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.     

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.     

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.     

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.     

The occurrence of proliferative kidney disease (PKD) in cultured and wild fish: further investigations

Abstract. In an investigation of the occurrence of proliferative kidney disease (PKD) in freshwater fish other than rainbow trout, 18 species of wild fish and seven species of fish raised in cultivation wore sampled from waters where the disease occurred annually in rainbow trout, Oncorhynchus mykiss (Richardson). Results revealed that certain wild stocks of brown trout. Salmo trutta L., grayling, Thymallus thymallus L., and pike, Esox lucius L., were infected with PKD, as were cultivated Atlantic salmon, Salmo salar L., parr, brown trout and char, Salvelinus alpinus (L.). Microscopical examination revealed the presence of the PKX cell in these species and also intraluminal protozoa possibly related to the PKX cell, which were not found in the rainbow trout. Other species of freshwater fish had myxosporidan infections but, unlike PKD infection, there was little host/parasite tissue response. The PKX cell as a myxosporidan stage is discussed and the presence of the disease in wild fish is reported.     

Ceratomyxa shasta genotypes cause differential mortality in their salmonid hosts

Ceratomyxa shasta is a myxozoan parasite of salmonid fish. In natural communities, distinct genotypes of the parasite are associated with different salmonid hosts. To test the hypothesis that genotypes of C. shasta cause differential mortality, the polychaete host was experimentally infected with different parasite genotypes. Genotype I was obtained from Chinook salmon, Oncorhynchus tshawytscha, and genotype II from either coho salmon, O. kisutch, or rainbow trout, O. mykiss, We then challenged four salmonid strains: Chinook and coho salmon that occur in sympatry with the parasite and allopatric Chinook salmon and rainbow trout. Parasite genotype I caused mortality only in Chinook strains, although mortality in the allopatric strain also occurred from exposure to genotype II. A second experiment demonstrated that genotype II could be separated into two biotypes based on differential mortality in rainbow trout and coho salmon. These differential patterns of mortality as a result of infection by certain genotypes of C. shasta support field observations and suggest a co‐evolutionary relationship between these parasites and their hosts.     

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.     

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.     

Proliferative kidney disease in brown trout, Salmo trutta L., and Atlantic salmon, Salmo salar L., parr: histopathological and epidemiological observations

Abstract Proliferative kidney disease (PKD) has been described in cultivated rainbow trout but major outbreaks have not been recognized in other fish species. This paper describes light and electron microscope studies and presents epidemiological data of a disease condition in first summer Salmo salar parr and S. trutta , which is associated with an organism apparently identical to the aetiological agent of PKD. The pathology was found to be similar to that described for PKD in rainbow trout. The epidemiology in the brown trout suggests that exposure after May does not result in infection. The data from the outbreaks in two salmon hatcheries appear complex and suggest different susceptibilities to PKD among salmon originating from different sources.     

Pancreas disease in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in Norway

The present paper describes, for the first time, clinical signs and pathological findings of pancreas disease (PD) in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in sea water in Norway. Similarities and differences with reports of PD from Ireland and Scotland are discussed. Samples of 68 rainbow trout from disease outbreaks on 14 farms and from 155 Atlantic salmon from outbreaks on 20 farms collected from 1996 to 2004 were included in the present study. The histopathological findings of PD in Atlantic salmon and rainbow trout in sea water were similar. Acute PD, characterized by acute necrosis of exocrine pancreatic tissues, was detected in nine Atlantic salmon and three rainbow trout. Salmonid alphavirus (SAV) was identified in acute pancreatic necroses by immunohistochemistry. Most fish showed severe loss of exocrine pancreatic tissue combined with chronic myositis. Myocarditis was often but not consistently found. Kidneys from 40% and 64% of the rainbow trout and Atlantic salmon, respectively, had cells along the sinusoids that were packed with cytoplasmic eosinophilic granules. These cells resembled hypertrophied endothelial cells or elongated mast cell analogues. Histochemical staining properties and electron microscopy of these cells are presented. SAV was identified by RT-PCR and neutralizing antibodies against SAV were detected in blood samples.     

Sleeping disease and pancreas disease: comparative histopathology and acquired cross-protection

Sleeping disease (SD), a disease of freshwater-reared rainbow trout, Oncorhynchus mykiss (Walbaum), was histologically compared to pancreas disease (PD), a disease of sea water-reared Atlantic salmon, Salmo salar L. Some weeks after injection of kidney homogenates from SD- or PD-affected fish in rainbow trout, the fish successively developed pancreatic, heart and muscular lesions in both populations, although the SD-injected population was the most affected. An acquired protection was observed against PD and SD after an initial injection with PD- or SD-infective material. This acquired cross-protection and the similar histopathologies in PD and SD suggest the two diseases could be caused by a similar or identical agent.     

Duration of immunity in salmonids vaccinated by direct immersion with Yersinia ruckeri and Vibrio anguillarum bacterins

Abstract. The level of protective immunity was determined for several salmonid species following vaccination by the direct immersion method with commercial Vibrio anguillarum (two serotypes) and Yersinia ruckeri (Hagerman strain) bacterins. The duration of protective immunity varied with the bacterin concentration, size and species of fish, but the duration between the two bacterins was comparable. In fish under 1 g duration of protective immunity was longest when the most concentrated bacterin was used. Generally, immunity lasted in 1-g fish for about 120 days, in 2-g fish for about 180 days, but in 4-g fish and larger immunity lasted for a year or longer. Coho salmon ( Oncorhynchus kisutch ) and sockeye salmon ( O. nerka ) retained immunity for a longer time and pink salmon ( O. gorbuscha ) the shortest time. Chinook salmon ( O. tshawytscha ) and rainbow trout ( Salmo gairdneri ) were intermediate. Field data generally followed the laboratory tests, but the duration appeared somewhat shortened. In one test 20-g rainbow trout were vaccinated by the shower method and no loss of immunity occurred after 311 days.     

Microsporidium rhabdophilia n. sp. from rodlet cells of salmonid fishes

Abstract. A microsporidian parasite, Microsporidium rhabdophilia n. sp. is described from rodlet cells of salmonid fishes. Chinook salmon, Oncorhynchus tshawytscha , coho salmon, Oncorhynchus kisutch , steelhead rainbow trout, Salmo gairdnerii gairdnerii , and various strains of domesticated rainbow trout were found infected. In living wet mount phase-contrast observations of various tissues, 16 mature spores could be seen infecting the nucleus of rodlet cells. The number of spores infecting each nucleus generally numbered 16 and spores were not found in any cell or organelle other than the nucleus of the rodlet cell. All observations were limited to California hatchery-reared 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.     

Competitive interactions among multiple non‐native salmonids and two populations of Atlantic salmon

Competitive interactions with non‐native species can have negative impacts on the conservation of native species, resulting in chronic stress and reduced survival. Here, juvenile Atlantic salmon (Salmo salar) from two allopatric populations (Sebago and LaHave) that are being used for reintroduction into Lake Ontario were placed into semi‐natural stream tanks with four non‐native salmonid competitors that are established in Ontario streams: brown trout (S. trutta), rainbow trout (Oncorhynchus mykiss), Chinook salmon (O. tshawytscha) and coho salmon (O. kisutch). Brown trout and rainbow trout reduced the survival and fitness‐related traits of Atlantic salmon, whereas Chinook salmon and coho salmon had no impact on these traits. These data support theories on ecological niche overlap and link differences in observed aggression levels with competitive outcomes. Measurements of circulating hormones indicated that the Atlantic salmon were not chronically stressed nor had a change in social status at the 10‐month time point in the semi‐natural stream tanks. Additionally, the Sebago population was better able to coexist with the non‐native salmonids than the LaHave population. Certain populations of Atlantic salmon may thus be more suitable for some environments of the juvenile stream phase for the reintroduction into Lake Ontario.     

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.     

Pathology and immunohistochemistry in three species of salmonids after experimental infection with Flavobacterium psychrophilum

Rainbow trout, Oncorhynchus mykiss (Walbaum), sea trout, Salmo trutta L., and Atlantic salmon, Salmo salar L., were experimentally infected with Flavobacterium psychrophilum in order to evaluate any species differences in susceptibility to the bacterium. Furthermore, differences in pathological changes and distribution of the bacteria in internal organs were studied. The bacteria were injected intraperitoneally in two doses, high dose (Hd) 1 x 10(7) colony forming units (CFU) fish(-1) and low dose (Ld) 1 x 10(6) CFU fish(-1). The mortalities in the Ld groups varied between 0 and 7.5% and in the Hd groups between 55-70%. No significant differences in mortality between the species were recorded. Clinical signs and pathological findings were similar in the three species and in accordance with those of rainbow trout fry syndrome. Rainbow trout showed more pronounced lesions in the spleen compared with the other species. Necrosis of renal tubular epithelium and haematopoietic tissue was most prominent in rainbow trout and Atlantic salmon. Intracellular eosinophilic droplets in the kidney tubular epithelium were a prominent finding in rainbow trout and sea trout surviving the infection. The distribution of the bacteria in internal organs was similar in the three species, as studied with immunohistochemistry.     

Alphavirus infections in salmonids--a review

The first alphavirus to be isolated from fish was recorded in 1995 with the isolation of salmon pancreas disease virus from Atlantic salmon, Salmo salar L., in Ireland. Subsequently, the closely related sleeping disease virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), in France. More recently Norwegian salmonid alphavirus (SAV) has been isolated from marine phase production of Atlantic salmon and rainbow trout in Norway. These three viruses are closely related and are now considered to represent three subtypes of SAV, a new member of the genus Alphavirus within the family Togaviridae. SAVs are recognized as serious pathogens of farmed Atlantic salmon and rainbow trout in Europe. This paper aims to draw together both historical and current knowledge of the diseases caused by SAVs, the viruses, their diagnosis and control, and to discuss the differential diagnosis of similar pathologies seen in cardiomyopathy syndrome and heart and skeletal muscle inflammation of Atlantic salmon.     

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.     

Appearance of Infectious Hematopoietic Necrosis Virus in Rainbow Trout,Oncorhynchus mykiss,During Seawater Adaptation

About 7% mortality occurred in rainbow trout, Oncorhynchus mykiss, during seawater adaptation at a marine farm in the South Sea of Korea during the winter of 2014. Most diseased fish showed petechial hemorrhaging of gills and internal fat with enlarged spleen. Although no parasites or bacteria were isolated from the diseased fish, all tissue filtrates produced cytopathic effects (CPEs) in fathead minnow and Chinook salmon embryo‐214 cells. The cell culture supernatant showing CPE contained specific 1527‐bp fragment for the infectious hematopoietic necrosis virus (IHNV) glycoprotein gene by polymerase chain reaction. Their nucleotide sequences shared 98.1–98.2% identities with IHNV RtUi02 isolated from rainbow trout in Korea. This isolate (RtGoH14) was closely related to Korean IHNV isolates of genogroup JRt rather than to those of North American and European genogroups. These results suggest that this IHNV isolate might have been introduced to rainbow trout farm (land‐based culture system) in Korea. This is the first report of IHNV infection in rainbow trout during seawater adaptation in Korea.