Identification of fish species through tRNA-based primer design.

BACKGROUND: The correct establishment of the barcode classification system for fish can facilitate biotaxonomists to distinguish fish species, and it can help the government to verify the authenticity of the ingredients of fish products or identify unknown fish related samples. The Cytochrome c oxidation I (COI) gene sequence in the mitochondria of each species possesses unique characteristics, which has been widely used as barcodes in identifying species in recent years. Instead of using COI gene sequences for primer design, flanking tRNA segments of COI genes from 2618 complete fish mitochondrial genomes were analyzed to discover suitable primers for fish classification at taxonomic family level. The minimal number of primer sets is designed to effectively distinguish various clustered groups of fish species for identification applications. Sequence alignment analysis and cross tRNA segment comparisons were applied to check and ensure the primers for each cluster group are exclusive. RESULTS: Two approaches were applied to improve primer design and re-cluster fish species. The results have shown that exclusive primers for 2618 fish species were successfully discovered through in silico analysis. In addition, we applied sequence alignment analysis to confirm that each pair of primers can successfully identify all collected fish species at the taxonomic family levels. CONCLUSIONS: This study provided a practical strategy to discover unique primers for each fishery species and a comprehensive list of exclusive primers for extracting COI barcode sequences of all known fishery species. Various applications of verification of fish products or identification of unknown fish species could be effectively achieved.

Applying Modified VP53A Recombinant Protein as an Anti-White Spot Syndrome Virus Biological Agent in Litopenaeus vannamei Farming.

Shrimp farming is an important economic activity. However, due to the spread of pathogens, shrimp aquaculture is becoming increasingly difficult. Many studies have confirmed that white spot syndrome virus (WSSV) recombinant proteins can inhibit viral infection. Among them, VP53 recombinant protein has been found to reduce mortality upon WSSV challenge. This study was conducted in Kaohsiung, Taiwan and reports the first field feeding trial to demonstrate that WSSV recombinant proteins can improve shrimp survival rates at a farming scale. Prior to the feeding trial, the shrimp were confirmed to be slightly infected with WSSV, Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND), non-AHPND V. parahaemolyticus strains, and Enterocytozoon hepatopenaei (EHP), which are common pathogens that shrimp farmers often face. The shrimp were then divided into two groups: a control group (C group) fed with a commercial diet and a protein group (P group) fed with the same commercial feed with VP53 recombinant protein. Our findings indicated that the survival rate and expression of immune genes of the P group were higher than those of the C group. The intestinal microbiota of the two groups were also analysed. Collectively, our results confirmed that the recombinant WSSV envelope protein derivative can be used as an effective anti-virus biological agent in shrimp farms.

Comprehensive Linear Epitope Prediction System for Host Specificity in Nodaviridae.

BACKGROUND: Nodaviridae infection is one of the leading causes of death in commercial fish. Although many vaccines against this virus family have been developed, their efficacies are relatively low. Nodaviridae are categorized into three subfamilies: alphanodavirus (infects insects), betanodavirus (infects fish), and gammanodavirus (infects prawns). These three subfamilies possess host-specific characteristics that could be used to identify effective linear epitopes (LEs). METHODOLOGY: A multi-expert system using five existing LE prediction servers was established to obtain initial LE candidates. Based on the different clustered pathogen groups, both conserved and exclusive LEs among the Nodaviridae family could be identified. The advantages of undocumented cross infection among the different host species for the Nodaviridae family were applied to re-evaluate the impact of LE prediction. The surface structural characteristics of the identified conserved and unique LEs were confirmed through 3D structural analysis, and concepts of surface patches to analyze the spatial characteristics and physicochemical propensities of the predicted segments were proposed. In addition, an intelligent classifier based on the Immune Epitope Database (IEDB) dataset was utilized to review the predicted segments, and enzyme-linked immunosorbent assays (ELISAs) were performed to identify host-specific LEs. PRINCIPAL FINDINGS: We predicted 29 LEs for Nodaviridae. The analysis of the surface patches showed common tendencies regarding shape, curvedness, and PH features for the predicted LEs. Among them, five predicted exclusive LEs for fish species were selected and synthesized, and the corresponding ELISAs for antigenic feature analysis were examined. CONCLUSION: Five identified LEs possessed antigenicity and host specificity for grouper fish. We demonstrate that the proposed method provides an effective approach for in silico LE prediction prior to vaccine development and is especially powerful for analyzing antigen sequences with exclusive features among clustered antigen groups.

Macrolide resistance genes and mobile genetic elements in waterways from pig farms to the sea in Taiwan.

OBJECTIVES: Macrolides have a long history of use in animals and humans. Dynamics of macrolide-antibiotic resistance genes (ARGs) in waterways from the origin to the sea has not been reported. METHODS: Resistant bacterial rate was measured by culture method, and copy numbers of macrolide-ARGs, mef(A), erm(B), mph(B), mef(C)-mph(G), and mobile genetic elements (MGEs) traI and IntI1 were quantitated in environmental DNA. Community composition in each site was investigated by 16S rRNA gene metagenomic sequencing. In Yilan area, antibiotics were quantitated. RESULTS: Surface water samples from pig farms to the sea in southern and northern areas in Taiwan were monitored. Macrolide-resistant bacteria accounted for 3%-28% of total colony-forming bacteria in aquaculture ponds and rivers, whereas in pig farm wastewater it was 26%-100%. Three common macrolide-ARGs mef(A), erm(B), and mph(B) and the relatively new mef(C)-mph(G) were frequently detected in pig farms, but not in aquaculture ponds and the sea. Rivers receiving pig wastewater showed ARG contamination similar to the pig farms. Among the MGEs, IntI1 was frequently distributed in all sites and was positively related to mef(A), erm(B), and mph(B) but not to mef(C)-mph(G). CONCLUSION: Pig farms are the origin of macrolide-ARGs, although macrolide contamination is low. Since lincomycin was detected in pig farms in the northern area, the increase of macrolide-ARGs is a future concern due to cross-resistance to lincomycin. ARGs abundance in aquaculture ponds was low, though MGEs were detected. Relation of IntI1 to ARG suggests convergence of ARGs to specific MGEs might be time/history dependent.

Conformational epitope matching and prediction based on protein surface spiral features.

BACKGROUND: A conformational epitope (CE) is composed of neighboring amino acid residues located on an antigenic protein surface structure. CEs bind their complementary paratopes in B-cell receptors and/or antibodies. An effective and efficient prediction tool for CE analysis is critical for the development of immunology-related applications, such as vaccine design and disease diagnosis. RESULTS: We propose a novel method consisting of two sequential modules: matching and prediction. The matching module includes two main approaches. The first approach is a complete sequence search (CSS) that applies BLAST to align the sequence with all known antigen sequences. Fragments with high epitope sequence identities are identified and the predicted residues are annotated on the query structure. The second approach is a spiral vector search (SVS) that adopts a novel surface spiral feature vector for large-scale surface patch detection when queried against a comprehensive epitope database. The prediction module also contains two proposed subsystems. The first system is based on knowledge-based energy and geometrical neighboring residue contents, and the second system adopts combinatorial features, including amino acid contents and physicochemical characteristics, to formulate corresponding geometric spiral vectors and compare them with all spiral vectors from known CEs. An integrated testing dataset was generated for method evaluation, and our two searching methods effectively identified all epitope regions. The prediction results show that our proposed method outperforms previously published systems in terms of sensitivity, specificity, positive predictive value, and accuracy. CONCLUSIONS: The proposed method significantly improves the performance of traditional epitope prediction. Matching followed by prediction is an efficient and effective approach compared to predicting directly on specific surfaces containing antigenic characteristics.

Identification and characterization of l-amino acid oxidase 2 gene in orange-spotted grouper (Epinephelus coioides).

Recently, l-amino acid oxidases (LAAOs) have been identified in several fish species as first-line defense molecules against bacterial infection. Here, we report the cloning and characterization of a fish LAAO gene, EcLAAO2, from orange-spotted grouper (Epinephelus coioides). The full-length cDNA is 3030 bp, with an ORF encoding a protein of 511 amino acids. EcLAAO2 is mainly expressed in the fin, gill, and intestine. Its expression is upregulated in several immune organs after challenge with lipopolysaccharide (LPS) and poly (I:C). The recombinant EcLAAO2 protein (rEcLAAO2), expressed and purified from a baculovirus expression system, was determined to be a glycosylated dimer. According to a hydrogen peroxide-production assay, the recombinant protein was identified as having LAAO enzyme activity with substrate preference for L-Phe and L-Trp, but not L-Lys as other known fish LAAOs. rEcLAAO2 could effectively inhibit the growth of Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis while exhibiting less effective inhibition of the growth of Escherichia coli. Finally, protein models based on sequence homology were constructed to predict the three-dimensional structure of EcLAAO2 as well as to explain the difference in substrate specificity between EcLAAO2 and other reported fish LAAOs. In conclusion, this study identifies EcLAAO2 as a novel fish LAAO with a substrate preference distinct from other known fish LAAOs and reveals that it may function against invading pathogens.

Omega-3 polyunsaturated fatty acids suppress metastatic features of human cholangiocarcinoma cells by suppressing twist.

Cholangiocarcinoma (CCA) is a highly malignant cancer of the bile duct, which has a five-year survival rate less than 5% due to a high metastasis rate and lack of therapeutic options. Although omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to inhibit the proliferation of CCA cells, the effects on CCA metastasis have not been previously reported. In this study, we first assessed the proliferation, migration and invasion effects of n-3 PUFA-based fish oil on human CCA cells. Then, we investigated PUFA effects on metastasis in vivo by xenografting CCA cells into zebrafish larvae that overexpress a critical n-3 PUFA synthesis gene, Δ6 fatty acid desaturase. The results indicated that n-3 PUFA-based fish oil suppresses CCA cell growth, potentially by blocking the cell cycle at G2/M phase, and it inhibits migration and invasion potential with coincident downregulation of migration-related genes. Furthermore, zebrafish endogenous n-3 PUFAs appear to suppress CCA metastasis by inhibiting the expression of twist, a key regulator of tumor metastasis. Interestingly, only long chain n-3 PUFAs could inhibit the expression of twist in CCA cells. Together, our results suggest that n-3 PUFAs, especially DHA, may inhibit proliferation and metastasis of CCA cells by inhibiting the expression of twist.

Granulin peptide GRN-41 of Mozambique tilapia is a novel antimicrobial peptide against Vibrio species.

In our previous study, the novel GRN-41 peptide generated from alternative splicing of the Mozambique tilapia PGRN1 gene was identified to be a potent peptide that protected against V. vulnificus in the transgenic zebrafish model by modulating innate immune-related genes. In this study, the anti-bacterial activities of synthetic Mozambique tilapia GRN-41 peptide (OmGRN-41) against various bacterial pathogens were investigated. The results showed that OmGRN-41 had bactericidal activity against Vibrio species, including V. vulnificus, V. alginolyticus, and V. harveyi, but exhibited bacteriostatic activity against V. parahaemolyticus. OmGRN-41 maintained bactericidal activity (64 µM) against V. vulnificus at pH 2 to pH 10 or after heat treatment for 1 h at high temperatures between 40 °C and 100 °C. TEM observations revealed that the outer membrane of V. vulnificus was disrupted by OmGRN-41, leading to morphological rupture and loss of cytoplasmic contents. Additionally, little hemolytic activity against tilapia and sheep erythrocytes was detected after treatment with 128 µM OmGRN-41. OmGRN-41 can effectively enhance the survival of Nile tilapia infected by V. vulnificus. Our results suggest that the OmGRN-41 is a novel antimicrobial peptide possessing bactericidal activity, especially against Vibrio species. These results indicate that OmGRN-41 can be applied in human Vibriosis treatment and has the potential to defend against Vibrio spp. infection in critical aquaculture organisms.

A voting mechanism-based linear epitope prediction system for the host-specific Iridoviridae family.

BACKGROUND: The Iridoviridae family is categorized into five genera and clustered into two subfamilies: Alphairidovirinae includes Lymphocystivirus, Ranavirus (GIV), and Megalocystivirus (TGIV), which infect vertebrate hosts and Betairidovirinae includes Iridovirus and Chloriridovirus, which infect invertebrate hosts. Clustered Iridoviridae subfamilies possess host-specific characteristics, which can be considered as exclusive features for in-silico prediction of effective epitopes for vaccine development. A voting mechanism-based linear epitope (LE) prediction system was applied to identify and endorse LE candidates with a minimum length requirement for each clustered subfamily RESULTS: The experimental results showed that four conserved epitopes among the Iridovirideae family, one exclusive epitope for invertebrate subfamily and two exclusive epitopes for vertebrate family were predicted. These predicted LE candidates were further validated by ELISA assays for evaluating the strength of antigenicity and cross antigenicity. The conserved LEs for Iridoviridae family reflected high antigenicity responses for the two subfamilies, while exclusive LEs reflected high antigenicity responses only for the host-specific subfamily CONCLUSIONS: Host-specific characteristics are important features and constraints for effective epitope prediction. Our proposed voting mechanism based system provides a novel approach for in silico LE prediction prior to vaccine development, and it is especially powerful for analyzing antigen sequences with exclusive features between two clustered groups.

Occurrence of sul and tet(M) genes in bacterial community in Japanese marine aquaculture environment throughout the year: Profile comparison with Taiwanese and Finnish aquaculture waters.

The use of antibiotics in aquaculture causes selection pressure for antibiotic-resistant bacteria (ARB). Antibiotic resistance genes (ARGs) may persist in ARB and the environment for long time even after stopping drug administration. Here we show monthly differences in the occurrences of genes conferring resistance to sulfonamides (i.e. sul1, sul2, sul3), and tetracyclines (tet(M)) in Japanese aquaculture seawater accompanied by records of drug administration. sul2 was found to persist throughout the year, whereas the occurrences of sul1, sul3, and tet(M) changed month-to-month. sul3 and tet(M) were detected in natural bacterial assemblages in May and July, but not in colony-forming bacteria, thus suggesting that the sul3 was harbored by the non-culturable fraction of the bacterial community. Comparison of results from Taiwanese, Japanese, and Finnish aquaculture waters reveals that the profile of sul genes and tet(M) in Taiwan resembles that in Japan, but is distinct from that in Finland. To our knowledge, this work represents the first report to use the same method to compare the dynamics of sul genes and tet(M) in aquaculture seawater in different countries.

Identification and expression analysis of 19 CC chemokine genes in orange-spotted grouper (Epinephelus coioides).

In this study, we describe 19 different CC chemokine genes from the orange-spotted grouper, Epinephelus coioides, identified by the analysis of the spleen transcriptome. Multiple sequence alignment of the 19 CC chemokines showed that although two genes, EcSCYA115 and EcSCYA117, shared 80% amino acid similarity (72% identity), the majority exhibited low similarity to each other. Phylogenetic analysis divided the 19 CC chemokines into six major groups. Tissue distribution analysis by RT-PCR showed that most of these chemokines were ubiquitously expressed in the 9 examined tissues, whereas some exhibited tissue-preferential expression patterns. For example, EcSCYA103 was preferentially expressed in fin and gill; EcSCYA109 in head kidney and spleen; EcSCYA114 in fin, gill, and liver; and EcSCYA119 in fin and stomach. Quantitative RT-PCR showed that after challenge with grouper iridovirus (GIV), four of the 19 CC chemokine genes, EcSYCA102, EcSYCA103, EcSYCA116, and EcSYCA118, were highly induced in the spleen. The expression of these four genes could also be upregulated by LPS and poly (I:C) challenges, suggesting that these four genes might be involved in immune response against invading pathogens.

Comparison of grouper infection with two different iridoviruses using transcriptome sequencing and multiple reference species selection.

Due to high-density aquafarming in Taiwan, groupers are commonly infected with two different iridoviruses: Megalocytivirus (grouper iridovirus of Taiwan, TGIV) and Ranavirus (grouper iridovirus, GIV). Iridoviral diseases cause mass mortality, and surviving fish retain these pathogens, which can then be horizontally transferred. These viruses have therefore become a major challenge for grouper aquaculture. In this study, comparisons of the biological responses of groupers to infection with these two different iridoviruses were performed. A novel approach for transcriptomic analysis was proposed to enhance the discovery of differentially expressed genes and associated biological pathways. In this method, suitable and available reference species are selected from the NCBI taxonomy tree and the Ensembl and KEGG databases instead of either choosing only one model species or adopting the NCBI non-redundant dataset as references. Our results show that selection of multiple appropriate model species as references increases the efficiency and performance of analyses compared to those of traditional approaches. Using this method, 17 shared pathways and 5 specific pathways were found to be significantly differentially expressed following infection with the two iridoviruses, among which 11 pathways were additionally identified based on the proposed method of multiple reference species selection. Among the pathways responsive to infection with a specific iridovirus, the spliceosomal pathway (ko03040; p-value = 0.0011) was exclusively associated with TGIV infection, while the glycolysis/gluconeogenesis pathway (ko00010; p-value = 0.0032) was associated with GIV infection. These findings and designed corresponding biological experiments may facilitate a deeper understanding of the mechanisms by which both TGIV and GIV cause fatal infections, as well as the ways in which they induce different pathologies and symptoms. We believe that the proposed novel mechanism for de novo transcriptomic analysis provides superior and comprehensive functional annotations and that the resulting shared and specific pathways identified may help immunologists develop specific vaccines against various types of iridovirus in the near future.

The novel mef(C)-mph(G) macrolide resistance genes are conveyed in the environment on various vectors.

BACKGROUND: The novel tandem genes mef(C) and mph(G) have been reported in marine bacteria in Japan. This paper aimed to characterise the extent of environmental distribution of mef(C) and mph(G) as well as their dissemination and persistence in aquatic bacterial communities. METHODS: Erythromycin-resistant bacteria were isolated from Japan, Taiwan and Thailand aquaculture sites. The mef(C)-mph(G) genes were detected by PCR. The size of mobile genetic elements conveying mef(C) and mph(G) was examined by Southern blotting. The conjugation rate was assessed by filter mating. RESULTS: The mef(C)-mph(G) tandem genes were distributed in erythromycin-resistant isolates from aquaculture seawater in Japan and northern Taiwan and in animal farm wastewater in Thailand. A total of 29 bacterial isolates were positive for mef(C)-mph(G). The genes were found on vectors of various sizes. Partial sequencing of the traI relaxase gene revealed homology with a pAQU1-like plasmid, an IncA/C-type plasmid and an SXT/R391 family integrative conjugative element (SRI) as vectors. Thirteen isolates (45%) were positive for traI(pAQU-IncA/C-SRI), whereas the others were negative. The traI(pAQU-IncA/C-SRI)-positive isolates exhibited a higher transfer frequency (10-4-10-5 transconjugants/donor) than traI(pAQU-IncA/C-SRI)-negative isolates (<10-9). CONCLUSIONS: These results suggest that mef(C)-mph(G) are coded on various vectors and are distributed among marine and wastewater bacteria in Asian countries. Vectors with traI(pAQU-IncA/C-SRI) play a role in the spread of mef(C)-mph(G).

Complete Genome Sequence of Streptococcus iniae 89353, a Virulent Strain Isolated from Diseased Tilapia in Taiwan.

Streptococcus iniae 89353 is a virulent strain isolated from diseased tilapia in Taiwan. The full-genome sequence of S. iniae 89353 is 2,098,647 bp. The revealed genome information will be beneficial for identification and understanding of potential virulence genes of Streptococcus iniae and possible immunogens for vaccine development against streptococcosis.

A member of the immunoglobulin superfamily, orange-spotted grouper novel immune gene EcVig, is induced by immune stimulants and type I interferon.

A novel grouper immune gene, EcVig was identified in orange-spotted grouper (Epinephelus coioides). We recently determined that EcVig expression can be induced by infection with nervous necrosis virus (NNV, an RNA virus), whereas NNV replication may be suppressed when EcVig was overexpressed. Although EcVig appeared to be involved in grouper antiviral activity, its immune effects have not been well characterized. In the present study, two PAMPs (pathogen-associated molecular patterns; lipopolysaccharides [LPS] and synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acid [poly(I:C)]), as well as fish DNA virus (red sea bream iridovirus, RSIV; grouper iridovirus, GIV), were used to study EcVig responses in orange-spotted grouper. In addition, groupers were given recombinant type I interferon to determine whether EcVig expression was induced. Poly(I:C) rapidly induced substantial expression of EcVig, whereas LPS stimulation did not appear to have any effect in grouper intestine. Expression levels of total EcVig and other IFN-stimulated genes (ISGs) were all significantly increased after RSIV and GIV infection. Furthermore, stimulation of recombinant type I IFN also increased EcVig expression. We conclude that EcVig may be a novel IFN-stimulated gene that demonstrates an antiviral immune response.

Cloning of the Major Capsid Protein (MCP) of Grouper Iridovirus of Taiwan (TGIV) and Preliminary Evaluation of a Recombinant MCP Vaccine against TGIV.

Fish iridoviruses cause systemic diseases with high mortality in various species of wild and farm-raised fish, resulting in severe economic losses. In 1998, we isolated a new epizootic iridovirus in cultured grouper (Epinephelus sp.) in Taiwan, thus named as grouper iridovirus of Taiwan (TGIV). We report here the cloning of TGIV major capsid protein (MCP). Phylogenetic analysis of the iridoviral MCPs confirmed the classification of TGIV into the Megalocytivirus genus. Recombinant TGIV MCP and GIV MCP were then generated to produce polyclonal antibodies. Western blot analysis revealed that the two antisera were species-specific, indicating no common epitope shared by the MCPs of the two viruses. We further assayed the potency of a subunit vaccine containing recombinant TGIV MCP. The vaccine effectively protected grouper from TGIV infection. The result demonstrated that MCP is a suitable antigen for anti-TGIV vaccines.

A novel quantitative immunomagnetic reduction assay for Nervous necrosis virus.

Rapid, sensitive, and automatic detection platforms are among the major approaches of controlling viral diseases in aquaculture. An efficient detection platform permits the monitoring of pathogen spread and helps to enhance the economic benefits of commercial aquaculture. Nervous necrosis virus (NNV), the cause of viral encephalopathy and retinopathy, is among the most devastating aquaculture viruses that infect marine fish species worldwide. In the present study, a highly sensitive magnetoreduction assay was developed for detecting target biomolecules with a primary focus on NNV antigens. A standard curve of the different NNV concentrations that were isolated from infected Malabar grouper (Epinephelus malabaricus) was established before experiments were conducted. The test solution was prepared by homogeneous dispersion of magnetic nanoparticles coated with rabbit anti-NNV antibody. The magnetic nanoparticles in the solution were oscillated by magnetic interaction with multiple externally applied, alternating current magnetic fields. The assay's limit of detection was approximately 2 × 10(1) TCID(50)/ml for NNV. Moreover, the immunomagnetic reduction readings for other aquatic viruses (i.e., 1 × 10(7) TCID(50)/ml for Infectious pancreatic necrosis virus and 1 × 10(6.5) TCID(50)/ml for grouper iridovirus) were below the background noise in the NNV solution, demonstrating the specificity of the new detection platform.