Effectiveness of dietary heat-killed Bacillus subtilis harboring plasmid containing 60 copies of CpG-ODN 1668 against Vibrio harveyi in Penaeus vannamei.

The cost of the purification process hinders the extensive use of cytosine phosphate guanosine-oligodeoxynucleotides (CpG-ODNs) for shrimp culture. Therefore, this study used a shuttle vector plasmid to carry 60 copies of CpG-ODN 1668 (pAD43-25_60CpG), which can replicate in Escherichia coli and Bacillus subtilis strain RIK1285. The first experiment used a reverse gavage procedure to deliver a substance (PBS [CK], pAD43-25 [P0], and pAD43-25_60CpG [P60], respectively) directly into the anterior midgut of Penaeus vannamei and transcriptome sequence analysis with a reference genome was performed to examine the expression of well-known immune-related genes. The results showed that the expression levels of immune-related genes in P60 group were significantly increased, particularly those associated with AMPs. In addition, using RT‒qPCR, the expression levels of AMP genes (LvALF, LvPEN-2, and LvPEN-3) in the P60 group may vary depending on the tissue and time point. The second experiment used dietary supplementation with three kinds of heat-killed B. subtilis (HKBS, HKBS-P0, and HKBS-P60) in 28 days of feeding experiments. The results showed that dietary supplementation with HKBS-P60 did not significantly improve shrimp growth performance and survival. However, on days 14 and 28 of the feeding regimens, alkaline phosphatase (AKP) and acid phosphatase (ACP) activity were considerably higher than in other treatments. In addition, following infection with Vibrio harveyi, AKP and ACP activity in the HKBS-P60 group was significantly higher than in other treatments, particularly at the early stage of bacterial infection. Moreover, HKBS-P60 was found to be better protected against V. harveyi infection with lower cumulative mortality (60%) compared to HKBS (90%) and HKBS-P0 (100%) at 7 days after infection. Overall, these findings confirmed that P60 could increase immunological responses in the shrimp midgut, and HKBS-P60 could be used as an effective tool to enhance the immune response and disease resistance in shrimp.

The moonlighting protein fructose 1,6-bisphosphate aldolase as a potential vaccine candidate against Photobacterium damselae subsp. piscicida in Asian sea bass (Lates calcarifer).

Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish.

Protective efficacy of four heat-shock proteins as recombinant vaccines against photobacteriosis in Asian seabass (Lates calcarifer).

Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of photobacteriosis in marine fish and is responsible for huge losses to marine aquaculture worldwide. Efforts have been made to develop a vaccine against this disease. Heat-shock proteins (HSPs) are a family of proteins that are ubiquitous in cellular life. Bacteria produce elevated levels of HSPs as a survival strategy when exposed to stressful environments in a host during infection. This group of proteins are also important antigens that can induce both humoral and cellular immune responses. In this study, four HSPs of Phdp, HSP90, HSP33, HSP70, and DnaJ, were selected for cloning and recombinant expression. Western blotting with rabbit anti-Phdp helped identify rHSP70 and rHSP33 as immunogenic proteins. Asian seabass (Lates calcarifer) immunised with rHSP90, rHSP33, rHSP70, and rDnaJ showed 48.28%, 62.07%, 51.72%, and 31.03% relative percent survival, respectively, after being challenged with Phdp strain AOD105021. High expression levels of immune-related genes and high antibody titres were observed in the rHSP33 group, and the sera of this group also exhibited a high level of bactericidal activity against Phdp. Collectively, our results suggest that HSP33 is a potential candidate for vaccine development against Phdp infection.

Genotypic diversity, and molecular and pathogenic characterization of Photobacterium damselae subsp. piscicida isolated from different fish species in Taiwan.

Photobacteriosis, caused by Photobacterium damselae subsp. piscicida (Phdp), is a serious disease in marine fish species worldwide. To date, the epidemiological characterization of this pathogen in Taiwan remains limited. In this study, we collected 39 Phdp isolates obtained from different farmed fish for phenotypic and genotypic analysis. Phenotype bioassays using API-20E and API-20NE systems showed that the Phdp is a homogeneous group. However, genotyping using the pulsed-field gel electrophoresis (PFGE) technique revealed genetic variability among Phdp isolates when 13 and 11 different PFGE band patterns were obtained with SmaI and NotI as restriction enzymes, respectively. Phylogenetic analysis using 16S rDNA and the Fur gene clustered Taiwanese isolates and other species of P. damselae in the same clade. In contrast, the ToxR phylogenetic tree, a powerful discriminatory marker, separated the two subspecies. Furthermore, the virulence-associated genes, AIP56, P55, PDP_0080, Sod and Irp1, were detected from all isolates. Virulence testing with nine representative isolates in cobia (Rachycentron canadum) and Asian sea bass (Lates calcarifer) showed that some were highly pathogenic with 80%-100% mortality rates. This study provides epidemiological data of Phdp infections in farmed fish in Taiwan, which is necessary to develop comprehensive prevention and control strategies for the disease.

Molecular characterization of cobia (Rachycentron canadum) CD4 homologues revealed the first evidence of soluble CD4 in fish.

The co-receptor CD4 plays an important role in distinguishing between helper T-cell (Th) and cytotoxic T lymphocyte (CTL). In the present study, we investigated the molecular features of CD4-2 cDNA to facilitate understanding of their roles in cobia (Rachycentron canadum). Two CD4-2 molecules have been identified and exhibited 16.10% amino acids identity with each other. The cDNA of CD4-2A consists of a 993 bp ORF encoding 330 aa with long intracytoplasmic tail containing conserved protein tyrosine kinase p56Lck binding (C-X-C) motif, a transmembrane region, and two extracellular Ig-like (Ig-like) domains are predicted. Comparatively, the cDNA of cobia CD4-2B consists of a 990 bp ORF encoding 329 aa without a transmembrane domain as well as C-X-C motif, and three Ig-like domains are present. Homology comparison showed that the CD4-2A aa sequence of cobia showed high similarity and similar structural features to CD4-2 from other species, while the deduced CD4-2B protein shares higher structural similarity to CD4-1 group. Phylogenetic analysis indicated that cobia CD4-2A was closer with CD4-2 molecules in other fish species, distant from the clade formed by fish CD4-1 and mammalian CD4 sequences. However, cobia CD4-2B grouped with other known teleost CD4-1 sequences. The expression pattern of CD4-2A and CD4-2B mRNA during the embryonic development followed the trend of an initial increase after fertilized, providing evidence of maternal transfer of CD4-2 homologues to the developing cobia embryos and larvae. All of these results are useful for better understanding of cell-mediated immunity of cobia.

Molecular cloning of IL-6, IL-10, IL-11, IFN-ɤ and modulation of pro- and anti-inflammatory cytokines in cobia (Rachycentron canadum) after Photobacterium damselae subsp. piscicida infection.

Photobacterium damselae subsp. piscicida (P. damselae subsp. piscicida) is the agent of Photobacteriosis, a serious fish disease that produces an acute infection and high mortality in farmed cobia. It has been proved that regulation of pro- and anti-inflammatory cytokines play a central role in initiation of proper inflammatory responses against bacterial infection. Here we have analyzed the expression of pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8 and IFN-ɤ) and anti-inflammatory cytokines (IL-10 and IL-11) in spleen and head kidney during acute P. damselae subsp. piscicida infection of cobia. Our data revealed that cytokines tested showed distinct patterns of expression. While TNF-α and IL-8 showed a decay pattern of expression, IL-1ß response was quite late. Moreover, P. damselae subsp. piscicida infection induced the simultaneous expressions of pro-inflammatory (IL-6, IFN-ɤ) and anti-inflammatory (IL-10, IL-11) cytokines. Together these results indicate the innate immunity of cobia is actively suppressed by P. damselae subsp. piscicida.

De novo transcriptome analysis of immune response on cobia (Rachycentron canadum) infected with Photobacterium damselae subsp. piscicida revealed inhibition of complement components and involvement of MyD88-independent pathway.

Cobia, Rachycentron canadum, one of the most important aquatic species in Taiwan, has suffered heavy losses from Photobacterium damselae subsp. piscicida, which is the causal agent of photobacteriosis. In this study, the transcriptomic profiles of livers and spleens from Pdp-infected and non-infected cobia were obtained for the first time by Illumina-based paired-end sequencing method with a focus on immune-related genes. In total, 164,882 high quality unigenes were obtained in four libraries. Following Pdp infection, 7302 differentially expressed unigenes from liver and 8600 differentially expressed unigenes from spleen were identified. Twenty-seven of the differently expressed genes were further validated by RT-qPCR (average correlation coefficient 0.839, p-value <0.01). Results indicated a negative regulation of complement components and increased expression of genes involved in MyD88-independent pathway. Moreover, a remarkable finding was the increased expression of IL-10, implying an inadequacy of immune responses. This study not only characterized several putative immune pathways, but also provided a better understanding of the molecular responses to photobacteriosis in cobia.

The effect of TLR9 agonist CpG oligodeoxynucleotides on the intestinal immune response of cobia (Rachycentron canadum).

Cytosine-guanine oligodeoxynucleotide (CpG ODN) motifs of bacterial DNA are recognized through toll-like receptor 9 (TLR9) and are potent activators of innate immunity. However, the interaction between TLR9 and CpG ODN in aquatic species has not been well characterized. Hence, cobia TLR9 isoform B (RCTLR9B) was cloned and its expression and induction in intestine were investigated. RCTLR9B cDNA consists of 3113bp encoding 1009 amino acids containing three regions, leucine rich repeats, transmembrane domain, and toll/interleukin-1 receptor (TIR) domain. Intraperitoneal injection of CpG ODN 2395 upregulated RCTLR9 A and B and MyD88 and also induced the expressions of Mx, chemokine CC, and interleukin IL-1 ß . Cobia intraperitoneally injected with CpG ODN 1668 and 2395 had increased survival rates after challenge with Photobacterium damselae subsp. piscicida. In addition, formulation of CpG ODN with formalin-killed bacteria (FKB) and aluminum hydroxide gel significantly increased expressions of RCTLR9 A (50 folds) and B (30 folds) isoforms at 10 dpi (CpG ODN 1668) and MyD88 (21 folds) at 6 dpv (CpG ODN 2395). Subsequently, IL-1 ß increased at 6 dpv in 1668 group. No histopathological damage and inflammatory responses were observed in the injected cobia. Altogether, these results facilitate CpG ODNs as an adjuvant to increase bacterial disease resistance and efficacy of vaccines in cobia.

Identification and expression analysis of cobia (Rachycentron canadum) Toll-like receptor 9 gene.

Cobia culture is hindered by bacterial infection (Photobacterium damselae subsp. piscicida) and in order to study the effect of P. damselae subsp. piscicida challenge and CpG ODN stimulation on cobia Toll like receptor 9 (RCTLR9), we used PCR to clone RCTLR9 gene and qRT-PCR to quantify gene expression. The results indicated that RCTLR9 cDNA contains 3141 bp. It encodes 1047 amino acids containing 16 typical structures of leucine-rich repeats (LRRs) including an LRRTYP, LRRCT and a motif involved in PAMP binding was identified at position 240-253 amino acid. Broad expression of RCTLR9 was found in larval, juvenile and adult stages irrespective of the tissues. In larval stage, RCTLR9 mRNA expression decreased at 5 d and then increased at 10 dph. At juvenile stage cobia, the expression was significantly high (p < 0.05) in spleen and intestine compared to gill, kidney, liver and skin. However, at adult stage, the significant high expression was found in gill and intestine. Cobia challenged with P. damselae subsp. piscicida showed significant increase in RCTLR9 expression at 24 h post challenge in intestine, spleen and liver, while in kidney the expression was peak at 12 h and later it decreased at 24 h. The highest expression was 40 fold increase in spleen and the lowest expression was ∼3.6 fold increase in liver. Cobia stimulated with CpG oligonucleotides showed that the induction of these genes was CpG ODN type and time dependent. In spleen and liver, CpG ODNs 1668 and 2006 injected group showed high expression of RCTLR9, IL-1ß, chemokine CC compared to other groups. Meanwhile, CpG ODN 2006 has induced high expression of IgM. The CpG ODNs 2395 have induced significant high expression of Mx in spleen and liver. These results demonstrates the potential of using CpG ODN to enhance cobia resistance to P. damselae subsp. piscicida infection and use as an adjuvant in vaccine development.

Simultaneous detection of multiple fish pathogens using a naked-eye readable DNA microarray.

We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 10(3) CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens.

Establishment of activity indicator of TiO2 photocatalytic reaction--hydroxyl radical trapping method.

In this study, a new, low cost and easy method, hydroxyl radical trapping method, was employed to investigate the photo-activity of UV/TiO2 photocatalytic reaction. The Taguchi method was utilized to optimize the preparation of titanium dioxide (TiO2) thin-film reactor through the modified chemical vapor deposition (CVD) method. The optimal yield of hydroxyl radicals was then evaluated by calculating the conversion ratio of salicylic acid under the optimal conditions. In the experiments, salicylic acid was used as the free-radical scavenger and the formation of three different intermediates were examined to shed light on the trend and kinetics of reaction of hydroxyl radical with organic substance under different operation conditions. The results indicated that the yield of hydroxyl radicals increased with increasing irradiation intensity and dissolved oxygen level. The optimal experimental conditions obtained in this study were irradiation with intensity of 2.9 mW cm(-2) on salicylic acid at concentration of 250 mg L(-1) by both agitation and aeration processes (dissolved oxygen level=8.2 mg O(2)L(-1)) at pH 5. Such conditions could achieve the optimal hydroxyl radical yield of 5.1 x 10(-17)M.

Effect of pH on Fenton process using estimation of hydroxyl radical with salicylic acid as trapping reagent.

This study estimates the yield of hydroxyl radical using salicylic acid as the trapping reagent and investigates the relationship between hydroxyl radical and pH value. The formation and variation of hydroxyl radical under different pH values were evaluated using reaction products, 2,3-DHBA, 2,5-DHBA, and catechol. The formation rate of hydroxyl radical was dependent on the ratio of ferrous ion to hydrogen peroxide and pH values. The difference between various pH values was explored. The kinetics and mechanisms of hydroxyl radical reactions were established in the Fenton process. Experimental results showed that the best reaction conditions were 8.5 mM H(2)O(2), 1.25 mM Fe(2 + ), Fe(2 + )/H(2)O(2) = 0.147 at pH 3 and the formation rate constant of hydroxyl radical was 1.12 x 10(11) M(-1) s(-1).