Impact of Sel-Plex® dietary supplementation on growth performance, physiological response, oxidative status, and immunity-linked gene expression in Nile tilapia (Oreochromis niloticus) fingerlings challenged with Aeromonas hydrophila.

Background: Organic selenium (Sel-Plex®) supplementation holds considerable promise for improving the effectiveness of fish production. Aim: This experiment was accomplished to judge the potential benefits of Sel-Plex® nutritional additive on growth outcomes, physiological response, oxidative status, and immunity-linked gene expression in Nile tilapia (Oreochromis niloticus) fingerlings exposed to bacterial infection with Aeromonas hydrophila. Methods: Utilizing a basal diet of 30% protein, four experimental diets were prepared, each of which contained Sel-Plex® at concentrations of 0.0, 0.5, 1, and 2 mg/kg, respectively. Three replicates of 20 fish/treatment were used using 240 healthy Nile tilapia fingerlings. Fish were placed in 12 glass aquariums and separated into 4 groups at random. For the entire span of 8 weeks, diets were admitted to fish at a 3% rate of fish biomass/aquarium. After the feeding trial, pathogenic A. hydrophila was intraperitoneally injected into fish of each treatment, and fish were observed for 15 days to track the survival rate (SR) after the challenge. Results: Growth performance, physiological response, immunological parameters (phagocytic activity, phagocytic index, and lysozyme), and antioxidant parameters [catalase, superoxide dismutase (SOD), malondialdehyde, and glutathione peroxidase (GPx)] were noticeably improved in Sel-Plex® treated groups. Moreover, Sel-Plex® increased gene expression linked with the immune system in the liver (tumor necrosis factor-alpha and interleukin 1ß), to growth (insulin-like growth factor 1 and growth hormone receptor), and antioxidants (SOD and GPx). Under pathogen-challenge conditions, the employed dietary Sel-Plex® supplementation could successfully lower fish oxidative stress, offering a potential preventive additive for Nile tilapia instead of antibiotics. On the other hand, Sel-Plex® significantly enhanced each of three intestinal morphological measurements (villus width, villus length, and crypt depth), demonstrating the greatest influence on the improvement of intestinal structure overall. In the Nile tilapia control group, the infection with A. hydrophila caused noticeable degenerative alterations in the gut, hepatopancreas, spleen, and posterior kidney. The severity of the lesion was significantly reduced and significantly improved with higher Sel-Plex® concentrations. Sel-Plex® supplemented groups had 100% SRs among the A. hydrophila-challenged groups. Conclusion: It could be advised to enrich the diets of Nile tilapia fingerlings with 1-2 mg.kg-1 of Sel-Plex® to enhance growth rate, physiological response, immunological reaction, and intestinal absorptive capacity.

Ameliorative effect of BIO-MOS® as a dietary supplementation on growth performance, physiological response, oxidative status, and immunity-linked gene expression in Nile tilapia (Oreochromis niloticus) fingerlings challenged with Aeromonas hydrophila.

Background: Mannanoligosaccharides (MOS) usage in fish production has drawn more attention because of their positive benefits on disease resistance and fish performance. Aim: The ongoing research was executed to assess the potential advantages of Bio-Mos® dietary supplementation regarding the growth outcomes, physiological response, oxidative biomarkers, and immunity-linked gene expression in Nile tilapia (Oreochromis niloticus) fingerlings exposed to bacterial infection with Aeromonas hydrophila. Methods: Four experimental diets were developed using a 30% protein baseline diet, with Bio-Mos® added at variable levels; 0.0, 0.5, 1, and 2 g/kg, respectively. 240 healthy Nile tilapia fingerlings were split into 4 groups at random and assigned to 12 glass aquariums (three replicates of 20 fish/treatment). Diets were admitted at a 3% rate of fish biomass/aquarium for 8 weeks. Following the feeding trial, fish from every treatment were intraperitoneally injected with pathogenic A. hydrophila, and then observed for 15 days to record the survival rate percent (SR%) post challenge. Results: Results revealed significant improvement in growth performance, physiological response, immunological parameters (phagocytic index, phagocytic activity, and lysozyme), and antioxidant parameters [catalase, malondialdehyde, glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD)] among Bio-Mos® treated groups. Moreover, Bio-Mos® increased the expression of tumor necrosis factor alpha and Interleukin 1ß, genes linked to the liver immune system. Growth-related genes (GHr), antioxidant-related genes (SOD and GSH-Px). In fish subjected to pathogens, dietary MOS supplementation could significantly lower oxidative stress, showing promise as a preventative supplement for Nile tilapia in place of antibiotics. On the other hand, Bio-Mos® considerably improved each of the three intestinal morphological measures (villus width, villus length, and crypt depth), showing the best overall intestinal structure-improving impact. The challenge with A. hydrophila caused marked degenerative alterations in the intestine, hepatopancreas, spleen, and posterior kidney of Nile tilapia, in the control group. However, lesion severity was greatly decreased and showed marked amelioration with an increased concentration of Bio-Mos®. The A. hydrophila-challenged groups revealed a 100% SR% mainly among the Bio-Mos® supplemented groups. Conclusion: It is recommended to enrich the Nile tilapia fingerlings diets with 2 g.kg-1 of MOS for better results on the growth rate, physiological response, immunological response, and intestinal absorptive capacity.

Aeromonas hydrophila induced necrotizing fasciitis following laparoscopic cholecystectomy.

Necrotizi ng fasciit is [NF] i s a m ultifaceted disease of the muscle fascia and body tissues which demands the earliest intervention. Past reviews have documented ver y few cases of Aeromonas Hydroph ila [AH] induced N F fol lo wing abdominal surgery. AH can cause fatal NF as seen in a 72 year old female patient reported at Liaquat National Hospital &Medical College; a ter tiary care center in Karachi, Pakistan on 2nd April, 2022. She had a k nown comorbidity of hypertension and presented with the chief complaint of symptomatic gallstones for which she unde rwent Laparoscopic Cholecystectomy (LAPCHOL). She developed NF of the lower ab domen post- oper atively. Following uneventful Laparoscopic Cholecystectomy our pati ent presented to the ER two days later with severe lower abdo minal pain and overlyi ng celluliti s. Fasc io to my revealed extensive myonecrosis with necrotizing soft tissue in fe ction. Despite u ndergoing extensive surgical debr idement and broad spectr um antibi ot ic administration; the patient died in the ICU on the fifth postoperat ive day followi ng septic complications. Histopathologica l an alysis, confirmed i nflammat ion and necrosis. Culture sensitivity of the debrided tissue revealed AH. Approach should lie towards analyzing the behaviour of such microbes in high risk patients through collective case studies. This is the first clinical case showcasing such parameters e ncountered in the General Surger y Department.

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.

Emergence and clonal expansion of Aeromonas hydrophila ST1172 that simultaneously produces MOX-13 and OXA-724.

BACKGROUND: Aeromonas hydrophila infections can cause gastrointestinal symptoms such as diarrhea; however, deep infections are rarely reported. Outbreaks of A. hydrophila are reported more frequently in fish, poultry, and snakes than in humans. This study aimed to track clonal relatedness of deep infections caused by A. hydrophila using whole genome sequencing (WGS). METHODS: We collected three isolates of A. hydrophila in July 19 to August 29, 2019, from patients that underwent spine surgery. Accurate species identification was performed using whole-genome average nucleotide identity (ANI). Antimicrobial susceptibility testing was performed using a VITEK 2 automated AST-N334 Gram-negative susceptibility card system. Antimicrobial resistance and virulence genes were identified using the Comprehensive Antibiotic Resistance Database and Virulence Factor Database VFanalyzer. RESULTS: All three isolates were identified as A. hydrophila based on ANI and multilocus sequence typing analysis revealed that A. hydrophila belonged to a novel sequence type (ST1172). All three isolates were susceptible to amikacin and levofloxacin; however, they were resistant to piperacillin/tazobactam, ceftriaxone, cefuroxime, cefoxitin, and imipenem. Isolate 19W05620 (patient 3) showed increased ceftazidime resistance (minimum inhibitory concentration ≥ 64 µg/mL). All three isolates possessed the same chromosomally encoded ß-lactamases, including blaOXA-724 (ß-lactamase), imiH (metallo-ß-lactamase), and blaMOX-13 (AmpC) in plasmids. CONCLUSIONS: Our study validated the transmission of a novel carbapenem-resistant A. hydrophila sequence type (ST1172) in patients that underwent spine surgery. Control measures should be developed to prevent dissemination of A. hydrophila in the hospital setting.

The impact of Lactococcus lactis KUST48 on the transcription profile of Aeromonas hydrophila-infected zebrafish spleen.

Aeromonas hydrophila, an aquatic pathogenic bacterium, has been found to infect many fish species and cause huge aquaculture losses. Antibiotics are the most common drugs used to treat these infections. However, antibiotic abuse can lead to the development of antibiotic resistance. Probiotics have the potential to replace antibiotics for preventing infections. Zebrafish (Danio rerio) is a model organism used to study the innate immune system and host-pathogen interactions. Currently, there is little information on how the fish immune system responds to A. hydrophila and probiotic treatment. To increase the understanding of the molecular mechanisms behind the zebrafish defense against A. hydrophila and provide evidence that antibiotics can be replaced by probiotics, a transcriptome analysis of the zebrafish spleen was conducted 48 hours after infection by A. hydrophila, as well as after treatment using Lactococcus lactis KUST48 4 hours after infection. A total of 36,499 genes were obtained. There were 3,337 genes found to have significant differential expression between treatment and control groups. According to further annotation and enrichment analysis, differentially expressed genes (DEGs) were involved in signal transduction, endocrine system cancer, and the immune system. Insulin resistance disappeared in the zebrafish after treatment. Quantitative real-time PCR was performed to confirm the significant regulation of immune defense DEGs, the results of which were consistent with the RNA-sequencing data. These results could serve as a basis for future studies on the immune response to A. hydrophila and provide suggestions for probiotic alternatives to antibiotics, which will be of great significance to aquaculture and environmental protection.IMPORTANCEIn recent years, the unreasonable use of antibiotics has led to the emergence of drug-resistant pathogenic bacteria, antibiotic residues, cross infection, toxic side effects, and so on, which has caused a serious threat to human food safety and life health. In recent years, many studies have demonstrated the potential of probiotics as a substitute for antibiotics, but there is still a lack of understanding of the molecular mechanisms underlying probiotic therapy. We conduct a research on the impact of Lactococcus lactis KUST48 on the transcription profile of Aeromonas hydrophila-infected zebrafish spleen. Mortality of zebrafish infected with A. hydrophila was significantly reduced after treatment with L. lactis KUST48. Our results can help to strengthen our understanding of the pathogenic mechanisms of zebrafish and provide a valuable reference for the molecular mechanisms of probiotic therapy.

Immune and endocrine alterations at the early stage of inflammatory assemblage in toads after stimulation with heat-killed bacteria (Aeromonas hydrophila).

The red-leg syndrome in amphibians is a condition commonly associated with the bacteria Aeromonas hydrophila and has led to population declines. However, there is little information concerning the inflammatory assemblage in infected anurans. We evaluated immune and endocrine alterations induced by stimulation with heat-killed A. hydrophila injected in Rhinella diptycha toads. Control animals were not manipulated, while the others were separated into groups that received intraperitoneal injection of 300 µl of saline or heat-killed bacteria: groups A1 (3 × 107 cells), A2 (3 × 108 cells), and A3 (3 × 109 cells). Animals were bled and euthanized six hours post-injection. We evaluated neutrophil: lymphocyte ratio (NLR), plasma bacterial killing ability (BKA), testosterone (T), melatonin (MEL), and corticosterone (CORT) plasma levels. Heat-killed A. hydrophila increased CORT and NLR, and decreased MEL, especially at higher concentrations. There was no effect of treatment on T and BKA. We then selected the saline and A3 groups to conduct mRNA expression of several genes including glucocorticoid receptor (GR), toll-like receptor-4 (TLR-4), interferon-γ (IFN-γ), interleukin (IL)-1ß, IL-6, and IL-10. We found higher expression of IL-6, IL-1ß, IL-10, and IFN-γ in group A3 compared to the saline group. These results indicate the beginning of an inflammatory assemblage, notably at the two highest concentrations of bacteria, and give a better understanding of how anurans respond to an infection within an integrated perspective, evaluating different physiological aspects. Future studies should investigate later phases of the immune response to elucidate more about the inflammation in amphibians challenged with A. hydrophila.

Excessive dietary iron exposure increases the susceptibility of largemouth bass (Micropterus salmoides) to Aeromonas hydrophila by interfering with immune response, oxidative stress, and intestinal homeostasis.

Iron is an essential cofactor in the fundamental metabolic pathways of organisms. Moderate iron intake can enhance animal growth performance, while iron overload increases the risk of pathogen infection. Although the impact of iron on the pathogen-host relationship has been confirmed in higher vertebrates, research in fish is extremely limited. The effects and mechanisms of different levels of iron exposure on the infection of Aeromonas hydrophila in largemouth bass (Micropterus salmoides) remain unclear. In this study, experimental diets were prepared by adding 0, 800, 1600, and 3200 mg/kg of FeSO4∙7H2O to the basal feed, and the impact of a 56-day feeding period on the mortality rate of largemouth bass infected with A. hydrophila was analyzed. Additionally, the relationships between mortality rate and tissue iron content, immune regulation, oxidative stress, iron homeostasis, gut microbiota, and tissue morphology were investigated. The results showed that the survival rate of largemouth bass infected with A. hydrophila decreased with increasing iron exposure levels. Excessive dietary iron intake significantly increased iron deposition in the tissues of largemouth bass, reduced the expression and activity of antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, increased the content of lipid peroxidation product malondialdehyde, and thereby induced oxidative stress. Excessive iron supplementation could influence the immune response of largemouth bass by upregulating the expression of pro-inflammatory cytokines in the intestine and liver, while downregulating the expression of anti-inflammatory cytokines. Additionally, excessive iron intake could also affect iron metabolism by inducing the expression of hepcidin, disrupt intestinal homeostasis by interfering with the composition and function of the gut microbiota, and induce damage in the intestinal and hepatic tissues. These research findings provide a partial theoretical basis for deciphering the molecular mechanisms underlying the influence of excessive iron exposure on the susceptibility of largemouth bass to pathogenic bacteria.

Chlorella vulgaris extract conjugated magnetic iron nanoparticles in nile tilapia (Oreochromis niloticus): Growth promoting, immunostimulant and antioxidant role and combating against the synergistic infection with Ichthyophthirius multifiliis and Aeromonashydrophila.

Nile tilapia reared under intensive conditions was more susceptible for Ichthyophthirius multifilii (I. multifiliis) infection eliciting higher mortality, lower productive rate and further bacterial coinfection with Aeromonas hydrophila (A. hydrophila). The higher potency of magnetic field of iron oxide nanoparticles (NPs) can kill pathogens through inhibiting their viability. Herein, coating of Chlorella vulgaris extract (ChVE) with magnetic iron oxide NPs (Mag iron NPs) can create an external magnetic field that facilitates their release inside the targeted tissues. Thus, the current study is focused on application of new functionalized properties of Mag iron NPs in combination with ChVE and their efficacy to alleviate I. multifiliis and subsequent infection with A. hydrophila in Nile tilapia. Four hundred fingerlings were divided into: control group (with no additives), three groups fed control diet supplemented with ChVE, Mag iron NPs and ChVE@Mag iron NPs for 90 days. At the end of feeding trial fish were challenged with I. multifiliis and at 9 days post challenge was coinfected by A. hydrophila. A remarkable higher growth rate and an improved feed conversion ratio were detected in group fed ChVE@Mag iron-NPs. The maximum expression of antioxidant enzymes in skin and gills tissues (GSH-Px, CAT, and SOD) which came in parallel with higher serum activities of these enzymes was identified in groups received ChVE@Mag iron-NPs. Furthermore, group fed a combination of ChVE and Mag iron-NPs showed a boosted immune response (higher lysozyme, IgM, ACH50, and MPO) prior to challenge with I. multifiliis. In contrast, fish fed ChVE@Mag iron-NPs supplemented diet had lower infection (decreased by 62%) and mortality rates (decreased by 84%), as well as less visible white spots (decreased by 92 % at 12 dpi) on the body surfaces and mucous score. Interestingly, post I. multifiliis the excessive inflammatory response in gill and skin tissues was subsided by feeding on ChVE@Mag iron-NPs as proved by down regulation of IL-1ß, TNFα, COX-2 and iNOS and upregulation of IL-10, and IgM, IgT and Muc-2 genes. Notably, group exposed to I. multifiliis-showed higher mortality when exposed to Aeromonas hydrophilia (increased by 43 %) while group fed ChVE@Mag iron-NPs exhibited lower morality (2%). Moreover, the bacterial loads of A. hydrophilia in fish infected by I. multifiliis and fed control diet were higher than those received dietary supplement of ChVE, Mag iron-NPs and the most reduced load was obtained in group fed ChVE@Mag iron-NPs at 7 dpi. In conclusion, ChVE@Mag iron-NPs fed fish had stronger immune barrier and antioxidant functions of skin and gills, and better survival following I. multifiliis and A. hydrophilia infection.

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.

Molecular characterization of Aeromonas hydrophila isolates from diseased fishes in district Kasur, Punjab, Pakistan / Caracterização molecular de isolados de Aeromonas hydrophila de peixes doentes no distrito de Kasur, Punjab, Paquistão

Abstract Pakistan is an agricultural country and fisheries play a very important role in the economic development of the country. Different diseases are prevalent in Pakistani fish but information related to the causative agents is not well-known. Keeping in view the significance of bacterial pathogens as the causative agents of multiple fish diseases, the present study was conducted for identification, characterization and analysis of virulence genes of Aeromonas spp. isolated from diseased fishes. A total of fifty fish samples having multiple clinical indications were collected from different fish farms of district Kasur, Punjab Pakistan. For isolation of Aeromonas spp. samples were enriched and inoculated on Aeromonas isolation medium. Isolates were identified and characterized by different biochemical tests, Analytical Profile Index (API) 20E kit and Polymerase Chain Reaction (PCR) assays. All isolates were screened for three putative virulence genes including aerolysin (aer), haemolysin (hyl) and heat labile cytotonic enterotoxin (alt). Seven isolates of Aeromonas (A.) hydrophila were retrieved and identified based on API 20E. These isolates were further confirmed as A. hydrophila on the basis of PCR assays. Three isolates were detected positive for the presence of virulence genes (alt and hyl). Whereas aerolysin (aer) gene was not present in any of A. hydrophila isolates. The present study confirmed A. hydrophila as the causative agent of epizootic ulcerative syndrome and motile Aeromonas septicemia in fish farms of district Kasur, Punjab Pakistan. Moreover, detection of two virulence genes (alt and hyl) in A. hydrophila isolates is a threat for fish consumers of study area.

Resumo O Paquistão é um país agrícola, onde a pesca desempenha um papel muito importante para o desenvolvimento econômico. Diferentes doenças são prevalentes em peixes do Paquistão, mas as informações relacionadas aos agentes causadores não são bem conhecidas. Tendo em vista a importância dos patógenos bacterianos como agentes causadores de múltiplas doenças em peixes, o presente estudo foi conduzido para identificação, caracterização e análise de genes de virulência de isolados de Aeromonas spp. de peixes doentes. Foram coletadas 50 amostras de peixes com múltiplas indicações clínicas em diferentes fazendas do distrito de Kasur, Punjab, Paquistão. Para isolar Aeromonas spp., as amostras foram enriquecidas e inoculadas em meio de isolamento. Os isolados foram identificados e caracterizados por diferentes testes bioquímicos, kit Analytical Profile Index (API) 20E, e ensaios de reação em cadeia da polimerase (PCR). Todos os isolados foram selecionados para três genes de virulência putativos, incluindo aerolisina (aer), hemolisina (hyl) e enterotoxina citotônica termolábil (alt). Sete isolados de Aeromonas hydrophila foram recuperados e identificados com base no API 20E. Esses isolados foram posteriormente confirmados como A. hydrophila de acordo com ensaios de PCR. Três isolados indicaram a presença de genes de virulência (alt e hyl), enquanto o gene aerolisina (aer) não esteve presente em nenhum dos isolados de A. hydrophila. O presente estudo confirmou A. hydrophila como o agente causador da síndrome ulcerativa epizoótica e septicemia móvel por Aeromonas em fazendas de peixes, no distrito de Kasur, Punjab, Paquistão. Além disso, a detecção de dois genes de virulência (alt e hyl) em isolados de A. hydrophila é uma ameaça para os consumidores de peixes da área de estudo.

The infection of Aeromonas hydrophila activated Multiple programmed cell death pathways in red blood cells of Clarias fuscus.

In contrast to mammalian red blood cells (RBCs), Osteichthyes RBCs contain a nucleus and organelles, suggesting the involvement of more intricate mechanisms, particularly in the context of ferroptosis. In this study, we utilized RBCs from Clarias fuscus (referred to as Cf-RBCs) as a model system. We conducted RNA-seq analysis to quantify gene expression levels in Cf-RBCs after exposure to both Aeromonas hydrophila and lipopolysaccharides. Our analysis unveiled 1326 differentially expressed genes (DEGs) in Cf-RBCs following 4 h of incubation with A. hydrophila, comprising 715 and 611 genes with upregulated and downregulated expression, respectively. These DEGs were further categorized into functional clusters: 292 related to cellular processes, 241 involved in environmental information processing, 272 associated with genetic information processing, and 399 linked to organismal systems. Additionally, notable changes were observed in genes associated with the autophagy pathway at 4 h, and alterations in the ferroptosis pathway were observed at 8 h following A. hydrophila incubation. To validate these findings, we assessed the expression of cytokines (DMT1, TFR1, LC3, and GSS). All selected genes were significantly upregulated after exposure to A. hydrophila. Using flow cytometry, we evaluated the extent of ferroptosis, and the group incubated with A. hydrophila for 8 h exhibited higher levels of lipid peroxidation compared with the 4-h incubation group, even under baseline conditions. An evaluation of the glutathione redox system through GSSG/GSH ratios indicated an increased ratio in Cf-RBCs after exposure to A. hydrophila. In summary, our data suggest that A. hydrophila may induce ferroptosis in Cf-RBCs, potentially by triggering the cystine/glutamate antiporter system (system XC-), while Cf-RBCs counteract ferroptosis through the regulation of the glutathione redox system. These findings contribute to our understanding of the iron overload mechanism in Osteichthyes RBCs, provide insights into the management of bacterial diseases in Clarias fuscus, and offer potential strategies to mitigate economic losses in aquaculture.

Characteristics of CD4-1 gene and its immune responses against Aeromonas veronii infection by activating NF-κB signaling in Qihe crucian carp Carassius auratus.

CD4-1 found in bony fish contains four extracellular immunoglobulin (Ig)-like domains similar to that of mammalian CD4, which is crucial for the activation of CD4+ helper T-cell. However, there is limited knowledge regarding the molecular markers, immune functions and regulation mechanism of CD4-1 in teleosts due to their vast diversity. In this study, we cloned and characterized two isoforms of Qihe crucian carp CD4-1, designated as CaCD4-1.1 and CaCD4-1.2. We further explored their expression responses upon stimulation with Aeromonas veronii, and the regulation of their immune responses against A. veronii by NF-κB. The ORF of CaCD4-1.1 and CaCD4-1.2 cDNA encoded 477 and 466 amino acids, respectively. Both proteins contained seven conserved cysteine residues in the extracellular domain, and a CCC motif in their cytoplasm, respectively. However, CaCD4-1.1 exhibited a relatively limited similarity with CaCD4-1.2 in the ectodomain. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the mRNA expression of CaCD4-1.1 and CaCD4-1.2 exhibited differential constitutive expression across all examined tissues. Furthermore, the expression level of CD4-1.2 was higher than that of CD4-1.1 in the gills, head kidney, and spleen of Qihe crucian carp subjected to A. veronii challenge, while it was lower in the trunk kidney. Inhibition of NF-κB activity resulted in a decrease in the expression levels of CD4-1.1 and CD4-1.2 mRNA in the gill, while inducing an increase in expression levels in the spleen, in accordance with the observed ultrastructural changes in both organs. Interestingly, the impact of NF-κB on the mRNA expression level of CD4-1.1 appears to be stronger than that of CD4-1.2. Our results suggest that CaCD4-1.1 and CaCD4-1.2 could be expressed on T cells and antigen-sampling cells that exhibit similar characteristics to mammalian M cells, respectively, and differentially regulated by NF-κB in adaptive immune responses against bacterial infection. This research contributes to a better understanding of the crucial role of CD4-1 in the immune response of Qihe crucian carp and provide novel insights for the prevention and treatment of fish diseases in aquaculture.

IL-6 Mutation Attenuates Liver Injury Caused by Aeromonas hydrophila Infection by Reducing Oxidative Stress in Zebrafish.

Interleukin-6 (IL-6), a pleiotropic cytokine, plays a crucial role in acute stress induced by bacterial infection and is strongly associated with reactive oxygen species (ROS) production. However, the role of IL-6 in the liver of fish after Aeromonas hydrophila infection remains unclear. Therefore, this study constructed a zebrafish (Danio rerio) il-6 knockout line by CRISPR/Cas9 to investigate the function of IL-6 in the liver post bacterial infection. After infection with A. hydrophila, pathological observation showed that il-6-/- zebrafish exhibited milder liver damage than wild-type (WT) zebrafish. Moreover, liver transcriptome sequencing revealed that 2432 genes were significantly up-regulated and 1706 genes were significantly down-regulated in il-6-/- fish compared with WT fish after A. hydrophila infection. Further, gene ontology (GO) analysis showed that differentially expressed genes (DEGs) were significantly enriched in redox-related terms, including oxidoreductase activity, copper ion transport, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were significantly enriched in pathways such as the PPAR signaling pathway, suggesting that il-6 mutation has a significant effect on redox processes in the liver after A. hydrophila infection. Additionally, il-6-/- zebrafish exhibited lower malondialdehyde (MDA) levels and higher superoxide dismutase (SOD) activities in the liver compared with WT zebrafish following A. hydrophila infection, indicating that IL-6 deficiency mitigates oxidative stress induced by A. hydrophila infection in the liver. These findings provide a basis for further studies on the role of IL-6 in regulating oxidative stress in response to bacterial infections.

The potential of shallot skin powder and actinomycetes metabolites as antimicrobe and antibiofilm in the treatment of eel (Anguilla bicolor bicolor) infected with Aeromonas hydrophila.

BACKGROUND: Eel (Anguilla bicolor bicolor) is an Indonesian export commodity. However, it is facing a problem related to Aeromonas hydrophila, which can cause motile aeromonas septicemia (MAS) and produce biofilm formation. Problem with antibiotic resistance challenges the need of an alternative treatment. Therefore, it is important to explore a solution to treat infection and the biofilm formed by A. hydrophila. OBJECTIVES: In this study, we used shallot skin powder and actinomycetes metabolite 20 PM as antimicrobe and antibiofilm to treated eels infected with A. hydrophila. RESULTS: Shallot skin powder (6.25 g 100 g-1 feed) and Actinomycetes 20 PM metabolite (2 mL 100 g-1 feed) were found to be effective as antimicrobe and antibiofilm agent in treating eels infected with A. hydrophila. Eel treated with antibiotic, shallot skin powder, and actinomycetes metabolite had 80%, 66%, and 73% survival rates, respectively. Other indicators such as red blood cell count, hemoglobin, and hematocrit were increased, but white blood cell count and phagocytic activity were dropped. Biofilm destruction were analyzed using scanning electron microscopy to determined antibiofilm activity of actinomycetes metabolite against biofilm of A. Hydrophila. CONCLUSIONS: Shallot skin powder and actinomycetes metabolite were potential to treat infection of A. hydrophila in eel as an alternative treatment to antibiotics.

Neem oil against Aeromonas hydrophila infection by disrupting quorum sensing and biofilm formation.

Aeromonas hydrophila is an opportunistic pathogen that can cause a number of infectious diseases in fish and is widely distributed in aquatic environments. Antibiotics are the main approach against A. hydrophila infections, while the emergence of resistant bacteria limits the application of antibiotics. Here, quorum-sensing (QS) was defined as the target and the inhibitory effects of neem oil against QS of A. hydrophila was studied. The results showed that neem oil could dose-dependently reduce aerolysin, protease, lipase, acyl-homoserine lactones (AHLs), biofilm and swarming motility at sub-inhibitory concentrations. Results of real-time PCR demonstrated that neem oil could down-regulate the transcription of aerA, ahyI and ahyR. Moreover, neem oil showed significant protections to A549 cells and a fish infection model. Taken together, these results indicated that neem oil could be chosen as a promising candidate for the treatment of A. hydrophila infections.

Hepcidin from Onychostoma macrolepis: Response to Aeromonas hydrophila infection by down-regulating expression of inflammatory factor genes and regulating iron homeostasis potentially.

Hepcidin, as an antimicrobial peptide, is associated with innate immunity and is considered a potential antibiotic substitute. In the present study, the hepcidin gene from the cavefish - Onychostoma macrolepis was identified and analyzed. The recombinant hepcidin protein (rOmhepc) was obtained by prokaryotic expression, evaluating the inhibitory effect of 5 pathogenic bacteria in vitro. Sixty O. macrolepis injected with 100 µL A. hydrophila (1.5 × 108 CFU/mL) were randomly divided into the therapeutic group and infection group, and therapeutic group was injected with 100 µL rOmhepc (100 µg/mL) at 6 and 18 h. The survival rates of O. macrolepis and bacterial load in liver were measured at 24 h. The liver tissues were collected at 0, 6, 12, and 24 h after A. hydrophila injection for investigating expression levels of immune-related, inflammatory factor genes and FPN1 gene. The results demonstrated that the hepcidin CDS contained 279 bp and encoded 93 aa. Hepcidin protein has a hydrophobic surface formed by multiple hydrophobic residues (CCGCCYC), and the theoretical pI was 7.53. Omhepc gene was expressed at varying levels in tested tissues, with the liver showing the highest expression, followed by the spleen. The expression of hepcidin gene following A. hydrophila infection was up-regulated and then down-regulated in liver, and the highest expression level was found at 12 h with a 10.93-fold. The rOmhepc remarkably inhibited the growth of A. hydrophila, Staphylococcus aureus, and Streptococcus agalactiae, with inhibition rates reaching 69.67 %, 42.97 %, and 65.74 % at 100 µg/mL. The mortality rates of O. macrolepis and bacterial load in liver were significantly decreased in the therapeutic group than that of infection group (p < 0.05). After the rOmhepc therapeutic, interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were significantly down-regulated with 14.4-fold and 106.07-fold at 24 h. Furthermore, the expression of immune-related genes (C3, TNF-α, IFN-γ) and Ferroportin gene (FPN1) significantly decreased (p < 0.05). The integrated analyses indicated that the rOmhepc could significantly inhibit the growth of A. hydrophila both in vitro and in vivo, attenuating the over-expression of inflammatory factor, FPN1 and immune-related genes.

Vitamin D ameliorates Aeromonas hydrophila-induced iron-dependent oxidative damage of grass carp splenic macrophages by manipulating Nrf2-mediated antioxidant pathway.

Aeromonas hydrophila (A. hydrophila) is one of major pathogenic bacteria in aquaculture and potentially virulent to grass carp (Ctenopharyngodon idella). As an essential nutrient for fish, vitamin D3 (VD3) has been reported to play a role against oxidative stress, but the exact mechanism remains to be elusive. In this study, we found that A. hydrophila induced ferrugination and macrophage aggregation in the spleen of grass carp. Along this line, using the splenic macrophages as the model, the effects of VD3 on A. hydrophila-caused iron deposition and subsequent injuries were determined. In the context, 1,25D3 (the active form of VD3) significantly reduced cellular free Fe2+, lipid peroxidation and lactic dehydrogenase (LDH) release induced by A. hydrophila in the splenic macrophages, indicating the protective effects of VD3 on A. hydrophila-led to ferroptosis-related injuries. In support of this notion, 1,25D3 was effective in hindering ferroptosis inducers-stimulated LDH release in the same cells. Mechanically, 1,25D3 enhanced iron export protein (ferroportin1) and glutathione peroxidase 4 (GPX4) protein levels, and glutathione (GSH) contents via vitamin D receptor (VDR). Moreover, NF-E2-related factor 2 (Nrf2) pathway mediated the regulation of 1,25D3 on GPX4 protein expression and GSH synthesis. Meanwhile, 1,25D3 maintained the stability of Nrf2 proteins possibly by attenuating its ubiquitination degradation. Furthermore, in vivo experiments showed that 1,25D3 injection could not only improve the survival of fish infected by A. hydrophila, but also enhance GSH amounts and decrease malonaldehyde (MDA) contents and iron deposition in the spleen. In summary, our data for the first time suggest that VD3 is a potential antioxidant in fish to fight against A. hydrophila induced-ferroptotic damages.

Effects of the combination of chitosan and Acinetobacter KU011TH on the growth and health performances and disease resistance of juvenile hybrid catfish (Clarias gariepinus × C. macrocephalus).

Aquatic animal health management has become a crucial component in the goal of increasing catfish aquaculture productivity. Additionally, hybrid catfish (Clarias gariepinus × C. macrocephalus) has been promoted as a highly profitable freshwater fish in Asia. Interestingly, the crucial diseases induced by Aeromonas hydrophila have been reported to greatly impede catfish production. To overcome this challenge, the aim was to investigate the effects of the oral administration of potentially synbiotic chitosan (CH) and Acinetobacter KU011TH (AK) on the growth performance, immunological responses, and disease resistance of hybrid catfish against A. hydrophila. The control group was fed a basal diet (A), the diet fed to treatment group B was supplemented with 20 mL of CH/kg diet (B), and the experimental feed fed to groups C-D was mixed with 1 × 108, 1 × 109 and 1 × 1010 CFU/mL AK coated with 20 mL of CH/kg diet. Five different groups of juvenile hybrid catfish were continuously fed the 5 formulated feeds for 4 weeks. The results revealed that all tested feeds did not significantly enhance the hybrid catfish's average daily gain, specific growth rate, feed conversion ratio, hematocrit and erythrocyte counts. Interestingly, the application of CH and AK significantly increased the leukocyte counts, respiratory burst, lysozyme activity, alternative complement pathway hemolytic activity, and bactericidal activity (P < 0.05). The expression levels of the immune-related genes in the whole blood, head kidney, and spleen were significantly increased after CH-AK application (P < 0.05), but this finding was not observed in the liver (P > 0.05). Additionally, after 14 days of A. hydrophila peritoneal injection, the fish in group C showed significantly higher survival rates of approximately 70.0 % compared with the control fish in groups B, D, and E (52.5 %, 40.0 %, 45.0 %, and 45.0 %, respectively) (P < 0.05). These results collectively suggest that short-term application of the diet fed to group C effectively boosted the immune responses and disease resistance of hybrid catfish against A. hydrophila.