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Aims Study the relationship between antibiotic resistance patterns of Pseudomonas isolated from farmed rainbow trout fillets and farm or transformation process locations. Methods and Results Pseudomonas strains were isolated from rainbow trout sampled in two differently located farms and filleted in laboratory or in a processing factory. One hundred and twenty-five isolates were confirmed as belonging to Pseudomonas using CFC selective media, Gram staining, oxidase test and quantitative polymerase chain reaction methods. Fifty-one isolates from separate fish fillets were further identified using MALDI-TOF mass spectrometry, and the minimal inhibitory concentrations (MIC) of 11 antibiotics were also determined by microdilution method. Most of the isolates belonged to the Pseudomonas fluorescens group (94.1%), and no relationship was established between antibiotic resistance patterns and sampling locations (farms or filleting areas). Multiple resistance isolates with high MIC values (from 64 µg ml−1 to more than 1024 µg ml−1) were identified. Conclusions Antibiotic resistance patterns found in Pseudomonas isolates were not influenced by farms or transformation process locations. Seven isolates were found highly resistant to four different antibiotic classes. Significance and Impact of the Study This study does not provide evidence of a relationship between farm or transformation process locations on antibiotic resistance patterns of Pseudomonas population.

Cryptocaryon irritans, a holotrichous ciliate parasitic protozoan, can trigger marine white spot disease and cause substantial economic losses in mariculture. However, methods of preventing and curing the disease have negatively affect fish, human, other organisms, and the natural environment. The antiparasitic activity of some antimicrobial peptides (AMPs) has garnered extensive attention of scholars. In this study, we identified and characterised a novel antiparasitic peptide, named So-pis, from Sciaenops ocellatus. The sequence analysis, structural features, and tissue distribution suggested that So-pis is genetically related to the piscidins family. However, So-pis showed a relatively low overall conservation compared with other known piscidins. So-pis is abound in glycine residues (22.7 %) and it has a neutral isoelectric point, weak amphipathicity, relatively long α-helix, and high hydrophobicity. These key elements are responsible for its biological activity. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicated that So-pis is a typically gill-expressed peptide. The expression of So-pis in the gill, skin, spleen, and head kidney could be regulated during C. irritans infection, thereby implicating a role of So-pis in immune defence against C. irritans. The synthetic So-pis had limited or no antimicrobial activity against bacterial and yeasts but exhibited potent antiparasitic activity against C. irritans in vitro. The activity of synthetic So-pis against erythrocytes was less potent than its antiparasitic activity against C. irritans. These results indicated that So-pis might be one of the crucial defence cytokines against C. irritans in the red drum. Cumulatively, our data suggested that So-pis might be a potential candidate for developing a novel, effective, and safe therapeutic agent against marine white spot disease.

The study was executed to find out the potential effects spent coffee ground (SCG) on Nile tilapia's skin mucosal and serum immunities, disease prevention, and growth rate reared in a biofloc system. Nile tilapia fingerlings (average weight 15.25 ± 0.07 g) were disseminated into 15 aquaria (150 L tank−1) at a density of 20 fish per aquarium and treated five diets: SCG1 (control), SCG2 (10 g kg−1), SCG3 (20 g kg−1), SCG4 (40 g kg−1), and SCG5 (80 g kg−1) for eight weeks. A Completely Randomized Design (CRD) with three replications was applied. Growth rate, skin mucus, and serum immunities were quantified every 4 weeks; whereas the challenge study was conducted at the termination of the feeding trial. The outputs indicated that dietary incorporation of SCG give rise to the enhancement of SGR and FCR in comparison with the control, with best levels noted in fish fed SCG2 diet. Similarly, significant enhancements in skin mucosal and serum immunities were revealed in fish treated SCG2 over the control and other SCG diets. Likewise, higher survival rates against Streptococcus agalactiae were displayed in fish fed SCG, with the maximum level displayed in the fish treated SCG2. In conclusion, dietary supplementation of SCG2 (10 g kg−1) can be potential used as immunostimulants in tilapia aquaculture.

The purpose of the current study was to explore the immunomodulatory effects of different adjuvants combined with inactivated vaccines under Aeromonas veronii TH0426 infection in crucian carp. This study explored the best conditions for A. veronii as an inactivated vaccine, and included an animal safety test. Furthermore, we expressed the flagellin FlaA of the A. veronii TH0426 strain for use as an adjuvant supplemented in the diet. Crucian carp were fed 12 different experimental diets for 35 days, including the administration of 10 different adjuvants and inactivated vaccine combinations (50% aluminum hydroxide gel and inactivated vaccine combination, and inactivated vaccine with 20%, 30%, or 50% glucan, astragalus polysaccharide or flagellin), inactivated vaccine alone, and PBS control without adjuvant and inactivated vaccine. After the 42 day feeding trials, the fish were challenged with A. veronii TH0426, and the survival rate over 14 days was recorded. In addition, flagellin FlaA can be expressed normally in large amounts. All experimental groups produced higher levels of IgM serum titres than the control group in the different feeding cycles. Moreover, the activity of serum ACP, AKP, SOD, and LZM, and the expression of inflammatory factors were significantly increased in the experimental groups compared with the control group. The results of qRT-PCR analysis showed that the transcription levels of the IL-10, IL-1β, IFN-γ and TNF-α genes in heart, liver, spleen and kidney tissues were significantly enhanced by adjuvant treatment, indicating that the addition of adjuvants can significantly promote the body's inflammatory response. In addition, the phagocytic activity of leukocytes in each adjuvant treated group was significantly enhanced compared to that in the groups without adjuvant. After the A. veronii challenge, the survival rate of all adjuvant-treated groups was significantly higher than that of the control group, and the 50% flagellin adjuvant group had the highest rate of 78.37%. Overall, our findings strongly indicate that adjuvants not only significantly improve the body's immunity, but also exhibit a strong anti-infection ability. Importantly, this work provides a new perspective for the prevention and control of aquaculture diseases.

Striped catfish (Pangasianodon hypophthalmus) is one of the important fish species in the world, which is widely cultured in several countries worldwide including Egypt. Aeromonas hydrophila infection showed a high prevalence in the farmed striped catfish negatively affecting its production causing economic loss. Therefore, the finding of functional feed supplements with antimicrobial activity is urgently needed. The use of marine seaweeds as feed supplements has produced satisfactory results as antibacterial agents. Accordingly, the present study was designed to evaluate the dietary effects of seaweeds mixture (Ulva lactuca, Jania rubens, and Pterocladia capillacea) extract (SME) on the growth performance, haemato-chemical parameters, antioxidant capacity, immune indices, and resistance of striped catfish fry against A. hydrophila infection. Four isonitrogenous (30% crude protein) diets were prepared to contain 0.0% (control), 1%, 2%, and 3% SME and were fed to striped catfish fry (1.7 ± 0.1 g) up to apparent satiety three times a day for 60 days. After the feeding trial, fish of each treatment were intraperitoneally injected with a virulent A. hydrophila strain and the relative percentage of survival (RPS) was recorded. The growth-stimulating activity of dietary SME was observed especially at 2% and 3% SME treatments. Similarly, haematological parameters (red blood cells, haemoglobin, haematocrit, and white blood cells) and serum protein profile (total protein, albumin, and globulin) were significantly (P 0.05) improved, meanwhile serum alanine and aspartate aminotransferases were significantly (P 0.05) decreased in fish fed with 2 and 3% SME diets. Serum creatinine and uric acid levels revealed no significant (P 0.05) differences among the experimental groups. Hepatic malondialdehyde and serum nitrous acid levels were significantly decreased; meanwhile hepatic superoxide dismutase, catalase, and glutathione peroxidase levels were significantly increased in all SME-treated groups than the control one. Also, SME supplementations significantly increased the serum lysozyme, total immunoglobulin, and complement C3 activities with highest values at 2% and 3% SME treatments. Interestingly, after bacterial challenge, fish fed SME-supplemented diet were more resistant with the lowest fish mortality (15%) at the 3% SME diet, which exhibit the highest RPS (81.9%). Meanwhile the control group showed highest mortality (85%). According to the regression fitting curve, the current study recommends using the dietary SME with optimum levels of 2.25-2.5% to improve the growth performance, welfare status, and resistance of striped catfish fry against A. hydrophila infection.

The present study investigated the virulence and expression of innate immunity genes in isolates of infectious hematopoietic necrosis virus (IHNV) in Gangwon province, South Korea, by challenging rainbow trout, Atlantic salmon, and coho salmon. Eight IHNV isolates were used to infect RTG-2 cells for viral replication using plaque assays. Three isolates with the highest replication rates, the RtPc0314g and RtPc0314c isolates of the JRt-Shizuoka type and the RtPc0816g isolate of the JRt-Nagano type, were experimentally infected into the fish. In rainbow trout, both RtPc0314c and RtPc0314g isolates showed 100% cumulative mortality while the RtPc0816g isolate showed 60% cumulative mortality for 14 days. In contrast, all three isolates showed

Short-chain bioactive peptides are new and promising antimicrobial, immune moderating, and antioxidant agents. Therefore, the present study was conducted to evaluate in vitro antibacterial activity of CM11, a short antimicrobial peptide (AMP), against Streptococcus iniae and Yersinia ruckeri as fish pathogenic bacteria using standard disk diffusion and microdilution assays. In addition, in vivo effects of CM11 on fish growth, immunity, antioxidant activity, and disease resistance were evaluated using zebrafish (Danio rerio) as an animal model. For in vivo study, based on in vitro susceptibility results, four diets were designed to include zero (as control), 10, 20, and 50 μg of CM11 per g diet referred to as control, P1, P2, and P3 treatments, respectively. After eight weeks of dietary trial, fish were challenged with Streptococcus iniae, and the survival rate was calculated for a period of two weeks. Results showed that CM11 effectively inhibited the growth of S. iniae and Y. ruckeri on agar plates at a concentration of eight μg/ml. Minimum inhibitory and minimum bactericidal concentrations of CM11 were measured at 8 and 32 μg/ml for S. iniae and 16 and 64 μg/ml Y. ruckeri, respectively. In vivo results showed no noticeable effects on fish growth parameters, however, feed conversion ratio (FCR) was found lower in P3 and P2 compared to control (P 0.05). Immunological and antioxidant responses were found strongly affected by CM11 in all treatment groups in which the highest values were found in the P3 treated group. Key immune and antioxidant genes were up-regulated particularly in fish receiving the highest level of CM11 (P3). Fish receiving the CM11 peptide showed better survival when challenged with S. iniae. These findings suggest the potential of CM11 for use in aquaculture as an antibacterial and immunostimulant agent.

Toll-like receptors (TLRs) are evolutionarily conserved proteins of pattern recognition receptors (PRRs) and play a crucial role in innate immune systems recognition of conserved pathogen-related molecular samples (PAMPs). We identified and characterized TLR18 from Nile tilapia (Oreochromis niloticus), OnTLR18, to elucidate its role in tissue expression patterns, modulation of gene expression after microbial challenge and TLR ligands, subcellular localization in fish and human cells, and the possible effectors TLR18 induces in a melanomacrophage-like cell line (tilapia head kidney (THK) cells). OnTLR18 expression was detected in all tissues examined, with the highest levels in the intestine and the lowest in the liver. OnTLR18 transcript was up-regulated in immune-related organs after bacterial and polyinosinic-polycytidylic acid (poly I:C) challenges and in the THK cells after lipopolysaccharide (LPS) stimulation. In transfected THK and human embryonic kidney (HEK) 293 cells, OnTLR18 localizes in the intracellular compartment. OnMyD88 and OnTRIF, but not OnTIRAP, were co-immunoprecipitated with OnTLR18, suggesting that the former two molecules are recruited by OnTLR18 as adaptors. The constitutively active form of OnTLR18 induced the production of pro-inflammatory cytokines, type I interferon (IFN), and antimicrobial peptides such as tumor necrosis factor α, interferon (IFN) d2.13, tilapia piscidin (TP)2, TP3, TP4, and hepcidin in THK cells. Our results suggest that OnTLR18 plays an important role in innate immunity through initiating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and IFN signaling pathways via OnMyD88 and OnTRIF and induces the production of various effectors in melanomacrophages.

The pleiotropic cytokine IL -1 is involved in important immune responses such as thymocyte proliferation and B cell growth and differentiation. Activation of the IL -1 pathway requires its functional receptor IL -1RI, making IL -1RI the critical point of the IL -1 pathway. In-depth study of IL -1RI will help to understand the immune mechanism involved in IL -1. In this study, we identified the cDNA of the IL -1RI gene of olive flounder (PoIL-1RI). The total length of the PoIL-1RI cDNA is 2490 bp, the open reading frame is 1689 bp long and encodes a protein of 562 amino acids. The protein has three Ig domains and a typical TIR domain, as in other mammals and fish. We found that PoIL-1RI is widely expressed in the tissues studied and shows a significant immune response after stimulation with bacteria and pathogen-associated molecular patterns (PAMPs) both in vitro and in vivo. After PoIL-1RI was overexpressed in olive flounder embryonic cell line (FEC), pro-inflammatory cytokines (IL -1β, IL -6, IL -8, TNF-α) and interferon (IFN-α, IFN-γ) were significantly upregulated. And we found that after overexpressing PoIL-1RI in FEC, the antibacterial ability of FEC was significantly stronger than that of the control group, and we found that overexpression of PoIL-1RI gene significantly increased the activity of NF-κB signaling pathway. These results suggest that PoIL-1RI plays an important role in innate immune response.

An eight-week feeding trial was performed to assess the effect of different dietary levels (0, 0.5, 1.0, and 1.5%) of β-glucan (sourced from Saccharomyces cerevisiae) on growth, survival, immunological parameters (immune gene expression, lysozyme, and antiprotease), total antioxidant status, thermal tolerance, and disease resistance of Tor putitora fry. Feeding of moderate doses (0.5 and 1.0%) of β-glucan significantly improved survival but not weight gain percentage as compared to that received unsupplemented control and highest dose (1.5%) of glucan. Supplementation of β-glucan in diets differentially influenced the mRNA expression of cytokine and other immune genes. For instance, transcripts of cytokines such as tnf-α and il-1β were significantly upregulated, while ifn-γ and il-10 were unaffected by β-glucan intake. Also, the relative mRNA expression of tlr-5 and hepcidin1 along with lysozyme and antiprotease activities were remained largely unchanged by dietary glucan administration. In contrast, β-glucan induced mRNA expression of defensin1 and c3 while decreased the transcript level of mhc-1. On the other hand, dietary inclusion of β-glucan markedly improved total antioxidant levels and extended the thermal tolerance limits at both the ends, as shown by increased CTmax and lower CTmin than the control group. After feeding β-glucan for eight weeks, the fish were bath challenged with a bacterial pathogen, Aeromonas salmonicida. The challenge study results revealed that β-glucan intake improved most of the studied immune parameters, resulting in lower mortality. Overall, dietary inclusion of β-glucan (0.5-1.0%) was efficient in improving the immune responses, thermal tolerance, and disease resistance of T. putitora fry.

The nervous necrosis virus (NNV) infection is generally observed in aquafarms when the seawater temperature is higher than 24 °C and the fishes seem to be refractory to disease at suboptimal temperatures below 20 °C suggesting a role of thermoregulation in NNV pathogenesis. The present study profiled the temperature-dependent regulation of cytokines (TNF-α, IL-1β and IFN-γ), innate antiviral factors (IFN-1, Mx, ISG-15), adaptive immune factors (CD-4, CD-8, IgM), signaling regulators (SOCS-1, SOCS-3), transcription factors (STAT-1, STAT-3) and microglial and NCC/NK specific cell markers (TMEM-119 and NCCRP-1) during NNV challenge in seven-band grouper, Hyporthodus septemfasciatus. The co-habitation challenge at 17 °C with showed a sustained expression of proinflammatory cytokines and following rechallenge with a dose of 104 TCID50/100μL/fish at optimal temperature, the survivors also exhibited a stable expression of immune factors. The 100% survival following the challenge at sub-optimal (17 °C) and rechallenge at optimal (25 °C) was due to the stable and sustained activation of the immune response. However, at 25 °C, the rechallenge displayed a priming effect with hyperactivation of the immune system evident from the immune gene expression profile. The mortality pattern observed is co-related with the cytokine storm as is evident from the gene expression profile. Whereas, neither of the adaptive immune markers was suggestive of humoral immune response in the 17 °C groups. Also, the data suggest a possible role of NK cell and microglia in mediating antiviral immune response following infection in the brain at different temperatures, where, former is beneficial in restricting viral infection with higher host tolerance.

Chinese rice-field eels rhabdovirus (CrERV), belonging to the genus Perhabdovirus in the family Rhabdoviridae, is the causative agent of the haemorrhagic disease of Chinese rice-field eels, Monopterus albus. The present study aims to establish a cell line derived from the kidney of Chinese rice-field eel (CrEK) for the further study of the pathogenic virus. CrEK cells were epithelioid-like and grew well in M199 medium supplemented with 10% foetal bovine serum at 28°C, and the cell line has been subcultured for more than 80 times. Karyotyping analysis of CrEK cells at 25th passage indicated a modal chromosome number of 24. Significant cytopathic effect (CPE) was observed in CrEK cells after infection with CrERV, and the virus titre reached 107.8 ± 0.45 TCID50 /mL. The transmission electron microscopy revealed that there were a large number of virus particles in the cytoplasm of cells. The virus infection in cells was also assayed by using indirect immunofluorescence assay (IFA), fluorescence in situ hybridization (FISH), reverse transcription PCR (RT-PCR) and quantitative real-time reverse transcription-PCR (qRT-PCR). In experimental infection, CrERV cultured by cells could cause over 90% mortality in fish. CrEK represents the first kidney cell line originated from Chinese rice-field eels and be a potential material for investigating the mechanism of virus infection in this fish and the control methods for the disease.

Renibacterium salmoninarum, a slow-growing facultative intracellular pathogen, is the causative agent of bacterial kidney disease, a chronic, progressive and granulomatous infection that threatens farmed and wild salmonids worldwide. Pathogenic R. salmoninarum colonizes tissues and invades the host through cell surface-associated and secreted proteins. While correlations between iron acquisition genes and virulence have been demonstrated in vitro, these mechanisms have not undergone proteomic characterization. The present study applied a proteomic approach to elucidate the differences between the virulent Chilean R. salmoninarum H-2 strain and the type strain ATCC 33209T . Analyses were conducted under normal (control) and iron-limited conditions (DIP) emulating the host environment. Interestingly, strain H-2 apparently responded better to the iron-limited condition-for example, only this strain presented a significantly enriched iron ion homeostasis pathway. Furthermore, key virulence factors related to an iron-limited environment were more abundant in strain H-2. Importantly, the lack of iron favoured the expression of the 57-kDa protein in strain H-2, the principal virulence factor for R. salmoninarum. Our findings can be employed in the design and development of treatments targeted to iron uptake mechanisms (e.g. siderophore synthesis or haem uptake), which represents a promising therapeutic approach for treating this persistent fastidious bacterium.

Cardiac abnormalities may pose a threat to salmonid aquaculture due to their potential detrimental effect on fish health and welfare. The teleost heart is an extremely plastic organ with important morphological differences between wild and farmed fish that include ventricular shape, alignment of the bulbus arteriosus and epicardial fat deposition. However, little is known about how different factors and interactions among them may affect cardiac morphology of Atlantic salmon. To determine whether rearing temperature could induce cardiac malformations in large Tasmanian Atlantic salmon, we examined a range of cardiac morphology indicators and growth parameters in a population of 1-2 kg seawater salmon (n = 60 temperature-1 diet-1 ) exposed to control and elevated temperatures of 15 and 19°C, respectively, while fed one of two commercial feeds with different dietary energy levels. Most fish possessed conspicuous fat around the heart with a tendency towards a rounded ventricle and a more obtuse angle of the bulbus arteriosus. However, fish showed no significant differences in heart shape and bulbus alignment in relation to water temperature and dietary energy. These results suggest that cardiac morphology of large Atlantic salmon is unlikely to be affected by rearing temperature and dietary energy during the grow-out phase.

Interleukin 8 (IL-8) is a critical chemokine regulating immune cells chemotaxis as well as their physiological or pathological activations. In fish cells, recombinant IL-8 proteins induced transcriptions of pro-inflammatory cytokines. However, the exact mechanisms underlying the function of fish IL-8 as a pro-inflammatory cytokine are still unclear. In this paper, we first prepared recombinant grass carp IL-8 (rgcIL-8) using an E. coli expression system, and later confirmed rgcIL-8 increased gene expression of il8, il1β and tumor necrosis factor alpha (tnfα) in grass carp head kidney leukocytes (HKLs). Using signaling pathway inhibitors, we demonstrated that rgcIL-8 regulated transcriptions of pro-inflammatory cytokines via MAPK and/or NF-κB signaling pathways. We cloned gcIL-8 specific receptor CXCR1 and subsequently discovered that gcIL-8 could increase the activity of NF-κB and the transcription of IL-1β via CXCR1. Simultaneously, antibody neutralization assay showed that endogenous IL-8 is partially relevant to the self-regulation of IL-1β. Moreover, rgcIL-8 led to the expression of inducible nitric oxide synthase gene, causing an accumulation of nitric oxide in the culture medium of HKLs, suggesting the potential of gcIL-8 to mediate inflammatory response. This study not only enriched the function of IL-8 in teleost, but also revealed it as a potential target for the inflammatory control in grass carp. This article is protected by copyright. All rights reserved.

Interferon regulatory factor 3 (IRF3) is activated by IκB kinase ε (IKKε) and Tank-binding kinase 1 (TBK1), which plays a crucial role in the interferon signaling in vertebrates. However, the regulation of teleost IRF3 by IKKε remains largely unknown. In this study, the IRF3 homologue (bcIRF3) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The transcription of bcIRF3 was detected to increase in host cells in response to different stimuli. bcIRF3 distributed predominantly in the cytosolic area; however, translocated into nuclei after virus infection. bcIRF3 showed IFN-inducing ability in reporter assay and EPC cells expressing bcIRF3 showed enhanced antiviral ability against both grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). Moreover, knockdown of bcIRF3 reduced the antiviral ability of the host cells, and the transcription of antiviral-related cytokines was obviously lower in bcIRF3-deficient host cells than that of control cells. The data of reporter assay and plaque assay demonstrated that bcIKKε obviously enhanced bcIRF3-mediated IFN production and antiviral activity. Immunofluorescent staining and co-immunoprecipitation assay revealed that bcIKKε interacted with bcIRF3. It was interesting that the nuclear translocation of bcIRF3 and bcIKKε was enhanced by each other when these two molecules were co-expressed in the cells, however, the protein levels of bcIRF3 and bcIKKε were decreased mutually. Thus, our data support the conclusion that bcIKKε interacts with bcIRF3 and enhances bcIRF3-mediated antiviral signaling during host innate immune activation.

The profitability of the olive flounder (Paralichthys olivaceus) aquaculture industry in Korea depends on high production and maintenance of flesh quality, as consumers prefer to eat raw flounders from aquaria and relish the raw muscles as ‘sashimi’. For sustaining high production, easy-to-deliver and efficient vaccination strategies against serious pathogens, such as viral hemorrhagic septicemia virus (VHSV), is very important as it cause considerable losses to the industry. Whereas, a safe and non-invasive vaccine formulation that is free from unacceptable side-effects and does not devalue the fish is needed to maintain flesh quality. We previously developed a squalene–aluminium hydroxide (Sq + Al) adjuvanted VHSV vaccine that conferred moderate to high protection in flounder, without causing any side effects when administered through the intraperitoneal (IP) injection route. However, farmers often demand intramuscular (IM) injection vaccines as they are relatively easy to administer in small fishes. Therefore, we administered the developed vaccine via IP and IM routes and investigated the safety and persistency of the vaccine at the injection site. In addition, we conducted a comparative analysis of vaccine efficacy and serum antibody response. The clinical and histological observation of the IM and IP groups showed that our vaccine remained persistence at the injection sites for 10–17 weeks post vaccination (wpv), without causing any adverse effects to the fish. The relative percentage of survival were 100% and 71.4% for the IP group and 88.9% and 92.3% for the IM group at 3 and 17 wpv, respectively. Thus, considering the persistency period (24 wpv) and both short and long-term efficacy of our vaccine, the present study offers an option to flounder farmers in selecting either IM or IP delivery strategy according to their cultured fish size and harvesting schedule — IM vaccination for small-sized fish and IP vaccination for table-sized fish.

Made available in DSpace on 2022-04-28T19:44:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Group B Streptococcus (GBS) causes meningitis in neonates and Nile tilapia (Oreochromis niloticus). The molecular mechanisms regulating the intracellular survival of this pathogen in the host cell are complex and crucial for the progression of infection. Thus, we propose the use of GBS-infected Nile tilapia microglia as an in vitro model system simulating infection caused by homologous bacteria in humans. We used this model to evaluate the phagocytic activity, as well as the functional aspects of the capsular proteins A, B, C, and D and the major redox enzymes, and the synergistic role of mechanisms/proteins involved in blocking phagocytic process. We observed that in the intracellular phase, GBS showed enhanced synthesis of the polysaccharide capsule and used superoxide dismutase, thioredoxin, NADH oxidase, and alkyl hydroperoxide reductase to scavenge reactive oxygen species and reactive nitrogen species produced by the host cell. Furthermore, although these virulence mechanisms were effective during the initial hours of infection, they were not able to subvert microglial responses, which partially neutralized the infection. Altogether, our findings provided important information regarding the intracellular survival mechanisms of GBS and perspectives for the production of new drugs and vaccines, through the druggability analysis of specific proteins. In conclusion, tilapia microglia serve as a potent in vitro experimental model for the study of meningitis. Department of Postgraduate in Health Sciences-PROCISA Federal University of Roraima (UFRR) Immunochemistry Laboratory Butantan Institute (CeTICs/FAPESP), Vital Brazil Avenue, 1500 Department of Technology School of Agrarian and Veterinary Sciences Sao Paulo State University (Unesp) Immunoregulation Unit of the Laboratory of Applied Toxinology (CeTICs/FAPESP) Butantan Institute, Vital Brazil Avenue, 1500 Department of Technology School of Agrarian and Veterinary Sciences Sao Paulo State University (Unesp)

Hirame rhabdovirus (HIRRV), a member of the genus Novirhabdovirus, causes morbidity and mortality in farmed olive flounder (Paralichthys olivaceus). As no information is available on the role of the NV gene of HIRRV, we produced a recombinant HIRRV with the NV gene deleted (rHIRRV-ΔNV) using reverse genetic technology and investigated whether the NV gene knockout affected HIRRV replication and the type I interferon response of the host cell. The rescue of rHIRRV-ΔNV was successful only when IRF9-gene-knockout Epithelioma papulosum cyprini (ΔIRF9-EPC) cells were used, suggesting that the NV protein of HIRRV might be involved in inhibition of the type I interferon response of the host cell. This conclusion was also supported by the significantly higher level of Mx gene induction in EPC cells infected with rHIRRV-ΔNV than in cells infected with recombinant HIRRV without the deletion. When cells were coinfected with rHIRRV-ΔNV and either wild-type HIRRV or wild-type viral hemorrhagic septicemia virus (VHSV), there was a decrease in the growth rate of not only wild-type HIRRV but also wild-type VHSV in a concentration-dependent manner. Further studies are required to investigate the role of HIRRV NV in virulence and its possible importance for the development of attenuated vaccines.

Five novel permanent cell lines have been established from gill, heart, kidney, eye and fin of snubnose pompano, Trachinotus blochii. They were designated as snubnose pompano gill (SPG), snubnose pompano heart (SPH), snubnose pompano kidney (SPK), snubnose pompano eye (SPE) and snubnose pompano fin (SPF), respectively. All these cell lines were characterized and cryopreserved successfully at different passage levels. Cell lines were passaged every alternate day; SPG, SPH, SPK, SPE and SPF cell lines attained passage levels of 68, 74, 82, 79 and 106, respectively, since the initiation of their development in 2019. The cell lines grew well in Leibovitz's 15 medium containing 15% foetal bovine serum at 28°C. Immunophenotyping of the cell lines revealed the presence of fibronectin and pancytokeratin. No mycoplasma contamination was found. The transfection study revealed the gene expression efficiency of these cell lines by expressing the green fluorescent protein (GFP). The authentication on origin of cell lines from T. blochii was confirmed by amplification of species-specific mitochondrial cytochrome oxidase I gene. The results showed the susceptibility of these cell lines to fish nodavirus (FNV) and tilapia lake virus (TiLV) and resistance to cyprinid herpesvirus 2 (CyHV-2). The FNV infection in the cell lines was confirmed by RT-PCR, Western blot, ELISA and immunocytochemistry, while TiLV infection was confirmed by RT-PCR assay. These results revealed that these cell lines are suitable for virological and foreign gene expression studies.

This study investigated the immunomodulatory effects of Sargassum polycystum extract administration in rainbow trout (Oncorhynchus mykiss). S. polycystum methanolic extract was administered orally using feeding needles to individual rainbow trout at the dose of 0 (control), 1 (S1), 3 (S3) and 5 (S5) mg/100 μl/per fish twice a day for 7 days. On 1st, 5th, 3rd and 7th day, blood and tissues were collected from the fish and changes in humoral immune responses and immune-related gene expressions were determined. The result of oxidative radical production showed no difference during early stage of the experiment and was lately decreased (P 0.05). Lysozyme activity increased on 3rd and 7th day of the study in S5 fish group and on 5th day in S3 group compared to control (P 0.05). Myeloperoxidase activity had an increased level on the 1st and 3rd day in S1, S5 and S5 fish groups, respectively. IL-1β gene was significantly up-regulated in kidney and intestine in all experimental groups (except on the 1st day, in the intestine of S5 fish group) compared to control (P 0.05). IL-8 gene expression was elevated on 1st and 3rd day in kidney of all experimental fish groups. IL-6 transcript enhanced in a dose-dependent manner on 3rd and 7th day. IL-10 and IL-12 genes were also up-regulated. Survival in all treated fish groups challenged with Aeromonas hydrophila was significantly increased compared to that of control. The highest survival rate was recorded in S5 fish group (83.65%) followed by S3 fish group (82.62%). Our results suggest that S. polycystum aqueous methanolic extract is an effective immunostimulant and provide protection against A. hydrophila infection in rainbow trout at a dose of 3-10 mg/20 g body weight/day.

The polymeric immunoglobulin receptor (pIgR) plays an important role in mediating the transcytosis of polymeric immunoglobulins (pIgs) to protect organisms against pathogen invasion. Here, a polyclonal antibody against grass carp (Ctenopharyngodon idellus) recombinant pIgR was developed by immunizing New Zealand white rabbit, and the responses of pIgR, IgM and IgZ were analyzed after bath immunization and intraperitoneal administration with Flavobacterium columnare. The results showed that pIgR transcription level was similar to IgM and IgZ, but pIgR rose much faster and peaked earlier than IgM and IgZ; the pIgR mRNA levels were higher in the skin and spleen for both immunized groups, while IgM and IgZ mRNA expression were higher in skin, gills, and intestines in bath immersion group, or spleen and head kidney in intraperitoneal immunization group. ELISA revealed that the IgM, IgZ and pIgR protein levels were up-regulated in skin mucus, gill mucus, gut mucus and bile, reaching a higher peak level earlier in skin mucus and gill mucus in bath immersion group, but a higher peak level in bile in injection group. Moreover, secretory component molecules were detected in grass carp's skin, gill and intestine mucus and bile, but not in serum, which molecular mass was near the theoretical mass obtained from the sequence of grass carp pIgR. These results demonstrated that bath and intraperitoneal immunization up-regulated pIgR and secretory Ig expression in secretions, which provided more insights into the role of pIgR in immunity and offer insight into ways of protecting teleost against pathogen invasion.

Infectious diseases are one of the main causes of social and economical losses in world aquaculture. Senegalese sole (Solea senegalensis) is an important species for aquaculture in southern Europe, whose production is affected by the appearance of bacterial diseases such as photobacteriosis, a septicemia caused by Photobacterium damselae subsp. piscicida (Phdp). The aim of this study was to obtain an oral DNA nanovaccine and to evaluate its efficacy against Phdp in S. senegalensis juveniles. For this purpose, the amplified product corresponding to the protein inosine-5′-monophophate dehydrogenase (IMPDH) from Phdp, was cloned into the expression vector pcDNA™6.2/C-EmGFP-GW obtaining the DNA vaccine named as pPDPimpdh. The correct transcription and protein expression was verified at 48 h post tansfection in HEK293 cells. Chitosan nanoparticles (CS-TPP NPs) were prepared by ionotropic gelation and their features were appropriate for use as oral delivery system. Therefore, pPDPimpdh was protected with chitosan CS-TPP NPs throughout complex coacervation method giving as a result a DNA nanovaccine referred as CS-TPP+pPDPimpdh NPs. Sole juveniles were vaccinated orally with CS-TPP NPs, pPDPimpdh and CS-TPP+pPDPimpdh NPs followed by a challenge with Phdp at 30 days post vaccination (dpv). The relative percentage survival (RPS) for pPDPimpdh vaccinated groups was 6.25%, probably due to its degradation in the digestive tract. RPS value obtained for CS-TPP NPs and CS-TPP+pPDPimpdh NPs was 40% and antibodies were observed in both cases. However, a delay in mortality was observed in sole juveniles vaccinated orally with CS-TPP+pPDPimpdh NPs. In fact, an upregulation of tf, mhcII, cd8a and igm in the posterior gut and c3, hamp1, tf and cd4 in spleen was observed in juveniles vaccinated with CS-TPP+pPDPimpdh NPs. After challenge, a modulation of cd8a and cd4 expression levels in the posterior gut and c3, tf, lyg, cd4, igm and igt expression levels in spleen was observed. Moreover, the concentration of lysozyme in skin mucus significantly increased in fish vaccinated orally with CS-TPP+pPDPimpdh NPs at 11 dpc. These data indicate that oral vaccination with CS-TPP+pPDPimpdh NPs could be acting through the non-specific immune responses as well as the specific humoral and cell mediated immunity and provide the first step toward a development of an oral DNA nanovaccine against Phdp in sole.

It is known that iron transporter proteins and their regulation can modulate the fish's immune system, suggesting these proteins as a potential candidate for fish vaccines. Previous studies have evidenced the effects of Atlantic salmon immunized with the chimeric iron-related protein named IPath® against bacterial and ectoparasitic infections. The present study aimed to explore the transcriptome modulation and the morphology of the sea louse Caligus rogercresseyi in response to Atlantic salmon injected with IPath®. Herein, Atlantic salmon were injected with IPath® and challenged to sea lice in controlled laboratory conditions. Then, female adults were collected after 25 days post-infection for molecular and morphological evaluation. Transcriptome analysis conducted in lice collected from immunized fish revealed high modulation of transcripts compared with the control groups. Notably, the low number of up/downregulated transcripts was mainly found in lice exposed to the IPath® fish group. Among the top-25 differentially expressed genes, Vitellogenin, Cytochrome oxidases, and proteases genes were strongly downregulated, suggesting that IPath® can alter lipid transport, hydrogen ion transmembrane transport, and proteolysis. The morphological analysis in lice collected from IPath® fish revealed abnormal embryogenesis and inflammatory processes of the genital segment. Furthermore, head kidney, spleen, and skin were also analyzed in immunized fish to evaluate the transcription expression of immune and iron homeostasis-related genes. The results showed downregulation of TLR22, MCHII, IL-1β, ALAs, HO, BLVr, GSHPx, and Ferritin genes in head kidney and skin tissues; meanwhile, those genes did not show significant differences in spleen tissue. Overall, our findings suggest that IPath® can be used to enhance the fish immune response, showing a promissory commercial application against lice infections.

Ecologically and economically valuable Pacific salmon and trout (Oncorhynchus spp.) are widespread and susceptible to the ectoparasite Salmincola californiensis (Dana). The range of this freshwater copepod has expanded, and in 2015, S. californiensis was observed in Blue Mesa Reservoir, Colorado, USA, an important kokanee salmon (O. nerka, Walbaum) egg source for sustaining fisheries. Few S. californiensis were detected on kokanee salmon in 2016 (

To date, the mechanisms of inflammation have been poorly studied in fish of commercial interest, due to the lack of development of appropriate experimental models. The current study evaluated a local inflammation triggered by a polymeric carrageenin mixture (a mucopolysaccharide derived from the red seaweed Chondrus crispus) in the skin of gilthead seabream (Sparus aurata). Fish were injected subcutaneously with phosphate-buffered saline (as control) or λ/κ-carrageenin (1%), and skin samples from the injection sites were collected 1.5, 3 and 6 hr post-injection, processed for inclusion in paraplast and stained with haematoxylin-eosin, Alcian blue or periodic acid-Schiff. Furthermore, immunohistochemistry and expression analyses of several cells' markers and proinflammatory genes were also analysed in samples of the injected sites. Microscopic results indicated an increased number of skin mucus-secreting cells and acidophilic granulocytes in the skin of fish studied at 1.5 hr and 3 hr post-injection with carrageenin, respectively, with respect to the data obtained in control fish. Otherwise, both the gene expression of the non-specific cytotoxic cell marker (granzyme B, grb) and the proinflammatory cytokine (interleukin-1β, il-1β) were up-regulated at 1.5 hr in the skin of fish injected with carrageenin compared with the control fish, whilst the gene expression of acidophilic granulocyte markers (NADPH oxidase subunit Phox22 and Phox40, phox22 and phox40) was up-regulated at 3 and 6 hr in the carrageenin group, compared with the control group. In addition, the gene expression of myeloperoxidase (mpo) was also up-regulated at 6 hr in the skin of fish injected with carrageenin in comparison with control samples. The present results indicate the chronological participation of two important immune cells involved in the resolution of the inflammation in the skin of gilthead seabream.

Effects of a novel dietary mixture of selenium nanoparticles (Se-NPs) and omega-3-fatty acids i.e., Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mitigating arsenic pollution, high-temperature stress and bacterial infection were investigated in Pangasianodon hypophthalmus. To aim this, four isocaloric and iso-nitrogenous diets were prepared: control feed (no supplementation), Se-NPs at 0.2 mg kg−1 diet with EPA + DHA at 0.2, 0.4 and 0.6% as supplemented diets. Fish were reared under normal condition or concurrent exposure to arsenic (2.65 mg L−1), and temperature (34 °C) (As + T) stress for 105 days. The experiment was conducted with eight treatments in triplicates. Response to various stresses i.e., primary (cortisol), secondary (oxidative stress, immunity, and stress biomarkers) and tertiary stress response (growth performance, bioaccumulation and mortality due to bacterial infection) were determined. Supplementation of dietary Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.2 and 0.4% reduced the primary stress level. Exposure to arsenic and temperature (As + T) and fed with control diet and EPA + DHA at 0.6% aggravated the cortisol level. Anti-oxidative enzymes (Catalase, superoxide dismutase, glutathione peroxidase and glutathione-s-transferase) and immunity (Nitroblue tetrazolium, total protein, albumin, globulin, A:G ratio, total immunoglobulin and myeloperoxidase) of the fish were augmented by supplementation of Se-NPs and EPA + DHA at 0.2 and 0.4%. Neurotransmitter enzyme, HSP 70, Vitamin C were significantly enhanced (p −1 and EPA + DHA at 0.2 and 0.4%. Whereas total lipid, cholesterol, phospholipid, triglyceride and very low-density lipoprotein (VLDL) were reduced (p −1 diet and EPA + DHA at 0.2 and 0.4%. Tertiary stress response viz. growth performance was also significantly enhanced with supplementation of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4% reared under As + T. Whereas arsenic bioaccumulation in fish tissues was significantly reduced with dietary supplementation of Se-NPs and EPA + DHA. Cumulative mortality and relative percentage survival were reduced with Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4%. The investigation revealed that a novel combination of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.4% followed by 0.2% has the potential to alleviate temperature stress, bacterial infection and arsenic pollution. Whereas diet containing Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.6% was noticeably enhanced the stress in P. hypophthalmus.

Tilapia lake virus (TiLV) is a notable contagious agent that causes massive economic losses in the tilapia industry globally. Evaluations of the histological changes associated with TiLV infection are not only crucial for diagnosis, but also to gain an understanding of the disease. We therefore synthesized a rabbit polyclonal immunoglobulin G antibody against TiLV and developed an immunohistochemical (IHC) procedure to detect TiLV localization in the tissues of infected fish for comparison with in situ hybridization (ISH) testing. A total of four different sample cohorts derived from TiLV-infected fish was used to validate the IHC procedure. The TiLV IHC application was successfully developed and facilitated nuclear and cytoplasmic immunolabelling in the intestines, gills, brain, liver, pancreas, spleen, and kidneys that corresponded with the ISH results. Apart from the ISH results, TiLV-IHC signals were clearly evident in the endothelial cells of various organs, the circulating leukocytes in the blood vessels, and the areas of tissue inflammation. Among the tested sample cohorts, the intestines, gills, and brain had IHC-positive signals, highlighting the possibility of these organs as common TiLV targets. Immunological staining pattern and distribution corresponded with the TiLV viral load but not the inoculation route. The TiLV IHC was also capable of detecting TiLV infection in the experimentally challenged ornamental cichlids, Mozambique tilapia, giant gourami, and naturally infected tilapia, indicating the dynamic range of IHC for TiLV detection. Overall, our study delivers the first IHC platform to detect TiLV infection and provides novel evidence of cellular tropism during TiLV infection. Our findings also reveal the TiLV distribution pattern of infected fish and propose the endotheliotropism and lymphotropism of this virus, which requires further elaboration. Importantly, this new IHC procedure could be applied to study the pathogenesis and interaction of TiLV in future research.

Interleukin-16 (IL-16), as a lymphocyte chemoattractant cytokine, plays a crucial role in regulating cellular activities and anti-pathogen immunity. In teleost, the information about the antibacterial effect of IL-16 is scarce. In our study, we examined the immune functions of an IL-16 homologue (CsIL-16) from tongue sole Cynoglossus semilaevis. The CsIL-16 precursor (proCsIL-16) is comprised of 1181 amino acid residues, sharing 21.1%-67.3% identities with IL-16 precursor from invertebrate and vertebrate. The C-terminal proCsIL-16 containing two PDZ domains was designated as mature CsIL-16 which was released into the supernatant of peripheral blood leukocytes (PBLs). CsIL-16 was expressed in various tissues and regulated by bacterial invasion. Recombinant CsIL-16 (rCsIL-16), as a homodimer, was able to bind to the membrane of PBLs and played essential roles in regulating chemotaxis and activation of PBLs, which in vitro inhibited intracellular survival of E. tarda. Under in vivo condition, rCsIL-16 could dramatically regulate the induction of inflammatory genes, and suppress the bacterial dissemination in fish tissues. Collectively, our results reveal that CsIL-16 plays positive roles in antibacterial immunity, and provide insights into the immune function of CsIL-16.

NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection.

Teleost fish skin serves as the first line of defense against pathogens. The interaction between pathogen and host skin determines the infection outcome. However, the mechanism(s) that modulate infection remain largely unknown. A proper tissue culture model that is easier to handle but can quantitatively and qualitatively monitor infection progress may shed some lights. Here, we use striped catfish (Pangasius hypophthalmus) to establish an ex vivo skin explant tissue culture model to explore host pathogen interactions. The skin explant model resembles in vivo skin in tissue morphology, integrity, and immune functionality. Inoculation of aquatic pathogen Aeromonas hydrophila in this model induces epidermal exfoliation along with epithelial cell dissociation and inflammation. We conclude that this ex vivo skin explant model could serve as a teleost skin infection model for monitoring pathogenesis under various infection conditions. The model can also potentially be translated into a platform to study prevention and treatment of aquatic infection on the skin in aquaculture applications.

Infectious spleen and kidney necrosis virus (ISKNV) is a fish-pathogenic virus belonging to the genus Megalocytivirus of the family Iridoviridae. In 2018, disease occurrences (40-50% cumulative mortality) associated with ISKNV infection were reported in grown-out Asian sea bass (Lates calcarifer) cultured in an inland freshwater system in Thailand. Clinical samples were collected from seven distinct farms located in the eastern and central regions of Thailand. The moribund fish showed various abnormal signs, including lethargy, pale gills, darkened body, and skin hemorrhage, while hypertrophied basophilic cells were observed microscopically in gill, liver, and kidney tissue. ISKNV infection was confirmed on six out of seven farms using virus-specific semi-nested PCR. The MCP and ATPase genes showed 100% sequence identity among the virus isolates, and the virus was found to belong to the ISKNV genotype I clade. Koch's postulates were later confirmed by challenge assay, and the mortality of the experimentally infected fish at 21 days post-challenge was 50-90%, depending on the challenge dose. The complete genome of two ISKNV isolates, namely KU1 and KU2, was recovered directly from the infected specimens using a shotgun metagenomics approach. The genome length of ISKNV KU1 and KU2 was 111,487 and 111,610 bp, respectively. In comparison to closely related ISKNV strains, KU1 and KU2 contained nine unique genes, including a caspase-recruitment-domain-containing protein that is potentially involved in inhibition of apoptosis. Collectively, this study indicated that inland cultured Asian sea bass are infected by homologous ISKNV strains. This indicates that ISKNV genotype I should be prioritized for future vaccine research.

Intensive fish farming at high densities results in a wide range of adverse consequences on fish welfare, including pathogen spreading, stress and increased mortality rates. In this work, we have assessed whether the survival of Senegalese sole infected with the nervous necrosis virus (NNV), a pathogen responsible for severe disease outbreaks, is affected by rearing density. Based on the different fish ratios per surface area (g cm−2) and water volume (g L−1), our research showed an earlier mortality onset in the tanks containing NNV-infected fish reared at medium density (MD: 0.071 g cm−2/5 g L−1) and high density (HD: 0.142 g cm−2/10 g L−1), as well as higher cumulative mortality values. However, transcription analysis of hsp70, gr1 and pepck genes, well-known stress biomarkers, seems to indicate that none of the challenged fish were under high stress conditions. NNV load was slightly higher both in dead and in sampled fish from MD and HD groups, and especially in the rearing water from these groups, where peaks in mortality seemed to correlate with increasing NNV load in the water. In conclusion, our results suggest that rearing NNV-infected Senegalese sole at high densities resulted in an earlier mortality onset and higher cumulative values and viral load L. Vázquez-Salgado would like to thank Interreg VA Spain–Portugal cooperation Program (POCTEP) 2014-2020, 0474_BLUEBIOLAB project, co-funded by FEDER for her research contract SI

Grouper nervous necrosis virus (GNNV) infection causes mass grouper mortality, leading to substantial economic loss in Taiwan. Traditional methods of controlling GNNV infections involve the challenge of controlling disinfectant doses; low doses are ineffective, whereas high doses may cause environmental damage. Identifying potential methods to safely control GNNV infection to prevent viral outbreaks is essential. We engineered a virus-binding bacterium expressing a myxovirus resistance (Mx) protein on its surface for GNNV removal from phosphate buffered saline (PBS), thus increasing the survival of grouper fin (GF-1) cells. We fused the grouper Mx protein (which recognizes and binds to the coat protein of GNNV) to the C-terminus of outer membrane lipoprotein A (lpp-Mx) and to the N-terminus of a bacterial autotransporter adhesin (Mx-AIDA); these constructs were expressed on the surfaces of Escherichia coli BL21 (BL21/lpp-Mx and BL21/Mx-AIDA). We examined bacterial surface expression capacity and GNNV binding activity through enzyme-linked immunosorbent assay; we also evaluated the GNNV removal efficacy of the bacteria and viral cytotoxicity after bacterial adsorption treatment. Although both constructs were successfully expressed, only BL21/lpp-Mx exhibited GNNV binding activity; BL21/lpp-Mx cells removed GNNV and protected GF-1 cells from GNNV infection more efficiently. Moreover, salinity affected the GNNV removal efficacy of BL21/lpp-Mx. Thus, our GNNV-binding bacterium is an efficient microparticle for removing GNNV from 10‰ brackish water and for preventing GNNV infection in groupers.

Infestation by parasitic copepods is a substantial problem in the cage culture of European sea bass (Dicentrarchus labrax). The Copepoda parasite, Lernanthropus kroyeri (Lernanthropidae), is one of the threats to the mariculture of sea bass. In the present study, we evaluated the data of abundance for L. kroyeri pre-adults and adult males (PAAM) and ovigerous adult females (AFo ) to estimate the internal infection pressure (IIP) in the same cage environment. The sea bass infested by L. kroyeri was collected from grow-out sea cages located in Gulluk Bay (Turkey) in September and October 2019. Mean fish weight and length of sea bass were 75.79 ± 1.66 g and 21.40 ± 0.56 cm, respectively. The fixed lag method was used to predict the abundance of L. kroyeri adult females. The overall prevalence of L. kroyeri was 60%. The mean abundance of PAAM and AFo varied from 0.8 ± 0.24 to 2.5 ± 0.67 and 2.9 ± 0.40 to 4.3 ± 0.55, respectively. The abundance of AF was strongly correlated with PAAM. The pattern of AFo and PAAM was interpreted as an indication of the continuous infestation of L. kroyeri on sea bass. Our results showed that the correlation of AFo abundance for five consecutive weeks was significant, representing the main determinative factor for the continuity of the parasitic load. In our approach, internal infestation pressure is the quantitative estimation of the potential infective copepodids, which are mainly characterized by AF abundance and the prevalence. We predicted that the internal infestation pressure could be high, even exceeding the 50.000 × 106 potential infective copepodids for one sea cage with the fish density of 20 sea bass/m3 .

Megalocytivirus cause diseases that have serious economic impacts on aquaculture, mainly in East and South-East Asia. Five primary genotypes are known: infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV) and scale drop disease virus (SDDV). ISKNV-mediated infectious spleen and kidney necrosis disease (ISKND) is a major viral disease in both freshwater and marine fish species. In this study, we report the isolation of ISKNV from diseased giant gourami, Osphronemus goramy, in India. Transmission electron microscopy of ultrathin sections of kidney and spleen revealed the presence of numerous polygonal naked viral particles having an outer nucleocapsid layer within the cytoplasm of enlarged cells (115-125 nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362 bp) gene in the infected fish had a high similarity to the other ISKNV-I isolates. Moreover, ISKNV was propagated in the Astronotus ocellatus fin (AOF) cell line and further confirmed genotypically. A high mortality rate (60%) was observed in gourami fish injected with ISKNV-positive tissue homogenate through challenge studies. Considering the lethal nature of ISKNV, the present study spotlights the implementation of stringent biosecurity practices for the proper control of the disease in the country.

Several biocides are widely used in rainbow trout aquaculture against various ectoparasites and ectobionts, but the inflammation induced in treated fish is less well described. Dose-response studies were conducted to elucidate the effects on rainbow trout (gills and fins) induced by a series of biocides including formalin, hydrogen peroxide (H2O2), peracetic acid (PAA) and the surfactant SPH6, which was isolated from the bacterium Pseudomonas H6. The compounds have documented antiparasitic effects, but the specific effects on fish needs further documentation. This study was performed over 24 h, and inflammatory reactions were evaluated in gills and fins. A dose-dependent effect was noted for expression of immune genes encoding for IL-1β, TNFα, IFNγ, IL-10, IL-8, lysozyme, serum amyloid A (SAA), hepcidin, precerebellin and complement factor C3. PAA induced the strongest upregulation of cytokine and acute phase reactant genes followed by H2O2 and formalin. SPH6 showed a lower effect, and in several cases the compound induced downregulation of several genes. Gills showed a stronger response compared to fins. The mucous cell density in fins showed a range of changes which varied by compound. PAA, and to a lesser degree H2O2 and formalin, initially induced mucous cell hyperplasia, whereas SPH6 immediately decreased the number of cells containing mucus.

The interactive applications of immunization route, vaccine type and delivery vectors are emerging as a key area of research within the field of mass immunization in fishery production. In an effort to improve DNA vaccine's immune efficiency in large-scale immunization, a promising bacterial ghost-loaded DNA vaccine was constructed based on Escherichia coli DH5α. In common carp was investigated the immune response to immersion immunization via related indicator analysis, and the challenge test of spring viraemia of carp virus (SVCV) was carried out. The result indicated that BG-loaded DNA vaccine induced higher serum antibody level than naked pEG-G. Simultaneously, the immunophysiological indicators and genes change at the more advanced levels in the BG/pEG-G immune group. At the treatment concentration of 20 mg/L of the BG/pEG-G group, IgM and IgZ expressions in vivo were markedly increased by 21.62 times and 6.91 times, respectively, and the relative percentage survival reached the peak of 59.57%. This study paves the way for future aquatic animal vaccine research, which aimed to develop the highly effective immersion vaccine system by delivery vectors, with the ultimate aim to prevent and restrict SVCV in actual production.

This study investigated the distribution of nematode larvae of Anisakidae and Raphidascarididae (genera Anisakis and Hysterothylacium) in Trachurus trachurus (Linnaeus, 1758) in the Ligurian and central-northern Tyrrhenian Seas. The relationship between the number of parasites and the length and weight parameters of the fish was assessed, and the possible effect of the parasites on the condition factor was evaluated. A total of 190 T. trachurus specimens were collected in July 2019. Parasites were found in 70 individuals. A total of 161 visible larvae were collected in the viscera. Morphological analysis revealed the presence of Anisakis spp. in 55 fish and Hysterothylacium spp. in 15 fish, while 5 fish showed coinfection with both genera. The specimens subjected to PCR (n = 67) showed that 85% of the Anisakis larvae analyzed belonged to the species A. pegreffii, while the remaining 15% belonged to hybrids of A. pegreffii-A. simplex (s.s.). A total of 58% (n = 7) of the Hysterothylacium larvae analyzed belonged to the species H. fabri, while 42% belonged to the species H. aduncum. Our results support the hypothesis that infection with these parasites does not affect the condition of the fish host analyzed, and that body size and depth are major drivers in determining infection levels with Anisakid and Raphidascaridid nematodes.

The mechanisms enabling fish tapeworms to avoid proteolytic attacks by digestive enzymes of their fish host have been studied in less detail compared with mammalian cestodes. This study aimed to assess the inhibitory ability towards trypsin and chymotrypsin in Eubothrium rugosum, an intestinal parasite of burbot Lota lota, and establish its localization in the tapeworm. To this end, the worms were treated with Triton X-100 followed by differential centrifugation to isolate the tegumental brush border membrane. The protease inhibitory abilities of the worms were mostly determined by their excretory/secretory products released into the incubation medium. These inhibitory abilities proved to be linked mainly with the brush border fractions. Notably, the per cent inhibition of both studied digestive enzymes (trypsin and chymotrypsin) hardly depended on the duration of the parasite exposure in the incubation medium, probably due to intermittent glycocalyx renewal. Improved knowledge on functions of the excretory/secretory proteins produced by fish tapeworms may contribute to a better understanding of host-parasite relations and development of new approaches to the treatment and prevention of diseases caused by pathogenic helminths.

Parasite infections in fish require constant surveillance and strategies for efficient treatments which guarantee the fish health, their sale value and the non-propagation of pathogens in new environments. Fish treatments based on nanotechnology become of increasing interest since nanoparticles have been shown as efficient materials for optimizing administration of bioactives. In this study a chitosan derivative, alginate and praziquantel conjugated nanobioparticle of effective action for oral treatment of digenetic trematodes in highly infected Corydoras schwartzi was evaluated in terms of histological and hematological safety. The inherent absence of alterations in intestinal tissue and the reversible blood cells counting during a period up to 35 days showed the safety of the drug delivery nanobioparticles, which thus represent a promising strategy for effective applications in pathogens treatments by oral administration.

Both the activation and attenuation of MAVS/IFN signaling are critical for host defensing against viral infection and thus lead to an elaborate regulation of MAVS-mediated signaling. However, the regulatory mechanisms concerning MAVS/IFN signaling in teleost fish are not well understood. RIPK3 has been identified as a key regulator of necroptosis, apoptosis, and inflammatory signaling in human and mammals. Here we report the identification of the RIPK3 homologue from black carp Mylopharyngodon piceus (bcRIPK3) and describe its role in regulating MAVS/IFN signaling. qPCR results demonstrated that bcRIPK3 was transcriptionally activated in response to poly (I:C) or LPS stimulation. Immunoblot assay and immunofluorescent staining assay showed that bcRIPK3 was a cytosolic protein with molecular weights of 47 kDa. Like its mammalian counterparts, bcRIPK3 exhibited a conserved function in inducing cell death. The reporter assay and plaque assay showed that overexpression of bcRIPK3 restricted bcMAVS-activated transcription of the interferon promoters of black carp and zebrafish, and suppressed bcMAVS-mediated antiviral activity. Notably, EPC cells co-expressing bcRIPK3, bcRIPK1 and bcMAVS presented much attenuated antiviral activity than the cells co-expressing bcRIPK3 and bcMAVS; and the subsequent co-IP assay identified the interaction between bcRIPK3 and bcRIPK1. Our findings collectively elucidate for the first time in teleost that black carp RIPK3 interacts with RIPK1 to inhibit MAVS-mediated antiviral signaling.

Background In Norway, x-cell parasites associated with disease in farmed salmonids have been known as a rare phenomenon for two decades. These parasites cause systemic infections in farmed rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar), but have so far not been characterized and described. Methods The x-cells from several cases of diseased fish were studied using light and electron microscopy, and by phylogenetic analysis based on small subunit ribosomal RNA (SSU rRNA) gene sequences. Results We describe here the x-cell parasite as a new species in a new genus, Salmoxcellia vastator n. gen., n. sp. Phylogenetic analyses placed Salmoxcellia n. gen. together with Gadixcellia among the xcelliids, a group of perkinsozoan alveolates. The new genus and species were found to have vacuolate plasmodial x-cells filled with lipid droplets, and an electron-dense alveolar pellicle. Electron-dense cytoplasmic inclusions, which are characteristic of the other xcelliid genera Xcellia and Gadixcellia, are lacking in Salmoxcellia n. gen. These x-cell plasmodia divide by plasmotomy and occur as aggregates in the host tissues, particularly in blood-rich tissues such as those of the kidney, red musculature, heart and liver. Host reaction and the refractive lipid droplets in the x-cells result in S. vastator n. gen., n. sp. aggregates appearing as white patches in the tissues. Conclusions We describe a new genus and species of xcelliid protist parasites from two very important farmed fish species and provide molecular methods for detection. The new parasite is associated with disease, but more importantly it has a spoiling effect on farmed salmonid fillets, rendering them unsuitable for sale. Consequently, this parasite represents a threat to the aquaculture industry. Graphical abstract

Recently, the culture of American eels (Anguilla rostrate) in China has been impacted by emergence of a disease with signs of haemorrhagic gill necrosis. The gills of diseased eels are covered with petecchia and they bleed when the operculum is pressed. In this study, a novel American eel adomavirus (AEAdoV) was isolated from the diseased eels using the eel ovary cell line (EO). The virus proliferated in the EO cells with a maximum TCID50 /ml of 106.29 ± 0.23 at 6 days post-infection. The virions were non-enveloped with a diameter of 75-85 nm and shown to be a DNA virus upon 5-iodo-2'-deoxyuridine (IDU) treatment. PCR assays showed that AEAdoV encodes a superfamily 3 helicases (S3H) replicase and shared high similarities with Anguilla marmorata adomavirus (MEAdoV). Although no clinical signs or mortality was observed among the eels injected with AEAdoV, the virus was reisolated from livers, kidneys and gills of injected eels at 35 days post-injection. Our results suggested that AEAdoV exhibited a latent infection in A. rostrata. The pathogenicity of the AEAdoV needs to be confirmed further.

AIM The aims of this study were to screen an antagonistic probiotic for the prevention and control of bacterial diseases in snakehead fish and to evaluate the antimicrobial activities, biosafety and biocontrol effect of the antagonistic strain. METHODS AND RESULTS In this study, the WLYS23 strain exhibiting the most effective antagonistic properties against several fish pathogens was selected from the intestine of healthy snakehead fish. The strain was identified as Bacillus velezensis based on morphological, physiological and biochemical characteristics, and phylogenetic analysis. This strain showed amylase, cellulase, protease and lipase activities according to extracellular enzyme activity assays. Moreover, the cell-free extract of the WLYS23 strain exhibited strong antibacterial activity, with MICs of ≤64 μg ml-1 for most fish pathogens. Additionally, the cell-free extract was heat and pH stable, and resistant to protease, whereas their antimicrobial activities were significantly influenced by metal ions at high concentration. The WLYS23 strain and its cell-free extract were safe for snakehead and zebrafish. The disease resistance of snakehead fish was significantly increased following administration of the WLYS23 strain and its cell-free extract respectively. The complete genome of the WLYS23 strain was sequenced and found to harbour seven gene clusters encoding secondary metabolites with antimicrobial activity. CONCLUSION The WLYS23 strain possesses great potential as a biocontrol agent, which can be commercially developed to improve disease control in freshwater aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY Snakehead fish are important commercially farmed fish worldwide. However, the bacterial diseases caused by Aeromonas sp. and Nocardia seriolae in farmed snakehead fish lead to huge economic losses. Safe, economical and efficient probiotics are limited to prevent and control these diseases. Here, we provide a promising biocontrol agent with antagonistic activity against bacterial diseases of snakehead.