Residual tissue magnesium concentration in jellyfish (Aurelia aurita and Cassiopea andromeda) following magnesium chloride euthanasia.

Magnesium chloride in high concentration is used for euthanasia of jellyfish to limit overpopulation and for predatory species consumption, but its use could lead to magnesium bioaccumulation and subsequent negative effects in consumers. Two species of scyphozoan jellyfish (Cassiopea andromeda and Aurelia aurita) were subjected to freezing (control), or magnesium chloride baths (144 g/L), with subsequent 30 min baths (one or two) in fresh artificial saltwater and submitted for inductively coupled plasma analysis to determine tissue concentration. Frozen jellyfish consistently yielded the lowest magnesium concentrations, while magnesium chloride euthanized individuals contained the highest concentrations in both species. C. andromeda displayed a significantly higher (p < .05) magnesium absorption capacity than A. aurita in both trials. Single and double baths significantly decreased magnesium concentrations (p < .05) in both species, however, magnesium remained consistently elevated compared to frozen specimens. This study demonstrated species-specific magnesium accumulation in jellyfish posteuthanasia and that rinsing was an effective method to limit excessive magnesium that could be deleterious to animals in public display aquaria. Magnesium concentrations of tissue and receiving water should be tested if magnesium chloride euthanasia is utilized for dietary supplementation in small bodies of water.

Autophagy modulation in rainbow trout Oncorhynchus mykiss L. and resistance to experimental infection with Flavobacterium psychrophilum.

Previously, rainbow trout fed deoxynivalenol (DON) or partially fed (pair-fed) for 4 weeks before and during experimental infection with Flavobacterium psychrophilum had significantly decreased mortality rates. Similar results were obtained in the present study after 12 days, but not after 6 days, feeding 5 ppm DON or pair-fed before infection. Furthermore, feeding 250 ppm chloroquine (CQ) also reduced mortality (p = .052) compared with controls and may have promise for treatment of some fish disease. Parallel groups of fish were maintained on the respective treatments for 15 days, with an additional group that was fasted, but were not infected to monitor autophagy. Fish that were fasted or fed DON had significantly increased LC3II in the liver and fasted fish had significantly decreased LC3II in muscle compared with controls using western blot. There was no difference in LC3II signal in the spleen of any treatment group. Fish that were fasted or pair-fed had significant up-regulation of the Atg genes atg4, atg7, lc3, gabarap and atg12 in muscle using quantitative PCR. Less alteration of Atg expression was seen in liver. Fish treated with CQ had significantly increased expression of atg4, becn1, lc3 and atg12 in the liver. Fish fed DON for 15 days had few alterations of Atg genes in either the liver or muscle. It is still not clear if autophagy is responsible for the resistance of rainbow trout fed DON, CQ or pair-fed before F. psychrophilum infection.

Cyanotoxins within and Outside of Microcystis aeruginosa Cause Adverse Effects in Rainbow Trout (Oncorhynchus mykiss).

The global expansion of toxic Microcystis blooms, and production of cyanotoxins including microcystins, are an increasing risk to freshwater fish. Differentiating intracellular and extracellular microcystin toxicity pathways (i.e., within and outside of cyanobacterial cells) in fish is necessary to assess the severity of risks to populations that encounter harmful algal blooms in pre-to-postsenescent stages. To address this, adult and juvenile Rainbow Trout (Oncorhynchus mykiss) were, respectively, exposed for 96 h to intracellular and extracellular microcystins (0, 20, and 100 µg L-1) produced by Microcystis aeruginosa. Fish were dissected at 24 h intervals for histopathology, targeted microcystin quantification, and nontargeted proteomics. Rainbow Trout accumulated intracellular and extracellular microcystins in all tissues within 24 h, with greater accumulation in the extracellular state. Proteomics revealed intracellular and extracellular microcystins caused sublethal toxicity by significantly dysregulating proteins linked to the cytoskeletal structure, stress responses, and DNA repair in all tissues. Pyruvate metabolism in livers, anion binding in kidneys, and myopathy in muscles were also significantly impacted. Histopathology corroborated these findings with evidence of necrosis, apoptosis, and hemorrhage at similar severity in both microcystin treatments. We demonstrate that sublethal concentrations of intracellular and extracellular microcystins cause adverse effects in Rainbow Trout after short-term exposure.

Impact of feed restriction, chloroquine and deoxynivalenol on viral haemorrhagic septicaemia virus IVb in fathead minnow Pimephales promelas Rafinesque.

Autophagy can markedly alter host response to infectious disease, and several studies have demonstrated that a restricted diet or deoxynivalenol modulates autophagy and reduces mortality of fish due to bacterial disease. The picture is less clear for viral diseases of fish. Duplicate tanks of fathead minnow, Pimephales promelas Rafinesque, were fed a replete diet (control), 100 µM chloroquine, 5 µM deoxynivalenol, 10% (fasted) or 40% of a replete diet (pair-fed) for 2 weeks and then experimentally infected by intraperitoneal injection with 2 × 105 viral haemorrhagic septicaemia virus IVb. Survival from highest to lowest for the different treatments was as follows: deoxynivalenol (average 43.3%); control (40.0%); pair-fed (35.0%); fasted (33.3%); and chloroquine (21.7%). No treatment significantly altered the survival rate of fathead minnow after VHSV IVb infection when compared to controls; however, the fish fed with chloroquine had significantly lower survival rate than the fish fed deoxynivalenol (p < .05).

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.

Magnesium concentration influences size and pulse rate in the upside-down jellyfish, Cassiopea andromeda.

Magnesium is involved in a variety of physiological processes in marine animals and is known to be deleterious in both excess and deficiency. The effects of magnesium concentration ranging from 700 mg/L (low), 1344 mg/L (control), and 2000 mg/L (high) on size and pulse rate in upside-down jellyfish (Cassiopea andromeda) medusae were examined in two separate 28-day trials. Exposure to low magnesium resulted in significantly (p < .05) higher pulse rates and decreased bell diameter and also produced oral arm degradation. Exposure to high magnesium resulted in significantly (p < .05) lower pulse rates and decreased bell diameter as well as oral arm cupping. In both low and high magnesium, almost all specimens changed color from pale blue on Day 1, to brown by Day 28, suggesting a loss of zooxanthellae. The decrease in bell diameter and color change was more pronounced and occurred more rapidly in low magnesium. The results of both trials demonstrate the deleterious effects of high and low magnesium on C. andromeda and emphasize the importance of monitoring magnesium concentration to maintain healthy display animals in public aquaria.

Understanding the pathogenesis of Flavobacterium psychrophilum using the rainbow trout monocyte/macrophage-like cell line, RTS11, as an infection model.

The life cycle of Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), appears to involve interactions with spleen and head kidney macrophages. To develop an in vitro model for studying this, F. psychrophilum was incubated with a rainbow trout splenic monocyte/macrophage-like cell line (RTS11) and fundamental macrophage functions evaluated. The animal cell basal medium, L15, supplemented with bovine serum (FBS) supports RTS11 maintenance, and surprisingly, L15 with 2% FBS (L15/FBS) also supported F. psychrophilum growth. L15/FBS in which the bacteria had been grown is referred to as F. psychrophilum conditioned medium (FpCM). Adding FpCM to RTS11 cultures caused a small, yet significant, percentage of cells to die, many cells to become more diffuse, and phagocytosis to be temporarily reduced. FpCM also significantly stimulated transcript expression for pro-inflammatory cytokines (IL-1ß, TNFα and IL-6) and the anti-inflammatory cytokine (IL-10) after one day of exposure but this upregulation rapidly declined over time. Adding live F. psychrophilum to RTS11 cultures also altered the cellular morphology and stimulated cytokine expression more profoundly than FpCM. Additionally, the phagocytic activity of RTS11 was also significantly impaired by live F. psychrophilum, but not to the same extent as when exposed to FpCM. Adding heat-killed bacteria to RTS11 cultures elicited few changes. These bacteria/RTS11 co-cultures should be useful for gaining a deeper understanding of the pathogenesis of F. psychrophilum and may aid in the development of effective measures to prevent infection and spread of this troublesome disease.

VHSV IVb infection and autophagy modulation in the rainbow trout gill epithelial cell line RTgill-W1.

Autophagy modulation influences the success of intracellular pathogens, and an understanding of the mechanisms involved might offer practical options to reduce the impact of infectious disease. Viral haemorrhagic septicaemia virus (VHSV) can cause high mortality and economic loss in some commercial fish species. VHSV IVb was used to infect a rainbow trout gill cell line, RTgill-W1, followed by the treatment of the cells with different autophagy-modulating reagents. LC3II protein using Western blot was significantly (p < .05) decreased for two days following VHSV infection, and immunofluorescence confirmed that LC3II-positive intracytoplasmic puncta were also decreased. Infection with VHSV resulted in significantly decreased expression of the autophagy-related (Atg) genes atg4, at12, atg13 and becn1 after one day using quantitative PCR. Both viral gene copy number and VHSV N protein were significantly decreased by treating the cells with autophagy-blocking (chloroquine) and autophagy-inhibiting reagents (deoxynivalenol and 3-methyladenine) after three days, while autophagy induction (restricted nutrition and rapamycin) had limited effect. Only treatment of RTgill-W1 with deoxynivalenol resulted in a significant increase in expression of type I interferon. Therefore, the suppression of autophagy initially occurs after VHSV IVb infection, but the modulation of autophagy can also inhibit VHSV IVb infection in RTgill-W1 after three days.

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum.

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 108 cfu/fish and after mortality had begun, fish were given erythromycin- and florfenicol-medicated feed at a rate of 75 mg kg- 1 day- 1 and 10 mg kg- 1  day- 1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30-day trial. Relative to antibiotic-free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.

Autophagy-related genes in rainbow trout Oncorhynchus mykiss (Walbaum) gill epithelial cells and their role in nutrient restriction.

Autophagy is primarily an adaptive response to provide nutrients and energy following exposure to stress and starvation but can also regulate muscle mass and impact infectious disease susceptibility. Expression of 10 autophagy-related (Atg) genes in rainbow trout was monitored throughout the autophagosome formation cycle. The Atg gene sequences of rainbow trout were compared to other species to identify highly conserved regions and to generate primers. Phylogeny trees created with rainbow trout and 14 other species demonstrate that rainbow trout Atg gene sequences have greatest similarity to Atlantic salmon and other fish species. RTgill-W1 cells were subjected to nutrient restriction and compared to cells in normal nutrient conditions using quantitative reverse transcriptase polymerase chain reaction to assess changes in Atg gene expression. Nutrient restriction had a direct impact on Atg gene expression, with atg4, atg9, atg12, lc3, gabarap and becn1 undergoing the greatest differential expression (p < 0.05), most dramatically on Day 3. This was corroborated by Western blot detection of LC3, which also showed a peak of autophagy activity at Day 3 post-nutrient restriction. Atg gene expression revealed autophagy flux in RTgill-W1, as well as, those genes that were most significantly altered by nutrient restriction.

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.

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum).

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD) in freshwater-reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 108  cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 106 , 107 or 108  cfu/fish of F. psychrophilum FPG 101, the 108  cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R2  = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety-seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log10 gene/copies and >3.0 log10 gene copies/reaction, respectively, following infection with 108  cfu/fish.

Atlantic salmon endothelial cells from the heart were more susceptible than fibroblasts from the bulbus arteriosus to four RNA viruses but protected from two viruses by dsRNA pretreatment.

Heart diseases caused by viruses are major causes of Atlantic salmon aquaculture loss. Two Atlantic salmon cardiovascular cell lines, an endothelial cell line (ASHe) from the heart and a fibroblast cell line (BAASf) from the bulbus arteriosus, were evaluated for their response to four fish viruses, CSV, IPNV, VHSV IVa and VHSV IVb, and the innate immune agonist, double-stranded RNA mimic poly IC. All four viruses caused cytopathic effects in ASHe and BAASf. However, ASHe was more susceptible to all four viruses than BAASf. When comparing between the viruses, ASHe cells were found to be moderately susceptible to CSV and VHSV IVb, but highly susceptible to IPNV and VHSV IVa induced cell death. All four viruses were capable of propagating in the ASHe cell line, leading to increases in virus titre over time. In BAASf, CSV and IPNV produced more than one log increase in titre from initial infection, but VHSV IVb and IVa did not. When looking at the antiviral response of both cell lines, Mx proteins were induced in ASHe and BAASf by poly IC. All four viruses induced Mx proteins in BAASf, while only CSV and VHSV IVb induced Mx proteins in ASHe. IPNV and VHSV IVa suppressed Mx proteins expression in ASHe. Pretreatment of ASHe with poly IC to allow for Mx proteins accumulation protected the culture from subsequent infections with IPNV and VHSV IVa, resulting in delayed cell death, reduced virus titres and reduced viral proteins expression. These data suggest that endothelial cells potentially can serve as points of infections for viruses in the heart and that two of the four viruses, IPNV and VHSV IVa, have mechanisms to avoid or downregulate antiviral responses in ASHe cells. Furthermore, the high susceptibility of the ASHe cell line to IPNV and VHSV IVa can make it a useful tool for studying antiviral compounds against these viruses and for general detection of fish viruses.

Development of a walleye spleen stromal cell line sensitive to viral hemorrhagic septicemia virus (VHSV IVb) and to protection by synthetic dsRNA.

A cell line, WE-spleen6, has been developed from the stromal layer of primary spleen cell cultures. On conventional plastic, WE-spleen6 cells had a spindle-shaped morphology at low cell density but grew to become epithelial-like at confluency. On the commercial extracellular matrix (ECM), Matrigel, the cells remained spindle-shaped and formed lumen-like structures. WE-spleen6 cells had intermediate filament protein, vimentin and the ECM protein, collagen I, but not smooth muscle α-actin (SMA) and von Willebrand factor (vWF) and lacked alkaline phosphatase and phagocytic activities. WE-spleen6 was more susceptible to infection with VHSV IVb than a fibroblast and epithelial cell lines from the walleye caudal fin, WE-cfin11f and WE-cfin11e, respectively. Viral transcripts and proteins appeared earlier in WE-spleen6 cultures as did cytopathic effect (CPE) and significant virus production. The synthetic double-stranded RNA (dsRNA), polyinosinic: polycytidylic acid (pIC), induced the antiviral protein Mx in both cell lines. Treating WE-spleen6 cultures with pIC prior to infection with VHSV IVb inhibited the early accumulation of viral transcripts and proteins and delayed the appearance of CPE and significant viral production. Of particular note, pIC caused the disappearance of viral P protein 2 days post infection. WE-spleen6 should be useful for investigating the impact of VHSV IVb on hematopoietic organs and the actions of pIC on the rhabdovirus life cycle.

Development and characterization of an endothelial cell line from the bulbus arteriosus of walleye, Sander vitreus.

A cell line has been developed from the bulbus arteriosus (BA) of the walleye (WE), Sander vitreus (Mitchill), and is termed WEBA. WEBA produced collagen I, and when held at confluency for days or weeks, spontaneously formed capillary-like tubes. WEBA cells bound fluorescently-labeled Ulex europaeus lectin agglutinin I (UEA-1), took up acetylated low density lipoprotein (Ac-LDL), were stained for von Willebrand factor (vWF), and produced nitric oxide (NO). The cytoskeleton consisted at least of α- and ß-tubulin, vimentin, and actin, with the actin organized into circumferential bundles. Immunofluorescence staining revealed at least two tight junction proteins, zonula occludens-1 (ZO-1) and claudin 3. Together these results suggest that WEBA is an endothelial cell line. Relatively high doses of 2,3,7,8-tetrachlorodibenzodioxin (TCDD) induced cytochrome P4501A (CYP1A) protein and 7-ethoxyresorufin o-deethylase (EROD) activity in WEBA. As one of the first fish endothelial and BA cell lines, WEBA should be useful in many disciplines in which the teleost cardiovascular system is a focus.

The effects of Pediococcus acidilactici MA18/5M on growth performance, gut integrity, and immune response using in vitro and in vivo Pacific salmonid models.

Microbial management is central to aquaculture's efficiency. Pediococcus acidilactici MA18/5M has shown promising results promoting growth, modulation of the immune response, and disease resistance in many fishes. However, the mechanisms through which this strain confers health benefits in fish are poorly understood, particularly in Pacific salmonid models. Briefly, the aims of this study were to i) assess the protective effects of P. acidilactici MA18/5M by examining gut barrier function and the expression of tight junction (TJ) and immune genes in vitro and in vivo, and ii) to determine the protective effects of this strain against a common saltwater pathogen, Vibrio anguillarum J382. An in vitro model of the salmonid gut was employed utilizing the cell line RTgutGC. Barrier formation and integrity assessed by TEER measurements in RTgutGC, showed a significant decrease in resistance in cells exposed only to V. anguillarum J382 for 24 h, but pre-treatment with P. acidilactici MA18/5M for 48 h mitigated these effects. While P. acidilactici MA18/5M did not significantly upregulate tight junction and immune molecules, pre-treatment with this strain protected against pathogen-induced insults to the gut barrier. In particular, the expression of ocldn was significantly induced by V. anguillarum J382, suggesting that this molecule might play a role in the host response against this pathogen. To corroborate these observations in live fish, the effects of P. acidilactici MA18/5M was evaluated in Chinook salmon reared in real aquaculture conditions. Supplementation with P. acidilactici MA18/5M had no effect on Chinook salmon growth parameters after 10 weeks. Interestingly, histopathological results did not show alterations associated with P. acidilactici MA18/5M supplementation, indicating that this strain is safe to be used in the industry. Finally, the expression pattern of transcripts encoding TJ and immune genes in all the treatments suggest that variation in expression is more likely to be due to developmental processes rather than P. acidilactici MA18/5M supplementation. Overall, our results showed that P. acidilactici MA18/5M is a safe strain for use in fish production, however, to assess the effects on growth and immune response previously observed in other salmonid species, an assessment in adult fish is needed.

Impact of Infection with Flavobacterium psychrophilum and Antimicrobial Treatment on the Intestinal Microbiota of Rainbow Trout.

The diversity and composition of intestinal microbiota in rainbow trout have been studied using next-generation sequencing (NGS), although few studies have examined the effects of antimicrobials. We evaluated the effect of antibiotics florfenicol and erythromycin and infection with or without Flavobacterium psychrophilum on the intestinal microbiota in rainbow trout juveniles (30-40 g) using NGS. Prophylactic oral antibiotic treatments were administered for 10 days before groups of fish were injected intraperitoneally with virulent F. psychrophilum. Intestinal content (allochthonous bacteria) was collected at day -11, 0, 12, and 24 p.i., and the v3-v4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Before prophylactic treatment, Tenericutes and Proteobacteria were the most abundant phyla identified and Mycoplasma was the most abundant genus. Fish infected with F. psychrophilum had decreased alpha diversity and a high abundance of Mycoplasma. Fish administered florfenicol had increased alpha diversity compared to the control at day 24 p.i., although both florfenicol and erythromycin-treated fish had a higher abundance of potential pathogens, specifically Aeromonas, Pseudomonas, and Acinetobacter. Mycoplasma disappeared after treatment but appeared again after day 24. This study demonstrates that prophylactic oral treatment with antibiotics florfenicol and erythromycin as well as F. psychrophilum infection changed the composition of intestinal microbiota in rainbow trout juveniles that did not recover by day 24 p.i. and further long-term effects on the host need to be investigated.

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.