A Fish Mystery

Between 1966 and 1975, more than one million young steelhead smolts from Oregon coastal hatchery stock were released into Oregon's Willamette River system. No returning adults were ever recorded. At the time, no one really knew why.

In retrospect, most researchers now agree that Ceratomyxa shasta, a microscopic parasite, killed the steelhead stocked in the Willamette three decades ago. And they know C. shasta has probably caused the demise of millions more wild and stocked salmon and trout over the past century in the Pacific Northwest.

For more than three decades researchers in OSU's Department of Microbiology and the Oregon Department of Fish and Wildlife (ODFW) have worked together, with funding from the OSU Agricultural Experiment Station, the Oregon Sea Grant program, the Bonneville Power Administration, the U.S. Fish and Wildlife Service and other agencies, to learn more about Ceratomyxa shasta. Native to some Pacific Northwest watersheds from Northern California to British Columbia, C. shasta continues to have profound impacts on salmon and trout management in the Pacific Northwest.

OSU microbiologist Jerri Bartholomew with a trout killed by the microscopic parasite she's studying. Photo: Lynn Ketchum

Despite 30-some years of study, scientists were stumped about the basic biology and life cycle of the tiny parasite-until OSU microbiologist Jerri Bartholomew and her colleagues solved the mystery.

Since its discovery by scientists in northern California in 1948, certain basic questions about C. shasta lay largely unanswered for many years. Where did the organism reside when it wasn't living in salmon and trout? Why did it fail to spread from fish to fish or drainage to drainage as do other diseases? Why was it found in certain rivers such as the Willamette and Deschutes, but not the Siletz? Why was it only in some areas within a river? Why were some fish stocks from certain areas more susceptible to infection than others? Was the resistance of fish to C. shasta due to genetics or acquired immunity, or both?

C. shasta has a strangely complicated life cycle. No one has yet been able to raise it through its complete life cycle in the laboratory. And until the life cycle of an organism can be completed in the lab, it's almost impossible to develop control strategies or to predict where the parasite may exist or spread.

The key piece of the life cycle puzzle lay on the river bottom. It resided in a bed of freshwater mussels. On the algae that grows on mussels and rocks lives a tiny wormlike organism called a polychaete. In only one of every hundred polychaetes is found a tiny microorganism that unlocked the half-century-long mystery. No wonder it took almost five decades to find it.

Research assistant Don Stevens gets ready to immerse a trout in the Willamette River. The fish will be kept at OSU's Salmon Disease Laboratory to see if it's infected by Ceratomyxa shasta, a microscopic parasite. This is one way to learn if the parasite is in a steam. Photo: Lynn Ketchum

This is the story of how OSU microbiologists and their colleagues at ODFW solved a difficult biological mystery. They used old-fashioned patience, perseverance and teamwork, combined with new tools in immunobiology and molecular genetics.

The story begins in 1948, when Ceratomyxa shasta was discovered when massive numbers of trout died at a newly completed fish hatchery near Mt. Shasta in northern California. This "new" organism infected the intestine of the fish and was released from the fish as a spore. Scientists assumed, based on its spore structure, that C. shasta belonged to the "myxosporean parasites," which include the organism that causes whirling disease in salmon and trout. Myxosporeans are now thought to fit somewhere between the jellyfish and primitive worms in the evolutionary scheme of things.

By the 1960s, researchers detected Ceratomyxa shasta in Oregon. Juvenile coho salmon at the Bonneville Hatchery on the Columbia River, as well as juvenile chinook and steelhead at Pelton Dam on the Deschutes River and returning adult spring chinook at Dexter Dam on the Middle Fork of the Willamette River, were succumbing to C. shasta. Eventually C. shasta was discovered in parts of Washington, Idaho and British Columbia as well as northern California and Oregon, in both adult and young fish, and in both hatchery and wild trout and salmon.

"What was intriguing is that C. shasta was found only in certain areas of the Pacific Northwest, including parts of the Klamath, Rogue, Columbia and Fraser River drainages," explained Bartholomew, who has studied C. shasta for the past 16 years. She earned master's and doctoral degrees working on the parasite with John Fryer, former chair of OSU's Department of Microbiology, now a professor emeritus in the department.

"C. shasta does not spread easily outside its geographic range," said Bartholomew. "Within this region, the areas where fish actually became infected were limited. And we believe the range of the parasite hasn't changed much with time. C. shasta has probably always been here, but we only recognized it when we started moving fish between watersheds."

Nor does the disease spread from fish to fish.

Watch out for flying fish: Bob Dahlberg of the Oregon Department of Fish and Wildlife's Roaring River Fish Hatchery near Scio stocks trout in Roaring River. The Ceratomyxa shasta parasite causes more problems for non-native, than native, fish. Some Oregon streams contain the parasite. Others don't. And only certain stretches of some rivers have the parasite. For decades researchers at OSU and elsewhere couldn't figure out why. Photos: Lynn Ketchum

"Each fish is an individual target-each has to come in contact with the parasite," she continued. "They can't catch it from each other like in an infectious disease such as the flu or a cold."

This lack of mobility also became an important clue. By the late 1960s, scientists began to suspect that something else must be involved in the life cycle of C. shasta, something that was relatively immobile in the rivers where fish were getting sick. Researchers suspected an "intermediate host"-that there was another yet unknown organism or stage to C. shasta's life cycle.

"Originally, we thought maybe the parasite might have to 'age' in the mud on river bottoms for a while before it would infect trout or salmon," recalled Fryer. "We'd bring in mud, add spores and allow them to 'age' before exposing fish to them. But we couldn't infect fish. However, when sediments were brought from a lake where the parasite existed, fish became infected. We started looking around for a second living or intermediate host."

In 1984, another big clue came along. Another research group, working on whirling disease, had found an intermediate host-an aquatic "oligochaete" worm that hosted another, previously unknown stage in the life cycle of whirling disease. This parasite lived part of its life in fish and another part in an oligochaete worm. It needed to go through all stages to complete a generation before it could infect another fish.

The OSU researchers thought C. shasta might have a similar life history, going through a second spore stage in an intermediate host. Don Stevens, a research assistant in OSU's Department of Microbiology, brought items such as mud, rocks and fresh water mussels-basically any substrate that might support aquatic worms-into the lab. He exposed fish in tanks to these materials.

Eureka! When researchers incubated fish in tanks containing mussels, the fish became infected with C. shasta.

"We knew the other previously unknown stage of C. shasta was present," said Bartholomew. "But we didn't know what the other life stage looked like or what organism it lived in.

OSU's Don Stevens gathers mussels and other river bottom materials from a stretch of the Willamette River that contains the Ceratomyxa shasta parasite. Photo: Lynn Ketchum

"We set up a tank with live mussels and filtered the water to search for the infectious stage of C. shasta," she recalled. "We saw parts of oligochaetes. So, for 12 months we looked at oligochaetes, searching for possible life stages of C. shasta."

Bartholomew and her colleagues hit a dead end. They had no luck infecting fish using oligochaetes as intermediate hosts. So they began to search again, this time for something that might be smaller.

They broadened their search to other invertebrates. And finally, they observed a tiny polychaete worm infected with spores similar to those seen in other myxosporean life cycles. As these tiny spores were released from the worm, they collected them and fed them directly to a fish.

For about three months they waited anxiously, checking daily to see if the test fish were still alive. One deserted day in the lab over Christmas break, Bartholomew and technician Margo Whipple found the fish dead.

"I never thought I could be excited by a fish dying," Bartholomew recalled. "We examined the fish and the intestine was full of C. shasta spores. After 16 years of looking for the alternate host of C. shasta, I was really thrilled! We celebrated over champagne."

The final proof that the polychaete was the intermediate host for C. shasta came by using genetic probes on the tiny spores the polychaete contained.

"The only way we could be sure the parasite in the fish and in the polychaete were the same was to compare their DNA," said Bartholomew.

She and her colleagues sequenced a gene from both spore stages. Indeed, they were one and the same species-Ceratomyxa shasta.

"This long search is a good example of how much work it sometimes takes to make scientific breakthroughs," she said. "It is not always easy or quick."

Bartholomew and her colleagues are anxious to put their knowledge of C. shasta's life cycle to work. They are learning to raise the polychaete in the lab and are trying to develop ways of treating the disease in salmon and trout.

"Knowing the complete life cycle may offer insights into ways of controlling C. shasta, and understanding the biology of the polychaete may help us predict where the parasite may exist or spread," she said.

After decades, OSU researchers discovered that algae, which grows on mussels like this, and on rocks and other river-bed materials, was home for a tiny wormlike organism that's an "intermediate host" for Ceratomyxa shasta. Photo: Lynn Ketchum

After 16 years of frustrating study, Bartholomew is still captivated.

"This organism is exquisitely adapted to both its hosts, the salmonid fish and the polychaete worm," she said, excitedly. "No one knows how these life cycles evolved, but I find it really intriguing."

The work on Ceratomyxa by OSU and ODFW researchers is helping fish biologists make better decisions when stocking hatchery fish in Oregon streams and rivers.

"Back in the good old days, we used to move fish from outside a river drainage all around through our hatchery programs," said ODFW research biologist Dave Buchanan. "It was a classic case of naiveté. We didn't consider the life history and evolutionary biology of local stocks. Ceratomyxa is a good example of why local stocks are important. You can't just introduce any old stock from one stream into another and expect good results. Once you lose your local stocks, you lose your evolutionary history, including resistance to things such as Ceratomyxa."

Although there is still no "cure" for the disease, fisheries biologists in the Pacific Northwest are learning to manage around it. They now consider whether salmon or trout are resistant or susceptible to C. shasta before they introduce them into a river or stream where the parasite lives.