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.     

Dispersal and growth rate of O-group salmon (Salmo salar L.) from point-stocking together with some information from scatter-stocking

Abstract– Experiments on the dispersal of O-group salmon from a 'point-stocking' were made in 1988, 1989, 1990 and 1991 in Bollihope Burn, Co. Durham. Experiments on 'scatter-stocking' were made in 1992 and 1993, but the 1992 experiment was aborted for technical reasons. In each year the fish were stocked as fry in spring and their distribution in September was ascertained by electrofishing. Some fish from point-stocking dispersed up to 50 m upstream or 500 m downstream of the stocking point, but the majority remained close to the stocking point. This gave rise to considerable spatial variation in September population density and fish weight, which could be modelled by semi-logarithmic curves. Survival from point-stocking was 19% and 14% in 1988 and 1989, respectively. Approximately 20 to 25% of the September survivors from point-stocking were found upstream of the release point. Scatter-stocking gave relatively little variation in population density or fish weight in September, and estimated survival was 27%.     

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.     

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.     

Multiple Induced Spawnings of the Indian Catfish Heteropneustes fossilis (Bloch) within a Prolonged Spawning Season

Abstract Singhi, the Indian catfish Heteropneustes fossilis , attained gonadal maturity repeatedly in a single spawning cycle by photothermal treatment. After the first spawning, maintenance at 30 C and 14 h light/10 h dark for 3 wk induced recruitment of the next batch of mature oocytes for subsequent spawnings. The number of eggs released declined with each spawning. However, fertilizability of the eggs was maintained until the fifth spawning. Mature fish were induced to spawn by D-Lys⁶ salmon gonadotropin-releasing hormone agonist administration. The dose of salmon gonadotropin-releasing hormone agonist for the second and subsequent spawnings was determined to be 25-fold less compared to the dose administered for the first spawning. Fish released the maximum number of eggs upon induction when they were maintained continuously at 30 C and 14 h light/10 h dark for 6 wk. The result obtained in the present study points to a possibility of obtaining multiple spawnings from those species of farmed fish which spawn once a year.     

Detection of Renibacterium salmoninarum in salmonid kidney samples: a comparison of results using double-sandwich ELISA and isolation on selective medium

Abstract A total of 1239 kidney samples from four species of salmonid fish, Atlantic salmon, Salmo salar L., brown trout, Salmo trutta L., Arctic charr, Salvelinus alpinus (L.), and rainbow trout, Oncorhynchus mykiss (Walbaum), were screened for Renibacterium salmoninarum using double-sandwich ELISA and bacterial isolation. For bacterial isolation, samples were homogenized, washed, plated onto S-KDM and incubated for 12 weeks. Samples for ELISA were kept frozen until tested. After thawing, 25% homogenates in PBS were heated at 100°C for 15 min in the presence (2.5% v/v) of HemoDe solvent (terpene and butylated hydroxyanisole) and then centrifuged. The supernatant was tested with polyclonal antibodies against whole bacterium in a double-sandwich ELISA. In seven out of 12 groups tested, all samples were negative in both tests. Positive ELISA results occurred in five groups. Renibacterium salmoninarum was isolated on SKDM from samples in four out of these five groups. The ELISA test gave significantly higher numbers of positive samples in three out of the four groups showing positive results in both tests.     

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.     

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.     

Immunofluorescence screening of Renibacterium salmoninarum in the tissues and eggs of farmed chinook salmon spawners

A simple and rapid on site-test method was used to screen farmed chinook salmon (Oncorhynchus tschawytscha) broodstock for bacterial kidney disease (BKD) at the time of spawning. Kidney tissue and ovarian fluid smears were examined, using the indirect fluorescent antibody technique (IFAT). To check the validity of our field screening procedure, intra-ovum fluid, taken from random samples of 300 surface-sterile, fertilized eggs, was examined in the laboratory for the presence or absence of Renibacterium salmoninarum, using the IFAT. Prior to sampling, eggs were incubated at 15°C for 40 days in enrichment KDM-2 broth. R. salmoninarum cells were found in one out of 10 samples of pooled contents of eggs from fish showing no R. salmoninarum in the kidney tissue (these fish were assumed to have R. salmoninarum-free ovarian fluid); three out of 10 pooled samples from fish in which R. salmoninarum was detected in the kidney but not the ovarian fluid; all of the 10 pooled samples from fish with R. salmoninarum in both the kidney tissue and the ovarian fluid. The number of R. salmoninarum in the pooled egg contents was low (2–23 per 100 fields). Using the IFAT technique it was not possible to determine whether the BKD-bacteria in the eggs were alive or dead. Methods of BKD treatment and screening are discussed as is the validity of using these techniques to prevent vertical transmission of BKD in farmed salmon.     

Monitoring of the in-river migration of smolts from two groups of spring chinook salmon, Oncorhynchus tshawytscha (Walbaum), with different profiles of Renibacterium salmoninarum infection

Abstract Broodstock segregation based on the measurement of maternal Renibacterium salmoninarum infection levels by the enzyme-linked immunosorbent assay (ELISA) and the membrane filtration-fluorescent antibody technique (MF-FAT) was previously shown to affect the prevalence and levels of bacterial kidney disease (BKD) in progeny of chinook salmon, Oncorhynchus tshawytscha (Walbaum), during hatchery rearing. Subgroups of fish from that study were marked with passive integrated transponder (PIT) tags, and monitored by PIT-tag detectors during the first 342km of their migration to the Pacific Ocean. Differences in the recovery of tagged fish were significant (P≤ 0·01) at each detection point and became more pronounced as the fish moved downstream. Cumulative recoveries of fish from the low-BKD group and the high-BKD group, respectively, were 31% and 28% after 116km, 44% and 37% after 176km, and 51% and 42% after 342km. There were no apparent differences in the migration timing of the two groups to the first detection point. The data suggested that in-river survival was higher in the progeny group from parents that had low R. salmoninarum infection levels or tested negative for R. salmoninarum (low-BKD group) than in the group female parents with high infection levels (high-BKD group).     

Jaw ulcers in Atlantic tomcod, Microgadus tomcod (Walbaum), from the St Lawrence River

A survey of external lesions on Atlantic tomcod from the St Lawrence River was carried out between mid-December 1992 and the end of January 1993 ( n = 1674). Jaw ulcerations were observed in 3.8% of the fish. These ulcers were usually single, and located at the junction between the skin epidermis and oral mucosa. They progressed from shallow lesions to deep ulcers with pathological fracture of the facial bone secondary to osteitis. The aetiology of these lesions could not be determined. Fish with ulcers were generally older than those of the monitored spawning population. The reason for a higher prevalence in older tomcods is unknown, but decreased immune functions and healing capacity caused by ageing are suggested as potential predisposing factors.     

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.     

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。     

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.     

Annually twice induced spawnings provide multiple benefits: Experimental evidence from an Indian major carp (Catla catla,Hamilton 1822)

Multiple spawnings are special reproductive tactics that many fish evolved to obtain different types of direct and/or indirect spawning benefits. This has led to two pertinent questions: (a) what types of benefit does a commercially important fish provide during multiple induced spawnings annually? and (b) is there any trade‐off between traits during these spawnings? To address these questions, the present study was carried out with two separate experiments during two spawning events of an Indian major carp (Catla catla) to assess: (a) male's spawning performances and (b) female's spawning performances. The male's spawning data revealed that they produced significantly higher amount of milt and hatchlings during the first spawning, while they significantly reduced hatchling deformation rate, enhanced offspring survival rate and enlarged offspring body size during the second spawning. The female's spawning information unveiled no significant differences in total egg weight, hatching rate and hatchling deformation rate between the first and second spawning. However, females had significantly higher offspring survival rate during the first spawning and enhanced offspring body size during the second spawning. Interestingly, the analysis of trait associations revealed that males allocated energy with a trade‐off between milt weight and offspring survival rate, whereas females showed a trade‐off between hatching rate and offspring survival rate. Thus, the study provides some important information for carp breeders and associated stakeholders to know which spawning season is preferable for the higher production of eggs, milt and good quality larvae.     

Diagnosis of bacterial kidney disease by detection of Renibacterium salmoninarum by real-time PCR

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), is a serious threat to salmon in aquaculture as well as to wild populations. We have developed a real-time polymerase chain reaction (PCR) for detection of Rs in kidney samples. The PCR is based on detection of unique parts of the 16S rRNA gene of Rs and DNA equivalent to 1-10 Rs genomes was detected per reaction. No cross-reactivity with other fish pathogenic or related bacteria could be demonstrated. Analysis of individual kidney samples collected from BKD classified populations identified 39.9% of the fish as positive by real-time PCR compared with 28.0% by polyclonal enzyme-linked immunosorbent assay (ELISA). The real-time PCR assay was found to be well suited for complementary use with ELISA for diagnosis of BKD, with the ability to detect clinical as well as covert Rs infections. The infection level determined by the polyclonal ELISA and by real-time PCR was significantly correlated.     

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 disease status of Australian salmonids: bacteria and bacterial diseases

Abstract. Eleven freshwater salmonid hatcheries in southern Australia were surveyed for bacterial pathogens and diseases between 1981 and 1985, Twenty-five populations of fish were examined in the study, representing a total of 2755 fish, from which kidney, liver, spleen, and in some cases peritoneum, blood and faeces were cultured. Bacteria of pathogenic significance isolated included Aeromonas hvdrophila, Streptococcus sp., Lactobacillus piscicola, Yersinia ruckeri, Mycobacterium fortuitum, M. chelonei and a filamentous acid-fast organism of uncertain taxonomic position. Lacto-bacillus piscicola and Streptococcus sp. were associated with clinical and subclinical peritonitis. Mycobacterium chelonei was isolated from visceral granulomas in an externally normal fish. Aeromonas salmonicida, Renibacterium salmoninarum and Edwardsiella tarda were not isolated, indicating that the diseases furunculosis, bacterial kidney disease and edwardsiellosis are exotic to Australian salmonids. Similarly, while Y. ruckeri was isolated, enteric redmouth disease had not been recorded and is considered an exotic disease.