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1.
Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS), causes high mortality in cultured salmonids. The present study was designed to determine the role antibody plays in conferring protection to rainbow trout fry, Oncorhynchus mykiss (Walbaum), by passive immunization with convalescent serum or serum from adult rainbow trout immunized with F. psychrophilum, and goat anti-F. psychrophilum serum. In each experiment, rainbow trout fry were injected intraperitoneally with antiserum and challenged by subcutaneous injection with a virulent strain (CSF-259-93) of F. psychrophilum 24-h post-immunization. Relative percentage survival (RPS) ranged from 9-42% when rainbow trout fry (mean weight 1.3 g) were injected with a 1:2 dilution of 25 microL of convalescent serum ranging in enzyme-linked immunosorbent assay antibody titres from 1600-102400. Rainbow trout fry (mean weight 1.0 g) passively immunized with 25 microL of serum from immunized adult fish exhibited RPS values of up to 57%. In each of these experiments, RPS increased with increasing antibody titres against F. psychrophilum. Passive immunization with 25 or 50 microL goat anti-F. psychrophilum serum, however, did not confer protection to fry (mean weight 1.3 g). These results suggest that trout antibody plays a role in conferring protection to F. psychrophilum, but antibody alone is unable to provide complete protection.  相似文献   

2.
Efficacy of mineral oil-based experimental injection vaccines against Flavobacterium psychrophilum were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), under laboratory and field conditions. The vaccines consisted of formalin- or heat-inactivated whole bacterium cell preparations of two different serotypes (Fd and Th) or a combination of serologically different F. psychrophilum (Fd and/or Th and/or Fp(T);Th). Specific antibody responses against the bacterium in plasma and skin mucus were evaluated post-vaccination with enzyme-linked immunosorbent assay. Efficacy of the vaccinations was determined by challenge trials to F. psychrophilum with the vaccinated rainbow trout. Significantly higher antibody levels in plasma were detected in vaccinated fish compared with mock-vaccinated fish. Injection vaccination did not trigger specific antibody production in the skin mucus. Significantly higher survival of i.p. vaccinated fish compared with non-vaccinated fish was observed during the challenge. The results suggest that mineral oil-based injectable vaccines containing formalin- or heat-inactivated virulent cells of F. psychrophilum effectively triggered specific antibody production and protected the fish against bacterial cold water disease.  相似文献   

3.
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.  相似文献   

4.
Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome has become a widespread fish pathogen in freshwater aquaculture worldwide. In this study, a low molecular mass fraction (P25-33), with an approximate weight of 25-33 kDa, was identified among F. psychrophilum strains in an immunoblotting analysis with anti-F. psychrophilum sera. The immunogenic efficacy of the isolated and extracted P25-33 was investigated in two intraperitoneal immunization trials with rainbow trout, Oncorhynchus mykiss (Walbaum). The first trial included immunizations using P25-33 with Freund's complete adjuvant (FCA) and the second trial included immunizations using P25-33, formalin-inactivated whole and sonicated F. psychrophilum cell preparations without FCA. In both trials, antibody titres against F. psychrophilum were analysed with an enzyme-linked immunosorbent assay and the efficacy of the immunizations was determined by a challenge with F. psychrophilum. The P25-33 was shown to give rise to a protective immune response in rainbow trout after immunization with FCA, but not without FCA when a low concentration of P25-33 was used. Instead formalin-inactivated whole and sonicated cells of F. psychrophilum were able to protect the immunized fish more effectively when immunized without FCA. The results suggest that whole or sonicated F. psychrophilum cells could be better candidates for a cost-effective water-based injection vaccine than the immunogenic fraction.  相似文献   

5.
Flavobacterium psychrophilum heat shock proteins (Hsp) 60 and 70 are highly immunogenic and were therefore investigated as potential vaccine candidates. Recombinant Hsps were purified from Escherichia coli and rainbow trout ( Oncorhynchus mykiss ) were intraperitoneally injected with phosphate buffered saline/Freunds complete adjuvant (FCA), 8 μg of rHsp60/FCA, rHsp70/FCA or a combination of 4 μg each of rHsp60 and rHsp70/FCA. Antibody responses against recombinant Hsp60 and Hsp70 8 weeks post-immunization were observed, but only fish immunized with rHsp70 exhibited highly elevated antibody levels against F. psychrophilum whole cell lysate. Some cross reactivity occurred, which may have been due to the V5 tag common to both proteins. Protection against F. psychrophilum challenge was not observed in any treatments at 8 weeks post-immunization. To further investigate any protective effect of these proteins, hsps were polymerase chain reaction amplified and cloned into pVAX1. Rainbow trout were intramuscularly injected with 8 μg of pVAX1hsp60, pVAX1hsp70 or a combination of 4 μg each of pVAX1hsp60 and pVAX1hsp70. Antibody responses at 4 weeks post-immunization were low and protection was not observed following challenge at 6 or 10 weeks post-immunization. Although Hsps of F. psychrophilum have been shown to be immunodominant, these antigens do not appear to be good vaccine candidates when delivered alone or in combination.  相似文献   

6.
Flavobacterium psychrophilum causes rainbow trout fry syndrome (RTFS) and cold water disease (CWD) in salmonid aquaculture. We report characterization of F. psychrophilum strains and their bacteriophages isolated in Chilean salmonid aquaculture. Results suggest that under laboratory conditions phages can decrease mortality of salmonids from infection by their F. psychrophilum host strain. Twelve F. psychrophilum isolates were characterized, with DNA restriction patterns showing low diversity between strains despite their being obtained from different salmonid production sites and from different tissues. We isolated 15 bacteriophages able to infect some of the F. psychrophilum isolates and characterized six of them in detail. DNA genome sizes were close to 50 Kbp and corresponded to the Siphoviridae and Podoviridae families. One isolate, 6H, probably contains lipids as an essential virion component, based on its chloroform sensitivity and low buoyant density in CsCl. Each phage isolate rarely infected F. psychrophilum strains other than the strain used for its enrichment and isolation. Some bacteriophages could decrease mortality from intraperitoneal injection of its host strain when added together with the bacteria in a ratio of 10 plaque-forming units per colony-forming unit. While we recognize the artificial laboratory conditions used for these protection assays, this work is the first to demonstrate that phages might be able protect salmonids from RTFS or CWD.  相似文献   

7.
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.  相似文献   

8.
Flavobacterium psychrophilum is the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. This study examined the genetic diversity of F. psychrophilum isolates retrieved from multiple epizootics at rainbow trout, Oncorhynchus mykiss, rearing facilities and from spawning coho salmon, O. kisutch. A total of 139 isolates were confirmed as F. psychrophilum by PCR assay and were further typed using pulsed-field gel electrophoresis (PFGE). Multiple epizootics at three proximally located rainbow trout rearing facilities were numerically dominated by three PFGE profiles, which accounted for 76% of all trout isolates. In coho salmon, 19 PFGE profiles were differentiated by PFGE and four numerically dominant PFGE profiles represented 56% of all coho salmon isolates. PFGE analysis also indicated that the average similarity of macrorestriction patterns of F. psychrophilum isolates was greater in rainbow trout than in coho salmon (88% vs. 70%). Furthermore, it was not unusual to isolate multiple PFGE profiles from a single coho salmon sample whereas only two PFGE profiles were shared between two sample dates separated by 1 month. It is clear that the domestic rainbow trout aquaculture facilities studied here were primarily affected by a complex of genetically related strains whereas spawning coho salmon supported a much more genetically diverse collection of F. psychrophilum.  相似文献   

9.
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.  相似文献   

10.
During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .  相似文献   

11.
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12.
The occurrence of Flavobacterium psychrophilum at four rainbow trout hatcheries was investigated to provide more knowledge about the reservoirs and transmission of this bacterium. Broodstock were sampled at stripping (including both unfertilized and fertilized eggs), and the offspring were then sampled at the eyed egg and fry stages. Water and surface samples (e.g. hatchery trays) were also sampled. Flavobacterium psychrophilum was found in ovarian fluid and milt, indicating that broodstock may serve as a reservoir and are latent carriers of the pathogen. Flavobacterium psychrophilum was not found on or inside eggs, but further egg studies will be necessary to elucidate the possibility of vertical transmission of the pathogen. Flavobacterium psychrophilum was isolated from water samples, but only from water that had been in close contact with farmed rainbow trout or eggs. Flavobacterium psychrophilum isolates were characterized and compared with well-characterized strains, using degradation of elastin, serotype and ribotype profiles. Different ribotypes of F. psychrophilum were found between hatcheries, but a common ribotype A was found at all four hatcheries. Different ribotypes were found in broodstock without clinical disease, whereas only a few ribotypes (mostly ribotype A) were found in diseased fry. The same ribotype A was found in broodstock, in water samples from hatchery trays and in fry, which suggests the possibility of transmission of F. psychrophilum between broodstock and offspring.  相似文献   

13.
A crude extracellular preparation (CEP) from a strain of Flavobacterium psychrophilum recovered from a case of necrotic myositis affecting rainbow trout was capable of causing severe muscle necrosis in rainbow trout following intramuscular injection. Cell wall-associated preparations, however, were unable to produce similar lesions in experimentally injected fish. The CEP degraded gelatin and type II collagen but not type I or type IV collagen. Furthermore, the CEP did not degrade 2-furanacryloyl- l -leucylglycyl- l -prolyl-alanine (FALGPA), chondroitin sulphates A, B or C, heparan sulphate, keratan sulphate, hyaluronic acid, elastin or rainbow trout erythrocytes. The addition of the protease inhibitors 1,10-phenanthroline, ethylenediamine-tetraacetic acid (EDTA) and EGTA to the CEP halted its ability to degrade gelatin in vitro and to produce muscle necrosis in rainbow trout in vivo . In vitro and in vivo activity was restored following the addition of 1 m m zinc chloride to the protease inhibitor-treated CEP, suggesting that this strain of F . psychrophilum secretes a protein complex with zinc metalloprotease-like activity. This protein complex, therefore, appears to be involved in the pathogenesis of necrotic myositis in rainbow trout.  相似文献   

14.
A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.  相似文献   

15.
The bacterial pathogen Flavobacterium psychrophilum was successfully identified from formalin-fixed, wax-embedded tissue blocks of infected rainbow trout heart and spleen tissues, using a polymerase chain reaction (PCR)-based assay. Filamentous bacteria were observed in haematoxylin and eosin and Giemsa-stained sections but no bacteria were recovered from the diseased fish using standard bacteriology isolation techniques. All infected fish had histopathological evidence of myocarditis or rainbow trout fry syndrome. Immunohistochemistry was attempted using three different anti-F. psychrophilum sera but the results were inconclusive, and an alternative molecular approach was therefore attempted. This paper describes the use of a PCR-based assay to help identify bacteria present in formalin-fixed, wax-embedded tissue samples. This is the first time that this technique has been used for the detection of fish bacteria from diagnostic samples.  相似文献   

16.
The intracellular behaviour of a Flavobacterium psychrophilum strain, ingested by spleen phagocytes of rainbow trout, Oncorhynchus mykiss , of different ages, was assessed in vivo . Three groups of rainbow trout weighing 1 g (aged 10 weeks), 25 g (aged 20 weeks) and 300 g (aged 15 months), respectively, were injected intraperitoneally with 1 × 106 cfu of a F. psychrophilum strain. It was found that only fry, aged 10 weeks, displayed clinical signs and suffered mortality. Bacteriological colony plating of different organs demonstrated that the spleen and to a lesser extent the kidney of only the fry were affected. The number of colony forming units per gram of spleen tissue increased with time. No bacteria were found in the trout aged 5 months and older. Light microscopical examination and epifluorescence microscopy revealed that the fry spleen phagocytes contained an increasing number of viable intracellular F. psychrophilum bacteria over time. Again, no bacteria were encountered in the phagocytes collected from older fish.  相似文献   

17.
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.  相似文献   

18.
Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout fry syndrome of salmonids. The pathogen has been reported from all regions in the world involved in salmonid aquaculture, but also from natural fresh-water environments. We established a quantitative loop-mediated isothermal amplification of DNA (LAMP) method to estimate quantities of F. psychrophilum . LAMP primers were designed based on the sequence of the DNA topoisomerase IV subunit B gene, parE , of F. psychrophilum. parE LAMP exhibited a high specificity for the parE gene of F. psychrophilum but not for other related species. parE LAMP detected the gene in a wide range of concentrations from 2.0 × 101 to 2.0 × 109 copies/reaction within 70 min and revealed a good correlation between threshold times and gene copy number.  相似文献   

19.
Subcellular components of the probiotics Aeromonas sobria GC2 and Bacillus subtilis JB-1, when administered to rainbow trout, Oncorhynchus mykiss , conferred protection against a new biogroup of Yersinia ruckeri . Thus, intraperitoneal or intramuscular injection of rainbow trout with cell wall proteins (CWPs), outer membrane proteins (OMPs), lipopolysaccharides (LPS), whole cell proteins (WCPs) and live cells followed by challenge on day 8 with Y. ruckeri led to 80–100% survival compared with 10% survival in the controls. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of WCPs and OMPs from GC2 had 10 and 5 variable protein bands in comparison to 11 and 5 bands in the WCPs and CWPs from JB-1. Proteomic analyses were employed following SDS-PAGE to categorize one dominant protein of 104.7 kDa from the CWPs of JB-1 and equated it with ' Bacillus spp. endoglucanase' with a Mascot score >69. These results point to the potential of using cellular components of probiotics for protection of fish against bacterial diseases.  相似文献   

20.
The protective effect in rainbow trout (Oncorhynchus mykiss) of an experimental subunit vaccine targeting antigens in the parasite Ichthyophthirius multifiliis has been evaluated and compared to effects elicited by a classical parasite homogenate vaccine. Three recombinant parasite proteins (two produced in E. coli and one in insect cells) were combined and injected i.p., and subsequently, protection and antibody responses were analysed. Both the experimental and the benchmark vaccine induced partial but significant protection against I. multifiliis when compared to control fish. Specific antibody responses of vaccinated trout (subunit vaccine) were raised against one neurohypophysial n‐terminal domain protein #10 of three recombinant proteins, whereas the benchmark vaccine group showed specific antibody production against all three recombinant proteins. The immunogenic parasite protein #10 may be a potential vaccine candidate supplementing the protective I‐antigen in future vaccine trials.  相似文献   

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