Publications by authors named "Shih-Chu Chen"

47 Publications

First report on genetic characterization, cell-surface properties and pathogenicity of Lactococcus garvieae, emerging pathogen isolated from cage-cultured cobia (Rachycentron canadum).

Transbound Emerg Dis 2021 Mar 23. Epub 2021 Mar 23.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.

The diseased cage-cultured cobia (Rachycentron canadum) displayed clinical signs, haemorrhagic eyes, dorsal darkness and gross pathological lesions, enlargement of spleen and liver. Haemorrhages were found in brain, heart and liver with cumulative mortality rates ranging from 20% to 50%. Extensive congestion in the heart, liver, spleen, kidney and brain was observed histopathologically. Epicarditis and meningitis were also revealed in diseased cobia. All isolates recovered from the organs (liver, spleen, head kidney, posterior kidney, brain and muscle) of cobia were found to be gram-positive, non-motile, ovoid cocci, short-chain-forming (diplococci) and α-haemolytic. The API 32 strep system together with the polymerase chain reaction assay for species-specific primers (pLG1 and pLG2) and the internal transcribed spacer (ITS) region (G1 and L1 primers) confirmed all four selected isolates as Lactococcus garvieae. Partial 16S rDNA nucleotide sequence (~1,100 bp) of one representative L. garvieae isolate AOD109191 (GenBank accession number, MW328528.1) shared 99.9% identities with the 16S rDNA nucleotide sequence of L. garvieae (GenBank accession numbers: MT604790.1). Transmission electron microscopy (TEM) evaluation of one representative L. garvieae isolate (AOD109191) and the results of multiplex PCR did not reveal the presence of the capsular gene cluster (CGC), thus categorizing the isolate as the KG+ phenotype. Capsule staining and TEM observations confirmed the presence of a hyaluronic acid-like capsule, a possible virulence factor in KG+ phenotype L. garvieae isolates. The pathogenic potential of the representative isolate (AOD109191) was assessed through intraperitoneal injection challenges in cobia. The gross lesions and histopathological changes found in experimentally infected cobia were similar to those seen in naturally infected fish. This is the first report that confirms L. garvieae-induced 'warm water lactococcsis' can cause outbreaks of diseases in cage-cultured cobia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tbed.14083DOI Listing
March 2021

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

Fish Shellfish Immunol 2021 Apr 6;111:179-188. Epub 2021 Feb 6.

International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan; Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan; Southern Taiwan Fish Diseases Research Centre, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan; Research Centre for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2021.02.002DOI Listing
April 2021

Recombinant resuscitation-promoting factor protein of Nocardia seriolae, a promissing vaccine candidate for largemouth bass (Micropterus salmoides).

Fish Shellfish Immunol 2021 Apr 2;111:127-139. Epub 2021 Feb 2.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1 Shuefu Road, Neipu, Pingtung, 91201, Taiwan; Southern Taiwan Fish Disease Centre, National Pingtung University of Science and Technology, No. 1 Shuefu Road, Neipu, Pingtung, 91201, Taiwan; International Degree Program of Ornamental Fish Science and Technology, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung, 91201, Taiwan; Research Centre for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan. Electronic address:

Nocardia seriolae is an important pathogenic bacterium that causes nocardiosis in various fish species and leads to economic losses in the fish industry. To develop an effective subunit vaccine against nocardial infection, the truncated resuscitation-promoting factor (tRPF) of N. seriolae was selected and recombinantly produced using the Escherichia coli expression system. Western blotting results indicated that the recombinant protein could be strongly recognised by largemouth bass anti-N. seriolae antibodies. The protective efficacy of tRPF recombinant protein was assessed in combination with the commercial adjuvant Montanide™ ISA 763 A VG. The results showed that emulsified tRPF + ISA significantly induced high serum antibody response and serum lysozyme activity in the vaccinated fish. Quantitative reverse transcription polymerase chain reaction analysis indicated that tRPF + ISA could notably enhance the expression of immune-related genes in both the head kidney and spleen of the vaccinated fish. Finally, vaccinated largemouth bass displayed higher immuno-protection with a relative percent survival of 69.23% compared to the control groups. Taken together, the combination of tRPF + ISA is an ideal vaccine candidate against N. seriolae infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2021.01.015DOI Listing
April 2021

Transcriptome analysis of immune- and iron-related genes after Francisella noatunensis subsp. orientalis infection in Nile tilapia (Oreochromis niloticus).

Fish Shellfish Immunol 2021 Apr 11;111:36-48. Epub 2021 Jan 11.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan; International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, Pingtung, Taiwan. Electronic address:

Francisella noatunensis subsp. orientalis (Fno) is a gram-negative intracellular bacterium identified in many fish species worldwide, including cultured Nile tilapia (Oreochromis niloticus) in Taiwan. To investigate the gene expression responses to Fno infection, we performed transcriptome analysis of the head kidney and spleen in Nile tilapia using RNA-seq. Total RNA was extracted from the head kidney and spleen of infected (Fno-injected) and uninfected (control) tilapia at 1-day and 2-days post-infection, and RNA-seq was performed using the Illumina HiSeq™ 4000 platform. After de novo assembly, a total of 106,534 transcripts were detected. These transcripts were annotated and categorized into a total of 7171 genes based on the KEGG pathway database. Differentially expressed genes (DEGs) were significantly (2-fold difference comparing Fno and PBS groups at each time point) enriched in the immune-related pathways, including the following: complement and coagulation cascades, cytokine-cytokine receptor interaction, hematopoietic cell lineage, lysosome, phagosome. We identified the upregulation of inflammatory cytokine-, apoptosis-, and neutrophil-related genes, and downregulation of complement- and lymphocyte-related genes. Additionally, we found the induction of natural resistance-associated macrophage protein 1 (NRAMP1) and heme responsive gene-1 (HRG1). Anemia of inflammation, caused by intracellular iron storage in spleen after Fno infection, was also observed. This study provides natural disease control strategies against Fno infection in tilapia. It is suggested that intercellular iron storage is a host protection strategy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2020.11.009DOI Listing
April 2021

Phenotype, genotype and pathogenicity of Streptococcus agalactiae isolated from cultured tilapia (Oreochromis spp.) in Taiwan.

J Fish Dis 2021 Jun 3;44(6):747-756. Epub 2020 Dec 3.

International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Tilapia (Oreochromis spp.) is globally used as an aquaculture fish species due to its high growth rate and disease resistance. However, it faces an increased risk of streptococcosis. Streptococcus agalactiae, also known as group B streptococcus (GBS), is the most important tilapia pathogen in Asia. Studies of Str. agalactiae infection in Taiwan are still unclear. Thus, this study aimed to explore the phenotype, genotype and pathogenicity of Str. agalactiae isolated from cultured tilapia in Taiwan in 2016-2018. The analysis revealed that 85% of the strains displayed β-haemolysis and 15% showed γ-haemolysis, with the same capsule level, and were positive for the CAMP test. The Rapid ID 32 Strep test showed a similarity of Rapid ID 32 Strep is more than 99.5% to GBS. Genotypic distribution by molecular serotyping detected only serotype Ia from all isolates, despite the regional differences. Pulsed-field gel electrophoresis (PFGE) was categorized into 3 and 10 clusters by restriction enzymes SmaI and ApaI, respectively. Virulence genes and antimicrobial resistance genes presented the same profile in all isolates. The challenge test with 10  CFU/fish (LD ), administered intraperitoneally, showed that the β-haemolysis strains had a higher mortality rate than γ-haemolysis, although they were from the same cluster from PFGE, year and region.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jfd.13296DOI Listing
June 2021

Comparison of the pathogenicity of Francisella orientalis in Nile tilapia (Oreochromis niloticus), Asian seabass (Lates calcarifer) and largemouth bass (Micropterus salmoides) through experimental intraperitoneal infection.

J Fish Dis 2020 Sep 23;43(9):1097-1106. Epub 2020 Jul 23.

International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Francisella orientalis is a highly virulent, emerging bacterium that causes mass mortalities in tilapia. This pathogen also affects numerous other warm-water fish species, including three-line grunt, hybrid striped bass and various ornamental fish. This study sheds light on two new species of fish that are susceptible to F. orientalis. Asian seabass and largemouth bass showed variable levels of susceptibility in a bacterial challenge experiment. After intraperitoneally injected with a dose of 10  CFU/fish, a total of 64.28% and 21.42% mortalities were obtained in Asian seabass and largemouth bass, respectively. Meanwhile, Nile tilapia showed acute mortality of 100%. All fish showed typical lesions of francisellosis, including multifocal granulomas in the spleen and head kidney. Immunohistochemical analysis revealed strong positive signals inside the granulomas of all fish. The bacterial recovery in solid media from infected fish was highest in Nile tilapia (85.71%), followed by Asian seabass (35.71%) and largemouth bass (21.42%). PCR results tested 100% positive for Nile tilapia, and 78.57% and 21.42% for Asian seabass and largemouth bass, respectively. In conclusion, Asian seabass and largemouth bass are susceptible to this pathogen, which warrants new management strategies when employing predation polyculture systems of these species with tilapia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jfd.13217DOI Listing
September 2020

Group C Streptococcus dysgalactiae infection in fish.

J Fish Dis 2020 Sep 13;43(9):963-970. Epub 2020 Jul 13.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Streptococcus dysgalactiae subsp. dysgalactiae (GCSD) is a Gram-positive, facultative anaerobic bacterium and mostly non-β-haemolytic with Lancefield group C antigen. GCSD infection has been identified in various vertebrates. From 2002 to the present, GCSD infection of fish has been reported to cause severe economic losses in aquaculture farms around the world. Moreover, GCSD isolates from teleosts have been identified in patients with ascending upper limb cellulitis. Therefore, the economic and clinical significance of GCSD has increased in aquaculture, livestock and human health. Many studies have been presented, from the first report of isolated GCSD in fish, to the pathogenesis, characterization, immune responses and vaccine development. In this review, we present the current knowledge of GCSD in teleosts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jfd.13211DOI Listing
September 2020

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

J Fish Dis 2020 Jul 17;43(7):757-774. Epub 2020 May 17.

International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, Pingtung, 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jfd.13173DOI Listing
July 2020

Immune Responses and Protective Efficacy of a Formalin-Killed Subsp. Vaccine Evaluated through Intraperitoneal and Immersion Challenge Methods in .

Vaccines (Basel) 2020 Apr 3;8(2). Epub 2020 Apr 3.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.

subsp. (), an intracellular bacterium, causes systemic granulomatous diseases, resulting in high mortality and huge economic losses in Taiwanese tilapia farming. In this study, we tested the efficacy of a formalin-killed vaccine in cultured tilapia. was isolated from diseased tilapia, inactivated with formalin, and mixed with the mineral oil base adjuvant (Montanide ISA 763 AVG). A total of 300 tilapia were divided into two groups. The experimental group was intraperitoneally injected with 0.1 mL of vaccine, which was substituted with phosphate-buffered saline (PBS) in the control group. A booster was administered at 2 weeks post-immunization. Tilapia were challenged at 6 weeks post primary immunization by intraperitoneal (IP) injection and immersion methods. Mortality was recorded at 21 and 60 days. The results revealed that the vaccine induced a greater antibody titer and led to 71% and 76% of relative percent survival (RPS) after the IP and immersion challenge. The transcripts of proinflammatory cytokines and immune-related genes, including interleukin-1β (IL-1β), tumor necrosis factor alpha (TNFα), C-X-C motif chemokine ligand 8 (CXCL8), and interleukin-17C (IL-17C), were significantly upregulated after vaccination. Additionally, vaccinated fish had lower bacterial loads in the blood and lower granuloma intensities in the kidney, spleen, liver, and gill than control fish. The results in this study demonstrate that the inactivated vaccine could be an essential resource in Taiwanese tilapia farming.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/vaccines8020163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348880PMC
April 2020

Interleukin 34 Serves as a Novel Molecular Adjuvant against Infection in Largemouth Bass ().

Vaccines (Basel) 2020 Mar 28;8(2). Epub 2020 Mar 28.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan.

DNA vaccines have been widely employed in controlling viral and bacterial infections in mammals and teleost fish. Co-injection of molecular adjuvants, including chemokines, cytokines, and immune co-stimulatory molecules, is one of the potential strategies used to improve DNA vaccine efficacy. In mammals and teleost fish, interleukin-34 (IL-34) had been described as a multifunctional cytokine and its immunological role had been confirmed; however, the adjuvant capacity of IL-34 remains to be elucidated. In this study, IL-34 was identified in largemouth bass. A recombinant plasmid of IL-34 (pcIL-34) was constructed and co-administered with a DNA vaccine encoding hypoxic response protein 1 (Hrp1; pcHrp1) to evaluate the adjuvant capacity of pcIL-34 against infection. Our results indicated that pcIL-34 co-injected with pcHrp1 not only triggered innate immunity and a specific antibody response, but also enhanced the mRNA expression level of immune-related genes encoding for cytokines, chemokines, and humoral and cell-mediated immunity. Moreover, pcIL-34 enhanced the protection of pcHrp1 against challenge and conferred the relative percent survival of 82.14%. Collectively, IL-34 is a promising adjuvant in a DNA vaccine against nocardiosis in fish.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/vaccines8020151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349345PMC
March 2020

The protective efficacy of recombinant hypoxic response protein 1 of Nocardia seriolae in largemouth bass (Micropterus salmoides).

Vaccine 2020 03 27;38(14):2925-2936. Epub 2020 Feb 27.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan; Southern Taiwan Fish Disease Centre, National Pingtung University of Science and Technology, No. 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan; International Degree Program of Ornamental Fish Science and Technology, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan; Research Centre for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan. Electronic address:

Nocardia seriolae has become one of the major pathogens affecting the aquaculture industry and causes Nocardiosis, a highly devastating disease of marine and freshwater fish that leads to severe economic losses. Therefore, research efforts towards developing efficacious vaccines to control this disease are of high importance. In this study, the hypoxic response protein 1 (HRP1) cloned into pET32a vector was expressed, and produced in Escherichia coli strain BL21 (DE3). The antigenicity of purified recombinant TRX-tagged HRP (rHRP1) was analysed by western blotting using largemouth bass anti-N. seriolae sera. The results showed that largemouth bass anti-N. seriolae sera could specifically detect a 33 kDa rHRP1 protein. Further, the vaccine efficacy of rHRP1 was evaluated in a largemouth bass fish model by calculating the relative percent survival (RPS). rHRP1 incurred an RPS of 73.33% as compared to the control group. Immunological analysis showed that rHRP1 could produce significantly higher serum concentrations of anti-N. seriolae antibodies and serum lysozyme activity as compared to the control groups. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that rHRP1 significantly enhanced the expression of immune-related genes, such as IL-12p40, IL-8, IL-1β, TNFα, IFNγ, NKEF, MHCIα, MHCIIα, CD4-1, CD8α, IgM, NF-κβ, STAT3, IRF4, RORα, and CCL20. These results indicate that rHRP1 may be a promising vaccine candidate against nocardiosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vaccine.2020.02.062DOI Listing
March 2020

α-Enolase as a novel vaccine candidate against Streptococcus dysgalactiae infection in cobia (Rachycentron canadum L.).

Fish Shellfish Immunol 2020 Mar 22;98:899-907. Epub 2019 Nov 22.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung, 91201, Taiwan, ROC; International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung, 91201, Taiwan, ROC; Southern Taiwan Fish Disease Centre, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung, 91201, Taiwan, ROC; Research Center for Animal Biologics, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung, 91201, Taiwan, ROC; Research Center for Fish Vaccines and Diseases, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung, 91201, Taiwan, ROC. Electronic address:

Streptococcus dysgalactiae is an important pathogenic bacterium that has caused economic loss for the cobia industry in Taiwan, ROC. This study presents a highly effective subunit vaccine composed of a moonlight protein, α-enolase, for the prevention of S. dysgalactiae infection. First, α-enolase was cloned, transformed, and expressed in E. coli for production of recombinant protein. Then, the protective efficacies of α-enolase recombinant protein were evaluated in combination with either a pro-inflammatory cytokine, TNF-α, or an oil adjuvant, ISA 763 AVG. The results showed that the combination of α-enolase and ISA 763 AVG was highly protective (RPS = 88.89%), while a negative effect was found in the group immunised with α-enolase adjuvanted with TNF-α (RPS = 22.22%). A further study was conducted with double dose of ISA 763 AVG, which led to an increased RPS value of 97.37%. Moreover, immunised cobia exhibited significantly greater lysozyme activity, antibody responses, and expression of certain immune-related genes post-challenge. Altogether, our results demonstrated that a combination of α-enolase recombinant protein with ISA 763 AVG adjuvant is a promising vaccine that can be employed for protection of cobia against S. dysgalactiae infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2019.11.050DOI Listing
March 2020

Establishment of loop-mediated isothermal amplification for rapid and convenient detection of Mycobacterium marinum complex.

J Microbiol Methods 2019 09 18;164:105671. Epub 2019 Jul 18.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Neipu, Pingtung, Taiwan, Republic of China. Electronic address:

Mycobacterium marinum is a zoonotic pathogen that can cause dermatological infection mainly from contaminated water or fish. Some well-known genetically similar species and subspecies are M. lifrandii and M. pseudoshottsii from amphibians and fish in aquaculture, and M. ulcerans, a causative agent of a neglected tropical disease (NTD), Buruli ulcer. They are believed to survive in water as their major niche, which might be related to their source of infection, but detailed ecological surveillance of the species complex remains to be done. Herein, we present a new detection system for M. marinum complex based on isothermal DNA amplification that can be conducted conveniently with high sensitivity and specificity. The target was a chromosomal gene, mrsA, including a restriction polymorphism between M. ulcerans (except for the most ancestral subspecies, M. ulcerans subsp. shinshuense) and the other species. The system was able to detect less than 500 fg (approximately 70 copies) of genomic DNA of M. marinum, within 60 min, and caused no amplification from mycobacterial species other than M. marinum complex species. It was also verified that restriction of the amplified DNA fragments was able to discriminate M. ulcerans as expected. This easy, quick, and convenient system is expected to facilitate detection of M. marinum complex from various resources.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mimet.2019.105671DOI Listing
September 2019

Differential expression of immune-related genes in head kidney and spleen of cobia (Rachycentron canadum) having Streptococcus dysgalactiae infection.

Fish Shellfish Immunol 2019 Sep 6;92:842-850. Epub 2019 Jul 6.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan; International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, Pingtung, Taiwan; Southern Taiwan Fish Disease Centre, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan; Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan; Research Center for Fish Vaccines and Diseases, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan. Electronic address:

Streptococcus dysgalactiae is a gram-positive bacterium and a harmful aquaculture pathogen. To investigate the immune response against S. dysgalactiae, we performed transcriptome analysis of the head kidney and spleen of cobia (Rachycentron canadum) using RNA-seq. Total RNA was extracted from the head kidney and spleen of cobia, 1 and 2 days after treatment with S. dysgalactiae or control PBS. After RNA purification and cDNA library generation, sequencing was performed using the Illumina HiSeq™ 4000 platform. The filtering and de novo assembling transcripts were annotated using several databases. To identify differentially expressed genes (DEGs) between the S. dysgalactiae and PBS groups, the mapped values of fragments per kilobase of transcripts per million fragments were calculated. After de novo assembly, a total of 106,984 transcripts were detected, with an N50 of 3020 bp. These transcripts were annotated and categorised into a total of 7608 genes based on the KEGG pathway database. DEGs (2-fold difference) were calculated by comparing the S. dysgalactiae and PBS control group gene expression levels at each time point. The DEGs were mainly annotated into signal transduction and immune system categories, based on the KEGG database. The DEGs were significantly enriched in the immune-related pathways - "cytokine-cytokine receptor interaction", "complement and coagulation cascades", and "hematopoietic cell linage". In this study, immune-related genes responding to S. dysgalactiae were detected, and several immune system pathways were categorized. We identified the IL17C-related pathway for inducing the expression of pro-inflammatory cytokine genes (IL-1β, IL-6, and IFNγ). Additionally, neutrophil-related genes (CSF3, CD121, and CD114) were induced in the spleen after S. dysgalactiae infection. It was suggested that these pathways contribute to immune responses against S. dysgalactiae infection. The data revealed in this study may offer improved strategies against S. dysgalactiae infection in cobia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2019.07.009DOI Listing
September 2019

Genotypic and phenotypic characterization of Edwardsiella isolates from different fish species and geographical areas in Asia: Implications for vaccine development.

J Fish Dis 2019 06 8;42(6):835-850. Epub 2019 Mar 8.

Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Oslo, Norway.

The genus Edwardsiella is one of the major causes of fish diseases globally. Herein, we examined 37 isolates from ten different fish species from India, South Korea and Taiwan to gain insight into their phenotypic and genotypic properties, of which 30 were characterized as E. tarda with phenotypic homology estimated at 85.71% based on API-20E biochemical tests. Genotyping using 16S rRNA put all isolates together with E. anguillarum, E. hoshinae, E. tarda, E. piscicida and E. ictaluri reference strains in a monophyletic group. In contrast, the gyrB phylogenetic tree clearly separated E. ictaluri, E. tarda and E. hoshinae reference strains from our isolates and put our isolates into two groups with group I being homologous with the E. anguillarum reference strain while group II was homologous with the E. piscicida reference strain. Hence, our findings point to E. piscicida and E. anguillarum as species infecting different fish species in Asia. Homology of the ompW protein suggested that strains with broad protective coverage could be identified as vaccine candidates. This study underscores the importance of combining genotyping with phenotyping for valid species classification. In addition, it accentuates the importance of phylogenetic comparison of bacterial antigens for identification of potential vaccine candidates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jfd.12984DOI Listing
June 2019

Comparative Study of Immune Reaction Against Bacterial Infection From Transcriptome Analysis.

Front Immunol 2019 5;10:153. Epub 2019 Feb 5.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Transcriptome analysis is a powerful tool that enables a deep understanding of complicated physiological pathways, including immune responses. RNA sequencing (RNA-Seq)-based transcriptome analysis and various bioinformatics tools have also been used to study non-model animals, including aquaculture species for which reference genomes are not available. Rapid developments in these techniques have not only accelerated investigations into the process of pathogenic infection and defense strategies in fish, but also used to identify immunity-related genes in fish. These findings will contribute to fish immunotherapy for the prevention and treatment of bacterial infections through the design of more specific and effective immune stimulants, adjuvants, and vaccines. Until now, there has been little information regarding the universality and diversity of immune reactions against pathogenic infection in fish. Therefore, one of the aims of this paper is to introduce the RNA-Seq technique for examination of immune responses in pathogen-infected fish. This review also aims to highlight comparative studies of immune responses against bacteria, based on our previous findings in largemouth bass () against , gray mullet () against , orange-spotted grouper () against , and koi carp () against , using RNA-seq techniques. We demonstrated that only 39 differentially expressed genes (DEGs) were present in all species. However, the number of specific DEGs in each species was relatively higher than that of common DEGs; 493 DEGs in largemouth bass against , 819 DEGs in mullets against , 909 in groupers against , and 1471 in carps against . The DEGs in different fish species were also representative of specific immune-related pathways. The results of this study will enhance our understanding of the immune responses of fish, and will aid in the development of effective vaccines, therapies, and disease-resistant strains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.00153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370674PMC
January 2020

Molecular characterization and pathogenicity of Francisella noatunensis subsp. orientalis isolated from cultured tilapia (Oreochromis sp.) in Taiwan.

J Fish Dis 2019 May 4;42(5):643-655. Epub 2019 Feb 4.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Francisella noatunensis subsp. orientalis is a causative agent of systemic granulomatous disease in tilapia. The present study was designed to understand the genetic and phenotypic diversities among Taiwanese Fno isolates obtained from tilapia (n = 17) and green Texas cichlid (Herichthys cyanoguttatus) (n = 1). The enzymatic profiles of the isolates were studied using the API ZYM system. Phylogenetic tree analysis of the 16S rRNA and housekeeping gene and pulsed-field gel electrophoresis (PFGE) were carried out to determine the genotypic characters of all isolates. The phylogenetic tree showed similarity of 99%-100% nucleotide sequences of 16S rRNA and housekeeping genes compared to the Fno references genes from GenBank database. Comparatively, the results revealed an identical profile of enzymatic and PFGE pattern which was distincted from that of F. philomiragia. To understand the pathogenicity, the isolates were intraperitoneal injected to tilapia the gross lesions were observed concomitant with natural outbreak. Median lethal dose upon Nile tilapia and red tilapia were 9.06 × 10 CFU/fish and 2.08 × 10 CFU/fish, respectively. Thus, our data provide understanding the epidemiology of Taiwanese Fno isolates, and help in development of future control and prevention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jfd.12964DOI Listing
May 2019

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.

Comp Biochem Physiol B Biochem Mol Biol 2019 Apr 18;230:10-18. Epub 2019 Jan 18.

Laboratory of Molecular Fish Immunology and Genetics, Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology,Pingtung 91201, Taiwan; Research Center for Animal Biologics, National Pingtung University of Science and Technology,Pingtung 91201, Taiwan. Electronic address:

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-1β, 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-1β 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbpb.2019.01.004DOI Listing
April 2019

Immune-Related Functional Differential Gene Expression in Koi Carp () after Challenge with .

Int J Mol Sci 2018 Jul 20;19(7). Epub 2018 Jul 20.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.

In order to understand the molecular basis underlying the host immune response of koi carp (), Illumina HiSeq 2000 is used to analyze the muscle and spleen transcriptome of koi carp infected with (). assembly of paired-end reads yielded 69,480 unigenes, of which the total length, average length, N50, and GC content are 70,120,028 bp, 1037 bp, 1793 bp, and 45.77%, respectively. Annotation is performed by comparison against various databases, yielding 42,229 (non-redundant protein sequence (NR): 60.78%), 59,255 (non-redundant nucleotide (NT): 85.28%), 35,900 (Swiss-Prot: 51.67%), 11,772 (clusters of orthologous groups (COG): 16.94%), 33,057 (Kyoto Encyclopedia of Genes and Genomes (KEGG): 47.58%), 18,764 (Gene Ontology (GO): 27.01%), and 32,085 (Interpro: 46.18%) unigenes. Comparative analysis of the expression profiles between bacterial challenge fish and control fish identifies 7749 differentially expressed genes (DEGs) from the muscle and 7846 DEGs from the spleen. These DEGs are further categorized with KEGG. Enrichment analysis of the DEGs and unigenes reveals major immune-related functions, including up-regulation of genes related with Toll-like receptor signaling, complement and coagulation cascades, and antigen processing and presentation. The results from RNA-Seq data are also validated and confirmed the consistency of the expression levels of seven immune-related genes after 24 h post infection with qPCR. Microsatellites (11,534), including di-to hexa nucleotide repeat motifs, are also identified. Altogether, this work provides valuable insights into the underlying immune mechanisms elicited during bacterial infection in koi carp that may aid in the future development of disease control measures in protection against .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms19072107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073842PMC
July 2018

Enhanced immune responses and effectiveness of refined outer membrane protein vaccines against Vibrio harveyi in orange-spotted grouper (Epinephelus coioides).

J Fish Dis 2018 Sep 8;41(9):1349-1358. Epub 2018 Jun 8.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK-OmpU fusion protein in addition to the metabolizable Montanide ISA 763 A VG adjuvant were developed and evaluated in the orange-spotted grouper. The results indicate that recombinant V. harveyi protein-based vaccines resulted in a remarkably higher expression of IL-1β and IL-8 at 24 hr, and greater antibody production, as early as 2 weeks postimmunization. Notably, enhanced immune responses and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine-injected fishes with relative per cent survival value of 81.8%. Additionally, the rOmpK-OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also Vibrio parahaermolyticus and Vibrio alginolyticus. Our results demonstrated that the fusion protein rOmpK-OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jfd.12828DOI Listing
September 2018

Identification of protective protein antigens for vaccination against Streptococcus dysgalactiae in cobia (Rachycentron canadum).

Fish Shellfish Immunol 2018 Sep 30;80:88-96. Epub 2018 May 30.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung, 91201, Taiwan; International Degree Program of Ornamental Fish Science and Technology, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung, 91201, Taiwan; Southern Taiwan Fish Diseases Research Center, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung, 91201, Taiwan; Research Center for Animal Biologics, National Pingtung University of Science and Technology, No. 1 Shuefu Road, Neipu, Pingtung, 91201, Taiwan. Electronic address:

Streptococcus dysgalactiae is considered a causative agent of severe infection and economic loss for the cobia industry in Taiwan. In this study, protective antigens of this pathogenic bacterium were identified and screened in cobia (Rachycentron canadum). Outer surface proteins (OMPs) of this pathogen were extracted using mutanolysin digestion. Immunogenic targets were detected by western blot and then subjected to peptide sequencing using NanoLC-MS/MS. Two surface proteins, namely phosphoenolpyruvate protein phosphotransferase (PtsA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), showed strong reactions with cobia antisera against S. dysgalactiae. Recombinant proteins were produced in Escherichia coli cells and their protective efficacies were investigated in cobia. Fish immunised with recombinant proteins, rPtsA + ISA (ISA 763 AVG) and rGAPDH + ISA, elicited higher levels of specific antibody responses against the recombinant proteins and had high levels of lysozyme activity. Notably, vaccinated fish were protected from lethal challenge with relative percentage of survival (RPS) values for rPtsA + ISA and rGAPDH + ISA groups being 91.67% and 83.33%, while 0% RPS value was found in both ISA injected and control groups. The results presented in the study demonstrate that the GAPDH and PtsA are promising vaccine candidates for preventing S. dysgalactiae disease in cobia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2018.05.052DOI Listing
September 2018

Efficacy of recombinant protein vaccines for protection against Nocardia seriolae infection in the largemouth bass Micropterus salmoides.

Fish Shellfish Immunol 2018 Jul 14;78:35-41. Epub 2018 Apr 14.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan, ROC; Research Center for Animal Biologics, National Pingtung University of Science and Technology, No. 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan, ROC. Electronic address:

A reverse vaccinology-based survey of potent antigens associated with fish nocardiosis was conducted using the largemouth bass, Micropterus salmoides, with an aim to develop subunit vaccines. The antigens selected from the virulent strain Nocardia seriolae 961113 include the gene products of NGL2579 (GAPDH), NGL5701 (MMP), NGL4377 (OCTase), NGL4486 (ABC transporter), NGL3372 (LLE), NGL3388 (GHf10), NGL6627 (Antigen-85), NGL6696 (Esterase), and NGL6936 (CBP). These antigens were heterologously expressed in E. coli BL21 (DE3) for recombinant protein production. Then fish were vaccinated was these antigens, boosted at 2 weeks, and challenged with N. seriolae at 6 weeks after vaccination. The relative protection survival assay revealed high and significant protection efficacies of 94.45, 50.00, and 44.45 in fish that received the NGL3388 (GHf10), NGL6936 (CBP), and NGL3372 (LLE) vaccines, respectively. There were no apparent relationships or differences in tissue lesions among the administered vaccines. The serum titers against the bacterial preparations were higher for all vaccinated groups than for the control group at 4 weeks after immunization. However, no significant difference in serum titer was found at 6 weeks after immunization. The results of this study demonstrate that subunit vaccines against fish nocardiosis have differential effects, but are highly promising for nocardial prophylaxis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2018.04.024DOI Listing
July 2018

Transcriptome analysis of immune response against Vibrio harveyi infection in orange-spotted grouper (Epinephelus coioides).

Fish Shellfish Immunol 2017 Nov 20;70:628-637. Epub 2017 Sep 20.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan; International Degree Program of Ornamental Fish Science and Technology, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan. Electronic address:

Vibrio harveyi is a gram-negative bacterium reported as found in many aquaculture species. To increase knowledge of the immune response against V. harveyi, in this study we performed transcriptome analysis of head kidney and spleen in orange-spotted grouper (Epinephelus coioides) at 1 and 2 days post-infection (dpi), using the Illumina sequencing platform. After de novo assembly, a total of 79,128 unigenes was detected with an N50 of 2511 bp. After alignments with sequences recorded in the major databases (NT, NR, Swiss-Prot COG, KEGG, Interpro and GO), based on sequence similarity, 61,208 (77.4%) of the unigene total could be annotated using at least one database. Comparison of gene expression levels between V. harveyi and a control group at each time point revealed differentially expressed genes (DEGs) (P < 0.05): a total of 7918 (5536 upregulated and 2282 downregulated genes) from head kidney at 1 day post infection (dpi), 4260 (1444 upregulated and 2816 downregulated genes) from head kidney at 2 dpi, 7887 (4892 upregulated and 2995 downregulated genes) from spleen at 1 dpi, and 8952 (7388 upregulated and 1564 downregulated genes) from spleen at 2 dpi. The DEGs were mainly annotated into signal transduction and immune system categories, based on the KEGG database. The DEGs were enriched in immune-related pathway functions, NOD-like receptor signaling pathways, Toll-like receptor signaling pathways, NF-κB signaling pathways, and Jak-STAT signaling pathways. Additionally, we selected several DEGs and validated their expression level by RT-qPCR. The data generated in this study may provide a valuable resource for further immune response research and offer improved strategies against V. harveyi infection in teleost fishes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2017.09.052DOI Listing
November 2017

Effectiveness of formalin-killed vaccines containing CpG oligodeoxynucleotide 1668 adjuvants against Vibrio harveyi in orange-spotted grouper.

Fish Shellfish Immunol 2017 Sep 8;68:124-131. Epub 2017 Jul 8.

Department of Veterinary Medicine, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan; Southern Taiwan Fish Diseases Research Center, College of Veterinary Medicine, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan; International Degree Program of Ornamental Fish Science and Technology, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan. Electronic address:

Vibrio harveyi is a major bacterial pathogen that causes serious vibriosis in cultured groupers, leading to massive deaths. In this study, we evaluated the immune responses and protective efficacy of vaccines containing V. harveyi formalin-killed cells (FKC) formulated with CpG ODN 1668-enriched plasmids (p30CpG and p60CpG) in the orange-spotted grouper. Results indicated that antibody titres were remarkably increased in vaccinated fish 2 weeks post-immunisation. Expression level of major histocompatibility complex (MHC) class II, CD 8, and toll-like receptor 9 was significantly upregulated in the spleen of fish immunised with CpG ODN 1668-adjuvanted vaccines, as recorded at 6 weeks after immunisation. Additionally, the FKC + p60CpG-vaccinated fish displayed greater mRNA levels of MHC I and tumor necrosis factor-alpha. Of note, the relative percent survival after V. harveyi challenge was significantly higher in FKC + p60CpG-vaccinated fish (96.2%) than in FKC + p30CpG-vaccinated (79.8%) and FKC-vaccinated fish (59.9%). These results demonstrate that the FKC + CpG ODN 1668 vaccines are promising candidates that could enhance both innate and adaptive immune responses, conferred remarkable protection, and CpG ODN 1668 is a potential adjuvant for vaccines against V. harveyi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2017.07.018DOI Listing
September 2017

Identification and expression analysis of two pro-inflammatory cytokines, TNF-α and IL-8, in cobia (Rachycentron canadum L.) in response to Streptococcus dysgalactiae infection.

Fish Shellfish Immunol 2017 Aug 12;67:159-171. Epub 2017 Jun 12.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan; International Degree Program of Ornamental Fish Science and Technology, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan. Electronic address:

Tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8/CXCL8) play pivotal roles in mediating inflammatory responses to invading pathogens. In this study, we identified and analyzed expressions of cobia TNF-α and IL-8 during Streptococcus dysgalactiae infection. The cloned cDNA transcript of cobia TNF-α comprised of 1281 base pairs (bp), with a 774 bp open reading frame (ORF) encoding 257 amino acids. The deduced amino acid sequence of cobia TNF-α showed a close relationship (84% similarity) with TNF-α of yellowtail amberjack. The cloned IL-8 cDNA sequence was 828 bp long, including a 300-bp ORF encoding 99 amino acids. The deduced amino acid sequence of cobia IL-8 shared 90% identity with IL-8 of striped trumpeter. Cobia challenged with a virulent S. dysgalactiae strain displayed an early significant up-regulation of TNF-α and IL-8 in head kidney, liver, and spleen. Notably, IL-8 expression level increased dramatically in the liver at the severe stage of infection (72 h). In conclusion, a better understanding of TNF-α and IL-8 allows more detailed investigation of immune responses in cobia and furthers study on controlling the infectious disease caused by S. dysgalactiae.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2017.06.014DOI Listing
August 2017

A formalin-inactivated vaccine provides good protection against Vibrio harveyi infection in orange-spotted grouper (Epinephelus coioides).

Fish Shellfish Immunol 2017 Jun 15;65:118-126. Epub 2017 Apr 15.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan; International Degree Program of Ornamental fish Science and Technology, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan. Electronic address:

Vibrio harveyi is one of the most common threats to farmed grouper, so considerable efforts are in practice to control the pathogen. This study presents a highly effective vaccine against V. harveyi in the orange-spotted grouper with the help of a single intraperitoneal immunization. The vaccine candidate was in form of whole, formalin-inactivated V. harveyi cells combined with a metabolizable ISA763 AVG adjuvant. Our results indicated that the vaccine triggered a remarkably higher expression level of interleukin (IL)-1β, IL-6, IL-8, and IL-10 in the groupers' kidneys and spleens, as recorded after 1 and 3 days of immunization. Antibody titers were significantly elevated in the vaccinated fish. A pivotal observation was that the vaccine highly protected the grouper from a homologous V. harveyi strain challenge with relative percentage survival values of 100% and 91.7% at 6 and 12 weeks post-immunization, respectively. Vaccinated fish also demonstrated strong cross-protection against a heterologous bacterial isolate challenge. Therefore, the inactivated V. harveyi vaccine is a promising candidate that could stimulate good immune responses and confer remarkable protection in farmed groupers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2017.04.008DOI Listing
June 2017

Transcriptome analysis of grey mullet (Mugil cephalus) after challenge with Lactococcus garvieae.

Fish Shellfish Immunol 2016 Nov 5;58:593-603. Epub 2016 Oct 5.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan, ROC. Electronic address:

Grey mullet (Mugil cephalus) is an economically important fish species in Taiwan mariculture industry. Moreover, grey mullet are common hosts of a bacterial infection by Lactococcus garvieae. However, until now the information related to the immune system of grey mullet is unclear. Therefore, to understand the molecular basis underlying the host immune response to L. garvieae infection, Illumina HiSeq™ 2000 was used to analyse the head kidney and spleen transcriptome of infected grey mullet. De novo assembly of paired-end reads yielded 55,203 unigenes. Comparative analysis of the expression profiles between bacterial challenge fish and control fish identified a total of 7192 from head kidney and 7280 in spleen differentially expressed genes (P < 0.05), including 4211 upregulated genes and 2981 downregulated genes in head kidney, while in spleen 3598 genes were upregulated and 3682 downregulated. A significant enrichment analysis of these differentially expressed genes (DEG) in spleen and head kidney revealed major immune-related pathways, including complement and coagulation cascades, Toll-like receptor signalling, and antigen processing and presentation. Moreover, selected DEGs were validated using qPCR. Altogether, the results obtained on immune-related genes may allow for a better understanding of immunity in grey mullet to Lactococcus garvieae, carrying out detailed functional analysis of these genes and developing strategies for efficient immune protection against infections in grey mullet.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2016.10.006DOI Listing
November 2016

Identification, Molecular Cloning of IL-1β and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides.

Int J Mol Sci 2016 Oct 1;17(10). Epub 2016 Oct 1.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 912, Taiwan.

In the present study, IL-1β cDNA was identified and analyzed from largemouth bass (). Full length IL-1β mRNA was obtained using Rapid Amplification of cDNA Ends (RACE), which contains 78 bp 3'-UTR, a 455 bp 5'-UTR, and an open reading frame (ORF) of 702 bp coding for 233 amino acid residues. The molecular weight and theoretical isoelectric point of largemouth bass IL-1β protein was predicted to be 26.7 kDa and 6.08 respectively. A largemouth bass IL-1β phylogenetic analysis showed a close relation to the IL-1βs of striped trumpeter (), Chinese perch (), and Japanese sea bass (). Peptidoglycan upregulated IL-1β in the spleen and head kidney, while lipopolysaccharide upregulated detectable levels of IL-1β in the spleen only. Largemouth bass, challenged with (1.0 × 10⁶ cfu/mL), showed a significant increase in IL-1β at 3 and 5 days post infection (dpi) in the spleen, while in the head kidney significant expression was found at 2 and 3 dpi, peaking at 3 dpi. Furthermore, tumor necrosis factor α (TNF-α) showed significantly higher expression in the spleen at 3 and 5 dpi, and in the head kidney at 1 and 3 dpi, with expression decreasing at 5 dpi in both tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms17101670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085703PMC
October 2016

De Novo Transcriptome Analysis of Differential Functional Gene Expression in Largemouth Bass (Micropterus salmoides) after Challenge with Nocardia seriolae.

Int J Mol Sci 2016 Aug 11;17(8). Epub 2016 Aug 11.

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.

Largemouth bass (Micropterus salmoides) are common hosts of an epizootic bacterial infection by Nocardia seriolae. We conducted transcriptome profiling of M. salmoides to understand the host immune response to N. seriolae infection, using the Illumina sequencing platform. De novo assembly of paired-end reads yielded 47,881 unigenes, the total length, average length, N50, and GC content of which were 49,734,288, 1038, 1983 bp, and 45.94%, respectively. Annotation was performed by comparison against non-redundant protein sequence (NR), non-redundant nucleotide (NT), Swiss-Prot, Clusters of Orthologous Groups (COG), Kyoto Encyclopaedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Interpro databases, yielding 28,964 (NR: 60.49%), 36,686 (NT: 76.62%), 24,830 (Swissprot: 51.86%), 8913 (COG: 18.61%), 20,329 (KEGG: 42.46%), 835 (GO: 1.74%), and 22,194 (Interpro: 46.35%) unigenes. Additionally, 8913 unigenes were classified into 25 Clusters of Orthologous Groups (KOGs) categories, and 20,329 unigenes were assigned to 244 specific signalling pathways. RNA-Seq by Expectation Maximization (RSEM) and PossionDis were used to determine significantly differentially expressed genes (False Discovery Rate (FDR) < 0.05) and we found that 1384 were upregulated genes and 1542 were downregulated genes, and further confirmed their regulations using reverse transcription quantitative PCR (RT-qPCR). Altogether, these results provide information on immune mechanisms induced during bacterial infection in largemouth bass, which may facilitate the prevention of nocardiosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms17081315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000712PMC
August 2016

Isolation and genetic characterization of Nocardia seriolae from snubnose pompano Trachinotus blochii in Vietnam.

Dis Aquat Organ 2016 Jul;120(2):173-7

Department of Biotechnology, Central Vietnam Veterinary Institute, km4 Dong De Street, Nha Trang, Vietnam.

A total of 480 cage-cultured fish were collected from 4 coastal provinces in central Vietnam to investigate the causative agent of nocardiosis. Fish displayed unique characteristics such as paleness and lethargy and exhibited haemorrhages and ulcers on the skin. Prominent white nodules varying in size were observed in the spleen, kidney, and liver. Furthermore, histopathological sections showed typical granulomatous lesions in these organs. Using the Ziehl-Neelsen staining method, isolated bacteria exhibited acid-fast, bead-like filament morphology when cultured in brain-heart infusion medium or Ogawa medium. Phylogenetic analysis of 16S rDNA confirmed that the isolated bacterium was Nocardia seriolae. This study demonstrates for the first time an outbreak of N. seriolae in snubnose pompano in central Vietnam.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3354/dao03023DOI Listing
July 2016