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Normal intestinal microbiota play an essential role in host health, whereas intestinal dysbiosis is closely associated with a variety of inflammatory diseases. It has been confirmed that the use of antibiotics can induce intestinal dysbiosis in fish. However, it is not clear whether intestinal dysbiosis affects the susceptibility of farmed fish to bacterial infection. In this study, a grass carp model of antibiotic-induced intestinal dysbiosis (AID) was used in challenge experiments to determine the susceptibility to bacterial infection. AID fish were challenged by either immersion or anal intubation with a virulent strain of Aeromonas hydrophila. After immersion challenge, AID fish developed prominent inflammatory symptoms and showed high mortality compared with controls. In immersion-challenged AID fish, the bacterial load in blood and intestinal contents was significantly higher. The immersion challenge also resulted in a significant reduction in the spleen index of AID fish. Histological examination showed severe injury in the intestine, liver, and spleen occurred in immersion-challenged AID fish. Furthermore, AID fish had significantly higher mRNA levels of il1β , il6 and il17a/f1 , and lower levels of il17n , il23r and tnfα in intestinal tissue than control fish. Overall, our results indicate that the susceptibility of grass carp to A. hydrophila infection increased due to intestinal dysbiosis. Therefore, intestinal dysbiosis can be proposed as a risk factor for severe bacterial infection in cultured fish. • Susceptibility to A. hydrophila infection increased in grass carp with intestinal dysbiosis. • Immersion challenge caused high mortality in fish with intestinal dysbiosis. • Immersion and anal intubation evoked different inflammatory responses, regardless of microbiota status. [ABSTRACT FROM AUTHOR]

Normal intestinal microbiota play an essential role in host health, whereas intestinal dysbiosis is closely associated with a variety of inflammatory diseases. It has been confirmed that the use of antibiotics can induce intestinal dysbiosis in fish. However, it is not clear whether intestinal dysbiosis affects the susceptibility of farmed fish to bacterial infection. In this study, a grass carp model of antibiotic-induced intestinal dysbiosis (AID) was used in challenge experiments to determine the susceptibility to bacterial infection. AID fish were challenged by either immersion or anal intubation with a virulent strain of Aeromonas hydrophila. After immersion challenge, AID fish developed prominent inflammatory symptoms and showed high mortality compared with controls. In immersion-challenged AID fish, the bacterial load in blood and intestinal contents was significantly higher. The immersion challenge also resulted in a significant reduction in the spleen index of AID fish. Histological examination showed severe injury in the intestine, liver, and spleen occurred in immersion-challenged AID fish. Furthermore, AID fish had significantly higher mRNA levels of il1β , il6 and il17a/f1 , and lower levels of il17n , il23r and tnfα in intestinal tissue than control fish. Overall, our results indicate that the susceptibility of grass carp to A. hydrophila infection increased due to intestinal dysbiosis. Therefore, intestinal dysbiosis can be proposed as a risk factor for severe bacterial infection in cultured fish. • Susceptibility to A. hydrophila infection increased in grass carp with intestinal dysbiosis. • Immersion challenge caused high mortality in fish with intestinal dysbiosis. • Immersion and anal intubation evoked different inflammatory responses, regardless of microbiota status. [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]

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]

Abstract Interleukin-12 receptor β2 (IL-12Rβ2) is a signaling subunit of heterodimeric receptors for IL-12 and IL-35. It plays important regulatory functions in the development of Th1 cells and in the expression of inflammatory cytokines in mammals and other higher vertebrates. However, little is known about IL-12Rβ2 in teleost fish. In this work, we have cloned and characterized IL-12Rβ2 from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp IL-12Rβ2 is 2875 bp, which encodes a mature protein with 741 amino acids. This mature protein contains three fibronectin type III domains, a transmembrane helix, and CXW and WSXWS-like motifs that are characteristic of the type I cytokine receptor family. Phylogenetic analysis revealed that cyprinid fish IL-12Rβ2 formed a single branch, clearly separated from those of other vertebrates. We expressed and purified a recombinant grass carp IL-12Rβ2 protein containing major antigenic regions, which was used to raise a polyclonal antibody. The specificity of the antibody was assessed by Western blotting analysis of whole cell lysates from Escherichia coli cells expressing the recombinant IL-12Rβ2, grass carp intestinal intraepithelial lymphocytes, and cultured C. idella kidney cells. To explore the potential regulatory role of IL-12Rβ2 in inflammation, we generated an intestinal inflammation model by anal intubation of fish with Aeromonas hydrophila. Immunohistochemical staining of the inflamed intestines revealed that IL-12Rβ2 expression is consistent with inflammatory cell recruitment during intestinal inflammation. Real-time quantitative PCR revealed that IL-12Rβ2 is widely expressed in normal tissues and is up-regulated in most tissues after infecting with A. hydrophila. We found that IL-12Rβ2, IL-12p35, and interferon-γ were expressed in similar patterns in the intestines during inflammation. Taken together, our results suggest that IL-12Rβ2 is involved in the regulation of intestinal inflammation. Highlights • The full-length cDNA encoding fish IL-12Rβ2 was cloned by RACE techniques. • IL-12Rβ2 is involved in Aeromonas hydrophila -induced intestinal inflammation. • IL-12Rβ2 is up-regulated in most tissues one day after A. hydrophila infection. • IL-12Rβ2, IL-12p35, and IFN-γ are expressed in similar patterns during inflammation. [ABSTRACT FROM AUTHOR]