Hydrophila Infection Research Articles

Enriched Diet With Orange Essential Oil Citrus sinensis for Tambaqui Colossoma macropomum Promotes Growth Performance and Resistance Against Aeromonas hydrophila.

The objective of this study was to evaluate the effect of dietary supplementation with Citrus sinensis (L.) Osbeck essential oil on the growth, immune system, and resistance to Aeromonas hydrophila infection in Colossoma macropomum fingerlings. The experiment was conducted with five treatments (control diet, Tween80 diet, and diets supplemented with 200, 400, and 800 mg L-1 of C. sinensis essential oil) with four replicates. At the end of the experimental period, growth parameters were measured, and blood samples were collected for thrombogram, leukogram, and phagocytic activity analysis. A bacterial challenge with A. hydrophila was conducted for 96 h. C. macropomum fingerlings that were fed with 400 and 800 mg L-1 of C. sinensis essential oil had the highest growth parameters, with final weights of 533.18 ± 2.03 mg and 531.91 ± 2.67 mg, respectively, and an increase in the number of thrombocytes, lymphocytes, monocytes, and neutrophils, as well as higher phagocytosis rates compared to the control group. Regarding the challenge, fish in the 400 and 800 mg L-1 treatments also exhibited the lowest cumulative mortality rate (26.66% ± 3.33%). Therefore, supplementation with C. sinensis essential oil promotes growth, improves health, and enhances resistance to A. hydrophila in C. macropomum fingerlings.

In-vitro and in-vivo assessment of the bactericidal potential of peracetic acid and hydrogen peroxide disinfectants against A. hydrophila infection in Nile tilapia and their effect on water quality indices and fish stress biomarkers

This study aimed to assess the in vitro and in vivo disinfectant potential of peracetic acid (PAA) (1 mg/L) and hydrogen peroxide (H2O2) (20 mg/L) on the physicochemical and microbiological water quality parameters of fish aquaria, the microbial density of Nile tilapia muscular tissue, fish hepatic cortisol levels, and antioxidant biomarkers. In vitro, PAA and H2O2 reduced A. hydrophila colony viability by 5 log units after 30 and 5 min of contact time, respectively. PAA and H2O2 were added to aquaria water twice a week for the three-week experiment. Increased fish escape reflexes were observed only in the PAA group, which returned to normal within 10 min. No mortalities were reported in either the PAA or H2O2 groups. An in vivo experimental challenge with a pathogenic strain of A. hydrophila revealed a 20% reduction in mortality in the PAA group, with no mortalities in the H2O2 group. Cortisol levels and antioxidant markers were measured to assess the impact of PAA and H2O2 on fish health. Cortisol levels in the PAA and H2O2 groups were significantly higher than in the control group after disinfectant exposure, but they progressively returned to normal. A significant reduction in superoxide dismutase (SOD) and catalase (CAT) activity, along with considerably higher glutathione peroxidase (GPx) and malondialdehyde (MDA) enzymatic activity, was observed in the PAA and H2O2 groups compared to the control group. A substantial increase in total antioxidant capacity (TAC) was recorded in the PAA group. Physicochemical analyses revealed reduced pH and increased dissolved oxygen levels in the PAA and H2O2 groups. Microbiological analyses showed a significant reduction in bacterial density in water by 64% and 76% after 30 min of exposure to PAA and H2O2, respectively, with a non-significant increase in microbial count after bacterial challenge. Additionally, aerobic bacterial count, Aeromonas spp., and psychotropic bacterial count in fish muscle showed a significant reduction in the H2O2 group compared to the PAA and control groups before and after infection. The study concludes that regular application of PAA and H2O2 can temporarily reduce bacterial load in aquaria and fish muscle, regulate stress responses, and improve fish health by reducing A. hydrophila-induced infections and improving survival.

Effect of Aeromonas hydrophila infection on leptin receptor overlapping transcript expression in Rana amurensis

The leptin receptor overlapping transcript (LepROT) has been suggested to play several roles in immunomodulatory mechanisms; however, the understanding of its role in Rana amurensis immunity is still very limited. Here, we performed hematoxylin-eosin staining, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), immunofluorescence and western blotting to investigate the roles of LepROT in the immunomodulatory mechanism and the influence of its expression on the nuclear factor-kappa B (NF-κB) signaling pathway, such as the activation of IκB kinase and NF-кB, in amphibian resistance to infection with Aeromonas hydrophila (Ah). After Ah infection, the liver, lung, kidney, skin, muscle, and stomach of R. amurensis showed cell structure disturbance, bleeding, and texture abnormalities. In addition, the relative expression levels of LepROT, NF-кB, IKKα, and IKKβ were all upregulated after Ah infection; however, they showed time-dependent differential expression. The NF-кB signaling pathway exhibited robust expression levels, which might be explained by the positive feedback regulation function of LepROT. Overall, this study provides a basis for further assessment of the biological functions of LepROT and highlights its role in the regulation of immune mechanisms.

The Effects of Dietary Orange Peel Fragments Enriched with Zinc and Vitamins C and E on the Antioxidant and Immune Responses of Nile Tilapia under Stress Conditions.

This study aimed to evaluate the effect of dietary orange peel fragments (OPFs) enriched with vitamins C (C) and E (E), as well as zinc (Zn) on the growth performance, hematological profile, immunological parameters, antioxidant capacity, and fillet lipid peroxidation of Nile tilapia subjected to heat/dissolved oxygen-induced stress (HDOIS), transport-induced stress (TIS), and Aeromonas hydrophila infection (BC). A group of 500 male Nile tilapia (2.7 ± 0.03 g) was randomly distributed in twenty-five 250 L aquaria (20 fish/aquarium) and fed diets containing OPFs (6 g kg-1), OPFs/C (6 g kg-1/1.8 g kg-1), OPFs/E (6 g kg-1/0.4 g kg-1), OPFs/Zn (6 g kg-1/0.21 g kg-1), or OPFs/C/E/Zn (6 g kg-1/1.8 g kg-1/0.4 g kg-1/0.21 g kg-1) for 100 days. The diets were formulated to contain 30% crude protein and 17 MJ kg-1 gross energy. After the feeding period, three groups of fish were independently subjected to a different type of stress: HDOIS (34 °C) for two days; TIS for four hours, or BC for 15 days. The hematological profile, antioxidant capacity, and fillet lipid peroxidation were determined before and after all the stress treatments, along with immunological parameters, which were investigated only for the fish subjected to bacterial infection. In summary, the results showed that growth was not affected by the OPFs, nor by the OPFs enriched with C, E, and Zn; bacterial infection determined anemia for the fish fed any of the experimental diets; the OPFs did not prevent lipid peroxidation under TIS and BC; on the other hand, when enriched with C/E/Zn, lipid peroxidation decreased under HDOIS and TIS. In conclusion, the OPFs enriched with C/E/Zn showed a synergistic effect that promoted an increase in antioxidant enzyme activity, a decrease in lipid peroxidation, and the maintenance of the hematological profile under HDOIS and TIS, but they were not able to maintain the health status under BC.

Two homologous genes encoding interleukin (IL)-34 in the common carp (Cyprinus carpio L.): Roles in inflammatory modulation and anti-bacterial defense

In mammals, interleukin 34 (IL-34) is a ligand for macrophage colony-stimulating factor receptor (M-CSFR), promoting inflammatory responses and inducing the synthesis and secretion of various cytokines. However, studies on its function in lower vertebrates is limited, and its evolutionary relationship with homologous molecules in mammals remains unclear. In this study, two IL-34-encoding genes were cloned and identified in common carp (Cyprinus carpio L.), designated as CcIL-34A and CcIL-34B, with an amino acid sequence similarity of 77.7 %. Gene synteny analysis revealed that the IL-34 gene loci are relatively conserved, and both are located downstream of SF3B3. The expression patterns of CcIL-34s were analyzed using qRT-PCR, and this showed that they are expressed across all tested tissues, with higher levels in the liver, spleen, and head kidney and lower levels in the gills and intestines. Following infection with Aeromonas hydrophila, the mRNA expression levels of CcIL-34s in the gills, head kidney, intestines, and spleen were significantly upregulated. Immunofluorescence was also employed to assess changes in CcIL-34 protein expression, showing a significant increase in carp spleens 24 h after A. hydrophila infection, suggesting that CcIL-34s contribute to host defense against this bacterium. To investigate the immunological function of IL-34 in vivo, pc-CcIL-34A and pc-CcIL-34B eukaryotic expression plasmids were constructed and injected intramuscularly into fish. Five days after injection, the expression levels of inflammation-related cytokines in the head kidney and spleen were significantly altered. Furthermore, 24 h post-A. hydrophila infection, the bacterial loads in the liver, spleen, and kidneys were significantly reduced. Ten days post-infection, the survival rates in the groups with CcIL-34A and CcIL-34B overexpression were 40 % and 36.7 %, respectively, compared to 16.7 % in the control group. These findings suggest that CcIL-34s are involved in modulating inflammatory responses, enhancing the immune response, and improving survival rates in fish following bacterial infection, thus supporting the potential use of IL-34 molecules in aquaculture.

Immunomodulatory and growth promotors effects of rosemary (Rosmarinus officinalis) and/or Spirulina with respect to some gene expression on Nile tilapia (Oreochromis niloticus) in Aswan Governorate

The purpose of this work trial was to determine the true impact of spirulina and rosemary in a combination treatment on Nile tilapia performance before experimental infection. Moreover, the effects of these treatments on the liver and kidney functions, the histology of certain organs, and the expression of certain antioxidant and immune-related genes in the liver tissue both before and after Aeromonas hydrophila (A. hydrophila) infection are also included. To achieve this purpose, four diets were prepared: the first was free of additives as a control group (Con), the second contained (SP) spirulina at 7.5 g/kg of diet, the third contained (RM) rosemary at a rate of 10 g/kg of diet, and the fourth one contained rosemary and spirulina as a SP + RM group. The results of the different parameters indicated the growth-stimulant effects of rosemary and spirulina either alone or in a combination when compared with the control group (P ≤ 0.05), with a superior effect to combination treatment. In addition, improved hematological parameters, immunological assays, phagocytic activity and index, and expression of antioxidant and immune-related genes occurred before and after infection. Moreover, there were positive effects of spirulina and rosemary without pathological lesions in the different organs before infection and fewer lesions after infection.

Biochemical and Molecular Detection of Aeromonas Hydrophila Isolated from Infected Cyprinus Carpio in Salah Al-Din Governorate

This study aimed to identify the presence of Aeromonas hydrophila bacteria in diseased common carp (Cyprinus carpio) fish collected from Salah al-Din Governorate in Iraq. For this purpose, 128 swabs were obtained from skin, gill and fin lesions of 70 fish showing symptoms of hemorrhagic sepsis, skin ulcers and boils, and fin and gill rot. Through different biochemical tests, the isolates showed negative results for Gram stain, methyl red, lactose fermentation, H2S, and string test, while positive results were recorded for glucose fermentation, oxidase, motility test, catalase, Voges-Proskauer, indole, citrate utilization, and gelatin hydrolysis. Eleven isolates were confirmed as A. hydrophila by amplification of the hemolysin gene fragment (hlyA) by conventional PCR. This study represents the first comprehensive study to characterize A. hydrophila bacteria from infected common carp fish in Salah al-Din Governorate. The findings of this study provide valuable insights for developing genetic diagnostic strategies for A. hydrophila infections in this region.

Selenomethionine alleviates Aeromonas hydrophila-induced oxidative stress and ferroptosis via the Nrf2/HO1/GPX4 pathway in grass carp

Aeromonas hydrophila infection is a severe, acute, and life-threatening disease affecting grass carp (Ctenopharyngodon idella) in aquaculture. Ferroptosis is a novel form of cell death characterized by the accumulation of free iron and harmful lipid peroxides within cells. While selenomethionine (Se-Met) is known to effectively inhibit ferroptosis and alleviate cell damage, its ability to counteract oxidative stress and ferroptosis induced by A. hydrophila remains unclear. The objective of this study is to reveal the possible mechanism behind the ferroptosis phenomenon during A. hydrophila infection. We established a macrophage model of A. hydrophila invasion to monitor the dynamic changes in iron metabolism markers to evaluate the correlation between ferroptotic stress and A. hydrophila infection. A. hydrophila infection induces cytotoxicity and mitochondrial membrane damage via ferroptosis. This damage is attributed to the accumulation of lipid peroxides due to intracellular ferrous ion overload and glutathione depletion. Supplementation of Se-Met reduced mitochondrial damage, enhanced antioxidant enzyme activity and reduced ferroptosis by activating the Nrf2/HO1/GPX4 axis. These findings provide new insights into the regulatory mechanisms of A. hydrophila-induced ferroptosis in teleosts and suggest that targeted inhibition of ferroptosis may offer a novel therapeutic strategy for managing A. hydrophila infections.

Transcriptomic and Proteomic Analyses of the Immune Mechanism in Pathogenetic and Resistant Chinese Soft-Shelled Turtle (Pelodiscus sinensis) Infected with Aeromonas hydrophila

Background: As intensive aquaculture practices have progressed, the prevalence of bacterial diseases in the Chinese soft-shell turtle (Pelodiscus sinensis) has escalated, particularly infections caused by Aeromonas hydrophila, such as ulcerative dermatitis and abscess disease. Despite this, little is known about their immune defenses against this pathogen. Methods: Our study pioneers an integrated analysis of transcriptomics and proteomics to investigate the immune responses of Chinese soft-shelled turtles to A. hydrophila infection. Results: The investigation revealed significant differences in immune-related pathways between groups susceptible and resistant to A. hydrophila infection after 4 days. A total of 4667 and 3417 differentially expressed genes (DEGs), 763 and 568 differentially expressed proteins (DEPs), and 13 and 5 correlated differentially expressed genes and proteins (cor-DEGs-DEPs) were identified in susceptible and resistant Chinese soft-shelled turtles, respectively. In the resistant group, upregulation of immune-related genes, such as CD3ε and CD45, enhanced T-cell activation and the immune response. The proteomic analysis indicated that immune proteins, such as NF-κB1, were significantly upregulated in the resistant group. The correlation analysis between transcriptomics and proteomics demonstrated that the CD40 gene and protein, differentially expressed in the resistant group compared to the control group, were commonly upregulated within the Toll-like receptor signaling pathway. Conclusions: The transcriptomic and proteomic data obtained from this study provide a scientific foundation for understanding the immune mechanisms that enable the Chinese soft-shelled turtle to resist A. hydrophila infection.

Galectin-9 activates host immune response and improve immunoprotection of Onychostoma macrolepis against Aeromonas hydrophila infection

Galectin-9 (Gal-9) belongs to a family of the glycan-binding proteins (GBPs) and is known to restrict bacterial activity via interacting with pathogen associated molecular pattern (PAMPs). However, the underlying immune mechanism of endogenous Gal-9 on fish against bacterial infection is still unclear. In this study, effect of Gal-9 from Onychostoma macrolepis (OmGal-9) on expression of immune related-genes were measured by HEK293T. The immune response of O. macrolepis with OmGal-9 overexpression to Aeromonas hydrophila (A. hydrophila) infection (1.65×108 CFU/mL) were evaluated by tissue bacterial load, fish survival rate and transcriptome analysis. The results showed that OmGal-9 displayed a punctate distribution in the nucleus and cytoplasm of HEK293T cells. Compared to cells transfected with the empty vector (EV) group, recombinant plasmid pEGFP-Gal9 treatment (Gal9) significantly down-regulated the expression of immune-related genes TNFα, STAT3, MyD88, LCK, and p52 of HEK293T cells stimulated with LPS at 24 h, while up-regulated IκBα and caspase1 (P < 0.05). The activities of catalase (CAT), superoxide dismutase (SOD), the total antioxidant capacity (T-AOC), alkaline phosphatase (AKP), acid phosphatase (ACP), and lysozyme (LZM) of O.macrolepis were significantly increased on 7 days in Gal9 group compared to EV group (P < 0.05). The bacterial load of liver, spleen, and kidney of O.macrolepis infected with A. hydrophila in Gal9 group at 24 h significantly lower than that in EV group (P < 0.05), and the survival rate has increased from 15% to 35%. A comparative transcriptome analysis between the Gal9 and EV group identified 305 differentially expressed genes (DEGs). The analysis showed that OmGal-9 might play important regulatory in glycolysis / gluconeogenesis, fatty acid degradation, and ascorbate and aldarate metabolism. Moreover, the immune-related DEGs were predominantly enriched in eleven pathways, with the most important three of them being linked to innate immunity: NOD-like, C-type lectin and Toll-like receptor signaling pathway. Taking together, OmGal-9 can enhance the resistance of fish to bacterial diseases by improving immune system function and activating immune-related pathways.

Innate and adaptive immunity gene expression profiles induced by virulent Aeromonas hydrophila infection in the immune-related organs of channel catfish

Aeromonas hydrophila causes motile Aeromonas septicemia (MAS) in freshwater fish. In recent years, MAS outbreaks due to virulent Aeromonas hydrophila (vAh) have been responsible for large-scale losses within commercial catfish farms in Mississippi and Alabama. The aim of this study was to evaluate immune gene expression in catfish immune-competent tissues during infection with vAh strain ML09-119. Specific pathogen-free catfish fingerlings were intraperitoneally infected with vAh strain ML09-119, and relative expression of thirteen immune-related genes was evaluated from head kidney, spleen, and liver. Our results revealed that vAh was detected 2 h post-infection (hpi) in the head kidney, liver, and spleen. The highest concentration of vAh was detected at 12 hpi, from which point concentrations decreased until clearance at 5 days post-infection (dpi). Gene expression analysis revealed upregulation of pro-inflammatory cytokines and innate immune response (TLR 4 and 5) in the first 24 hpi. Adaptive immune-related genes were upregulated at 7 dpi in the spleen and 14 dpi in the head kidney. Furthermore, immunoglobulin M showed significant upregulation at 14 dpi in the head kidney and 21 dpi in the spleen. In summary, vAh ML09-119 infection induced a strong inflammatory response involving multiple innate immunity genes, proinflammatory cytokines, and chemokines. Surviving catfish were able to clear the infection and produce antibodies and memory cells. Assessment of the immunological response to vAh infection is critical for understanding the pathogen's mechanisms of pathogenesis and developing means for MAS control, including vaccine development and improved treatments.

Baicalin enhances antioxidant, inflammatory defense, and microbial diversity of yellow catfish (Pelteobagrus fulvidraco) infected with Aeromonas hydrophila.

The aim of this research was to clarify the mechanism through which baicalin exerts its inhibitory effects on Aeromonas hydrophila infection. The antibacterial efficacy of baicalin was assessed by determining its minimum inhibitory concentration (MIC) against A. hydrophila. Various parameters, including the growth curve, cell wall integrity, biofilm formation, AKP content, and morphological alterations of A. hydrophila, were analyzed. In vivo experiments involved the administration of A. hydrophila 4 h postintraperitoneal injection of varying doses of baicalin to induce infection, with subsequent monitoring of mortality rates. After a 3 d period, liver, spleen, and intestinal tissues were harvested to evaluate organ indices, antioxidant and immune parameters, as well as intestinal microbial composition. The findings indicated that baicalin treatment resulted in the disruption of the cell wall of A. hydrophila, leading to the loss of its normal structural integrity. Furthermore, baicalin significantly inhibited biofilm formation and facilitated the release of intracellular proteins (P < 0.05). In vivo, baicalin enhanced the survival rates of yellow catfish infected with A. hydrophila. Compared to the control group, the liver index of yellow catfish was elevated, while the spleen and intestinal indices were reduced in the baicalin-treated group (P < 0.05). Additionally, baicalin at an appropriate dosage was found to increase levels of SOD, GSH, CAT, ACP, and AKP in yellow catfish (P < 0.05), while simultaneously decreasing MDA accumulation and the mRNA expression of inflammatory markers such as Keap1, IL1, IFN-γ, and TNF-α, (P < 0.05). Moreover, baicalin significantly enhanced the operational taxonomic unit (OTU) count in A. hydrophila-infected yellow catfish (P < 0.05), restoring the abundance of Barnesiellaceae, Enterobacteriaceae, Plesiomonas, and UBA1819 (P < 0.05). In summary, baicalin demonstrates the potential to improve the survival rate of yellow catfish subjected to A. hydrophila infection, augment antioxidant and immune responses, mitigate inflammation, and enhance intestinal microbial diversity.

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

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

Toll-like Receptor Expression in Pelodiscus sinensis Reveals Differential Responses after Aeromonas hydrophila Infection.

Background: Toll-like receptor (TLR), as an important pattern recognition receptor, is a bridge between non-specific immunity and specific immunity, and plays a vital role in the disease resistance of aquatic animals. However, the function of TLR in Pelodiscus sinensis is still unclear. Methods and Results: The sequence characteristics and homology of three TLRs (PsTLR2, PsTLR3 and PsTLR5) were determined in this investigation. Their annotation and orthologies were supported by phylogenetic analysis, functional domain prediction, and sequence similarity analysis. qPCR showed that the identified TLRs were expressed in all tissues, among the high expression of PsTLR5 in the brain and liver and the high expression of PsTLR2 and PsTLR3 in the liver. PsTLR2 mRNA expression increased 6.7-fold in the liver 12 h after Aeromonas hydrophila infection, while the mRNA expression of PsTLR3 was down-regulated by 0.29 times in liver and 0.31 times in spleen. The mRNA expression of PsTLR5 was significantly up-regulated in four immune tissues, and it was up-regulated by 122.8 times in the spleen after 72 h infection. Finally, the recombinant proteins of extracellular LRR domains of these three TLRs were obtained by prokaryotic expression technology, and the binding tests were performed to discover their ability of binding pathogenic microorganisms. Microbial binding test showed that rPsTLR2, rPsTLR3 and rPsTLR5 can combine A. hydrophila, Edwardsiella tarda, Vibrio parahaemolyticus, Staphylococcus aureus, Streptococcus agalactiae and Candida albicans, while rPsTLR3 can bind A. hydrophila, E. tarda, V. parahaemolyticus and C. albicans. Conclusions: Our findings suggested that TLRs may be crucial to turtles' innate immune response against microbes.

Proteomic insights into extracellular matrix dynamics in the intestine of Labeo rohita during Aeromonas hydrophila infection.

The study underscores the critical role of the extracellular matrix (ECM) and its associated proteins in the immune response of aquatic organisms during bacterial infections like Aeromonas hydrophila. Understanding the intricate interplay between ECM alterations and immune response pathways provides crucial insights for developing effective disease control strategies in aquaculture. By identifying key proteins and pathways involved in host defense mechanisms, this research lays the groundwork for targeted interventions to mitigate the impact of bacterial infections on fish health and aquaculture production.

Immune response for acute Aeromonas hydrophila infection in two distinct color morphs of northern snakehead, Channa argus

To compare and analyze the differences in immunological response between the two color morphs of Channa argus, a fish cohort was divided into four groups: black C argus + PBS (B-PBS), black C argus + Aeromonas hydrophila (B-Ah), white C. argus + PBS (W-PBS), and white C. argus + A hydrophila (W-Ah). The B-PBS and W-PBS groups received 100 μL PBS, while the B-Ah and W-Ah groups received 3.6 × 105 CFU/mL A. hydrophila in the same volume. The death rate in each group was noted, changes in plasma biochemical indicators and the expression of liver immune-related genes were examined, and transcriptome techniques were used to compare the differences between the two colors of C. argus following stress. No mortality occurred in the B-PBS and W-PBS groups. Mortality in the W-Ah and B-Ah groups showed an upward and then downward trend after A. hydrophila injection. The highest mortality occurred within 24 h and was higher in the W-Ah group than in the B-Ah group. MDA levels in the B-Ah and W-Ah groups increased and then decreased, while SOD and T-AOC showed the reverse tendency. The W-Ah and W-PBS groups differed significantly in MDA at 3, 12, and 24 h, SOD from 6 to 96 h, and T-AOC between 6 and 48 h. Plasma MDA and T-AOC levels at 12 h and SOD levels at 24 and 48 h differed significantly between the B-PBS and B-Ah groups. In both the W-Ah and B-Ah groups, the expression levels of IL-1β and IL-8 in the liver showed a temporal pattern with an initial increase followed by a decrease, reaching peak levels after 24 h, while IL-10 showed the reverse pattern. Transcriptome analysis of the liver revealed significant differences between the two C. argus colors. Differential genes in black C. argus were mainly enriched in steroid biosynthesis, glycolysis/gluconeogenesis, and glutathione and propanoate metabolism pathways 24 h after infection. In contrast, differential genes in white C. argus were mainly enriched in pathways such as oxidative phosphorylation, pancreatic secretion, and protein digestion and absorption 24 h after infection. After A. hydrophila infection, white C. argus had higher mortality, more severe oxidative stress and inflammatory responses, and lower antioxidant capacity than black C. argus.

Effects of dietary immunostimulants on the growth and non-specific immune responses of hybrid sturgeons (Huso huso♀ × Acipenser ruthenus♂)

Herein, we investigated the effects of immunostimulants on the growth, immune responses, and post-infection survival of hybrid sturgeons (Huso huso♀ × Acipenser ruthenus♂). Therefore, we examined the effects of dietary supplementation of β-glucan, vitamin C, and Radix Bupleuri (Chaihu) extract (RBE) at various concentrations (0.2, 0.4, and 0.6% incorporated through feed) on the growth rate and non-specific immune responses of hybrid sturgeon. When supplemented individually, these immunostimulants significantly reduced the feed conversion ratio while promoting weight gain, with the vitamin C-administered groups showing the most pronounced differences. Further, we observed that dietary β-glucan, vitamin C, and RBE, when fed at 0.4 and 0.6% concentrations, significantly enhanced the rates of superoxide anion production and phagocytosis by head kidney leukocytes. Moreover, vitamin C and RBE supplementation at 0.4 and 0.6% and β-glucan supplementation at 0.6% concentration significantly increased the serum lysozyme activity. Further, vitamin C and β-glucan supplementation at 0.6% and RBE supplementation at 0.4 and 0.6% concentrations showed the highest survival rates after Aeromonas hydrophila infection. In conclusion, our findings highlight that the dietary supplementation of β-glucan, vitamin C, or RBE in appropriate concentrations can significantly enhance superoxide anion production and phagocytic activity of leukocytes, serum lysozyme activity, and the survival of hybrid sturgeons after A. hydrophila infection. Our results suggest that incorporating immunostimulants such as Chinese medicinal herbs at optimal levels can effectively boost the growth, immunity, and disease resistance of hybrid sturgeons in aquaculture. These results provide valuable insights for future studies on the inclusion of immunostimulants in aquaculture feed.

Single-cell transcriptome landscape of the kidney reveals potential innate immune regulation mechanisms in hybrid yellow catfish after Aeromonas hydrophila infection

Aeromonas hydrophila, the pathogen that is the causative agent of motile Aeromonas septicemia (MAS) disease, commonly attacks freshwater fishes, including yellow catfish (Pelteobagrus fulvidraco). Although the kidney is one of the most important organs involved in immunity in fish, its role in disease progression has not been fully elucidated. Understanding the cellular composition and innate immune regulation mechanisms of the kidney of yellow catfish is important for the treatment of MAS. In this study, single-cell RNA sequencing (scRNA-seq) was performed on the kidney of hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂) after A. hydrophila infection. Nine types of kidney cells were identified using marker genes, and a transcription module of marker genes in the main immune cells of hybrid yellow catfish kidney tissue was constructed using in-situ hybridization. In addition, the single-cell transcriptome data showed that the differentially expressed genes of macrophages were primarily enriched in the Toll-like receptor and Nod-like receptor signaling pathways. The expression levels of genes involved in these pathways were upregulated in macrophages following A. hydrophila infection. Transmission electron microscopy and TUNEL analysis revealed the cellular characteristics of macrophages before and after A. hydrophila infection. These data provide empirical support for in-depth research on the role of the kidney in the innate immune response of hybrid yellow catfish.

Toll-like receptor 21 in Labeo rohita recognizes double-stranded RNA and lipopolysaccharides by engaging the critical motifs in the LRR domain and gets activated against bacterial assaults

TLR (Toll-like receptor)-21 is a non-mammalian TLR and exhibits a unique function within the innate immune systems of fishes, birds, and amphibians. Despite its important role as PRR (pattern recognition receptor), research on TLR21 in many fish species, as well as in rohu (Labeo rohita), remains relatively limited. This article describes the molecular cloning of LrTLR21 (TLR21 in L. rohita), 3D (3-dimensional) modelling of its LRR (leucine-rich repeat)-regions, prediction of LRR18 to LRR20 as the LPS (lipopolysaccharide)-binding site, and LRR1 to LRR5 and LRR21 to LRR23 as the poly I:C (polyinosinic-polycytidylic acid) binding sites. It also describes the response of LrTLR21 in response to Aeromonas hydrophila and Edwardsiella tarda infections and PAMPs (pathogen-associated molecular patterns) (LPS and poly I:C)-stimulations. The ORF (open reading frame) of the LrTLR21 comprises 2955 nucleotides, encoding 984 aa (amino acid) residues with molecular weight and isoelectric point of 113.791 kDa and ∼8.79, respectively. Domain analysis of the deduced LrTLR21 displayed the existence of a signal peptide (residues 1–24), 26 LRR regions (residues 61–685), a TM (transmembrane) domain (residues 736–758), and a TIR (Toll/interleukin-1 receptor) domain (residues 790–937). The 3D model of LrTLR21-LRR regions has parallel β-sheets and few α-helices. Phylogenetically, LrTLR21 is closely related to the Onychostoma macrolepis and Carassius gibelio TLR21, and during ontogenesis, it is expressed in most of the developmental stages. In rohu fingerlings, it is consistently expressed in all examined tissues viz., skin, liver, heart, blood, eye, muscle, kidney, intestine, brain, gill, and spleen. Upon exposure to E. tarda and A. hydrophila infections, as well as LPS and poly I:C stimulations, the expression of LrTLR21 is significantly (p < 0.05) increased in the blood, kidney, liver, and gill. In the RBCs (red blood cells), PBLs (peripheral blood leukocytes), and kidney macrophages, LrTLR21 is also significantly induced following in-vivo and in-vitro LPS and poly I: C-stimulations. These findings on LrTLR21 are the first ones showing its structural insights and PAMPs binding motifs and its key role in recognizing pathogens and their PAMPs in RBCs, PBLs, and macrophages.

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

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