobagrus fulvidraco) in high-density aquaculture. Understanding the interactions between host and intestinal bacteria is helpful to intestinal inflammatory disease control. Here, we constructed a model of intestinal inflammation after Aeromonas hydrophila infection in yellow catfish, and characterized variations in gene expression and microbiome in the gut through high-throughput sequencing. Furthermore, host gene-microbiome interactions were identified. Histology observation showed disordered distribution of columnar epithelial cells and decrease of goblet cells in intestine. A total of 4741 genes showed differentially expression, mostly in comparisons between 12 hpi group with each other groups respectively, including control, 24 hpi and 48 hpi groups. These genes were enriched in immune-related pathways including the IL-17 signaling pathway, triggering strong inflammatory response at the invading stage within 12 h. Subsequently, the host strengthened energy consumption by activating carbohydrate and lipid metabolism pathways to repair the intestinal mucosal immune defense line. In addition, fish with A. hydrophila infection show decreased richness of gut microbial, reduced relative abundance of probiotics including Akkermansia, and elevated pathogenic bacteria such as Plesimonas. An integrative analysis identified A. hydrophila-related genes, such as il22 and stat3, for which expression level is close associated with the shift of A. hydrophila-related bacteria relative abundance, such as Akkermansia and Cetobacterium. Aside from picturing the variations of intestine gene expression and mucosal microbiome of yellow catfish coping with A. hydrophila infection, our study probed the underlying host-microbe interactions in A. hydrophila infection induced intestinal inflammatory, providing new insights for disease control in aquaculture.

Acrylamide exposure induces growth retardation, neurotoxicity, stress, and immune/antioxidant disruption in Nile tilapia (Oreochromis niloticus): The alleviative effects of Chlorella vulgaris diets.

This study looked at the toxic impacts of water-born acrylamide (ACR) on Nile tilapia (Oreochromis niloticus) in terms of behaviors, growth, immune/antioxidant parameters and their regulating genes, biochemical indices, tissue architecture, and resistance to Aeromonas hydrophila. As well as the probable ameliorative effect of Chlorella vulgaris (CV) microalgae as a feed additive against ACR exposure was studied. The 96-h lethal concentration 50 of ACR was investigated and found to be 34.67 mg/L for O. niloticus. For the chronic exposure study, a total of 180 healthy O. niloticus (24.33 ± 0.03 g) were allocated into four groups in tri-replicates (15 fish/replicate), C (control) and ACR groups were fed a basal diet and exposed to 0 and 1/10 of 96-h LC50 of ACR (3.46 mg/L), respectively. ACR+ CV5 and ACR+ CV10 groups were fed basal diets with 5 % and 10 % CV supplements, respectively and exposed to 1/10 of 96-h LC50 of ACR for 60 days. After the exposure trial (60 days) the experimental groups were challenged with A. hydrophila. The findings demonstrated that ACR exposure induced growth retardation (P˂0.01) (lower final body weight, body weight gain, specific growth rate, feed intake, protein efficiency ratio, final body length, and condition factor as well as higher feed conversion ratio). A substantial decrease in the immune/antioxidant parameters (P˂0.05) (lysozyme, serum bactericidal activity %, superoxide dismutase, and reduced glutathione) and neurotransmitter (acetylcholine esterase) (P˂0.01) was noticed with ACR exposure. A substantial increase (P˂0.01) in the serum levels of hepato-renal indicators, lipid peroxidation biomarker, and cortisol was noticed as a result of ACR exposure. ACR exposure resulted in up-regulation (P˂0.05) of the pro-inflammatory cytokines and down-regulation (P˂0.05) of the antioxidant-related gene expression. Furthermore, the hepatic, renal, brain, and splenic tissues were badly affected by ACR exposure. ACR-exposed fish were more sensitive to A. hydrophila infection and recorded the lowest survival rate (P˂0.01). Feeding the ACR-exposed fish with CV diets significantly improved the growth and immune/antioxidant status, as well as modulating the hepatorenal functions, stress, and neurotransmitter level compared to the exposed-non fed fish. In addition, modulation of the pro-inflammatory and antioxidant-related gene expression was noticed by CV supplementation. Dietary CV improved the tissue architecture and increased the resistance to A. hydrophila challenge in the ACR-exposed fish. Noteworthy, the inclusion of 10 % CV produced better results than 5 %. Overall, CV diets could be added as a feed supplement in the O. niloticus diet to boost the fish's health, productivity, and resistance to A. hydrophila challenge during ACR exposure.

Effects of phoxim on antibacterial infection of silver carp.

The efficacy of phoxim in treating bacterial sepsis in silver carp is significant, yet its underlying mechanism remains elusive. This study aimed to establish a model of Aeromonas veronii infection in silver carp and subsequently treat the infected fish with 10 µg/L phoxim. Kidney and intestine samples from silver carp were collected for transcriptome analysis and assessment of intestinal microbial composition, with the aim of elucidating the mechanism underlying the efficacy of phoxim in treating bacterial sepsis in silver carp. The results of transcriptome and intestinal microbial composition analysis of silver carp kidney indicated that A. veronii infection could up-regulate the expression of il1ß, il6, nos2, ctsl, casp3 et al., which means, signifying that the kidney of silver carp would undergo inflammation, induce apoptosis, and alter the composition of intestinal microorganisms. Phoxim immersion might enhance the energy metabolism of silver carp and change its intestinal microbial composition, potentially elevating the antibacterial infection resistance of silver carp. These findings may contribute to an understanding of how phoxim can effectively treat bacterial sepsis in silver carp.

Immunoprotective efficacy evaluation of OmpTS subunit vaccine against Aeromonas hydrophila infection in Megalobrama amblycephala.

Bacterial septicemia in freshwater fish is mainly caused by Aeromonas hydrophila infection, which affects the development of aquaculture industry. In the context of sustainable aquaculture, subunit vaccines are of great values because they play positive roles in reducing the overuse of antibiotics and protecting aquatic animals against bacterial infection. In this study, the recombinant outer membrane protein OmpTS of A. hydrophila were used as subunit vaccine to immunize Megalobrama amblycephala, and its immunoprotective effect and host immune responses were evaluated. The survival rates of the vaccinated groups after bacterial infection were significantly higher than that of the control group, especially of the OmpTS high-dose vaccinated group. The better protective effects of vaccinated groups might be attributed to the increased levels of serum IgM-specific antibody titer, the reduced relative abundance of A. hydrophila in various tissues, the increased number of immune-positive cells with different epitopes, the up-regulated expression levels of immune-related genes, and the enhanced activities of antibacterial enzymes. In conclusion, OmpTS subunit vaccine could strongly induce host immune responses in M. amblycephala, thereby enhancing both cellular and humoral immunity, which exhibited excellent and effective immunoprotective efficacy.

Immune protection of grass carp by oral vaccination with recombinant Bacillus methylotrophicus expressing the heterologous tolC gene.

In the field of aquaculture, the enhancement of animal health and disease prevention is progressively being tackled using alternatives to antibiotics, including vaccines and probiotics. This study was designed to evaluate the potential of a recombinant Bacillus methylotrophicus, engineered to express the outer membrane channel protein TolC of Aeromonas hydrophila AH3 and the green fluorescent protein GFP, as an oral vaccine. Initially, the genes encoding tolC and GFP were cloned into a prokaryotic expression system, and anti-TolC mouse antiserum was generated. Subsequently, the tolC gene was subcloned into a modified pMDGFP plasmid, which was transformed into B. methylotrophicus WM-1 for protein expression. The recombinant B. methylotrophicus BmT was then administered to grass carp via co-feeding, and its efficacy as an oral vaccine was assessed. Our findings demonstrated successful expression of the 55 kDa TolC and 28 kDa GFP proteins, and the preparation of polyclonal antibodies with high specificity. The BmT exhibited stable expression of the GFP-TolC fusion protein and excellent genetic stability. Following oral immunization, significant elevations were observed in serum-specific IgM levels and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in grass carp. Concurrently, significant upregulation of immune-related genes, including IFN-I, IL-10, IL-1ß, TNF-α, and IgT, was noted in the intestines, head kidney, and spleen of the grass carp. Colonization tests further revealed that the BmT persisted in the gut of immunized fish even after a fasting period of 7 days. Notably, oral administration of BmT enhanced the survival rate of grass carp following A. hydrophila infection. These results suggest that the oral BmT vaccine developed in this study holds promise for future applications in aquaculture.

Comparative safety and efficacy of autogenous vaccine administrated by different routes against furunculosis caused by Aeromonas salmonicida sub. salmonicida in large Rainbow trout (Oncorhynchus mykiss).

The development and growth of fish farming are hindered by viral and bacterial infectious diseases, which necessitate effective disease control measures. Furunculosis, primarily caused by Aeromonas salmonicida, stands out as a significant bacterial disease affecting salmonid fish farms, particularly rainbow trout. Vaccination has emerged as a crucial tool in combating this disease. The objective of this experiment was to assess and compare the efficacy and duration of different vaccine protocols against furunculosis in large trout under controlled rearing conditions, utilizing single and booster administrations via intraperitoneal, oral, and immersion routes. Among the various vaccination protocols tested, only those involving intraperitoneal injection, administered at least once, proved truly effective in preventing the expression of clinical signs of furunculosis and reducing mortality rates. A single intraperitoneal administration provided protection for up to 2352°-days, equivalent to approximately 5 months in water at 16 °C. However, intraperitoneal vaccination may lead to reduced growth in the fish due to resultant intraperitoneal adhesions. Additionally, protocols incorporating booster doses via intraperitoneal injection demonstrated efficacy regardless of the administration route of the primary vaccination. Nevertheless, the use of booster vaccinations via the intraperitoneal route did not confer any significant advantage over a single intraperitoneal injection in terms of efficacy.

Changes of intestinal barrier in the process of intestinal inflammation induced by Aeromonas hydrophila in snakehead (Channa argus).

Bacterial intestinal inflammation frequently occurs in cultured fish. Nevertheless, research on intestinal barrier dysfunction in the process of intestinal inflammation is deficient. In this study, we explored the changes of intestinal inflammation induced by Aeromonas hydrophila (A. hydrophila) in snakehead and the relationship between intestinal barrier and inflammation. Snakehead [(13.05 ± 2.39) g] were infected via anus with A. hydrophila. Specimens were collected for analysis at 0, 1, 3, 7 and 21 d post-injection. The results showed that with the increase of exposure time, the hindgut underwent stages of normal function, damage, damage deterioration, repair and recovery. Relative to 0 d, the levels of IL-1ß and TNF-α in serum, and the expression of nod1, tlr1, tlr5, nf-κb, tnf-α and il-1ß in intestine were significantly increased, and showed an upward then downward pattern over time. However, the expression of tlr2 and il-10 were markedly decreased, and showed the opposite trend. In addition, with the development of intestinal inflammation, the diversity and richness of species, and the levels of phylum and genus in intestine were obviously altered. The levels of trypsin, LPS, AMS, T-SOD, CAT, GPx, AKP, LZM and C3 in intestine were markedly reduced, and displayed a trend of first decreasing and then rebounding. The ultrastructure observation showed that the microvilli and tight junction structure of intestinal epithelial cells experienced normal function initially, then damage, and finally recovery over time. The expression of claudin-3 and zo-1 in intestine were significantly decreased, and showed a trend of first decreasing and then rebounding. Conversely, the expression of mhc-i, igm, igt and pigr in intestine were markedly increased, and displayed a trend of increasing first and then decreasing. The above results revealed the changes in intestinal barrier during the occurrence and development of intestinal inflammation, which provided a theoretical basis for explaining the relationship between the two.

In vivo and ex vivo studies support the immunostimulant and immunoprotective effect of Damiana (Turnera diffusa Willd) in Almaco Jack (Seriola rivoliana).

Damiana (Turnera diffusa Willd) was evaluated in vitro for antioxidant and antibacterial activities against Staphylococcus aureus and Streptococcus pyogenes (as a preliminary screening assessment) by high-performance thin-layer chromatography (HPTLC)-Direct bioautography. A study was performed in vivo to evaluate the effects of Damiana enriched diets at 0.5 % on immune parameters in mucus and serum and gene expression in Almaco Jack (Seriola rivoliana) intestine after two and four weeks; an infection with Aeromonas hydrophila at 1x107 colony forming units (CFU) followed and an ex vivo study was carried out using head-kidney leukocytes. Ferric reducing ability of plasma (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays showed high antioxidant activities in Damiana leaves; even in the ABTS assay, Damiana at 300 µg/mL showed similar activity to ascorbic acid - the standard control. Damiana exhibited strong in vitro antimicrobial activity against S. aureus and S. pyogenes. In vivo studies showed a strong enhancement of myeloperoxidase, nitric oxide, superoxide dismutase, and catalase activities in mucus and serum of S. rivoliana supplemented with Damiana; their immunological response enhanced after infection with A. hydrophila. IL-1ß, TNF-α, and IL-10 gene expressions upregulated in the fish intestine challenged with the bacterium. Piscidin and macrophage (MARCO) receptor gene expression up-regulated at week 4 and down-regulated after infection. Intestinal histology results confirm that Damiana not cause inflammation or damage. Finally, the ex vivo study confirmed the immunostimulant and protective effects of Damiana through increased phagocytic, respiratory burst, myeloperoxidase activities and nitric oxide generation before and upon the bacterial encounter. These results support the idea that Damiana has the potential as an immunostimulant additive for diets in aquaculture by enhancing immune parameters and protecting Almaco Jack against A. hydrophila infections upon four weeks of supplementation.

Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

Fish arginase constrains excessive production of nitric oxide and limits mitochondrial damage during Aeromonas hydrophila infection.

Bacteria-enhanced inducible nitric oxide synthase (iNOS) overproduces nitric oxide (NO) leading to mitochondrial and cellular damage. In mammals, arginase (ARG), the enzyme consuming the same substrate l-arginine with iNOS, was believed to inhibit iNOS activity by competing the substrate. But in fish, this conception has been widely challenged. In this study, the gene expression using real-time quantitative PCR (RT-qPCR) technology showed that when stimulated by Aeromonas hydrophila (A. hydrophila), grass carp (gc) iNOS was up-regulated in head kidney monocytes/macrophages (M0/MФ), and its changes were not detected in the whole tissue of liver or spleen, showing a high degree of cell-specific expression pattern. At the same time, gcARG2 had a high basal expression in tissues and was up-regulated by A. hydrophila stimulation. Next, phthalaldehyde-primaquine reaction was first used in the determination of intracellular urea in fish cells. It was found that the induced gcARG2 led to an increase in the intracellular urea content. Moreover, urea and NO production in M0/MФ were increased in a substrate dose-dependent manner from 30 to 100 µM of l-arginine and reached the highest yield at 300 and 3000 µM of l-arginine, respectively. Furthermore, head kidney M0/MФ was cultured in RPMI1640 medium containing physiological concentration (500 µM) of l-arginine to evaluate the effect of ARG. Under A. hydrophila stimulation, treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) showed that inhibition of arginase could further enhance the NO production stimulated by A. hydrophila. This in turn led to a cumulation in peroxynitrite (ONOO-) content and an injury of the mitochondrial membrane potential. Our study showed for the first time that fish ARG in head kidney M0/MФ can limit excessive production of NO and harmful products by iNOS to maintain mitochondrial and cellular homeostasis.

Effect of dietary heat-killed Lactiplantibacillus plantarum VSG3 on growth, immunity, antioxidant status, and immune-related gene expression in pathogen-aggravated Cyprinus carpio.

Heat-killed probiotics offer an alternative approach to enhance growth and disease resistance in farmed fish. In this study, we isolated Lactiplantibacillus plantarum VSG3 from the gut of Labeo rohita to investigate the effects of heat-killed L. plantarum (HK-LP) on the health and growth performance of Cyprinus carpio fingerlings. Different concentrations of HK-LP (0, 50, 100, 200, 300, and 400 mg/kg) were administered to the fish, followed by a challenge with Aeromonas hydrophila after 8 weeks of feeding. Notably, the LP200 group exhibited significantly improved percentage weight gain and specific growth rate, accompanied by the lowest feed conversion ratio. Post-challenge survival rates were considerably enhanced in the LP200 group, reaching 60.65%. Moreover, serum analysis indicated significantly higher levels of total protein and albumin in the LP200 group than in the control group. Although HK-LP had no substantial impact on certain serum parameters (glucose, total cholesterol, cortisol, and alanine aminotransferase), aspartate aminotransferase levels were considerably low in the LP200 group. Intestinal protease and trypsin activities significantly increased in the LP200 group, while no significant changes were observed in lipase and amylase activities post-pathogen challenge. Serum immunological indices, including lysozyme, alternative complement pathway, and phagocytic activity, improved considerably in the LP200 group. Additionally, serum antioxidant enzyme activities (superoxide dismutase [SOD], glutathione peroxidase [GPx], catalase [CAT], and myeloperoxidase) were significantly elevated in the LP200 group, while malondialdehyde level was reduced. Gene expression analysis in liver tissue indicated strong upregulation of antioxidant-related genes (SOD, CAT, nuclear factor erythroid 2 [NFE2]-related factor 2 [Nrf2], Kelch-like ECH-associated protein 1[Keap1]) in the LP100 and LP200 groups. Pro-inflammatory cytokines (IL-1ß and TNF-α) were considerably downregulated in the kidneys of the LP200 post-challenged fish, although the anti-inflammatory cytokine IL-10 showed an increased expression. Quadratic regression analysis identified the optimal dietary HK-LP level for maximizing growth and immune performance (200.381-270.003 mg/kg). In summary, our findings underscore the potential of HK-LP as a valuable dietary supplement for enhancing carp aquaculture, particularly at the appropriate concentration.

Differential polyvalent passive immune protection of egg yolk antibodies (IgY) against live and inactivated Vibrio fluvialis in fish.

Egg yolk antibodies (IgY) can be prepared in large quantities and economically, and have potential value as polyvalent passive vaccines (against multiple bacteria) in aquaculture. This study prepared live and inactivated Vibrio fluvialis IgY and immunized Carassius auratus prior to infection with V. fluvialis and Aeromonas hydrophila. The results showed that the two IgY antibodies hold effective passive protective rates against V. fluvialis and A. hydrophila in C. auratus. Further, the serum of C. auratus recognized the two bacteria in vitro, with a decrease in the bacteria content of the kidney. The phagocytic activity of C. auratus plasma was enhanced, with a decrease in the expression of inflammatory and antioxidant factors. Pathological sections showed that the kidney, spleen, and intestinal tissue structures were intact, and apoptosis and DNA damage decreased in kidney cells. Moreover, the immunoprotection conferred by the live V. fluvialis IgY was higher than that of the inactivated IgY. Addition, live V. fluvialis immunity induced IgY antibodies against outer membrane proteins of V. fluvialis were more than inactivated V. fluvialis immunity. Furthermore, heterologous immune bacteria will not cause infection, so V. fluvialis can be used to immunize chickens to obtain a large amount of IgY antibody. These findings suggest that the passive immunization effect of live bacterial IgY antibody on fish is significantly better than that of inactivated bacterial antibody, and the live V. fluvialis IgY hold potential value as polyvalent passive vaccines in aquaculture.

A bacterial ghost vaccine against Aeromonas salmonicida infection in turbot (Scophthalmus maximus).

Aeromonas salmonicida is one of the most prevalent pathogens that causes huge economic losses to aquaculture. Effective vaccination is the first choice for preventing infection. Bacterial ghost (BG), an empty bacterial shell devoid of cytoplasm, is a promising vaccine antigen with distinct advantages. Herein, we established strategies for producing a substantial yield of A. salmonicida ghost (ASG) and investigated the immune-protective properties of it. As a result, 2.84 mg/ml NaOH was discovered to be capable of inducing considerable amounts of ASG. Furthermore, the ASG vaccine elicited adaptive immunity in turbots after rapid activation of innate immunity. Even though formalin-killed cells (FKC) produced a few more antibodies than ASG, ASG ultimately provided a much stronger immune protection effect because it strengthened cellular immunity, with a relative percentage survival (RPS) of 50.1 % compared to FKC. These findings demonstrated that ASG effectively activated cell-mediated immunity, which helped get rid of microorganisms inside cells. Therefore, this study presented novel perspectives for future research on furunculosis vaccine products based on ASG as an antigen.