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.     

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.     

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.     

Isolation,identification and character analysis of Streptococcus dysgalactiae from Megalobrama terminalis

Pure bacterial cultures were isolated from different tissues of moribund Megalobrama terminalis from a high mortality event that occurred at a farm in Foshan, China. Two isolates (F2 and F3) were identified as Streptococcus dysgalactiae subsp. dysgalactiae based on morphological and biochemical detection as well as molecular analysis. In brain heart infusion broth, the best growth conditions of isolate F3 were 35ºC, salinity 5‰ and pH 7. Furthermore, infection with isolate F3 (1.2 × 10⁶ CFU/fish) led to the death of M. terminalis and zebrafish (Danio rerio). However, isolate F3 had no obvious pathogenicity to tilapia (GIFT, Oreochromis niloticus). When the water temperature was 29ºC, the corresponding mortality rates for zebrafish infected by isolate F3 were higher than those at 23ºC. Culture for 24 and 72 hr with isolate F3 resulted in the same mortality rates for zebrafish. The antimicrobial susceptibility assay revealed that isolate F3 was susceptible to ampicillin, florfenicol and several other antibiotics but resistant to nalidixic acid, streptomycin, sulfamethoxazole/trimethoprim, neomycin and amikacin. To our knowledge, this is the first report that S. dysgalactiae infected the subtropical freshwater fish M. terminalis, which indicates that this bacterium is a potential threat to subtropical freshwater fish.     

Spatiotemporal distribution of Flavobacterium psychrophilum and ayu Plecoglossus altivelis in rivers revealed by environmental DNA analysis

Outbreaks of bacterial cold-water disease (BCWD), caused by Flavobacterium psychrophilum, are widespread in Japan, especially among ayu Plecoglossus altivelis. There are few investigations of F. psychrophilum in river water, and its seasonal distribution has not been clarified. We aimed to identify the spatiotemporal dynamics of F. psychrophilum and ayu to provide information that is useful for establishing a countermeasure for BCWD. Quantitative analysis of environmental DNA (eDNA) was used to clarify the year-round dynamics of ayu and F. psychrophilum. We sampled river water from the Nagara and Ibi rivers in Japan, and conducted monthly water sampling and eDNA quantification. Changes in the eDNA concentration of ayu were consistent with the known life histories of the fish. There was a strong negative correlation between the eDNA concentration of F. psychrophilum and water temperature, suggesting a strong dependence of F. psychrophilum dynamics in the river on water temperature. Furthermore, relatively high eDNA concentrations were recorded for both organisms in early summer and fall, suggesting that ayu is infected with F. psychrophilum during these seasons when experiencing up- and downmigration, respectively.

     

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.     

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.     

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.     

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.     

Heat‐inactivated Mycobacterium bovis protects zebrafish against mycobacteriosis

Control of mycobacterial infection constitutes a priority for human and animal health worldwide. However, effective vaccines are needed for the control of human and animal tuberculosis (TB). Adult zebrafish have become a useful model for studying the pathophysiology of mycobacterial infection and for the development of novel interventions for TB control and prevention. Recently, parenteral and oral immunization with the heat‐inactivated Mycobacterium bovis vaccine (M. bovis IV) protected wild boar against TB. The objectives of this study were to provide additional support for the role of M. bovis IV in TB control using the zebrafish model and to conduct the first trial with this vaccine for the control of fish mycobacteriosis. The results showed that M. bovis IV protected zebrafish against mycobacteriosis caused by low and high infection doses of Mycobacterium marinum and provided evidence suggesting that the protective mechanism elicited by M. bovis IV in zebrafish as in other species is based on the activation of the innate immune response through the C3 pathway, with a role for the regulatory protein Akr2 in this process. These results encourage the use of M. bovis IV for TB control in different species.     

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.     

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.     

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.     

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.     

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.     

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.