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Herpesviral hematopoietic necrosis caused by cyprinid herpesvirus-2 (CyHV-2) is highly prevalent in gibel carp (Carassius gibelio Bloch) and is associated with high mortality and great economic loss. Currently, no efficient methods of prevention and control of CyHV-2 infection are available. Increasing evidence suggests that antigen-specific egg yolk immunoglobulins (IgY) are highly effective against various pathogens in farmed fish. Therefore, the aim of this study was to develop a specific IgY against CyHV-2 infection in gibel carp. We first produced a recombinant D4ORFs protein that encodes four open reading frames of CyHV-2 and then generated specific IgY by immunizing laying hens with this protein. The eggs laid by immunized hens were processed into spray-dried egg powder containing D4ORFs IgY. The D4ORFs IgY showed high neutralizing activity in vitro against CyHV-2, as evidenced by a significant reduction in cytopathic effect in gibel carp brain cells cultured in the presence of the IgY antibodies. Whole-egg powder containing IgY specific to D4ORFs protein was incorporated into the basal diet at levels of 0, 0.1, 0.3, and 0.6% to prepare experimental diets for use in intervention studies in gibel carp. Another group received non-specific IgY. For preventive intervention, all fish were fed these experimental diets for 25 days before being immersion challenged with CyHV-2, after which the fish were switched to the basal diet for 21 days. For therapeutic intervention, fish were first challenged with CyHV-2 and then fed the diet with 0.6% D4ORFs IgY. Fish that received D4ORFs IgY prior to CyHV-2 challenge had significantly higher survival rates and significantly lower viral load in the trunk kidney compared to those that received non-specific IgY. Improved survival and reduced viral load were also observed in CyHV-2-challenged fish that received dietary supplementation of D4ORFs IgY. Hematological and serum biochemical analysis also showed improved health and enhanced antiviral defense in fish that received D4ORFs IgY. Overall, these findings demonstrated both preventive and therapeutic effects of anti-D4ORFs IgY against CyHV-2 infection. • D4ORFs encoded by four tandem ORFs of CyHV-2 showed high immunogenicity. • Anti-D4ORFs IgY can effectively neutralize CyHV-2 in vitro. • Dietary inclusion of anti-D4ORFs IgY improves survival in CyHV-2-infected gibel carp. • Anti-D4ORFs IgY is prophylactically and therapeutically effective against CyHV-2 infection. [ABSTRACT FROM AUTHOR]

The genomic and coding sequences of four members of grass carp interleukin (IL)-17 cytokine family, IL-17A/F2, IL-17A/F3, IL-17C and IL-17N, were isolated using rapid amplification of cDNA ends and conventional PCR techniques. A phylogenetic analysis showed that grass carp IL-17 family genes, including previously identified IL-17A/F1 and IL-17D, clustered with their counterparts from other fish species. IL-17D had two exons and one intron, while all the other members were composed of three exons and two introns. A multiple sequence alignment revealed that grass carp IL-17 family cytokines shared a cystine-knot motif that was highly conserved in mammals. A quantitative real-time PCR analysis revealed that these genes were expressed in the different examined tissues at varying levels. Moreover, they displayed different tissue expression patterns in fish at 24 h after intraperitoneal injection (IP) or anal intubation (AI) of Aeromonas hydrophila , implying roles in inflammatory responses. Next, we compared disease activity status and histopathological changes in intestinal tissues over a 7 day period after IP or AI, and then examine the potential differential effects of different infection routes on the expression profiles of IL-17 family genes in inflamed intestinal tissues. The intraperitoneal route resulted in more pronounced pathological symptoms than the anal route in terms of the disease activity index (DAI). Changes in the DAI highly coincided with the severity of intestinal inflammation. The two infection routes resulted in differential IL-17 family gene expression patterns over time in grass carp intestine, which suggests that IL-17 family members may play differential roles in mucosal and systemic immune responses and that IL-17N and IL-17A/F1 might possibly function as pro-inflammatory cytokines that provide defense against bacterial infection in grass carp intestine. • Grass carp IL-17A/F2, IL-17A/F3, IL-17C and IL-17 N sequences were isolated. • Most grass carp IL-17 family genes exhibited an identical exon-intron structure. • Grass carp IL-17 family cytokines contained a cystine-knot motif found in mammals. • Tissue expression patterns of IL-17 family genes differed after bacterial infection. • IL-17 family members had unique roles in mucosal and systemic immune responses. [ABSTRACT FROM AUTHOR]

Intestinal microbiota has increasingly been recognized as a major determinant of host health due to its critical role in the maintenance of immune homeostasis. An experimental fish model with disrupted intestinal microbiota allows us to identify potential strategies for restoring or modulating microbiota composition. The aim of this study was to develop a reliable grass carp model with disrupted intestinal microbiota by treatment with an antibiotic cocktail consisting of vancomycin, enrofloxacin, florfenicol, and metronidazole at different concentrations. To identify the appropriate concentration and exposure time of antibiotic treatment required to disrupt intestinal microbiota, five antibiotic regimens were compared in terms of their effects on intestinal microbiota composition, growth and health status, and histological changes in the intestine, hepatopancreas, and spleen. Antibiotic-induced changes in the expression of inflammation- and apoptosis-related genes in grass carp intestines were also examined. The results revealed that the highest antibiotic concentration over a 28 day period was required to disrupt both luminal and mucosal microbiota through significant expansion of Proteobacteria and suppression of Fusobacteria. Treatment with this regimen induced significant down-regulation of caspase-8, MLCK, ZO-1, IL-17N, and IL-23R, and up-regulation of IL-1β and IL-6 in the intestine, suggesting possible effects on intestinal epithelial tight junction and inflammatory conditions. This treatment did not produce substantial histopathological changes, and was not directly detrimental to growth and health. Therefore, this antibiotic regimen may provide a suitable option to disrupt intestinal microbiota in grass carp. In addition, our study also implies that fish may serve as a promising model for investigating growth-promoting effects of antibiotics, when treated with antibiotics in low doses. • Antibiotic treatment did not affect alpha diversity of the luminal microbiota. • Antibiotic exposure duration matters more than concentration in disrupting microbiota. • The microbiota composition differed substantially between intestinal content and mucosa. • The growth-promoting effect of low-dose antibiotics was confirmed in grass carp. [ABSTRACT FROM AUTHOR]