Validation of diagnostic assays to screen broodstock for Flavobacterium psychrophilum infections

It is hypothesized that the frequency of bacterial coldwater disease outbreaks can be reduced through the detection of the aetiologic agent, Flavobacterium psychrophilum, in broodstock followed by culling of eggs from heavily infected broodstock. Before a culling programme can be instituted, however, it is necessary to determine the sensitivity and specificity of existing assays for the detection of F. psychrophilum. In this study, tissue and ovarian fluid samples were collected from 224 fish at five hatcheries and screened using an enzyme‐linked immunosorbent assay (ELISA), a membrane‐filtration fluorescent antibody test (MF‐FAT), bacteriological culture and nested PCR. Latent class analysis was used to estimate sensitivity and specificity of kidney culture, kidney ELISA, nested PCR and MF‐FAT. Analytical sensitivity of the ELISA varied but was greatest when bacteria were cultured under iron‐limiting conditions. Diagnostic sensitivity estimates ranged from 0.02 (kidney culture) to 0.97 (kidney ELISA). Specificity estimates ranged from 0.02 (MF‐FAT) to 0.98 (kidney ELISA). In a separate challenge experiment, the ELISA confirmed the presence of F. psychrophilum in sub‐clinically infected fish. Results from this study demonstrate that the ELISA is an appropriate tool to screen broodstock and provides an indication of infection severity, which is crucial for implementation of a screening/culling programme.     

Development and validation of a real‐time PCR assay for the detection of Aeromonas salmonicida

A real‐time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10⁴ ± 1 × 10⁴ CFU g^?1 (without enrichment) and 40 ± 10 CFU g^?1 (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real‐time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real‐time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real‐time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.     

High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 10⁰ amplicon copies per assay (equivalent to 2 × 10^?11 ng/µl, C_q value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 10⁰ gene copies in tissues artificially infected and 0.02 × 10⁰ in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.     

Iron acquisition and siderophore production in the fish pathogen Renibacterium salmoninarum

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty‐two Chilean isolates and the DSM20767^T type strain grew in the presence of 2,2′‐Dipyridyl at varying concentrations (250–330 μm ), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200 μm of 2,2′‐Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron‐limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100 μm ), it did facilitate growth at lower iron concentrations (10 μm and 1 μm ). An exception was the H‐2 isolate, which only grew with 100 μm of iron sulphide. In turn, 100 μm of haemin was toxic when isolates were grown in normal KDM‐2. In silico R. salmoninarumATCC 33209^T genome analysis detected various genes coding iron uptake‐related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.     

Renibacterium salmoninarum iron‐acquisition mechanisms and ASK cell line infection: Virulence and immune response

Renibacterium salmoninarum is the aetiological agent of bacterial kidney disease (BKD) in salmonid farms. This pathogen possesses at least three iron‐acquisition mechanisms, but the link between these mechanisms and virulence is unclear. Therefore, this study used RT‐qPCR to assess the effects of normal and iron‐limited conditions on iron‐uptake genes controlled by IdeR and related to iron acquisition in Chilean R. salmoninarum strain H‐2 and the type strain DSM20767^T. Further evaluated was the in vitro immune‐related response of the Atlantic Salmon Kidney (ASK) cell line, derived from the primary organ affected by BKD. R. salmoninarum grown under iron‐limited conditions overexpressed genes involved in haemin uptake and siderophore transport, with overexpression significantly higher in H‐2 than DSM20767^T. These overexpressed genes resulted in higher cytotoxicity and an increased immune response (i.e., TNF‐α, IL‐1β, TLR1 and INF‐γ) in the ASK cell line. This response was significantly higher against bacteria grown under iron‐limited conditions, especially H‐2. These observations indicate that iron‐acquisition mechanisms are possibly highly related to the virulence and pathogenic capacity of R. salmoninarum. In conclusion, treatments that block iron‐uptake mechanisms or siderophore synthesis are attractive therapeutic approaches for treating R. salmoninarum, which causes significant aquaculture losses.     

Identification and typing of Vagococcus salmoninarum using genomic and proteomic techniques

This study reports on the characterization of Vagococcus salmoninarum using phenotypic, serological, antigenic, genetic and proteomic methods. All strains of V. salmoninarum were resistant to most of the antimicrobials tested, and only 10% of strains were sensitive to florfenicol. Serological analysis demonstrated a high antigenic homogeneity within the species. No cross‐reaction was detected with other fish pathogenic species causing streptococcosis (Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae, Streptococcus agalactiae, Carnobacterium maltaromaticum) using serum against V. salmoninarum CECT 5810. Electrophoretic analysis of cell surface proteins and immunoblot supported the antigenic homogeneity within V. salmoninarum strains. Moreover, limited diversity was detected using genomic (RAPD, ERIC‐PCR and REP‐PCR) and MALDI‐TOF‐MS analyses. The phenotypic, genomic and proteomic methods tested allowed the rapid differentiation of V. salmoninarum from the other species causing streptococcosis. However, MALDI‐TOF‐MS is the most promising method for typing and characterization of V. salmoninarum.     

Detection of Herpesvirus anguillae (AngHV‐1) in European eel Anguilla anguilla (L.) originating from northern Poland—assessment of suitability of selected diagnostic methods

The Community Action Plan requests EU member states to implement measures that ensure the recovery of the severely depleted European eel stocks. One of the main threats is posed by Anguillid herpesvirus 1 (AngHV‐1) leading to increased mortality in both wild and farmed eels. Following recommendations of the OIE to minimize the risk of obtaining false‐negative results, the main aim of the study was to optimize diagnostic methods for AngHV‐1 detection using conventional PCR, nested PCR and in situ hybridization assay. While 53.3% of the individual organ samples were tested positive for AngHV‐1 by PCR, the additional virus analysis via nested PCR revealed that the actual prevalence was 93.3%. In the cell cultivation passages, a cytopathic effect was hardly found in the first two rounds. In the third passage onto cell cultures, a lytic CPE was detected. The identification and confirmation of the viruses obtained from cell cultures as well as directly from the organ tissues were proceeded by PCR, nested PCR and sequencing of the PCR products. While no positive signal was detectable in the first round by PCR using samples from the third cell culture passages, the nested PCR provided weak but visible positive signals.     

Effects of temperature on Renibacterium salmoninarum infection and transmission potential in Chinook salmon,Oncorhynchus tshawytscha (Walbaum)

Renibacterium salmoninarum is a significant pathogen of salmonids and the causative agent of bacterial kidney disease (BKD). Water temperature affects the replication rate of pathogens and the function of the fish immune system to influence the progression of disease. In addition, rapid shifts in temperature may serve as stressors that reduce host resistance. This study evaluated the effect of shifts in water temperature on established R. salmoninarum infections. We challenged Chinook salmon with R. salmoninarum at 12 °C for 2 weeks and then divided the fish into three temperature groups (8, 12 and 15 °C). Fish in the 8 °C group had significantly higher R. salmoninarum‐specific mortality, kidney R. salmoninarum loads and bacterial shedding rates relative to the fish held at 12 or 15 °C. There was a trend towards suppressed bacterial load and shedding in the 15 °C group, but the results were not significant. Bacterial load was a significant predictor of shedding for the 8 and 12 °C groups but not for the 15 °C group. Overall, our results showed little effect of temperature stress on the progress of infection, but do support the conclusion that cooler water temperatures contribute to infection progression and increased transmission potential in Chinook salmon infected with R. salmoninarum.     

A tentative new species Aphanomyces fennicus sp. nov. interferes with molecular diagnostic methods for crayfish plague

Several isolates of an unknown oomycete resembling the genus Aphanomyces were obtained into laboratory culture from samples of noble crayfish (Astacus astacus) in 2016–2017. The crayfish were kept in cages in connection with a study on an eventually persistent crayfish plague infection in a small Finnish lake, following an acute episode of the disease in 2010. Despite the close resemblance of the isolates to the causative agent of crayfish plague, Aphanomyces astaci, and the positive results obtained in OIE recommended A. astaci‐specific ITS‐based conventional PCR and qPCR molecular assays, the isolates can be distinguished from A. astaci by morphological features concerning hyphal structure and chlamydospore formation, as well as using the randomly amplified polymorphic DNA‐polymerase chain reaction (RAPD‐PCR) method, microsatellite‐based genotyping, the pathogenicity test and phylogenetic analysis based on ITS sequencing. The name Aphanomyces fennicus sp. novum is proposed for this close relative of A. astaci. The detection of this tentative novel species giving false‐positive results in existing diagnostic assays for the crayfish plague highlights the importance of careful interpretation of the results from molecular methods, especially concerning crayfish with low‐level infections, excluding the possibility to verify the results from clinical or sequencing data.     

Utility of genetically based health indicators for selection purposes in captive-reared chinook salmon, Oncorhynchus tshawytscha, Walbaum

Three health indicators, plasma lysozyme activity, PCR‐based detection of Renibacterium salmoninarum (a causative agent of bacterial kidney disease), and a necropsy‐based Health Assessment Index (HAI), were used to examined genetically based variation in a captive population of chinook salmon (Oncorhynchus tshawytscha W.). The study group consisted of four distinct genetic cross‐types: two purebred cross‐types originating from mating wild parents (Big Qualicum River, BC, Canada) and domestic parents (Yellow Island Aquaculture, Ltd, Quadra Island, BC, Canada) and two reciprocal hybrid cross‐types from the mating of wild and domestic parents. Narrow‐sense heritability estimates for plasma lysozyme activity and the incidence of R. salmoninarum were calculated, and the genetic correlation of health indicator response with survival and growth was estimated. Significant differences among cross‐types were found for plasma lysozyme activity, HAI, survival after a natural outbreak of vibriosis (but not after a vibriosis disease challenge), relative growth rate, size‐at‐age (420 and 615 days post fertilization), and R. salmoninarum presence. Despite a significant sire component of heritability for plasma lysozyme activity, the lack of significant heritability estimates for R. salmoninarum presence, and non‐significant genetic correlations with performance variables indicates that selection to improve the health status of fish stock using the three health indicators examined here would likely not result in a measurable correlated response in survival or growth.     

Development and validation of a real‐time PCR assay for the detection of anguillid herpesvirus 1

Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody‐based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real‐time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real‐time PCR is faster, less labour‐intensive and has a reduced risk of cross‐contamination. The real‐time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r²‐value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real‐time PCR. The developed real‐time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels.     

Development of real‐time PCR for detection and quantitation of Streptococcus parauberis

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real‐time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real‐time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra‐ and interassay coefficient of variation (CV) values ranged from 0.42–1.95%, demonstrating that the assay has good reproducibility. There was not any cross‐reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real‐time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real‐time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples.     

Identification and characterization of lactic acid bacteria isolated from rainbow trout (Oncorhynchus mykiss,Walbaum 1792), with inhibitory activity against Vagococcus salmoninarum and Lactococcus garvieae

In this study, a total of 98 lactic acid bacteria isolated from rainbow trout intestines were screened for their probiotic properties. The isolates were tested for their ability to inhibit growth of Vagococcus salmoninarum and Lactococcus garvieae. Based on in vitro antagonism, 10 isolates were selected and evaluated pathogenicity in rainbow trout. Isolates were further investigated for hydrophobicity, bile salts and acid tolerance. These isolates were able to survive low pH and high bile concentrations and showed good adherence characteristics. Isolates were characterized phenotypically, and then, 16S rRNA gene sequence analysis was used for confirmation. Selected strains were administered orally at 10⁸ cfu/g feed, and fish were challenged with V. salmoninarum and L. garvieae. The fish fed with lactic acid bacteria supplemented diets did not improve protection against V. salmoninarum. However, administration of Lactococcus lactis subsp. lactis M17 2‐2 and Lactobacillus sakei 2‐3 resulted in a significant reduction in mortality due to L. garvieae when compared to the control fish. RPS values were calculated as 80 and 53% in fish fed with L. sakei 2‐3 and L. lactis subsp. lactis M17 2‐2, respectively. Our results suggest that these strains could provide an alternative for lactococcosis control in aquaculture.     

Assay performance during validation of freezing channel catfish Ictalurus punctatus (Rafinesque) infected with a Gram‐negative bacterium

Recovery of bacteria from infected fish during population sampling can be affected by factors including the type of assay, method of specimen preservation and concentration of bacteria present. Consequently, before use in field sampling, methods should be validated. The three objectives of this study were, first, to determine whether a channel catfish Ictalurus punctatus (Rafinesque) fingerling classified as positive for Gram‐negative Edwardsiella ictaluri infection according to bacterial culture before freezing was also classified as positive after freezing, second, to determine how direct culture from the kidney (DIRECT), culture of homogenate (HOMOG) and standard PCR (PCR) agree with bacterial culture in terms of classifying fish as positive or negative and third, to estimate diagnostic sensitivity (dSe) and diagnostic specificity (dSp) for DIRECT, HOMOG and PCR. In fresh and frozen fish, as bacterial concentration decreased, the ability of each assay to detect positive fish also decreased, especially when there were <10⁴ colony‐forming units per gram (CFU g^?1) tissue. HOMOG proved to be the most reliable at correctly classifying catfish, whether they were subclinically or clinically infected. PCR assay was the least reliable. Overall, values for this study population for dSe were 0.66, 0.92 and 0.43, and for dSp were 0.86, 0.91 and 0.95, for DIRECT, HOMOG and PCR respectively.     

Resistance of pearlspot larvae,Etroplus suratensis,to redspotted grouper nervous necrosis virus by immersion challenge

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty‐day‐old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post‐infection, and the infected larvae were subjected to nested RT‐PCR and histology. The virus was isolated from infected larvae using SSN‐1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT‐PCR and the virus was isolated using SSN‐1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT‐PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.     

Detection of Cyprinid herpesvirus 2 in association with an Aeromonas sobria infection of Carassius carassius (L.), in Italy

Sixteen specimens of female crucian carp, Carassius carassius (L.), during the breeding season, were investigated for post‐mortem and full diagnostic examination during a mortality outbreak in a tributary stream of the Arno River in Tuscany in 2011. Necropsy highlighted the presence of a swollen anus and widespread haemorrhages in the body, fins, gills and eyes. Haemorrhages in internal organs and spleen granulomas were also observed. Bacteria isolated from the brain, kidney and spleen of affected fish were identified as A. sobria. Microscopic lesions observed in gills were characterized by necrosis of the secondary lamellae, congestion and multifocal lamellar fusion. The kidney showed necrosis, oedema, fibrin exudation and areas of haemorrhages, while in the spleen the main lesions were by multifocal necrosis of the lymphoid tissue. In the gills, transmission electron microscopy revealed herpesvirus‐like particles, subsequently identified as Cyprinid herpesvirus‐2 (CyHV‐2) with a nested PCR protocol. Although it was not possible to attribute a pathogenic role to CyHV‐2 in this mortality event, the identification of this herpesvirus in crucian carp increases the concern about its potential role in this species.     

Development and application of molecular methods (PCR) for detection of Tasmanian Atlantic salmon reovirus

Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general, and Tasmanian Atlantic salmon reovirus (TSRV) specifically, were developed, and their diagnostic sensitivity and specificity were determined and compared with virus isolation in cell culture. Intralaboratory and interlaboratory comparison of PCR (conventional hemi‐nested RT‐PCR & RT‐qPCR) and virus isolation in cell culture using finfish cell lines, CHSE‐214 and EPC, was carried out for the detection and identification of TSRV using field samples of farmed Atlantic salmon Salmo salar, L. from various aquaculture sites around Tasmania. The interlaboratory comparison of diagnostic methods was carried out between two laboratories, AAHL‐CSIRO and DPIPWE‐Tasmania. A total of 144 fish from nine sites (12–33 fish per site) were sampled from two regions of Tasmania (Tamar River estuary in the north and Huon River estuary in the south‐east) during late spring to early summer of 2009, and the data were analysed using different statistical approaches. The prevalence of TSRV ranged from 6% to 22% in both regions. All the diagnostic methods (data from both laboratories) had high specificity, while the estimated sensitivity varied between tests with RT‐qPCR being the most sensitive (95.2%) method followed by virus isolation and then conventional hemi‐nested RT‐PCR.     

Case definition for clinical and subclinical bacterial kidney disease (BKD) in Atlantic Salmon (Salmo salar L.) in New Brunswick,Canada

Bacterial kidney disease (BKD) is considered an important cause of loss in salmon aquaculture in Atlantic Canada. Causative agent of BKD is the Gram‐positive bacteria Renibacterium salmoninarum. Infected salmon are often asymptomatic (subclinical infection), and the disease is considered chronic. One of the challenges in quantifying information from farm production and health records is the application of a standardized case definition. Case definitions for farm‐level and cage‐level clinical and subclinical BKD were developed using retrospective longitudinal data from aquaculture practices in New Brunswick, Canada, combining (i) industry records of weekly production data including mortalities, (ii) field observations for BKD using reports of veterinarians and/or fish health technicians, (iii) diagnostic submissions and test results and (iv) treatments used to control BKD. Case definitions were evaluated using veterinarians’ expert judgements as reference standard. Eighty‐nine and 66% of sites and fish groups, respectively, were associated with BKD at least once. For BKD present (subclinical or clinical), sensitivity and specificity of the case definition were 75–100% varying between event, fish group, site cycle and level (site pen). For clinical BKD, sensitivities were 29–64% and specificities 91–100%. Industry data can be used to develop sensitive case definitions.