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In November 2017, a group of farmed rainbow trout (Oncorhynchus mykiss) died in Yunnan Province, China, likely due to infection. These rainbow trout exhibited slow movement, no feeding, dark body color, exophthalmia, and occasional ulcers on the body surface. Pathogens were isolated from diseased rainbow trout livers, head kidneys, spleens, and eyes, and the strain was preliminarily identified as Acinetobacter johnsonii based on morphology, biochemical tests, and 16S rRNA and rpoB sequences. The isolated strains were highly sensitive to florfenicol, ciprofloxacin, oxolinic acid, and norfloxacin but resistant to ampicillin, doxycycline, sulfadiazine, thiamphenicol, and sulfamethazine. The selected isolate was performed for the experimental infection of rainbow trout to confirm its pathogenicity. Experimentally infected fish showed disease symptoms similar to those observed in fish naturally infected with these bacteria. Vacuolar degeneration was prevalent in the liver and spleen of diseased fish. Cytoplasm volume was very high in the lymphocytes of the head kidney. The protein structure and topology of monomeric outer membrane protein A (OmpA), outer membrane protein 34 (Omp34), and a nucleoside-specific outer membrane transporter protein Tsx (OmpTsx) of A. johnsonii were predicted by AlphaFold 2. The predicted local distance difference test (pLDDT) score was used for the assessment of structure prediction. Comprehensive analysis of antigenic determinants of the OMP family member, OmpA, contains three antigenic determinants, which are 90% similar with epitopes of A. baumannii. OmpA is a recombinant protein with 35 kDa expressed in the Escherichia coli system. Based on these findings, A. johnsonii is regarded as an emerging opportunistic pathogen in farmed rainbow trout. OmpA protein can be used as a subunit vaccine candidate molecule of A. johnsonii. [ABSTRACT FROM AUTHOR]

TLR22 exists in nearly all the poikilothermic vertebrates and plays a central role in the initiation of innate immunity and activation of adaptive immunity. TLR22 signaling pathway has been characterized in detail in fugu (Takifugu rubripes). Here, we thoroughly remold the localization and signaling pathways of TLR22. We characterized TLR22a and TLR22b in grass carp (Ctenopharyngodon idella), designated as CiTLR22a and CiTLR22b, and explored the ligand(s), adaptor(s), and signaling pathway(s). Results show that both CiTLR22a and CiTLR22b localize to lysosome, acidic compartment. Correspondingly, CiTLR22a and CiTLR22b directly bind and respond to dsRNA analog poly(I:C) at pH 5, but not at pH 7.4, the physiological pH. Moreover, CiTLR22a and CiTLR22b exhibit antagonistic function in signal transmission, wherein CiTLR22a facilitates the protein and phosphorylation levels of IRF7 and enhances the promoter activities of major IFNs and NF-κBs, while CiTLR22b downregulates IRF7 phosphorylation and IRF3 protein level and suppresses the IFN and NF-κB pathways. Further investigations revealed that CiTLR22a restrains grass carp reovirus (GCRV) replication and protects cells from GCRV infection, whereas CiTLR22b plays a negative role in response to GCRV infection. This is the first time to systematically clarify the signaling pathways of two isotype TLR22s; especially, subcellular localization and adaptor are different from previous TLR22 report, which results from technical limitations. The results will serve the antiviral immune mechanisms in poikilothermic vertebrates and evolutionary immunology. [ABSTRACT FROM AUTHOR]

Prophylactic administration of immunopotentiators has been tested and practiced as one of the most promising disease prevention methods in aquaculture. Chitosan oligosaccharide (COS), as an ideal immunopotentiator, is mainly used as feed additives in aquaculture, and the antimicrobial and immune enhancement effects are highly correlated with molecular weight (MW), but little is known about the mechanisms in teleost. Here, we isolated and purified macrophages in head kidney from blunt snout bream (Megalobrama amblycephala), stimulated them with three different MW (~500 Da, ~1000 Da and 2000~3000 Da) COSs, performed RNA-sequencing, global transcriptional analyses, and verification by quantitative real-time PCR (qRT-PCR) and immunofluorescent staining methods. Differential expression gene (DEG) analysis indicated that gene expression patterns are different and the proportion of unique genes are relatively high in different treatment groups. Biological process and gene set enrichment analysis (GSEA) demonstrated that all three COSs activate resting macrophages, but the degrees are different. Weighted gene co-expression network analysis (WGCNA) reflected gene modules correlated to MW, the module hub genes and top GO terms showed the activation of macrophage was positively correlated with the MW, and larger MW COS activated cell death associated GO terms. Further use of the screening and enrichment functions of STRING and Pfam databases discovered that apoptosis-related pathways and protein families were activated, such as the P53 pathway and caspase protein family. qRT-PCR results showed that as the stimulation time extends, the innate immune-related and P53 pathways are gradually activated, and the degree of activation is positively correlated with the stimulation time. In addition, apoptosis was detected by immunofluorescent staining in three groups. Therefore, the use of COS has two sides—it can activate the immune system against pathogen invasion, but with the increase in stimulation time and MW, macrophage apoptosis is induced, which may be caused by abnormal replication of DNA and excessive inflammation. This study provides a theoretical basis for the rational use of COS as an immunopotentiator in aquaculture. [ABSTRACT FROM AUTHOR]

Mannose receptor (MR) plays a significant role in innate immune responses to pathogens in vertebrates. Here we characterized the first teleost MR from Megalobrama amblycephala, named maMR and its expression patterns were investigated. The full-length maMR consists of 5,295 bp encoding a putative protein of 1,433 amino acids. The predicted amino acid sequences showed that maMR contained a signal peptide, a cysteine-rich domain, a single fibronectin type II domain, eight tandemly arranged C-type lectin-like domains, a transmembrane domain and a C-terminal cytoplasmic domain. Phylogenetic analysis revealed the highest similarity of maMR with Danio rerio MR predicted by computational analysis. The maMR-mRNAs were ubiquitously transcribed in different tissues, However the highest transcripts were observed in head kidney. Transcripts of maMR significantly increased at the late stages of embryo and continued to be at the high levels after hatching. The maMR transcripts were significantly increased in M. amblycephala after stimulation with killed Aeromonas hydrophila. [ABSTRACT FROM AUTHOR]

Microcystins (MCs) are the toxic molecules produced by common cyanobacterium in freshwater blooms. Their toxicities raise severe health issues in livestock and human beings. In current study, the immunotoxic effects of MC-LR were investigated in rabbit through evaluating the dynamics of white blood cell (WBC) numbers and cytokine production such as interleukin-3 (IL-3), IL-4, IL-6, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α). MCs at the high dose (50 μg MC-LReq kg−1) significantly induced increase in the WBC number but decrease in the Th1 (IFN-γ, TNF-α) and Th2 (IL-3, IL-4, IL-6) production. In the low dose group(12.5 μg MC-LReq kg−1), the number of WBC and the production of IFN-γ, IFN-α, IL-4, IL-3, and IL-6 increased gradually in first 12 h, reach the peaks at 12 h, and dropped after 24 h. Significantly positive correlations were found between the cytokines production of IL-4 and IL-6, IFN-γ and IFN-α, or IL-4 and IFN-γ. In conclusion, MC-LR is able to disturb the rabbit immune system and there exists time-dose response relationship in the MC-LR-eliciting perturbation, which probably give a better insight into investigating the immunotoxicity mechanisms of MCs in vivo. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012. [ABSTRACT FROM AUTHOR]

Hematological abnormalities or derangements have been demonstrated in patients suffering form microcystins (MCs) in hemodialysis unit in Caruaru, Brazil, 1996. While experimental study on hematological effect of microcystins has been rare and the underlying mechanisms are still puzzling. In the present study, microcystins were repeatedly intraperitoneally injected with a dose of 6 μg/kg/day in rabbits ( Oryctolagus cuniculus) for 14 days, and the prolonged effects of extracted microcystins on hematotoxicology were investigated. Significant decreases were observed in the hematological indices red blood cell counts, hematocrit, hemoglobin, and platelet count, while an obvious anemia occurred in rabbits after 14-day exposure. Moreover, red blood cell volume distribution width, mean corpuscular volume, and mean corpuscular hemoglobin did not vary significantly, indicating that rabbits suffered from normocytic anemia. In bone marrow, on the 14th day after toxin exposure, the frequency of micronucleus increased significantly, and the viability of bone marrow cells decreased markedly compared with the control. Serum erythropoietin levels declined on the 7th and 14th day, which suggested that the ability to regulate differentiation and maturation of erythrocytes was impaired. These results indicate that repeated exposure of microcystins can result in normocyte anemia, and the bone marrow injures and the sharp decreases of erythropoietin levels were responsible for the anemia. © 2011 Wiley Periodicals, Inc.Environ Toxicol, 2011. [ABSTRACT FROM AUTHOR]

Grass carp reovirus (GCRV) is a severe virus that causes great losses to grass carp culture every year, and GCRV-II is the current popular and fatal strain. VP56, fibrin on the outer surface of GCRV-II, mediates cell attachment. In this study, we firstly divided the VP56 gene into four fragments to screen the optimal antigen by enzyme-linked immunosorbent assay and neutralizing antibody methods. The second fragment VP56-2 demonstrates the optimal efficiency and was employed as an antigen in the following experiments. Bacillus subtilis were used as a carrier, and VP56-2 was expressed on the surface of the spores. Then, we performed the oral immunization for grass carp and the challenge with GCRV-II. The survival rate was remarkably raised, and mRNA expressions of IgM were significantly up-regulated in spleen and head kidney tissues in the B. s-CotC-VP56-2 group. Three crucial immune indexes (complement C3, lysozyme and total superoxide dismutase) in the sera were also significantly enhanced. mRNA expressions of four important genes (TNF-α, IL-1β, IFN1 and MHC-II) were significantly strengthened. Tissue lesions were obviously attenuated by histopathological slide examination in trunk kidney and spleen tissues. Tissue viral burdens were significantly reduced post-viral challenge. These results indicated that the oral recombinant B. subtilis VP56-2 subunit vaccine is effective for controlling GCRV infection and provides a feasible strategy for the control of fish virus diseases. [ABSTRACT FROM AUTHOR]

Aeromonas veronii (A. veronii) is one of the main pathogens causing bacterial diseases in aquaculture. Although previous studies have shown that hepcidin as an antimicrobial peptide can promote fish resistance to pathogenic bacterial infections, but the mechanisms remain unclear. Here, we expressed and purified recombinant yellow catfish (Pelteobagrus fulvidraco) hepcidin protein (rPfHep). rPfHep can up-regulate the expression of ferritin and enhance the antibacterial activity in primary hepatocytes of yellow catfish. We employed berberine hydrochloride (BBR) and Fursultiamine (FSL) as agonists and antagonists for hepcidin, respectively. The results indicated that agonist BBR can inhibit the proliferation of pathogenic bacteria, and the antagonist FSL shows the opposite effect. After gavage administration, rPfHep and the agonist BBR can enhance the accumulation of iron in liver, which may hinder the iron transport and limit the amount of iron available to pathogenic bacteria. Moreover, rPfHep and the agonist BBR can also reduce the mortality rate, bacterial load and histological lesions in yellow catfish infected with A. veronii. Therefore, hepcidin is an important mediator of iron metabolism, and it can be used as a candidate target for prevent bacterial infections in yellow catfish. Hepcidin and BBR have potential application value in preventing anti-bacterial infection. [ABSTRACT FROM AUTHOR]

Hepcidin is an antimicrobial peptide and regulator of iron homeostasis which has two isoforms in most fishes and some mammals. Previous studies have reported that the two hepcidin isoforms have different roles. Hamp type-1 plays a regulatory role in iron metabolism and hamp type-2 mostly performs an antimicrobial role. In this study, we found that Ctenopharyngodon idella (C. idella) have only one hepcidin isoform (hamp type-1), which showed both broad-spectrum antibacterial and iron regulatory functions. C. idella hepcidin mature peptide (hepcidin-25) and truncated peptide (hepcidin-20) exhibited bactericidal activities against both Gram-positive and Gram-negative bacteria in a dose-dependent manner in part through membrane rupture and binding to bacterial genomic DNA. The data from challenge tests demonstrated that the administration of hepcidin-25 significantly reduced mortality rates of C. idella by A. hydrophila infection, probably due to direct bactericidal activities of the peptide and a reduction of iron content in the fish serum. In addition, a comparison between hepcidin-20 and -25 suggests that the N terminal 5 amino acids play a critical role in reducing iron content in fish serum. Our findings revealed an important role of hamp type-1 in maintaining iron homeostasis and fighting against bacterial infections, suggesting the hepcidin has implications for the prevention and control of bacterial infection in aquaculture. [ABSTRACT FROM AUTHOR]

Macrophages are very versatile immune cells, with the characteristics of a proinflammatory phenotype in response to pathogen-associated molecular patterns. However, the specific activation marker genes of macrophages have not been systematically investigated in teleosts. In this work, leukocytes (WBC) were isolated using the Percoll gradient method. Macrophages were enriched by the adherent culture of WBC, then stimulated with lipopolysaccharide (LPS). Macrophages were identified by morphological features, functional activity and authorized cytokine expression. Subsequently, we collected samples, constructed and sequenced transcriptomic libraries including WBC, resting macrophage (Mø) and activated macrophage (M(LPS)) groups. We gained a total of 20.36 Gb of clean data including 149.24 million reads with an average length of 146 bp. Transcriptome analysis showed 708 differential genes between WBC and Mø, 83 differentially expressed genes between Mø and M(LPS). Combined with RT-qPCR, we proposed that four novel cell surface marker genes (CD22-like, CD63, CD48 and CD276) and two chemokines (CXCL-like and CCL39.3) would be emerging potential marker genes of macrophage in grass carp. Furthermore, CD69, CD180, CD27, XCL32a.2 and CXCL8a genes can be used as marker genes to confirm whether macrophages are activated. Transcriptome profiling reveals novel molecules associated with macrophages in C. Idella, which may represent a potential target for macrophages activation. [ABSTRACT FROM AUTHOR]