In response to critically low levels of winter run Chinook salmon in the Sacramento River during 1991, a captive broodstock program was initiated at the Bodega Marine Laboratory (University of California) in cooperation with the Coleman National Fish Hatchery (CNFH) and several state and federal agencies. Now in its fifth year, the project has reached a significant achievement with the production of gametes from this endangered species and subsequent supplementation of the propagation program at CNFH. However, a systemic protist, the rosette agent, continues to be isolated from multiple year classes of the captive broodstock, and it has contributed to morbidity and mortality in sub adult and adult fish. Recent evidence suggests that the organism first infects these anadromous fish while in freshwater, but infestation and disease progress until the time of spawning. In 1995, workers at the California-Nevada Fish Health Center conducted a survey of over 220 adult salmonids from the Upper Sacramento River for the rosette agent (Scott Foott, pers. comm.). Both histological specimens of various tissues and stained kidney imprints from adult Chinook comprising 3 different Sacramento River Chinook stocks (fall, late-fall, and winter run) were examined. The rosette agent was detected in 25 of 78 kidney imprints (32%) of adult late-fall run Chinook as well as histological specimens from these fish, although no obvious signs of infection were observed in these post-spawning adults. The parasite has not been detected in either fall run or winter run Chinook adults in 1994 or 1995. Despite the identification of the parasite in other stocks of Chinook in the Sacramento River, movement of infected winter run Chinook adults from the captive broodstock program remains restricted to reduce the risk of spreading the disease. The parasite is susceptible to iodophor at 100 ppm, so surface disinfection of eggs has been used to allow for the movement of eggs from the captive broodstock program to CNFH.

Histological specimens of infected winter run Chinook reveal systemic dissemination of the rosette agent. The parasite can be found in a variety of cell types: hemopoietic, epithelial, and mesenchymal. Two populations of the rosette agent can be detected in an infected fish; one population is composed of small, round, eosinophilic organisms 2-4 microns in diameter, while the other is comprised of larger, round, eosinophilic organisms 4-6 microns in diameter. Affected kidney tissue is characterized by widespread, marked tubular necrosis, membranous glomerulonephritis, and necrotizing interstitial nephritis. Multifocal hepatocellular necrosis with multifocal granuloma formation is evident. The parasite is found both intra - and extracellularly within the tissues of most visceral organs, often forming focal aggregates of organisms. In advanced infections, the rosette agent can be detected in subserosal aggregates in the swim bladder, and may be associated with granulomatous meningitis. In early infections (subadult fish) the rosette agent can be found within vessels of the gill. In the blood, intra monocytic protozoa are found and this may be evidence of vascular dissemination of organisms. Finally, the parasite is evident within biliary and renal tubular epithelium and is identified intraluminally within bile ductules and renal tubules; this suggests that bile and urine are routes of excretion and potential sources of horizontal transmission.

To evaluate the relative susceptibility of other salmonids to infection by the rosette agent? Five species: Chinook (Oncorhynchus tshawytscha), Coho (O. kisutch), rainbow trout (O. mykiss), brook trout (Salvelinus fontinalis), and brown trout (Salmo trutta), were exposed to in vitro cultures of the rosette agent by intraperitoneal injection. Fish were challenged with 1.6 x 10⁷ organisms in 0.1 mL of minimal essential media (MEM) while fish injected with MEM alone served as a control. Fish were sacrificed at 6 months post-exposure, and both stained kidney imprints and histological specimens were examined for the presence of the parasite. Prevalence was estimated by counting the number of rosette agent parasites in 40 fields of stained kidney imprints at 1250x magnification. All of the Chinook salmon were infected with the rosette agent, with 220.5 ± 47.4 (mean ± SE) parasites detected. Ninety-eight percent of the Coho were infected, with 53.2 ± 8.1 parasites counted. The parasite was detected in 42.5% (2.2 ± 0.8) and 43.3% (2.1 ± 0.6) of the rainbow trout and brown trout sampled, respectively. The rosette agent was detected in only 0.03% (0.2 ± 0.2) of the brook trout. Prevalence varied with the species tested, and pathological changes associated with the presence of the parasite were only detected in the Chinook and Coho salmon. Focal aggregates of the parasite could be detected in renal, hepatic, and splenic tissue of both the infected Chinook and Coho salmon. Most notably, challenged fish of these species suffered multifocal hepatocellular necrosis. In the other species, the parasite was often found within melano macrophages, suggesting limited spread or perhaps clearance of the parasite.

A similar organism to the rosette agent found in the captive winter run Chinook has been reported from other salmon species as well as other Chinook populations (Harrell et al.1986, Hedrick et al. 1989). Our recent sequence analyses offer compelling evidence that the rosette agent isolated from the captive winter run Chinook and the other two isolates are the same organism. Evidence of these natural outbreaks, along with the results of experimental infection of Chinook and Coho salmon, suggest that the rosette agent is a significant pathogen of salmon, but that trout species may be at a lower risk of infection. While the parasite remains detectable 6 months post-infection, the susceptibility of the rainbow, brook, and brown trout to disease appears limited. However, the potential role of these species as carriers of the pathogen remains unknown.

References

1.  Harrell, L.W., Elston, R.A., Scott, T.M., and M.T. Wilkinson. 1986. A significant new systemic disease of net-pen chinook salmon (Oncorhynchus tshawytscha) brood stock. Aquaculture 55:249-262.

2.  Hedrick, R.P., Friedman, C.S., and J. Modin. 1989. Systemic infection in Atlantic salmon Salmo salar with a Dermocystidium-like species. Diseases of Aquatic Organisms 7: 171 177.