Effect of hydrogen peroxide and/or Flavobacterium psychrophilum on the gills of rainbow trout,Oncorhynchus mykiss (Walbaum)

The immune response and morphological changes in the gills of rainbow trout fry after immersion in hydrogen peroxide (H₂O₂), Flavobacterium psychrophilum or combined exposure were examined. The gills were sampled 4, 48, 125 and 192 h after exposure, and the regulation of expression of the following genes was investigated using qPCR: IgT, IgM, CD8, CD4, MHC I, MHC II, IL-4/13A, TcR-β, IL-10, IL-1β, IL-17, SAA and FoxP3. Bacteria were not observed in haematoxylin-and-eosin-stained gill tissue, but the presence of F. psychrophilum 16S rRNA was detected using qPCR. The 16S rRNA levels were correlated with gene expression. Although pretreatment with H₂O₂ before immersion in F. psychrophilum did not significantly alter the amount of bacteria found in the gill, the immune response was influenced: exposure to F. psychrophilum resulted in a negative correlation with expression of IL-17c1, MHC I and MHC II, while pretreatment with H₂O₂ resulted in a positive correlation with IL-4/13A and IgM. Exposure to either H₂O₂ or F. psychrophilum influenced the regulation of gene expression and damaged tissue. Exposure to both combined altered the immune response to infection and postponed healing of gill tissue.     

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.     

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.     

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.     

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.     

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.     

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.     

Identification and pathogenicity study of emerging fish pathogens Acinetobacter junii and Acinetobacter pittii recovered from a disease outbreak in Labeo catla (Hamilton, 1822) and Hypophthalmichthys molitrix (Valenciennes, 1844) of freshwater wetland in West Bengal,India

The disease outbreaks in aquaculture system of wetlands are the major cause of fish mortality. Among various bacterial septicaemic diseases, fish mortality caused by Acinetobacter spp. is recently reported in different fish species. Fish disease outbreak was investigated in a wetland of West Bengal, India to identify the aetiological factors involved. The moribund fish were examined and subjected to bacterial isolation. Two bacterial causative agents were identified as Acinetobacter junii and Acinetobacter pittii by biochemical characterization and 16S rRNA gene amplification. Both the isolates were oxidase‐negative, nitrate‐negative, catalase‐positive and indole‐negative. The molecular identification using 16S rRNA gene sequencing and phylogenetic tree analysis further confirmed the two Acinetobacter spp. with 97%–99% similarity. The antibiotic resistance patterns of these two bacteria revealed that both of them were resistant to β‐lactam, cefalexin, cephalothin, amoxyclav, cefuroxime, cefadroxil, clindamycin, vancomycin and penicillin. In addition, A. pittii was also resistant to other antibiotics of cephams group such as ceftazidime and cefotaxime. In the challenge experiment, both A. junii and A. pittii were found to be pathogenic with LD₅₀ of 1.24 × 10⁵ and 1.88 × 10⁷ cfu/fish respectively. Histopathological examination of gill, liver and kidney revealed prominent changes supporting bacterial septicaemia. The investigation reports for the first time on concurrent infection by A. junii and multidrug‐resistant (MDR)‐A. pittii as emerging fish pathogens to cause severe mortality in Labeo catla and Hypophthalmichthys molitrix in a freshwater wetland.     

Zebrafish (Danio rerio) as an animal model for bath infection by Flavobacterium psychrophilum

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout syndrome in freshwater salmonid fish worldwide, generating injuries and high mortality rates. Despite several studies on this bacterium, the infection mechanism remains unknown due to limitations in the employed animal models. In this work, we propose using zebrafish (Danio rerio) as a model for studying bacterial pathogenicity. To substantiate this proposal, zebrafish infection by F. psychrophilum strain JIP 02/86 was characterized. Zebrafish larvae were infected using the bath method, and morphological changes and innate immune system activation were monitored using transgenic fish. Salmonid-like infection phenotypes were observed in 4.74% of treated larvae, as manifested by fin, muscle and caudal peduncle damage. Symptomatic and dead larvae accounted for 1.35% of all challenged larvae. Interestingly, infected larvae with no infection phenotypes showed stronger innate immune system activation than specimens with phenotypes. A failure of function assay for myeloid factor pu.1 resulted in more infected larvae (up to 43.5%), suggesting that low infection rates by F. psychrophilum would be due to the protective actions of the innate immune system against this bacterium in zebrafish larvae. Our results support the use of zebrafish as an infection model for studying F. psychrophilum. Furthermore, the percentage of infected fish can be modulated by disturbing, to varying extents, the differentiation of myeloid cells. Using this evidence as a starting point, different aspects of the infection mechanism of F. psychrophilum could be studied in vivo.     

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.     

Dynamics of piscine francisellosis differs amongst tilapia species (Oreochromis spp.) in a controlled challenge with Francisella noatunensis subsp. orientalis

A 25‐week immersion challenge was conducted exposing Oreochromis mossambicus, Oreochromis aureus and Oreochromis urolepis hornorum to Francisella noatunensis subsp. orientalis (Fno). Two populations were compared for each fish species; ‘resident fish’ were defined as fish maintained in tanks since week 0 of challenge, whereas ‘naïve fish’ were defined as fish added to tanks once temperature in water reached <26 °C at 21 weeks post‐challenge. Fno genome equivalents (GEs) in water were similar in all treatments 1 h post‐challenge; however, significantly lower Fno GEs were detected 2 weeks post‐challenge in all tanks, and the only treatment with detectable Fno GE after 4 weeks of challenge were the O. mossambicus tanks. Twenty‐one weeks post‐challenge, naïve fish were stocked with ‘resident’ cohorts. Over a 4‐week period, mortalities occurred consistently only in O. mossambicus naïve cohorts. Overall presence of granulomas in spleen of survivors was similar (>55%) in all resident populations; however, in naïve populations, only O. mossambicus presented granulomas. Similarly, only O. mossambicus presented viable Fno in the spleen of survivors, and Fno GEs were only detected in O. mossambicus, and in resident O. aureus. In conclusion, the results of this study suggest different susceptibility of tilapia species to piscine francisellosis.     

Identification,annotation of Mucin genes in channel catfish (Ictalurus punctatus) and their expression after bacterial infections revealed by RNA‐Seq analysis

Mucins are large glycoproteins that cover epithelial surfaces of the body and play important roles in prevention of inflammatory and various infectious diseases. In this study, five membrane‐bound and seven secreted mucin genes in the channel catfish were identified. All these identified mucin genes possess at least one PTS, von Willebrand D (VWD) or SEA domains. The expression of the 12 mucin genes in channel catfish was first studied with infection of Edwardsiella ictaluri and Flavobacterium columnare. Expression difference in MUC13a, MUC13, MUC2 and MUC5b was found in the intestine after E. ictaluri infection. Eight mucin gene expressions (except MUC3a, MUC2, MUC4 and MUC5f) were up‐regulated at 4 hr and down‐regulated after 24 hr in the gill with F. columnare infection. Expression level of MUC2 gene was up‐regulated in the intestine with E. ictaluri infection without no significant change in the gill under the F. columnare infection, which indicate that MUC2 is tissue‐specific gene expression and has different immune respond to two bacterial challenge. Taken together, the study showed mucin from the gill by F. columnare challenge induced an obvious response than mucin from the intestine with E. ictaluri infection.     

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.     

In vivo adherence of Flavobacterium psychrophilum to mucosal external surfaces of rainbow trout (Oncorhynchus mykiss) fry

The adherence of Flavobacterium psychrophilum to surfaces of epithelial tissues has been inconclusively suggested as a mechanism, which enables the bacterium to invade the host. Hence, the present study aimed to examine the adherence of the cells of two colony phenotypes, smooth and rough, of F. psychrophilum to mucosal tissues of rainbow trout fry and to test the skin mucus as a nutrient for the growth of F. psychrophilum. Fish were immersed in water containing 10⁶ CFU mL^?1 F. psychrophilum for each colony phenotype. Mucosal tissue samples from fins, gills, skin and eyes, and swab samples from spleen and kidney were taken and inoculated onto TYES agar plates. Colony phenotypes of F. psychrophilum were identified and number of colonies counted. The results showed that cells of both phenotypes initially (0 h) adhered to all mucosal surfaces, but only the rough cells were still present on tissues 1 h post‐immersion. Both phenotypes showed a tissue tropism with the fin tissue being the most adhered. Furthermore, skin mucus promoted the growth of both colony phenotypes. We suggest that the growth of F. psychrophilum cells in skin mucus apparently facilitates the bacterial adherence to mucosal surfaces, and the subsequent invasion into the host.     

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.     

Preliminary success using hydrogen peroxide to treat Atlantic salmon,Salmo salar L., affected with experimentally induced amoebic gill disease (AGD)

Currently, the only effective and commercially used treatment for amoebic gill disease (AGD) in farmed Tasmanian Atlantic salmon is freshwater bathing. Hydrogen peroxide (H₂O₂), commonly used throughout the aquaculture industry for a range of topical skin and gill infections, was trialled in vitro and in vivo to ascertain its potential as an alternative treatment against AGD. Under in vitro conditions, trophozoites of Neoparamoeba perurans were exposed to three concentrations of H₂O₂ in sea water (500, 1000 and 1500 mg L^?1) over four durations (10, 20, 30 and 60 min) each at two temperatures (12 and 18 °C). Trophozoite viability was assessed immediately post‐exposure and after 24 h. A concentration/duration combination of 1000 mg L^?1 for >10 min demonstrated potent amoebicidal activity. Subsequently, Atlantic salmon mildly affected with experimentally induced AGD were treated with H₂O₂ at 12 and 18 °C for 15 min at 1250 mg L^?1 and their re‐infection rate was compared to freshwater‐treated fish over 21 days. Significant differences in the percentage of filaments affected with hyperplastic lesions (in association with amoebae) and plasma osmolality were noted between treatment groups immediately post‐bath. However, the results were largely equivocal in terms of disease resolution over a 3‐week period following treatment. These data suggest that H₂O₂ treatment in sea water successfully ameliorated a clinically light case of AGD under laboratory conditions.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

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).     

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.