Gut mucosal immune responses and protective efficacy of oral yeast Cyprinid herpesvirus 2 (CyHV-2) vaccine in Carassius auratus gibelio.

Cyprinid herpesvirus 2 (CyHV-2) causes herpesviral hematopoietic necrosis (HVHN) disease outbreaks in farmed Cyprinid fish, which leads to serious economic losses worldwide. Although oral vaccination is considered the most suitable strategy for preventing infectious diseases in farmed fish, so far there is no commercial oral vaccine available for controlling HVNN in gibel carp (C. auratus gibelio). In the present study, we developed for the first time an oral vaccine against CyHV-2 by using yeast cell surface display technology and then investigated the effect of this vaccine in gibel carp. Furthermore, the protective efficacy was evaluated by comparing the immune response of a single vaccination with that of a booster vaccination (booster-vaccinated once 2 weeks after the initial vaccination). Critically, the activities of immune-related enzymes and genes expression in vaccine group, especially in the booster vaccine group, were higher than those in the control group. Moreover, strong innate and adaptive immune responses could be elicited in both mucosal and systemic tissues after receipt of the oral yeast vaccine. To further understand the protective efficacy of this vaccine in gibel carp, we successfully developed the challenge model with CyHV-2. Our results showed the relative percent survival was 66.7% in the booster vaccine group, indicating this oral yeast vaccine is a promising vaccine for controlling CyHV-2 disease in gibel carp aquaculture.

Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish.

Mammalian studies have demonstrated that B cell immune responses are regulated by mechanistic target of rapamycin complex 1 (mTORC1) signaling. Teleost fish represent the oldest living bony vertebrates that contain bona fide B cells. So far, whether the regulatory mechanism of mTORC1 signaling in B cells occurred in teleost fish is still unknown. In this study, we developed a fish model by using rapamycin (RAPA) treatment to inhibit mTORC1 signaling and demonstrated the role of mTORC1 signaling in teleost B cells. In support, we found inhibition of mTORC1 signaling by RAPA decreased the phagocytic capacity, proliferation, and Ig production of B cells. Critically, Flavobacterium columnare induced specific IgM binding in serum, and these titers were significantly inhibited by RAPA treatment, thus decreasing Ab-mediated agglutination of F. columnare and significantly increasing the susceptibility of fish upon F. columnare reinfection. Collectively, our findings elucidated that the mTORC1 pathway is evolutionarily conserved in regulating B cell responses, thus providing a new point for understanding the B cells functions in teleost fish.

Molecular cloning and expression analysis of CD79a and CD79b in rainbow trout (Oncorhynchus mykiss) after bacterial, parasitic, and viral infection.

CD79a and CD79b heterodimers are important components that consist of B cell receptor compound, which play a crucial role in transduction activation signal of the antigen binding BCR, and B cell development and antibody production. In order to investigate the characters and potential functions of CD79a and CD79b in rainbow trout (Oncorhynchus mykiss), we firstly cloned and analyzed the expression of CD79a and CD79b and found that the cDNA sequences of CD79a and CD79b both contained open reading frame of 711 and 645 bp in length for encoding the protein of 237 and 215 amino acid residues, respectively. The predicted amino acid sequences from trout were highly conserved with those of other teleost fishes in structure. Phylogenetic tree was constructed to analyze the evolutionary relationship between the trout and other known species, the result indicated that CD79a and CD79b of trout clustered at high bootstrap values with Salmo salar. Moreover, three trout infection models with F. columnare G4, I. multifiliis and infectious hematopoietic necrosis virus (IHNV) were constructed, which resulted in morphological changes and serious lesions in skin and gills. Importantly, the high expression of CD79a and CD79b occurred in skin, gills, and followed by head kidney in response to bacterial, parasitic, and viral infection, as its expression was closely related to that of Igs. Our findings indicated that CD79a and CD79b play vital roles in both systemic and mucosal immune responses of rainbow trout during bacterial, parasitic, and viral infection, which will contribute to explore the roles of CD79 subunits in B cell signaling during ontogeny and disease.

Molecular Characterization and Expression Analysis of Intercellular Adhesion Molecule-1 (ICAM-1) Genes in Rainbow Trout (Oncorhynchus mykiss) in Response to Viral, Bacterial and Parasitic Challenge.

The intercellular adhesion molecule-1 (ICAM-1), known as CD54, is a transmembrane cell surface glycoprotein that interacts with two integrins (i.e., LFA-1 and Mac-l) important for trans-endothelial migration of leukocytes. The level of ICAM-1 expression is upregulated in response to some inflammatory stimulations, including pathogen infection and proinflammatory cytokines. Yet, to date, our knowledge regarding the functional role of ICAM-1 in teleost fish remains largely unknown. In this study, we cloned and characterized the sequence of ICAM-1 in rainbow trout (Oncorhynchus mykiss) for the first time, which exhibited that the molecular features of ICAM-1 in fishes were relatively conserved compared with human ICAM-1. The transcriptional level of ICAM-1 was detected in 12 different tissues, and we found high expression of this gene in the head kidney, spleen, gills, skin, nose, and pharynx. Moreover, upon stimulation with infectious hematopoietic necrosis virus (IHNV), Flavobacterium columnare G4 (F. columnare), and Ichthyophthirius multifiliis (Ich) in rainbow trout, the morphological changes were observed in the skin and gills, and enhanced expression of ICAM-1 mRNA was detected both in the systemic and mucosal tissues. These results indicate that ICAM-1 may be implicated in the mucosal immune responses to viral, bacterial, and parasitic infections in teleost fish, meaning that ICAM-1 emerges as a master regulator of mucosal immune responses against pathogen infections in teleost fish.

Interactions Between Commensal Microbiota and Mucosal Immunity in Teleost Fish During Viral Infection With SVCV.

The mucosa of vertebrates is a particularly complex but dynamic environment in which the host constantly interacts with trillions of commensal microorganisms and pathogens. Although the internal and external mucosal microbiomes with immune defense of mammals have been well investigated, the relationship between mucosal microbes and their host's immune responses has not been systematically understood in the early vertebrates. In this study, we compared the composition and distribution of mucosal microbiota in common carp (Cyprinus carpio), and found that there were significant differences of microbiota between in the internal (gut) and external mucosal (buccal mucosa, gills and skin) tissues. Next, we successfully constructed an infection model with spring viremia of carp virus (SVCV). Specifically, following viral infection, the immune and antiviral related genes showed different up-regulation in all selected mucosal tissues while significant morphological changes were only found in external tissues including buccal mucosa, gills and skin. Using 16S rRNA gene sequence, we revealed that the abundance of Proteobacteria in mucosal tissues including buccal mucosa, gills and gut showed increased trend after viral infection, whereas the abundance of Fusobacteria significantly decreased in gut. In addition, the loss of dominant commensal microorganisms and increased colonization of opportunistic bacteria were discovered in the mucosal surfaces indicating that a secondary bacterial infection might occur in these mucosal tissues after viral infection. Overall, our results firstly point out the distribution of internal and external mucosal microbiota and analyze the changes of mucosal microbiota in common carp after SVCV infection, which may indicated that the potential role of mucosal microbiota in the antiviral process in early vertebrates.

Mediation of Mucosal Immunoglobulins in Buccal Cavity of Teleost in Antibacterial Immunity.

The buccal mucosa (BM) of vertebrates is a critical mucosal barrier constantly exposed to rich and diverse pathogens from air, water, and food. While mammals are known to contain a mucosal associated lymphoid tissue (MALT) in the buccal cavity which induces B-cells and immunoglobulins (Igs) responses against bacterial pathogens, however, very little is known about the evolutionary roles of buccal MALT in immune defense. Here we developed a bath infection model that rainbow trout experimentally exposed to Flavobacterium columnare (F. columnare), which is well known as a mucosal pathogen. Using this model, we provided the first evidence for the process of bacterial invasion in the fish BM. Moreover, strong pathogen-specific IgT responses and accumulation of IgT+ B-cells were induced in the buccal mucus and BM of infected trout with F. columnare. In contrast, specific IgM responses were for the most part detected in the fish serum. More specifically, we showed that the local proliferation of IgT+ B-cells and production of pathogen-specific IgT within the BM upon bacterial infection. Overall, our findings represent the first demonstration that IgT is the main Ig isotype specialized for buccal immune responses against bacterial infection in a non-tetrapod species.

Impact of poly dimethyldiallylammonium chloride on membrane fouling mitigation in a membrane bioreactor.

Poly dimethyldiallylammonium chloride (PDMDAAC) was applied in a membrane bioreactor (MBR) to study its effects on mitigation of MBR membrane fouling. Floc size, zeta potential, soluble microbial substances (SMP) and extracellular polymeric substances (EPS) secretion were studied with respect to PDMMAAC-dosing operations. Results demonstrated that a sustainable filtration cycle extended 3.3 times with the optimal PDMDAAC dosage of 90 mg L-1. The addition of PDMDAAC could increase zeta potential of sludge floc, which led to the decrease in repulsive electrostatic interactions between flocs, as well as the facilitation of flocs-to-flocs aggregation. With the optimal dosage of PDMDAAC, the mean size of sludge was 3.23 ± 0.55 times higher than the control group, resulting in higher impact resistance and better adaptive capacity to the changing environment, which led to less SMP secretion. Moreover, a high contaminants removal rate was achieved in the reactor that was dosed with PDMDAAC. The average effluent concentrations of chemical oxygen demand and total nitrogen were less than 45.6 ± 2.85 and 5.23 ± 0.61 mg L-1, respectively, and the corresponding removal rates were 93.1 ± 5.81% and 89.1 ± 9.61%.

Effects of Lactobacillus delbrueckii on immune response, disease resistance against Aeromonas hydrophila, antioxidant capability and growth performance of Cyprinus carpio Huanghe var.

The aim of the present study was to investigate effects of dietary Lactobacillus delbrueckii (L. delbrueckii) on immune response, disease resistance against Aeromonas hydrophila (A. hydrophila), antioxidant capability and growth performance of Cyprinus carpio Huanghe var. 450 fish (mean weight of 1.05 ± 0.03 g) were randomly distributed into five groups that fed diets containing different levels of L. delbrueckii (0, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 CFU g-1) for 8 weeks. The results showed that intestinal immune parameters such as lysozyme, acid phosphatase, and myeloperoxidase activities, immunoglobulin M content, and the survival rate were improved in fish fed with 1 × 106 and 1 × 107 CFU g-1L. delbrueckii. In addition, 1 × 107 CFU g-1L. delbrueckii supplementation down-regulated mRNA levels of TNF-α, IL-8, IL-1ß and NF-κBp65, and up-regulated IL-10 and TGF-ß mRNA levels in the intestine. The survival rate was significantly (P < 0.05) higher (68.33%) in fish fed 1 × 106 CFU g-1L. delbrueckii than the control diet-fed group (40%) after challenge by A. hydrophila. Fish fed with diet containing 1 × 106 CFU g-1L. delbrueckii showed higher antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and total antioxidant capacity (T-AOC) and lower MDA concentrations than those of the control group (P < 0.05). The relative gene expression (SOD, CAT, GPX) showed the same trend with their activities. In addition, the growth performance was significantly improved in fish fed with the diet containing 1 × 106 and 1 × 107 CFU g-1L. delbrueckii (P < 0.05). These results demonstrated that dietary optimal levels of L. delbrueckii enhanced immunity, disease resistance against A. hydrophila antioxidant capability and growth performance in Cyprinus carpio Huanghe var.