FISH DISEASE LEAFLET 78 BACTERIAL KIDNEY DISEASE OF SALMONID FISHES CAUSED BY RENIBACTERIUM SALMONINARUM G. L. Bullock and R. L. Herman U.S. Fish and Wildlife Service, National Fisheries Research Center-Leetown, National Fish Health Research Laboratory, Box 700, Kearneysville, West Virginia 25430 UNITED STATES DEPARTMENT OF THE INTERIOR, Fish and Wildlife Service, Research and Development, Washington, D. C. 20240, 1988 Revision of Fish Disease Leaflet 60 (1980), same title, by G. L. Bullock. Introduction Bacterial kidney disease (BKD) is a systemic infection that commonly causes high mortality in populations of wild and propagated salmonids. The disease course is typically chronic, but acute outbreaks sometimes occur, especially at moderate temperatures (1318°C). The first report of BKD described an infection in Atlantic salmon (Salmo salar) in Scotland. In the United States, the disease was first reported by Belding and Merrill (1935), who described gross external and internal pathology in brook trout (Salvelinus fontinalis). The bacterium from trout was morphologically identical with the organism described from salmon. Because focal abscesses occur in the kidneys, spleen, and liver, BKD has also been called white boil disease. Etiology and Disease The causative agent is a small (0.4x0.8 mm), nonmotile, asporogenous, grampositive diplobacillus. Ordal and Earp (1956) made the initial isolation and, on the basis of morphology, classified it as a Corynebacterium. A taxonomic study of 25 isolates by Austin and Rodgers (1980) showed diversity among strains. One group of 6 strains was related to Corynebacterium pyogenes, a second group of 12 represented a new taxon, and 7 strains did not fall in either group. On the basis of biochemical characteristics of strains of the causative bacterium, Sanders and Fryer (1980) proposed the name Renibacterium salmoninarum. Studies of the fatty acid profiles of R. salmoninarum strains, as well as numerical taxonomic studies, have supported the integrity of the genus Renibacterium (Goodfellow et al. 1985). Immunoelectrophoretic analyses of R. salmoninarum strains indicate seven common components (AG), among which antigen F is the major surface antigen (Getchell et al. 1985). Immunoblot techniques revealed the presence of antigenic proteins attached to the cell wall (Fiedler and Draxl 1986). Because R. salmoninarum is a slowgrowing organism that typically requires 1 to 3 weeks to isolate, culture techniques are not usually used for diagnosis. Presumptive diagnosis is based on the presence of typical clinical signs and small, grampositive diplobacilli in infected tissues. Several serological procedures have been developed for definitive diagnosis. Immunodiffusion procedures have been used for diagnosis of overt kidney disease (Chen et al. 1974; Kimura et al. 1978), but the more rapid direct and indirect fluorescent antibody tests (FAT) are more commonly used for diagnosis of both overt and subclinical infections (Bullock and Stuckey 1975; Bullock et al. 1980). Ochiai et al. (1985) adapted the direct FAT test for detection of R. salmoninarum in fixed and paraffin-embedded tissue sections. Kimura and Yoshimizu (1981) used staphylococci specifically sensitized with antibody against R salmoninarum to develop a coagglutination test. The most recently developed serological procedures for diagnosis and detection of BKD are an enzymelinked immunosorbent assay (ELISA) procedure that detects soluble antigen of R. salmoninarum (Pascho and Mulcahy 1987) and a filtration FAT technique (Elliott and Barila 1987) that concentrates the organism and facilitates its detection in ovarian fluids. Austin et al. (1985), who isolated a pathogenic gram-positive bacterium from rainbow trout (Salmo gairdneri) that gave a falsepositive FAT, suggested that confirmation of BKD should include cultivation. Three media are now used: kidney disease medium-two (KDM2; Evelyn 1977); charcoal agar (Daly and Stevenson 1985); and the selective medium developed by Austin et al. (1983). Clinical Signs and Pathology Kidney disease is systemic and progresses slowly. Clinical signs may not be evident until the disease is well established. External signs typically include exophthalmos and small closed blebs or open lesions. The unruptured blebs contain fluid (cream colored to red) composed of blood cells, necrotized tissue, and large numbers of R. salmoninarum. In advanced cases the lesions sometimes coalesce and form large shallow ulcers. Internally, the kidneys are the organs most often affected; they become swollen and show discrete white areas that contain leucocytes, bacteria, and host cell debris. In advanced cases much kidney tissue is destroyed and hematopoietic and excretory functions are both affected. Hemorrhages occur in the body wall and testes, and peritoneal fluid is commonly present. The hind gut can be hemorrhagic and filled with white or yellow viscous fluid. Petechia were common in the peritoneum of infected Atlantic salmon in Scotland (Smith 1964); the fish also showed white false-membranes over liver, spleen, and gonads when infection occurred at temperatures below 8.3°C (but not at higher temperatures). Similar membranes occur in trout at a higher temperature (1213°C). In an outbreak of BKD in chinook salmon (Oncorhynchus tschawyscha) the only external sign was exophthalmos. Bacteria were initially present only in tissue behind the affected eyes. Later, the disease became systemic (Hendricks and Leek 1975). Recently we have seen severe eye lesions in coho salmon (O. kisutch) in which massive numbers of R. salmoninarum could be demonstrated but neither bacteria nor signs of pathology were present in the visceral organs. Histologically, the lesions are characteristically a diffuse, granulomatous inflammatory response to a bacteremia affecting, but not limited to, the kidneys. Early lesions of the internal organs are necrotic foci in which bacteria occur both intracellularly and extracellularly. These expand into the more characteristic diffuse granulomas with necrotic centers. The granulomas may be relatively discrete but are rarely encapsulated. Bacteria tend to be only in the periphery of the granulomas and may be difficult to find. When brook trout were infected intraperitoneally, bacteria first colonized the outer surfaces of organs and later invaded deeper tissues. Changes in the fine structure of glomeruli and renal tubules resemble those of glomerulonephritis and nephritic syndrome in mammals (Young and Chapman 1978). Transmission Bacterial kidney disease may be transmitted both horizontally and vertically. Subclinically infected or latent carrier salmonids are reservoirs of infection. Mitchum and Sherman (1981) reported that naturally infected feral brook trout transmitted BKD to newly stocked rainbow trout, brown trout (Salmo trutta), and brook trout, and that newly stocked trout began dying within 9 months. Austin and Rayment (1985) reported that R. salmoninarum is excreted in the feces of clinically diseased trout, and that the organism can survive up to 21 days in feces or pond sediments. Results from several studies have shown that R. salmoninarum can be transmitted in the egg and that a 15min disinfection with 100 250 ppm povidoneiodine does not prevent transmission (Evelyn et al. 1984; Bruno and Munro 1986). Additionally, the pathogen can be sequestered internally by immersing eggs before fertilization in coelomic fluid containing high numbers of R. salmoninarum (Evelyn et al. 1986b). Occurrence Although BKD occurs mainly in freshwater, significant mortality also occurs in saltwater (Banner et al. 1983). As a consequence of infection, juvenile anadromous salmonids are unable to acclimate to seawater and die of BKD. When infected smolts of coho salmon were held in freshwater or saltwater, 17.2% and 4%, respectively, died of BKD (Fryer and Sanders 1981). Even if salmon are lightly infected when they enter saltwater, the disease continues to progress and deaths occur. Among samples of seven species of anadromous salmonids captured in the Pacific Ocean, Banner et al. (1986) reported a 311% prevalence of BKD. Infected salmonids that survive in the ocean can infect other salmonids. Incubation Period Although BKD develops slowly, progress of the disease is affected by water temperature. At 1520°C experimentally infected juvenile salmon and trout died 2134 days after inoculation, but at 6.7°C none died until 60 to 71 days after inoculation (Sanders et al. 1978). Host and Geographic Range Bacterial kidney disease has been reported only in salmonids: Pacific and Atlantic salmon, grayling (Thymallus sp.), and several species of trout. In recent years the severity of BKD in most species of cultured trout has decreased dramatically, for unknown reasons. Some fishery workers attribute the reduction to improved diets and culture practices. However, BKD continues to cause serious mortality in lake trout (Salvelinus namaycush) and Pacific and Atlantic salmon. The known range of BKD includes Canada, England, France, Germany, Iceland, Italy, Japan, Scotland, Spain, Turkey, United States, and Yugoslavia. However, the disease can occur wherever susceptible populations of salmonids are present. Control Because BKD is vertically transmitted and R. salmoninarum occurs intracellularly, the prevention or treatment of epizootics is difficult. However, the results of recent studies have indicated that the severity and number of epizootics can be reduced by diet modification or use of the antibiotic erythromycin. Bell et al. (1984) reported that survival of experimentally infected sockeye salmon was inversely proportional to dietary levels of vitamin C in rations that also contained low levels of zinc and manganese. The prevalence of BKD was reduced in Atlantic salmon that were fed a diet high in iron, copper, manganese, iodine, cobalt, and fluorine, or a diet low in calcium (Patterson et al. 1981). Lall et al. (1985) also noted that increased iodine and fluorine reduced the prevalence of BKD when dietary zinc and manganese concentrations were low. In an attempt to reduce or prevent vertical transmission of BKD, Amos (1977) waterhardened salmon eggs 1 h in 2 ppm erythromycin. However, erythromycin was rapidly eliminated and dropped below detectable levels within 24 h after waterhardening (Groman 1983; Evelyn et al. 1986a; Bullock and Leek 1986). Considering that R. salmoninarum grows slowly, it is doubtful whether short exposure to erythromycin would be effective. Monthly subcutaneous injections of adult female Pacific salmonids with 11 mg/kg erythromycin reduced prespawning mortality from BKD (Klontz 1983). Erythromycin injection also resulted in the deposition of erythromycin in eggs that persisted at effective concentrations for as long as 70 days postspawning (Bullock and Leek 1986; Evelyn et al. 1986a). Injection of adults with erythromycin can reduce or possibly prevent vertical transmission. There is evidence that resistance to BKD has a genetic basis. In coho salmon three transferrin types (AA, AC, and CC) were experimentally exposed to R. salmoninarum. Type AA was consistently the most susceptible and CC the most resistant (Suzumoto et al. 1977). Winter et al. (1980) reported similar results in coho salmon and steelhead. No effective bacterin has been developed for control of BKD. Injection of R. salmoninarum emulsion in Freund's complete adjuvant produced elevated humoral antibody titers, but Evelyn (1971) reported a variable response in chinook salmon: titers in salmon 100 days after injection ranged from a low of 1:10 to a high of 1:2,560. Underyearling and older Atlantic salmon parr produced high agglutinating antibody titers after they were injected with R. salmoninarum and complete adjuvant (Paterson et al. 1981). However, the duration of immunity in underyearlings was not sufficient to provide protection from BKD 2 years later, when the fish underwent smoltification. Rainbow trout were protected from BKD by intraperitoneal injection of a lysed bacterin but not by a 2min immersion or twostep hyperosmotic infiltration (McCarthy et al. 1984). Treatment Kidney disease is among the most difficult of the fish bacteremias to treat, possibly because the bacterium occurs intracellularly and is thus beyond the reach of some antimicrobials. Under laboratory conditions, erythromycin given orally at the rate of 910 g per 100 kg of fish per day for 3 weeks gave the best, albeit partial, control (Wolf and Dunbar 1959). Field applications have given similar results; control was effected in some lots, but in others the disease recurred after drug administration was stopped. All published reports of treatment with sulfonamides indicate that mortality recurred after treatment ceased. In a recent study in which Austin (1985) tested more than 70 antimicrobial compounds, clindamycin, erythromycin, kitasamycin, penicillin, and spiramycin effectively controlled early clinical cases of BKD. Annotated Bibliography Amos, K. H. 1977. The control of bacterial kidney disease in spring chinook salmon. M.S. thesis, University of Idaho, Moscow. 20 pp. Multiple injections of erythromycin were used to control BKD in prespawning adult Pacific salmon, and eggs were waterhardened in erythromycin phosphate to control vertical transmission. Austin, B. 1985. Evaluation of antimicrobial compounds for the control of bacterial kidney disease in rainbow trout, Salmo gairdneri. J. Fish Dis. 8:209220. A comparison of more than 70 antimicrobial compounds by in vitro and in vivo methods showed that clindamycin, erythromycin, kitasamycin, penicillin G, and spiramycin could be used successfully to combat early clinical cases of BKD in rainbow trout. Austin, B., D. Bucke, S. Feist, and J. N. Rayment. 1985. A false positive reaction in the fluorescent antibody test for Renibacterium salmoninarum with a "coryneform" organism. Bull. Eur. Assoc. Fish Pathol. 5:89. An organism with some of the key characteristics of R. salmoninarum was isolated from apparently healthy rainbow trout being examined for the presence of BKD. This organism of the coryneform group gave a false positive reaction with the indirect fluorescent antibody test for BKD. It killed rainbow trout within 6 days, even though the inoculum used was equivalent to only 10% of the lethal dose of R. salmoninarum. Austin, B., T. M. Embley, and M. Goodfellow. 1983. Selective isolation of Renibacterium salmoninarum. Fed. Eur. Microbiol. Soc. Microbiol. Lett. 17:111114. A selective medium was developed that allowed the isolation of R. salmoninarum from sources such as river water and fish feces, which support fast developing bacteria that usually overgrow it. Austin, B., and J. N. Rayment. 1985. Epizootiology of Renibacterium salmoninarum, the causal agent of bacterial kidney disease in salmonid fish. J. Fish Dis. 8:505509. The use of a selective medium enabled recovery of R. salmoninarum from rainbow trout. In asymptomatic fish, small numbers of colonies were recovered only from the anterior part of the kidney; in clinically diseased fish, dense pure culture growth was obtained from kidney, spleen, heart, blood, ascitic fluid, and feces. The bacterium was not recovered from the water or sediment of 56 infected or uninfected freshwater fish farms in England and Wales. Laboratory experiments showed that the pathogen was excreted in the feces of clinically diseased fish and that bacterial cells survived for as long as 21 days in sediment or fecal material. Austin, B., and C. J. Rodgers. 1980. Diversity among strains causing bacterial kidney disease in salmonid fish. Curr. Microbiol. 3:231235. The biochemical, cultural, morphological, physiological, and serological characters were studied in 25 grampositive bacterial isolates from BKD. Two distinct homogenous groups and seven singlemember clusters were defined as a result of overall similarity, based on analyses with the Jaccard coefficient. One phenon was equated with Corynebacterium pyogenes, but the second was a novel taxon. Banner, C. R., J. J. Long, J. L. Fryer, and J. S. Rohovec. 1986. Occurrence of salmonid fish infected with Renibacterium salmoninarum in the Pacific Ocean. J. Fish Dis. 9:273275. Kidney material from 3,680 salmonids of 7 species from the Pacific Ocean near Washington and Oregon was examined for R. salmoninarum by the fluorescent antibody test. From 1 to 11% of the fish were infected, and the authors speculated that BKD causes significant mortality of salmonids in the open ocean. Banner, C. R., J. S. Rohovec, and J. L. Fryer. 1983. Renibacterium salmoninarum as a cause of mortality among chinook salmon in salt water. J. World Maricult. Soc. 14:236239. In a study of the contribution of R. salmoninarum to saltwater mortality of chinook salmon, a direct fluorescent antibody technique was used to examine dead fish for the presence of the bacterium. Fall and spring release groups from each fish population were examined. The capacity of R. salmoninarum to cause mortality of chinook salmon in saltwater was demonstrated, and there were indications of horizontal transmission. Belding, D. L., and B. Merrill. 1935. A preliminary report upon a hatchery disease of the Salmonidae. Trans. Am. Fish. Soc. 65:76-84. Gross external and internal pathology was described from this first reported outbreak of kidney disease in the United States, and the causative agent was cultured. The isolated bacterium was morphologically identical to R. salmoninarum, although it was reported to be motile. Bell, G. R., D. A. Higgs, and G. S. Traxler. 1984. The effect of dietary ascorbate, zinc, and manganese on the development of experimentally induced bacterial kidney disease in sockeye salmon (Oncorhynchus nerka). Aquaculture 36:293311. In sockeye salmon infected with R. salmoninarum, survival time was inversely related to dietary ascorbate, but only when dietary zinc and manganese were also low. There was no significant effect on antibody production in vaccinated fish. Bruno, D. W. 1986. Histopatbology of bacterial kidney disease in laboratory infected rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L., with reference to naturally infected fish. J. Fish Dis. 9:523537. Bacterial kidney disease was established in rainbow trout and Atlantic salmon after the intraperitoneal injection of viable R. salmoninarum. The disease was characterized by a chronic systemic infection and signs were similar to those seen in natural outbreaks. Colonization and multiplication of the bacterium occurred on serosal surfaces and within connective tissue elements. Bruno, D. W., and A. L. S. Munro. 1986. Observations on Renibacterium salmoninarum and the salmonid egg. Dis. Aquat. Org. 1:8387. Cells of R. salmoninarum were seen entering the developing eggs of experimentally infected juvenile rainbow trout. The bacterium was cultured from the surface and the insides of eggs of a mature Atlantic salmon, even though the eggs had been bathed in erythromycin phosphate, penicillin, or proflavin during waterhardening. No bacteria were detected in the fry hatched from these infected eggs. Bullock, G. L., B. R. Griffin, and H. M. Stuckey. 1980. Detection of Corynebacterium salmoninus by direct fluorescent antibody test. Can. J. Fish. Aquat. Sci. 37:719721. Description of a rapid procedure suitable for screening large numbers of tissue samples. Bullock, G. L., and S. L. Leek. 1986. Use of erythromycin in reducing vertical transmission of bacterial kidney disease. Vet. Hum. Toxicol. 28 (Suppl. 1):1820. Studies were conducted in 198082 to determine uptake and retention of erythromycin when eggs of chinook salmon were waterhardened for 1 h in 2 ppm of the antibiotic as a means of preventing vertical transmission of BKD. Although eggs absorbed up to 1 ppm of the drug during waterhardening, they retained it less than 24 h. Injection of adult female chinook salmon with 11 mg of erythromycin per kilogram of body weight to prevent prespawning mortality from BKD resulted in deposition of drug in the eggs. Erythromycin persisted in the eggs at concentrations as high as 0.6 ppm for 30 to 60 days postinjection-a period that would allow prolonged contact of drug with the causative agent of BKD. Injection of prespawning female chinook salmon with erythromycin is potentially more effective than the waterhardening of eggs in erythromycin in preventing the vertical transmission of BKD. Bullock, G. L., and H. M. Stuckey. 1975. Fluorescent antibody identification and detection of the Corynebacterium causing kidney disease of salmonids. J. Fish. Res. Board Can. 32:22242227 An indirect fluorescent antibody test described can be completed in 2 h. The procedure was superior to Gram staining in detecting small numbers of the organism. Chen, P. K., G. L. Bullock, H. M. Stuckey, and A. C. Bullock. 1974. Serological diagnosis of corynebacterial kidney disease of salmonids. J. Fish. Res. Board Can. 31:19391940. Clinical kidney disease was serologically diagnosed in less than 24 h by the use of infected tissues, cultures, and rabbit antiserum in an agar gel diffusion test. Bands of identity were formed where the antiserum reacted with the culture of the kidney disease organism or with the antigen from infected tissues. This test provides a rapid reliable diagnosis of kidney disease. Cipriano, R. C., C. E. Starliper, and J. H. Schachte. 1985. Comparative sensitivities of diagnostic procedures used to detect bacterial kidney disease in salmonid fishes. J. Wildl. Dis. 21:144148. Kidney and spleen homogenates from each of 60 coho salmon and steelhead were examined for detection of R. salmoninarum. The proportions of positives differed widely with the detection procedures used: in coho salmon, 5% were positive by the Gram stain procedure, 10% by the direct fluorescent antibody test, 48% by bacteriological isolation, 65% by staphylococcal coagglutination, and 73% by counterimmunoelectrophoresis. In steelhead, 3% were positive by Gram stain, 8.3% by fluorescent antibody, 17% by bacteriological isolation, and 67% by counterimmunoelectrophoresis. Renibacterium salmoninarum was not detected in either species by immunodiffusion analysis. Daly, J. G., and R. M. W. Stevenson. 1985. Charcoal agar, a new growth medium for the fish disease bacterium Renibacterium salmoninarum. Appl. Environ. Microbiol. 50:868871. Charcoal is an effective replacement for serum in culture media for R. salmoninarum. Eight strains of R. salmoninarum from dilute inocula grew as well on the charcoal medium as on a standard medium containing serum. The medium was effective for both primary isolations from fish and repeated transfers and may be useful for antigen preparation and physiological studies. Elliott, D. G., and T. Y. Barila. 1987. Membrane filtration fluorescent antibody staining procedure for detecting and quantifying Renibacterium salmoninarum in coelomic fluid of chinook salmon (Oncorhynchus tshawytscha). Can. J. Fish. Aquat. Sci. 44:206210. A method was developed to rapidly detect and quantify R. salmoninarum in coelomic fluid of spring chinook salmon by concentrating the bacteria on 0.2mm polycarbonate filters and staining them with specific fluoresceinlabeled antibody. Centrifugation of samples and resuspension of the sedimented material in phosphatebuffered saline containing Triton X100 increased the ease of filtration. Background fluorescence was reduced by counterstaining filters with Eriochrome black T. Postfiltration staining, rinsing, and counterstaining were done in the syringemounted filter holders, reducing the handling of the filters and possible loss of bacteria. When the filtrationfluorescent antibody technique was used, the bacterium was detected in the coelomic fluid of 85% of spawning female spring chinook salmon sampled from a hatchery population. Evelyn, T. P. T. 1971. The agglutinin response in sockeye salmon vaccinated intraperitoneally with a heatkilled preparation of the bacterium responsible for salmonid kidney disease. J. Wildl. Dis. 7:328335. Injection of various amounts of killed BKD bacteria into sockeye salmon produced a wide response in antibody production. Titers in salmon 100 days after injection ranged from 1:10 to 1:2,560. The migration of antibody present in the serum resembled that of human beta and gamma globulins. Evelyn, T. P. T. 1977. An improved growth medium for the kidney disease bacterium and some notes on using the medium. Bull. Off. Int. Epizoot. 87:511513. Growth of the BKD bacterium on several media is described and the formulation for an improved medium is given. Evelyn, T. P. T., J. E. Ketcheson, and L. ProsperiPorta. 1981. The clinical significance of immunofluorescencebased diagnoses of the bacterial kidney disease carrier. Fish. Pathol. 15:293300. Experiments were undertaken to determine whether diagnoses of the R. salmoninarum carrier based on the fluorescent antibody test could be corroborated by using a culture method. Preliminary experiments showed that culture could be used for corroborative purposes because it was more sensitive than the FAT in detecting R. salmoninarum. Maximum sensitivity of the culture method was achieved only when the tissue samples were first freed of antiR. salmoninarum activity. Evelyn, T. P. T., J. E. Ketcheson, and L. ProsperiPorta. 1984. Further evidence for the presence of Renibacterium salmoninarum in salmonid eggs and for the failure of povidoneiodine to reduce the intraovum infection rate in waterhardened eggs. J. Fish Dis. 7:173-182. Unfertilized waterhardened eggs, obtained from a coho salmon with coelomic fluid containing R. salmoninarum (4 x 109 cells/mL) were examined by cultural and histological methods to determine whether an earlier finding of the pathogen in coho salmon eggs could be corroborated. Also the ability of iodine (500 mg/L, in the form of povidoneiodine) to kill R. salmoninarum in the egg was examined. The results confirmed that the pathogen occurs within the egg (11.6-15.1 % of the eggs were infected), and suggested a location in the yolk for the pathogen. Although iodine was effective in killing the pathogen on the surface of the eggs, it was ineffective against intraovum infections. The study pointed out the need for a method for treating eggs internally and confirmed that the use of eggs from salmonids having cloudy coelomic fluid is inadvisable. Evelyn, T. P. T., J. E. Ketcheson, and L. ProsperiPorta. 1986a. Use of erythromycin as a means of preventing vertical transmission of Renibacterium salmoninarum. Dis. Aquat. Org. 2:711. The salmonid pathogen R. salmoninarum is now known to be transmitted from parent to progeny within the salmonid egg. Two groups of experiments were conducted to evaluate the possible efficacy of the antibiotic erythromycin in preventing this mode of transmission. The first evaluated the widely practiced disinfection technique of waterhardening eggs in aqueous erythromycin. Eggs so treated rapidly accumulated the antibiotic, but only in the perivitelline fluid, not in the yolk where the intraovum pathogen is believed to occur. Further, the erythromycin was leached from the egg too rapidly to permit bactericidal activity. The second group of experiments investigated erythromycin levels attained in eggs when the brood fish were injected with the antibiotic before spawning. Fish injected with erythromycin (20 mg antibiotic per kilogram of fish) 30 to 56 days before spawning yielded eggs containing levels of the antibiotic that exceeded the minimum lethal concentration determined for R. salmoninarum. Moreover, the drug did not leach and it was in the yolk, where it would contact the intraovum pathogen. This encouraging result indicated that further studies to evaluate the procedure for injecting brood stock are clearly warranted. Evelyn, T. P. T., L. ProsperiPorta, and J. E. Ketcheson. 1986b. Experimental intraovum infection of salmonid eggs with Renibacterium salmoninarum and vertical transmission of the pathogen with such eggs despite their treatment with erythromycin. Dis. Aquat. Org. 1:197202. Studies with eggs of steelhead and coho salmon showed that the eggs can be infected internally with R. salmoninarum by immersing them before fertilization and waterhardening into coelomic fluid containing large numbers of R. salmoninarum cells. These results supported the hypothesis that, in vivo, salmonid eggs acquire their intraovum R. salmoninarum infections from heavily infected coelomic fluid that surrounds them after their release into the body cavity. Whether eggs are also infected early in oogenesis is not known. Intraovum infection rates were low (3.55.0%) and did not increase when eggs were fertilized while still immersed in the infected coelomic fluid. These and other data lead to the conclusion that the male salmonid plays a relatively unimportant role in vertical transmission. Waterhardening of experimentally infected coho salmon eggs in erythromycin did not appear to affect the intraovum infection rate, and the fry that hatched from the treated eggs carried the pathogen. Evelyn, T. P. T., L. ProsperiPorta, and J. E. Ketcheson. 1986c. Persistence of the kidneydisease bacterium, Renibacterium salmoninarum, in coho salmon, Oncorhynchus kisutch (Walbaum), eggs treated during and after waterhardening with Povidoneiodine. J. Fish Dis. 9:461464. Unfertilized eggs from a fish with a natural intraovum R. salmoninarum infection were immersed in an iodine-povidone solution (250 ppm iodine) at Ph 7.0 for 15 min or 2 h after waterhardening, or for 1 or 3 h during water-hardening. Half the treated eggs were incubated in flowing water (1012°C) to detect perivitelline membrane damage and the rest were examined for intraovum R. salmoninarum. All eggs survived treatment well except those treated for 2 h during waterhardening. Infection rates were similar in all treatment groups (3.37.6%). None of the treatments reduced the risk of vertical transmission of R. salmoninarum. Fiedler, F.,and R. Draxl. 1986. Biochemical and immunochemical properties of the cell surface of Renibacterium salmoninarum. J. Bacteriol. 168:799804. The biochemical composition of the cell envelope of R. salmoninarum was investigated in 13 strains isolated from different salmonid species at various locations in the United States, Canada, and Europe. Both peptidoglycan and the cell wall polysaccharide were marked similarly to the type strain R. salmoninarum ATCC 33209. The purified cell wall polysaccharide showed antigenic activity with antiserum obtained by immunization of rabbits with heatinactivated trypsinized cells of R. salmoninarum. Immunoblotting experiments with nontrypsinized cell walls and antisera against R. salmoninarum cells revealed that antigenic proteins were attached to the cell walls. Fryer, J. L., and J. E. Sanders. 1981. Bacterial kidney disease of salmonid fish. Annu. Rev. Microbiol. 35:273298. A comprehensive review of BKD-its history, etiological agent, epizootiology, pathology, diagnosis, and control. Getchell, R. G., J. S. Rohovec, and J. L. Fryer. 1985. Comparison of Renibacterium salmoninarum isolates by antigenic analysis. Fish Pathol. 20:149159. Antigens of seven isolates of R. salmoninarum were compared serologically by using immunoelectrophoretic mobilities in each antigenic profile. One of these common components, designated antigen F, was partly purified by ammonium sulphate precipitation and gel filtration. Cross adsorption analysis showed antigen F to be the major surface antigen of R salmoninarum; it was heat stable, and its estimated molecular weight was 57,000. Goodfellow, M., T. M. Embley, and B. Austin. 1985. Numerical taxonomy and amended description of Renibacterium salmoninarum. J. Gen. Microbiol. 131:27392752. Numerical taxonomic techniques based on 86 unit characters were used to compare 44 strains of R. salmoninarum and 12 representative cultures of Actinomyces, Arthrobacter, Lactobacillus, Microbacterium, Micrococcus, Planococcus, Rothia, and Listeria denitrificans. An amended description of R. salmoninarum was developed, based on the numerical data and the results of recent chemical and microbiological studies. Groman, D. B. 1983. Studies examining the identification, epizootiology and control of Renibacterium salmoninarum infections in chinook salmon (Oncorhynchus tshawytscha). Ph.D. thesis, University of Idaho, Moscow. 159 pp. The use of counterimmunoelectrophoresis to identify precipitating antigen and antibody in spring chinook salmon infected with R. salmoninarum was examined, as well as the effects of screening six specific organ systems on the efficiency of the direct FAT to identify R. salmoninarum carriers. The chronological distribution of the pathogen in carriers for BKD epizootics during both freshwater and initial seawater rearing was described and the efficacy of erythromycin phosphate therapy and prophylaxis in the control of BKD was evaluated. Hendricks, J., and S. L. Leek. 1975. Kidney disease postorbital lesions in spring chinook salmon (Oncorhynchus tshawytscha). Trans. Am. Fish. Soc. 104:805807. Histological description of BKD in which bacteria were found predominantly in postorbital tissues. Kettler, S., C. PfeilPutzien, and R. Hoffman. 1986. Infection of grayling (Thymallus) with the agent of bacterial kidney disease (BKD). Bull. Eur. Assoc. Fish Pathol. 6:6971. Fifteen of 20 grayling died within 8 months after injection of R. salmoninarum. All infected fish were anemic and had pale empty guts with hemorrhages, and large swollen kidneys. None of 20 fish placed in contact with the experimentally infected grayling developed clinical signs, although the bacterium was isolated from the kidneys of five of them. Renibacterium salmoninarum was also isolated from two of five wild grayling from a river in which wild trout and trout in a connecting fish farm had BKD. Kimura, T., Y. Ezura, K. Tajima, and M. Yoshimizu. 1978. Serological diagnosis of bacterial kidney disease of salmonid (BKD): immunodiffusion test by heat stable antigen extracted from infected kidney. Fish Pathol. 13:103108. An immunodiffusion test with heat stable antigen extracted from the kidney of infected fish is described as a method of BKD diagnosis. Specific precipitin bands were formed within 24 h when the rabbit antiserum reacted with the extract of affected parts of the kidney containing heat stable specific BKD antigen. No precipitin bands were formed with heat extract of the kidney of healthy fish or of cells of other fish pathogenic bacteria, such as Aeromonas salmonicida or Vibrio anguillarum. Kimura, T., and M. Yoshimizu. 1981. Rapid method for detection of bacterial kidney disease of salmonid (BKD) by coagglutination of antibody sensitized protein Acontaining staphylococci. Bull. Jpn. Soc. Sci. Fish. 47:11731183. A coagglutination test, in which staphylococci specifically sensitized with antibody against R. salmoninarum was used for diagnosis, proved to be simple, rapid, and reliable for use in the laboratory or field and required no special apparatus. It was highly specific and more sensitive than the immunodiffusion test. Klontz, G. W. 1983. Bacterial kidney disease in salmonids an overview. Pages 177200 in D. P. Anderson, M. Dorson, and P. H. Dubourget, eds. Antigens of fish pathogens. Collection Foundation Marcel Merieux, Lyon, France. A comprehensive discussion of BKD that includes characteristics of the bacterium, epidemiology, pathology, immunology, diagnosis, and control. Lall, S. P., W. D. Paterson, J. A. Hines, and N. J. Adams. 1985. Control of bacterial kidney disease in Atlantic salmon, Salmo salar L., by dietary modification. J. Fish Dis. 8:113124. The effects of dietary treatments were investigated as a prophylactic measure to reduce the occurrence and severity of BKD infections in Atlantic salmon at Margaree Fish Culture Station, Nova Scotia, Canada. Six diets containing various levels of calcium, magnesium, zinc, iron, copper, manganese, cobalt, and iodine, as well as one commercial diet, were fed to postyearling Atlantic salmon in two consecutive experiments. Natural infection was used to examine the effects of each diet on the prevalence of BKD. Diet containing high levels of iodine (4.5 mg/kg feed) and fluorine (4.5 mg/kg) reduced the BKD rate to 3 and 5%, respectively, compared to 95 and 38% with commercial feed. Some other experimental diets also reduced prevalence of BKD, but to a lesser degree than the diet with additional iodine and fluorine. Lehmann, J., and D. Mock. 1985. Identification of bacterial kidney disease in Westphalia. Fischwirtschaft 35:2122. Bacterial kidney disease was identified in fry of rainbow trout in November 1984 in North RhineWestphalia (Federal Republic of Germany); R. salmoninarum was abundant in the kidneys of fish showing exophthalmus, dark skin pigmentation, and anemia. McCarthy, D. H., T. R. Croy, and D. F. Amend. 1984. Immunization of rainbow trout, Salmo gairdneri Richardson, against bacterial kidney disease: preliminary efficacy evaluation. J. Fish Dis. 7:6571. A bacterin for immunization of salmonid fishes against BKD is described. Cultures were grown in Evelyn's KDM2 medium containing 10% calf serum in a fermenter under the following conditions: Ph 7.2, temperature 15°C, air 8 mL/min, agitation 200 revolutions/min, and incubation 515 days. Possible substitutes for calf serum were 10% horse serum, 0.15% starch, and leptospira medium. The bacterins were inactivated with 0.3% formalin; no adjuvants were used. A Phlysed bacterin, 50% concentrated bacterin, and 50% concentrated Phlysed bacterin were evaluated in other tests. Juvenile rainbow trout were vaccinated by intraperitoneal injection, 2min immersion, or twostep hyperosmotic infiltration. Fish were held for 4 to 6 weeks at 11 °C. then challenged by intraperitoneal injection with the homologous virulent bacterium. Fish died 19-40 days after challenge. The best preparation was pHlysed bacterin given by a single injection; hyperosmotic and immersion vaccination were not effective. Typically, 80% of unvaccinated controls and 10% of the vaccinated fish were infected (as detected by Gram stain). Mitchum, D. L., and L. E. Sherman. 1981. Transmission of bacterial kidney disease from wild to stocked hatchery trout. Can. J. Fish. Aquat. Sci. 38:547551. Natural horizontal transmission of BKD from infected wild brook trout to newly stocked hatchery brook trout, brown trout, and rainbow trout was shown in a small lake and stream system in southeastern Wyoming. Stocked trout died in 9 months or less after exposure to infected wild fish. Dead and live fish collected from each of three stations were necropsied. Fluorescent antibody techniques were used to detect the BKD organism in all samples. Lowseverity infections were often detected by FAT at a higher rate when feces, rather than kidney tissues, were examined. Because other known pathogens were essentially lacking, BKD was diagnosed as the cause of all deaths in both stocked hatchery fish and wild fish. The rainbow trout was the most refractory species. Mitchum, D. L., L. E. Sherman, and G. T. Baxter. 1979. Bacterial kidney disease in feral populations of brook trout (Salvelinus fontinalis), brown trout (Salmo trutta), and rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can. 36: 13701376. Prevalence and effects of BKD were determined in trout in a small lake and stream system in southeastern Wyoming, where yearly epizootics were observed in 197278. During 1976, dead fish collected at 3 upstream stations and 60 live fish collected from each of 11 stations were necropsied, and virological, bacteriological, and parasitological examinations were conducted by standard methods. All of the dead brook trout collected were diagnosed as having BKD; prevalence in live fish ranged from 83% at an upstream station to only 3 % at the most downstream location, and was highest in brook trout and lowest in rainbow trout. Two longnose suckers (Catostomus catostomus), the only nonsalmonids collected, were negative for BKD. Clinical signs of infection were found only in brook trout, the species with the most severe infections. Five agegroups of feral brook trout were involved in the epizootics. Because other known pathogens were essentially absent, all deaths were probably due to BKD. Relations between susceptibility to BKD, and species, agegroups, water chemistry, water temperatures, and certain ecological conditions are discussed. Ochiai, T., W. T. Yasutake, and R. W. Gould. 1985. Direct fluorescent antibody technique for the detection of bacterial kidney disease in paraffinembedded tissues. Fish Pathol. 19:271272. Direct FAT was successfully used to detect the causative agent of BKD, in fixed and paraffinembedded egg and tissue sections. This method was superior to the use of the Gram stain and could be particularly useful in detecting the BKD organism in fish with lowgrade infections. Pascho, R. J., and D. Mulcahy. 1987. Enzymelinked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Can. J. Fish. Aquat. Sci. 44:183191. A doubleantibody enzymelinked immunosorbent assay (ELISA) for detection of a soluble fraction of R. salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. When antigen was present, absorbances were higher when Tween 80 was in the wash buffer than when Tween 20 was used. Background absorbance did not change when Tween 80 was added to the wash buffer, but increased when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37°C and 16 h at 4°C. Antigen attachment to antibody coated microplate wells depended more on incubation temperature than on duration; a 3h incubation at 25°C was adopted. Conjugate incubation for longer than 1 h at 37°C or 3 h at 25°C resulted in unacceptable background levels. No crossreactions resulted from heatextracted antigens of 10 other species of bacteria. The optimized ELISA is a 6h test that enables detection of levels of soluble antigen as low as 2 to 20 mg. Paterson, W. D., D. Desautels, and J. M. Weber. 1981. The immune response of Atlantic salmon, Salmo salar L., to the causative agent of bacterial kidney disease, Renibacterium salmoninarum. J. Fish Dis. 4:99111. Both underyearling and older parr of Atlantic salmon produced high agglutinating antibody titers in response to a single intraperitoneal injection of killed R. salmoninarum cells emulsified in Freund's complete adjuvant. Little or no response was observed in animals injected with cells in saline or in animals vaccinated by hyperosmotic immersion. Immunological duration was insufficient in fish vaccinated as underyearling parr to provide protective immunity 2 years later, when the fish had become smolts. Older Atlantic salmon parr injected with cells in Freund's complete adjuvant had fewer lesions than did control fish when both were observed as smolts 1 year after vaccination. Paterson, W. D., C. Gallant, D. Desautels, and L. Marshall. 1979. Detection of bacterial kidney disease in wild salmonids in the Margaree River system and adjacent waters using an indirect fluorescent antibody technique. J. Fish. Res. Board Can. 36:14641468. The indirect fluorescent antibody technique demonstrated the enzootic nature of BKD in 224 of 605 wild salmonids sampled from the Margaree River system and some adjacent waters. Asymptomatic infections were observed in 33% of 456 Atlantic salmon parr and 35% of 37 returning adults in samples taken from the Margaree River downstream to and including saltwater areas off the coast. Paterson, W. D., S. P. Lall, and D. Desautels. 1981. Studies on bacterial kidney disease in Atlantic salmon (Salmo salar) in Canada. Fish Pathol. 15:283292. Renibacterium salmoninarum was widespread in trout and salmon sampled over a 20year period from freshwater streams and saltwater sites on Cape Breton Island, Nova Scotia, Canada. Although BKD is ubiquitous in this area, major mortalities have been reported from only a few isolated locations. Heavy losses have occurred, however, when infected fish were acclimated to seawater before their saltwater phase of life. Vaccination and nutrition were examined as possible prophylactic methods of reducing the occurrence and severity of BKD. In vaccination trials, yearling Atlantic salmon parr that received a 0.1mL intraperitoneal injection of formalinkilled cells emulsified in Freund's complete adjuvant showed an elevated agglutinating antibody response and almost complete absence of lesions in the kidneys. In the nutritional studies, a reduction in the incidence of infections with lesions was observed in a July sample in fish fed diets with a high concentration of trace elements or a low calcium content. Sanders, J. E., and R. M. J. Barros. 1986. Evidence by the fluorescent antibody test for the occurrence of Renibacterium salmoninarum among salmonid fish in Chile. J. Wildl. Dis. 22:255257. Infection with R. salmoninarum was demonstrated in salmonid stocks at hatcheries in Chile in 198385. All Chilean stocks were from eggs imported after the early 1970's. Sanders, J. E., and J. L. Fryer. 1980. Renibacterium salmoninarum gen. nov., sp. nov., the causative agent of bacterial kidney disease in salmonid fishes. Int. J. Syst. Bacteriol. 30:496502. The bacterium causing BKD was considered a single species that should be placed in a new genus, on the basis of the guanine plus cytosine ratio of DNA, cell wall sugar composition, and amino acid composition of the peptidoglycan cell wall layer. Sanders, J. E., K. S. Pilcher, and J. L. Fryer. 1978. Relation of water temperature to bacterial kidney disease in coho salmon (Oncorhynchus kisutch), sockeye salmon (O. nerka), and steelhead trout (Salmo gairdneri). J. Fish. Res. Board Can. 35:811. The effects of seven temperatures (3.9 to 20.2°C) on BKD were tested. Juvenile coho salmon and rainbow trout were most sensitive to R. salmoninarum at 6.7 to 12.2°C. As temperature increased, mortality declined; at 20°C the disease was suppressed. Temperature had no apparent effect on sockeye salmon, which sustained essentially 100% mortality at temperatures of 6.7 to 20.2°C. Smith, I. W. 1964. The occurrence and pathology of Dee disease. Freshwater Salmon Fish. Res. 34:112. A comprehensive description of Dee disease (BKD) in Atlantic salmon in Scotland. External and internal clinical signs are described and illustrated in color. The influence of temperature on host membrane formation is discussed. Isolation procedures and some characteristics are given. Specker, J. L., and C. B. Schreck. 1980. Stress responses to transportation and fitness for marine survival in coho salmon (Oncorhynchus kisutch) smolts. Can. J. Fish. Aquat. Sci. 37:765-769. Smolting coho salmon were transported at low and high densities (12 and 120 g/L) for short and long periods (4 and 12 h). Because smolts can be transplanted directly to seawater, half the fish in each treatment were transported to tanks containing seawater and half to tanks containing freshwater. Plasma corticosteroids and glucose were elevated at unloading in all groups, and corticosteroids were still above the resting level 24 h later. Potential smoltification indicators such as plasma thyroxin concentration and gill (Na+/K+)ATPase activity were not affected by transportation. Increased corticosteroids were correlated to increased mortality in transported salmon compared to acclimated control fish. The various transport regimes did not increase the expression of latent bacterial kidney disease. Suzumoto, B. K., C. B. Schreck, and J. D. McIntyre. 1977. Relative resistances of three transferrin genotypes of coho salmon (Oncorhynchus kisutch) and their hematological responses to bacterial kidney disease. J. Fish. Res. Board Can. 34:18. Juvenile salmon of three transferrin genotypes (AA, AC, and CC) were experimentally infected with the causative agent of BKD and mortalities were observed. Six experimental and control groups were used: bacteriainfected + no iron, bacteriainfected + low iron; bacteriainfected + high iron; saline control; highiron control; and lowiron control. In all experimental groups, the AA genotype was the most susceptible and CC the most resistant to BKD. Addition of exogenous iron did not appear to increase pathogenicity. Wedemeyer, G. A. 1976. Physiological response of juvenile coho salmon (Oncorhynchus kisutch) and rainbow trout (Salmo gairdneri) to handling and crowding stress in intensive fish culture. J. Fish. Res. Board Can. 33:26992702. Changing the loading density of coho salmon 4 to 5 in. long, held in soft (20 ppm CaCO3) water, from a relatively light 0.5 lb to 1.2 or 4.0 lb/ft3 caused stress as indicated by loss of feeding behavior, but only minor physiological disturbances, as indicated by lack of hyperglycemia or hypochloremia. Increasing the density to 6 or 12 lb/ft3 caused physiological stress that required at least a week for recovery. Smolting coho salmon were physiologically stressed by population densities of I lb/ft3 or more and a subclinical kidney infection was activated. Rainbow trout (45 in. long) were physiologically stressed when moved and held at I lb/ft or more but retained normal feeding behavior. This stress indicated that handling and crowding stress can be reduced in softwater areas if densities in fish distribution trucks or in ponds or raceways during disease treatments are held to 0.10.5 lb/gal. Winter, G. W., C. B. Schreck, and J. D. McIntyre. 1980. Resistance of different stocks and transferrin genotypes of coho salmon, Oncorhynchus kisutch, and steelhead trout, Salmo gairdneri, to bacterial kidney disease and vibriosis. U.S. Natl. Mar. Fish. Serv., Fish. Bull. 77:795802. Juvenile coho salmon and steelhead of different stocks and three transferrin genotypes (AA, AC, and CC), all reared in identical or similar environments, were experimentally infected with the causative agent of BKD or with Vibrio anguillarum. Mortality due to the pathogens was compared among stocks within a species and among transferrin genotypes within a stock to determine whether there was a genetic basis for resistance to disease. Differences in resistance to BKD among coho salmon stocks had a genetic basis, whereas stock susceptibility to vibriosis was strongly influenced by environmental factors. Coho salmon or steelhead of a particular stock were sometimes resistant to one disease but susceptible to the other. The importance of the transferrin genotype of coho salmon in resistance to BKD was stock specific; in stocks that showed differential resistance of genotypes, AA was the most susceptible. No differences in resistance to vibriosis were observed among transferrin genotypes. Wolf, K., and C. E. Dunbar. 1959. Test of 34 therapeutic agents for control of kidney disease in trout. Trans. Am. Fish. Soc. 88:117124. In vitro tests of 16 strains of the BKD bacterium were tested for their sensitivity to 34 chemotherapeutic agents. The results aided in selecting 11 agents for in vivo trials. Recommendations for therapy with erythromycin were given. Wolke, R. E. 1975. Pathology of bacterial and fungal diseases affecting fish. Pages 33116 in W. E. Ribelin and G. Migaki, eds. The pathology of fishes. University of Wisconsin Press, Madison. A concise review and description of the pathology of BKD. Young, C. L., and G. B. Chapman. 1978. Ultrastructural aspects of the causative agent and renal histopathology of bacterial kidney disease in brook trout (Salvelinus fontinalis). J. Fish. Res. Board Can. 35:12341248. The ultrastructural changes in renal tissues of naturally and artificially infected brook trout are described and reported to be similar to those occurring in mammalian renal disease.

U.S. Department of the Interior || U.S. Geological Survey 11649 Leetown Road, Kearneysville, WV 25430, USA URL: http://www.lsc.usgs.gov Maintainer: lsc_webmaster@usgs.gov Last Modified: February 26, 2004 dwn Privacy Policy and Disclaimers || FOIA || Accessibility