Preparation of Porous Composite Materials through Silicon Component Foaming and Special Heat Treatment.

This study proposes a foaming method along with calcination to produce silica-based porous materials. The high-silicon-content waste residue reacts with sodium hydroxide for hydrogen evolution foaming reactions. Then, the foaming green bodies are embedded and calcined in the calcination powder consisting of silicon, silicon carbide, graphite, and activated carbon at 1200 °C. The calcination powder creates a reducing atmosphere and prevents adhesion of the foaming green bodies to the crucible during calcination. Furthermore, the addition of activated carbon and calcination improve the macroporosity issue of the prepared sample through a gas-liquid-solid transformation. The BET surface area measurement is 64.197 m2/g, and the mercury intrusion porosimetry measurement indicates a porosity of 56.48%, with an average pore diameter of 396.48 nm. The porous composite materials exhibit the capability to adsorb methylene blue in solution. The porous materials possessing functional groups suggest promising potential for future development and application prospects.

IL-20 is produced by CD3γδ T cells and induced in the mucosal tissues of grass carp during infection with Aeromonas hydrophila.

Interleukin (IL) 20 is a multifunctional cytokine and plays a vital role in regulating autoimmune diseases, inflammation, and immune responses. IL-20 homologs have been described in fish. However, due to the lack of antibodies, cellular sources and immunological functions of fish IL-20 in response to infections have not been fully characterized. In this study, a monoclonal antibody (mAb) was generated against the recombinant grass carp (Ctenopharyngodon idella) IL-20 protein and characterized by immunoblotting, immunofluorescent microscopy and flow cytometry. It was shown that the IL-20 mAb specifically recognized recombinant IL-20 proteins expressed in the E. coli cells and HEK293 cells. Using confocal microscopy, the IL-20+ cells were identified in the head kidney, gills and intestine of grass carp, and induced after infection with Aeromonas hydrophila. Moreover, the IL-20 protein was found to be secreted mainly by CD3γδ T cells which were located predominantly in the gill filaments and intestinal mucosa. Taken together, our results suggest that IL-20 producing T cells are required for the mucosal immunity against bacterial infection in fish.

Distinct antiviral activities of IFNφ1 and IFNφ4 in zebrafish.

Interferons (IFNs) are a group of secreted cytokines that play a crucial role in antiviral immunity. Type I IFNs display functional disparities. In teleosts, type I IFNs are categorized into two subgroups containing one or two pairs of disulfide bonds. However, their functional differences have not been fully elucidated. In this study, we comparatively characterized the antiviral activities of zebrafish IFNφ1 and IFNφ4 belonging to the group I type I IFNs. It was found that ifnφ1 and ifnφ4 were differentially modulated during viral infection. Although both IFNφ1 and IFNφ4 activated JAK-STAT signaling pathway via CRFB1/CRFB5 receptor complex, IFNφ4 was less potent in inducing phosphorylation of STAT1a, STAT1b and STAT2 and the expression of antiviral genes than IFNφ1, thereby conferring weaker antiviral resistance of target cells. Taken together, our results provide insights into the functional divergence of type I IFNs in lower vertebrates.

CBFß is induced by spring viremia of carp virus and promotes virus replication in zebrafish.

The core binding factor subunit beta (CBFß) is a transcription factor that forms a complex with virial proteins to promote viral infection. In this study, we identified a CBFß homolog from zebrafish (zfCBFß) and characterized the biological activity. The deduced zfCBFß protein was highly similar to orthologs from other species. The zfcbfß gene was constitutively expressed in tissues and was induced in immune tissues after infection with spring viremia carp virus (SVCV) and stimulation with poly(I:C). Interestingly, zfcbfß is not induced by type I interferons. Overexpression of zfcbfß induced tnfα expression but inhibited isg15 expression. Also, overexpression of zfcbfß significantly increased SVCV titer in the EPC cells. Co-immunoprecipitation assay revealed that zfCBFß interacts with SVCV phosphoprotein (SVCVP) and host p53, resulting in the increased stability of zfCBFß. Our results provide evidence that CBFß is targeted by virus to suppress host antiviral response.

Zebrafish SETD3 mediated ubiquitination of phosphoprotein limits spring viremia of carp virus infection.

Lysine methylation is a post-translational modification of histone and non-histone proteins and affects numerous cellular processes. The actin histidine methyltransferase SET domain containing 3 (SETD3) is a member of the protein lysine methyltransferase (PKMT) family which catalyse the addition of methyl groups to lysine residues. However, the role of SETD3 in virus-mediated innate immune responses has rarely been investigated. In this study, zebrafish SETD3 was shown to be induced by poly(I:C) and spring viremia of carp virus (SVCV) and inhibited virus infection. Further, it was found that SETD3 directly interacted with SVCV phosphoprotein (SVCV P) in the cytoplasm of EPC cells, initiating ubiquitination to degrade the SVCV P protein via proteasomal pathway. Interestingly, mutants lacking the SET and RSB domains were able to promote degradation of SVCV P, indicating that they are not required for SETD3 mediated degradation of SVCV P. Taken together, our study demonstrates that SETD3 is an antiviral factor which limits virus replication by promoting ubiquitination of viral phosphoprotein and subsequent protein degradation.

IL-4/13 expressing CD3γ/δ+ T cells regulate mucosal immunity in response to Flavobacterium columnare infection in grass carp.

Interleukin (IL) 4 and 13 are signature cytokines orchestrating Th2 immune response. Teleost fish have two homologs, termed IL-4/13A and IL-4/13B, and have been functionally characterized. However, what cells express IL-4/13A and IL-4/13B has not been investigated in fish. In this work, the recombinant IL-4/13A and IL-4/13B proteins of grass carp (Ctenopharyngodon idella) were produced in the Escherichia coli (E. coli) cells and purified. Monoclonal antibodies (mAbs) against the recombinant CiIL-4/13A and CiIL-4/13B proteins were prepared and characterized. Western blotting analysis showed that the CiIL-4/13A and CiIL-4/13B mAbs could specifically recognize the recombinant proteins expressed in the E. coli cells and HEK293T cells and did not cross-react with each other. Confocal microscopy revealed that the CiIL-4/13A+ and CiIL-4/13B+ cells were present in the gills, intestine and spleen and could be upregulated in fish infected with Flavobacterium columnare (F. columnare). Interestingly, the cells expressing CiIL-4/13A and CiIL-4/13B were mostly CD3γ/δ+ cells. The CD3γ/δ+/IL-4/13A+ and CD3γ/δ+/IL-4/13B+ cells were significantly upregulated in the gill filaments and the intestinal mucosa after F. columnare infection. Our results imply that the CD3γ/δ+/IL-4/13A+ and CD3γ/δ+/IL-4/13B+ cells are important for homeostasis and the regulation of mucosal immunity.

The expanded ISG12 family in zebrafish: ISG12.1 suppresses virus replication via targeting viral phosphoprotein.

In mammals, interferon (IFN)-stimulated genes (ISGs) play important roles in restricting the replication of viruses. However, the functions of many ISGs have not been investigated in fish. In this study, eight isg12 homologs (termed isg12.1-8) were identified in zebrafish and all contain a typical ISG12 family domain rich of hydrophobic amino acid residues. Isg12.1-7 were significantly induced in the ZF4 cells by poly(I:C) and IFNφ1, and in the kidney and spleen after infection with spring viremia of carp virus (SVCV). In the EPC cells, overexpression of isg12.1 inhibited SVCV replication. Further, it was found that zebrafish ISG12.1 interacted with SVCV phosphoprotein (SVCV-P) and promoted SVCV-P degradation which could be attenuated by 3-MA and CQ (autophagy inhibitors). Our results indicate that zebrafish ISG12.1 restricts viral replication by targeting viral phosphoprotein for degradation.

IL-27 suppresses spring viremia of carp virus replication in zebrafish.

Interleukin (IL) 27 is a member of the IL-12 family and is a heterodimeric cytokine composed of IL-27A and Epstein-Barr virus-induced 3 (EBI3). It plays an important role in regulating inflammation and cancer progression. IL-27A not only functions by dimerizing with EBI3 but also acts alone. Here, we report that IL-27A and EBI3 suppress spring viremia of carp virus (SVCV) replication in zebrafish. Expression analysis reveals that il-27a and ebi3 were significantly upregulated in the ZF4 cells by SVCV and poly(I:C), and in the zebrafish caudal fin (ZFIN) cells overexpressed with SVCV genes. Interestingly, il-27a and ebi3 were not modulated by IFNφ1, indicating that they are not IFN stimulated genes (ISGs). Furthermore, overexpression of IL-27A and EBI3 alone inhibited SVCV replication in the EPC cells, but less potent than co-expression of IL-27A and EBI3. Intriguingly, IL-27A could not induce the expression of irf3, ifn, isg15 and mx1. Taken together, our results demonstrate that IL-27A and EBI3 activate innate antiviral response in an IFN independent manner in zebrafish.

Grass carp IL-20 binds to IL-20R2 but induces STAT3 phosphorylation via IL-20R1.

IL-20 is a pleiotropic cytokine that belongs to the IL-10 family and has a variety of biological functions in tissue homeostasis and regulation of host immune defenses. It signals through a heterodimeric receptor composed of a subunit with a long intracellular domain (R1 type receptor) and a subunit with a short intracellular domain (R2 type receptor). In this study, the R1 type receptor (CiIL-20R1/CRFB8) and the R2 type receptor (CiIL-20R2/CRFB16) were identified in grass carp Ctenopharyngodon idella. Expression analysis revealed that IL-20R2 was highly expressed in the gills and skin in healthy fish. Infection with Flavobacterium columnare resulted in the downregulation of both receptors in the gill at 48 and 72 h, whilst infection with grass carp reovirus induced their expression in the head kidney and spleen at 72 h. In the primary head kidney leucocytes, the expression levels of IL-20R1 and IL-20R2 were decreased after stimulation with 250 ng/mL IL-1ß but not affected by IFN-γ. Co-immunoprecipitation analysis showed that CiIL-20R2/CRFB16 but not CiIL-20R1/CRFB8 bound to CiIL-20L. Furthermore, it was shown that CiIL-20R1/CRFB8 was responsible for activating the phosphorylation of STAT3, whilst CiIL-20R2/CRFB16 was not involved. Structural modeling analysis showed that key residues involved in the interaction between IL-20 and receptors were highly conserved between grass carp and humans, suggesting that the signal transduction and functions of IL-20/IL-20R axis are evolutionarily conserved.

Novel Dimeric Architecture of an IFN-γ-Related Cytokine Provides Insights into Subfunctionalization of Type II IFNs in Teleost Fish.

Gene duplication leads to subfunctionalization of paralogs. In mammals, IFN-γ is the sole member of the type II IFN family and binds to a receptor complex consisting of IFN-γR1 and IFN-γR2. In teleost fish, IFN-γ and its receptors have been duplicated due to the teleost-specific whole-genome duplication event. In this study, the functions of an IFN-γ-related (IFN-γrel) cytokine were found to be partially retained relative to IFN-γ in grass carp (Ctenopharyngodon idella [CiIFN-γrel]). CiIFN-γrel upregulated the expression of proinflammatory genes but had lost the ability to activate genes involved in Th1 response. The results suggest that CiIFN-γrel could have been subfunctionalized from CiIFN-γ. Moreover, CiIFN-γrel induced STAT1 phosphorylation via interaction with duplicated homologs of IFN-γR1 (cytokine receptor family B [CRFB] 17 and CRFB13). Strikingly, CiIFN-γrel did not bind to the IFN-γR2 homolog (CRFB6). To gain insight into the subfunctionalization, the crystal structure of CiIFN-γrel was solved at 2.26 Å, revealing that it forms a homodimer that is connected by two pairs of disulfide bonds. Due to the spatial positions of helix A, loop AB, and helix B, CiIFN-γrel displays a unique topology that requires elements from two identical monomers to form a unit that is similar to IFN-γ. Further, mutagenesis analyses identified key residues interacting with CiIFN-γrel receptors and those required for the biological functions. Our study can help understand the subfunctionalization of duplicated IFN-γ paralogs in fish.

IRF2 Cooperates with Phosphoprotein of Spring Viremia of Carp Virus to Suppress Antiviral Response in Zebrafish.

IFN regulatory factor (IRF) 2 belongs to the IRF1 subfamily, and its functions are not yet fully understood. In this study, we showed that IRF2a was a negative regulator of the interferon (IFN) response induced by spring viremia of carp virus (SVCV). Irf2a-/- knockout zebrafish were less susceptible to SVCV than wild-type fish. Transcriptomic analysis reveals that differentially expressed genes (DEGs) in the irf2a-/- and irf2a+/+ cells derived caudal fins were mainly involved in cytokine-cytokine receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and transforming growth factor-beta (TGF-beta) signaling pathway. Interestingly, the basal expression levels of interferon stimulating genes (ISGs), including pkz, mx, apol, and stat1 were higher in the irf2a-/- cells than irf2a+/+ cells, suggesting that they may contribute to the increased viral resistance of the irf2a-/- cells. Overexpression of IRF2a inhibited the activation of ifnφ1 and ifnφ3 induced by SVCV and poly(I:C) in the epithelioma papulosum cyprini (EPC) cells. Further, it was found that SVCV phosphoprotein (SVCV-P) could interact with IRF2a to promote IRF2a nuclear translocation and protein stability via suppressing K48-linked ubiquitination of IRF2a. Both IRF2a and SVCV-P not only destabilized STAT1a but reduced its translocation into the nucleus. Our work demonstrates that IRF2a cooperates with SVCV-P to suppress host antiviral response against viral infection in zebrafish. IMPORTANCE Interferon regulatory factors (IRFs) are central in the regulation of interferon-mediated antiviral immunity. Here, we reported that IRF2a suppressed interferon response and promoted virus replication in zebrafish. The suppressive effects were enhanced by the phosphoprotein of the spring viremia of carp virus (SVCV) via inhibition of K48-linked ubiquitination of IRF2a. IRF2a and SVCV phosphoprotein cooperated to degrade STAT1 and block its nuclear translocation. Our work demonstrated that IRFs and STATs were targeted by the virus through posttranslational modifications to repress interferon-mediated antiviral response in lower vertebrates.

PLAAT1 inhibits type I interferon response via degradation of IRF3 and IRF7 in Zebrafish.

PLAAT1 is a member of the PLAAT protein family and plays important roles in tumor suppression, transglutaminase activation and peroxisomal biogenesis. Recently, PLAAT1 has been shown to promote degradation of p53 protein and cellular organelles such as mitochondria, endoplasmic reticulum and lysosome. In this study, we show that PLAAT1 inhibits the production of type I interferon and promotes virus replication in zebrafish. Overexpression of Plaat1 in zebrafish cells suppresses antiviral responses and promotes virus replication. Mechanistically, PLAAT1 interacts with IRF3 and IRF7 to initiate degradation of IRF3 and IRF7, which can be attenuated by 3-methyladenine, an inhibitor of autophagosome. Our study provides novel insights into the functions of PLAAT1 in host immune response to viral infection.

PLAAT1 promotes p53 degradation via autophagy-lysosome pathway in zebrafish.

PLAAT1 belongs to the PLAAT family and plays regulatory roles in cell growth, tumor suppression and phospholipid metabolism. However, whether PLAAT1 is involved in p53 mediated signaling has not been investigated. Here, we report that PLAAT1 promotes degradation of p53 in zebrafish. We found that the plaat1 gene was constitutively expressed in tissues including liver, kidney, spleen, intestine, eye and brain, with relative higher expression levels detected in the brain and eye. Overexpression of plaat1 led to inhibition of p53 and tnfα mRNA expression. Furthermore, it was shown that PLAAT1 interacted with p53 to facilitate p53 degradation via autophagy-lysosome dependent pathway. Our work indicates that PLAAT1 is involved in the interplay between p53 mediated cellular responses and autophagy.

Structural and Functional Analyses of Type I IFNa Shed Light Into Its Interaction With Multiple Receptors in Fish.

Teleost type I interferons (IFNs) are categorized into group I and II subgroups that bind to distinct receptors to activate antiviral responses. However, the interaction between ifn ligands and receptors has not fully been understood. In this study, the crystal structure of grass carp [Ctenopharyngodon idella (Ci)] IFNa has been solved at 1.58Å and consists of six helices. The CiIFNa displays a typical structure of type I IFNs with a straight helix F and lacks a helix element in the AB loop. Superposition modeling identified several key residues involved in the interaction with receptors. It was found that CiIFNa bound to cytokine receptor family B (CRFB) 1, CRFB2, and CRFB5, and the three receptors could form heterodimeric receptor complexes. Furthermore, mutation of Leu27, Glu103, Lys117, and His165 markedly decreased the phosphorylation of signal transducer and activator of transcription (STAT) 1a induced by CiIFNa in the Epithelioma papulosum cyprini (EPC) cells, and Glu103 was shown to be required for the CiIFNa-activated antiviral activity. Interestingly, wild-type and mutant CiIFNa proteins did not alter the phosphorylation levels of STAT1b. Our results demonstrate that fish type I IFNs, although structurally conserved, interact with the receptors in a manner that may differ from mammalian homologs.

Molecular identification of Klebsiella pneumoniae and expression of immune genes in infected spotted gar Lepisosteus oculatus.

Spotted gar (Lepisosteus oculatus) is a primitive ray-finned fish which has not undergone the third round whole genome duplication and commonly used as a model to study the evolution of immune genes. In this study, a pathogenic strain of Klebsiella pneumoniae (termed KPY01) was isolated from a diseased spotted gar, based on the Gram-stain and phylogenetic analysis of the 16S rDNA and khe genes. Further, the virulence genes and drug resistance genes were determined and drug sensitivity tests were performed to explore the virulence and drug resistance of the KPY01. Putative biosynthetic gene clusters (BGCs) for the biosynthesis of secondary metabolites were predicted using the anti-SMASH5.0 online genome mining platform. Histopathological analysis revealed that the immune cells were significantly decreased in the white pulp of spleen of fish infected with K. pneumonia and tissue inflammation became apparent. Besides, the expression of cytokines including interleukin (il) -8, il-10, il-12a, il-18 and interferon γ (ifn-γ) were shown to be modulated in the spleen, gills and kidney. Our work provides useful information for further investigation on the virulence of K. pneumoniae and host immune responses to K. pneumoniae infection in fish.

Lipopolysaccharide-induced TNFα factor (LITAF) promotes inflammatory responses and activates apoptosis in zebrafish Danio rerio.

Lipopolysaccharide-induced TNFα factor (LITAF) is an important transcription factor which activates the transcription of TNFα and regulates cell apoptosis and inflammatory response. In the present study, a LITAF gene homologue was identified in zebrafish (Danio rerio) and was shown to be well conserved in the protein sequence, genomic organization and synteny with human LITAF. DrLITAF was constitutively expressed in tissues, with the highest expression detected in the gills. Its expression could be modulated by LPS, poly(I:C), and infection with Edwardsiella tarda, Aeromonus hydrophila and septicemia viremia of carp virus (SVCV). DrLITAF, when overexpressed, was shown to be located on the cellular membrane and nuclear membrane of HEK293T and ZF4 cells and was associated with the endoplasmic reticulum. Stimulation with LPS resulted in rapid translocation of DrLITAF into the nucleus. In addition, DrLITAF was able to induce cell apoptosis and the expression of caspase 3. The results demonstrate that DrLITAF is involved in the immune defence against bacterial and viral infection and plays a role in regulating inflammation and apoptosis.

[Effects of bergapten on damages of osteocytes MLO-Y4 induced by TCP wear particles and its mechanism].

OBJECTIVE: To study the effects of bergapten (BP) on damages of osteocytes MLO-Y4 induced by tricalcium phosphate (TCP) wear particles and its mechanism. ;Methods: MLO-Y4 cells were treated with TCP wear particles for 48 h to establish the model of osteocytes injuries in vitro. The MLO-Y4 cells were divided into the following five groups: control group, TCP wear particles treated (0.1 mg/ml) group, bergapten (1, 5 and 20 µmol/L) treated groups. MTT assay and Calcein-AM staining were used to determine the viability of MLO-Y4 cells; Hoechst 33342 staining and the flow cytometry were applied to detect the apoptosis of MLO-Y4; real-time PCR was performed to examine the mRNA levels of dentin matrix protein1 (DMP-1), sclerostin (SOST) and fibroblast growth factor23 (FGF23); Western blot was performed to examine protein expressions of glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK) phospho-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α (p-eIF2α), activating transcription factor 4 (AFT4), C/EBP homologous protein (CHOP) and caspase-3 in MLO-Y4 cells. ;Results: Compared with control group, the MLO-Y4 viability and DMP-1 mRNA level in TCP group were decreased significantly (P＜0.05), while the percentage of apoptosis and mRNA levels of SOST and FGF23 were obviously increased (P＜0.05), and protein expressions of GRP78, AFT4, CHOP, p-PERK/PERK and p-eIF2α/eIF2α were up-regulated significantly in MLO-Y4 cells (P＜0.05). Compared with TCP group, the damages of MLO-Y4 and cell apoptosis in bergapten treated groups were decrease obviously (P＜0.05), the expressions of GRP78, AFT4, CHOP, p-PERK/PERK and p-eIF2α/eIF2α were down-regulated remarkably (P＜0.05). ;Conclusion: Bergapten can inhibit osteocytes damages induced by TCP wear particles, which may be related to reducing ER stress and PERK pathway activation.

Hematological and Immunological plasma assays for grass carp (Ctenopharyngodon idella) infected with Aeromonas hydrophila as an immune model in carp aquaculture.

Aeromonas hydrophila is the causative agent of bacterial septicemia, a common disease observed in grass carp, Ctenopharyngodon idella. In our study, C. idella specimens were infected with A. hydrophila, and parameters of Hematological and Immunological plasma parameters were monitored. At blood cell level, levels of red blood cells (RBCs), hematocrit (HCT), and mean corpuscular volume (MCV) showed no differences between the treatment and control groups, but levels of white blood cells (WBCs) increased. The monocyte and neutrophil varied significant according to stimulation by A. hydrophila at 1 DPI, the thrombocyte and lymphocyte at 14 and 21 DPI. At serum level, total protein, lysozyme, and IgM increased at the early infection phase and then decreased at other time points; however, peroxidase levels were significantly lower in the treatment group than that in the control group during the early infection phase. ACH50 was significantly higher in the treatment group than that in the control group during the late infection phase. On the basis of the results, we suggest that innate and adaptive immune mechanisms of C. idella are able to neutralize the virulence factors secreted by A. hydrophila. Our findings would help in understanding the mechanisms underlying resistance to infection by A. hydrophila.

Identification and functional analysis of the toll-like receptor 20.2 gene in grass carp, Ctenopharyngodon idella.

We characterized and identified the cDNA sequence of Toll-like receptor 20.2 in Ctenopharyngodon idella (gctlr20.2); it consisted of 3197 bp, with an open reading frame of 2835 bp that encoded a 944 amino acid polypeptide. Relatively, high expression levels of gctlr20.2 were observed in the spleen, head kidney, liver and brain tissues, with lower expression levels in the trunk kidney, intestine and heart tissues. In vivo and in vitro, after being challenged with Aeromonas hydrophila or grass carp reovirus (GCRV), gctlr20.2 expression was induced in C. idella kidney cells stimulated with lipopolysaccharide, flagellin or polyinosinic-polycytidylic acid. Overexpression of gctlr20.2 increased the expression of il1ß, il8 and tnf-α, but not ifn, and also increased the activity of the nf-κB signal pathway. Silencing, via siRNA-tlr20.2, inhibited gctlr20.2 transcription by 65.7% and down-regulated the expression of inflammatory cytokine genes, but not tnf-α. This study increases understanding of the immune system in C. idella.

MicroRNA-induced negative regulation of TLR-5 in grass carp, Ctenopharyngodon idella.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in antibacterial defence in fish has not been fully determined. Here, we identified that nine miRNAs are differentially expressed in kidney between susceptible and resistant grass carp strains. Analysis of spatial and temporal miRNA expression patterns suggests that cid-miRn-115 and miR-142a-3p are potential regulators of anti-bacterial activity. Overexpressing of cid-miRn-115 and miR-142a-3p results in a visible change in Ctenopharyngodon idella kidney (CIK) cells immune effector activity. Bioinformatics analysis and overexpressing assay shows that cid-miRn-115 and miR-142a-3p directly regulate tlr5 expression. cid-miRn-115 and miR-142a-3p overexpressing leads to a significant decrease in tlr5 expression in CIK, thereby repressing its downstream genes, such as il-1ß, il-8 and tnf-α. These findings provide a novel insight into the determination of anti-bacterial compounds in grass carp.