Fish and shellfish immunology reports最新文献

This study aims to immunize the koi fingerlings immersion using the formalin-killed and freeze-dried E. coli DH5α carrying plasmid for the KHV DNA vaccine. 200 fish on each tank in a total water volume of 20 L. Each tanks consists of different vaccination group: PBS as control (10 mL; C), empty E. coli DHα (10 mL at 108 CFU mL-1; E), formalinkilled E. coli DHα:ORF81 (10 mL at 108 CFU mL-1; KE), freezedried E.coli DHα:ORF81 without formalin inactivation (10 mL at 108 CFU mL-1; FE), and formalin-killed and then freeze-dried E. coli DHα:ORF81 (10 mL at 108 CFU mL-1; KFE). The bath vaccination was conducted for 1 × 30 min. For the challenged test, fish were immersed with the 100 mL of LD50 dose of KHV (10–2 dilution from the KHV stock) for 30 min. The vaccination using E. coli DH5α:ORF81 could significantly modulate the innate and adaptive immunity of the fish and result in higher fish survival after KHV infection. The vaccination using formalin-killed or formalin-killed and freezedried E. coli DH5α:ORF81 will be further developed as an alternative to the costefficient koi or carp fingerlings vaccination method.

Global temperature is increasing due to anthropogenic activities and the effects of elevated temperature on DNA lesions are not well documented in marine organisms. The American oyster (Crassostrea virginica, an edible and commercially important marine mollusk) is an ideal shellfish species to study oxidative DNA lesions during heat stress. In this study, we examined the effects of elevated temperatures (24, 28, and 32 °C for one-week exposure) on heat shock protein-70 (HSP70, a biomarker of heat stress), 8‑hydroxy-2′-deoxyguanosine (8-OHdG, a biomarker of pro-mutagenic DNA lesion), double-stranded DNA (dsDNA), γ-histone family member X (γH2AX, a molecular biomarker of DNA damage), caspase-3 (CAS-3, a key enzyme of apoptotic pathway) and Bcl-2-associated X (BAX, an apoptosis regulator) protein and/or mRNA expressions in the gills of American oysters. Immunohistochemical and qRT-PCR results showed that HSP70, 8-OHdG, dsDNA, and γH2AX expressions in gills were significantly increased at high temperatures (28 and 32 °C) compared with control (24°C). In situ TUNEL analysis showed that the apoptotic cells in gill tissues were increased in heat-exposed oysters. Interestingly, the enhanced apoptotic cells were associated with increased CAS-3 and BAX mRNA and/or protein expressions, along with 8-OHdG levels in gills after heat exposure. Moreover, the extrapallial (EP) fluid (i.e., extracellular body fluid) protein concentrations were lower; however, the EP glucose levels were higher in heat-exposed oysters. Taken together, these results suggest that heat shock-driven oxidative stress alters extracellular body fluid conditions and induces cellular apoptosis and DNA damage, which may lead to increased 8-OHdG levels in cells/tissues in oysters.

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 early immune-related events arising from the interaction of antigen and innate immune cells are central to modulating the acquired immune response. Ideally, the immunizing antigen should elicit immunological changes similar to that observed after infection with the wild type pathogen. Here, we evaluated early changes on the expression of selected proinflammatory genes (TNF-α, IL-1β, IRAK4 and myeloperoxidase) and innate immune parameters (serum myeloperoxidase, lysozyme and complement hemolytic activity) in silver catfish vaccinated or infected with Aeromonas hydrophila, the etiological agent of hemorrhagic septicemia. The humoral immune response and resistance to challenge were also evaluated in vaccinated and placebo inoculated fish. We found that the expression of TNF-α and IL-1β genes was higher (p<0.05) in vaccinated or infected fish at 24 h post inoculation (p.i) compared to the control group but returned to basal levels at 72 h p.i. The expression of IRAK4 gene, however, was not altered by vaccination or infection. In addition, the natural hemolytic activity of complement was higher (p<0.05) at 24 h and 72 h p.i. in the vaccinated and infected groups; serum myeloperoxidase was higher (p<0.05) in these groups but only at 24 h p.i. and lysozyme activity was higher (p<0.05) only in the infected group at 72 h p.i. Furthermore, vaccination induced the production of IgM-like antibodies and protection to challenge with the A. hydrophila. Our results indicate that the vaccine formulation induces an immune response similar to that induced by the infecting pathogen and might be a valuable tool in the prophylaxis of hemorrhagic septicemia in silver catfish.

The use of Poly (lactic acid) (PLA) as a slow-release vehicle for vaccines has attracted the attention of researchers, since its insertion improves the uptake of them, and reduces side effects or by stimulating recruited defense cells, assisting immunity without the need for booster vaccine doses. Seeking to develop new strategies for the administration of drugs and vaccines in aquaculture, we evaluated the biocompatibility and biodegradation of polymeric PLA devices and PLA plus vitamin E devices, implanted through subcutaneous (SC) and intraperitoneal (IP) routes in Nile tilapia. To carry out this study, 84 male tilapia (initial 243.82 ± 56.74 g; final 400.71 ± 100.54 g) were randomly distributed in 3 tanks (n = 28 fish per treatment/tank). The devices were prepared in two formulations: neat PLA (containing 100% PLA) and PLAVE (PLA plus vitamin E) implanted using a commercial AnimalTag® applicator, and non-implanted fish (control). Fish were sampled 15, 30, 60, and 120 days post-implantation (DPI). Blood analysis was used to access blood cells and blood smear for differential leucocytes count. Serum biochemistry to evaluated changes in serum proteins and glycemia. Histopathological investigation using hematoxylin-eosin (H&E) was used to assess polymer-tissue interaction. Histochemistry and immunohistochemistry was used to detection immune cells and phagocytes in capsule, and analyses of melanomacrophage centers (MMCs) to morphometric evaluation and percentage amount of melanin, hemosiderin and lipofucsin pigments. Histopathological study revealed an increase of capsular formation and inflammatory cell infiltration in PLAVE-implanted tilapia through SC route (15 DPI). Tilapia implanted with PLAVE and PLA (SC) presented mast cells and eosinophilic granular cells during 15, 30, and 60 DPI, with a decrease in these cells in the fibrous capsule around the polymer at 120 DPI. PLAVE implanted tilapia SC at 60 DPI showed significantly phagocytosis points than other groups. Phagocytic cells (F4/80+) were observed near to biopolymers in phagocytosis sites. Lipofuscin at 120 DPI in spleen melanomacrophage centers were significantly high in PLAVE implanted tilapias when compared to fish with PLA implants and control. The serum biochemical study of tilapia did not reveal changes in cytotoxicity and liver function in implanted fish. The absence of side effects in hematological and biochemical findings, including the absence of mortality after device implantation, proves its clinical safety. PLA implants in tilapia have demonstrated biocompatibility, biodegradation, clinical safety, and excellent evolution of foreign body inflammatory responses.

A feeding trial was conducted to investigate the effects of dietary yeast culture (YC) on health status in digestive tract of juvenile Pacific white shrimp Litopenaeus Vannamei. Shrimps (initial weight: 3.33 ± 0.06 g) were fed with graded levels of dietary YC (control, 0.3%, 0.5% and 1.0%). Results of the present study showed that villus height and the ratio between villus height and crypt depth in the digestive tract of juvenile shrimp was significantly increased by dietary 0.5% and 1.0%YC (P < 0.05). Besides, dietary 0.5% and 1.0%YC significantly activities of phenoloxidase (PO), lysozyme (LZ), acid phosphatase (ACP) and alkaline phosphatase (AKP) (P < 0.05), significantly up-regulated mRNA levels of prophenoloxidase (propo), lysozyme (lz), anti-lipopolysaccharide factor (alf), crustin and penaienadin (P < 0.05) and down-regulated mRNA levels of caspase-1, nuclear factor κB p65 (nf-κbp65) myeloid differentiation primary response protein (myd88) and toll like receptor (tlr) in the digestive tract of juvenile shrimp (P < 0.05). Compared with the control, dietary 0.5%YC increased Chao1 index in the digestive tract of juvenile shrimp. In addition, compared with the control, dietary 0.5% and 1.0%YC significantly increased relative abundance of Lactobacillus (P < 0.05). It can be concluded that dietary YC made positive contribution to health status in digestive tract of juvenile shrimp through improving morphology and microbiota, enhancing immune function, and inhibiting inflammation of digestive tract.

Here we show that selected nutritional feed additives reduce the adverse effects of transport stress on the immune system and hematology of tambaqui. We formulated a control diet to contain normal dietary levels of vitamin E (21.6 mg/ kg diet) and C (143 mg/kg), then we added supra levels of these vitamins (vit E – 264 mg/kg and vit C – 1000 mg/kg) to a second diet. Finally, a third diet was produced to contain similar levels of vitamins from diet 2 with 0.1% beta-glucan supplementation. Four hundred thirty-two tambaquis (20.91 g ± 0.27 g) were randomly assigned to 12 aquaria and fed the diets for 15 days; then, all fish were transported for five h and then returned to the aquaria. Blood samples were collected before and after the transport and at the end of the trial (60 days). Transportation significantly increased blood glucose that returned to baseline levels at the end of the trial. However, cortisol seemed to be unresponsive to the stress. Surprisingly, the stress significantly increased the immunoglobulin level after transport. Additionally, the transport markedly reduced the red blood cell count and leukocyte and lymphocytes counts while increasing the control group's neutrophil number. These effects lasted until the end of the trial in the control group. Supra levels of the vitamins and glucan supplementation prevented the decrease in red blood cell and leukocyte count after the stress. Additionally, beta-glucan supplementation induced lower cortisol levels in all the sampling points. However, the effect on the immune parameters was limited, increasing only the lysozyme activity and serum protein levels in the beta-glucan supplemented group and the group fed only the supra levels of vitamins, respectively. In sum, our results indicated that transport for five h induced a limited effect on stress biomarkers. The use of supra levels of antioxidant vitamins alone or in combination with beta-glucan could restore or prevent the adverse effects of stress on hematology and the immune system.

Although human gC1qR is a multi-ligand binding protein with diverse biological functions, the functions of invertebrate gC1qR homologues remain largely unknown. In the present study, we characterized a novel gC1qR homologue, namely SpgC1qR, from mud crab Scylla paramamosain. SpgC1qR shared high identity and similar three-dimensional structure with human gC1qR. After challenge with White spot syndrome virus (WSSV), the transcripts of SpgC1qR were significantly increased, suggesting that SpgC1qR may be involved in antiviral immune response. To reveal the likely antiviral activity of SpgC1qR, the proliferation profile of WSSV in SpgC1qR-silenced crabs was examined. The result showed that knockdown of SpgC1qR by RNAi facilitated viral proliferation in vivo. This result was further confirmed by a SpgC1qR pre-incubation assay, in which pre-incubating WSSV particles with rSpgC1qR dramatically suppressed viral replication. Moreover, a GST pull-down assay revealed that SpgC1qR specifically bound to the viral envelope protein VP28. These findings clearly demonstrated that SpgC1qR specifically interacted with viral envelope protein VP28 and restricted WSSV replication, suggesting that it played a crucial role in anti-WSSV immune response of mud crab. This study provided new insights into the antiviral mechanism mediated by SpgC1qR and the biological functions of invertebrate gC1qR homologues.

Nucleotide-binding oligomerization domain-like receptors (NOD-like receptors or NLRs) are key members of the immune system that act as intracellular sentinels. These pathogen recognition receptors are essentially characterized by a central nucleotide binding domain and a C-terminal leucine rich repeat domain responsible for recognition of pathogens. Over the past decade, our understanding of teleosts' NLRs has enhanced significantly although the signaling pathways remain to be elucidated. In this brief review, we have tried to decipher the structural and functional aspects of NLRs in teleost. The review also engages in illustrating the various downstream signaling pathways/molecules reported so far in fishes that enable the NLRs to act as important players in immune responses and defense mechanisms against pathogens. Importantly, we try to explore the lacunae in structural and mechanistic details of NLRs in the teleost that would help in identifying key areas in which research is needed to complete our understanding of NLRs and their structural and functional evolution.

Gills reportedly play a crucial role in induction of an antiviral immune response in fish. We investigated the expression of innate response genes in the rainbow trout gill epithelial cell line RTgill-W1 36 h after pretreatment with ultraviolet-inactivated viral hemorrhagic septicemia virus (UV-VHSV), flagellin C protein from Edwardsiella tarda (FliC), VHSV and SVCV using an Agilent 4 × 44k cGRASP salmonid microarray. RTgill-W1 cells pretreated with UV-VHSV, triggered an independent gene expression profile from those treated with a recombinant flagellin C protein from Edwardsiella tarda. In addition, exposure of RTgill-W1 cells to live viruses spring viremia of carp virus and viral hemorrhagic septicemia virus induced a less robust transcriptional change of 24 and 22 gene probes, respectively, when compared to 123 genes for UV-VHSV. Further the pretreatment of RTgill-W1 cells with (UV-VHSV) significantly reduced VHSV genome copy number at 6 d post infection (dpi) relative to the FliC-treated and untreated control. A quantitative PCR was used to study the transcriptional modulation of a set of 25 innate immune-related genes highlighted by the microarray data and a panel of 7 established antiviral genes in the protected cells. Notably, the expression of ifn1, ifn2, mx1 and mx3 were expressed more in untreated cells than in UV-VHSV-treated cells where virus replication was inhibited. The results from this study shed light on the mechanisms and pathways used by teleost gill epithelium innate immunity in combating viral and bacterial infection.