Co‐infection of rainbow trout (Oncorhynchus mykiss) with infectious hematopoietic necrosis virus and Flavobacterium psychrophilum

Co‐infection of rainbow trout with infections haematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum is known to occur, and it has been speculated that a combined infection can result in dramatic losses. Both pathogens can persist in fish in an asymptomatic carrier state, but the impact of co‐infection has not been well characterized or documented. In this study, it was hypothesized that fish co‐infected with F. psychrophilum and IHNV would exhibit greater mortality than fish infected with either pathogen alone. To test this, juvenile rainbow trout were co‐infected with low doses of either IHNV or F. psychrophilum, and at 2 days post‐initial challenge, they were given a low dose of the reciprocal pathogen. This combined infection caused high mortality (76.2%–100%), while mortality from a single pathogen infection with the same respective dose was low (5%–20%). The onset of mortality was earlier in the co‐infected group (3–4 days) when compared with fish infected with F. psychrophilum alone (6 days) or IHNV (5 days), confirming the synergistic interaction between both pathogens. Co‐infection led to a significant increase in the number of F. psychrophilum colony‐forming units and IHNV plaque‐forming units within tissues. This finding confirms that when present together in co‐infected fish, both pathogens are more efficiently recovered from tissues. Furthermore, pathogen genes were significantly increased in co‐infected groups, which parallel the findings of increased systemic pathogen load. Extensive tissue necrosis and abundant pathogen present intracellularly and extracellularly in haematopoietic tissue. This was pronounced in co‐infected fish and likely contributed to the exacerbated clinical signs and higher mortality. This study provides novel insight into host–pathogen interactions related to co‐infection by aquatic bacterial and viral pathogens and supports our hypothesis. Such findings confirm that mortality in fish exposed to both pathogens is greatly elevated compared to a single pathogen infection.     

Assessment of cross‐protection to heterologous strains of Flavobacterium psychrophilum following vaccination with a live‐attenuated coldwater disease immersion vaccine

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, remains one of the most significant bacterial diseases of salmonids worldwide. A previously developed and reported live‐attenuated immersion vaccine (F. psychrophilum; B.17‐ILM) has been shown to confer significant protection to salmonids. To further characterize this vaccine, a series of experiments were carried out to determine the cross‐protective efficacy of this B.17‐ILM vaccine against 9 F. psychrophilum isolates (representing seven sequence types/three clonal complexes as determined by multilocus sequence typing) in comparison with a wild‐type virulent strain, CSF‐259‐93. To assess protection, 28‐day experimental challenges of rainbow trout (Oncorhynchus mykiss) fry were conducted following immersion vaccinations with the B.17‐ILM vaccine. F. psychrophilum strains used in challenge trials were isolated from several fish species across the globe; however, all were found to be virulent in rainbow trout. The B.17‐ILM vaccine provided significant protection against all strains, with relative percent survival values ranging from 51% to 72%. All vaccinated fish developed an adaptive immune response (as measured by F. psychrophilum‐specific antibodies) that increased out to the time of challenge (8 weeks postimmunization). Previous studies have confirmed that antibody plays an important role in protection against F. psychrophilum challenge; therefore, specific antibodies to the B.17‐ILM vaccine strain appear to contribute to the cross‐protection observed to heterologous strain. The ability of such antibodies to bind to similar antigenic regions for all strains was confirmed by western blot analyses. Results presented here support the practical application of this live‐attenuated vaccine, and suggest that it will be efficacious even in aquaculture operations affected by diverse strains of F. psychrophilum.     

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.)

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country‐wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin‐inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin‐killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate‐buffered saline or vaccine formulated with Montanide by intra‐peritoneal (i.p.) injection and challenged by intra‐muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post‐vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un‐adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.     

Evaluation of the immune response in rainbow trout fry,Oncorhynchus mykiss (Walbaum), after waterborne exposure to Flavobacterium psychrophilum and/or hydrogen peroxide

The immune response in rainbow trout fry against Flavobacterium psychrophilum was elucidated using an immersion‐based challenge with or without prior exposure to hydrogen peroxide (H₂O₂). Samples were taken from the head kidney 4, 48, 125 and 192 h after immersion, and the regulation of several genes was examined. Bacterial load was assessed based on the presence of 16S rRNA and correlated with gene expression, and the levels of specific antibodies in the blood were measured 50 days post‐infection. Separately, both H₂O₂ and F. psychrophilum influenced gene expression, and pre‐treatment with H₂O₂ influenced the response to infection with F. psychrophilum. Pre‐treatment with H₂O₂ also affected correlation between gene regulation and pathogen load for several genes. A delay in antibody production in H₂O₂‐treated fish in the early phase of infection was indicated, but H₂O₂ exposure did not affect antibody levels 50 days post‐infection. An increasing amount of F. psychrophilum 16S rRNA was found in the head kidneys of infected fish pre‐treated with H₂O₂ relative to the F. psychrophilum group. The results show that a single pre‐treatment with H₂O₂ impairs the response against F. psychrophilum and may intensify infection.     

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.     

Purified deoxynivalenol or feed restriction reduces mortality in rainbow trout,Oncorhynchus mykiss (Walbaum), with experimental bacterial coldwater disease but biologically relevant concentrations of deoxynivalenol do not impair the growth of Flavobacterium psychrophilum

Diets containing deoxynivalenol (DON) were fed to rainbow trout Oncorhynchus mykiss (Walbaum) for 4 weeks followed by experimental infection (intraperitoneal) with Flavobacterium psychrophilum (4.1 × 10⁶ colony‐forming units [CFU] mL⁻¹). Mortality of rainbow trout fed either 6.4 mg kg⁻¹ DON or trout pair‐fed the control diet was significantly reduced (P < 0.05) in comparison with trout fed the control diet to apparent satiation (<0.1 mg kg⁻¹ DON). In a second experiment, trout were fed one of three experimental diets; a control diet, a diet produced with corn naturally contaminated with DON (3.3 mg kg⁻¹ DON) or a diet containing purified DON (3.8 mg kg⁻¹); however, these fish were not experimentally infected. The presence of DON resulted in significant reduction (P < 0.0001) in feed intake as well as weight gain after 4 weeks. Respiratory burst of head‐kidney leucocytes isolated from rainbow trout fed diets containing purified DON (3.8 mg kg⁻¹) was significantly higher (P < 0.05) at 35 day post‐exposure compared with controls. The antimicrobial activity of DON was examined by subjecting F. psychrophilum in vitro to serial dilutions of the chemical. Complete inhibition occurred at a concentration of 75 mg L⁻¹ DON, but no effect was observed below this concentration (0–30 mg L⁻¹).     

Immunization of rainbow trout Oncorhynchus mykiss (Walbaum) with a crude lipopolysaccharide extract from Flavobacterium psychrophilum

Control methods for Flavobacterium psychrophilum are limited and oftentimes ineffective; hence, research efforts have focused on vaccine development. This study tested the hypothesis that a crude lipopolysaccharide (LPS) extract from F. psychrophilum will elicit a protective immune response in rainbow trout Oncorhynchus mykiss (Walbaum) against F. psychrophilum challenge. Rainbow trout (mean weight, 3 g) were immunized intraperitoneally with the following treatment and control preparations: 10 μg of crude LPS with or without Freund's complete adjuvant (FCA), 25 μg of crude LPS with or without FCA and saline with or without FCA. Immunization of fish with 10 or 25 μg of crude LPS/FCA resulted in significant antibody responses against F. psychrophilum using ELISA with a whole‐cell lysate as the coating antigen, but only minimal levels of protection were conferred following F. psychrophilum challenge at 14 weeks post immunization. Western blot analyses demonstrated that fish exhibited antibodies specific for low‐molecular mass proteins present in the crude LPS extract, but did not exhibit antibodies specific for F. psychrophilum LPS. The results indicate that higher immunization doses and/or the use of an alternative extraction method that yields larger LPS molecules (23–70 kDa) may be necessary to elicit specific antibody responses against F. psychrophilum LPS.     

Diets containing corn naturally contaminated with deoxynivalenol reduces the susceptibility of rainbow trout (Oncorhynchus mykiss) to experimental Flavobacterium psychrophilum infection

The objective of this study was to determine if deoxynivalenol (DON) exposure alters the susceptibility of rainbow trout to bacterial coldwater disease caused by Flavobacterium psychrophilum. Rainbow trout were fed a nutritionally complete diet containing corn that was naturally contaminated with DON at a desired concentration of <0.5 (control and pair‐fed treatments), 4 or 6 ppm over 7 weeks to apparent satiation. After 4 weeks, fish were infected by intraperitoneal injection with F. psychrophilum (3.03x10⁶ CFU mL^?1) via intraperitoneal injection and monitored for morbidity and mortality. A significant linear reduction in feed intake was associated with increasing dietary levels of DON contamination over the initial 4 weeks. There was a significant reduction (P < 0.05) in cumulative per cent mortality in DON‐fed groups (4.1 ppm, 11%; 5.9 ppm, 7%) in comparison to control (46%) and pair‐fed (25%) groups at 21 days post infection. Mortality of trout pair‐fed the control diet was also significantly lower (P < 0.05) than the control group fed to apparent satiation. A replicate trial using genetically similar fish and the same experimental design produced similar results. These results suggest that DON exposure and restricted feed intake provided a protective effect for rainbow trout infected with F. psychrophilum.     

Multilocus sequence typing (MLST) analysis of California Flavobacterium psychrophilum reveals novel genotypes and predominance of CC‐ST10 in California salmonid hatcheries

The Gram‐negative bacterium, Flavobacterium psychrophilum, is endemic to California, USA, where it is an important pathogen in salmonid aquaculture, especially in rainbow trout (Oncorhynchus mykiss). Disease outbreaks caused by F. psychrophilum in rainbow trout fingerlings can approach 90% mortality, resulting in millions of dollars of economic losses annually. The focus of this study was to investigate the genetic diversity of 49 F. psychrophilum isolates collected from disease outbreaks in 17 salmonid hatcheries in California, USA, from 2015 to 2018 using multilocus sequence typing. Results suggest California F. psychrophilum isolates are diverse, representing 11 distinct sequence types (STs), three of which were previously undescribed. Still, the majority of genotyped isolates (n = 41) belonged to a single clonal complex (CC), CC‐ST10, which is the largest CC worldwide and has been linked to disease outbreaks on several continents. Results of this study provide evidence of marked intraspecific genetic diversity of F. psychrophilum from California. The biological significance of this genetic variability is unclear but could have implications for future vaccine development and treatments. Further studies investigating the virulence, antigenic, and antimicrobial susceptibility profiles of F. psychrophilum are warranted to better understand the epizootiology of this pathogen in the Western United States.     

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.     

Effects of dietary yeast inclusion and acute stress on postprandial plasma free amino acid profiles of dorsal aorta‐cannulated rainbow trout

In a 4‐week experiment, 15 cannulated rainbow trout were fed three diets based on fish meal (FM), Saccharomyces cerevisiae yeast (SC) and Wickerhamomyces anomalus and S. cerevisiae yeast mix (WA). Fish were fed daily, and blood samples were collected on day 7 of each week at 0, 3, 6, 12 and 24 hr after feeding. In the final week, fish were exposed to a 1‐min netting stressor. All essential and non‐essential plasma amino acid levels except methionine were similar between fish fed diets FM, SC and WA. Plasma methionine and sarcosine were significantly higher in fish fed diets SC and WA, possibly due to the crystalline methionine level, form or feeding regime. Hydroxy‐proline and 3‐methyl‐histidine were higher in fish fed diet FM, which can be explained by the higher levels present in fish meal compared with yeast. In stressed fish, there were no dietary effects on plasma amino acid levels, but significant increases in taurine and cystathionine were found in stressed compared with unstressed fish. These results demonstrate that yeast‐based diets produce similar plasma amino acid profiles to fish meal and suggest that yeast may be a suitable fish meal replacement in diets for rainbow trout.     

Isolation of bacterial probiotic candidates from the gastrointestinal tract of rainbow trout,Oncorhynchus mykiss (Walbaum), and screening for inhibitory activity against Flavobacterium psychrophilum

In this study, 318 bacterial strains were isolated from the gastrointestinal (GI) tracts of 29 rainbow trout, Oncorhynchus mykiss (Walbaum). These bacteria were screened in vitro for their ability to inhibit growth of Flavobacterium psychrophilum, the causative agent of coldwater disease. Bacteria observed to inhibit F. psychrophilum growth were further screened against rainbow trout bile, as an indicator of their ability to survive in the GI tract. This screening resulted in narrowing the pool to 24 bacterial isolates. Those 24 isolates were then tested for pathogenicity in rainbow trout by intraperitoneal injection. Following a 28‐day challenge, eight isolates were shown to cause direct mortality and were eliminated from further study. As a result, 16 bacterial isolates were identified as probiotic candidates with the potential to control or reduce disease caused by F. psychrophilum.     

Effects of oral passive immunization against somatostatin‐14 on growth performance,body composition and IgY delivery in rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio)

Egg‐yolk antibodies (IgY) against somatostatin‐14 (anti‐SST‐14) were evaluated as orally administered, growth promotants in gastric rainbow trout (Oncorhynchus mykiss) and agastric common carp (Cyprinus carpio) in an 8‐week feeding trial. Feeding groups were compared with fish which did not receive anti‐SST‐14 IgY. Growth responses and IgY plasma contents of the blood were assessed. In contrast to rainbow trout, oral anti‐SST‐14 addition to carp significantly improved protein efficiency ratio (PER, 1.02 ± 0.04) and protein productive value (PPV, 26.7 ± 0.91) after 56 days (P < 0.05) compared to the negative control (PER = 0.91 ± 0.02; PPV = 22.9 ± 0.66). IgY was undetectable in plasma of rainbow trout after oral administration, indicating that gastric degradation of the fed IgY makes this application route for growth promotion in gastric trout challenging.     

Effect of combination of dietary Bacillus subtilis and trans‐cinnamic acid on innate immune responses and resistance of rainbow trout,Oncorhynchus mykiss to Yersinia ruckeri

The present study investigated the effects of combination of dietary Bacillus subtilis and trans‐cinnamic acid on serum biochemical parameters, innate immune responses and resistance of rainbow trout, Oncorhynchus mykiss to Yersinia ruckeri. Six experimental groups of fish with mean weights of 20.58 ± 0.35 g were used in the study. Five experimental groups of fish were fed diets containing Bacillus subtilis (10⁷ per gram) or a mix of the Bacillus subtilis (BS) and trans‐cinnamic acid (25 mg/kg‐25trcBS, 50 mg/kg‐50trcBS, 75 mg/kg‐75 trcBS, 150 mg/kg‐150 trcBS), whereas an additive‐free basal diet served as the control (Cont). In this study, an increase was observed in granulocyte percentage, respiratory burst activity, phagocytic activity, phagocytic index, myeloperoxidase activity and total antiprotease activity especially in fish fed with mix of the BS and trans‐cinnamic acid‐supplemented diets (p < .05). Moreover, at the end of the 20‐day challenge period the survival rates and antibody titre (p < .05), and relative per cent survival were higher in the BS group and all trcBS groups compared with control group. As a conclusion, the results in the present study show that feeding rainbow trout with diets containing a mix of B. subtilis and trans‐cinnamic acid over a 60‐day period might be sufficient for improving fish immune responses and disease resistance against Y. ruckeri.     

First identification and characterization of Streptococcus iniae obtained from tilapia (Oreochromis aureus) farmed in Mexico

This is the first study to isolate, identify and characterize Streptococcus iniae as the causative disease agent in two tilapia (Oreochromis aureus) populations. The populations were geographically isolated, of distinct origins, and did not share water sources. Affected fish showed various external (e.g., exophthalmia and cachexia, among others) and internal (e.g., granulomatous septicaemia and interstitial nephritis, among others) signs. All internal organ samples produced pure cultures, two of which (one from each farm, termed S‐1 and S‐2) were subjected to biochemical, PCR and 16S rRNA sequencing (99.5% similarity) analyses, confirming S. iniae identification. The two isolates presented genetic homogeneity regardless of technique (i.e., RAPD, REP‐PCR and ERIC‐PCR analyses). Pathogenic potentials were assessed through intraperitoneal injection challenges in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). Rainbow trout mortalities were respectively 40% and 70% at 10⁴ and 10⁶ CFU per fish with the S‐1 isolate, while 100% mortality rates were recorded in zebrafish at 10² and 10⁴ CFU per fish with the S‐2 isolate. The obtained data clearly indicate a relationship between intensified aquaculture activities in Mexico and new disease appearances. Future studies should establish clinical significances for the tilapia industry.     

Experimental infection by Yersinia ruckeri O1 biotype 2 induces brain lesions and neurological signs in rainbow trout (Oncorhynchus mykiss)

Pathological manifestations in rainbow trout (Oncorhynchus mykiss) following experimental waterborne infection with Yersinia ruckeri serotype O1 biotype 2 (strain 07111224) were investigated. Rainbow trout were exposed to 8 × 10⁷ CFU/ml of Y. ruckeri by bath for 6 hr, and mortality was then monitored for 22 days post‐infection (dpi). Organs were sampled at 3 dpi and also from moribund fish showing signs of severe systemic infection such as bleeding, exophthalmia or erratic swimming behaviour. Y. ruckeri was observed in the meninges and diencephalon of the brain, and lamina propria of olfactory organ at 3 dpi. At 12 dpi, Y. ruckeri had spread throughout the brain including cranial connective tissues and ventricles and the infection was associated with haemorrhages and an infiltration with leucocytes. Y. ruckeri infection and associated with leucocyte infiltration were observed at 13 dpi. In conclusion, Y. ruckeri strain 07111224 causes encephalitis in the acute phase of infection, which could explain why Y. ruckeri‐affected fish show exophthalmia and erratic swimming known as signs of ERM.     

Partial replacement of soybean meal with Methylobacterium extorquens single‐cell protein in feeds for rainbow trout (Oncorhynchus mykiss Walbaum)

A feeding trial was conducted with juvenile rainbow trout (15–16 g initial weight) to assess the effects of including single‐cell protein (SCP) produced from Methylobacterium extorquens in trout feeds. Three isonitrogenous and isoenergetic diets were produced: a control diet and two experimental diets containing 5% or 10% bacterial protein meal replacing soybean meal. Triplicate tanks, each containing 35 fish, were fed each diet to apparent satiation in a constant‐temperature (15°C), flow‐through tank system for 12 weeks. No statistically significant differences in final fish weight or other fish growth parameters were observed. Similarly, feed efficiency parameters showed no significant differences among groups. Nutrient retention indices (protein, fat, energy) were relatively high and similar among fish in each dietary treatment group, as were whole body proximate compositions. Fish survival was high, with a small but statistically significant increase for the 10% SCP diet. Overall, results demonstrate that SCP from M. extorquens is a safe and effective alternative protein for rainbow trout diets at the low inclusion levels tested. Slightly lower weight gain in fish fed the 10% SCP diet was largely due to lower feed intake, suggesting that adding palatability‐enhancing ingredients to feeds may allow higher levels of M. extorquens SCP to be used without compromising fish growth.     

Recovery of Bacillus mycoides,B. pseudomycoides and Aeromonas hydrophila from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) with gill disease

During a 3‐month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B. pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.