Flavobacterium psychrophilum, invasion into and shedding by rainbow trout Oncorhynchus mykiss

The infection route of Flavobacterium psychrophilum into rainbow trout Oncorhynchus mykiss was studied using bath and cohabitation challenges as well as oral challenge with live feed as a vector. Additionally, the number of bacterial cells shed by infected fish into the surrounding water was determined in the cohabitation experiment and in challenge experiments at 3 different water temperatures. The experiments showed that skin and skin mucus abrasion dramatically enhanced the invasion of F. psychrophilum into the affected fish in bath and cohabitation challenges. Disruption of the skin is discussed as an important invasion route for F. psychrophilum into the fish. The shedding rate of F. psychrophilum by infected fish was associated with water temperature and the mortality of the infected fish. High numbers of F. psychrophilum cells were released into the water by dead rainbow trout during a long time period compared to the numbers of cells shed by live fish. The results emphasise the importance of removing dead and moribund fish from rearing tanks in order to diminish the infection pressure against uninfected fish in commercial fish farms. In immunohistochemical examinations of organs and tissues of orally infected fish, F. psychrophilum cells were detected in only 1 fish out of 31 studied. Mortality of the orally challenged fish was not observed in the experiment.     

Reproducible methods for experimental infection with Flavobacterium psychrophilum in rainbow trout Oncorhynchus mykiss.

Experiments were done in order to achieve a reproducible method that can be used to infect rainbow trout Oncorhynchus mykiss with Flavobacterium psychrophilum, the causal agent of coldwater disease and rainbow trout fry syndrome. The main method investigated was intraperitoneal injection, and this method was tested using isolates with different elastin-degrading profiles and representing different serotypes. Injecting trout, average weight 1 g, with 10(4) CFU (colony-forming units) per fish caused cumulative mortalities around 60 to 70%. The virulent strains belonged to certain serotypes and degraded elastin. The intraperitoneal injection challenge method could be used on larger fish, but the infection dose was 10(7) CFU per fish before mortalities occurred. Bath infection and bath infection in combination with formalin treatment (stress) seemed to be reproducible methods that could be used as alternatives to the intraperitoneal method, although the mortalities among infected trout were lower. The results of investigated methods were influenced by parameters such as the challenge isolate, number of fish in the tank affecting the infection pressure, origin of fish and weight of fish.     

Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification

Rainbow trout fry syndrome and cold-water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex(R) 100 chelating resin. The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl. psychrophilum. The obtained detection limit of Fl. psychrophilum seeded into rainbow trout brain tissue was 0.4 cfu in the PCR tube, corresponding to 17 cfu mg-1 brain tissue. The PCR-assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non-sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR-assay was 1.7 cfu in the PCR tube, corresponding to 110 cfu ml-1 water. The PCR-assay detected Fl. psychrophilum in water samples taken from a rainbow trout farm, but Fl. psychrophilum could not be isolated using inoculation on selective agar. The method presented here has the potential to detect low levels of Fl. psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl. psychrophilum.     

Standardization of experimental infection with Flavobacterium psychrophilum, the agent of rainbow trout Oncorhynchus mykiss fry syndrome

Rainbow trout fry syndrome (RTFS) is a septicaemic infection of young rainbow trout Oncorhynchus mykiss occurring at low temperatures and responsible for severe economic losses in European fish farming. The causative agent, Flavobacterium psychrophilum, is a gliding bacterium, and difficulties in culturing it have long been an impediment to investigations on pathogenesis and immunity. Successful attempts at experimentally inducing the disease have been reported, but no experimental model resulting in well-controlled and quantitatively reproducible effects has been described. Recent improvements in F. psychrophilum cultivation made it possible to produce bacterial suspensions with nearly constant viability and to complete challenge injections in rainbow trout fingerlings, using accurately adjusted infective doses. Parenteral injection resulted in significant mortality, which was higher when administered intramuscularly (IM) than intraperitoneally (IP). Lethal doses 50 % lower than 10(3) colony forming units were consistently obtained in trout weighing 3 to 5 g, and the regular shape of the cumulative mortality curves appeared to lend itself to quantitative analyses. Bath experiments produced milder effects, although mortality ranging between 45 and 60 % was obtained in 6 g trout when skin lesions or stressors were induced along with bacterial exposure. Temperature, salinity and the process of preserving isolates (at least over short periods of time) did not seem to be associated with the severity of infection. Nevertheless, infection trials performed at 2 different locations differing both in water quality and in the system of fish maintenance resulted in different mortalities. These findings notwithstanding, the proposed IM model appears easy to apply under standardized experimental conditions and should contribute to effective advances in the study of the disease.     

Occurrence of Flavobacterium psychrophilum in fish-farming environments

Occurrence of Flavobacterium psychrophilum in fish farms and fish-farming environments was studied using agar plate cultivation, the immunoflourescence antibody technique (IFAT) and nested PCR. Characteristics of 64 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss, fish farm rearing water, ovarian fluid and wild fish were serotyped, ribotyped and compared biochemically. Virulence of F. psychrophilum isolates from different sources was compared by injection into rainbow trout. Additionally, the number of F. psychrophilum cells shed by naturally infected rainbow trout was determined. F. psychrophilum was detected and isolated from skin mucus, skin lesions and internal organs of diseased rainbow trout and from fish without clinical disease. The pathogen was also present in wild perch Perca fluviatilis, roach Rutilus rutilus, and ovarian fluids of farmed rainbow trout brood fish. Isolates were biochemically homogenous, excluding the capability to degrade elastin. Five different agglutination patterns with different antisera against F. psychrophilum were found among the isolates studied. Although several different ribopatterns were found (ClaI: 12 ribopatterns and HaeIII: 9 ribopatterns), ribotype A was the most dominant. Farmed rainbow trout brood fish carried a broad-spectrum of serologically and genetically different F. psychrophilum in ovarian fluids. Virulence of the tested isolates in rainbow trout varied and naturally infected rainbow trout shed 10(4) to 10(8) cells fish(-1) h(-1) of F. psychrophilum into the surrounding water.     

Association of Flavobacterium psychrophilum with rainbow trout (Oncorhynchus mykiss) kidney phagocytes in vitro

The capacity of virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes to associate with isolated rainbow trout (Oncorhynchus mykiss, 300-500 g) kidney phagocytes was evaluated in vitro. The results showed that F. psychrophilum was associated with the phagocytes but large differences in association were observed between the different bacterial strains examined. These differences in association with the phagocytes was not clearly related to the serotype or virulence of the bacteria, although all strains tested of the non-virulent serotype FpT showed strong association with the isolated phagocytes. A competitive association assay with treatment of the phagocytes with seven different carbohydrates, suggested a role for N-acetylneuraminic acid (sialic acid) in the binding of F. psychrophilum to phagocytes. A significant dose dependent inhibition of the association was observed with sialic acid. Treatment of F. psychrophilum with sodium-metaperiodate showed that carbohydrate components play a role in the adhesion of the bacteria to the phagocytes. The results indicate that the binding of F. psychrophilum to rainbow trout kidney phagocytes can be mediated by opsonin independent cell-receptor adhesion. All tested strains seemed to be non-cytotoxic for rainbow trout kidney phagocytes in vitro suggesting that a phagocyte toxin is not necessary for the virulence of F. psychrophilum     

Ultraviolet irradiation inactivates the waterborne infective stages of Myxobolus cerebralis: a treatment for hatchery water supplies

The effects of ultraviolet (UV) irradiation on the viability of the waterborne triactinomyxon stages of Myxobolus cerebralis were evaluated by vital staining and the infectivity for juvenile rainbow trout Oncorhynchus mykiss. A dose of 1300 mWs cm-2 was required to inactivate 100% of the triactinomyxons held under a static collimated beam of UV as determined by vital staining. Juvenile rainbow trout were protected from infections with M. cerebralis when exposed to 14,000 or 1400 triactinomyxon spores per fish that had been treated with the collimating beam apparatus (1300 mWs cm-2). Among all fish receiving UV-treated triactinomyxons, none had clinical signs of whirling disease, or evidence of microscopic lesions or spores of M. cerebralis after 5 mo at water temperatures of 15 degrees C. In contrast, 100% of the fish receiving the higher dose of untreated triactinomyxons developed clinical signs of whirling disease and both microscopic signs of infection and spores were detected in all of the high and low dose trout receiving untreated triactinomyxon exposures. Two additional trials evaluated the Cryptosporidium Inactivation Device (CID) for its ability to treat flow-through 15 degrees C well water to which triactinomyxons were added over a 2 wk period. CID treatments of a cumulative dose exceeding 64,000 triactinomyxons per fish protected juvenile rainbow from infections with M. cerebralis. Rainbow trout controls receiving the same number of untreated triactinomyxons developed both microscopic lesions and cranial spore concentrations up to 10(4.6) per 1/2 head, although no signs of clinical whirling disease were observed. UV (126 mWs cm-2, collimated beam apparatus) was also effective in killing Flavobacterium psychrophilum, the agent causing salmonid bacterial coldwater disease, as demonstrated by the inability of bacterial cells to grow on artificial media following UV treatment.     

Spleen size predicts resistance of rainbow trout to Flavobacterium psychrophilum challenge

Selective breeding of animals for increased innate resistance offers an attractive strategy to control disease in agriculture. However, this approach is limited by an incomplete knowledge of the heritability, duration, and mechanism(s) of resistance, as well as the impact of selection on the immune response to unrelated pathogens. Herein, as part of a rainbow trout broodstock improvement program, we evaluated factors involved in resistance against a bacterial disease agent, Flavobacterium psychrophilum. In 2005, 71 full-sibling crosses, weighing an average of 2.4 g, were screened, and resistant and susceptible crosses were identified. Naive cohorts were evaluated at 10 and 800 g in size, and most maintained their original relative resistant or susceptible phenotypes, indicating that these traits were stable as size increased >300-fold. During the course of these studies, we observed that the normalized spleen weights of the resistant fish crosses were greater than those of the susceptible fish crosses. To test for direct association, we determined the spleen-somatic index of 103 fish crosses; created high, medium, and low spleen-index groups; and determined survival following challenge with F. psychrophilum or Yersinia ruckeri. Consistent with our previous observations, trout with larger spleen indices were significantly more resistant to F. psychrophilum challenge; however, this result was pathogen-specific, as there was no correlation of spleen size with survival following Y. ruckeri challenge. To our knowledge, this is the first report of a positive association between spleen size and disease resistance in a teleost fish. Further evaluation of spleen index as an indirect measure of disease resistance is warranted.     

Electrophoretic and Western blot analyses of the lipopolysaccharide and glycocalyx of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the aetiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS) and it has emerged as one of the most significant bacterial pathogens in salmonid aquaculture worldwide. Previous studies have suggested that the O-polysaccharide (O-PS) component of the lipopolysaccharide (LPS) of F. psychrophilum is highly immunogenic and may be involved in eliciting a protective immune response in rainbow trout (Oncorhynchus mykiss Walbaum). In the present study, SDS-PAGE and Western blotting techniques were used to analyse the carbohydrate antigens of F. psychrophilum. Our analysis identified two distinct carbohydrate-banding patterns. One banding pattern corresponds with LPS, and we hypothesise that the other carbohydrate-banding pattern is that of the loosely associated glycocalyx of F. psychrophilum. Electron microscopy of F. psychrophilum cells immunogold labelled with a monoclonal antibody specific for this banding pattern supports this hypothesis as the outermost layer of the bacterium was heavily labelled. This is a significant finding because the immunogenic antigens that have been referred to as the O-PS of LPS, and implicated as potential vaccine candidate antigens, appear to be components of the glycocalyx of F. psychrophilum. This research suggests that the glycocalyx of F. psychrophilum may be an important antigen to consider for the development of a vaccine to control CWD and RTFS.     

Virulence and nucleotide sequence analysis of marine viral haemorrhagic septicaemia virus following in vivo passage in rainbow trout Onchorhynchus mykiss

A marine isolate of viral haemorrhagic septicaemia virus (VHSV) (860/94) was passaged in triplicate through sequential batches of rainbow trout via an intra-peritoneal infection route, without amplification in tissue culture. Following 5 passages, the VHSV glycoprotein gene was amplified directly from fish tissue homogenates and the consensus sequence compared to that of the original tissue culture isolate. Virus was also recovered directly from pools of kidney and spleen material after 5 passage events, and its virulence compared to that of unpassaged material by intra-peritoneal infection. Following passage in rainbow trout, isolate 860/94 exhibited a higher virulence for rainbow trout than unpassaged material. Sequence comparisons identified no difference in the consensus sequence of the glycoprotein gene following in vivo passage. The mechanisms responsible for the observed increase in virulence of isolate 860/94 following passage in rainbow trout thus remain unknown. The possibility that viral isolates may exhibit an increased virulence following passage in novel host species does, however, have important implications with regard to the epidemiology of this important fish pathogen.     

Involvement of a sialic acid-binding lectin with hemagglutination and hydrophobicity of Flavobacterium psychrophilum

Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and Fp(T) exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5 degrees C, compared to that at 15 degrees C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65 degrees C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.     

Cytokine expression in the intestine of rainbow trout (Oncorhynchus mykiss) during infection with Aeromonas salmonicida

Gene expression of a number of cytokines in the intestine of rainbow trout (Oncorhynchus mykiss) was investigated after challenge with a pathogenic strain of Aeromonas salmonicida. Fish were exposed to A. salmonicida by immersion in a bacterial suspension (bath challenge) and tissue samples of the distal and proximal intestine were collected at days 0, 2, 4, 6 and 8 post-exposure. Head kidney tissue was also collected to assess the effect in a systemic immune tissue. A classic profile of pro-inflammatory cytokine upregulation was observed in the proximal intestine of fish infected by bath challenge, as determined by semi-quantitative RT-PCR. Expression of IL-1beta, IL-8, TNF-alpha and IFN-gamma was increased in the proximal intestine. TGF-beta was significantly decreased in the distal intestine. In the head kidney, infection with A. salmonicida by bath challenge caused decreased expression levels of IL-1beta, IL-8, TNF-alpha and TGF-beta. The results are discussed in the context of potential immune mechanisms in the gut to prevent infection.     

Development and application of a nested PCR to monitor brood stock salmonid ovarian fluid and spleen for detection of the fish pathogen Flavobacterium psychrophilum

AIMS: To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain). This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only. The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid. Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl. psychrophilum. The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O. kisutch) samples were contaminated. CONCLUSIONS: The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl. psychrophilum in the ovarian fluid. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Fl. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.     

The outer membrane fraction of Flavobacterium psychrophilum induces protective immunity in rainbow trout and ayu

Flavobacterium psychrophilum is the causative agent of coldwater disease, which is responsible for serious losses in fish aquaculture in several parts of the world. No commercial vaccines are currently available for the prevention of coldwater disease. The present study sought to assess the efficacy of a F. psychrophilum vaccine based on the antigenic outer membrane fraction (OMF). This fraction induced significantly higher protection against coldwater disease in both rainbow trout (Oncorhynchus mykiss) and ayu (Plecoglossus altivelis) compared to inactivated whole cell F. psychrophilum bacterin. Coincident with higher protection, sera of fish immunised with the OMF vaccine had higher agglutination titres than those of fish immunised with inactivated whole cell F. psychrophilum.     

Adhesion of the fish pathogen Flavobacterium psychrophilum to unfertilized eggs of rainbow trout (Oncorhynchus mykiss) and n-hexadecane

AIMS: The ability of Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome (RTFS) in fish, to attach to unfertilized rainbow trout (Oncorhynchus mykiss) eggs and to hydrocarbon n-hexadecane was examined in the present study. METHODS AND RESULTS: Five different isolates of Fl. psychrophilum obtained from a variety of origins were compared. The effect of the age of the bacterium and conditions of starvation on the ability of the bacterium to adhere, were also evaluated. CONCLUSION: The different isolates were found to exhibit a similar ability to attach to both substrates. Increased surface hydrophobicity and a greater ability to attach to the surface of the eggs were observed with bacteria aged for one month, compared to bacteria cultured in Cytophaga agar for only three days. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide useful information regarding the pathogenicity of RTFS, especially during the initial steps of infection.     

Effect of Flavobacterium psychrophilum strains and their metabolites on the oxidative activity of rainbow trout oncorhynchus mykiss phagocytes

The oxidative activity of rainbow trout phagocytes was studied using a chemiluminescence technique using 12 different Flavobacterium psychrophilum strains and their metabolites. Phagocytes were obtained from the head kidney of rainbow trout Oncorhynchus mykiss. The addition of viable F. psychrophilum or their metabolites to the phagocytes resulted in an immediate chemiluminescence response. The stimulating effects of both the F. psychrophilum and their metabolites on the phagocytes were found to be heat stable. No significant differences in stimulation capacity were found between the strains tested. To investigate the nature of the stimulating agent, both the bacteria and the supernatant were treated with either sodium metaperiodate or polymyxin B. Adding polymyxin B to the bacterial cells and supernatant did not change the chemiluminescence pattern, suggesting that the capacity of F. psychrophilum to stimulate the phagocytes probably is not due to lipopolysaccharides (LPS). However, following incubation of the bacteria and their metabolites with sodium metaperiodate, the capacity to stimulate phagocytes was significantly impaired. This suggests that a carbohydrate component most likely plays an important role in the ability of F. psychrophilum to stimulate phagocytes. Opsonisation of the bacteria with native trout serum or with rabbit anti-F. psychrophilum serum resulted in an additional chemiluminescence peak which was significantly higher than the first peak. This extra peak disappeared following heat treatment of the trout serum and the rabbit anti-F. psychrophilum serum, pointing towards the involvement of heat labile complement in opsonisation.     

Comparative susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease.

The susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease, was assessed following dosed exposures to the infectious stages (triactinomyxons). Parallel groups of age-matched brown trout and rainbow trout were exposed to 10, 100, 1000 or 10,000 triactinomyxons per fish for 2 h and then placed in aquaria receiving single pass 15 degrees C well water. Severity of infection was evaluated by presence of clinical signs (whirling and/or black tail), prevalence of infection, severity of microscopic lesions, and spore counts 5 mo after exposure. Clinical signs of whirling disease, including a darkened caudal region (black tail) and radical tail chasing swimming (whirling), occurred first among rainbow trout at the highest dose at 6 to 7 wk post exposure. Black tail and whirling occurred among rainbow trout receiving 1000 and 100 triactinomyxons per fish at 8 to 9 wk post exposure. Only 1 of 20 fish had a black tail among rainbow trout receiving 10 triactinomyxons per fish, although 30% of the fish were infected at 5 mo post exposure. Black tails were observed in brown trout at 1000 and 10,000 triactinomyxons per fish beginning at 11 and 7 wk post exposure, respectively. There was no evidence of the tail chasing swimming (whirling) in any group of brown trout. The prevalence of infection, spore numbers, and severity of microscopic lesions due to M. cerebralis among brown trout were less at each exposure dose when compared to rainbow trout. Infections were found among rainbow trout at all doses of exposure but only among brown trout exposed to doses of 100 triactinomyxons per fish or greater. Risk of infection analyses showed that rainbow trout were more apt to be infected at each exposure dose than brown trout. Spore counts reached 1.7 x 10(6) per head among rainbow trout at the highest dose of exposure compared to 1.7 x 10(4) at the same exposure dose among brown trout. Spore numbers increased with dose of exposure in rainbow trout but not in brown trout. As microscopic lesion scores increased from mild to moderate, spore numbers increased in rainbow trout but not brown trout. The mechanisms by which brown trout resist infections with M. cerebralis were not determined. Cellular immune functions, including those of eosinophilic granular leukocytes that were more prominent in brown trout than rainbow trout, may be involved.     

Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction

We evaluated a polymerase chain reaction (PCR) method for detecting Yersinia ruckeri, the bacterial pathogen causing enteric redmouth disease (ERM), in blood of rainbow trout Oncorhynchus mykiss. Identification of the PCR product was confirmed by Southern blot hybridization with a 32P-labeled oligonucleotide probe matching a sequence within the small subunit ribosomal RNA gene of Y. ruckeri. Following a 1 h immersion of rainbow trout in water with 4.5 x 10(6) colony-forming units of Y. ruckeri l(-1), the PCR was positive for all blood samples from 1 h (first sample) to 5 d and was negative from 9 to 30 d (last sample). Fish in this experiment did not show signs of disease, probably because they had been vaccinated against Y. ruckeri. To test this method with naturally infected fish, 42 rainbow trout from hatcheries were examined. Four of these fish had clinical signs of ERM and were infected with Y. ruckeri based on bacteriological culture. The PCR method detected Y. ruckeri in blood, intestine, liver, and trunk kidney from the 4 fish with ERM and from 5 additional rainbow trout that were bacteriologically negative for Y. ruckeri. Three of 5 rainbow trout from streams receiving effluent from hatcheries were positive for Y. ruckeri when tested with PCR, although there was no growth of Y. ruckeri on culture plates inoculated with the same samples. Samples were successfully stored for 1 wk in lysis buffer at 25 degrees C. This study demonstrated that a non-lethal blood sample can be used with PCR to detect Y. ruckeri.     

A Saprolegnia parasitica challenge system for rainbow trout: assessment of Pyceze as an anti-fungal agent for both fish and ova.

A reproducible Saprolegnia parasitica spore delivery system was developed and demonstrated to be effective in providing a sustained spore challenge for up to 10 d. Treatment of rainbow trout with slow-release intraperitoneal implants containing cortisol resulted in chronically elevated blood cortisol levels and rendered the fish susceptible to infection by S. parasitica when exposed to the spore challenge. Sham-implanted fish were not susceptible to infection. Bronopol (2-bromo-2-nitro-propane-1,3-diol), formulated as Pyceze, was effective in protecting predisposed fish from infection by S. parasitica when administered as a daily bath/flush treatment at concentrations of 15 mg l-1 and greater. Pyceze was also demonstrated to protect fertilised rainbow trout ova from S. parasitica challenge when administered as a daily bath/flush treatment at concentrations of between 30 and 100 mg l-1. Pyceze appears to qualify as a safe and effective replacement for malachite green and formalin in the prevention of fungal infections in the aquaculture environment.