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.     

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.     

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.     

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.     

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.     

Cross-protection of a live-attenuated Flavobacterium psychrophilum immersion vaccine against novel Flavobacterium spp. and Chryseobacterium spp. strains

For salmonid producers, a common threat is Flavobacterium psychrophilum. Recent advancements in bacterial coldwater disease (BCWD) management include the development of a live-attenuated immersion vaccine that cross-protects against an array of F. psychrophilum strains. Emerging family Flavobacteriaceae cases associated with clinical disease have been increasing, including pathogenic isolates of Flavobacterium spp. and Chryseobacterium spp. The cross-protective ability of a live-attenuated F. psychrophilum vaccine was determined against three virulent Flavobacteriaceae isolates. Juvenile rainbow trout were vaccinated, developed high F. psychrophilum-specific antibody titres and were challenged with Chryseobacterium spp. isolates (S25 and T28), a Flavobacterium sp. (S21) isolate, a mixed combination of S21:S25:T28, and a standard virulent F. psychrophilum CSF259-93 strain. Results demonstrated strong protection in the CSF259-93 vaccinated group (relative per cent survival (RPS)=94.44%) when compared to the relevant CSF259-93 controls (p < .001). Protection was also observed for vaccinated fish challenged with the S21:S25:T28 mix (RPS = 85.18%; p < .001). However, protection was not observed with the S21, S25 or T28 isolates alone. Analysis of whole-cell lysates revealed differences in protein banding by SDS-PAGE, but conserved antigenic regions by Western blot in S25 and T28. Results demonstrate that this live-attenuated vaccine provided protection against mixed flavobacterial infection and suggest further benefits against flavobacteriosis.     

Comparative proteomic analysis of virulent and rifampicin-attenuated Flavobacterium psychrophilum

Flavobacterium psychrophilum is the aetiologic agent of bacterial coldwater disease and rainbow trout fry syndrome. In this study, we compared a wild‐type strain (CSF 259‐93) with a rifampicin‐resistant strain and virulence‐attenuated strain of F. psychrophilum (CSF 259‐93B.17). The attenuated strain harboured a mutation in the rpoB gene consistent with resistance to rifampicin. Two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and mass spectrometry demonstrated an altered proteome with eight proteins characteristic for the parent strain and six that were unique to the attenuated strain. Immunoblotting with a diagnostic monoclonal antibody (FL‐43) identified a putative antigen (FP1493) that was subsequently cloned, expressed as a recombinant protein and confirmed as recognized by FL‐43. 2D‐PAGE, immunoblotting with rainbow trout, Oncorhynchus mykiss (Walbaum), convalescent antisera and mass spectrometry of bacterial whole‐cell lysates revealed several uniquely expressed immunoreactive proteins including FP1493. An FP1493 recombinant subunit vaccine was tested, but did not provide protection against challenge with the CSF259‐93 strain. While the exact mechanism responsible for altered protein synthesis and attenuation of CSF 259‐93B.17 is still unknown, the differentially expressed immunoreactive proteins are a valuable resource to develop subunit vaccines and to identify proteins that are potentially involved in disease.     

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.     

Characterization of serum and mucosal antibody responses and relative per cent survival in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization and challenge with Flavobacterium psychrophilum

Serum and mucosal antibody responses of juvenile rainbow trout, Oncorhynchus mykiss, were characterized by enzyme‐linked immunosorbent assay (ELISA) following immunization with various preparations of formalin‐killed Flavobacterium psychrophilum cells. The protective nature of these preparations was then determined by immunizing rainbow trout fry and challenging with the bacterium. Juvenile rainbow trout immunized intraperitoneally (i.p.) with formalin‐killed F. psychrophilum emulsified with Freund's complete adjuvant (FCA), and i.p. with formalin‐killed F. psychrophilum either with or without culture supernatant generated significant serum antibody responses by 6 and 9 weeks, respectively. Significant mucosal antibody responses were detected by 9 weeks only in fish immunized i.p. with killed F. psychrophilum/FCA. Following immunization and bacterial challenge of rainbow trout fry, protective immunity was conferred in F. psychrophilum/FCA and saline/FCA groups with relative per cent survival values of up to 83 and 51, respectively. Significant protection was not observed in treatment groups immunized by immersion or i.p. without adjuvant at the challenge doses tested. Results suggest that stimulation of non‐specific immune factors enhances the ability of fish to mount a protective immune response, but specific antibody appears necessary to provide near complete protection. In this study, an ELISA was developed to monitor anti‐F. psychrophilum antibody production in trout. The relationship of such responses to protective immunity suggests that future vaccination strategies against coldwater disease may require stimulation of both the innate and adaptive arms of the immune response.     

Serological diversity in Flavobacterium psychrophilum: A critical update using isolates retrieved from Chilean salmon farms

Chile is currently the second largest producer of farmed salmon worldwide, but Flavobacterium psychrophilum, as one of the most detrimental pathogens, is responsible for major losses during the freshwater culturing step in salmonid fish farms. An antigenic study conducted 10 years ago reported four serological groups using 20 F. psychrophilum Chilean strains. To reduce disease outbreaks and to develop vaccine candidates, antigenic knowledge needs to be regularly updated using a significant number of additional recent F. psychrophilum isolates. The present study aimed at investigating the serological diversity of 118 F. psychrophilum isolates collected between 2006 and 2018 from farmed Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch). The current study supports an expansion of the known antigenic groups in Chile from 4 to 14. However, the use of the slide-agglutination technique for serotyping is costly, is labour-intensive and requires significant technical expertise. Addressing these points, the mPCR-based procedure was a very useful tool for serotyping the collected Chilean F. psychrophilum isolates. This technique revealed the presence of diverse mPCR serotypes (i.e. types 0, 1, 2 and 4). Therefore, mPCR should be employed to select the bacterial strain(s) for vaccine development and to conduct follow-up, selective breeding or epidemiological surveillance in Chilean fish farms. Given the presented findings, changes to Chilean fish-farming practices are vital for ensuring the continued productivity and well-being of farmed salmonids.     

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.     

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.     

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).     

Are brown trout Salmo trutta fario and rainbow trout Oncorhynchus mykiss two of a kind? A comparative study of salmonids to temperature‐influenced Tetracapsuloides bryosalmonae infection

Proliferative kidney disease (PKD) of salmonids caused by Tetracapsuloides bryosalmonae causes high mortalities of wild brown trout (Salmo trutta fario) and farmed rainbow trout (Oncorhynchus mykiss) at elevated water temperatures. Here the aim was to compare the temperature‐dependent modulation of T. bryosalmonae in the two salmonid host species, which display different temperature optima. We used a novel experimental set‐up in which we exposed brown trout and rainbow trout to an identical quantified low concentration of T. bryosalmonae for a short time period (1 hr). We followed the development of the parasite in the fish hosts for 70 days. PKD prevalence and parasite kinetics were assessed using qPCR. Exposures were performed at temperatures (12°C and 15°C) that reflect an environmental scenario that may occur in the natural habitat of salmonids. T. bryosalmonae infection was confirmed earliest in brown trout kept at 15°C (day 7 post‐exposure) while, in all other groups, T. bryosalmonae was not confirmed until day 15 post‐exposure. Moreover, significantly greater infection prevalence and a faster increase of parasite intensity were observed in brown trout kept at 15°C than in all other groups. These results indicate that PKD is differentially modulated by water temperature in related host species.     

Radiological examination of the spinal column in farmed rainbow trout Oncorhynchus mykiss (Walbaum): experiments with Flavobacterium psychrophilum and oxytetracycline

Flavobacterium psychrophilum and oxytetracycline have both been associated with spinal deformities in salmonids. Experiments were carried out to investigate whether infection with F. psychrophilum or medication with oxytetracycline (OTC) at the fry stage would result in an increased occurrence of vertebral column deformities in rainbow trout, Oncorhynchus mykiss (Walbaum). Fish were on‐grown for 9 months and examined by radiology at the end of the experiments. There was a relationship between infection by F. psychrophilum and deformities of the spinal column, if fish with more than 10 affected vertebrae were classified as deformed. The deformities found among infected fish were often visible externally and were more severe than those seen among control fish (most deformities foun among controls were only seen on X‐ray photographs). Deformities were evenly spread along the vertebral column of infected fish. OTC treatments of up to 200 mg of OTC (kg fish)^?1 day^?1 for 10 days and repeated three times did not result in increased spinal deformities relative to untreated control groups; therefore, medication of rainbow trout with oxytetracycline did not cause deformities of the spinal column under our treatment conditions.     

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.     

Development and validation of glycoprotein‐based native‐subunit vaccine for fish against Aeromonas hydrophila

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein‐based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA‐specific lectin and size‐exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)₃] and Group IV [with Al(OH)₃ and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.     

Water recirculation and good management: potential methods to avoid disease outbreaks with Flavobacterium psychrophilum

Flavobacterium psychrophilum infections cause high mortality among rainbow trout, Oncorhynchus mykiss, fry in Danish fish farms and hatcheries. Hatcheries based entirely on bore‐hole water recirculation systems have been suggested as a possibility for eliminating F. psychrophilum or at least keeping the amount of this bacterium low. The occurrence of the bacterium in a bore‐hole water recirculation system was compared with a combined bore‐hole water and stream water flow‐through system in a hatchery where outbreaks of rainbow trout fry syndrome caused by F. psychrophilum often occurred. Broodfish, unfertilized and fertilized eggs, eyed eggs and fry, as well as water samples from the tanks/troughs with broodfish/fry, were examined. Suspect yellow bacterial colonies were either confirmed or rejected as F. psychrophilum by growth characteristics and by PCR. As both virulent and less virulent F. psychrophilum isolates are known, isolates were characterized. The isolates were ribotyped and grouped according to ribotyping patterns. Representatives of the groups were serotyped. Fry isolates were very homogeneous whereas isolates from broodfish were heterogeneous, whether the isolates originated from external surfaces of the fish (mucus from skin and gills, haemorrhages and ulcers) or internal organs. Flavobacterium psychrophilum was isolated from broodfish in both water systems; 56% of investigated broodfish from the borehole/flowthrough system and 36% from the recirculation facility harboured the bacterium. In the recirculation system, the bacterium was isolated from fish (ulcers, milt, liver, abdominal cavity) kept in the system for 11 months. Flavobacterium psychrophilum was found in milt and ovarian fluid as well as on the surface of fertilized eggs, but not inside the eggs. Fry also harboured F. psychrophilum, but in the water recirculation system the bacterium was first isolated from the fry after they had been graded. Flavobacterium psychrophilum was found regularly in other parts of the hatchery (outside the recirculation facility), including at the time of grading, suggesting that the occurrence of F. psychrophilum in the fry recirculation facility was due to contamination from the borehole/flowthrough hatchery. It is suggested that the combination of bore‐hole water recirculation systems and good management procedures (including egg disinfection) is a possible method for hatcheries to avoid disease outbreaks due to F. psychrophilum.