Oral vaccination of Nile tilapia (Oreochromis niloticus) against francisellosis elevates specific antibody titres in serum and mucus.

Although Nile tilapia (Oreochromis niloticus) is a well-established aquaculture species globally, there are a limited number of commercial vaccines available or are used for this species. The majority of diseases affecting farmed tilapia are bacterial, with antibiotics frequently used to treat fish. The current study was performed to optimise the use of mucosal vaccines for tilapia by adapting an existing bacterin vaccine against Francisella noatunensis subsp. orientalis (Fno) as a proof of concept. This vaccine has previously provided excellent protection by injection, however, the preference for tilapia farmers would be to vaccinate fish by immersion or orally, due to the lower cost and ease of application. These vaccination routes, however, are often less efficacious probably due to the lack of adjuvants in immersion and oral vaccines. The aims of this study, therefore, were to optimise the formulation and dose of the Fno vaccine with mucosal adjuvants for oral and immersion delivery. Tilapia fry (av. 6 g) were given three concentrations (high, medium, low; i.e. 1×109, 1×108 and 1×107 CFU mL-1) of antigen combined with the oral adjuvant by oral gavage, to optimise the dose needed to induce an immune response to Fno, and the immune response obtained compared with fish vaccinated by immersion (with and without an immersion adjuvant). Fry were boosted by the same route at 420 degree days (DD), and samples (serum, mucus ) taken at 840 DD for specific antibody responses measured by ELISA and western blotting. Specific IgM titres were significantly elevated in serum and mucus of fish given the high dose adjuvanted vaccine by gavage. In addition, by western blotting with serum, a significant immunogenic reaction was evident between 20 and 37 kDa in the fish given the high dose oral vaccine by gavage. As protection against Fno provided by the injection vaccine was correlated with specific antibody responses these findings suggest the oral vaccine also has potential to provide protection. Further studies are needed to optimise delivery of the vaccine via feed.

Development and evaluation of a quantitative polymerase chain reaction for aquatic Streptococcus agalactiae based on the groEL gene.

AIMS: The aim of this study was to develop a TaqMan quantitative polymerase chain reaction (qPCR), based on the Streptococcus agalactiae groEL gene, to specifically quantify levels of bacteria within samples derived from aquatic sources, particularly aquaculture. Enumeration of bacteria by qPCR was compared with culture-based methods. METHODS AND RESULTS: The qPCR was sensitive to 33 isolates of S. agalactiae, representing 11 clonal complexes from aquatic, bovine and human hosts. The specificity of the assay was 92·5% at a threshold Cq value of 35. No cross-reaction with Streptococcus iniae was noted and of the 22 comparator species screened to test assay specificity, Streptococcus porcinus had a Cq value of 33·7 S, while Streptococcus gallolyticus subsp. macedonicus and Streptococcus ictaluri had one replicate value above the Cq threshold of 35 (34·5 and 34·4 respectively), while only S. agalactiae were detected with a Cq value of 30. The limit of detection of the assay was 1·7 copies per µl at Cq 35. Discrepancies between molecular and culture-based methods of enumeration were noted. CONCLUSIONS: The qPCR was able to detect a diverse range of S. agalactiae isolates from different clonal complexes (CCs) and could distinguish between S. agalactiae and closely related species, notably S. iniae. The results suggest that a Cq 30 would be a very meaningful cut-off, allowing the detection of infected fish while ruling out all false positives. SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid and sensitive qPCR assay is useful to quantify DNA copy number in the laboratory and could prove useful for detecting low levels of S. agalactiae in aquaculture systems, including Oreochromis niloticus culture.

Efficacy and safety of a non-mineral oil adjuvanted injectable vaccine for the protection of Atlantic salmon (Salmo salar L.) against Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the causative agent of Rainbow Trout Fry Syndrome which has had a major impact on global salmonid aquaculture. Recent outbreaks in Atlantic salmon in Scotland and Chile have added to the need for a vaccine to protect both salmon and trout. At present no licensed vaccines are available in Europe, leaving antibiotics as the only course of action to contain disease outbreaks. Outbreaks generally occur in fry at temperatures between 10 and 15 °C. Recently outbreaks in larger fish have given added impetus to the development of a vaccine which can provide long term protection from this highly heterogeneous pathogen. Most fish injectable vaccines are formulated with oil emulsion adjuvants to induce strong and long lasting immunity, but which are known to cause side effects. Alternative adjuvants are currently sought to minimise these adverse effects. The current study was performed to assess the efficacy of a polyvalent, whole cell vaccine containing formalin-inactivated F. psychrophilum to induce protective immunity in Atlantic salmon. The vaccine was formulated with an adjuvant containing squalene and aluminium hydroxide, and was compared to a vaccine formulated with a traditional oil adjuvant, Montanide ISA 760VG, and a non-adjuvanted vaccine. Duplicate groups of salmon (23.5 ± 6.8 g) were vaccinated with each of the vaccine formulations or phosphate buffered saline by intraperitoneal injection. Fish were challenged by intramuscular injection with F. psychrophilum six weeks post-vaccination to test the efficacy of the vaccines. Cumulative mortality reached 70% in the control salmon, while the groups of salmon that received vaccine had significantly lower mortality than the controls (p = 0.0001), with no significant difference in survival between vaccinated groups. The squalene/alum adjuvant was safe, more readily metabolised by the fish and induced less histopathological changes than the traditional oil adjuvant.

Identification of aquatic mycobacteria based on sequence analysis of the 16S-23S rRNA internal transcribed spacer region.

PURPOSE: Mycobacteria are common causative agents of bacterial infections in many species of freshwater and marine fish. Identification of mycobacteria to the species level based on phenotypic tests is inappropriate and time consuming. Molecular methods such as partial or entire gene sequence determination in mycobacteria have been employed to resolve these problems. The objective of this study was to assess the use of sequence analysis of the mycobacterial 16S-23S internal transcribed spacer (ITS) region for the identification of different aquatic mycobacteria species. METHODOLOGY: Using published primers, the ITS sequences of 64 field and reference strains were determined.Results/Key findings. The identity of all isolates previously identified as Mycobacterium marinum by RFLP was confirmed as M. marinum by sequence analysis. With the exception of five rapidly growing mycobacteria isolates, all other mycobacteria were easily identified by sequencing of the ITS region. Using this spacer region, it was possible to differentiate between slowly growing and rapidly growing mycobacteria, even before sequence analysis, by the size of the PCR product, although species identification could not be made by size alone. CONCLUSION: Overall, direct sequencing of this genetic element following PCR has been shown to be useful in the identification of aquatic mycobacteria species. With regard to the variability of the ITS region for different mycobacteria isolates, this may be a useful tool in epidemiological studies.

Complex Gill Disease: an Emerging Syndrome in Farmed Atlantic Salmon (Salmo salar L.).

Gill disorders have become a significant problem during the marine phase of farming Atlantic salmon (Salmo salar L.). The term complex gill disease (CGD) includes a wide range of clinical gill disease presentations generally occurring from the end of summer to early winter on marine Atlantic salmon farms. The gross and histological lesions observed are the resultant culmination of exposure to a mixture of environmental insults, pathogenic organisms and farm management practices. None of the three principal agents purportedly associated with CGD (Desmozoon lepeophtherii, salmon gill poxvirus or Candidatus Branchiomonas cysticola) have been cultured successfully in-vitro, so individual in-vivo challenge studies to identify their pathogenesis have not been possible. Studies of cohabitation of single pathogen-infected fish with naïve fish, and epidemiological investigations are required urgently to elucidate the roles of these pathogens and other factors in CGD.

Evaluation of PCR primers targeting the groEL gene for the specific detection of Streptococcus agalactiae in the context of aquaculture.

AIMS: The aim of this study was to design a set of primers for specific detection and identification of Streptococcus agalactiae in polymerase chain reaction (PCR) that can detect a diverse range of S. agalactiae isolates from different hosts and that it is capable of discriminating between S. agalactiae and other species that are closely related or potentially present in aquaculture environments, notably Streptococcus iniae. METHODS AND RESULTS: Primers, based on the groEL2 gene of S. agalactiae, were shown to be epidemiologically sensitive to 97 isolates of S. agalactiae, representing 11 clonal complexes derived from piscine, terrestrial and aquatic mammalian host species. The primers were tested with 10 S. iniae isolates and 22 other comparator species with no cross-reaction observed after optimization of reaction conditions. They have a high analytical sensitivity, detecting as few as 10 copies of S. agalactiae genomic DNA per reaction and are capable of detecting the target in DNA extracted from the brains of infected fish. CONCLUSIONS: The primers proved suitable for the sensitive and specific detection of S. agalactiae from dairy-, human- and fish-related origins by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the importance of S. agalactiae as a pathogen, many PCR primers have been published for this bacterium, designed largely for its detection in dairy and human samples, but many cross-reacting with S. iniae. The ability to differentiate between S. agalactiae and S. iniae in aquaculture derived samples is important as both infect fish, causing similar disease symptoms and are phenotypically similar, yet control strategies and zoonotic risk are species specific.

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

AIMS: 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. METHODS AND RESULTS: The composition of the OMPs of a virulent strain of Fno (STIR-GUS-F2f7), isolated from diseased red Nile tilapia in the United Kingdom, was examined. The sarcosine-insoluble OMPs fraction was screened with tilapia hyperimmune sera by western blot analysis following separation of the proteins by 1D SDS-PAGE. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was used to identify the various proteins present in the OMP profile. Two hundred and thirty-nine proteins were identified, of which 44 were found in the immunogenic band recognized by the tilapia hyperimmune serum. In silico analysis was performed to predict the function and location of the OMPs identified by MS. CONCLUSIONS: Using a powerful proteomic-based approach in conjugation with western immunoblotting, proteins comprising the outer membrane fraction of Fno STIR-GUS-F2f7 were identified, catalogued and screened for immune recognition by tilapia sera. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study is the first report on the characterization of Fno-OMPs. The findings here provide preliminary data on bacterial surface proteins that exist in direct contact with the host's immune defences during infection and offer an insight into the pathogenesis of Fno.

Antimicrobial susceptibility of Flavobacterium psychrophilum isolates from the United Kingdom.

Routine application of antimicrobials is the current treatment of choice for rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum. In this study, the antimicrobial susceptibilities of 133 F. psychrophilum isolates, 118 of which were from the UK, were evaluated by broth microdilution and disc diffusion methods following VET04-A2 and VET03-A guidelines of Clinical and Laboratory Standards Institute (CLSI), respectively. Isolates were categorized as wild type (fully susceptible, WT) or non-wild type (NWT) using normalized resistance interpretation (NRI)-determined cut-off values (COWT ). Broth microdilution testing showed that only 12% of UK isolates were WT to oxolinic acid (MIC COWT  ≤ 0.25 mg/L) and 42% were WT for oxytetracycline (MIC COWT  ≤ 0.25 mg/L). In contrast, all the isolates tested were WT (MIC COWT  ≤ 2 mg/L) for florfenicol, the main antimicrobial for RTFS control in the UK. Disc diffusion-based COWT values were ≥51 mm for 10 µg amoxicillin, ≥44 mm for 30 µg florfenicol, ≥30 mm for 2 µg oxolinic acid and ≥51 mm for 30 µg oxytetracycline. There was a high categorical agreement between the classifications of the isolates by two testing methods for florfenicol (100%), oxytetracycline (93%) and oxolinic acid (99%).

Application of low-frequency sonophoresis and reduction of antibiotics in the aquatic systems.

A major concern in aquaculture is the use of chemical therapeutics, such as antibiotics, because of their impact on the environment as well as on the fish product. As a potential tool for reducing antibiotic use, we tested the application of low-frequency ultrasound as a method for enhancing antibiotic uptake. Rainbow trout juveniles (Oncorhynchus mykiss) were exposed to two different concentrations of oxytetracycline (OTC), flumequine (FLU) and florfenicol (FLO), administered by bath after the application of ultrasound. After exposure, concentrations of these substances were measured in the liver and blood of treated fish. Results showed that the ultrasound treatment can significantly increase the uptake for all three antibiotics. The uptake of OTC for example, in fish exposed to an OTC concentration of 20 mg L-1 after prior treatment with ultrasound, was similar to the OTC concentrations in their liver and blood to fish exposed to 100 mg L-1 without sonication. For FLU and FLO, the use of ultrasound caused significant differences of uptake in the liver at high antibiotic concentrations. This suggests that the use of ultrasound as a technique to deliver antibiotics to fish can ultimately reduce the amount of antibiotics discharged into the aquatic environment.

Ultrastructural analysis of sequential cyprinid herpesvirus 3 morphogenesis in vitro.

Cyprinid herpesvirus 3 (CyHV-3) is an alloherpesvirus, and it is the aetiological agent of koi herpesvirus disease. Although the complex morphogenic stages of the replication cycle of CyHV-3 were shown to resemble that of other members of the Herpesvirales, detailed analysis of the sequence and timing of these events was not definitively determined. This study describes these features through a time course using cyprinid cell cultures (KF-1 and CCB) infected with CyHV-3 (KHV isolate, H361) and analysed by transmission electron microscopy. Rapid viral entry was noted, with high levels of intracellular virus within 1-4 h post-infection (hpi). Intranuclear capsid assembly, paracrystalline array formation and primary envelopment of capsids occurred within 4 hpi. Between 1 and 3 days post-infection (dpi), intracytoplasmic secondary envelopment occurred, as well as budding of infectious virions at the plasma membrane. At 5-7 dpi, the cytoplasm contained cytopathic vacuoles, enveloped virions within vesicles, and abundant non-enveloped capsids; also there was frequent nuclear deformation. Several morphological features are suggestive of inefficient viral assembly, with production of non-infectious particles, particularly in KF-1 cells. The timing of this alloherpesvirus morphogenesis is similar to other members of the Herpesvirales, but there may be possible implications of using different cell lines for CyHV-3 propagation.

The effects of increasing dietary levels of soy protein concentrate (SPC) on the immune responses and disease resistance (furunculosis) of vaccinated and non-vaccinated Atlantic salmon (Salmo salar L.) parr.

Juvenile salmon, with an initial weight of 9 g, were fed three experimental diets, formulated to replace 35 (SPC35), 58 (SPC58) and 80 (SPC80) of high quality fishmeal (FM) with soy protein concentrate (SPC) in quadruplicate tanks. Higher dietary SPC inclusion was combined with increased supplementation of methionine, lysine, threonine and phosphorus. The experiment was carried out for 177 days. On day 92 salmon in each tank were bulk weighed. Post weighing eighty salmon from each tank were redistributed in two sets of 12 tanks. Salmon from the first set of tanks were vaccinated, while the second group was injected with phosphate buffer saline (PBS). Salmon were sampled on day 92 (pre-vaccination), day 94 (2 days post vaccination [dpv]/PBS injection [dpPBSinj]) and day 154 (62 dpv/dpPBSinj) of the trial for the assessment of their immune responses, prior to the performance of salmon bulk weights for each tank. On day 154, fish from each tank were again bulk weighed and then seventeen salmon per tank were redistributed in two sets of twelve tanks and intra-peritoneally infected with Aeromonas salmonicida. At Day 154, SPC80 demonstrated lower performance (weight gain, specific growth rate and thermal growth coefficient and feed conversion ratio) compared to SPC35 salmon. Reduced classical and total complement activities for salmon fed diets with over 58% of protein from SPC, were demonstrated prior to vaccination. Reduced alternative complement activity was detected for both SPC58 and SPC80 salmon at 2 dpv and for the SPC80 group at 62 dpv. Total and classical complement activities demonstrated no differences among the dietary groups after vaccination. Numerical increases in classical complement activity were apparent upon increased dietary SPC levels. Increased phagocytic activity (% phagocytosis and phagocytic index) was exhibited for the SPC58 group compared to SPC35 salmon at 62 dpPBSinj. No differences in serum lysozyme activity, total IgM, specific antibodies, protein, glucose and HKM respiratory burst were detected among the dietary groups at any timepoint or state. Mortalities as a result of the experimental infection only occurred in PBS-injected fish. No differences in mortality levels were demonstrated among the dietary groups. SPC58 diet supported both good growth and health in juvenile Atlantic salmon while SPC80 diet did not compromise salmon' immunity or resistance to intraperitoneally inflicted furunculosis.

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.

The effects of increasing dietary levels of amino acid-supplemented soy protein concentrate and constant dietary supplementation of phosphorus on growth, composition and immune responses of juvenile Atlantic salmon (Salmo salar L.).

Diets with 50 (SPC50), 65 (SPC65) and 80 % (SPC80) substitution of prime fish meal (FM) with soy protein concentrate (SPC) were evaluated against a commercial type control feed with 35 % FM replacement with SPC. Increases in dietary SPC were combined with appropriate increases in methionine, lysine and threonine supplementation, whereas added phosphorus was constant among treatments. Diets were administered to quadruplicate groups of 29 g juvenile Atlantic salmon were exposed to constant light, for 97 days. On Day 63 salmon were subjected to vaccination. Significant weight reductions in SPC65 and SPC80 compared with SPC35 salmon were observed by Day 97. Linear reductions in body cross-sectional ash, Ca/P ratios, and Ca, P, Mn and Zn were observed at Days 63 (prior vaccination) and 97 (34 days post-vaccination), while Mg presented a decrease at Day 63, in salmon fed increasing dietary SPC. Significant reductions in Zn, Ca, P and Ca/P ratios persisted in SPC65 and SPC80 compared with SPC35 salmon at Day 97. Significant haematocrit reductions in SPC50, SPC65 and SPC80 salmon were observed at Days 63, 70 and 97. Enhanced plasma haemolytic activity, increased total IgM, and a rise in thrombocytes were demonstrated in SPC50 and SPC65 salmon on Day 97, while increased lysozyme activity was demonstrated for these groups on Days 63, 70 and 97. Leucocyte and lymphocyte counts revealed enhanced immunostimulation in salmon fed with increasing dietary SPC at Day 97. High SPC inclusion diets did not compromise the immune responses of salmon, while SPC50 diet also supported good growth without compromising elemental concentrations.

Quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass Dicentrarchus labrax.

Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red-spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture-based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 µg mL(-1) (3 × 10(5) TCID50 per mL), whereas for capture-based ELISA, the sensitivity for the antigen in solution was 17 µg mL(-1) (35 × 10(5) TCID50 per mL). The capture-based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 10(8)  TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv-specific IgM is required, or for use in samples where PCR is difficult to perform.

A validated web-based tool to display individualised Crohn's disease predicted outcomes based on clinical, serologic and genetic variables.

BACKGROUND: Early treatment for Crohn's disease (CD) with immunomodulators and/or anti-TNF agents improves outcomes in comparison to a slower 'step up' algorithm. However, there remains a limited ability to identify those who would benefit most from early intensive therapy. AIM: To develop a validated, individualised, web-based tool for patients and clinicians to visualise individualised risks for developing Crohn's disease complications. METHODS: A well-characterised cohort of adult patients with CD was analysed. Available data included: demographics; clinical characteristics; serologic immune responses; NOD2 status; time from diagnosis to complication; and medication exposure. Cox proportional analyses were performed to model the probability of developing a CD complication over time. The Cox model was validated externally in two independent CD cohorts. Using system dynamics analysis (SDA), these results were transformed into a simple graphical web-based display to show patients their individualised probability of developing a complication over a 3-year period. RESULTS: Two hundered and forty three CD patients were included in the final model of which 142 experienced a complication. Significant variables in the multivariate Cox model included small bowel disease (HR 2.12, CI 1.05-4.29), left colonic disease (HR 0.73, CI 0.49-1.09), perianal disease (HR 4.12, CI 1.01-16.88), ASCA (HR 1.35, CI 1.16-1.58), Cbir (HR 1.29, CI 1.07-1.55), ANCA (HR 0.77, CI 0.62-0.95), and the NOD2 frameshift mutation/SNP13 (HR 2.13, CI 1.33-3.40). The Harrell's C (concordance index for predictive accuracy of the model) = 0.73. When applied to the two external validation cohorts (adult n = 109, pediatric n = 392), the concordance index was 0.73 and 0.75, respectively, for adult and pediatric patients. CONCLUSIONS: A validated, web-based tool has been developed to display an individualised predicted outcome for adult patients with Crohn's disease based on clinical, serologic and genetic variables. This tool can be used to help providers and patients make personalised decisions about treatment options.

Sensitivity of seven PCRs for early detection of koi herpesvirus in experimentally infected carp, Cyprinus carpio L., by lethal and non-lethal sampling methods.

Koi herpesvirus (KHV) causes an economically important, highly infectious disease in common carp and koi, Cyprinus carpio L. Since the occurrence of mass mortalities worldwide, highly specific and sensitive molecular diagnostic methods have been developed for KHV detection. The sensitivity and reliability of these assays have essentially focused at the detection of low viral DNA copy numbers during latent or persistent infections. However, the efficacy of these assays has not been investigated with regard to low-level viraemia during acute infection stages. This study was conducted to compare the sensitivity of seven different polymerase chain reaction (PCR) assays to detect KHV during the first hours and days post-infection (hpi; dpi), using lethal and non-lethal sampling methods. The results highlight the limitations of the assays for detecting virus during the first 4 dpi despite rapid mortality in experimentally infected carp. False-negative results were associated with time post-infection and the tissue sampled. Non-lethal sampling appears effective for KHV screening, with efficient detection in mucus samples obtained from external swabs during this early infection period (<5 dpi), while biopsies from gills and kidney were negative using the same PCR assays. Non-lethal sampling may improve the reliability of KHV detection in subclinical, acutely infected carp.

Examination of the early infection stages of koi herpesvirus (KHV) in experimentally infected carp, Cyprinus carpio L. using in situ hybridization.

Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody-based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin-embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8 h and following 10 days post-infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1 hpi. Respiratory epithelial cells were positive as early as 2 hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp.

Ultrastructural morphogenesis of salmonid alphavirus 1.

Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 (SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells (CHSE-214). Different stages of viral replication were observed under electron microscopy. Virus-like particles were observed inside membrane-bound vesicles as early as 1 h following contact of the virus with the cells. Membrane-dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome-like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive-sense single-stranded RNA (+ssRNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by 'fuzzy'-coated membranes was greater in virus-infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post-infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences.

Pseudomonas M162 confers protection against rainbow trout fry syndrome by stimulating immunity.

AIMS: To evaluate the antagonistic effect of Pseudomonas M162 against Flavobacterium psychrophilum. METHODS AND RESULTS: The antagonistic activity of M162 was tested in vivo and in vitro, and its mode of action examined by siderophore production and immunological responses of rainbow trout (Oncorhynchus mykiss) fry. Pseudomonas M162 inhibited the growth of Fl. psychrophilum in vitro and increased the resistance of the fish against the pathogen, resulting in a relative per cent survival (RPS) of 39·2%. However, the siderophores produced by M162 did not have an inhibitory effect on Fl. psychrophilum. In fish fed with M162, the probiotic colonized the gastrointestinal tract and stimulated peripheral blood leucocyte counts, serum lysozyme activity and total serum immunoglobulin levels after 3 weeks from the start of feeding. CONCLUSIONS: This study showed the potential of Pseudomonas M162 as a probiotic by reducing the mortalities that occurred during an experimental Fl. psychrophilum infection, resulting mainly through the immunostimulatory effects of the bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: Rainbow trout fry syndrome (RTFS) causes high mortalities during the early life stages of the fish's life cycle, partly because their adaptive immunity has not yet fully developed. Thus, immunomodulation by probiotics could be an effective prophylactic method against RTFS.

Pseudomonas sp. M174 inhibits the fish pathogen Flavobacterium psychrophilum.

AIMS: To study the antagonic affect of probiotic Pseudomonas M174 on the fish pathogen Flavobacterium psychrophilum. METHODS AND RESULTS: The ability of Pseudomonas M174 to inhibit the growth of Fl. psychrophilum was examined in iron-sufficient and -deficient media. Possible siderophore production was also investigated. Antagonistic activity was confirmed in disease challenge experiments using a rainbow trout (Oncorhynchus mykiss) model. Adhesion of Pseudomonas M174 to fish surfaces and its ability to stimulate innate immunity was also investigated in vivo. Pseudomonas M174 antagonized Fl. psychrophilum and produced siderophores in vitro. In challenge experiments with Fl. psychrophilum, fish fed with Pseudomonas M174 had lower levels of mortalities than the controls. It was possible to find Pseudomonas M174 in the intestinal content of these fish after feeding and bathing with the probiotic, but probiotic was obtained from the gills only after feeding. Respiratory burst activity was also found to be enhanced in the M174 fed fish. CONCLUSIONS: These results suggest that M174 is a potential probiotic against Fl. psychrophilum and has several modes of action. SIGNIFICANCE AND IMPACT OF THE STUDY: Probiotics are a promising alternative to the use of antibiotics in aquaculture and could be a more sustainable disease control method.