Salmonid novirhabdovirus (IHNV) causes infectious haematopoietic necrosis (IHN) in salmonid species. Despite an injectable plasmid-based DNA vaccine of the glycoprotein (G) gene is effective, there are no oral vaccines for mass vaccination of rainbow trout (Oncorhynchus mykiss) fry. Recombinant baculoviruses were generated, used in cabbage looper (Trichoplusia ni) insect larvae to produce IHNV G and IHNV G-C5a proteins. Western blotting and chemiluminescence assays confirmed the expression of recombinant proteins, which were added to the fish feeding and top-coated with unflavored gelatin binder. Commercial rainbow trout were fed with experimental diets containing either IHNV G or IHNV G-C5a proteins for 2 weeks, and boosted 4 weeks after. Four weeks post-booster, fish were challenged with IHNV by immersion. Survival upon the infection challenge was evaluated. Spleen were sampled at 7 and 14 days post infection (dpi). Non-vaccinated and IHNV G fed trout reached a mortality of 91.7 and 97.6%, and 70.9 and 88.4%, respectively at 8 and 15 dpi. The IHNV G-C5a fed group exhibited a reduced mortality of 51.2% at 8 dpi, reaching 81.7% at 15 dpi, suggesting some level of antiviral protection. The individual viral load was measured by RT-qPCR detection of IHNV N gene, showing no significant difference across experimental groups. The transcription modulation of selected immune response markers was evaluated across experimental groups, including Type I IFN-a, Mx-1, CD4, and IgM. Further study is needed to assess how new oral vaccines may become effective to mitigate IHNV pathogenesis in juvenile trout by modulating the host immune response to protect towards IHNV exposure.
As one of the mucosal barriers, fish gills represent the first line of defense against pathogen infection. However, the exact mechanism of gill mucosal immune response to bacterial infection still needs further investigation in fish. Here, to investigate pathological changes and molecular mechanisms of the mucosal immune response in the gills of crucian carp (Carassius auratus) challenged by Aeromonas hydrophila, the transcriptomics and proteomics were performed by using multi-omics analyses of RNA-seq coupled with iTRAQ techniques. The results demonstrated gill immune response were mostly related to the activation of complement and coagulation cascades, antigen processing and presentation, phagosome, NOD-like receptor (NLR) and nuclear factor κB (NFκB) signaling pathway. Selected 21 immune-related DEGs (ie., Clam, nfyal, snrpf, acin1b, psme, sf3b5, rbm8a, rbm25, prpf18, g3bp2, snrpd3l, tecrem-2, cfl-A, C7, lysC, ddx5, hsp90, α-2M, C9, C3 and slc4a1a) were verified for their immune roles in the A. hydrophila infection via using qRT-PCR assay. Meanwhile, some complement (C3, C7, C9, CFD, DF and FH) and antigen presenting (HSP90, MHC Ⅱ, CALR, CANX and PSME) proteins were significantly participated in the process of defense against infections in gill tissues, and protein-protein interaction (PPI) network displayed the immune signaling pathways and interactions among these DEPs. The correlation analysis indicated that the iTRAQ and qRT-PCR results was significantly correlated (Pearson's correlation coefficient = 0.70, p < 0.01). To our knowledge, the transcriptomics and proteomics of gills firstly identified by multi-omics analyses contribute to understanding on the molecular mechanisms of local mucosal immunity in cyprinid species.
The effects of Photobacterium damselae ssp. piscicida (Phdp) on immune responses and intestinal ultrastructure of Artemia franciscana following infection and their amelioration by the probiotic bacteria Bacillus subtilis, Lactobacillus plantarum and Lactococcus lactis were evaluated. Pathogen growth inhibition in coculture with each probiotic and its virulence against Artemia were confirmed with an LC50 of 105 CFU mL−1. Phdp administration to Artemia at sublethal levels resulted in depletion of superoxide dismutase, glutathione reductase, glutathione transferase and phenoloxidase activities, extensive lipid peroxidation and reduced survival. Following a combined administration of each probiotic and the pathogen, enzyme activities and survival were significantly higher, while lipid peroxidation was reduced, compared to the infected group with no probiotic treatment (P < 0.05). The transmission electron microscopy study revealed that pathogen infection resulted in disarranged and fragmented microvilli, formation of empty or pathogen containing cytoplasmic vacuoles and damaged mitochondria. In the probiotic-treated and Phdp-infected series, intestinal cells showed normal appearance, except for the presence of pathogen-containing vacuoles and highly ordered but laterally stacked microvilli. The results of the present study indicate that Phdp induces cell death through an oxidative stress response and probiotics enhance Artemia immune responses to protect it against the Phdp induced damage.
The skin mucus of fish is an important part of the innate immune system, which is poorly understood at the proteomic level. The study established a complete map of the proteins in the skin mucus of Ctenopharangdon idella (C. idella) and discussed the Differentially Expressed Proteins (DEPs) after Aeromonas hydrophila (A. hydrophila) infection. Using Label Free Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis, a total of 126 proteins were identified as differentially expressed, 89 proteins of which were upregulated, and 37 proteins were downregulated. Functional annotations of DEPs showed that the upregulated proteins in the skin mucus of the treated group were mostly associated with complement system and cytoskeleton proteins, whereas downregulated proteins were associated with metabolism. The key upregulated immune proteins were transferrin variant C, lysozyme g, annexin A11, 26S proteasome non-ATPase regulatory subunit 8, hypothetical protein ROHU_000884, 60S ribosomal L7a, calpain-2 catalytic subunit-like protein, calpain-9-like protein, complement component C9, complement C3, cathepsin S, cathepsin Z, 14 kDa apolipo, heat shock protein and intelectin, whereas, leukocyte elastase inhibitor, annexin A11, C-factor-like protein, biotinidase isoform X1 and epidermal growth factor receptor substrate 15-like were the downregulated proteins. Moreover, we for the first-time report proteins such as coactosin, lamin-B2 and kelch 12, which were never reported in fish. Our study directly pointing out the possible immunological biomarkers in the skin mucus of C. idella after A. hydrophila treatment. Each of the protein we report in this study could be used as base to establish their mechanism of action during bacterial infection that may contribute to the strategies against bacterial prevention and control in fishes.
Interleukins (ILs) are a subgroup of secreted cytokines, which are molecules involved in the intercellular regulation of the immune system. In this study, 12 IL homologs were cloned and functionally identified from obscure puffer Takifugu obscurus, and they were termed as ToIL-1β, ToIL-1, ToIL-6, ToIL-10, ToIL-11, ToIL-12, ToIL-17, ToIL-18, ToIL-20, ToIL-24, ToIL-27, and ToIL-34. Multiple alignment results showed that except for ToIL-24 and ToIL-27, other deduced ToIL proteins shared typical characteristics and structure with other known fish ILs. Phylogenetic analysis revealed that 12 ToILs were evolutionarily closely related to their counterparts in other selected vertebrates. Tissue distribution assay demonstrated that the mRNA transcripts of most ToIL genes were constitutively expressed in all tissues examined, with relatively high expression in immune tissues. Following Vibrio harveyi and Staphylococcus aureus infection, the expression levels of 12 ToILs in the spleen and liver were significantly upregulated, and their response over time varied. Taken together, these data were discussed accordingly with the ToIL expression and the immune response under the different situations tested. The results suggest that the 12 ToIL genes are involved in the antibacterial immune response in T. obscurus.
