[Show abstract][Hide abstract] ABSTRACT: The protective efficacy of oral delivery and intramuscular (i.m.) injection of a recombinant protein, rVP28 derived from white spot syndrome virus (WSSV) and synthesized using wheat germ cell-free technology was investigated in kuruma shrimp (Marsupenaeus japonicus). Shrimps were administered with the unpurified rVP28 and challenged with WSSV. Expression of innate immune-related genes was examined in intestine, heart, and lymphoid organ at 1, 3 and 7 days after oral- administration. Shrimps received rVP28 protein through i.m. injection or oral route and then challenged with WSSV displayed higher survival (90 and 85.7%, respectively) compared to the respective control groups. A significant up-regulation (P<0.01) of innate immune-related genes, such as Rab7, penaeidin, lysozyme and crustin, was noticed in orally treated shrimps. Our results indicate that this recombinant protein could elevate immune responses and protection in kuruma shrimp and therefore would have potential utility against WSSV infection in shrimp aquaculture.
Full-text · Article · Jun 2014 · Turkish Journal of Fisheries and Aquatic Sciences
[Show abstract][Hide abstract] ABSTRACT: Fifty-one lactic acid bacteria (LAB) strains were isolated and identified based on 16S ribosomal DNA sequence from the intestinal tracts of 142 kuruma shrimps (Marsupenaeus japonicus) collected from Kanmon Strait, Fukuoka and Tachibana Bay, Nagasaki, Japan. Cellular immunomodulatory function of 51 isolated LAB strains was assessed by measuring the level of interferon (IFN)-γ induction in mouse spleen cell culture. The strain Lactococcus lactis D1813 exhibited the highest amount of IFN-γ production and also bactericidal activity and was selected for testing its immunomodulatory role as a probiotic in kuruma shrimp. We also assessed the effect of dietary incorporation of this probiotic on resistance to Vibrio penaeicida infection in the kuruma shrimp. Our results demonstrate that probiotic L. lactis D1813-containing diet-fed (10(5) cfu g(-1)) shrimps displayed a significant up-regulation of lysozyme gene expressions in the intestine and hepatopancreas. However, insignificantly higher expression of anti-lipopolysaccharide factor, super oxide dismutase, prophenoloxidase, and toll-like receptor 1 was recorded in the intestine of shrimps fed the probiotic diet. Moreover, significantly increased (P < 0.01) resistance to the bacterial pathogen in term of better post-infection survival (61.7 %) was observed in the shrimps fed with the probiotic-incorporated diet compared with the control diet-fed group (28.3 %). The present study indicates the immunomodulatory role of the LAB L. lactis D1813 on the kuruma shrimp immune system and supports its potential use as an effective probiotic in shrimp aquaculture.
[Show abstract][Hide abstract] ABSTRACT: Vaccines (subunit and DNA) targeting major envelope proteins VP19 and/or VP28 of white spot syndrome virus (WSSV) in penaeid shrimp were developed and elicited good protection against white spot disease (WSD). However, the immune responses in shrimp after administration of these vaccines are not well understood. In this study, we developed a DNA vaccine encoding the VP28 envelope protein in kuruma shrimp (Marsupenaeus japonicus) and confirmed the potentiality of protection against WSSV infection. The efficacy of the DNA vaccine against WSSV infection was confirmed by WSSV artificial challenge at 7 days post vaccination in kuruma shrimp. However, the efficacy of the vaccine did not last 30 days post vaccination. The transcript of VP28 gene derived from expression vector in tissues of vaccinated shrimp was analyzed by RT-PCR. The transcript of VP28 gene was detected in various tissues including muscle, hemolymph, gill, intestine, stomach, heart, hepatopancreas and lymphoid organ tested at 1, 3 and 7 days post vaccination. Subsequently, the expression of innate immune-related genes in intestine and lymphoid organ was analyzed at 1, 3 and 7 days post vaccination. The expression of innate immune-related genes such as Rab7, penaeidin, lysozyme, and crustin was up-regulated upon DNA vaccination. These results suggest that DNA vaccination induces significant protection against WSSV by stimulating innate immune responses in kuruma shrimp.
[Show abstract][Hide abstract] ABSTRACT: The important role played by cytokines in host innate immunity and the interaction of subsets of immune and inflammatory cells through cytokines offer avenues for immune interventions. We investigated 16 cytokine gene responses in the Japanese pufferfish, Takifugu rubripes orally treated with a heat-killed lactic acid bacterium (LAB), Lactobacillus paracasei spp. paracasei (strain 06TCa22) (Lpp) isolated from a Mongolian dairy product at 1mgg(-1)body weightd(-1) for 3days. Additionally, we assessed superoxide anion production (SAP) and phagocytic activity (PA) of head kidney cells and resistance to Vibrio harveyi infection in treated fish. Significant up-regulation of pro-inflammatory (IL-1β, IL-6, IL-17A/F-3, TNF-α and TNF-N), cell-mediated immunity inducing (IL-12p35, IL-12p40 and IL-18), antiviral/intra-cellular pathogen killing (I-IFN-1 and IFN-γ), anti-inflammatory (IL-10) and peripheral T cell expansion and survival controlling (IL-2, IL-7, IL-15, IL-21 and TGF-β1) cytokines was observed in the treated fish. Furthermore, significantly increased SAP, PA and pathogen resistance were observed in the treated fish compared to untreated fish. Our results indicate the enhancement of cytokine mediated immunity in T. rubripes by the use of the heat-killed Lpp as a potential immunostimulant and would be of great use in immunomodulation trials with the possibility to monitor positive immune response.
Full-text · Article · Oct 2013 · International immunopharmacology
[Show abstract][Hide abstract] ABSTRACT: With the aim of evaluating the effect of a Mongolian dairy product derived Lactobacillus paracasei spp. paracasei (strain 06TCa22) (Lpp) on the cytokine-mediated immune responses to Vibrio harveyi infection, we examined 16 cytokine expressions in the Japanese pufferfish, Takifugu rubripes. Fish were orally treated with the heat-killed Lpp at 1 mg g(-1) body weight d(-1) for 3 days. At 24 h post treatment, fish were infected by an intramuscular injection of 0.1 mL V. harveyi bacterial suspension (10(8) cfu mL(-1)). Additionally, superoxide anion production (SAP) and phagocytic activity (PA) of head kidney cells were assessed during 120 h post infection period. Significant up-regulation of pro-inflammatory (IL-1β, IL-6, IL-17A/F-3, TNF-α and TNF-N), cell-mediated immune inducing (IL-12p35, IL-12p40 and IL-18), antiviral/ intra-cellular pathogen killing (I-IFN-1 and IFN-γ), anti-inflammatory (IL-10) and lymphocyte agonistic (IL-2, IL-7, IL-15, IL-21 and TGF-β1) cytokines was observed in the treated fish compared to control ones during the pathogen infection. Furthermore, significantly increased SAP and PA (P<0.01; 0.05) were recorded in the treated fish compared to untreated fish. These results suggest the beneficial role of Lpp in enhancement of cytokine-mediated immunity in the Japanese pufferfish against V. harveyi infection and application of this product as a potential fish immunostimulant.
[Show abstract][Hide abstract] ABSTRACT: Monocytes are antigen-presenting cells (APCs) in mammals. Antigen presentation by monocytes via costimulatory molecules was recently confirmed in the Japanese pufferfish Fugu rubripes (also known as Takifugu rubripes) (Sugamata et al., 2009). However, no detailed investigations examining how cytokine gene expression regulates the activation/differentiation of these cells have been conducted to date. Therefore, in this study, the expression of cytokine genes in Fugu monocytes stimulated with TLR agonists (LPS, polyI:C, and IMQ) was profiled. First, the morphological changes and phagocytic activity of stimulated monocytes were examined. At 5days post-stimulation, the ratio of activated to inactivated monocytes increased (as determined by FCM analysis), and the phagocytic activity of the cells was higher (5-15%) than that of nonactivated cells. Analysis of cytokine gene expression in stimulated monocytes revealed that the genes encoding CSF-1b and IFNγrel are important cytokines in activation/differentiation of monocytes/macrophages, and that genes encoding inflammatory cytokines such as IL-1β, IL-6, IL-18, and TNF-α are upregulated by stimulation. The present study revealed the types of cytokines expressed in Fugu monocytes stimulated with TLR agonists.
Full-text · Article · Jul 2013 · International immunopharmacology
[Show abstract][Hide abstract] ABSTRACT: CD4(+) T (Th) cells are a central component of the adaptive immune response and are divided into distinct sets based on their specific cytokine production pattern. Several reports have suggested that fish possess Th subset activity similar to that of mammals. The aim of the present study was to isolate CD4(+) T cells from the blood of Japanese pufferfish, Fugu rubripes, and to characterize their cytokine expression profile. We produced a specific antibody against Fugu CD4 and performed cell sorting with the magnetic activated cell sorting system. Sorted Fugu CD4(+) cells were characterized by morphology and expression analysis of cell marker genes. Fugu CD4(+) cells expressed T-cell marker genes but not macrophage or B-cell marker genes. In addition, peripheral blood lymphocytes were stimulated with lipopolysaccharide (LPS), polycytidylic acid (polyI:C), concanavalin A (ConA) prior to sorting, and then Multiplex RT-PCR was used to examine the expression of Th cytokines by the stimulated Fugu CD4(+) cells. LPS and polyI:C stimulation upregulated the expression of Th1, Th17 and Treg cytokines and downregulated the expression of Th2 cytokines. ConA stimulation upregulated the expression of all Th cytokines. These results suggest that fish exhibit the same upregulation of Th-specific cytokine expression as in mammals.
[Show abstract][Hide abstract] ABSTRACT: Cytokine responses in the Japanese pufferfish (Takifugu rubripes) head kidney (HK) cells to heat-killed lactic acid bacteria probiotics isolated from the Mongolian dairy products were investigated by transcriptomic examination. The HK cells were incubated with two heat-killed bacteria, namely Lactobacillus paracasei spp. paracasei (strain 06TCa22) and L. plantarum (strain 06CC2) and the responses of 16 cytokine genes at 0 (control), 1, 4, 8, 12, 24 and 48h post-stimulation were assayed by multiplex RT-PCR analysis (GenomeLab Genetic Analysis System, GeXPS; Beckman Coulter, Inc.). The 16 genes included in the assay were pro-inflammatory cytokines (IL-1β, IL-6, IL-17A/F-3, TNF-α and TNF-N), cell-mediated immune regulators (IL-12p35, IL-12p40 and IL-18), antiviral (I-IFN-1 and IFN-γ) and other regulatory (IL-2, IL-7, IL-15, IL-21, IL-10 and TGF-β1) cytokines. Despite the differences in the transcriptional profiles, expression of all the cytokines tested here was significantly elevated by both the probiotic bacterial stimulants compared with the unstimulated control. Therefore, this in vitro study has demonstrated the modulation of cytokine defense mechanisms in the HK cells by the two heat-killed probiotics indicating their potentiality as novel immunostimulants to fish. However, strain-dependent varied expression of important cytokines (cell-mediated immune regulators, antiviral and anti-inflammatory cytokines) suggests better efficacy of L. paracasei spp. paracasei strain as fish immunostimulant. Further in vivo studies to elucidate the cytokine regulation networks will validate our present observations. A careful evaluation of ant-inflammatory properties may be undertaken using single strain to affirm the immunostimulatory capability. Moreover, application timings and frequency to assess the longevity of immunostimulant effects and to make the application cost-effective need to be evaluated before any practical use in aquaculture.
Full-text · Article · May 2013 · Fish & Shellfish Immunology
[Show abstract][Hide abstract] ABSTRACT: In fish, T cell lineage commitment has not been studied, although there are reports related to CD4 and CD8 positive cells. This study describes the cloning and analysis of a master regulator involved in this process, the Th-POK gene in Japanese pufferfish, Takifugu rubripes. The fugu Th-POK cDNA was composed of 1901bp, with a 75bp 5'-UTR, a 131bp 3'-UTR, and a 1692bp open reading frame which translates into a peptide of 564 amino acid residues. The deduced fugu Th-POK protein contained a BTB / POZ domain, krüppel motif (H/C linker) and krüppel-like zinc finger DNA binding domain with C2H2 structure. Homology analysis of fugu Th-POK (ZBTB7B) with other known ZBTB7 members (ZBTB7A, 7C) showed low identity, and the phylogenetic tree analysis showed the fugu Th-POK clustered with the mammalian Th-POK, away from other ZBTB7 members. The analysis of transcriptional control region of Th-POK gene suggested that the 5'-flanking region and intron 1 includes numerous canonical binding motifs for transcription factors regulating T cells development. The genomic organization of the fugu Th-POK gene was composed of three exons and two introns, and its structure was identical to that of its human counterpart. Comparison of the fugu and human genomes showed that high levels of conserved synteny existed around the Th-POK gene. The high expression of the fugu Th-POK gene in unstimulated tissues was seen in head kidney, muscle, skin and gills. Moreover, the expression of the fugu Th-POK gene in thymic cell s was increased by LPS, polyI:C and PHA stimulation.
No preview · Article · Nov 2012 · Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology
[Show abstract][Hide abstract] ABSTRACT: To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1β, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-β1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.
No preview · Article · Nov 2012 · Veterinary Immunology and Immunopathology
[Show abstract][Hide abstract] ABSTRACT: Immune stimulant effects of a nucleotide-rich baker's yeast extract (Vertex IG20) were investigated in the kuruma shrimp, Marsupenaeus japonicus by examining expression of anti-microbial peptides/proteins (AMPs) such as penaeidin (MjPen), crustin (MjCrus) and lysozyme (MjLyz) genes. Furthermore, to confirm that the baker's yeast extract-induced AMPs were functional, we also assessed the effect of its oral administration on resistance to Vibrio nigripulchritudo infection in the kuruma shrimp. Our results demonstrate that baker's yeast extract-injected and fed shrimps displayed a significant up-regulation of MjPen, MjCrus and MjLyz gene expression in the lymphoid organ. Moreover, significantly increased (P<0.01) resistance to the bacterial pathogen in term of better post infection survival (66.6%) was observed in the shrimp fed with the yeast extract-incorporated diet compared with the control diet fed group (8.3%). The present study indicates the immunostimulatory effects of the nucleotide-rich baker's yeast extract on the kuruma shrimp immune system and supports its potential use in aquaculture.
[Show abstract][Hide abstract] ABSTRACT: The mononuclear phagocyte system is composed of monocytes, macrophages and dendritic cells and has crucial roles in inflammation, autoimmunity, infection, cancer, organ transplantation and in maintaining organismal homeostasis. Interleukin-34 (IL-34) and macrophage colony stimulating factor (MCSF), both signalling through the MCSF receptor, regulate the mononuclear phagocyte system. A single IL-34 and MCSF gene are present in tetrapods. Two types of MCSF exist in teleost fish which is resulted from teleost-wide whole genome duplication. In this report, we first identified and sequence analysed six IL-34 genes in five teleost fish, rainbow trout, fugu, Atlantic salmon, catfish and zebrafish. The fish IL-34 molecules had a higher identity within fish group but low identities to IL-34s from birds (27.2-33.8%) and mammals (22.2-31.4%). However, they grouped with tetrapod IL-34 molecules in phylogenetic tree analysis, had a similar 7 exon/6 intron gene organisation, and genes in the IL-34 loci were syntenically conserved. In addition, the regions of the four main helices, along with a critical N-glycosylation site were well conserved. Taken together these data suggest that the teleost IL-34 genes described in this report are orthologues of tetrapod IL-34.
Full-text · Article · Oct 2012 · Molecular Immunology
[Show abstract][Hide abstract] ABSTRACT: Teleost Interleukin-17 (IL-17) was reported to consist of seven ligands includingchracterisitic member IL-17N in teleost fish. However, IL-17 receptors and their signaling molecules (or transcription factors related to IL-17 signaling pathway) are few reported in telsosts. In mammals, IL-17 receptors lead to the recruitment of Act1 through homotypic interactions between SEFIR domains. This is turn allows the incorporation of TRAF6 into the signaling complex and activates the NF-kB transcription factor pathway. In this report, we identified seven IL-17 receptors (RA, RC-1, -2, RD-1, -2, -3 and RE), Act1 and TRAF6 from Japanese pufferfish, Takifugu rubripes using comparative genomics based on chromosomal synteny and characterized their biological activities.
[Show abstract][Hide abstract] ABSTRACT: Cytokines are important regulators of the immune system and investigation of their functions may prove useful for the development of vaccines and immunostimulants for aquaculture. We therefore investigated the cytokine [interleukin (IL)-1β, IL-10, IL-12 p35 and p40, tumor necrosis factor (TNF)-α, CXC-chemokine and interferon (IFN)-α and γ] responses of the common carp, Cyprinus carpio L., upon treatment with a commercial
baker's yeast extract (CW-I) that contained nucleotides and β-glucan. Additionally, to confirm that the CW-I-induced cytokines were functional, we also assessed the effect of CW-I administration on superoxide anion production and phagocytic activities of head kidney leucocytes and resistance to Aeromonas hydrophila infection in the common carp. Our results demonstrate that baker's yeast extract-treated fish displayed a significant up-regulation of pro-inflammatory cytokine (IL-1β, IL-12 p35 and p40, TNF-α, CXC-chemokine, IFN-γ2) gene expression and a down-regulation of anti-inflammatory cytokine (IL-10) gene expression. Furthermore, significantly increased phagocytic activity and superoxide anion production in kidney cells, and resistance to a bacterial pathogen, were observed in the yeast extract-treated fish compared to nontreated fish. The current study indicates the immunostimulatory effects of a baker's yeast extract rich in nucleotides and β-glucan on the carp immune system and supports its potential use in aquaculture.
[Show abstract][Hide abstract] ABSTRACT: A multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay can detect expression of 30 genes simultaneously within a reaction tube, which is advantageous to the conventional analytical methods where only one gene per single reaction is detected. Therefore, to economize the cost, labor, time, sample number and experiment frequency, a novel platform for quantitative multiplexed analysis was developed for the expression study of cytokine genes in Japanese pufferfish, Takifugu rubripes. Using a custom designed multiplex RT-PCR assay and the GenomeLab Genetic Analysis System (GeXPS), expression profiles were analyzed for 19 cytokine genes including pro-inflammatory [Interleukin (IL)-1β, IL-6, IL-17A/F3, IL-18, Tumor Necrosis Factor (TNF)-α, and TNF-N], anti-inflammatory (IL-4/13A, IL-4/13B, and IL-10), T-cell proliferation/differentiation [IL-2, IL-12p35, IL-12p40, IL-15, IL-21, and Transforming Growth Factor (TGF)-β1], activation/differentiation of B-cells (IL-7, IL-6, IL-4/13A, and IL-4/13B), induction of anti-viral activity [type I-Interferon (IFN)-1 and IFN-γ], and proliferation of monocytes/macrophage progenitor cells [M-Colony Stimulating Factor (CSF)-1β] in tissues under immune stimulatory conditions. We observed that immune stimulants such as LPS and polyI:C altered cytokine gene expression. The expression profiles were different in the un-stimulated control and each immune-stimulated fish. The expression profile following the pathogenic bacterial injection was similar to that for LPS stimulation. Notably, the type I-IFN-1 and IFN-γ genes showed significant up-regulation by polyI:C stimulation but not by stimulation with LPS or bacteria. Based on the results, this technique allows rapid and sensitive analysis of cytokine genes transcribed at different conditions to evaluate the health status of fish. The constructed multiplex RT-PCR assay scopes further understanding of cytokine network complex in immune regulation in fish.