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Made available in DSpace on 2022-10-10T13:56:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-06-06. Added 1 bitstream(s) on 2022-10-10T15:39:51Z : No. of bitstreams: 1 S1519-69842024000100325.pdf: 683252 bytes, checksum: 60c423322185e736c29136f6cb27ce89 (MD5) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) A infecção por micobactérias não tuberculosas é uma das doenças bacterianas crônicas mais comuns em peixes ornamentais de aquário e parece estar diretamente relacionada a práticas de manejo estressantes. Além disso, também representa potencial zoonótico. Aqui, apresentamos o isolamento e a caracterização de micobactérias não tuberculosas de peixes-anjo de água doce doentes (Pterophyllum scalare) em São Paulo, Brasil. Foram avaliadas nove fêmeas reprodutoras descartadas com sinais de doença. Os peixes exibiam letargia, perda de apetite, caquexia, úlceras cutâneas e exoftalmia. Na necropsia, quatro peixes apresentaram granulomas macroscópicos no baço. Mycobacterium chelonae, M. fortuitum, M. gordonae, M. intracellulare e M. peregrinum foram isolados e identificados por análise de restrição por PCR de hsp65. A análise histopatológica revelou lesões microscópicas compatíveis com micobacteriose, e o bacilo de Mycobacterium foi observado pela coloração de Ziehl-Neelsen. Notavelmente, todas as espécies de Mycobacterium identificadas neste estudo já foram relatadas em pacientes humanos; portanto, animais doentes podem ser uma fonte de infecção para pessoas que manipulam peixes e aquários. Nontuberculous mycobacteria infection is one of the most common chronic bacterial diseases in ornamental aquarium fish and appears to be directly related to stressful husbandry practices. Furthermore, it also represents zoonotic potential. Here we present the isolation and characterization of non-tuberculous mycobacteria from diseased freshwater angelfish (Pterophyllum scalare) in São Paulo, Brazil. Nine discarded breeding females with signs of disease were evaluated. The fish exhibited lethargy, loss of appetite, cachexia, skin ulcers, and exophthalmia. At necropsy, four fishes presented macroscopic granulomas in the spleen. Mycobacterium chelonae, M. fortuitum, M. gordonae, M. intracellulare and M. peregrinum were isolated and identified by hsp65 PCR restriction analysis. Histopathological analysis revealed microscopic lesions compatible with mycobacteriosis, and Mycobacterium bacillus were observed by Ziehl-Neelsen stain. Notably, all Mycobacterium species identified in this study have already been reported in human patients; therefore, diseased animals may be a source of infection for people who handle fish and aquariums. Universidade de São Paulo, Departamento de Medicina Veterinária Preventiva e Saúde Animal Universidade Estadual Paulista (UNESP), Instituto de Biotecnologia Universidade Estadual Paulista (UNESP), Instituto de Biotecnologia CAPES: 001 CAPES: 1808006 CNPq: 154900/2018-4 CNPq: 310736/2018-8

Fish possess a powerful IFN system to defend against aquatic virus infections. Nevertheless, spring viremia of carp virus (SVCV) causes large-scale mortality in common carp and significant economic losses to aquaculture. Therefore, it is necessary to investigate the strategies used by SVCV to escape the IFN response. In this study, we show that the SVCV nucleoprotein (N protein) negatively regulates cellular IFN production by degrading stimulator of IFN genes (STING) via the autophagy-lysosome–dependent pathway. First, overexpression of N protein inhibited the IFN promoter activation induced by polyinosinic-polycytidylic acid and STING. Second, the N protein associated with STING and experiments using a dominant-negative STING mutant demonstrated that the N-terminal transmembrane domains of STING were indispensable for this interaction. Then, the N protein degraded STING in a dose-dependent and autophagy-lysosome–dependent manner. Intriguingly, in the absence of STING, individual N proteins could not elicit host autophagic flow. Furthermore, the autophagy factor Beclin1 was found to interact with the N protein to attenuate N protein–mediated STING degradation after beclin1 knockdown. Finally, the N protein remarkably weakened STING-enhanced cellular antiviral responses. These findings reveal that SVCV uses the host autophagic process to achieve immune escape, thus broadening our understanding of aquatic virus pathogenesis.

Changes in the thermal optima of fish impacts changes in the physiology and immune response associated with infections. The present study showed that at suboptimal temperatures (17 °C), the host tries to evade viral infection by downregulating the inflammatory response through enhanced neuronal protection. There was significantly less abundance of IgM + B cells in the 17 °C group compared to that in the 25 °C group. An increased macrophage population (Iba1+) during the survival phase in fish challenged at 25 °C demonstrated inflammation. Optimal temperature challenge activated virus-induced senescence in brain cells, demonstrated with a heterochromatin-associated H3K9me3 histone mark. There was an abundant expression of anti-inflammatory cytokines in the brain of fish at the suboptimal challenge. Besides the cytokines, the expression of BDNF was significantly higher in the suboptimally challenged group, suggesting that its neuronal protection activity following NNV infection is mediated through TGFβ. The suboptimal challenge resulted in H3k9ac displaying transcriptional competency, activation of trained immunity H3K4me3, and enrichment of H3 histone-lysine-4 monomethylation (H3K4me1), resulting in a robust re-stimulatory immune response. The observations from the H4 modifications showed that besides H4K12ac and H4K20m3, all the assayed modifications were significantly higher in suboptimal convalescent fishes. The suboptimally challenged fish acquired more methylation along cytosine residues than the optimally infected fish. Together, these observations suggest that optimal temperature results in an immune priming effect, whereas the protection enabled in suboptimal convalescent fishes is operated through epigenetically controlled trained immune functions.

Silver pomfret has been widely cultured in China due to its high economic value. Photobacterium damselae subsp. damselae (PDD) is a Gram-negative bacterium that has been shown to infect many fish species. To increase knowledge of the molecular mechanisms of the host defense against PDD, we conducted transcriptome analysis of head kidney in silver pomfret at 24 h and 72 h post-infection (hpi) via Illumina sequencing. The de novo assembly resulted in the identification of 79,063 unigenes, with 59,386 (75.11%) successfully annotated in public databases (NR, NT, KO, Swiss-Prot, Pfam, GO, and KOG databases). Comparison of gene expression profiles between PBS-injected fish (sham control) and PDD-challenged fish revealed 329 and 570 differentially expressed genes (DEGs) were screened at 24 hpi and 72 hpi, respectively. The DEGs were enriched in multiple immune-related pathways such as Hepatitis C, Gastric acid secretion, CAMs and Leukocyte transendothelial migration pathways, Primary immunodeficieny, ECM-receptor interaction, PI3K-Akt signaling pathway. The data obtained in the present study offers valuable information for acute immune response of silver pomfret challenged with PDD, which will facilitate further investigations on strategies against Photobacterium spp. infection in teleosts.

In mammals, nemo-like kinase 2 (NLK2) is a conservative protein kinase involved in Wnt/β-catenin signaling pathway and immune response. However, the role of NLK2 in immune response in teleost remain unclear. In this study, we identified an ortholog of mammalian NLK from grass carp (Ctenopharyngodon idellus) named CiNLK2. CiNLK2 shares a high level of homology with the counterparts, especially with that of Cyprinus carpio. CiNLK2 was ubiquitously expressed in all tested tissues (liver, brain, spleen, gill, kidney and eye) and its expression was up-regulated under the treatment with poly I:C or GCRV. Overexpression of CiNLK2 suppressed the production of IFN I in CIK cells whether or not treated with poly I:C. However, knockdown of CiNLK2 increased the expression level of IFN I. The analysis of subcellular localization showed that CiNLK2 protein was scattered throughout the cytoplasm and nucleus. In terms of mechanism, CiNLK2 can directly interact with MAVS and inhibit MAVS-induced IFN I response. Moreover, CiNLK2 increased the phosphorylation level of MAVS, which led to the degradation of MAVS protein. On the other hand, CiNLK2 suppressed the phosphorylation and nuclear translocation of IRF3. In general, CiNLK2 served as an inhibitor for IFN I response by targeting MAVS-IRF3 signal axis.

Krüppel-like factor 2a (KLF2A), a transcription factor of the krüppel-like family, is involved in regulating the immune molecules and is associated with viral infection. However, the function of KLF2A during viral infections in fish remains unclear. In this study, grass carp (Ctenopharyngodon idellus) was used to predict the target genes regulated by KLF2A. The results showed that the candidate target genes included four members of the serpin gene family (serpinb1l2, serpinc1, serpinh1a, and serpinh1b). Dual-luciferase experiments showed that klf2a positively regulates serpinc1 expression. Dose-dependent klf2a overexpression in C. idellus kidney (CIK) cells significantly upregulated the expression of serpinc1. Overexpressing klf2a or serpinc1 in CIK cells activated interferon responses and suppressed grass carp reovirus (GCRV) replication. Klf2a and serpinc1 co-expression inhibited GCRV replication. These results show that klf2a upregulates serpinc1 mRNA expression, promotes type 1 interferon responses, and suppresses GCRV infection. This study provides insights into the regulatory role and biological functions of KLF2A in host-virus interactions in fish.

Interleukin (IL)-22 is an IL-10 family cytokine secreted by CD4

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signaling pathway. To better understand the functions of STAT2 in fish immune response, a STAT2 gene from orange-spotted grouper (Epinephelus coioides) (EcSTAT2) was cloned and characterized in this study. EcSTAT2 encoded a 802-amino acid peptide which shared 99.5% and 91.5% identity with giant grouper (Epinephelus lanceolatus) and leopard coral grouper (Plectropomus leopardus), respectively. Amino acid alignment analysis showed that EcSTAT2 contained five conserved domains, including N-terminal protein interaction domain, coiled coil domain (CCD), DNA binding domain (DBD), Src-homology 2 (SH2) domain, and C-terminal transactivation domain (TAD). Phylogenetic analysis indicated that EcSTAT2 clustered into fish STAT2 group and showed the nearest relationship to giant grouper STAT2. In healthy grouper, EcSTAT2 was distributed in all tissues tested, and the expression of EcSTAT2 was predominantly detected in spleen, kidney and gill. In vitro, EcSTAT2 expression was significantly increased in response to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization showed that EcSTAT2 was located in the cytoplasm in a punctate manner. EcSTAT2 overexpression significantly inhibited RGNNV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral genes and protein. Consistently, knockdown of EcSTAT2 using small interfering RNA (siRNA) promoted RGNNV replication. Furthermore, EcSTAT2 overexpression increased both interferon (IFN) and interferon stimulated genes (ISGs) expression. In addition, EcSTAT2 knockdown decreased the transcription levels of IFN and ISGs. Together, our data demonstrated that EcSTAT2 exerted antiviral activity against RGNNV through up-regulation of host interferon response.

IRF4 is a master member of the interferon regulatory factor (IRF) family playing vital regulatory roles in immune system development and function. Tetrapods have a single-copy IRF4 gene, while teleosts harbor duplicated IRF4 genes. This work describes three IRF4 paralogs from yellow catfish (Pelteobagrus fulvidraco), designated PfIRF4A, PfIRF4B and PfIRF4B-like. These genes all contain a typical IRF structural architecture. Phylogenic and synteny analyses indicate that they should arise from the teleost-specific whole-genome duplication. PfIRF4 genes are abundantly expressed in the immune-related tissues and upregulated by PolyI:C, LPS, and Edwardsiella ictaluri. Ectopic expression of these genes inhibits the activation of fish type Ⅰ IFN promoters and downregulates the transcription levels of IFN-responsive genes, thus allowing the efficient replication of a fish rhabdovirus, spring viremia of carp virus (SVCV). PfIRF4s possess a repressive effect on MyD88-mediated activation of IFN and NF-κB. Some differences are observed between each individual paralog. PfIRF4B is the main form expressed across the tissues and the most up-regulated one after pathogen induction. It exerts a stronger inhibitory effect on IFN antiviral response than the other two paralogs. PfIRF4A and PfIRF4B-like are primarily present in the nucleus, while PfIRF4B displays colocalization and direct associations with MyD88 in the cytoplasm. Overall, the data demonstrate that three PfIRF4 paralogs show shared and individual functional properties in the negative regulation of type Ⅰ IFN response.

Rainbow trout (Oncorhynchus mykiss), an economically important cold-water fish cultured worldwide, suffers from infectious hematopoietic necrosis virus (IHNV) infection, resulting in huge financial losses. In order to understand the immune response of rainbow trout during virus infection, we explored trout intestine transcriptome profiles following IHNV challenge, and identified 3355 differentially expressed genes (DEGs) and 80 differentially expressed miRNAs (DEMs). Transcriptome analysis revealed numerous DEGs involved in immune responses, such as toll-like receptor 3 (TLR3), toll-like receptor 7/8 (TLR7/8), tripartite motif-containing 25 (TRIM25), DExH-Box helicase 58 (DHX58), interferon-induced with helicase C domain 1 (IFIH1), interferon regulatory factor 3 (IRF3/7), signal transducer and activator of transcription 1 (STAT1) and heat shock protein 90-alpha 1 (HSP90A1). Integrated analysis identified five key miRNAs (miR-19-y, miR-181-z, miR-203-y, miR-143-z and miR-206-y) targeting at least two important immune genes (TRIM25, DHX58, STAT1, TLR7/8 and HSP90A1). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that DEGs and target genes were significantly enriched in various immune-related terms including immune system process, binding, cell part and pathways of Toll-like receptor signalling, RIG-I-like receptor signalling, NOD-like receptor signalling, JAK-STAT signalling, PI3K-Akt signalling, NF-kappa B signalling, IL-17 signalling and AGE-RAGE signalling. In addition, protein-protein interaction networks (PPI) was used to display highly interactive DEG networks involving eight immune-related pathways. The expression trends of 12 DEGs and 10 DEMs were further verified by quantitative real-time PCR, which confirmed the reliability of the transcriptome sequencing results. This study expands our understanding of the immune response of rainbow trout infected with IHNV, and provides valuable resources for future studies on the immune molecular mechanism and disease resistance breeding.

The goal of this paper is to investigate the influence of the waning immunity on the dynamics of Tilapia Lake Virus (TiLV) transmission in wild and farmed tilapia within freshwater. We formulate a model for which susceptible individuals can contract the disease in two ways: (i) direct mode caused by contact with infected individuals; (ii) indirect mode due to the presence of pathogenic agents in the water. We obtain an age-structured model which combines both age since infection and age since recovery. We derive an explicit formula for the reproductive number $ \mathcal {R}_0 $ and show that the disease-free equilibrium is locally asymptotically stable when, $ \mathcal {R}_0 \lt 1 $ . We discuss on the form of the waning immunity parameter and show numerically that a Hopf bifurcation may occur for suitable immunity parameter values, which means that there is a periodic solution around the endemic equilibrium when, $ \mathcal {R}_0 \gt 1 $ .

Aeromonas veronii is a pathogen which can induce diseases in humans, animals and aquatic organisms, but its pathogenic mechanism and virulence factors are still elusive. In this study, we successfully constructed a mutant strain (ΔascP) by homologous recombination. The results showed that the deletion of the ascP gene significantly down-regulated the expression of associated effector proteins in A. veronii compared to its wild type. The adhesive and invasive abilities of ΔascP to EPC cells were 0.82-fold lower in contrast to the wild strain. The toxicity of ΔascP to cells was decreased by about 2.91-fold (1 h) and 1.74-fold (2 h). Furthermore, the LD

OBJECTIVE To evaluate the effect of a multidose acyclovir protocol on koi herpesvirus (KHV) viral load and mortality in a cohabitation challenge. ANIMALS 180 koi fish. PROCEDURES Forty fish (shedders) were immersed in a 0.5 KHV plaque-forming units/mL static bath for 8 hours. Mock shedders were treated similarly but exposed to cell culture media. KHV shedders were then transferred into 8 tanks (5 shedders per tank) containing 10 naïve fish (cohabitants) each. Fish in the acyclovir group (AT) received a 10 mg/kg acyclovir intracoelomic injection 1, 3, and 6 days after the first confirmed KHV mortality. Positive controls (PC) were treated similarly but received sterile saline injections. Negative controls (NC) were exposed to mock shedders. Morbidity and mortality were evaluated daily for 50 days post-challenge. Quantitative PCR was used to determine viral load in the gill biopsies of shedders and cohabitants collected at days 19 (T1), 22 (T2), 25 (T3), 34 (T4), and 50 (T5) post-challenge. RESULTS Survival curves analyzed by the Gehan-Breslow-Wilcoxon method revealed a delayed onset of mortalities and a significantly lower KHV load at T2 and T3 detected in AT cohabitant fish (P = .042) compared to PC group. However, there were no significant differences in overall mortality or viral loads at T5. CLINICAL RELEVANCE The acyclovir protocol used in this study did not control viral infection or mortality at the end of the 50-day trial. Shorter intervals between injections could improve outcomes, but the additional stress inflicted by handling should be considered. Exploring other therapeutic alternatives and doses is warranted.

Pyroptosis is an inflammatory and programmed cell death initiated by the formation of the inflammasome, which consists of NLR, ASC, and Caspase. Pyroptosis has received growing attention due to its association with innate immunity and various diseases. However, the involvement and induction of the NLRCs and pyroptosis-related genes in fish immunity remain poorly studied. In this study, several NLRCs and pyroptosis-related genes in Nile tilapia (Oreochromis niloticus) were identified and characterized. Their involvement in bacterial infection and expression profiles in Nile tilapia lymphocyte responses were also assessed. Overall, three NLRC members (NOD1, NOD2, and NLRC3) and five pyroptosis-related genes (ASC1, Caspase1, Gsdme, NLRP3, and NLRP14) in Nile tilapia were cloned and characterized. The transcript levels of these molecules were broadly distributed in various tissues with comparatively high expression in the gills, intestine, and spleen. Their transcripts were also induced during Streptococcus agalactiae or Aeromonas hydrophila infection. Moreover, they were primarily expressed in T cells, NCCs, and Mo/Mφ and showed antibacterial and partially antiviral responses. The present study lays a theoretical foundation for further investigation of the pyroptosis mechanisms in fish as well as the evolution of the antiviral roles of pyroptosis in vertebrates.

Transcription factor ATF1 is a member of the ATF/CREB family of the CREB subfamily and is involved in physiological processes such as tumorigenesis, organ development, reproduction, cell survival, and apoptosis in mammals. However, studies on ATF1 in fish have been relatively poorly reported, especially on its role in antiviral immunity in fish. In this study, ATF1 from orange-spotted grouper (named EcATF1) were cloned and characterized. Molecular characterization analysis showed that EcATF1 encodes a 307-amino-acid protein, containing PKID and bZIP_CREB1 domains. Homology analysis showed that had the highest homology with E. lanceolatus(88.93%). Tissue expression pattern showed that EcATF1 was extensively distributed in twelve selected tissues, with higher expression in the skin, gill, liver and spleen. Subcellular localization analysis showed that EcATF1 was distributed in the nucleus of GS cells. EcATF1 overexpression inhibits Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by a diminished degree of CPE induced by SGIV and RGNNV and a reduction in the level of viral gene transcription and viral capsid protein expression. Furthermore, EcATF1 overexpression upregulated interferon pathway-related genes and proinflammatory factors, and increased the promoter activities of IFN, IFN stimulated response element (ISRE), and nuclear factor κB(NFκB). Meanwhile, EcATF1 overexpression positive regulate the MHC-I signaling pathway, and upregulated the promoter activity of MHC-I. Collectively, these data demonstrate that EcATF1 plays an important role during the host antiviral immune response. This study provides insights into the function of ATF1 in the immune system of lower vertebrates.

In mammals, six interleukin-17 (IL-17) genes, as potent pro-inflammatory cytokines, all accelerate the inflammatory responses. In teleosts, seven IL-17 genes have been found in various species, but little is known about the function of teleost-specific IL-17N. In this study, teleost IL-17N and IL-17A/F2 genes all had six conserved cysteine residues forming three intrachain disulfide bridges, the length of three exons of teleost IL-17N gene was similar to that of teleost IL-17A/F2 gene, and the neighbor-joining (NJ) phylogenetic tree showed that teleost IL-17N was clustered with vertebrate IL-17A/F, implying that teleost IL-17N gene may be a paralog of teleost IL-17A/F gene. Pelteobagrus fulvidraco (Pf) IL-17N gene was highly expressed in the blood, brain and kidney of healthy yellow catfish. Pf_IL-17N transcript and protein were notably up-regulated in the spleen, head kidney, gill and kidney detected after Edwardsiella ictaluri infection. Lipopolysaccharides (LPS), polyinosinic-polycytidylic acid (Poly I:C) and peptidoglycan (PGN) also remarkably induced the expression of Pf_IL-17N in the isolated peripheral blood leucocytes (PBLs) of yellow catfish. These results reveal that Pf_IL-17N may play important roles in preventing the invasion of pathogens. Furthermore, the recombinant (r) Pf_IL-17N protein could significantly induce the mRNA expressions of inflammatory cytokines, chemokines and antimicrobial peptide genes in yellow catfish in vivo and in vitro, and it also notably promoted the phagocytosis of myeloid cells in the PBLs and the chemotaxis of the PBLs and gill leucocytes (GLs) in yellow catfish. Besides, though the rPf_IL-17N protein could induce and aggravate inflammation infiltration in the kidney of yellow catfish, it did not effectively and notably increase the survival rate of yellow catfish after E. ictaluri infection. Furthermore, the rPf_IL-17N protein could induce the mRNA expressions of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signal pathways related genes, and the inhibitor of NF-κB and MAPK signal pathways could restrain the rPf_IL-17N protein-induced inflammatory response. This study provides crucial evidence that the Pf_IL-17N may mediate inflammatory response to eliminate invasive pathogens.

Teleosts have a unique immune system because their head kidney (HK) and trunk kidney (TK) are sites for hematopoiesis. However, the immune functions of the HK and TKs require further elucidation in yellow catfish (Pelteobagrus fulvidraco). In the present study, imprints of the HK and TK were examined using the Wright's-Giemsa staining method. Morphological characteristics of the blood cell lineages revealed that the HK and TK were hematopoietic organs. To explore its immune function, transcriptome sequencing was performed after infection with Aeromonas hydrophila. A total of 1139 genes showed significant alterations in their expression in the kidney; these genes included 737 upregulated and 402 downregulated genes. Furthermore, 1117 differentially expressed genes were observed in the HK, which included 784 upregulated and 333 downregulated genes. Both organs showed 357 upregulated genes and 85 downregulated genes. Some immune-related genes were only expressed in the TK, such as ATP-dependent RNA helicase DDX58, the gene encoding the immunoglobulin heavy chain and light chain. The immune responses in the HK and TK were differential and the TK played a critical role in the mechanism underlying the immune response. The purpose of the present study was to facilitate the elucidation of the immune defense mechanism of yellow catfish and other teleosts.

Dual-specificity Phosphatases (DUSPs) are not only the key regulators of dephosphorylating and inactivating mitogen-activated protein kinases (MAPKs), but play a crucial role in the immune response. However, the role of DUSP genes in Japanese flounder (PoDUSPs) is still unclear. In this study, 28 DUSP genes in Japanese flounder were identified and classified based on the whole genome database. Phylogenetic analysis and protein structure analysis revealed that DUSPs had highly conserved domains in teleosts. Molecular evolution analysis indicated that the PoDUSP genes were conservative during evolution and were functional-constrained. Meanwhile, PoDUSP genes were found to express in different embryonic and larval stages which might play the role of sentinel in healthy organisms. Furthermore, PoDUSP genes' expression profiles after temperature stress and Edwardsiella tarda (E. tarda) infection were determined in Japanese flounder without precedent, and the results demonstrated that Podusp1, Podusp2 and Podusp16 were more respective to temperature variation whereas Podusp1 and Podusp6 were more respective to E. tarda infection. In summary, our results provide useful resources for understanding the immune responsibilities of DUSP genes in flatfish.

Emerging pathogen, carp edema virus (CEV) causes koi sleepy disease (KSD) in Koi and common carp causing severe mortalities worldwide. In the present study, a total of 150 fish species belonging to eight different families were sampled from the ornamental fish retailers and farms, located in Karnataka, India. The OIE protocol viz., level-I, II and III diagnoses confirmed the infection of CEV in 10 koi fish. Interestingly, other fish species belonging to different fish family including cyprinidae family were negative to CEV. Further, CEV infection was confirmed by sequencing (partial 4a gene); it showed the similarity with that of CEV reported from India and Germany strains with similarity of 97.4-99.94% and belonged to genogroup IIa. TEM analysis of purified CEV, in vivo cohabitation and tissue infection experiments confirmed the CEV infection. In addition, viral load was significantly higher (10

Alginate oligosaccharide (AOS) is widely used in agriculture because of its many excellent biological properties. However, the possible beneficial effects of AOS and their underlying mechanisms are currently not well known in grass carp (Ctenopharyngodon idellus). Here, grass carp were fed diets supplemented with 5, 10, or 20 g/kg AOS for six weeks. HE and PAS staining showed that the diets of AOS significantly increased the number of goblet cells in the intestinal. According to transcriptome and quantitative real-time PCR (qRT-PCR) data, AOS-supplemented diets activated the expression of fat metabolism-related pathways and genes. The 16S rRNA sequencing results showed that supplementation with AOS affected the distribution and abundance of the gut bacterial assembly. qRT-PCR and activity assays revealed that the AOS diets significantly increased the antioxidant resistance in gut of grass carp, and down-regulated the expression of inflammatory and up-regulated anti-inflammatory cytokines. Finally, the Aeromonas hydrophila infection assay suggested that the mortality in the groups fed dietary AOS was slightly lower than that in the control. Therefore, supplementing the diet of grass carp with an appropriate amount of AOS can improve fat metabolism and immune responses and alter the intestinal bacterial community, which may help to fight bacterial infection.

Amphiprion clarkii is increasingly being used as a captive-bred ornamental fish in South Korea. However, its breeding has recently been greatly hindered by destructive diseases due to pathogens. B-cell lymphoma-2 (Bcl2), a mitochondrial apoptosis regulatory gene involved in immune responses, has not been investigated in anemonefish, including A. clarkii. Herein, we aimed to annotate Bcl2 in the A. clarkii transcriptome and examined its role against virus infections. Sequence analysis indicated that Bcl2 in A. clarkii (AcBcl2) contained all four Bcl-2 homology domains. The structure of AcBcl2 closely resembled those of previously analyzed anti-apoptotic Bcl2 proteins in mammals. Expression analysis showed that the highest level of AcBcl2 was expressed in blood. AcBcl2 expression in the blood was downregulated within 24 hpi when challenged with immune stimulants poly I:C and lipopolysaccharides. AcBcl2 reduced poly I:C-induced cell death. The propagation of viral hemorrhagic septicemia virus (VHSV) was higher in the presence of AcBcl2. Cell mortality was higher in AcBcl2 when transfected cells were infected with VHSV, and a higher viral transcript was observed compared to their respective controls. In conclusion, AcBcl2 is an anti-apoptotic protein, and its activity may facilitate the propagation of VHSV.

Long non-coding RNA (lncRNA) play vital regulatory roles in various biological processes. Intestine is one of the most sensitive organs to environmental and homeostatic disruptions for fish. However, systematic profiles of lncRNAs in the intestine of teleost in responses to pathogen infections is still limited. Turbot (Scophthalmus maximus L.), an important commercial fish species in China, has been suffering with Vibrio anguillarum infection, resulted in dramatic economic loss. Hereinto, the intestinal tissues of turbot were sampled at 0 h, 2 h, 12 h, and 48 h following V. anguillarum infection. The histopathological analysis revealed that the pathological trauma was mainly present in intestinal tunica mucosal epithelium. After high-throughput sequencing and bioinformatic analysis, a total of 9722 lncRNAs and 21,194 mRNAs were obtained, and the average length and exon number of lncRNAs were both less than those of mRNAs. Among which, a set of 158 lncRNAs and 226 mRNAs were differentially expressed (DE-lncRNAs and DEGs) in turbot intestine at three time points, related to many immune-related genes such as complement, interleukin, chemokine, lysosome, and macrophage, indicating their potential critical roles in immune responses. In addition, 2803 and 1803 GO terms were enriched for DEGs and co-expressed target genes of DE-lncRNAs, respectively. Moreover, 127 and 50 KEGG pathways including cell adhesion molecules (CAMs), phagosome, JAK-STAT signaling pathway, cytokine-cytokine receptor interaction, and intestinal immune network for IgA production, were enriched for DEGs and co-expressed target genes of DE-lncRNAs, respectively. Finally, qRT-PCR was conducted to confirm the reliability of sequencing data. The present study will set the foundation for the future exploration of lncRNA functions in teleost in response to bacterial infection.

The present study investigated the involvement of key molecular regulators of oxidative stress in amoebic gill disease (AGD), a parasitic infestation in Atlantic salmon. In addition, the study evaluated how these molecular biomarkers responded when AGD-affected fish were exposed to a candidate chemotherapeutic peracetic acid (PAA). Atlantic salmon were experimentally infected with the parasite Neoparameoba perurans, the causative agent of AGD, by bath exposure and after 2 weeks, the fish were treated with three commercial PAA products (i.e., Perfectoxid, AquaDes and ADDIAqua) at a dose of 5 ppm. Two exposure durations were evaluated - 30 min and 60 min. Sampling was performed 24 h and 2 weeks after PAA treatment (equivalent to 2- and 4-weeks post infection). At each sampling point, the following parameters were evaluated: gross gill pathology, gill parasitic load, plasma reactive oxygen species (ROS) and total antioxidant capacity (TAC), histopathology and gene expression profiling of genes with key involvement in oxidative stress in the gills and olfactory organ. AGD did not result in systemic oxidative stress as ROS and TAC levels remained unchanged. There were no clear patterns of AGD-mediated regulation of the oxidative stress biomarkers in both the gills and olfactory organ; significant changes in the expression were mostly related to time rather than infection status. However, the expression profiles of the oxidative stress biomarkers in AGD-affected salmon, following treatment with PAA, revealed that gills and olfactory organ responded differently - upregulation was prominent in the gills while downregulation was more frequent in the olfactory organ. The expression of catalase, glutathione S-transferase and thioredoxin reductase 2 was significantly affected by the treatments, both in the gills and olfactory organ, and these alterations were influenced by the duration of exposure and PAA product type. Parasitic load in the gills did significantly increase after treatment regardless of the product and exposure duration; the parasite was undetectable in some fish treated with AquaDes for 30 mins. However, PAA treated groups for 30 min showed lower macroscopic gill scores than the infected-untreated fish. Histology disclosed the classic pathological findings such as multifocal hyperplasia and increased number of mucous cells in AGD-affected fish. Microscopic scoring of gill injuries showed that AGD-infected-PAA-treated fish had lower scores, however, an overall trend could not be established. The morphology and structural integrity of the olfactory organ were not significantly altered by parasitism or PAA treatment. Collectively, the results indicate that AGD did not affect the systemic and mucosal oxidative status of Atlantic salmon. However, such a striking profile was changed when AGD-affected fish were exposed to oxidative chemotherapeutics. Moreover, the gills and olfactory organ demonstrated distinct patterns of gene expression of oxidative stress biomarkers in AGD-infected-PAA-treated fish. Lastly, PAA treatment did not fully resolve the infection, but appeared not to worsen the mucosal health either.

Membrane-associated RING-CH (MARCH) family, as Ring-type E3 ligases, have attracted extensive attention to their immune functions. MARCH4 plays an essential role in regulating immune response in mammal. In the present study, it is the first to report on MARCH4 characteristics and signal pathway in fish. MARCH4 in large yellow croaker Larimichthys crocea (named as LcMARCH4) encodes a RING-CH domain and two TM domains, as well as other function domains, including an N-terminal proline rich domain, an AxxxG-motif in TM1, a tyrosine-based YXXØ motif, and a C-terminal PDZ-binding domain. LcMARCH4 is a tissue-specific protein with highly significant expression in brain. The mRNA transcripts of LcMARCH4 were significantly induced in the main organs (skin, gill, spleen, and head-kidney) by C. irritans infection. Consistently, significant increase was observed in spleen and head-kidney after LPS, Poly I:C stimulation and V. parahaemolyticus infection. Subcellular localization analysis showed that LcMARCH4 was localized in the cytoplasm and membrane. Moreover, we found 46 DEGs in a comparative transcriptome analysis between the LcMARCH4 overexpression group and control vector group. The analysis showed that HSPA6, HSPA1B and DNAJB1 might play important regulatory roles to MARCH4 in fish. Notably, two noncoding RNA, both RN7SL1 and RN7SL2, the expression levels went up in MARCH4 overexpression cells. Taken together, this study will provide new insights into finfish MARCH4 and its potential regulatory signaling pathway as well.

The tripartite motif (TRIM) proteins play critical roles in viral infection by modulating innate immunity. However, the molecular and antiviral activity of TRIM59 in mandrain fish is not fully understood. In present study, we cloned and sequenced the TRIM59 core sequence and explored its characteristics in Mandarin fish. The Siniperca chuatsi TRIM59 (scTRIM59) showed relatively high expression in immune-related organs. scTRIM59 expression was significantly down-regulated post ISKNV infection in vivo and vitro, but up-regulated at the early stages of SCRV infection in CPB cells. The overexpression of scTRIM59 inhibited ISKNV and SCRV infection, but decreased the expression of IRF3/IRF7-mediated signal genes. However, knockdown of scTRIM59 promoted the ISKNV and SCRV infection, but increased the expression of IRF3/IRF7-mediated signal genes. Those results indicated that scTRIM59 negatively regulated ISKNV, SCRV infection and IRF3/IRF7-mediated signal genes. This study provided new ideas about the function of scTRIM59.

To identify activation pathways and effector mechanisms of innate immunity in fish has become relevant for the sanitary management of intensive fish farming. However, little is known about the blocking of cysteinyl leukotrienes receptors (CysLTRs) and their effects in teleost fish. Our study evaluated the anti-inflammatory effect of 250 and 500 μg zafirlukast (antagonist of CysLTRs)/kg b.w., administered orally in the diet, during acute inflammatory reaction induced by Aeromonas hydrophila bacterins in Oreochromis niloticus. 80 tilapia were distributed in 10 aquariums (100L of water each, n = 8) to constitute three treatments: Control (inoculated with A. hydrophila bacterin and untreated); Treated with 250 μg or 500 μg of zafirlukast/kg b.w. and inoculated. To be evaluated in three periods: 6, 24 and 48 h post-inoculation (HPI), totaling nine aquariums. A tenth group was sampled without any stimulus to constitute reference values (Physiological standards). Tilapia treated with zafirlukast demonstrated dose-response effect in the decrease of accumulated inflammatory cells, strongly influenced by granulocytes and macrophages. Zafirlukast treated-tilapia showed decrease in blood leukocyte counts (mainly neutrophils, and monocytes) and reactive oxygen species production. Treatment with zafirlukast resulted in down-regulation of ceruloplasmin, complement 3, alpha2-macroglobulin, transferrin and apolipoprotein A1, as well as up-regulation of haptoglobin. Our study provided convincing results in the pathophysiology of tilapia inflammatory reaction, considering that treatment with zafirlukast, antagonist of cysteinyl leukotriene receptors, resulted in a dose-response effect by suppressing the dynamics between leukocytes in the bloodstream and cell accumulation in the inflamed focus, as well as modulated the leukocyte oxidative burst and the acute phase protein response.

In this study, we conducted functional analyses for two ISG15 homologues of Larimichthys crocea (LcISG15-1 and LcISG15-2). Our results of qRT-PCR showed that both LcISG15-1 and LcISG15-2 were significantly changed in head kidney and peripheral blood, after poly (I:C) stimulation. Western blot analyses with prepared polyclonal antibodies suggested that LcISG15-1 and LcISG15-2 both could be secreted by primary head kidney lymphocytes into the extracellular milieu. The purified recombinant LcISG15-1 (rLcISG15-1) and LcISG15-2 (rLcISG15-2) could both activate primary macrophages as extracellular cytokines and significantly enhance macrophage respiratory burst, NO production and bactericidal activity and induce the expression of proinflammatory cytokine genes of the cells. Moreover, rLcISG15-2 exhibited much stronger cytokine-like activities than those of rLcISG15-1, indicating the ISG15-2 gene copy evolved enhanced activity after gene duplication of ISG15 in sciaenid fishes. These results indicated important roles of LcISG15-1 and especially LcISG15-2 in immune regulation and host immune defense of large yellow croaker against viral and bacterial infection.

In the present study, the zinc finger aspartate-histidine-histidine-cysteine (DHHC)-type containing 1 (ZDHHC1) gene was identified in a commercial fish, the Chinese perch Siniperca chuatsi. The ZDHHC1 has five putative transmembrane motifs and conserved DHHC domain, showing high amino-acid identity with other teleost fish, and vertebrate ZDHHC1 loci are conserved from fish to human. In vivo expression analysis indicated that ZDHHC1 gene was constitutively transcribed in all the examined organs/tissues, and was induced following infectious spleen and kidney necrosis virus (ISKNV) infection. It is further observed that ZDHHC1 interacts with MITA and the overexpression of ZDHHC1 in cells resulted in the upregulated expression of ISGs, such as Mx, RSAD2, IRF3 and type I IFNs such as IFNh and IFNc, exhibiting its antiviral function in fish as reported in mammals.

The weissellosis agent bacterium (WS08

Miamiensis avidus is a parasitic pathogen that causes the disease scuticociliatosis in teleost fish species. It is a ciliate and a free-living marine protozoan belonging to the order Philasterida, subclass Scuticociliatida, class Oligohymenophorea, and phylum Ciliophora. The complete mt-genome of M. avidus was linear and 38,695 bp in length with 47 genes, including 40 protein-coding genes, two ribosomal RNA (rRNA) genes, and five transfer RNA (tRNA) genes. Of these, 20 genes typically belong to the clusters of orthologous groups, playing roles in energy production and conversion, translation, ribosomal structure and biogenesis, and defense mechanisms. This is the first report of sequencing and characterization of the mt-genome of M. avidus, which was observed to be linear and possessing the typical ciliate mitochondrial genome organization and phylogenetic relationships. Remarkable differences were observed between M. avidus and other ciliates in the mitochondrially encoded rRNAs, extensive gene loss in ribosomal genes and tRNAs, terminal repeat sequences, and stop codon usage. A comparative and phylogenetic analysis of M. avidus and Uronema marinum of the order Hymenostomatida, which is most closely related to the order Philasterida, signified the promise of the mitogenome data of M. avidus as a valuable genetic marker in species detection and taxonomic research. The present study has potential applications in epidemiological studies and host-parasite interaction investigations facilitating disease control.

The nervous necrosis virus (NNV) causes the viral nervous necrosis (VNN) disease in aquatic animals and has been a major threat in aquaculture. Thus, it is essential for the development of a prevention method to minimize economic losses caused by NNV such as the identification of NNV resistance genes and application of these genes in molecular breeding to increase disease resistance. gab3 is an important NNV resistance gene in Asian seabass. However, the mechanism of gab3 in NNV resistance has not been elucidated. In this study, knockdown of gab3 in NNV-infected Asian seabass cells resulted in a significant decrease in viral RNA and virus titers. Knockout of gab3 in zebrafish led to an increased survival rate and resistant time after NNV infection. Cellular localization of the GAB3 and NNV by immunofluorescence staining showed that the GAB3 was translocated from the nucleus to the cytoplasm, and finally reached the cell membrane of SB cells after 48 h post NNV infection. Our study suggests that gab3 plays an important role in NNV replication and silencing gab3 can inhibit virus replication.

This study determines the occurrence and molecular characterisation of Monogenea from three commercially important Australian fish: Australian sardine Sardinops sagax (Jenyns), Australian anchovy Engraulis australis (White), and eastern school whiting Sillago flindersi McKay. Earlier studies have provided only morphological species identification, whereas this study combines both morphological and molecular methods. A total of 247 fish across 3 species, sourced from the New South Wales and Victorian coasts, were examined for Monogenea. A total of 187 monogenean parasites were recovered from the gills. The overall prevalence, mean intensity, and mean abundance were 34%, 2.23, and 0.78, respectively. The parasites were initially classified morphologically as three species across two families. Family Mazocraeidae was represented by Mazocraes australis Timi et al. J Parasitol 85:28–32, 1999, and family Microcotylidae by Polylabris sillaginae (Woolcock, Parasitology 28:79–91, 1936) Dillon, Hargis, and Harrises, 1983 and P. australiensis Hayward, 1996. Molecular identification of parasites was conducted through sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The fish hosts in the present study were also barcoded (mitochondrial cox1 gene) to confirm specific identities. There was no comparable cox1 sequence available in GenBank for the parasites found in the present study. However, the phylogenetic tree clustered the monogenean species identified in this study according to their familial groups of Mazocraeidae and Microcotylidae. The presence of M. australis on E. australis and S. sagax was confirmed in this study. Polylabris australiensis was only found on S. sagax but Si. flindersi was found to be a host for both Polylabris species. This study is the first to explore the mitochondrial cox1 genes of these three-monogenean species. These findings will serve as a foundation for future monogenean research in Australian waters and elsewhere.

Edwardsiella tarda represents one of the most important pathogens that infects a variety of hosts including aquatic animals and humans. The outbreak of E. tarda infection is frequently reported in aquaculture that causes huge economic loss. Due to the widespread of antibiotic resistance, available antibiotics to treat bacterial infection are limited. Therefore, enhancing aquatic animals to survive upon E. tarda infection become an urgent issue. In this study, we profiled the metabolomic change of tilapia in-between the dying and survival fish upon E. tarda infection. The dying and survival fish mounts differential metabolic response, from which we identify a key metabolite, taurine, whose abundance is increased in both the survival group and the dying group but is more significant in the survival group. Exogenous taurine increases tilapia survival rate by 37.5% upon E. tarda infection. Further quantitative PCR analysis demonstrate taurine increases the expression of immune genes in liver, spleen and head kidney. Therefore, our study shows a new strategy to enhance fish immune response against bacterial infection.