Rowena Hoare

The interrelation of growth and disease resistance of different populations of juvenile Atlantic halibut (Hippoglossus hippoglossus L.)

Abstract Growth of juvenile Atlantic halibut from three areas of the North Atlantic (Canada, Iceland and Norway) was studied in an experiment using individual tagged fish reared at 15°C for 85 days. Fish from each population were subsequently split into two groups and acclimatised to either 12°C or 18°C. The fish were then injected intra-peritoneally with a Vibrio anguillarum bacteria suspension and mortality monitored for 4 weeks. Growth rates of the Canadian population ranked lowest, whereas the Norwegian population had the highest mean growth rates (SGR=1.70% day −1 , 1.62% day −1 and 1.53% day −1 for the Norwegian, Icelandic and Canadian populations, respectively). The halibut from Norway had the best survival following bacterial challenge (80%, 50% and 55% survival for the Norwegian, Icelandic and Canadian populations, respectively). Mortality was higher at 18°C than at 12°C in the Icelandic (62% at 12°C and 27% at 18°C) and Canadian (56% at 12°C and 32% at 18°C) fish, whereas a smaller difference between temperatures was observed in the Norwegian fish (25% at 12°C and 13% at 18°C). Fish that survived the challenge test were those that had grown fastest in the growth trial. Low, but significant, correlations between survival and size and growth were seen, but these correlations varied between populations. In the Canadian population, no correlation between size and growth and survival were seen; only size was correlated ( r =0.27) with survival in the Icelandic population, whereas both size ( r =0.18) and growth ( r =0.17) were correlated with survival in the Norwegian population.

Identification of DLD, by immunoproteomic analysis and evaluation as a potential vaccine antigen against three Vibrio species in Epinephelus coioides.

Vibrio spp. represent a serious threat to the culture of Epinephelus coioides (Orange-spotted Grouper) in Southeast Asia. In this study we used two-dimensional electrophoresis (2-DE) and Western blotting to identify common immunogenic proteins of Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus. Membranes were probed with orange-spotted grouper anti-V. alginolyticus sera and accordingly 60, 58 and 48 immunogenic protein spots were detected. By matching analysis for the three Western blotting membranes, 6 cross immunogenic spots for the three Vibrio species were identified. They were Outer membrane protein W (OmpW), dihydrolipoamide dehydrogenase (DLD), succinate dehydrogenase flavoprotein subunit(SDHA), elongation factor Ts(Ts), peptide ABC transporter periplasmic peptide-binding protein and phosphoenolpyruvate carboxykinase(PEPCK). One of the proteins, DLD, was used to evaluate the cross protective function for E. coioides with a bacterial immunization and challenge method. The relative percent survival rate of E. coioides against V. alginolyticus, V. harveyi and V. parahaemolyticus was 90%, 86% and 80%, respectively. This work may provide potential cross protective vaccine candidate antigens for three Vibrio species, and DLD may be considered as an effective cross-protective immunogen against three Vibrio species.

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

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)5-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)5-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.

Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs) were developed against Segment 3 (encoding the viral nucleoprotein, NP) of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique (IFAT) and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex) bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L.) smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p.) injection of virus at 50% Tissue Culture Infective Dose (TCID50) = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point), was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections). To our knowledge this is the first report of the use of Luminex (Bio-Plex) technology for the detection of a fish pathogen.

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

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.

Construction of a Vibrio alginolyticus hopPmaJ (hop) mutant and evaluation of its potential as a live attenuated vaccine in orange-spotted grouper (Epinephelus coioides)

&NA; Vibrio alginolyticus, a bacterial pathogen in fish and humans, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. However, little is known about the associated effectors (T3SEs) and their physiological role. In this study, the T3SE gene hopPmaJ (hop) was cloned from V. alginolyticus wild‐type strain HY9901 and the mutant strain HY9901&Dgr;hop was constructed by the in‐frame deletion method. The results showed that the deduced amino acid sequence of V. alginolyticus HopPmaJ shared 78–98% homology with other Vibrio spp. In addition, the HY9901&Dgr;hop mutant showed an attenuated swarming phenotype and a 2600‐fold decrease in the virulence to grouper. However, the HY9901&Dgr;hop mutant showed no difference in morphology, growth, biofilm formation and ECPase activity. Finally, grouper vaccinated via intraperitoneal (IP) injection with HY9901&Dgr;hop induced a high antibody titer with a relative percent survival (RPS) value of 84% after challenging with the wild‐type HY9901. Real‐time PCR assays showed that vaccination with HY9901&Dgr;hop enhanced the expression of immune‐related genes, including MHC‐I&agr;, MHC‐II&agr;, IgM, and IL‐1&bgr; after vaccination, indicating that it is able to induce humoral and cell‐mediated immune response in grouper. These results demonstrate that the HY9901&Dgr;hop mutant could be used as an effective live vaccine to combat V. alginolyticus in grouper. HighlightsThe biological functions of HopPmaJ in alginolyticus were investigated.HY9901&Dgr;hop suppressed swarming motility, adhesion and virulence.The RPS of grouper vaccinated with HY9901&Dgr;hop was 84%.HY9901&Dgr;hop could stimulate innate and acquired immune responses in E. coioides.

Characterization of the outer membrane proteome of Francisella noatunensis subsp. orientalis

The aims of the current study were to characterize the outer membrane proteins (OMPs) of Francisella noatunensis subsp. orientalis (Fno) STIR‐GUS‐F2f7, and identify proteins recognized by sera from tilapia, Oreochromis niloticus, (L) that survived experimental challenge with Fno.