Comparison of the effectiveness of three different growth media for primary isolation of Renibacterium salmoninarum from Atlantic salmon, Salmo salar L., broodfish

Abstract. The effectiveness of three different growth media KDM-2, S-KDM and S-KDM-C for primary isolation of Renibacterium salmoninarum was examined over a 14-week incubation period. Kidney samples were taken from Atlantic salmon, Salmo salar L., broodfish returning to a sea ranch after 2 years at sea. Homogenized samples were inoculated onto two selective media, S-KDM supplemented with lamb serum and S-KDM-C supplemented with activated charcoal. The third medium was the non-selective serum supplemented KDM-2. Renibacterium salmoninarum was isolated from 112 samples on one or more of the media used. Of all positive samples, 91% were positive on S-KDM, 60% on S-KDM-C and 35% on KDM-2. These results demonstrate that selectivity significantly enhances the isolation capacity of the medium and that supplementing with serum is significantly more effective than supplementing with charcoal.     

Incubation time for the cultivation of Renibacterium salmoninarum from Atlantic salmon, Salmo salar L., broodfish

Abstract. Cultivation plates for the primary isolation of Renibacterium salmoninarum were incubated for 12 or more weeks over 3.5 years. The incubation time for sample of two groups of Atlantic salmon, Salmo solar L., broodfish on a selective agar medium is reported. Samples turned positive over a long time-span. but the longest incubation time recorded for growth of the bacterium was 19 week. In one group, 79% of all positive samples form fish with macroscopic signs of bacterial kidney disease were positive after 6 weeks of incubation and the highest incidence of positive samples occured during the sixth week. From covertly infected fish within the same group.42% of sample were positive after 6 weeks of incubation, and the highest incidence of positive sample occured during the sixth and the ninth weeks of incubation. The growth pattern of the bacterium at primary isolation was characterized by a prolonged lag phase followed by a relatively growth in one week from microscopic to macroscopic size of the colonies.     

Epizootiology of Renibacterium salmoninarum, the causal agent of bacterial kidney disease in salmonid fish

Abstract. By use of a selective medium, Renibacterium salmoninarum was recovered from rainbow trout, Salmo gairdneri Richardson. With asymptomatic animals, small numbers of Renibacterium colonies were recovered only from the anterior part of the kidney; whereas in clinically diseased fish, dense pure culture growth was obtained from kidney, spleen, heart, blood, ascitic fluid and faeces. There was no evidence for the presence of R. salmoninarum in the water or sediment of 56 freshwater fish farms in England and Wales. Nevertheless, laboratory–based experiments showed that the pathogen was excreted in the faeces of clinically diseased fish. Moreover, there was survival of the bacterial cells for up to 21 days in sediment/faecal material.     

An investigation into the prevalence of Renibacterium salmoninarum in farmed rainbow trout, Oncorhynchus mykiss (Walbaum), and wild fish populations in selected river catchments in England and Wales between 1998 and 2000

A cross-sectional survey of Renibacterium salmoninarum infection in farmed rainbow trout (RBT) and wild fish populations was carried out in 10 farms and six river catchments, respectively, in England and Wales. The majority of the wild fish were sampled in 1998 and the farmed fish in 2000. Grayling, Thymallus thymallus, and brown trout, Salmo trutta, were the main wild species sampled. Two fish, one grayling and one salmon, Salmo salar, were R. salmoninarum culture-positive, compared with 40 confirmed polymerase chain reaction-positive wild fish. The highest prevalence of R. salmoninarum infection was found in grayling in rivers with RBT farms with a history of R. salmoninarum infection. One hundred and fifty fish were sampled from each RBT farm, but none of the fish was found to be R. salmoninarum-positive. Evidence was found, for the first time, for the presence of R. salmoninarum in an eel, Anguilla anguilla.     

Comparative sensitivity of carp, Cyprinus carpio L. and rainbow trout, Salmo gairdneri Richardson, to Renibacterium salmoninarum

Abstract. The pathogenicity of Renibacterium salmoninarum to carp, Cyprinus carpio L., and rainbow trout, Salmo gairdneri Richardson, was investigated. All carp injected with 4·8 × 10⁸ cells/fish, or 4·8 × 10⁷ cells/fish survived for 38 days. R. salmoninarum was isolated from all moribund fish, but not from the kidney of surviving fish, although R. salmoninarum antigen was detected in several of these fish by the dot blot assay. On the other hand, mortality in rainbow trout was 95% in the fish injected with 4·8 × 10⁸ cells/fish, and 15% in those which received 4·8 × 10⁷ cells/fish. R. salmoninarum antigen was detected by the dot blot assay in all surviving rainbow trout. The number of R. salmoninarum cells was immediately decreased by carp or rainbow trout serum, and the serum bactericidal activity of carp was higher than that of rainbow trout. Carp blood leucocytes had higher phagocytic activity than those of rainbow trout.     

Haematological assessment of rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L., infected with Renibacterium salmoninarum

Abstract. Groups of rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, S. salar L., were infected with Renibacterium salmoninarum , injected with killed bacteria or starved. Selected red and white cell parameters were measured over a 35–day period. As a result of successful infection a typical granulomatous disease developed during which haematocrit, red cell count, haemoglobin, red cell diameter and the ratio of mature to immature erythrocytes all decreased. Red cell depletion was shown to be caused by their retention in the spleen and to be the cause of the observed splenomegaly. Increases in monocytes, thrombocytes and neutrophils were recorded in both infected groups.     

Relevance of Renibacterium salmoninarum in an asymptomatic carrier population of brook trout, Salvelinus fontinalis (Mitchill)

Abstract. These studies were done to determine the prevalence of infection, over time, of progeny from a population of 1988 year class brook trout, Salvelinus fontinalis (Mitchill), that was asymptomatically infected with Renibacterium salmoninarum , the causative agent of bacterial kidney disease. This population and its progeny from the 1991 and 1992 spawnings were monitored for approximately 3 years for prevalence of R. salmoninarum and development of bacterial kidney disease. Ovarian fluid samples from the 1991 and 1992 spawnings, and from a 1993 spawning of the 1991 progeny, were analysed for R. salmoninarum by enzyme linked immunosorbent assay (ELISA) and the membrane filtration fluorescent antibody technique. Kidney and spleen tissues of the 1988 year class were assayed by ELISA following the 1992 spawning. The progeny of the 1991 and 1992 spawnings were followed for increased prevalence of R. salmoninarum by the ELISA. Kidney tissues were assayed and sampling began once the fish were large enough. Each population of progeny was sampled quarterly for one year. Overall, the progeny displayed decreasing prevalence of R. salmoninarum and all ovarian fluids of the 1991 progeny (in 1993) were negative. Brood fish and progeny remained asymptomatic during the course of the study.     

A sensitive loop-mediated isothermal amplification (LAMP) method for detection of Renibacterium salmoninarum, causative agent of bacterial kidney disease in salmonids

Loop-mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification with high specificity, sensitivity and rapidity and does not require expensive equipment or reagents. In the present study, we developed and evaluated a LAMP method for the rapid detection of Renibacterium salmoninarum causing the bacterial kidney disease in salmonids. This method was more sensitive than quantitative real-time polymerase chain reaction (qPCR). Using DNA template extracted from cultured R. salmoninarum , the LAMP method gave an amplification signal from template diluted to 10⁻⁸ while the limit of detection of qPCR was10⁻⁷. The LAMP method was also highly specific and did not amplify DNA purified from five other Gram-positive and -negative bacterial fish pathogens. The method also worked well using extracts of macrophages infected with R. salmoninarum and kidney material from rainbow trout, which were positive for R. salmoninarum by qPCR and crude R. salmoninarum culture. There was some evidence for inhibitors of the LAMP reaction in the kidney samples, which was overcome by diluting the sample.     

Estimation of variation of virulence of Renibacterium salmoninarum by survival analysis of experimental infection of salmonid fish

The variation of virulence of Renibacterium salmoninarum , the causative agent of bacterial kidney disease (BKD) in salmonid fish, was studied by infecting rainbow trout, Oncorhynchus mykiss (Walbaum), with two isolates (strains 325 and 932) from diseased Atlantic salmon, Salmo salar L., and one isolate (strain 4366) from an apparently healthy Atlantic salmon. Coho salmon, Oncorhynchus kisutch (Walbaum), were injected with the strain 932 to estimate difference in fish species resistance. Fish were removed by random sampling for other study purposes, a study design possible with analysis of lifetime distributions incorporating both sampling-, death- and survival-times. At the end of the experiment, the rainbow trout infected with strains 325, 932 and 4366 had a survival probability of 33%, 51% and 72%, respectively. The coho salmon infected with strain 932 had a 26% survival probability. The strain differences were significant according to the log-rank test, and the risk ratio between the strains ranged from 1·8 to 5·4. The strain from the apparently healthy fish was least virulent. The survival of the fish species was different over time. Rainbow trout were more likely to die early in the time course, but high numbers of coho died later, resulting in an overall risk of mortality of 1·4 in favour of rainbow trout. Differences in virulence may reflect changed selective pressure on R. salmoninarum when introduced from feral stocks into the environment of fish farms.     

The use of Western blot and electroimmunotransfer blot assays to monitor bacterial kidney disease in experimentally challenged Atlantic salmon, Salmo salar L.

Abstract. Western blot (WB) and electroimmunotransfer blot (EITB) methods were used to monitor bacterial soluble antigens and scroconversion profiles, respectively, in Atlantic salmon, Salmo salar L., experimentally challenged with Renibacterium salmoninarum. The two challenged populations exhibited differences in susceptibility to R. salmoninarum permitting an evaluation of the methods in fish populations harbouring different levels of the pathogen, WB detection of R. salmoninarum soluble antigens in serum provided a sensitive method for detecting pathogen presence during the active infection stages. For comparison, results for drop-plate culture and direct fluorescent antibody techniques were obtained. During the recovery phase, surviving fish were negative for the pathogen by these methods. However, >90% of challenged fish sampled from early infection stages to the end of the experimental period (141 days post-infection) were scropositive by the EITB assay for Atlantic salmon immunoglobulin reactive with R. salmoninarum soluble antigens. The results suggest that these methods for detecting R. salmoninarum or its soluble antigens may be useful during active infections. However, asymptomatic fish populations exposed to the pathogen, perhaps having recovered from an active infection and representing potential disease carrier sources, may be more readily identified by antigen-specific antibodies using methods such as the EITB assay.     

Atypical growth of Renibacterium salmoninarum in subclinical infections

Two growth types of Renibacterium salmoninarum were isolated from subclinically infected rainbow trout, one producing the smooth colonies typical of R. salmoninarum and the other forming a thin film on the surface of the agar with no separate colonies. The atypical growth was present on kidney disease medium agar in primary cultures of the kidney but not on selective kidney disease medium (SKDM). Fluorescent antibody staining of the fresh isolate and polymerase chain reaction amplification were the most reliable techniques to identify the atypical growth of R. salmoninarum. The condition was reversible, with growth reverting from atypical to the smooth colony form in experimentally infected rainbow trout and under laboratory conditions. There was no mortality, or any clinical signs of bacterial kidney disease (BKD) in the fish challenged with the atypical growth, although small numbers of smooth colonies of R. salmoninarum were isolated from 8% of these fish. The atypical growth reported here may explain some of the failures of culture, when SKDM agar alone is used for the detection of BKD in subclinically infected fish.     

Efficacy of Elevated Dietary Fluoride for the Control of Renibacterium salmoninarum Infection in Rainbow Trout Salmo gairdneri

Laboratory studies were performed to evaluate the efficacy of dietary fluoride for the control of Renibacterium salmoninarum infection in rainbow trout and to determine potential dietary fluoride toxicity to the fish. Fish were fed diets containing fluoride (F), as NaF, to give doses of 0, 2, 4, 8, or 16 mg F/kg fish weight per day. Efficacy of fluoride for the control of bacterial kidney disease (BKD) was observed at 4, 8, and 16 mg/kg doses. No signs of toxicity due to dietary fluoride were observed in the fish.     

Can smolting and maturation of hatchery-reared brown trout Salmo trutta L. be affected by food deprivation during the first and second years of rearing?

Smolting and maturation of 2+ brown trout Salmo trutta L. were evaluated after exposing the groups of trout to different feeding regimes during the summers at 0+ and 1+ ages. The hypothesis tested was based on the theory of smolting of a congeneric species, Atlantic salmon Salmo salar L., in which the physiological smolting decision is expected to be taken at the end of July or beginning of August. During the first summer, the growth of the trout was restricted in two groups out of four by low feeding frequency. During the second summer, food was totally withheld for 3 weeks in June–July (i.e. before the expected sensitive period), in August or not at all (control). The proportion of sexually mature males in November was 5.2% in the groups fasted during August, but somewhat lower in the groups fasted in June–July (average 2.3%) or in control fish (3%). The tendency for smolting was evaluated during the following spring in an artificial stream with the help of PIT-tag technology, which allowed monitoring of the movements of individually tagged trout. Seawater challenge tests were also carried out in April and June. Differences in osmoregulatory ability in seawater indicated that feeding treatments had a slight effect on the timing of smolting, but no differences were observed in movement behaviour between treatment groups. Mature and maturing males moved less at the peak migration time (mid-May) but more in October than immature fishes. These results suggest that the smolting decision in brown trout may be taken at a different time than in Atlantic salmon and that periodic poor growth conditions during the summer will not prevent smolting of trout during the following spring.     

Association between initial weight and weight gain in brook trout, Salvelinus fontinalis Mitchill, and brown trout, Salmo trutta L.

Abstract. Individual weighings of samples of brook trout, Salvelinus fontinalis Mitchill, and brown trout, Salmo trutta L., al the initiation and termination of their growth period, enabled estimating the association of weight gain on initial weight. In both cases the regressions were positive and the correlations significant. Variation in initial weight among tested individuals and groups is expected to bias growth estimates of these groups in programmes of genetic testing, as a result of this association. Obviating this bias requires an adequate correcting technique, involving the estimation of an environmentally generated correction factor. Phenotypic regression coefficients, as estimated in this investigation, cannot serve as correction factors, but may be regarded as maximum estimates.     

用nested—PCR方法快速检测鲑鱼肾杆菌

描述了用嵌套式聚合酶链式反应(nested PCR)快速检测鲑鱼细菌性肾病病原鲑鱼肾杆菌的方法。以BKDR和BKDF为引物,扩增鲑肾杆菌编码57kDa主要可溶性蛋白基因中501bp的DNA片段,再用引物BKDR2和BKDF2扩增其中长度为314bp的DNA片段。用其它15种常见鱼类致病菌验证这两组引物的特异性,结果没有非特异性的DNA片段被扩增出来。用酚抽提法和煮沸加冻融的方法获得的细菌裂解产物,PCR检测的灵敏度均可达到1.8×103CFU·mL-1,用Nested PCR进一步扩增PCR扩增的产物,检测灵敏度可再提高100倍。检测鲑鱼肾杆菌菌悬液与鲟卵的混合物,结果表明,该方法能准确、可靠、快速地检测鲑肾杆菌。     

Surveillance of health status on eight marine rainbow trout, Oncorhynchus mykiss (Walbaum), farms in Denmark in 2006

The health status of eight marine rainbow trout farms was followed from mid-June to mid-September 2006 by sampling both dead and healthy fish approximately every 2 weeks for bacteriological and virological investigation. No fish pathogenic viruses were detected, but all farms experienced disease and mortality as a result of various bacterial infections. Yersinia ruckeri was found on four and Renibacterium salmoninarum on five of the farms, but only during the first part of the surveillance period. This indicates that the fish carried the infection from fresh water, and cleared the infection in salt water. Aeromonas salmonicida subsp. salmonicida caused mortality on five farms, but persisted throughout the sampling period. Although A. salmonicida was probably carried from fresh water, the fish were not able to clear the infection in the sea. Vibrio anguillarum caused mortality on six of the farms throughout the sampling period, O1 being the dominant serovar, and Photobacterium damselae subsp. damselae was found on seven farms as a cause of disease. During the period of highest water temperatures Vibrio parahaemolyticus and Vibrio vulnificus were detected in dead fish in five and two farms, respectively, although their significance as causative pathogens is questionable. Vibrio vulnificus has not previously been found in rainbow trout in Denmark. Both mortality and number of antimicrobial treatments during the period were considerably higher in unvaccinated compared with vaccinated fish. Resistance to commonly used antimicrobials was low or absent.     

Reduction of brown trout, Salmo trutta L., salmon, S. salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), smolt bycatches in eel pound nets

Two types of bycatch reduction devices (BRDs) were tested in the Randers Fjord, Denmark, aimed at deflecting the surface-oriented smolts of brown trout, Salmo trutta L., salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), before they became trapped, while retaining the catches of eel, Anguilla anguilla (L.). The methods tested were: a) placing a floating guard net between the wings; and b) submerging the pot net (i.e. last enclosure before fyke net) 55–100 cm below mean sea level. After each haul the pound net-setups were changed from the control to one of the types of BRD, and vice versa. Both methods significantly reduced the bycatch of brown trout smolts, while catches of legal-sized eels were not affected. Submerging the pot net reduced smolt catches of all species; mean reductions were 91.1% for brown trout, 74.5% for rainbow trout, and 86.1% for salmon.     

An immune-complex glomerulonephritis of Chinook salmon, Oncorhynchus tshawytscha (Walbaum)

Chinook salmon from New Zealand were shown to have a generalized membranous glomerulonephritis that was most severe in large fish. Marked thickening of the glomerular basement membrane was the most consistent lesion, with the presence of an electron-dense deposit beneath the capillary endothelium.Severely affected glomeruli also had expansion of the mesangium and loss of capillaries,synechiae of the visceral and parietal epithelium and mild fibrosis of Bowmans capsule. Chinook salmon from British Columbia, Canada with bacterial kidney disease caused by Renibacterium salmoninarum had similar histological lesions. They also had thickened glomerular basement membranes that were recognized by rabbit antiserum to rainbow trout immunoglobulin. This was true only when frozen sections of kidney were used and not formalin-fixed tissue. An attempt to experimentally produce a glomerulopathy in rainbow trout by repeated immunization with killed R. salmoninarum was not successful. Case records from the Fish Pathology Laboratory at the University of Guelph over a 10-year period revealed that a range of species were diagnosed with glomerulopathies similar to those seen in Chinook salmon. The majority of these cases were determined to have chronic inflammatory disease. This report has identified the presence of immunoglobulin within thickened basement membranes of Chinook salmon with glomerulonephritis and supports the existence of type III hypersensitivity in fish.     

Renibacterium salmoninarum bar forms: characterization, occurrence, and evidence of a host response to a R. salmoninarum infection

Unique-staining Renibacterium salmoninarum (Rs) cells, termed bar forms, first observed in a coho salmon, Oncorhynchus kisutch (Walbaum), in 1983, could not be cultured, making their characterization difficult and significance obscure. They can be detected only by the fluorescent-antibody technique (FAT) and their numbers estimated only by a quantitative FAT (QFAT). Data collected over a 10-year period showed that bar forms were observed only in vivo and appeared associated with a host response. Bar forms were observed in 10 salmonid species from five countries and in fish from < 1 g to spawning adults. They were observed in 50.1% of kidney smears prepared from 10,061 Rs positive chinook, O. tshawytscha (Walbaum), coho, and Atlantic salmon, Salmo salar L. Bar forms were shown to be Rs cells based on absorption studies, their reaction with an Rs-specific FAT and enzyme-linked immunosorbent assay, and a transition from 'typical' Rs cells to bar forms in naturally and experimentally infected fish. Bar forms were determined to be non-virulent, damaged or dead Rs cells, based on fluorescence and electron microscopy observations, the inability to culture them, and mortality data. Bar forms appeared to represent visual markers of recovery from an Rs infection.