Quantitative PCR for Tenacibaculum dicentrarchi and T. finnmarkense.

Numerous Tenacibaculum species, including T. dicentrarchi, T. maritimum and T. finnmarkense, are associated with tenacibaculosis in finfish; however, quantitative identification techniques are limited. Quantitative PCR assays were developed to detect T. dicentrarchi and T. finnmarkense. TaqMan assays using 16S rDNA demonstrated low detection limits (0.07-269 bacteria), suitable amplification efficiencies (>86%) and moderate specificity. However, the amplification of isolates with 100% sequence similarity to T. finnmarkense AY7486TD using both the T. finnmarkense and T. dicentrarchi assays indicates that other genes should be investigated. Both assays may help describe the pathogenesis of tenacibaculosis and may aid management practices for the aquaculture industry.

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice-structuring protein LS-12 in the acute-phase response to Flavobacterium psychrophilum infection.

A previous proteomic study examining the plasma acute-phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5-kDa spot using 2D-PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443-bp nucleotide sequence that was 98.6% similar to type-4 ice-structuring protein LS-12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS-12 following experimental intraperitoneal injection of rainbow trout with either 106 or 108 colony-forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS-12 up to 15 days post-infection in any group. Hepatic LS-12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post-infection in fish injected with 108 CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute-phase response. Under the conditions used, LS-12 is not a positive acute-phase protein in rainbow trout.

Genomics of Tenacibaculum Species in British Columbia, Canada.

Tenacibaculum is a genus of Gram-negative filamentous bacteria with a cosmopolitan distribution. The research describing Tenacibaculum genomes stems primarily from Norway and Chile due to their impacts on salmon aquaculture. Canadian salmon aquaculture also experiences mortality events related to the presence of Tenacibaculum spp., yet no Canadian Tenacibaculum genomes are publicly available. Ribosomal DNA sequencing of 16S and four species-specific 16S quantitative-PCR assays were used to select isolates cultured from Atlantic salmon with mouthrot in British Columbia (BC), Canada. Ten isolates representing four known and two unknown species of Tenacibaculum were selected for shotgun whole genome sequencing using the Oxford Nanopore's MinION platform. The genome assemblies achieved closed circular chromosomes for seven isolates and long contigs for the remaining three isolates. Average nucleotide identity analysis identified T. ovolyticum, T. maritimum, T. dicentrarchi, two genomovars of T. finnmarkense, and two proposed novel species T. pacificus sp. nov. type strain 18-2881-AT and T. retecalamus sp. nov. type strain 18-3228-7BT. Annotation in most of the isolates predicted putative virulence and antimicrobial resistance genes, most-notably toxins (i.e., hemolysins), type-IX secretion systems, and oxytetracycline resistance. Comparative analysis with the T. maritimum type-strain predicted additional toxins and numerous C-terminal secretion proteins, including an M12B family metalloprotease in the T. maritimum isolates from BC. The genomic prediction of virulence-associated genes provides important targets for studies of mouthrot disease, and the annotation of the antimicrobial resistance genes provides targets for surveillance and diagnosis in veterinary medicine.

Application of Quantitative-PCR to Monitor Netpen Sites in British Columbia (Canada) for Tenacibaculum Species.

Tenacibaculum are frequently detected from fish with tenacibaculosis at aquaculture sites; however, information on the ecology of these bacteria is sparse. Quantitative-PCR assays were used to detect T. maritimum and T. dicentrarchi at commercial Atlantic salmon (Salmo salar) netpen sites throughout several tenacibaculosis outbreaks. T. dicentrarchi and T. maritimum were identified in live fish, dead fish, other organisms associated with netpens, water samples and on inanimate substrates, which indicates a ubiquitous distribution around stocked netpen sites. Before an outbreak, T. dicentrarchi was found throughout the environment and from fish, and T. maritimum was infrequently identified. During an outbreak, increases in the bacterial load in were recorded and no differences were recorded after an outbreak supporting the observed recrudescence of mouthrot. More bacteria were recorded in the summer months, with more mortality events and antibiotic treatments, indicating that seasonality may influence tenacibaculosis; however, outbreaks occurred in both seasons. Relationships were identified between fish mortalities and antimicrobial use to water quality parameters (temperature, salinity, dissolved oxygen) (p < 0.05), but with low R2 values (<0.25), other variables are also involved. Furthermore, Tenacibaculum species appear to have a ubiquitous spatial and temporal distribution around stocked netpen sites, and with the potential to induce disease in Atlantic salmon, continued research is needed.

Experimental Induction of Tenacibaculosis in Atlantic Salmon (Salmo salar L.) Using Tenacibaculum maritimum, T. dicentrarchi, and T. finnmarkense.

There is a limited understanding of the pathogenesis of tenacibaculosis in Atlantic salmon (Salmo salar L.) and there are few reproducible exposure models for comparison. Atlantic salmon were exposed via bath to Tenacibaculum maritimum, T. dicentrarchi, or T. finnmarkense, and were then grouped with naïve cohabitants. Mortalities had exaggerated clinical signs of mouthrot, a presentation of tenacibaculosis characterized by epidermal ulceration and yellow plaques, on the mouth and less frequently on other tissues. Histopathology showed tissue spongiosis, erosion, ulceration, and necrosis ranging from mild to marked, locally to regionally extensive with mats of intralesional bacteria on the rostrum, vomer, gill rakers, gill filaments, and body surface. Exposure to T. maritimum resulted in less than a 0.4 probability of survival for both exposed and cohabitants until Day 21. Exposures to T. dicentrarchi resulted in 0 and 0.55 (exposed), and 0.8 and 0.9 (cohabitant) probability of survival to Day 12 post-exposure, while T. finnmarkense had a 0.9 probability of survival to Day 12 for all groups. This experimental infection model will be useful to further investigate the pathogenesis of tenacibaculosis, its treatment, and immunity to Tenacibaculum species.

Advancements in Characterizing Tenacibaculum Infections in Canada.

Tenacibaculum is a genus of gram negative, marine, filamentous bacteria, associated with the presence of disease (tenacibaculosis) at aquaculture sites worldwide; however, infections induced by this genus are poorly characterized. Documents regarding the genus Tenacibaculum and close relatives were compiled for a literature review, concentrating on ecology, identification, and impacts of potentially pathogenic species, with a focus on Atlantic salmon in Canada. Tenacibaculum species likely have a cosmopolitan distribution, but local distributions around aquaculture sites are unknown. Eight species of Tenacibaculum are currently believed to be related to numerous mortality events of fishes and few mortality events in bivalves. The clinical signs in fishes often include epidermal ulcers, atypical behaviors, and mortality. Clinical signs in bivalves often include gross ulcers and discoloration of tissues. The observed disease may differ based on the host, isolate, transmission route, and local environmental conditions. Species-specific identification techniques are limited; high sequence similarities using conventional genes (16S rDNA) indicate that new genes should be investigated. Annotating full genomes, next-generation sequencing, multilocus sequence analysis/typing (MLSA/MLST), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), and fatty acid methylesters (FAME) profiles could be further explored for identification purposes. However, each aforementioned technique has disadvantages. Since tenacibaculosis has been observed world-wide in fishes and other eukaryotes, and the disease has substantial economic impacts, continued research is needed.

Screening of Fish Cell Lines for Piscine Orthoreovirus-1 (PRV-1) Amplification: Identification of the Non-Supportive PRV-1 Invitrome.

Piscine reovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), which is detrimental to Atlantic Salmon (AS) aquaculture, but so far has not been cultivatable, which impedes studying the disease and developing a vaccine. Homogenates of head kidney and red blood cells (RBC) from AS in which PRV-1 had been detected were applied to fish cell lines. The cell lines were from embryos, and from brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, and had the shapes of endothelial, epithelial, fibroblast, and macrophage cells. Most cell lines were derived from the Neopterygii subclass of fish, but one was from subclass Chondrostei. Cultures were examined by phase contrast microscopy for appearance, and by quantitative polymerase chain reaction (qPCR) for PRV-1 RNA amplification and for the capacity to transfer any changes to new cultures. No changes in appearance and Ct values were observed consistently or transferable to new cultures. Therefore, 31 cell lines examined were unable to support PRV-1 amplification and are described as belonging to the non-supportive PRV-1 invitrome. However, these investigations and cell lines can contribute to understanding PRV-1 cellular and host tropism, and the interactions between virus-infected and bystander cells.

Development of an Atlantic salmon heart endothelial cell line (ASHe) that responds to lysophosphatidic acid (LPA).

As diseases and abnormalities of the heart can interfere with the aquaculture of Atlantic salmon, the heart was investigated as a source of cell lines that could be used to study the cellular basis of these conditions. An Atlantic salmon heart endothelial cell line, ASHe, was developed and characterized for growth properties, endothelial cell characteristics, and responsiveness to lysophosphatidic acid (LPA). AHSe cells stained negative for senescence associated ß-galactosidase and grew well in 10 and 20% FBS/L15 at high cell density, but not in L15 medium supplemented with calf serum. It displayed many endothelial cell-like characteristics including a cobblestone morphology, capillary-like structures formation on Matrigel, and expression of von Willebrand factor and endothelial cell-related tight junction proteins ZO-1, claudin 3, and claudin 5. ASHe cells responded to the cardiovascular modulator, LPA, in two contrasting ways. LPA at 5 and 25 µM inhibited the ability of ASHe cells to heal a wound but stimulated their proliferation, especially as evaluated by colony formation in low-density cultures. The enhancement of proliferation by LPA parallels what has been observed previously in mammalian endothelial cell cultures exposed to LPA, whereas the LPA slowing of ASHe cell migration contrasted with the LPA-enhanced migration of some mammalian cells. Therefore, this cell line is a potentially useful model for future comparative studies on piscine and mammalian cardiovascular cell biology and for studies on diseases of Atlantic salmon in aquaculture.

Function and heterogeneity of fish lectins.

Lectins are primordial molecules with multiple known functions. They have been known to exist in fish and other animals for decades and were initially identified as (hem)agglutinins. Demonstration of the importance of vertebrate lectins in innate immunity is a recent effort and is still largely unrealised for fish. This mini-review will tabulate those fish lectins identified since the last major review. In addition, particular lectins for which either functional relevance or functional or structural heterogeneity has been demonstrated are discussed in greater detail.

Application of a multiplex PCR for the detection of protozoan pathogens of the eastern oyster Crassostrea virginica in field samples.

Populations of eastern oysters Crassostrea virginica along the east coast of North America have repeatedly experienced epizootic mass mortality due to infections by protozoan parasites, and molecular diagnostic methodologies are fast becoming more widely available for the diagnosis of protozoan diseases of oysters. In this study we applied a modified version of an existing multiplex polymerase chain reaction (PCR) for detection of the eastern oyster parasites Haplosporidium nelsoni, H. costale and Perkinsus marinus from field-collected samples. We incorporated primers for DNA quality control based on the large subunit ribosomal RNA (LSU rRNA) gene of C. virginica. The multiplex PCR (MPCR) simultaneously amplified genomic DNA of C. virginica, and cloned DNA of H. nelsoni, P. marinus and H. costale. In field trial applications, we compared the performance of the MPCR to that of the conventional diagnostic techniques of histopathological tissue examination and the Ray/Mackin fluid thioglycollate medium (RMFT) assay. A total of 530 oysters were sampled from 18 sites at 12 locations along the east coast of the United States from the Gulf of Mexico to southern New England. The modified MPCR detected 21% oysters with H. nelsoni, 2% oysters with H. costale, and 40% oysters with P. marinus infections. In comparison, histopathological examination detected H. nelsoni and H. costale infections in 6 and 0.8% oysters, respectively, and the RMFT assay detected P. marinus infection in 31% oysters. The MPCR is a more sensitive diagnostic assay for detection of H. nelsoni, H. costale, and P. marinus, and incorporation of an oyster quality control product limits false negative results.

Effects of anionic polyacrylamide products on gill histopathology in juvenile rainbow trout (Oncorhynchus mykiss).

Anionic polyacrylamide (PAM) products are commonly used to remove suspended materials from turbid waters and to help mitigate soil erosion. In the present study, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to 3 mg/L to 300 mg/L of 10 commercially available PAM products (Clearflow Water Lynx Polymer Log and Clearflow Soil Lynx Granular Polymer; Clearflow Enviro Systems Group), and gill histological parameters were measured following either 7 d or 30 d of polymer exposure. A cationic polymer product (≤0.38 mg/L MagnaFloc 368; Ciba Specialty Chemical) was also tested for comparison. Mild gill lesions were observed in fish exposed to polymer products. Lamellar fusion, interlamellar hyperplasia, epithelial lifting, mucous cell metaplasia, and cell counts of epithelial swelling and necrosis/apoptosis were minimal in fish exposed to environmentally relevant concentrations of anionic polymer (≤30 mg/L). Gill morphology was largely unaffected by exposure to concentrations up to 300 mg/L of many PAM products. Several anionic polymer products noticeably affected gill tissue by increasing epithelial hypertrophy, interlamellar hyperplasia, mucous cell metaplasia, and the frequency of necrotic cells. The severity of the lesions lessened with time, suggesting that fish may have experienced a short-term irritant effect. Similar levels of gill pathology were frequently observed in fish exposed to cationic polymer MagnaFloc 368 despite the concentration being 1000-fold lower than that of the PAM products. These observations highlight the increased toxicity of cationic polymers to aquatic life compared with anionic PAMs.

The influence of oocyte cortisol on the early ontogeny of intelectin and TLR-5, and changes in lysozyme activity in rainbow trout (Oncorhynchus mykiss) embryos.

The ontogeny of lysozyme activity, intelectin, TLR-5M and TLR-5S gene expression and intelectin localization was examined in rainbow trout (Oncorhynchus mykiss) reared from oocytes immersed for 3h prior to fertilization in either ovarian fluid alone (CC) or cortisol-enriched ovarian fluid at either 100 ng mL(-1) (C1) or 1000 ng mL(-1) (C2) [final oocyte cortisol concentrations were ~3, ~5, and ~7.5 ng oocyte(-1) for the CC, C1 and C2 treatment groups, respectively]. Lysozyme activity was elevated in the cortisol-treated groups from the zygote until 13-days post fertilization (dpf), but was not affected at 21-dpf. Intelectin levels were elevated in both cortisol treatment groups at 12-hpf (2-cell stage) and then suppressed between 36- and 48-hpf. Intelectin mRNA transcript levels were elevated in both cortisol treatment groups in oocytes; there were no differences among treatment groups at 1- and 5-dpf, and suppressed in the C2 treatment group in 13-dpf and 26-dpf. TLR-5 mRNA transcripts were higher in cortisol-treated oocytes prior to fertilization; TLR-5S mRNA was more abundant than TLR-5M mRNA. The ontogeny of the gene expression patterns, and the gene, lectin and lysozyme responses to oocyte cortisol adjustments suggest an important role of innate immune systems in the early cleavage stages of embryonic cells.

Mass mortality associated with koi herpesvirus in wild common carp in Canada.

Koi herpesvirus (KHV) was identified as being associated with more than one mortality event affecting common carp in Canada. The first was an extensive mortality event that occurred in 2007 in the Kawartha Lakes region, Ontario, affecting Lakes Scugog and Pigeon. Fish had branchial necrosis and hepatic vasculitis with an equivocal interstitial nephritis. Several fish also had branchial columnaris. Subsequent mortality events occurred in 2008 in additional bodies of water in south-central Ontario, such as Lake Katchewanooka and outside of Ontario in Lake Manitoba, Manitoba. Koi herpesvirus was detected in fish submitted for examination from all of these lakes by polymerase chain reaction (PCR), and sequence of the PCR product revealed 100% homology to KHV strains U and I. Real-time PCR analysis of KHV-infected wild carp revealed viral loads ranging from 6.02×10(1) to 2.4×10(6) copies µg(-1) host DNA. This is the first report of KHV in Canada.

Bacterial-binding activity and plasma concentration of ladderlectin in rainbow trout (Oncorhynchus mykiss).

Soluble, defense lectins bind conserved microbial patterns leading to pathogen opsonization, enhanced phagocytosis and activation of complement. These immune functions, however, vary widely among individuals due to genetic and acquired differences affecting binding capacity or plasma concentration. Most evidence for the defensive function of soluble lectins is based on mammals, but several functionally homologous, but less well-characterized, lectins have been identified in fish. In this study, we compared binding of rainbow trout plasma ladderlectin to relevant, intact bacterial targets. A polyclonal antiserum raised against a synthetic peptide identical to the 20 N-terminal amino acids of the reduced 16 kDa rainbow trout ladderlectin subunit was used to detect plasma ladderlectin in immunoblots and indirect enzyme-linked immunosorbent assay (ELISA). Ladderlectin binding to Aeromonas salmonicida subsp. salmonicida, Aeromonas hydrophila, Yersinia ruckeri and Pseudomonas sp. was detected by PAGE and immunoblots of saccharide elutions from intact bacteria incubated in the presence of normal trout plasma. Although plasma concentrations of immunoreactive ladderlectin were low in the majority of trout, significant (P < 0.0001) variation between individual fish was observed in two separate populations. In addition, one population demonstrated a subset of individuals whose ladderlectin levels were approximately seven-fold higher than the population median. These findings indicate that rainbow trout have variable amounts of plasma ladderlectin capable of binding to the surfaces of several relevant bacterial targets.