Publications by authors named "Kim D Thompson"

52 Publications

Modulation of the mucosal immune response of red tilapia (Oreochromis sp.) against columnaris disease using a biomimetic-mucoadhesive nanovaccine.

Fish Shellfish Immunol 2021 May 4;112:81-91. Epub 2021 Mar 4.

Wildlife Exotic Aquatic Animal Pathology-Research Unit, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand. Electronic address:

Columnaris, a highly contagious bacterial disease caused by Flavobacterium columnare, is recognized as one of the most important infectious diseases in farmed tilapia, especially during the fry and fingerling stages of production. The disease is associated with characteristic lesions in the mucosa of affected fish, particularly their skin and gills. Vaccines delivered via the mucosa are therefore of great interest to scientists developing vaccines for this disease. In the present study, we characterized field isolates of F. columnare obtained from clinical columnaris outbreaks in red tilapia to select an isolate to use as a candidate for our vaccine study. This included characterizing its colony morphology, genotype and virulence status. The isolate was incorporated into a mucoadhesive polymer chitosan-complexed nanovaccine (CS-NE), the efficacy of which was determined by experimentally infecting red tilapia that had been vaccinated with the nanoparticles by immersion. The experimental infection was performed 30-days post-vaccination (dpv), which resulted in 89% of the unvaccinated control fish dying, while the relative percentage survival (RPS) of the CS-NE vaccinated group was 78%. Histology of the mucosal associated lymphoid tissue (MALT) showed a significantly higher presence of leucocytes and a greater antigen uptake by the mucosal epithelium in CS-NE vaccinated fish compared to control fish and whole cell vaccinated fish, respectively, and there was statistically significant up-regulation of IgT, IgM, TNF α, IL1-β and MHC-1 genes in the gill of the CS-NE vaccinated group. Overall, the results of our study confirmed that the CS-NE particles achieved better adsorption onto the mucosal surfaces of the fish, elicited great vaccine efficacy and modulated the MALT immune response better than the conventional whole cell-killed vaccine, demonstrating the feasibility of the mucoadhesive nano-immersion vaccine as an effective delivery system for the induction of a mucosal immune response against columnaris disease in tilapia.
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http://dx.doi.org/10.1016/j.fsi.2021.02.017DOI Listing
May 2021

Development and evaluation of colloidal gold immunochromatography test strip for rapid diagnosis of nervous necrosis virus in golden grey mullet (Chelon aurata).

J Fish Dis 2021 Feb 1. Epub 2021 Feb 1.

Aquaculture Research Group, Moredun Research Institute, Penicuik, UK.

A lateral flow immunochromatography strip test, based on antibody-gold nanoparticles specific for nervous necrosis virus (NNV), was developed for rapid, on-site detection of the virus in fish stocks. A monoclonal antibody against NNV was conjugated with colloidal gold as the detector antibody. A rabbit anti-NNV polyclonal antibody and goat anti-mouse IgG antibody were blotted onto the nitrocellulose membrane as the capture antibodies on the test line and control line, respectively. The reaction could be seen by the eye within 15 min and did not cross-react with the other viruses tested. The detection limit of the strip was approximately 10 TCID /ml and had good stability after storage at 4°C for 8 months. When brains of 70 naturally infected golden grey mullet, Chelon aurata, were tested with the strip test, the diagnostic specificity and sensitivity of the test compared to real-time RT-PCR were 100% and 74%, respectively. Therefore, the one-step test strip developed here had high specificity, reproducibility, and stability. This, together with its simplicity to use and rapid detection, without the requirement of sophisticated equipment or specialized skills, makes the strip suitable for pond-side detection of NNV in farmed fish.
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http://dx.doi.org/10.1111/jfd.13302DOI Listing
February 2021

Elucidating the Functional Roles of Helper and Cytotoxic T Cells in the Cell-Mediated Immune Responses of Olive Flounder ().

Int J Mol Sci 2021 Jan 15;22(2). Epub 2021 Jan 15.

Laboratory of Aquatic Animal Diseases, Research Institute of Natural Science, College of Veterinary Medicine, Gyeongsang National University, 501-201, 501, Jinju-daero, Jinju-si 52828, Korea.

In higher vertebrates, helper and cytotoxic T cells, referred to as CD4 and CD8 T lymphocytes, respectively, are mainly associated with adaptive immunity. The adaptive immune system in teleosts involves T cells equivalent to those found in mammals. We previously generated monoclonal antibodies (mAbs) against olive flounder () CD4 T cells, CD4-1 and CD4-2, and used these to describe the olive flounder's CD4 Tcell response during a viral infection. In the present study, we successfully produced mAbs against CD8 T lymphocytes and their specificities were confirmed using immuno-blotting, immunofluorescence staining, flow cytometry analysis andreverse transcription polymerase chain reaction (RT-PCR). The results showed that these mAbs are specific for CD8 T lymphocytes. We also investigated variations in CD4 and CD8 T cells populations, and analyzed the expression of immune-related genes expressed by these cells in fish infected with nervous necrosis virus or immunized with thymus dependent and independent antigens. We found that both CD4 and CD8 T lymphocyte populations significantly increased in these fish and Th1-related genes were up-regulated compared to the control group. Collectively, these findings suggest that the CD4 and CD8 T lymphocytes in olive flounder are similar to the helper and cytotoxic T cells found in mammals, and Th1 and cytotoxic immune responses are primarily involved in the early adaptive immune response against extracellular antigens.
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http://dx.doi.org/10.3390/ijms22020847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829854PMC
January 2021

Passive Immunization with Recombinant Antibody VLRB-PirA/PirB-Enriched Feeds against Infection in Shrimp.

Vaccines (Basel) 2021 Jan 16;9(1). Epub 2021 Jan 16.

Laboratory of Aquatic Animal Diseases, Research Institute of Natural Science, College of Veterinary Medicine, Gyeongsang National University, 501-201, 501, Jinju-daero, Jinju-si, Gyeongsangnam-do 52828, Korea.

The causative agent of acute hepatopancreatic necrosis disease (AHPND) is the bacterium, , which secretes toxins into the gastrointestinal tract of its host. toxins A and B (PirA/PirB) have been implicated in the pathogenesis of this disease, and are, therefore, the focus of studies developing treatments for AHPND. We previously produced recombinant antibodies based on the hagfish variable lymphocyte receptor B (VLRB) capable of neutralizing some viruses, suggesting that this type of antibody may have a potential application for treatment of AHPND. Here, recombinant PirA/PirB, produced using a bacterial expression system, were used as antigens to screen a hagfish VLRB cDNA library to obtain PirA/PirB-specific antibodies. A cell line secreting these antibodies was established by screening and cloning the DNA extracted from hagfish B cells. Supernatants collected from cells secreting the PirA/PirB antibodies were collected and concentrated, and used to passively immunize shrimp to neutralize the toxins PirA or PirB associated with AHPND. Briefly, 10 μg of PirA and PirB antibodies, 7C12 and 9G10, respectively, were mixed with the shrimp feed, and fed to shrimp for three days consecutive days prior to experimentally infecting the shrimp with (containing toxins A and B), and resulting mortalities recorded for six days. Results showed significantly higher level of survival in shrimp fed with the PirB-9G10 antibody (60%) compared to the group fed the PirA-7C12 antibody (3%) and the control group (0%). This suggests that VLRB antibodies may be a suitable alternative to immunoglobulin-based antibodies, as passive immunization treatments for effective management of AHPND outbreaks within shrimp farms.
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http://dx.doi.org/10.3390/vaccines9010055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829966PMC
January 2021

Efficacy of Feed-Based Formalin-Killed Vaccine of Stimulates the Gut-Associated Lymphoid Tissues and Immune Response of Red Hybrid Tilapia.

Vaccines (Basel) 2021 Jan 14;9(1). Epub 2021 Jan 14.

Faculty of Marine Science, Lasbela University of Agricultural, Water and Marine Science, Uthal District Lasbela, Balochistan 90150, Pakistan.

Red hybrid tilapia were fed a formalin-killed oral vaccine (FKV) in the present study was assessed. Three hundred Red hybrid tilapia 80 ± 10 g were divided into five groups (1A, 1B, 2A, 2B, and Cx), each consisting of 60 fish. Fish from Groups 1A, 1B, 2A, and 2B were fed with FKV over different periods of administration, while Group 2B was the only group of fish to receive an oral booster vaccination on day 14- and 21-days post-vaccination (dpv). Group Cx was fed with normal pellets containing no vaccine as a control group. At four weeks post-vaccination (wpv), all fish were experimentally infected with . Groups 2A and 2B had the lowest level of mortalities following vaccination (45% and 30%, respectively) compared to Groups 1A and 1B (80% and 55%, respectively), while the level of mortalities in Group Cx was 100%. All vaccinated groups showed a significant increase in anti- IgM levels ( < 0.05) in serum, mucus, and gut-lavage, while Group Cx did not ( > 0.05) and all fish in this group died by five weeks post-infection. In conclusion, fish fed with the FKV had a greater level of protection against , with increased specific antibody response to the vaccine and there was also evidence of GALT stimulation by the vaccine.
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http://dx.doi.org/10.3390/vaccines9010051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830294PMC
January 2021

Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder () to Nervous Necrosis Virus.

Int J Mol Sci 2020 Jun 11;21(11). Epub 2020 Jun 11.

Laboratory of Aquatic Animal Diseases, Research Institute of Natural Science, College of Veterinary Medicine, Gyeongsang National University, 501 Jinju, Gyeongnam 52828, Korea.

The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3ε and CD4-1 T cells in olive flounder () in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3ε T cell populations contain both types of CD4 cells, with the majority of the CD4 T cell subpopulations being CD4-1/CD4-2 cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.
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http://dx.doi.org/10.3390/ijms21114180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312829PMC
June 2020

The Importance of Porins and β-Lactamase in Outer Membrane Vesicles on the Hydrolysis of β-Lactam Antibiotics.

Int J Mol Sci 2020 Apr 17;21(8). Epub 2020 Apr 17.

Laboratory of Aquatic Animal Diseases, Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea.

Gram-negative bacteria have an outer membrane inhibiting the entry of antibiotics. Porins, found within the outer membrane, are involved in regulating the permeability of β-lactam antibiotics. β-lactamases are enzymes that are able to inactivate the antibacterial properties of β-lactam antibiotics. Interestingly, porins and β-lactamase are found in outer membrane vesicles (OMVs) of β-lactam-resistant and may be involved in the survival of susceptible strains of in the presence of antibiotics, through the hydrolysis of the β-lactam antibiotic. In this study, OMVs isolated from β-lactam-resistant and from mutants, lacking porin or β-lactamase, were evaluated to establish if the porins or β-lactamase in OMVs were involved in the degradation of β-lactam antibiotics. OMVs isolated from deficient in β-lactamase did not show any degradation ability against β-lactam antibiotics, while OMVs lacking OmpC or OmpF showed significantly lower levels of hydrolyzing activity than OMVs from parent . These data reveal an important role of OMVs in bacterial defense mechanisms demonstrating that the OmpC and OmpF proteins allow permeation of β-lactam antibiotics into the lumen of OMVs, and antibiotics that enter the OMVs can be degraded by β-lactamase.
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http://dx.doi.org/10.3390/ijms21082822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215730PMC
April 2020

Reclassification of subsp. Ottem . 2009 as sp. nov., subsp. subsp. nov. and emended description of .

Int J Syst Evol Microbiol 2020 Mar;70(3):2034-2048

Fish Health Research Group, Norwegian Veterinary Institute, Oslo, Pb 750 Sentrum, N-0106 Oslo, Norway.

is a fastidious facultative intracellular bacterial pathogen that causes 'piscine francisellosis', a serious disease affecting both marine and fresh water farmed and wild fish worldwide. Currently two subspecies are recognized, i.e. subsp. and subsp. . In the present study, the taxonomy of was revisited using a polyphasic approach, including whole genome derived parameters such as digital DNA-DNA hybridization, whole genome average nucleotide identity (wg-ANIm), whole genome phylogenetic analysis, whole genome G+C content, metabolic fingerprinting and chemotaxonomic analyses. The results indicated that isolates belonging to subsp. represent a phenotypically and genetically homogenous taxon, clearly distinguishable from subsp. that fulfils requirements for separate species status. We propose, therefore, elevation of subsp. to the species rank as sp. nov. with the type strain remaining as Ehime-1 (DSM 21254=LMG 24544). Furthermore, we identified sufficient phenotypic and genetic differences between subsp. recovered from diseased farmed Atlantic salmon in Chile and those isolated from wild and farmed Atlantic cod in Northern Europe to warrant proposal of the Chilean as a novel subspecies, i.e. subsp. subsp. nov. with strain PQ1106 (CECT 9798=NCTC14375) as the type strain. Finally, we emend the description of by including further metabolic information and the description of atypical strains.
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http://dx.doi.org/10.1099/ijsem.0.004009DOI Listing
March 2020

Characterization of Hagfish () Variable Lymphocyte Receptor-Based Antibody and Its Potential Role in the Neutralization of Nervous Necrosis Virus.

J Immunol 2020 02 13;204(3):718-725. Epub 2019 Dec 13.

Laboratory of Aquatic Animal Diseases, Research Institute of Natural Science, College of Veterinary Medicine, Gyeongsang National University, Jinju, Gyeongnam 52828, South Korea;

The variable lymphocyte receptor (VLR) mediates the humoral immune response in jawless vertebrates, including lamprey () and hagfish (). Hagfish VLRBs are composed of leucine-rich repeat (LRR) modules, conjugated with a superhydrophobic C-terminal tail, which contributes to low levels of expression in recombinant protein technology. In this study, we screened Ag-specific VLRBs from hagfish immunized with nervous necrosis virus (NNV). The artificially multimerized form of VLRB was constructed using a mammalian expression system. To enhance the level of expression of the Ag-specific VLRB, mutagenesis of the VLRB was achieved in vitro through domain swapping of the LRR C-terminal cap and variable LRR module. The mutant VLRB obtained, with high expression and secretion levels, was able to specifically recognize purified and progeny NNV, and the Ag binding ability of this mutant was increased by at least 250-fold to that of the nonmutant VLRB. Furthermore, preincubation of the Ag-specific VLRB with NNV reduced the infectivity of NNV in E11 cells in vitro, and in vivo experiment. Our results suggest that the newly developed Ag-specific VLRB has the potential to be used as diagnostic and therapeutic reagents for NNV infections in fish.
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http://dx.doi.org/10.4049/jimmunol.1900675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971507PMC
February 2020

Comparison of histologic methods for the detection of spores in the gills of Atlantic salmon.

J Vet Diagn Invest 2020 Jan 18;32(1):142-146. Epub 2019 Nov 18.

Moredun Research Institute, Pentlands Science Park, Scotland, UK (Herrero, Dagleish, Thompson).

is a microsporidian associated with gill disease in farmed Atlantic salmon (). Detection of the parasite in histologic tissue sections is challenging using common histochemical stains given that the small, widely distributed parasite spores typically occur individually or in small clusters. We compared the ability of 4 histologic methods to detect spores in serial sections of Atlantic salmon gill tissue: hematoxylin and eosin (H&E), Gram-Twort (GT), calcofluor white (CW), and immunohistochemistry (IHC). Using CW as a benchmark to calculate a relative ratio, IHC consistently detected more spores than CW (median: 1.3), followed by GT (median: 0.2) and H&E (median: 0.1). IHC detected significantly more spores than GT ( < 0.05) and H&E ( < 0.05), and GT more than H&E ( < 0.05). We found significant underestimation of numbers of microsporidia spores in gill disease in Atlantic salmon using conventional histochemical stains and recommend the use of CW or IHC to detect the parasite in tissue sections.
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http://dx.doi.org/10.1177/1040638719887707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003232PMC
January 2020

Involvement of CD4-1 T cells in the cellular immune response of olive flounder (Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and nervous necrosis virus (NNV) infection.

Dev Comp Immunol 2020 02 9;103:103518. Epub 2019 Oct 9.

Lab. of Aquatic Animal Diseases, Research Institute of Natural Science, College of Veterinary Medicine, Gyeongsang National University, 501 Jinju, Gyeongnam, 52828, South Korea; Centre for Marine Bioproducts Development, Flinders University, Bedford Park, Adelaide, SA, 5042, Australia. Electronic address:

The occurrence of CD4 helper T cells has already been established for a number of teleost species, though, it has not been possible to analyze these responses at a cellular level due to a large lack of appropriate monoclonal antibodies (mAbs). In the present study, we produced a mAb against olive flounder (Paralichthys olivaceus) CD4-1 lymphocyte to investigate the functional activity of the cells to improve our understanding of the T cell response in this species. This mAb is specifically able to detect CD4-1 lymphocytes in olive flounder proved by immunofluorescence staining and RT-PCR analysis. In flow cytometry analysis, the number of CD4-1-positive lymphocytes was observed to gradually increase from 3 days post infection (dpi) and then reach peak at 7 dpi against two viruses challenge. As a conclusion, both the basic properties of CD4-1 T cells and its response to viral infections in olive flounder are very similar to the helper T cells in terrestrial animals.
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http://dx.doi.org/10.1016/j.dci.2019.103518DOI Listing
February 2020

Whole cell inactivated autogenous vaccine effectively protects red Nile tilapia (Oreochromis niloticus) against francisellosis via intraperitoneal injection.

J Fish Dis 2019 Aug 11;42(8):1191-1200. Epub 2019 Jun 11.

Faculty of Natural Sciences, Institute of Aquaculture, University of Stirling, Stirling, UK.

Francisella noatunensis subsp. orientalis is a pathogen of tilapia and other warm-water fish for which no vaccines are commercially available. In this study, a whole cell formalin-inactivated vaccine was developed for the first time using the highly virulent isolate STIR-GUS-F2f7 and the oil-based adjuvant Montanide™ ISA 763A VG. The efficacy of the vaccine was assessed in red Nile tilapia via intraperitoneal (i.p.) injection using homologous experimental infection and correlates of protection such as seral antibody production and bacterial loads in the spleen. For immunization, fish were i.p. injected with 0.1 ml of the vaccine, the adjuvant alone or PBS. At 840 degree days post-vaccination, all fish were i.p. injected with 4.0 × 10 CFU/fish of pathogenic bacteria. The RPS at the end of the trial was 100% in the vaccinated group with significantly higher survival than in the adjuvant and control groups. The RPS in the adjuvant group was 42%, and no significant difference was seen in survival between this and the PBS group. Moreover, significantly higher antibody titres in the serum and significantly lower bacterial loads in the spleen were detected in the vaccinated fish by ELISA and qPCR, respectively. These findings highlight the potential of autogenous vaccines for controlling francisellosis in tilapia.
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http://dx.doi.org/10.1111/jfd.13041DOI Listing
August 2019

Pattern Recognition by Melanoma Differentiation-Associated Gene 5 (Mda5) in Teleost Fish: A Review.

Front Immunol 2019 26;10:906. Epub 2019 Apr 26.

Laboratory of Aquatic Animal Diseases, College of Veterinary Medicine, Gyeongsang National University, Jinju, South Korea.

Teleost fish, as with other vertebrates, rely on their innate immune system as a first line of defense against invading pathogens. A very important characteristic of the innate immune response is its ability to recognize conserved molecular structures, such as viral dsRNA and ssRNA. Mda5 is one of the three pattern recognition receptors (PRRs) that recognize cytoplasmic viral ligands. Teleost Mda5 is widely conserved among several fish species and possesses the same structural domains as those seen in their mammalian counterparts. Fish Mda5 has been shown to be capable of initiating an inflammatory response both (in different fish cell lines) and using synthetic viral analogs or virus. The interferon (IFN) pathway is triggered as a result of Mda5 activation, leading to the expression of type I IFNs, IFN- stimulated genes and pro-inflammatory cytokines. Although it is known that Mda5 acts as a receptor for virally-produced ligands, it has been shown more recently that it can also initiate an immune response against bacterial challenges. This review discusses recent advances in the characterization of teleost Mda5 and its potential role in antiviral and antibacterial immunity in teleost fish.
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http://dx.doi.org/10.3389/fimmu.2019.00906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497758PMC
September 2020

Efficacy of an inactivated whole-cell injection vaccine for nile tilapia, Oreochromis niloticus (L), against multiple isolates of Francisella noatunensis subsp. orientalis from diverse geographical regions.

Fish Shellfish Immunol 2019 Jun 2;89:217-227. Epub 2019 Apr 2.

Institute of Aquaculture, Faculty of Natural Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, UK.

Francisellosis, induced by Francisella noatunensis subsp. orientalis (Fno), is an emerging bacterial disease representing a major threat to the global tilapia industry. There are no commercialised vaccines presently available against francisellosis for use in farmed tilapia, and the only available therapeutic practices used in the field are either the prolonged use of antibiotics or increasing water temperature. Recently, an autogenous whole cell-adjuvanted injectable vaccine was developed that gave 100% relative percent survival (RPS) in tilapia challenged with a homologous isolate of Fno. In this study, we evaluated the efficacy of this vaccine against challenge with heterologous Fno isolates. Healthy Nile tilapia, Oreochromis niloticus (∼15 g) were injected intraperitoneally (i.p.) with the vaccine, adjuvant-alone or phosphate buffer saline (PBS) followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection in all groups of vaccinated tilapia, with a significantly higher RPS of 82.3% obtained against homologous challenge, compared to 69.8% and 65.9% with the heterologous challenges. Protection correlated with significantly higher specific antibody responses, and western blot analysis demonstrated cross-isolate antigenicity with fish sera post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by qPCR in conjunction with significantly greater expression of IgM, IL-1 β, TNF-α and MHCII, 72 h post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to fish injected with adjuvant-alone and PBS. The Fno vaccine described in this study may provide a starting point for development a broad-spectrum highly protective vaccine against francisellosis in tilapia.
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http://dx.doi.org/10.1016/j.fsi.2019.03.071DOI Listing
June 2019

Development of a modified yeast display system for screening antigen-specific variable lymphocyte receptor B in hagfish (Eptatretus burgeri).

J Immunol Methods 2019 03 3;466:24-31. Epub 2019 Jan 3.

Laboratory of Aquatic Animal Diseases, Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, 501 Jinju-daero, Jinju, Gyeongnam 660-701, South Korea. Electronic address:

The variable lymphocyte receptor B (VLRB) of jawless vertebrates has a similar function to the antibodies produced by jawed vertebrates, and has been considered as an alternative source to mammalian antibodies for use in biological research. We developed a modified yeast display vector system (pYD8) to display recombinant hagfish VLRB proteins on the extracellular surface of yeast for the isolation of antigen-specific VLRBs. After observing an up-regulation in the VLRB response in hagfish immunized with hemagglutinin 1 of avian influenza virus H9N2 subtype (H9N2-HA1), the antigen-specific VLRBs decorated on the yeast's surface were selected by quantitative library screening through magnetic-activated cell sorting (MACS) and fluorescent-activated cell sorting (FACS). We also demonstrated a strong specificity of the antigen-specific VLRBs, when expressed as a secreted protein using a mammalian expression system. Together, our findings suggest that the pYD8 vector system could be useful for screening antigen-specific hagfish VLRBs, and the specificity of secreted VLRB may have potential for a variety of biological applications.
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http://dx.doi.org/10.1016/j.jim.2019.01.001DOI Listing
March 2019

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.).

J Fish Dis 2019 Feb 6;42(2):229-236. Epub 2018 Dec 6.

Institute of Aquaculture, University of Stirling, Stirling, UK.

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country-wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin-inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin-killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate-buffered saline or vaccine formulated with Montanide by intra-peritoneal (i.p.) injection and challenged by intra-muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post-vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un-adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.
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http://dx.doi.org/10.1111/jfd.12919DOI Listing
February 2019

Dual functionality of lamprey VLRB C-terminus (LC) for multimerization and cell surface display.

Mol Immunol 2018 12 5;104:54-60. Epub 2018 Nov 5.

Laboratory of Aquatic Animal Diseases, College of Veterinary Medicine, Gyeongsang National University 900 Gajwadong, Jinju, Gyeongnam, 660-701, South Korea. Electronic address:

Lamprey, one of the living representatives of jawless vertebrates, uses variable lymphocyte receptors B (VLRB) for antigen recognition, rather than immunoglobulin (Ig) based receptors as used by higher vertebrates. The C-terminus of lamprey VLRB (LC) possess a glycosylphosphatidylinositol (GPI) signal sequence and seven cysteine residues providing dual functionality of the VLRB antibody in the form of a humoral agglutinin and cell membrane receptors. Here, we show that the LC can be either secreted or be membrane anchored as a heterologous fused protein in a multimeric form comprising of eight or ten monomeric units. Using serially truncated LC variants, we showed that the LC, in which the last three amino acid "RKR" were deleted, referred to as LC7, was the most suitable domain for multimeric construction, whereas, the intact LC is more tailored for applications involving membrane anchorage. We show that an antibody specific for viral hemorrhagic septicemia virus (VHSV) (VLR43), displayed on HEK-293F cells using a PiggyBac (PB) transposase system, exhibited a dose-dependent reaction with its antigen, verifying that the LC can be applied in antibody display technology. Therefore, the present report provides valuable insight into the structure of the lamprey VLRB and highlights its potential use as a novel fusion partner for multimerization and membrane anchorage of chimeric proteins.
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http://dx.doi.org/10.1016/j.molimm.2018.10.020DOI Listing
December 2018

Expression and characterization of monomeric variable lymphocyte receptor B specific to the glycoprotein of viral hemorrhagic septicemia virus (VHSV).

J Immunol Methods 2018 11 16;462:48-53. Epub 2018 Aug 16.

Laboratory of Aquatic Animal Diseases, College of Veterinary Medicine, Gyeongsang National University, 900 Gajwadong, Jinju 660-701, South Korea. Electronic address:

Monomeric variable lymphocyte receptor B (VLRB) is one of the smallest binding scaffold (20-25 kDa) from jawless vertebrates, hagfish and lamprey. This relatively new class of binding scaffold has various advantages: i) it has a single peptide composition, amenable to molecular engineering for enhancing its stability and affinity; ii) it has a small size, contributing better tissue penetration and easier production using microorganism expression system. Monomeric arVLRB142, which can specifically bind to the glycoprotein of viral hemorrhagic septicemia virus (VHSV), was expressed in Pichia pastoris. High quantity recombinant monomeric arVLRB142 (rVLR142) was purified from 100 ml of culture with a resulting yield of 2.6 ±1.3 mg of target protein. Functional studies revealed that the purified rVLR142 can specifically recognize low levels of the target antigen (recombinant glycoprotein) (i.e. as low as 0.1 nM), but also the native glycoprotein of VHSV. The expressed rVLR142 exhibited high levels of stability and it retained it binding capacity over broad temperature (4 °C ~ 60 °C) and pH ranges (pH 1.5-12.5). We developed an effective expression system for mass production of monomeric VLRB based on P. pastoris. The recombinant protein that was obtained offers promising binding avidity and biophysical stability and its potential use in various biotechnological applications.
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http://dx.doi.org/10.1016/j.jim.2018.08.006DOI Listing
November 2018

Globular-shaped variable lymphocyte receptors B antibody multimerized by a hydrophobic clustering in hagfish.

Sci Rep 2018 Jul 17;8(1):10801. Epub 2018 Jul 17.

Laboratory of Aquatic Animal Diseases, Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, 501 Jinju-daero, Jinju, 52828, South Korea.

In hagfish and lampreys, two representative jawless vertebrates, the humoral immunity is directly mediated by variable lymphocyte receptors B (VLRBs). Both monomeric VLRBs are structurally and functionally similar, but their C-terminal tails differ: lamprey VLRB has a Cys-rich tail that forms disulfide-linked pentamers of dimers, contributing to its multivalency, whereas hagfish VLRB has a superhydrophobic tail of unknown structure. Here, we reveal that VLRBs obtained from hagfish plasma have a globular-shaped multimerized form (approximately 0.6 to 1.7 MDa) that is generated by hydrophobic clustering instead of covalent linkage. Electron microscopy (EM) and single-particle analysis showed that the multimerized VLRBs form globular-shaped clusters with an average diameter of 28.7 ± 2.2 nm. The presence of VLRBs in the complex was confirmed by immune-EM analysis using an anti-VLRB antibody. Furthermore, the hydrophobic hagfish C-terminus (HC) was capable of triggering multimerization and directing the cellular surface localization via a glycophosphatidylinositol linkage. Our results strongly suggest that the hagfish VLRB forms a previously unknown globular-shaped antibody. This novel identification of a structurally unusual VLRB complex may suggest that the adaptive immune system of hagfish differs from that of lamprey.
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http://dx.doi.org/10.1038/s41598-018-29197-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050320PMC
July 2018

Streptococcus agalactiae infection kills red tilapia with chronic Francisella noatunensis infection more rapidly than the fish without the infection.

Fish Shellfish Immunol 2018 Oct 11;81:221-232. Epub 2018 Jul 11.

AquaAcademy Farm, Tha Chana, Surat Thani, 84170, Thailand; Department of Anatomy, Faculty of Science, Prince of Songkla University, Hatyai, Songkla, 90112, Thailand.

In this study we examined the effect that a Francisella noatunensis (Fno) infection had on hybrid red tilapia (Oreochromis niloticus × Oreochromis mossambicus) subsquently infected with Streptococcus agalactiae. A variety of hemato-immunological parameters (haematocrit, total red blood cell count, mean corpuscular volume, total white blood and differential cell counts, total plasma protein, plasma lysozyme and plasma peroxidase activities, and respiratory burst and phagocytic activities of head-kidney macrophages) were measured in hybrid red tilapia that had been previously exposed to an Fno outbreak in a tilapia grow-out farm. The head-kidneys of these apparently healthy survivors, when checked by PCR were found to be Fno-positive with hemato-immunological parameters that were similar to fish without an a priori infection. The only exception was the percentage lymphocyte count in the peripheral blood, which was slightly, but significantly, lower in the Fno-infected fish, compared to those without the infection. When experimentally infected with S. agalactiae, the Fno-infected fish died more rapidly and at a significantly higher rate than fish without the infection. During the challenge, the hemato-immunological parameters of both groups of fish were very similar, although the Fno-infected fish, challanged with S. agalactiae expressed significantly higher plasma lysozyme and peroxidase activities, and their head kidney macrophages had significantly higher respiratory burst activity compared to non-Fno-infected fish challanged with S. agalactiae. The only two parameters for which Fno-infected fish showed significantly lower expressions than that of their non-infected counterparts were haematocrit and total red blood cell count. The cause of the rapidity and higher rates of mortality observed in the Fno-infected fish when challenged with S. agalactiae is unknown; but it may be due to a reduced erythropoiesis capability within the head-kidney because of the presence of Fno.
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http://dx.doi.org/10.1016/j.fsi.2018.07.022DOI Listing
October 2018

Complete Genome Sequences of Three Fish-Associated Isolates.

Genome Announc 2018 Feb 8;6(6). Epub 2018 Feb 8.

Aquaculture Research Group, Moredun Research Institute, Pentlands Science Park, Penicuik, United Kingdom.

The whole-genome sequences are described here for three group B (GBS) () serotype Ib isolates obtained from tilapia () farmed at sites in Honduras, Costa Rica, and the United States. The bacteria were isolated from the brains of fish displaying signs of streptococcosis.
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http://dx.doi.org/10.1128/genomeA.00025-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805872PMC
February 2018

A Polyphasic Approach for Phenotypic and Genetic Characterization of the Fastidious Aquatic Pathogen subsp. .

Front Microbiol 2017 12;8:2324. Epub 2017 Dec 12.

Faculty of Natural Sciences, Institute of Aquaculture, University of Stirling, Stirling, United Kingdom.

subsp. () is the causative agent of piscine francisellosis, an emerging infectious disease in Asia and Latin America. In this study two outbreaks of francisellosis were diagnosed in the UK on the basis of histopathology, electron microscopy, PCR, bacterial isolation and fulfillment of Koch's postulates. Furthermore, a phenotypic fingerprint based on biochemical analyses, metabolic activity, chemotaxonomic composition, and antimicrobial assays was generated for the novel isolates, the type strain Ehime-1 from Asia and other from Latin America. The genetic relatedness between the novel and other species was investigated by sequencing and comparing the 16SrRNA gene, 8 housekeeping genes (individually and concatenated) and the 16SrRNA-ITS-23SrRNA sequence. The phenotypic profiling indicated a high degree of similarity among the strains as all were able to metabolize dextrin, N-acetyl-D glucosamine, D-fructose, α-D-glucose, D-mannose, methyl pyruvate, acetic acid, α-keto butyric acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-glutamic acid, L-proline, L-serine, L-threonine, inosine, uridine, glycerol, D L-α-glycerol phosphate, glucose-1-phosphate, and glucose-6-phosphate. The chemotaxonomic analyses indicated that 24:1 (20.3%), 18:1n-9 (16.9%), 24:0 (13.1%) 14:0 (10.9%), 22:0 (7.8%), 16:0 (7.6%), and 18:0 (5.5%) were the predominant structural fatty acids in . The antimicrobial assays showed little variation between the isolates and high susceptibility to enrofloxacin, gentamicin, neomycin, streptomycin, amikacin, ciprofloxacin, gatifloxacin, nitrofurantoin, tobramycin, kanamycin, tetracycline, oxytetracycline, florfenicol, oxolinic acid, and streptomycin in all the analyzed. In all the phylogenetic trees the strains clustered together in independent branches confirming a high degree of homogeneity. Interestingly in five of the 11 trees i.e., , 16SrRNA-ITS-23SrRNA, and concatenated sequence the two ssp. diverged more from each other than from the closely related (). The phenotypic and genetic characterization confirmed the isolates represent a solid phylo-phenetic taxon that in the current context of the genus seems to be misplaced within the species . We propose the use of the present polyphasic approach in future studies to characterize strains of and and verify their current taxonomic rank of and other aquatic spp.
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http://dx.doi.org/10.3389/fmicb.2017.02324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733052PMC
December 2017

Complete Genome Sequences of Three Serotype Ia Isolates Obtained from Disease Outbreaks in Nile Tilapia ().

Genome Announc 2018 Jan 4;6(1). Epub 2018 Jan 4.

Aquaculture Research Group, Moredun Research Institute, Bush Loan, Penicuik, United Kingdom.

This paper describes the whole-genome sequences for three serotype Ia isolates. The isolates were recovered from the brains of clinically sick tilapia, , that were suffering from streptococcosis. One isolate was from tilapia in the United States and the other two from fish in China.
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http://dx.doi.org/10.1128/genomeA.01432-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754503PMC
January 2018

Impact of Salmonid alphavirus infection in diploid and triploid Atlantic salmon (Salmo salar L.) fry.

PLoS One 2017 26;12(9):e0179192. Epub 2017 Sep 26.

Institute of Aquaculture, University of Stirling, Stirling, United Kingdom.

With increasing interest in the use of triploid salmon in commercial aquaculture, gaining an understanding of how economically important pathogens affect triploid stocks is important. To compare the susceptibility of diploid and triploid Atlantic salmon (Salmo salar L.) to viral pathogens, fry were experimentally infected with Salmonid alphavirus sub-type 1 (SAV1), the aetiological agent of pancreas disease (PD) affecting Atlantic salmon aquaculture in Europe. Three groups of fry were exposed to the virus via different routes of infection: intraperitoneal injection (IP), bath immersion, or cohabitation (co-hab) and untreated fry were used as a control group. Mortalities commenced in the co-hab challenged diploid and triploid fish from 11 days post infection (dpi), and the experiment was terminated at 17 dpi. Both diploid and triploid IP challenged groups had similar levels of cumulative mortality at the end of the experimental period (41.1% and 38.9% respectively), and these were significantly higher (p < 0.01) than for the other challenge routes. A TaqMan-based quantitative PCR was used to assess SAV load in the heart, a main target organ of the virus, and also liver, which does not normally display any pathological changes during clinical infections, but exhibited severe degenerative lesions in the present study. The median viral RNA copy number was higher in diploid fish compared to triploid fish in both the heart and the liver of all three challenged groups. However, a significant statistical difference (p < 0.05) was only apparent in the liver of the co-hab groups. Diploid fry also displayed significantly higher levels of pancreatic and myocardial degeneration than triploids. This study showed that both diploid and triploid fry are susceptible to experimental SAV1 infection. The lower virus load seen in the triploids compared to the diploids may possibly be related to differences in cell metabolism between the two groups, however, further investigation is necessary to confirm this and also to assess the outcome of PD outbreaks in other developmental stages of the fish when maintained in commercial production systems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179192PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614425PMC
October 2017

Development of a monoclonal antibody against the CD3ε of olive flounder (Paralichthys olivaceus) and its application in evaluating immune response related to CD3ε.

Fish Shellfish Immunol 2017 Jun 20;65:179-185. Epub 2017 Apr 20.

Laboratory of Aquatic Animal Diseases, College of Veterinary Medicine, Gyeongsang National University, 900 Gajwa-dong, Jinju, Gyeongnam, 660-701, South Korea. Electronic address:

The T cell receptor (TCR) is the binding site of antigen and is responsible for specifically activating the adaptive immune response. CD3, an essential component of the CD3-TCR complex, is known to be composed of γδ and ε chains in teleost. However, there are few monoclonal antibodies (mAb) available to identify these molecules on T cells, so we aimed to produce a mAb against CD3ε to improve our understanding of T cell immune response in olive flounder (Paralichthys olivaceus). CD3ε recombinant protein was expressed in yeast, the expression of which was confirmed by SDS-PAGE, MALDI-TOF/TOF MS and Western blot analysis. A CD3ε-specific mAb 4B2 was selected, the specificity of which was examined by confocal microscopy, flow cytometry and RT-PCR, and the mAb was subsequently used to examine the CD3ε lymphocyte population in several different immune organs, with relatively high percentages of these cells seen in trunk-kidney and spleen, while lower percentages were seen in the liver and peripheral blood of olive flounder. During a viral hemorrhagic septicemia virus (VHSV) infection in olive flounder, the number of CD3ε lymphocytes was seen to gradually increase in the liver, spleen and trunk-kidney of infected fish until 7 days post infection (dpi). In peripheral blood, on the other hand, the increase in CD3ε lymphocyte numbers peaked by 3 dpi. These results suggest that CD3ε lymphocytes might be involved in the immune response against VHSV.
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http://dx.doi.org/10.1016/j.fsi.2017.04.016DOI Listing
June 2017

Genetic and serological diversity of Flavobacterium psychrophilum isolates from salmonids in United Kingdom.

Vet Microbiol 2017 Mar 31;201:216-224. Epub 2017 Jan 31.

Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, UK.

Flavobacterium psychrophilum is one of the most important bacterial pathogens affecting cultured rainbow trout (Oncorhynchus mykiss) and is increasingly causing problems in Atlantic salmon (Salmo salar L.) hatcheries. Little is known about the heterogeneity of F. psychrophilum isolates on UK salmonid farms. A total of 315 F. psychrophilum isolates, 293 of which were collected from 27 sites within the UK, were characterised using four genotyping methods and a serotyping scheme. A high strain diversity was identified among the isolates with 54 pulsotypes, ten (GTG)-PCR types, two 16S rRNA allele lineages, seven plasmid profiles and three serotypes. Seven PFGE groups and 27 singletons were formed at a band similarity of 80%. PFGE group P (n=75) was found to be numerically predominant in eight sites within the UK. Two major PFGE clusters and 13 outliers were found at the band similarity of 40%. The predominant profileobserved within the F. psychrophilum isolates examined was PFGE cluster II - (GTG)-PCR type r1-16S rRNA lineage II - serotype Th (70/156 isolates examined, 45%). Co-existence of genetically and serologically heterogeneous isolates within each farm was detected, confounding the ability to control RTFS outbreaks. The occurrence over time (up to 11 years) of F. psychrophilum pulsotypes in three representative sites (Scot I, Scot III and Scot V) within Scotland was examined, potentially providing important epidemiological data for farm management and the development of site-specific vaccines.
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http://dx.doi.org/10.1016/j.vetmic.2017.01.032DOI Listing
March 2017

Detection of the florfenicol resistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule?

FEMS Microbiol Ecol 2017 04;93(4)

Moredun Research Institute, Pentlands Science Park, Penicuik EH26 0PZ, UK.

Bacteria from the family Flavobacteriaceae often show low susceptibility to antibiotics. With the exception of two Chryseobacterium spp. isolates that were positive for the florfenicol resistance gene floR, no clinical resistance genes were identified by microarray in 36 Flavobacteriaceae isolates from salmonid fish that could grow in ≥ 4 mg/L florfenicol. Whole genome sequence analysis of the floR positive isolates revealed the presence of a region that contained the antimicrobial resistance genes floR, a tet(X) tetracycline resistance gene, a streptothricin resistance gene and a chloramphenicol acetyltransferase gene. In silico analysis of 377 published genomes for Flavobacteriaceae isolates from a range of sources confirmed that well-characterised resistance gene cassettes were not widely distributed in bacteria from this group. Efflux pump-mediated decreased susceptibility to a range of antimicrobials was confirmed in both floR positive isolates using an efflux pump inhibitor (phenylalanine-arginine β-naphthylamide) assay. The floR isolates possessed putative virulence factors, including production of siderophores and haemolysins, and were mildly pathogenic in rainbow trout. Results support the suggestion that, despite the detection of floR, susceptibility to antimicrobials in Flavobacteriaceae is mostly mediated via intrinsic mechanisms rather than the horizontally acquired resistance genes more normally associated with Gram-negative bacterial pathogens such as Enterobacteriaceae.
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http://dx.doi.org/10.1093/femsec/fix015DOI Listing
April 2017

A comparison of the response of diploid and triploid Atlantic salmon (Salmo salar) siblings to a commercial furunculosis vaccine and subsequent experimental infection with Aeromonas salmonicida.

Fish Shellfish Immunol 2016 Oct 25;57:301-308. Epub 2016 Aug 25.

Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, UK.

Sterile triploid fish represent a solution to the problems associated with sexual maturation and escapees in aquaculture. However, as disease outbreaks continue to cause significant economic losses to the industry, it is essential that the response of triploids to disease and disease treatments be characterised. The aim of this study was to compare the response of triploid Atlantic salmon to a commercial furunculosis vaccine with that of diploid fish, and to assess the vaccine efficacy in the two ploidies through an experimental infection with Aeromonas salmonicida. Diploid and triploid Atlantic salmon were injected intraperitoneally with either phosphate buffered saline, liquid paraffin adjuvant or a commercial furunculosis vaccine. Following vaccination, growth, adhesion scores and a variety of assays to assess immune function, such as respiratory burst and antibody response, were measured. Vaccination did not have a significant effect on the weight of either ploidy prior to challenge at 750° days. Adhesion scores were significantly higher in vaccinated fish compared to unvaccinated fish, although no effect of ploidy was observed. Ploidy significantly affected respiratory burst activity following vaccination, however, with triploids exhibiting higher activity than diploids. Combined with lower white blood cell numbers observed in the triploids, it may be that this low cell number is compensated for by increased cellular activity. Ploidy however, did not have a significant effect on complement activity or antibody response, with significantly higher antibody levels detected in all vaccinated fish compared to unvaccinated controls. In addition, both ploidy groups were well protected following challenge with no difference in the relative percentage survival. Based on these results, it appears that ploidy does not affect the severity of adhesions that result post-vaccinate or in the fish's immune response following vaccination, and the furunculosis vaccine performs equally well in both diploid and triploid Atlantic salmon.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042121PMC
http://dx.doi.org/10.1016/j.fsi.2016.08.049DOI Listing
October 2016

Understanding the interaction between Betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy.

Fish Shellfish Immunol 2016 Jun 17;53:35-49. Epub 2016 Mar 17.

Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, United Kingdom.

Over the last three decades, the causative agent of viral encephalopathy and retinopathy (VER) disease has become a serious problem of marine finfish aquaculture, and more recently the disease has also been associated with farmed freshwater fish. The virus has been classified as a Betanodavirus within the family Nodaviridae, and the fact that Betanodaviruses are known to affect more than 120 different farmed and wild fish and invertebrate species, highlights the risk that Betanodaviruses pose to global aquaculture production. Betanodaviruses have been clustered into four genotypes, based on the RNA sequence of the T4 variable region of their capsid protein, and are named after the fish species from which they were first derived i.e. Striped Jack nervous necrosis virus (SJNNV), Tiger puffer nervous necrosis virus (TPNNV), Barfin flounder nervous necrosis virus (BFNNV) and Red-spotted grouper nervous necrosis virus (RGNNV), while an additional genotype turbot betanodavirus strain (TNV) has also been proposed. However, these genotypes tend to be associated with a particular water temperature range rather than being species-specific. Larvae and juvenile fish are especially susceptible to VER, with up to 100% mortality resulting in these age groups during disease episodes, with vertical transmission of the virus increasing the disease problem in smaller fish. A number of vaccine preparations have been tested in the laboratory and in the field e.g. inactivated virus, recombinant proteins, virus-like particles and DNA based vaccines, and their efficacy, based on relative percentage survival, has ranged from medium to high levels of protection to little or no protection. Ultimately a combination of effective prophylactic measures, including vaccination, is needed to control VER, and should also target larvae and broodstock stages of production to help the industry deal with the problem of vertical transmission. As yet there are no commercial vaccines for VER and the aquaculture industry eagerly awaits such a product. In this review we provide an overview on the current state of knowledge of the disease, the pathogen, and interactions between betanodavirus and its host, to provide a greater understanding of the multiple factors involved in the disease process. Such knowledge is needed to develop effective methods for controlling VER in the field, to protect the various aquaculture species farmed globally from the different Betanodavirus genotypes to which they are susceptible.
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http://dx.doi.org/10.1016/j.fsi.2016.03.033DOI Listing
June 2016

Expression of immunogenic structural proteins of cyprinid herpesvirus 3 in vitro assessed using immunofluorescence.

Vet Res 2016 Jan 8;47. Epub 2016 Jan 8.

Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, UK.

Cyprinid herpesvirus 3 (CyHV-3), also called koi herpesvirus (KHV), is the aetiological agent of a fatal disease in carp and koi (Cyprinus carpio L.), referred to as koi herpesvirus disease. The virus contains at least 40 structural proteins, of which few have been characterised with respect to their immunogenicity. Indirect immunofluorescence assays (IFAs) using two epitope-specific monoclonal antibodies (MAbs) were used to examine the expression kinetics of two potentially immunogenic and diagnostically relevant viral antigens, an envelope glycoprotein and a capsid-associated protein. The rate of expression of these antigens was determined following a time-course of infection in two CyHV-3 susceptible cell lines. The results were quantified using an IFA, performed in microtitre plates, and image analysis was used to analyse confocal micrographs, enabling measurement of differential virus-associated fluorescence and nucleus-associated fluorescence from stacks of captured scans. An 8-tenfold increase in capsid-associated protein expression was observed during the first 5 days post-infection compared to a ≤ 2-fold increase in glycoprotein expression. A dominant protein of ~100 kDa reacted with the capsid-associated MAb (20F10) in western blot analysis. This band was also recognised by sera obtained from carp infected with CyHV-3, indicating that this capsid-associated protein is produced in abundance during infection in vitro and is immunogenic to carp. Mass spectrometry carried out on this protein identified it as a previously uncharacterised product of open reading frame 84. This abundantly expressed and immunogenic capsid-associated antigen may be a useful candidate for KHV serological diagnostics.
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http://dx.doi.org/10.1186/s13567-015-0297-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705813PMC
January 2016